DE102022004596A1 - Novel cannabinoid oligosaccharides - Google Patents

Abstract

The invention relates to methods for the preparation and isolation of cannabinoid oligosaccharides, demonstrated by cannabidiol oligosaccharides, based on the use of specific plant UDP-glucose-dependent glycosyltransferases of the UGT73a class in a first step and of cyclomaltodextrin glucanotransferase(s) and/or glucansucrases in a second, to enable the formation of a cannabinoid oligosaccharide with an amphiphilic structure typical of water-soluble compounds and of emulsifiers/solubilizers. Preferred embodiments are methods based on heterologous expression in microorganisms such as E. coli or in Cannabis sativa; processes based on heterologous expression in Cannabis sativa can use the glucosyl residues as protecting groups to prevent tetrahydrocannabinol biosynthesis and offer the option of aqueous extraction as a first production step. The invention further relates to compositions obtained by these methods, methods for isolating cannabidiol oligosaccharides from such compositions and the use of these cannabinoid oligosaccharides as active ingredients. Concentrated solutions of active ingredients as pharmaceuticals or as veterinary medicine are also an advantageous embodiment of the invention. Glycosidases, in particular β-glucosidases, and amylases can be used together with cannabinoid oligosaccharides in various forms to trigger the release of aglycone. The differences in the enzymatic degradability of α- versus β-D-glucopyranosyl residues in cannabinoid oligosaccharides by amylases and β-glucosidases enable targeted release of active ingredients.

Description

The invention describes novel oligosaccharides of cannabidiol and other cannabinoids and specific processes for their preparation, isolation and applications facilitated by their specific properties.

State of the art

Cannabinoids are secondary metabolites of hemp (Cannabis sativa) and are prenylated phenols. The most important cannabinoid compounds are Δ ⁹ -tetrahydrocannabinol (THC), cannabidiol (CBD) ( ), cannabichromene (CBC) and cannabigerol (CBG). Cannabinol (CBN) is known from some hemp varieties and is derived from THC. These lead cannabinoid compounds vary in their respective relative proportions in different hemp varieties. In addition to neutral cannabinoids, cannabinoid acids with carboxyl groups occur and dominate in fresh plant material. Acidic cannabinoids are precursors of cannabinoids and can also be converted into the latter non-enzymatically. Up to 60 different cannabinoid compounds are known from hemp varieties. Non-acidic cannabinoids are lipophilic compounds, often with a phenolic structure and mainly C ₂₁ compounds. Parts of the structure come from fatty acid and isoprenoid biosynthesis as well as the polyketide pathway.

Various forms of cannabinoids are consumed around the world, either as herb (marijuana) or in resin form (hash oil), by an estimated 2.6 - 5.0% of the world's population (UNODC, 2012). Cannabinoid-containing pharmaceutical products are available for medicinal use in several countries and contain either natural cannabis extracts (Sativex ^® ) or synthetic cannabinoids (dronabinol, nabilone). THC from Cannabis sativa has been approved by the Food and Drug Administration (FDA) to treat nausea and vomiting associated with chemotherapy and, more recently, to stimulate appetite in AIDS patients. In Germany, cannabis flowers ("cannabis flos") are used pharmaceutically, dronabinol can be prescribed as a formulation with certain treatments. Other biological activities allow therapeutic applications such as in the treatment of glaucoma, migraine, spasticity, anxiety and as an analgesic.

It is well documented that active ingredients such as cannabinoids and endocannabinoids, which activate cannabinoid receptors in the body, modulate appetite and reduce nausea, vomiting, pain ( Martin BR and Wiley, JL, Mechanism of action of cannabinoids: how it may lead to treatment of cachexia, vomiting and pain, Journal of Supportive Oncology 2: 1-10, 2004 ), Multiple sclerosis ( Pertwee, RG, Cannabinoids, and multiple sclerosis, Pharmacol. Ther. 95, 165-174, 2002 ) and relieve epilepsy ( Wallace, MJ, Blair, RE, Falenski, KWW, Martin, BR, and DeLorenzo, RJ, Journal Pharmacology and Experimental Therapeutics, 307: 129-137, 2003 ) .

In addition, CB2 receptor agonists have been shown to be effective in treating pain ( Clayton N., Marshall FH, Bountra C., O'Shaughnessy CT, 2002. CB1 and CB2 cannabinoid receptors are implicated in inflammatory pain. 96, 253 - 260 ; Malan TP, Ibrahim MM, Van derah TW, Makriyannis A., Porreca F., 2002. Inhibition of pain responses by activation of CB (2) cannabinoid receptors. Chemistry and Physics of Lipids 121, 191 - 200 ; Malan TP, Jr., Ibrahim MM, Deng H., Liu Q., Mata HP, Vanderah T., Porreca F., Makriyannis A., 2001. CB2 cannabinoid receptor - mediated peripheral antinociception. 93, 239 - 245 .; Quartilho A., Mata HP, Ibrahim MM, Van derah TW, Porreca F, Makriyannis A, Malan TP, Jr., 2003. Inhibition of inflammatory hyperalgesia by activation of peripheral CB2 cannabinoid receptors. Anesthesiology 99, 955 - 960 ) and multiple sclerosis ( Pertwee, RG Cannabinoids and multiple sclerosis, Pharmacol. Ther. 95, 165 174, 2002 ) in animal models.

Recently, the approval of cannabis and cannabinoid products for both recreational and medicinal purposes has been expanded. The development of more efficient production and isolation of cannabinoid compounds is needed. Traditional methods of cannabinoid production typically focus on the extraction and purification of cannabinoids from raw harvested cannabis. However, conventional cannabinoid extraction and purification processes face technical and practical problems, mainly the need for non-aqueous solvents (organic solvents ( US Patent No. 6,403,126 (Webster et al.); US Patent App. No. . 20160326130 (Lekhram et al.); butane) or supercritical CO ₂ ( CA2424356 (Muller et al.)) for the extraction and purification of lipophilic cannabinoids. An analogous demand for water-soluble forms of cannabinoids arises in their application, mainly due to instability (short shelf life) and turbidity of emulsions of such water-based products.

Glycosides of cannabinoids were investigated.

Tanaka et al. (Journal of Natural Products (1993) 56(12): 2068-2072 ) described glucosides of cannabinol by biotransformation with in vitro plant tissue.

Tanaka et al. (Plant Cell Reports (1996) 15: 819-823) described β-D-glucopyranosides of cannabidiol and cannabidiolic acid obtained by biotransformation with plant tissue of Pinellia ternata.

The US20190085347A1 disclosed “high level in vivo biosynthesis and isolation of water-soluble cannabinoids in plant systems”. The water solubility of cannabinoids is realized in a final biosynthetic step via the glucosylation of cannabinoids by heterologous expression of suitable plant UDP-dependent glycosyl- or glucuronosyltransferases, which produce β-D-glucopyranosides or β-D-glucuronopyranosides. WO2020239784A1 describes “genetically modified host cells producing glycosylated cannabinoids”, which are based on UDP-dependent glycosyltransferases and therefore always produce β-D-glycosides, with the exception of one example of an α-L-rhamnoside, the rare representative of L-carbohydrates.

The WO2021146687A1 claims “a cannaboside composition and method to produce” by feeding it to animals (particularly insects/orthopterans, crickets). Their description includes β-D-glucopyranosides of cannabinoid compounds obtained with up to six glucosidic residues.

WO2021173130A1 discloses “novel cannabinoid glycosides and uses thereof”. The cannabinoid glucopyranoside structures given are all β-anomers with up to four glucopyranoside residues.

The WO2022099078A1 describes the “production of glycosylated cannabinoids” via UDP-dependent glycosyltransferases from Arabidopsis thaliana and Helianthus annuus.

WO2022126028A1 reports on a “continuous enzymatic perfusion reactor system” for the production of cannabinoid glycosides.

In summary, all reports focus on the production and/or handling of β-D-glycosides, mostly β-D-glucopyranosides of cannabinoids with mainly one to four carbohydrate units and various connections between carbohydrates (1-3, 1-4, 1-6).

A general and important requirement for cannabinoid glycosides is their degradability by enzymatic hydrolysis, such as by the widely used β-glucosidases, after consumption of the cannabinoid-containing product.

Object of the invention

The object of the invention was to find an efficient process for the production of highly glucosylated cannabidiol derivatives that show superior water solubility, but also have a structure typical of surfactants/solubilizers, designed to enable solubilization or emulsification of less glycosylated or free cannabinoids and also with a structure that allows enzymatic cleavage. This would enable the development of superior isolation processes and facilitate new and improved forms of application of cannabinoids. This should be illustrated with cannabidiol as the leading cannabinoid alongside THC.

The invention had to solve the problem of producing such highly glucosylated cannabinoid derivatives that should also be degradable by enzymes when consumed by humans. Beyond the state of the art, the requirement for water-soluble cannabinoid derivatives would also be met by α-D-glucopyranosides of cannabinoids. In addition, α-D-glucopyranosides can be hydrolyzed by amylases, typical enzymes of the human digestive tract. With the aim of very good water solubility and a structure typical of surfactants/solubilizers, a production process for highly glucosylated cannabinoid derivatives had to be found. Cyclomaltodextrin glucanotransferases are a well-known class of enzymes. They not only produce α-, β-, γ- (6-, 7-, 8-membered rings) or higher cyclodextrins from starch, but also carry out transglycosylation reactions with carbohydrate acceptors and also hydrolysis with water as an acceptor ( https:/ /www.cazypedia.org/index.php/Glycoside_Hydrolase_Family_13 ). However, this is mainly reported for the transfer to other carbohydrates/oligosaccharides/polysaccharides, with only low activities reported for the direct transglycosylation of non-carbohydrate acceptor substrates. In contrast to UDP-α-glucose-dependent glycosyltransferases, the stereochemistry at the carbohydrate is not converted by the substitution mechanism to build up -glucopyranosides, but is maintained by a double substitution with an enzyme-linked intermediate. Consequently, with (1-4)-α-D-glucopyranosyl residues as co-substrate, α-D-glucopyranosides are formed by cyclomaltodextrins by cyclomaltodextrin glucanotransferases. Some reactions with these enzymes such as partial transglycosylation, partial re-transglycosylation (disproportionation) or partial hydrolysis (e.g. Ara et al. Glycobiology, 2015, Vol. 25, No. 5, 514-523, doi: 10.1093/glycob/cwu182 ) can lead to a smaller number of (1-4)-α-D-glucopyranosyl residues than expected by simple transfer of a cyclodextrin residue. Such a smaller number of α-D-glucopyranosyl residues can also be obtained by transglycosylation reactions of glucansucrases (https://www.cazypedia.org/index.php/Glycoside_Hydrolase_Family_70) that use disaccharides, mainly sucrose, instead of cyclodextrins as co-substrate.

For steviol glycosides and rebaudioside A, transglycosylation of natural glucosides as substrates has been shown to improve the taste quality of the derived compounds ( Muñoz-Labrador et al. Foods 2020, 9, 1753; doi:10.3390/foods9121753 ). Given this substrate acceptance and that of UDP-dependent glycosyltransferases (once an enzyme with a suitable specificity for the aglycone was identified), a possible strategy seemed to be to obtain highly glycosylated cannabinoid derivatives via an initial and direct parent glycosylation by UDP-dependent glycosyltransferases to β-D-glucopyranosides and a subsequent and stepwise transfer of six to eight units (by cyclomaltodextrin glucanotransferases) or of single (by glucansucrases) (1-4) α-D-glucopyranoside residues. The glucansucrases from Liquorilactobacillus nagelii and Liquorilactobacillus hordei used for the invention are known as dextransucrases ( Bechtner et al. 2022, Gels 2022, 8, 171. https://doi.org/10.3390/gels8030171 ) .

This strategy would enable the formation of cannabinoid oligosaccharides with the intended mixed β/α structure.

Description of the invention

Surprisingly, it was found that cannabinoids such as cannabidiol ( above) with only small numbers of β-D-glucopyranosyl residues, for example CBD-1βG or CBD-2βG ( below), are very good substrates for cyclodextrin glucanotransferases. CBD-2βG was chosen for demonstration because it carries β-D-glucopyranosyl residues on its two hydroxy functions and is therefore the simplest representative structure of this type. This allows the specific transfer of groups of six, seven or eight (or higher number) of (1-4)-α-D-glucopyranosyl residues from either cyclodextrins, substituted cyclodextrins (such as hydroxypropyl, methyl) or a mixture of such residues from linear substrate with polymeric (1-4)-α-D-glucopyranosyl structure such as starch or glycogen ( ). Therefore, highly glycosylated forms of cannabinoids are to be obtained that have an inherent and intended amphiphilic structure typical of surfactants/solubilizers and a mixed anomeric structure (β/α). For such, not only very good water solubility is to be expected, but also solubilizing effects on compounds with lower water solubility such as cannabinoids or cannabinoids with a lower glycosylation pattern, but also a variation in the mechanisms of aglycone release. Like the β-D-glucopyranosyl residues by means of β-D-glucosidases, α-D-glucopyranosyl residues can be cleaved by the activity of amylases, such as those found in the human digestive tract (saliva, small intestine), which enables a gradual and presumably more targeted degradation or release of the aglycone for the purpose of targeted drug delivery. A positive effect on the taste profile of products also seems to be achievable. Such derivatives with long hydrophilic residues (such as well-known polysorbates) have optimal properties for use in pharmaceuticals and other areas for various water-based products.

Definitions

• Amylase Spezifische Glycosidasen, die (1-4)-α-D-Oligo- oder Polysaccharide wie Stärke oder Verbindungen mit (1-4)-α-verknüpften Oligosaccharidresten hydrolysieren. Das griechische Präfix bezieht sich auf Enzymunterklassen, nicht auf Anomere von Glycosiden. α-Amylasen spalten an zufälligen Positionen und produzieren Dextrine ((1-4)α-verknüpfte Oligosaccharide), Maltotriose (Trisaccharideinheiten aus drei 1-4-verknüpften α-D-Glucosemolekülen), Maltose (Disaccharideinheiten aus zwei 1-4-verknüpften α-D-Glucosemoleküle) und Glucose. β-Amylasen spalten terminal Maltose und γ-Amylasen terminal Glucosemonosaccharideinheiten ab. α-Amylasen kommen in Tieren (Speicheldrüsen, Bauchspeicheldrüse), Pflanzen, Mikroben vor. β-Amylasen kommen in Pflanzen (Samen, Früchte) und Mikroben vor, γ-Amylasen kommen in Tieren (Dünndarm) und Mikroben vor.
• Anomere Diastereomere von Kohlenhydraten, hier insbesondere von D-Glucose, bezogen auf die acetalische Hydroxyfunktion am C-Atom 1 in einem Glucosid, entsprechend der α- oder β-Form des Glucosids.
• Amphiphil Moleküle mit sowohl hydrophilen als auch lipophilen Anteilen werden als amphiphil bezeichnet.
• Cannabidiol 2-[(1R,6R)-3-Methyl-6-prop-1-en-2-yl-1-cyclohex-2-enyl]-5-pentylbenzo-1,3-diol
• Cannabinoide Wie hier verwendet, bezieht der Begriff sich auf für Hanf (Cannabis sativa) spezifische Sekundärmetaboliten, bei denen es sich um prenylierte Phenole handelt.
• Cannabinoid-Oligosaccharide Der hier verwendete Begriff bezieht sich auf Cannabinoide mit einem Muster aus einem anfänglichen Stamm von null bis vier β-D-Glucopyranosidylresten und einem oder mehreren zusätzlichen ((1-4)-α-D-Glucopyranosyl)5,6, 7 oder > 7(1-4)-α-D-Glucopyranosid-Oligosaccharidrest(en). Eine zentrale beispielhafte Struktur ist in dargestellt.
  • Gestaffelte Cannabinoid-Oligosaccharide Cannabinoid-Oligosaccharide können eine geringere Anzahl von (1-4)-α-D-Glucopyranosylresten besitzen, als durch einfachen Cyclodextrinrest-Transfer erwartet, durch teilweise Transglykosylierung, teilweise Re-Transglykosylierung (Disproportionierung) oder teilweise Hydrolyse. Sie können auch durch die Verwendung von Glucansucrase-Enzymen und Disacchariden wie Saccharose als Co-Substrat erhalten werden und können in diesem Fall nicht nur (1-4)-α-verknüpft sein, sondern auch (1-2)-, (1 -3)- oder (1-6)-α-verknüpft.
• Cyclomaltodextringlucanotransferase Transferasen, die ((1-4)-α-D-Glucopyranosyl)5,6,7 oder >7(1-4)-α-D-Glucopyranosid-Oligosaccharide von α-, β-, γ- oder höheren Cyclodextrinen übertragen (griech Präfix bezieht sich hier auf die Art des Cyclodextrins), durch Ringöffnung oder aus Stärke durch Helixöffnung. Die Definition folgt https://www.cazypedia.org/index.php/Glycoside_Hydrola se_Family_13
• Glycosid Chemische Struktur, die aus einem Kohlenhydrat mit einem Alkohol, einem Amin oder einem Thiol gebildet wird (entspricht O-, N- oder S-Glycosiden). Hier werden nur O-Glykoside betrachtet.
• Glucansucrase Familie GH70-Glucansucrase (GS)-Enzyme katalysieren die Synthese von α-Glucanen aus Saccharose. Die Definition folgt https://www.cazypedia.org/index.php/Glycoside_Hydrola se_Family_7 Glucansaccharasen können Alterane α-(1-3), (1-6), Dextrane α-(1-6), Reuterane α-(1-4) oder Mutane α-(1-3) bilden.
• Glucosid Spezifisches Glycosid mit Glucose als gebundenem Kohlenhydrat. Wie bei Glykosiden werden α- und β-D-Glucoside unterschieden, abhängig von der Position des die glykosidische Bindung bildenden Sauerstoffatoms in Bezug auf das Kohlenhydrat in Standardprojektion (α-Glucosid: Sauerstoff nach oben in Bezug auf Ring, bei β-Glucosiden nach unten) (andere Definitionen beziehen sich auf die Konfiguration von C5).
• Glycosidase Enzym mit hydrolytischer Aktivität für Glycoside, dies ist eine Spaltung von Glycosiden unter Aufnahme eines Wassermoleküls. β-Glycosidasen können β-Glycoside wie β-Glucoside spalten. Glucosidasen sind spezielle Glycosidasen oder spezifische Aktivitäten von solchen.
• Lipophil Mit Affinität zu Fett bzw. Öl. Der Begriff beschreibt Moleküle mit geringer Polarität.
• Transglycosylierung Die Übertragung von Kohlenhydratresten von einem Glycosid auf ein anderes.

For the description of the invention, the terms are understood as follows:

• Amylase Specific glycosidases that hydrolyze (1-4)-α-D-oligo- or polysaccharides such as starch or compounds with (1-4)-α-linked oligosaccharide residues. The Greek prefix refers to enzyme subclasses, not to anomers of glycosides. α-Amylases cleave at random positions and produce dextrins ((1-4)α-linked oligosaccharides), maltotriose (trisaccharide units made up of three 1-4-linked α-D-glucose molecules), maltose (disaccharide units made up of two 1-4-linked α-D-glucose molecules) and glucose. β-amylases break down terminal maltose and γ-amylases break down terminal glucose monosaccharide units. α-amylases are found in animals (salivary glands, pancreas), plants and microbes. β-amylases are found in plants (seeds, fruits) and microbes, γ-amylases are found in animals (small intestine) and microbes.
• Anomeric diastereomers of carbohydrates, here in particular of D-glucose, based on the acetal hydroxy function at C-atom 1 in a glucoside, corresponding to the α- or β-form of the glucoside.
• Amphiphilic Molecules with both hydrophilic and lipophilic parts are called amphiphilic.
• Cannabidiol 2-[(1R,6R)-3-methyl-6-prop-1-en-2-yl-1-cyclohex-2-enyl]-5-pentylbenzo-1,3-diol
• Cannabinoids As used here, the term refers to hemp (Cannabis sativa) specific secondary metabolites, which are prenylated phenols.
• Cannabinoid oligosaccharides As used herein, cannabinoids with a pattern of an initial stem of zero to four β-D-glucopyranosidyl residues and one or more additional ((1-4)-α-D-glucopyranosyl)5,6, 7 or > 7(1-4)-α-D-glucopyranoside oligosaccharide residues are described. A key exemplary structure is shown in shown.
  • Staggered Cannabinoid Oligosaccharides Cannabinoid oligosaccharides can possess a smaller number of (1-4)-α-D-glucopyranosyl residues than expected by simple cyclodextrin residue transfer, by partial transglycosylation, partial re-transglycosylation (disproportionation) or partial hydrolysis. They can also be obtained by using glucansucrase enzymes and disaccharides such as sucrose as co-substrate and in this case can be not only (1-4)-α-linked, but also (1-2)-, (1-3)- or (1-6)-α-linked.
• Cyclomaltodextrin glucanotransferase Transferases that transfer ((1-4)-α-D-glucopyranosyl)5,6,7 or >7(1-4)-α-D-glucopyranoside oligosaccharides from α-, β-, γ- or higher cyclodextrins (Greek prefix here refers to the type of cyclodextrin) by ring opening or from starch by helix opening. The definition follows https://www.cazypedia.org/index.php/Glycoside_Hydrola se_Family_13
• Glycoside Chemical structure formed from a carbohydrate with an alcohol, an amine or a thiol (corresponds to O-, N- or S-glycosides). Only O-glycosides are considered here.
• Glucansucrase family GH70-glucansucrase (GS) enzymes catalyze the synthesis of α-glucans from sucrose. The definition follows https://www.cazypedia.org/index.php/Glycoside_Hydrola se_Family_7 Glucansucrases can form alterans α-(1-3), (1-6), dextrans α-(1-6), reuterans α-(1-4) or mutans α-(1-3).
• Glucoside Specific glycoside with glucose as the bound carbohydrate. As with glycosides, α- and β-D-glucosides are distinguished depending on the position of the oxygen atom forming the glycosidic bond in relation to the carbohydrate in standard projection (α-glucoside: oxygen upwards in relation to the ring, in β-glucosides downwards) (other definitions refer to the configuration of C5).
• Glycosidase Enzyme with hydrolytic activity for glycosides, this is a cleavage of glycosides with the addition of a water molecule. β-glycosidases can cleave β-glycosides like β-glucosides. Glucosidases are special glycosidases or specific activities of such.
• Lipophilic With affinity to fat or oil. The term describes molecules with low polarity.
• Transglycosylation The transfer of carbohydrate residues from one glycoside to another.

Specific chemicals used

• β-Amylase was purchased from Roth (8717.1), Germany. Cyclomaltodextrin glucanotransferase (CGTase) was obtained from Amano, Japan.
• Glucansucrases were obtained directly from cultures of Lactobacillus sp.
• Rapidase (mixture of four β-glucosidases) was purchased from DSM Foodspecialities B.V., The Netherlands, under the trade name Rapidase AR 2000.

Abbreviations

CBD

Cannabidiol

CBD-1βG

Cannabidiol-2'-β-D-glucopyranoside

CBD-2βG

Cannabidiol-2',6'-β-D-diglucopyranoside

CBD-2βG-6αG

Cannabidiol-2'-β-D-glucopyranosyl-((1-4)-α-D-glucopyranosyl)) ₅ -(1-4)-α-D-glucopyranoside,6'-β-D-glucopyranoside

CBD-(βG-6aG)2

Cannabidiol-2'-β-D-glucopyranosyl-((1-4)-α-D-glucopyranosyl)) ₅ -(1-4)-α-D-glucopyranoside,6'-β-D-glucopyranosyl-((1-4)-α-D-glucopyranosyl)) ₅ -(1-4)-α-D-glucopyranoside

CBD-2βG-12αG

Cannabidiol-2'-β-D-glucopyranosyl-((1-4)-α-D-glucopyranosyl)) ₁₁ -(1-4)-α-D-glucopyranoside,6'-β-D-glucopyranoside

CBD-2βG-7αG

Cannabidiol-2'-β-D-glucopyranosyl-((1-4)-α-D-glucopyranosyl)) ₆ -(1-4)-α-D-glucopyranoside,6'-β-D-glucopyranoside

CBD-(βG-7αG)2

Cannabidiol-2'-β-D-glucopyranosyl-((1-4)-α-D-glucopyranosyl)) ₆ -(1-4)-α-D-glucopyranoside,6'-β-D-glucopyranosyl-((1-4)-α-D-glucopyranosyl)) ₆ -(1-4)-α-D-glucopyranoside

CBD-2βG-14αG

Cannabidiol-2'-β-D-glucopyranosyl-((1-4)-α-D-glucopyranosyl)) ₁₃ -(1-4)-α-D-glucopyranoside,6'-β-D-glucopyranoside

CBD-2βG-8αG

Cannabidiol-2'-β-D-glucopyranosyl-((1-4)-α-D-glucopyranosyl)) ₇ -(1-4)-α-D-glucopyranoside,6'-β-D-glucopyranoside ( 2 )

CBD-(βG-8αG)2

Cannabidiol-2'-β-D-glucopyranosyl-((1-4)-α-D-glucopyranosyl)) ₇ -(1-4)-α-D-glucopyranoside,6'-β-D-glucopyranosyl-((1-4)-α-D-glucopyranosyl)) ₇ -(1-4)-α-D-glucopyranoside

CBD-2βG-16αG

Cannabidiol-2'-β-D-glucopyranosyl-((1-4)-α-D-glucopyranosyl)) ₁₅ -(1-4)-α-D-glucopyranoside,6'-β-D-glucopyranoside

CBD-2βG-(6/7/8αG)n

Cannabidiol-2'-β-D-glucopyranosyl-((1-4)-α-D-glucopyranosyl)) _5,6,7 -(1-4)-α-D-glucopyranoside,6'-β-D-glucopyranoside (mixture of compounds with variable chain length obtained by using starch, glycogen, dextrins or mixtures of cyclodextrins as substrates), including compounds having several (n) such substituents in different positions.

CGTase

Cyclomaltodextrin glucanotransferase

GT

Glycosyltransferase

mg

milligram

min

minute

mL

milliliters

mM

millimolar

rpm

rotations per minute, revolutions per minute, centrifugation speed

w/v

weight/volume; weight per volume, concentration value

Examples

example 1

Formation of CBD oligosaccharides via CBD-2ßG with cyclomaltodextrin glucanotransferase

The glycosyltransferase UGT73A gene from Nicotiana benthamiana (sequence 1 ( )) in an expression vector was used for the glucosylation of cannabidiol to CBD-2βG by biotransformation in E. coli BL21 (DE3) pLysS (Novagen, Schwalbach, Germany). Preparatory cultures were grown overnight at 37°C in Luria Bertani (LB) medium containing 100 pg/ml ampicillin and 23 pg/ml chloramphenicol. The next day, 50 ml of M9 minimal medium containing 1% glucose and the mentioned antibiotics were inoculated with 1 ml of the preparatory cultures and cultured at 37°C and 160 rpm until an OD600 (optical density at 600 nm) of 1 was reached. Subsequent cultivation was carried out at 18°C and induced by addition of 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside). After 6 hours, 5 mg of the substrate cannabidiol (CBD isolate 99.034%, DB Labs, Las Vegas, NV, USA) was added. At regular intervals, 200 µL samples were taken from the culture. Cells were removed from the samples by centrifugation and the latter were diluted 1:2 for HPLC analysis, thereby monitoring the progress of biotransformation.

CBD-2βG was purified by adsorption to a polystyrene matrix and elution with methanol.

For the cyclodextrin glucantransferase reaction, 0.0175 mg CBD-2ßG from a stock solution was incubated with 2.25 U CGTase (Amano) in 25 mM sodium acetate buffer pH 5.5 and with either 0.056 mg α, β, γ-cyclodextrin or starch from stock solutions in a final volume of 0.14 ml, each overnight at room temperature. Aliquots of the respective reactions were analyzed by HPLC ( 4 ).

Example 2

Synthesis of CBD-2βG-derived oligosaccharides

CBD-2βG, the intermediate from Example 1, was used for a small preparative planned synthesis of CBD-2βG-8αG. For this purpose, 5 mg CBD-2βG was incubated with 450 U CGTase (Amano) in 25 mM sodium acetate buffer pH 5.5 with 15 mg γ-cyclodextrin in a final volume of 5 mL and incubated overnight at room temperature. An aliquot of the respective reactions was analyzed by HPLC ( 5 ).

Example 3

LC-MS-MS2 analysis of CBD-2ßG-derived oligosaccharides

Liquid chromatography coupled to mass spectroscopy analyses were performed using a Bruker esquire 3000plus mass spectrometer (Bruker Daltonics, Bremen, Germany) with an Agilent 1100HPLC system, an Agilent 1100/1200 Micro Wellplate Autosampler 1 and an Agilent 1100/1200 Diode Array Detector SL 1. For mass spectroscopy, a dual ion source (DUIS) was used and operated in a negative ionization mode. Separation was performed using a LUNA C18 100A 150 x 2 mm reversed phase column (Phenomenex, Aschaffenburg, Germany). Milli-Q purified water (A) and methanol (B), each with 0.1% formic acid, were used as mobile phases. The flow rate was 0.2 ml min ^-1 , the gradient was: 0 min 40% B, 15 min 100% B, 20 min 40% B, 30 min 40% B. The sample injection volume was 5 µl. The flow to mass spectroscopy was regulated to 0.2 ml by a flow separator. The mass spectroscopic profiles were recorded from mass/charge ratios from 50 m/z to 1000 m/z at negative ionization and a scan speed of 4000 V and an interfacial tension of -500 V using nitrogen as collision gas. shows an LC-MS analysis of the products of the biotransformation of cannabidiol to CBD oligosaccharides from Example 2. The results show that the staggered cannabinoid oligosaccharides CBD-2βG-1αG, CBD-2βG-2aG and CBD-2βG-3αG were obtained as a mixture ( ). The structure with regard to the anomeric character of glucopyranosyl residues is further supported by hydrolytic cleavage experiments (Example 4).

Example 4

Hydrolytic cleavage of the staggered oligosaccharides CBD-2ßG-1αG, CBD-2βG-2αG and CBD-2βG-3αG by saliva

0.2 ml of a solution of 0.5 mg/ml staggered oligosaccharides CBD-2βG-1αG, CBD-2βG-2αG, CBD-2βG-3αG and 0.5 mg/ml CBD-2βG (substrate residues) from Example 2 were mixed with 0.1 mL saliva and incubated for 24 hours at 30°C. The samples were analyzed by HPLC ( upper panel, control in lower panel). The result shows the faster hydrolysis of the α-glucopyranosyl residues of this cannabinoid oligosaccharide composition with saliva compared to its parent β-glucopyranosyl glycosylation (remaining as CBD-2βG in this assay).

Example 5

Hydrolytic cleavage of the staggered oligosaccharides CBD-2ßG-1αG, CBD-2βG-2αG and CBD-2βG-3αG by β-glucosidase

0.2 ml of a solution of 0.5 mg/ml staggered oligosaccharides CBD-2βG-1αG, CBD-2βG-2αG, CBD-2βG-3αG and 0.5 mg/ml CBD-2βG (residual substrate) from Example 2 were dissolved in 0.1 ml water together with 5 mg Rapidase (a mixture of β-glucosidases) and incubated for 24 hours at 30 °C. The control was carried out with 0.1 ml water instead of enzyme solution. The samples were analyzed by HPLC ( 7 , middle graph, lower control graph). The result shows that the degradation of CBD-2βG at the parent glycosylation or the removal of glucopyranosyl residues is possible, as intended in applications of cannabinoid oligosaccharide compounds and compositions with release of the aglycone. The free aglycone is water-insoluble and cannot be determined with this assay; only the still water-soluble intermediate product of the degradation CBD-1βG is observable.

Example 6

Formation of CBD oligosaccharides via CBD-2ßG with glucansucrases 50 ml of MRS medium each were inoculated with the glucansucrase (dextransucrase) producing strains Liquorilactobacillus hordei TM 1.1822 and Liquorilactobacillus nagelii TM 1.1827 and incubated overnight at 35°C and 200 rpm. Both cultures, each with 50 ml, were centrifuged for 15 min and 4,700 rpm. Each cell pellet was resuspended in 1 ml of 100 mM citrate buffer pH 6, supplemented with 0.25 mg CBD-2ßG and 10 mg sucrose, and incubated at 30°C and shaken at 300 rpm. After 1, 2 and 4 hours, 0.2 ml samples were taken for analysis and centrifuged at 15,000 rpm for 15 minutes.

HPLC analysis of both reaction mixtures shows the formation of staggered CBD oligosaccharides from CBD-2βG by glucan(dextran)sucrases from Liquorilactobacillus hordei TM 1.1822 and Liquorilactobacillus nagelii TM 1.1827. The products were CBD-2βG-1αG, CBD-2βG-2αG and CBD-2βG-3αG ((1-6)-α-linked) ( ); the efficiency was lower than that of CGTase, resulting in α-(1-4)-linked products. Due to the known product specificity of Liquorilactobacillus hordei TM 1.1822 and Liquorilactobacillus nagelii TM 1.1827 as dextrasucrases, the linkage of α-glucopyranosyl residues must be α-(1-6), in contrast to those of CGTase products with α-(1-4) linkages.

Illustrations

: Chemical structure of cannabidiol (top). Note that positions 2' and 6' are equivalent by rotation around the resorcinol-cyclohexene linkage. Chemical structure of CBD-2βG (bottom) ("β-stem glucosylation").

: Chemical structure of the cannabinoid oligosaccharide cannabidiol-2'-β-D-glucopyranosyl-((1-4)-α-D-glucopyranosyl)) ₇ -(1-4)-α-D-glucopyranoside, 6'-ß-D-glucopyranoside (CBD-2β-8αG), a central cannabinoid oligosaccharide obtained via cannabidiol-2'6'-di-β-D-glucopyranoside by reaction with γ-cyclodextrin catalyzed by cyclomaltodextrin glucanotransferase. It consists of a 2',6'-ß-stem glucosylation and a modular α-glucosylation by chains of 6, 7 or 8 or > 8 α-glucopyranosyl residues, 8 in this example (n=7, n+1 for the glucopyranosyl end group). Smaller numbers of (1-4)-α-D-glucopyranoside residues are often used due to known disproportionation and hydrolysis reactions. Cyclomaltodextrin glucanotransferase (staggered cannabinoid oligosaccharides) sequestration activities were observed. R1 and R2 can also each be such modular α-glucopyranosyl chains. The modular α-glucopyranosyl chains have a helical structure like starch. Cannabinoid oligosaccharides obtained with glucansucrase can have linkages other than (1-4)-α;, as in the dextransucrases of Example 6, which give (1-6)-α; linkages.

Seq1 (DNA), NbGT-fc6

Seq2 (AA), NbGT-fc6

Seq3 (DNA), CaGT2

Seq4 (DNA), CaGT2

: GT73A gene (NbGT-fc6, Seq1 (DNA), Seq2 (AA)), which was used in Example 1 for heterologous expression and biotransformation of cannabidiol to CBD-2βG, and second gene GT73A gene (CaGT2, Seq3 (DNA), Seq4 (AA) from Catharanthus roseus), which also enables the production of CBD-2βG.

: Synthesis of CBD oligosaccharides from CBD-2βG (Example 1), analysis of aliquots by HPLC. Enzymatic conversion of CBD-2βG with CGTase and (graphs from top to bottom) α-, β-, γ-cyclodextrin and starch as co-substrates and control with CBD-2ßG only. Products are observed with all co-substrates. The peak eluting before the CBD-2βG peak (4.2 min) is CBD-2βG-1αG (3.9 min). The peaks eluting before it (at about 3.6 min) are CBD-2βG-2αG and CBD-2βG-3αG (small peak at about 3.4 min).

: Synthesis of staggered CBD oligosaccharides via CBD-2βG (Example 2), analysis of aliquots by HPLC. Lower curve: Control: CBD-2βG and γ-cyclodextrin substrates, reaction assay without CGTase enzyme. Upper curve: The peaks formed eluting before CBD-2βG (at about 4.1 min) are CBD-2βG-1αG (at about 3.9 min), CBD-2βG-2αG (at about 3.6 min) and CBD-2βG-3αG (small peak at about 3.4 min), which represent partial products of the planned CBD2βG-8αG.

: LC-MS analysis of staggered CBD oligosaccharides (Example 3). Upper graph: Upper box: total ion current chromatogram. Lower boxes: chromatograms of individual compounds at their respective m/z in negative mode, CBD-2ßG m/z 683 [M+HCOOH-H] ^- (adduct with formic acid from solvent) (second box), CBD-2βG-1αG m/z 799 [MH] ^- (third box), CBD-2βG-2αG m/z 961 [MH] ^- (fourth box), CBD-2βG-3αG m/z 1123 [MH] ^- (fifth box). Lower panel: MS/MS2 spectra of CBD-2βG m/z 683 [M+HCOOH-H] ^- (first box), CBD-2βG-1αG m/z 799 [MH] ^- (second box), CBD-2βG-2αG m/z 961 [MH] ^- (third box), CBD-2βG-3αG m/z 1123 [MH] ^- (fourth box). The MS2 spectra of all compounds show the expected fragmentation with loss of one or more glucopyranosyl residues (multiples of (-162 D)).

: HPLC-Analyse der Bildung von gestaffelten CBD-Oligosacchariden aus CBD-2βG durch Glucansucrasen von Lactobacillus hordei TM 1.1822 (oberer Kasten) und Lactobacillus nagelii TM 1.1827 (unterer Kasten) (Beispiel 6). Kurven sind jeweils für Kontrolle (0), 1, 2 und 4 Stunden Inkubation der enzymatischen Reaktion, von unten nach oben. Das Substrat CBD-2ßG eluiert bei ca. 4,2 min, die Produkte CBD-2βG-1αG, CBD-2βG-2αG und CBD-2βG-3αG (alle mit (1-6)-α-Bindungen) eluieren davor mit abnehmender Elutionszeit. : Hydrolytic cleavage of the staggered CBD oligosaccharides CBD-2βG-1αG, CBD-2βG-2αG and CBD-2βG-3αG composition by saliva (upper graph, example 4) or β-glucosidase (middle Graph, Example 5) and control without hydrolyzing enzyme (substrate of the reaction shown, lower graph). The CBD-2βG peak elutes at 4.2 min, the peaks eluting before are CBD-2βG-1αG (3.9 min), CBD-2βG-2αG (3.6 min) and CBD-2βG-3αG (minor amounts elute shortly before). Hydrolysis products are CBD-2βG and CBD-1ßG (5.5 min).

: HPLC analysis of the formation of staggered CBD oligosaccharides from CBD-2βG by glucansucrases from Lactobacillus hordei TM 1.1822 (upper box) and Lactobacillus nagelii TM 1.1827 (lower box) (Example 6). Curves are for control (0), 1, 2 and 4 hours of incubation of the enzymatic reaction, from bottom to top. The substrate CBD-2ßG elutes at ca. 4.2 min, the products CBD-2βG-1αG, CBD-2βG-2αG and CBD-2βG-3αG (all with (1-6)-α-bonds) elute before this with decreasing elution time.

This is followed by a sequence protocol as an electronic document. This can be accessed both in DEPATISnet and in the DPMA register.

QUOTES INCLUDED IN THE DESCRIPTION

This list of documents listed by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions.

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Claims (19)

A process for the preparation of cannabinoid oligosaccharide(s), for example cannabidiol (CBD) oligosaccharide(s), comprising the step of contacting a cannabinoid such as CBD with an isolated UDP carbohydrate-dependent glycosyltransferase, preferably of the class UGT73A, preferably with the glycosyltransferases identified as [Seq1 (DNA), Seq2 (AA) = NbGT-fc6] and [Seq3 (DNA), Seq4 (AA) = CaGT2], or with UDP carbohydrate-dependent glycosyltransferases which or a nucleic acid exhibit acid sequence identity of at least 70%, of at least 80%, of at least 90%, of at least 95%, of at least 98% with [Seq1 = CaGT2] or with an amino acid sequence identity of at least 70%, of at least 80%, of at least 90%, of at least 95% to [Seq2 = CaGT2], in a Reaction mixture under conditions including all co-substrates and co-factors required for glucosyltransferase activity effective to produce cannabinoid glycoside(s) and for subsequently or in parallel, or with cannabinoid β-D-glycosides from other sources, contacting them with a cyclomaltodextrin glucanotransferase with at least one of its substrates cyclodextrin or substituted cyclodextrin (for example hydroxypropyl, methyl), linear maltodextrins, starch or glycogen, and a further step of purifying the cannabinoid oligosaccharide(s) from the reaction mixture to obtain the isolated cannabinoid oligosaccharide(s). Procedure according to Claim 1 , wherein the isolated UDP carbohydrate-dependent glycosyltransferase uses UDP glucose or an activated form of fructose, glucose, galactose, mannose, rhamnose, ribose, arabinose, xylose or an activated oligosaccharide or an activated aldonic or uronic acid, for example gluconic acid, glucuronic acid or galacturonic acid, or N-substituted derivatives of said carbohydrates, for example 2-N-acetylglucosamine, as co-substrate and a cannabinoid such as CBD, and wherein a cyclomaltodextrin glucanotransferase is used and the intermediate (or added starting material from other sources) uses cannabinoid β-D-glycoside as a substrate to form higher glycosylated oligosaccharides (the latter with α-anomeric glucopyranosyl residues) and the products are the respective cannabidiol oligosaccharides with parent β-D-glycosidic structures and branched α-glucopyranosyl oligomers. Method according to one of the Claims 1 , 2 , wherein the isolated UDP-carbohydrate-dependent glycosyltransferase and the cyclomaltodextrin glucanotransferase are obtained by heterologous expression in one or two organisms of species different from the species of origin of the glycosyltransferase or the cyclomaltodextrin glucanotransferase; preferably, the conversion of cannabinoids to cannabinoid oligosaccharide(s) relies on the cellular supply of the co-substrate identified as UDP-glucose or other activated carbohydrates and putatively on the provision by the cells or import of at least one of the substrates cyclodextrin or substituted cyclodextrins (e.g. hydroxypropyl, methyl), linear maltodextrins, starch or glycogen, and takes place within the same heterologous organism(s), which may be optimized for this purpose by genetic and/or recombinant means. Method according to one of the Claims 1 - 3 , wherein instead of or in addition to cyclomaltodextrin glucanotransferase and its substrates (cyclodextrin or substituted cyclodextrin (e.g. hydroxypropyl, methyl), linear maltodextrins, starch or glycogen), glucansucrase(s) and its substrate sucrose (or other compatible disaccharides) are used for the addition of (1-2)-, (1-3)-, (1-4)- or (1-6)-linked α-D-glucopyranoside residues to produce staggered cannabinoid oligosaccharides. The procedure according to one of the Claims 1 - 4 according to which cannabinoid oligosaccharides can be obtained, for example but not limited to, CBD-2βG-8αG and/or higher reaction products or staggered cannabinoid oligosaccharides from cannabidiol with GT40/UDP-glucose and with cyclomaltodextrin glucanotransferase/γ-cyclodextrin or with glucansucrases and sucrose (or other disaccharides). Cannabinoid oligosaccharides obtained by processes according to claims (1-5) with a glycosylation pattern comprising no or one to four β-D-glycopyranosyl parent residue(s) and one or more additional ((1-4)-α-D-glucopyranosyl)5,6,7, or>7-(1-4)-α-D-glucopyranoside)n-oligosaccharide residues or staggered cannabinoid oligosaccharides, wherein the staggered cannabinoid oligosaccharides are also obtained by glycosyltransferase or transglycosylation reactions other than cyclomaltodextrin glucanotransferase such as by glucansucrases and in this case not only (1-4)- but may also be (1-2)-, (1-3)- or (1-6)-linked, and the (1-4)-α-D-glucopyranoside residue(s) may be linked directly to the cannabinoid, to its β-D-glycopyranosyl residue(s) or, if more than one, to each other. CBD oligosaccharides contain according to (6) comprising zero or one to four β-D-glucopyranosyl parent residue(s) and one or more additional ((1-4)-α-D-glucopyranosyl)5,6,7or>7-(1-4)-α-D-glucopyranoside)n-oligosaccharide residues or staggered cannabinoid oligosaccharides which may be (1-2)-, (1-3)-, (1-4)- or (1-6)-α-linked. The compounds CBD-2βG-6αG, CBD-(βG-6αG)2, CBD-2βG-12αG, CBD-2βG-7αG, CBD-(βG-7αG)2, CBD-2βG-14αG, CBD-2βG -8αG, CBD-(βG-8αG)2, CBD-2βG-16αG, CBD-2βG-(6/7/8αG)n and staggered cannabinoid oligosaccharides, the latter may be (1-2)-, (1-3)-, (1-4)- or (1-6)-α-linked, or esters thereof, or acetal or hemiacetal derivatives thereof, or an ester of the latter. Method according to one of the Claims 1 - 5 , whereby strains of Escherichia coli or yeasts, for example Saccharomyces cerevisiae or Pichia pastoris, are used as microorganisms for production. Method according to one of the Claims 1 - 5 , whereby the UDP-carbohydrate-dependent glycosyltransferase and the cyclomaltodextrin glucanotransferase and/or a glucansucrase are heterologously expressed in Cannabis sativa and cannabinoids such as cannabidiol from the secondary metabolism of the plant are used as a substrate to convert them into the respective cannabinoid oligosaccharides and thereby obtain them. Method according to one of the Claims 1 - 5 , 9 , wherein the UDP-carbohydrate-dependent glycosyltransferase is heterologously expressed in Cannabis sativa and wherein the glucose residues on the 2'- and 6'-hydroxy groups of a cannabinoid such as cannabigerolic acid act as protecting groups that prevent or reduce metabolism and cyclization to tetrahydrocannabinol(s). Compositions obtained by processes according to any of the Claims 1 - 11 , comprising - cannabinoid oligosaccharides, for example cannabidiol oligosaccharides, - components of the reaction medium according to the Claims 1 - 3 , or - components of an organism according to Claim 3 , 9 , 10 , 11 , and/or - components of a culture medium according to Claims 3 , 9 and their degradation products; the compositions may optionally contain unconverted cannabinoid substrates such as cannabidiol and by-products derived from cannabinoid oligosaccharides, for example acetates with variable substitution sites at the glucopyranosyl residues. A further process comprising the step of obtaining cannabinoid oligosaccharides obtained by the process according to any one of the Claims 1 - 11 obtained, in a second step from resulting compositions according to Claim 12 enriched and purified, and wherein extractions with organic solvents, preferably ethyl acetate, and adsorption on a lipophilic solid phase, preferably on a polystyrene matrix, are carried out for enrichment and purification and an organic solvent, preferably methanol, is used for elution from this matrix. Method according to one of the Claims 1 - 8th , 10 - 13 , wherein the first step for obtaining the cannabinoid oligosaccharides from the organism Cannabis sativa for subsequent enrichment and purification is an extraction with an aqueous phase or a hot aqueous phase (water or steam) or a polar solvent or a polar solvent mixture. Kit according to (1-14) for the production, purification and analysis of cannabinoid oligosaccharides, for example cannabidiol oligosaccharides, comprising the gene and/or the enzyme of [Seq 1] and a gene and/or enzyme of a cyclomaltodextrin glucanotransferase and/or a glucansucrase in the form of a DNA sequence, or a codon-optimized DNA sequence, or an amino acid sequence, or an isolated DNA, or a DNA in a vector or an expression vector, or the enzyme or a composition comprising the enzyme or a transgenic microorganism or a transgenic plant, as well as specific means and instructions for carrying out the production and purification processes and for the analysis and quality control of the products. Cannabinoid oligosaccharides according to one of the Claims 1 - 14 , for example cannabidiol oligosaccharides, optionally substituted at glycosidic residues, for example acetylated or peracetylated, for use as biologically active compounds, as medicine or veterinary medicine, which may have an improved water solubility compared to the respective free cannabinoid, which may have a solubilizing or emulsifying effect on other glycosides of cannabidiol or on other cannabinoids with a smaller number of carbohydrate residues (e.g. CBD-1ßG, CBD-2βG) or on free cannabidiol or on other free cannabinoids or on other components with low solubility within a composition, for example on flavorings or other biologically active compounds in a beverage, a different taste or mouthfeel, a different absorption into the body, an altered metabolism, an altered activity, or an altered pharmacokinetics. Composition comprising - cannabinoid oligosaccharides, for example cannabidiol oligosaccharides; and - at least one glycosidase, preferably at least one β-glucosidase, or a composition of glycosidases, preferably β-glucosidases; and/or - microorganisms producing glycosidases, preferably β-glucosidases; and/or - amylase(s) and/or - encapsulated forms of individual, some or all of these components of the composition for use as an active pharmaceutical principle, medicament or veterinary medicine. A process using cannabinoid oligosaccharides, for example cannabidiol oligosaccharides and/or substituted derivatives according to Claim 16 or compositions with these Claim 17 in concentrated aqueous solution to obtain a concentrate, thereby producing an intermediate for the Claims 16 and 17 described purposes, which can advantageously be used to produce the respective end product, which as such concentrates are more durable or are easier to store or transport due to their smaller volume and weight; such a concentrate has at least twice the concentration of cannabinoid oligosaccharides or their derivatives compared to the concentration in the respective end product (for example a beverage), more preferably at least five times the concentration in the concentrate. The use of cannabinoid oligosaccharides with mixed α- and β-glucopyranosyl structure for the differential release of the aglycones depending on the organ-specific (e.g. oral cavity, stomach, small and large intestine) presence of hydrolytic enzymes/enzymes with glycosidase activity (e.g. various human amylases, e.g. microbial β-glucosidases) for the purpose of targeted drug release.