WO2024042226A2

WO2024042226A2 - Procedure for production of opioid-antagonist-releasing compounds and their use as a medicine

Info

Publication number: WO2024042226A2
Application number: PCT/EP2023/073401
Authority: WO
Inventors: Thilo Fischer; Fong-Chin Huang; James Ferri
Original assignee: Biosynth Gmbh; Virginia Commonwealth University
Priority date: 2022-08-25
Filing date: 2023-08-25
Publication date: 2024-02-29
Also published as: WO2024042226A3

Abstract

A method for producing a purified morphinan-glycoside conjugate. The method includes the steps of contacting a morphinan compound with an activated saccharide or activated oligosaccharide and a glycosyltransferase in a reaction mixture under conditions, including any co-factors necessary for glycosyltransferase activity, effective to produce a morphinan-glycoside conjugate; and purifying the morphinan-glycoside conjugate from the reaction mixture to obtain the isolated morphinan-glycoside conjugate. Compounds, compositions, conjugates, and methods for their use are also provided.

Description

Procedure For Production of Opioid-Antagonist-Releasing Compounds and Their Use as a Medicine

FIELD

[001] The subject matter disclosed herein relates to glycoside conjugate compounds, methods for preparing them, and methods for their use.

BACKGROUND

[002] Morphinan compounds, including opiates from the opium poppy (Papaver somniferum), have been used medicinally as analgesics and antidiarrheal drugs for a long time (Dhawan et al. 1996, International Union of Pharmacology XII. Classification of Opiate Receptors, Pharmacological Reviews 48 (4), 567-592).

[003] Based on the basic structure of morphine, numerous derivatives such as codeine (3-

O-methyl-morphine, analgesic, antitussive) and diacetylmorphine (heroin) have been developed for medical purposes (Dhawan et al. 1996).

[004] Physiologically, codeine is activated by the Cyp450 enzyme and oxidative demethylation to morphine (SH Snyder and GW Pasternak, 2003, Trends in Pharmacological Sciences, 24 (4), 198-205).

[005] Opiates have a specific effect on cellular opioid receptors in the peripheral and central nervous system. Endogenous agonists of these receptors, initially identified and characterized via non- endogenous agonists, are endorphins, enkephalins and dynorphins (Snyder and Pasternak 2003, Historical review: Opioid receptors, TRENDS in Pharmacological Sciences 24 (4), 198-205). Synthetic analgesics such as fentanyl and methadone also bind to opioid receptors (Dhawan et al. 1996).

[006] A distinction is made between delta (8), kappa (K), and mu (p) receptor classes

(Dhawan et al. 1996), as well as epsilon (s) and ORL. The opioid receptors are G-protein coupled and the binding of agonists is synergistically positively influenced by GTP and Na⁺ ion (Snyder and Pasternak 2003).

[007] The different classes of opioid receptors are localized differently in the body and occur both pre- and postsynaptic. The physiological effects when they are activated are correspondingly diverse. These include the pharmacologically in the foreground analgesic and narcotic effects, but also euphoria, hypothermia, miosis and respiratory depression as effects.

[008] The natural function of this receptor system is seen in a protective function when the organism is overloaded (Schaumann: Analgesika und “protective” system. Die Naturwissenschaften . Volume 41, No. 4, 1954, pp. 96-96).

[009] The event of a medical or abusive overdose of opioid receptor agonists (analgesics, anesthetics, drugs such as heroin), respiratory depression, which can lead to respiratory paralysis, is particularly dangerous.

[0010] Opioid receptor antagonists such as naloxone and naltrexone were synthesized early on and played a major role in the discovery and analysis of the various classes of receptors (Dhawan et al. 1996, Snyder and Pasternak 2003). Naloxone mainly binds to p-opioid receptors.

[0011] Medically, antagonists such as naloxone are of great importance for the treatment after fentanyl anesthesia or opioid overdoses in the case of drug abuse and primarily counteract respiratory paralysis. A disadvantage of these antagonists is their rapid metabolism. Naloxone, for example, must therefore be administered intravenously repeatedly, or is used briefly as an emergency nasal spray in the event of an overdose of opiates. More stable antagonist forms would also be useful in opiate withdrawal therapy.

[0012] Naloxone is metabolized to B-D-glucuronide and excreted as such (https://pubchem.ncbi.nlm.nih.gov/compound/Naloxone-3-glucuronide and citations therein).

[0013] One problem solved by the present disclosure is to find new forms of opioid antagonists such as naloxone and naltrexone that physiologically exhibit a depot or delayed- or extended- release effect. For example, codeine, as 3-O-methyl-morphine, has hardly any effect as an agonist on opioid receptors, but develops such activity after demethylation.

[0014] Most B-D-glucosides can be cleaved by B-glycosidases. These enzymes occur in humans and their microbiome. The present disclosure offers a solution to the problem above; the present inventors have found that, of many possible substitutions which could achieve a releaseretarding effect, the selection of a glycosyl group is particularly favorable.

[0015] Without being bound by any theory of the invention, glycosylation of a morphinan compound (MC) is thought to inhibit or prevent the binding of the MC to its receptor, and so control of such derivatization by enzymatic synthesis of the conjugate by a cell-free system or in cell culture, or hydrolysis of the conjugate in situ for release of the active agent MC, can be used to modulate the timing of release or pharmacologic profile of a MC active agent.

[0016] Thus, the compounds and methods disclosed herein can be applied to, for instance opioid receptor agonists like codeine or morphine, and also with opioid receptor antagonists like naloxone and naltrexone.

[0017] The selection of glucoside residues is also particularly favorable, since such glycosides can be physiologically cleaved (hydrolyzed) and, moreover, the cleaved residue, glucose, can be metabolized without any problems.

[0018] Furthermore, it was unexpectedly found that plant UDP-glycosyltransferases could accept morphinan compounds, such as naloxone and naltrexone, as substrates and that, due to steric hindrance, the enzymatic glycosylation is not at the 6-O- or 17-N-position, but selectively in the 3- 0 position when a morphinan substrate, for example codeine, is used to synthesize the conjugate.

[0019] Further derivatization with half acetal or full acetal or ester residues on the glycoside residue expands the options for slowing down metabolism and improving the depot effect.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The invention will now be described in more detail with reference to working examples of the invention, given only by way of example, and with reference to the accompanying drawings, in which:

[0021] FIG. 1 shows LC-MS analysis of the synthesis approach for naloxone-3-O-B-D- glucoside (NaG) and a nal oxone-3 -O-B-D-glucoside acetate (NaGAc), prepared in Example 1. Top: UV chromatogram, 232 nm. Bottom: mass spectrum at m / z 490 in positive mode, molecular peak of [M + H] ⁺.

[0022] FIG. 2 shows MS / MS2 analysis of naloxone-3-O-B-D-glucoside (example 2). As expected, the MS (top) shows the protonated naloxone-3-O-B-D-glucoside at m / z 490, the MS2 (bottom) shows a protonated fragment after dehydration at m / z 472 and another protonated fragment after the glucoside residue has been split off at m / z 328, as well as another fragment with m / z 310 due to elimination of water from the latter. Mass and fragmentation agree with those to be expected from naloxone-3-O-B-D-glucoside. [0023] FIG. 3 shows partial cleavage of naloxone-3-O-B-D-glucoside (NaG) by 13- glucosidase with naloxone (N) as product (upper part of the diagram) in the HPLC analysis (UV chromatogram, 232 nm) (example 3), the lower part of the diagram shows the control without prior 13-glucosidase treatment.

[0024] FIG. 4 shows NMR analysis of naloxone-3-O-13-D-glucoside as described in Example 4. Top: 1H-NMR, middle: 13C-NMR, bottom: 1H-13NMR-2D-NMR and structural formula of naloxone-3-O-13-D-glucoside.

[0025] FIG. 5 shows LC-MS analysis of naltrexone-3-O-[3-D-glucoside (NtG) synthesized and analyzed as described in Example 5 from naltrexone (Nt). Top: UV chromatogram, 232 nm. Bottom: mass spectrum at m / z 504 in positive mode, molecular peak of [M + H] ⁺.

[0026] FIG. 6 shows MS / MS2 analysis of naltrexone-3-O-13-D-glucoside as described in Example 6. Top: MS shows the protonated naltrexone-3-O-13-D-glucoside at m / z 504. Bottom: The MS2 shows a protonated fragment with m / z 486 of naltrexone-3-O-13-D-glucoside after elimination of water and another protonated fragment after elimination of the glucoside residue from naltrexone at m / z 342, as well as another fragment with m / z 324 due to elimination of water from the latter. Mass and fragmentation agree with those to be expected from naltrexone-3- O-13-D-glucoside.

[0027] FIG. 7 shows enzymatic cleavage of naltrexone-3-O-13-D-glucoside (NtG) by 13- glucosidase with naltrexone (Nt) as product. Top: Partial cleavage as shown by the HPLC analysis (UV chromatogram, 232 nm) performed in the manner of Example 3). Bottom: HPLC analysis of the NtG product from Example 6 used as the control without prior 13-glucosidase treatment.

[0028] FIG. 8 shows the purification of nal oxone-3 -O-[3-D-glucoside (NaG) (example 7). Top: the final purified naloxone-3-O-[3-D-glucoside analyzed by HPLC (UV chromatogram, 232 nm). Bottom: HPLC analysis of the biosuspension after fermentation that contained nal oxone-3 - O-P-D-glucoside (NaG), nal oxone-3 -O-[3-D-glucoside acetate (NaGAc), low amount of naloxone (N), and some unidentified peaks.

[0029] FIG. 9 shows the UGT cDNA 4G-GT6, which was used in Example 1 for the heterologous expression and biotransformation of naloxone to naloxone-3-O-13-D-glucoside. Fig. 9a) shows the cDNA sequence (without introns). Fig. 9b) shows the encoded amino acid sequence.

[0030] FIG. 10 shows antagonism of antinociceptive effects of the mu opioid receptor agonist fentanyl by naloxone (NLX) as a function of time in a warm water tail-withdrawal antinociception assay. Ordinate is the latency measure, expressed as percent maximum effect (%MPE), and abscissa is the time in min after administration of NLX at TO ("time post-antagonist admin"). Fentanyl was administered 15 min after NLX-pretreatment at TO. Symbols: open triangles represent administration of 10 mg/kg NLX + 0.56 mg/kg fentanyl; open squares represent administration of saline + 0.56 mg/kg fentanyl; open circles represent saline + saline (control). Filled squares denote p<0.05 vs. saline at respective time point (Fisher’s LSD multiple comparison test).

[0031] FIG. 11 shows antagonism of antinociceptive effects of the mu opioid receptor agonist fentanyl by naloxone-3-O-P-D-glucoside (NaG) as a function of time in a warm water tailwithdrawal antinociception assay. Ordinate is the latency measure, expressed as percent maximum effect (%MPE), and abscissa is the time in min after administration of NaG at TO ("time postantagonist admin"). Fentanyl was administered 15 min after NaG-pretreatment at TO. Symbols: open triangles represent administration of 10 mg/kg NaG + 0.56 mg/kg fentanyl; open squares represent administration of saline + 0.56 mg/kg fentanyl; open circles represent saline + saline (control). Filled squares denote p<0.05 vs. saline at respective time point (Fisher’s LSD multiple comparison test).

DETAILED DESCRIPTION

[0032] Definitions - the following terms are defined as set forth below:

[0033] As used herein, the terms "about" or "approximately" for any numerical values or ranges indicate a suitable dimensional tolerance that allows the part or collection of components to function for its intended purpose as described herein. More specifically, "about" or "approximately" may refer to the range of values ±10% of the recited value, e.g., "about 90%" may refer to the range of values from 81% to 99%.

[0034] ‘Anomers” are diastereomers of sugars, for example D-glucose, with regard to their acetalic hydroxyl function on carbon atom 1 in a glucoside, i.e., the a- or B-form of the glucoside.

[0035] A “morphinan compound” is one having a core bridged heteropoly cyclic structure, as in Formula I.

(Formula I).

In Formula I, -(Ra)_n generally means that any (and one or more) hydrogen (H) atom of Formula I, including the hydrogen at position 14 and the hydrogen attached to the nitrogen atom at position

17, may be substituted by Ra. Further, in Formula I, Ra can be any group, which group can include additional rings, or any two Ra groups can also join to form additional rings, wherein said additional rings may contain at least one further heteroatom. For example, two Ra groups might form a 5- or 6-member epoxy group, a 5- or 6- member thioexpoxy group, a pyrrolidine or a piperidine group. An Ra group might contain a cyclopropyl ring or an oxirane ring, n can be from

0 to 8. Each Ra can be the same or different. Each Ra can be further specified as R1 , R2, R3, etc. Ra can be selected from among alkyl, such as methyl or ethyl, alkenyl, alkynl, allyl, azine, benzoyl, hydroxy, methoxy, unsubstituted, monosubstituted, or di-substituted amine, thiol, keto, Me(C=O)O- and Et(C=O)O. The 7 and 8 carbons can be joined by a double bond. In some embodiments, the 4 and 5 positions are joined by an epoxy ring. A “morphinan compound” can also include additional heteroatoms, either in the polycyclic bridged core, or in substituents appended to the core, or in both.

[0036] A “morphinan group” is a radical of a morphinan compound.

[0037] A “glycoside” is a chemical combination of an alcohol with a carbohydrate. Glycosides can also be joined via N, S and C atoms (thus also “N-“, “S-“, and “C-glycosides”). Typically, when forming a glycoside conjugate, a morphinan group is attached to a glycoside via an oxygen atom, but morphinan compounds can also be conjugated to glycosides via N or S atoms. A distinction can be made between a- and B-D-glycosides, depending on the position of the oxygen bridge atom in relation to the carbohydrate in the normal projection (a-gly cosides: oxygen is from the ring downwards, with B-glycosides upwards).

[0038] ‘Glucosides” are particular glycosides in which glucose is the bound carbohydrate. As with glycosides (in biology commonly of the D-series) in general, a- and B-D-glucosides can be distinguished. [0039] A “glycosidase” is an enzyme having a biological activity of hydrolysis of glycosides, i.e., a cleavage of glycosides with the uptake of a water molecule.

[0040] A “glycosyl transferase” or “glycosyltransferase” is an enzyme having a biological activity of attaching the saccharide part of an activated saccharide or oligosaccharide to a nucleophilic acceptor group on an organic compound, typically via an oxygen atom on the substrate compound provided by a hydroxyl group. However, the nucleophile can be carbon-, nitrogen-, or sulfur-based.

[0041] ‘Naloxone” is a specific compound; trivially named N-allyl-oxymorphone; IUPAC name (5R, 9R, 13S, 14S) -17-allyl-3,14-dihydroxy-4,5-epoxymorphinan-6-one.

[0042] “Naltrexone” is a specific compound; IUPAC name (5R, 9R, 13S, 14S) -17- Cyclopropylmethyl-3,14-dihydroxy-4,5-epoxymorphinan-6-one.

[0043] As used herein, the terms "patient," "host," "user," and "subject" refer to any human or animal subject and are not intended to limit the systems or methods to human use.

EXEMPLARY MODES OF CARRYING OUT THE INVENTION

[0044] A first aspect of the present disclosure relates to a method for producing a purified morphinan-glycoside conjugate (m-g conjugate) comprising: a) contacting a morphinan compound with an activated saccharide or activated oligosaccharide and a glycosyltransferase in a reaction mixture under conditions, including any co-factors necessary for glycosyltransferase activity, effective to produce a m-g conjugate; and b) purifying the conjugate m-g conjugate from the reaction mixture to obtain the isolated m-g conjugate.

[0045] A morphinan compound used in such method (or released as an “active agent”) can be one having a core bridged heteropoly cyclic framework, as in Formula I.

(Formula I) [0046] In Formula I, Ra can be any group, which group can include additional rings, or any two Ra groups can also join to form additional rings, which additional rings may contain at least one further heteroatom. For example, two Ra groups might form a 5- or 6-member epoxy group, a 5- or 6- member thioexpoxy group, a pyrrolidine or a piperidine group, n can be from 0 to 8. Each Ra can be the same or different. Each Ra can be further specified as Rl, R2, R3, etc. as convenience or specificity of description might require. Each Ra can be a group selected from among alkyl, such as methyl or ethyl, alkenyl, alkynl, allyl, azine, benzoyl, hydroxy, methoxy, unsubstituted, monosubstituted, or di-substituted amine, thiol, keto, Me(C=O)O- and Et(C=O)O-. The 7 and 8 carbons can be joined by a double bond. In some embodiments, the 4 and 5 positions are joined by an epoxy ring.

[0047] A “morphinan compound” can also include additional heteroatoms, either in the polycyclic bridged core, or in substituents appended to the core, or in both. Furthermore, a “morphinan compound” is preferably one that has appended a hydroxyl group that is sterically accessible to a glycosyl transferase enzyme. The morphinan group can be one that is substituted at least one position by a hydroxyl, thiol, or amine group, for example a morphinan compound wherein at least the 3 -position of the morphinan compound is so substituted.

[0048] Additionally, or alternatively the morphinan compound can be one wherein neither of the 9 or 13 positions are substituted.

[0049] A morphinan compound used to prepare a conjugate (or released as an “active agent”) can be one such as Naloxone, Naltrexone, Naloxonazine, Naloxonbenzoylhydrazone, or a derivative of one of these.

[0050] In the instance of Naloxone, (Formula II),

Formula II (naloxone), n = 5; two of Ra are hydroxyl groups, two Ra groups form a 5 -member epoxy ring joining the 4 and 5 positions, one Ra is a keto group and one

Ra is an allyl group.

[0051] In the instance wherein a morphinan compound is naltrexone (Formula III),

Formula III (naltrexone), n = 6, two of Ra are hydroxyl groups, two Ra groups form a 5 -member epoxy ring joining the 4 and 5 positions, one Ra is a keto group and one

Ra is a methyl-cylcopropyl group.

[0052] In the instance wherein a morphinan compound is codeine (Formula IV)

Formula IV, n = 5, one Ra is a methoxy group, one Ra is a hydroxyl group, two Ra groups form a 5-member epoxy ring joining the 4 and 5 positions, one Ra is a methyl group, and the 7 and 8 carbons are joined by a double bond.

[0053] In the instance wherein a morphinan compound is morphine (Formula V),

5-member epoxy ring joining the 4 and 5 positions, one Ra is a methyl group, and the 7 and 8 carbons are joined by a double bond.

[0054] The morphinan compound can further be one of Formula VI

wherein:

Ri is a group selected from among hydrogen, alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, alkenyl, preferably C2-C4 alkenyl, more preferably allyl, Cs-Ce-cycloalkyl, C3-C6- cycloalkyl-Ci-C4-alkyl, preferably cyclopropylmethyl or cyclopropylethyl, C3-Ce-cycloalkyl-C2- C4-alkylene, Cs-Ce-aryl, C5-Ce-aryl-Ci-C4-alkyl, C5-Ce-aryl-C2-C4-alkenyl benzoyl, hydroxy, methoxy, Me(C=O)-, Et(C=O)-, Me(C=O)O- and Et(C=O)O-; and

R2 is OH, -OR or -O(C=O)R with R being alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, or alkenyl, preferably C2-C4 alkenyl, more preferably allyl; particularly, Ri is as defined above and R2 is OH, or Ri is as defined above and R2 is CH3-O-, or Ri is as defined above and R2 is CH3-CH2-O-.

[0055] Preferably, the morphinan compound is one of Formula VII

Formula VII wherein:

Ri is a group selected from among hydrogen, alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, alkenyl, preferably C2-C4 alkenyl, more preferably allyl, Cs-Ce-cycloalkyl, C3-C6- cycloalkyl-Ci-C4-alkyl, preferably cyclopropylmethyl or cyclopropylethyl, C3-Ce-cycloalkyl-C2- C4-alkylene, Cs-Ce-aryl, C5-Ce-aryl-Ci-C4-alkyl, C5-Ce-aryl-C2-C4-alkenyl benzoyl, hydroxy, methoxy, unsubstituted, monosubstituted, or di-substituted amine, Me(C=O)-, Et(C=O)-, Me(C=O)O- and Et(C=O)O-, preferably Ri is a group selected from among alkyl, preferably Ci- C4 alkyl, more preferably methyl or ethyl, alkenyl, preferably C2-C4 alkenyl, more preferably allyl, C3-Ce-cycloalkyl, C3-Ce-cycloalkyl-Ci-C4-alkyl, preferably cyclopropylmethyl or cyclopropylethyl, C3-C6-cycloalkyl-C2-C4-alkylene, Cs-Ce-aryl, C5-Ce-aryl-Ci-C4-alkyl, C5-C6- aryl-C2-C4-alkenyl, or Ri is more preferably a group selected from among alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, alkenyl, preferably C2-C4 alkenyl, more preferably allyl, C3-Ce-cycloalkyl, C3-Ce-cycloalkyl-Ci-C4-alkyl, preferably cyclopropylmethyl or cyclopropylethyl, and C3-Ce-cycloalkyl-C2-C4-alkylene;

R2 is OH, -OR or -O(C=O)R with R being alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, or alkenyl, preferably C2-C4 alkenyl, more preferably allyl, preferably R2 is OH or -OR, with R being alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, or alkenyl, preferably C2-C4 alkenyl, more preferably allyl, and more preferably R2 is OH, CH3-O- or CH3-CH2-O-; and R3 is H or OH; and

Rj is H, OH or O (i.e., an oxygen atom bound by a double bond to the carbon atom).

[0056] The morphinan compound of formula VII is more preferably a compound of formula VIII

Formula VIII wherein:

Ri is a group selected from among hydrogen, alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, alkenyl, preferably C2-C4 alkenyl, more preferably allyl, Cs-Ce-cycloalkyl, C3-C6- cycloalkyl-Ci-C4-alkyl, preferably cyclopropylmethyl or cyclopropylethyl, C3-Ce-cycloalkyl-C2- C4-alkylene, Cs-Ce-aryl, C5-Ce-aryl-Ci-C4-alkyl, C5-Ce-aryl-C2-C4-alkenyl benzoyl, hydroxy, methoxy, unsubstituted, monosubstituted, or di-substituted amine, Me(C=O)-, Et(C=O)-, Me(C=O)O- and Et(C=O)O-, preferably Ri is a group selected from among alkyl, preferably Ci- C4 alkyl, more preferably methyl or ethyl, alkenyl, preferably C2-C4 alkenyl, more preferably allyl, C3-Ce-cycloalkyl, C3-Ce-cycloalkyl-Ci-C4-alkyl, preferably cyclopropylmethyl or cyclopropylethyl, C3-C6-cycloalkyl-C2-C4-alkylene, Cs-Ce-aryl, C5-Ce-aryl-Ci-C4-alkyl, C5-C6- aryl-C2-C4-alkenyl, or Ri is more preferably a group selected from among alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, alkenyl, preferably C2-C4 alkenyl, more preferably allyl, Cs-Ce-cycloalkyl, C3-Ce-cycloalkyl-Ci-C4-alkyl, preferably cyclopropylmethyl or cyclopropylethyl, and C3-C6-cycloalkyl-C2-C4-alkylene;

R2 is OH, -OR or -O(C=O)R with R being alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, or alkenyl, preferably C2-C4 alkenyl, more preferably allyl, preferably R2 is OH or -OR, with R being alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, or alkenyl, preferably C2-C4 alkenyl, more preferably allyl, and more preferably R2 is OH, CH3-O- or CH3-CH2-O-; and R3 is H or OH.

[0057] The morphinan compound of formula VIII is still more preferably a compound of formula IX

wherein:

Ri is a group selected from among hydrogen, alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, alkenyl, preferably C2-C4 alkenyl, more preferably allyl, Cs-Ce-cycloalkyl, C3-C6- cycloalkyl-Ci-C4-alkyl, preferably cyclopropylmethyl or cyclopropylethyl, C3-Ce-cycloalkyl-C2- C4-alkylene, Cs-Ce-aryl, C5-Ce-aryl-Ci-C4-alkyl, C5-Ce-aryl-C2-C4-alkenyl benzoyl, hydroxy, methoxy, unsubstituted, monosubstituted, or di-substituted amine, Me(C=O)-, Et(C=O)-, Me(C=O)O- and Et(C=O)O-, preferably Ri is a group selected from among alkyl, preferably Ci- C4 alkyl, more preferably methyl or ethyl, alkenyl, preferably C2-C4 alkenyl, more preferably allyl, C3-Ce-cycloalkyl, C3-Ce-cycloalkyl-Ci-C4-alkyl, preferably cyclopropylmethyl or cyclopropylethyl, C3-C6-cycloalkyl-C2-C4-alkylene, Cs-Ce-aryl, C5-Ce-aryl-Ci-C4-alkyl, C5-C6- aryl-C2-C4-alkenyl, or Ri is more preferably a group selected from among alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, alkenyl, preferably C2-C4 alkenyl, more preferably allyl, C3-Ce-cycloalkyl, C3-Ce-cycloalkyl-Ci-C4-alkyl, preferably cyclopropylmethyl or cyclopropylethyl, and C3-Ce-cycloalkyl-C2-C4-alkylene; and R2 is OH, -OR or -O(C=O)R with R being alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, or alkenyl, preferably C2-C4 alkenyl, more preferably allyl, preferably R2 is OH or -OR, with R being alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, or alkenyl, preferably C2-C4 alkenyl, more preferably allyl, and more preferably R2 is OH, CH3-O- or CH3-CH2-O-; particularly, Ri is as defined above and R2 is OH, or Ri is as defined above and R2 is -OR with R being alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, or alkenyl, preferably C2-C4 alkenyl, more preferably allyl, or Ri is as defined above and R2 is CH3-O- or CH3-CH2-O-.

[0058] The morphinan compound of formula IX is yet more preferably a compound of formula X or XI

wherein:

Ri is a group selected from among hydrogen, alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, alkenyl, preferably C2-C4 alkenyl, more preferably allyl, Cs-Ce-cycloalkyl, C3-C6- cycloalkyl-Ci-C4-alkyl, preferably cyclopropylmethyl or cyclopropylethyl, C3-Ce-cycloalkyl-C2- C4-alkylene, Cs-Ce-aryl, C5-Ce-aryl-Ci-C4-alkyl, C5-Ce-aryl-C2-C4-alkenyl benzoyl, hydroxy, methoxy, unsubstituted, monosubstituted, or di-substituted amine, Me(C=O)-, Et(C=O)-, Me(C=O)O- and Et(C=O)O-, preferably Ri is a group selected from among alkyl, preferably Ci- C4 alkyl, more preferably methyl or ethyl, alkenyl, preferably C2-C4 alkenyl, more preferably allyl, C3-Ce-cycloalkyl, C3-Ce-cycloalkyl-Ci-C4-alkyl, preferably cyclopropylmethyl or cyclopropylethyl, C3-C6-cycloalkyl-C2-C4-alkylene, Cs-Ce-aryl, C5-Ce-aryl-Ci-C4-alkyl, C5-C6- aryl-C2-C4-alkenyl, or Ri is more preferably a group selected from among alkyl, preferably C1-C4 alkyl, more preferably methyl or ethyl, alkenyl, preferably C2-C4 alkenyl, more preferably allyl, C3-Ce-cycloalkyl, C3-Ce-cycloalkyl-Ci-C4-alkyl, preferably cyclopropylmethyl or cyclopropylethyl, and C3-C6-cycloalkyl-C2-C4-alkylene.

[0059] The morphinan compound can have various biochemical activities, for example as an opioid receptor antagonist or as an opioid receptor agonist. A morphinan compound “active agent,” for instance released from a m-g conjugate as described elsewhere herein, can have such agonist or antagonist activity selectively for one of the mu, kappa, delta, or epsilon subtype of opioid receptor. For instance, in treating acute opioid overdose, it might be preferable that the opioid receptor antagonist have selectivity for the mu receptor subtype.

[0060] In any embodiment of this first aspect of the disclosure, the activated saccharide can be an activated form of fructose, glucose, galactose, mannose, ribose, or another monosaccharide, or an activated oligosaccharide of one or more of these. For example, the saccharide or oligosaccharide can be one that is activated by an O-linked ester, for example a methyl or ethyl ester, or that is activated by an O-linked phosphoester such as a uridine diphosphate phosphoester (O-UDP).

[0061] In any embodiment of this first aspect of the disclosure, the glycosyltransferase can be an isolated UDP-carbohydrate-dependent glycosyltransferase.

[0062] For example, an embodiment can be one wherein the glycosyltransferase uses UDP-glucose as the activated saccharide and the m-g conjugate product is the corresponding B-D- glucoside.

[0063] In any embodiment of this first aspect of the disclosure, the glycosyltransferase can be one that, when employed as in Example 1 using naloxone as a morphinan substrate or using naltrexone as a substrate, has a turnover rate such that at least about 89% of a naloxone substrate or about 17% of a naltrexone substrate are consumed in the conjugation after 900 minutes. Additionally, or alternatively, the glycosyltransferase can be one that provides a product glycoside of which at least about 5% is the 0-D-O-gly coside of a naloxone substrate and/or at least about 5% is the 0-D-O-glycoside of a naltrexone substrate.

[0064] Any embodiment of this first aspect of the disclosure can be one in which the contacting step is performed in a cultured medium in which the glycosyltransferase is heterologously expressed by a host cell, and wherein said host cell also produces the activated saccharide or activated oligosaccharide as a metabolic product.

[0065] There are different elements in the biochemical pathways of gene expression. Accordingly, a gene encoding a glycosyl transferase is “heterologously expressed” if one or more of the following are true in respect of the system by which the glycosyl transferase gene is expressed: a) the promoter of the expression cassette has a nucleotide sequence of a promoter of a gene in a first species whereas the nucleotide sequence of the structural gene of the expression cassette is of a gene of a second species; or b) the host cell used in the expression system is of a species different from the species from which at least one nucleotide sequence of an element of the expression cassette (e.g., promoter, structural gene, enhancer, and the like) are derived.

[0066] An expression cassette that contains one or more elements (e.g., a promoter or structural gene) that include artificially constructed nucleotide sequences, including gene fusions, are included among genes that are deemed “heterologously expressed.”

[0067] As a non-limiting example, the glycosyl transferase structural gene might be obtained from a plant of the genus Arabidopsis, Fragaria or Mentha, or from a bacterium of the genus Bacillus or Lactobacillus, whereas this structural gene is expressed in a host cell that is one of a strain of Escherichia coli.

[0068] In a heterologous expression embodiment, preferably the activated saccharide is UDP-glucose, or the activated oligosaccharide is an oligosaccharide comprising UDP-glucose.

[0069] In such an embodiment, then the m-g conjugate product can be the corresponding B-D-glucoside.

[0070] In any embodiment of the first aspect of the disclosure, the glycosyltransferase can be a UDP-glucose-dependent glycosyltransferase from a plant or a bacterium.

[0071] For example, the UDP-glucose-dependent glycosyltransferase can be one derived from a plant of the genus Arabidopsis, Fragaria or Mentha, or from a bacterium of the genus Bacillus or Lactobacillus.

[0072] In some embodiments utilizing heterologous expression the host cell is one of a strain of Escherichia coli.

[0073] In the morphinan-glycoside conjugate (m-g conjugate) described in relation to the first aspect of the present invention (and also in relation to all other aspects of the present invention), the morphinan group is preferably conjugated at the 3-position with the glycoside.

[0074] A second aspect of the disclosure lies in a method for purifying a glycoside conjugate of a morphinan compound (“m-g conjugate”) from a reaction mixture comprising: a) adjusting the pH of the reaction mixture to from 7 to 10.5, or preferably to from 8 to 10 or from 9 to 10, to convert m-g conjugate acetate in the reaction mixture to m-g conjugate; b) neutralizing the reaction mixture and contacting it with a lipophilic solid phase to obtain immobilized m-g conjugate and washing the immobilized m-g conjugate; c) eluting the m-g conjugate from the solid phase with an alcohol; d) drying the eluate, redissolving the dried eluate in water or a buffer to obtain an aqueous solution; e) optionally back extracting the aqueous solution with a polar organic solvent to remove any remaining morphinan compound and recovering the aqueous solution; f) drying the aqueous solution to obtain the solid m-g conjugate; g) optionally redissolving the m-g conjugate in an alcohol and treating the alcohol solution with activated carbon to remove colored and other impurities, recovering the alcohol solution, and drying the alcohol solution to obtain the solid m-g conjugate.

[0075] A third aspect of the disclosure is a method for purifying a morphinan compound O-glycoside conjugate from a biological culture comprising: a) removing cells from the culture to obtain a supernatant containing m-g conjugate and m-g conjugate acetate; b) adjusting the pH of the supernatant to from 7 to 10.5, preferably to from 8 to 10 or from 9 to 10, to convert m-g conjugate acetate in the supernatant to m-g conjugate; c) neutralizing the supernatant and contacting it with a lipophilic solid phase to obtain immobilized m-g conjugate and washing the immobilized m-g conjugate; d) eluting the m-g conjugate from the solid phase with an alcohol; e) drying the eluate, redissolving the dried eluate in water or a buffer to obtain an aqueous solution; f) optionally back extracting the aqueous solution with a polar organic solvent to remove any remaining morphinan compound and recovering the aqueous solution; g) drying the aqueous solution to obtain the solid m-g conjugate; h) optionally redissolving the m-g conjugate in an alcohol and treating the alcohol solution with activated carbon to remove colored or other impurities, recovering the alcohol solution, and drying the alcohol solution to obtain the solid m-g conjugate.

[0076] In any embodiments of this third aspect cells can be removed from the culture by any methods know in the art, including centrifugation or filtration. [0077] In any embodiments of these second and third aspects, “drying” can be performed under reduced pressure, such as in a “rotovap” apparatus or the like.

[0078] In any embodiments of these second and third aspects, the solid phase can be a lipohilic solid phase, an Amberlite™ polymeric adsorbent resin, a Purolite™ polymeric adsorbent resin, or a mixture of any two or more of these.

[0079] In any embodiments of these second and third aspects, the alcohol can be methanol or ethanol. Furthermore, the polar organic solvent can be ethyl acetate, a C4 alcohol, a C5 alcohol, for example n-butanol, or 2-methyl-l -butanol.

[0080] A fourth aspect of the disclosure is a compound Nal oxone-3 -O-B-D-glucoside, Naltr exone-3 -O-B-D-glucoside, or an ester thereof, or an acetal, semiacetal derivative thereof or an ester of any of these.

[0081] A fifth aspect of the disclosure is a morphinan-glycoside conjugate (m-g conjugate) compound that comprises a radical of a morphinan compound of Formula I:

wherein Ra can be any group, which group can include additional rings, or any two Ra groups can also join to form additional rings, which additional rings may contain at least one further heteroatom. For example, two Ra groups might form a 5- or 6-member epoxy group, a 5- or 6- member thioexpoxy group, a pyrrolidine or a piperidine group; n can be from 0 to 8, for example from 1 to 8, or from 2 to 8, or from 4 to 8 or from 4 to 6; each Ra can be the same or different, and can be selected from among alkyl, such as methyl or ethyl, alkenyl, alkynl, allyl, azine, benzoyl, hydroxy, methoxy, unsubstituted, monosubstituted, or di-substituted amine, thiol, keto, Me(C=O)O- and Et(C=O)O-; additionally or alternatively, the 7 and 8 carbons can be joined by a double bond; additionally or alternatively to Ra groups can join the 4 and 5 positions by an epoxy ring.

[0082] A “morphinan compound” can also include additional heteroatoms, either in the polycyclic bridged framework, or in substituents appended to the framework, or in both. Such a m-g conjugate can be one wherein the morphinan compound is Naloxone, Naltrexone, Naloxonazine or Naloxonbenzoylhydrazone, or a derivative of any of these.

[0083] A m-g conjugate of the fifth aspect of the disclosure can be one wherein the morphinan group further comprises at least one additional N, O or S heteroatom.

[0084] A m-g conjugate can be one wherein the morphinan group comprises a radical of a substituted morphinan compound of Formula I:

(Formula I), wherein: n can be from 1 to 8, or from 2 to 8, or from

4 to 8, or from 4 to 6; each Ra can be the same or different, and can be selected from among alkyl, such as methyl or ethyl, alkenyl, alkynl, allyl, azine, benzoyl, hydroxy, methoxy, unsubstituted, monosubstituted, or di-substituted amine, thiol, keto, Me(C=O)O- and Et(C=O)O-; additionally or alternatively, the 7 and 8 carbons can be joined by a double bond; additionally or alternatively to Ra groups can join the 4 and 5 positions by an epoxy ring. Further, the morphinan compound of Formula I can be as defined above.

[0085] Further, the morphinan compound can be of any one of Formulas II to XI described and defined above.

[0086] The glycoside in the m-g conjugate is as defined above and can be, for example, fructose, glucose, galactose, mannose, ribose, or an oligosaccharide of one or more of these.

[0087] Such a m-g conjugate can be one wherein the morphinan group is conjugated via an O, N or S atom. For example, the morphinan group can be conjugated at the 3-position, e.g., as a 3-O-glycoside. Additionally, or alternatively, a m-g conjugate can comprise a morphinan group in which neither of the 9 or 13 positions of the morphinan group are substituted. [0088] The glycoside conjugates of morphinan compounds described herein in most embodiments, in comparison with the starting morphinan compound, will show improved water solubility, a changed taste, a changed uptake to the body, a changed metabolization, a changed activity or a change in pharmacokinetics.

[0089] A sixth aspect of the disclosure is a composition comprising a) at least one morphinan-glycoside conjugate as described above, or an acetal, semiacetal or ester-derivative thereof; and b) at least one glycosidase, and/or at least one microorganism that produces a one or more glycosidases.

[0090] Such a composition can be one in which the m-g conjugate is Naloxone- or Naltr exone-3 -O-gly coside or an acetal, semiacetal or ester-derivative thereof.

[0091] For example, such a composition can comprise Naloxone- or Naltrexone-3-O-B-D- glucoside as the m-g conjugate.

[0092] In any embodiment of this sixth aspect of the disclosure, the glycosidase can be a B-glucosidase, or a mixture of B-glucosidases. As a non-limiting example, the glycosidase can be a B-glucosidase, or a mixture of B-glucosidases, derived from bacteria of genus Lactobacillus.

[0093] In any embodiment of the sixth aspect of the disclosure, the composition a), and optionally the composition b), is provided in a dosage form of a transdermal patch; a solid oral formulation; a liquid oral formulation; a topical formulation for application to skin; a spray or powder formulation for inhalation; or an enteric formulation. In some embodiments of the sixth aspect of the disclosure both of the compositions a) and b) are included in the dosage form.

[0094] For example, a topical formulation can be a cream, an unguent, or gel, or an enteric formulation can be a solid formulation for oral administration to the intestinal tract, or as a solid formulation for suppository administration to the colon or vagina.

[0095] A composition of this sixth aspect of the disclosure can be formulated for intravenous, subcutaneous, or intramuscular injection, oral, nasal, by inhalation, dermal, or rectal or vaginal administration, or for administration by a transdermal patch. Application forms for dermal or transdermal patch administration can be, for example, in the form of a “band-aid”, all dermal application forms may contain the m-g conjugate and a cleaving glycosidase either in a dry form, or layered dry forms, or as a dispersion in non-aqueous phase (salve, unguent) and become activated by hydration by application on the skin, or, alternatively may contain the mixture in an aqueous formulation with a pH inhibiting the cleaving enzyme and which becomes activated by application on the skin (neutralization by skin pH-active compounds (such as millimolar ammonia solution) and/or evaporation of a pH-active or enzyme-denaturing volatile compound (such as isopropanol) from the formulation).

[0096] Such a composition can be an encapsulated formulation. An encapsulated formulation can include, one or more, or all, of the ingredients in encapsulated form. An encapsulated composition can include a plurality of capsules differing in the ingredients they contain. A capsule material will preferably be water soluble or breakable by applied pressure.

[0097] A seventh aspect of the disclosure lies in a pharmaceutical composition comprising at least one morphinan-glycoside conjugate as described above, or an acetal, semiacetal or esterderivative thereof; and one or more pharmaceutically acceptable carriers and/or excipients. Such a pharmaceutical composition can be formulated for oral administration, including as an encapsulated formulation as described above, administration by transdermal patch, or for administration by injection or infusion, or by an inhaler. General considerations for preparing pharmaceutical compositions for each of these administration routes can be found, for example, in Remington: The Science and Practice of Pharmacy,” 23^rd Ed., c. 2021 by University of the Sciences in Philadelphia, publ. Elsevier, Inc., hereby incorporated by reference in its entirety and for all purposes.

[0098] A pharmaceutical composition of this seventh aspect of the disclosure can be used in either human patients or in veterinary medicine.

[0099] An eighth aspect of the disclosure is a method for increasing the rate of hydrolysis of a morphian-glycoside conjugate as described above, or an acetal, semiacetal or ester-derivative thereof; comprising contacting said morphinan-glycoside conjugate or an acetal, semiacetal or ester-derivative thereof with at least one glycosidase, and/or at least one microorganism that produces one or more glycosidases.

[00100] A ninth aspect of the disclosure is a method for increasing the release of morphinan active agent, for example, as an opioid agonist or opioid antagonist from a morphinan-glycoside conjugate (m-g conjugate) in situ on a surface to provide the free morphinan active agent comprising: a) applying a composition comprising the m-g conjugate as described above to the surface; and b) contacting the composition with at least one glycosidase, and/or at least one microorganism, which can be a microorganism that is indigenous to the surface to which the composition is applied, that produces one or more glycosidases.

[00101] A microorganism is “indigenous” to a surface if it is a species that can typically be found living on that surface, e.g., upon a skin or mucosal surface of a human. One way to identify a microorganism as indigenous to a surface is one that can be cultured in a known culture medium from a scrape biopsy of the surface. Another way to identify a microorganism as indigenous to a surface is to apply 16S rRNA gene sequence or metagenomics DNA sequence analysis to a biological sample obtained from the surface (see, e.g. “Recent advances in genomic DNA sequencing of microbial species from single cells,” R.S. Lasken and J.S. McLean, Nat Rev Genet. 2014 Sep; 15(9): 577-584). Species identified in the sample are taken as “living” on the surface. [00102] In some embodiments of this ninth aspect of the disclosure the composition applied in step a) comprising the m-g conjugate can be a dry composition; and the composition applied in step b) comprising the at least one glycosidase and/or at least one microorganism is also a dry composition, and an aqueous solution comprising any co-factors required for activity of the glycosidase, are applied after step b).

[00103] A tenth aspect of the disclosure is a method for producing a purified morphinan-O- gly coside conjugate comprising: a) contacting a morphinan compound with an activated saccharide or activated oligosaccharide and a glycosyltransferase in a reaction mixture under conditions, including any co-factors necessary for glycosyltransferase activity, effective to produce a morphinan- O-glycoside conjugate (m-g conjugate); and b) purifying the m-g conjugate from the reaction mixture to obtain the isolated m-g conjugate.

[00104] In some embodiments of this tenth aspect of the disclosure, the contacting step is performed in a cultured medium in which the glycosyltransferase is heterologously expressed by a host cell, and the host cell also produces the activated saccharide or activated oligosaccharide as a metabolic product.

[00105] In some embodiments of the tenth aspect of the disclosure, the activated saccharide can be UDP-glucose, or the activated oligosaccharide can be an oligosaccharide comprising UDP- glucose. [00106] In some embodiments of the tenth aspect of the disclosure, the m-g conjugate product is the corresponding B-D-glucoside.

[00107] In any embodiments of the tenth aspect of the disclosure, the glycosyltransferase can be a UDP-glucose-dependent glycosyltransferase from a plant or a bacterium. For example, the UDP-glucose-dependent glycosyltransferase can be derived from a plant of the genus Arabidopsis, Fragaria ox Mentha, or from a bacterium of the genus Bacillus or Lactobacillus .

[00108] An eleventh aspect of the disclosure is a process for preparing a medicament, comprising mixing at least morphinan compound-glycoside conjugate or composition thereof, of any one of the first through seventh aspects above, or an acetal, semiacetal or ester-derivative thereof, with one or more pharmaceutically acceptable carriers and/or excipients.

[00109] A twelfth aspect of the disclosure is use of the compounds and composition as described above, e.g., Naloxone, Naltrexone, Naloxonazine or Naloxonbenzoylhydrazone, or a derivative of any of these, in treatment of acute opioid overdose or chronic treatment of opioid addiction. Such a use for addiction treatment can comprise transdermal administration of the m-g conjugate or release of the morphinan compound to the skin of a subject from a m-g conjugate via a transdermal patch dosage form.

[00110] A method of treatment for opioid addiction can comprise administering a pharmaceutical composition comprising a m-g conjugate that comprises a morphinan group as described above to a subject presenting with opioid addiction. In such a method the composition can be administered topically, by injection, e.g., by intramuscular or subcutaneous injection, or by oral administration. In such a method, the composition can be administered via a transdermal patch. In such a method, the dosage form can be one that provides a morphinan compound active agent having activity as an opioid antagonist, e.g., as measured by an opioid receptor binding assay typical of the art, in an amount to provide a therapeutic concentration of the antagonist vs. said opioid receptor, in the blood of the subject. A minimum such therapeutic concentration is considered to be at least the IC50 for the antagonist binding to the target opioid receptor.

[00111] A method of treatment for opioid overdose can comprise administering a pharmaceutical composition comprising a m-g conjugate that comprises a morphinan group as described above to a subject presenting with opioid overdose. In such a method the composition can be administered by injection, e.g., by inhalation or by intravenous injection, intramuscular or subcutaneous injection, or by oral administration. In such a method, the dosage form can be one that provides a morphinan compound active agent having activity as an opioid antagonist, e.g., as measured by an opioid receptor binding assay typical of the art, in an amount to provide a therapeutic concentration of the antagonist vs. said opioid receptor, in the blood of the subject. A minimum such therapeutic concentration is considered to be at least the IC50 for the antagonist binding to the target opioid receptor.

[00112] In embodiments in which the morphinan compound is Naloxone, Naltrexone, Naloxonazine or Naloxonbenzoylhydrazone, a therapeutically effective dose is known in the art.

[00113] Any of the examples or embodiments described herein may include various other features in addition to or in lieu of those described above. The teachings, expressions, embodiments, examples, etc., described herein should not be viewed in isolation relative to each other. Various suitable ways in which the teachings herein may be combined should be clear to those skilled in the art in view of the teachings herein.

EXAMPLES

[00114] Naloxone Hydrochloride 98% used in the examples below was obtained from Alfa Aesar, CAS number 357-08-4. The free substance naloxone was obtained by adding aqueous NaOH and back-extraction over a lipophilic column and used for comparative NMR analysis.

[00115] Naltrexone Hydrochloride 95% minimum was purchased from Alfa Aesar, CAS number 16676-29-2.

[00116] Rapidase (P-glucosidase) was obtained from DSM Food specialties BV under the trade name Rapidase AR 2000.

[00117] Abbreviations mg milligram min Minutes mL Milliliters nm Nanometer

OD600 Optical density at 600 nm wavelength pg Microgram w / v weight / volume; Weight based on volume as a concentration Example 1 - Synthesis of Naloxone-3-O-B-D-Glucoside

[00118] The nucleotide sequence encoding the glycosyltransferase 4G-GT6 from Mentha piperata, having the amino acid sequence shown in Figure 9, was cloned into the expression vector pET29a and the construct was transformed into E. coli BL21 (DE3) pLysS (Novagen, Schwalbach, Germany). The glycosylase expression strain was used to glucosylate naloxone to naloxone B-D- glucoside by a biotransformation in culture. Precultures were grown overnight at 37°C in Luria Bertani (LB) medium with 100 pg / ml ampicillin and 23 pg / ml chloramphenicol. The next day 50 mL M9 minimal medium with 1% glucose and the antibiotics mentioned were inoculated with 1 mL of the preculture and grown at 37°C and 160 rpm until an OD600 (optical density at 600 nm) of 1 was reached. The culture was continued at 18°C. and 1 mM IPTG (isopropyl-0-D- thiogalactopyranoside) was added for induction. After 6 hours, 10 mg of the substrate naloxone hydrochloride (98%, Alfa Aesar, Ward Hill, Massachusetts, United States) was added. A sample of 200 pL of the culture was taken at regular intervals, centrifuged to remove the cells, and diluted 1 : 2 for an LC-MS analysis in order to monitor the progress of the biotransformation.

Example 2 - LC-MS-MS2 Analysis of Naloxone- 3-O-B-D-Glucoside

[00119] The analysis by means of liquid chromatography with mass spectroscopy coupling was conducted with a Bruker Esquire 3000plus (Bruker Daltonics, Bremen, Germany) mass spectrometer coupled to an Agilent 1100 HPLC system that used an Agilent 1100/1200 Micro Wellplate Autosampler 1, and an Agilent 1100/1200 Diode Array Detector SL 1. The separation was conducted with a reversed-phase column LUNA Cl 8 100A 150 * 2 mm (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 “ the gradient was: 0 min 10% B, 7 min 50% B, 10 min 50% B, 15 min 100% B, 20 min 100% B, 30 min 10% B. The injection volume of the sample was 5 pL. The flow for mass spectroscopy was set to 0.2 mL by a flow divider. The mass spectroscopy profiles were recorded from mass / charge ratios of 50 m / z to 1000 m / z with negative ionization with a scan speed of 4000 V and an interface voltage of -500 V with nitrogen as the collision gas.

[00120] FIG. 1 shows the LC-MS analysis of the product of the biotransformation of naloxone to naloxone-3-O-B-D-glucoside with the glycosyltransferase 4G-GT6 in E. coli from Example 1. [00121] FIG. 1 (top) shows the HPLC chromatogram (232 nm) with the corresponding peak of naloxone-3-O-B-D-glucoside at about 9 min and nal oxone-3 -O-B-D-glucoside acetate at 10 min. [00122] FIG. 1 (bottom) shows the MS in positive mode with a mass / charge ratio of 490 m / z, which corresponds to the protonated nal oxone-3 -O-B-D-glucoside.

[00123] FIG. 2 shows the MS and MS2 analyzes of the peak at about 9 min. The MS (top) shows the protonated naloxone-3-O-B-D-glucoside, as expected, at m / z 490, the MS2 (bottom), a protonated fragment after dehydration at m / z 472 and a further protonated fragment after splitting off the glucoside residue at m / z 328, as well as another fragment with m / z 310 due to dehydration from the latter. Masses and fragmentation agree with nal oxone-3 -O-B-D-glucoside as an expected product.

Example 3 - Enzymatic Cleavage of Naloxone-3-O-B-D-Glu coside by B-Glucosidase

[00124] 1 mL of a solution of 1 mg / mL naloxone-3-O-B-D-glucoside from Example 1 was mixed with 1 mg / ml of the B-glucosidase Rapidase, incubated for 330 min at 30 ° C, and analyzed by means of HPLC for naloxone (cleavage product) and remaining nal oxone-3 -O-B-D-glucoside (FIG. 3).

[00125] The cleavage by a B-glucosidase confirms the B-anomerism of the glucoside (FIG. 3). It also demonstrates the possibility of releasing the aglycone again by hydrolysis, as is intended in some embodiments.

Example 4 -NMR Analysis of Naloxone- 3-O-B-D-Glu coside

[00126] Furthermore, an NMR analysis of the synthesized naloxone-3-O-B-D-glucoside (30 mg in 600 pL of deuterated DMSO-d6) was conducted with a Bruker DRX 500 at 500, 13 MHz. The ’H and ¹³C-NMR- Spectra and a 2D representation confirm the structure with the glucoside residue in position 3 (FIG. 4).

Example 5 - Determination of the Water Solubility of Naloxone-3-O-B-D-Glucoside

[00127] The water solubility at 25 ° C for naloxone is approximately 1.4 g / L, for naloxone- 3-O-B-D-glucoside it is 623 g / L. Nal oxone-3 -O-B-D-glucoside is 445 times more soluble in water than naloxone itself.

Example 6 - Synthesis and Confirmation of Naltrexone-3-O-B-D-Glucoside

[00128] Naltr exone-3 -O-B-D-glucoside was synthesized using the same method as in

Example 1, but using Naltrexone hydrochloride for the substrate morphinan. LC-MS/MS2 analysis and biochemical characterization of the resulting conjugate were performed as in Examples 2 (results in FIGS. 5 and 6) and 3 above (results in FIG. 7).

Example 7 - Purification of Naloxone-3-O-B-D-Glucoside

[00129] After fermentation conducted as in Example 1, the cells were removed from the biosuspension by centrifugation (5000 rpm for 20 min). The supernatant containing nal oxone-3 - O-P-D-glucoside and naloxone-3-O-P-D-glucoside acetate was adjusted to pH 10 with NaOH. After the complete conversion of naloxone-3-O-P-D-glucoside acetate into naloxone-3-O-P-D- glucoside, the supernatant was neutralized with phosphoric acid and further incubated with a lipophilic solid phase. The resin was then thoroughly washed with water and the glucosides were eluted with 5 times of methanol (the volume of methanol to cover 3 mm above the resin) by vacuum filtration. The methanol was evaporated, and the residue was dissolved in water, followed by a liquid-liquid extraction with ethyl acetate to remove remaining naloxone. After extraction, the water phase was dried by rotation evaporator, and the residue was dissolved in methanol and treated with activated carbon to remove colored impurities. Finally, the glucosides were dried by rotation evaporator and analyzed by HPLC to check its purity. The result (FIG. 8) showed that more than 97 % purity of naloxone-3-O- P-D-glucoside was obtained.

Example 8 - Antagonistic Effects of Naloxone-3-O-[3-D-Glucoside

[00130] To assess the ability of naloxone-3-O-P-D-glucoside (NaG) to antagonize antinociceptive effects of opioid receptor agonists, the effects of the mu opioid receptor agonist fentanyl (SC injection) were determined alone and after SC pretreatment with NaG and the classical antagonist naloxone (NLX). Swiss Webster mice were randomly assigned to groups (N=8), and each group received (a) 10 mg/kg NLX antagonist at TO and the agonist fentanyl after 15 min (T15), (b) 10 mg/kg NaG antagonist at TO and the agonist fentanyl at T15, (c) saline at TO and 0.56 mg/kg fentanyl at T15, and (d) saline at TO and at T15 (control).

[00131] A warm water tail-withdrawal antinociception assay was used to determine antinociception at T15 and then in 30-min intervals up to 270 min. The distal tip of the mouse’s tail was placed in a 50°C warm water bath and the tail withdrawal latency was measured. To prevent tissue damage, a cut-off latency of 10 seconds was imposed. Latencies were expressed as percentage of maximum possible effect (%MPE) by the formula: test latency — baseline latency

%MPE = - — - - - - - - - - - — X100 cutoff latency — baseline latency [00132] A comparison of the results obtained for NLX (FIG. 10) and NaG (FIG. 11) according to the invention shows that glycosylated naloxone (NaG) exhibits a delayed onset of action compared to naloxone (NLX). This delayed onset (or delayed effect or release of the glucoside) could help in an opioid overdose situation to treat patients with NaG doses that lasts longer than NLX (which has a fast action and short circulation half-life). In particular, the data shown in the figures suggest that the present invention is useful in prophylaxis. Also, law enforcement could benefit from this delayed onset to mitigate unintentional or weaponized exposure to opioid narcotics. The present invention therefore provides promising options in the prophylaxis or prevention of the opioid overdose state.

[00133] Having shown and described exemplary embodiments of the subject matter contained herein, further adaptations of the methods and systems described herein may be accomplished by appropriate modifications without departing from the scope of the claims. In addition, where methods and steps described above indicate certain events occurring in certain order, it is intended that certain steps do not have to be performed in the order described but in any order as long as the steps allow the embodiments to function for their intended purposes. Therefore, to the extent there are variations of the invention, which are within the spirit of the disclosure or equivalent to the inventions found in the claims, it is the intent that this patent will cover those variations as well. Some such modifications should be apparent to those skilled in the art. For instance, the examples, embodiments, geometries, materials, dimensions, ratios, steps, and the like discussed above are illustrative. Accordingly, the claims should not be limited to the specific details of structure and operation set forth in the written description and drawings.

ENUMERATED EXAMPLES

[00134] Further illustrative, non-exclusive enumerated examples of compounds, compositions, methods and uses according to the present disclosure are described below. The scope of the invention disclosed herein is not to be limited by these listed examples, but only by the claims appended to this disclosure.

[00135] EE1: A method for producing a purified morphinan-glycoside conjugate (m-g conjugate) comprising: a) contacting a morphinan compound of Formula I,

(Formula I) wherein:

Ra can be any group, which group can include additional rings, or any two Ra groups can also join to form additional rings, which additional rings may contain at least one further heteroatom. n is from 0 to 8; each Ra can be the same or different, and is selected from among alkyl, such as methyl or ethyl, alkenyl, alkynl, allyl, azine, benzoyl, hydroxy, methoxy, unsubstituted, monosubstituted, or di-substituted amine, thiol, keto, Me(C=O)O- and Et(C=O)O-; optionally, the 7 and 8 carbons are joined by a double bond; optionally, two Ra groups join the 4 and 5 positions by an epoxy ring; and optionally the morphinan compound includes additional heteroatoms in the morphinan framework, or in substituents appended to the framework, or in both; with an activated saccharide or activated oligosaccharide and a glycosyltransferase in a reaction mixture under conditions, including any co-factors necessary for glycosyltransferase activity, effective to produce a morphinan-glycoside conjugate; and b) purifying the morphinan-glycoside conjugate (m-g conjugate) from the reaction mixture to obtain the isolated m-g conjugate.

[00136] EE2: The method of EE1, wherein the morphinan compound further comprises at least one additional N, O or S heteroatom.

[00137] EE3: The method of EE1 or 2, wherein the morphinan compound is a substituted morphinan compound, wherein n is from 4 to 6.

[00138] EE4: The method of EE3, wherein the morphinan compound is Naloxone, Naltrexone, or a derivative of one of these.

[00139] EE5: The method of EE4, wherein the morphinan compound is Naloxone, Naltrexone, Naloxonazine or Naloxonbenzoylhydrazone. [00140] EE6: The method of any one of EEsl-3, wherein the morphinan compound is substituted at at least one position by a hydroxyl, thiol, or amine group.

[00141] EE7: The method of any one of EEs 1-6, wherein at least the 3 -position of the morphinan compound is substituted.

[00142] EE8: The method of EE3, 6 or 7, wherein neither of the 9 or 13 position of the morphinan compound is substituted.

[00143] EE9: The method of any one of EEs 1-8, wherein the morphinan compound has biochemical activity as an opioid receptor antagonist.

[00144] EE10: The method of any one of EEsl-9, wherein the activated saccharide is an activated form of fructose, glucose, galactose, mannose, ribose, or another monosaccharide, or an activated oligosaccharide of one or more of these.

[00145] EE11: The method of any one of EEs 1-10, wherein the saccharide or oligosaccharide is activated by an O-linked ester.

[00146] EE12: The method of EE11, wherein the O-linked ester is a phosphate ester.

[00147] EE13: The method of EE12, wherein the phosphate ester is of uridine diphosphate

(UDP).

[00148] EE14: The method of any one of EEsl-13, wherein the glycosyltransferase is an isolated UDP-carbohydrate-dependent glycosyltransferase.

[00149] EE15: The method of any one of EEsl-14, wherein the glycosyltransferase uses

UDP-glucose as the activated saccharide and the m-g conjugate product is the corresponding B-D- glucoside.

[00150] EE16: The method of any one ofEEsl-15 in which the contacting step is performed in a cultured medium in which the glycosyltransferase is heterologously expressed by a host cell, and wherein said host cell also produces the activated saccharide or activated oligosaccharide as a metabolic product.

[00151] EE17: The method of EE16, wherein the activated saccharide is UDP-glucose, or the activated oligosaccharide is an oligosaccharide comprising UDP-glucose.

[00152] EE18: The method of EE17, wherein the m-g conjugate product is the corresponding B-D-glucoside.

[00153] EE19: The method of any one of EEsl-18 in which the glycosyltransferase is a

UDP-glucose-dependent glycosyltransferase from a plant or a bacterium. [00154] EE20: The method of EE19, wherein the UDP-glucose-dependent glycosyltransferase is derived from a plant of the genus Arabidopsis, Fragaria ox Mentha, or from a bacterium of the genus Bacillus or Lactobacillus.

[00155] EE21 : The method of any one of EEs 16-20 in which the host cell is one of a strain of Escherichia coli.

[00156] EE22: A method for purifying an O-glycoside conjugate of a morphinan compound

(m-g conjugate) from a reaction mixture comprising: a) adjusting the pH of the reaction mixture to from 7 to 10.5, or to from 8 to 10 or from 9 to 10, to convert m-g conjugate acetate in the reaction mixture to m-g conjugate; b) neutralizing the reaction mixture and contacting it with a lipophilic solid phase to obtain immobilized m-g conjugate and washing the immobilized m-g conjugate; c) eluting the m-g conjugate from the solid phase with an alcohol; d) drying the eluate, redissolving the dried eluate in water or a buffer to obtain an aqueous solution; e) optionally back extracting the aqueous solution with a polar organic solvent to remove any remaining morphinan compound and recovering the aqueous solution; f) drying the aqueous solution to obtain the solid m-g conjugate; and g) optionally redissolving the m-g conjugate in an alcohol and treating the alcohol solution with activated carbon to remove colored or other impurities, recovering the alcohol solution, and drying the alcohol solution to obtain the solid m-g conjugate.

[00157] EE23: A method for purifying a morphinan compound O-glycoside conjugate (m- g conjugate) from a biological culture comprising: a) removing cells from the culture to obtain a supernatant containing m-g conjugate and m-g conjugate acetate; b) adjusting the pH of the supernatant to from 7 to 10.5, or to from 8 to 10 or from 9 to 10, to convert m-g conjugate acetate in the supernatant to m-g conjugate; c) neutralizing the supernatant and contacting it with a lipophilic solid phase to obtain immobilized m-g conjugate and washing the immobilized m-g conjugate; d) eluting the m-g conjugate from the solid phase with an alcohol; e) drying the eluate, redissolving the dried eluate in water or a buffer to obtain an aqueous solution; f) optionally back extracting the aqueous solution with a polar organic solvent to remove any remaining morphinan compound and recovering the aqueous solution; g) drying the aqueous solution to obtain the solid m-g conjugate; h) optionally redissolving the m-g conjugate in an alcohol and treating the alcohol solution with activated carbon to remove colored or other impurities, recovering the alcohol solution, and drying the alcohol solution to obtain the solid m-g conjugate.

[00158] EE24: The method of EE22 or 23, wherein the solid phase is a lipohilic solid phase, an Amberlite™ polymeric adsorbent resin, a Purolite™ polymeric adsorbent resin, or a mixture of any two or more of these.

[00159] EE25 : The method of any one of EEs22-24, wherein the alcohol is methanol or ethanol.

[00160] EE26: The method of any one of EEs22-25, wherein the polar organic solvent is ethyl acetate, a C4 alcohol, a C5 alcohol, among the latter n-butanol, or 2-methyl-l -butanol.

[00161] EE27: A compound Naloxone-3-O-B-D-glucoside, Naltrexone-3-O-B-D-glucoside, or an ester thereof, or an acetal, semiacetal derivative thereof or an ester of these.

[00162] EE28: A morphinan-glycoside conjugate (m-g conjugate) compound that comprises a radical of a morphinan compound of Formula I,

(Formula I) wherein:

Ra can be any group, which group can include additional rings, or any two Ra groups can also join to form additional rings, which additional rings may contain at least one further heteroatom. n is from 0 to 8; each Ra can be the same or different, and is selected from among alkyl, such as methyl or ethyl, alkenyl, alkynl, allyl, azine, benzoyl, hydroxy, methoxy, unsubstituted, monosubstituted, or disubstituted amine, thiol, keto, Me(C=O)O- and Et(C=O)O-; optionally, the 7 and 8 carbons are joined by a double bond; optionally, two Ra groups join the 4 and 5 positions by an epoxy ring; and optionally the morphinan compound includes additional heteroatoms in the morphinan framework, or in substituents appended to the framework, or in both; conjugated to a glycoside.

[00163] EE29: The m-g conjugate of EE28, wherein the morphinan group is a radical of

Naloxone, Naltrexone, or a derivative of one of these.

[00164] EE30: The m-g conjugate of EE29, wherein the morphinan group is a radical

Naloxone, Naltrexone, Naloxonazine or Naloxonbenzoylhydrazone.

[00165] EE31 : The m-g conjugate of EE28, wherein the morphinan group further comprises at least one additional N, O or S heteroatom.

[00166] EE32: The m-g conjugate of EE28 or 31, wherein the morphinan group comprises a substituted morphinan group of the formula I:

(Formula I), wherein Ra can be any group, which group can include additional rings, or any two

Ra groups can also join to form additional rings, which additional rings may contain at least one further heteroatom. For example, two Ra groups might form a 5- or 6-member epoxy group, a 5- or 6- member thioexpoxy group, a pyrrolidine or a piperidine group; n can be from 1 to 8; each Ra can be the same or different, and can be selected from among alkyl, such as methyl or ethyl, alkenyl, alkynl, allyl, azine, benzoyl, hydroxy, methoxy, unsubstituted, monosubstituted, or di-substituted amine, thiol, keto, Me(C=O)O- and Et(C=O)O-; additionally or alternatively, the 7 and 8 carbons can be joined by a double bond; additionally or alternatively to Ra groups can join the 4 and 5 positions by an epoxy ring; and optionally the morphinan group can also include additional heteroatoms, either in the morphinan framework, or in substituents appended to the framework, or in both.

[00167] EE33: The m-g conjugate of any one of EEs28-32, wherein the morphinan group is conjugated by an O, S or N atom.

[00168] EE34: The m-g conjugate of EE33, wherein the 3-position of the morphinan group is conjugated.

[00169] EE35: The m-g conjugate of any one of EEs28-34, wherein neither of the 9 or 13 position of the morphinan group is conjugated.

[00170] EE36: A composition comprising a) at least one morphinan-gly coside conjugate of any one of EEs27-35 or an acetal, semiacetal or ester-derivative thereof; and b) at least one glycosidase, and/or at least one microorganism that produces a one or more glycosidases.

[00171] EE37: The composition of EE36, in which the m-g conjugate is Naloxone- or

Naltrexone-3-O-gly coside or an acetal, semiacetal or ester-derivative thereof.

[00172] EE38: The composition of EE36 or 37, wherein the m-g conjugate is a Naloxone- or Naltrexone-3-O-B-D-glucoside.

[00173] EE39: The composition of any one of EEs36-38, wherein the glycosidase is a 13- glucosidase, or a mixture of 13-glucosidases.

[00174] EE40: The composition of any one of EEs36-39 in which the glycosidase is a 13- glucosidase, or a mixture of 13-glucosidases, derived from bacteria of genus Lactobacillus.

[00175] EE41: The composition of any one of EEs36-40 formulated for oral, dermal, or rectal administration, or administration by a transdermal patch.

[00176] EE42: The composition of EE41 that is an encapsulated formulation.

[00177] EE43 : A pharmaceutical composition comprising at least one morphinan-gly coside conjugate of any one of EEs27-35, or an acetal, semiacetal or ester-derivative thereof; and one or more pharmaceutically acceptable carriers and/or excipients.

[00178] EE44: The pharmaceutical composition of EE43, that is formulated for oral administration, administration by transdermal patch, or for administration by intravenous injection or infusion, for administration by intramuscular injection or for administration for subcutaneous injection. [00179] EE45: A method for increasing the rate of hydrolysis of a morphinan-gly coside conjugate of any one of EEs27-35, or an acetal, semiacetal or ester-derivative thereof; comprising contacting said morphinan compound-glycoside conjugate or an acetal, semiacetal or esterderivative thereof with at least one glycosidase, and/or at least one microorganism that produces one or more glycosidases.

[00180] EE46: A method for increasing the release of a morphinan compound active agent from a morphinan-glycoside conjugate in situ on a surface to provide the free morphinan compound comprising: a) applying a composition comprising the morphinan-glycoside conjugate of any one of EEs27-35 to the surface; and b) contacting the composition with at least one glycosidase, and/or at least one microorganism, which can be a microorganism that is indigenous to the surface to which the composition is applied, that produces one or more glycosidases.

[00181] EE47 : The method of EE46, in which the composition comprising the morphinan- glycoside is a dry composition; and the composition comprising the at least one glycosidase and/or at least one microorganism is a dry composition, and an aqueous solution comprising any cofactors required for activity of the glycosidase are applied after step b).

[00182] EE48: A method for producing a purified morphinan compound-O-glycoside conjugate comprising: a) contacting a morphinan compound with an activated saccharide or activated oligosaccharide and a glycosyltransferase in a reaction mixture under conditions, including any co-factors necessary for glycosyltransferase activity, effective to produce a morphinan O-glycoside conjugate (m-g conjugate); and b) purifying the m-g conjugate from the reaction mixture to obtain the isolated m-g conjugate.

[00183] EE49: The method of EE48 in which the contacting step is performed in a cultured medium in which the glycosyltransferase is heterologously expressed by a host cell, and wherein said host cell also produces the activated saccharide or activated oligosaccharide as a metabolic product.

[00184] EE50: The method of EE48 or 49, wherein the activated saccharide is UDP- glucose, or the activated oligosaccharide is an oligosaccharide comprising UDP-glucose. [00185] EE51 : The method of EE50, wherein the m-g conjugate product is the corresponding B-D-glucoside.

[00186] EE52: The method of any one of EEs49-51, in which the glycosyltransferase is a

UDP-glucose-dependent glycosyltransferase from a plant or a bacterium.

[00187] EE53: The method of EE52, wherein the UDP-glucose-dependent glycosyltransferase is derived from a plant of the genus Arabidopsis, Fragaria ox Mentha, or from a bacterium of the genus Bacillus or Lactobacillus.

[00188] EE54: The method of EEs46 or 47, wherein the composition a), and optionally the composition b), is provided in a dosage form of a transdermal patch; a solid oral formulation; a liquid oral formulation; a topical formulation for application to skin; a spray or powder formulation for inhalation; or an enteric formulation.

[00189] EE55: The method of EE54, wherein a topical formulation is a cream or gel, or an enteric formulation is a solid formulation for oral administration to the intestinal tract, or as a solid formulation for suppository administration to the colon or vagina.

[00190] EE56: The method of EE54 or EE55, wherein both of the compositions a) and b) are included in the dosage form.

[00191] EE57: Use of the compound of any one of EEs27-35 or of the composition of any one of EEs36-44, in treatment of acute opioid overdose or chronic treatment of opioid addiction.

[00192] EE58: The use of EE57, that includes transdermal administration of the m-g conjugate or release of the morphinan compound active agent from a m-g conjugate to the skin of a subject via a transdermal patch dosage form.

[00193] EE59: The use of EE57, that comprises administering the m-g conjugate or a composition comprising the m-g conjugate, to a subject via inhalation or intravenous injection or intramuscular injection or subcutaneous injection.

[00194] EE60: Use of the compound of any one of EEs27-35 or of the composition of any one of EEs36-44 to prepare a medicament comprising mixing the compound or composition with one or more pharmaceutically acceptable carriers and/or excipients.

[00195] EE61 : The use of EE60, wherein the medicament is formulated for administration to a subject via inhalation or intravenous injection or intramuscular injection or subcutaneous injection, or for application to the skin of a subject as a topical formulation or via a transdermal patch. [00196] While the subject invention has been illustrated and described in detail in the drawings and foregoing description, the disclosed EEsare illustrative and not restrictive in character. All changes and modifications that come within the scope of the invention are desired to be protected.

Claims

What is claimed is:

1. A method for producing a purified morphinan-glycoside conjugate (m-g conjugate) comprising: contacting a morphinan compound of the formula I:

wherein:

Ra can be any group, which group can include additional rings, or any two Ra groups can also join to form additional rings, which additional rings may contain at least one further heteroatom; n is from 0 to 8; each Ra can be the same or different, and is selected from among alkyl, alkenyl, alkynl, allyl, azine, benzoyl, hydroxy, methoxy, unsubstituted, monosubstituted, or di-substituted amine, thiol, keto, Me(C=O)O- and Et(C=O)O-; optionally, the 7 and 8 carbons are joined by a double bond; optionally, two Ra groups join the 4 and 5 positions by an epoxy ring; and optionally the morphinan compound includes additional heteroatoms in the morphinan framework, or in substituents appended to the framework, or in both; a) with an activated saccharide or activated oligosaccharide and a glycosyltransferase in a reaction mixture under conditions, including any co-factors necessary for glycosyltransferase activity, effective to produce a morphinan-glycoside conjugate; and b) purifying the morphinan-glycoside conjugate (m-g conjugate) from the reaction mixture to obtain the isolated m-g conjugate.

2. The method of claim 1, wherein the morphinan compound comprises at least one further N, O or S heteroatom. The method of claim 1 or 2, wherein the morphinan compound comprises a substituted morphinan group wherein n is from 4 to 6. The method of claim 3, wherein the morphinan compound is Naloxone, Naltrexone, or a derivative of one of these. The method of claim 4, wherein the morphinan compound is Naloxone, Naltrexone, Naloxonazine or Naloxonbenzoylhydrazone. The method of any one of claims 1-3, wherein the morphinan compound is substituted at at least one position by a hydroxyl, thiol, or amine group. The method of any one of claims 1-6, wherein at least the 3 -position of the morphinan compound is substituted. The method of claim 3, 6 or 7, wherein neither of the 9 or 13 position of the morphinan compound is substituted. The method of any one of claims 1-8, wherein the morphinan compound has biochemical activity as an opioid receptor antagonist. The method of any one of claims 1-9, wherein the activated saccharide is an activated form of fructose, glucose, galactose, mannose, ribose, or another monosaccharide, or an activated oligosaccharide of one or more of these. The method of any one of claims 1-10, wherein the saccharide or oligosaccharide is activated by an O-linked ester. The method of claim 11, wherein the O-linked ester is a phosphate ester. The method of claim 12, wherein the phosphate ester is of uridine diphosphate (UDP). The method of any one of claims 1-13, wherein the glycosyltransferase is an isolated UDP- carbohydrate-dependent glycosyltransferase. The method of any one of claims 1-14, wherein the glycosyltransferase uses UDP-glucose as the activated saccharide and the m-g conjugate product is the corresponding B-D-glucoside. The method of any one of claims 1-15 in which the contacting step is performed in a cultured medium in which the glycosyltransferase is heterologously expressed by a host cell, and wherein said host cell also produces the activated saccharide or activated oligosaccharide as a metabolic product. The method of claim 16, wherein the activated saccharide is UDP-glucose, or the activated oligosaccharide is an oligosaccharide comprising UDP-glucose. The method of claim 17, wherein the m-g conjugate product is the corresponding B-D- glucoside. The method of any one of claims 1-18, in which the glycosyltransferase is a UDP-glucose- dependent glycosyltransferase from a plant or a bacterium. The method of claim 19, wherein the UDP-glucose-dependent glycosyltransferase is derived from a plant of the genus Arabidopsis, Fragaria or Mentha, or from a bacterium of the genus Bacillus or Lactobacillus. The method of any one of claims 16-20 in which the host cell is one of a strain of Escherichia coli. A method for purifying an O-glycoside conjugate of a morphinan compound (m-g conjugate ) from a reaction mixture comprising: a) adjusting the pH of the reaction mixture to from 7 to 10.5, or to from 8 to 10 or from 9 to 10, to convert m-g conjugate acetate in the reaction mixture to m-g conjugate ; b) neutralizing the reaction mixture and contacting it with a lipophilic solid phase to obtain immobilized m-g conjugate and washing the immobilized C-O-conjugate; c) eluting the m-g conjugate from the solid phase with an alcohol; d) drying the eluate, redissolving the dried eluate in water or a buffer to obtain an aqueous solution; e) optionally back extracting the aqueous solution with a polar organic solvent to remove any remaining morphinan compound and recovering the aqueous solution; f) drying the aqueous solution to obtain the solid m-g conjugate; and g) optionally redissolving the m-g conjugate in an alcohol and treating the alcohol solution with activated carbon to remove colored or other impurities, recovering the alcohol solution, and drying the alcohol solution to obtain the solid M-O- conjugate. A method for purifying a Morphinan-O-glycoside conjugate (m-g conjugate) from a biological culture comprising: a) removing cells from the culture to obtain a supernatant containing m-g conjugate and m-g conjugate acetate; b) adjusting the pH of the supernatant to from 7 to 10.5, or to from 8 to 10 or from 9 to 10, to convert m-g conjugate acetate in the supernatant to m-g conjugate; c) neutralizing the supernatant and contacting it with a lipophilic solid phase to obtain immobilized m-g conjugate and washing the immobilized m-g conjugate; d) eluting the m-g conjugate from the solid phase with an alcohol; e) drying the eluate, redissolving the dried eluate in water or a buffer to obtain an aqueous solution; f) optionally back extracting the aqueous solution with a polar organic solvent to remove any remaining morphinan compound and recovering the aqueous solution; g) drying the aqueous solution to obtain the solid m-g conjugate; h) optionally redissolving the m-g conjugate in an alcohol and treating the alcohol solution with activated carbon to remove colored or other impurities, recovering the alcohol solution, and drying the alcohol solution to obtain the solid m-g conjugate.

24. The method of claim 22 or 23, wherein the solid phase is a lipohilic solid phase, an Amberlite™ polymeric adsorbent resin, a Purolite™ polymeric adsorbent resin, or a mixture of any two or more of these.

25. The method of any one of claims 22-24, wherein the alcohol is methanol or ethanol.

26. The method of any one of claims 22-25, wherein the polar organic solvent is ethyl acetate, a C4 alcohol, a C5 alcohol, among the latter n-butanol, or 2-methyl-l -butanol.

27. A compound Naloxone-3-O-B-D-glucoside, Naltrexone-3-O-B-D-glucoside, or an ester thereof, or an acetal, semiacetal derivative thereof or an ester of these.

28. A morphinan-glycoside conjugate (m-g conjugate) compound that comprises a morphinan group that is a radical of a morphinan compound of the formula I:

(Formula I), wherein:

Ra can be any group, which group can include additional rings, or any two Ra groups can also join to form additional rings, which additional rings may contain at least one further heteroatom. For example, two Ra groups might form a 5- or 6-member epoxy group, a 5- or 6- member thioexpoxy group, a pyrrolidine or a piperidine group; n is from 0 to 8; each Ra can be the same or different, is selected from among alkyl, , alkenyl, alkynl, allyl, hydroxy, methoxy, unsubstituted, monosubstituted, or di-substituted amine, thiol, keto, Me(C=O)O- and Et(C=O)O-; optionally, the 7 and 8 carbons are joined by a double bond; optionally, two Ra groups join the 4 and 5 positions by an epoxy ring; and optionally the morphinan compound includes additional heteroatoms in the morphinan framework, or in substituents appended to the framework, or in both; conjugated to a glycoside.

29. The m-g conjugate of claim 28, wherein the morphinan compound is Naloxone, Naltrexone, or a derivative of one of these.

30. The m-g conjugate of claim 29, wherein the morphinan compound is Naloxone, Naltrexone, Naloxonazine or Naloxonbenzoylhydrazone.

31. The m-g conjugate of claim 28, wherein the morphinan compound further comprises at least one additional N, O, or S heteroatom.

32. The m-g conjugate of claim 28 or 31, wherein the morphinan compound is a substituted morphinan group wherein n = 4 to 6.

33. The m-g conjugate of any one of claims 28-32, wherein the morphinan group is conjugated via an O, N, or S atom.

34. The m-g conjugate of claim any one of claims 28-33, wherein the morphinan group is conjugated at the 3-position.

35. The m-g conjugate of any one of claims 28-34, wherein neither of the 9 or 13 position of the morphinan group is substituted.

36. A composition comprising at least one morphinan-glycoside conjugate of any one of claims 27-35 or an acetal, semiacetal or ester-derivative thereof; and at least one glycosidase, and/or at least one microorganism that produces one or more glycosidases. The composition of claim 36, in which the m-g conjugate is Naloxone- or Naltrexone-3-O- glycoside or an acetal, semiacetal or ester-derivative thereof. The composition of claim 36 or 37, wherein the m-g conjugate is a Naloxone- or Naltrexone- 3-O-B-D-glucoside. The composition of any one of claims 36-38, wherein the glycosidase is a B-glucosidase, or a mixture of B-glucosidases. The composition of any one of claims 36-39 in which the glycosidase is a B-glucosidase, or a mixture of B-glucosidases, derived from bacteria of genus Lactobacillus. The composition of any one of claims 36-40, formulated for oral, dermal, or rectal administration, administration by inhalation or administration by a transdermal patch. The composition of claim 41, that is an encapsulated formulation. A pharmaceutical composition comprising at least one morphinan-glycoside conjugate of any one of claims 27-35, or an acetal, semiacetal or ester-derivative thereof; and one or more pharmaceutically acceptable carriers and/or excipients. The pharmaceutical composition of claim 43 that is formulated for oral administration, administration by transdermal patch, or for administration by inhalation or administration by injection or infusion. A method for increasing the rate of hydrolysis of a morphinan-glycoside conjugate of any one of claims 27-35, or an acetal, semiacetal or ester- derivative thereof; comprising contacting said morphinan-glycoside conjugate or an acetal, semiacetal or ester-derivative thereof with at least one glycosidase, and/or at least one microorganism that produces one or more glycosidases. A method for increasing the release of a morphinan compound from a m-g conjugate in situ on a surface to provide the free morphinan compound comprising: a) applying a composition comprising the m-g conjugate of any one of claims 27-35, to the surface; and b) contacting the composition with at least one glycosidase, and/or at least one microorganism, which can be a microorganism that is indigenous to the surface to which the composition is applied, that produces one or more glycosidases. The method of claim 46, in which the composition comprising the m-g conjugate is a dry composition; the composition comprising the at least one glycosidase and/or at least one microorganism is a dry composition, and an aqueous solution comprising any co-factors required for activity of the glycosidase are applied after step b). A method for producing a purified morphinan-O-glycoside conjugate (m-g conjugate conjugate) comprising: a) contacting a morphinan compound with an activated saccharide or activated oligosaccharide and a glycosyltransferase in a reaction mixture under conditions, including any co-factors necessary for glycosyltransferase activity, effective to produce a m-g conjugate; and b) purifying the m-g conjugate from the reaction mixture to obtain the isolated m-g conjugate. The method of claim 48, in which the contacting step is performed in a cultured medium in which the glycosyltransferase is heterologously expressed by a host cell, and wherein said host cell also produces the activated saccharide or activated oligosaccharide as a metabolic product. The method of claim 48 or 49, wherein the activated saccharide is UDP-glucose, or the activated oligosaccharide is an oligosaccharide comprising UDP-glucose. The method of claim 50, wherein the m-g conjugate product is the corresponding B-D- glucoside. The method of any one of claims 48-51, in which the glycosyltransferase is a UDP-glucose- dependent glycosyltransferase from a plant or a bacterium. The method of claim 52, wherein the UDP-glucose-dependent glycosyltransferase is derived from a plant of the genus Arabidopsis, Fragaria or Mentha, or from a bacterium of the genus Bacillus or Lactobacillus. The method of claim 46 or 47, wherein the composition a), and optionally the composition b), is provided in a dosage form of a transdermal patch; a solid oral formulation; a liquid oral formulation; a topical formulation for application to skin; an enteric formulation, a formulation for inhalation or a formulation for intravenous or intramuscular injection. The method of claim 54, wherein a topical formulation is a cream or gel, or an enteric formulation is a solid formulation for oral administration to the intestinal tract, or as a solid formulation for suppository administration to the colon or vagina. The method of claim 54 or claim 55, wherein both of the compositions a) and b) are included in the dosage form. A process for the preparation of a naloxone glycoside and a naltrexone glycoside, or glycosides of naloxone or naltrexone derivatives such as, for example, naloxonazine or naloxonbenzoylhydrazone, selected from other possible substitutions at the 3-0 position, comprising the steps of converting naloxone, naltrexone, or their derivatives with an isolated glycosyltransferase and their cosubstrate(s) to glycosides as well as their enrichment and purification. The process according to claim 57, using an activated form of fructose, glucose, galactose, mannose, ribose, or another monosaccharide or an activated oligosaccharide as cosubstrate(s). The process according to claim 57 or 58, in which the glycosyltransferase is an isolated UDP- carbohydrate-dependent glycosyltransferase. The process according to any one of claims 57-59, in which the isolated glycosyltransferase uses UDP-glucose as a co-substrate and converts naloxone, naltrexone or a derivative to their P-D-glucosides and these can be obtained. The process according to any one of claims 57-60, in which the isolated glycosyltransferase is obtained by heterologous expression in an organism other than that from which the glycosyltransferase gene originates; the conversion of naloxone, naltrexone or a derivative to the respective P-D-glucoside can particularly preferably also take place in this other organism using the cell's own co-substrate UDP-glucose. The process according to any one of claims 57-61, in which the glycosyltransferase is expressed by heterologous expression in a microorganism and can thus be used for the method. The process according to any one of claims 57,-62, in which living microorganisms are used and these express both the glycosyltransferase and a co-substrate UDP-glucose as part of their metabolism, possibly optimized for this with genetic and / or genetic engineering make, and also carry out the conversion to naloxone, naltrexone or derivative B-D-glucoside in their cells and this can be obtained in this way. The process according to any one of claims 57-63, in which a vegetable or bacterial UDP- glucose-dependent glycosyltransferase is used, preferably from the vegetable genera Arabidopsis (thale cress), Fragaria (strawberry) or Mentha (mint), or the bacterial genus Bacillus. The process according to any one of claims 57-64, in which strains of Escherichia coli are used as the microorganism.

6. A composition obtained by the method according to any one of claims 57-65, comprising naloxone and/or naltrexone-B-D-glycoside, preferably naloxone and naltrexone-3-O-B-D- glucoside; components of the reaction medium according to any one of claims 57-60, or components of an organism according to claim 61, or, components of a culture medium according to any one of claims 62-64, and their degradation products. 7. The composition of claim 66, that further comprises unreacted naloxone or naltrexone substrate and also optionally by-products formed from naloxone or naloxone glycosides, for example naloxone-3-O-P-D-glucoside acetate with various substitution positions on the glucoside residue. 8. The process of any one of claims 57-65, in which naloxone and naltrexone glycosides produced are enriched and purified in a second step from a resulting composition according to and in which extractions with organic solvents, preferably ethyl acetate, as well as adsorption a lipophilic solid phase, preferably on a polystyrene matrix, is used for the enrichment and purification and an organic solvent, preferably methanol, is used for the elution from this matrix. 9. Naloxone and naltrexone glycosides, optionally substituted on the glycoside residue, for example acetylated, for use as a medicament or animal medicament, preferably of naloxone and naltrexone-3-O-B-D-glucoside as a human medicament or animal medicament, which compared to the naloxone or naltrexone exhibits at least one property of better water solubility, optionally a different taste, a changed absorption in the body, a changed metabolism, optionally a changed effect and / or optionally a changed kinetics of action and in different forms of administration, especially non-intravenous forms of administration such as inhalation, nasal, percutaneous or can be used as a suppository. 0. A process for the derivatization of naloxone and naltrexone-P-D-glycosides and especially their P-D-glucosides by semi-acetalic, fully acetalic or ester groups on hydroxyl groups of the glycoside and especially the glucoside residues. 1. A composition comprising at least one naloxone or naltrexone glycoside, preferably naloxone and naltrexone-3-O-P-D- glucoside or semi- or fully acetal or ester derivatives of these; and at least one glycosidase, preferably at least one 0-glucosidase, or a composition of glycosidases, preferably B-glucosidases; and/or microorganisms which produce glycosidases, preferably 0-glucosidases, preferably Lactobacillus ; and/or encapsulated forms of individual, several, or all of the components of the composition. The composition of claim 71, for use as a human medicament or an animal medicament. A process for preparing a medicament, comprising mixing at least one morphinan-glycoside conjugate of any one of claims 27-35, or an acetal, semiacetal or ester-derivative thereof, with one or more pharmaceutically acceptable carriers and/or excipients. The process of claim 73, wherein the medicament is formulated for administration by inhalation, topical administration, administration via a transdermal patch, administration by intravenous injection, intramuscular injection, or subcutaneous administration by oral administration. Use of a m-g conjugate of any one of claims 27-35, or of a composition of any one of claims36- 44, for treatment of opioid addiction or of acute opioid overdose in a subject. A method of treatment for opioid addiction comprising administering a pharmaceutical composition comprising a m-g conjugate according to any one of claims 27-35, to a subject presenting with opioid addiction. The method of claim 76, wherein the pharmaceutical composition is administered topically, by intramuscular injection, by subcutaneous injection or by oral administration. The method of claim 76, wherein the composition is administered via a transdermal patch. The method of any one of claims 75-78, wherein the morphinan compound is Naloxone, Naltrexone, Naloxonazine or Naloxonbenzoylhydrazone. A method of treatment of acute opioid overdose comprising administering a pharmaceutical composition comprising a m-g conjugate according to any one of claims 27-35, to a subject presenting with acute opioid overdose. The method of claim 80, wherein the pharmaceutical composition is administered by inhalation or by intravenous injection. The method of claim 80 or 81, wherein the morphinan compound is Naloxone, Naltrexone, Naloxonazine or Naloxonbenzoylhydrazone.