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WO2008152669A2 - Vecteurs colloïdaux à structure polyaminoacide pour une libération orale de peptides et de protéines et leur méthode de production - Google Patents

Vecteurs colloïdaux à structure polyaminoacide pour une libération orale de peptides et de protéines et leur méthode de production Download PDF

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Publication number
WO2008152669A2
WO2008152669A2 PCT/IT2008/000376 IT2008000376W WO2008152669A2 WO 2008152669 A2 WO2008152669 A2 WO 2008152669A2 IT 2008000376 W IT2008000376 W IT 2008000376W WO 2008152669 A2 WO2008152669 A2 WO 2008152669A2
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groups
functionalities
polymer
phea
insulin
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WO2008152669A3 (fr
Inventor
Mariano Licciardi
Gaetano Giammona
Gennara Cavallaro
Giovanna Pitarresi
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Universita degli Studi di Palermo
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Universita degli Studi di Palermo
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention concerns colloidal vectors with polyaminoacid structure for oral release of peptides and proteins and a method for their production.
  • the invention concerns systems for the release of active substances, specifically peptides and proteins, by means of their incorporation in nanoparticles, nano-aggregates or complexes based on properly derivat- ized synthetic polyaminoacids.
  • These polymeric systems are proposed to release peptide drugs or proteins from oral dosage forms in an effective manner, besides increasing the physicochemical stability of proteins in liquid or solid pharmaceutical dosage forms.
  • the oral route is the administration route that offers the most significant advantages, including accuracy of dosage, an easy use and a high patients' compliance.
  • some drawbacks due to the administration of peptides and proteins by oral route are difficult to solve.
  • the transit in the gastrointestinal tract causes the con- tact of these molecules with physiological media whose pH value or enzymatic content are not compatible with their active form and their potential absorption.
  • the oral administration could be performed only by using gastro-resistant dosage forms and possibly with co-administration of peptidase enzyme inhibitors.
  • it must be considered that generally proteins and peptides have a high molecular weight and an adequate hydrophilic character. As a consequence they possess a reduced permeability across cell membranes and a low absorption.
  • insulin is entrapped into nanoparticles based on a polycyanoacrylate, prepared by a polymerization of the cyanoacry- late monomer at low pH and in the presence of insulin.
  • a polycyanoacrylate prepared by a polymerization of the cyanoacry- late monomer at low pH and in the presence of insulin.
  • the mucoadhesion of a polymer i.e. its ability to adhere to mucous membranes of the organism (and, in particular, to gastrointestinal mucosa) is an important property for the development of drug delivery systems, especially for peptides and proteins, since thanks to mucoadhesive properties it is possible to obtain a close contact between the polymeric systems and the mucous membrane, thus preventing the alteration of peptides and proteins in the gas- - A -
  • trointestinal tract In addition, it is possible to increase the residence time of peptides and proteins in the adsorption site, where the high concentration obtained will promote their absorption.
  • Thiolated polymers thiomers or thiopolymers
  • polymers where thiol groups are linked to the backbone represent an example of mucoadhe- sive polymers useful for the preparation of solid oral dosage forms [Bernkop- Schnurch A., Krauland A.H., Leitner V.M., Palmberger T., Thiomers: potential excipients for non-invasive peptide delivery systems, Eur. J. Pharm. Bio- pharm. 58 (2004), 253-263].
  • both ionic and neutral polymers can interact with proteins, thus forming macromolecular aggregates: the former polymers act through the formation of hydrogen bonds, van der Waal's and electrostatic interactions with the polar groups of the proteins; the latter polymers act mainly through hydrophobic interactions.
  • the presence of hydrophobic regions in a polymer can promote the interaction of a hydrophobic nature with the lipophilic portions of biological membranes, thus increasing the permeability of polymer-protein complex.
  • the preferred polyaminoacids re- ported in the patent are poly(Leu/Glu), with a ratio Leu/(Glu+l_eu) of not less than 6% for block polyaminoacids, and not less than 20% for statistical polyaminoacids, with a molecular weight not less than 4000 Da.
  • the fraction of leucine must be adequately high to avoid the complete water solubility of the polymer and to give it suit- able hydrophobic properties, in order to cause an association of polymeric chains to form nanoparticulate systems.
  • the obtained nanoparticles form a colloidal dispersion in aqueous medium, that can be loaded with the peptide drug.
  • particles containing the active ingredient can be obtained by dispersion of ⁇ -polyaminoacids in the solution containing the peptide drug, before aggregation.
  • the European patent EP 1131056 (Flamel Technologies), which corresponds to the US patent US 6630171 , discloses a similar release system, that has been called “Medusa ® " probably to remind the action mechanism of polymeric vectors in the incorporation of polypeptide substances, i.e. the ability to interact with the active ingredient and to "wrap up" the active ingredient, thus protecting it from degradation phenomena and carrying it as particulate form (nano or micro) until the action site.
  • the polyaminoacidic carriers that form the principal backbone of the particles are homopolymers or copolymers of glu- tamic acid (AcGIu) and/or aspartic acid (AcAsp) and their salts (recurring ami- noacids, both ionizable), and a percentage of them present a hydrophobic side chain, linked to the carboxyl group of AcGIu or AcAsp by ester (or amide) linkages.
  • the length in the polymeric chain ranges from 200 to 1000 aminoac- ids, i.e. with a molecular weight equal to 26,000-65,000 for an AcGIu ho- mopolymer and 23,000-57,000 for an AcAsp homopolymer.
  • the hydrophobic groups (R) linked to the polymeric backbone can be linear or branched carbon chains or they can contain double bonds, and preferably they are linear groups C 2 -C 18 .
  • the molar percentage of recurring ami- noacids derivatized with hydrophobic functions ranges from 3 to 70%.
  • Some examples of derivatized polymers reported in the patent are: sodium polyglu- tamate-ib/oc/c-exadecyl glutamate; sodium polyglutamate-co-ethyl glutamate; sodium polyaspatic acid-Jb/oc/c-propyl aspatate; sodium polyaspatic acid-b/oc/c- benzyl aspartate.
  • the preparation of the release particles based on the above polymers occurs by a decrease in solubility of the hydrophobic fraction, by adding a saline solution that causes the precipitation of the polymer with a consequent formation of nanoparticles.
  • a further aggregation step is made to occur, by using an aggregation agent, such as an electrolyte, an acid, a base or a ionic polymer (polylisine, polyethyleneimine).
  • the particles can be loaded with the active protein by performing their formation in a medium containing the active ingredient, or by dispersion and incubation of empty lyophilized particles in a medium where the active ingredient is dissolved or dispersed.
  • a main object of the present invention is thus to increase the oral bioavailability of peptide and protein active ingredients, e.g. insulin, by exploiting the ability of carrier copolymers to interact with the protein molecules, to reduce their degradation by digestive enzymes and gastric pH and to promote their oral absorption.
  • Another object of this invention is to employ the proposed copolymers as excipients for the production of oral or liquid dosage forms containing peptides or proteins in order to prolong their stability both in the dosage form and in physiological environment.
  • colloidal polymeric vectors both to increase the oral bioavailability thereof and to increase the physicochemical stability of said peptides and proteins in liquid or solid dos- age forms
  • the approach employed in the present invention is the synthesis of copolymers based on a poly-hydroxyethyl-aspartamide or PHEA, more exactly ⁇ , ⁇ - poly(N-2-hydroxyethyl)-D,L-aspartamide, having the following structure:
  • copolymers that present, in the side chains, hydrophobic, ionizable and thiol functionalities, covalently linked through the hydroxyl groups that are present one in each repeating unit of these polymers.
  • the obtained copolymers are able to form pharmaceutical systems for the release of peptides and proteins, through their incorporation into nanoparticles or nano-aggregates or complexes, formed by the interaction between these copolymers and the proteins or peptides at issue.
  • PHEA is a derivative of a high molecular weight polysuc- cinimide (PSI), obtained by aminolysis of the latter, by reaction with ethanola- mine.
  • PSI high molecular weight polysuc- cinimide
  • the coupling of ethanolamine can occur in such a way as to leave a methylene group either in the polymeric backbone or in the pendent functional group. Therefore, the repeating unit can have a structure slightly different in the first or in the second case, but its molecular weight is the same.
  • PHEA is a polymer with excellent biopharmaceutical properties, such as biocompatibility, in fact in the past it has been proposed as plasma expander [Neri P., Antoni G., Benvenuti F., Cocola F., Gazzei G., Synthesis of ⁇ , ⁇ -poly[(2-hydroxyethyl)-DL-aspartamide], a new plasma expander, J. Med.
  • the technology proposed by the present invention consists in incorporating peptides or proteins into nanoparticles, nano-aggregates or complexes based on synthetic linear polyaminoacids, specifically polyaspartamide-like polyaminoacids, properly derivatized with thiol, hydrophobic and ionizable groups.
  • the suitable combination of these functionalities in side chains of the copolymer confers to it the property to interact with the protein of interest, through non-covalent interactions, such as hydrophobic, electrostatic and hydrogen bonds, and to form supramolecular systems with colloidal size, having a high kinetic stability and mucoadhesive properties, besides all advantageous properties of thiomers, such as ability to act as protease inhibitors and permeation enhancers.
  • thiomers such as ability to act as protease inhibitors and permeation enhancers.
  • other polymers with polyaspartamide structure with nucleophilic pendent groups along the chain can be also used and properly derivatized with hydrophobic, ionizable and thiol functionalities, in such a way to result in compositions and materials according to this invention, each time with specific properties.
  • the present invention specifically provides a colloidal polymeric vector for the formulation and the release of peptides and proteins from nanoparticle, aggregate or complex dosage forms, which vector consists of a polymer or copolymer with polyaspartamide structure, carrying hydrophobic functionalities, ionisable functionalities and thiol functionalities in side chains, each individually or in combination with each other.
  • the proposed polymeric vectors are obtained starting from macro- molecules with polypeptide structure (specifically, polyaspartamide) having nucleophilic groups in each repeating unit and from their copolymers.
  • the moieties or functionalities reported above are covalently linked to these nu- oleophilic groups.
  • these moieties or functionalities are either of a hydrophobic, or ionizable or thiol type, and each of such functionalities can be linked to the polymeric backbone individually or in combination with other different functionalities.
  • said polymer or co- polymer with polyaspartamide structure is ⁇ , ⁇ -poly(N-2-hydroxyethyl)-D,L- aspartamide (PHEA) and its copolymers, carrying in side chains hydrophobic, ionizable and thiol functionalities, linked to the hydroxyl groups of the starting polymer through amide, enamine, urethane or ester bonds, as a function of the conjugated group.
  • PHEA poly(N-2-hydroxyethyl)-D,L- aspartamide
  • the length of the polyaminoacid chain is such that the molecular weight ranges from 10,000 to 60,000 dalton. In the preferred case of PHEA, this corresponds to a number of repeating units of hydroxyethyl- aspartamide ranging from 63 to 315.
  • the hydrophobic groups linked to the polymer backbone are preferably chosen in the group consisting of: linear, branched or cyclic alkyl with up to 18 carbon atoms; linear, branched or cyclic alkenyl with up to 18 carbon at- oms; linear, branched or cyclic alkynyl with up to 18 carbon atoms; an aromatic or isocyclic arylalkyl group with up to 18 carbon atoms.
  • these hydrophobic groups are chosen among: linear ethyl (C 2 ), propyl (C 3 ), butyl (C 4 ), dodecyl (C1 2 ) or hexa- decyl (Ci 6 ) groups; branched propyl (C 3 ), butyl (C 4 ), dodecyl (Ci 2 ) or hexade- cyl (Ci ⁇ ) groups; propyl (C 3 ), butyl (C 4 ), dodecyl (C12) or hexadecyl groups containing double bonds and aromatic groups
  • the molar ratio of molecules carrying the hydrophobic groups to moles of repeating units of the polymer ranges from 0.01 to 0.6.
  • the ionizable functionalities that preferably are linked to the polyas- partamide structure of this invention are made of: linear or branched alkyl, alkenyl or alkynyl groups, with a number of carbon atoms ranging from 2 to 20, carrying carboxyl functionalities (-COOH); groups carrying phosphoric functionalities (-PO 3 Ha), such as O-phosphocolamine; groups carryng sulfuric (-SO 4 H) or sulfonic (-SO 3 H) functionalities, such as taurine; groups carrying primary amines (-NH 2 ), groups carrying secondary amines (-NHR) or groups carrying tertiary amines (-NR 1 R") or groups carrying ammonium quaternary salts [- + N(R) 3 ].
  • the molar ratio between moles of molecules carrying the ionizable group and the moles of repeating units of the polymer ranges from 0,01 to 0,6.
  • the thiol functionalities linked to the polymeric backbone can be derived from thiolated molecules, such as cysteine, homocysteine, cysteamine and their derivatives (preferably cysteine).
  • the molar ratio between moles of molecules carrying the thiol group and moles of repeating units of the polymer ranges from 0.01 to 0.5.
  • the colloidal polymeric vector carrying, in the polyaspartamide chain, hydrophobic functionalities such as linear alkyl, ionizable functionalities such as carboxyl groups and thiol functionalities deriving from cysteine is made of the polymer ⁇ , ⁇ -poly(N-2-hydroxyethyl)-co-[N-2-(butylcarbamate)ethylen]-co-[N-2(2'car- boxysuccinyl)ethylen]-co-[N-2-(2'-L-cysteine-succinylamide)ethylen]aspart- amide).
  • This polymer has been called PHEA-C 4 -CS-CySt.
  • the present invention concerns a procedure for the production of the above-defined polymeric colloidal vectors, including the following sequence of steps: a) activation of reactive groups of a starting polymer with polyaspartamide structure by means of activating agents; b) reaction of the polymer thus activated with amines corresponding to desired hydrophobic functionalities in the side chains, by using a molar ratio between moles of amine and moles of repeating units of polymer ranging from 0.01 to 0.6; c) conjugation of the polymer obtained from the previous reaction with molecules carrying ionizable functionalities, by using a molar ratio between moles of ionizable functionalities and moles of repeating units of polymer ranging from 0.01 to 0.6; d) conjugation of the polymer obtained from the previous reaction with molecules carrying thiol groups, in the presence of activating agents, by using a molar ratio between moles of thiol groups and moles of repeat- ing units of polymer ranging from 0.1
  • the starting polyaspartamide is ⁇ , ⁇ -poly(N- 2-hydroxyethyl)-D,L-aspartamide or PHEA
  • the activating agents of step a) consist of bis (4- nitrophenyl)carbonate (4-NPBC) in dimethylformamide (DMF) solution, and this reaction is carried out at constant temperature, ranging from 25 to 60 0 C.
  • the activators of step b) consist of N-hydroxy- succinimide (NHS) and N-3(3-dimethylaminopropyl)-N-ethyl-carbodiimide hydrochloride (EDC-HCI) in aqueous solution, and this reaction is carried out at a temperature ranging from 15 and 40 0 C.
  • NHS N-hydroxy- succinimide
  • EDC-HCI N-3(3-dimethylaminopropyl)-N-ethyl-carbodiimide hydrochloride
  • the hydroxyl groups of PHEA have been activated by reaction with bis(4-nitrophenyl)carbonate (4-NPBC) in dimethylformamide (DMF) solution, at constant temperature (preferably between 25 and 60 °C).
  • DMF dimethylformamide
  • the reaction with butylamine by using a molar ratio between moles of butylamine and moles of repeating units of PHEA ranging from 0.01 to 0.6, allowed to derivatize the polymer with butyl groups in side chain, thus obtaining the copolymer PHEA-C 4 ( ⁇ , ⁇ -poly(N-2-hydroxyethyl)-co-[N-2- (butylcarbamate)ethylen]-aspartamide)
  • PHEA-C 4 copolymer after exhaustive dialysis against distilled water and Iy- ophilization.
  • the percentage of alkylamine functionalities linked to PHEA in the PHEA-C 4 copolymer is in the range between 1 and 60 mol % (by preference between 15 and 20 mol%).
  • the weight average molecular weight of the PHEA-C 4 derivative is comprised between 30 and 50 kDa, depending on the degree of derivatization, as determined by using a calibration curve obtained with PEO/PEG standards.
  • the PHEA-C 4 copolymer obtained as previously reported, reacted with succinic anhydride (SA) in DMF solution, in the presence of triethylamine (TEA) for a time period ranging from 4 to 24 hours, depending on the molar ratio between SA and PHEA repeating units, and at constant temperature, preferably between 15 and 40 0 C.
  • SA succinic anhydride
  • TAA triethylamine
  • the molar ratio between SA and PHEA repeating units can be comprised in the range between 0.01 and 0.6. In such a condition the still free hydroxyl groups of the PHEA-C 4 copolymer react easily with succinic anhydride, according to the scheme reported scheme below.
  • the resulting PHEA-C 4 -CS copolymer ( ⁇ , ⁇ -poly(N-2-hydroxyethyl)-co-[N-2- (butylcarbamate)ethylen]-co-[N-2(2'carboxysuccinyl)ethylen]-aspar- tamide) has been characterized by FT-IR e 1 H-NMR. Analytical and spectral data of copolymer confirmed the introduction of succinic group into the copolymer.
  • the percentage of succinic functionalities linked to PHEA-C 4 -CS copolymer is in the range between 1 and 60 mol% (preferably between 30 and 50 mol%).
  • the weight average molecular weight of PHEA-C 4 -CS copolymer, determined by size exclusion chromatography (SEC) is included between 30 and 50 kDa, depending on the degree of derivatization, by using a calibration curve obtained with PEO/PEG standards.
  • the obtained PHEA-C 4 -CS copolymer reacted with cysteine, in the presence of N-hydroxysuccinimide (NHS) and N-3(3-dimethylaminopropyl)-N-ethyl-carbodiimmide hydrochloride (EDC-HCI) in aqueous solution, for a period time ranging from 1 to 18 hours, depending on the molar ratio between cysteine and succinic groups linked to PHEA, at constant temperature, preferably between 15 and 40 0 C.
  • NHS N-hydroxysuccinimide
  • EDC-HCI N-3(3-dimethylaminopropyl)-N-ethyl-carbodiimmide hydrochloride
  • the molar ratio between cysteine and succinic acid groups linked to PHEA can be in the range between 0.5 and 2, corresponding to a molar ratio between thiol- bearing molecules and copolymer repeating units in the range between 0.1 and 0.8.
  • the PHEA-C 4 -CS-CySt copolymer After purification by extaustive dialysis against distilled water, the PHEA-C 4 -CS-CySt copolymer has been characterized by FT-IR and 1 H-NMR.
  • the percentage of thiol groups linked in the PHEA-C 4 -CS-CySt copolymer (expressed as moles of linked cysteine/moles of copolymer repeating units x 100), has been determined by Ellman assay, allowing the evaluation of thiol group amount (SH) and then the percentage of cysteine linked to copolymer. This percentage is from 1 to 50 mol % (the most preferred being 10 % of repeating units of PHEA i.e. 30 % of succinic groups).
  • the weight average molecular weight of PHEA-C 4 -CS-CySt copolymer, determined by size exclusion chromatography (SEC) is between 30 and 50 kDa, depending on the degree of derivatization, by using a calibration curve obtained with PEO/PEG molecular standards.
  • the present invention concerns pharmaceutical products obtained by loading the proposed polymeric vectors with active ingredients preferably, but not exclusively, of protein or peptide nature. Therefore, the invention concerns a pharmaceutical composition for the delivery of one or more active ingredients, specifically with protein or pep- tide structure, in the form of nanoparticles, aggregates or complexes, wherein said one or more active ingredients are carried by a polymeric colloidal vectors as defined above.
  • the proposed pharmaceutical formulation can advantageously be used for oral administration.
  • the said one or more active ingredients incorpo- rated into polymeric vectors according to the present invention can be formulated together with one or more pharmaceutical excipients acceptable for oral administration.
  • the present invention further concerns pharmaceutical formulations containing one or more active agents with peptide or protein structure as above defined, administered as liquid or solid dosage forms.
  • copolymers object of the present invention allows to protect them from both chemical and enzymatic degradation and to release them in an intact form in the administration site, thus offering the advantages of a minor degradation of the protein drug and of a greater absorption.
  • Polymeric delivery systems according to this invention have shown to be able to increase oral bioavailability of insulin, used as model protein, and to induce a decrease of the plasmatic glucose levels in the rats equal to 30 % of the ipoglicemic effect obtained after subcutaneous administration of insulin. Moreover, these vectors have not evidenced any in vitro citotoxicity and showed a very good in vivo tolerability on the animal models used. In addition, the proposed systems have been able to increase chemical stability of insulin in the presence of proteolytic enzymes such as ⁇ -chimitripsin, and in media mimicking gastrointestinal fluids.
  • the use of the vectors according to the present invention therefore, allows to increase the oral bioavailability of peptide and protein drugs, such as for example insulin, and to prolong the stability of peptide and protein molecules when administered in the presence of these copolymers as excipients, used in either liquid or solid formulations.
  • peptide and protein drugs such as for example insulin
  • Figure 1 shows the cell availability profile, evaluated by MTS test, on intestinal epithelial cells after 24 hours of incubation with increasing concentration (0,1 , 0,5 e 1 mg/ml) of PHEA-C 4 -CS and PHEA-C 4 -CS-CySt copolymers;
  • Figure 2 shows the transmittance profile as a function of time of insulin, PHEA-C 4 and insulin/PHEA-C 4 complex sample solutions at different weight ratios (1/1 , 1/3, 1/6);
  • Figure 3 shows the transmittance profile as a function of time of insulin, PHEA-C 4 -CS and insulin/PHEA-C 4 -CS complex sample solutions at different weight ratios (1/1, 1/3, 1/6);
  • Figure 4 shows the transmittance profile as a function of time of insu- lin, PHEA-C 4 -CS-CySt and insulin/PHEA-C 4 -CS-Cyst complex sample solutions at different weight ratios (1/1 , 1/3, 1/6);
  • Figure 5 shows the agarose gel electrophoresis of insulin and PHEA- C 4 /insulin, PHEA-C 4 -CS/insulin, PHEA-C 4 -CS-Cyst/insulin complexes to confirm the structural integrity of complexed protein;
  • Figure 6 shows the percentage of intact insulin released as a function of time from insulin/copolymer complexes after incubation in the presence of ⁇ -chymotrypsin;
  • Figure 7 shows the percentage of insulin released from tablets containing different insulin/copolymer complexes in media mimicking gastrointestinal fluids (pH 1 , up to 120 min, pH 6.8 from 121 to 360 min);
  • Figure 8 shows the glycemic profile of rats after administration of: insulin/PHEA-C 4 -CS complex (insulin/P1 os), not complexed insulin (free oral, insulin os); free copolymer (P1 os) and subcutaneous insulin (insulin sc) as positive control;
  • Figures 9 a) and b) show the observation under UV transilluminator of rat intestin segments after oral administration of FITC-insulin/PH EA-C 4 - CS complexes;
  • Figures 10 a), b) and c) show confocal laser scanning microscope images of rat intestinal tissues after oral administration of FITC- insulin/PHEA-C 4 -CS complexes.
  • the copolymers object of the present invention have been subjected to in vitro biocompatibility studies on human intestinal epithelial cells to evaluate the possible toxic effects by MTS test.
  • cell availability data after 24 hours of incubation with copolymer concentration of 0,1 , 0,5 and 1 mg/ml have not shown any cytotoxic effects on the cellular model used.
  • Turbidimetric studies have been performed by recording the transmit- tance at 500 nm of insulin, single copolymers and insulin/copolymer complexes at different weight ratios in buffer solution at pH 7.4.
  • the insulin/ PHEA-C 4 or insulin/PHEA-C 4 -CS complexes show a transmittance decrease as a function of time. This result is much more pronounced in the case of the insulin/ PHEA- C 4 -CS-Cyst complex, with a strong trasmittance decrease as a function of time (up to 24 hours) and as a function of the insulin/copolymer ratio. Transmittance results as a function of time are reported in Figures 2, 3 and 4.
  • Insulin/PHEA-C 4 and insulin/ PHEA-C 4 -CS complexes showed a smaller size than free insulin, thus suggesting the occurrence of protein condensation processes due to the interaction with copolymers. Such a behaviour seems to be depending on the time for PHEA-C 4 -CS-CySt copolymer. In fact the complex size decreases strongly as a function of time, probably due to the occurrence of S-S bridges between copolymer chains.
  • zeta potential measurements show a decrease of the complex surface potential in comparison with naked insulin; that confirms a screening effect on the surface charge of the insulin molecule after the com- plexation with the copolymer.
  • copolymers according with the present invention to improve the stability of protein molecules to intestinal degradation, has been evaluated after incubation of the protein/copoiymer complexes, previously prepared and lyophilised, in the presence of ⁇ -chymotrypsin and evaluating, at regular time intervals the percentage of intact protein in the reaction medium.
  • the copolymers object of the present invention show a protection effect reducing the enzymatic degradation of insulin in the presence of ⁇ -chymotrypsin.
  • This effect of protection appears to increase in the order PHEA-C4 ⁇ PHEA-C 4 -CS ⁇ PHEA-C 4 -CS-CySt.
  • the presence of insulin in the intact form is recorded until 140 min from the beginning of the enzymatic incubation.
  • naked insulin under- goes complete degradation in the presence of ⁇ -chymotrypsin within 40 min.
  • results show a slow release of intact insulin from the tablets containing the insulin/copolymer complexes.
  • a delayed release is particularly evident in the cases of lnsulin/PHEA-C 4 -CS and insulin/PHEA-C-rCS-Cyst.
  • the animals were divided in four groups of 3 animals each; each group was treated separately with different dosage forms, upon sedative administration of medetomidine (Domitor) at the dose suggested by the pharmaceutical company.
  • Domitor medetomidine
  • the dosage form containing just inert excipients without insulin has been administered
  • insulin sc a sterile physiological solution containing 2 mg (56 USP units) ( ⁇ 8 mg/kg) of bovine insulin (spleen, Sigma) has been given by subcutaneous administration (positive control).
  • insulin/Pi os the formulation insuline/PHEA-C 4 -CS complex, having 2 mg (56 USP units) ( ⁇ 8 mg/kg) of bovine insulin (pancreas, Sigma) has been given.
  • insulin os 200 ⁇ l of an aqueous solution of insulin containing 2 mg (56 USP units) ( ⁇ 8 mg/kg) of bovine insulin (pancreas, Sigma), has been administered by the oral route (negative control).
  • Fluorescein(FITC)-labeled insulin complexed with PHEA-C 4 -CS was filled into capsules to yield 2.0 mg of FITC-insulin per capsule and administered by placing the capsules deeply into the throat, in order to initiate the swallow-reflex to Wistar rats fasted overnight (3 rats).
  • the rats were sacrificed 3 h later and intestinal segments localized after laparotomy by observation under UV transilluminator. After washing with isotonic saline, intestinal epithelial cell membranes were stained with formalin (10% paraformaldehyde in PBS pH 7.4) for 24 h. Tissues were rinsed and vertical sections were prepared, mounted on glass slides and observed using a confocal laser scanning micro- scope.
  • Control rats (3 rats) were dosed with FITC-insulin or PHEA-C 4 - CS containing capsules.

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  • Crystallography & Structural Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Nutrition Science (AREA)
  • Physiology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Optics & Photonics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des systèmes d'administration pour la libération de principes actifs, en particulier des peptides et des protéines, par incorporation de ceux-ci dans des nanoparticules, des nano-agrégats ou des complexes à base de polyaminoacides synthétiques dérivés de manière appropriée, comprenant des copolymères et un squelette polyaspartamide (en particulier, alpha bêta-poly-hydroxyéthyl-aspartamide ou PHEA) pour lesquels les fonctions hydrophobes, ionisables et thiol dans les chaînes latérales sont liées par covalence, seules ou en combinaison. Lesdits systèmes polymères permettent de libérer des médicaments peptidiques ou des protéines, comme de l'insuline, à partir de formes posologiques orales de manière efficace, et afin d'augmenter la stabilité physicochimique de protéines dans des formes posologiques liquides ou solides.
PCT/IT2008/000376 2007-06-11 2008-06-04 Vecteurs colloïdaux à structure polyaminoacide pour une libération orale de peptides et de protéines et leur méthode de production Ceased WO2008152669A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITRM20070327 ITRM20070327A1 (it) 2007-06-11 2007-06-11 Vettori colloidali a struttura poliamminoacidica per il rilascio orale di peptidi e proteine e relativo metodo di produzione.
ITRM2007A000327 2007-06-11

Publications (2)

Publication Number Publication Date
WO2008152669A2 true WO2008152669A2 (fr) 2008-12-18
WO2008152669A3 WO2008152669A3 (fr) 2009-02-12

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PCT/IT2008/000376 Ceased WO2008152669A2 (fr) 2007-06-11 2008-06-04 Vecteurs colloïdaux à structure polyaminoacide pour une libération orale de peptides et de protéines et leur méthode de production

Country Status (2)

Country Link
IT (1) ITRM20070327A1 (fr)
WO (1) WO2008152669A2 (fr)

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19810965A1 (de) * 1998-03-13 1999-09-16 Aventis Res & Tech Gmbh & Co Nanopartikel, Verfahren zu ihrer Herstellung und ihre Verwendung
MXPA01010751A (es) * 1999-04-23 2002-05-14 Alza Corp Enlace liberable y composiciones que contienen el mismo.
HUP0400171A2 (en) * 2001-06-01 2007-02-28 Astellas Pharma Europ B V Lipid-polymer-conjugates
WO2003002096A1 (fr) * 2001-06-28 2003-01-09 Wisconsin Alumni Research Foundation Procedes et compositions d'antibiotiques de la famille des polyenes a toxicite reduite
WO2007034479A2 (fr) * 2005-09-20 2007-03-29 Yissum Research Development Company Nanoparticules pour administration ciblee de principes actifs
ITMI20060494A1 (it) * 2006-03-17 2007-09-18 Sifi Spa Composizione farmaceutica oftalmica contenente copolimeri anfifilici della poliaspartammide

Also Published As

Publication number Publication date
ITRM20070327A1 (it) 2008-12-12
WO2008152669A3 (fr) 2009-02-12

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