AU2024203121B1 - Validation of natural matrices for therapeutic use on humans, animals and plants on bio-physical bases as an alternative to validation through chemically definable substances derived from said matrices by isolation and/or synthesis processes and also as an alternative to products comprising natural matrices validated on the basis of their traditional use. - Google Patents
Validation of natural matrices for therapeutic use on humans, animals and plants on bio-physical bases as an alternative to validation through chemically definable substances derived from said matrices by isolation and/or synthesis processes and also as an alternative to products comprising natural matrices validated on the basis of their traditional use. Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
#$%^&*AU2024203121B120250626.pdf#####
97
VALIDATION OF NATURAL MATRICES FOR THERAPEUTIC USE ON
HUMANS, ANIMALS AND PLANTS ON BIO-PHYSICAL BASES AS AN
ALTERNATIVE TO VALIDATION THROUGH CHEMICALLY DEFINABLE
SUBSTANCES DERIVED FROM SAID MATRICES BY ISOLATION AND/OR
SYNTHESIS PROCESSES AND ALSO AS AN ALTERNATIVE TO PRODUCTS 5
COMPRISING NATURAL MATRICES VALIDATED ON THE BASIS OF THEIR
TRADITIONAL USE.
ABSTRACT
The present invention relates to a new method for defining acceptability values
of the spectroscopy or spectrophotometry spectra for the compliance validation of 10
batches by means of spectroscopy or spectrophotometry analysis of therapeutic or
beneficial products, the products comprising or consisting of one or more natural
matrices. The invention also relates to a new process for the compliance validation of
one or more batches of said products.
97
VALIDATION OF NATURAL MATRICES FOR THERAPEUTIC USE ON
HUMANS, ANIMALS AND PLANTS ON BIO-PHYSICAL BASES AS AN
ALTERNATIVE TO VALIDATION THROUGH CHEMICALLY DEFINABLE
SUBSTANCES DERIVED FROM SAID MATRICES BY ISOLATION AND/OR
5 SYNTHESIS PROCESSES AND ALSO AS AN ALTERNATIVE TO PRODUCTS
COMPRISING NATURAL MATRICES VALIDATED ON THE BASIS OF THEIR
TRADITIONAL USE.
ABSTRACT
The present invention relates to a new method for defining acceptability values
10 of the spectroscopy or spectrophotometry spectra for the compliance validation of
batches by means of spectroscopy or spectrophotometry analysis of therapeutic or
beneficial products, the products comprising or consisting of one or more natural
matrices. The invention also relates to a new process for the compliance validation of
one or more batches of said products.
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Description
VALIDATION OF VALIDATION NATURAL MATRICES OF NATURAL MATRICES FOR FOR THERAPEUTIC THERAPEUTIC USE USEONON HUMANS, ANIMALS HUMANS, ANIMALS AND AND PLANTS PLANTS ON ONBIO-PHYSICAL BIO-PHYSICAL BASES BASES AS AS AN AN ALTERNATIVE TO ALTERNATIVE VALIDATION THROUGH TO VALIDATION CHEMICALLY DEFINABLE THROUGH CHEMICALLY DEFINABLE SUBSTANCES DERIVED SUBSTANCES DERIVED FROM FROMSAID SAIDMATRICES MATRICESBYBY ISOLATIONAND/OR ISOLATION AND/OR 5 5 SYNTHESIS PROCESSES SYNTHESIS PROCESSES AND AND ALSO AS AN ALSO AS ALTERNATIVE TO AN ALTERNATIVE TO PRODUCTS PRODUCTS COMPRISING NATURAL COMPRISING NATURAL MATRICES MATRICES VALIDATED VALIDATED ONBASIS ON THE THE BASIS OF THEIR OF THEIR TRADITIONALUSE. TRADITIONAL USE. 2024203121
Thepresent The presentinvention inventionrelates relates to to aa new newmethod methodforfor defining defining acceptabilityvalues acceptability values 10 10 of the of the spectroscopy spectroscopyororspectrophotometry spectrophotometry spectra spectra for compliance for the the compliance validation validation of of batches bybymeans batches means of spectroscopy of spectroscopy or spectrophotometry or spectrophotometry analysisanalysis of therapeutic of therapeutic or or beneficial products, beneficial products, the the products productscomprising comprising or consisting or consisting of orone of one or natural more more natural matrices. Products matrices. Productscomprising comprising or consisting or consisting of oneof orone ornatural more more matrices natural matrices are are characterised by characterised bythe thefact factthat thatthethe matrix matrix or matrices or matrices themselves themselves show distinctive show distinctive
15 15 emerging emerging properties, properties, i.e.i.e. properties properties that that are different are different from represented from those those represented by the sumby the sum
of the of the properties properties of of their their individual individual components. The components. The invention invention also also relates relates to to a new a new
process for process forthethecompliance compliance validation validation (batches (batches compliance compliance control control in in industrial industrial
production processes production processesof of the the product) product) of of one or more one or batchesof more batches of said said products. products.
The present The present invention invention relates relates to to new batch control new batch control methods methodsfor forproducts products 20 20 comprisingororconsisting comprising consistingof of a natural a natural matrix matrix or mixtures or mixtures of vegetal of vegetal matrices, matrices, said said products being products beingcharacterised characterisedbybythethefact factthat thatthe thematrix matrixorormatrices matricesthemselves themselvesshowshow
distinctive emerging distinctive propertiesdifferent emerging properties differentfrom fromthose those represented represented by sum by the the of sumthe of the
propertiesofoftheir properties theirindividual individual components. components.
STATE OF STATE OF THE THE ART ART 25 25 Thehistorical The historical concept conceptunder underwhich which patents patents are are granted granted for for the the benefit benefit of of the the public,particularly public, particularlyin inmatters matters of health of health and safety, and safety, has rootshasthat roots datethat backdate back centuries. centuries.
Theunderlying The underlyingprinciple principleis isthat, that,while while patents patents provide provide inventors inventors with with a temporary a temporary
monopoly monopoly on their on their creations, creations, the ultimate the ultimate aim isaim is to the to serve serve the greater greater good of good of society. society.
In contemporary In contemporarytimes,times,thisthishistorical historical concept conceptisisreflected reflectedinin various variouslegal legalprovisions provisions 30 30 and policies and policies that that govern patents. It govern patents. It underscores underscores the the understanding understandingthat that while whileinventors inventors deserve recognition deserve recognitionand andprotection protectionforfortheir theircontributions, contributions,society societyasasa awhole whole should should
ultimatelybenefit ultimately benefit from from these these innovations, innovations, particularly particularly in areasin areas critical critical to publicto public health health
andsafety. and safety. In other In other words, words,the thehumanitarian humanitarianbasis basisof ofthethepatent patent system system lieslies in in itsits goalto to goal
35 35 strike aa balance strike betweenfostering balance between fosteringinnovation innovation andand ensuring ensuring that that the benefits the benefits of that of that
innovationareare innovation shared shared for for the betterment the betterment of society of society as aInwhole. as a whole. In particular, particular, the patent the patent
2 10 May 2024
systemshould system shouldensure ensurea aknowledge knowledge sharing sharing and and the the promotion promotion of progress of progress for scope for the the scope indicated above. indicated above.
In particular, In particular, the the patent patent system can play system can playa acrucial crucialrole rolein inaddressing addressing humanitarianand humanitarian andglobal globalchallenges. challenges.For Forinstance, instance,itit can can incentivize incentivize the the development developmentofof 5 5 sustainable medicines, sustainable medicines,environmentally environmentally sustainable sustainable technologies, technologies, and solutions and solutions for for pressing issues like clean energy and water scarcity. pressing issues like clean energy and water scarcity.
Fromthe From thebeginning beginningof of thethe 16th 16th century century until until today, today, paritcularlyininthethefield paritcularly fieldofof medicalandandbeneficial beneficial products, products, has has been beenpossible possibletoto standardise, standardise, andand hence hencetotovalidate validate 2024203121
medical
only artificial only artificial substances substancesproduced produced with with chemically definable alchemical chemically definable alchemical processes. processes. 10 10 This path, This path, which whichalthough althoughvery very reductionisthashas reductionist proven proven to beto be of great of great value, value, allowing allowing
manydiseases many diseasestotobebeeradicated eradicatedininthethepast past5 5centuries, centuries,isisnow nowencountering encountering itsits limits, limits,
whichderive which derivefrom fromthe theextraneous extraneousnature natureofofchemical chemicalsubstances substancestotovital vital processes. processes. Concerningthe Concerning thedevelopment development of of newnew sustainable sustainable medicaments, medicaments, it isit now is now alsoalso ascertained ascertained
that artificial that artificial (in (in particular, particular, chemically chemicallysynthesised) synthesised) therapeutical therapeutical products products are are 15 15 generating harmful generating harmfulimpacts impactsononbiodiversity biodiversityand andnative nativeimmune immune systems. systems.
It is It is well knownthat well known that synthetic synthetic APIs APIs can enter can enter ecosystems ecosystems throughthrough various various routes, primarily through routes, throughthe thedischarge dischargeofofpharmaceutical pharmaceutical waste waste from from manufacturing manufacturing
plants and plants andimproper improper disposal disposal of unused of unused or expired or expired medications. medications. This canThis lead can to lead to
bioaccumulationof of bioaccumulation artificialandand artificial poorly poorly biodegradable biodegradable substances substances in aquatic in aquatic and and 20 20 terrestrial organisms, terrestrial potentially organisms, potentially disrupting disrupting food food chainschains and threatening and threatening biodiversity. biodiversity.
Studies have Studies shownadverse have shown adverseeffects effects on on aquatic aquatic organisms, organisms, such such asas altered altered behaviour, reproduction, behaviour, reproduction,and andeven even mortality, mortality, because because of exposure of exposure to synthetic to synthetic APIs.APIs.
(Boxall, A. (Boxall, A. B.B. etet alal (2012). (2012). Pharmaceuticals Pharmaceuticals and and personal personal carecare products products inin the the environment:what environment: what areare thethe bigbig questions?. questions?. Environmental Environmental health health perspectives, perspectives, 120(9), 120(9),
25 25 1221-1229); 1221-1229); Fick, Fick,J.,J., &&Lindberg, Lindberg, R. R. H. H. (2015). (2015). Tysklind, Tysklind, M. Larsson, M. and and Larsson, D. G. D.J. G. J.
(2015). Predicted (2015). Predictedcritical criticalenvironmental environmental concentrations concentrations for 500forpharmaceuticals. 500 pharmaceuticals. RegulatoryToxicology Regulatory Toxicology andand Pharmacology, Pharmacology, 73(1),73(1), 607-616.) 607-616.)
It isiswell It wellknown known thatthat many syntheticAPIs many synthetic APIsarearedesigned designedtotobebebiologically biologicallyactive active andparticularly and particularlystable, stable,which, which,as aasresult, a result, can can hinder hinder theirtheir natural natural degradation degradation processes. processes.
30 30 Consequently, these Consequently, these molecules molecules persist persist in in the the environment environment for for extended extendedperiods, periods, potentially accumulating potentially accumulatingininsoils soilsandand waters. waters. ThisThis reduced reduced biodegradability biodegradability raises raises
concerns about concerns aboutlong-term long-termenvironmental environmental impacts impacts and and the the potential potential for for bioaccumulation bioaccumulation
in organisms in organisms[Kasprzyk-Hordern,
[Kasprzyk-Hordern, B., B., et alet (2008). al (2008). The removal The removal of pharmaceuticals, of pharmaceuticals,
personal care personal careproducts, products,endocrine endocrine disruptors disruptors and illicit and illicit drugs drugs during during wastewaterwastewater 35 35 treatment and treatment and its its impact impact on on the the quality quality ofof receiving receiving waters. waters. Water research, 43(2), Water research, 43(2), 363- 363- 380; Verlicchi P., 380; Verlicchi P., etet alal (2012). (2012).Occurrence Occurrence of pharmaceutical of pharmaceutical compounds compounds in urbanin urban
wastewater:Removal, wastewater: Removal, mass mass load load andand environmental environmental riskrisk after after a secondary a secondary treatment—A treatment-A
review. Science review. Scienceof of the the total totalenvironment, environment, 429,429, 123-155]. 123-155].
3 10 May 2024
Furthermore,there Furthermore, thereisis growing growingconcern concern about about the the potential potential effects effects of of synthetic synthetic
APIson APIs onhuman humanandand animal animal immune immune systems. systems. Some Some pharmaceuticals pharmaceuticals have have been beentofound found to interfere with interfere with immune activity, either immune activity, either directly directly or or indirectly, indirectly,leading leadingtotoaltered altered immune immune
responses or responses or increased increased susceptibility susceptibility to infections. This to infections. This cancan have havesignificant significant 5 5 implications for implications for both both individual individualhealth healthandandpopulation-level population-level immunity immunity [Vos [Vos T. et T. al et al
(2016). Global,regional, (2016). Global, regional,and andnational national incidence, incidence, prevalence, prevalence, and and yearsyears lived lived with with
disability for disability for 310 310diseases diseasesandand injuries, injuries, 1990–2015: 1990-2015: a systematic a systematic analysis analysis for the Global for the Global
BurdenofofDisease DiseaseStudy Study2015. 2015.TheThe Lancet, 388(10053), 1545-1602; Calabrese, E. &J., & 2024203121
Burden Lancet, 388(10053), 1545-1602; Calabrese, E. J.,
Baldwin,L.L.A.A.(2003). Baldwin, (2003).Toxicology Toxicology rethinks rethinks itsits centralbelief. central belief. Nature, Nature, 421(6924), 421(6924),691- 691- 10 10 692]. 692].
In conclusion, In conclusion, while while synthetic synthetic APIsAPIshave have undoubtedly undoubtedly contributed contributed to to advancementsininhealthcare, advancements healthcare,their their environmental environmentaland andhealth healthimpacts impactsshould should be be carefully carefully
considered. Efforts considered. Efforts toto develop developgreener greenerpharmaceuticals, pharmaceuticals, improve improve wastewaste management, management,
and monitor and monitorenvironmental environmental contamination contamination are crucial are crucial stepssteps towards towards mitigating mitigating these these
15 15 concerns. concerns. In addition, In addition, itit has has to to be be noted that, while noted that, while being chemicallyanalogous being chemically analogousto to their their
synthetic counterparts if taken in isolation, natural molecules within a natural matrix are synthetic counterparts if taken in isolation, natural molecules within a natural matrix are
likely toto possess likely possessdistinct distinct fingerprints fingerprints with with respect respect to theirto synthetic their synthetic analogues,analogues, due to due to the totally the totally different different synthetic synthetic pathway pathwayin in terms terms of primary of primary metabolites, metabolites, reactants, reactants,
20 20 reaction temperatures, reaction temperatures,energy energy sources, sources, catalysts catalysts etc., etc., potentially potentially influencing influencing theirtheir
physicochemical physicochemical behaviour behaviour and reactivity, and reactivity, thereforetherefore their biological their biological function.function.
Accordingtotothe According theconventional conventionalparadigm, paradigm, fromfrom a chemical-structural a chemical-structural viewpoint, viewpoint,
the identity the identity of of a a molecule molecule is is embedded embedded in its in its atomic atomic composition composition andgeometric and the the geometric arrangementthereof. arrangement thereof.ByBywayway of of example, example, estragol estragol (1-allyl-4-methoxybenzene), (1-allyl-4-methoxybenzene), which which
25 25 in nature in nature is is prominently identified in prominently identified in essential essentialoils such oils such asasthose thosederived derived from fromOcimum Ocimum
basilicum and basilicum andArtemisia Artemisiadracunculus dracunculus is is known known in thein the art art forforitsitspotential potentialaromatic aromaticand and medicinal application. medicinal application.The The molecular molecular constitution constitution and associated and associated energyenergy states ofstates of estragol, contingent estragol, contingent upon upon its its origin, origin, havehave been been a subject a subject of robust of robust scientific scientific deliberation. deliberation.
Whiletraditional While traditional perspectives perspectives postulate postulate uniform uniformmolecular molecular attributes,aamore attributes, morerigorous rigorous 30 30 scrutiny suggests scrutiny suggests nuanced differences. nuanced differences.
Giventhis Given thispremise, premise,estragol, estragol,whether whether procured procured from botanical from botanical sourcessources via via distillation oror synthesized distillation synthesized in in laboratory confines, should laboratory confines, shouldideally ideallybebecongruent congruent in in itsits
inherent physicochemical inherent attributes. physicochemical attributes.
However,it's However, it's paramount paramounttotodistinguish distinguishbetween between thethe pathways pathways of production. of production. In In
35 35 botanical matrices, botanical matrices, biosynthesis biosynthesis ofofestragol estragolisis orchestrated orchestratedbybya aseries seriesofofenzymatic enzymatic reactions, commencing reactions, commencing withwith primary primary metabolites, metabolites, and culminating and culminating in this specific in this specific
secondary secondary metabolite. metabolite. It isIt known is knownin theinart thethat art that each each of these of these enzymatic enzymatic transformations transformations
operates within operates within aa distinct distinct energy energylandscape, landscape,potentially potentiallyconferring conferringtotothethemolecule molecule a a
4 10 May 2024
unique energy state. unique energy state.
Conversely,the Conversely, thelaboratory laboratorysynthesis synthesisofofestragol estragolhinges hingeson on chemical chemical reactions reactions
steered by steered by different different precursors precursors and andconditions conditions(such (suchas as temperatures temperatures not not compatible compatible
with the with the life lifeofofa aplant). plant). The energy The energydynamics dynamics of of such such synthetic synthetic routes, routes,governed governed by the by the
5 5 thermodynamics thermodynamics and kinetics and kinetics intrinsic intrinsic to the to the reactions, reactions, arelikely are highly highlytolikely deviatetofrom deviate from the plant-mediated the enzymaticpathways. plant-mediated enzymatic pathways. In addition, In addition,itit isis evident evidentthat thatalso alsothetheisotopic isotopic abundances abundances resulting resulting from from the two the two
different pathways pathways(natural (naturalandand synthetic) are are unlikely to betothebesame. the Isotopic same. Isotopic 2024203121
different synthetic) unlikely
abundances,even abundances, evenifif subtly subtly varied, varied, areare known known to toexert exert tangible tangible influences influences on on vibrational vibrational 10 10 frequencies,bond frequencies, bond strengths, strengths, and and consequentially, consequentially, the states the energy energyofstates of the molecule the molecule itself itself
[Bigeleisen,
[Bigeleisen, J. J. (1996). Nuclearspin (1996). Nuclear spinconversion conversion in in polyatomic polyatomic molecules. molecules. Journal Journal of of ChemicalPhysics, Chemical Physics,105(18), 105(18),8121-8129]. 8121-8129]. Given Given the likely the likely isotopic isotopic disparities disparities between between
botanical sources botanical sources andandsynthetic syntheticreagents, reagents, the the resultant resultant estragol estragol molecules molecules are are likely likely toto harbourdifferential harbour differential energy energy imprints imprints and biological and biological activities. activities. In the In the light of light of the above, the above,
15 15 while being while beingchemically chemicallyanalogous, analogous, molecules molecules from from naturalnatural and synthetic and synthetic originsorigins are are reasonably likely to possess distinct energetic fingerprints, potentially influencing their reasonably likely to possess distinct energetic fingerprints, potentially influencing their
physicochemicalproperties, physicochemical properties,reactivity reactivity andandtherefore thereforetheirtheirbiological biologicalfunction. function.Indeed, Indeed, the difference the differencebetween between the the activity activity of synthetic of synthetic and natural and natural estragolestragol has beenhas been in reported reported in the art the art (Suzanne (SuzanneM.F. M.F. et al. et al. “Basil "Basil extractextract inhibits inhibits the sulfotransferase the sulfotransferase mediated mediated
20 20 formation of formation of DNA DNA adducts adducts of the of the procarcinogen procarcinogen 1′-hydroxyestragole l'-hydroxyestragole by ratby and rat human and human liver S9 liver S9 homogenates homogenates and and inin HepG2 HepG2 humanhuman hepatoma hepatoma cells" cells” FoodFood and Chemical and Chemical Toxicology,2008, Toxicology, 2008,4646(6)(6)2296-2302, 2296-2302,https://doi.org/10.1016/j.fct.2008.03.010.). https://doi.org/10.1016/j.fct.2008.03.010.). In addition, In addition, synthetic synthetic molecules molecules(intended (intended as molecules as molecules obtained obtained through through a a production carried production carried out out by by manmanthrough through chemical chemical synthesis synthesis laboratory/industrial laboratory/industrial 25 25 processes)arearedesigned processes) designed in order in order to provide to provide the desired the desired interaction interaction with a given with a specific specific given target molecule, target said design molecule, said designnot nottaking takingintointoaccount accountallallthe theinteractions interactionsthat thatthe thesaid said moleculemay molecule mayandand willhave will have within within a a naturalmatrix, natural matrix,withwiththetheenvironment, environment,andand with with the the
wholereceiving whole receivingnetwork networkofofthe theorganism organismininwhich whichtheythey willbebeused. will used.OnOn thethe otherhand, other hand, nativebiosynthesised native biosynthesised molecules, molecules, being being produced produced in natural,in non-artificial natural, non-artificial settings, willsettings, will 30 30 intrinsically carry intrinsically carry all all the the essential essential features features to to exist exist and exert their and exert their functions functions in in anan epigenetically determined epigenetically determined contestcontest whose whosedescription descriptionis isinaccessible inaccessiblewhen when a a conventionaldeterministic conventional deterministic chemical chemicalapproach approachisisused. used.AApossible possibledeciphering decipheringofofnatural natural matrices and matrices and biosynthesised biosynthesisedmolecules moleculesmay may be be provided provided applying applying quantum quantum biology. biology.
Products obtained Products obtainedfromfromnatural naturalsources sourceshave havebeen beenused used forforthousands thousands of of yearstoto years
35 35 prevent and prevent andcure curehuman human diseases. diseases. In In thisthis context, context, manymany studies studies havehave been been limited limited to to characterizing their characterizing theirchemical chemical composition composition at at thethe monomolecular monomolecularlevel levelandand thethe monomolecularactivities, monomolecular activities, while while the thespontaneous spontaneousassemblies, assemblies,interactions interactionsandand supramolecularorganisation supramolecular organisationofofallallthe thecomponents components in said in said natural natural products products have have not not
5 10 May 2024
been fully been fully investigated investigated and thus understood. and thus understood. Since Since the the development developmentofofmodern modern chemistry, the chemistry, the reductionist reductionist approach focusingononthe approach focusing theisolation isolation of of single single molecules from molecules from
natural products natural products and andthethesubsequent subsequent artificialsynthesis artificial synthesis of of molecules molecules of therapeutic of therapeutic
interest, the aim has been to develop selected active principles that act on a given target interest, the aim has been to develop selected active principles that act on a given target
5 5 following the following the key-lock key-lock paradigm. paradigm. Thishad This hadledledto tothethe conviction conviction that that research research in thein the field field of life ofsciences life sciences was to bewas to be aimedatatsubstances aimed substances thatthatcan can be chemically be chemically validated, validated, with datawith such data such as quantities as quantities of the of the individual substances substancesatata amolecularmolecular level, generatingveryvery powerful and effective 2024203121
individual level, generating powerful and effective
artificial products, artificial products, withwithlinear linear dynamics. dynamics. It is Itnow is becoming now becoming evident evident that that this direction this direction
10 10 is also is alsogenerating generating harmful harmful impacts on biodiversity impacts on biodiversity and native immune and native systems. immune systems.
Theinability The inabilitytotostandardise standardise products products deriving deriving from natural from natural sources, sources, including,including, in in particular, products particular, products that that areare prepared prepared by byhumans, humans, butbut thatthat consist consist mainly mainly or only or only of of componentsoriginating components originatingfrom from natural natural raw raw (starting) (starting) sources sources i.e.i.e. that that consist consist 100%100% of of non-artificial matter, non-artificial matter, has has been oneofofthethemajor been one major difficultiesforfortechnology, difficulties technology, thereby thereby
15 15 openingthe opening thedoors doorsto to thethe current current API-based, API-based, pharmacological pharmacological approach approach in orderin to order to
ensure the ensure the batch batch to to batch batch validation validation of of products deriving from products deriving fromnatural naturalsources sourcesintended intended for medical or beneficial application. for medical or beneficial application.
By way By wayofofexample, example,natural naturalmatrices, matrices, suchsuch as as plant plant matrices, matrices, are are complex complex systems characterized systems characterized by by many many molecular molecular components components belonging belonging to different to different 20 20 phytochemicalclasses phytochemical classesthat thatinteract interactwith witheach each other other already already in the in the plantplant in order in order to to determinethe determine theplant's plant’s biology. biology. ThisThisinteraction interaction continues continuesalso also in in the the processing processingphases phases and different and different processing processingtechniques techniques affect affect the the post-processing post-processing interactions interactions of said of said components.These components. These compounds compounds can interact can interact at the functional at the functional and structural and structural level. level. Supramolecular aggregates Supramolecular aggregates asaswell well as their as their chemical-physical chemical-physical and structural and structural 25 25 characteristics that characteristics result in that result in both both structural structural andandfunctional functional networks networks are dynamic are dynamic
interactions and interactions and can can bebe modulated modulatedbybyenvironmental environmental conditions conditions and,and, as one as one can can expect, expect,
theseinteractions these interactionsaffect affectthethe reactivity reactivity of the of the individual individual components components and,thethrough and, through SO the so called "matrix called “matrix effect”, effect", result result in properties in properties typical typical of the of the distinct distinct entity represented entity represented by by the matrix the matrix and andare aredifferent differentfrom fromthethe sumsum of properties of the the properties of itsof single its single molecular molecular
30 30 components.Such components. Such properties properties areare defined defined as “emerging as "emerging properties”. properties". This This phenomenon phenomenon
hasbeen has beendescribed describedand and attributed attributed specifically specifically to living to living matter,matter, which haswhich a drivehastoaself- drive to self- assembleand assemble andself-organize self-organizetotoform formsupramolecular supramolecular complex complex entities entities [Jean-Marie
[Jean-Marie Lehn Lehn
Towardcomplex Toward complex matter: matter: Supramolecular Supramolecular chemistry chemistry and self-organization. and self-organization. PNAS,PNAS, 2002, 2002,
99 (8) 99 (8) 4763-4768 4763-4768 https://doi.org/10.1073/pnas.072065599]. https://doi.org/10.1073/pnas.072065599] This inherent This inherent complexity complexity
35 35 leads to leads to the the fact factthat thatindividual individualmolecules molecules within within aa natural naturalmatrix matrix cannot cannot be be considered considered
as to as to be be contained contained in in isolated isolated andand fixed fixed packages, packages, as as mutual mutualnon-covalent non-covalentand and dynamic dynamic
interactions continuously interactions continuouslyoccur occur between them. Such between them. Suchinteractions interactions are are intra- intra- andand intermolecular and intermolecular andoccur occurbothboth among among molecules molecules of theofsame the type sameastype well asas well amongas among
6 10 May 2024
moleculesbelonging molecules belongingtotodifferent different chemical chemicalclasses. classes. This Thisintroduces introducesthe theneed needtotoconsider consider that the that the ability ability of of aa natural natural matrix matrix to to exert exert aa therapeutic therapeutic activity activity on on the the human body human body
dependsnot depends notonly onlyononthethequali-quantitative quali-quantitativecomposition composition of the of the matrix, matrix, which which is byis its by its ownnature own natureprone proneto tobebe variable variable perper se,se, butbut also also on on thethe presence presence of such of such interactions interactions
5 5 betweensame between same andand different different molecules, molecules, including including small small molecules molecules as well asas well more as more
complexones complex onessuch suchasasproteins, proteins,polysaccharides, polysaccharides,lipids, lipids, RNA, etc. RNA, etc.
In this In this context, context, the the classical classical validation validation of of therapeutic therapeutic products productsbased basedon on the the
pharmacologicalrelationship relationshipbetween between structure andand activity (SAR) which is theis most the most 2024203121
pharmacological structure activity (SAR) which
relevant relationship relevant relationshipininclassical classical pharmacological pharmacological activities activities betweenbetween an activean active
10 10 pharmaceuticalingredient pharmaceutical ingredient(API)(API)and and the receptor the receptor targeted targeted by said byAPI, saidwhich API,is which is considered considered at at thethe level level of single of single molecules, molecules, is unlikely is unlikely to be respected to be respected across different across different
batchesofofthe batches thesame same natural natural matrix. matrix.
These considerations These considerations appearappear toto profoundly profoundlydistinguish distinguish the thestudy studyof ofthethe interaction between interaction between a aself-assembled self-assemblednatural naturalmatrix matrix andand a biological a biological system, system, imbuedimbued 15 15 with complexity with complexityatatthe themolecular molecularand andsupramolecular supramolecular level, level, fromfrom the the interaction interaction which which
wouldbebeestablished would establishedbybyan an APIAPI and and its target its target receptor receptor cellular cellular structure.In In structure. fact,the fact, the latter isisunequivocally latter unequivocally defined by the defined by the exquisitely exquisitely deterministic deterministic canons canonsofofthe thekey-lock key-lock mechanismbybyfixing, mechanism fixing,also alsoand andabove above allall in in structural terms structural termsdefined definedininboth botha a quantitative and quantitative qualitative manner and qualitative mannerthetheinteraction interactionbetween between the the API API andspecific and its its specific 20 20 target. This target. conceptisisSOsopervasive This concept pervasive fromfrom a conceptual a conceptual point point of view ofthat view it that then it then
translates into translates intothe thepossibility possibilityofofcontrolling controlling the the reproducibility reproducibility of theofbiological the biological functionfunction of the of the API APIthrough through thethe solesole control control of of thethe reproducibility reproducibility of its of its molecular molecular structure structure
basedonontheir based their structure-activity structure-activity relationship relationship (SAR).(SAR). This is This is evidently evidently not applicable not applicable to to natural matrices natural matrices ororproductsproductscomprising comprising them,them, due toduethetocharacteristics the characteristics discussed discussed
25 25 above. above.
It therefore It therefore appears appears necessary necessary to to note note fundamental fundamentaldifferences differencesbetween between natural natural
self-assembled matrices and self-assembled matrices andAPIs: APIs: - the - the first, first, which are eubiotic which are eubiotic withwithrespect respecttotoman manandand the the environment, environment, are are
characterized bybya aphysiological characterized physiological interconnection interconnection at theat molecular the molecular level with levelthewith the 30 30 receiver’s biological receiver's systemand, biological system and,precisely preciselybybyvirtue virtueofoftheir theircomplexity, complexity, areare clearly clearly
inappropriately described inappropriately described and andcharacterised characterisedthrough throughthe theuse useofofdeterministic deterministictoolstoolssuch such as the as the key-lock key-lock model;model;
- the - the latter, latter,whichwhich are arexenobiotes xenobiotes withwith respect respect toto man andthe man and theenvironment, environment, areare
instead characterized instead characterized by bythetheclear clearpossibility possibility ofofdescribing describingtheirtheirinteraction interactionwithwiththe the 35 35 receiver’s biological receiver's biological systemsystem according according to totally to totally deterministic deterministic canons,canons, typically typically summarizedbyby summarized thethe"key "keyandand lock" lock" model. model.
Therefore, knowledge Therefore, knowledge of of thetheidentity identityand andamount amount of of each each andand everyevery molecule molecule in in
a natural a naturalmatrix matrixisisnot notsufficient sufficientto to predict predict the the dynamic dynamic and kinetic and kinetic properties, properties, as well asas well as
7 10 May 2024
the therapeutic the therapeutic effectiveness, effectiveness, of of the the matrix matrix itself. itself.The Theopposite opposite happens whenselected happens when selected single molecules, single molecules, such as APIs, such as APIs, are are considered, considered, whereby whereby the the SAR, SAR, the the pharmacodynamic pharmacodynamic and pharmacokinetic and pharmacokinetic properties properties are intrinsically are intrinsically related related to the to the chemicalidentity chemical identity ofof the theactive activeprinciple, principle, andandtotothe thepharmacodynamic pharmacodynamic inertia inertia of the of the
5 5 excipients. For excipients. this reason, For this reason, the canonical concepts the canonical concepts ofof pharmacodynamics pharmacodynamics and and pharmacokineticsmake pharmacokinetics makesensesense solely solely whenwhen referring referring to a single to a single molecule molecule (the active (the active
principle), or principle), oraa representative representative thereof thereof (a functional (a functional marker). marker).
The network network established established among amongall all components componentsofofthe thematrix matrix yielding yielding "the “the 2024203121
The matrix effect" matrix effect” makes makesitit impossible impossibletotoidentify identify aa single single marker markerasasrepresentative representativeofofthethe 10 10 network, because network, becausenonosingle singlecomponent component is capable is capable of conveying of conveying alonealone all the all the properties properties
specific to specific to the the matrix, matrix, since since no single component no single componentreflects reflectsthe theinteraction interactionbetween between thethe
matrixand matrix andthethe target target living living organism. organism.
Thematrix The matrixeffect effect confers confersthe the specific specific and and unique uniqueproperties propertiesofofthe the matrix matrixitself itself or of or of aa mixture mixtureofofmatrices matrices that that result result in in a new a new different different matrix, matrix, calledcalled emergent emergent
15 15 properties, which properties, cannotbebereconducted which cannot reconducted to the to the properties properties of any of any of components of the the components taken inin isolation. taken isolation. This Thisperfectly perfectlyreflects reflects the theimpossibility impossibilitytotocorrectly correctlystudy study such such
properties through properties through deterministic deterministic chemical chemical methods, commonlyused methods, commonly used in in classical classical pharmacologicalchemistry, pharmacological chemistry, which, which, as said as said above, above, are well are well adequate adequate only only for for single single
active principles and excipients in pharma settings. active principles and excipients in pharma settings.
20 20 Rather, the Rather, the dynamic dynamicandand kinetic kinetic behaviour behaviour of matrix of the the matrix is the is result the result of theof the dynamicnetwork dynamic network ofof interactionstaking interactions takingplaceplacewithin withinthethe matrix, matrix, showing: showing: • the the presence presence of of aa great great number number of of components, components, • the the inability inability to to reconduct reconductthe theproperties propertiesofofthethe matrix matrix to the to the sumtheof the sum of
propertiesofofthe properties thesingle singlesubstances substances 25 25 • the the impossibility impossibility totodescribe describethetheinteraction interactionbetween between the matrix the matrix and the and the
receiving organism receiving according toto the organism according thekey-lock key-lockparadigm paradigm (model), (model), which which is the is the foundationof foundation of SAR. SAR. At present, At present, human beingsare human beings arebecoming becomingaware awarethat that thethe response response to to most most problemslies problems lies inin nature natureitself, itself, and that there and that there isis the the need need ofof developing developingprocesses processesandand 30 30 methodsthat methods thatallow allowtotounderstand, understand,and andtherefore thereforesomehow somehow standardise, standardise, natural natural complex complex
entities self-organizing entities self-organizing their theirsupramolecular supramolecular networks, networks, suchsuchasas natural natural matrices. matrices. These These entities are entities are thethe only onlyonesones physiologically physiologically compatible compatible with everything with everything that forms that forms
creation. Therefore, creation. Therefore, the the need needofofmoving moving fromfrom the Enlightenment-reductionist the Enlightenment-reductionist based based
validation to validation to aa probabilistic probabilistic approach approachinspired inspiredto tothethe latestevolutions latest evolutions in scientific in scientific
35 35 thinking, thus thinking, eventually opposing thus eventually opposing linear linear dynamics dynamicstotocircularcircular dynamics. dynamics.TheThe deterministic chemical deterministic chemicalapproach approach is is thereforenotnotadequate therefore adequate to investigate to investigate andand monitor monitor
matrix properties matrix properties and andquality. quality. Approaches Approaches thatassess that assessthetheinteractions interactionswithin withincomplex complex systemsareare systems necessary, necessary, to allow, to allow, as required, as required, the identification the identification of the relevant of the features features relevant
8 10 May 2024
for the reproducibility of the therapeutic properties of the matrix. for the reproducibility of the therapeutic properties of the matrix.
Hencethe Hence theneed needtototurn turn to to approaches inspired by approaches inspired by Systems SystemsTheory. Theory. Thestudy The studyofofsuchsuchproperties, properties,previously previouslynotnotachievable, achievable,requires requirestools toolssuch suchas as the “omics” the sciences, among "omics" sciences, amongwhich which transcriptomics transcriptomics andand metabolomics metabolomics can can be be identified identified
5 5 as of as of pivotal pivotalimportance. importance. Concerningproducts Concerning products comprising comprising or consisting or consisting of natural of natural matrices, matrices, havinghaving a a therapeutic effect, therapeutic effect, ititisisherein hereinreminded reminded that thatthe theMedical Medical Device (MD) Device (MD) EU EU Regulation Regulation
2017/745 (Regulation) (Regulation) was wasofficially officially published published inin Europe EuropeMayMay on 2017 5th, 2017 2024203121
2017/745 on 5th,
[REGULATION (EU) (EU) 2017/745 2017/745OFOF THETHEEUROPEAN EUROPEAN PARLIAMENT PARLIAMENT AND ANDOF OFTHETHE 10 10 COUNCIL COUNCIL of 5ofApril 5 April 20172017 on medical on medical devices, devices, amending amending Directive Directive 2001/83/EC, 2001/83/EC, Regulation (EC) Regulation (EC) No No178/2002 178/2002andand Regulation Regulation (EC) (EC) No 1223/2009 No 1223/2009 and repealing and repealing Council Directives Council Directives 90/385/EEC 90/385/EEC and and 93/42/EEC] 93/42/EEC]and andintroduced introduceda acompletely completelynewnew governance governance into into all all aspects aspects of the of the lifecycle lifecycle of a of MD.a MD.
The term The term Medical MedicalDevice, Device,according accordingtotothe the Regulation, Regulation, comprises comprises products products 15 15 whichdodoachieve which achievea atherapeutic therapeuticeffect effectbutbutnot notwith witha apharmacological, pharmacological, immunological, immunological,
or metabolic or (Ph.IM)mode metabolic (Ph.IM) modeof of action action (MOA). (MOA). The Ph.I.M The Ph.I.M MOA is MOA is the the mode mode of of action action characterized by characterized by aa key-lock key-lockmodelmodelwherewherethethe selected selected APIAPI obeys obeys the the rulesrules of SAR of SAR and and
acts on acts on its itstarget targetreceptor. receptor. Thus, Thus,products productscomprising comprising or or consisting consisting of of complex systems complex systems
such as such as natural natural matrices matrices cancan comply comply with with thethe Regulation. Regulation. In In particular,the particular, theRegulation Regulation 20 20 also indicates also indicates thatthat aa product product whichwhichmodifies modifies a pathological a pathological or physiological or physiological statestate or or
process through process throughaa non-Ph-IM non-Ph-IM mechanism mechanism of action of action is aisMD. a MD. Therefore, the Therefore, the Regulation Regulationposes poses a double a double problem problem to betosolved: be solved: on oneon one side, side,
MDsconsisting MDs consistingofofmaterials materialsofofnatural naturalorigin, origin, such such as as plant plant matrices matrices and andthethe like, like, need need
to undergo to undergo a quality a quality control control validation validation in order in order to be available to be available for therapy, for therapy, on the other on the other
25 25 side, side, there there is is the the need need to to demonstrate demonstratethat thatthethetherapeutic therapeuticeffecteffectofofsaidsaidproducts productsis is
achieved by achieved bymeans meansofofnon-Ph-IM non-Ph-IM mechanisms mechanisms of action. of action.
Thecurrent The currentvalidation validationofofproducts products forfor therapeutic therapeutic use,use, based based exclusively exclusively on on reproducibility of reproducibility of their their chemical chemicalcomposition, composition, is is applicable applicable exclusively exclusively to products to products
acting with acting with aa Ph-IM mechanisms Ph-IM mechanisms of of action. action.
30 30 Althoughtaking Although takingininaccount accountthethecomplexity complexity of the of the natural natural matrices, matrices, thethe present present
state of state of the the art art is is based based on onthethevalidation validation of products of products comprising comprising or consisting or consisting of natural of natural matricesforforthethetreatment matrices treatment of aof a pathological pathological condition condition (i.e., a (i.e., producta product falling falling within thewithin the
definition of definition of MD accordingtotoEUEU MD according Regulation) Regulation) at atthethechemical chemical level level as as forAPIs. for APIs.This Thisisis in striking in striking contrast contrastwithwiththethe intimate intimate nature nature of such of such products products for allfortheall the reasons reasons provided provided
35 35 above.There above. Thereis is hence hence the the need need in theinart thetoart to develop develop validation validation proceduresprocedures that are based that are based
on the on the acknowledgement acknowledgement of of saidsaid complexity complexity and and thatthat arearenotnot limited limited to to theevaluation the evaluationofof the reproducibility the reproducibility of of such such products based uniquely products based uniquelyonontheir theirmolecular molecularcomposition. composition. In In
addition,the addition, thepresent present state state of of thethe artart doesdoes not not provide provide methodsmethods for the assessment for the assessment of the of the
9 10 May 2024
mechanism mechanism of of actionofofsaid action saidproduct. product. There is at present a strongly felt need in finding alternatives to the traditional There is at present a strongly felt need in finding alternatives to the traditional
pharmacology pharmacology butbut at at thesame the same timetime guaranteeing guaranteeing the the validation validation and and standardization standardization of of
products, comprising products, comprisingororconsisting consisting of of one one oror more morenatural naturalmatrices, matrices, used usedforfor therapeutic therapeutic 5 5 purposes. purposes. Theprovision The provisionofofmethodsmethods validating validating the the quality quality of a of a product product comprising comprising or or consisting of consisting of natural natural matrices matricesthat that are are not notbased basedon onthethe mere mere chemical chemical composition composition
thereof, would allowthe theuseuseinintherapy therapyofofproducts productsofofnatural naturalorigin, origin, having havingaacircular circular 2024203121
thereof, would allow
dynamic dynamic rather rather than than a linear a linear one,one, i.e.,i.e., acting acting on theonoverall the overall physiological physiological state altered state altered by by 10 10 a pathological a pathological condition conditionratherratherthanthanonona single a single alteration.This alteration. Thiswould would result result in the in the
developmentofofnewnew development research research fields fields and and in the in the possibility possibility of using of using a whole a whole networknetwork (such as (such as aa natural natural matrix) matrix) in intherapy, therapy,said saidnetwork network operating operating onon the the network network represented represented
by the by thereceiving receivingtreated treatedsubject subjecti.e., i.e.,with witha circular a circulardynamic dynamicratherrather than than a single a single
compound, compound, acting acting on a on a single single point point of theof the receiving receiving network network i.e., withi.e., with dynamic. a linear a linear dynamic. 15 15 Thepresent The present invention invention solves solves the the first first problem problem summarised summarised ininthe theparagraph paragraphabove. above.
Thepresent The presentinvention inventionprovides provides a new a new method method for defining for defining the acceptability the acceptability
values of values of aa spectroscopy or spectrophotometry spectroscopy or spectrophotometryanalysis analysisfor forthe the compliance compliancevalidation validationofof one orormore one more batches batches of aofproduct, a product, fortreatment for the the treatment of a pathological of a pathological condition, condition,
20 20 comprisingororconsisting comprising consistingofofone oneorormore more natural natural matrices; matrices; comprising comprising a step a step in which in which
said acceptability said acceptability values valuesare arecalculated calculatedon on the the spectroscopy spectroscopy or spectrophotometry or spectrophotometry
spectra of spectra of aa reference referencestandard standardof of said said product, product, saidsaid reference reference standard standard having having an an ascertainedtherapeutic ascertained therapeutic effect effect in the in the treatment treatment of pathological of said said pathological condition, condition, and of one and of one or more or moredifferent different batches batches of said of said product, product, wherein wherein said spectra said spectra are asdefined are defined as acceptable acceptable
25 25 or not or not acceptable acceptable ononthe thebasis basisofofselected selectedbiological biologicalactivities activities exerted exerted in in at at least least one one
cellular-based assay cellular-based assay byby said said reference reference standard standardandandsaid saidoneoneorormore more differentbatches different batches on one on one or or more morehallmarks hallmarks of of saidpathological said pathologicalcondition condition andand notnot on on thethe mere mere chemical chemical
compositionthereof. composition thereof. In other In other words, words,the theacceptability acceptability values valuesarearedefined definedbased based on on thethe deviation deviation of of
30 30 said aforementioned said spectrafrom aforementioned spectra fromthe theaverage averagespectrum spectrum obtained obtained from from them. them.
The products The products that that cancan be be validated validated with with the the method methodofofthe theinvention inventionare are preferably prepared preferably prepared according according to to good good manufacturing manufacturingpractices, practices, hence hence following following standardised procedures standardised proceduresto toobtain obtain a priori a priori a high a high degreedegree of homogeneity of homogeneity between between
batchesdespite batches despitethethe fact fact that that theythey contain contain or consist or consist of natural of natural matrices matrices and are and are therefore therefore
35 35 obtained from obtained fromnatural natural products. products. As disclosed As disclosedabove, above,duedue to to thethe complex complex interactions interactions within within natural natural matrixmatrix or or matrices and matrices and therapeutic therapeutic or or beneficial beneficial (for(for health) health) products products comprising comprising or or consisting consisting ofof
10 10 May 2024
said matrices and the impossibility to ascribe the therapeutical or beneficial effects to said matrices and the impossibility to ascribe the therapeutical or beneficial effects to
specific APIs, specific APIs, there thereisisa aclear clearneed need in the in the art, art, not only not only for a for besta practice best practice and and standardisation standardisation through through the whole process the whole process ofofpreparation preparationofofeubiotic eubioticproducts products comprisingororconsisting comprising consistingofofnatural natural matrices, matrices, butbut also also for for aa batch batch control control model model of of said said 5 5 products that products that is is not not based basedononthetheidentification, identification,quantification quantificationand andevaluation evaluation of of thethe
individualchemical individual chemical entities entities contained contained inmatrix in said said matrix or matrices or matrices as their therapeutic as their therapeutic or or beneficial emerging beneficial propertiesare emerging properties arenot notascribable ascribabletotothe the mere meresum sum of of thethe properties properties of of
their individual components of of said matrix or matrices. This This control model model should should 2024203121
their individual components said matrix or matrices. control
therefore consider therefore consider thethewhole whole system, system, allowing allowing batchbatch assessment assessment within context within context of of 10 10 mutual interactions mutual interactions among among allall components. components.This Thisdifferent different assessment assessment method methodis is necessary because necessary becausethethe properties properties (emerging (emerging properties) properties) of products of products comprising comprising or or consisting consisting ofof aa natural natural matrix matrixorormatrices, matrices,ininparticular particulara avegetal vegetalmatrix, matrix,an an animal animal
matrix or matrix or mixtures mixtures thereof thereof are are due duetotothe theveryveryinterconnection interconnectionamongamong all all the the componentsofofthe components thenatural naturalmatrices matricespresent presentininthis this kind kind ofof products. products. This This means meansthat, that, as as 15 15 explained above, explained above,asasopposed opposed to classical to classical pharmaceutical pharmaceutical products, products, whosewhose activityactivity is is defined by a specific API, it is not possible to ascribe the emerging properties typical of defined by a specific API, it is not possible to ascribe the emerging properties typical of
natural matrices natural matrices toto aasinglesingleororfewfew components components functional functional interactions. interactions. Indeed,Indeed, the the properties of properties of aa natural natural matrix matrixstem stemnotnotonly only fromfromeacheach and every and every singlesingle component component
therein but therein but also also from from thethe supramolecular supramolecular interconnection interconnectionamong said components among said components 20 20 including the including the way waysaidsaid components components self-assemble self-assemble themselvesthemselves at the at the supramolecular supramolecular
level, which level, results inin aanetwork which results network of of interactions interactions amongamong all theall components the componentsof the of the
matrix.InInother matrix. otherwords, words, in products in products comprising comprising or consisting or consisting of amatrix of a natural natural therematrix is there is not an not an "active “activeprinciple" principle”responsible responsibleforforthethetherapeutic therapeutic features features of of thethe product, product, or or
“excipients” responsible "excipients" responsible for for not not interfering interfering with with the features the features of the of the active active principle, principle, as in as in 25 25 a typical pharmaceutical a pharmaceutical product,product,but butthere thereareare multiple multipleinterconnected interconnectedand andinteracting interacting componentswhich components which are allare responsible all responsible for the foremergent the emergentpropertiesproperties of the matrix. of the matrix.
“Emergent” "Emergent" isisthe theterm termmostmostoften oftenused usedtotodescribe describethe theobserved observedintegrated integratedfeatures featuresof of aa system. system.
Characteristicisisalso Characteristic alsothetheinteraction interaction between between the natural the natural matrixmatrix and the and the receiving receiving
30 30 organism,bybyway organism, way of example, of example, the human the human bodyinteractions body i.e., i.e., interactions betweenbetween the donor the donor network(the network (the matrix matrix or natural or natural material material according according to the specification) to the specification) and the receiving and the receiving
network(the network (thebody bodyofofthe thesubject subjecttotowhom whom the the product product is administered is administered e.g.:e.g.: thethe human human
body). Such body). Suchinteractions interactionsbring bringto tothethe modification modification of aofmultitude a multitude of interconnected of interconnected
biologicalpathways biological pathways in ain a way way that that is is distinctive distinctive ofbiological of each each biological matrix. matrix. As opposedAstoopposed to 35 35 specific APIs, specific APIs, thethe effect effect of of aa therapeutical therapeutical productproduct comprising comprising or orconsisting consistingofofone oneoror morenatural more natural matrices matrices is is broad broad andand encompasses encompasses SO somanymany aspects aspects of the of the physiology, physiology, thatthat the result the result isis that that the theproduct product affects affects thethe overall overall pathological pathological state rather state rather than modifying than modifying
a single a singlefunction function contributing contributing to pathological to the the pathologicalstate. state. The modification The modification of the overall of the overall
11 10 May 2024
pathological state is the result of the multitude of biological components constituting the pathological state is the result of the multitude of biological components constituting the
natural material natural material consisting consisting ofofororcomprising comprising oneone or more or more natural natural matrices matrices (i.e.,(i.e., the the
product), acting product), acting in in aa coordinated coordinatedway way (i.e.,emergent (i.e., emergent properties properties or or matrix matrix effect) effect) by by
meansofof means both both functional functional and structural and structural interactions. interactions.
5 5 As discussed As discussedabove, above,it itisisherein hereinalso alsoreminded reminded that,that, while while beingbeing chemically chemically
analogous analogous to to their their synthetic synthetic counterparts counterparts if taken if taken in isolation, in isolation, natural natural molecules molecules within a within a
naturalmatrix natural matrix areare likely likely to possess to possess distinct distinct fingerprints fingerprints with to with respect respect to their synthetic their synthetic
analogues, due due to to the the totally totally different different synthetic synthetic pathway in terms terms ofofprimary primary 2024203121
analogues, pathway in metabolites,reactants, metabolites, reactants, reaction reaction temperatures, temperatures, energy energy sources, sources, catalysts catalysts etc., potentially etc., potentially
10 10 influencing their influencing their physicochemical physicochemicalbehaviourbehaviour and and reactivity, reactivity, therefore therefore their their biological biological
activity. activity.
For the For the reasons reasonsdepicted depictedabove, above, a preferred a preferred standardised standardised eubiotic eubiotic protocol protocol is is used toto produce used producethethe natural natural matrices, matrices, in particular in particular plantplant matrices matrices andtheoffinal and of the final products toto bebevalidated products validatedwith with thethe methods methods of the ofinvention. the invention. The eubiotic The eubiotic protocolprotocol
15 15 preferably starts preferably starts from from the theagricultural agricultural production productiontotothethefinal finaltransformation transformation of of rawraw
sources designed sources designedsuch suchasastotopreserve preservethe thebasic basicnatural naturalprogrammatic programmatic rules,which rules, which have have
allowedthe allowed theinterconnection interconnectionbetween betweenallall thethecomponents components of living of living things, things, organic organic and and inorganic, for millions of years is provided herein. inorganic, for millions of years is provided herein.
TheApplicant's The Applicant’s research research disclosed disclosed herein herein demonstrates demonstrates that thethat the classical classical
20 20 approach used approach usedforfor standard standard pharmaceutical pharmaceutical products products (i.e.,(i.e., quali-quantitative quali-quantitative characterisationofofthethe characterisation matrix) matrix) is not is not applicable applicable to products to products providing providing therapeutic therapeutic and/or and/or health benefits, health benefits, when the product when the comprisesororconsists product comprises consists of of complex complexnatural naturalsystems systems(i.e.(i.e. natural matrices). natural matrices). As Asknown known in art, in the the art, one ofonetheofmajor the major problems problems linked tolinked the to the validation of validation of therapeutical therapeutical product productcomprising comprising or or consisting consisting of one of one or more or more naturalnatural 25 25 matrices,lies matrices, liesininthat thata anatural naturalmatrix matrix extracted, extracted, e.g.,e.g., from from a given a given plant, plant, is neveristotally never totally identical to identical to “the "the same” natural matrix same" natural extracted in matrix extracted in the the same same waywayfrom from another another plant plant of of
the same the species or same species or even even variety, variety, from the point from the point ofof view view of of the the molecular molecular components. components.
Duetotothe Due thevery very nature nature of of natural natural matrices, matrices, contrary contrary to classical to the the classical quality quality
control acceptance control acceptanceparameters parametersused usedforfor classical classical pharmaceutical pharmaceutical products products basedbased on a on a
30 30 specific API specific APIrule,rule,a adegree degree of of variability variability in the in the quali-quantitative quali-quantitative composition composition of of products comprising products comprising or or consisting consisting of of natural natural matrices, matrices, must must bebetolerated tolerated asasa a manifestation of manifestation of the the most mostintimate intimatenature natureofofsuch suchentities entitiesandandtheir theirmode mode of of action action on on
the receiving the receiving organism; organism; the the problem problem isis however howevertotoidentify identify howhowto toassess assesssaid said acceptabledegrees acceptable degrees of variability. of variability.
35 35 The present The present application application provides provides aa new new andandreliable reliable method method for for assessing assessing spectroscopyororspectrophotometry spectroscopy spectrophotometry acceptability acceptability values values suitablesuitable for the forqualitythe quality processes of processes of products, products, comprising comprisingororconsisting consistingofofcomplex complex natural natural systems, systems, having having a a therapeutic or therapeutic or healthy healthyeffect effectthetheacceptability acceptabilityvalues valuesbeing being based based on biological on the the biological
12 18 Dec 2024 2024203121 18 Dec 2024
activities of said activities of saidkind kindofofproducts products on hallmarks on hallmarks of a pathological of a given given pathological state state rather rather than than
on thequali-quantitative on the quali-quantitative analysis analysis of specific of specific chemical chemical substances substances in said products. in said products.
Theauthors The authorsof of the the present present invention invention surprisingly surprisingly found that, notwithstanding found that, the notwithstanding the
quali-quantitative differences quali-quantitative differences among natural matrices among natural matricesobtained obtainedfrom fromdifferent differentmembers members 55 of the same of the samesource source (even (even through through the production the same same production procedures), procedures), the different the different
molecularentities molecular entities in in said said matrices matrices appear appear to to act act in inaaredundant redundant manner witheach manner with eachother other 2024203121
both functionally and structurally. This redundance results in a clear maintenance of the both functionally and structurally. This redundance results in a clear maintenance of the
biological activity biological activity exercised exercised by products comprising by products comprisingor orconsisting consisting of of naturalmatrices natural matrices even when even when thethe quali-quantitative quali-quantitative composition composition of different of different batches batches of products of said said products 10 10 wouldnot would notbebeconsidered considered acceptable acceptable using using classical classical batch batch control control protocols protocols at present at present
demanded demanded by by the the legislations legislations designed designed to regulate to regulate deterministically deterministically acting APIs. acting APIs.
Without beingbound Without being boundto to theories,the theories, theobserved observedmaintenance maintenance of the of the biological biological activityisis activity
likely duetotothe likely due thefact factthat, that,asassaid saidabove, above, the the emerging emerging properties properties of a natural of a natural matrix are matrix are
due to the due to the matrix matrix network networkacting actingasasa awhole wholeentity entitywith withdistinctive distinctiveproperties propertiesand andmay may 15 15 not be ascribable to each single molecule as if it were in isolation. not be ascribable to each single molecule as if it were in isolation.
The inventors The inventors have havehence hencediscovered discovered thatdifferent that differentbatches batchesof ofproducts products comprisingororconsisting comprising consistingofofcomplex complex natural natural systems, systems, that, that, according according to the to the common common
standard qualitycontrol standard quality controlvalidation validation techniques techniques based based onquali-quantitative on their their quali-quantitative compositions resultedinina adetermination compositions resulted determination of of non-compliance, non-compliance, surprisingly surprisingly maintained maintained
20 20 equivalent biological effects equivalent biological effects resulting resulting inintheir theirdesired desired biological biological activity activity
notwithstandingtheir notwithstanding their different different composition. composition.
Therefore, for Therefore, for therapeutic therapeutic products productscomprising comprising or consisting or consisting of or of one onemore or more natural matrices, instead of basing the acceptability values of the quality control on the natural matrices, instead of basing the acceptability values of the quality control on the
identification andevaluation identification and evaluation of selected of selected individual individual chemical chemical entities,entities, and/or performing and/or performing
25 25 statistical statisticalpreclearing preclearing of spectroscopydata of spectroscopy datain inorder order to discard to discard outliers outliers classically classically
accepted for APIs, accepted for APIs, the the applicant applicant developed developeda anew new method method thatthat bases bases the the assessment assessment of of quality controlparameters, quality control parameters, for for validating validating different different batches batches said product, said product, on the analysis on the analysis
of selected parameters that are representative of its biological effect, which is the pivotal of selected parameters that are representative of its biological effect, which is the pivotal
feature of aa medical feature of medicaldevice device constituted constituted by by natural natural matrices. matrices. The The assessment assessment of the of the
30 30 acceptability acceptability values values and and validation validation method hereinprovided, method herein provided,are areadequate adequatefor forthe the quality quality control of biological control of biologicalmaterial materialwith with therapeutic therapeutic activity activity as defined as defined by Regulation by Regulation
2017/745, reflect 2017/745, reflect product product conformity conformity to to GSPR GSPR 11ofofAnnex Annex I of I of said said Regulation, Regulation,
specifically inthe specifically in thefirst firstlines linesreciting: reciting:"Devices “Devices shallshall achieve achieve the performance the performance intended intended
by their by their manufacturer manufacturerandand shall shall be be designed designed and manufactured and manufactured in suchina such a way way that, that, 35 35 during normal during normal conditions conditions of use, of use, they they are suitable are suitable for intended for their their intended purpose”. purpose".
13
spectra spectra of of a reference standard standard having havinga aknownknown therapeutic or beneficial effectin in thethe 30 May 2025 2024203121 30 May 2025
a reference therapeutic or beneficial effect
treatment of treatment of said said pathological pathologicalconditionconditionororininadjuvating adjuvating homeostasis homeostasis in said in said altered altered
physiological state physiological state and andofofone oneor ormoremore batches batches of said of said product,product, said spectra said spectra being being
defined defined asasacceptable acceptable or non-acceptable or non-acceptable on the on theofbasis basis of the biological the biological activityinexerted in activity exerted
55 said at said at least least one cell-based assay one cell-based assaybybysaid saidreference reference standard standard and and said said one or onemoreor more batches on batches onone oneorormore more hallmarks hallmarks of said of said pathological pathological condition condition or ofora of a pathological pathological
conditionthat condition thatcancan derive derive fromfrom said said altered altered physiological physiological state. state.
Also herein Also hereindisclosed disclosedisisa amethod method for for selecting, selecting, among among differentdifferent batches, batches, a a referencestandard reference standard of of a product a product for treatment for the the treatment of a pathological of a pathological condition,condition, comprisingcomprising 2024203121
10 10 one one oror more morenaturalnaturalmatrices, matrices,asasdefineddefined in in thethe claims. claims. Further Further disclosed disclosed herein herein is ais a
process for process for the the validation validation (i.e. (i.e.quality qualitycontrol control compliance) compliance) of of one one oror more batches of more batches of aa productfor product forthethetreatment treatment of aof a pathological pathological condition condition or of a or of a beneficial beneficial product, product, wherein wherein said said product product comprises comprises one oneorormore morenatural naturalmatrices, matrices,comprising comprisingthe thefollowing followingsteps: steps: a. carrying a. carrying out out aaspectroscopy spectroscopy or or spectrophotometry spectrophotometry analysis analysis of of each batch, each batch,
15 15 b. validating b. validating each each batch wherein the batch wherein the obtained obtained parameters parameters fall fall within within thethe acceptability acceptability values values defined defined according according to to the the method providedforforsaid method provided saiddefinition definitioninin the the presentinvention. present invention. According According totoa afirst first embodiment embodiment of the of the present present invention, invention, therethere is provided is provided a a methodfor method fordefining definingthetheacceptability acceptabilityvalues values of of a spectroscopy a spectroscopy or spectrophotometry or spectrophotometry
20 20 analysis for analysis for thethe compliance compliancevalidation validationof of oneone or more or more batches batches of a product of a product for thefor the
treatment ofofa apathological treatment pathologicalcondition condition or for or for adjuvating adjuvating homeostasis homeostasis in an altered in an altered
physiological state, physiological state, thethe product productcomprising comprising or consisting or consisting of oneoforone moreornatural more natural matricesselected matrices selected from: from: cut orcutpulveri or pulverized plantplant ed plant parts, parts, plant ,extracts extracts fractions, of fractions said of said extracts, extracts, such such as as for for example example the thefractions fractions obtained obtainedbybyfiltration filtration onon aa semi-permeable semi-permeable 25 25 membrane membrane (microfiltration, (microfiltration, ultrafiltration, ultrafiltration, nanofiltration), nanofiltration), or thoseor those obtainedobtained by treatmentby treatment on adsorption resins, on adsorption resins, or or microorganisms, microorganisms, honey, honey,propolis, propolis,silk,silk,wax, wax,plantplantresins, resins, plant plant gums, plantexudates, gums, plant exudates,vegetable vegetable oils,vegetable oils, vegetable essentialoils, essential oils,animal animal tissueslysates, tissues lysates, extract fromanananimal extract from animal tissue, tissue, plantplant or animal or animal fluids;fluids; said method said method comprising: comprising:
performing at performing at least least oneoneininvitro vitrocell-based cell-basedassay assayandand calculatingsaid calculating said 30 30 acceptability acceptability values values on on aa spectroscopy spectroscopy or or spectrophotometry spectrophotometry spectra spectraof: of: aa reference reference standard standardhavinghaving a known a known therapeutic therapeutic or beneficial or beneficial effecteffectin thein the
treatment of treatment of said said pathological pathologicalconditionconditionororininadjuvating adjuvating homeostasis homeostasis in said in said altered altered
physiological physiological state;andand state;
one or one or more batchesofof said more batches said product; product; 35 35 said spectra said being defined spectra being definedasasacceptable acceptableorornon-acceptable non-acceptable on onthe the basis basis of the of the
biological function biological function exerted exertedininsaid saidat atleast leastoneone cell-based cell-based assay assay by reference by said said reference standard standard and andsaidsaid oneoneorormoremore batches batches on onone one or more or more hallmarks hallmarks of saidof pathological said pathological condition or condition or ofof aa pathological condition that pathological condition that can derive from can derive fromsaidsaid altered altered physiological physiological state; and state; and
40 40 defining the defining the acceptability acceptabilityvalues valuesof of thethe spectroscopy spectroscopy or spectrophotometry or spectrophotometry
spectra spectra as as the the variability variabilityrange rangeofofthe thespectroscopy spectroscopy or orspectrophotometry spectrophotometry spectra spectra values values of all of of all of said acceptablebatches said acceptable batchesand and of said of said reference reference standardstandard batch, preferably batch, preferably refined refined by the by thespectra spectraofofsaid saidnon-acceptable non-acceptable batches. batches.
Accordingtotoanother According anotherembodiment embodiment of theof the presentpresent invention, invention, there there is provided is provided a a
13a 13a process for for the the compliance compliance validation of of one one or more batches of a product for the for the 07 Mar 2025 2024203121 07 Mar 2025
process validation or more batches of a product
treatmentofofa apathological treatment pathological condition condition or of or of a beneficial a beneficial product, product, said product said product comprisingcomprising
one or more one or natural matrices, more natural matrices, said said method comprisingthe method comprising thefollowing followingsteps: steps: a. a. performing performing aa spectroscopy spectroscopy or or spectrophotometry spectrophotometryanalysis analysisofofeach eachbatch; batch; 55 b. validating b. validating each batch for each batch for which whichthetheobtained obtained spectrum spectrum satisfiesthethe satisfies acceptability values acceptability values defined defined according according to to the the method accordingtoto the method according the first first embodiment. embodiment.
Also herein Also hereindisclosed disclosedisisa amethod method for for determining determining a reference a reference standard standard of a of a therapeutic product therapeutic product for for the the treatment treatment ofof aa pathological pathologicalcondition, condition,wherein whereinthetheproduct product 2024203121
comprises comprises one oneorormore morenatural naturalmatrices; matrices;saidsaid method methodcomprising: comprising: 10 10 (1) performingatatleast (1) performing leastoneone in vitro in vitro cell-based cell-based assayassay designed designed to simulate to simulate
conditions related conditions related to to said said pathological pathological condition condition on several on several batches batches of saidand of said product, product, and on one or on one or more morereference referencedrug drugfor forthe thetreatment treatmentofofsaid saidpathological pathologicalcondition, condition, wherein wherein the read-out the read-out of of said said cell-based cell-based assayassay is isrepresentative representativeofofthe themodulation modulation ofof one one or or more more
biological activity biological activityassociated associatedwith with one one oror more hallmarks of more hallmarks of said said pathological pathological condition condition 15 15 and determining and determiningfor foreach eachofofsaid saidoneoneorormore morebiological biologicalactivities activities the the modulation modulationtrendtrend thereoffor thereof forrestoring restoringa ahealthy healthy state; state;
(2) (2) quantifying quantifying in in terms terms ofof numerical values the numerical values the modulation modulationofofsaidsaidone oneorormore more biologicalactivities biological activitiesinduced induced by by eacheach batchbatch andofdrug and drug step of (1)step for (1) eachfor each cell-based cell-based assay assay read-out, and read-out, defining as and defining as reference reference value valuefor for each eachbiological biologicalactivity activity modified modifiedbybysaid said 20 20 one one oror more morereference referencedrug drugaccording according to to saidsaid modulation modulation trend trend for for restoring restoring a healthy a healthy
state, state, the the value associated value associated to to the the weakest weakest performance; performance;
(3) (3) selecting selecting the the batches batches whose valuescalculated whose values calculatedinin(1) (1) induce induceaamodulation modulation of of
each measured each measuredbiological biologicalactivity activityininsaid saidcell-based cell-basedassay assaywhose whose modulus modulus is toisthe ≥ to the modulusofofthethereference modulus reference value value calculated calculated in (2) in (2) and and defining defining as product as product reference reference
25 25 standard batchesthe standard batches theoneonewithwiththethe highest highest modulus modulus valuesvalues on theon the larger larger number number of of biologicalactivities. biological activities.
GLOSSARY GLOSSARY A "natural matrix" in the present application refers to a material consisting of a A "natural matrix" in the present application refers to a material consisting of a
networkrepresented network representedby by a broad a broad numbernumber of components/constituents of components/constituents obtained (e.g. obtained (e.g.
30 30 extracted) directly from extracted) directly from aamember member of the of the natural natural kingdom kingdom or a naturally or a naturally occurring occurring
portion thereof portion thereof (i.e. (i.e. from froma anatural naturalrawraw source), source), without without significant significant processing processing or or synthetic alteration, wherein “without significant processing or synthetic alteration” is synthetic alteration, wherein "without significant processing or synthetic alteration" is
intended intended as asprocessed processedonly only by manual, by manual, mechanical mechanical or gravitational or gravitational means means e.g. by e.g. by
dissolution in dissolution in water water oror other other naturally naturally occurring occurring solvents, solvents, such such as as water, water, water-alcohol water-alcohol
35 35 solutions etc.; by solutions etc.; by flotation; flotation; bybyextraction extractionwith with water water or other or other naturally naturally occurring occurring
solvents; solvents; by by steam steamdistillation distillation ororbybyheating heating solely solely to remove to remove water water or any or any other other
naturally occurring naturally solvent; or occurring solvent; or extracted extracted from air by from air any means by any meansand andwith withthethe provision provision
that "natural that “natural matrix" matrix”excludes excludessaidsaid member member of the ofnatural the natural kingdomkingdom or a naturally or a naturally
occurring portion thereof occurring portion thereofasassuch. such.InInother otherwords, words, a natural a natural matrix, matrix, or aormixture a mixture of of 40 40 natural matrices, natural matrices, are are materials materials obtained fromentities obtained from entities that that are are self-assembled self-assembled in in nature nature and processed and processedwhile whilepreserving preservingtheir their native native bio-physical bio-physical characteristics characteristics which which
14 10 May 2024
determinetheir determine their physiological physiological interaction interaction with with other otherliving livingorganisms, organisms,such suchas asthe thehuman human
organism. Their organism. Their emerging emergingproperties properties can canbebeexpressed expressed by by contributing contributing to to the the rebalancing of rebalancing of metabolic metabolicprocesses processesororstates states of of the the receiving receiving organism organismand/or and/orofofsome some organs oror tissues organs tissues alongside alongsidethethephysiological physiological actions actions that that will will be be activated activated in each in each
5 5 specific context. specific context. According According to to the the present presentinvention inventionthe thenatural naturalmatrix matrixcan canbebefrom froma a
material obtained material obtained from fromanyanysource source in in thethe lifekingdoms life kingdoms i.e.,Monera, i.e., Monera, Protista, Protista, Fungi, Fungi,
Plantae and Plantae and Animalia. Animalia.The The term term hence hence encompasses encompasses a plant a plant natural natural matrix, matrix, an animal an animal
natural matrix, matrix, a a fungi natural matrix matrix aa protista protista (archaea or bacteria) bacteria) natural natural matrix matrix a 2024203121
natural fungi natural (archaea or a
moneranatural monera naturalmatrix. matrix.AAnatural naturalmatrix matrixmay may also also comprise comprise natural natural inorganic inorganic materials materials
10 10 such as such as minerals minerals extracted extracted from fromnatural natural raw rawmaterials. materials. AAsynonym synonym of natural of natural matrix matrix or or
one or one or more morenatural naturalmatrices matricesinin the the present present description description is is “natural "natural material” material" as as defined defined
below. below.
Anexample An example of of naturallyoccurring naturally occurring portion portion of of an an organism organism may may be represented be represented
by e.g. by e.g. ,roots, ,roots, leaves, leaves,bark, bark,fruit, fruit, flower, flower,ofofa aplant plantororsections sections thereof, thereof, organs, organs, tissues. tissues.
15 15 In any In part of any part of the the description description thethe general general term term natural natural matrix matrix can can be be substituted substituted
with: with:
a plant natural matrix or a natural matrix obtained from a plant, a plant natural matrix or a natural matrix obtained from a plant,
an animal an natural matrix animal natural matrix oror aa natural naturalmatrix matrixobtained obtained from from an an animal, animal,
a fungi a naturalmatrix fungi natural matrixor or a natural a natural matrix matrix obtained obtained from afrom a fungus, fungus,
20 20 a protista a naturalmatrix protista natural matrixorora anatural natural matrix matrix obtained obtained from from a protista, a protista,
a monera a natural matrix monera natural matrix oror aa natural natural matrix matrix obtained obtained from from aa monera, monera,
or with or witha aplant plantmaterial material and/or and/or extract, extract, an extract an extract from from an an tissue animal animalortissue organ, or organ, fungi fungi
and/or aa fungi and/or fungi extract, extract, or or aa mixture thereof. In mixture thereof. In addition, addition, aa natural naturalmatrix matrix may contain may contain
minerals or minerals or components components obtainedobtainedfromfrom minerals. minerals.
25 25 Plant is Plant is synonymous synonymous with withherb. herb. For example, For example,ininthe the context contextofof products productscomprising comprisingororconsisting consistingofofmaterial materialofof plant origin plant origin (obtained (obtained from plants) such from plants) such asas herbal herbal supplements supplementsa anaturalnaturalmatrix matrixwould would comprisea apart comprise part of the of the original, original, not intentionally not intentionally altered, altered, preferably preferably unaltered, unaltered,
constituentsthat constituents thatarearenaturally naturally present present in the in the source source materials materials (e.g.,(e.g., extracts extracts of theof the source source
30 30 material)and material) andtherefore therefore contain contain various various organic organic and inorganic and inorganic constituents constituents that are found that are found
in the in the aforementioned kingdoms. aforementioned kingdoms.
Theterm The term"natural" "natural”matrix matrixemphasizes emphasizes thetheretaining retainingthe theintegrity integrity andand complexity complexity of aa network of networkofofconstituents/components constituents/components as as in in thethe originalnatural original naturalsource, source,rather ratherthan than isolating, purifying, isolating, purifying, or extracting specific or extracting specific molecules moleculesor or molecular molecular classes classes through through
35 35 extensive processing extensive processing or or chemical chemicalmodification. modification. Duetotothe Due thesupramolecular supramolecular self-assembly self-assembly of the of the constituents/components constituents/components of a of a
natural matrix, natural matrix, thethe whole matrixbehaves whole matrix behavesasasa acomplex complex network network that that doesdoes not interact not interact
with aa single with singletarget target molecule moleculebutbut thatthat interactswith interacts with a network a network of recipients of recipients (also (also
15 10 May 2024
organised asas aanetwork) organised network)in inthethereceiving receiving organism. organism. Therefore, Therefore, the the interaction interaction natural natural
matrix-receiving organism matrix-receiving organismisisnot, not,asasfor forcommon common pharmaceutical pharmaceutical APIsresult APIs the the result of a of a point-to-point interaction, point-to-point interaction, but but the the result result ofof an “interactor” networks an "interactor" (i.e. the networks (i.e. the matrix) matrix) “receiver”network "receiver" network (i.e. (i.e. thethe organism organism to the to whom whom theismatrix matrix is administered) administered) interaction.interaction.
5 5 Theterm The term natural natural matrix matrix canalso can be be substituted also substituted in any in partany of part of the description the description and and claims with claims with complex complexnatural naturalsystem. system. Nowherein in Nowhere thethe description description andand in the in the claims claims the term the term naturalnatural matrixmatrix can becan be
interpretedasas"a"aproduct productof of nature”, rather, a natural matrix is a product obtained from a 2024203121
interpreted nature", rather, a natural matrix is a product obtained from a
natural organism natural organismand andprocessed processed (e.g.,extracted) (e.g., extracted)therefrom therefrom by by techniques techniques that that do not do not
10 10 substantially alter substantially alterbiological biologicalstructure structureand andthethesupramolecular interconnections among supramolecular interconnections among the components the withinthe components within thematrix. matrix. Emerging Emerging properties properties according according to present to the the present description description and toand thetoart, the the art, the termdefines term definesthethe properties properties of a of a natural natural matrixmatrix or of aor of a natural natural material material according according to the to the present specification, present specification, i.e.i.e. properties properties thatthat are are not notrepresented representedby by the the meremere sum of sum of 15 15 properties of properties of each singled out each singled out constituent/component constituent/componentofofsaid saidmatrix/material matrix/materialbut butbybythe the intermolecular interactions intermolecular interactions among among allall constituents/components constituents/components of matrix/material of the the matrix/material that are that are the the result resultofofthe thesupramolecular supramolecular self-assembly of said self-assembly of said components/constituents components/constituents
within the matrix/material itself. within the matrix/material itself.
"Emerging properties"hence "Emerging properties" hence refer refer to to technical technical effects,such effects, suchas as therapeutic therapeutic or or
20 20 homeostasis- -adjuvating homeostasis adjuvatingproperties properties(i.e., (i.e., beneficial beneficial effect), effect), that that the the interactions interactions and and relationships among relationships among thethe constituents/components constituents/components of of aa natural natural matrix matrix exert exert onon aa receiving living receiving living system. system. Emergent propertiesare Emergent properties arenotnot immediately immediatelyevident evidentororpredictable predictable based solely based solelyononthetheindividual individual characteristicsof of characteristics eacheach constituent/component constituent/component of theof the
matrix. Instead, matrix. Instead, they they “emerge” "emerge" as asall all the the constituents/component constituents/component ofofthe thematrix matrixnetwork network 25 25 interact with interact with one one another another and and with with the the living living system system receiving receiving network network inin aa dynamic dynamic andand complexway. complex way. Emerging Emerging properties properties have have been been broadly broadly discusseddiscussed in the inartthe in art in various various
scientific and scientific systems-orientedfields, and systems-oriented fields,including including physics, physics, chemistry, chemistry, biology, biology, and and complexsystems complex systemstheory. theory. Keypoints Key points about aboutemerging emergingproperties propertiesinclude: include: 30 30 System Complexity:Emerging System Complexity: Emergingproperties propertiesare areassociated associated withwithsystems systemsthatthat exhibit aa certain exhibit certain level level ofofcomplexity. complexity. In In simple simple systems, systems, the interactions the interactions betweenbetween constituents/componentsare constituents/components arelimited, limited,andandproperties propertiesare aremore moreeasily easilydeducible deducible from from thethe
properties of properties of individual individual constituents. constituents. In In complex complex systems, systems, however, however, the interactions the interactions
betweencomponents between components and and theirtheir supramolecular supramolecular organisation organisation can rise can give give to risenovel to novel and and
35 35 unexpectedfeatures. unexpected features. Nonlinearity: Emergent Nonlinearity: Emergent properties properties oftenoften result result from from nonlinear nonlinear interactions, interactions,
wherethetherelationship where relationship between between cause cause and is and effect effect is not proportional. not proportional.
Holism:The Holism: Theconcept concept of of emerging emerging properties properties emphasizes emphasizes a holistic a holistic perspective, perspective,
16 10 May 2024
recognizing that the whole system is more than the sum of its parts. recognizing that the whole system is more than the sum of its parts.
AA product productcomprising comprising or consisting or consisting of one of one or more or more natural natural matrices matrices (alias(alias "a “a product comprising product comprisingororconsisting consistingofofcomplex complexnatural naturalsystem/s") system/s”)according accordingto to thepresent the present inventionisisa aproduct invention product that that comprises comprises or consists or consists of one of or one more or morematrices, natural natural matrices, herein herein 5 5 also defined also defined as as aa "natural “natural material" material”i.e., i.e., aa “material obtained/manufactured/processed "material obtained/manufactured/processed
from aa natural from natural raw source(raw raw source (rawmaterial) material) or or from fromaamember member of of thethe naturalkingdom" natural kingdom”cfr.cfr.
below. below.
In any part ofof the the description descriptionand and the theclaims claims “a"a product product comprising or consisting consisting 2024203121
In any part comprising or
of one of or more one or natural matrices" more natural matrices” can can be be replaced replacedby by"a“aproduct productcomprising comprisingororconsisting consisting 10 10 of one of one orormore moreplant plantmatrix" matrix”or or by by “a product "a product comprising comprising or consisting or consisting of complex of complex
naturalsystem/s" natural system/s” "a “a natural natural material material or a or a material material of natural of natural origin”. origin".
Furthermore,the Furthermore, theterm term"product “productcomprising comprising or or consisting consisting of of oneone or or more more natural natural
matrices” according matrices" accordingtotothethepresent present description description can can be anbeintermediate, an intermediate, or theorfinal the final formulation for formulation for intended intendeduse use(e.g. (e.g. resuspended resuspendeddrydryproduct) product)or,or,inin particularwhen particular whenthethe
15 15 formulation formulation forfor intended intended useinisliquid use is in liquid form,form, the termthecan term canadefine define dry or aa dry or a lyophilised lyophilised
or aa concentrated or concentratedform formthereof thereof to to which which water water will will be added be added by theby theoruser user or by the by the physician in order to prepare the formulation for administration. physician in order to prepare the formulation for administration.
Nowhereininthe Nowhere thedescription description and andininthetheclaims claims"a “a product product comprising comprising or or consisting of consisting of one one or or more morenatural naturalmatrices" matrices”cancan be be intended intended as as a product a product of nature as of nature as 20 20 such. such. InInaddition, addition,when when a single a single natural natural matrixmatrix is present is present in or consists in or consists of said product, of said product,
said natural said natural matrix matrix isis obtained (e.g. extracted) obtained (e.g. extracted) fromfrom anan organism organismasasdefined defined above above by by
technological means; technological means;whenwhen a mixture a mixture of of natural natural matrices matrices is is comprised comprised in consists in or or consists of of
the product, the product, saidsaid mixture mixtureisis aa mixture mixtureofofselected selectednatural naturalmatrices matricesmade madeby by man,man,and and
said mixture said cannotbebefound mixture cannot found as as such such in in anyany of of thethe natural natural products products of origin of origin of each of each
25 25 matrix containedtherein. matrix contained therein.Therefore, Therefore, whenwhen the product the product comprises comprises or consists or consists of a of a plurality of plurality of natural naturalmatrices, matrices,saidsaidnatural naturalmatrices matriceshave havebeen been combined combined by byman man andandthethe
resulting product resulting product isisendowed endowed of of new newemergent emergent properties. properties.
Accordingto tothethe According present present description, description, the expression the expression “biological "biological activitiesactivities relatedtotoa apathological related pathological condition” condition" refers refers to a settoofa biological set of biological activities activities associatedassociated to to 30 30 a distancing/deviation a distancing/deviation from from homeostasis homeostasis whichwhich may may orormay maynotnot reach reach thethe onset, onset, progression, worsening, progression, worsening,ofofa apathological pathologicalcondition. condition.Hence, Hence, thetheexpression expression "biological "biological
activities modifications activities related to modifications related to a pathological condition" a pathological condition"refers refers totomodulations modulations or or
changesinin the changes the normal normal(healthy) (healthy)physiological physiologicalstate stateofofbiological biologicalactivities activities (processes) (processes) within an within an organism organism(preferably (preferablya ahuman) human)that that are directly are directly associated associated to anto an 35 35 alteration/impairment alteration/impairment of of homeostasis, homeostasis,upup to to a pathological a pathological statestate or or disease. disease. In other In other
words,ititdescribes words, describesthethe specific specific adjustments adjustments or deviations or deviations from from the the physiological healthy healthy physiological state of state of aa set set or or "network" “network” of biological of biological activities activities that that occuroccur as a result as a result or thatorconcur that concur to to the onset, the onset,progression, progression, worsening, worsening, of a of a pathological pathological conditioncondition or disease. or disease.
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Byway By wayof of example, example, in the in the case case of diabetes, of diabetes, biological biological activities activities related related to glucose to glucose
metabolism,insulin metabolism, insulinproduction productionand andmanagement management are modified are modified in ways in ways thatdirectly that are are directly associated to associated to the thepathological pathologicalcondition condition of diabetes of diabetes and hence and are are hence relatedrelated to the to the pathological condition or state of diabetes according to the present description. pathological condition or state of diabetes according to the present description.
5 5 Syntheticaccording Synthetic accordingtotothe thepresent presentdescription descriptionhashasthethemeaning meaning conventionally conventionally
accepted in accepted in chemistry. chemistry.
Conventionally,inin chemistry, Conventionally, chemistry,the theterm term"synthetic" "synthetic"refers refers toto the the origin origin or or source source
of aa material material ororsubstance. substance.Synthetic Synthetic substances or materials are are produced by manby man 2024203121
of substances or materials produced
through artificial through artificial synthesis synthesis i.e., i.e., through throughlaboratory laboratorychemical chemical reactions reactions usually usually by by 10 10 reacting simpler reacting simpler chemicals chemicalstotocreate createmore more complex complex onesones through through processes processes that often that often
use different use different pathways, pathways,temperature temperature conditions, conditions, pressure pressure conditions, conditions, energy energy sources sources
and/or catalysers and/or catalysers from from those those used used byby living living organisms. organisms.
Examples:Synthetic Examples: Synthetic substances substances or materials or materials include include plastics, plastics, pharmaceutical pharmaceutical
drugs, and drugs, and many manyindustrial industrialchemicals. chemicals.For Forexample, example, nylon nylon is is a synthetic a synthetic polymer polymer mademade
15 15 through chemical through chemicalsynthesis, synthesis,and andaspirin aspirinisisa asynthetic syntheticdrug drugproduced produced through through specific specific
chemicalreactions. chemical reactions. An inin vitro An vitro cell-based cell-based assay, assay, in in the the present present description description has has the the meaning meaning conventionally used in the art, in particular, it refers to an analytical procedure based on conventionally used in the art, in particular, it refers to an analytical procedure based on
cells, for cells, evaluatingthethe for evaluating cell cell behaviour behaviour and reaction and reaction to insults to insults or stimuli, or stimuli, in the context in the context
20 20 of aa disease of diseaseororofofa apathological pathological or pre-pathological or pre-pathological condition condition or of a condition or of a condition wherein wherein homeostasis homeostasis is is altered. altered. This This typetype of assay of assay is designed is designed to study tothe study the biological biological response of response of
cells in cells in aa controlled controlledenvironment, environment, oftenoften in a petri in a petri dish ordish or aplate. a well well According plate. According to the to the invention, suitable invention, suitable in in vitro vitro cell-based cell-based assays are assays assays are assays whose whoseread read outout is is associated associated
with the with the modulation modulationofofbiological biologicalactivities activitiesassociated associatedtotoone oneor or more more hallmark hallmark of a of a
25 25 given pathological given pathological or or pre-pathological pre-pathological condition conditionor or ofof aa condition condition wherein whereinhomeostasis homeostasis is altered is (altered physiological altered (altered physiological state). state).
Thecells The cells can can bebe derived derived from fromhumans, humans, animals, animals, or or celllines cell linesthat that mimic mimicspecific specific tissues or tissues or organs. organs. In the In the context context of of aa disease diseaseororpathological/pre-pathological pathological/pre-pathologicalcondition, condition,ororofofa a 30 30 condition wherein condition whereinhomeostasis homeostasis is is altered,aacell-based altered, cell-basedassay assayisis specifically specifically designed to designed to
simulate or simulate or mimic mimic conditions conditions related related to the to the disease disease or pathological/pre-pathological or pathological/pre-pathological
condition or condition or to to the the condition conditionwherein whereinhomeostasis homeostasis is altered.In In is altered. particular,cell-based particular, cell-based assays can assays canbebeusedusedto toevaluate evaluate in in vitro vitro thethe therapeutic, therapeutic, adjuvating adjuvating and/or and/or beneficial beneficial
activity of activity of different different compounds compounds oror products. products. It It cancan involve involve exposing exposing cellscells to factors to factors
35 35 knownto to known be be associated associated with with the disease the disease or pathological or pathological condition, condition, to potential to potential
therapeutic or therapeutic or adjuvating productsadjuvating adjuvating products adjuvatingthe therestoration restorationofof the the physiological physiologicalstate state or using or usingcells cellsthat thatareareororhave have been been genetically genetically modified modified to carrytodisease carry disease or pathological or pathological
condition-specific condition-specific traits. traits.
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Theselected The selected cells cells can can be be treated treated to to induce the pathological, induce the pathological, pre-pathological pre-pathological or or altered homeostasis altered homeostasiscondition conditionororcancan be be cells cells already already presenting presenting the desired the desired altered altered
phenotype. phenotype.
A healthy A healthyphysiological physiological state state refers refers to to thethe condition condition of organism's of an an organism's body,body,
5 5 organ, apparatus, organ, apparatus, system systemor or body body district,andand district, its its internal internal processes processes whenwhen they they are are functioningoptimally functioning optimally and within and within normal normal parameters parameters for that individual, for that individual, i.e.tothe state to i.e. the state
whichhomeostasis which homeostasis tends. tends. A healthy A healthy physiological physiological state,state, in the in the context context of biological of biological
activities known known to to contribute to hallmarks of a disease given disease or pathological condition, condition, 2024203121
activities contribute to hallmarks of a given or pathological
refers to refers to the the condition conditionin inwhich whichsaidsaid various various biological biological activities activities are operating are operating optimallyoptimally
10 10 and within and withinnormal normal(healthy) (healthy)parameters. parameters. This This state state is is characterized characterized by by thethe absence absence of of
significantaberrant significant aberrantcellular cellularor or molecular molecular processes processes associated associated with the with thedisease specific specific disease under consideration. under consideration. Theterm The term cancan refer refer to the to the hallmarks hallmarks of a particular of a particular disease, disease, which arewhich are distinctive distinctive
features or features or characteristics characteristics that that are are typically typically observed observed in in individuals individualsaffected affectedbybythatthat 15 15 disease. These disease. hallmarkscan These hallmarks caninclude includespecific specificcellular cellular behaviours, behaviours,molecular molecularpathways, pathways, or physiological or responses that physiological responses that play play a a key role in key role in the thedevelopment or progression development or progressionofof the the disease. disease.
In summary, In summary, a ahealthy healthyphysiological physiologicalstate stateininthe thecontext contextofofaa specific specific disease disease oror pathologicalcondition pathological conditionis ais a state state in which in which the biological the biological activitiesactivities related related to to the known the known
20 20 hallmarksofof that hallmarks that disease disease oror pathological pathological condition condition are are modulated modulatedinina adirection directionthatthatisis consistent with consistent with aa non-diseased non-diseased(non-pathological) (non-pathological)state, state, in in other other words, words, opposed opposedtotothe the diseased(pathological) diseased (pathological) state. state.
A healthy A healthyphysiological physiological state,therefore, state, therefore,also also indicates indicates the the direction direction of the of the
modulationofofbiological modulation biologicalactivities activities that thatareareknown hallmarksof known hallmarks of aa pathological pathological condition condition 25 25 in homeostasis, in homeostasis, i.e., i.e., before the onset before the onset ofof aa pathological pathologicalcondition, condition,ininotherotherwords words thethe
homeostaticdirection homeostatic directionofofthe themodulation modulation of of a seta set of of biological biological activitiesascribed activities ascribedtotoa a specific system, specific system, district, district, apparatus apparatusorororganorgan ofhealthy of a a healthy subject. subject. In theInpresent the present descriptionhealthy description healthy physiological physiological statestate and healthy and healthy state state are areasused used as aliases. aliases.
Altered physiological Altered physiological statesstates andand altered altered homeostasis homeostasisare areclosely closely related related 30 30 concepts that concepts that describe describe deviations deviations from the normal from the normalfunctioning functioningand andbalance balanceofofthe thebody's body's internal environment. internal Whilethey environment. While theyoverlap, overlap,there therearearesome some distinctionsbetween distinctions between thethe twotwo
terms: terms:
AlteredPhysiological Altered PhysiologicalStates: States:This Thistermterm encompasses encompasses a broad a broad range range of changes of changes
in the in the body's normalfunctioning, body's normal functioning,including includingdisruptions disruptionsininorgan organsystems, systems, biochemical biochemical
35 35 processes, and processes, and cellular cellular functions. functions. Altered Altered physiological physiologicalstatesstates can canresult result from fromvarious various factors such factors such asas disease, disease, injury, injury, medication, medication,environmental environmental factors,andand factors, psychological psychological
stress. Examples stress. include fever, Examples include fever, inflammation, hormonalimbalances, inflammation, hormonal imbalances, and and impaired impaired organ organ
function. function.
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AlteredHomeostasis: Altered Homeostasis: Homeostasis Homeostasis refersrefers tobody's to the the body's ability ability to maintain to maintain a a stable internal stable internal environment despite external environment despite external changes. changes. This Thisstability stability is is achieved achieved through through
regulatory mechanisms regulatory mechanisms thatcontrol that controlvariables variablessuch suchasasbody body temperature, temperature, blood blood pressure, pressure,
pHbalance, pH balance,andandblood bloodglucose glucoselevels levelswithin withinnarrow narrow ranges. ranges. Altered Altered homeostasis homeostasis occurs occurs
5 5 whenthese when theseregulatory regulatorymechanisms mechanisms failfail to to maintain maintain balance, balance, leading leading to to deviations deviations from from
the body's the body's normal normalset setpoints. points.These Thesedeviations deviationscancanbe be temporary temporary or chronic or chronic and may and may
involve compensatory involve compensatorymechanisms mechanisms to restore to restore balance. balance.
In summary, alteredphysiological physiologicalstates statesdescribe describethe theobservable observablechanges changesin in thethe 2024203121
In summary, altered
body's normal body's normalfunctioning, functioning,while whilealtered altered homeostasis homeostasisrefers refersto to the the underlying underlying disruption disruption 10 10 of the of the body's body'sregulatory regulatory mechanisms mechanisms that maintain that maintain internal internal stability.stability. Analtered An altered homeostasis homeostasisunderlies underliesaltered alteredphysiological physiologicalstates,states,asasdisruptions disruptionsinin homeostaticmechanisms homeostatic mechanisms thatthat cancan leadlead to physiological to physiological imbalances imbalances and manifestations and manifestations
of illness of illness or or dysfunction. dysfunction. Hallmark Hallmark of of a a diseaseororofofaapathological disease pathologicaloror medical medicalcondition conditionaccording accordingtotothethe 15 15 present description present description hashas the the meaning meaning conventionally conventionally usedused in thein the art.art. According According to theto the state of state of the the art, art,hallmarks hallmarks of of aa disease disease are are indicators indicatorsthat thatcan canmark the progression mark the or progression or
control of control of a a given disease or given disease or pathological pathological condition. condition. These Thesehallmarks hallmarks(also (alsocalled called'key ‘key indicators’) are indicators') are typically typically aa set setof offeatures featuresor orpatterns patternsthat thata aphysician physicianwould monitor, would monitor,
over time, over time, to to track track the the progression or regression progression or regression of of aa particular particular illness. illness.In Insummary, summary, aa
20 20 hallmarkofofa adisease hallmark diseaseis isa defining a defining feature feature or characteristic or characteristic whose whose modification modification is is indicative of indicative of a agiven given medical medical condition, condition, aiding aiding in its identification, in its identification, diagnosis,diagnosis, monitoringand monitoring andunderstanding. understanding. By of By way way of example, example, for neurodegenerative for neurodegenerative diseases diseases
(NDDs)atatleast (NDDs) least the the following following eight eight hallmarks hallmarksofofNDD NDDare are known known in theinart: the art: (pathological protein) aggregation, (pathological protein) aggregation,synaptic synapticand and neuronal neuronal networknetwork (dysfunction), (dysfunction),
25 25 (aberrant) proteostasis, cytoskeleton (aberrant) proteostasis, cytoskeleton(abnormalities), (abnormalities),(altered) (altered)energy energy homeostasis, homeostasis,
DNA DNA and and RNARNA (defects), (defects), inflammation inflammation (increase), (increase), and and neuronal neuronal cell cell death death (increase). (increase). In In
cancerresearch, cancer research, thethe hallmarks hallmarks of cancer of cancer areofa distinctive are a set set of distinctive characteristics characteristics that are that are commonly commonly found found in cancer in cancer cells.cells. TheseThese hallmarks hallmarks include include (sustained) (sustained) proliferative proliferative
signalling, (evasion signalling, (evasion of) of)growth growth suppressors, suppressors, (resistance (resistance to) cell to) cell death,death, (enabling) (enabling)
30 30 replicative immortality, replicative immortality,(inducing) (inducing) angiogenesis, angiogenesis, and (activating) and (activating) invasioninvasion and and metastasis. metastasis.
Hallmarksofofa disease, Hallmarks a disease, biological biological activities activities associated associated to hallmarks, to said said hallmarks, parameterswhose parameters whose analysis analysis allowsallows to assess to assess the modifications the modifications of said biological of said biological
activities etc. activities etc. are areaaframework framework to study to study a disease a disease or a pathological or a pathological or condition or medical medical condition 35 35 using an using an integrated/holist: integrated/ holistic approach. approach. Theexpression The expression"natural "natural material," material," according according to the to the present present description, description, refers refers
to materials to materials consisting consisting of of one oneorormore more natural natural matrices matrices withwith the provision the provision that that said said
material/s are material/s are notnot found foundininnature natureas as suchsuch but but are are the result the result of a of a technical technical humanhuman
20 10 May 2024
intervention (such as, e.g., extraction processes, filtration, and the like, i.e., elaborates). intervention (such as, e.g., extraction processes, filtration, and the like, i.e., elaborates).
Thesematerials These materialsare aretypically typically obtained obtainedfrom fromplants, plants,animals, animals,fungifungiorormicroorganisms, microorganisms, and minerals and mineralsthrough throughpreparation preparation processes processes thatthat aim aim to preserve to preserve the integrity the integrity of the of the
networkswithin networks withinthe thenatural natural raw rawsources sourcesfrom fromwhich whichthethe naturalmaterial natural materialisisprepared. prepared.InIn 5 5 the present the present description description "natural “natural material" material” is is considered considered as as aa synonym synonym of ofoneone or more or more
naturalmatrices. natural matrices.A A natural natural material material according according to the to the present present description description can therefore can therefore be be a product, a product, such such asas aa product with aa therapeutic product with therapeutic effect, effect, consisting consisting onlyonly of of or or comprising comprising
different natural natural matrices matricesselectively selectivelyassembled assembled (that(that are combined not combined in such in such 2024203121
different are not
combinationininnature) combination nature) inin order order toto produce produce aa given therapeutic effect given therapeutic effect thereby thereby forming forming anan 10 10 “interactor network” "interactor whoseadministration network" whose administrationtotoa asubject subject(i.e. (i.e. aa receiving receiving living living network) network)
showsemerging shows emerging properties properties providing providing a therapeutic a therapeutic effect effect or or a homeostasis a homeostasis adjuvating adjuvating
effect ,, i.e. effect i.e.anan effect effect beneficial beneficial toto the the health healthofofsaid saidsubject. subject. “HavingTherapeutic "Having Therapeutic effect”: effect": according according to theto present the present description description a product a product
havinga atherapeutic having therapeutic effect effect is aisproduct a product which, which, upon administration upon administration to a subjectto aaffected subject affected 15 15 by aa pathological by pathologicalcondition, condition,reduces reducesthethe severity severity of ofthethe subject'scondition subject's condition (i.e.,the (i.e., the severity is severity is at at least least partially partiallyreduced reduced or or mitigated), mitigated), and/or provides some and/or provides somealleviation, alleviation, mitigation oror decrease mitigation decreasein inat atleast leastoneone clinical clinical symptom symptom and/orand/or in aindelay in a delay the in the progression of progression of said said condition condition oror reinstates reinstates (completely (completely or or inin part) part) a ahealthy healthy physiological physiological condition condition in thein the district district affected affected by said by said pathological pathological condition. condition.
20 20 Havingbeneficial Having beneficialeffecteffectaccording according to to thethe present present description description encompasses encompasses a a product whose product whoseadministration administrationtotoa ahealthyhealthysubject subjectorora asubject subjectwhich whichisishealthy healthybutbutnot not in homeostasis, in homeostasis, or or to to an aninin vitro vitro cell cell assay assay representing representing an anadequately adequatelyhealthy healthystatus, status, results in results in an anininvitro vitroororininvivo vivoevidence evidenceof aof a reinstatement reinstatement or adjuvation or adjuvation of homeostasis of homeostasis
upon upon administration administration in in said said cell cell assay assay or or in in the the recipient’s recipient's
25 25 system/district/apparatus/organ system/district/apparatus/organ of interest. of interest.
The terms The terms "prevent," “prevent,” "preventing," “preventing,” and and "prevention “preventionof" of”(and(andgrammatical grammatical variations thereof) variations thereof) refer refer to toreduction reduction and/or and/or delay delay of of the the onset onset and/or and/or progression progression of of aa disease, disorder disease, and/or aa clinical disorder and/or clinical symptom(s) symptom(s)inina subject a subjectand/or and/or a reduction a reduction in the in the
severity of severity of the theonset onsetand/or and/or progression progression of the of disease, the disease, disorder disorder and/or clinical and/or clinical
30 30 symptom(s)relative symptom(s) relativetoto what whatwouldwouldoccur occurininthetheabsence absenceofofthe themethods methodsofofthe theinvention. invention. Theprevention The preventioncan canbebecomplete, complete, e.g.,the e.g., thetotal total absence absenceofofthe thedisease, disease,disorder disorderand/or and/or clinical symptom(s). clinical symptom(s). The Theprevention preventioncan canalso alsobebepartial, partial, such such that that the the occurrence occurrence of of the the disease, disorder disease, disorder and/or and/or clinical clinical symptom(s) symptom(s) in in the the subject subject and/or and/or thethe severity severity ofof onset onset and/or the and/or the progression progression isis lessless than whatwould than what wouldoccuroccurin in thethe absence absence of of a composition a composition
35 35 accordingtotothethepresent according present invention. invention.
Having Having a “beneficial/healthy/beneficial a "beneficial/healthy/beneficial to health” to health" effect effect accordingaccording to the present to the present
description encompasses description encompassesa producta product whose whose administration administration to a to a healthy healthy subject subject or to or a to a
healthy subject healthy subject whowhoisisnot notininhomeostasis homeostasis or or to to an an in in vitrocell vitro cellassay assayrepresenting representinganan
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adequately healthy status, results in an in vitro or in vivo evidence of a reinstatement or adequately healthy status, results in an in vitro or in vivo evidence of a reinstatement or
adjuvation of adjuvation of homeostasis homeostasisupon upon administration administration in in said said cell cell assay assay or or in in thethe recipient’s recipient's
system/district/apparatus/organ system/district/apparatus/organ of interest. of interest.
The term The term "product “product having having(ascertained) (ascertained) therapeutic therapeutic properties properties comprising comprising 5 5 one or one or more morenatural naturalmatrices" matrices” or or “product "product forforthethe treatmentofofa apathological treatment pathological condition, said condition, said product productcomprising comprising oneone or more or more naturalnatural matrices” matrices" according according to the to the
present invention present invention is is aa product product as as defined defined above whereinthe above wherein the emerging emergingproperties propertiesofofsaid said productprovide provide a therapeutic effect as defined below below in this in this glossary. 2024203121
product a therapeutic effect as defined glossary.
Theterm The termcancanbebereplaced replaced in in anyany partpart of of thethe description description andand of the of the claims claims by by
10 10 "product havingtherapeutic "product having therapeuticactivities activitiescomprising comprising or consisting or consisting of complex of complex naturalnatural systems" "formulation systems" "formulationhaving having therapeutic therapeutic properties properties comprising comprising (or consisting (or consisting of) of) complex natural complex natural systems" systems"or orwith with "composition "composition having having therapeutic therapeutic properties properties comprising (or comprising (or consisting consisting of) of) complex natural systems” complex natural systems" or or with “mixture having with "mixture having therapeutic properties therapeutic properties comprising comprising (or (or consisting consisting of) of) complex complexnatural naturalsystems" systems” wherein wherein
15 15 the term the term "complex “complex natural natural systems” systems" can becansubstituted be substituted withor“one with "one more or more natural natural
matrices” or with “natural material”. matrices" or with "natural material".
The definition The definition applies, applies, mutatis mutatis mutandis, mutandis, to to thethe term term "product “producthaving having beneficial/healthy/beneficial beneficial/healthy/beneficial to to health healthproperties propertiescomprising comprising one one or more or more natural natural matrices”. matrices".
20 20 Accordingtotothethepresent According presentdescription description thethetermterm homeostasis homeostasis has thehasmeaning the meaning conventionally conventionally accepted accepted in art, in the the art, and refers and refers therefore, therefore, to the to the physiological physiological process byprocess by
whichliving which living organisms organismsmaintain maintaina astable stable internal internal environment environment despitedespiteexternal external changes. changes. Thisstability This stabilityisis crucial crucialforforthe theproper proper functioning functioning of cells, of cells, tissues, tissues, and organs. and organs. The goalThe goal of homeostasis of homeostasisisis to to ensure ensurethat that the the internal internal conditions conditions of of an an organism organismremainremain within within
25 25 optimal ranges optimal ranges for for survival survival and proper physiological and proper physiological functioning functioning(healthy (healthyphysiological physiological state). Homeostasis state). Homeostasis is is obtained obtained by byorganisms organismsbyby thethe modulation modulation of aofset a set of of biological biological
activities and activities processes and processes aimed aimed to maintain to maintain a healthy a healthy physiological physiological state. state. A product A product adjuvating adjuvating homeostatic homeostatic processes processes isis a aproductproductthat thatmodulates modulates biologicalactivities biological activitiesininthethe direction direction of aof a healthy healthy physiological physiological state,a hence, state, hence, product a product
30 30 that is that is suitable suitablefor fora ahealthy healthyindividual individualand and that thatsupports supportsthe thehomeostatic homeostatic mechanisms mechanisms
that contribute that contribute to to the the healthy healthy physiological physiologicalstate state and andthatthatcancanbebeused usedby by a healthy a healthy
individualtotoadjuvate individual adjuvate the the homeostatic homeostatic regulations regulations of biological of biological activitiesactivities associatedassociated to to hallmarks of hallmarks of aa given given pathological pathological condition. condition.
Medicaldevice, Medical device,alsoalsoMD, MD, according according to theto the present present description description is aisproduct a product as as
35 35 defined above, defined above,according accordingto tothethedefinition definitionininEUEU Regulation Regulation 2017/745 2017/745 Article Article 2 (1) 2 (1)
indents 1-3, indents 1-3, necessarily necessarily used used forfor therapeutic therapeutic purposes purposeshence, hence,'medical ‘medical device’ device' means means
any … [omissis] any [omissis] ...material materialintended intended by bythethe manufacturer manufacturer to to bebe used, used, alone alone oror in in combination,for combination, forhuman human beings beings for or for one onemoreor of morethe of the following following specific specific medical medical
22 10 May 2024
purposes: purposes: —treatment - treatment oror alleviationofofdisease, alleviation disease, — treatment, alleviation of, or compensation for, an injury or disability, - treatment, alleviation of, or compensation for, an injury or disability,
—modification - modification of of a a physiologicalororpathological physiological pathologicalprocess processororstate, state, 5 5 and which and whichdoes doesnotnot achieve achieve its its principalintended principal intendedaction actionby by pharmacological, pharmacological, immunological, or immunological, or metabolic metabolic means, means, in in or or on on the the human humanbody,body,butbutwhich whichmay maybe be assistedinin its assisted its function functionbybysuchsuch means. means.
‘Performance’ 'Performance' ofofa amedicalmedical device means the ability of a of a medical device device as 2024203121
device means the ability medical as
hereindefined herein defined to to achieve achieve its its intended intended purpose purpose as stated as stated by the by the manufacturer. manufacturer.
10 10 ‘Clinical 'Clinical performance’ performance' of of aa medical devicemeans medical device meansthetheability ability of of aa medical medicaldevice device as herein as herein defined, defined, resulting resulting from from any any direct direct or or indirect indirectmedical medical effects effectswhich which stem stem from from
its technical its technical ororfunctional functionalcharacteristics, characteristics, including including diagnostic diagnostic characteristics, characteristics, to achieve to achieve
its intended its purposeasasclaimed intended purpose claimed by the by the manufacturer, manufacturer, therebythereby leadingleading to a clinical to a clinical
benefitfor benefit forpatients, patients,when whenusedused as intended as intended by theby the manufacturer. manufacturer.
15 15 ‘Clinical 'Clinical benefit’ benefit'of ofthe themedical medicaldevice device means the positive means the positive impact impact of of aa device device asas herein defined herein definedononthethehealth health of of an an individual, individual, expressed expressed in terms in terms of a meaningful, of a meaningful,
measurable,patient-relevant measurable, patient-relevantclinical clinicaloutcome(s), outcome(s), including including outcome(s) outcome(s) related related to to diagnosis, or diagnosis, or aa positive positiveimpact impact on on patient patientmanagement orpublic management or publichealth. health. Also, if Also, if aa composition, (e.g. aa contrivance, composition, (e.g. contrivance, such such asasproduct producthaving havingtherapeutic therapeutic 20 20 properties comprising properties comprisingone oneor or more more natural natural matrices), matrices), is intended is intended to have to have a medical a medical
purpose, such as diagnosis, treatment, mitigation, or prevention of a disease or to affect purpose, such as diagnosis, treatment, mitigation, or prevention of a disease or to affect
the structure the structureororfunction function of of the the body, body, and itand it meets meets the criteria the criteria outlinedoutlined in the definition, in the definition,
it may it be classified may be classified asasa amedical medical device device byby the theUS US FDA. FDA.
Per Section Per Section 201(h)(1) 201(h)(1) of of the the Food, Drug, and Food, Drug, andCosmetic CosmeticAct, Act,a adevice deviceis: is: 25 25 Aninstrument, An instrument,apparatus, apparatus,implement, implement, machine, machine, contrivance, contrivance, implant, implant, in vitro in vitro
reagent, or reagent, or other other similar similar or or related related article, article, including including a componentpart, a component part,ororaccessory accessory whichis:is: which
(A) recognized (A) recognized in in thethe official official National National Formulary, Formulary, or or the the United UnitedStates States Pharmacopoeia,ororany Pharmacopoeia, anysupplement supplement to to them, them,
30 30 (B) intended (B) intendedfor for use useininthe thediagnosis diagnosisofofdisease diseaseororother otherconditions, conditions,or orin inthethe cure, mitigation, cure, mitigation,treatment, treatment, or or prevention prevention of disease, of disease, in maninorman otheroranimals, other animals, or or (C) intended (C) intended toto affect affect the structure structure or or any any function function ofof the the body body ofof man manororother other animals, and animals, andwhich whichdoes doesnotnot achieve achieve its its primary primary intended intended purposes purposes through through chemicalchemical action within action within oror on the body on the bodyofof manmanororother otheranimals animalsandand which which is not is not dependent dependent uponupon
35 35 being metabolized being metabolizedforforthethe achievement achievement of primary of its its primary intended intended purposes. purposes. The term The term "device" does not "device" does not include include software software functions functions excluded excludedpursuant pursuanttotosection section 520(o). 520(o). Theclassification The classification of of medical medicaldevices deviceswithin withina risk a riskclass classisistypically typicallybased basedonon factors such factors suchasasintended intended use,use, indications indications for use, for use, andassociated and risk risk associated with thewith the device. device.
23 10 May 2024
A product A productconsidered consideredasashaving having an ascertained an ascertained or known or known therapeutic therapeutic effect effect accordingto tothethe according present present invention invention meansmeans that itthat is aitproduct is a product for whichforatwhich at least least one batch one batch
(hereinafter: (hereinafter: reference standard) of reference standard) of said saidproduct productthat thatshows shows the the desired desired therapeutic therapeutic
effect at effect at least least in in vitro, vitro, e.g., e.g., in in laboratory settingsusing laboratory settings using cells,organoids, cells, organoids, tissues tissues and/or and/or in in 5 5 vivo vivo ononanimal animal models models or clinical or clinical trials trials (i.e. (i.e. a batch a batch whose therapeutic whose desired desired therapeutic activity activity has been has beenvalidated validatedat at least least in in vitro) vitro) exists. exists.
Theexpression The expressionhavinghaving an an ascertained ascertained or known or known beneficial/healthy/beneficial beneficial/healthy/beneficial
effect according according to to thethe present invention means means that it that is a itproduct is a product foratwhich at least one 2024203121
effect present invention for which least one
batch (hereinafter: batch (hereinafter: reference reference standard) standard) of of said said product product that that shows shows thethedesired desiredeffect effect of of 10 10 reinstatement/adjuvation ofofhomeostasis reinstatement/adjuvation homeostasis in the in the at least at least in vitro, in vitro, e.g., e.g., in laboratory in laboratory
settings using settings usingcells, cells,organoids, organoids, tissues tissues and/or and/or in vivo in vivo on animal on animal models ormodels clinical ortrials clinical trials (i.e. (i.e. aa batch batch whose desired whose desired beneficial beneficial activity activity has been has been validated validated at leastatinleast in vitro) vitro) exists. exists.
A subject A subject"in “inneed need thereof” thereof" as as used used herein herein refers refers to to a subject a subject thatthatcancan benefit benefit
from the from the therapeutic therapeutic and/or and/or prophylactic prophylactic effects effects of of the the therapeutical therapeuticalcompositions. compositions. Such Such
15 15 a subject a subject can be diagnosed can be diagnosedwith witha adisease diseaseorordisorder, disorder,aa subject subject suspected suspectedofofhaving havingoror developing a disorder or, and/or a subject determined to be at increased risk of having or developing a disorder or, and/or a subject determined to be at increased risk of having or
developing developing a disease a disease or disorder. or disorder.
By the By the terms terms"treat," “treat,”"treating," “treating,”oror"treatment “treatment of" of” (and(and grammatical grammatical variationsthereof) variations thereof)it itisismeant meantthatthat the the severity severity of theofsubject's the subject's condition condition is reduced, is reduced, at at 20 20 least partially least partially improved improved ororameliorated, ameliorated,and/orand/orthat thatsomesome alleviation, alleviation, mitigation mitigation or or decrease in decrease in at at least least one one clinical clinical symptom symptom is is achieved achieved and/or and/or therethere is aisdelay a delay in the in the
progression progression of of thethe disease disease or disorder. or disorder.
Compliance validation Compliance validation refers refers to to thevalidation the validationofofthe the batch-to-batch batch-to-batchcompliance compliance in the in the production productionprocess processofofa given a given product, product, i.e.,i.e.,thethe differentbatches different batches obtained obtained in in
25 25 productionmaintain production maintaintheir their compliance compliancewith withthe thereference referencestandard standard(also(alsodefined definedasas"gold “gold standard”ininindustrial standard" industrialproduction production processes) processes) desireddesired therapeutic therapeutic or beneficial or beneficial activities. activities.
Acceptabilityvalues Acceptability values(also(alsoknown known as acceptability as acceptability ranges ranges or cutoffs) or cutoffs) of the of the
spectroscopyororspectrophotometry spectroscopy spectrophotometry spectra spectra are are defined defined as the asvariability the variability acceptable acceptable
range from range fromthe theaverage averagespectrum spectrum obtained obtained fromfrom the the reference reference standard standard batchbatch spectrum spectrum
30 30 of aa product of productandand thethe spectra spectra of the of the acceptable acceptable batchesbatches of said of said product product and are preferably and are preferably
refined by refined by spectra spectraofofnon-acceptable non-acceptable batches batches according according to (c)to of (c)the of method the method of the of the
invention, under invention, underwhich which thethe deviation deviation valuevalue calculated calculated by overlapping by overlapping spectrumspectrum of of unknown unknown batch batch ofof product product tested(not tested (notvalidated validatedthrough throughcell-based cell-basedassay) assay)ononthetheaverage average spectrumisisdeemed spectrum deemed to maintain to maintain its compliance its compliance with a with a reference reference standard standard of said of said
35 35 product, i.e. product, i.e. toto maintain maintain the the desired desired formulation formulation and andtherapeutic/beneficial therapeutic/beneficial activity.As activity.As already stated already stated in in the the description description above, they are above, they are defined based on defined based onthethe deviation deviationof of said said aforementionedspectra aforementioned spectra(of (ofc)c) from fromthethe average averagespectrum spectrumobtained obtainedfromfrom them. them.
The'weakest The ‘weakestperformance' performance’ of a of a reference reference standard standard VS. a vs.reference a referencedrugdrug (i.e., (i.e., a a
24 10 May 2024
drug commonly drug commonly prescribed prescribed for for thethe treatment treatment of the of the same same pahtological pahtological condition) condition) refers refers
to the least therapeutic or least beneficial modulation value of a given biological activity to the least therapeutic or least beneficial modulation value of a given biological activity
calculated when calculated comparing when comparing thethe same same modulation modulation valuevalue in a in a cell-based cell-based assayassay according according
to the to the invention. invention. 5 5 A pathophysiological A pathophysiological ororphysio physiopathological pathologicalstatestaterefers refers to to an an abnormal abnormal physiological condition physiological conditionororprocess processwithin withinthethe body body that that is typically is typically associated associated with with
disease oror dysfunction. disease dysfunction.In In the the present present description description and claims and claims it isdefined it is also also defined “pathological state”.ItItinvolves involves thethe study of functional the functional changes thatasoccur as a result 2024203121
"pathological state". study of the changes that occur a result
of disease of diseaseororinjury injuryandand how how thesethese alterations alterations manifest manifest at the cellular, at the cellular, tissue,and tissue, organ, organ, and 10 10 systemic levels. systemic levels. Pathophysiology encompasses Pathophysiology encompasses thethe understanding understanding of both of both thethe underlying underlying
mechanisms mechanisms of of disease disease andand thethe body's body's response response to these to these disruptions disruptions to diagnose, to diagnose, treat, treat,
and manage and manage various various healthhealth conditions. conditions. Understanding Understanding pathophysiological pathophysiological states isstates is
crucial in crucial in medicine medicine forfor bothboth research research and clinical and clinical practice. practice.
Pathophysiologyinvolves Pathophysiology involves thethe study study of various of how how various factors,factors, such as such as genetic genetic
15 15 abnormalities, environmental abnormalities, environmental influences, influences, or disease or disease processes, processes, disruptdisrupt the normal the normal
physiological functions physiological functions of of the the body, body,leading leadingtotothe thedevelopment developmentof of health health disorders disorders or or diseases.Understanding diseases. Understanding physiophysio pathological pathological states isstates is essential essential for diagnosing, for diagnosing, treating, treating, and managing and managing a wide a wide range range of medical of medical conditions conditions across different across different specialties specialties in in healthcare. healthcare.
20 20 Different batches Different batchesororlotslotsofofaaproduct product ininthethepresent presentdescription descriptionasasininthethestate state of the of theart, art, refer refer totodistinct distinctgroups groupsof of items items produced produced or manufactured or manufactured at times at different different times or under or underdifferent different conditions, conditions, but still but still belonging belonging to the to samethe sameline. product product line. In different In different
batches,the batches, thestarting startingrawraw materials materials canfrom can be be the from thestocks same sameorstocks or fromstocks. from different different stocks. Eachbatch Each batchor or lotlot typically typically receives receives a unique a unique identifier identifier which distinguishes which distinguishes it from other it from other
25 25 batches. These batches. identifiers help These identifiers help inin quality quality control, control, inventory management,and inventory management, and traceability throughout traceability throughout the the production production process process and supply chain. and supply chain. Batchesof Batches of products productscomprising comprisingoneone or or more more natural natural matrix matrix or consisting or consisting of of oneone
or more or morenatural naturalmatrix matrixare are expected expected to varyto in vary theirin qualitative their qualitative and quantitative and quantitative
chemicalcomposition chemical composition due due to factors to factors like like raw material raw material variations. variations. In addition, In addition, the the 30 30 batches can batches can vary vary as as any any other other product productbatch, batch, due due to to production productionconditions, conditions, or or equipment equipment used. used.
DETAILEDDESCRIPTION DETAILED DESCRIPTIONOF OFTHE THEFIGURES FIGURES Legendofofthe Legend thebatches: batches: 35 35 Figures2-7 Figures 2-7and and12: 12: Product Arté-Gx Product Arté-Gxininlyophilised lyophilisedform form(see (seedetailed detailed composition compositionofofthe theproduct productinin Example1): Example 1):
25 10 May 2024
Batch 20B1955 Batch 20B1955 alsoL L also 20B1955 20B1955 reference reference standard standard (indicated (indicated in the in the figuresasasGold figures Gold Standard) Standard)
Batch 20B0596 Batch also LL 20B0596 20B0596 also 20B0596
Batch 2011297 Batch 20I1297also alsoLL2011297 20I1297 5 5 Batch 21E1640 Batch also LL 21E1640 21E1640 also 21E1640
Batch 20J1770 Batch 20J1770also alsoL L20J1770 20J1770 Batch Dest Dest 21E1640 2024203121
Batch 21E1640
Figure99Product Figure ProductBBreference referencestandard standard(indicated (indicatedin in the the figures figures as asGold Gold Standard) Standard)
Figure1111Product Figure ProductC Creference referencestandard standard(indicated (indicatedinin the the figures figures as as Gold Gold Standard) Standard)
10 10 (see detailed (see detailed composition of the composition of the products products in in Example 1). Example 1).
In the figures 2-4, 9 and 11, the modulations values were calculated as Z-scores In the figures 2-4, 9 and 11, the modulations values were calculated as Z-scores
as described as describedininthethespecification specification andand in the in the examples). examples).
Figure 1 Exemplification of hallmarks of osteoarthritis including the trend of the Figure 1 Exemplification of hallmarks of osteoarthritis including the trend of the
hallmarkrepresenting hallmark representingananimprovement improvement of diseased of the the diseased state,state, (column (column 1), biological 1), biological
15 15 activities concurring activities to each concurring to eachdisease disease (column (column 2) modulations 2) and and modulations thereof thereof in the in the pathological conditions pathological conditions (column (column3)3)and and modulation modulation of each of each of said of said biological biological activity activity
representative of representative of aa healthy healthy physiological physiologicalstate state(column (column4) 4) dark dark grey: grey: up modulation up modulation
light grey: light grey:down modulation. down modulation.
Figure2 2Modulation Figure Modulation of selected of selected biological biological activities activities in in a chondrocytes a chondrocytes cell-cell-
20 20 based assay based assayinin osteoarthritis: osteoarthritis: column column 11hallmarks hallmarksincluding including thethe trend trend of of thethe hallmark hallmark
representing an representing animprovement improvement of diseased of the the diseased state,state, column column 2 biological 2 biological activities, activities,
column3 3predicted column predictedmodulation modulation of of biological biological activitiesininpathological activities pathologicalstate, state, column column4 4 desiredmodulation desired modulation of biological of biological activities activities in healthy in healthy physiological physiological state,5column state, column cell- 5 cell- based assay based assaywithout withouttherapeutic therapeutictreatment, treatment, representative representative of of thethe biological biological activities activities
25 25 modulationofofthe modulation thepathological pathologicalstate, state,column column 6 modulation 6 modulation induced induced by thebyreference the reference standard of standard of the the tested tested product product .TheThe cell-basedassay cell-based assay shows shows thatthat thethe reference reference standard standard
modulatesthe modulates theselected selectedactivities activitiesaccording accordingto to thethe healthy healthy physiological physiological state. state. The The
numbersreported numbers reportedinineach eachsquare squarerepresent representa aZ-score Z-scorecalculated calculatedwith withthethemethod method of the of the
inventionrepresenting invention representing the the modulation modulation of eachofbiological each biological activity activity observed.observed.
30 30 Figure3 3Modulation Figure Modulation of selected of selected biological biological activities activities in in a chondrocytes a chondrocytes cell-cell-
based assay based assay representative representative of of osteoarthritis: osteoarthritis: column column 11 hallmarks hallmarksincluding includingthe thetrend trendofof the hallmark the hallmarkrepresenting representingananimprovement improvement of the of the diseased diseased state, state, column column 2 biological 2 biological
activities, column 3 predicted modulation of biological activities in pathological state, activities, column 3 predicted modulation of biological activities in pathological state,
column4 4desired column desired modulation modulation of biological of biological activities activities in healthy in healthy physiological physiological state, state,
35 35 column column 5 cell-based 5 cell-based assay assay without without therapeutic therapeutic treatment, treatment, representative representative of the biological of the biological
activities modulation activities modulation ofof the thepathological pathologicalstate, state,column column 6 modulation 6 modulation induced induced by a by a
26 10 May 2024
reference drug reference (Triamcinolone Acetonide), drug (Triamcinolone Acetonide), column column 7 7modulation modulationinduced induced by by the the reference standard reference standardofofthe thetested testedproduct, product,columns columns 8-118-11 modulation modulation induced induced by fourby four different batches different batches of of the thetested testedproduct. product.Column Column 11 represents the 11 represents the modulation inducedbyby modulation induced
an intentionally an intentionally destabilised destabilised batch batch(DEST (DEST 21E1640) showingweaker 21E1640) showing weaker therapeutic therapeutic 5 5 activity. The activity. cell-based assay The cell-based assayshows shows thatthat thethe reference reference standard standard andadditional and the the additional batchessimilarly batches similarly modulate modulate the selected the selected activities activities according according to the physiological to the healthy healthy physiological state. The state. numbers The numbers reported reported in each in each squaresquare represent represent the Z-score the Z-score value calculated value calculated
representingthethemodulation modulation of each biological activity observed. 2024203121
representing of each biological activity observed.
Figure44Corresponds Figure Correspondstoto figure3 3plus figure pluscolumn column1212 showing showing the the control control analysis analysis of of
10 10 the modulation the inducedbyby modulation induced thethe nonnon destabilised destabilised batch batch (21E1640) (21E1640) that that was was validated validated as as compliantaccording compliant accordingtotothethe process processofof the the invention. invention. The cell-based assay The cell-based assay shows showsthat that the the reference standard reference standard and andthe theadditional additional batches batchessimilarly similarly modulate modulatethetheselected selectedactivities activities according toto the according the healthy healthyphysiological physiologicalstate stateand andtherefore thereforeconfirms confirmsthethecompliance complianceof of
the batch the that was batch that validated with was validated with the the method methodofofthe theinvention. invention.The Thenumbers numbers reported reported in in
15 15 each square each squarerepresent representthethe Z-score Z-scorevalue value(modulation (modulation value) value) calculated calculated representing representing thethe
modulationofofeach modulation each biological biological activityobserved. activity observed. TheThe reference reference Z score Z score valuesvalues were were calculated according calculated according toto the the invention invention considering considering thethe Reference standardZ-score Reference standard Z-scorevalues values (column (column 8 8of offigure figure4)4) and andthe thedrug drugZ-score Z-scorevalues values(column (column 9 figure 9 of of figure4) 4) selecting selecting as as
reference Z-score reference Z-score value value(column (column1313 of of figure4)4)forforeach figure eachmodulated modulated biological biological activity activity
20 20 the Z-score the value associated Z-score value associated to to the the weakest weakestperformance performance as as depicted depicted in in thetable the tablebelow below (gold (gold standard=reference standard): standard=reference standard):
Reference drug Triamcinolone Gold standard Hallmarks of the pathology Biological functions Reference Z-score values acetonide Z-score values
Drug Z-score values
Skeletal and muscular system
development and function
Induction of proliferation articular dysfunction joint -0.31 0.15 0.15 space Formation of cartilage tissue
Inflammatory disease
Reduction of inflammation inflammation and nociceptions -0.26 -0.24 -0.24 Inflammation of joint
Skeletal and muscular system
function difficulty moving a
joint Non-traumatic -0.17 -0.32 -0.17 arthropathy Protection from anatomical Organismal injury and damage abnomalities joint inflammation and swelling, -0.15 -0.31 -0.15 difficulty moving a joint
Osteoarthritis
As the As the therapeutic therapeutic effect effect is is ascertained ascertained both both for for the the product productasaswell wellasasfor forthe the drug, it drug, it isis correct correctto toaccept, accept,when both the when both the reference reference drug drugand andthethereference referencestandard standard 25 25 provide modulation of a selected biological activity in the same direction of the healthy provide modulation of a selected biological activity in the same direction of the healthy
27 10 May 2024
physiological state, physiological state, the the weakest weakestperformance performance z-score z-score as reference as reference cut-offcut-off for for that that modulation. modulation.
Figure55Targeted Figure Targetedmetabolomics metabolomics of five of five batches batches (including (including reference reference standard) standard)
of Arté of Arté GxGxmain main chemical chemical classes. classes. The The figure figure clearly clearly showsshows thatbatch that each each tested batch tested 5 5 differs from differs eachother from each otherandand fromfrom the reference the reference standardstandard in the in the quali-quantitative quali-quantitative
composition.Comparison composition. Comparison withwith teststests of the of the selected selected biological biological activities activities of of the the samesame
batches on batches onthe thecell-based-assay cell-based-assay(figures (figures3 and 3 and4), 4), shows shows that that the quali-quantitative the quali-quantitative 2024203121
analysisofofdifferent analysis differentbatches batches of aof a product product fortreatment for the the treatment of a pathological of a pathological condition, condition,
the product the comprisingororconsisting product comprising consistingofofone oneorormore morenatural naturalmatrices matricesdoes does not not allow allow to to
10 10 correctlyestimate correctly estimateitsitsactivity activityprofile. profile. Figure666a. Figure 6a. NIR NIRspectrum spectrumof of thereference the referencestandard standardandand threedifferent three differentpositive positive batches selected batches selected in iv) iv) in inthe thewavelength wavelength range range of interest. interest.6b. 6b.NIR NIRaverage average spectrum of spectrum of
the four the four batches batches ofof 6a. 6a. and andNIR NIR spectrum spectrum of negative of negative batchbatch 21E1640 21E1640 Dest, Dest, 6c. NIR6c. NIR average spectrum average spectrumofofthe thefour four batches batches of of 6a.and 6a.and two twoaa priori priori undesired spectra (undesired undesired spectra (undesired
15 15 A Centella A Centella asiatica asiatica extract, extract,undesired undesiredBB Echinacea extract) 6d. Echinacea extract) 6d. NIR average spectrum NIR average spectrumofof the four the four batches batches of of 6a. 6a. and andNIR NIR spectrum spectrum of unassessed of unassessed batch batch 21E1640 21E1640 (not (not destabilised). destabilised).
Figure7 7ROS Figure ROS scavenging scavenging activity activity of 4 of 4 validated validated batches batches including including reference reference
standard of standard of Arté-Gx. Arté-Gx.The Thequantity quantityofofROS ROS detected detected in in samples samples treated treated with with AAPH AAPH was was 20 20 considered as considered as the the maximum release maximum release of of ROS ROS (100%) (100%) andquantity and the the quantity of ROS of ROS detected detected in in the other the other samples sampleswaswascalculated calculatedas aspercentage percentage with with respect respect to the to the maximum maximum releaserelease
induced by induced by AAPH. AAPH. One-way One-way ANOVAANOVA and Dunnett's and Dunnett's post-test post-test were applied. were applied. Only Only values capable values capableofofreturning returningp<0.05 p<0.05were were considered considered significant significant (*p<0.05, (*p<0.05, **p<0.01, **p<0.01,
***p<0.001 , ****p<0.0001). ***p<0.001 , ****p<0.0001). Statistical Statistical significance significance of all samples of all samples vs untreated VS untreated cells. cells. 25 25 The figure The figure demonstrates demonstrates that that the the validated validated batches batches are, are, indeed, indeed, inducing inducing ROS ROS scavenging activity as desired for the intended therapeutic use. scavenging activity as desired for the intended therapeutic use.
Figures8a8aand Figures and8b 8b Exemplification Exemplification of hallmarks of hallmarks of mild of mild cognitive cognitive impairment impairment
(MCI),(column (MCI), (column1),1), biological biological activitiesconcurring activities concurring to to each each disease disease (column (column 2) and2) and modulationsthereof modulations thereofininthe thepathological pathologicalconditions conditions(expressed (expressed as as alterationofofhealthy alteration healthy 30 30 physiological state) physiological state) (column (column3)3)andand modulation modulation of of of each each saidofbiological said biological activies activies representative of representative of aa healthy healthy physiological physiologicalstate state(column (column 4) 4) darkdark grey: grey: up modulation up modulation
light grey: light grey:down modulation. down modulation.
Figures9a9aand Figures and9b9b MCI. MCI. Modulation Modulation of selected of selected biological biological activitiesinina ahuman activities human neuronal cell-based neuronal cell-based assay assay(cells (cells SH-SY5Y): SH-SY5Y): column column 1 hallmarks, 1 hallmarks, column column 2 biological 2 biological
35 35 activities, column 3 predicted modulation of biological activities in pathological state, activities, column 3 predicted modulation of biological activities in pathological state,
column4 4desired column desired modulation modulation of biological of biological activities activities in healthy in healthy physiological physiological state,state,
column5 5modulation column modulation induced induced by reference by the the reference standard standard of tested of the the tested product product B. TheB. The
28 10 May 2024
cell-based assay cell-based assay shows showsthat thatthethe reference reference standard standard modulates modulates the selected the selected activities activities
according totothe according thehealthy healthyphysiological physiological state.TheThe state. numbers numbers reported reported insquare in each each square represent aa Z-score represent Z-scorecalculated calculated with with the the method method of the of the invention invention representing representing the the modulationofofeach modulation eachbiological biologicalactivity activity observed. observed.
5 5 In figures 9a and 9b the tested product B used is described in example 1. In figures 9a and 9b the tested product B used is described in example 1.
Figures 10a Figures 10aand and10b10b Exemplification Exemplification of of hallmarks hallmarks of osteoporosis of osteoporosis (OP) (OP) including the including the trend trend of of the the hallmark hallmark representing representingananimprovement improvement of the of the diseased diseased state, state, 2024203121
(column (column 1),1), biological biological activities activities concurring concurring to to each hallmark (column each hallmark (column2)2)andand modulationsthereof modulations thereofininthe thepathological pathologicalconditions conditions(expressed (expressed as as alteration alteration of of healthy healthy
10 10 physiological state) physiological state) (column (column3)3)andand modulation modulation of each of each of biological of said said biological activities activities
representative of representative of aa healthy healthyphysiological physiologicalstate state(column (column 4) 4) dark dark grey: grey: up modulation up modulation
light grey: light grey:down modulation. down modulation.
Figures 11a Figures 11a and and11b11b OP.OP. Modulation Modulation of selected of selected biological biological activitiesinin activities
adipocyte-derived, mesenchymal adipocyte-derived, mesenchymal stem stem cellcell lines lines (hADMSC), (hADMSC), capable capable of differentiating of differentiating
15 15 into osteoblasts into osteoblasts and and mineralize mineralizethe theextracellular extracellular matrix matrix(ECM): (ECM): column column 1 hallmarks, 1 hallmarks,
column 2 biological activities, column 3 predicted modulation of biological activities in column 2 biological activities, column 3 predicted modulation of biological activities in
pathological state, pathological state, column column4 desired 4 desired modulation modulation of biological of biological activities activities in healthy in healthy
physiological state, physiological state, column column 55 modulation modulationinduced inducedbybythethereference referencestandard standardofofthe thetested tested product C.C.TheThe product cell-based cell-based assayassay showsshows thatreference that the the reference standard standard modulatesmodulates the the 20 20 selectedactivities selected activitiesaccording according to the to the healthy healthy physiological physiological state. state. The The reported numbers numbersinreported in each square each square represent represent aa Z-score calculated with Z-score calculated the method with the method of of the the invention invention representing representing the modulation the modulation of each of each biological biological activity activity observed. observed.
In figures In figures 11a 11a and and 11b the tested 11b the tested product product C C used is described used is described in in example example 11
Figure1212modulation Figure modulation of biological of the the biological activity activity oxidative oxidative stress stress (hallmark (hallmark
25 25 inflammation)ofof44validated inflammation) validated batches batchesincluding includingreference referencestandard standardofofArté-Gx. Arté-Gx.Ascorbic Ascorbic acid has acid has been beenused used as as positive positive control control and and insult insult with with AAPH AAPH as as negative negative control control representativeofofpathological representative pathological state. state. As clear As clear from from the the figure, figure, also thealso thea use use of of a different different
parameter parameter forfor monitoring monitoring the modulation the modulation of the biological of the biological activity related activity causally causally to related the to the pathologicalhallmark pathological hallmark of osteoarthritis of osteoarthritis (OA) (OA) is is suitable suitable for validating for validating the batchestheasbatches an as an 30 30 alternativetototranscriptomics alternative transcriptomics parameters. parameters.
Figure13 Figure 13Biophysical Biophysicalcharacterization characterizationofofbiological biological vegetal vegetal material material total total RNA. RNA.
Figure 13 Figure 13 shows showsthe theelectropherogram electropherogramof of theproduction the production intermediate intermediate of of product product A, A, Arté Arté
GX, i.e., the Centella asiatica and Echinacea water coextract in the proportions depicted GX, i.e., the Centella asiatica and Echinacea water coextract in the proportions depicted
in example in example 1,1,before beforeultrafiltration, ultrafiltration, panel panel “A” showsthethesize "A" shows sizedistribution distributionofofthe thetotal total 35 35 RNA RNA and and panel panel "B"“B” shows shows the the RNA RNA size distribution size distribution between between 4 and4150 andnt. 150 nt. Figure141414a Figure 14aRAMAN RAMAN spectrum spectrum of the reference of the reference standardstandard and threeanddifferent three different positive batches positive batches selected selectedininiv) iv)ininthe thewavelength wavelength range range of interest. of interest. 14b.14b. RAMANRAMAN
29 10 May 2024
average spectrum average spectrumofofthe thefour fourbatches batchesofof14a. 14a.and andRAMAN RAMAN spectrum spectrum of negative of negative batch batch 21E1640Dest, 21E1640 Dest, 14c. 14c. RAMAN average RAMAN average spectrum spectrum of ofthethe fourbatches four batchesofof 14a.and 14a.and two two aa priori undesired priori spectra (undesired undesired spectra (undesired AACentella Centellaasiatica asiatica extract, extract, undesired undesired BBEchinacea Echinacea extract) 14d. extract) 14d. RAMAN average RAMAN average spectrum spectrum of theoffour the four batches batches of 14a. of 14a. andspectrum and NIR NIR spectrum 5 5 of unassessed of batch 21E1640 unassessed batch 21E1640 (notdestabilised). (not destabilised).
Thepresent presentinvention inventionprovides provides methods and and processes forbatch the batch to batch 2024203121
The methods processes for the to batch
validation of validation of the the quality quality compliance complianceofofproducts products comprising comprising or consisting or consisting of natural of natural
matrices, such matrices, as products such as consisting of products consisting of 100% naturalmaterial 100% natural materialand andexerting exertingtherapeutic therapeutic 10 10 or beneficial or beneficial activity activity (( the the latter latterbyby allowing allowing the the homeostatic rebalancingofofphysio- homeostatic rebalancing physio- pathological states pathological states or or slight slightimbalances imbalances inin living living beings beings such as the such as the human organism), human organism),
through the through the provision provision of of spectroscopy spectroscopyororspectrophotometry spectrophotometry acceptability acceptability values values based based
on measurements on measurements of of thethe modification modification of of specific specific biologicalactivities biological activitiesconcurring concurringtotothe the therapeuticororbeneficial therapeutic beneficial effect. effect. TheThe Applicant Applicant hereinherein hence provides hence provides for time for the first the first a time a 15 15 method and method anda aprocess processwhichwhichwillwillallow allowtotostandardise standardise said said kind kind ofof products products and and therefore allow therefore to comply allow to complywith withthetheregulatory regulatoryframeworks frameworks for for granting granting a viable a viable space space
for innovative for uses of innovative uses of said said products productsinin therapy. therapy. InIn the the past past decades decadesthe theApplicant Applicantdiddid pursue an pursue an intersectoral intersectoral research path which, research path which, along alongwith withscientific scientific innovations, innovations, resulted resulted in the in the development development ofofeubiotic eubioticprotocols protocolsfor for agricultural agricultural production and transformation production and transformation 20 20 processes of processes of raw rawsources sources(starting (starting materials) materials) totopreserve preservetheir theirbasic basicnatural natural programmaticrules, programmatic rules,which whichhave haveallowed allowed interconnection interconnection forfor millionsofofyears millions yearsamong amongallall
the components the components ofofliving living things, things, organic organic and inorganic. and inorganic.
Followingthe Following theteachings teachingsofofthethepresent present invention invention it isnownow it is possible possible to assess to assess
precise parameters precise to validate parameters to validate the the batch-to-batch batch-to-batch compliance of products compliance of productswith withemerging emerging 25 25 properties resulting properties resulting in in aa physiological physiologicaltherapeutic therapeuticor or beneficial beneficial activity,when activity, when the the product comprises product comprisesororconsists consists of of one or more one or natural matrices. more natural matrices. This is of particular interest for all the products for therapeutic/beneficial use This is of particular interest for all the products for therapeutic/beneficial use
that are that not based are not basedononclassical classicalpharmacological pharmacological formulation formulation "APIs“APIs plus excipients”, plus excipients",
such as such as products productsthatthat comprise compriseororconsists consistsofofoneoneorormore more natural natural matrices. matrices. TheThe mainmain
30 30 problem with problem withthisthiskind kind of products of products is that,is that, notwithstanding notwithstanding the important the important therapeutic/beneficial therapeutic/beneficial effects effectsexerted, exerted,ininthetheabsence absenceofofrepeatable repeatableandandprecise precisemethods methods
for aa batch for batch to to batch batch validation, validation,ensuring ensuringthethecompliance compliance of of the the product product with with the the claimed claimed
therapeutic/beneficial effect, therapeutic/beneficial effect, which, which, due due to their to their very very originorigin (nature) (nature) presentpresent an intrinsic an intrinsic
variability variability inin their their quali-quantitative quali-quantitative composition, cannotbe be composition, cannot correctly correctly assessed assessed by by
35 35 classical validation classical validation processes based onona amere processes based mere quali-quantitative quali-quantitative composition. composition. Up toUp to now,in now, in fact, fact, itithas hasnot notbeen been possible possible to tobring bringproducts products comprising comprising oror consisting consisting ofof one one
or more or morenatural naturalmatrices matricesinina aregulatory regulatoryframework framework thatthat would would take take this this intointo account account
30 10 May 2024
and allow for a high degree of innovation. At present, this kind of products are validated and allow for a high degree of innovation. At present, this kind of products are validated
following standard following standardprocedures proceduressuch suchasasquantification quantificationofofone oneorormore more chemical chemical classclass of of
compounds compounds which which doesdoes not not represent represent the the complexity complexity and full and full effectiveness effectiveness of products of products
with emerging with emergingproperties properties suchsuch as natural as natural matrices-based matrices-based products. products. This problem This problem is is 5 5 solvedbybythethepresent solved present invention. invention.
As the As the method methodandand process process of the of the invention invention are are designed designed for products for products comprising comprising or or consisting consisting of natural of natural matrices, matrices, it is preferred it is preferred that thethat the production production of these of these
products isiscarried carriedoutout standardisingthe the entire production processprocess both in both in the 2024203121
products standardising entire production the
agricultural and agricultural and manufacturing processes. manufacturing processes.
10 10 The inventors The inventors herein herein demonstrate demonstrate thatthat an an approach approachbasedbasedexclusively exclusivelyonon reproducibility assays reproducibility assayswith with classical classical targeted targeted metabolomics, metabolomics, such assuch as the quali- the quali-
quantitative analysis quantitative analysisofof aa number number of of chemical chemicalclasses classes ororofofspecific specificchemical chemical compounds compounds of of a product a product with with therapeutical therapeutical or beneficial or beneficial effects,judged effects, judged using using criteria criteria
that are that are designed designed for for the the managementof managementof single single APIsAPIs acting acting via via the the deterministic deterministic key-key-
15 15 lock mechanism lock mechanism thanks thanks to the to the existence existence of aofclear a clear SAR,SAR, S not ssuitable not suitable for a for a quality quality
control when control whenthe theproduct productcomprises comprises or consists or consists of of oneone or more or more natural natural matrices matrices i. Ini. In fact, the fact, the results resultsobtained obtainedby by the the inventors inventors from from the analysis of of the the chemical classes of chemical classes of different batches different batches ofofthe thesame same product, product, when when confronted confronted with selected with selected biologicalbiological
activities (underlying activities (underlying thethe therapeutic therapeutic or beneficial or beneficial effect)effect) of eachof eachshow batch, batch, that show the that the 20 20 quantitativefluctuations quantitative fluctuations of of thethe individual individual chemical chemical classesclasses of substances of substances that are that are present present
in said in said different differentbatches, batches, if if used used as the as the means means for assessing for assessing the ofquality the quality of said batches said batches
according toto criteria according criteria applied applied to to APIs wouldlead APIs would leadto toan an a prioriassumption a priori assumption thatthat these these
batches have batches haveaadifferent different biological biological activity activity from oneanother from one another(see (seeexample example 3.13.1 second second
table and table andfigure figure5).5).TheThe experiments experiments carriedcarried out by out the by the inventors inventors on different on different batches of batches of
25 25 a therapeutic a therapeutic product product comprising comprising one oneorormore morenatural naturalmatrices matrices(cfr. (cfr. example example3.1 3.1and, and,inin particular, figures particular, figures3,3,4 4and and 5)5) onon the the contrary, contrary, demonstrate demonstrate that,that, notwithstanding notwithstanding their their variable quali-quantitative variable quali-quantitative chemical composition,said chemical composition, saidbatches batches nevertheless nevertheless entail entail thethe
sametherapeutic same therapeuticactivity, activity, with withaaresilient resilient behaviour behaviourpossibly possiblydue dueto to thethe presence presence of of
redundancyamong redundancy among thethe single single components components at both at both structural structural andand functional functional level. level.
30 30 Thus,the Thus, theexperiments experiments carried carried out out by theby the inventors inventors (in particular, (in particular, cfr. figures cfr. figures 3, 4 3, 4 and 5) and 5) show showthat,that,when when considered considered alone, alone, the the quali-quantitative quali-quantitative analysis analysis of different of different
batches of batches of aa product productfor forthe thetreatment treatmentofofa apathological pathologicalcondition, condition,does doesnotnot allowallow to to
correctly estimate correctly estimate the the activity activityprofileprofilewhen when the the product product comprises comprises or or consists consists of of one or one or
morenatural more naturalmatrices. matrices.Hence, Hence,thethedata data provided provided herein herein demonstrate demonstrate that,that, contrary contrary to to
35 35 pharmaceuticalproducts pharmaceutical productsbased basedonon traditional(i.e., traditional (i.e., synthetic chemical) APIs, the chemical) APIs, the quali- quali- quantitativeanalysis quantitative analysis of of thethe individual individual constituents constituents of a therapeutical of a therapeutical productproduct comprisingcomprising or consisting or consisting of of one one or or more morenatural natural matrices matrices(i.e.(i.e. providing providing aa therapeutic therapeutic effect effect based based
on aa network on networktotonetwork network interaction),when interaction), when performed performed as a as a method method for assessing for assessing the the
31 10 May 2024
therapeutic and therapeutic and quality quality reproducibility reproducibility ofofa anon-SAR-based non-SAR-based entity, entity, is not is not suitable suitable to to evaluate the correct compliance batch to batch of said product. evaluate the correct compliance batch to batch of said product.
In fact, In fact, given given the very nature the very nature of of aa natural natural matrices, matrices, the batch to the batch to batch batch quantitativelyvariable quantitatively variable chemical chemical profiles, profiles, wouldwould prompt prompt the validator the validator to considerto various consider various 5 5 batches of batches of aa product productthatthatcomprises comprises or or consists consists of of oneone or ormoremore natural natural matrices matrices as a as a
“different material” "different with respect material" with respecttotoa agiven givenreference reference standard standard of said of said product, product, and and
therefore to therefore to discard discard said said batches, batches, while, while, ononthethecontrary, contrary,the theresults resultsprovided providedbyby thethe
inventors herein, herein, demonstrate demonstratethat thatininbiological biologicalsystems, systems,quali-quantitatively quali-quantitativelydifferent different 2024203121
inventors
batchesofofa agiven batches given product product exertexert the same the same relevant relevant effect effect for theirforintended their intended therapeutical therapeutical
10 10 use. This use. This isis another anotherdemonstration demonstration thatthat the the therapeutic therapeutic or beneficial or beneficial activity activity of a of a
complexmatrix complex matrix cannot cannot be traced be traced back back to thetosumtheof sum of the activities the activities of each of each single single
moleculewithin molecule withinthethe matrix matrix itselfitself and that, and that, when natural when natural matricesmatrices are involved, are involved,
differently from differently classical APIs, from classical APIs,thethereproducibility reproducibilityof of thethe therapeutic therapeutic or beneficial or beneficial
activity of activity of the thematrix matrixdoes does not not exclusively exclusively dependdepend on reproducibility on reproducibility of theandidentity and of the identity
15 15 quantity of quantity of the the molecular molecular components components which which constituteit. constitute it. Theresults The results obtained obtained by bythe theinventors inventorsdemonstrate demonstrate thatthere that thereare areboth bothstructural structural and functional and functionalredundancy redundancy mechanisms mechanisms withinwithin naturalnatural matrices matrices that to that confer confer said to said matrices aa particular matrices particular functional functional resilience, resilience, and that that and that that matrix matrix per perseseshould shouldnotnot be be
perceived asas aacompilation perceived compilationofofmolecules molecules acting acting independently independently from from one another one another as if as if
20 20 they were they werestill still subject subject to tothe theSAR that is SAR that is commonly attributedtotosuch commonly attributed suchmolecules molecules when when
theyare they arestudied studiedininisolation. isolation. As shown As shownininthe thepresent presentdescription description (see (see examples examplesand andfigures), figures), different different batches batches
of aa tested of tested product product that that comprises comprisesororconsists consistsofofoneoneor or more more natural natural matrices, matrices, with with
ascertained differences ascertained differences in in their their quali-quantitative quali-quantitative composition composition one onefrom fromthetheother otherandand 25 25 from aareference from referencestandard standardofofsaidsaidproduct, product,cancan retainthethe retain abilitytotomediate ability mediate thethe same same
therapeutic ororbeneficial therapeutic beneficial activity activity notwithstanding notwithstanding their different their different quantitative quantitative compositions compositions at the at the molecular molecular level. level.
Theauthors The authorsof of the the invention invention therefore, therefore, developed developed aa newnewmethod method forfor defining defining thethe
acceptability values of acceptability of aa spectroscopy spectroscopyororspectrophotometry spectrophotometry analysis analysis thatthat areare suitable suitable
30 30 for the for the compliance validation of compliance validation of one one oror more batchesofofaa product more batches productfor for the thetreatment treatmentofof aa pathological condition, pathological condition, wherein whereinthe theproduct productcomprising comprising or consisting or consisting of one of one or more or more
natural matrices; natural matrices; andand aaprocess processfor forthe thecompliance compliance validation validation (e.g. (e.g. quality quality control control in in
industrial production industrial production processes) processes)of of different different batches batches of a of a product product comprising comprising or or consistingofofoneone consisting or or more more natural natural matrices, matrices, that isthat is on based based on the conservation the conservation of selectedof selected
35 35 intrinsic characteristics intrinsic characteristicsofofthethematrices matrices(the(the modulation modulation of selected of selected biological biological features),features),
rather than rather than on the absolute on the absolute identity identity of their their molecular components.The molecular components. The method method of the of the
invention allows invention allows the the definition definition of of validation validation acceptability acceptability values values based based onon the the analysis analysis of selected of selected parameters parameters to to which whichthe themaintenance maintenance of of a a desiredoverall desired overallbiological biologicalactivity activity
32 10 May 2024
is, as demonstrated in the present description, effectively correlated. is, as demonstrated in the present description, effectively correlated.
Thepresent The presentdescription description discloses discloses in in the the examples examplesexperimental experimentaldata dataonon a model a model
product herein product herein indicated indicated as as Arté-GX Arté-GXororproduct productA A (see (see examples examples for for thethe composition composition of of
the product), the product), which which is is for for the thetreatment treatmentof ofosteoarthritis osteoarthritis (disclosed (disclosedin WO in WO2018/138678) 2018/138678)
5 5 and substantially and substantially consists consists of of plant plant derived derived natural natural matrices. matrices. Faced with the Faced with the problem problemofof satisfying satisfying the regulatory requirements the regulatory requirementsforforbringing bringing thethe aforementioned aforementioned productproduct into into
therapy, the therapy, the inventors inventors tested tested thetheclassical classical validation validationmethods methods andand realised realised thatthat saidsaid
methods(based (basedononthe theanalysis analysisofof the the quali-quantitative quali-quantitative chemical composition)were werenotnot 2024203121
methods chemical composition)
suitablefor suitable forcorrectly correctlyassessing assessing the the therapeutic therapeutic effecteffect of theof the product product (compare(compare figures 3, figures 3,
10 10 4 and 4 and5).5). Theinventors The inventorstherefore therefore developed developedthethemethod method andand process process herein herein disclosed disclosed and and
found results found results consistent consistent with the ones with the ones reported reported herein hereinalsoalso on onother otherdifferent different products, products, withtherapeutical with therapeutical effects, effects, comprising comprising or consisting or consisting of one of or one more or morematrices. natural natural matrices. All the All the products products tested tested disclosed disclosed in the in the present present description description consistconsist of or comprise of or comprise
15 15 natural matrices natural matrices andandare aretherapeutic therapeuticororbeneficial beneficialproducts. products. TheThe matrices matrices comprised comprised
therein were therein obtainedfrom were obtained fromplant plantbiological biologicalmaterial materialsuitably suitably processed processedand andformulated formulated to obtain to a final obtain a final natural natural material material (as (as defined defined in in the the glossary) glossary) which is able which is able to to modify, modify,
uponadministration, upon administration,a pathological a pathological state state or altered or an an altered physiological physiological state state and toand to promote promote thethe restoration restoration of healthy of healthy physiological physiological conditions. conditions. For all theFortested all the tested products, products,
20 20 a reference a reference standard standardwith witha aknown known and and verified verified therapeutic therapeutic oir beneficial oir beneficial effect effect was was
available. A available. detailed description A detailed description ofofthe thetested testedproducts productsisisprovided provided in in thethe examples examples
section. section.
In the In the tested tested products, products, the the hundreds hundreds of of components components comingcomingfromfrom the the coarse coarse raw raw
plant parts plant partshave havenotnot beenbeen deprived deprived of the of the ability, ability, characteristic characteristic of materials of materials of natural,of natural,
25 25 biological biological origin, origin, to to establish establishmultiple multiple combinations combinations of of molecular molecularand andsupramolecular supramolecular interactions among interactions amongthemselves themselves and and with with the target the target tissuestissues and therefore and therefore maintainmaintain
therapeutic emerging therapeutic properties. emerging properties.
In particular, In particular, the theproducts products tested tested by by thethe Applicant Applicant havehavebeen beenprepared, prepared,starting starting fromthe from thepreparation preparation of the of the soilsoil to the to the production production of theof the product, final final product, following following eubiotic eubiotic
30 30 protocols that protocols that have beenselected have been selectedand andstandardised standardisedbybyApplicant Applicant throughout throughout moremore than than
40 years 40 yearsofofexperience experience to render, to render, a priori, a priori, the thefinalfinal products products as homogeneous as homogeneous as as possible and possible and therefore therefore increase increasethe theefficiency efficiencyinintermstermsofofyield yieldofofvalid validbatches. batches.TheThe eubiotic protocol eubiotic protocol developed developed by bythetheapplicant, applicant, has hasbeen beendesigned designedininorder ordertotostandardise standardise as much as muchas as possible possible eacheachstepstep leading leading to the todesired the desired final product final product respecting respecting the the 35 35 eubioticsofofeach eubiotics each step, step, SO so to to provide provide a final a final product product which which is 100% is 100%asnatural natural opposedastoopposed to
a synthetic a syntheticororpartially partiallysynthetic synthetic product product (i.e.(i.e. a product a product thatnot that does does not comprise comprise a single a single
component obtained component obtained bybyartificial artificial chemical synthesis) wherein chemical synthesis) wherein each componentisis each component producedunder produced under eubiotic eubiotic conditions. conditions. It isItpossible is possible to develop to develop a fully eubiotic, a fully eubiotic,
33 10 May 2024
standardised, production standardised, processwith production process withthe theintegration integration of of inter-sectoral inter-sectoral technologies technologies and and
research in several different fields, from the agricultural one to -omics and mathematical research in several different fields, from the agricultural one to -omics and mathematical
sciences ones sciences ones bybycomparing, comparing, or or ratherintegrating, rather integrating,the thereductionist reductionist approach approachdiscussed discussed abovewith above withthat thatofofsystems systemstheory theoryandand quantum quantum biology. biology. Preferably, Preferably, according according to theto the
5 5 invention, the invention, the tested tested products products comprising comprising or or consisting consisting ofof one one or or more morenatural naturalmatrices matrices do not do not comprise comprisemolecules molecules of chemical of chemical synthesis synthesis nor comprise nor comprise natural natural matrices matrices that that have been have been putput inin contact contact andandthat that maymayhavehave internalised molecules internalised moleculesofofchemical chemical synthesis, thereby thereby allowing to provide provide natural natural matrices matrices and and final final products consisting ofof 2024203121
synthesis, allowing to products consisting
100% naturalingredients. 100% natural ingredients. 10 10 Anobject An object ofof the the invention invention is is aa method for defining method for defining the the acceptability acceptability values values of of aa
spectroscopyororspectrophotometry spectroscopy spectrophotometry analysis analysis for for the the compliance compliance validation validation of oneoforone or
morebatches more batchesof of a product a product for treatment for the the treatment of a pathological of a pathological conditioncondition or for adjuvating or for adjuvating
homeostasisininananaltered homeostasis alteredphysiological physiologicalstate,state, the the product productcomprising comprising or or consisting consisting of of
one or one or more morenatural naturalmatrices, matrices,comprising comprising performing performing at least at least one one in vitro in vitro cell-based cell-based
15 15 assay and assay and calculating calculating said said acceptability acceptability values values on a spectroscopy on a spectroscopy or or spectrophotometry spectrophotometry spectra of spectra of a reference standard a reference standard having havinga aknown known therapeutic therapeutic or or beneficial beneficial effect effect in in the the
treatment of treatment of said said pathological pathologicalcondition conditionororininadjuvating adjuvatinghomeostasis homeostasis in said in said altered altered
physiological state physiological state and andofofone oneor ormore more batches batches of said of said product, product, said spectra said spectra being being definedasasacceptable defined acceptable or non-acceptable or non-acceptable on the on theofbasis basis of the biological the biological activityinexerted in activity exerted
20 20 said at said at least least one cell-based assay one cell-based assaybybysaid saidreference reference standard standard and and saidsaid onemore one or or more batches on batches ononeoneorormore more hallmarks hallmarks of said of said pathological pathological condition condition or of or aofpathological a pathological conditionthat condition thatcan canderive derive from from said said altered altered physiological physiological state. state.
Dependingononthethepathological Depending pathologicalcondition condition of of interest, one interest, oneorormore morehallmarks hallmarks cancan
be selected. be selected. Preferably Preferably aaplurality plurality ofof hallmarks hallmarksisisselected, selected,although although in in thethe casecase of of
25 25 cancers, for cancers, for example, example,the theskilled skilledperson personskilled skilledperson person is is well well awareaware that that the most the most
relevant hallmark relevant hallmarkisis the the proliferatin proliferatin of of cancer cells. Therefore, cancer cells. Therefore, the the elected elected cell-based cell-based assaycan assay canbebeininthis thiscase, case,anan assay assay verifying verifying the viability the viability of neoplastic of neoplastic cells orcells or of tumour of tumour
massesupon masses upon administration administration of theof the product product of interest. of interest. Still, according Still, according to the invention to the invention it it is preferred is preferred to touseusea asingle single cell-based cell-based assayhowever, assayhowever,more more than than one one cell-based cell-based assayassay can can
30 30 also be also used. In be used. In aa non-limiting non-limiting example, example,when when for for thethe analysis analysis of of different different hallmarks hallmarks
different cell-based different cell-based assays assays areare deemed deemed more more suitable,additional suitable, additionalcell-based cell-basedassaysassayscancan be carried be carriedout. out. According to According to thethe invention invention the the cell-based cell-based assay assay isis designed designed totosimulate simulate conditionsrelated conditions relatedtotosaid said pathological pathological condition condition or to or saidto altered said altered physiological physiological state. state.
35 35 Themain The maindifference differencebetweenbetween the the product product validation validation procedures procedures of the of present the present inventionandand invention those those of the of the statestate of theof art, the art, is based is based on theon the demonstration demonstration that, although that, although
informative, the informative, thequali-quantitative quali-quantitativecharacterisation characterisation of aofproduct a product comprisingcomprising or or consisting of consisting of one one oror more morenatural naturalmatrices, matrices,duedue to to thethe dynamic dynamic interactions interactions of all of all itsits
34 10 May 2024
constituents that result in new emerging properties, is not suitable for assessing the real constituents that result in new emerging properties, is not suitable for assessing the real
effectiveness of effectiveness of these these kind kind of of products and therefore products and therefore the the validation validation procedures procedureshave havetoto be carried be carriedout outonon a basis a basis that that differs differs from from that that which which underlies underlies the validation the validation proceduresprocedures used for used forclassical classicalAPIAPI basedtherapeutical basedtherapeutical products. products. Indeed,Indeed, a quali-quantitative a quali-quantitative
5 5 validation, in validation, in case case of of products products comprising comprising or orconsisting consistingofofnatural natural matrices, matrices, as as shown shown also by also by the the data dataprovided provided in in thethe examples examples andtheinfigures, and in the figures, is sufficient is not not sufficient nor nor
suitabletoto ensure suitable ensurethethetherapeutic therapeutic effectiveness effectiveness ofproduct. of the the product. As aa result, result, the theapplicant applicanthas hasdeveloped developed newnew methods andprocesses processesthat thatbase basethe the 2024203121
As methods and
validation procedures validation procedureson on the the definition definition of validation of validation parameters parameters that are that trulyare truly
10 10 representativeofofthethetherapeutic representative therapeutic effectiveness effectiveness of theofproduct. the product. Additional metabolomics Additional metabolomics analysis analysis may may nevertheless nevertheless still still be desirable be desirable for thefor the manufacturers manufacturers e.g., e.g., to to evaluate evaluate the the toxicological toxicological profile profile of theof the product. product.
Dependingononthethepathological Depending pathologicalcondition conditionofofinterest, interest, the the hallmarks hallmarks cancan bebe one oneoror more, when more, whenavailable, available,preferably preferablymore morehallmarks hallmarks areare selected,however, selected, however, e.g.ininthe e.g. thecase case 15 15 of cancers, of cancers, thethe skilled skilled person personisiswellwellaware aware that that thethe most most relevant relevant hallmark hallmark is theis the
proliferation of the cancer cells, therefore the skilled person can limit the method of the proliferation of the cancer cells, therefore the skilled person can limit the method of the
invention to invention to this this single single hallmark hallmark and and thethe elected elected cell-based cell-based assay assaywillwill be beinin this this case, case, assays verifying assays verifying the the viability viability of neoplastic cells of neoplastic cells or or ofof tumour tumourmassesmasses uponupon administration administration of of thethe product product of interest. of interest.
20 20 According to According to an an embodiment embodimentofofthe theinvention, invention, the the method method isis aa method methodfor for definingthe defining theacceptability acceptability values values of aof a spectroscopy spectroscopy or spectrophotometry or spectrophotometry analysis foranalysis the for the compliancevalidation compliance validationofofoneone or more or more batchesbatches of a product of a product for thefor the treatment treatment of a of a pathological condition, pathological condition, wherein whereinthe theproduct product comprises comprises one one or more or more naturalnatural matrices; matrices;
said method said comprising: method comprising:
25 25 (a) (a) performing performing at at leastoneone least in in vitro vitro cell-based cell-based assayassay on a on a reference reference standard standard batch batch of said of said product, product, wherein whereinthethereference reference standard standard has has a known a known therapeutic therapeutic effect effect for for treatment ofof said treatment saidpathological pathologicalcondition, condition,andand on one on one or test or more morebatches test batches of saidof said
product, wherein product, whereinthethe read-out read-out of cell-based of said said cell-based assay is assay is representative representative of the of the modulationofofone modulation oneorormore morebiological biologicalactivity activityassociated associatedwith withone oneorormore morehallmarks hallmarks of of
30 30 said pathological said pathologicalcondition; condition; (b) (b) quantifying quantifying in in terms terms of of numerical numerical values values the the modulation (“modulation modulation ("modulation values”) values") ofof said said one or more one or biological activities more biological activities induced induced by by each eachbatch batchofofstep step (a) (a) for for eachcell-based each cell-based assay assay read-out read-out defining defining as reference as reference values thevalues valuesthecalculated values calculated for the for the reference standard reference batch; standard batch;
35 35 (c) (c) defining defining as as acceptable acceptable the the test testbatches batcheswhosewhose values values calculated calculated in in (b) (b) induce induce
a modulation a modulationof ofeach each measured measured biological biological activity activity in saidin cell-based said cell-based assay whose assay whose
modulusisis> ≥totothe modulus themodulus modulus of ofthethe reference reference valuevalue calculated calculated in (b) in (b) and and defining defining as as
non-acceptablethe non-acceptable thebatches batcheswhosewhose values values calculated calculated in (b) in (b) induce induce a modulation a modulation of at of at
35 10 May 2024
least one of said biological activities in said cell-based assay whose modulus is < to the least one of said biological activities in said cell-based assay whose modulus is < to the
modulus modulus of of thethe reference reference valuevalue calculated calculated in (b);in (b);
(d) (d) performing performing aaspectroscopy spectroscopyororspectrophotometry spectrophotometry on said on said reference reference standard standard
batchand batch andonon said said oneone or more or more different different test batches; test batches; and and 5 5 (e) defining (e) defining thethe acceptability acceptability values values ofof the the spectroscopy spectroscopyororspectrophotometry spectrophotometry spectra as spectra as the the variability variabilityrangerangeofofthethespectroscopy spectroscopy or orspectrophotometry spectra values spectrophotometry spectra values of all of all of said acceptable of said acceptable batches batches and and of said of said reference reference standard standard batch, preferably batch, preferably refined refined bythe thespectra spectraofofsaidsaidnon-acceptable non-acceptable batches. 2024203121
by batches.
Theexpression The expression"value/s “value/scalculated calculatedinin(b)"(b)”cancanbebesubstituted substitutedininanyanypart partofofthe the 10 10 description and description and of the claims of the claims withwith "modulation “modulation value/s" value/s” or or "modulation “modulationvalue/s value/s calculatedinin(b). calculated (b). Whenthethesame When same hallmark hallmark can can be associated be associated withwith biological biological activities activities thatcan that canbebe monitored monitored through through different different biological biological parameters, parameters, the person the skilled skilledcan person decidecan decide to carry to carry
out more out thanone more than onecell-based cell-basedassayassaytotomonitor monitorsaidsaidactivity. activity. In In the the example sectionand example section and 15 15 in the in the figures figures thethe definition definition of of the the acceptability acceptability values valueswith withdifferent differentparameters parameters is is
provided, the results show that in each case, although different parameters for analysing provided, the results show that in each case, although different parameters for analysing
the modulation the modulation of the of the biological biological activity/ies activity/ies were selected, were selected, the resulttheisresult is consistent consistent and, and, when the when thesamesame spectroscopy spectroscopy technique technique is used, is used, the the resulting resulting spectroscopy spectroscopy or or spectrophotometryspectra spectrophotometry spectravariability variabilityrangerange defining defining the the acceptability acceptability values values is theis the 20 20 same. same. Theskilled The skilled person person will will be be able able toto select select the themore more suitable suitable parameters parameters depending depending
on the on theselected selectedhallmarks hallmarks of the of the pathological pathological condition condition of interest. of interest.
Said parameters Said parameterscan canbe, be,by byway wayofofexample, example, genes genes andand theirtheir expression expression patterns, patterns,
ROS,oxidative ROS, oxidative stress, stress, cellcell viability viability and and others. others.
25 25 Themethod The method can can be be applied, applied, mutatis mutatis mutandis, mutandis, to beneficial to beneficial products products (adjuvants (adjuvants
of homeostasis) of homeostasis)comprising comprising oneone or more or more natural natural matrices. matrices. Hence Hence the invention the invention also also encompassesa method encompasses a method according according to claim to claim 1 for1 defining for defining the acceptability the acceptability valuesvalues of a of a
spectroscopyororspectrophotometry spectroscopy spectrophotometry analysis analysis for for the the compliance compliance validation validation of oneoforone or
more batches more batches ofofa abeneficial beneficial product productfor foradjuvating adjuvatinghomeostasis homeostasisininan an altered altered 30 30 physiological state, physiological state, wherein wherein the the product productcomprises comprisesoneone or or moremore natural natural matrices; matrices; saidsaid
methodcomprising: method comprising: (a) (a) performing performing at at leastoneone least in in vitro vitro cell-based cell-based assayassay on a reference on a reference standard standard batch batch of said of said product, product,wherein wherein thethe reference reference standard standard has ahas a known known beneficial beneficial effect foreffect for adjuvating homeostasis adjuvating homeostasisininananaltered alteredphysiological physiologicalcondition, condition,and andon on oneone or more or more test test
35 35 batches of batches of said said product, product, wherein whereinthe theread-out read-outofofsaid saidcell-based cell-basedassayassayisis representative representative of the of the modulation modulationofofoneone or or more more biological biological activities activities associated associated withwith one oronemore or more hallmarksofofaapathological hallmarks pathologicalcondition conditionthat thatcancandevelop develop fromfrom saidsaid altered altered physiolgical physiolgical
condition; condition;
36 10 May 2024
(b) quantifying (b) quantifying in in terms terms of of numerical values the numerical values the modulation ofsaid modulation of said one oneor or more more biological activities induced by each batch of step (a) for each cell-based assay read-out biological activities induced by each batch of step (a) for each cell-based assay read-out
definingasasreference defining reference values values the the values values calculated calculated for theforreference the reference standardstandard batch; batch; (c) defining (c) defining as as acceptable acceptable the the test testbatches batcheswhose whose values values calculated calculated in in (b) (b)induce induce
5 5 a modulation a modulationof ofeach each measured measured biological biological activity activity in said in cell-based said cell-based assay whose assay whose
modulusisisto≥ the modulus to the modulus modulus of theof reference the reference valuevalue calculated calculated in (b)inand (b)defining and defining as as non-acceptable the non-acceptable the batches batches whosewhose modulation modulationvaluesvaluescalculated calculated inin (b)(b) induce inducea a modulationofofatatleast least one oneofofsaid saidbiological biological activities activities in in said said cell-based cell-based assay assay whose 2024203121
modulation whose
modulus modulus is is < to < to thethe modulus modulus of theofreference the reference value calculated value calculated in (b);. in (b);.
10 10 (d) (d) performing performing aa spectroscopy spectroscopyororspectrophotometry spectrophotometry on on saidsaid reference reference standard standard
batchand batch andonon said said oneone or more or more different different test batches; test batches; and and (e) (e) defining defining the the acceptability acceptability valuesvalues ofof the the spectroscopy spectroscopyororspectrophotometry spectrophotometry spectra as spectra as the the variability variabilityrangerangeofofthe thespectroscopy spectroscopyor orspectrophotometry spectrophotometry spectra spectra values values
of all of all of of said acceptablebatches said acceptable batchesand and of said of said reference reference standardstandard batch, preferably batch, preferably refined refined
15 15 by the by thespectra spectraofofsaid saidnon-acceptable non-acceptable batches. batches.
Accordingtotothe According the invention, invention, step step a) a) can can comprise: comprise:
a. retrieving from the state of the art a list of the hallmarks of said pathological a. retrieving from the state of the art a list of the hallmarks of said pathological
condition; condition;
b. identifying b. identifying for for each each of of said said hallmarks hallmarks a aset setofofbiological biologicalactivities activities 20 20 modifications detectable modifications detectable in in said said pathological pathological condition condition andand determining determiningthe themodulation modulation of each of eachofofsaid saidactivities activities concurring concurring to desired to the the desired therapeutic therapeutic effect designing effect thereby thereby designing the modulation the modulationpatternpatternofofeach eachofofsaid saidactivities activitiesrepresenting representinga ahealthy healthyphysiological physiological state; state;
c. identifying C. identifying the the parameters parameters and the modulation and the modulationpattern patternthereof thereof(such (suchasasgenes genes 25 25 and expression and expression pattern pattern thereof) thereof) underlying underlying the the modification modification detectable detectable in in said said pathologicalcondition pathological conditionfor for eacheach of biological of said said biological activities activities and for and setting setting each for each of said of said
parametersthe parameters themodulation modulation pattern pattern opposite opposite to the to the one identified one identified as the as expression the expression patternindicative pattern indicativeofofsaidsaidhealthy healthy physiological physiological state;state; step b) step b) can can comprise: comprise:
30 30 analysing the analysing the modulation modulationpattern patternofofeach eachofofthetheparameters parameters (such (such as as genes genes and and
expression thereof) expression thereof) identified identified in in a) a) c. C.induced induced by a reference by a reference standard standard of of the the analysed analysed product inina asuitable product suitablein invitro vitrocell-based cell-based assay, assay, determining determining the quali-quantitative the quali-quantitative
modulationofof++each modulation eachofofsaid said parameters parametersinduced inducedbybysaidsaidreference referencestandard standardwith withrespect respect to the to the pathophysiological pathophysiological state state control control of in of said said in vitro vitro cell-based cell-based assay, assay, and and calculating calculating
35 35 the reference the referencestandard standard modulation modulation value value for eachforofeach said of said biological biological activitiesactivities induced by induced by said reference said reference standard standard and selecting each and selecting each of said said reference reference standard standard modulation values modulation values
as reference as referencevalues values (cut-offs) (cut-offs) indicative indicative of said of said desired desired therapeutic therapeutic effect;effect;
and analysing and analysingthe themodulation modulation pattern pattern of the of the parameters parameters (such (such as genesas and genes and
37 10 May 2024
expression thereof) expression thereof) identified identified inina)a)C.c.induced inducedby by further further different different batches batches of said of said
product, inin said product, saidininvitro vitrocell-based cell-based assay assay and determining and determining the quali-quantitative the quali-quantitative
modulation of modulation of each each of of said said parameters parameters induced induced bybyeach eachofofsaid saidbatches batchesthereby thereby calculating the calculating the modulation modulationvalue value induced induced by each by each of batches of said said batches for ofeach for each saidof said 5 5 biological activities; biological activities;
step c) step c) can cancomprise: comprise: comparingthe comparing themodulation modulation value value of of eacheach of of said said biologicalactivities biological activities induced inducedbyby each ofof said saidbatches batchescalculated calculatedin in b) b) with the the corresponding reference valuesvalues and 2024203121
each with corresponding reference and
defining as defining as acceptable acceptablethe thetest test batches batches(preferably (preferablyatatleast leastthree) three)whose whose modulation modulation
10 10 value calculated value calculated ininb)b)complies complies withwith the corresponding the corresponding reference reference valuesthe(i.e. the values (i.e.
reference values reference values areare intended intended as as cut-offs cut-offs and and aa batch is defined batch is defined asas acceptable acceptable when the when the
reference cut-off reference cut-off is is reached reached forfor each eachbiological biologicalactivity) activity) and andpreferably preferablyatatleast leastone one negative batch negative batch for for which which atat least least one one modulation value calculated modulation value calculated in in b) does does not not comply comply
(hence does (hence does not not reach reachthethe desired desired cut-off) cut-off) with the corresponding referencemodulation corresponding reference modulation 15 15 value; the value; the method also comprises method also comprisesstepssteps d) carrying d) carrying out out aa spectroscopy spectroscopyororspectrophotometry spectrophotometry analysis analysis of of said said reference reference
standard and of the batches selected in c) and standard and of the batches selected in c) and
e) defining e) defining the the acceptability acceptability valuesvalues of ofthe thespectroscopy spectroscopy or or spectrophotometry spectrophotometry as as the variability the variabilityrange rangeofofthethespectra spectra values values resulting resulting byacceptable by the the acceptable batches, batches, preferably preferably
20 20 refined by refined by thethe spectra spectraofofsaid saidnon-acceptable non-acceptable batches. batches. In In fact, fact, whenwhen non-acceptable non-acceptable
batches are batches are present, present, their their spectra spectra will will define define non-acceptable non-acceptable valuesvalues when whendetermining determining the compliance the compliance variability variability range. range. therefore, therefore, the method the method providesprovides the acceptability the acceptability values values of the of the spectroscopy spectroscopy or or spectrophotometry spectrophotometry spectra. spectra. As stated As stated above, above,the the reference referencemodulation modulation value value is is intended intended as as a cut-off a cut-off value value
25 25 (positive in (positive in case case ofofup-modulation up-modulation and andnegative negativeinin case case ofof down-modulation) down-modulation) that that must must
be reached be reached forfor each each selected selected biological biological activity activity by abatch by a test test in batch orderintoorder to beasdefined as be defined
acceptable. acceptable.
Therange The rangeofofvariability variability in in each each point pointofofthe the spectra spectra ofof the the acceptable acceptablebatches batches and of and of the the reference standard standard batchbatch cancan then then bebe used used as as acceptability acceptability rangerange ofof values of 30 30 newbatches new batchesof ofthethe product product in compliance in compliance validation validation processesprocesses at the industrial at the industrial level. level. Accordingtotothe According the invention inventionthe the product productsubjected subjectedto to the the batch batch to to batch batch validation validation
process of process of the the invention invention and andforfor which whichsuitable suitableacceptability acceptabilityvaluesvaluesare areprovided providedwith with the method the methodofofthetheinvention invention is is a product, a product, comprising comprising or consisting or consisting of one of orone moreor more natural matrices, natural matrices, for for thethe treatment treatment ofofa apathological pathologicalcondition condition i.e.a aproduct i.e. product withwith a a
35 35 therapeuticeffect therapeutic effectverified verified at least at least preclinically preclinically in vitro in vitro on cellson and/or cells tissues and/or and/or tissues and/or organoids and/or organoids and/orinin vivovivo on onanimal animalmodels, models,andand therefore therefore a product a product expected expected to, to, whenwhen administered totoa apatient administered patientsuffering sufferingfromfrom a given a given pathological pathological condition, condition, reduce reduce the the
severity of severity of thethe subject's subject's condition (i.e. the condition (i.e. the severity severity is is at at least leastpartially partiallyimproved improved oror
38 10 May 2024
ameliorated), and/or ameliorated), and/or provide providesome some alleviation,mitigation alleviation, mitigation or or decrease decrease in least in at at least oneone
clinical symptom of said condition and/or delay in the progression of the said condition. clinical symptom of said condition and/or delay in the progression of the said condition.
Thesubject The subjecttreated, treated, according accordingtoto thethe invention, invention, is is an an animal animalincluding includinghumans humans (hence (hence
the product the productisisfor forhuman human or for or for veterinary veterinary use), use), or aeven or even a plant. plant.
5 5 The methods The methodsand andprocesses processesprovided providedininthethepresent presentinvention inventionare, are, indeed, indeed, processes that processes that can canbebe carried carried outout withwith the goal the goal of validating of validating batchesbatches of a given of a given
therapeutic product, therapeutic product, comprising comprisingor or consisting consisting of of natural natural matrices, matrices, in the in the production production
chain. 2024203121
chain.
Theproduct, The product,asasalready alreadystated stated above, above,isis aa product productthatthat comprises comprisesororconsists consistsofof 10 10 one or one or more morenatural natural matrices, matrices, i.e. i.e. ofofcomplex complex natural natural systems; systems; aa non-limiting non-limiting example example ofof said natural said natural matrices matrices is is represented represented by byoneoneorormore moreof:of: cutcutor or pulverized pulverized plant plant parts, parts,
plant extracts, plant extracts,processed processed plant plant parts, parts, fractions fractions of plant of plant extracts extracts such such as, foras, for example, example, the the fractions obtained fractions obtainedbyby filtrationon on filtration a semi-permeable a semi-permeable membranemembrane (microfiltration, (microfiltration,
ultrafiltration, nanofiltration) ultrafiltration, nanofiltration) ororbybytreatment treatment on adsorptionresins, on adsorption resins, microorganisms, microorganisms, 15 15 honey, propolis, honey, propolis, silk, silk, wax, wax,plant plantresins, resins,plant plantgums, gums, plant plant exudates, exudates, vegetable vegetable oils,oils,
vegetable essential oils, animal tissues lysates, plant or animal fluids. vegetable essential oils, animal tissues lysates, plant or animal fluids.
Preferably, said Preferably, said microorganisms microorganisms are are inactivated inactivated microorganisms microorganismssuch suchas as tyndallized organisms. tyndallized organisms.
In aa most In preferred embodiment, most preferred embodiment, thethe therapeuticproduct therapeutic product is is a a product product consisting consisting
20 20 of 100% of 100% ofofnatural naturalcomponents, components, intended intended as components as components that not that are are obtained not obtained by manby man through chemical through chemicalsynthesis synthesis reactions,therefore, reactions, therefore,when whenthe the product product comprises comprises one orone or
morenatural more natural matrices, matrices, it can it can alsoalso comprise comprise minerals, minerals, and in any and in general general other any other organic or organic or
inorganic material inorganic material found foundin innature. nature.Preferably, Preferably, thethe product product subject subject to methods to methods and and processes of processes of thethe invention, invention, is is aa product product obtained obtained or or obtainable obtainable according according to to aa 25 25 standardised protocol, standardised protocol, more preferablythrough more preferably throughananeubiotic eubioticstandardised standardisedprotocol. protocol.When When no standardised no standardised protocol protocolisis available, available, thethe skilled skilled person person can nevertheless minimise can nevertheless minimisethe the differences between differences betweenbatches batches of of thethesamesame product product by using, by using, for each fornatural each natural matrix matrix
comprised comprised in in thethe product, product, a pool a pool of a of a starting starting raw sources raw sources or of intermediate or of intermediate materials materials or or of natural of naturalmatrices. matrices.InInthisthisway, way,the the inherent inherent variability variability of natural of natural matrices matrices derived derived from from 30 30 different samples different samples of of thethe samesame kind ofkind of source source (e.g., (e.g., same same plant plant from fromcultivars), different different cultivars), can be can be diminished diminishedbybysaid saidpooling. pooling. Accordingtotothe According theinvention inventionsaid said product product can can be e.g., be e.g., a, aa,food a food supplement, supplement, a a medicaldevice, medical device, or or aa medicament. medicament.
In an In an embodiment embodiment said saidproduct product is is a a medical medical device device as as defined defined in in EU EU Regulation Regulation
35 35 2017/745Article 2017/745 Article2 2(1)(1)indents indents 1-31-3 wherein wherein the the medical medical purposepurpose is the istreatment the treatment or or alleviationofofaadisease alleviation diseaseororthethe modification modification of a of a pathological pathological processprocess or state. or state. Article 22 (1) Article (1)indents indents1-3 1-3of ofEUEU Regulation 2017/745recite: Regulation 2017/745 recite: Forthe For thepurposes purposes of of thisthis Regulation, Regulation, the following the following definitions definitions apply: apply:
39 10 May 2024
(1) ‘medical (1) device’means 'medical device' meansanyany […]
[...] material material or or other other articleintended article intended by by the the
manufacturertotobebeused, manufacturer used,alone aloneororinin combination, combination,for forhuman human beings beings forfor oneone or or more more of of the following the specific medical following specific purposes: medical purposes:
—diagnosis, - diagnosis, prevention, prevention, monitoring, monitoring, prediction, prediction, prognosis, prognosis, treatment, treatment, or or 5 5 alleviation of disease, alleviation of disease,
-—diagnosis, diagnosis, monitoring, monitoring, treatment, treatment, alleviation alleviation of, orof, compensation or compensationfor, anfor, an
injury orordisability, injury disability, — investigation, investigation, replacement, replacement,orormodification modificationofof the the anatomy or of of a a 2024203121
anatomy or
- physiological physiological or or pathological pathological process process or state, or state,
10 10 and which and whichdoesdoesnotnot achieve achieve its its principal principal intended intended action action by pharmacological, by pharmacological,
immunological, or immunological, or metabolic metabolic means, means, in in or or on on the the human humanbody,body,but butwhich whichmay may be be assistedininits assisted its function functionbybysuch such means. means. The product The product may alsomay be a also be aclassified product product as classified a as a medicaldevice medical devicebybythe theFDA. FDA. Per Section Per Section 201(h)(1) 201(h)(1) of of the the Food, Drug, and Food, Drug, andCosmetic CosmeticAct, Act,a adevice deviceis: is: 15 15 Aninstrument, An instrument,apparatus, apparatus,implement, implement, machine, machine, contrivance, contrivance, implant,implant, in vitro in vitro
reagent, or reagent, or other other similar similar or or related related article, article, including including a componentpart, a component part,ororaccessory accessory whichis: which is: (A) recognized (A) recognized in in the the official official National National Formulary, Formulary, or or the the United UnitedStates States Pharmacopoeia,ororany Pharmacopoeia, anysupplement supplement to to them, them,
20 20 (B) intended for (B) intended for use useininthethediagnosis diagnosisofofdisease diseaseororotherotherconditions, conditions,ororin inthethe cure, mitigation, cure, mitigation,treatment, treatment, or or prevention prevention of disease, of disease, in maninorman otheroranimals, other animals, or or (C) intended (C) intended to to affect affect thethe structure structure or or any any function function of of the the body body of of man manororother other animals, and animals, andwhich whichdoes does notnot achieve achieve its its primary primary intended intended purposes purposes throughthrough chemical chemical
action within action within or or on the body on the bodyofof man manororotherotheranimals animalsandand which which is not is not dependent dependent uponupon
25 25 being metabolized being metabolizedforforthethe achievement achievement of its of primary its primary intendedintended purposes. purposes. The term The term "device" does not "device" does not include include software software functions functions excluded excludedpursuant pursuanttotosection section 520(o). 520(o). Thespectroscopy The spectroscopyororspectrophotometry spectrophotometry analysis analysis of method of method of theofinvention the invention is is preferablycarried preferably carriedoutouton on a dry a dry formform of theofproduct the product of interest, of interest, SO when sothewhen final the final product product
intendedforforuseuse intended is is a solution, a solution, a suspension, a suspension, or liquid or other other liquid forms, itforms, can beitcarried can beoutcarried out 30 30 on the on thelyophilised lyophilised product product prior prior to its to its rehydration. rehydration.
In its In its final final form form forforadministration administration the the product product may bemay be in in the formtheof form of a apowder, a a powder,
granulate,a atablet, granulate, tablet,a asyrup, syrup,a solution, a solution, a suspension, a suspension, a harda or hard orgelatine, soft soft gelatine, a capsule, aa a capsule,
spray,aacream, spray, cream,ororthethe like. like.
Accordingtotothe According theinvention, invention,a apathological pathologicalcondition condition is is a specificdisease a specific diseaseor ora a 35 35 pathological state. pathological state. Non-limiting examplesofofpathological Non-limiting examples pathologicalconditions conditions include include mildmild mildmild
cognitive impairment cognitive impairment(MCI), (MCI), osteoporosis osteoporosis (OP),(OP), including including post or postperiormenopausal peri menopausal osteoporosis (PMO), osteoporosis (PMO),osteoarthritis osteoarthritis (OA), (OA),cancer. cancer. Withoutlimitation Without limitationthereto,thereto,cancers cancersofofmajormajor interest interest suchsuch as head as head and neck and neck
40 10 May 2024
cancer, melanoma, cancer, breastcancer, melanoma, breast cancer,bladder bladderand andosteosarcoma osteosarcomaareare included. included.
Hallmarksofofpathological Hallmarks pathologicalconditions conditions as as well well as as of of specific specific diseases diseases are are well- well-
known in the art. The skilled person can easily retrieve in the scientific literature the known in the art. The skilled person can easily retrieve in the scientific literature the
hallmarksofofpathological hallmarks pathologicalconditions conditions of of interest interest as as wellwell as biological as the the biological activities activities
5 5 underlyingsaid underlying saidhallmarks. hallmarks.ItIt evident evidentthatthat the theskilled skilled person personwillwillselect select the the biological biological activities underlying activities underlying a given a given hallmark hallmark considering considering the specific the specific pathological pathological condition of condition of
interest. interest.
Bywaywayofofexample, example,thethe skilled personcan can investigatethe the patho-physiological 2024203121
By skilled person investigate patho-physiological
state-of-the-artofofthe state-of-the-art thedisease diseaseofofinterest interest using using different different prior prior art sources. art sources.
10 10 Theskilled The skilled person personwishing wishingtotodefine definehallmarks hallmarks of of a disease a disease of of interestwill interest willbebe able to able to retrieve retrieve the thedesired desiredinformation information fromfrom the scientific the scientific literature. literature. By ofway of By way
example, non-limiting example, non-limiting example example of of sources sources that that can can bebeused usedRobbinsRobbins & Cotran & Cotran Pathologic Pathologic Basis Basis of of Disease Disease(Robbins(Robbins Pathology) Pathology) 10th 10th Edition; Edition; https://calgaryguide.ucalgary.ca/; Biomedical https://calgaryguide.ucalgary.ca/; Biomedicalliterature literaturefrom PubMedCentral from PubMed Central 15 15 (https://pubmed.ncbi.nlm.nih.gov/) and the like. (https://pubmed.ncbi.nlm.nih.gov/) and the like.
For each For each ofof said said hallmarks hallmarksseveral severalbiological biological activities activities underlying underlying them them areare also also known known in in thethe art, art, thethe skilled skilled person person can select can select said functions said functions from thefrom the ones indisclosed in ones disclosed
the art. the art. When When moremore biological biological activities activities are ascribed are ascribed in to in the art thea art to hallmark given a given ofhallmark a of a givenpathology given pathology in the in the method method of the of the invention, invention, preferablypreferably at least at least two, two,three at least at least or three or
20 20 moreofofsaid more saidactivities activitiesareare selected. selected.
Whenwishing When wishing to to useuse specific specific software software available available forfor easily easily carrying carrying outout certain certain
steps of steps of the the method method ofofthe theinvention inventionsuch suchasasfinding findingbiological biologicalactivities activities underlying underlyinga a hallmarkofofa adisease, hallmark disease,the theskilled skilledperson personmaymay wishwish to adapt to adapt the retrieved the retrieved hallmarks hallmarks
adaptingtheir adapting theirdefinitions definitions to to better better interrogate interrogate the software the software used. used. By way By way ofinexample, in of example,
25 25 case the case the software software used usedis is Qiagen QiagenIPA IPA(IPA(IPA version version 94302991 94302991 Qiagen), Qiagen), the information the information
foundininthe found theaforementioned aforementioned resources resources can becanused, be used, if necessary, if necessary, to redefine to redefine said said hallmarkstotointerrogate hallmarks interrogateIPA IPA if if thethe IPAIPAusesuses a different a different internal internal definition definition of said of said
hallmarks. ToTorapidly hallmarks. rapidlyidentify identifythethe biological biological activitiesunderlying activities underlying a hallmark a hallmark of a of a
disease, when disease, when IPA IPAisis used, used, the the following following procedure procedurecan canbebefollowed: followed: 30 30 Hallmarkscan Hallmarks canbebewritten, written,oneonebybyone,one,ininthe the"disease "diseaseand andfunctions" functions"queryqueryboxbox
andthe and thesearch searchisisthen then launched. launched.
Theobtained The obtainedresuming resuming table table allows allows thethe skilled skilled person person to filterdisease/activity to filter disease/activity that come that come fromfrom many manylines linesofofevidence. evidence.ByBywayway of example, of example, the the source source for for the the relationship can relationship can bebethetheIngenuity IngenuityKnowledge Knowledge Base,Base, including including curation curation from journal from journal
35 35 articles, OMIM, articles, OMIM, JAXJAX andand ClinicalTrials.gov. ClinicalTrials.gov
Thetool The toolisis therefore therefore ableabletotoassociate associatewith witheach eachbiological biologicalactivity activitya adefined defined numberofofgenes number genes whosewhose regulation regulation can influence can influence the modulation the modulation of the biological of the biological
activityitself. activity itself.
41 10 May 2024
Examples ofofknown Examples known hallmarks hallmarks associatedwith associated with pathologicalconditions pathological conditionsare are shown in figures 1-4, 8-12. shown in figures 1-4, 8-12.
Merely by Merely by way wayofofexample, example, hallmarks hallmarks of ofOA OA known known in the in art the comprise: art comprise: Proliferation (Skeletal Proliferation (Skeletal andand muscular systemdevelopment muscular system development andand function function suchsuch as articular as articular
5 5 dysfunction and dysfunction joint space); and joint space); Inflammation; Inflammation; Anatomical Anatomical damage. Still by damage. Still by way way ofof example,suitable example, suitable biological biological activities activitiesunderlying underlying the theOA hallmarksabove OA hallmarks abovecan cancomprise comprise the following: the following: Proliferation: thethe biological biological activities activities ofofinterest interestbeing being e.g.,skeletal, skeletal,andand 2024203121
Proliferation: e.g.,
muscularsystem muscular system development development and function and function such assuch as articular articular dysfunction dysfunction and jointand joint
10 10 space,articular space, articulardisfunction, disfunction, joint joint space space and and formation formation of the of the cartilage cartilage tissue,tissue,
Inflammation:the Inflammation: thebiological biologicalactivities activities interested interested being beinginflammatory inflammatory disease, disease,
inflammationand inflammation andnociception, nociception,inflammation inflammation of of thethejoint, joint, Protection from Protection fromanatomical anatomical damage: damage: the biological the biological activities activities interested interested beingbeing organismalinjury organismal injury and andabnormalities abnormalitiessuchsuchasasjoint jointinflammation inflammation andand swelling, swelling, difficulty difficulty
15 15 moving moving a joint: a joint: osteoarthritis. osteoarthritis.
Possible state of the art keywords defining said activities are indicated in figures Possible state of the art keywords defining said activities are indicated in figures
1-4. 1-4.
Further, by Further, wayofofexample, by way example,known known hallmarks hallmarks of MCI of MCI may comprise may comprise Cognition Cognition
(impairment), activation (impairment), activation and andviability viability (neuronal, (neuronal, decrease), decrease), Myelination Myelinationand andbranching branching 20 20 (decrease), Inflammation (decrease), (increase), Skeletal and Inflammation (increase), and muscular systemfunction muscular system function(decrease). (decrease). Cognition: the biological function interested being cognition and learning; Cognition: the biological function interested being cognition and learning;
Activation and Activation andviability: viability: the the biological biological activity activity interested interested being being development, development, differentiationofofneurons, differentiation neurons, proliferation proliferation of neuronal of neuronal cells;cells;
Myelinationand Myelination andbranching: branching:thethebiological biologicalactivity activity interested interested being being branching branchingofof 25 25 neurons, neuronal neurons, neuronalsprouting; sprouting; Inflammation:thethebiological Inflammation: biologicalactivity activityinterested interested being being chronic chronic inflammatory inflammatory
disorder; disorder;
Skeletal and Skeletal and muscular muscularsystem system function: function: thethe biological biological activityinterested activity interestedbeing being proliferationofofmuscle proliferation muscle cells cells andand necrosis necrosis of muscle. of muscle.
30 30 Possiblestate Possible stateofofthetheartartkeywords keywords defining defining said activities said activities are indicated are indicated in figures in figures
8 and 8 and9.9. Still by Still by way way of example, example, known known hallmarks hallmarks of of OP,OP, including including PMOPMO may comprise may comprise
mineralisation, Inflammation mineralisation, Inflammation (increase), (increase), functionality functionality adiposeadipose tissue (increase), tissue (increase),
remodelling remodelling of of bone, bone, osteoporosis, osteoporosis, differentiation differentiation of osteoblasts of osteoblasts (decrease). (decrease).
35 35 Mineralisation: the Mineralisation: the biological biological activity activity interested interested being being mineralisation mineralisationofofbone bone cell lines cell andosteoblasts, lines and osteoblasts,formation formation of bone; of bone;
Inflammation:the Inflammation: thebiological biologicalactivity activity interested interested being being inflammation inflammationofofadipose, adipose, connective and connective andwhite whiteadipose adiposetissue;tissue;
42 10 May 2024
Functionality of Functionality of adipose adiposetissue: tissue: the the biological biological activity activity interested interested being weight being weight
gain, transdifferentiation, differentiation of adipocytes; gain, transdifferentiation, differentiation of adipocytes;
Remodellingofofbone: Remodelling bone:thethe biological biological activity activity interestedbeing interested being remodelling remodelling and and
resorptionofofbone; resorption bone; 5 5 Osteoporosis: Osteoporosis: thethe biological biological activity activity interested interested beingbeing osteoporosis osteoporosis itself; itself;
Differentiationofof Differentiation osteoblasts: osteoblasts: the the biological biological activity activity interested interested being activation being activation
of alkaline of alkalinephosphatase, phosphatase, differentiation differentiation of bone of bone cells,cells, differentiation differentiation of osteoblasts. of osteoblasts.
Possiblekeywords keywords linked to said activities are indicated in figures 10 and 11. 2024203121
Possible linked to said activities are indicated in figures 10 and 11.
For each For eachofofsaid saidhallmarks hallmarksbiological biologicalactivities activities underlying underlyingthem them areare known known in in
10 10 theart. the art. For each For eachofofsaid saidbiological biologicalactivities activitiesthe themodulation modulation causally causally related related to the to the
pathologicalcondition pathological condition is also is also known known in thein thetherefore art, art, therefore the opposite the opposite modulationmodulation can be can be considered as considered as the themodulation modulationconcurring concurring to desired to the the desired therapeutical therapeutical effect, effect, consequently, consequently, a panel a panel indicating indicating the desired the desired modulation modulation pattern pattern of each ofofsaid each of said activities activities
15 15 can bebedesigned, can designed,saidsaid panel panel being being representative representative of “the of "the modulation modulation trend of trend said of said
activities in a healthy physiological state” i.e., said panel being representative of the activities in a healthy physiological state" i.e., said panel being representative of the
wayinin which way whichsaid saidactivities activities would wouldneed needtotobebemodulated modulated in in order order to to restorethe restore thehealthy healthy physiological state physiological state (see (see figures figures 1-4,1-4, 8-11). 8-11).Modulation Modulation in in the the present present description description is is
intendedasasupup intended or or down down modulation modulation of a givenof aactivity given activity and of a and of a parameter specific specific that parameter is that is 20 20 associated to associated to the the development development or or presence presence of particular of particular condition condition (e.g.,(e.g., pathological, pathological,
altered but altered butnotnotyetyetpathological, pathological, or healty). or healty). Up modulation Up modulation refers torefers to the modification the modification in in the activity, the activity, expression, expression, of of particular particular genes, genes,proteins, proteins,cellular cellular pathways pathways or or cellular cellular
componentsresulting components resultingininananenhancement enhancement of a ofgiven a given biological biological activity.Downregulation activity. Downregulation is the is oppositeofofupregulation. the opposite upregulation. It involves It involves a modification a modification in the activity, in the activity, expressionexpression of of 25 25 genes, proteins, genes, proteins, cellular cellular pathways pathways or or cellular cellular components, components,leading leading to to a reduction a reduction in in a a
givenbiological given biological activity. activity. A modulation A modulation trend trend for for restoring restoring a healthya physiological healthy physiological state state is therefore is therefore intended intended as as the the directionality directionality(up (uporordown) down) ofof the the modulation modulation of of aa selected selected biological activity biological activity that thatisisobservable observable or or expected expected when passingfrom when passing from a pathological a pathological or or
altered condition altered condition totoa ahealthy healthystate.state. Therefore, Therefore, if a ifgiven a given activity activity underlying underlying a a 30 30 pathological condition pathological conditionisisup-modulated up-modulated in ain a pathological pathological or altered or altered condition, condition, the the modulationtrend modulation trendforforrestoring restoringa healthy a healthy physiological physiological state state is down-modulation. is down-modulation. In In other words, other words, the the modulation modulationtrend trendfor forrestoring restoringaa healthy healthyphysiological physiologicalstate state ofof the the one one or more or moreactivities activities underlying underlying aa pathological pathologicalororalteredaltered condition conditionselected selectedaccording accordingtoto the method the method of of the the invention, invention, is theis opposite the opposite to the toonethe one detectable detectable in the pathological in the pathological or or 35 35 altered condition. altered condition. Non-limitingexamples Non-limiting examples of theof modulations the modulations of biological of biological activities activities that are that are causally linked causally linked to to pathological pathological conditions conditions (OA ... PMO) (OA PMO) are depicted are depicted in Figures in Figures 1-4 and 1-4 and 8-12. 8-12.
43 10 May 2024
Therefore, conversely, Therefore, conversely, restoring restoring the the healthy physiological state healthy physiological state from an existing from an existing pathologyofofinterest pathology interest can be designed can be designedasas requiring requiring the the inverse inverse modulation modulationofofthe theone oneoror moreselected more selectedbiological biological activities. activities. By By way of example way of examplebut butnot notlimited limitedthereto, thereto, for for OA, OA, MCI,and MCI, andOP, OP, in in particularPMO, particular PMO, the the modulation modulation leading leading to healthy to the the healthy physiological physiological
5 5 state is depicted in figures 1-4 and 8-12 state is depicted in figures 1-4 and 8-12
Whenthethe When modulation modulation patternpattern leading leading to thetohealthy the healthy physiological physiological state is state is
observedupon observed upontreatment treatment with with a given a given product, product, for for one one or more, or more, preferably preferably most most and and ideally all, all, of of the the biological activities causally causally related related to to the hallmarksofofa agiven given 2024203121
ideally biological activities the hallmarks
disease, then disease, then the the product product cancan bebeidentified identified asas aa product product having havingthethedesired desiredtherapeutic therapeutic 10 10 effect. Therefore, effect. Therefore,a aproduct product inducing inducing the modulation the modulation of the biological of the biological activitiesactivities causally causally
related to related to the the hallmarks hallmarks of of aa given givenpathological pathologicalcondition conditionininthe thedirection directiontoward towardthethe
healthyphysiological healthy physiological state, state, can can be identified be identified as a as a product product having having the therapeutic the desired desired therapeutic effect on effect the overall on the overall pathological pathological state. state. When Whenonlyonlysomesome of biological of the the biological activities activities
causally related causally related toto the thehallmarks hallmarks of of a disease a disease are altered are altered in direction in the the direction of theof the
15 15 physiological physiological state state as as defined defined above, above, has aonly has only a partial partial therapeutic therapeutic effectdoes effect which which not does not
result in an entirely healthy physiological state. Nevertheless, a partial therapeutic effect result in an entirely healthy physiological state. Nevertheless, a partial therapeutic effect
is considered is considered toto fallwithin fall within the the scope scope of the of the invention. invention.
Whengene When gene expression expression is is selected selected as asa a parameter, parameter, thethe genes, genes, andand thethe expression expression
pattern of pattern of each each ofof said said genes genesunderlying underlying thethe modification modification of each of each biological biological activity activity
20 20 causallylinked causally linkedtotoeach each selected selected hallmark hallmark of a pathological of a pathological condition condition can be by can be retrieved retrieved by the skilled the skilledperson person from from the the state state of art, of the the art, this this work work can becan be facilitated facilitated by usingby ad using hoc ad hoc bioinformatics tools. bioinformatics tools. The sameQiagen The same QiagenIPAIPA indicated indicated above above is is suitableforfora afast suitable fast sorting sorting out of out of said said information informationfrom from scientificliterature, scientific literature, as as it it is is an aggregatorofofscientific an aggregator scientific references that references that allows allows toto search search forfor information information on on genes/proteins genes/proteinsand andthetheconstruction construction 25 25 of networks of networksthat thatpredict predictthe thebehaviour behaviourof of biological biological systems systems according according to their to their genegene
expressionstatus. expression status. Thepathophysiological The pathophysiological features features (hallmarks) (hallmarks) availableavailable in the state-of-the-art in the state-of-the-art for a for a given pathological given pathological condition condition are are used used to to interrogate interrogate IPA via the IPA via the "IPA “IPABioprofiler" Bioprofiler”tool, tool, using them using themasaskey key words, words, additional additional keywords keywords related related to said to said hallmarks hallmarks can also canbealso be 30 30 added. added.
Theuse The useofof "IPA "IPABioprofiler" Bioprofiler"allows allowsthe theskilled skilledperson persontotoidentify identify thethe expressed expressed genes causally genes causallylinked linkedtotoeach eachof ofthethe identified identified biological biological activitiesandand activities thethe specific specific
molecular pathway molecular pathway underpinning underpinning them. them. Information Information concerning concerning the the measured measuredgene gene expression data expression data(by(bywaywayof of example, example, following following the manufacturer’s the manufacturer's instructions instructions Fold Fold
35 35 changevalue change valuecut-off cut-off<-2≤-2and and> ≥+2+2 andand p-value≤0.05) p-value<0.05) induced induced by each by each batch batch was thenwas then superimposedonon superimposed thethenetworks networks obtained, obtained, to define to define influenced influenced genes genes and and modulation modulation of of the connected the biofunction. connected biofunction.
Oncethe Once themost mostrelevant relevantgenes genesandand thethe expression expression pattern pattern thereof thereof underlying underlying thethe
44 10 May 2024
modification detectable in the pathological condition of interest for each of the selected modification detectable in the pathological condition of interest for each of the selected
biologicalactivities biological activitiesareareidentified, identified,forforeach each of of saidsaid genes genes the expression the expression patternpattern oppositeopposite
to the to the one oneidentified identifiedis is setset as expression as the the expression pattern pattern indicative indicative of the of the healthy healthy
physiological physiological state state of of said said biological biological activities. activities.
5 5 As stated As stated above, above, modulation modulationofofbiological biologicalactivity activity isis divided divided inindown-down- modulationororup-modulation modulation up-modulation depending depending on theonregulation the regulation of the of the related related genes The genes The expression pattern expression pattern induced inducedbybythe thereference referencebatchbatchofofthethe product productofofinterest interest can be used can be used to calculate calculate thethe expected expectedimpactimpactonon thetheoneone or more connected biological activities. 2024203121
to or more connected biological activities.
Specifically, the Specifically, the resulting resulting expected calculated impact, expected calculated impact,based basedononthethe literature,ononthe literature, the 10 10 related biofunctions related biofunctions can canbebedetermined determined by “IPA by "IPA MoleculeMolecule ActivityActivity Predictor" Predictor” tool tool (MAP) (MAP) and and resumed resumed in ainheatmap a heatmap visualization visualization usingusing a colour a colour code can code that thateasily can easily be be transformedinto transformed into numeric numericvaluesvaluesbybythe theuser. user. In vitro In vitro cell-based cell-based assays assays are are well-known laboratorytechniques well-known laboratory techniquesthat thatinvolve involvethethe use ofofisolated use isolatedcells cellstotostudy study biological biological processes processes or the or test testeffects the effects of drugs, of drugs, chemicals, chemicals,
15 15 or other or othersubstances. substances. Accordingtotothe According theinvention, invention,cell cell culture culture models modelssuch such as as monolayer monolayer cultures, cultures, or three- or three-
dimensional(3D) dimensional (3D)systems systemscancan bebe used.When used. When appropriate, appropriate, disease-specific disease-specific celllines cell linescan can be used, depending on the disease of interest (e.g., cancer) also proliferation assays can be used, depending on the disease of interest (e.g., cancer) also proliferation assays can
be used. be used. 20 20 AAsuitable suitableininvitro vitrocell-based cell-based assayassay is anisassay an assay designed designed to mimictoa mimic disease a disease e.g., e.g.,
duetotothe due thenature natureof of thethe cells cells used, used, or by orinducing by inducing a diseased a diseased phenotypephenotype in cells treated in cells treated
with specific with specific compounds compounds i.e., i.e., aa "disease "disease model assay" or model assay" or "disease-in-a-dish" "disease-in-a-dish" model.model.
Cell Type Cell TypeSelection: Selection:a acell cellline line ororprimary primarycells cellsrelevant relevanttotothe thedisease diseasebeing being modelledisisselected. modelled selected. By Byway way of of example, example, if studying if studying OA, a OA, a suitable suitable and recognised and recognised
25 25 cell-based assay cell-based assay is is with withchondrocytes chondrocytes that,upon that, upon treatment treatment withwith IL1B,IL1B, are a are a disease disease
model ofofOAOA model acknowledged acknowledged in the in art. the art. StillStill by of by way wayexample, of example, if studying if studying neurodegenerativediseases, neurodegenerative diseases,neuronal neuronalcell cell lines lines like like SH-SY5Y, primary SH-SY5Y, primary neurons neurons may may be be
selected or selected or primary cells in primary cells in which which the the desired desired phenotype phenotypecan canbebeinduced induced by by an an “insult” "insult"
with aa given with compound given compound cancan be be used. used.
30 30 For the For the study study ofof Osteoporosis, Osteoporosis, in in particular particularPMO, PMO, aa suitable suitable cell-based cell-based assay assay can can
be prepared be prepared bybyusing usingHuman, Human, adipocyte-derived, adipocyte-derived, mesenchymal mesenchymal stem lines stem cell cell lines (hADMSC), (hADMSC), capable capable of differentiating of differentiating intointo osteoblasts osteoblasts andand mineralize mineralize the the extracellular extracellular
matrix (ECM) matrix (ECM) asas describedininthe described theexamples. examples. Forthe For thestudy studyofofcancers, cancers, viability viability teststests can can be carried be carried out onout on suitable suitable (depending (depending
35 35 onthe on thecancer cancerofofinterest) interest) cell-based cell-based assays. assays.
In case, In case, e.g., e.g., ROS ROS scavenging scavenging activity activity is a selected is a selected biological biological activity,activity, a suitable a suitable
cell-based assay cell-based assay cancanbebecarried carriedout outwithwitha ahuman human fibroblast fibroblast (HuDe) (HuDe) cell cell line line insulted insulted
with aa generator with generatorofofROSROS (e.g., (e.g., AAPH AAPH 2,2’-azobis-2-methyl-propanimidamide, 2,2'-azobis-2-methyl-propanimidamide,
45 10 May 2024
dihydrochloride) as known in the art. dihydrochloride) as known in the art.
For cell-based For cell-based assays assayswherein wherein thethe pathological pathological statestate mustmust be induced be induced by an by an
insult, the insult, the assay assaycells cellscan can also also be be directly directly tested tested with with the product the product of interest of interest in orderin toorder to
verify that verify thatthe themodulation modulation of selected of the the selected biological biological activities activities has thehassamethe same trend trend of the of the
5 5 modulation modulation expected expected to lead to to lead to a healthy a healthy physiological physiological state. Thisstate. This is particularly is particularly useful useful for assessing for assessingthe thebeneficial beneficial (adjuvating (adjuvating homeostasis) homeostasis) effect effect of a product. of a product.
Thecell-based The cell-basedassays assayspreferably preferably include include appropriate appropriate control control groups, groups, such such as as untreated cells, cells, vehicle-treated vehicle-treatedcells, andandcells treated with compounds knowntotohave have 2024203121
untreated cells, cells treated with compounds known
no impact no impactononthe thedisease diseasephenotype. phenotype.These These controlshelp controls helpdistinguish distinguishthe thespecific specificeffects effects 10 10 of the of the tested testedcompounds. compounds.
In those In those embodiments embodiments ininwhich which thethe expression expression of of oneoneor or more more genes, genes, e.g.,a agene e.g., gene expressionprofile, expression profile,is isused usedas as a parameter a parameter of oneoforone moreorbiological more biological activitiesactivities connected connected
to aa pathological to pathological condition condition of interest, of interest, a transcriptomics a transcriptomics analysisanalysis on a insuitable on a suitable vitro in vitro cell-basedassay cell-based assay mimicking mimicking the pathological the pathological conditioncondition of interestof can interest can be be carried carried out, and out, and 15 15 the genes, the genes, and the genes and the genes and andthethe expression expressionpattern pattern thereof thereof underlying underlyingthe themodification modification detectable in the pathological condition for each of the selected biological activities can detectable in the pathological condition for each of the selected biological activities can
be identified. be identified.InInthethecase caseininwhich which the the disease disease phenotype phenotype is caused is by caused by the administration the administration
of aa specific of specificagent agentto to thethe cultured cultured cells,cells, the modification the modification representing representing the pathological the pathological
condition is condition is the the modification modificationinsultedinsultedcellscellsVS.vs.pre-insulted, pre-insulted,untreated untreatedcellscellsand and thethe
20 20 modification induced modification inducedbyby thethe reference reference standard standard is the is the modification modification insulted insulted cells cells VS. vs.
insultedcells insulted cells++reference reference standard. standard.
Thetranscriptomic The transcriptomicanalysisanalysiscancan be carried be carried out with out with any suitable any suitable technique technique knownininthe known theart, art, including including nextnext generation generation sequencing sequencing and andgenegeneexpression expressionmicroarrays microarrays and the and the transcriptomic transcriptomicexpression expression profilein in profile thethe basal basal cellscells as as opposed opposed to the to cells the cells 25 25 treated to treated to mimic mimic the thepathological pathologicalcondition conditioncancan be be evaluated evaluated in order in order to identify to identify the the
significantly differentially significantly differentiallyexpressed expressedgenesgenes andand their their expression expression patterns. patterns. According According to to the method the methodofofthetheinvention, invention,once oncethethe expression expression pattern pattern of of the the significantly significantly differentially expressed differentially expressed genes genes of of the the cells cells representing representing the the disease diseasephenotype phenotype VS.vs. thethe
cells before cells before thethe insult insult inducing inducingsaid saiddiseased diseased phenotype phenotype is assessed, is assessed, the opposite the opposite
30 30 expression pattern expression patternis isconsidered considered representative representative of theofrestoration the restoration of a healthy of a healthy
physiological state physiological state (herein (herein also abberviated as also abberviated as "representative “representative of of thethe healthy healthy physiological physiological state). state).
When When genegene expression expression is theisselected the selected parameter, parameter, for each for each biological biological activity inactivity a) in a) the genes the genes andandthetheexpression expressionpattern patternthereof thereofunderlying underlyingthethemodification modification detectable detectable in in
35 35 said pathological said pathological condition condition for each for each ofbiological of said said biological activities activities can be identified can be identified from from the state the state of of the the art art using usingappropriate appropriate tools. tools.
Byway By wayof ofexample, example, the the skilled skilled person person can can derivederive this this information information usingusing any any
suitable approach suitable includingusing approach including usingsoftware softwarespecifically specificallydesigned designedfor forthis this scope scopesuchsuchasas
46 10 May 2024
Ingenuity Pathway Ingenuity PathwayAnalysis Analysis(IPA (IPA version version 94302991 94302991 Qiagen). Qiagen).
The interpretation The interpretation of of high-throughput gene-expressiondata high-throughput gene-expression dataisisgreatly greatlyfacilitated facilitated by the by the consideration consideration ofof prior prior biological biological knowledge. knowledge.This This cancan be be done done using using statistical statistical
gene-set-enrichment methods gene-set-enrichment methods where where differentially differentially expressed expressed genes genes are are intersected intersected withwith 5 5 sets of sets of genes genesthat thatare areassociated associated withwith a particular a particular biological biological activity activity or pathway or pathway
(Abatangelo, (Abatangelo, L. L. et et al. al. (2009) (2009) Comparative studyofofgene Comparative study geneset setenrichment enrichmentmethods. methods. BMCBMC
Bioinform., 10, Bioinform., 10,275). 275).OneOne more more recent recent approach approach involves involves the application the application of causal of causal
networksthatthat integrate integrate previously previouslyobserved observedcause-effect cause–effect relationshipsreported reported in in thethe 2024203121
networks relationships
literature (Chindelevitch, literature (Chindelevitch, L. L. et et al. al. (2012a) Causalreasoning (2012a) Causal reasoningonon biological biological networks: networks:
10 10 interpreting interpreting transcriptional transcriptionalchanges. changes. Bioinformatics, Bioinformatics, 28,28, 1114–1121; Felciano,R.M. 1114-1121; Felciano, R.M.et et
al. (2013) al. (2013) Predictive systemsbiology Predictive systems biologyapproach approach to to broad-spectrum, broad-spectrum, host-directed host-directed drugdrug
target discovery target discovery in in infectious infectiousdiseases. diseases.Pac. Symp. Pac. Symp. Biocomput., 2013, 17-28; Biocomput., 2013, 17–28;Kumar, Kumar, R.R.
et al. et al. (2010) (2010) Causal reasoningidentifies Causal reasoning identifies mechanisms mechanisms of sensitivity of sensitivity forfor a novel a novel AKT AKT
kinase inhibitor, kinase inhibitor, GSK690693. GSK690693. BMC BMC Genom., Genom., 11, 419; 11, 419; Martin,Martin, F. etF.al. et (2012) al. (2012) 15 15 Assessmentofofnetwork Assessment network perturbation perturbation amplitudes amplitudes by applying by applying high-throughput high-throughput data to data to
causal networks. causal networks. BMC BMC Syst. Syst. Biol., Biol., 6, 6, 54;54; Pollard,J.J.Jr. Pollard, Jr. et et al. al. (2005) (2005) AAcomputational computational modeltotodefine model definethe themolecular molecular causes causes of type of type 2 diabetes 2 diabetes mellitus. mellitus. Diabetes Diabetes Technol. Technol.
Ther., 7, 323–336). While still depending on statistics, this is more powerful than gene- Ther., 7, 323-336). While still depending on statistics, this is more powerful than gene-
set enrichment set sinceitit leverages enrichment since leverages knowledge knowledge about about thethe direction direction of of effectsrather effects ratherthan than 20 20 mereassociations. mere associations. In aa preferred In preferred embodiment, embodiment, the theskilled skilledperson personcan canfollow followthetheprotocol protocolprovided provided in the in the publication publication by by Kramer Krameret etal,al,Bioinformatics Bioinformatics volvol 30 30 no no 4 2014, 4 2014, pagespages 523-530523-530 “Causal analysis approaches "Causal analysis approachesininIngenuity IngenuityPathway Pathway Analysis Analysis provides provides andand discuss discuss a suite a suite
of algorithms of algorithms andandtools tools for for inferring inferring andand scoring scoring regulator regulator networks networksupstream upstream of of genegene 25 25 expression data expression databased basedon on a large-scale a large-scale causal causal network network derivedderived from thefrom the Ingenuity Ingenuity
KnowledgeBase" Knowledge Base”or or thethe manufacturer’s manufacturer's instructionsofof(IPA instructions (IPA version version 94302991 94302991 Qiagen). The Qiagen). Themethod method andand algorithms algorithms disclosed disclosed in in thethe paper paper enable enable thethe skilledperson skilled personto to
predictdownstream predict downstream effects effects on biological on biological activities activities and diseases. and diseases.
In the In the article, article, thethe authors authors describe describe causal analysis approaches causal analysis approachesthat thathave havebeenbeen 30 30 implementedininIngenuity implemented IngenuityPathway Pathway Analysis Analysis (IPA)(IPA) with with particular particular focusfocus on theondetails the details of the of the underlying underlyingalgorithms, algorithms,and andthetheapplication application to to several several real-world real-world useuse cases. cases. In In
particular, points particular, pointsa)a)b.b.and anda)a)C.c.can can be be readily readily carried carried outthe out by byskilled the skilled personperson by using by using Ingenuity Pathway Ingenuity Analysis (IPA Pathway Analysis (IPA version version 94302991 94302991Qiagen)Qiagen)whichwhich is iswell-known well-known pathwayanalysis pathway analysisapplication applicationamongamong thethelife lifescience science research research community community cited cited inin tensofof tens
35 35 thousandsofof articles thousands articles that that allows allows to to understand causal connections understand causal connectionsbetween between andand among among
diseases, genes diseases, genes and and networks networks of of upstream upstreamregulators. regulators. Step b) Step b) of of the the method method ofof the the invention invention comprises: comprises:
quantifying in quantifying in terms termsofofnumerical numericalvaluesvaluesthethe modulation modulation of said of said one one or moreor more
47 10 May 2024
biological activities induced by each batch of step a) for each cell-based assay read-out biological activities induced by each batch of step a) for each cell-based assay read-out
definingasasreference defining reference values values the the values values calculated calculated for theforreference the reference standardstandard batch; batch; moreinin detail more detail the the step stepcan can comprise comprise analysing analysing the the modulation modulation of ofeach eachofof the the one one of more of biological activities more biological activities and/or and/or parameters thereof induced parameters thereof induced by byaareference referencestandard standard 5 5 of said of said product productininthethein in vitro vitro cell-based cell-based assayassay performed performed in step in a) step a) and determining and determining the the quali-quantitative modulation quali-quantitative modulationofofeach each of one of one of biological of more more biological activities activities and/or and/or
parametersthereof parameters thereofinduced inducedbybysaid saidreference reference standard standard andand calculating calculating thethe modulation modulation
value for for each each of of said said biological biological activities activities induced induced by said reference reference standard standard thereby thereby 2024203121
value by said
providingthethe providing reference reference standard standard modulation modulation values values for each for eachbiological of said of said biological activities activities
10 10 indicativeofofsaid indicative saiddesired desired therapeutic therapeutic effect effect (cut-off (cut-off values). values).
Fromthetheresults From results obtained obtained withwith the cell the cell basedbased assay assay it is possible it is possible to determine to determine the the quali-quantitative modulation quali-quantitative modulation ofofeach eachof ofthethe activitiesand/or activities and/or parameters parameters previously previously
identified, induced identified, inducedby by each each product product batchbatch samplesample with to with respect respect to thegroup the control control group and it and it
is therefore is possible to therefore possible to obtain obtain values valuesrepresenting representingthethequalitative qualitative(which (which activity activity is is
15 15 modulated and modulated andininwhich whichdirection, direction,i.e. i.e. up up modulation modulationpositive positive value valueorordown- down- modulationnegative modulation negativevalue) value)and andquantitative quantitative(how (howmuch much is is each each activitymodulated activity modulated with with
respect to respect to the the control controlgroup) group) modulation modulation induced induced by by each batch on each batch on each each ofof said said biological activity; the magnitude of the value representing the distance (in terms of %, biological activity; the magnitude of the value representing the distance (in terms of %,
folds etc.) folds etc.) with with respect respect to to the the control control group. group. The Themodulation modulation of of eacheach activity activity cancan be be
20 20 expressedasasa numerical expressed a numerical value, value, such such a statistical a statistical numerical numerical value. value.
Dependingonon Depending thethe parameters parameters selected, selected, thethe modulation modulation of a of a biological biological activity activity
can be can bedefined definedandand characterized characterized by the by the skilled skilled person person by commonly by commonly used variousused various quantitativevalues quantitative values depending depending on theon the specific specific contextcontext and the typeand the type of biological of biological activity activity being studied. being studied. Some common Some common values values usedused to define to define modulation modulation include: include: foldfold change change i.e. i.e.
25 25 the ratio the ratio of of the thevalue valueofofthethe biological biological activity activity underunder a particular a particular condition condition or treatment or treatment
to its to itsvalue valueunder under aacontrol controlor orreference referencecondition; condition;bybyway way ofof example, example, a a fold fold change change ofof 11 indicates indicates no no change, change, while values greater while values greater thanthan 1 1 indicate indicate upregulation upregulation and values less and values less than 11 indicate than indicate downregulation; downregulation;loglogfold foldchange change i.e. i.e.thethelogarithm logarithm (usually (usually base base 2 or 2 or
base 10) base 10) of of the the fold fold change; change; percentage percentagechange,change,i.e. i.e. the the percentage percentagedifference differencebetween between 30 30 the value the value ofof the the biological biological activity activity under under aa particular particular condition condition and andits its value value under undera a control or control or reference reference condition; condition; z-score, z-score, which whichz-score z-scorerepresents representsthethedeviation deviationof ofthethe
observedvalue observed valueof ofthethe biological biological activity activity from from its mean its mean value,value, normalized normalized by the by the
standarddeviation standard deviation (a positive (a positive z-score z-score indicates indicates an increase an increase in activity, in activity, while a while negativea negative z-score indicates z-score indicates aadecrease); decrease);effect effectsize,size,i.e.i.e. aameasure measure of the of the magnitude magnitude of theof the
35 35 difference between difference betweentwotwo groups, groups, oftenoften standardized standardized to facilitate to facilitate comparisons comparisons across across
studies or studies or datasets datasets (Cohen's (Cohen'sd dis isa acommoncommon effecteffect size measure, size measure, calculatedcalculated as the as the
difference in difference in means dividedby means divided bythe the standard standard deviation); deviation); area area under the curve under the (AUC),for curve (AUC), for dynamicbiological dynamic biologicalactivities, activities, such such as as signalling signalling pathways pathwaysororphysiological physiologicalresponses, responses,
48 10 May 2024
the AUC can be used to quantify the overall activity or response over a period of time. the AUC can be used to quantify the overall activity or response over a period of time.
These values These values can canbebeused used individuallyor or individually in in combination combination to provide to provide a a comprehensivecharacterization comprehensive characterization of modulation of the the modulation of a biological of a biological activity activity under under different conditions different conditions or or treatments. treatments. TheThechoice choiceof of whichwhich value(s) value(s) the skilled the skilled person person 5 5 decides to decides to use use depends dependsonon thethe specificresearch specific research question, question, thethe nature nature of of thethe biological biological
activity, and activity, andthetheavailable availabledata. data. In the In the context contextofofbiological biologicalactivities, activities, aa z-score z-scorecan canbebe usedused to express to express the the
modulation or or alteration of aof a specific biological activity relative to itsto its typical or baseline 2024203121
modulation alteration specific biological activity relative typical or baseline
behaviour. This behaviour. Thisisisoften oftenemployed employed in fields in fields like like systems systems biology, biology, wherewhere researchers researchers
10 10 analyse high-dimensionaldatasets analyse high-dimensional datasetstoto understand understandcomplex complex biologicalprocesses. biological processes. In this In this case, case, the the z-score z-score represents represents howhowmuch much a particular a particular biological biological activity activity
deviates from deviates fromitsitsexpected expected or or average average behaviour behaviour withinwithin a givena context, given context, often in often in
response to response to some somestimulus, stimulus,treatment, treatment, oror condition. condition. For example, For example,inin gene geneexpression expressionanalysis, analysis,aa z-score z-score might mightbebecalculated calculated to to assess assess 15 15 howmuch how much thethe expression expression level level of of a a gene gene changes changes in in response response to to a therapeutic a therapeutic treatment treatment
compared totoits compared its expression expression inincontrol controlconditions. conditions. A Apositive positivez-score z-scoreindicates indicates upregulation, while upregulation, a negative while a negative z-score z-score indicates indicates downregulation. downregulation.
Mathematically,a asuitable Mathematically, suitableformula formula forfor calculating calculating thethe z-score z-score of of a biological a biological
activity modulation activity might modulation might involve involve comparing comparing its observed its observed valuea specific value under under a specific 20 20 condition to condition to the the mean andstandard mean and standarddeviation deviationofofits its values values across across multiple multiple conditions conditions or or replicates: replicates:
z=x−μ/σ z=x-u/o WhereX xrepresents Where representsthethe observed observed value value of biological of the the biological activity activity (e.g.,(e.g., gene gene
expression level), μ is the mean value of the activity across all conditions, and σ is the expression level), u is the mean value of the activity across all conditions, and o is the
25 25 standarddeviation standard deviationof of thethe activity activity across across all conditions. all conditions.
This z-score This z-score approach approachallows allows researchers researchers to identify to identify and and prioritize prioritize biological biological
activities that activities that are aresignificantly significantlyaltered altered under under certain certain experimental experimental conditions, conditions, providing providing
insights into insights intothe theunderlying underlying mechanisms mechanisms of ofbiological biological systems. systems. Oncethe Once thedesired desiredmodulation modulation of of the the biological biological activity activity is is defined defined (i.e.the (i.e. theone one 30 30 representingthethe representing healthy healthy physiological physiological state)state) and optionally and optionally the regulation the desired desired regulation of the of the related parameter related parameter(e.g. (e.g.ROSROS scavenging scavenging activity, activity, genes, genes, etc.) etc.) is identified is identified for thefor the pathological condition pathological conditionofofinteres,t, interes,t, an ananalysis analysisofofthethemodulation modulation of biological of the the biological activity activity induced induced by bythethereference referencestandard standardof of thethe therapeutic therapeutic product product (optionally (optionally the the
modulationofofone modulation oneorormoremore parameters parameters thereof, thereof, e.g.,gene e.g., gene expression expression as determined as determined by by
35 35 transcriptomics analysis) transcriptomics analysis)isiscarried carriedoutout on in on the thevitro in vitro cell-based cell-based assayis that is assay that
representative of representative of the thepathological pathological condition. condition. As already As already clarified clarified previously, previously, the the referencestandard reference standardhas has a previously a previously assessed assessed therapeutic therapeutic or beneficial or beneficial effect. Theeffect. quali- The quali-
quantitativemodulation quantitative modulation of selected of the the selected biological biological activity/ies activity/ies and/or parameter/s and/or parameter/s thereof thereof
49 10 May 2024
(e.g. of (e.g. of the the expression of the expression of the genes genesofofinterest) interest) induced inducedbybythethereference reference standard standard is is determinedand determined andthethemodulation modulation values values forfor each each of the of the selected selected biological biological activitiesare activities are calculated and calculated subsequentlyused and subsequently usedasasreference referencemodulation modulation values values or or “cut-offvalues" "cut-off values”forfor eachofofsaid each saidbiological biological activities activities indicative indicative of said of said desired desired therapeutic therapeutic effect.effect.
5 5 Thereference The referencestandard standardofofthe theproduct producttested testedis,is, asas stated stated above, above,aabatch batchofofthethe productofofinterest product interestforfor which which a therapeutic a therapeutic or beneficial or beneficial effect haseffect been has beeneither assessed assessed either in vitro in vitro and/or and/orininvivo, vivo,preclinically, preclinically, or or clinically. clinically. In In anyany casecase it isit aisbatch a batch of product of the the product of interest interest for for which thetherapeutic therapeuticororbeneficial beneficialeffectiveness effectivenesshashasbeen been previously 2024203121
of which the previously
verified and verified andisishence hence defined defined as aas a batch batch with with a known a known therapeutic therapeutic or beneficial or beneficial effect. effect. 10 10 Accordingtotoananembodiment, According embodiment, thethe reference reference standard standard modulation modulation values values for each for each
biologicalactivity biological activityare aretaken takenas as reference reference modulation modulation values values that arethat are representative representative of the of the desired therapeutic desired therapeutic ororbeneficial beneficialeffect. effect.TheThe reference reference modulation modulation values values can be can be
adjusted, as adjusted, as explained below,when explained below, when thethe cell-based cell-based assay assay is is carried carried outout alsoalso on on oneone or or
morereference more reference drugs drugs (i.e.(i.e. drugs drugs that that are known are known in the in the art art as indicate as indicate for the treatment for the treatment of of 15 15 the pathological the pathological condition conditionofofinterest). interest). Said drugsare Said drugs areexpected expectedtotomodulate modulate withwith the the
desired trend desired trend at at least least some ofthe some of theselected selectedbiological biologicalactivities activities although, although, due duetototheir their different mechanism different mechanism ofofaction action(SAR), (SAR), saiddrugs said drugs areare notexpected not expected to to modulate modulate withwith the the desiredtrend desired trendallallofofsaid said selected selected biological biological activities. activities. Therefore, Therefore, as explained as explained in more in more detail below, detail the adjustment below, the adjustmentofofthe thereference referencemodulation modulation values values considering considering also also the the
20 20 drug modulation drug modulationvalues values willbe be will carried carried outout onlyonly forfor saidsaid biological biological activitiesthat activities thatareare modulatedbybythethetested modulated testeddrugdrugin in thethewithwith thethesamesame trendtrend of the of the healthy healthy physiological physiological
state. state.
Themodulation The modulation values values in in step step b) b) areare calculated calculated to to represent represent thethe directionality directionality
and magnitude and magnitudeofofthe themodulation modulation forforeach eachbiological biologicalactivity activity exerted exerted byby aa given given batch batch ofof 25 25 product. For product. For transcriptomics, transcriptomics, the the Core Core analysis analysis of of the the IPA IPA version version 94302991 Qiagen 94302991 Qiagen cancan
be used be used by bythe the skilled skilled person person toto readily readily obtain obtain thethe aforementioned modulationvalues aforementioned modulation valuesinin termz of termz of Z-score Z-score values values following followingthe the manufacturer's manufacturer’sinstructions. instructions. In case In casethe theCore Core analysis analysis does does not sufficient not yield yield sufficient relevantrelevant information, information,
alternative approaches alternative approaches can canbebe employed. employed. An example An example of an alternative of an alternative approachapproach is is 30 30 providedbelow provided below(overlay (overlay analysis).Step analysis). Stepb) b) of of thethe method method of theof invention the invention comprises comprises
quantifying inin terms quantifying termsofofnumerical numerical values values the the modulation modulation pattern pattern of the of the parameters parameters
identifiedinina)a)C.c.induced identified induced by further by further different different batchesbatches of said of saidinproduct product said in in said in vitro vitro
cell-based assay cell-based assaybybydetermining determining the the quali-quantitative quali-quantitative modulation modulation of eachof ofeach said of said
parametersinduced parameters inducedbybyeach eachof of saidbatches said batches thereby thereby calculating calculating theZ-score the Z-score modulation modulation
35 35 valuefor value foreach eachofofsaid said biological biological activities activities induced induced byofeach by each saidof said batches. batches.
Preferably at Preferably at least least three three batches that are batches that acceptable and are acceptable andoneonebatch batchthat thatisisnot not acceptable with acceptable withare aredesired, desired,andand so the SO the skilled skilled personperson must analyse must analyse at leastatfour least four additionalbatches additional batches of ofthethe product product of interest. of interest.
50 10 May 2024
As stated As stated above, above,the thebatches batchesofofthat thatare areconsidered considered acceptable acceptable areare batches batches for for
whichthe which themodulation modulation value value forfor each each biological biological is is activity activity > ≥ thereference the reference modulation modulation
value when value whenthe thedesired desiredmodulation modulationof of thebiological the activityisis an biologicalactivity an up upmodulation modulationandand < ≤ the reference the reference modulation valuewhen modulation value when thethedesired desiredmodulation modulation of ofthethe biologicalactivity biological activityisis 5 5 a down a modulation. down modulation. Conversely, Conversely, whenwhen the modulation the modulation value value for atfor at least least one biological one biological
activity activity does does not fulfil the not fulfil therequirements requirements above, the batch above, the batch is is considered considerednon-compliant non-compliant (not acceptable) and (not acceptable) andcancan be be usedused as a as a reference reference of notofacceptability not acceptability for thefor the final final
assessmentofof thethe acceptability acceptability values values ofof spectroscopy spectroscopy or or spectrophotometry. 2024203121
assessment spectrophotometry.
Accordingtotoa preferred According a preferred embodiment embodiment of theof the invention, invention, additionaladditional different different 10 10 batches of batches of the the product product ofof interest interest can can bebe analysed. analysed. InIn particular, particular,whenwhen a a batch batch X X of of the the product ofofinterest, product interest, that that has has not notbeen beenanalysed analysedwithwith the the cell-based cell-based assayassay described described
herein, does herein, not fall does not fall within within the the spectroscopy spectroscopy or or spectrophotometry spectrophotometryacceptability acceptabilityvalues values in the in the process processofofbatch-to-batch batch-to-batch validation validation of theof the invention, invention, steps a)steps to c) a) of to thec)method of the method for determining for determiningthetheacceptability acceptabilityvalues values of the of the spectroscopy spectroscopy or spectrophotometry or spectrophotometry
15 15 analysis can analysis be performed can be performedononsaidsaidbatch. batch.InIncase casethe thebatch batchcomplies complies with with thethe reference reference
modulationvalues modulation values (i.e.thethebatch (i.e. batch exerts exerts the the desired desired modulation modulation of the of the biological biological
activities which are indicative of the desired therapeutic effect), batch X is considered activities which are indicative of the desired therapeutic effect), batch X is considered
acceptable and acceptable and the the acceptability acceptability values values ofof the the spectroscopy spectroscopy or or spectrophotometry spectrophotometrytotouse use in the in the process processofofthe theinvention invention can can be adjusted be adjusted accordingly. accordingly.
20 20 Additionally, if Additionally, if desired, desired,steps stepsa)a)and andb)b)can canbebeperformed performed also also with with one or more one or more reference drug reference drug(i.e., (i.e., aadrug drugconventionally conventionally administered administered fortreatment for the the treatment of the of the
pathological condition pathological condition ofof interest). interest). For each biological For each biological activity activity modified modifiedalsoalsobybysaid said one oror more one more drug drug in the in the direction direction of healthy of the the healthy physiological physiological state,state, the reference the reference
modulationvalue modulation valuecancanbe be adjusted adjusted by comparing by comparing the drugthemodulation drug modulation value andvalue the and the
25 25 reference standard reference standard modulation modulationvalue, value,andand selecting selecting as as reference reference modulation modulation valuevalue the the
one associated one associated to to the the weakest performance. weakest performance.
As conventional As conventionaldrugs drugs areare expected expected to modulate to modulate in thein desired the desired direction direction only only
someofofthe some theselected selected biological biological activities activities due due to to their theirmechanism mechanism of of action action (SAR), (SAR),only only the modulation the modulation values values related related to thetoactivities the activities thatsignificantly that are are significantly modulatedmodulated with the with the 30 30 samedirectionality same directionality of of thethe healthy healthy physiological physiological statebewill state will be considered. considered.
As known As knownby by thethe skilledperson, skilled person,reference referencedrugsdrugsforforthethetreatment treatmentofofa a pathological condition pathological condition may maybebe known known a priori a priori to provide to provide a therapeutic a therapeutic effect effect onlyonly on on
oneororfew one few hallmarks hallmarks of disease. of the the disease. In thisIncase, this case, the information the information that willthat will be integrated be integrated
in the in the method method ofofthe theinvention inventionwill willrelate relate only onlytotothe theinterested interested hallmarks hallmarksand andrelated related 35 35 biologicalactivities. biological activities. As stated As stated above, above, thethe method methodofofthe theinvention inventionmaymay comprise comprise determining determining one orone or moreparameter more parameterand andthethemodulation modulation trend trend thereof thereof underlying underlying thethe modification modification detectable detectable
in said in said pathological pathological condition condition for each for each of biological of said said biological activities activities in both in both diseased diseased and and
51 10 May 2024
healthy physiological healthy physiological state. state. In In aa preferred preferred embodiment saidparameters embodiment said parametersandand areare genes. genes.
Preferably, gene Preferably, gene modulation is assessed modulation is assessed by by transcriptomic transcriptomic analysis. analysis.
In aa not In not limiting limitingembodiment the method embodiment the methodofofthe the invention invention can can be be carried carried with with the the
aid of aid of IPA IPA version version 94302991 Qiagenoutoutasasfollows: 94302991 Qiagen follows: 5 5 Definition of Definition pathophysiological state of pathophysiological state of the art of the art of of the the disease disease and andof ofthethe pathophysiologicalhallmarks pathophysiological hallmarksofofthe the disease disease with with which to interrogate which to interrogate IPA IPA
The pathophysiological state-of-the-art of the disease of interest is investigated The pathophysiological state-of-the-art of the disease of interest is investigated
usingdifferent differentspecific specificsources: sources: 2024203121
using
- Robbins - Robbins && Cotran CotranPathologic Pathologic Basis Basis ofof Disease Disease (Robbins (Robbins Pathology) Pathology) 10th 10th 10 10 Edition Edition
- https://calgaryguide.ucalgary.ca/ - https://calgaryguide.ucalgary.ca/
- Biomedical - literature from Biomedical literature from PubMed Central(https://pubmed.ncbi.nlm.nih.gov/) PubMed Central (https://pubmed.ncbi.nlm.nih.gov/) The information The informationfound foundininthe the aforementioned aforementionedresources resourcesareareused usedtotoidentify identify hallmarks to hallmarks to
interrogate IPA interrogate IPA by by the following following procedure: procedure:
15 15 • Hallmarksare Hallmarks arewritten, written, one one bybyone, one,inin the the "disease "disease and and functions" functions"query query box and box and the the search search is is then thenlaunched. launched.
• The obtained The obtainedresuming resuming table table allows allows you you to filter to filter disease/activity disease/activity thatthat comefrom come frommany manylineslines of evidence. of evidence. The The sourcesource for the for relationship the relationship is theis Ingenuity the Ingenuity KnowledgeBase, Knowledge Base,including includingcuration curationfrom from journal journal articles,OMIM, articles, OMIM,JAX andJAX and 20 20 ClinicalTrials.gov. ClinicalTrials.gov.
• In silico In silicomodel model will willbe belimited limitedtotogenes genesandand mRNAs. mRNAs.
The tool The tool cancan associate associate with with each eachbiological biological activity activity aa defined defined number number ofofgenesgenes whosemodulation whose modulation is able is able to influence to influence the modulation the modulation of the biological of the biological activity activity itself. itself.
The following The followingapplies appliestotoany anystep stepofofthethemethod method of ofthethe invention invention in in any any embodiment embodiment
25 25 herein described, herein described, wherein whereinananininvitrovitro cell-based cell-based assay assay isis carried carried out out treating treating thethe cells cells
with the with the disease disease phenotype phenotypewith with a given a given compound, compound, be it be theitreference the reference standard, standard, a a different batch different batchofofthetheproduct product of of interest, interest, a reference a reference drug, drug, or the or the like. like.
A whole A wholetranscriptome transcriptomeraw rawdatadataanalysis analysis Wholetranscriptome Whole transcriptome expression expression profile profile is evaluated is evaluated in in in the thevitro in vitro cellular cellular
30 30 model representing model representing the the disease disease for forcontrol controlcells cells andandforfor model modelcells. cells.A AHumanHuman Clariom™S SPico ClariomM PicoAssay Assay HT HT (Applied (Applied Biosystems, Biosystems, ThermoFisher ThermoFisher Scientific) Scientific) on on a a
GeneTitanMCMC GeneTitan Instrument Instrument (Applied (Applied Biosystems, Biosystems, ThermoFisher ThermoFisher Scientific), Scientific), can becanused be used following the following themanufacturer's manufacturer'sinstructions. instructions.CEL CEL Intensity Intensity filesfiles cancan be generated be generated by by Affymetrix GeneChip Affymetrix Command GeneChip Command Console Console Software(AGCC, Software (AGCC, ThermoFisher ThermoFisher Scientific). Scientific). 35 35 Data analyses Data analyses cancan bebe performed performed using using Transcriptomic Transcriptomic Analysis Analysis Console Console Software Software (TAC,ThermoFisher (TAC, ThermoFisher Scientific) Scientific) that that provides provides qualityquality controlcontrol analysis, analysis, performsperforms
normalization and normalization andsummarization, summarization, based based on the on the SignalSignal SpaceSpace Transformation-Robust Transformation-Robust
Multi-Chip Analysis Multi-Chip Analysis (SST-RMA) (SST-RMA) analysis analysis algorithm, algorithm, and provides and provides a lista oflist of
52 10 May 2024
differentially expressed differentially expressed genes (Limma genes (Limma Bioconductor Bioconductor package). package). This This phase phase allows allows the the user totoobtain user obtaina list a listofofdifferentially differentially expressed expressed genesgenes (DEGs),(DEGs), identified identified based on their based on their
expression fold expression fold changes changeswith with respect respect to to a relevant a relevant control control experimental experimental condition, condition, in in
this case this onethat case one thatreproduces reproduces the the pathological pathological state state in vitro. in vitro.
5 5 Thetranscriptional The transcriptionalmodifications modifications profile profile thusthus obtained obtained is subjected is subjected to a to a functional pathway functional enrichmentanalysis. pathway enrichment analysis.One One ofof thecommercial the commercial tools tools thatcan that canbebeused usedisis
Ingenuity Pathway Ingenuity PathwayAnalysis Analysis (IPA(IPA version version 94302991, 94302991, Qiagen) Qiagen)
[Krämer[Krämer et al. (2014)]. et al. (2014)].
Theuse useofofIPA IPAallows allows thethe userto to estimate howhowand and to what extent the modulation of 2024203121
The user estimate to what extent the modulation of
gene expression gene expressionininthethe cellularsystem cellular system (cell-based (cell-based assay)assay) influences influences the biological the biological
10 10 activities related activities related toto the thepathology pathology of of interest. interest.
IPApre-analysis IPA pre-analysis filtering filtering (contextual (contextual data data analysis) analysis) of transcriptional of transcriptional profile profile
In preparation In preparationforforsubsequent subsequent analyses, analyses, transcriptomic transcriptomic profilesprofiles undergoundergo a a filtration process filtration process to to identify identify relevant relevant genes genesandand theirtheir corresponding corresponding measurement measurement
values.This values. Thisfiltration filtrationaimsaims to select to select onlyonly significantly significantly perturbed perturbed genes, asgenes, as indicated indicated by by 15 15 their fold their fold change comparedtotothe change compared thepathological pathologicalcondition. condition.Typically Typically(e.g., (e.g.,asasaccording according to the manufacturer’s instructions), the fold change threshold is set to encompass values to the manufacturer's instructions), the fold change threshold is set to encompass values
≤ -2 < -2 and ≥ +2, and +2, accompanied accompanied by a by a statistical statistical significance significance denoted denoted by by a p-value a p-value of of ≤ 0.05. < 0.05.
However,thetheskilled However, skilledoperator operatorhashas thethe flexibilitytotoadjust flexibility adjustthethecut-off cut-offbased basedon on their their
expertise, considering expertise, considering the thesuccessful successfulperformance performance of negative of negative controls controls (a sample(a sample 20 20 representingthethe representing pathological pathological state) state) or reference or reference standard standard known to known be abletotobe able to counteract counteract
fully or fully or partially partially the thepathological pathological state. state.
Thepossible The possiblemethodological methodological approaches approaches to extract to extract biological biological meaningfulness meaningfulness
from aalist from list of of modifications modificationsofofgene geneexpression expression profiles profiles through through IPA IPA can becanof be twoof two
types: “Core types: analysis” and "Core analysis" and"Overlay “Overlayanalysis analysisofofininsilico silicomodel modelofofpatho-physiological patho-physiological 25 25 state” (abbreviated state" (abbreviated as as overlay overlay analysis). analysis).
At this At this stage stage ofof the the procedure, hence, two procedure, hence, twodifferent, different, alternative, alternative, options options can can be be
pursued. pursued.
CoreAnalysis Core Analysis Thelist The list of of Differentially Differentially expressed expressedgenes genes(DEGs) (DEGs) afterafter administration administration of the of the
30 30 reference standard reference standard or or ofof any any other other product productbatch/reference batch/referencedrug drugororother othersubstance, substance,and and correspondingdata corresponding datameasurement measurement values values (fold (fold change change withwith respect respect to pathological to pathological state) state)
identified in identified in the the different different experimental conditionsare experimental conditions areuploaded uploaded intointo thethe application. application.
Available identifiers Available identifiers are aremapped mapped toto their their corresponding entity in corresponding entity in QIAGEN's Knowledge QIAGEN's Knowledge
Base. Base.
35 35 Bylaunching By launchinga a"Core“Core Analysis”, Analysis", significantlyperturbed significantly perturbed DEGs, DEGs, called called Network Network
Eligible molecules, Eligible molecules,are areoverlaid overlaidonto onto a global a global molecular molecular networknetwork developed developed from from information contained information containedinin the the QIAGEN QIAGEN Knowledge Knowledge Base. Base. NetworksNetworks of Networkof Network Eligible Eligible
Moleculesare Molecules arethen thenalgorithmically algorithmicallygenerated generatedbased basedonontheir theirconnectivity. connectivity.
53 10 May 2024
The core The core analysis analysis provides provides aa comprehensive comprehensive list list of of approximately approximately top top 500 500 biological activities biological activities derived derived from the generated from the generatednetworks. networks.TheThe program’s program's association association
”Biological functions-genes" "Biological functions-genes”is isalways always supported supported by annotations by annotations corresponding corresponding to to scientific peer scientific peer review publications that review publications that substantiate, substantiate, through through the the automatic association automatic association
5 5 of aa Z-score of Z-score[Krämer
[Krämer et al. et al. (2014)], (2014)], the the calculated calculated directionality directionality and and magnitude magnitude of of modulationofofthethebiological modulation biologicalactivities. activities. InInessence, essence,this thisvalue valuerepresents representsa statistical a statistical metric that metric that assesses assessesthe thesimilarity similaritybetween between the observed the observed pattern pattern of Differentially of Differentially
ExpressedGenesGenes (DEGs) andexpected the expected patternpattern based on existing literature for a for a 2024203121
Expressed (DEGs) and the based on existing literature
given annotation. given annotation. 10 10 It is It is the the responsibility responsibility ofofthetheproficient proficientoperator operator to carefully to carefully choosechoose the the biologicalactivities biological activitiesthat thatarearerelevant relevant to the to the specific specific pathology pathology under investigation. under investigation. The The selectionofofbiological selection biological activities activities will will be structured be structured based based on the on the identified identified hallmarkshallmarks of of the pathology the pathology of of interest. interest.
Theassociated The associated Z-score Z-score values values will bethen will then usedbe toused to indicate indicate the directionality the directionality and and 15 15 magnitudeofofmodulation magnitude modulation for foreach eachbiological biologicalactivity. activity. OverlayAnalysis Overlay Analysis If the core analysis does not yield sufficient relevant information, an alternative If the core analysis does not yield sufficient relevant information, an alternative
approachcalled approach called"Overlay "Overlay analysis" analysis" can can be employed. be employed. This analysis This analysis focuses focuses on the on the
biologicalactivities biological activitiesidentified identified by by the the "in “in silico silico model model of theof the patho-physiological patho-physiological state". state". 20 20 Theselection The selection of of biological biological activities activities is structured is structured based based on on the identified the identified hallmarks hallmarks of of the pathology the pathology of of interest. interest.
The"Overlay The "Overlay analysis" analysis" is structured is structured by establishing by establishing a relationship a relationship betweenbetween patterns of patterns of differentially differentially expressed expressed genes genesandand selected selected biological biological activities activities (always (always
supported supported bybyannotations annotations corresponding corresponding to scientific to scientific peer peer reviewreview publications publications that that 25 25 substantiatethe substantiate thedirectionality directionality andand magnitude magnitude of modulation of modulation of the biological of the biological activities) activities)
using the using the following procedure: following procedure:
Importthethesetsetofofbiological Import biological activities activities selected selected from from the in-silico the in-silico model of model of
the patho-physiological the patho-physiological state state into intoaanew new sheet sheet called called"my"my pathway." pathway."
Utilize the Utilize the "Build "Buildtool" tool"andand"Grow "Grow tool" totool" to identify identify Differentially Differentially
30 30 ExpressedGenes Expressed Genes (DEGs) (DEGs) belonging belonging to thetotranscriptomic the transcriptomicprofileprofile under under investigation investigation
andare and arelinked linkedtotoregulation regulation of the of the biological biological activities activities selected selected in theinprevious the previous step. step.
Modulationofofthethe Modulation identifiedDEGs identified DEGs is represented is represented using using green green colour colour
(indicating down-modulation) (indicating down-modulation) and and redcolour red colour(indicating (indicatingup-modulation). up-modulation). To determine To determinethethe expected expected calculated calculated impact impact of suchof experimentally such experimentally 35 35 observedmodulations observed modulationsofofgene gene expression expression on on thethe biological biological activities, the activities, the "Overlay" "Overlay"and and "Molecule ActivityPredictor" "Molecule Activity Predictor"tool tool(MAP) (MAP) are are employed. employed. The "Prediction" The "Prediction" function function is is activated within activated within MAPMAP tooltotocalculate tool calculatethe theresulting resultingexpected expectedmodulation modulation of of biological biological
activities. Colour activities. coding Colour coding is is thusthus established: established:
54 10 May 2024
- orange: increase in activity - orange: increase in activity
- blue: - decreaseininactivity blue: decrease activity - white: - white: notnotachievable/not achievable/not predictable predictable
As the As the"Overlay "Overlay analysis" analysis" doesdoes not directly not directly calculate calculate the Z-score the Z-score for eachfor each 5 5 biologicalactivity biological activitybut butprovides provides results results in terms in terms of colours of colours indicating indicating the direction the direction of the of the modification and modification andintensity intensityofofthe thecolour coloursignal signalproportional proportional to to thethe magnitude magnitude of theof the modulation modulation of interest, of interest, it is it is necessary necessary to translate to translate the intensity the intensity of the of the modulation modulation signal signal (graphically represented ininthe the"my"my pathway" tab) tab) into a numerical value. value. This is This is 2024203121
(graphically represented pathway" into a numerical
achievedbybyconverting achieved convertingthe thecolour colourintensity intensityobtained obtainedfor foreach eachbiological biologicalactivity activityinto into aa 10 10 Z-score. Z-score.
Oneofofthe One thepossible possibletools tools that that can be used can be usedforfor this this purpose purpose is is "IPAmap_Parser" “IPAmap_Parser” app, aa web app, webport portappapp of Pipeline of Pipeline PilotPilot that that aims aims to assign to assign a score,a score, called z-score, called z-score, to genes to genes
and biofunctions and biofunctionsbased basedonon theircoloration their colorationwithin within a biological a biological pathway pathway generated generated by by QIAGEN's QIAGEN's Ingenuity Ingenuity Pathway Pathway Analysis Analysis software. software. The keyThe keyofstep step the of the algorithm algorithm is the is the
15 15 conversionfrom conversion fromthe theRGBRGB colour colour modelmodel to thetoLAB themodel LAB(https://www.xrite.com/it- model (https://www.xrite.com/it- it/blog/lab-color-space posted it/blog/lab-color-space posted on October2018 on October 2018by by TimTim Mouw), Mouw), a colorimetric a colorimetric codingcoding
that also that also allows allowsthe theintensity intensityofofthethecolour colour to to be recorded be recorded andjust and not not the justRGBthe RGB composition.This composition. Thisconversion conversion occurs occurs within within a Pipeline a Pipeline PilotPilot “component” "component" that auses a that uses
procedurewritten procedure written using usingRRsoftware softwarethat thatisisbased basedononspecific specificfeatures featuresofofthe thecolorspace colorspace 20 20 packagee(https://cran.r-project.org/web/packages/colorspace/index.html, package (https://cran.r-project.org/web/packages/colorspace/index.html, details details cancanbebe foundononZeileis found Zeileis et et al al 2020journal 2020journal of statistical of statistical software, software, doi:10.18637/jss.v096.i01). doi:10.18637/jss.v096.i01).
TheZ-score The Z-scoreallowsallowsforforan an objective objective comparison comparison of theof the impacts impacts of different of different
treatments. treatments.
Whenthe When theabove aboveprotocol protocolis iscarried carriedout outwithwiththethereference referencestandard, standard, and and 25 25 optionally with optionally with one one oror more morereference referencedrugs, drugs,the themodulation modulation (Z-score) (Z-score) values values obtained obtained
for each for biological activity each biological activity as as disclosed disclosed above above are are considered consideredthe thereference referencecut-off cut-offZ-Z- score values score values forfor the the desired desiredmodulation modulation of of saidsaid activity,therefore, activity, therefore,when whenthe the desired desired
modulationisisananupup modulation modulation modulation the cut-off the cut-off Z-scoreZ-score value value will correspond will correspond to > the to ≥ the reference Z-score reference Z-score value value obtained obtainedaccording accordingtotothe the present present description description ,, when when thethe desired desired 30 30 modulationisisa adown modulation down modulation modulation the cut-off the cut-off Z-score valuevalue Z-score will correspond will correspond to < the to ≤ the reference Z-score reference Z-score value value obtained obtainedaccording accordingtotothe thepresent presentdescription. description. In case In case one one oror more morereference referencedrugs drugs is is alsotested also testedininthe theselected selectedininvitro vitro cell- cell- basedassay, based assay,thethemodulation modulation Z-score Z-score values values observedobserved for the biological for the biological activities activities that are that are knowntotobebemodulated known modulated by by said said reference reference drugs, drugs, cancan be be considered considered in order in order to establish to establish
35 35 the compliance the compliance criteria criteria (acceptable (acceptable or notoracceptable not acceptable batch).batch).
A reference A referencedrug drugisis aa drug drugrecommended recommended by theby clinical the clinical guidelines guidelines for for treating treating
the pathology the pathology or or thethe hallmark hallmark of interest. of interest.
Whena reference When a reference drug drug tested tested in in vitro vitro in ina) a) modulates modulates one one or more or more biological biological
55 10 May 2024
activity associated with hallmarks of the pathological condition of interest in the desired activity associated with hallmarks of the pathological condition of interest in the desired
direction (healthy direction (healthy physiological physiological state), state), both both the the Z-score Z-scoreobtained obtained with with the the reference reference
standard and standard andwith withthethereference referencedrugdrug are are considered considered effective effective for said for said one oronemore or more biological activities, biological activities, therefore, therefore, thethe value associated totothe value associated theweakest weakest performance performance is is 5 5 assumed to assumed to identify identify thethe lower limit of lower limit of compliance compliance for for each each ofof said said one one or or more more biologicalactivities biological activitiesand andwillwill be be considered considered the reference the reference (cut-off) (cut-off) Z-score Z-score to refer to to refer for to for definingacceptable defining acceptable or not or not acceptable acceptable the batches the test test batches of the of the product. product.
Step d) d) of of the the method comprises: 2024203121
Step method comprises:
(d) performing (d) performing aaspectroscopy spectroscopyororspectrophotometry spectrophotometry on said on said reference reference standard standard
10 10 batch and batch andononsaid saidoneone or more or more different different test batches; test batches; A non-limiting A non-limiting example example of of spectroscopyanalysis spectroscopy analysisaccording accordingtotothe theinvention inventionisis Near NearInfraRed InfraRedSpectroscopy Spectroscopy (NIR), (NIR),
Fourier Transformed Fourier TransformedInfraRed InfraRed (FTIR), (FTIR), RamanRaman Spectroscopy, Spectroscopy, Spectrophotometry, Spectrophotometry, such such as, e.g. as, e.g. UV-VIS (UV-Visible), UV-VIS (UV-Visible), fluorescence fluorescence spectroscopy, spectroscopy, lightlight scattering. scattering. TheThe skilled skilled
person, depending person, dependingononthe theformulation formulationofofthetheproduct productanalysed analysed (e.g.,dry (e.g., drypowder, powder, liquid, liquid,
15 15 etc.) will etc.) willreadily readilyselect selectthe spectroscopy the spectroscopyor orspectrophotometry techniquemore spectrophotometry technique moresuitable suitable for the analysis of the selected product. for the analysis of the selected product.
According to According to aapreferred preferred embodiment embodimentof of thethe invention invention said said spectroscopy spectroscopy analysis is analysis is carried carriedout outbybyNIR. NIR. Indeed, Indeed, as as the theskilled skilled person personknows, knows, NIR spectroscopyisis NIR spectroscopy
a vibrational a vibrational spectroscopy spectroscopytechnique technique thatthatprovides provides qualitative qualitative information information about about the the
20 20 chemicalspecies chemical speciespresent presentininthethematerial materialanalysed analysed together together withwith information information about about its its
physical state. physical state. Performing PerformingNIRNIR analysis analysis means means subjecting subjecting the material the material to lighttooflight of
different wavelengths different wavelengths and andmeasuring measuring thethe vibration vibration of ofthethe material material at at each each wavelength. wavelength.
Thevibrations The vibrations detected detected depend dependononthethecomposition composition andand on the on the interactions interactions between between the the
components,which components, which affectthethevibrational affect vibrationalcapabilities capabilities of of each eachcomponent component placed placed within within
25 25 the matrix. the matrix.This Thisanalysis analysis generates generates a vibrational a vibrational footprint footprint specificspecific to thatto that material. material.
Theanalysis The analysisofofthe theNIR NIR Fingerprint Fingerprint allows allows the the reconstruction reconstruction of a of a chemical- chemical-
physical profile physical profile characteristic characteristic of of the the molecular compositionof ofeach molecular composition each analysed analysed sample sample
that is that is influenced influenced bybythe thechemical chemical surroundings. surroundings. Indeed,Indeed, the molecules the molecules within within the the sample can sample can formform bonds bondsbetween betweenthem, them,in in particular hydrogen particular hydrogen bonds, bonds, bothboth 30 30 intermolecular and intermolecular andintramolecular. intramolecular. ThisThisleadsleads to an to an alteration alteration of the vibration of the vibration
frequencies of frequencies of both both stretching stretching and bendingofofthe and bending the hydrogen hydrogenatom atom andand result result in in a a shiftinin shift
vibrationalfrequencies vibrational frequencies withwith respect respect to theto single the single isolated isolated molecule. molecule. For thisFor this NIR reason, reason, NIR spectrometrycan spectrometry canbe bean an excellent excellent means means of characterizing of characterizing complexcomplex matrices.matrices. This This technique isis therefore technique therefore particularly particularly suitable suitable for for the the analysis analysis ofofnatural naturalmatrices matricesasasit it 35 35 provides aa fingerprint provides fingerprint of of the the whole matrixnetwork whole matrix networkandand of of theinteractions the interactionsbetween between thethe
componentsofofthe components thematrix. matrix. Anexample An exampleofofthe theNIRNIR spectralprofiles spectral profilesisis shown shownFigureFigure6.6. Step e) Step e) ofof the themethod comprises: method comprises:
56 10 May 2024
defining the defining the acceptability acceptability values values of of the the spectroscopy spectroscopyororspectrophotometry spectrophotometry spectra spectra as as the variability the variability range range ofof the the spectroscopy spectroscopy or or spectrophotometry spectrophotometry spectra spectravalues valuesofofall allofof said acceptable said batches and acceptable batches andofof said said reference reference standard standardbatch,batch, preferably preferablyrefined refined by bythe the spectraofofsaid spectra saidnon-acceptable non-acceptable batches. batches.
5 5 The variability The variability range range obtained obtained will will depend depend on on thethe spectroscopy spectroscopy or or spectrophotometrytechnique spectrophotometry techniqueused usedandandonon theproduct the product tested. tested.
Independentlyofofthe Independently thespectroscopy spectroscopy or or spectrophotometry spectrophotometry techniques techniques used, used, the the common featureis isthat thateach eachofofthethe"acceptable" “acceptable”batches batchesand andthethereference referencestandard standardareare 2024203121
common feature
set aa priori set priori asasvalid validandandacceptable acceptable products products due todue theirto assessed their assessed effect oneffect on the selected the selected
10 10 biologicalactivities. biological activities. WhenNIR When NIRis is used used thethe NIRNIR acceptability acceptability values values obtained obtained withwith thethe method method of the of the
invention are invention are suitable suitable forfor use use in in NIR NIRconformity conformity testsforforbatch-to-batch tests batch-to-batch compliance compliance
(i.e., validation (i.e., validation of of the batchfor the batch forproduction). production). It is It is evident evident thatthat the the conformity conformity testsmust tests must be carried be carried out the out with withsame the same 15 15 spectroscopy or spectroscopy or spectrophotometry spectrophotometry techniquetechniqueused usedforfor thethe assessment assessment of the of the acceptability values. acceptability values.
In order In order toto define define the the acceptability acceptabilityvalues, values,the thecomplete completeNIR NIR spectrum spectrum of of all all the the
acceptable batches acceptable batchessamples samplesandand of of thethe reference reference standard standard is acquired, is acquired, thethe spectra spectra areare
aligned and aligned and normalised normalised(Standard (Standard Normal Normal Variate) Variate) the wavelength and wavelength and the (λ) regions (2) regions of of 20 20 interest of interest of the thespectra spectraare arehence hence defined defined and and the the average spectrumofofthe average spectrum the spectra spectra of of all all the compliant the compliant (positive) (positive) samples samples + reference + reference standardstandard is generated. is generated.
Theaverage The averagespectrum spectrum obtained obtained willwill be bethe the reference reference spectrum spectrum over over the whole the whole
procedure. procedure.
In the In the execution execution of of aa NIR NIRconformity conformitytest testfor forpharmaceutical pharmaceuticalAPI-based API-based 25 25 products, pre-processing products, pre-processingcomprises comprises (SNV) (SNV) as indicated as indicated above, above, definition definition of the 2 of the λ regions of regions of interest, interest, and and thetheMaxMax Conformity Conformity IndexIndex Value Value is normally is normally used in used the in the conformitytests conformity tests and andisis conventionally conventionallyimposedimposed as 3.5 as 3.5 whichwhich meansmeans that the thathighest the highest value of value of standard standard deviations deviations accepted acceptedfor forpharmaceutical pharmaceutical products products in in anyany point point of of thethe
spectrumisis normally spectrum normally 3.5. 3.5. 30 30 In the In the method methoddisclosed disclosedherein herein thethe acceptability acceptability values values areare notnot based based on the on the
quali-quantitative analysis quali-quantitative analysis of of specific specific chemical substancesinin said chemical substances said product productbut butononthethe modulation modulation activity activity exerted exerted by product by said said product on selected on selected biologicalbiological activities,activities, therefore, therefore,
the Max the Conformity Max Conformity Index Index (MCI) (MCI) ValueValue conventionally conventionally imposed imposed in the in the quality quality controlscontrols for classical for classical APIs, whichisisrelated APIs, which relatedtotothethequali-quantitative quali-quantitativechemical chemical composition, composition,
35 35 cannot be cannot be considered consideredas as aa priori priori acceptable acceptable and and the the value value isishence hence assigned assigned based based onon the the
spectraofofthe spectra thebatches batches selected selected in in iv)iv) andand the the reference reference standard. standard.
In addition, In addition, Sum Sum 22 (which (whichderives derives from fromCICIbut butalso alsoconsiders considersNIR NIRspectra spectraofofoneone or more or moreundesired undesired samples), samples), which which is moreis more suitablesuitable for heterogeneous for heterogeneous samples,samples, is is
57 10 May 2024
preferably used as an acceptability parameter according to the present invention. preferably used as an acceptability parameter according to the present invention.
Accordingtotothis According this embodiment embodiment of of thethe invention,thetheCICI(Conformity invention, (Conformity Index) Index) limit limit
to which to the conformity which the conformitytest test will will be be pre-processed, pre-processed, corresponds correspondstotothe themaximum maximum valuevalue of CI of CI MAX MAX (also(also defined defined herein herein as CIaslimit) CI limit) defined defined by theby the spectra spectra of the of the batches batches
5 5 selectedininiv) selected iv)and andthe thereference reference standard. standard.
Therefore, according Therefore, accordingtotothethepresent present invention invention the the acceptability acceptability values values of the of the
spectroscopy or spectroscopy spectrophotometry analysis or spectrophotometry analysis are are defined defined basedbased on the modulation on the modulation activity on eachselected selectedbiological biologicalactivity activityexerted exertedby by the the reference standard and and 2024203121
activity on each reference standard
batchesofofthe batches theproduct productthatthat areare compliant compliant withreference with the the reference standardstandard in said modulation. in said modulation.
10 10 In the In the case caseofofa aNIRNIR spectroscopy, spectroscopy, hence,hence, the CI the limitCI limit i.e., thei.e., the acceptability acceptability cut- cut- off is off is calculated according calculated according to to thethe following following formula: formula:
CI = (A reference, i - A sample, i) / s reference, i CI = (A reference, i - A sample, i) / S reference, i
Where Where 15 15 A A reference, = average absorbance at a given wavelength(i) of the reference (average reference, i i = average absorbance at a given wavelength(i) of the reference (average
spectrum) spectrum)
Asample, A sample, i i==absorbance absorbance atataagiven givenwavelength(i) wavelength(i) of test of the the test samplesample s reference, S reference, i= standard i= standarddeviation deviation at at aa given wavelength(i) given wavelength(i) of of thethe reference reference (average (average
spectrum) spectrum)
20 20 In conformity In conformityteststests related related to to heterogeneous heterogeneoussamples,samples, as as indicated indicated above, above, the the
Sum2parameter Sum2 parameter isismore more suitable. suitable.
Sum2= Sum2 (Sum(Sum of Allof CIs All >CIs CI > CI limit limit - CI -limit)/(Sum CI limit)/(Sum of the ofnumber the number of points of points in in the spectrum the spectrum withwith CICI>>CI CIlimit) limit) Selecting the Selecting the appropriate appropriate parameter parameterinina aconformity conformity testtest depends depends on the on the user-user- 25 25 specific control specific control problem that can problem that can be be easily easily addressed addressedbybythe theskilled skilled person, person, in in the the case case
of products of products comprising comprisingororconsisting consistingofofoneone or or moremore natural natural matrices, matrices, i.e.,i.e., extremely extremely
heterogeneoussamples, heterogeneous samples,Sum Sum2 is 2 isa asuitable suitableparameter. parameter. In one In embodiment one embodiment of ofthetheinvention, invention,Sum2 Sum2 parameter parameter is selected is selected forfor determining determining
the acceptability the acceptabilitycut-off cut-offofofthethe conformity conformity test.test.
30 30 Dependingon on Depending thethe spectroscopy spectroscopy or spectrophotometry or spectrophotometry used, the used, the acceptability acceptability
values are values are calculated calculated mutatis mutatis mutandis, mutandis, with with the the same sameratio ratio used usedforfor NIR NIRspectroscopy, spectroscopy, i.e., by i.e., defining asas acceptable by defining acceptablethethesapectra sapectra obtained obtained from from the acceptable the acceptable batchesbatches
accordingtotothetheinvention according invention and and the reference the reference standard. standard.
Whencarrying When carryingoutoutthetheprocess process forfor thethecompliance compliance validation validation process process of oneof one or or
35 35 morebatches more batchesofofaa product product(conformity (conformitytest) test) comprising comprisingone oneorormore morenatural naturalmatrices matricesfor for the treatment the treatment of of aa pathological pathologicalcondition, condition,according accordingtotothe thepresent presentinvention, invention,a abatch batch mayresult, may result,inina afirst firstanalysis, analysis,"not “not compliant” compliant" as itsasspectroscopy its spectroscopy or spectrophotometry or spectrophotometry
spectrumdoes spectrum doesnotnotfall fallwithin withinthe theacceptability acceptability values valuesdefined definedwith withthethemethod method of the of the
58 10 May 2024
invention. invention.
In that In that case, case,itit may maybe be of of interest interest to verify to verify whether whether the non-compliance the non-compliance result is result is
caused by caused byananeffective effective non-compliance non-compliance of of thethe batch batch with with thethe desired desired modulation modulation of the of the
biological activities biological activities (i.e., (i.e., the therapeutic effect) the therapeutic effect) ororisisduedue to fact to the the fact that the that the
5 5 acceptability parameters acceptability parameterscan can be broadened, in be broadened, in that that case, case, an an adjustment adjustment ofof saidsaid parameterscan parameters canbebemade. made. Accordingtotothe According themethod method ofof thetheinvention, invention,steps stepsa)a)toto c) c) can can be be repeated repeated for for said said batchand, and,ififthe thebatch batch results as as acceptable, the acceptability valuesvalues of the spectroscopy 2024203121
batch results acceptable, the acceptability of the spectroscopy
or spectrophotometry or spectrophotometry can canbeberecalculated recalculatedalso alsoincluding includingthe the spectrum spectrumofofthe the new newbatch. batch. 10 10 The higher The higherthe thenumber number of acceptable of acceptable batches, batches, the accurate the more more accurate the the spectroscopyor spectroscopy or spectrophotometry spectrophotometry acceptabilityvalues acceptability valuesobtained obtainedwithwiththethemethod method of of thethe
inventionwill invention willbe.be. Furthermore,the Furthermore, the method methodofofthe theinvention inventionmay may alsofurther also furthercomprise: comprise: in step in step d) d) performing performinga aspectroscopy spectroscopy or or spectrophotometry spectrophotometry onorone on one or amore a more
15 15 priori undesired priori batchesofofsaid undesired batches saidproduct, product,and andverifying verifyingthatthatthethespectra spectraofofsaid saidone oneoror moreundesired more undesiredbatches batchesdoes does notnot fallwithin fall withinthe theacceptability acceptabilityvalues valuesdefined definedinine), e), and, and, in the negative (i.e., when the batch results within the acceptability values defined in e)) in the negative (i.e., when the batch results within the acceptability values defined in e))
e’) defining e') defining new, new, narrower, narrower, acceptability acceptability valuesvalues of of thethe spectroscopy spectroscopy or or spectrophotometry spectrophotometry spectra spectra resulting resulting by considering by considering the variability the variability range ofrange the of the 20 20 spectroscopyororspectrophotometry spectroscopy spectrophotometry spectra spectra values values of of allall ofofsaid saidacceptable acceptablebatches batchesandand of said of said reference referencestandard standard batchbatch andallofsaid and of all said undesired undesired batchesbatches as not acceptable. as not acceptable.
Theadditional The additionalfeatures featuresabove abovecancan be be desired desired by product by the the product manufacturer manufacturer to to makesure make surethat thatbatches batchesthat thatmaymay result result fromfrom a formulative a formulative errorerror expectable expectable from from the the productionchain, production chain, such suchasasthethe absence absenceofofananingredient ingredientororthe thelike like are are excluded excludedaapriori priori 25 25 from the from theacceptability acceptability values valuesofofthe thespectroscopy spectroscopyororspectrophotometry spectrophotometry spectra spectra in the in the
compliancebatch-to-batch compliance batch-to-batchcontrol. control. Theinvention The inventionalso alsoprovides provides a method a method determining determining a reference a reference standardstandard of a of a therapeutic product therapeutic product for for the the treatment treatment ofofaapathological pathologicalcondition, condition,wherein whereinthetheproduct product comprisesone comprises oneorormore morenatural naturalmatrices; matrices;said said method methodcomprising: comprising: 30 30 (1) performing (1) performingatatleast leastoneone in vitro in vitro cell-based cell-based assayassay designed designed to simulate to simulate
conditionsrelated conditions related to to said said pathological pathological condition condition on several on several batches batches of saidand of said product, product, and on one on one or or more morereference referencedrug drugfor forthe thetreatment treatmentofofsaid saidpathological pathologicalcondition, condition,wherein wherein the read-out the read-out of of said said cell-based cell-based assayassay isisrepresentative representativeof ofthe themodulation modulation of of one one or or more more
biological activity biological activityassociated associatedwith with one one oror more hallmarksof more hallmarks of said said pathological pathological condition condition 35 35 and determining and determiningfor foreach eachofofsaidsaidoneoneorormore morebiological biologicalactivities activities thethe modulation modulationtrendtrend thereoffor thereof forrestoring restoringa ahealthy healthy state; state;
(2) quantifying (2) quantifying in in terms terms of of numerical values the numerical values the modulation modulationofofsaid saidone oneorormore more biologicalactivities biological activitiesinduced induced by each by each batchbatch and drugandofdrug step of (1)step for (1) eachfor each cell-based cell-based assay assay
59 10 May 2024
read-out, and read-out, defining as and defining as reference reference value valuefor for each eachbiological biologicalactivity activity modified modifiedbybysaid said one or one or more morereference referencedrug drugaccording according to to said said modulation modulation trend trend for for restoring restoring a healthy a healthy
state, the value associated to the weakest performance; state, the value associated to the weakest performance;
(3) selecting (3) selecting the the batches batches whose valuescalculated whose values calculatedinin(1)(1) induce induceaamodulation modulation of of
5 5 each measured each measuredbiological biologicalactivity activityininsaid saidcell-based cell-basedassay assaywhose whose modulus modulus ≥ to the is toisthe
modulusofofthethereference modulus reference value value calculated calculated in (2) in (2) and and defining defining as product as product reference reference
standard batche standard batchethe theoneone with with the the highest highest modulus modulus valuesvalues on the on the number larger larger of number of biologicalactivities. activities. 2024203121
biological
Steps (1) Steps (1) and and(2)(2)areare carried carried out, out, mutatis mutatis mutandis, mutandis, as steps as steps a) anda)b)and as b) as
10 10 described above described aboveand andininthe the examples. examples. Theparameters The parametersandand modulation modulation patterns patterns thereof thereof as described as described above above for the for the
methodfor method fordefining definingthetheacceptability acceptabilityvalues valuesof of a spectroscopy a spectroscopy or spectrophotometry or spectrophotometry
analysis for analysis for the thecompliance validation of compliance validation of one one or or more batches of more batches of aa product apply mutatis product apply mutatis mutandistotothe mutandis themethod method forfor defining defining a reference a reference standard. standard. Hence, Hence, in aninembodiment, an embodiment, 15 15 said parameters said parameters areare genes genes andmodulation and the the modulation thereof thereof is their is their expression expression pattern. pattern.
Finally, the Finally, invention provides the invention providesa aprocess process forfor thethe compliance compliance validation validation (i.e., (i.e.,
productionquality production quality control) control) of of one oneorormore morebatches batches of of a product a product forfor thethe treatment treatment of of a a pathological condition pathological condition oror ofof aa beneficial beneficial prodcut, prodcut, saidsaid product comprisingone product comprising oneorormore more natural matrices, natural matrices, said saidmethod comprisingthe method comprising thefollowing followingsteps: steps: 20 20 a. performing a. performing aa spectroscopy spectroscopyor or spectrophotometry spectrophotometryanalysisanalysisofofeach eachbatch, batch, b. validating b. validating each batch for each batch for which whichthetheobtained obtained spectrum spectrum satisfiesthethe satisfies acceptability values acceptability defined according values defined accordingtotothe themethod method of of thethe invention. invention. Given Given the the factfact
that the that the method method ofofthetheinvention inventionprovides provides reliableparameters reliable parameters forfor useuse in the in the batch batch to to
batch validation batch validation for for the the compliance compliance of of products products comprising comprisingororconsisting consistingofof one oneoror more more 25 25 natural matrices, natural matrices, the theskilled skilledperson personwillwill readily readily understand understand thatthat the the method method of theof the
inventioncancan invention also also be be applied, applied, mutatis mutatis mutandis, mutandis, for defining for defining the acceptability the acceptability values of values of
a spectroscopy a spectroscopyororspectrophotometry spectrophotometry analysis analysis forfor thethe compliance compliance validation validation of one of one or or morebatches more batchesofofa aproduct productadjuvating adjuvating thethe homeostasis homeostasis of aofsubject's a subject’s system, system, organorgan or or apparatus comprising apparatus comprisingororconsisting consistingof of one one or or more morenatural naturalmatrices; matrices; comprising comprisinga astepstepin in 30 30 whichsaid which saidacceptability acceptability values valuesareare calculated calculated on ona aspectroscopy spectroscopyororspectrophotometry spectrophotometry spectra of spectra of aa reference referencestandard standardof ofsaidsaid product, product, saidsaid reference reference standard standard havinghaving an an ascertained homeostatic ascertained homeostaticadjuvant adjuvanteffect effectininthe themaintenance maintenance of of a healthy a healthy physiological physiological
state of state said system of said systemorgan organor or apparatus, apparatus, andandone one or more or more different different batches batches of saidof said
product; wherein product; whereinsaidsaidspectra spectraare aredefined definedasasacceptable acceptable on on thethe basis basis of of thethe biological biological
35 35 activity activity exerted exerted inin a cellular-based assay a cellular-based assay by bysaid saidreference referencestandard standardandandsaidsaid oneone or or
moredifferent more different batches batchesofofsaid said product productononone oneor or more more biological biological activitiesunderlying activities underlying said healthy said healthyphysiological physiological state. state.
In an In an embodiment embodiment ofof theinvention the inventionthe themethod method cancanbe be defined defined also also asas follows: follows:
60 10 May 2024
A method A methodfor for defining defining the acceptability the acceptability ranges ranges or cut-offs or cut-offs of a spectroscopy of a spectroscopy or or spectrophotometryanalysis spectrophotometry analysisforforthe thevalidation validation of of one oneoror more morebatches batchesofofa aproduct, product,for for use in use in the the treatment treatmentofofa apathological pathological condition, condition, comprising comprising or consisting or consisting of one or ofmore one or more natural matrices; natural matrices; comprising calculating said comprising calculating said acceptability acceptability ranges ranges oror cut-offs cut-offs from from the the 5 5 spectroscopyororspectrophotometry spectroscopy spectrophotometry spectra spectra of of a gold a gold standard standard (reference (reference standard) standard) of of
said product, said product,said saidgold gold standard standard having having an ascertained an ascertained therapeutic therapeutic effect ineffect in the treatment the treatment
of said of said pathological condition, and pathological condition, and ofof one oneorormore more differentbatches different batchesof ofsaid saidproduct; product; whereinsaid saidspectra spectraarearedefined definedasasacceptable acceptableor or not-acceptable on on the the basis of the 2024203121
wherein not-acceptable basis of the
biological activity biological activity exerted exerted inin atat least least one onecell-based cell-basedassay assay by said by said gold gold standard standard
10 10 (reference (reference standard) standard) andand said said one one oror more more different different batches batches on on one one or or more more hallmarks of hallmarks of
said pathological said pathologicalcondition. condition. Themethod The methodasasdefined definedabove abovecan cancomprise comprise thethe following following steps: steps:
i) i)
a. retrieving a. retrieving from from thethe state state of of the the art art aa list list of of the the hallmarks hallmarksofofsaidsaid 15 15 pathological condition; pathological condition;
b. identifying b. identifying for for each each of of said said hallmarks hallmarksa asetsetofofbiological biologicalactivities activities modifications detectable modifications detectableininsaid saidpathological pathological condition condition and and determining determining the the modulation modulation of of each each of said of said activities activities concurring concurring to the to the desired desired therapeutic therapeutic effect effect therebydesigning thereby designing the the modulation modulation patternpattern of eachofofeach said of said activities activities representing representing a a 20 20 healthyphysiological healthy physiological state; state;
c. identifying C. identifying the the parameters parameters and and thethe modulation modulationpattern patternthereof thereof underlying the underlying the modification modification detectable detectable in in said said pathological pathological condition condition for for each each of of
said biological said biological activities activitiesand andsetting setting forfor each eachofofsaidsaidparameters parametersthe modulation the modulation
pattern opposite pattern oppositetotothetheoneone identified identified as the as the modulation modulation patternpattern indicative indicative of said of said
25 25 healthyphysiological healthy physiological state; state;
ii) ii)
analysing the analysing the expression expressionpattern patternofofeach eachof ofthetheparameters parameters identified identified in in i) i) C. c.
induced bybya agold induced goldstandard standard(reference (referencestandard) standard)ofofsaid saidproduct product in in a suitableininvitro a suitable vitro cell-based assay, cell-based assay, determining determining the the quali-quantitative quali-quantitative modulation modulation of each ofofeach said of said
30 30 parametersinduced parameters inducedby by saidsaid gold gold standard standard (reference (reference standard) standard) with respect with respect to the to the
pathophysiologicalstate pathophysiological state control control of of said said inin vitro vitro cell cell based assay, and based assay, and calculating calculating the the gold standard gold standard(reference (referencestandard) standard) Z-score Z-score valuevalue (i.e.(i.e. a modulation a modulation value)value) of the of the
modulationofofeach modulation each of of saidsaid biological biological activitiesinduced activities induced by said by said goldgold standard standard and and
selecting each selecting of said each of said gold gold standard standard(reference (referencestandard) standard)Z-score Z-scorevalues values as as reference reference
35 35 cut-off Z-score cut-off Z-scorevalues values indicative indicative of said of said desired desired therapeutic therapeutic effect, effect, iii) iii)
analysing the analysing the modulation modulationpatternpattern ofof the the parameters parametersidentified identified in in i) i)c.C.induced inducedby by
further different further different batches batchesofofsaid said product product in said in said in vitro in vitro cell-based cell-based assay and assay and
61 10 May 2024
determiningthe determining thequali-quantitative quali-quantitative modulation modulationof of each each of said of said parameters parameters induced induced by by each of each of said said batches batches thereby thereby calculating calculating thethe Z-score Z-score value value ofof the the modulation modulationofofeacheachofof said biological said biologicalactivities activitiesinduced induced by each by each of said of said batches batches
iv) iv)
5 5 comparing the comparing the Z-score Z-score value value of of the the modulation modulation ofofeach eachofofsaidsaidbiological biological activities induced activities induced byby each eachofofsaidsaidbatches batches calculated calculated in iii) in iii) with with the the corresponding corresponding
reference cut-off reference cut-off Z-score values and Z-score values andselecting selectingatat least least three three positive positive batches batches for for which which
each Z-score Z-scorevalue valuecalculated calculatedininiii) iii) complies withthe the corresponding correspondingreference referencecut-off cut-off 2024203121
each complies with
Z-score values Z-score valuesandandatatleast leastone onenegative negative batch batch for for which which at least at least one one Z-score Z-score value value
10 10 calculated in calculated in iii) iii) does not comply does not complywith with thethe corresponding corresponding reference reference cut-off cut-off Z-score Z-score
value value
v) v)
carrying out carrying out aa spectroscopy spectroscopy or or spectrophotometry spectrophotometry analysisofofsaid analysis saidgold goldstandard standard andofofthe and thebatches batches selected selected in iv) in iv)
15 15 vi) vi)
defining the defining the variability variability spectroscopy or spectrophotometry spectroscopy or spectrophotometry ranges ranges resulting resulting by by considering each considering eachofofthe the results results obtained obtained in in v) v) as as acceptable acceptable for for each positive batch each positive and batch and
non-acceptable for non-acceptable for each eachnegative negativebatch batch thereby thereby providing providing said said acceptability acceptability spectroscopyor spectroscopy or spectrophotometry spectrophotometryranges ranges oror cut-offs. cut-offs.
20 20 Themethod The methodmaymay further further comprise comprise in step in step ii)ii) analysingthethemodulation analysing modulation pattern pattern of of
the parameters the parametersidentified identifiedinini)i)C.c.induced induced by or by one onemoreor reference more reference drug fordrugthe for the
treatment of treatment of said said pathological pathological condition condition in in said said in in vitro vitro cell-based cell-based assay assay and and determiningthe determining thequali-quantitative quali-quantitative modulation modulation of of eacheach of said of said parameters parameters induced induced by by each drug each drugand andcalculating calculatingthe thedrug drugZ-score Z-scorevalue value(modulation (modulation value) value) of of thethe modulation modulation
25 25 of each of eachofofsaid saidbiological biological activities activities induced induced by of by each each saidofdrug saidand, drugforand, eachfor each biological biological
function modified function modifiedalso alsoby by saidsaid one one or drug or more moreindrug in the direction the direction of the healthy of the healthy
physiological state, physiological state, comparing comparing the the drug drugZ-score Z-scorevalue valueandandthethe gold gold standard standard (reference (reference
standard) Z-score standard) Z-scorevalue, value, andandselecting selectingasasreference referencecut-off-Z cut-off-Zscorescorevalue, value,thetheZ-score Z-score value associated value associated to to the the weakest weakest performance. performance.
30 30 In an In embodiment an embodiment thethe parameters parameters and and modulation modulation patternpattern thereofthereof correspond correspond to to the genes the genes and andthe theexpression expressionpattern patternthereof thereofunderlying underlying themodification the modification detectable detectable in in
said pathological said pathological condition condition for for eacheach of said of said biological biological activities. activities.
Byway By wayofofexample, example, thetheproduct productanalysed analysed with with thethe method method of the of the invention invention cancanbe be in in drydry
or lyophilised or lyophilisedform.form. 35 35 In addition, In addition, in in the the method method as asdefined definedininthetheparagraphs paragraphs above, above, stepstep iii)can iii) canbebe carried out carried out onon one oneorormore more additional additional differentbatch different batch of of said said product product and and the the one orone or moreadditional more additional batch batch for for which whichthe theZ-score Z-score(modulation (modulationvalue)value)value valueofofthethemodulation modulation of each of each ofof said said biological biological activities activities complies complies withwithsaid saidcorresponding corresponding cut-offZ-score cut-off Z-score
62 10 May 2024
values is values is subjected subjected to to the the same spectroscopyororspectrophotometry same spectroscopy spectrophotometry analysis analysis carried carried outout
in v) and the acceptability ranges or cut-offs defined in vi) are recalculated by defining in v) and the acceptability ranges or cut-offs defined in vi) are recalculated by defining
also each also eachofofsaid saidbatches batches as acceptable. as acceptable.
Further, the Further, the method maycomprise method may comprise thethe stepsof: steps of: 5 5 vii) vii)
carrying out carrying out aaspectroscopy spectroscopyor or spectrophotometry spectrophotometry analysis analysis of orone of one moreor amore a priori undesired priori batch ofof said undesired batch said product productandandverifying verifying thatsaid that saidoneone or or more more undesired undesired
batchdoes does notnot result within the acceptability ranges ranges or cut-offs defined indefined ininvi), and, in 2024203121
batch result within the acceptability or cut-offs vi), and,
the negative the negative
10 10 viii) viii)
defining new, defining new, narrower, narrower,variability variability spectroscopy spectroscopy or or spectrophotometry spectrophotometry ranges ranges or or
cut-offsresulting cut-offs resultingbybyconsidering considering eacheach the results the results obtained obtained in vii)in asvii) as non-acceptable. non-acceptable.
In any In any part part of of the the description description and andofofthe theclaims claimsthe theterm term"comprising" “comprising” can can be be
replaced by replaced by "consisting “consisting of"of” and and"reference “referencemodulation modulation value/s” value/s" cancan be be substituted substituted with with
15 15 “modulationcut-off "modulation cut-offvalue/s" value/s” oror "reference “reference modulation modulationcut-off cut-offvalue/s". value/s”. Theexamples The examplesbelow below areare not not intended intended as as limitingthe limiting thepresent presentinvention. invention.
EXAMPLES EXAMPLES 1. 1. Tested Tested Products composition Products composition
Product AA(Arté Product (ArtéGX) GX) (figures2-6) (figures 2-6) 20 20 Centella Asiatica Centella Asiatica dry dry Leaves Leaves 90% w/w 90% w/w Echinaceapurpurea Echinacea purpureadry dryFlowers Flowers 10% w/w 10% w/w Coextractedin Coextracted in water water Product BB(figure Product (figure 9) 9) Melissaofficinalis Melissa officinalisleaves leavesdrydry extract extract 2.5% w/w 2.5% w/w 25 25 Royaljelly Royal jellylyophilised lyophilised 2.5% w/w 2.5% w/w Blueberrydry Blueberry dryextract extract 0.29% w/w 0.29% w/w Concentratedblueberry Concentrated blueberryjuicejuice 5% w/w 5% w/w Cynarascolymus Cynara scolymus L.L. leavesdry leaves dryextract extract 0.05% w/w 0.05% w/w Curcumalonga Curcuma longaL.L. radixdry radix dryextract extract 0.16% w/w 0.16% w/w 30 30 Medicagosativa Medicago sativaseeds seedsdry dryextract extract 1.6% w/w 1.6% w/w Panaxginseng Panax ginsengradix radixdry dryextract extract 1.6% 1.6% w/w w/w Honeycombdry Honeycomb dryextract extract 1.6% w/w 1.6% w/w Concentratedapple Concentrated applejuice juice 44.35% w/w 44.35% w/w Clarified lemon Clarified juice lemon juice 0.5% w/w 0.5% w/w 35 35 Honey Honey 30% w/w 30% w/w Deionisedwater Deionised water 6.45% w/w 6.45% w/w Malpighiaemarginata Malpighia emarginatajuicejuice 1% w/w 1% w/w
63 10 May 2024
Sambucus Sambucus nigrum nigrum juice juice 2% w/w 2% w/w Product CC(figure Product (figure 11) 11) Coral calcium Coral calciumpowder powder 32% w/w 32% w/w Eggshell Egg shell calcium calciumpowder powder 30.2% w/w 30.2% w/w 5 5 Coral calcium Coral calciumcitrate citrate powder powder 13% w/w 13% w/w Agaricus bisporum Agaricus bisporum powder powder 4.65% w/w 4.65% w/w Equisetumarvense Equisetum arvenseflowering flowering topstopsdrydryextract extract 2% w/w 2% w/w Malpighiaemarginata emarginatacarried carriedbybyinulin inulindrydryextract extract 1.50% w/w 2024203121
Malpighia 1.50% w/w Cetraria islandica Cetraria islandica powder powder 2% w/w 2% w/w 10 10 Agavesisalana Agave sisalanaleaves leavespowder powder 12% w/w 12% w/w Acaciasenegal Acacia senegalpowder powder 2.15% w/w 2.15% w/w In the In the examples, examples,thetheword word compliant, compliant, when when referred referred to the to the cell-based cell-based assay assay
samplesindicates samples indicatesthetheacceptability acceptabilityofofthethebatches batches with with reference reference to the to the modulation modulation
values of values of the the reference reference standard, standard, when when referred referred to to the the spectroscopy spectroscopy oror 15 15 spectrophotometryanalysis spectrophotometry analysisit itrefers referstotothe theconformity conformity of of thethe batch batch withwith the desired the desired
quality of the product for final commercialisation. quality of the product for final commercialisation.
2. In 2. vitro cell In vitro cell assay representative assay representative of of Osteoarthritis Osteoarthritis
Anininvitro An vitrocellular cellular model model capable capable of recapitulating of recapitulating features features of osteoarthritis of osteoarthritis [1-3] was [1-3] was
established by established by exposing primaryhuman exposing primary human chondrocytes chondrocytes (HC,(HC, Cell Cell Application Application INC 402K- INC 402K-
20 20 05) to 05) to IL1B IL1B[5[5ng/ml] ng/ml]for for 6 hours, 6 hours, onceonce the cellular the cellular modelmodel was established was established it was it was
exposedover exposed over2424hours hourstotofive five different different batches batches of of “Arté-GX” "Arté-GX" [1.4[1.4mg/ml]: mg/ml]: Batch 20B1955 Batch 20B1955 (reference (reference standard) standard)
Batch 20I1279 Batch 20I1279 Batch 20J1770 Batch 20J1770 25 25 Batch 20B0596 Batch 20B0596 in aa successive in successive moment, moment, Batch Batch 21E1640 21E1640 as wellas aswell as an aliquot an aliquot of saidof said
batchthat batch thatwaswas treated treated to induce to induce destabilisation destabilisation of the of the product, product, herein as herein referred referred Batch as Batch
DEST DEST 21E1640, 21E1640, werewere alsoalso analysed analysed separately separately (see(see figures figures 3 and 3 and 4).4).
Eachtime Each timeoneoneofofthethebatch batchsolutions solutionswas wasadded, added, fresh fresh IL1B IL1B [5 ng/ml]
[5 ng/ml] was was also also
30 30 addedto added to the the medium. medium.
2.1.1. Time 2.1.1. Time schedule schedule on on chondrocyte chondrocyte experiment experiment
Thetime The timeschedule schedule usedused for experimental for experimental setting setting is as follows: is as follows:
64 10 May 2024
6 hrs 24 hrs
IL1B pre-treatment Treatment administration + IL1B Gene expression
[5 ng/ml] [5 ng/ml] analysis
Vehicle administration + IL1B [5 IL1B pre-treatment Gene expression
[5 ng/ml] ng/ml] analysis 2024203121
2.1.2. Gene 2.1.2. Gene expression expression analysis analysis
At the At the end of described end of treatment periods, described treatment periods, cells cells were were washed with100 washed with 100ulµlPBS PBSandand lysed lysed
and collected and collected in in RLT buffer(Qiagen, RLT buffer (Qiagen,1053393) 1053393) withwith added added β-mercaptoethanol -mercaptoethanol (Sigma,(Sigma,
5 5 M3148)andand M3148) DX DX reagent reagent (Qiagen, (Qiagen, 19088)19088) forexpression for gene gene expression analysis analysis experiments. experiments.
Total RNA Total RNA was was extracted extracted from from cells cells lysatesusing lysates usingananQIAsymphony QIAsymphony RNA RNA Kit Kit (Qiagen,) (Qiagen,)
with the with the QIAsymphony SP instrument QIAsymphony SP instrument (Qiagen). (Qiagen).
The quality The quality and and quantity quantityofofRNA was determined RNA was determined by by A230, A230,A260, A260,A280 A280 and and A320measurements A320 measurementson on Varioskan™ VarioskanTM LUX LUX multimode multimode microplate microplate reader reader (Thermo (Thermo 10 10 Scientific™). Integrity ScientificTM). IntegrityofofRNA waschecked RNA was checkedusing usinga a2100 2100 expert_Eukaryote expert_Eukaryote Total Total RNA RNA
NanoKit Nano Kit(Agilent). (Agilent).Whole Whole transcriptome transcriptome expression expression profile profile was evaluated was evaluated using a using a Human Human Clariom™ ClariomTM S Pico S Pico AssayAssay HT (Applied HT (Applied Biosystems, Biosystems, ThermoFisher ThermoFisher Scientific)Scientific) on on a GeneTitan a GeneTitan MCMC Instrument Instrument (Applied (Applied Biosystems, Biosystems, ThermoFisher ThermoFisher Scientific), Scientific), following following
the manufacturer's the instructions. Briefly, manufacturer's instructions. Briefly,66ng ngof oftotal totalRNA RNA was usedto was used to generate generate cDNA, cDNA, 15 15 then fragmented then fragmented and and labelled labelledcDNA was hybridized cDNA was hybridized to to aa Human ClariomS S96-array Human Clariom 96-array plate for plate for 17 17 h hatat45°C. 45°C. Arrays Arrays werewere washed, washed, stained, stained, andscanned and then then scanned using theusing the
GeneTitan MC GeneTitan MCInstrument Instrument(Applied (AppliedBiosystems, Biosystems,ThermoFisher ThermoFisherScientific) Scientific) and and CELCEL Intensity files Intensity fileswere generated were generated by by Affymetrix Affymetrix GeneChip GeneChipCommand Console Software Command Console Software (AGCC, (AGCC, ThermoFisher ThermoFisher Scientific). Scientific).
20 20 2.1.3. Transcriptomics 2.1.3. Transcriptomicsdata dataanalysis analysis Data analysis Data analysis was wasperformed performed using using Transcriptomic Transcriptomic Analysis Analysis ConsoleConsole Software Software
(TAC,ThermoFisher (TAC, ThermoFisher Scientific) Scientific) that provides that provides qualityquality control control analysis,analysis, performs performs
normalizationand normalization andsummarization, summarization, based based on Signal on the the Signal Space Space Transformation-Robust Transformation-Robust
Multi-Chip Analysis Multi-Chip Analysis (SST-RMA) (SST-RMA) analysis analysis algorithm, algorithm, and provides and provides a list aof list of 25 25 differentially expressed differentially expressed genes genes (Limma Bioconductor (Limma Bioconductor package, package, p-value≤0.05). p-value<0.05).
2.1.4. Bioinformatic 2.1.4. Bioinformaticmodelling modelling ofof experimentally experimentally observed observed transcriptomics transcriptomics datadata Ingenuity Ingenuity Pathways Pathways Analysis Analysis (IPA) (IPA) (QIAGEN (QIAGEN \Inc., \Inc.,
https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis) [4] https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis) [4] was was used, for used, for each each investigated investigated batch batchtoto evaluate evaluatemodulations modulations of of gene gene expression expression relevant relevant
30 30 for effects for effects of of interest. interest. IPAisisananaggregator IPA aggregator of scientific of scientific references references that allows that allows to search to search for information for information
on genes/proteins on genes/proteinsandand thethe construction construction of networks of networks that predict that predict the behaviour the behaviour of of
65 10 May 2024
biological systems according to their gene expression status. biological systems according to their gene expression status.
Thepatho-physiological The patho-physiologicalfeatures featuresof of state-of-the-art"Osteoarthritis state-of-the-art “Osteoarthritiscondition" condition” were considered with particular attention to the following areas involved: were considered with particular attention to the following areas involved:
Keywords used to interrogate Chosen biological function IPA Osteoarthritis [osteoarthrosis] Arthralgia/arthritis Non-traumatic arthropathy;Osteoarthritis arthropathy Damage of cartilage tissue Formation of cartilage 2024203121
cartilage tissue tissue
Inflammation of joint Inflammation of joint Pain of joint
5 5 This knowledge This knowledgewaswas usedused to interrogate to interrogate IPAthe IPA via via"IPA theBioprofiler" “IPA Bioprofiler” tool, tool, using the using the above keywords. above key words. Theuse The useofof"IPA "IPA Bioprofiler" Bioprofiler" allowed allowed to identify to identify clusters clusters of expressed of expressed genesgenes
causally linked to each of the identified biological activities and the specific molecular causally linked to each of the identified biological activities and the specific molecular
pathwayunderpinning pathway underpinning them. them. Information Information concerning concerning the the measured measured gene gene expression expression data data
10 10 (Fold change (Fold changevaluevaluecut-off cut-off<-2 ≤-2and and+2≥ and+2 and p-value≤0.05) p-value<0.05) induced induced by eachbybatch each was batch was then superimposed then superimposed onon thenetworks the networks obtained, obtained, toto defineinfluenced define influencedgenesgenes and and modulation modulation
of the of the connected biofunction. connected biofunction.
Modulationof ofexpressed Modulation expressed genes genes were were shown shown in different in different intensities intensities of blue of blue
(signifying down-modulation) (signifying down-modulation) oror redred(signifying (signifyingup-modulation). up-modulation).TheThe resultingexpected resulting expected 15 15 calculatedimpact, calculated impact, based based on literature, on the the literature, onrelated on the the related biofunctions biofunctions was determined was determined by by “IPAMolecule "IPA MoleculeActivity ActivityPredictor" Predictor”tool tool(MAP) (MAP) andand resumed resumed in ain a heatmap heatmap visualization. visualization.
Thecolour The colourandandintensity intensity thereof thereof were transformedinto were transformed into numerical numericalvalues. values. 2.1.5. Results 2.1.5. Results Theresults The results ofof these these tests testsyielded yieldedaacomparative comparative study study ofof the the performance performance and andofof 20 20 the mechanism the mechanism of of action action of of fivedifferent five differentbatches batchesofofArtéArtéGX.GX. The analysis The analysis revealed revealed
that while that while allall the the different different batches batches werewerecapable capableofofreturning returningreproducible reproducible biological biological
effects, itit was effects, also possible was also possibletotoidentify identifybatch-specific batch-specific fluctuations fluctuations in in the the induced induced
transcriptional pattern. transcriptional Evidently the pattern. Evidently theinduction inductionof of slightly slightly different different transcriptional transcriptional
patternsstill patterns still results results inin the thesame same desirable desirable regulation regulation of biological of biological activities. activities. This isThis due is due
25 25 to aa functional to functional redundancy redundancy ininthe theinteractions interactionsbetweenbetweenthethecomponents components of product of the the product and the and thebody, body,whereby, whereby, by virtue by virtue of the of multifocal the multifocal mechanism mechanism of action, of slightly action, slightly different compositions different compositions elicit elicit thethe samesame effect effect (Figs.3(Figs.3 and 4).and 4). Thefive The five batches batches are are therefore therefore considered considered as as having equivalent biological having equivalent biological outputs outputs
sincethe since theinduction induction andand repression repression patterns patterns are conserved. are conserved.
30 30 From the From theresults results summarised summarisedininfigure figure4 4ititisisevident evidentthatthat the the observed observed transcriptionalpatterns transcriptional patternsof of thethe five five different different batches batches elicitelicit a verya very reproducible reproducible biological biological
effect leading effect leadingtotoa ageneral general modification modification of theof the pathological pathological processesprocesses and of theand of the overall overall
66 10 May 2024
pathological state equivalence for all the batches reported in Figure 4. pathological state equivalence for all the batches reported in Figure 4.
3.1. Targeted 3.1. Targetedmetabolomics metabolomics To appreciate To appreciatewhether whetherthethefinal finalmatrix matrixconstituting constituting"Arté “ArtéGX"GX” is characterized is characterized
by the by the matrix matrixeffect, effect, aa series series ofof analyses analyses toto grasp graspthetheproduct's product’sfeatures featuresonondifferent different 5 5 aspects was aspects carried out was carried out on the batches on the batches reported reported above. above. A A targeted targeted metabolomics metabolomics analysis capable analysis capable ofof identifying identifyingmost mostofofsuchsuch molecular molecular components, components, was carried was carried out out togetherwith together withthethe other other analysis analysis reported reported herein. herein.
Theproducts, products, asas described described above, above,consist consist ofof two twovegetal vegetal matrices matricesassembled assembledand and 2024203121
The
resulting in resulting in aa final finalnew new vegetal vegetal matrix. matrix. Several Several analytical analyticaltechniques techniques have have been usedto been used to 10 10 identify and identify and quantify quantifycompounds compounds belonging belonging to theto theclasses main main classes present inpresent plants.in plants. Although metabolomic Although metabolomicanalysisanalysis does does notnot allow allow toto appreciate appreciate the the dynamic changes dynamic changes within the within the components components of of thethe matrix, matrix, it it allows allows a “picture” a "picture" of the of the composition composition in the in the
moment moment the the analysis analysis is carried is carried out. out.
In the In the following followinganalysis analysis each each individual individual component component (plant metabolite) (plant metabolite) is is 15 15 specifically researched, specifically researched, forfor this this reason reason thethe analysis analysis isiscalled called“targeted "targetedmetabolomics”. metabolomics".
This analysis This analysis allows allowstotocapture capturea frame a frame on qualitative on the the qualitative data, data, by determining by determining the the chemicalcompounds chemical compounds present present in thein material, the material, and quantitative and quantitative data, data, by defining by defining the the concentrations of concentrations of each compound each compound in in thematerial. the material. For Arté For Arté GX, GX,a aqualitative qualitativeand andquantitative quantitative characterization characterization of of as as many manyprimary primary 20 20 and secondary and secondarymetabolites metabolitesas aspossible possible waswas carried carried outout using using an “omic” an "omic" approach, approach, the the targeted metabolomics targeted analysis,based metabolomics analysis, basedononthetheuse useofof multiple multiple analytical analytical methodologies. methodologies.
Theanalytical The analyticalmethods methods usedused for chemical for the the chemical characterization characterization of each of each batch arebatch are
described below. described below.The Themostmost appropriate appropriate analytical analytical techniques techniques have have beenbeen adopted adopted basedbased
on the on the chemical chemical nature nature of of the the classes classes of of compounds compoundspresent. present.TheTheanalysis analysiswithwith 25 25 chromatographicmethods chromatographic methods combined combined with different with different detection detection techniques techniques (e.g.,(e.g., GC andGC and LCeach LC eachcombined combinedwithwith a suitable a suitable detector), detector), made made it possible it possible to to identify identify andand quantify, quantify,
as appropriate, as appropriate, the the organic organic compounds. compounds. The Theinductively inductivelycoupled coupled plasma plasma analysis analysis using using a a
single quadrupole single quadrupolemass mass spectrometer spectrometer (ICP-MS) (ICP-MS) or an or an optical optical emissionemission spectrometer spectrometer
(ICP-OES) (ICP-OES) made made it it possibletotoestablish possible establishthethelevels levels of of elements present, while elements present, while the the anions anions 30 30 were determined were determinedbyby ionion chromatography chromatography and conductivity and conductivity detector. detector. Other Other gravimetric gravimetric
methodswere methods were used used forforthethe determination determination of classes of classes of substances of substances non-quantifiable non-quantifiable by by
meansofofchromatographic means chromatographic methods. methods.
Thetable The table below belowsummarizes summarizes allall themethods the methods used. used. Typeofof Type Class of Class of Compounds Compounds Characteristics Characteristics Short description Short description Method Method Fibres Fibres Insoluble Fibers Insoluble Fibers Gravimetric Gravimetric MethodAOAC Method AOAC991.42 (A1) 991.42(A1)
Soluble fibres Soluble fibres Gravimetric Gravimetric MethodAOAC Method AOAC (A2)(A2) 993.19 993.19
67 10 May 2024
Typeofof Type Class of Class of Compounds Compounds Characteristics Characteristics Short description Short description Method Method Extraction of Extraction of FOS FOSandand Fructans Fructans followed followed by by enzymatic digestion; analysis enzymatic digestion; analysis by ion by ion FOS, Fructans FOS, Fructans HPAEC-PAD HPAEC-PAD chromatographer equipped chromatographer equippedwithwitha pulsed a pulsed amperometric amperometric detector. detector. (A3) (A3)
Extraction ofofwater-soluble Extraction water-soluble polysaccharides polysaccharides
Polysaccharides Polysaccharides ≥ and analysis and analysis by by HPLC HPLCequipped equipped with with molecular molecular Polysaccharides Polysaccharides HPLC-RID HPLC-RID 20.000Dalton 20.000 Dalton exclusion column and refractive exclusion column and refractive index index detector. (B) detector. (B) 2024203121
Water Water Loss on Loss drying on drying Gravimetric Gravimetric Method ISTISAN Method ISTISAN 1996/34 1996/34 (pages (pages (C)(C) 7-10). 7-10).
Sample extraction Sample extraction and reverse phase and reverse phase chromatography chromatography analysis analysis by by UHPLC UHPLC coupled coupled to to UHPLC-qToF UHPLC-qToF a a quadrupole-time-of-flight quadrupole-time-of-flight mass mass Phenols Phenols Phenolspolar Phenols polar (D1) spectrometer.(D1) spectrometer.
HPLC-UV HPLC-UV Sample extraction Sample extraction and reverse phase and reverse phase chromatography chromatography analysis analysis by by HPLC HPLC coupled coupled to to UV-VIS (D2) detector. (D2) UV-VIS detector.
Sample extraction Sample extraction and reverse phase and reverse phase chromatography chromatography analysis analysis by by UHPLC UHPLC coupled coupled to to
UHPLC-qToF UHPLC-qToF a a quadrupole-time-of-flight quadrupole-time-of-flight mass mass Terpenes Terpenes Terpenespolar Terpenes polar (E1) spectrometer.(E1) spectrometer.
HPLC-UV HPLC-UV Sample extraction Sample extraction and reverse phase and reverse phase chromatography chromatography analysis analysis by by HPLC HPLC coupled coupled to to UV-VIS (E2) detector. (E2) UV-VIS detector.
Headspace analysis Headspace analysis of of the the sample sample bybygas gas Terpenesapolar Terpenes apolar GC-TQ GC-TQ chromatography coupled chromatography coupled to to a triple a triple quadrupolemass quadrupole mass spectrometer. spectrometer. (E3) (E3)
Analysis of Analysis of the samplebybygas the sample gaschromatograph chromatograph coupledtotoa aflame coupled flameionization ionizationdetector, detector, after after Sterols Sterols GC-FID GC-FID derivatization and derivatization transformationintointo and transformation trimethylsilyl ethers. (E4) trimethylsilyl ethers. (E4)
Organic acids Organic acids mono-, mono-, Sample extraction Sample extraction and andanalysis analysis bybyHPLC HPLC Organicacids Organic acids HPLC-UV HPLC-UV di-, tri-carboxylic di-, tri-carboxylic coupledto coupled to UV UVdetector. (F) detector.(F) NITROGEN Nitrogen-containing Nitrogen-containing Kjeldahl Method AOAC (G) (G) 920.53. NITROGEN Kjeldahl Method AOAC 920.53. Compounds Compounds substances, total substances, total Lipids Lipids Fats, total Fats, total Gravimetric Gravimetric Method ISTISAN Method ISTISAN 1996/34 1996/34 (pag(pag 39-40). 39-40). (H1) (H1)
Headspace analysis Headspace analysis of of the the sample sample bybygas gas Fatty acidsderivatives Fatty acids derivatives GC-TQ GC-TQ chromatograph chromatograph coupled coupled totriple to a a triplequadrupole quadrupole massspectrometer. mass (H2) spectrometer.(H2)
Sample extraction Sample extraction and andanalysis analysis bybyHPLC HPLC Fatty acids Fatty acids HPLC-UV HPLC-UV coupledto coupled to UV UVdetector. (H3) detector.(H3) Analysis of Analysis of the samplebybygas the sample gaschromatograph chromatograph Fatty acids Fatty acids GC-FID GC-FID coupledtotoa aflame coupled flameionization ionizationdetector, detector, after after
derivatization. (H4) derivatization. (H4)
Sample extraction Sample extraction and andanalysis analysisby by ion ion Sugarsand Sugars and Monosaccharides Monosaccharides IC-PAD IC-PAD chromatographer coupled to chromatographer coupled to pulsed pulsed Derivatives Derivatives amperometric amperometric detector. detector. (I) (I)
68 10 May 2024
Typeof Type of Class of Class of Compounds Compounds Characteristics Characteristics Short description Short description Method Method
Sample extraction Sample extraction and andanalysis analysisby by ion ion Disaccharides Disaccharides IC-PAD IC-PAD chromatographer coupled chromatographer coupled totopulsed pulsed amperometric amperometric detector. detector. (I) (I)
Headspace analysis of Headspace analysis of the the sample sample bybygasgas OtherOrganic Other Organic Otherorganic Other organic GC-TQ GC-TQ chromatographer coupled to chromatographer coupled to a triple a triple Compounds compounds, compounds, apolar apolar (L) (L) Compounds quadrupolemass quadrupole mass spectrometer. spectrometer.
Acid mineralization Acid mineralization of the sample of the samplein ina a 2024203121
microwave microwave oven oven andandanalysis analysis ofof the the ICP-MS, ICP-MS, conductively induced conductively inducedplasma plasma by means by means of a of a
Inorganic Inorganic ICP-OES ICP-OES single quadrupole mass spectrometer, oror single quadrupole mass spectrometer, Elements Elements Compounds Compounds optical emission optical emission spectrometer. (M1) spectrometer. (M1)
IC-CD IC-CD Sample extraction Sample extraction andandanalysis analysisby by ion ion chromatographer chromatographer coupled coupled to conductometric to conductometric
detector. (M2) detector. (M2)
Sample extraction Sample extraction andandanalysis analysisby by ion ion Anions Anions IC-CD IC-CD chromatographer chromatographer coupled coupled to conductometric to conductometric
detector. (M2) detector. (M2)
After extraction After extraction and anddigestion digestion of of RNARNA from from
the sample the samplea areverse reverse phase phase chromatography chromatography UHPLC-qToF UHPLC-qToF Total RNA Total RNA Nucleosides Nucleosides analysis was analysis appliedbybyUHPLC was applied UHPLC coupled coupled to a to a
quadrupole-time-of-flightmass quadrupole-time-of-flight massspectrometer. spectrometer. (N) (N)
The results, The results, that thatare aresummarised summarised in in the the table table below, below, show an appreciable show an appreciable compositionvariability composition variability between betweenthe thedifferent different batches batches and andunderline underlinethe theimpossibility impossibilityto to recapitulate the recapitulate the properties properties of ofthe thematrix matrix as asthe thesum sum of of its itssingle singlecomponents. components. The work The work
performedand performed and reported reported herein herein (see(see cell-based cell-based assay assay results) results) together together with with the the data data 5 5 below, demonstrates below, demonstrates thatthat thethe biological biological effect effect elicited elicited by by a product a product comprising or comprising or consisting of consisting of one one or or more morenatural naturalmatrices matricescannot cannot be be recapitulated recapitulated by by the the sum sum of the of the
effects elicited effects elicitedbybythe thesingle single molecular molecular components components butresult but is the is theofresult of interconnections interconnections
and interactions and interactions among among thethe components: components: the matrix the matrix effect.effect. This translates This translates into the into the
impossibilitytotoformally impossibility formally define define a structure-activity a structure-activity relationship relationship (SAR) according (SAR) according to to the the 10 10 principlescanonically principles canonically applied applied to APIs. to APIs.
Method COMPOUNDS Method COMPOUNDS 21E1640 21E1640 20B1955 20B1955 20I1279 2011279 20J1770 20J1770 20B0596 20B0596 21E1640 21E1640 20I1279 2011279 20J1770 20J1770 20B0596 20B0596 (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (d%) (d%) (d%) (d%) (d%) (d%) (d%) (d%)
NITROGEN- NITROGEN- CONTAINING G CONTAINING 5,1 5,1 5 4,7 0,76 0,00 1,96 7,84 85,10 SUBSTANCES, G 5,1 5,1 5 4,7 0,76 0,00 1,96 7,84 85,10 SUBSTANCES, Total Total
FIBERS, Total FIBERS, Total 5,4 5,4 2,3 2,3 1,5 1,5 1,7 1,7 1,8 1,8 134,78 134,78 34,78 34,78 26,09 26,09 22,26 22,26
SOLUBLEFIBERS, FIBERS, A2 SOLUBLE 5,4 2,3 1,5 1,1 1,5 1,5 134,78 34,78 52,17 34,78 Total A2 5,4 2,3 1,5 1,1 134,78 34,78 52,17 34,78 Total
69 10 May 2024
Method COMPOUNDS Method COMPOUNDS 21E1640 21E1640 20B1955 20B1955 20I1279 2011279 20J1770 20J1770 20B0596 20B0596 21E1640 21E1640 20I1279 2011279 20J1770 20J1770 20B0596 20B0596 (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (d%) (d%) (d%) (d%) (d%) (d%) (d%) (d%) FRUCTOLIGOSAC A3 FRUCTOLIGOSAC <LdQ <LdQ <LdQ 0,6 0,6 0,2880 // // // // CHARIDES,Total Total A3 <LdQ <LdQ <LdQ 0,2880 CHARIDES,
INSOLUBLE A1 INSOLUBLE <LdQ <LdQ <LdQ <LdQ <LdQ // // // // FIBERS, Total Total A1 <LdQ <LdQ <LdQ <LdQ <LdQ FIBERS,
MACROMOLECUL MACROMOLECUL ES ES > > 20000 B 20000 <LdQ <LdQ <LdQ <LdQ <LdQ // // // // B (POLYSACCHARID (POLYSACCHARID <LdQ <LdQ <LdQ <LdQ <LdQ 2024203121
ES), Total ES), Total
C C WATER,Total WATER, Total 3,4 3,4 5,4 5,4 4 4 4,10 4,10 4,50 4,50 37,04 37,04 25,93 25,93 24,07 24,07 16,67 16,67
PHENOLS, Total PHENOLS, Total 4,57 4,57 3,45 3,45 3,20 3,20 3,33 3,33 2,97 2,97 32,30 32,30 7,37 7,37 3,48 3,48 13,95 13,95
FLAVONOIDS, FLAVONOIDS, 1,80 1,49 1,49 1,74 1,18 20,48 0,47 16,39 20,81 Total 1,80 1,49 1,49 1,74 1,18 20,48 0,47 16,39 20,81 Total
D1 D1 Rottlerin Rottlerin <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // // //
D1 D1 Phloridzin Phloridzin nq nq nq nq nq nq // // // // // //
FLAVANONES, FLAVANONES, 0,01 0,01 0,0052 0,0052 0,0096 nq nq 92,31 84,62 84,62 // // Total 0,0096 nq nq 92,31 Total
D1 D1 Flavanomarein Flavanomarein 0,01 0,01 0,0052 0,0052 0,0096 0,0096 nq nq nq nq 92,31 92,31 84,62 84,62 // //
FLAVONES, FLAVONES, Total Total 0,0258 0,0258 0,02 0,02 0,0297 0,0297 0,023858 0,023858 0,0134 0,0134 29,00 29,00 48,50 48,50 19,29 19,29 33,00 33,00
3',4',5,5',6,7- 3',4',5,5',6,7-
D1 D1 Hexamethoxyflav Hexamethoxyflav <LdQ <LdQ 0,0002 0,0002 0,0002 0,0002 0,000119 0,000119 0,0002 0,0002 // 0,00 0,00 40,55 40,55 5,52 5,52 one one D1 D1 Diosmin Diosmin nd nd <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // // //
D1 D1 Homoorientin Homoorientin 0,009 0,009 0,0063 0,0063 0,0128 0,0128 0,003715 0,003715 0,0037 0,0037 42,86 42,86 103,17 103,17 41,03 41,03 41,65 41,65
D1 D1 Isoschaftoside Isoschaftoside 0,0008 0,0008 0,001 0,001 0,0009 0,0009 0,004852 0,004852 0,0022 0,0022 20,00 20,00 10,00 10,00 385,20 385,20 124,34 124,34
D1 D1 Isovitexin Isovitexin 0,0012 0,0012 0,001 0,001 0,0011 0,0011 0,000779 0,000779 0,0006 0,0006 20,00 20,00 10,00 10,00 22,10 22,10 40,89 40,89
D1 D1 Linarin Linarin nd nd nd nd nd nd <LdQ <LdQ // // // // //
Luteolin-7-O- Luteolin-7-0- D1 D1 beta-D-glucoside beta-D-glucoside nd nd nd nd nd nd <LdQ <LdQ // // // // // (Cynaroside) (Cynaroside)
Luteolin-7-O- Luteolin-7-0- D1 nd nd nd <LdQ <LdQ // // // // glucuronide D1 nd nd nd <LdQ <LdQ glucuronide
D1 D1 Nobiletin Nobiletin <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
D1 D1 Orientin Orientin 0,0025 0,0025 0,0026 0,0026 0,0018 0,0018 nq nq nq nq 3,85 3,85 30,77 30,77 // //
D1 D1 Schaftoside Schaftoside 0,0067 0,0067 0,0044 0,0044 0,0081 0,0081 0,008796 0,008796 0,0036 0,0036 52,27 52,27 84,09 84,09 99,91 99,91 19,06 19,06
D1 D1 Vicenin-2 Vicenin-2 0,0038 0,0038 0,0032 0,0032 0,0029 0,0029 0,004069 0,004069 0,0021 0,0021 18,75 18,75 9,38 9,38 27,16 27,16 33,32 33,32
D1 D1 Vitexin Vitexin 0,0018 0,0018 0,0013 0,0013 0,0019 0,0019 0,001528 0,001528 0,0010 0,0010 38,46 38,46 46,15 46,15 17,55 17,55 25,67 25,67
FLAVONOLS, FLAVONOLS, 1,7619 1,4669 1,4458 1,707743 1,1682 20,11 1,44 16,42 20,36 Total 1,7619 1,4669 1,4458 1,707743 1,1682 20,11 1,44 16,42 20,36 Total
Isorhamnetin-3- Isorhamnetin-3- D1 0,0022 0,0018 0,0018 0,002 0,002 0,001514 0,0016 0,0016 22,22 11,11 11,11 15,92 11,28 11,28 O-glucoside D1 0,0022 0,001514 22,22 15,92 O-glucoside
Isorhamnetin-3- Isorhamnetin-3- D1 <LdQ <LdQ <LdQ <LdQ // // // // // O-rutinoside D1 <LdQ <LdQ <LdQ <LdQ O-rutinoside
D1 D1 Kaempferol Kaempferol 0,0062 0,0062 0,0136 0,0136 0,0104 0,0104 0,002918 0,002918 0,0030 0,0030 54,41 54,41 23,53 23,53 78,54 78,54 77,76 77,76
70 10 May 2024
Method COMPOUNDS Method COMPOUNDS 21E1640 21E1640 20B1955 20B1955 20I1279 2011279 20J1770 20J1770 20B0596 20B0596 21E1640 21E1640 20I1279 2011279 20J1770 20J1770 20B0596 20B0596 (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (d%) (d%) (d%) (d%) (d%) (d%) (d%) (d%)
Kaempferol-3-O- Kaempferol-3-0- D1 0,0342 0,0342 0,1187 0,1187 0,0743 0,0743 0,032471 0,0729 0,0729 71,19 71,19 37,41 37,41 72,64 72,64 38,56 38,56 D1 glucoside glucoside 0,032471
Kaempferol-3-O- Kaempferol-3-0- D1 0,3409 0,3409 0,2751 0,2751 0,2855 0,2855 0,618309 0,5180 0,5180 23,92 23,92 3,78 3,78 124,76 124,76 88,31 88,31 glucuronide D1 0,618309 glucuronide
Kaempferol-3-O- Kaempferol-3-0- D1 0,0076 0,0119 0,0092 0,00195 0,0035 36,13 22,69 83,61 70,69 rutinoside D1 0,0076 0,0119 0,0092 0,00195 0,0035 36,13 22,69 83,61 70,69 rutinoside
D1 Quercetin 0,0201 0,0352 0,0159 0,012575 0,0192 42,90 54,83 64,27 45,36 2024203121
D1 Quercetin 0,0201 0,0352 0,0159 0,012575 0,0192 42,90 54,83 64,27 45,36
Quercetin-3-O- Quercetin-3-0- D1 D1 glucopyranoside glucopyranoside 0,0694 0,0694 0,0871 0,0871 0,0694 0,0694 0,025885 0,025885 0,0598 0,0598 20,32 20,32 20,32 20,32 70,28 70,28 31,36 31,36 (Isoquercetin) (Isoquercetin)
Quercetin-3-O- Quercetin-3-0- D1 1,2456 1,2456 0,8651 0,8651 0,9198 0,9198 0,985214 0,4558 0,4558 43,98 43,98 6,32 6,32 13,88 13,88 47,31 47,31 D1 glucuronide glucuronide 0,985214
D1 D1 Rutin Rutin 0,0357 0,0357 0,0584 0,0584 0,0593 0,0593 0,026907 0,026907 0,0313 0,0313 38,87 38,87 1,54 1,54 53,93 53,93 46,34 46,34
ISOFLAVONES, <LdQ <LdQ <LdQ <LdQ <LdQ / // // / ISOFLAVONES, / / Total <LdQ <LdQ <LdQ <LdQ <LdQ Total
D1 D1 Genistin Genistin <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ / / / / / / / /
PHENOLICACIDS ACIDS, 0,0745 0,0707 0,0923 0,077983 0,1091 5,37 30,55 10,30 54,35 PHENOLIC Total 0,0745 0,0707 0,0923 0,077983 0,1091 5,37 30,55 10,30 54,35 Total
Protocatechuic Protocatechuic D1 0,0745 0,0707 0,0923 0,077983 0,1091 5,37 30,55 10,30 54,35 acid D1 0,0745 0,0707 0,0923 0,077983 0,1091 5,37 30,55 10,30 54,35 acid
D1 D1 Vanillic acid Vanillic acid nd nd nd nd nd nd <LdQ <LdQ // / / / / / / / /
PHENYLPROPAN PHENYLPROPAN 2,682 1,872 1,606 1,490576 0,929 43,25 14,23 20,39 50,38 OIDS, Total Total 2,682 1,872 1,606 1,490576 0,929 43,25 14,23 20,39 50,38 OIDS,
COUMARINS, COUMARINS, 0,0149 0,0168 0,0164 0,007515 0,0022 11,31 2,38 55,27 86,90 Total 0,0149 0,0168 0,0164 0,007515 0,0022 11,31 2,38 55,27 86,90 Total
D1 D1 Aesculin Aesculin 0,0051 0,0051 0,0043 0,0043 0,0112 0,0112 0,006231 0,006231 0,0047 0,0047 18,60 18,60 160,47 160,47 44,90 44,90 8,63 8,63
D1 D1 Esculetin Esculetin 0,0098 0,0098 0,0125 0,0125 0,0052 0,0052 0,001284 0,001284 0,0053 0,0053 21,60 21,60 58,40 58,40 89,73 89,73 57,65 57,65
D1 D1 Fraxin Fraxin <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ / / <LdQ <LdQ // / / // / /
HYDROXYCINNA HYDROXYCINNA 2,667 1,856 1,590 1,442046 0,9068 0,9068 43,75 14,33 22,29 51,13 MIC ACIDS,Total Total 2,667 1,856 1,590 1,442046 43,75 14,33 22,29 51,13 MIC ACIDS,
3,5- 3,5-
D1 D1 Dicaffeoylquinic Dicaffeoylquinic 1,043 1,043 0,5573 0,5573 0,516 0,516 0,4833 0,4833 0,2254 0,2254 87,15 87,15 7,41 7,41 13,28 13,28 59,56 59,56 acid acid
4,5- 4,5-
D1 D1 Dicaffeoylquinic Dicaffeoylquinic 0,1822 0,1822 0,1601 0,1601 0,1568 0,1568 0,058033 0,058033 0,0547 0,0547 13,80 13,80 2,06 2,06 63,75 63,75 65,85 65,85 acid acid
D1 D1 Caffeic acid Caffeic acid 0,0219 0,0219 0,0168 0,0168 0,0274 0,0274 0,008865 0,008865 0,0089 0,0089 30,36 30,36 63,10 63,10 47,23 47,23 47,04 47,04
D2 D2 Caftaric acid Caftaric acid 0,2338 0,2338 0,2825 0,2825 0,2005 0,2005 0,1949 0,1949 0,2198 0,2198 17,24 17,24 29,03 29,03 31,01 31,01 22,19 22,19
D2 D2 Chicoric acid Chicoric acid 0,282 0,282 0,2948 0,2948 0,17 0,17 0,1748 0,1748 0,2142 0,2142 4,34 4,34 42,33 42,33 40,71 40,71 27,34 27,34
D2 D2 Chlorogenicacid Chlorogenic acid 0,8213 0,8213 0,4702 0,4702 0,4644 0,4644 0,4276 0,4276 0,2931 0,2931 74,67 74,67 1,23 1,23 9,06 9,06 37,66 37,66
D2 D2 Echinacoside Echinacoside <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // / / / / / /
D1 D1 Ferulic acid Ferulic acid 0,0081 0,0081 0,0047 0,0047 0,0055 0,0055 0,003602 0,003602 nq nq 72,34 72,34 17,02 17,02 23,36 23,36 / /
Neochlorogenic Neochlorogenic D1 0,0626 0,0626 0,0658 0,0658 0,0445 0,0445 0,090946 0,0598 0,0598 4,86 32,37 32,37 38,22 9,18 acid D1 0,090946 4,86 38,22 9,18 acid
D1 D1 Rosmarinicacid Rosmarinic acid 0,0082 0,0082 0,0034 0,0034 0,0026 0,0026 nq nq // 141,18 141,18 23,53 23,53 / / / /
D1 D1 Verbascoside Verbascoside 0,0043 0,0043 <LdQ <LdQ 0,0019 0,0019 // nq nq // / / / / / /
71 10 May 2024
Method COMPOUNDS Method COMPOUNDS 21E1640 21E1640 20B1955 20B1955 20I1279 2011279 20J1770 20J1770 20B0596 20B0596 21E1640 21E1640 20I1279 2011279 20J1770 20J1770 20B0596 20B0596 (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (d%) (d%) (d%) (d%) (d%) (d%) (d%) (d%)
PHLOROGLUCIN PHLOROGLUCIN nq nq nq <LdQ // // // // // OLS, Total OLS, Total ng nq nq <LdQ
Phloroglucinol Phloroglucinol D1 nq nq nq <LdQ // // // // // carboxylic acid D1 carboxylic acid nq nq nq <LdQ
SALICYLATES, 0,0118 0,0162 0,0136 0,0145 0,006522 27,16 16,05 10,50 59,74 SALICYLATES, 0,0118 0,0162 0,0136 0,0145 16,05 Total 0,006522 27,16 10,50 59,74 Total
D1 D1 Salicylic acid Salicylic acid 0,0118 0,0118 0,0162 0,0162 0,0136 0,0136 0,0145 0,0145 0,006522 0,006522 27,16 27,16 16,05 16,05 10,50 10,50 59,74 59,74 2024203121
TERPENES, TERPENES, Total Total 13,63 13,63 15,80 15,80 16,59 16,59 14,92 14,92 14,93 14,93 13,74 13,74 5,02 5,02 5,57 5,57 5,49 5,49
MONOTERPENES, MONOTERPENES, 0,000408 0,000329 0,000329 0,00042 0,000305 0,0008 0,0008 23,96 23,96 29,09 29,09 7,15 7,15 143,18 Total 0,000408 0,00042 0,000305 143,18 Total
MONOTERPENE MONOTERPENE 0,000355 0,00028 0,00037 0,000278 0,000309 26,59 32,76 0,95 10,17 ALCOHOLS,Total Total 0,000355 0,00028 0,00037 0,000278 0,000309 26,59 32,76 0,95 10,17 ALCOHOLS,
E3 E3 alpha-Terpineol alpha-Terpineol <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
E3 E3 Carvacrol Carvacrol 4,49E-05 4,49E-05 5,08E-05 5,08E-05 5,1E-05 5,1E-05 2,61E-05 2,61E-05 9,34E-05 9,34E-05 11,67 11,67 0,85 0,85 48,61 48,61 83,83 83,83
E3 E3 Eucalyptol Eucalyptol <LdQ <LdQ nd nd nd nd <LdQ <LdQ <LdQ <LdQ // // // //
E3 E3 Linalool Linalool 0,00031 0,00031 0,00023 0,00023 0,00032 0,00032 0,000252 0,000252 0,000216 0,000216 35,06 35,06 39,82 39,82 9,59 9,59 6,13 6,13
MONOTERPENE MONOTERPENE HYDROCARBONS, 5,27E-05 HYDROCARBONS, 5,27E-05 4,85E-05 4,85E-05 5,2E-05 5,2E-05 2,77E-05 2,77E-05 <LdQ <LdQ 8,70 8,70 7,90 7,90 42,97 42,97 // Total Total
E3 E3 4-Cymene 4-Cymene 5,27E-05 5,27E-05 4,85E-05 4,85E-05 5,2E-05 5,2E-05 2,77E-05 2,77E-05 <LdQ <LdQ 8,70 8,70 7,90 7,90 42,97 42,97 / /
E3 E3 alpha-Pinene alpha-Pinene nd nd nd nd <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // / /
E3 E3 alpha-Terpinene alpha-Terpinene <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
E3 E3 beta-Pinene beta-Pinene <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // / / // / /
E3 E3 Camphene Camphene <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
gamma- E3 E3 gamma- <LdQ <LdQ <LdQ <LdQ <LdQ // // // // Terpinene <LdQ <LdQ <LdQ <LdQ <LdQ Terpinene
E3 E3 Myrcene Myrcene <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // / /
E3 E3 Sabinene Sabinene <LdQ <LdQ nd nd <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
E3 E3 Terpinolene Terpinolene <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
TRITERPENES, TRITERPENES, 13,627 13,627 15,799 16,592 14,919 14,9314 13,74 5,02 5,57 5,49 Total 15,799 16,592 14,919 14,9314 13,74 5,02 5,57 5,49 Total
PHYTOSTEROLS, PHYTOSTEROLS, 0,001141 0,001289 <LdQ // // 11,45 // // // Total 0,001141 0,001289 <LdQ 11,45 Total
24- 24- E4 E4 Methylenecholest Methylenecholest 0,000056 0,000056 <LdQ <LdQ <LdQ <LdQ / / // // // // // erol erol
7- 7-
E4 E4 Dehydrocholester Dehydrocholester <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // // // // ol ol
E4 E4 beta-Sitosterol beta-Sitosterol 0,000435 0,000435 0,000578 0,000578 <LdQ <LdQ // // 24,65 24,65 // / / / /
E4 E4 Brassicasterol Brassicasterol <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // // // //
E4 E4 Campestanol Campestanol 0,000181 0,000181 6,19E-05 6,19E-05 <LdQ <LdQ // // 192,73 192,73 // // //
E4 E4 Campesterol Campesterol 0,000149 0,000149 0,000125 0,000125 <LdQ <LdQ // // 19,35 19,35 // / / //
E4 E4 Cholesterol Cholesterol 8,78E-05 8,78E-05 0,000075 0,000075 <LdQ <LdQ // // 17,32 17,32 // // //
72 10 May 2024
Method COMPOUNDS Method COMPOUNDS 21E1640 21E1640 20B1955 20B1955 20I1279 2011279 20J1770 20J1770 20B0596 20B0596 21E1640 21E1640 20I1279 2011279 20J1770 20J1770 20B0596 20B0596 (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (d%) (d%) (d%) (d%) (d%) (d%) (d%) (d%)
E4 E4 Clerosterol Clerosterol <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ / / / / // // // / /
delta-5- delta-5- E4 <LdQ 7,1E-05 <LdQ // // // / / // / / Avenasterol E4 <LdQ 7,1E-05 <LdQ Avenasterol
delta-5,23- delta-5,23- E4 <LdQ <LdQ <LdQ // / / // / / // // Stigmastadienol E4 <LdQ <LdQ <LdQ Stigmastadienol
delta-5,24- delta-5,24- E4 <LdQ <LdQ <LdQ // / / // // // // Stigmastadienol E4 <LdQ <LdQ <LdQ 2024203121
Stigmastadienol
delta-7- delta-7- E4 <LdQ <LdQ <LdQ // / / // / / // // Avenasterol E4 <LdQ <LdQ <LdQ Avenasterol
delta-7- delta-7- E4 <LdQ <LdQ <LdQ // / / // // // // Campesterol E4 <LdQ <LdQ <LdQ Campesterol
delta-7- delta-7- E4 0,000074 0,000107 <LdQ // // 30,79 // // // Stigmastenol E4 0,000074 0,000107 <LdQ 30,79 Stigmastenol
delta-7,9(11)- delta-7,9(11)- E4 <LdQ <LdQ <LdQ <LdQ // // // // // // Stigmastadienol E4 <LdQ <LdQ Stigmastadienol
E4 E4 Sitostanol Sitostanol 0,000041 0,000041 9,55E-05 9,55E-05 <LdQ <LdQ // / / 57,01 57,01 / / / / / /
E4 E4 Stigmasterol Stigmasterol 0,000116 0,000116 0,000175 0,000175 <LdQ <LdQ // // 33,72 33,72 // // //
SAPOGENINS, SAPOGENINS, 0,0091 0,0154 0,0176 <LdQ 0,0054 40,91 14,29 // 64,94 Total 0,0091 0,0154 0,0176 <LdQ 0,0054 40,91 14,29 64,94 Total
E1 E1 Asiatic acid Asiatic acid 0,001 0,001 0,0033 0,0033 0,0024 0,0024 <LdQ <LdQ <LdQ <LdQ 69,70 69,70 27,27 27,27 // //
E1 E1 Madecassic acid Madecassic acid 0,0081 0,0081 0,0121 0,0121 0,0152 0,0152 <LdQ <LdQ 0,005364 0,005364 33,06 33,06 25,62 25,62 // 55,67 55,67
SAPONINS, SAPONINS, Total Total 13,617 13,617 15,782 15,782 16,574 16,574 14,919 14,919 14,926 14,926 13,72 13,72 5,02 5,02 5,47 5,47 5,42 5,42
E2 E2 Asiaticoside Asiaticoside 5,526 5,526 7,121 7,121 6,935 6,935 6,143 6,143 6,801 6,801 22,40 22,40 2,61 2,61 13,73 13,73 4,49 4,49
E2 E2 Madecassoside Madecassoside 8,091 8,091 8,661 8,661 9,639 9,639 8,776 8,776 8,125 8,125 6,58 6,58 11,29 11,29 1,33 1,33 6,19 6,19
SESQUITERPENES SESQUITERPENES <LdQ nd <LdQ <LdQ nd // // // // , Total <LdQ nd <LdQ <LdQ nd , Total
E3 E3 Farnesol Farnesol <LdQ <LdQ nd nd <LdQ <LdQ // / / // // // / /
ORGANICACIDS, ACIDS, ORGANIC 10,37 14,48 9,94 9,03 15,07 28,38 31,31 37,66 4,07 Total 10,37 14,48 9,94 9,03 15,07 28,38 31,31 37,66 4,07 Total
MONOCARBOXYL MONOCARBOXYL <LdQ 2,03 <LdQ 1,431 1,627 // // 29,51 19,85 IC ACIDS, Total <LdQ 2,03 <LdQ 1,431 1,627 29,51 19,85 IC ACIDS, Total
FF Acetic acid Acetic acid <LdQ <LdQ 1,07 1,07 <LdQ <LdQ 0,151 0,151 0,177 0,177 // / / 85,89 85,89 83,46 83,46
FF Formic acid Formic acid <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LoQ <LoQ // // // //
FF Lactic acid Lactic acid <LdQ <LdQ 0,96 0,96 <LdQ <LdQ 1,28 1,28 1,45 1,45 // // 33,33 33,33 51,04 51,04
DICARBOXYLIC DICARBOXYLIC 8,2088 10,4075 7,6748 4,7048 10,05 21,13 26,26 54,79 3,44 ACIDS,Total Total 8,2088 10,4075 7,6748 4,7048 10,05 21,13 26,26 54,79 3,44 ACIDS,
FF Fumaric acid Fumaric acid 0,067 0,067 0,0446 0,0446 0,067 0,067 0,13 0,13 0,136 0,136 50,22 50,22 50,22 50,22 191,48 191,48 204,93 204,93
FF Malic acid Malic acid 8,08 8,08 2,2 2,2 7,54 7,54 4,55 4,55 5,75 5,75 267,27 267,27 242,73 242,73 106,82 106,82 161,36 161,36
FF Succinic acid Succinic acid <LdQ <LdQ 8,14 8,14 <LdQ <LdQ <LdQ <LdQ 4,13 4,13 // // // 49,26 49,26
FF Tartaric acid Tartaric acid 0,0618 0,0618 0,0229 0,0229 0,0678 0,0678 0,0248 0,0248 0,034 0,034 169,87 169,87 196,07 196,07 8,30 8,30 48,47 48,47
TRICARBOXYLIC TRICARBOXYLIC 2,16 2,04 2,27 2,89 3,39 5,88 11,27 41,67 66,18 ACIDS,Total Total 2,16 2,04 2,27 2,89 3,39 5,88 11,27 41,67 66,18 ACIDS,
FF Citric acid Citric acid 2,16 2,16 2,04 2,04 2,27 2,27 2,89 2,89 3,39 3,39 5,88 5,88 11,27 11,27 41,67 41,67 66,18 66,18
73 10 May 2024
Method COMPOUNDS Method COMPOUNDS 21E1640 21E1640 20B1955 20B1955 20I1279 2011279 20J1770 20J1770 20B0596 20B0596 21E1640 21E1640 20I1279 2011279 20J1770 20J1770 20B0596 20B0596 (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (d%) (d%) (d%) (d%) (d%) (d%) (d%) (d%)
SUGARS AND SUGARS AND DERIVATIVES, DERIVATIVES, 11,42 11,42 1,85 1,85 10,78 10,78 13,34 13,34 7,46 7,46 516,13 516,13 481,66 481,66 619,61 619,61 302,37 302,37 Total Total
MONOSACCHARI MONOSACCHARI 11,423 11,423 1,854 10,784 10,0247 10,0247 7,16 7,16 516,13 516,13 481,66 481,66 440,71 286,19 286,19 DES, Total 1,854 10,784 440,71 DES, Total
I I Fructose Fructose 5,707 5,707 1,854 1,854 5,38 5,38 6,20591 6,20591 4,5300 4,5300 207,82 207,82 190,18 190,18 234,73 234,73 144,34 144,34
I Galactose 1,369 <LdQ 1,315 1,30558 1,1000 / / / / 2024203121
I Galactose 1,369 <LdQ 1,315 1,30558 1,1000 / / / /
I I Glucose Glucose 4,347 4,347 <LdQ <LdQ 4,089 4,089 2,51321 2,51321 1,2900 1,2900 / / / / / / / /
DISACCHARIDES, DISACCHARIDES, <Ldq <Ldq <Ldq <Ldq 3,31694 0,30 / / / / / / / / Total <Ldq <Ldq 3,31694 0,30 Total
I I Lactose Lactose <Ldq <Ldq <Ldq <Ldq <Ldq <Ldq <LdQ <LdQ // / / / / / / / /
I | Maltose Maltose <Ldq <Ldq <Ldq <Ldq <Ldq <Ldq 1,54 1,54 // / / / / / / / /
Sucrose Sucrose nq nq nq nq nq nq 1,77694 1,77694 0,30 0,30 / / / / / / / /
LIPIDS, Total LIPIDS, Total 0,200 0,200 0,170 0,170 0,190 0,190 4,03E-05 4,03E-05 5,88E-05 5,88E-05 17,64 17,64 11,76 11,76 99,98 99,98 99,97 99,97
FATTYACIDS, ACIDS, 0,2 0,17 0,19 <LdQ 5,88E-05 17,65 11,76 / 99,97 FATTY / Total 0,2 0,17 0,19 <LdQ 5,88E-05 17,65 11,76 99,97 Total
H4 H4 Decanoicacid Decanoid acid nd nd nd nd 0,01 0,01 / / / / / / / / / / / /
H4 H4 Dodecanoic acid Dodecanoid acid 0,02 0,02 nd nd nd nd / / / / / / / / / / / /
H4 H4 Linoleic acid Linoleic acid nd nd nd nd nd nd / / / / / / / / / / / /
H4 H4 Linolenic acid Linolenic acid nd nd nd nd nd nd / / / / / / / / / / / /
H4 H4 Myristic acid Myristic acid nd nd nd nd nd nd / / / / / / / / / / / /
H4 H4 Octanoicacid Octanoic acid nd nd nd nd 0,01 0,01 / / / / / / / / / / / /
H4 H4 Oleic acid Oleic acid nd nd nd nd nd nd / / / / // / / / / / /
H4 H4 Palmitic acid Palmitic acid 0,1 0,1 0,1 0,1 0,09 0,09 / / / / 0,00 0,00 10,00 10,00 / / / /
H3 H3 Propionic acid Propionic acid <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ / / / / / / / / / / / /
H4 H4 Stearic acid Stearic acid 0,08 0,08 0,07 0,07 0,08 0,08 / / // 14,29 14,29 14,29 14,29 / / / /
FATTY FATTY ACID ACID DERIVATIVES, DERIVATIVES, 9,38E-05 9,38E-05 9,37E-05 9,37E-05 9,5E-05 9,5E-05 4,03E-05 4,03E-05 5,88E-05 5,88E-05 0,09 0,09 1,20 1,20 57,05 57,05 37,24 37,24 Total Total
FATTY ACID ACID 9,38E-05 9,37E-05 9,5E-05 4,03E-05 5,88E-05 0,09 1,20 57,05 37,24 FATTY ESTERS, Total 9,38E-05 9,37E-05 9,5E-05 4,03E-05 5,88E-05 0,09 1,20 57,05 37,24 ESTERS, Total
H2 H2 Ethyl Ethyl palmitate palmitate 9,38E-05 9,38E-05 9,37E-05 9,37E-05 9,5E-05 9,5E-05 4,03E-05 4,03E-05 5,88E-05 5,88E-05 0,09 0,09 1,20 1,20 57,05 57,05 37,24 37,24
OTHER ORGANIC OTHER ORGANIC COMPOUNDS, COMPOUNDS, 0,00069 0,00069 0,00048 0,00048 0,00069 0,00069 0,00064 0,00064 0,000361 0,000361 44,12 44,12 44,78 44,78 33,41 33,41 24,64 24,64 Total Total
AROMATIC AROMATIC COMPOUNDS, COMPOUNDS, 0,000691 0,000691 0,00048 0,00048 0,00069 0,00069 0,00064 0,00064 0,000361 0,000361 44,12 44,12 44,78 44,78 33,41 33,41 24,64 24,64 Total Total
AROMATIC AROMATIC 0,000665 // 0,00067 0,000622 0,000361 // / / / / ALCOHOLS, Total 0,000665 0,00067 0,000622 0,000361 / / ALCOHOLS, Total
para,alpha,alpha- para,alpha,alpha- L L Trimethylbenzyl 0,000665 Trimethylbenzyl 0,000665 0,000466 0,000466 0,00067 0,00067 0,000622 0,000622 0,000361 0,000361 42,67 42,67 44,25 44,25 -33,35 -33,35 22,46 22,46 alcohol alcohol
L L Benzaldehyde Benzaldehyde <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // / / / /
STYRENES,Total STYRENES, Total 2,62E-05 2,62E-05 1,35E-05 1,35E-05 2,2E-05 2,2E-05 1,82E-05 1,82E-05 <LdQ <LdQ 94,07 94,07 62,94 62,94 35,28 35,28 / /
74 10 May 2024
Method COMPOUNDS Method COMPOUNDS 21E1640 21E1640 20B1955 20B1955 20I1279 2011279 20J1770 20J1770 20B0596 20B0596 21E1640 21E1640 20I1279 2011279 20J1770 20J1770 20B0596 20B0596 (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (d%) (d%) (d%) (d%) (d%) (d%) (d%) (d%) L L Dimethylstyrene Dimethylstyrene 2,62E-05 2,62E-05 1,35E-05 1,35E-05 2,2E-05 2,2E-05 1,82E-05 1,82E-05 <LdQ <LdQ 94,07 94,07 62,94 62,94 35,28 35,28 //
INORGANIC INORGANIC COMPOUNDS, COMPOUNDS, 21,85 21,85 26,21 26,21 21,45 21,45 22,98 22,98 27,44 27,44 16,63 16,63 18,18 18,18 12,34 12,34 4,68 4,68 Total Total
ANIONS,Total ANIONS, Total 4,6313 4,6313 6,34 6,34 4,4114 4,4114 5,463733 5,463733 5,53 5,53 26,95 26,95 30,42 30,42 13,82 13,82 12,78 12,78
M2 Nitrate 0,0013 <LdQ 0,0014 0,092379 <ldQ // // // // 2024203121
Nitrate 0,0013 <LdQ 0,0014 0,092379 <ldQ M2 M2 M2 Nitrite Nitrite <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <ldQ <ldQ // // // //
M2 M2 Phosphate Phosphate 1,63 1,63 1,74 1,74 1,55 1,55 1,529767 1,529767 1,4700 1,4700 6,32 6,32 10,92 10,92 12,08 12,08 15,52 15,52
M2 M2 Sulfate Sulfate 3 3 4,6 4,6 2,86 2,86 3,841587 3,841587 4,0600 4,0600 34,78 34,78 37,83 37,83 16,49 16,49 11,74 11,74
MACROELEMENT MACROELEMENT 17,05 19,66 16,87 17,32551 21,9985 13,28 14,18 11,88 11,89 S, Total 17,05 19,66 16,87 17,32551 21,9985 13,28 14,18 11,88 11,89 S, Total
M1 M1 Calcium Calcium 1,745388 2,544027 1,745388 2,544027 1,75523 1,75523 1,847967 1,847967 2,8998 2,8998 31,39 31,39 31,01 31,01 27,36 27,36 13,98 13,98
M2 M2 Chloride Chloride 4,67 4,67 5,32 5,32 4,36 4,36 5,496178 5,496178 4,8700 4,8700 12,22 12,22 18,05 18,05 3,31 3,31 8,46 8,46
M1 M1 Magnesium Magnesium 1,270564 1,533499 1,270564 1,533499 1,25715 1,25715 1,233837 1,233837 1,6827 1,6827 17,15 17,15 18,02 18,02 19,54 19,54 9,73 9,73
M1 M1 Phosphorus Phosphorus 0,624007 0,624007 0,619255 0,619255 0,60815 0,60815 0,579524 0,579524 0,7223 0,7223 0,77 0,77 1,79 1,79 6,42 6,42 16,64 16,64
M1 M1 Potassium Potassium 5,728856 6,134733 5,728856 6,134733 5,80766 5,80766 5,657116 5,657116 7,6743 7,6743 6,62 6,62 5,33 5,33 7,79 7,79 25,10 25,10
M1 M1 Sodium Sodium 2,326488 2,326488 2,741546 2,741546 2,40561 2,40561 2,510884 2,510884 3,8194 3,8194 15,14 15,14 12,25 12,25 8,41 8,41 39,31 39,31
M1 M1 Sulfur Sulfur 0,683772 0,683772 0,767168 0,767168 0,6784 0,6784 1,56 1,56 <LdQ <LdQ 10,87 10,87 11,57 11,57 103,35 103,35 //
MICROELEMENTS MICROELEMENTS 0,137432 0,165006 0,12715 0,134181 0,1918 0,1918 16,71 22,94 18,68 16,24 , Total 0,137432 0,165006 0,12715 0,134181 16,71 22,94 18,68 16,24 , Total
M1 M1 Chromium Chromium 3,09E-05 3,09E-05 0,000102 0,000102 0,00011 0,00011 8,77E-05 8,77E-05 0,0007 0,0007 69,77 69,77 9,86 9,86 14,21 14,21 567,60 567,60
M1 M1 Cobalt Cobalt 0,000127 0,000127 0,000122 0,000122 0,00013 0,00013 0,000149 0,000149 0,0001 0,0001 4,08 4,08 8,87 8,87 22,19 22,19 7,45 7,45
M1 M1 Copper Copper 0,000718 0,000718 0,000226 0,000226 0,00091 0,00091 0,000945 0,000945 0,0009 0,0009 217,47 217,47 303,07 303,07 317,91 317,91 287,93 287,93
M2 M2 Fluoride Fluoride NQ NQ NQ NQ NQ NQ <Ldq <Ldq nq nq // // // //
M1 M1 Iron Iron 0,000372 0,000777 0,000372 0,000777 0,00326 0,00326 0,002209 0,002209 0,0027 0,0027 52,15 52,15 319,33 319,33 184,10 184,10 243,53 243,53
M1 M1 Manganese Manganese 0,128456 0,128456 0,156247 0,156247 0,11479 0,11479 0,121585 0,121585 0,1722 0,1722 17,79 17,79 26,53 26,53 22,18 22,18 10,20 10,20
M1 M1 Molybdenum Molybdenum <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
M1 M1 Nickel Nickel 0,000873 0,000873 0,000727 0,000727 0,00106 0,00106 0,00111 0,00111 0,0008 0,0008 20,11 20,11 45,89 45,89 52,65 52,65 3,32 3,32
M1 M1 Selenium Selenium <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
M1 M1 Tin Tin <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
M1 M1 Vanadium Vanadium <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
M1 M1 Zinc Zinc 0,006855 0,006855 0,006804 0,006804 0,00688 0,00688 0,008096 0,008096 0,0145 0,0145 0,75 0,75 1,09 1,09 18,99 18,99 112,71 112,71
OTHER OTHER 0,035826 0,049164 0,03758 0,056604 0,0508 27,13 23,57 15,13 3,33 ELEMENTS, Total 0,035826 0,049164 0,03758 0,056604 0,0508 27,13 23,57 15,13 3,33 ELEMENTS, Total
M1 M1 Aluminum Aluminum 0,00085 0,00085 0,001453 0,001453 0,00176 0,00176 <LdQ <LdQ 0,0021 0,0021 41,48 41,48 20,92 20,92 // 45,49 45,49
M1 M1 Antimony Antimony <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
M1 M1 Arsenic Arsenic <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
M1 M1 Barium Barium 0,00455 0,00455 0,005308 0,005308 0,00493 0,00493 0,005361 0,005361 0,0055 0,0055 14,29 14,29 7,16 7,16 0,98 0,98 4,28 4,28
M1 M1 Boron Boron 0,002366 0,002735 0,002366 0,002735 0,00244 0,00244 0,002466 0,002466 0,0028 0,0028 13,49 13,49 10,73 10,73 9,84 9,84 3,87 3,87
M3 M3 Bromide Bromide 0,00019 0,00019 0,00027 0,00027 0,00018 0,00018 0,016442 0,016442 // 29,63 29,63 33,33 33,33 5989,72 5989,72 //
M1 M1 Cadmium Cadmium <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
M1 M1 Gadolinium Gadolinium <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
75 10 May 2024
Method COMPOUNDS Method COMPOUNDS 21E1640 21E1640 20B1955 20B1955 20I1279 2011279 20J1770 20J1770 20B0596 20B0596 21E1640 21E1640 20I1279 2011279 20J1770 20J1770 20B0596 20B0596 (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (d%) (d%) (d%) (d%) (d%) (d%) (d%) (d%)
M1 M1 Gallium Gallium <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
M1 M1 Gold Gold <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // / /
M1 M1 Iridium Iridium <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
M1 M1 Lead Lead <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ nd nd // // // //
M1 M1 Lithium Lithium 2,79E-05 2,79E-05 4,26E-05 4,26E-05 3,6E-05 3,6E-05 3,27E-05 3,27E-05 0,0000 0,0000 34,59 34,59 16,02 16,02 23,32 23,32 12,05 12,05
M1 Lutetium Lutetium <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // // 2024203121
M1 M1 M1 Mercury Mercury <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
M1 M1 Rubidium Rubidium 0,016844 0,016844 0,022168 0,022168 0,01703 0,01703 0,019779 0,019779 0,0230 0,0230 24,02 24,02 23,20 23,20 10,78 10,78 3,70 3,70
M1 M1 Silver Silver <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
M1 M1 Strontium Strontium 0,008375 0,008375 0,013771 0,013771 0,00873 0,00873 0,01 0,01 0,0125 0,0125 39,19 39,19 36,62 36,62 27,38 27,38 9,23 9,23
M1 M1 Tellurium Tellurium <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
M1 M1 Thallium Thallium <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
M1 M1 Thorium Thorium <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
M1 M1 Titanium Titanium 0,002622 0,002622 0,003414 0,003414 0,00248 0,00248 0,002523 0,002523 0,0047 0,0047 23,20 23,20 27,35 27,35 26,09 26,09 39,02 39,02
M1 M1 Tungsten Tungsten <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
M1 M1 Uranium Uranium <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
M1 M1 Ytterbium Ytterbium <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ <LdQ // // // //
Note1. Note 1. Gray boxesindicate Gray boxes indicatechemical chemicalmacro macro classes. classes.
Note 2. Note 2. %= compound %= compound concentration concentration expressed expressed as percentage as percentage of theofcomposition. the composition. Note3. Note 3. (d%)= percentage (d%)= percentage deviation: deviation: (|Reference (|Reference standard standard (%)-Test(%)|/(Reference (%)-Test(%)I/(Reference standard standard (%) ) X (%) )x 100). 100). 5 5 Note4. Note 4. The term"Total" The term “Total”refers refersto to the the sum sumofofthe thevalues valuesofof the the various variouscompounds compounds which which forms forms the corresponding the corresponding
group. group. Note5. Note 5. <LdQ= below <LdQ= below thelimit the limitofofquantification. quantification. Note6. Note 6. nd = compound nd = compound notnot detected detected
Note7. Note 7. nq nq or or NQ= compound NQ= compound not not quantifiable. quantifiable.
10 10 Note 8. Note 8. /= /= compound compound notnot reported. reported.
The results The results show show that, that, between between the thefive five batches batches ofofco-extract, co-extract, there there are are quantitative fluctuations quantitative fluctuations(up (upor ordown) down) in in the theindividual individualchemical chemical classes classesof ofcomponents. components.
Thesefluctuations, These fluctuations, ifif used usedtotopredict predictthe theperformance performance
[or [or effect] effect] of the of the individual individual
15 15 batches, would batches, wouldlead leadtotoanana priori a priori view view thatthat these these batches batches havehave different different biological biological
function and function andtoto the the rejection rejection of of the the batches batchesthat thatare arenot notcomparable comparableto to thethe reference reference
standard, if standard, if the criteria criteria applied applied were those valid were those validfor forananAPI API acting acting viavia the the keylock keylock
paradigmthanks paradigm thankstotothe thepresence presenceofofananevident evidentSAR. SAR. TheThe analysis analysis herehere reported reported argues argues
the fact the fact that, that, despite despite the the biological biological function function being being maintained acrossall maintained across all the the different different 20 20 batches assessed, batches assessed, none none of of the the single single molecular components molecular components identifiedwould identified would respect respect thethe
criteria set for a single API, thus demonstrating that the matrix should not be considered criteria set for a single API, thus demonstrating that the matrix should not be considered
as aa compilation as compilation of of APIs. APIs.
Asseen As seenabove, above,thethe biological biological function function is preserved is preserved in batch. in every every batch. Thus, figure Thus, figure 55 shows showsthat thatititisisnot notrepresentative representativeand andtherefore thereforenotnotcorrect correcttoto
76 10 May 2024
entrust the estimation of the reproducibility of the activity profile of a complex matrix entrust the estimation of the reproducibility of the activity profile of a complex matrix
purelyononthethequantitative purely quantitative analysis analysis of theof individual the individual constituents. constituents. In fact,Ingiven fact,the given very the very
natureofofa acomplex nature complex matrix, matrix, quali-quantitatively quali-quantitatively different different chemicalchemical profiles, profiles, which, on which, a on a chemicalstandpoint chemical standpointshouldshould be considered be considered different, different, evoke instead, evoke instead, in biological in biological
5 5 systems,thethesame systems, same reaction reaction relevant relevant for theforintended the intended use. Thisuse. Thisnot should should not be a surprising be a surprising
observation but observation butanother anotherdemonstration demonstration thatthat biological biological function function of aofcomplex a complex matrixmatrix
cannot be cannot betraced tracedback backtotothethesumsum of the of the activity activity of of each each single single molecule molecule within within the the
matrixitself itself(i.e., (i.e., the chemical standpoint). Therefore, the activity of the of the cannot matrix cannot 2024203121
matrix the chemical standpoint). Therefore, the activity matrix
be predicted be predicted solely solely based based onon the the identity identity and and quantity quantity ofof the the molecules molecules which constitute which constitute
10 10 it. it.
This also This alsohighlights highlightsthethefact factthat that there there are are bothboth structural structural and functional and functional
redundancymechanisms redundancy mechanisms thatthat give give the the matrix matrix particular particular resilience,i.e., resilience, i.e., as as shown above, shown above,
the ability the ability to to mediate mediatethethesame same activity activity despite despite different different quali-quantitative quali-quantitative compositions. compositions.
In other In other words, words, thethe study study ofof aa matrix matrix from from anan exclusively exclusively molecular molecularpoint point of of view viewisis not not 15 15 correct because correct becausethe theidentity identity ofof itsits individual individual components components cannot cannot predict predict its its features. features.
This confirms the need for methods capable of describing the intrinsic characteristics of This confirms the need for methods capable of describing the intrinsic characteristics of
the matrix the rather than matrix rather than looking looking exclusively exclusively at at its itsmolecular molecular components. components. ThoseThosemethods, methods, as the as the one providedininthe one provided the present present invention, invention, should shouldmonitor monitorthethepreservation preservationof ofthose those parameterson parameters onwhich whichthe themaintenance maintenance of of biologicalfunction biological functionreally reallydepends. depends. 20 20 3.2. Near 3.2. Near Infrared Infrared Spectroscopy Spectroscopy(NIR) (NIR) 3.2.1. Introduction 3.2.1. Introduction To create To createthe thecontrol controlchart chartandand provide provide the the NIR NIR acceptability acceptability cut-offs, cut-offs, four four
batches (comprising batches (comprising the the reference referencestandard) standard)ofof ArtéArtéGXGX made made on on aa prototype prototype and and industrial scale industrial scale selected selected as as positive positive batches batches according according to to the the method methodofofthe theinvention, invention, 25 25 steps i) steps i) to toiv), iv),were wereanalysed analysed andand formed formed the thelibrary library ofof good goodquality qualitysamples samples(training (training set). The set). batchesused The batches used to build to build the training the training set selected set were were selected after passing after passing the the conformitytest conformity testperformed performed through through the biological the biological assayassay of theofinvention the invention (see also(see also examplesabove). examples above). Oncethe Once thelibrary library was wasdefined, defined,a abatch batchofofArté ArtéGXGX DEGRDEGR 21E1640 21E1640 selectedselected as as 30 30 negative batch negative batchaccording accordingto to thethe method method of theof invention, the invention,steps steps i) to i) iv),to was iv), also was also analysed. analysed.
Thefive The five batches batches werewereused usedtoto set set the the NIR acceptability parameters. NIR acceptability parameters.
Oncethe Once theacceptability acceptability parameters parameterswere were defined,twotwo defined, poor poor formulative formulative qualities qualities
as defined as defined above above (R19L4299 (R19L4299 and and R19L4298) R19L4298)asaswell wellasas anan unknown unknownArté ArtéGXGX batch batch 35 35 (21E1640) (21E1640) asastest test samples sampleswerewereanalysed analysed using using thethe samesame operating operating methods. methods. Following Following
the result the result obtained obtained after after analysis analysis with with conformity tests, the conformity tests, the test testsamples samples areare confirmed confirmed
as being as of good being of goodquality quality or or not not depending dependingononwhetherwhether they they havehave passed passed the the conformity conformity
test performed test performed viavia aa biological biological assay assay to to confirm confirmor, or,possibly, possibly, modify modifythe theacceptability acceptability
77 10 May 2024
criteria identified by the control chart” as above. criteria identified by the control chart" as above.
Theaim The aimofofthis this study study was wastoto build build aa control control map map of of NIR NIRspectra, spectra,defined definedaapriori priori throughevaluation through evaluation of conformity of conformity in biological in biological functionfunction of thethat of the batches batches thatofare are part it,part of it,
as aa fingerprint as fingerprint to to be be used used to to discriminate discriminate valid valid conformity conformitybatches batchesofofArtéArté GX GX fromfrom
5 5 samplesof samples of poor poorbiological biological and/or and/or formulation formulationquality. quality. Below Below is is thethe listofofthethe list four four batches batches used used forcreation for the the creation of the of NIR the NIRand library library and the list the list of of samples analysed samples analysed as tests: as tests:
44 batches batchesArtéArtéGX GX for construction the construction of the of the library (Tab. 1)dried freeze dried 2024203121
for the library (Tab. 1) freeze
11 poor qualitybatch poor quality batch of of Arté Arté GX (degraded, GX (degraded, poor biological poor biological activity quality) activity quality)
10 10 11 batch batch of ofArté ArtéGXGX as library as library testtest (Tab. (Tab. 2) freeze 2) freeze dried dried
22 poor poorformulative formulative qualities qualities (Tab. (Tab. 2) 2)
Table 1. (Training set). Table 1. (Training set).
Product Product Batch Batch
Arté GX Arté GXReference Reference standard standard 20B1955 20B1955
Arté GX Arté GX 20I1279 2011279
Arté GX Arté GX 20J1770 20J1770
Arté GX Arté GX 20B0596 20B0596
Arté GX Arté GX DEGR21E1640 DEGR 21E1640
Table 2. (Test set). Table 2. (Test set).
Product Product Batch Batch Arté GX Arté GX 21E1640 21E1640 Waterextract Water extract Centella Centella Asiatica Asiatica leaves leaves R19L4299 R19L4299
Waterextract Water extract Echinacea EchinaceaPurpurea Purpurealeaves leaves R19L4298 R19L4298
3.2.2. instruments 3.2.2. instruments 15 15 BrukerNIR Bruker NIRspectrometers, spectrometers,model model MPAMPA (Multi-Purpose (Multi-Purpose Analyzer): Analyzer):
• Resolution: Resolution: 16 16 cm-1 cm-1 • Wavenumber Wavenumber Reproducibility: Reproducibility: Better Better than than 0.04 0.04 cm-1 cm-1
• Wavenumber Wavenumber Accuracy: Accuracy: Better Better thanthan 0.1 0.1 cm-1cm-1
• Photometric PhotometricAccuracy: Accuracy:0.1% 0.1% T T
20 20 • Wavenumber Wavenumberrange: range:from from4000 4000toto 12500 cm-1 12500 cm-1 • Background Backgroundscans: scans:6464
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• Scans Scansforfor sample sampleacquisition: acquisition: 64 64 3.3. Statistics 3.3. Statistics tests tests Compliancetest Compliance test Compliancetesting Compliance testingisisa asimple simplemethod method forfor testing testing deviations deviations of of measured measured NIR NIR
5 5 spectrawithin spectra within certain certain limits. limits. To these To set set these limitslimits (acceptability (acceptability values ofvalues of NIR spectra), NIR spectra),
samplesofofgood samples goodandandbadbad qualitybelong quality belong to to atatleast leastone onebatch batchororproduction productioncyclecycleofofthe the final product final product to to be be identified identified as as reference referencespectra spectraare areneeded needed that.According that. According to the to the
present invention, invention, the thereference referencesamples samplesare are identified through a biological assay assay 2024203121
present identified through a biological
deemedappropriate deemed appropriate(cell-based (cell-basedassay)assay)and andarearestudied studiedviaviaNIR, NIR,to to assess assess thetheminimum minimum 10 10 range ofof specifications range specifications such suchasastotoinclude includethethe batches batches themselves. themselves. According According to theto the
invention, this invention, this range range isis imposed imposed as as acceptable acceptable in in order order to to recognize recognize whether whethera abatch batchofof unknownconformity unknown conformity(not (nottested tested in in the the cell-based cell-based assay assay hence hence notnot known whether known whether acceptableorornotnot acceptable acceptable acceptable on theonbasis the ofbasis its of its biological biological activity)activity) is compliant is compliant or not. or not. TheNIR The NIRspectra spectraofofthese thesesamples samples reflectthe reflect thedifferent differentvariations variations ofof the the sample samplecapable capable 15 15 of achieving of achieving compliant compliantand andnon-compliant non-compliant performance performance in terms in terms of modulation of modulation of one of one
or more or moreselected selectedbiological biologicalactivity activity(therapeutic (therapeuticor or beneficial beneficial activity) activity) andand formform a a confidenceband confidence bandininthe thespectral spectral range. range. To To pass pass the the NIR NIRconformity conformity test,the test, thespectrum spectrumofof a new a newsample samplemustmustfall fall within within this this confidence confidence band. band. First,First, the meanthe and mean and standard standard
deviation of deviation of the the absorbance absorbancevalues valuesforfor each each wavelength wavelength (i) must(i) must be calculated. be calculated. The The 20 20 meanvalue mean valueplus/minus plus/minus thethe standard standard deviation deviation determines determines the confidence the confidence band within band within
the spectral the spectral range range and defines what and defines whatamount amount of of variationover variation over each each spectral spectral wavelength wavelength
is acceptable is acceptable forforthe theproduct product analysed. analysed.
Secondly,it Secondly, it is is necessary necessary toto check check whether whether thethe spectrum spectrumofofaasample sampletotobebetested tested falls within falls within the the confidence confidence band banddefined definedininthe thespectral spectral range. range. TheThedifference differencebetween between 25 25 this sample this andthe sample and theaverage averageofofthethereference referencesamples samples is is calculatedatateach calculated each wavelength wavelength
(i). This (i). This absolute deviation absolute deviation is is then then weighted weighted by thebycorresponding the corresponding standard standard deviation “s” deviation "s"
onthe on therespective respective wavelength, wavelength, whichwhich resultsresults in a relative in a relative deviation deviation called thecalled the Conformity Conformity
Index(CI) Index (CI)equation equation (1).(1).
(1) CI = (A reference, i - A sample, i) / s reference, i (1) CI = (A reference, i - A sample, i) / S reference, i
30 30 A A reference, = average absorbance at a given wavelength(i) of the reference reference, i i = average absorbance at a given wavelength(i) of the reference
(average spectrum) (average spectrum) Asample, A sample, i i==absorbance absorbance atataagiven givenwavelength(i) wavelength(i) oftest of the the test sample sample
Ss reference, reference, i= standard iF standarddeviation deviation at at aa given wavelength(i) given wavelength(i) of of thethe reference reference (average (average
spectrum) spectrum)
35 35 In conformity In conformity teststests another another parameter parameter cancan be be used usedtoto evaluate evaluate the the batches batches against against the reference the referencelibrary libraryapplying applying the the limit limit of CI. of the the CI. This parameter This parametercalled called Sum2 Sum2isisrepresented representedbybyequation equation(2): (2):
79 10 May 2024
(2) Sum2= Sum2= (Sum (Sum of All of All CIsCIs > CI > CI limit limit - CIlimit)/(Sum - CI limit)/(Sumof of thenumber the numberof of points points in in the the
spectrumwith spectrum withCICI>>CICIlimit) limit)
5 5 Selecting the Selecting the appropriate appropriate parameter parameterinina aconformity conformity test test depends depends on user- on the the user- specific control specific control problem that can problem that can be be easily easily addressed addressedbybythe theskilled skilled person, person, in in the the case case of products of products comprising comprisingororconsisting consistingof ofoneone or or more more natural natural matrices, matrices, i.e., i.e., extremely extremely
heterogeneoussamples, samples,Sum Sum 2 is a a suitableparameter. parameter. 2024203121
heterogeneous 2 is suitable
Hence, in Hence, in the the present present case, case, Sum2 Sum2 parameter parameter was was selected selected for for determining determining the the 10 10 acceptability cut-off of the conformity test. acceptability cut-off of the conformity test.
3.3.1 Preparation 3.3.1 Preparationofofthe thesample sample Eachsample Each samplewaswas transferred transferred to to thesample the sample holder holder suitable suitable forfor thethe NIR NIR analysis analysis
of inhomogeneous of inhomogeneous solids. solids. Before Before the the analysis, analysis, it was it was checked checked that that the bottom the bottom of theof the
sampleholder sample holderwas wascompletely completely covered. covered.
15 15 3.3.2 Sample 3.3.2 Sample acquisition acquisition
TheNIR The NIRspectra spectrawere wereacquired acquired inin reflectionmode reflection mode using using a rotatingsample a rotating sample holder holder
suitable for suitable for the the analysis analysisof ofsamples samples such as inhomogeneous such as solids,asaswell inhomogeneous solids, wellasaspowders, powders, to ensure high reproducibility of the data. to ensure high reproducibility of the data.
Quality Control Quality Control and and background backgroundsubtraction subtractionwaswas performed performed before before each each 20 20 acquisition. acquisition.
Thesamples, The samples,alsoalsoreported reportedininthetheprevious previoustables tables (Tab. (Tab.11and andTabTab2),2),were were prepared prepared as as
described above described aboveand andanalysed analysedatatNIR. NIR. 3.3.3. Data 3.3.3. Datapre-processing pre-processing Pre-processingisis aa mathematical Pre-processing mathematicalmanipulation manipulation to extrapolate to extrapolate spectral spectral features features
25 25 andreduce and reduce sources sources of variability. of variability.
For the For the development development of of thethe preprocessing preprocessing method, method, the pre-processing the pre-processing methodmethod
involving the involving the use useofofSNV SNV normalization normalization was was selected selected andregion and the the region of theofspectrum the spectrum 1 from4200 from 4200toto9000 9000cm-cm-waswas selected selected as the as the region region of of thethe spectrum spectrum of of greatest greatest relevance relevance
for the for the model. model. 30 30 The OPUS The OPUS Software Software (Opus(Opus 8.5, 8.5, Bruker) Bruker) was for was used usedcarrying for carrying out theout the conformity conformity test. test.
Byapplying By applyingthe thepre-processing pre-processingmethod methodthe the following following parameters parameters in theinOPUS the OPUS ConformityIndex Conformity Indexmethod method were were set:set:
a) Pre-processing: a) Pre-processing: SNV; SNV;
35 35 b) Regions: b) Regions: FromFrom42004200toto 9000 9000 cm-1; cm-1;
c) Conformity c) Conformity Test Testparameters: parameters:Max Max Conformity Conformity Index Index Value; Value; Sum2 Sum2
3.3.4. Data 3.3.4. Dataacquisition acquisition Fourbatches Four batches indicated indicated in Tab in Tab 1 as 1 as as well well theas the batches batches in tab 2inwere tab analysed. 2 were analysed.
80 10 May 2024
Thespectra The spectra of of the the first first four fourbatches batches above above were usedasas reference were used referencespectra spectra in in the the creation of creation of the the Conformity Conformity Index Index (CI) (CI) as had as they theythehad the desired desired biological biological activity activity
(acceptable according (acceptable according to the to the cell-based cell-based assay). assay).
SNVnormalization SNV normalization pre-processing pre-processing waswas carried carried out out and and the the region region fromfrom 4200 4200 to to -1 selected as the region for data reprocessing. The CI MAX threshold and 5 5 9000cmcmwas 9000 was selected as the region for data reprocessing. The CI MAX threshold and Sum2 2values Sum valuesofofthe the batches batchesof of the the card card are are shown below: shown below: CIMAX experimental CIMAX experimental assay (interval assay (interval based onbased on the biological the biological data (steps data i) to(steps iv) of i) to iv) of the the
methodofofthe method the invention invention 2024203121
Cell-based Cell-based CI CI Max Max Sum22 Sum Batch type Batch type Batch ID- Batch ID- assay assay REFERENCE REFERENCE Reference Reference 20B1955 20B1955 STANDARD, STANDARD, 1.5 1.5 0.0 0.0 COMPLIANT COMPLIANT Reference Reference 20I1279 2011279 COMPLIANT COMPLIANT 1.5 1.5 0.0 0.0
Reference Reference 20J1770 20J1770 COMPLIANT COMPLIANT 1.5 1.5 0.0 0.0
Reference Reference 20B0596 20B0596 COMPLIANT COMPLIANT 1.3 1.3 0.0 0.0
(NIRspectra (NIR spectra in in Figure Figure 6a) 6a) 10 10 Whenreferred When referredtotothe thecell-based cell-basedassay, assay,the the tables tables indicate indicate "compliant" “compliant”when whenthethe
batch resulted acceptable according to step c) of the method of the invention. batch resulted acceptable according to step c) of the method of the invention.
As reported As reported above, above,the the Max MaxConformity Conformity Index Index Value Value is assigned is assigned based based on the on the maximum maximum
CI MAX CI MAX value value defined defined by theby the training training consisting consisting of compliant of compliant (positive) (positive) samples samples accordingto tothethe according cell-based cell-based assay. assay. From From thedata the first first(reported data (reported above), above), the thewas CI limit CI limit was 15 15 set to set to 1.5, this value 1.5, this washence value was hence usedused to calculate to calculate the2 Sum the Sum of the2training of the training set and ofsettheand of the test set. test set. To define To define the the conformity conformitylimitlimitofof Sum Sum2,2,the thenegative negativebatch batch(i.e., (i.e., non-compliant non-compliant
with the with the biological biological data data (cell-based (cell-based assay) assay) according accordingtotothe themethod method of the of the invention) invention)
DEST DEST 21E160 21E160 was was tested tested against against the the control control chart. chart. TheThe table table below below shows shows the the results results in in
20 20 terms of terms of CI and Sum CI and Sum2 2for foreach eachbatch. batch. Batch Batch Formulative Formulative Cell-based Cell-based CI CI Max Max Sum22 Sum Batch type Batch type conformity ID- ID- conformity assay assay
Reference Reference 20B1955 20B1955 COMPLIANT COMPLIANT COMPLIANT COMPLIANT 1.5 1.5 0.0 0.0
Reference Reference 20I1279 2011279 COMPLIANT COMPLIANT COMPLIANT COMPLIANT 1.5 1.5 0.0 0.0
Reference Reference 20J1770 20J1770 COMPLIANT COMPLIANT COMPLIANT COMPLIANT 1.5 1.5 0.0 0.0
81 10 May 2024
Reference Reference 20B0596 20B0596 COMPLIANT COMPLIANT COMPLIANT COMPLIANT 1.3 1.3 0.0 0.0
DEG DEG NOT NOT Reference Reference COMPLIANT COMPLIANT 5.8 5.8 1.3 1.3 21E1640 21E1640 COMPLIANT COMPLIANT (NIR averagespectrum (NIR average spectrum ofof compliant compliant batches batches plus plus NIRNIR spectrum spectrum of non-compliant of non-compliant batch batch
figure 6b) figure 6b) Basedononthe Based theSum Sum 2 values 2 values of of batch batch DEGDEG 21E1640 21E1640 and itsandnon-compliance its non-compliance with with the cell-based the cell-based assay, assay, the the threshold threshold SumSum2 2value valueto to define define a batch a batch as as notnot compliant compliant is is 2024203121
5 5 hence set hence set as as <1.2. ≤1.2. Once Oncethisthisacceptability acceptability cut-off cut-off was wassetset following followingthethemethod methodof of thethe
invention, the invention, the spectra spectra ofofaanew new unknown (notverified unknown (not verified through throughcell-based cell-basedassay) assay) batch batchof of Arté GX Arté GXandandtwotwo known known formulative formulative non-compliant non-compliant batchesbatches weretotested were tested to the verify verify the reliability ofofthe reliability the method method of of the theinvention inventionand anditsitssuitability suitability for for aaprocess processofofquality quality controlaccording control according to to thethe description description and and to thetoclaims. the claims. 10 10
Results: Results:
Formulative Formulative Sample Sample compliance Cell-based Cell-based CI Max CI Max Sum22 Sum Batch compliance Expected NIRResult Result (≤ 1.2) Batch Expected NIR type type assay assay (< 1.2)
Reference Reference 20B1955 20B1955 COMPLIANT COMPLIANT COMPLIANT COMPLIANT 1.5 1.5 0.0 0.0 COMPLIANT COMPLIANT COMPLIANT COMPLIANT
Reference Reference 20I1279 2011279 COMPLIANT COMPLIANT COMPLIANT COMPLIANT 1.5 1.5 0.0 0.0 COMPLIANT COMPLIANT COMPLIANT COMPLIANT
Reference Reference 20J1770 20J1770 COMPLIANT COMPLIANT COMPLIANT COMPLIANT 1.5 1.5 0.0 0.0 COMPLIANT COMPLIANT COMPLIANT COMPLIANT
Reference Reference 20B0596 20B0596 COMPLIANT COMPLIANT COMPLIANT COMPLIANT 1.3 1.3 0.0 0.0 COMPLIANT COMPLIANT COMPLIANT COMPLIANT
DEST DEST NOT NOT NOT NOT Reference Reference COMPLIANT NOT COMPLIANT 5.8 5.8 1.3 1.3 NOT 21E1640 21E1640 COMPLIANT COMPLIANT COMPLIANT COMPLIANT COMPLIANT COMPLIANT
Test Test 21E1640 21E1640 COMPLIANT Not COMPLIANT Notassessed assessed 3.6 3.6 0.6 0.6 UNKNOWN COMPLIANT COMPLIANT UNKNOWN
NOT NOT NOT NOT NOT NOT Test Test R19L4299 R19L4299 Notassessed Not assessed 5.9 5.9 1.6 1.6 COMPLIANT COMPLIANT COMPLIANT COMPLIANT COMPLIANT COMPLIANT
NOT NOT NOT NOT NOT NOT Test Test R19L4298 R19L4298 Notassessed Not assessed 49.4 49.4 8.1 8.1 COMPLIANT COMPLIANT COMPLIANT COMPLIANT COMPLIANT COMPLIANT
82 10 May 2024
Therefore, according Therefore, accordingtotothe theNIR NIR assay assay withwith the the acceptability acceptability cut-off cut-off assessed assessed
with the with the method methodofofthetheinvention, invention,batch batch 21E1640 21E1640 resulted resulted compliant compliant according according to theto the validation process of the invention. validation process of the invention.
In order In order to to confirm confirmthe thevalidity validity ofof the the validation validation process processofofthe theinvention, invention,thethe 5 5 modulationofofthethebiological modulation biologicalactivities activitiesidentified identified for for the the product productArtéArtéGX GX described described
abovewas above wascarried carriedout outalso alsofor forbatch batch21E1640 21E1640 andand the the product product resulted resulted acceptable, acceptable, i.e.i.e.
compliant with compliant with the the reference reference modulation modulation values values according according to to the the method methodofofthethe invention. 2024203121
invention.
Hence,processes Hence, processesandand methods methods have have been identified been identified that defining that allow allow defining the the 10 10 acceptability criteria acceptability criteria ofof aa product comprisingor or product comprising consisting consisting of of one one or more or more natural natural
matrices based matrices basedononthetheconservation conservation of of a number a number of selected of selected biological biological activities activities and and
regardlessofofthe regardless thenotion notionof of itsits composition composition atmolecular at the the molecular level. level.
3.4 3.4 RAMAN RAMAN spectroscopy spectroscopy Samplesofofthe Samples the same samebatches batchesofof3.2 3.2were wereused. used. 15 15 Eachsample Each samplewaswas deposited deposited between between two microscope two microscope slides slides SO thatsothethatpowder the powder created aa thickness created thickness of of approximately approximately1 mm. 1 mm. The The measurements measurements were conducted were conducted with with -1 Rigaku's Xantus-2 Rigaku's Xantus-2spectrometer spectrometerininthethe2000-200 2000-200 cminterval, cm-1 interval,using using theusing the usingthethe1064 1064 nm laser nm laser line line as as an an exciter, exciter, anan excitation excitationpowerpower of of approximately approximately 100 100 mW, mW, and and collectingthe collecting theradiation radiationscattered scattered at at 180° 180° withwith respect respect to thetoexcitation the excitation (back-scattering). (back-scattering).
20 20 3.4.1 Acquisition 3.4.1 Acquisition conditions conditions
Thefollowing The followingmeasurement measurement conditions conditions werewere selected selected for for the the acquisition acquisition of of each each
spectrum: spectrum:
Methodofofacquisition Method acquisitionofof the the sample samplebeing beinganalyzed. analyzed. Sample Sample Sample treatment Sample treatment Spectrum range Spectrum range N° scans N° scans Spectrum Spectrum
resolution resolution
Product AA Product Approximately 100mg Approximately 100mgwere were 2000-200 cm-¹ 2000-200 cm-1 30 x1s 30x1s 7 cm 7 -1 cm-¹
depositedand deposited and pressed pressed integration integration
between twomicroscope between two microscope time time
slides slides
For each For each sample, sample,three threesamplings samplings were were repeated repeated to check to check the the reproducibility reproducibility of of 25 25 the data: the data: the the three three replicates replicateswere were then then averaged averaged toto obtain obtain aa representative representative spectrum spectrum ofof the sample the sample toto be be characterized. characterized. Average spectrumandand Average spectrum standard standard deviation deviation were were obtained obtained
with Bruker with BrukerOptics OpticsOpus Opus8.1 8.1software. software. 3.4.2 Pre-processing 3.4.2 Pre-processing
Theaverage The averagespectra spectrathus thusobtained obtainedfor forall all samples wereprocessed samples were processedtotosubtract subtractthe the -1 30 30 baseline;for baseline; forthis thispurpouse, purpouse,thethe intensity intensity at 200, at 200, 790,910, 790,910, 1520 1520 and 1800and cm 11800 cm was set to was set to zero. Subsequently zero. Subsequently the thespectral spectral profiles profiles were werenormalized normalized to to theintensity the intensityofofthe thesignal signal -1 centered at centered at approximately 1610cm-1 approximately 1610 cm (Figure (Figure14). 14).These These manipulations manipulations were were carried carried out out
83 10 May 2024
using Opus using Opus8.1 8.1software softwarefrom fromBruker Bruker Optics. Optics.
As shown As shownininFigure Figure14,14,thethespectra spectrashow showan an enlarged enlarged signal signal of of notable notable intensity intensity
in the low frequency region: this signal could derive from a residual luminescence of the in the low frequency region: this signal could derive from a residual luminescence of the
samplecover sample coverglass glassforforwhich which in subsequent in subsequent processing processing it wasitdecided was decided to limittothe limit the 5 5 analysis range analysis range toto region region between 780and between 780 and2000 cm-1. 2000cm-1. 3.4.3 Statistical 3.4.3 Statistical analysis analysis Thestatistical The statistical analysis analysis ofof the thespectroscopic spectroscopic datadata was was conducted conducted using the using the
Origin2023Software Software from OriginLaband and following all the steps necessary to obtain thethe 2024203121
Origin2023 from OriginLab following all the steps necessary to obtain
parameterstoto be parameters be used usedinin the the conformity conformityteststests such suchas: as: the the CI CI Conformity ConformityIndex Index andand thethe
10 10 Sum2parameter Sum2 parameter using using equations equations (1)(1) andand (2)(2)inin3.3 3.3above. above. 3.4.4 Definition 3.4.4 Definitionofofthethe Control Control Chart Chart
Theconstruction The constructionofofthe the control control chart chart was wasdone doneusing usingthethesame same criterionadopted criterion adopted for the for the NIR and ATR-FTIR NIR and ATR-FTIR spectra. spectra.
Usingthe Using the Origin Origin20232023software, software,the thespectra spectraofof20B1955, 20B1955, 20I1279, 2011279, 20J1770 20J1770 and and
15 15 20B0596 20B0596 were were usedused as as reference reference spectra: spectra: from from these these thethe average average spectrum spectrum withwith relative relative
standard deviation standard deviation was wasobtained. obtained.The Theintensity intensity values values (normalized (normalizedRaman) Raman)andand standard standard
deviation as a function of frequency constituted the data indicated with I deviation as a function of frequency constituted the data indicated with I reference, reference, i and i and Ss reference, i ininequation reference, i (1) equation (1) of of 3.3.3.3. 3.4.5 Execution 3.4.5 Execution of of the the conformity test conformity test
20 20 Themaximum The maximum value value of CIof as CI the as the frequency frequency varied varied was was calculated calculated usingusing equation equation
(1) for (1) for each eachofofthe thereference reference spectra: spectra: in this in this way way it wasitpossible was possible to determine to determine the valuesthe values
of (CI limit) of limit) to tobe be used used for forthe thecalculation calculationofofSumSum 2 2 using using equation (2). This equation (2). This value value was was
set equal set equal to tothethemaximum maximum CICI valueobtained value obtainedforforthe thereference referencespectra: spectra: CI limit CI limit == CI CI Max Max ==1.50 1.50 25 25 Usingthis Using this value, value, viavia equation equation (2) (2) the the two twoconformity conformitytests testswere wereapplied appliedto to thethe
spectraofofall spectra all the theanalyzed analyzed samples. samples.
Thetable The table below belowsummarizes summarizes thethe values values found found forfor thethe Co-extract Co-extract samples samples used used as as
ReferenceSpectra Reference Spectra(R) (R)ininthe thecreation creationofofthe the Conformity Conformity Index Index method method since since theythey havehave
the desired the desired biological biological activity, activity, thethe degraded 21E1640 degraded 21E1640 sample sample necessary necessary to define to define the the
30 30 compliancelimit compliance limitof of Sum Sum2,2,and andthose thoseofofthe the samples samplesusedusedasasmethod method tests(T). tests (T). Type Type Batch Batch Formulative Formulative Cell-based Cell-based CI Max CI Max Sum22 Sum Expected Expected RAMAN RAMAN result result
complancei complancei assay assay (≤1.5) (<1.5)
R R _20B1955 20B1955 COMPLIANT COMPLIANT COMPLIANT COMPLIANT 1.47 1.47 0.0 0.0 COMPLIANT COMPLIANT COMPLIANT COMPLIANT
R R _20I1279 _2011279 COMPLIANT COMPLIANT COMPLIANT COMPLIANT 1.50 1.50 0.0 0.0 COMPLIANT COMPLIANT COMPLIANT COMPLIANT
R R _20J1770 _20J1770 COMPLIANT COMPLIANT COMPLIANT COMPLIANT 1.50 1.50 0.0 0.0 COMPLIANT COMPLIANT COMPLIANT COMPLIANT
R R _20B0596 _20B0596 COMPLIANT COMPLIANT COMPLIANT COMPLIANT 1.49 1.49 0.0 0.0 COMPLIANT COMPLIANT COMPLIANT COMPLIANT
Type Type ID ID Conformità Conformità Cell-based Cell-based CI Max CI Max Sum22 Sum Expected Expected RAMAN RAMAN result result
formulativa formulativa assay assay (≤2.09) (<2.09)
R R DEST_21E1640 DEST_21E1640 COMPLIANT COMPLIANT NON COMPLIANT NON COMPLIANT 4.95 4.95 2.10 2.10 NOT COMPLIANT NOT COMPLIANT NOT COMPLIANT NOT COMPLIANT
84 10 May 2024
T T _21E1640 21E1640 COMPLIANT COMPLIANT UNKNOWN UNKNOWN 5.34 5.34 2.06 2.06 UNKNOWN UNKNOWN COMPLIANT COMPLIANT
T T _R19L4299 R19L4299 NOT NOT NOTASSESSED NOT ASSESSED 6.98 6.98 2.43 2.43 NOT COMPLIANT NOT COMPLIANT NOT COMPLIANT NOT COMPLIANT COMPLIANT COMPLIANT
T T R19L4298 R19L4298 NOT NOT NOTASSESSED NOT ASSESSED 29.46 29.46 5.84 5.84 NOT COMPLIANT NOT COMPLIANT NOT COMPLIANT NOT COMPLIANT COMPLIANT COMPLIANT
In this In this case case the the value value of of Sum Sum 2 2for forthe thedegraded degradedsample sample is is slightlyhigher slightly higherthan than those obtained those obtainedfor forthe thenon-degraded non-degraded sample sample of same of the the same batch.batch. On theOn the ofbasis basis thisof this conformitycriterion, criterion, thethe unknown sample is compliant as required by the formulation 2024203121
conformity unknown sample is compliant as required by the formulation
of the of the co-extract.4. co-extract.4. 5 5 Radical scavenger Radical scavengeractivity activity As additional As additional control control of of the the validity validity of ofthe themethod method and and process process of of the the invention, invention, all the all the Arté Arté GX batchesabove GX batches above were were alsoalso tested tested for for their their radical radical scavenger scavenger activity activity as as
this activity this activity is isknown known to to have havea anotable notablepromoting promoting effect effect in osteogenesis. in osteogenesis. Figure Figure 12 12
summarises summarises the the hallmark hallmark and biological and biological activityactivity related related to this parameter. to this parameter.
10 10 4.1. Assay 4.1. Assay method method Theassay The assayinvolves involvesthe theuse useofofHuman Human fibroblast fibroblast (HuDe) (HuDe) cell cell linelineas aasmodel a model to to
test the test the ability ability of productstotohave of products havean an antioxidant antioxidant activity activity basedbased on a scavenger on a scavenger activity.activity.
Five different Five different batches batchesofofArté ArtéGX GX were were testedtested at theatconcentration the concentrationof 1,4 of 1,4 mg/ml, mg/ml,
calculatedbybyapplying calculated applying the dilution the dilution factorfactor of 2,8 of 2,8 reflecting reflecting product product dilution indilution synovialin synovial
15 15 fluid in fluid in an an in in vivo vivoscenario. scenario. TheROS The ROS scavenger scavenger activity activity testtest is based is based on use on the the ofuse theofreactive the reactive oxygenoxygen
species species (ROS) (ROS) generator generator AAPH AAPH (2,2’-azobis-2-methyl-propanimidamide, (2,2'-azobis-2-methyl-propanimidamide,
dihydrochloride) which dihydrochloride) whichcan cansimulate simulate the theappearance appearance of exogenous of exogenous pro-oxidant pro-oxidant damagedamage and thereby and thereby induces inducesproduction productionofofendogenous endogenous ROS. ROS. Fluorescent Fluorescent probeprobe 6-carboxy-2',7'- 6-carboxy-2',7'-
20 20 dichlorodihydrofluorescein diacetate dichlorodihydrofluorescein diacetate (H2DCFDA, (H2DCFDA, Life Life Technologies) Technologies) was wasused usedasas indicator of indicator of the the presence presence of of ROS ROS inin cells. cells. The The fluorescence emissionofof H2DCFDA fluorescence emission H2DCFDA was was
measuredatatregular measured regulartimetimeintervals intervals(every (every1010minutes minutes forfor a totalofof9090minutes) a total minutes) with aa with
fluorometer (Varioskan fluorometer (VarioskanLux, Lux, Thermo-Scientific) Thermo-Scientific) and quantitatively and quantitatively correlated correlated to theto the
production production of of free free radicals radicals in cells. in cells. To account To account for cellfornumber cell number at the endatofthe theend of the assay, assay,
25 25 all of all of the the calculated calculated fluorescence fluorescence valuesvaluesare arenormalized normalized withwith respect respect to relevant to relevant cellcell viability measured viability measured by byMTTMTT (tetrazolium (tetrazolium salt,salt, [3-(4,5-dimethylthiazol-2yl)-2,5-
[3-(4,5-dimethylthiazol-2y1)-2,5- diphenyltetrazolium bromide, diphenyltetrazolium bromide, Sigma Aldrich) assay Sigma Aldrich) assayperformed performedaccording according to tothe the manufacturer’sinstructions. manufacturer's instructions. The Thedegree degree of protection of protection from from the generation the generation of ROS of ROS
providedbybythe provided thetested tested products productsisis compared compared to to those those obtained obtained by by protecting protecting cells cells with with
30 30 ascorbic acid ascorbic acid (considered (considered as as benchmark benchmark ofofantioxidant antioxidantmolecule). molecule).The The resultsare results areshown shown as comparison as comparison ofofintegrated integrated areaarea underunderfluorescence fluorescenceversus versustimetimecurvecurve(AUC) (AUC) calculated versus calculated versus AAPH (considered AAPH (considered as as 100%100% of ROSof ROS production). production).
4.2. Results 4.2. Results
85 10 May 2024
Figure 77 shows Figure showsequivalent equivalentradical radical scavenger scavengeractivity activity of of all all five fivebatches batches of ofArté ArtéGX. GX. All
batches show an equivalently highly significant protective effect on fibroblasts. batches show an equivalently highly significant protective effect on fibroblasts.
Theabove The abovediscussed discusseddata datafurther furtherqualify qualifythe thefive five batches batches asas equivalent equivalent inin terms terms of induction of induction ofof the same biological effect, same biological effect, due due to influence influence on on gene expressionasas well gene expression well 5 5 as to as to radical scavenging radical scavenging activity. activity.
Themodulation The modulationvalue value(percentage) (percentage)was was calculatedasasininFigure calculated Figure12.12. All the batches All batches were acceptable with were acceptable withrespect respect to to the the modulation valuesidentified modulation values identified for the the reference referencestandard standard andand therefore theacceptability NIR acceptability values calculated as above as above 2024203121
for therefore the NIR values calculated
confirmedthe confirmed thesuitability suitabilityofofthethemethods methodsand and process process of theofinvention the invention also using also using
10 10 different parameters different parameters for monitoring the for monitoring the modulation modulationofofthetheselected selectedbiological biological activity/ies. activity/ies.
4. 4. ISOTOPIC ABUNDANCE ISOTOPIC ABUNDANCE 4.1. Introduction 4.1. Introduction Theanalysis The analysis of of isotopic isotopic abundance abundanceisisa away waytotodescribe describematter matterfromfrom an an atomic atomic
15 15 point of point of view. view. The Theisotopic isotopicdistribution distributioncharacterizing characterizingthe thestarting startingmaterials materialsmaymaybe be
influenced by influenced byphenomena phenomena of different of different nature nature whichwhich in may in turn turnlead mayto lead to significant significant
variations in variations in the the final finalproducts products[ISPRA,
[ISPRA, Quaderni Quaderni -–Laboratorio Laboratorio2/2018. 2/2018.ISBN ISBN 978-88- 978-88-
448-0873-0].The 448-0873-0]. Theisotopic isotopiccomposition composition ofsample of a a sample is equal is equal to ratio to the the ratio between between the the abundanceofofthe abundance theheavy heavy andand light light isotopic isotopic forms forms (example (example the relationship the relationship 13C/12C) 13C/12C)
20 20 andisis expressed and expressed as as a deviation, a deviation, in parts in parts per thousand, per thousand, from anfrom an internationally internationally identifiedidentified
standard reference standard reference material. material. AApositive positivevaluevalueofof8 δindicates indicatesthat thatthetheheavy heavyisotope isotopeis is
enriched in enriched in the the sample compared sample compared to to thethe standard,while standard, while a negative a negative value value indicates indicates that that
the heavy the isotope in heavy isotope in the the sample sample is is impoverished. impoverished.
A marked A markeddifference difference in in isotopic isotopic abundance abundance ratios ratiosofofa asamplesample compared compared to to 25 25 samplesknown samples known to tobebeofofgood good qualitycancanaccount quality account forfor differentintra- different intra- and and intermolecular intermolecular interactions between interactions between the the phytochemical phytochemical classes classes thatmake that make up the up the matrix matrix regardless regardless of of
the quantitative the quantitative profile profile of of the the individual individual species species and the different and the different chemical reactivity chemical reactivity
kinetics. In kinetics. In the thefirst firstcase case this phenomenon this phenomenon is is defined defined asas geometric geometric isotope effect effect (GIE) (GIE)
and is and is due due in in particular particular to to hydrogen binding. InIn fact, hydrogen binding. fact, the length length of thethe hydrogen bond hydrogen bond
30 30 with oxygen with oxygenisissmaller smallerthan thanthat thatbetween between deuterium deuterium and and oxygen.oxygen. This This may involve may involve a a different structural different structuralrearrangement rearrangement of both of both intra-intra- and intermolecular and intermolecular structures. structures.
Isotope abundance Isotope abundancealso also changes changes the the kinetics kinetics of reactions, of reactions, knownknown as Kinetic as Kinetic
isotope effect isotope effect (KIE), (KIE), which which can canbebeeither either primary primaryororsecondary, secondary,depending depending on on whether whether
the isotope the isotopechanges changes thethe reaction reaction making making it faster it faster or slower or slower than the than the process process of interest. of interest. It It 35 35 is therefore is clearthat therefore clear thatthetheKIE KIE establishes establishes a link a link between between a givena given isotopicisotopic abundance abundance of a of a materialand material anditsitsability abilityto tointeract interactin in a reproducible a reproducible mannermanner with biological with biological systems. It systems. It therefore seems therefore plausible that seems plausible that the the analysis analysis of isotopic isotopic abundance abundance is is aa possible possible tool tool for for monitoringthe monitoring theconformity conformity of of thethe product product fromfrom a physical-chemical a physical-chemical and potentially and potentially
86 10 May 2024
biological point of view. biological point of view.
In aa batch, In batch, the thepresence presenceofofalterations alterationsininthetheisotopic isotopicabundance abundance ratios ratios may may
indicate adulteration, indicate adulteration, poor poor quality qualityofofthetheproduct productand,and, when when samples samples representing representing
different intermediates different intermediates along the production along the production process processareareconsidered, considered,possibly possiblya ageneral general 5 5 loss of loss of its its desirable nativeconformation. desirable native conformation. In this In this context, context, the the acquisition acquisition ofof several several good-quality batchesofofthe good-quality batches theproduct, product, together with together with established established conformity conformityverification verification techniques techniquessuchsuchasasNIR, NIR,maymay leadlead to to
the generation generation of of aareference referencelibrary librarywhere whereindividual individual batchescancan be evaluated, and and 2024203121
the batches be evaluated,
isotopicreproducibility isotopic reproducibility verified verified within within established established ranges. ranges.
10 10 In addition, the In the 14C activity assessment 14C activity assessment can can define definethethe system systemasas100% 100% natural. natural.
This is This is because the 14C because the 14Cisisananunstable unstableisotope isotope(half-life (half-life of of 5730 5730years) years)and, and,therefore, therefore, tends to tends to accumulate accumulateininliving livingmaterial materialwhile while petroleum petroleum derivatives derivatives have have a verya low very low presence/absence presence/absence of this of this unstable unstable carbon carbon isotope. isotope.
4.1.1. Results 4.1.1. Results and anddiscussion discussion 15 15 Theanalysis The analysis of of isotopic isotopic abundance abundancewas was carriedoutoutonon carried thethefive fivebatches batchesincluded included in the in the development andvalidation development and validationofofthe the NIR NIRmethod methodandand thus thus constituting constituting good good quality quality
batches (20B0596, batches (20B0596,20B1955, 20B1955, 20I1279, 2011279, 20J1770, 20J1770, 21E1640), 21E1640), batches batches 20B0596, 20B0596, 20B1955 20B1955
were prepared were preparedfrom from different different batches batches of starting of starting materials materials with with respect respect to batches to batches
20I1279,20J1770, 2011279, 20J1770,21E1640. 21E1640. 20 20 Thesamples The sampleswere weresent senttotothe theChelab Chelab(Tentamus (Tentamus Company) Company) laboratory laboratory and tested and tested
for stable isotopes as follows: for stable isotopes as follows:
- - δ18O: Method 8180: Method IRMS, IRMS, UNIT UNIT ‰ %oV-SMOW. V-SMOW. - - δ13C: Method 813C: Method QMA-M-01, QMA-M-01, EA-IRMS, EA-IRMS, UNITUNIT %‰V-PDB. V-PDB. C14-activity C14- activitywaswas also also tested: tested:
25 25 - - 14C-activity: 14C-activity: Method MethodISO-16620-2;2015 ISO-16620-2;2015 (AMS), (AMS),UNIT UNIT % modern % modern carbon (pMC). carbon (pMC). Theresults The resultswere were as as follows. follows.
δ ratio of the main isotopes 8 ratio of the main isotopes of of thethe co-extract co-extract centella-echinacea. centella-echinacea.
Arté GX Arté GXBatch Batch δ18O 8180 δ13C 813C 14C-activity 14C-activity (% (% modern modern carbon Pmc) carbon Pmc)
20B0596 20B0596 22,6 22,6 -25,54 -25,54 102,35 102,35 20I1279 2011279 20,3 20,3 -28,64 -28,64 102,53 102,53 20B1955 20B1955 22,3 22,3 -28,83 -28,83 102,28 102,28 20J1770 20J1770 20,6 20,6 -28,54 -28,54 103,09 103,09 21E1640 21E1640 20,8 20,8 -28,51 -28,51 102,56 102,56 (the values (the values inin the thetable tableincludes includesthethepercent percent error error according according to official to the the official 30 30 methodused) method used)
87 10 May 2024
Themeasured The measured values values forfor thethe C14C14 activity activity of of thethe samples samples of Pmc of Pmc correspond correspond to to those for those for substances substances fromfrompurely purelybio-based bio-basedcarbon. carbon.There Thereis is nono evidence evidence of of a synthetic a synthetic
source inin the source theanalysed analysed material. material. TheThe C14-activity C14-activity value value perfectly perfectly overlapsoverlaps among among
batchesasasititisisnotnotaffected batches affected by biological by the the biological variability variability of the starting of the starting materials,materials, thus thus 5 5 identifying high identifying reproducibility of high reproducibility of the the production process according production process accordingtotoconservation conservationofof this parameter. this parameter.
On two On twobatches batchesofofco-extract co-extract (20J1770 (20J1770 ee 21E1640) 21E1640)a astudy studyofofthetheisotopic isotopic abundanceduringduringdifferent differentsteps steps ofof the the manufacturing processwaswasconducted. conducted. 2024203121
abundance manufacturing process
Theresults The resultsarearereported reported in the in the tabletable belowbelow depicting depicting δ ratio the of the 8 ratio of the the main mainofisotopes of isotopes
10 10 the coarse the coarseplant plantrawraw plant plant parts parts of the of the co-extract co-extract centella-echinacea. centella-echinacea.
Description Description δ18O/16O δ13C/12C 813C/12C
Centella leaves, Centella leaves, +20.8 +20.8 -28.57 -28.57 Batch Batch 20H1282 20H1282 Echinacea roots, Echinacea roots, +22.7 +22.7 -28.17 -28.17 Batch Batch 20F0840 20F0840 Mixture Mixture Centella Centella +20.9 +20.9 -28.86 -28.86 leaves leaves + Echinacea + Echinacea roots roots (batches (batches above) above) Batch 21E1640 Batch 21E1640 +20.8 +20.8 -28.51 -28.51 (Manufactured in (Manufactured in presently used asas presently used clinical clinical
experimental experimental batch) batch) Batch 20J1770 Batch 20J1770 +20.6 +20.6 -28.54 -28.54
Theassessment The assessmentofofthe theisotopic isotopicabundance abundanceof of thethe materials materials along along thethe production production
process shows process showsthat thatthetheproduction production process process doesdoes not alter not alter the abundance the abundance ratios,ratios, thus thus substantiatingthethe substantiating fact fact that that the the process process conserves conserves thebiophysical the native native biophysical characteristics characteristics
15 15 of the of the starting startingmaterials. materials. Analysis for Analysis for the the batches under study batches under studyshowed showed substantialsimilarity substantial similarityofofvalues valuesandand maintenanceofofratios maintenance ratios during duringthethe manufacturing manufacturingprocess. process.This Thisisisconsistent consistentwith withthe the NIR NIR results previously results described, according previously described, accordingtotowhich whichallallbatches batcheswere were similar similar andand merged merged
withoutoutliers without outlierstotosimilar similar spectra. spectra.
20 20 5. 5. RNA RNA evaluation evaluation inin production production intermediate intermediate
Biophysical characterization Biophysical characterization of biological vegetal of biological material includes vegetal material includes thethe evaluationofofbiological evaluation biological material material in production in production intermediates. intermediates.
“RIC199EL0, extract_BLEND CENT_ECH "RIC199EL0, extract_BLEND CENT_ECH EL, batch EL, batch R20I4716” R2014716" which which
88 10 May 2024
correspondstotoa aproduction corresponds production intermediate intermediate of of Arté Arté GX, GX, i.e.,i.e., the the Centella Centella asiatica asiatica and and Echinacea water Echinacea water coextract coextract ininthetheproportions proportionsdepicted depictedin in example example 1, before 1, before ultrafiltration. The ultrafiltration. The presence presence of ofRNARNA has evaluated has been been evaluated both quantitatively both quantitatively and and qualitatively. RNA qualitatively. wasextracted RNA was extractedusing usinga aplant plantmatrix matrixspecific specifickit kit (RNeasy (RNeasy PowerPlant PowerPlant
5 5 kit) after kit) afterhomogenization with the homogenization with the use use of of QIAshredder QIAshreddercolumns columns before before proceeding proceeding with with
the kit the kit extraction extraction protocol. protocol. The Thedimensional dimensional distribution distribution of of the the RNA RNA obtained obtained was was performed using performed using Bioanalyzer Bioanalyzer 2100 2100 withwith RNA 6000Nano, RNA 6000 Nano,RNARNA 60006000 Pico Pico andand small small RNA kits. 2024203121
RNA kits.
In Figure In 13, the Figure 13, the electropherogram electropherogram"A"“A” shows shows the the sizesize distribution distribution of of thethe total total
10 10 RNA RNA and and thethe electropherogram electropherogram "B" “B” showsshows the size the RNA RNAdistribution size distribution betweenbetween 4 and 4 and 150 150
nt. nt.
Then, to Then, to quantitatively quantitatively assess assess thethetotal RNA total RNA extracted extracted from from thethe sample, sample, aa nucleic nucleic acid digestion acid digestion was performedusing was performed usingthe theNew New England England Biolabs Biolabs nucleoside nucleoside digestion digestion Mix Mix
kit.The kit. .TheRNARNA concentration concentration was expressed as was expressed as total total nucleosides nucleosides by by UHPLC-qToF UHPLC-qT0F 15 15 analysis. The analysis. The Table belowreports Table below reports RNA RNA expressed expressed as as totalnucleosides. total nucleosides. BLEND CENT_ECH EL ug/g % Guanosine 9,20 41,39 Adenosine 3,51 15,79 Uridine 2,06 9,27 Cytidine 7,46 33,56
Total 22,23 100
These observations, These observations, in addition to in addition to providing providing a method for a method for validating validating the the biological origin biological origin of of a amatrix, matrix,identify identifyan an additional additional degree degree of both of both structural structural and and functional complexity functional to be complexity to be considered consideredin in product product management. management. 20 20
For aa prompt For promptreference referenceand andconfrontation, confrontation,the theexperimental experimentalprotocols protocolsused used forfor thethree the three different products different products described described in in example example 11 are are summarised summarised ininthe thetable table hereinbelow. hereinbelow.
Arté GX Arté GX PRODUCTBB PRODUCT PRODUCT C PRODUCT C (reference (reference model) model) CELL CELL 2. In 2. In vitro vitrocell cellassay assay Human Human SH-SY5Y SH-SY5Y Human, Human, adipocyte-derived, adipocyte-derived, BASED ASSAY BASED ASSAY representative representative of of (ATCC® (ATCC No. No. CRL- CRL- mesenchymal mesenchymal stemstem cell cell lines(hADMSC), lines (hADMSC), Osteoarthritis Osteoarthritis 2266™) 2266TM) capable of differentiating capable of differentiating into into neuroblastomacells neuroblastoma cells osteoblasts and mineralize osteoblasts and mineralize the the An ininvitro An vitrocellular cellular are aa commonly are commonly extracellular matrix extracellular matrix (ECM) (ECM) were usedinin were used model capable model capable ofof used asasa aneuronal used neuronal this study. this study. These Thesecellscells were wereobtained obtained recapitulating recapitulating model, as model, as theythey during general during general surgery surgery from fromthree three features features of of maintain maintain several several differentpatients different patients(PA42, (PA42,PA59PA59 and PA69) and PA69)
osteoarthritis osteoarthritis [1-3]
[1-3] neuron neuron markers markers (Romagnoli (Romagnoli etetal, al, "In "In vitro vitro Behavior Behavior ofof
89 10 May 2024
was established was established by by [Barbosa,
[Barbosa, D.J.; D.J.; Human AdiposeTissue-Derived Human Adipose Tissue-DerivedStem Stem exposing exposing primary primary Capela, J.P.; Capela, J.P.; dede Cells on Cells on Poly(e-caprolactone) Poly(ε-caprolactone)Film Filmforfor humanchondrocytes human chondrocytes Lourdes Bastos, Lourdes Bastos,M.; M.; Bone TissueEngineering Bone Tissue Engineering Applications", Applications", (HC, Cell Application (HC, Cell Application Carvalho,F.F.In In Carvalho, vitro vitro BioMedResearch BioMed Research International, International, vol. vol. INC 402K-05)totoIL1B INC 402K-05) IL1B models models for for 2015,Article 2015, ArticleIDID323571, 323571, 12 pages, 12 pages, 2015. 2015.
[5 ng/ml]for
[5 ng/ml] for6 6hours. hours. neurotoxicology neurotoxicology https://doi.org/10.1155/2015/323571). https://doi.org/10.1155/2015/323571). insulted cells insulted cells were were research. [Toxicol. research. [Toxicol. These cell These cell lines lineshavehave been characterized been characterized exposedfor exposed for 2424hours hours Res. 2015, Res. 2015, 4,4,801- 801– with respect with respect to to the the mainmainstemness stemness to five to fivedifferent different 842]. 842]. markers markers of of mesenchymal mesenchymalstem stemcells cells batches of batches of "Arté-Gx" “Arté-Gx” The cells The cells were were (CD44, CD105, and (CD44, CD105, and STRO1) and by STRO1) and by
[1.4 mg/ml]:
[1.4 mg/ml]: exposedfor exposed for 24 24 hours hours studying their multipotency studying their multipotency toward toward 2024203121
Batch Batch 20B1955 20B1955 to product to product BB at at 1.07 1.07 osteogenic osteogenic phenotypes phenotypes atat the the (referencestandard) (reference standard) mg/ml mg/ml in in the the DepartmentofofSurgery Department Surgeryand andTranslational Translational Batch 20I1279 Batch 2011279 appropriate cellcell appropriate medicineofofthethe medicine University University of Florence. of Florence.
Batch 20J1770 Batch 20J1770 culture medium. culture medium. hADMSCswere hADMSCs werecultured culturedinina agrowth growth Batch 21E1640 Batch 21E1640 medium (GM) medium (GM) and and grown grown to to 70-80% 70–80% Batch 20B0596 Batch 20B0596 confluence. Afterwards, confluence. Afterwards,the thecells cellswere were Batch 21E1640Dest Batch 21E1640 Dest seeded in 24-well plate at aa seeded in 24-well plate at Each time one Each time oneofofthethe concentration concentration of of 1 X1105 × 10 5 cells/well. cells/well. AfterAfter batch solutions batch solutionswas was a week, a week, the the GMGM waswas replaced replaced withwith added, fresh added, freshIL1BIL1B[5[5 osteogenic medium osteogenic medium (OM) (OM) containing containing thethe ng/ml] was ng/ml] was also also fluorophore calcein fluorophore calcein 1 μg/mL1 ug/mL and and added added to to the the incubated for incubated for 2828days dayswithwithor or without without medium. medium. product C. product C. The The medium medium withwithor or without without the product the product CC was was refreshed refreshed twice twice aa week. week. DEFINITIONOF DEFINITION OF The The patho- patho- The alteration The alteration ofof The alteration The alteration ofof healthy healthyphysiological physiological THE PATHO- THE PATHO- physiological features physiological features healthy physiological healthy physiological state features of state features of state-of-the-art state-of-the-art PHYSIOLOGICAL PHYSIOLOGICAL of of state-of-the-art state-of-the-art state features ofof state features “Osteoporosis” "Osteoporosis" were were considered considered with with HALLMARKSOF HALLMARKS OF “Osteoarthritis "Osteoarthritis state-of-the-art state-of-the-art particular attention to the following particular attention to the following THE DISEASE THE DISEASE condition” condition" were were “Mild "Mild Cognitive Cognitive areasinvolved: areas involved: WITH WHICH WITH WHICHTO TO considered with with Impairment” Impairment" were were - Remodelling Remodelling of of bone bone considered - INTERROGATEIPA INTERROGATE IPA particularattention particular attentiontoto considered considered with with - Osteoporosis Osteoporosis -
the following the followingareas areas particular attention particular attention - Differentiation - Differentiationofofosteoblasts osteoblasts involved: involved: to the to thefollowing following - Mineralization - Mineralization
-inflammatory -inflammatory areasinvolved: areas involved: - Inflammation - Inflammation process, process, -Cognition -Cognition - Funcitonality - Funcitonalityofofadiposeadipose tissue tissue
-cellular proliferation, -cellular proliferation, -Activation -Activation and and This knowledge This knowledge was wasused usedtotointerrogate interrogate -anatomical damage, -anatomical damage, viability viability IPA via the IPA via the"IPA “IPABioprofiler" Bioprofiler”tool, tool,using using -oxidativestress -oxidative stress -Myelination -Myelination and and the following the followingkeywords: keywords: osteoporosis, osteoporosis,
This knowledge This knowledge was was branching branching postmenopausal postmenopausal osteoporosis, osteoporosis, used used totointerrogate interrogate -Reduction -Reduction of of calcification of calcification of bone, bone,osteoblast osteoblast andand
IPA via IPA via the the "IPA“IPA inflammation inflammation osteoclastdifferentiation, osteoclast differentiation,bone bone mineral mineral
Bioprofiler” Bioprofiler" tool, tool, -Skeletal -Skeletal and and density. density.
using the using thefollowing following muscular muscular system system keywords: keywords: function function osteoarthritis, osteoarthritis, This knowledge This knowledgewas was arthropathy, arthropathy, used to interrogate used to interrogate formationof of formation cartilage cartilage IPA IPA via via thethe"IPA “IPA tissue, destruction tissue, destruction of of Bioprofiler” Bioprofiler" tool, tool,
cartilage cartilage tissue, tissue, using the using thefollowing following damageof ofcartilage, damage cartilage, keywords: keywords: connective tissue connective tissue neurodegeneration, neurodegeneration, disorder, disorder, cognitive cognitive deficit, deficit,
inflammation of joint, inflammation of joint, memory memory deficit, deficit,
immune immune cell cell function of function of muscle, muscle,
90 10 May 2024
trafficking. trafficking. oxidative stress and oxidative stress and
inflammatory inflammatory process. process.
Arté GX Arté GX PRODUCTBB PRODUCT PRODUCT C PRODUCT C (reference model) (reference model) GENE GENE 2.1.2. 2.1.2. Gene Gene expression expression At At the end the end of of 2.1.2. 2.1.2. Gene Geneexpression expression EXPRESSION EXPRESSION analysis analysis described treatment described treatment analysis analysis ANALYSIS ANALYSIS At the At the end end of of described described periods, cells were periods, cells were At the At the end endof of described described 2024203121
treatment periods, treatment periods,cellscellswere were washed with washed with 100100 ul µl treatment periods, treatment periods,cellscells washed with washed with 100100ulµlPBS PBSandand PBS PBS andand lysed lysed and and were washed were washed with with 100100 pl µl lysed andcollected lysed and collectedin in RLTRLT bufferbuffer collected collected in in RLT RLT PBS PBS and and lysed lysed and and (Qiagen, (Qiagen, 1053393) 1053393) addedaddedwith with- β- buffer buffer (Qiagen, (Qiagen, collected inin RLT collected RLT buffer buffer mercaptoethanol mercaptoethanol (Sigma, (Sigma, 1053393) addedwith 1053393) added with (Qiagen, 1053393) (Qiagen, 1053393)added added M3148) M3148) and andDXDX reagent reagent (Qiagen, (Qiagen, β-mercaptoethanol B-mercaptoethanol with β-mercaptoethanol with 3-mercaptoethanol 19088) 19088) for for genegeneexpression expression (Sigma, (Sigma, M3148) M3148) and and (Sigma, M3148) (Sigma, M3148) and andDXDX analysis experiments. analysis experiments. Total Total RNA RNA DX reagent(Qiagen, DX reagent (Qiagen, reagent (Qiagen,19088) reagent (Qiagen, 19088) was extracted was extractedfrom fromcells cellslysates lysates 19088) 19088) for for gene gene for gene for geneexpression expression using using an an QIAsymphony QIAsymphony RNA RNAKit Kit expression analysis expression analysis analysis analysis experiments. experiments. (Qiagen,) (Qiagen,) with with the the QIAsymphony QIAsymphony experiments. experiments. Total Total Total RNA Total RNAwaswas extracted extracted SP instrument SP instrument (Qiagen). (Qiagen). RNA RNA was wasextracted extracted fromcells from cellslysates lysatesusing using an an
The quality The quality and and quantity quantity of of RNA RNA from cells from cells lysates lysates QIAsymphony QIAsymphony RNA RNAKitKit was determined was determinedby by A230, A230, A260, A260, using using an an (Qiagen,) (Qiagen,) with thewith the A280 and A280 and A320 A320measurements measurements QIAsymphony QIAsymphony RNA RNA QIAsymphony QIAsymphony SP SP on Varioskan on Varioskan™LUX LUXmultimode multimode Kit Kit (Qiagen,) withthe (Qiagen,) with the instrument instrument (Qiagen). (Qiagen).
microplate microplate reader reader (Thermo (Thermo QIAsymphony QIAsymphony SP SP Thequality The qualityand and quantity quantity of of
Scientific™). Integrity ScientificTM). Integrity ofof RNA RNA was was instrument (Qiagen). instrument (Qiagen). RNA RNA was wasdetermined determinedbyby checked using checked using a 2100 a 2100 The quality The quality and and A230, A260, A230, A260, A280 A280 and and expert_Eukaryote expert_Eukaryote Total Total RNA RNA quantity quantity ofof RNA RNA was was A320 measurements A320 measurements on on Nano Kit (Agilent). Nano Kit (Agilent). Whole Whole determinedbybyA230, determined A230, Varioskan™ Varioskan LUX LUX transcriptome expression transcriptome expressionprofile profile A260, A280 A260, A280andandA320 A320 multimode multimode microplate microplate was evaluated using was evaluated using a Human a Human measurements measurements onon reader reader (Thermo (Thermo Clariom™ Clariom S S Pico PicoAssay AssayHT HT Varioskan™ Varioskan LUX LUX Scientific™). Scientific Integrity of of TM . Integrity
(Applied (Applied Biosystems, Biosystems, multimode multimode RNA RNA waswaschecked checked using using a a ThermoFisherScientific) ThermoFisher Scientific) onon a a microplate microplate reader reader 2100 expert_Eukaryote 2100 expert_Eukaryote GeneTitan GeneTitan MC MC Instrument Instrument (Thermo Scientific™). (Thermo ScientificTM) Total RNA Total RNA Nano Nano Kit Kit (Applied (Applied Biosystems, Biosystems, Integrity Integrityof ofRNARNA waswas (Agilent). (Agilent). Whole Whole ThermoFisher ThermoFisher Scientific), Scientific), checked using aa 2100 checked using 2100 transcriptome expression transcriptome expression following the following the manufacturer's manufacturer's expert_Eukaryote expert_Eukaryote profile profile was was evaluated evaluated instructions. Briefly, 66ngngofoftotal instructions. Briefly, total Total RNA Total Nano Kit RNA Nano Kit using using a a Human Clariom™ Human Clariom S S RNA wasused RNA was usedtotogenerate generatecDNA, cDNA, (Agilent). (Agilent). Whole Whole Pico Assay HT Pico Assay HT(Applied (Applied then fragmented and then fragmented andlabelled labelled transcriptome transcriptome Biosystems, ThermoFisher Biosystems, ThermoFisher cDNAwas cDNA washybridized hybridizedtotoaa HumanHuman expression expression profile profile Scientific) onon aa GeneTitan Scientific) GeneTitan ClariomS S96-array Clariom 96-array plate plate for for 17 h17 h was evaluated was evaluatedusing using MC Instrument(Applied MC Instrument (Applied at 45°C. at 45°C. Arrays Arrays were washed, were washed, RNA-Seq RNA-Seq data data Biosystems, ThermoFisher Biosystems, ThermoFisher stained and stained and thenthenscanned scanned using using obtained obtained with with Scientific), following Scientific), following thethe the GeneTitan the GeneTitan MC MCInstrument Instrument Illumina Illumina NextSeq, NextSeq, manufacturer's manufacturer's (Applied (Applied Biosystems, Biosystems, sequencedininpaired- sequenced paired- instructions. Briefly,6 6ng ng instructions. Briefly,
ThermoFisherScientific) ThermoFisher Scientific) and and CELCEL end mode. end mode. of total of total RNA wasused RNA was usedto to Intensity files were Intensity files weregenerated generated by by generate cDNA, generate cDNA, then then Affymetrix GeneChip Affymetrix GeneChip Command Command fragmented fragmented and and labelled labelled Console Console Software Software (AGCC, (AGCC, cDNA cDNA waswashybridized hybridizedto toa a ThermoFisher ThermoFisher Scientific). Scientific). Human Clariom Human Clariom S 96-array S 96-array plate for 1717h hat at plate for 45°C. 45°C.
91 10 May 2024
Arrays were Arrays were washed, washed, stained and stained and then thenscanned scanned using using the the GeneTitan GeneTitan MC MC Instrument Instrument (Applied (Applied
Biosystems, ThermoFisher Biosystems, ThermoFisher
Scientific) and Scientific) and CEL CEL Intensity files Intensity files were were generated bybyAffymetrix generated Affymetrix GeneChip GeneChip Command Command Console Software Console Software(AGCC, (AGCC, ThermoFisher ThermoFisher Scientific). Scientific). 2024203121
Arté GX Arté GX PRODUCTBB PRODUCT PRODUCT C PRODUCT C (reference model) (reference model) TRANSCRIPTOMICS TRANSCRIPTOMICS 2.1.3. Transcriptomics 2.1.3. Transcriptomicsdata data Sample was Sample was mapped mapped 2.1.3. Transcriptomics 2.1.3. Transcriptomics DATA ANALYSIS DATA ANALYSIS analysis analysis on reference data analysis data analysis on reference Data analysis was Data analysis was performed performed genome genome using using the the Data analysiswas Data analysis was using Transcriptomic using Transcriptomic Analysis Analysis bioinformatics tool bioinformatics tool performed using performed using Console Software Console Software(TAC,(TAC, STAR(version STAR (version 2.7.0f), 2.7.0f), Transcriptomic Transcriptomic Analysis Analysis
ThermoFisher ThermoFisher Scientific) Scientific) that that with the with the standard standard Console Software Console Software(TAC, (TAC, provides qualitycontrol provides quality control analysis, analysis, parameters parameters for for ThermoFisher ThermoFisher Scientific) Scientific)
performs normalization and performs normalization and paired reads. paired reads. The The that provides that providesquality quality summarization,based summarization, basedononthethe reference track was reference track was controlanalysis, control analysis,performs performs Signal Space Signal Space Transformation- Transformation- the assembly the assembly Human Human normalization normalization and and Robust Multi-Chip Robust Multi-Chip Analysis Analysis (SST-(SST- obtained obtained from from summarization,based summarization, basedonon RMA) analysis algorithm, RMA) analysis algorithm, and and GenCode GenCode (HG38). (HG38). the Signal the SignalSpace Space provides provides a alist list of of differentially differentially Thequantification The quantification of of Transformation-Robust Transformation-Robust expressed genes expressed genes(Limma (Limma transcriptsexpressed transcripts expressed Multi-Chip Analysis Multi-Chip Analysis (SST- (SST-
Bioconductor package,p- Bioconductor package, p- for each for sequenced each sequenced RMA) analysis RMA) analysis algorithm, algorithm,
value≤0.05). value<0.05). sample was andprovides and provides a listofof a list sample performed performed using was using differentially expressed differentially expressed featureCount featureCount genes (Limma genes (Limma algorithm. algorithm. Bioconductor package,p- Bioconductor package, p- R R was usedtoto create was used create value≤0.05). value<0.05).
a matrix of all genes a matrix of all genes
expressed expressed in in all all samples with samples with the the correspondingread- corresponding read- counts and counts and the the Bioconductor Bioconductor package package DESeq2 DESeq2 was was used used to to normalize normalize the data, the data, using usingthe the median median ofofratio,ratio,toto perform perform the the differential differential
expression analysis. expression analysis. Qualitycontrol Quality controlcheckcheck such as Euclidean such as Euclidean distances (Heatmap distances (Heatmap Distances) Distances) and and Principal Principal component component analysis (PCA) analysis (PCA) were were performed among performed among allall samples in samples in each each condition condition
92 10 May 2024
considered. considered.
5. 5. CONCLUSIONS CONCLUSIONS Takentogether, Taken together, the the data data here here reported reported allow allow to to identify identify acceptability acceptability parameters parameters
which are which are key keytotoguarantee guaranteethe thequality quality ofofthe theproduction productionprocess processofofa aproduct product comprisingororconsisting comprising consistingofofone oneor ormore more matrix matrix of biological of biological originorigin (natural (natural matrix). matrix).
5 5 Intriguingly, the Intriguingly, the data data shows showsthat thatmonitoring monitoring exclusively exclusively the reproducibility the reproducibility of theof the
compositionatatthe composition themolecular molecularlevel leveldoesdoesnotnot represent represent a rewarding a rewarding strategy, strategy, as such as such a a 2024203121
strategy ignores strategy ignores key keyfeatures features of matrix of the the matrix that impact that impact its ability its ability to induce to a induce a reproducible reproducible effect effect when when this this is used is used to treat to treat a biological a biological system.system. Presumably Presumably due to the due to the
presence ofof redundancy presence redundancy effects effects between between molecular molecular components components at bothatthe both the structural structural
10 10 and functional and functional levels, levels, and due to and due to the the presence presenceofofaanetworknetworkofofphysical physicalandand functional functional
interactions within interactions within the the complex complex matrix, matrix, itsits biological biological function function strongly strongly depends depends on on features better features better monitored monitored by byanalysing analysingcarefully carefullyselected selectedbiophysical biophysicalproperties propertiesofofthe the matrix. matrix.
Veryeloquently, Very eloquently,the theexclusive exclusiveuseuse of of targeted targeted metabolomics metabolomics data, data, especially especially
15 15 whenjudged when judgedaccording according to to thetheprinciples principlesusually usuallyreserved reservedtotosinglesingleAPIs, APIs,isisnotnotcapable capable of predicting of predictingthe thecapability capability of different of different batches batches of theofsame theproduct same to product elicit to elicit
reproducible biological reproducible biological effects effects (see (see figure figure 5 5 vs figures 33 and VS figures and 4). 4). By Byapplying applyingtargeted targeted metabolomicsalone, metabolomics alone,such such batches batches are are incorrectly incorrectly perceived perceived as unacceptably as unacceptably different different
fromone from oneanother. another.InIncontrast, contrast, the the application application of of techniques techniquesmonitoring monitoringfeatures featuresofofthe the 20 20 matrix that matrix that are are emergent emergent and andderiving derivingfromfromnetworks networks of of interactions,therefore interactions, thereforedifferent different from and from andonly onlypartially partially dependent dependentononconservation conservationofofthe thesingle singlemolecular molecularcomponents, components, as disclosed as disclosed inin the thepresent presentapplication, application,correctly correctlyidentifies identifiesa asatisfactory satisfactorydegree degree of of
similarity between similarity between the the different different batches analysed and batches analysed andexpress expressconsistency consistencywith withthethefact fact that they that theyindeed indeedelicit elicitreproducible, reproducible, desirable desirable biological biological effects. effects.
25
93 30 May 2025 2024203121 30 May 2025
CLAIMS: CLAIMS: 1. 1. A method A method forfor defining defining the the acceptability acceptability valuesvalues of a of a spectroscopy spectroscopy or or spectrophotometryanalysis spectrophotometry analysisfor forthethecompliance compliance validation validation of ofoneone or more or more batches batches of a of a
productfor product forthethetreatment treatment of aofpathological a pathological condition condition or for or for adjuvating adjuvating homeostasis homeostasis in an in an 55 altered altered physiological physiological state, state,the theproduct productcomprising comprising or or consisting consisting of of one one or or more natural more natural
matrices selected from: matrices selected from: cut cut ororpulverized pulverizedplant plantparts, parts, plant plant extracts extracts fractions , fractionsofofsaidsaid extracts, extracts, such such as as for for example example the thefractions fractions obtained obtainedbybyfiltration filtration on on aa semi-permeable semi-permeable 2024203121
membrane membrane (microfiltration, (microfiltration, ultrafiltration, ultrafiltration, nanofiltration), nanofiltration), or those or those obtained obtained by treatment by treatment
on adsorption resins, on adsorption resins, oror microorganisms, microorganisms, honey, honey,propolis, propolis,silk, silk,wax, wax,plant plantresins, resins, plant plant 10 10 gums, plantexudates, gums, plant exudates,vegetable vegetable oils,vegetable oils, vegetable essentialoils, essential oils,animal animal tissueslysates, tissues lysates, extract fromanananimal extract from animal tissue, tissue, plant plant or animal or animal fluids;fluids; said method said method comprising: comprising:
performing at performing at least least oneoneininvitro vitrocell-based cell-basedassay assayandand calculatingsaid calculating said acceptability acceptability values values on on aa spectroscopy spectroscopy or or spectrophotometry spectrophotometry spectra spectraof: of: aa reference reference standard standardhavinghaving a known a known therapeutic therapeutic or beneficial or beneficial effecteffect in thein the
15 15 treatment of treatment of said said pathological pathologicalcondition conditionororininadjuvating adjuvating homeostasis homeostasis in said in said altered altered
physiological physiological state;andand state;
one one oror more batchesofof said more batches said product; product; said said spectra spectra being definedasasacceptable being defined acceptableorornon-acceptable non-acceptable on on the the basis basis of the of the
biological function biological function exertedexertedininsaid saidat atleast leastoneone cell-based cell-based assay assay by said by said reference reference
20 20 standard standard and andsaidsaid oneoneorormore more batches batches on on oneone or more or more hallmarks hallmarks of saidof pathological said pathological condition condition or or ofof aa pathological pathological condition condition thatthat can derive from can derive from said said altered altered physiological physiological state; state; and and
defining the defining the acceptability acceptabilityvalues valuesof of thethe spectroscopy spectroscopy or spectrophotometry or spectrophotometry
spectra spectra as as the the variability variabilityrange rangeofofthe thespectroscopy spectroscopy or orspectrophotometry spectra values spectrophotometry spectra values 25 25 of all of of all of said acceptablebatches said acceptable batchesand and of said of said reference reference standard standard batch, preferably batch, preferably refined refined by the by thespectra spectraofofsaid saidnon-acceptable non-acceptable batches. batches.
2.2. The method The methodaccording according to to claim claim 1 for 1 for defining defining thethe acceptabilityvalues acceptability valuesofof aa spectroscopy spectroscopyororspectrophotometry spectrophotometry analysis analysis forfor thethe compliance compliance validation validation of one of one or or
morebatches more batchesofofa aproduct productforfor thethe treatment treatment of of a pathological a pathological condition, condition, wherein wherein the the
30 30 product comprises product comprisesone oneorormore morenatural naturalmatrices; matrices;said saidmethod method comprising: comprising:
(a) performing (a) performing at at least least oneone in vitro in vitro cell-based cell-based assayassay on: on:
(i) (i) aa reference reference standard batch of standard batch of said said product, product, wherein whereinthe thereference reference standard standard hashasa aknown known therapeutic therapeutic effecteffect for treatment for treatment of said of said pathological pathological
condition; condition; andand
35 35 (ii) (ii) one one orormore more test test batches batches of said of said product; product;
whereinthe wherein the read-out read-out of of said said cell-based cell-based assay assay isis representative representative of of the themodulation modulation
of of one or more one or biological activity more biological activity associated associated with with one or more one or hallmarksofofsaid more hallmarks said
94 07 Mar 2025 2024203121 07 Mar 2025
pathological condition; pathological condition; (b) (b) quantifying quantifying in in terms terms ofof numerical values the numerical values the modulation modulationofofsaid saidone oneorormore more biologicalactivities biological activitiesinduced induced by each by each batchbatch of(a) of step step for(a) forcell-based each each cell-based assay read-out assay read-out
defining defining asasreference reference values values the the values values calculated calculated for thefor the reference reference standardstandard batch; batch; 55 (c) (c) defining defining as as acceptable acceptable thethe test testbatches batcheswhose values calculated whose values calculated inin (b) (b) induce induce
aa modulation modulationof ofeach each measured measured biological biological activity activity in saidin cell-based said cell-based assay whose assay whose
modulusisis to modulus ≥ to thethe modulus modulus of theof reference the referencevaluevalue calculated calculated in (b)in and (b) defining and defining as as 2024203121
non-acceptablethe non-acceptable thebatches batcheswhose whose values values calculated calculated in (b) in (b) induce induce a modulation a modulation of at of at
least least one one ofofsaid saidbiological biological activities activities in in said said cell-based cell-based assayassay whosewhose modulusmodulus is less than is less than
10 10 the modulus the modulus of of thethe reference reference valuevalue calculated calculated in (b);in (b);
(d) (d) performing performing aaspectroscopy spectroscopyororspectrophotometry spectrophotometry on said on said reference reference standard standard
batchand batch andonon said said oneone or more or more different different test batches; test batches; and and (e) defining (e) defining the the acceptability acceptability values values ofof the the spectroscopy spectroscopyororspectrophotometry spectrophotometry spectra spectra as as the the variability variabilityrange rangeofofthethespectroscopy spectroscopy or orspectrophotometry spectra values spectrophotometry spectra values 15 15 of all of of all of said acceptablebatches said acceptable batchesand and of said of said reference reference standard standard batch, preferably batch, preferably refined refined bythe by thespectra spectraofofsaidsaidnon-acceptable non-acceptable batches. batches.
3. 3. Themethod The method according according to claim to claim 2 further 2 further comprising comprising determining determining for for each ofsaid each of saidoneone or or more more biological biological activities activities the modulation the modulation trendforthereof trend thereof for restoring restoring
aa healthy physiological healthy physiological state. state.
20 20 4. 4. The method The methodaccording according to to claim3 3further claim furthercomprising: comprising: in step in step (a) (a) performing performing saidsaid inin vitro vitro cell-based cell-based assay assay ononone oneorormore more reference reference
drug forthe drug for thetreatment treatment of of said said pathological pathological condition; condition;
in in step step (b) (b) quantifying quantifying in in terms terms of of numerical numericalvalues valuesthethemodulation modulation induced induced in in
step (a) by step (a) byeach eachreference reference drug drug for for each each cell-based cell-based assay assay read-out read-out and, forand, eachfor each
25 25 biological activity biological activity modified modifiedbyby said said reference reference standard standard and byand byone said said or one more or more
reference drug reference drugaccording according to said to said modulation modulation trend trend for for restoring restoring a healthy a state, healthy state, comparingthe comparing thevalue valuecalculated calculatedforforeacheachreference reference drug drug andand for for thethe reference reference standard standard
andselecting and selectingasasreference reference value, value, the the value value associated associated to the to the weakest weakest performance. performance.
5. 5. Themethod The methodaccording according to to claim claim 1 for 1 for defining defining thethe acceptabilityvalues acceptability valuesofof 30 30 aa spectroscopy spectroscopyororspectrophotometry spectrophotometry analysis analysis forfor thethe compliance compliance validation validation of one of one or or
more batches more batches ofofa abeneficial beneficial product productfor foradjuvating adjuvatinghomeostasis homeostasisininan an altered altered physiological state, physiological state, wherein wherein thethe product productcomprises comprisesoneone or or moremore natural natural matrices; matrices; saidsaid
methodcomprising: method comprising: (a) (a) performing performing at at least least one one in in vitro vitro cell-based cell-based assayassay on: on:
35 35 (i) (i) aa reference reference standard batch of standard batch of said said product, product, wherein whereinthe thereference reference standard standard hashas aa known known beneficialeffect beneficial effectfor foradjuvating adjuvatinghomeostasis homeostasis in in an an
95 07 Mar 2025 2024203121 07 Mar 2025
altered physiological altered physiological condition; condition; and and
(ii) (ii) on on oneoneor or more more test test batches batches of of saidproduct; said product; whereinthe wherein the read-out read-out of of said said cell-based cell-based assay assay is is representative representative of of the themodulation modulation
of of one one oror more morebiological biologicalactivities activities associated associated with one or with one or more morehallmarks hallmarks of of a a
55 pathological condition pathological condition that that can develop from can develop fromsaid saidaltered alteredphysiological physiological condition; condition;
(b) (b) quantifying quantifying in in terms terms ofof numerical values the numerical values the modulation modulationofofsaid saidone oneorormore more 2024203121
biologicalactivities biological activitiesinduced induced by each by each batchbatch of(a) of step step for(a) forcell-based each each cell-based assay read-out assay read-out
defining defining asasreference reference values values the the values values calculated calculated for thefor the reference reference standardstandard batch; batch; 10 10 (c) (c) defining defining as as acceptable acceptable thethe test testbatches batcheswhose values calculated whose values calculated inin (b) (b) induce induce
aa modulation modulationof ofeach each measured measured biological biological activity activity in said in cell-based said cell-based assay whose assay whose
modulusisis to modulus ≥ to thethe modulus modulus of reference of the the reference valuevalue calculated calculated in (b)in and (b) defining and defining as as non-acceptablethe non-acceptable thebatches batcheswhosewhose values values calculated calculated in (b) in (b) induce induce a modulation a modulation of at of at
least least one one ofofsaid saidbiological biological activities activities in in saidsaid cell-based cell-based assayassay whose whose modulus modulus is < to theis < to the
15 15 modulus modulus of of thethe reference reference valuevalue calculated calculated in (b);in (b);
(d) (d) performing performing aaspectroscopy spectroscopyororspectrophotometry spectrophotometry on said on said reference reference standard standard
batchand batch andonon said said oneone or more or more different different test batches; test batches; and and (e) (e) defining defining the the acceptability acceptability valuesvalues ofof the the spectroscopy spectroscopyororspectrophotometry spectrophotometry spectra spectra as as the the variability variabilityrange rangeofofthethespectroscopy spectroscopy or orspectrophotometry spectra values spectrophotometry spectra values 20 20 of all of of all of said acceptablebatches said acceptable batchesand and of said of said reference reference standard standard batch, preferably batch, preferably refined refined by the by thespectra spectraofofsaid saidnon-acceptable non-acceptable batches. batches.
6. 6. The method The method according according to claim to claim 5 further 5 further comprising comprising determining determining for for each ofsaid each of saidoneone or or more more biological biological activities activities the modulation the modulation trendforthereof trend thereof for restoring restoring
aa healthy physiological healthy physiological state. state.
25 25 7. 7. The method The method according according to any to any one one of claims of claims 2 to 62 further to 6 further comprising comprising
determining, for determining, for each eachofofsaid saidbiological biologicalactivities, activities,one oneorormore more parameters parameters and the and the
modulationtrend modulation trendthereof thereofunderlying underlyingthethe modification modification detectable detectable in said in said pathological pathological
conditionininboth condition both diseased diseased and and healthy healthy physiological physiological state. state.
8. 8. The method The methodaccording according to to claim claim 7 wherein 7 wherein saidsaid parameters parameters are are genes genes and and
30 30 modulationthereof. modulation thereof.
9. 9. The method The methodofofclaim claim6,6,wherein wherein thethe modulation modulation trend trend of said of said parameters parameters
is is assessed bytranscriptomic assessed by transcriptomic analysis. analysis.
10. 10. The method The methodofofanyany oneone of of claims claims 1 to1 to 9, 9, wherein wherein the the cell-based cell-based assay assay is is
designed designed toto simulate simulate conditions conditions related related to saidtopathological said pathological conditioncondition or to said or to said altered altered
35 35 physiological physiological state. state.
96 07 Mar 2025 2024203121 07 Mar 2025
11. 11. The The method method according according to anytoone anyofone of claims claims 1 to 101 wherein to 10 wherein said onesaid or one or
more testbatches more test batchesareare at at least least three three test test batches. batches.
12. 12. TheThe method method according according to one to any any ofoneclaims of claims 1 to 1 to 11 11 wherein wherein said said pathological condition pathological conditioncomprises comprises osteoarthritis, osteoarthritis, mildmild cognitive cognitive impairment, impairment, post post 55 menopausal osteoporosisororcancer. menopausal osteoporosis cancer.
13. 13. TheThe method method according according to to anyany oneone of of claimsfrom claims from1 1toto1212wherein whereinsaid said 2024203121
spectroscopy spectroscopyanalysisanalysis is is selected selected fromfromNIR,NIR, FTIR, FTIR, RAMAN RAMAN and and said said spectrophotometry analysisisisselected spectrophotometry analysis selectedfrom fromUltraviolet Ultravioletand andvisible visiblelight lightspectroscopy, spectroscopy, fluorescence spectroscopy, fluorescence spectroscopy, lightlight scattering. scattering.
10 10 14. A process 14. A process forforthethe compliance compliance validationofof one validation oneorormore morebatches batchesofofaa product for product for the the treatment treatmentofofa apathological pathologicalcondition conditionor or of of a beneficial a beneficial product, product, saidsaid product comprising product comprisingone oneorormore more naturalmatrices, natural matrices,said saidmethod method comprising comprising the the following following
steps: steps:
a. a. performing performing aa spectroscopy spectroscopy or or spectrophotometry spectrophotometryanalysis analysisofofeach eachbatch; batch; 15 15 b. validating b. validating each batch for each batch for which whichthetheobtained obtained spectrum spectrum satisfiesthethe satisfies acceptability acceptability values values defined defined according according toto the the method accordingtoto any method according anyone oneofofclaims claims11to to 13. 13.
1/19 1/19 10 May 2024
Healthy Hallmarks of the pathology Biological activities Pathological state physiological state
Skeletal and muscular system development and function Induction of proliferation articular dysfunction joint
space Formation of cartilage tissue
Inflammatory disease Reduction of inflammation inflammation and nociceptions 2024203121
Inflammation of joint
Skeletal and muscular system function difficulty moving a
joint Non-traumatic arthropathy Protection from anatomical damage Organismal injury and
abnomalities joint inflammation and swelling, difficulty moving a joint
Osteoarthritis
UP modulation DOWN modulation
Fig. Fig. 11
Healthy Gold standard batch Hallmarks of the pathology Biological activities Pathological state IL1B vs untreated physiological state 20B1955 vs IL1B
Skeletal and muscular system
development and function
Induction of proliferation articular dysfunction -> joint -0.20 0.15 space Formation of cartilage tissue
Inflammatory disease
inflammation and Reduction of inflammation nociceptions 0.24 -0.24 Inflammation of joint
Skeletal and muscular system
function difficulty moving 0.05 -0.32 a joint Non-traumatic arthropathy Protection from anatomical damage Organismal injury and
abnomalities joint inflammation and swelling, 0.04 -0.31 difficulty moving a joint
Osteoarthritis
UP modulation DOWN modulation
Fig. 22 Fig.
2/19 Reference Z-
score value
-0.24 -0.17 -0.15 10 May 2024
0.15
DEST.21E1640 vs IL1B -0.32 -0.08 -0.16 Batch 0.20
20J1770
vs IL1B -0.33 -0.31 -0.27 Batch 0.15 SET TRAINING 2024203121
20B0596
vs IL1B -0.33 -0.30 -0.29 Batch 0.15
2011279 vs IL1B -0.33 -0.33 -0.31 Batch 0.15
20B1955 vs
standard
-0.24 -0.32 -0.31 batch 0.15 Gold IL1B Triamcinolone acetonide vs
-0.31 -0.26 -0.17 -0.15 DRUG IL1B
untreated
MODEL IL1B vs -0.20 0.24 0.05 0.04
physiological
Healthy
state THE OF STATE Pathological
state ART
articular joint a moving difficulty of Formation space joint of Inflammation muscular and Skeletal muscular and Skeletal activities Biological joint abnomalities and injury Organismal development system joint disease Inflammatory difficulty swelling, Non-traumatic and inflammation and inflammation tissue cartilage Osteoarthritis function system arthropathy
moving a joint
nociceptions
dysfunction
and function OSTEOARTHRITIS damage anatomical modulation DOWN the of Hallmarks from Protection modulation UP inflammation Reduction of proliferation Induction of
pathology
Fig 3
3/19 Reference
Z-score -0.24 -0.17 -0.15 10 May 2024 value 0.15
21E1640
NEWLY TESTED vs IL1B -0.30 -0.25 -0.30 BATCH Batch 0.15
DEST.21E1640 vs IL1B -0.32 -0.08 -0.16 Batch 0.20 2024203121
20J1770
vsIL1B -0.33 -0.31 -0.27 Batch 0.15 SET TRAINING 20B0596
vs IL1B -0.33 -0.30 -0.29 Batch 0.15
2011279 vs IL1B -0.33 -0.33 -0.31 Batch 0.15
20B1955 vs
standard
-0.24 -0.32 -0.31 batch 0.15 Gold IL1B Triamcinolone vs acetonide REFERENCE
-0.31 -0.26 -0.17 -0.15 DRUG IL1B
untreated
MODEL IL1B vs -0.20 0.24 0.05 0.04
physiological
Healthy
state THE OF STATE Pathological
state ART
articular joint a moving difficulty activities Biological of Formation joint of Inflammation muscular and Skeletal muscular and Skeletal joint abnomalities and injury Organismal development system joint dysfunction disease Inflammatory difficulty swelling, Non-traumatic and inflammation and inflammation tissue cartilage Osteoarthritis function system arthropathy
moving a joint
nociceptions
and function OSTEOARTHRITIS space modulation DOWN damage anatomical the of Hallmarks from Protection modulation UP inflammation Reduction of Induction of proliferation
pathology
Fig 4
4/19
Fig. 5
5/19 5/19
0.1 2011279 10 May 2024
20B0596
20B1955 0.08 20J1770
0.06
0.04 2024203121
0.02
0
-0.02
-0.04
-0.06
4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 Wavenumber (cm^-1)
Fig. Fig. 6°6° o
0.1 Average Spectrum 21E1640 Dest
0.08
0.06
0.04
0.02
0
-0.02
-0.04
-0.06
4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 Wavenumber (cm^-1)
Fig. Fig. 6b 6b
6/19 6/19
0.1 Batch 21E640 Dest 10 May 2024
Undesired A
Undesired B
0.08 Average Spectrum
0.06
0.04 2024203121
0.02
0
-0.02
-0.04
-0.06
4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 Wavenumber (cm^-1) Fig. 6c Fig. 6c
0.1 Average Spectrum
21E1640
0.08
0.06
0.04
0.02
0
-0.02
-0.04
-0.06
4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 Wavenumber (cm^-1)
Fig. 6d Fig. 6d
7/19 7/19 10 May 2024
100 2024203121
80
ns 18% 21%
0
Fig. Fig. >
8/19 8/19 10 May 2024
MILD COGNITIVE IMPAIRMENT STATE OF THE ART Alteration of Healthy Biological activities 5 Hallmarks of the pathology healthy physiological state physiological state
Neurobiology Cognition and behavior Cognition 2024203121
Cognition
Neurobiology Cognition and behavior Learning and Learning memory
Neurobiology Central
nervous system Development of nervous
system Development of neurons
Neurobiology Development of nervous system Activation and viability Neurogenesis and neuronal differentiation
Neurogenesis Differentiation of neurons
Neurobiology Development of nervous system Neurogenesis and neuronal differentiation
Neurogenesis Proliferation
of neuronal cells
UP modulation DOWN modulation Fig. 8a Fig. 8a
9/19 9/19 10 May 2024
MILD COGNITIVE IMPAIRMENT STATE OF THE ART Alteration of Healthy Hallmarks of the pathology E Biological activities healthy physiological state physiological state
Neurobiology Development of nervous system Branching of neurons 2024203121
Myelination and branching
Cell Morphology Sprouting
Inflammatory disease inflammation Chronic inflammatory disorder
Skeletal and muscular system function Proliferation Proliferation of muscle cells
Skeletal and muscular system function
Skeletal and muscular
disorders necrosis Necrosis of muscle
UP modulation DOWN modulation Fig. Fig. 8b 8b
10/19 10/19
TREATMENT 10 May 2024 MILD COGNITIVE IMPAIRMENT STATE OF THE ART CONTROL CARD BATCH
Alteration of Healthy Hallmarks of the pathology Biological activities healthy Gold standard batch physiological state physiological state
Neurobiology -> Cognition and behavior Cognition 2,38
Cognition 2024203121
Neurobiology Cognition and behavior Learning and 2,60 Learning memory
Neurobiology Central nervous system Development of nervous 2,12 system Development of neurons
Neurobiology Development of nervous system Neurogenesis and neuronal Activation and viability differentiation 1,83 Neurogenesis Differentiation of neurons
Neurobiology Development of nervous system Neurogenesis and neuronal differentiation 2,04 Neurogenesis Proliferation of neuronal cells
UP modulation DOWN modulation
Fig. 9a Fig. 9a
11/19 11/19 10 May 2024
Alteration of Healthy Hallmarks of the pathology Biological activities healthy Gold standard batch physiological state physiological state
Neurobiology Development 2024203121
of nervous system 1,22 Branching of neurons
Myelination and branching
Cell Morphology Sprouting 2,03
Inflammatory disease inflammation -1,48 Chronic inflammatory disorder
Skeletal and muscular system function Proliferation 2,05
Skeletal and muscular system Proliferation of muscle cells T function
Skeletal and muscular
disorders necrosis -2,14 Necrosis of muscle
UP modulation
DOWN modulation
Fig. Fig. 9b 9b
12/19 12/19 10 May 2024
Alteration of Healthy Hallmarks of the pathology Biological activities healthy physiological state physiological state
Skeletal and Muscular System
Development and Function 2024203121
Remodelling Remodelling of bone
Remodelling of bone
Skeletal and Muscular System
Development and Function Remodelling Resorption of
bone
Skeletal and Muscular
Osteoporosis Disorders Osteoporosis Osteoporosis
Increased Levels of Alkaline
phosphatase Activation Increased activation of
alkaline phosphatase
Connective Tissue
Development and function Differentiation of osteoblasts Differentiation
Differentiation of bone cells
Skeletal and Muscular System
Development and Function Differentiation
Differentiation of osteoblasts
UP modulation DOWN modulation
Fig. 10a Fig. 10a
13/19 13/19 10 May 2024 OSTEOPOROSIS STATE OF THE ART
Alteration of Healthy Hallmarks of the pathology Biological activities healthy physiological state physiological state
Skeletal and Muscular System
Development and Function Mineralization
Mineralization of bone cell
lines
Skeletal and Muscular System
Development and Function 2024203121
Mineralization Mineralization
Mineralization of osteoblasts
Skeletal and Muscular System
Development and Function Formation -> Formation of
bone
Inflammatory Response Inflammation Inflammation of adipose tissue
Inflammatory Response inflammation Inflammation Inflammation of connective tissue
Inflammatory Response Inflammation Inflammation of white adipose tissue
Connective Tissue
Development and Function Quantity Quantity of adipose tissue
Nutritional Disease Weight gain Weight gain
Functionality of adipose tissue
Connective Tissue
Development and Function Transdifferentiation
Transdifferentiation
Connective Tissue
Development and Function Differentiation
Differentiation of adipocytes
UP modulation DOWN modulation
Fig. 10b Fig. 10b
14/19 14/19
TREATMENT 10 May 2024
Alteration of Healthy Hallmarks of the pathology Biological activities healthy Gold standard batch physiological state physiological state
Skeletal and Muscular System
Development and Function Remodelling Remodelling of -0,49 bone
Remodelling of bone
Skeletal and Muscular System 2024203121
Development and Function Remodelling Resorption of -0,49 bone
Skeletal and Muscular
Osteoporosis Disorders Osteoporosis -0,31 Osteoporosis
Increased Levels of Alkaline
phosphatase Activation Increased activation of 0,19 alkaline phosphatase
Connective Tissue
Development and function Differentiation of osteoblasts Differentiation 0,37 Differentiation of bone cells
Skeletal and Muscular System
Development and Function Differentiation 0,39 Differentiation of osteoblasts
UP modulation DOWN modulation
Fig. Fig. 11a 11a
15/19 15/19
TREATMENT OSTEOPOROSIS STATE OF THE ART 10 May 2024
CONTROL CARD BATCH Alteration of Healthy Hallmarks of the pathology Biological activities healthy Gold standard batch physiological state physiological state
Skeletal and Muscular System
Development and Function
Mineralization 0,29 Mineralization of bone cell
lines
Skeletal and Muscular System
Development and Function Mineralization 2024203121
Mineralization 0,34 Mineralization of osteoblasts
Skeletal and Muscular System
Development and Function
Formation Formation of 0,17 bone
Inflammatory Response Inflammation Inflammation -0,46 of adipose tissue
Inflammatory Response inflammation Inflammation Inflammation -0,46 of connective tissue
Inflammatory Response Inflammation Inflammation -0,38 of white adipose tissue
Connective Tissue
Development and Function Quantity -> Quantity of -0,29 adipose tissue
Nutritional Disease Weight
gain Weight gain -0,49
Functionality of adipose tissue
Connective Tissue
Development and Function
Transdifferentiation 0,28 Transdifferentiation
Connective Tissue Development and Function
Differentiation -0,45 Differentiation of adipocytes
UP modulation DOWN modulation
Fig. Fig. 11b 11b
16/19 AAPH (ROS %) 10 May 2024 20B0596 +
21
+ AAPH (ROS %)
20J1770 Batch 17
SET TRAINING + AAPH (ROS %)
2011279 Batch 2024203121
17
+ AAPH (ROS %) 20B1955 batch standard Gold 17
MODEL (ROS %) AAPH 100
acid + AAPH REFERENCE STANDARD
Ascorbic
17
Untreated
cells
18
physiological
Healthy
state THE OF STATE Pathological
state ART scavenging->biosynthesis-) activities Biological stress oxidative Free radical OSTEOARTHRITIS modulation DOWN the of Hallmarks modulation UP inflammation Reduction of
pathology
Fig. 12
17/19 17/19
A 10 May 2024
30
20
10
o 2024203121
[nt] 25 200 1000 4000
100
so
o
4 20 40 80 150 [nt]
Fig.13 Fig. 13
2011279 20B1955 20J1770 20B0596
1.5
1.0
0.5
0.0
400 600 800 1000 1200 1400 1600 1800
Raman shift (cm-1)
Fig. 14a Fig. 14a
18/19 18/19 10 May 2024
Average spectrum 21E1640 Dest
1.5
1.0 2024203121
0.5
0.0
400 600 800 1000 1200 1400 1600 1800 Raman shift (cm-1)
Fig. Fig. 14b 14b
Batch 21E1640 Dest Undesired A Undesired B Average spectrum 2.0
1.5
1.0
0.5
0.0
400 600 800 1000 1200 1400 1600 1800
Raman shift (cm-1)
Fig. Fig. 14c 14c
19/19 19/19 10 May 2024
Average spectrum 21E1640
1.5 2024203121
1.0
0.5
0.0
400 600 800 1000 1200 1400 1600 1800
Raman shift (cm-1
14d 14d Fig. 14 Fig. 14
Claims (14)
- CLAIMS: 1. A method for defining the acceptability values of a spectroscopy or spectrophotometry analysis for the compliance validation of one or more batches of a product for the treatment of a pathological condition or for adjuvating homeostasis in an altered physiological state, the product comprising or consisting of one or more natural matrices selected from: cut or pulverized plant parts, plant extracts , fractions of said extracts, such as for example the fractions obtained by filtration on a semi-permeable membrane (microfiltration, ultrafiltration, nanofiltration), or those obtained by treatment on adsorption resins, or microorganisms, honey, propolis, silk, wax, plant resins, plant gums, plant exudates, vegetable oils, vegetable essential oils, animal tissues lysates, extract from an animal tissue, plant or animal fluids; said method comprising: performing at least one in vitro cell-based assay and calculating said acceptability values on a spectroscopy or spectrophotometry spectra of: a reference standard having a known therapeutic or beneficial effect in the treatment of said pathological condition or in adjuvating homeostasis in said altered physiological state; and one or more batches of said product; said spectra being defined as acceptable or non-acceptable on the basis of the biological function exerted in said at least one cell-based assay by said reference standard and said one or more batches on one or more hallmarks of said pathological condition or of a pathological condition that can derive from said altered physiological state; and defining the acceptability values of the spectroscopy or spectrophotometry spectra as the variability range of the spectroscopy or spectrophotometry spectra values of all of said acceptable batches and of said reference standard batch, preferably refined by the spectra of said non-acceptable batches.
- 2. The method according to claim 1 for defining the acceptability values of a spectroscopy or spectrophotometry analysis for the compliance validation of one or more batches of a product for the treatment of a pathological condition, wherein the product comprises one or more natural matrices; said method comprising: (a) performing at least one in vitro cell-based assay on: (i) a reference standard batch of said product, wherein the reference standard has a known therapeutic effect for treatment of said pathological condition; and (ii) one or more test batches of said product; wherein the read-out of said cell-based assay is representative of the modulation of one or more biological activity associated with one or more hallmarks of said pathological condition; (b) quantifying in terms of numerical values the modulation of said one or more biological activities induced by each batch of step (a) for each cell-based assay read-out defining as reference values the values calculated for the reference standard batch; (c) defining as acceptable the test batches whose values calculated in (b) induce a modulation of each measured biological activity in said cell-based assay whose modulus is > to the modulus of the reference value calculated in (b) and defining as non-acceptable the batches whose values calculated in (b) induce a modulation of at least one of said biological activities in said cell-based assay whose modulus is less than the modulus of the reference value calculated in (b); (d) performing a spectroscopy or spectrophotometry on said reference standard batch and on said one or more different test batches; and (e) defining the acceptability values of the spectroscopy or spectrophotometry spectra as the variability range of the spectroscopy or spectrophotometry spectra values of all of said acceptable batches and of said reference standard batch, preferably refined by the spectra of said non-acceptable batches.
- 3. The method according to claim 2 further comprising determining for each of said one or more biological activities the modulation trend thereof for restoring a healthy physiological state.
- 4. The method according to claim 3 further comprising: in step (a) performing said in vitro cell-based assay on one or more reference drug for the treatment of said pathological condition; in step (b) quantifying in terms of numerical values the modulation induced in step (a) by each reference drug for each cell-based assay read-out and, for each biological activity modified by said reference standard and by said one or more reference drug according to said modulation trend for restoring a healthy state, comparing the value calculated for each reference drug and for the reference standard and selecting as reference value, the value associated to the weakest performance.
- 5. The method according to claim 1 for defining the acceptability values of a spectroscopy or spectrophotometry analysis for the compliance validation of one or more batches of a beneficial product for adjuvating homeostasis in an altered physiological state, wherein the product comprises one or more natural matrices; said method comprising: (a) performing at least one in vitro cell-based assay on: (i) a reference standard batch of said product, wherein the reference standard has a known beneficial effect for adjuvating homeostasis in an altered physiological condition; and (ii) on one or more test batches of said product; wherein the read-out of said cell-based assay is representative of the modulation of one or more biological activities associated with one or more hallmarks of a pathological condition that can develop from said altered physiological condition; (b) quantifying in terms of numerical values the modulation of said one or more biological activities induced by each batch of step (a) for each cell-based assay read-out defining as reference values the values calculated for the reference standard batch; (c) defining as acceptable the test batches whose values calculated in (b) induce a modulation of each measured biological activity in said cell-based assay whose modulus is > to the modulus of the reference value calculated in (b) and defining as non-acceptable the batches whose values calculated in (b) induce a modulation of at least one of said biological activities in said cell-based assay whose modulus is < to the modulus of the reference value calculated in (b); (d) performing a spectroscopy or spectrophotometry on said reference standard batch and on said one or more different test batches; and (e) defining the acceptability values of the spectroscopy or spectrophotometry spectra as the variability range of the spectroscopy or spectrophotometry spectra values of all of said acceptable batches and of said reference standard batch, preferably refined by the spectra of said non-acceptable batches.
- 6. The method according to claim 5 further comprising determining for each of said one or more biological activities the modulation trend thereof for restoring a healthy physiological state.
- 7. The method according to any one of claims 2 to 6 further comprising determining, for each of said biological activities, one or more parameters and the modulation trend thereof underlying the modification detectable in said pathological condition in both diseased and healthy physiological state.
- 8. The method according to claim 7 wherein said parameters are genes and modulation thereof.
- 9. The method of claim 6, wherein the modulation trend of said parameters is assessed by transcriptomic analysis.
- 10. The method of any one of claims 1 to 9, wherein the cell-based assay is designed to simulate conditions related to said pathological condition or to said altered physiological state.
- 11. The method according to any one of claims 1 to 10 wherein said one or more test batches are at least three test batches.
- 12. The method according to any one of claims 1 to 11 wherein said pathological condition comprises osteoarthritis, mild cognitive impairment, post menopausal osteoporosis or cancer.
- 13. The method according to any one of claims from 1 to 12 wherein said spectroscopy analysis is selected from NIR, FTIR, RAMAN and said spectrophotometry analysis is selected from Ultraviolet and visible light spectroscopy, fluorescence spectroscopy, light scattering.
- 14. A process for the compliance validation of one or more batches of a product for the treatment of a pathological condition or of a beneficial product, said product comprising one or more natural matrices, said method comprising the following steps: a. performing a spectroscopy or spectrophotometry analysis of each batch; b. validating each batch for which the obtained spectrum satisfies the acceptability values defined according to the method according to any one of claims 1 to 13.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IBPCT/IB2024/050280 | 2024-01-11 | ||
| IB2024050280 | 2024-01-11 | ||
| AU2024200219 | 2024-01-12 | ||
| AU2024200219 | 2024-01-12 | ||
| PCT/IB2024/054526 WO2025149786A1 (en) | 2024-01-11 | 2024-05-09 | Validation of natural matrices for therapeutic use on humans, animals and plants on bio-physical bases as an alternative to validation through chemically definable substances derived from said matrices by isolation and/or synthesis processes and also as an alternative to products comprising natural matrices validated on the basis of their traditional use. |
| AUPCT/IB2024/054526 | 2024-05-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2024203121B1 true AU2024203121B1 (en) | 2025-06-26 |
Family
ID=96094598
Family Applications (2)
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