(+)-cis-Tetrahydrocannabinol ((+)-CIS-THC) for use as a medicine

Abstract

The present invention relates to tetrahydrocannabinol (THC)-type cannabinoid compounds for pharmaceutical use. The THC-type cannabinoids are enantiomers of (-)-trans-tetrahydrocannabinol, a naturally occurring cannabinoid that can be found in cannabis plant varieties that have been bred to produce THC as the predominant cannabinoid. It has been discovered that the specific enantiomer (+)-cis-tetrahydrocannabinol has properties that differ from those of naturally occurring (-)-trans-THC. The cannabinoid (+)-cis-THC has been found to occur in low concentrations in certain cannabis plant varieties that have been bred to produce cannabidiol (CBD) as the predominant cannabinoid. Furthermore, the cannabinoid can be produced by synthetic methods.

Description

[Technical field]

[0001] The present invention relates to cannabinoid compounds of the tetrahydrocannabinol (THC) type for pharmaceutical use.

[0002] The THC-type cannabinoids are enantiomers of (-)-trans-tetrahydrocannabinol, a naturally occurring cannabinoid that can be found in cannabis plant varieties that have been bred to produce THC as the predominant cannabinoid. It has been discovered that the specific enantiomer (+)-cis-tetrahydrocannabinol has different properties than the naturally occurring (-)-trans-THC.

[0003] The cannabinoid (+)-cis-THC has been found to occur in low concentrations in certain cannabis plant varieties that have been bred to produce cannabidiol (CBD) as the primary cannabinoid, and can also be produced by synthetic means.

[0004] Disclosed herein are data demonstrating the efficacy of (+)-cis-THC in disease models, and methods for synthesizing (+)-cis-THC are also described. [Background technology]

[0005] Cannabinoids are natural and synthetic compounds structurally or pharmacologically related to components of the cannabis plant or to endogenous agonists of the cannabinoid receptors CB1 or CB2 (endocannabinoids). The only way nature produces these compounds is through production by the cannabis plant. Cannabis is a genus of flowering plants in the family Cannabaceae, including the species Cannabis sativa, Cannabis indica, and Cannabis ruderalis (sometimes considered part of Cannabis sativa).

[0006] The cannabis plant contains a highly complex mixture of compounds. At least 568 unique molecules have been identified. Among these compounds are cannabinoids, terpenoids, sugars, fatty acids, flavonoids, other hydrocarbons, nitrogenous compounds, and amino acids. With regard to cannabinoids, over 100 different cannabinoids have been identified (see, for example, Handbook of Cannabis, Roger Pertwee, Chapter 1, pages 3-15).

[0007] Cannabinoids exert their physiological effects through various receptors, including, but not limited to, adrenergic receptors, cannabinoid receptors (CB1 and CB2), GPR55, GPR3, or GPR5. The major cannabinoids present in the cannabis plant are the cannabinoid acids Δ9-tetrahydrocannabinolic acid (Δ9-THCA) and cannabidiolic acid (CBDA), each of which contains small amounts of neutral (decarboxylated) cannabinoids. In addition, cannabis may contain lower levels of other minor cannabinoids. "The chemical composition, pharmacological profiling, and full physiological efficacy of these medicinal plants, and of the more extensive extracts from cannabis, have not been fully elucidated." Lewis, MM, et al., ACS Omega, 2, 6091-6103 (2017).

[0008] The compound tetrahydrocannabinol (THC), the natural form of (-)-trans-THC, is psychoactive. Medical uses of (-)-trans-THC include for the treatment of chemotherapy-induced nausea and vomiting and in the treatment of HIV / AIDS-associated anorexia, and (-)-trans-THC is also a component of nabiximols (Sativex), which is approved in Europe and Canada as an approved treatment for multiple sclerosis-associated spasticity.

[0009] The tetrahydrocannabinol molecule is known to exist in four stereoisomers: (-)-trans-delta-9-tetrahydrocannabinol, (+)-trans-delta-9-tetrahydrocannabinol, (-)-cis-delta-9-tetrahydrocannabinol, and (+)-cis-delta-9-tetrahydrocannabinol. See Figure 1.

[0010] For synthetic drugs that have chiral centers and can therefore form such stereoisomers, it is important to understand the properties of the various enantiomers, since the synthesis often results in racemic mixtures, thereby producing both (-) and (+) enantiomers.

[0011] The pharmacological activity of THC is stereospecific, with the (-)-trans-THC isomer (dronabinol) being 6- to 100-fold more potent than the (+)-trans-THC isomer, depending on the assay (Dewey et al., 1984).

[0012] In other medicines, both enantiomers have similar activity, for example, both ibuprofen enantiomers have anti-inflammatory properties. Care must also be taken to ensure that one of these enantiomers is not toxic or harmful to the patient.

[0013] In the case of THC, in addition to having optical, or mirror image, (+) and (-) enantiomers, it also has geometric isomers called cis and trans isomers. The Food and Drug Administration (FDA) has determined that the geometric isomers are chemically distinct in nature and should be treated as separate drugs (https: / / www.fda.gov / regulatory-information / search-fda-guidance-documents / development-new-stereoisomeric-drugs). [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Handbook of Cannabis, Roger Pertwee, Chapter 1, pp. 3-15 [Non-Patent Document 2] Lewis, MM et al., ACS Omega, 2, pp. 6091-6103 (2017) [Non-Patent Document 3] https: / / www.fda.gov / regulatory-information / search-fda-guidance-documents / development-new-stereoisomeric-drugs Summary of the Invention [Problem to be solved by the invention]

[0015] The present invention demonstrates that the compound (+)-cis-THC has surprisingly been found to exhibit therapeutic efficacy in animal models of disease. Heretofore, this compound has not been found to have any therapeutic efficacy. [Means for solving the problem]

[0016] According to a first aspect of the present invention there is provided (+)-cis tetrahydrocannabinol ((+)-cis-THC) for use as a medicine.

[0017] Preferably, the (+)-cis-THC is in the form of a plant extract. More preferably, the (+)-cis-THC is in the form of a highly purified extract of cannabis.

[0018] Preferably, the highly purified extract contains at least 80% (w / w) (+)-cis-THC, more preferably, the highly purified extract contains at least 85% (w / w) (+)-cis-THC, more preferably, the highly purified extract contains at least 90% (w / w), more preferably, the highly purified extract contains at least 95% (w / w) (+)-cis-THC, and even more preferably, the highly purified extract contains at least 98% (w / w) (+)-cis-THC.

[0019] Alternatively, (+)-cis-THC exists as a synthetic compound.

[0020] Preferably, the dose of (+)-cis-THC is greater than 100 mg / kg / day. More preferably, the dose of (+)-cis-THC is greater than 250 mg / kg / day. More preferably, the dose of (+)-cis-THC is greater than 500 mg / kg / day. More preferably, the dose of (+)-cis-THC is greater than 750 mg / kg / day. More preferably, the dose of (+)-cis-THC is greater than 1000 mg / kg / day. More preferably, the dose of (+)-cis-THC is greater than 1500 mg / kg / day.

[0021] Alternatively, the dose of (+)-cis-THC is less than 100 mg / kg / day. More preferably, the dose of (+)-cis-THC is less than 50 mg / kg / day. More preferably, the dose of (+)-cis-THC is less than 20 mg / kg / day. More preferably, the dose of (+)-cis-THC is less than 10 mg / kg / day. More preferably, the dose of (+)-cis-THC is less than 5 mg / kg / day. More preferably, the dose of (+)-cis-THC is less than 1 mg / kg / day. More preferably, the dose of (+)-cis-THC is less than 0.5 mg / kg / day.

[0022] According to a second aspect of the present invention there is provided a composition for use as a medicament comprising (+)-cis-tetrahydrocannabinol ((+)-cis-THC) and one or more pharma- ceutically acceptable excipients.

[0023] According to a third aspect of the present invention there is provided (+)-cis tetrahydrocannabinol ((+)-cis-THC) for use in the treatment of epilepsy.

[0024] According to a fourth aspect of the present invention, there is provided a method for producing (+)-cis tetrahydrocannabinol ((+)-cis-THC).

[0025] Embodiments of the invention are further described below with reference to the accompanying drawings. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 shows the four stereoisomers of tetrahydrocannabinol. [Diagram 2] Superposition of the optimized conformations of (-)-trans-Δ9-THC (magenta), (+)-cis-Δ9-THC (dark pink), and (-)-cis-Δ9-THC (light pink), and (+)-cis-Δ9-THC (orchid), at the phenol ring level, in stick drawings. [Diagram 3] FIG. 1 shows representative skeletons from MD simulations of CB1R in complex with (−)-trans-THC (panel A), (−)-cis-THC (panel B), and (+)-cis-THC (panel C). [Figure 4] FIG. 1 shows representative scaffolds from MD simulations of CB2R in complex with (−)-trans-THC (panel A), (−)-cis-THC (panel B), and (+)-cis-THC (panel C). [Diagram 5] HPLC chromatograms showing separation of cis enantiomers on a semi-preparative column. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] definition "Cannabinoids" refers to a group of compounds that include endocannabinoids, phytocannabinoids, and cannabinoids that are neither endocannabinoids nor phytocannabinoids, hereinafter referred to as "syntho-cannabinoids."

[0028] "Endocannabinoids" are endogenous cannabinoids that are high affinity ligands for the CB1 and CB2 receptors.

[0029] "Phytocannabinoids" are cannabinoids that occur naturally and can be found in the cannabis plant. Phytocannabinoids can be present in extracts that contain isolated or resynthesized botanical drug substances.

[0030] "Synthocannabinoids" are those compounds that are not found endogenously or in the cannabis plant. Examples include WIN 55212 and rimonabant.

[0031] "Isolated phytocannabinoids" are phytocannabinoids that have been extracted from the cannabis plant and purified to such an extent that all additional components, such as secondary and minor cannabinoids, as well as non-cannabinoid fractions, have been removed.

[0032] "Synthetic cannabinoids" are cannabinoids produced by chemical synthesis. The term includes modifying isolated phytocannabinoids, for example by forming pharma- ceutically acceptable salts thereof.

[0033] A "substantially pure" cannabinoid is defined as a cannabinoid present in a purity of greater than 95% (w / w), more preferably greater than 96% (w / w), 97% (w / w), 98% (w / w), and 99% (w / w).

[0034] "Stereoisomers" are molecules that have identical atomic composition and bonding, but differ in the arrangement of their atoms in three dimensions.

[0035] "Geometric isomers" are chemically distinct and pharmacologically distinct enantiomers that are generally readily separated without the use of chiral techniques.

[0036] "Diastereoisomers" are isomers of a drug with more than one chiral center that are not mirror images of one another.

[0037] The present invention provides data demonstrating the distinct physicochemical properties of the claimed compound, (+)-cis-tetrahydrocannabinol, compared to its optical and geometric isomers. Additionally, data are presented demonstrating the efficacy of the compound in animal models of disease. In another aspect, a method for the synthetic production of (-)-cis-THC is provided. EXAMPLES

[0038] In silico virtual screening of phytocannabinoid isomers By using a combined molecular docking and molecular dynamics approach in a membrane environment, we were able to identify the putative binding mode of (+)-cis-THC to the CB1 and CB2 cannabinoid receptors in comparison with other stereoisomers of THC.

[0039] method Computer-based methods The geometry of the starting ligand was constructed using Ghemical 2.99.23 and then energy minimized (EM), first at the molecular mechanics level using Tripos 5.2 force field parameterization and then at the AM1 semiempirical level; fully optimized using the GAMESS program4 at the Hartree-Fock level with the STO-3G basis set; and HF / 6-31G* / STO-3G single-point calculations were performed to derive partial atomic charges with the RESP procedure5.

[0040] Docking studies were performed with AutoDock 4.2 distribution by using the crystal structures of CB1R complexed to agonist AM11542 (PDB id: 5XRA) and CB2R complexed to antagonist AM10257 (PDB id: 5ZTY).

[0041] Both proteins and ligands were processed with the AutoDock Tools (ADT) package version 1.5.6rc16 to add non-polar hydrogens, calculate Gasteiger charges, and select rotatable side chain bonds.

[0042] A grid for docking evaluation of 60 × 70 × 60 points with 0.375 Å spacing, centered on the ligand binding site, was generated using the program AutoGrid 4.2 included in the Autodock 4.2 distribution.

[0043] Different runs were performed by using different combinations of mobile residues.

[0044] Molecular docking was performed employing a Lamarckian genetic algorithm (LGA) with docking parameters of 100 individuals in a population with up to 15 million energy calculations and up to 37,000 generations, followed by 300 iterations of Solis-Wets local search. A total of 100 docking runs were performed for each calculation.

[0045] The missing loops in the crystal structure used in this study were modeled using the MODELLER v9.11 program.

[0046] Representative complexes for each ligand-receptor combination were completed by addition of all hydrogen atoms and energy minimized. The energy minimized complexes were embedded in a POPC bilayer using the CHARMM-GUI web-interface, and molecular dynamics (MD) simulations in the membrane environment were then performed using the pmemd.cuda module of the Amber16 package8, using the lipid14ff force field for lipids, the ff14SB force field for proteins, and the gaff parameters for the ligands. MD generation runs were performed for 100 runs.

[0047] result Comparison of THC isomers To compare the conformations of the polycyclic moiety, the 3D coordinates of the THC isomers, obtained as described in the Methods section, were superimposed at the level of the phenol ring and are shown in Figure 2.

[0048] The (-)-cis-THC isomer was found to have a tetrahydrobenzomethyl ring that fits onto the scaffold of (-)-trans-THC, at the level of the dimethyl-pyran moiety, and points in the same direction.

[0049] However, the conformation of the (+)-cis-THC isomer is significantly different from both the (-)-trans and (-)-cis isomers.

[0050] Theoretical Binding at the CB1 Receptor (CB1R) Since (−)-trans-THC is a known CB1R partial agonist as shown in Figure 3A, the X-ray structure of agonist-bound CB1R was chosen for docking studies.

[0051] For comparison, the cis isomer of THC was docked in the same X-ray structure: Figure 3B shows the docking of (-)-cis-THC, and Figure 3C shows the docking of (+)-cis-THC.

[0052] It can be seen that both (-)-trans-THC and (-)-cis-THC adopted the L-form conformation, with the pentyl chain pointing towards the tricyclic ring system forming hydrophobic interactions with Trp2795.43, π-π, on helix V, and Phe268, Phe3797.35, Phe1893.25, and Phe1772.64 on loop ECL2, and hydrogen bonding with Ser3837.39.

[0053] The pentyl chain also participates in hydrophobic interactions with Phe2003.36, a residue that is important in CB1R activation as it is part of a toggle switch with Trp3566.48. Indeed, π-π stacking between Trp3566.48 and Phe2003.36 stabilizes the inactive form of the receptor.

[0054] (-)-cis-THC has the same pose as (-)-trans-THC, except for the tetrahydro-methyl-benzene group, which is tilted compared to trans-THC.

[0055] However, (+)-cis-THC adopts an inverted orientation in the tricycle, with the pentyl chain pointing toward the N-terminus and away from Phe2003.36, toward Phe1772.64 (Fig. 3C).

[0056] Theoretical binding at the CB2 receptor The binding of (-)-trans-THC to CB2R is shown in Figure 4A.

[0057] For comparison, the cis isomer of THC was docked in the same X-ray structure: Figure 4B shows the docking of (-)-cis-THC, and Figure 4C shows the docking of (+)-cis-THC.

[0058] The overall arrangement of the studied compounds within the CB2R ligand binding site overlaps well with that already observed in the CB1R complex. The interaction between (-)-trans-THC and CB2 is mainly hydrophobic and aromatic, involving residues from ECL2 and helices II, III, V, and VI. The THC tricycle forms hydrophobic interactions with Phe183ECL2 and with Phe1063.25 and Phe942.64, whereas the pentyl chain forms hydrophobic interactions with Trp1945.43 and Phe1173.36. This last residue is part of the switch toggle together with Trp2586.48. The hydroxy group of the terpenoid ring of the THC tricycle is involved in an H-bond with Ser2857.39.

[0059] Similar to binding at the CB1R, the (-)-cis-THC isomer adopts a similar orientation to (-)-trans-THC in the ligand-binding site, whereas (+)-cis-THC is the opposite.

[0060] conclusion A combined molecular docking and molecular dynamics approach allowed us to determine the putative binding modes of (-)-trans-THC, (-)-cis-THC, and (+)-cis-THC isomers within the ligand-binding sites of the CB1 and CB2 receptors.

[0061] The binding modes of the three compounds to the two receptors are similar, as the residues and overall configurations of the helices, N-terminus, and ECL2 loop are all tightly conserved between the two subtypes.

[0062] The two cis isomers of THC differ significantly from each other in the conformation of the tricyclic scaffold, with (-)-cis-THC being more similar to (-)-trans-THC, whereas (+)-cis-THC has an opposite binding mode within the ligand-binding site of both receptors, predicting different functional profiles for this isomer. EXAMPLES

[0063] Evaluation of the anticonvulsant effects of (+)-CIS-THC in the mouse supramaximal electroshock seizure (MES) model of generalized seizures The efficacy of (+)-cis-THC was tested in a mouse model of seizures using the maximal electroshock (MES) test.

[0064] method Mice were administered MES (30 mA, square current: 0.6 ms pulse width, 0.2 s duration, 50 Hz) via corneal electrodes connected to a constant current shock generator (Ugo Basile: type 7801) to reliably induce tetanic hindlimb convulsions. The number of tonic convulsions was recorded.

[0065] Sixteen mice were studied per group. The study was performed in a blinded fashion.

[0066] The test substance, (+)-cis-THC, was evaluated at four doses (10, 50, 100, and 150 mg / kg), administered intraperitoneally 60 min before MES and compared with a vehicle control group (administered under the same experimental conditions).

[0067] Valproic acid (positive control) was administered intraperitoneally at 250 mg / kg 30 min prior to MES and used as a reference substance and compared with the vehicle group (administered intraperitoneally 60 min prior to MES).

[0068] Data were analyzed by comparing treatment groups with the appropriate vehicle controls using two-tailed Fisher's exact test (p<0.05 was considered significant).

[0069] result Table 1 shows the data obtained in this experiment.

[0070] In the positive control (valproic acid) group, a significant 100% change was observed in the number of tonic-clonic seizures the animals had compared to vehicle, demonstrating the expected anticonvulsant effect.

[0071] Mice treated with (+)-cis-THC showed a dose-related increase in the percentage change in the number of tonic-clonic seizures observed in animals compared to vehicle.

[0072] The highest dose of 150 mg / kg administered intraperitoneally 60 min before testing resulted in a significant (p<0.05) 37.5% reduction in tonic convulsions compared with vehicle controls.

[0073] At the lowest dose (10 mg / kg), no effect was observed.

[0074] [Table 1]

[0075] conclusion These data demonstrate that the enantiomer (+)-cis-THC produced significant anticonvulsant effects in the MES model. These data are the first to demonstrate a therapeutic effect of this enantiomer of THC. EXAMPLES

[0076] Evaluation of the antinociceptive potential of (+)-CIS-THC in mice using the hot plate method The efficacy of (+)-cis-THC was tested in a mouse model of pain using the hot plate test.

[0077] method Mice were allowed a minimum acclimation period of 7 days prior to the start of the study. Naive mice were acclimated to the procedure room in their home cages with food and water available ad libitum.

[0078] Animals were treated with either vehicle, 1, 15, 50, 100, 125, and 150 mg / kg (+)-cis-THC intraperitoneally at 10 ml / kg, or morphine at 10 mg / kg or morphine vehicle (saline) at 10 ml / kg intraperitoneally.

[0079] Animals were placed on a hot plate set at 52°C and the latency to withdrawal threshold (first response of lifting front or hind paws, licking, or attempting to escape) was determined 1 hour after treatment or 0.5 hours for positive controls.

[0080] Animals were culled according to Schedule 1 procedures immediately after measurement.

[0081] Data were analyzed by comparing withdrawal thresholds back to the vehicle treated group.

[0082] result Table 2 shows the data obtained in this experiment.

[0083] In the positive control (morphine) group, withdrawal thresholds were significantly elevated compared to vehicle, demonstrating the expected antinociceptive effect.

[0084] In (+)-cis-THC treated mice, no significant differences were observed compared to vehicle at any of the doses tested.

[0085] [Table 2]

[0086] conclusion These data demonstrate that the enantiomer (+)-cis-THC has no antinociceptive effects in animal models of pain. EXAMPLES

[0087] Method for the synthetic production of (+)-CIS-tetrahydrocannabinol As mentioned above, the compound (+)-cis-THC is produced as a minor cannabinoid by the cannabis plant, which primarily produces the cannabinoid cannabidiol (CBD). Assuming that in highly purified extracts of CBD the total amount of THC is less than about 0.1% (w / w) relative to the total amount of cannabinoids in the preparation, the amount of (+)-cis-THC remaining in the extract is extremely small.

[0088] THC in purified extracts obtained from CBD-producing cannabis plants is known to exist as both trans and cis-THC geometric isomers, and the ratio of trans-THC:cis-THC is known to vary during processing and purification of the extract from about 3.6:1 trans-THC:cis-THC to about 0.8:1 trans-THC:cis-THC.

[0089] Additionally, the cis-THC present in purified preparations has been found to exist as a mixture of the optical isomers (-)-cis-THC and (+)-cis-THC, with the ratio of (-)-cis-THC:(+)-cis-THC ranging from about 9:1 ((-)-cis-THC:(+)-cis-THC).

[0090] Considering that the compound (+)-cis-THC is found in nature at extremely low levels, a methodology that may be used to produce larger amounts of the cannabinoid (+)-cis-THC is detailed by the synthetic route set forth below as Scheme 1.

[0091] The compounds are numbered and their full names are given in boxes below the pathway.

[0092] [ka]

[0093] [Table 3]

[0094] The resulting racemate of cis-THC was separated using chiral separation of the enantiomers using an HPLC column Phenomenex Lux Cellulose 2 chiral column.

[0095] A reversed-phase gradient of MeCN / H2O (0.1% HCO2H) was used.

[0096] Each isolated material was analyzed by optical rotation, chiral HPLC, LCMS, and 1H NMR.

[0097] FIG. 5 shows the HPLC chromatogram, in which peak 2 was observed to be (+)-cis-THC with an enantiomeric excess of 99.4%, while peak 3 was identified as (−)-cis-THC with an enantiomeric excess of 96.8%.

Claims

1. A pharmaceutical composition comprising (+)-cis tetrahydrocannabinol ((+)-cis-THC) for use in the treatment of epilepsy.

2. A pharmaceutical composition as described in claim 1 for use as an anticonvulsant.

3. A pharmaceutical composition described in claim 1 or 2, wherein the (+)-cis-THC is in the form of a plant extract.

4. The pharmaceutical composition of claim 3, wherein the (+)-cis-THC is in the form of a purified extract of Cannabis.

5. The pharmaceutical composition of claim 4, wherein the purified extract contains at least 80% (w / w) (+)-cis-THC.

6. The pharmaceutical composition of claim 4, wherein the purified extract contains at least 95% (w / w) (+)-cis-THC.

7. The pharmaceutical composition of claim 1, wherein the (+)-cis-THC is in the form of a synthetic compound.

8. The pharmaceutical composition of claim 1 or 2, wherein the dose of (+)-cis-THC is greater than 100 mg / kg / day.

9. 3. The pharmaceutical composition of claim 1 or 2, wherein the dose of (+)-cis-THC is less than 100 mg / kg / day.

10. The pharmaceutical composition of claim 1 or 2, further comprising one or more pharma- ceutically acceptable excipients.

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