ABUSE-DETERRENT PHARMACEUTICAL COMPOSITIONS

Abstract

Disclosed are pharmaceutical compositions comprising a drug with a laccase-reactive functional group and a laccase (EC.10.3.2) and methods for manufacturing such compositions.

Claims

1. A pharmaceutical composition comprising a drug with a laccase-reactive functional group and a laccase (EC 1.10.3.2).

2. The pharmaceutical composition according to claim 1, wherein the drug with a laccase-reactive functional group is selected from the group consisting of a substance with a phenolic hydroxyl-group, aminophenol, benzenethiol, diphenol, polyphenols, methoxy-substituted phenol, amino phenol, diamine, aromatic amine, polyamine, ascorbate, hydroxyindols, aryl diamine, and anilin.

3. The pharmaceutical composition according to claim 1, wherein the laccase is selected from the group consisting of laccase from Trametes villosa, laccase from Myceliophthora thermophila, and laccase from Pleurotus ostreatus.

4. The pharmaceutical composition according to claim 1, wherein the drug is an opioid drug with a laccase-reactive functional group.

5. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is selected from the group consisting of a tablet, a mini-tablet, a coated tablet, a bi-layer tablet, a multi-layer tablet, a capsule, a pellet, a multiple unit pellet system, a granulate, and a powder.

6. The pharmaceutical composition according to claim 1, wherein the composition comprises a coated laccase.

7. The pharmaceutical composition according to claim 1, comprising the drug in an amount of 0.1 to 5 000 mg per dosage unit.

8. The pharmaceutical composition according to claim 1, comprising the laccase in an amount of 1 to 1,000 units.

9. The pharmaceutical composition according to claim 1, further comprising an abuse-deterrent feature.

10. The pharmaceutical composition according to claim 1, wherein the composition comprises a matrix containing 1 to 80 wt. % of one or more hydrophobic or hydrophilic polymers.

11. The pharmaceutical composition according to claim 1, wherein the composition is storage stable.

12. The pharmaceutical composition according to claim 1, further comprising a hydrogel-forming component and/or a crosslinker.

13. A method for manufacturing a pharmaceutical composition according to claim 1, comprising mixing the drug with the laccase and finishing the mixture to a pharmaceutical composition.

14. A method for manufacturing a pharmaceutical composition according to claim 1, comprising providing the drug and the laccase in separated form and finishing the separated drug and laccase to a pharmaceutical composition.

15. A method for treating pain and/or drug addiction, the method comprising administering the pharmaceutical composition according to claim 1 to a subject in need thereof to treat pain and/or drug addiction.

Description

[0078] The present invention is further illustrated by the following examples and the figures, yet without being restricted thereto.

[0079] FIG. 1 shows the strategy of the present invention, exemplified by the example of morphine as drug. The abuse deterrent morphine/laccase system according to the present invention either converts morphine into a precipitated product or creates in combination with additives a hydrogel with which it is not possible anymore to inject the drug with a syringe. If the drug is administered as foreseen, proteases from the body deactivate the laccase and the drug unfolds its effect.

[0080] FIG. 2 shows the opioids that are investigated in the examples (morphine (left), tapentadol (middle) and oxycodone (right)).

[0081] FIG. 3 shows laccase oxidation of morphine with Myceliophthora thermophila (MtL) (violet) and Trametes villosa (TvL) (green).

[0082] FIG. 4 shows laccase oxidation of tapentadol with MtL (violet) and TvL (green).

[0083] FIG. 5 shows laccase oxidation of oxycodone with MtL (green) and TvL (violet).

[0084] FIG. 6 shows a TLC of morphine after enzymatic conversion at different time points compared to morphine as reference (first lane).

[0085] FIG. 7 shows a TLC of tapentadol after enzymatic conversion at different time points compared to tapentadol as reference (first lane).

[0086] FIG. 8 shows an FTIR of morphine precipitate (red line: morphine reference, blue line: precipitated morphine polymer)

[0087] FIG. 9 shows HPLC measurement of the enzymatically oxidized morphine.

[0088] FIG. 10 shows GC-MS spectra of morphine after enzymatic conversion.

[0089] FIG. 11 shows GC-MS spectra of morphine out of a GC-MS database library; comparison of the measured spectrum to the database spectrum.

[0090] FIG. 12 shows the conversion of morphine by laccase, detected by GC-MS.

[0091] FIG. 13 shows the chitosan-catechol-morphine hydrogel.

[0092] FIG. 14 shows the carboxymethylchitosan-morphine hydrogel.

[0093] FIG. 15 shows oxygen concentration measurements with various opioids and laccase.

[0094] FIG. 16 shows oxygen concentration measurement for the laccase mediator system.

[0095] FIG. 17 shows oxygen concentration measurement for the laccase mediator system and oxycodone, with the mediators vanillin, ethylvanillin, gallic acid and syringic acid.

[0096] FIG. 18 shows oxycodone concentrations in percent for various LMS experiments with oxycodone and the respective controls.

[0097] FIG. 19 shows peak area [PA] found by MS-TOF analysis for the molecule formula C.sub.17H.sub.19NO.sub.4, which is oxymorphone.

[0098] FIG. 20 shows oxygen concentration measurement for the laccase mediator system and methadone, with the mediator ABTS.

[0099] FIG. 21 shows the reaction scheme for the enzymatic deacetylation of diacetylmorphine to morphine with esterases. Morphine can then in turn be oxidized by laccases.

[0100] FIG. 22 shows acetate yields over time with different enzymes and temperatures measured by HPLC/RI.

[0101] FIG. 23 shows concentrations of DAM and morphine for the assays with different enzymes and temperatures.

[0102] FIG. 24 shows inactivation of enzymes within the human body.

[0103] FIG. 25 shows SDS-Page gel showing the proteolytic degradation of different enzymes, namely Myceliophthora thermophila laccase MTL, Humicola insolens cutinase HiC and horse radish peroxidase HRP.

[0104] FIG. 26 shows pH activity profile of MTL using ABTS as substrate.

[0105] FIG. 27 shows the stability profile of MTL in different pH levels over time.

[0106] FIG. 28 shows elimination of various concentrations of morphine with 10 U/ml laccase.

[0107] FIG. 29 shows that increase of laccase concentration is linear to the decrease in morphine concentration (.fwdarw.10 times more laccase results in a 10 times faster degradation).

[0108] FIG. 30 shows the result with 60000 mg/L morphine, nearly at the solubility limit of morphine.

EXAMPLE

Laccase for Use in Abuse-Deterrent Opioid Formulations

[0109] Opioids are an important component of modern pain management, though the abuse and/or misuse of those drugs has created a growing public health problem. To counteract this problem, there is a strong demand for new technologies for preventing the abuse of opioids. This project therefore tries to approach this problem with a very unique way based on using enzymes.

[0110] The potential of enzymes to polymerize opioids, thereby preventing abuse were investigated. These enzymes will not be active when opioids are administered correctly. These possibilities are thoroughly investigated by the present project.

[0111] In the present example, the development of an appropriate enzyme system to eliminate opioids from solution is shown. Moreover, optimized reaction conditions for effective conversion of the opioid solution preventing administration through injection are provided. Finally, the function of the system is verified by showing the inactivation of the opioid destroying enzymes proteolytically when the drugs are administered as foreseen.

1. Materials

[0112] The opioids that are investigated were morphine, tapentadol and oxycodone as shown in FIG. 2.

[0113] Two different laccases were used to achieve elimination of opioidsone of them originated from Trametes villosa (TvL), the other one from Myceliophthora thermophila (MtL).

2. Oxygen Measurements

[0114] To determine whether the laccases act on any of the given opioids (morphine, tapentadol, oxycodone), an optical oxygen sensor was used. Laccases use oxygen as electron acceptor, consequently the oxygen concentration decreases upon substrate oxidation.

[0115] The conditions for the following reactions are shown in Table 1:

TABLE-US-00001 TABLE 1 Conditions for oxygen measurement of laccase catalysed oxidation of opioids Enzyme (Laccase) 10 U/ml Substrate 2 mg/ml Solvent Distilled water Temperature Room temperature

[0116] In FIG. 3 the oxidation of morphine by two different laccases namely from Myceliophthora thermophila (MtL) and Trametes villosa (TvL) is shown. The blue and red lines represent the two blanks, blue with distilled water and enzyme, and red distilled water with morphine. The violet and green lines represent the two reactions with the enzymes (violet: MtL, green: TvL). After approx. 5 minutes there is a clear decrease in the oxygen concentration in solution, meaning that the laccases are converting morphine to a reaction product. Additionally, the two solutions turned cloudy which means the reaction product precipitated. There was no significant difference between the two enzymes.

[0117] In FIG. 4 the oxidation of tapentadol by MtL (violet) and TvL (green) is shown. The decrease of oxygen with MtL is much slower compared to the decrease of oxygen with TvL. This could be because TvL has a much higher redox potential than the MtL. The different curves also show that the specificity of the two enzymes is very different on tapentadol. TvL converts tapentadol better which is indicated by the slope of the curve as well as the fact that the curve in the case of TvL drops to almost 0% oxygen.

[0118] In FIG. 5, the oxidation of oxycodone is shown. All of the curves look similar indicating that the two used enzymes are not capable of converting oxycodone. The cause for that is the missing phenolic OH group in oxycodone (shown in FIG. 2), which is needed by laccases.

3. Thin Layer Chromatography

[0119] The reactions of morphine and tapentadol were also analysed by thin layer chromatography and are shown in FIGS. 6 and 7. FIG. 6 shows the conversion of morphine. The first lane represents morphine as a reference, whereas the other lanes show samples that were taken at different time points (2, 10, 30, 60, 120 minutes). After only 2 minutes, 2 new dots are appearing on the TLC plate, indicating that a new product is formed out of the enzymatic reaction with morphine. The morphine reference dot disappears after 30 minutes, which indicates that all of the morphine is converted. The same procedure is shown in FIG. 7 for tapentadol. The conversion of tapentadol is much slower, but there is also a new dot appearing after 2 minutes and the tapentadol dot is disappearing after 120 minutes.

4. Analysis of the Precipitated Product

[0120] As mentioned above the reaction product of morphine precipitated, consequently the next part was the analysis of this precipitate. The following conditions were used for the reaction (Table 2).

TABLE-US-00002 TABLE 2 Conditions for the analysis of the morphine precipitate that is formed upon the enzymatic reaction with MtL Enzyme 20 U/ml Substrate 20 mg/ml Solvent Distilled water Temperature Room temperature

[0121] After 24 hours, the reaction mixture was centrifuged for 15 min at 16.100 rcf (relative centrifugal force). The supernatant was discarded and the remaining precipitate was lyophilized overnight. On the next day the dry precipitate was analyzed via FTIR as shown in FIG. 8. The red line shows morphine, the blue line the precipitated reaction product. It can be seen that there are new peaks arising and other peaks decreasing due to the polymerization reaction of the morphine.

[0122] For further analysis a solubility test of the precipitate was performed. As indicated in Table 3, the results for the solubility test were ambivalent. In none of the used solvents the precipitate was soluble, most likely meaning that a high molecular weight product was formed upon the enzymatic reaction. This is desirable for the main goal of the project, but for further analysis a soluble compound is necessary.

TABLE-US-00003 TABLE 3 Solubility tests of the precipitate formed upon the laccase-catalyzed oxidation of morphine Concentration of morphine precipitate Solvent [mg/ml] Solubility Acetonitrile 0.4 THF 0.3 Diethylether 0.4 Toluene 0.4 Hexane 0.3 100 mM Citrate buffer pH 4 1 ~ 100 mM Phosphate buffer pH 3 1 ~ 50 mM Ammonium formate buffer pH 3 0.001 ~

5. Analysis of the Precipitated Product

[0123] To address the second taskthe changing of the rheological properties of the reaction mixturedifferent additives were added to increase the viscosity of the solution. The tested additives are shown in Table 4 and were mixed to the reaction as stated. Most of the chosen additives are already used in pharmaceutical applications. The desired increase in viscosity was only achieved when using hydroxypropylmethylcellulose (HPMC).

TABLE-US-00004 TABLE 4 Additives for the enzymatic morphine reaction to increase the viscosity Ratio/ Trouble- Increased Additive concentration shooting viscosity Ferulic acid 1:1 Catechol 1:1 toxic Starch 1:1 solubility Polyvinylpyrrolidone (PVP) 50 mg/ml solubility Polyethylenglycol (PEG6000) 50 mg/ml HPMC 100 mg/ml time + Catechin 1:1 Neohesperidin dihydrochalcon 1:1

6. Measurements of Enzymatic Conversion Using HPLC & GC

[0124] The kinetic of the enzymatic conversion of morphine was analyzed via HPLC and GC analysis.

[0125] Samples were taken at different time points (0, 2, 5, 10, 15, 30 minutes) during the reaction. The starting solution was 0.2 mg/ml morphine in distilled water. To start the reaction, MtL was added to a final concentration of 78.3 U/ml. 100 L sample were taken and put into 900 L of methanol (MeOH) to precipitate the enzyme. The solution was centrifuged and the supernatant was transferred to an HPLC vial via a 0.2 m filter. Then the solution was measured with following HPLC conditions shown in Table 5.

TABLE-US-00005 TABLE 5 Conditions for the HPLC measurement of enzymatically oxidized morphine reaction rate determination Column Poroshell 120 EC-C18 3.0 0.5 mm Flow 0.8 ml/min Isocratic 85% mQ H.sub.2O, 5% MeOH, 10% formic acid Injection volume 5 L Column temperature 40 C. Signal wave length 240 nm

[0126] The result of the HPLC measurement is shown in FIG. 9. It is clearly visible that morphine is converted by the laccase. After 15 minutes no morphine signal was measured anymore. This means after approx. 15 minutes 100% of the morphine was converted to a partly precipitated product. Further analysis of the exact reaction mechanism and reaction product has to be investigated.

[0127] In addition to the HPLC method, a GC-MS method was established and the samples were analysed using the following conditions shown in Table 6.

TABLE-US-00006 TABLE 6 GC-MS conditions for enzymatically oxidized morphine reaction rate determination Column Agilent Technologies DB17MS Temperature program 120 C.-320 C. Run time 11.5 min

[0128] The MS spectrum in FIG. 10 shows the result of the GC-MS measurement of morphine after the reaction. The sharp peak to the right represents morphine, which is confirmed by the MS spectrum library shown in FIG. 11. This is the proof that morphine was detected.

[0129] In FIG. 12 the conversion rate of the reaction of morphine and laccase from Myceliophthora thermophila is shown. For this reaction a concentration of 2 mg/ml morphine in distilled water was used. To start the reaction MtL was added to a final concentration of 78.3 U/ml. For the sample preparation 100 L sample were taken and added to 900 L of methanol (MeOH) to precipitate the enzyme. The solution was centrifuged and the supernatant was transferred to an HPLC vial via a 0.2 m filter. In the vial there was NaSO.sub.4 to bind remaining water from the enzyme which could cause problems in the GC chromatograph.

[0130] The conversion rate, compared to the conditions of the ones mentioned above measured by HPLC, is much slower. After 30 minutes approx. 60% of the morphine is converted. The cause for this is most likely the different enzyme/substrate ratio. For the HPLC measurement, much more enzyme was used compared to the samples that were prepared for the GC measurement. This shows that it is possible to influence the conversion rate with the proportion of enzyme to substrate.

7. Hydrogel

[0131] According to a preferred embodiment, the compositions according to the present inventions are provided as hydrogels. This involves the inclusion of another abuse deterrent approach, namely to create a system which changes the rheological properties of the reaction solution, i.e. the formation of a hydrogel. Hydrogels are very viscous and as a consequence, the drawing up with a needle is not possible. For this purpose different set ups were tried as shown in Table 7. The substances were mixed together until a gel was formed.

[0132] The first trial with chitosan formed a hydrogel after approx. 15 minutes. The idea is that catechol crosslinks the chitosan molecules and that the morphine is covalently imbedded via Michael's type reactions. This trial was a successful proof of concept while catechol needs to be replaced with other molecules already used as drug additives.

[0133] The second trial with carboxymethylchitosan formed a hydrogel just with morphine after approx. 24 h. This system would be non-harmful and the reaction time can be optimized.

TABLE-US-00007 TABLE 7 Conditions for enzymatic crosslinked hydrogels Substances Viscosity increased Chitosan 2% (w/v) + 500 M Catechol + +(after 15 min) morphine 10 mg/ml + 2 U/ml MtL Carboxymethylchitosan 2% (w/v) + +(after 24 hours) morphine 10 mg/ml + 2U MtL

[0134] FIG. 13 shows the chitosan-catechol-morphine hydrogel. FIG. 14 shows the carboxymethylchitosan-morphine hydrogel. Both hydrogels are not injectable again and cannot be used anymore for administration.

Discussion:

[0135] This study demonstrates an entirely new enzymatic approach for the development of abuse deterrent opioids. Tapentadol and morphine were successfully converted by the enzyme laccase which was confirmed by oxygen consumption measurements, TLC, HPLC-MS, FTIR and GC-MS analysis.

[0136] Since the enzymatic conversion of morphine was quite straight forward, this reaction was chosen as a model for further analysis. The reaction rate of the laccase from Myceliophthora thermophila on morphine was analysed via HPLC and GC measurement. After approx. 15 minutes 100% of morphine was converted to a product which precipitated. The precipitated reaction product was analysed via FTIR and also solubility tests were conducted. The precipitate was hardly soluble in any of the used solvents, which is desired for abuse prevention.

[0137] Overall the desired abuse prevention system based on enzyme polymerization was successfully developed. According to the presented results it is plausible that the present system is extendable in principle to all drugs, especially all opioids that have a laccase-reactive functional group.

8. Additional Laccase O.SUB.2 .Measurements

[0138] Oxygen concentration measurements were done to confirm the results that were gathered in the previous experiments. The following additional opioids were tested: methadone, dihydrocodeine, diacetylmorphine and hydromorphone. [0139] Final reaction mixture: 1000 mg L.sup.1 opioid, 10 Units laccase per ml in NaPi Buffer pH 7 50 mM.

[0140] The results of the measurements can be seen in FIG. 15. The reactions confirm the previous measurements of oxycodone, tapentadol and morphine, where a drop in oxygen concentration and therefore laccase activity could only be witnessed in the presence of a phenolic group. For the newly tested opioids exclusively hydromorphone showed laccase activity, whereas with all other opioids the concentration remained steady.

9. Laccase Mediator System (LMS): Elimination of Opioids

[0141] Mediators can be used as electron shuttles to facilitate the oxidation of complex substrates that could otherwise not be oxidized by a laccase on its own. While opioids containing a phenolic group readily serve as substrate for laccases, other opioids such as oxycodone, methadone or dihydrocodeine are not as readily oxidized. The substrate range of laccases can be increased with mediators: These often small molecules are oxidized by the laccase in a first step and then react in their oxidized state with a broad variety of target substrates.

[0142] During the reaction oxygen acts as electron acceptor and is reduced to water by laccases while the mediators are oxidized. The concentration of oxygen was measured as described above. Oxygen is consumed until the mediator is fully oxidized. An open experimental setup was chosen; hence the oxygen concentration can recover to its starting value. The second step of the reaction is initiated by addition of the target substrate, which is consequently oxidized by the mediators. The now reduced mediators can again be targeted by the laccases, which leads to a decline in oxygen concentration once more (see FIG. 16).

[0143] In the present experiments the final reaction mixtures contained 0.2 to 6 mM mediator, 1-10 units of laccase per ml and 100-3000 mg L.sup.1 opioid.

9.1. LMS and Oxycodone

[0144] FIG. 17 shows the oxygen concentration levels for oxycodone in the laccase mediator system. The different mediators were used at concentrations of 2 mM with 10 units laccase ml.sup.1 and 1000 mgL.sup.1 oxycodone. The first drop in oxygen concentration represents the oxidation of the respective mediators with the laccase, the second dropwhich took place just after the addition of the opioidwas due to the subsequent oxidation of the opioid.

[0145] The mediators vanillin, ethylvanillin and syringic acid showed a drop in oxygen concentration after the addition of oxycodone, which can be seen in FIG. 17. This shows an oxidation of oxycodone.

[0146] To further confirm the oxidation and elimination of oxycodone HPLC-MS/TOF measurements were done.

[0147] The concentrations were determined with a liquid chromatography-electrospray ionization-time of flight (HPLC-ESI-TOF) mass spectrometer from Agilent (A1260 series, Agilent US). The substances contained in the samples were separated by a Zorbax Hilic Plus, 2.1100 mm, 3.5 m (Agilent, US) column. The gradient was set to 100% mobile phase A (65 mM ammonium formiate pH 3.2) with a flow rate of 0.4 mL min.sup.1 at 40 C. and changed to 100% mobile phase B (acetonitrile) in 15 minutes with an injection volume of 1 L. The spectra were acquired over the m/z range from 100 to 3000 at a scan rate of two spectra per second. The standard curve for each opioid was performed with corresponding standards ranging from 0.001 to 50 mg L.sup.1.

[0148] In addition to the reactions shown in FIG. 17, the mediator vanillin was used at a higher concentration (6 mM). The results can be seen in FIG. 18 and FIG. 19. The decrease in oxycodone concentration was confirmed. One of oxycodone's main degradation products, oxymorphone, was found in all samples. Oxymorphone is an impurity of oxycodone and is therefore present even in the controls where no laccase or mediator were used. However, an increase in peak area of oxymorphone over time can only be observed for oxycodone in combination with the laccase mediator system. The only mediator that had no apparent effect on oxycodone was gallic acid, an observation that was supported by both the oxygen concentration measurement and the MS-TOF analysis.

9.2. LMS and Methadone

[0149] For methadone, oxygen measurements were done, but with an extended palette of mediators: In addition to the aforementioned mediators, syringic acid, TEMPO, syringol and ABTS were tested. The best effect was observed with ABTS, as can be seen in FIG. 20. Again, the decline in oxygen concentration shows oxidation of the target substance.

10. Diacetylmorphine (DAM) Esterase API Enzyme Couple

[0150] As an example for a two-step reaction for an API-enzyme couple, diacetylmorphine was investigated. In a first reaction, an esterase is used to deacetylate diacetylmorphine to monoacetylmorphine and subsequently to morphine. The general reaction scheme can be seen in FIG. 21. Morphine can then be oxidized by laccases as described before.

[0151] In addition to the HPLC MS-TOF quantification the increase in acetate resulting from the deacetylation of DAM was measured by HPLC/RI.

10.1. Esterase Activity Assay

[0152] Esterase activity was determined photometrically in 50 mM sodium phosphate buffer (pH 7) using p-nitrophenol acetate (pNPA) as substrate. The method used was described by Huggins et al., J. Biol. Chem. 170 (1947) 467-482 with some modifications (Herrero Acero et al., Macromolecules 44 (2011), 4632-4640). Esterases can hydrolyze pNPA to acetic acid and p-nitrophenol. Further the increase of the absorbance of p-nitrophenol can be measured.

[0153] A stock solution of 8.3 mM pNPA was prepared in DMSO and diluted in sodium phosphate buffer. The final reaction mixture contained 200 l of pNPA solution and 20 l of appropriately diluted enzyme solution. The absorbance of liberated p-nitrophenol was measured at 30 C. and 405 nm (=90.32 mL mol.sup.1 cm.sup.1 at pH 7) using a multimode microplate reader. One unit of esterase activity was defined as the amount of enzyme releasing 1 mol p-nitrophenol per minute under assay conditions. For blanks the reaction mixture was prepared the same way except that the enzyme solution was substituted by water. A blank was measured using 20 l buffer instead of sample. Volumetric enzyme activity was calculated using an adaptation of the Lambert Beer law (Hughes et al., Anal. Chem. 24 (1952), 1349-1354).

10.2. Enzymatic conversion of Diacetylmorphine to Morphine

[0154] Humicola insolens cutinase (HiC) was purchased from Novozymes (Denmark) and Thermobifida cellulosilytica cutinase 1 (THC) was produced and purified as described by Herrero Acero et al., 2011. These enzymes have shown to be capable of hydrolyzing complex structures in the past (Perz et al., Nat. Biotechnol. 33, (2016), 295-304). Activities of both enzymes were determined by pNPA activity assay.

[0155] The enzymatic conversion of diacetylmorphine to morphine was carried out using the two different esterases, HiC and THC. Hydrolysis reaction was performed by incubating 15 mg diacetylmorphine in 50 mM sodium phosphate buffer (pH 7) with 10 U ml.sup.1 of the respective enzyme at room temperature and at 50 C. for 5 days. The final volume of the reaction mixture was 10 mL and was shaken at 400 rpm throughout the experiment. Since acetic acid is volatile the reaction had to be carried out in an airtight sealed glass tube. Samples of 1 mL at each timepoint were removed and filtered through a syringe and a membrane filter with a 0.45 m pore size and were subsequently precipitated using the Carrez precipitation to remove the enzyme. To 1 mL of sample 20 L of Carrez reagent 1 (10.6% w/v potassium hexacyanoferrate(II)-trihydrate) were added and vortexed. After 1 min 20 L of C2 (28.8% w/v zinc sulfate heptahydrate) were added, vortexed and incubated for another 5 minutes followed by centrifuging for 30 minutes and 13000 rpm. The supernatant was used for further analysis.

[0156] The amount of resulting acetic acid was detected by RI-detector after separation through high performance liquid chromatography (parameters are described below). The amount of resulting acetic acid was detected by HPLC-RI analysis. For quantification acetic acid standards were prepared in a range of 1 g L.sup.1 to 0.01 g L.sup.1 in ultrapure water. Before HPLC analysis the pH of each sample had to be adjusted to 4-6 through addition of 10-20 l of 1M HCl. All experiments were performed as duplicates. As a positive control diacetylmorphine was hydrolyzed chemically in 0.1M NaOH as described by Nakamura et al. (Nakamura et al., J. Chromatogr. 110 (1975), 81-89).

10.3. Quantification of Deacetylation by HPLC

[0157] The obtained products were separated by high performance liquid chromatography (HPLC) from Agilent (A1100 series, Agilent US). This was done by an ICSep ION 300, 7.8300 mm, 7 m column supplied from Transgenomic (US). As mobile phase 0.01 N H.sub.2SO.sub.4 was used with a flow rate of 0.325 mL min.sup.1 at 45 C. The injection volume was 40 L of sample at a runtime of 60 min. The amount of acetic acid was determined by the RI-detector of the instrument. The method was calibrated using acetic acid standards within a range of 10 mg L.sup.1 to 1000 mg L.sup.1.

10.4. Results:

[0158] The total acetate yield over time can be seen in FIG. 22. At a temperature of 50 C. both enzymes were able to hydrolyze more than 50% of the diacetylmorphine, indicating a complete deacetylation to morphine.

[0159] The direct measurement of opioid concentrations confirms this assumption, which can be seen in FIG. 23: While the DAM concentration declines in all samples, the morphine concentration is only increased in the samples that were incubated at 50 C. At lower temperatures, it seems that only monoacetylmorphine is produced.

11. Inactivation of EnzymesSGF and Proteases

[0160] The inactivation of the enzymes when the drugs are administered as foreseen is an integral part of the invention. Inactivation can be achieved both by acidic denaturation and proteolytic degradation (FIG. 24).

[0161] To assess the feasibility of the inactivation methods, protease digestion assays as well as enzyme stability trials in various pH levels were done.

11.1. Pepsin Digestion Assay

[0162] The method used to demonstrate pepsin digestion of MTL was described by Thomas et al. (Thomas et al., Regul. Toxicol. Pharmacol. 39 (2004), 87-98) and is a standardized protocol using simulated gastric fluid (SGF). SGF contains 0.084 M HCl, 0.035 M NaCl, 4000 U of pepsin per reaction mixture and has a pH level of 1.2 according to the United States Pharmacopeia (1995). A ratio of 10000 Units of pepsin activity to 1 mg of test protein was used throughout the assay, which is based on an evaluation of the average activity of pepsin recommended in the United States Pharmacopeia (24.sup.th edition, 2000) with some modifications. Pepsin (ref. #P7000) was purchased from Sigma-Aldrich (US). All proteins that were used were dissolved in 50 mM Tris-HCl (pH 9.5) at a concentration of 5 mg mL.sup.1. The reaction mixture contained 1.52 mL of SGF preheated to 37 C. before the addition of 0.08 mL of protein solution (Thomas et al., 2004). The mixture was placed into a thermomixer at 37 C. and was shaken at 400 rpm. For denaturating SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis samples of 160 l were removed at 0 minutes, 0.5 minutes, and 15 minutes after initiation.

11.2 SDS-PAGE Analysis

[0163] SDS-PAGE was performed to analyze and visually inspect stained protein bands whether the protein band is still intact or fragmented after the digestion assay. Precast tris-glycin gels (ref. #456-1086) with 4-15% polyacrylamide, 10 tris-glycine-SDS (TGS) buffer (ref. #1610772) and 4 Laemmli sample buffer (ref. #1610747), were purchased from Bio-Rad Laboratories (Hercules, USA). The 4 Laemmli buffer was diluted 10:1 with 2-mercaptoethanol before usage. The following sample preparation method was described by Thomas et al (Thomas et al. 2004).

[0164] Each 160 l of the digestion samples was quenched by adding 56 l of 200 mM NaHCO.sub.3 as neutralization step and 56 l 4 Laemmli (Laemmli 1970) buffer for electrophoresis. Samples were immediately heated up to 99 C. for 10 minutes and analyzed directly or stored at 20 C. Control samples for pepsin and test protein stability (SGF without pepsin but with test protein) were treated in the same way as described above. The zero time point digestion samples were prepared differently, since pepsin immediately starts to digest and auto-digest as soon as it is in solution. Before adding the test protein, pepsin was already denatured in quenching solution and heated up to 99 C. for 5 minutes. Afterwards the sample was heated again for 5 minutes to ensure proper denaturation of the test protein. 15 l of each sample and 5 l of prestained protein marker IV (ref. #27-2110, Peqlab) were loaded onto the gel and were subsequently run in tris-glycin running buffer for 30-45 minutes at 200 V. For visualization the gels were incubated with Coomassie-blue staining solution (0.1% w v.sup.1 Coomassie R250, 10% v v.sup.1 acetic acid, 45% v v.sup.1 methanol) for 30 minutes followed by a destaining step (10% v v.sup.1 acetic acid, 40% v v.sup.1 methanol) for 10-30 minutes.

[0165] The results of the proteolytic enzyme degradation can be seen in FIG. 25. MTL and HRP are very susceptible to proteolytic degradation, as their respective protein bands vanished within 30 seconds. The HiC displayed a moderate decrease in concentration.

11.3 Laccase Stability Assay

[0166] To establish a pH profile for MTL enzyme activities were measured at different pH levels from acidic to neutral (pH 2.5-pH 7) over time using ABTS as substrate. For every pH level MTL was diluted 1:100 in the respective buffer and was incubated for one hour and room temperature. Enzyme activity was measured at distinctive time points. Activity was measured and calculated by ABTS-assay (described above), using different extinctions coefficients for each pH level (pH7: =11.38 mL mol.sup.1 cm.sup.1, pH6: =23.34 mL mol.sup.1 cm.sup.1, pH5: =32.03 mL mol.sup.1 cm.sup.1, pH4: =35.49 mL mol.sup.1 cm.sup.1, pH3, 2: =36.00 mL mol.sup.1 cm.sup.1; master thesis (Laccases as effective siccatives in alkyd resins), Scholz, 2015.

[0167] The pH profile of the MTL can be seen in FIG. 26, whereas the stability profile in different pH levels over time is shown in FIG. 27. MTL has two activity maxima, one at pH 7 and the second at pH 3 and shows no activity at SGF medium conditions. The stability profile indicates that MTL remains most of its activity at a pH range from 4 to 7 but loses activity rather quickly at lower pHs.

12. Additional LaccaseMorphine Trials (High Concentrations)

[0168] Several additional experiments were done using various concentrations of morphine and laccase. The presented results in the following examples and figures support the claims that the rate of degradation correlates directly with amount of laccase as well as the opioid concentration. The present examples are also based on an even more reliable and improved analytics.

12.1. Polymerization of Morphine with Laccase from Myceliophthora thermophila

[0169] Morphine polymerization was carried out in different approaches, with morphine concentrations ranging from 1 mg l.sup.1 to 60000 mg l.sup.1. The reactions were carried out in 50 mM sodium phosphate buffer pH 7. The oxidation process was started by addition of the laccase MTL with a final activity of 10 U to 100 U. Various concentrations of morphine are shown in FIG. 28.

[0170] FIG. 29 shows that an increase of laccase concentration is linear to the decrease in morphine concentration (i.e. 10 times more laccase results in a 10 times faster degradation). FIG. 30 shows the conversion of 60000 mg/L morphine, nearly at the solubility limit of morphine.

[0171] These results are therefore fully in line with 1.6, above.

Preferred Embodiments

[0172] The present invention therefore relates to the following preferred embodiments:

[0173] 1. Pharmaceutical composition comprising a drug with a laccase-reactive functional group and a laccase (EC 1.10.3.2).

[0174] 2. Pharmaceutical composition according to embodiment 1, wherein the drug with a laccase-reactive functional group is selected from the group comprising substances with a phenolic hydroxyl-group, aminophenols, benzenethiols, di- or polyphenols, methoxy-substituted phenols, amino phenols, diamines, aromatic amines, polyamines, ascorbate, hydroxyindoles, aryl diamines, and anilins, preferably substances with a phenolic hydroxyl-group.

[0175] 3. Pharmaceutical composition according to embodiment 1 or 2, wherein the laccase is selected from the group of laccase from Trametes villosa, laccase from Myceliophthora thermophila, and laccase from Pleurotus ostreatus.

[0176] 4. Pharmaceutical composition according to any one of embodiments 1 to 3, wherein the drug is an opioid drug with a laccase-reactive functional group, preferably selected from the group morphine, tapentadol, hydromorphone, etorphine, desomorphine, oxymorphone, buprenorphine, opioid peptides comprising a phenylalanine residue, such as adrenorphin, amidorphin, casomorphin, DADLE ([D-Ala.sup.2, D-Leu.sup.5]-Enkephalin), DAMGO ([D-Ala.sup.2, N-MePhe.sup.4, Gly-ol]-enkephalin), dermorphin, endomorphin, morphiceptin, and TRIMU 5 (L-tyrosyl-N-{[(3-methylbutyl)amino]acetyl}-D-alaninamide); oripavine, 6-MDDM (6-methylenedihydrodesoxymorphine), chlornaltrexamine, dihydromorphine, hydromorphinol, methyldesorphine, N-phenethylnormorphine, RAM-378 (7,8-Dihydro-14-hydroxy-N-phenethylnormorphine), heterocodeine, 7-spiroindanyloxymorphone, morphinone, pentamorphone, semorphone, chloromorphide, nalbuphine, oxymorphazone, 1-iodomorphine, morphine-6-glucuronide, 6-monoacetylmorphine, normorphine, morphine-N-oxide, cyclorphan, dextrallorphan, levorphanol, levophenacylmorphan, norlevorphanol, oxilorphan, phenomorphan, furethylnorlevorphanol, xorphanol, butorphanol, 6,14-endoethenotetrahydrooripavine, BU-48 (N-Cyclopropylmethyl-[7,8,2,3]-cyclohexano-1[S]-hydroxy-6,14-endo-ethenotetrahydronororipavine), cyprenorphine, dihydroetorphine, norbuprenorphine, 5-guanidinonaltrindole, diprenorphine, levallorphan, meptazinol, methylnaltrexone, nalfurafine, nalmefene, naloxazone, naloxone, nalorphine, naltrexone, naltriben, naltrindole, 6-naltrexol-d4, pseudomorphine, naloxonazine, norbinaltorphimine, alazocine, bremazocine, dezocine, ketazocine, metazocine, pentazocine, phenazocine, cyclazocine, hydroxypethidine (bemidone), ketobemidone, methylketobemidone, propylketobemidone, alvimopan, picenadol and pharmaceutically acceptable salts, hydrates, solvates, esters, prodrugs and mixtures thereof, more preferred morphine, tapentadol, buprenorphine, oxymorphone, pentazocine, levorphanol, hydromorphone, especially morphine.

[0177] 5. Pharmaceutical composition according to any one of embodiments 1 to 4, wherein the pharmaceutical composition is selected from a tablet, a mini-tablet, a coat-core tablet (coated tablet), a bi-layer tablet, a multi-layer tablet, a capsule, a pellet, a MUPS (multiple unit pellet system), a granulate, a powder, especially coated, sugar-coated and/or functionally coated (e.g. enteric coated) forms thereof.

[0178] 6. Pharmaceutical composition according to any one of embodiments 1 to 5, wherein the composition comprises a coated laccase.

[0179] 7. Pharmaceutical composition according to any one of embodiments 1 to 6, wherein the drug is contained in an amount of 0.1 to 5 000 mg, preferably 0.5 to 1 000 mg, especially 1 to 500 mg, per dosage unit.

[0180] 8. Pharmaceutical composition according to any one of embodiments 1 to 7, wherein the laccase is contained in an amount of 1 to 1 000 units, preferably 10 to 100 units.

[0181] 9. Pharmaceutical composition according to any one of embodiments 1 to 8, wherein the composition comprises a further abuse-deterrent feature, preferably selected from the group a physical or chemical barrier, especially increased tablet hardness, a drug antagonist, an aversion component, an abuse-deterrent delivery system and a prodrug, especially a physical barrier or an aversion component, especially a gelling agent and/or a non-gelling viscosity-increasing agent.

[0182] 10. Pharmaceutical composition according to any one of embodiments 1 to 9, wherein the composition comprises a matrix containing 1 to 80 wt. % of one or more hydrophobic or hydrophilic polymers, preferably a matrix comprising agar, alamic acid, alginic acid, carmellose, carboxymethylcellulose sodium, carbomer, carrageenan, chitosan, especially carboxymethylchitosan, catechol, copovidone, dextrin, gelatin, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methacrylic acid copolymers, methylcellulose derivatives, microcrystalline cellulose, polyacrylic acid, polyalkylene oxide, especially polyethylene glycol, polyvinyl alcohol, polyvinyl acetate, povidone, propylene glycol alginate, a polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft co-polymer, pullulan, silicon dioxide, sodium alginate, starch, vinylpyrrolidone-vinyl acetate copolymers or of a non-polymer matrix former, preferably microcrystalline wax, fatty alcohols and fatty acids, especially stearyl alcohol, cetyl stearyl alcohol, stearic acid, palmitic acid or salts and mixtures thereof, mono-, di- and triglycerides of saturated fatty acids with a chain length between 16 and 22 carbon atoms and a mixture of such mono- di- and triglycerides.

[0183] 11. Pharmaceutical composition according to any one of embodiments 1 to 10, wherein the composition is storage stable, preferably by comprising less than 5%, especially less than 1%, laccase-processed drug after 6 month storage at 25 C. under dry conditions.

[0184] 12. Pharmaceutical composition according to any one of embodiments 1 to 11, further comprising a hydrogel-forming component and/or a crosslinker, preferably chitosan and/or catechol or carboxymethylchitosan and/or vanillin.

[0185] 13. Pharmaceutical composition according to any one of embodiments 1 to 12, wherein the composition is a modified release composition, especially a prolonged release composition.

[0186] 14. Pharmaceutical composition according to any one of embodiments 1 to 13, wherein the composition renders immediate release, modified release, or a combination thereof.

[0187] 15. Pharmaceutical composition according to any one of embodiments 1 to 14, wherein the composition comprises an opioid analgesic alone or in combination with a non-opioid analgesic, especially with ibuprofen, diclofenac, naproxen, paracetamol and acetyl-salicylic acid.

[0188] 16. Pharmaceutical composition according to any one of embodiments 1 to 15 for use in the treatment of drug addiction.

[0189] 17. Pharmaceutical composition according to any one of embodiments 1 to 15 for use in the treatment of pain.

[0190] 18. Pharmaceutical composition according to any one of embodiments 1 to 15, wherein the drug-processing enzyme essentially does not act on the drug in vivo when the composition is administered in the intended way and intact.

[0191] 19. Pharmaceutical composition according to any one of embodiments 1 to 15, wherein the drug-processing enzyme exists in the formulation in an essentially non-releasable form when the composition is administered in the intended way and intact.

[0192] 20. Pharmaceutical composition according to any one of embodiments 1 to 15, wherein the drug-processing enzyme is deactivated in vivo.

[0193] 21. Pharmaceutical compositions according to any one of embodiments 1 to 20, with a moisture content below 10%, preferably with a moisture content of below 5%, especially with a moisture content of below 2%.

[0194] 22. Method for manufacturing a pharmaceutical composition according to any one of embodiments 1 to 21 comprising the steps of mixing the drug with the laccase and finishing the mixture to a pharmaceutical composition.

[0195] 23. Method for manufacturing a pharmaceutical composition according to any one of embodiments 1 to 21 comprising the steps of providing the drug and the laccase in separated form and finishing the separated drug and laccase to a pharmaceutical composition.

[0196] 24. Method of administration the pharmaceutical composition according to any one of embodiments 1 to 21 to a patient in need thereof, wherein the pharmaceutical composition is orally administered to this patient in an effective amount and wherein the laccase is automatically deactivated in the course of this oral administration.