CANNABIDIOL-TYPE CANNABINOID COMPOUND

Abstract

The present invention relates to a cannabidiol (CBD) type cannabinoid compound for use as a medicament. The CBD-type cannabinoid, 6-hydroxy cannabidiol (6-0 H CBD), is a metabolite of CBD. The cannabinoid can be produced by synthetic means and a method for the production of 6-0 H CBD is described herein. In addition, disclosed herein are data which demonstrate the efficacy of 6-0 H CBD in a model of disease.

Claims

1. 6-hydroxy cannabidiol (6-OH CBD) for use as a medicament.

2. 6-OH CBD for use according to claim 1, wherein the 6-OH CBD is in the form of a synthetic compound.

3. 6-OH CBD for use according to claim 1, wherein the 6-OH CBD is in the form of a pure or isolated compound.

4. 6-OH CBD for use according to any of the preceding claims, wherein the dose of 6-OH CBD is greater than 100 mg/kg/day.

5. 6-OH CBD for use according to any of the preceding claims, wherein the dose of 6-OH CBD is less than 100 mg/kg/day.

6. A composition for use as a medicament comprising 6-hydroxy cannabidiol (6-OH CBD) and one or more pharmaceutically acceptable excipients.

7. 6-hydroxy cannabidiol (6-OH CBD) for use in the treatment of epilepsy.

8. 6-OH CBD for use according to claim 7, wherein the epilepsy treated is in a mammal.

9. 6-OH CBD for use according to claim 8, wherein the mammal is a human.

10. 6-OH CBD for use according to claim 8, wherein the mammal is a dog.

11. A process for the preparation of 6-hydroxy cannabidiol (6-OH CBD).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0037] FIG. 1 shows the effect of 6-OH CBD in the MEST test in mouse.

[0038] FIG. 2 shows the pharmacokinetics of 6-OH CBD in plasma and brain of mice following a single intravenous or intraperitoneal administration.

CANNABINOIDS AND THEIR ABBREVIATIONS

[0039] The cannabinoids described in the present application are listed below along with their standard abbreviations.

TABLE-US-00001 CBD Cannabidiol [00001] 6-OH CBD Alpha 6-hydroxy cannabidiol [00002]

DETAILED DESCRIPTION

Example 1: Synthetic Production Method for Alpha 6-Hydroxy Cannabidiol (6-OH CBD)

[0040] The compound 6-OH CBD is a known metabolite of cannabidiol.

[0041] The synthetic pathway described below details a methodology that can be used in order to produce the cannabinoid alpha 6-OH CBD.

[0042] On the scheme R=C.sub.5H.sub.11

[0043] To cannabidiol (5.00 g, 15.8 mmol) in anhydrous pyridine (20 mL) was added acetic anhydride (5.13 g, 4.75 mL, 50.2 mmol) and the solution was stirred for 4 h. Dichloromethane (300 mL) was added and the solution was washed with water (200 ml), 1M hydrochloric acid (200 mL), saturated aqueous sodium bicarbonate (200 mL), dried (MgSO4) and concentrated to give cannabidiol diacetate (5.72 g, quantitative), as a straw yellow oil which was used without further purification.

[0044] To cannabidiol diacetate (6.18 g, 15.5 mmol) in glacial acetic acid (14 mL) and acetic anhydride (7.12 g, 6.59 mL, 69.8 mmol) was added sodium dichromate (4.87 g, 18.6 mmol) and the mixture was stirred at room temperature for 4 days. The resulting solution was diluted with water (200 mL) and extracted with diethyl ether (200 mL followed by 150 mL). The combined organic layers were washed with saturated aqueous sodium bicarbonate (2×100 mL), dried (MgSO4) and concentrated to give a yellow oil that was purified using a Biotage Isolera automated chromatography system under normal phase conditions (silica column, gradient of 5.fwdarw.32% ethyl acetate in petrol) with detection at 254 nm to give 6-oxo-cannabidiol diacetate (2.03 g 33%), as a colourless oil.

Rf=0.45 (ethyl acetate-petrol, 1:4 v/v)

[0045] To lithium aluminium hydride (355 mg, 9.37 mmol) in diethyl ether (36 mL) at 0° C. was added 6-oxo-cannabidiol diacetate (0.92 g, 2.23 mmol) in diethyl ether (8 mL) and the mixture was stirred at room temperature for 4 h. The resulting mixture was cooled in an ice bath and cautiously quenched with water (10 mL) dropwise. 1 M Hydrochloric acid (60 mL) was added and the mixture was extracted with diethyl ether (100 mL). The organic layer was washed with saturated brine (80 mL), dried (MgSO4) and concentrated to give a pale yellow oil that was purified using a Biotage Isolera automated chromatography system under normal phase conditions (silica column, gradient of 7.fwdarw.47% ethyl acetate in petrol) with detection at 254 nm to afford 6-oxo-cannabidiol (0.51 g, 69%), as a white glassy solid.

[0046] Rf=0.34 (ethyl acetate-petrol, 3:7 v/v)

##STR00003##

[0047] The resultant material was confirmed to be alpha 6-hydroxy-cannabidiol (6-OH CBD). The compound is a yellow glassy semi-solid material with the chemical formula C.sub.21H.sub.30O.sub.3 and a molecular weight of 330.5 g/mol.

[0048] Purity of the compound was tested by HPLC which was shown to produce a 95.6% pure material.

[0049] 6-OH CBD was stored at −20° C. and protected from light until required for testing.

Example 2: Evaluation of 6-Hydroxy CannabidioL (6-OH CBD) for Anticonvulsant Activity Using the Supramaximal Electroshock Seizure (MES) Test in the Mouse

[0050] The efficacy of 6-OH CBD was tested in a mouse model of seizure, the supramaximal electroshock seizure (MES) test.

[0051] The supramaximal electroshock seizure (MES) test is widely utilized preclinically to evaluate anti-convulsant properties of molecules and standard anti-epileptic drugs (Loscher et al., 1991).

[0052] The MES test is a very stringent model in which mice receive a predetermined high-level electrical stimulus of sufficient intensity to reliably produce tonic hindlimb extensor seizures in 100% of control animals. As such the MES test is a rigorous evaluation of anticonvulsant activity (Swinyard, 1985).

Methods

[0053] Naïve mice were acclimatised to the procedure room in their home cages, with food and water available ad libitum.

[0054] Animals were dosed i.p. according to treatment group.

[0055] The vehicle (10 ml/kg i.p. 60 min pre-treatment time) was 1:1:18 vehicle 5% ethanol, 5% kolliphor EL, 90% saline.

[0056] The test compound, alpha 6-OH CBD was prepared according to the method described in Example 1.

[0057] The test compound, 6-OH CBD was administered at doses of 3, 30, 100 and 200 mg/kg given at 10 ml/kg i.p. 60 min pre-treatment time.

[0058] In addition, a dose of 100 mg/kg CBD was given at 10 ml/kg i.p. at 120 minutes pre-treatment time to assess the effect of the drug over a longer time course.

[0059] The positive control valproate was used at 250 mg/kg (10 ml/kg i.p. 30 min pre-treatment time).

[0060] Mice were individually assessed for the production of a tonic hind limb extensor seizure following a pre-determined high level (30 mA: 50 Hz) corneally delivered electroshock (0.2 sec duration) of sufficient intensity to reliably produce tonic hindlimb seizures in 100% of control animals.

[0061] Induction of seizure is measured as an all-or-nothing effect scored as either present (+) or absent (0) for each animal.

[0062] Data was collected by an observer unaware of the treatment for each animal and was expressed as the number of +'s or 0's for each treatment group.

[0063] The percentage inhibition of relevant vehicle treated group (the protection relative to vehicle treated controls) was then generated.

[0064] Significant differences between individual treatment groups and vehicle-treated groups were assessed using 2-tailed Fisher's Exact Probability test (p<0.05 considered significant).

Results

[0065] Table 1 below demonstrates the data produced in this experiment.

[0066] In the positive control valproate (250 mg/kg) treated group, administered i.p. 30 minutes before the test, all animals were scored as not having a seizure. This result was statistically significant (p<0.001) compared to the vehicle control.

[0067] In the 6-OH CBD treatment groups, administered i.p. 120 minutes before the test, the dose of 3 mg/kg 6-OH CBD was ineffective. However, the doses of 30, 100 and 200 mg/kg 6-OH CBD enabled all mice within the group to withstand seizures and produced a statistically significant effect compared to vehicle (p<0.001).

[0068] In addition, the dose of 6-OH CBD (100 mg/kg) given 120 minutes also produced a statistically significant reduction of seizures compared to the vehicle control.

TABLE-US-00002 TABLE 1 Evaluation of effect of 6-OH CBD in the MES test % change Dose Pre-treatment from Treatment (mg/kg) N time (mins) vehicle Significance Vehicle — 10 60 — — Valproate 250 10 30 100% P < 0.001 6-OH CBD 3 10 60  0% — 6-OH CBD 30 10 60 100% P < 0.001 6-OH CBD 100 10 60 100% P < 0.001 6-OH CBD 200 10 60 100% P < 0.001 6-OH CBD 100 10 120 100% P < 0.001

Conclusions

[0069] These data demonstrate for the first time a therapeutic effect for the compound 6-OH CBD.

[0070] The data showing the 6-OH CBD given 2 hours (120 minutes) before the mice received the electroshock demonstrates that the compound was able to have a long-lasting effect.

[0071] These data are significant as they provide heretofore unknown evidence that this cannabinoid may be of therapeutic value.

Example 3: Evaluation of 6-Hydroxy Cannabidiol (6-OH CBD) for Anticonvulsant Activity Using the Maximal Electroshock Seizure Threshold (MEST) Test in the Mouse

[0072] The efficacy of 6-OH CBD was tested in a mouse model of generalised seizure, the maximal electroshock seizure threshold (MEST) test.

[0073] The maximal electroshock seizure threshold (MEST) test is widely utilized preclinically to evaluate pro- or anti-convulsant properties of test compounds (Loscher et al., 1991).

[0074] In the MEST test the ability of a drug to alter the seizure threshold current required to induce hind limb tonic extensor convulsions is measured according to an “up and down” method of shock titration (Kimball et al., 1957). An increase in seizure threshold is indicative of anti-convulsant effect. Antiepileptic drugs including the sodium channel blockers (e.g. lamotrigine) with clinically proven efficacy against generalised tonic-clonic seizures all exhibit anti-convulsant properties in this test in the mouse.

[0075] Conversely, a reduction in seizure threshold is indicative of a pro-convulsant effect as observed with known convulsant agents such as picrotoxin.

[0076] The ability of a test compound to alter the stimulus intensity, expressed as current (mA), required to induce the presence of tonic hind limb extensor convulsions, is assessed in the MEST. The outcome of the presence (+) or absence (0) of tonic hind limb extensor convulsions observed from a current to produce tonic hind limb extension in 50% of animals in the treatment group (CC.sub.50) determines the seizure threshold for the treatment group and the effects were then compared to the CC.sub.50 of the vehicle control group.

Methods

Study Details

[0077] Naïve mice were acclimatised to the procedure room in their home cages for up to 7 days, with food and water available ad libitum.

[0078] All animals were weighed at the beginning of the study and randomly assigned to treatment groups based on a mean distribution of body weight across groups. All animals were dosed at 10 mL/kg via intraperitoneal (i.p) injection, with either vehicle, test compound at 3, 10 or 30 mg/kg or diazepam at 2.5 mg/kg.

[0079] Animals were individually assessed for the production of a tonic hind limb extensor convulsion at 30 min post-dose for vehicle, 30, 15 and 60 min post-dose for 6-OH CBD at 3, 10 and 30 mg/kg respectively, and 30 min post-dose for diazepam, from a single electroshock.

[0080] The first animal within a treatment group was given a shock at the expected or estimated CC.sub.50 current. For subsequent animals, the current was lowered or raised depending on the convulsions outcome from the preceding animal.

[0081] Data generated from each treatment group were used to calculate the CC.sub.50±SEM values for the treatment group.

Test Compounds

[0082] Vehicle: (5% ethanol, 5% solutol, 90% Saline) was prepared as follows: 2 mL of ethanol, 2 mL of solutol were warmed to 60° C., in 36 mL of saline (1:1:18).

[0083] Positive control: diazepam was used at 2.5 mg/kg.

[0084] The test compound, alpha 6-OH CBD was prepared according to the method described in Example 1. 6-OH CBD was administered at 3, 10 and 30 mg/kg (i.p.) in a 1:1:18 ethanol:solutol:0.9% saline formulation.

Sample Collection

[0085] Each animal was humanely killed immediately after production of a convulsion by destruction of the brain from striking the cranium, followed by the confirmation of permanent cessation of the circulation from decapitation under The Humane Killing of Animals under Schedule 1 to the Animals (Scientific Procedures) Act 1986. Terminal blood and brain collection were performed following decapitation.

[0086] Blood was collected in Lithium-heparin tubes and centrifuged at 4° C. for 10 minutes at 1500×g. The resulting plasma was removed (>100 μL) and split into 2 aliquots of 0.5 mL Eppendorf tubes containing 100 μL of ascorbic acid (100 mg/mL) for stabilisation. Brains were removed, washed in saline and halved. Each half was placed into separate 2 mL screw cap cryovials, weighed and frozen on cardice.

Statistical Analysis

[0087] The data for each treatment group were recorded as the number of +'s and 0's at each current level employed and this information is then used to calculate the CC.sub.50 value (current required for 50% of the animals to show seizure behaviour)±standard error.

[0088] 6-OH CBD effects were also calculated as percentage change in CC.sub.50 from the vehicle control group.

Significant difference between drug-treated animals and controls were assessed according to Litchfield and Wilcoxon (1949).

Results

[0089] Table 2 below demonstrates the data produced in this experiment, and FIG. 1 illustrates these results.

[0090] In the vehicle group, the CC.sub.50 value was calculated to be 24.0 mA.

[0091] In the positive control diazepam (2.5 mg/kg) treated group, administered i.p. 30 minutes before the test, the CC.sub.50 value was 48.8 mA. This result was statistically significant (p<0.001) compared to the vehicle control.

[0092] In the 6-OH CBD treatment groups, administered i.p. 30, 15, and 60 minutes before the test, the doses of 3, 10 and 30 mg/kg 6-OH CBD produced a statistically significant CC.sub.50 value compared to vehicle at all three doses of the compound.

[0093] Such data are indicative that this compound will be of therapeutic benefit.

TABLE-US-00003 TABLE 2 Evaluation of effect of 6-OH CBD in the MEST test Pre- Dose treatment % change Treatment (mg/kg) N time (mins) CC.sub.50 ± SEM from vehicle Significance Vehicle — 12 30 24.0 ± 0.4 — — Diazepam 2.5 12 30 48.8 ± 1.1 103% P < 0.001 6-OH CBD 3 12 30 30.0 ± 1.1  25% P < 0.001 6-OH CBD 10 12 15 56.5 ± 2.1 135% P < 0.001 6-OH CBD 30 12 60 102.5 ± 2.2  327% P < 0.001

Conclusions

[0094] 6-OH CBD produced a dose-related increase in MEST, which provides evidence that this compound exhibits anticonvulsive properties. Significant effects were observed at 3, 10, and 30 mg/kg, when compared to vehicle.

[0095] These data are significant as they provide heretofore unknown evidence that this cannabinoid may be of therapeutic value.

Example 4: Pharmacokinetics of 6-Hydroxy Cannabidiol (6-OH CBD)

[0096] The objective of this study was to determine brain and plasma pharmacokinetic parameters of 6-OH CBD following a single intravenous or intraperitoneal administration of 6-OH CBD to the mouse; and to determine brain: plasma concentration ratios for 6-OH CBD.

Methods

[0097] Ninety-nine male mice each received a single intravenous or intraperitoneal dose of 6-OH CBD, as detailed below in Table 3.

TABLE-US-00004 TABLE 3 Details of dosing Dose level Number of Dose route (mg/kg) animals intravenous 1 27 intraperitoneal 3 27 intraperitoneal 10 27 intraperitoneal 30 27

[0098] For each formulation, 6-OH CBD was formulated at the required concentration in ethanol: Kolliphor EL (Cremophor EL): 0.9% (w/v) Saline (1:1:18, v/v/v).

[0099] Each animal received a single intravenous administration, via a tail vein, at a nominal dose volume of 2 mL/kg or a single intraperitoneal administration at a nominal dose volume of 5 mL/kg.

[0100] Following dosing, terminal blood samples were collected from each animal via cardiac puncture, and brains were excised, at each of the following time points: [0101] Intravenous groups: 7, 15 and 30 minutes, 1, 2, 4, 8, 12 and 24 hours; [0102] Intraperitoneal groups: 15 and 30 minutes, 1, 2, 4, 8, 12 and 24 hours.

[0103] Blood was collected into tubes containing lithium heparin anticoagulant and centrifuged to prepare plasma. Brain samples were frozen on dry ice, then weighed and stored deep frozen prior to bioanalysis. The remaining carcasses were discarded. Plasma was stabilised with an equal volume of ascorbic acid solution, then stored deep frozen prior to bioanalysis.

[0104] Stabilised plasma samples were analysed to determine the concentrations of 6-OH CBD, using qualified LC-MS/MS methods. The results of these analyses were evaluated to determine non-compartmental pharmacokinetic parameters.

Results

[0105] Tables 4 and 5 demonstrate the results obtained in this study, and FIG. 2 illustrates the results. The following parameters were determined:

[0106] The following parameters were determined: [0107] C.sub.max Maximum concentration observed. [0108] T.sub.max Time of maximum observed concentration. [0109] AUC.sub.0-t Area under the concentration-time curve from hour 0 to the last quantifiable concentration, estimated by the linear trapezoidal rule. [0110] AUC.sub.0-24 Area under the concentration-time curve from hour 0 to hour 24, estimated using the linear trapezoidal rule. [0111] t.sub.1/2 Elimination half-life, determined as In(2)/λz. [0112] Cl Clearance, calculated (for intravenous doses only) as Dose/AUC.sub.0-inf. [0113] Vss Volume of distribution, based on the terminal elimination phase, calculated (for intravenous doses only) as Cl/λz. [0114] F.sub.abs Absolute bioavailability of the intraperitoneal dose.

TABLE-US-00005 TABLE 4 Composite pharmacokinetic parameters of 6-OH CBD in the plasma and brain following a single intravenous or intraperitoneal administration Dose Level C.sub.max T.sub.max AUC.sub.0-t AUC.sub.0-24 t.sub.l/2 F.sub.abs CI Vss Matrix Treatment (mg/kg) (ng/mL) (h) (h * ng/mL) (h * ng/mL) (h) (%) (mL/min/kg) (L/kg) Plasma IV 1 775 0.117 342 343 0.451 NA 48.7 1.90 IP 3 658 0.250 533 535 0.993 52.1 NA NA IP 10 2550 0.250 2590 2600 1.08 75.8 NA NA IP 30 11000 0.250 17700 17700 0.959 173 NA NA Brain IV 1 1580 0.117 679 690 0.312 NA NA NA IP 3 851 0.500 756 768 0.569 NA NA NA IP 10 3680 0.250 3750 3940 0.706 NA NA NA IP 30 13500 1.00 25000 25000 0.919 NA NA NA

TABLE-US-00006 TABLE 5 Brain: plasma ratios for 6-OH CBD C.sub.max and AUC.sub.0-24 values following a single intraperitoneal administration of 6-OH CBD Dose Dose level Brain: Plasma Route (mg/kg) C.sub.max AUC.sub.0-24 IV 1 2.03 2.01 IP 3 1.29 1.44 IP 10 1.45 1.52 IP 30 1.23 1.41

[0115] As can be seen from Table 4, following intravenous (IV) administration of 6-OH CBD, at a dose level of 1 mg/kg, T.sub.max was observed at 0.117 hours (7 minutes) post dose. After reaching C.sub.max, 6-OH CBD concentrations declined, with a half-life (t.sub.1/2) of 0.451 hours. Total plasma clearance was 48.7 mL/min/kg and the volume of distribution was 1.90 L/kg.

[0116] After intraperitoneal (IP) administration at 3, 10 or 30 mg/kg, 6-OH CBD was absorbed, with T.sub.max values of 0.250 hours (15 minutes) at each dose level. After reaching C.sub.max, plasma concentrations declined, with half-life (t.sub.1/2) values ranging between 0.993 and 1.08 hours and similar across each dose level assessed.

[0117] Following IP administration, 6-OH CBD exposure in plasma (as assessed by C.sub.max and AUC.sub.0-24 values) increased with each increase in dose level between 3 and 30 mg/kg. The observed increases in exposure were greater than dose-proportional, with increases of 16.7-fold for C.sub.max and 33.2-fold for AUC.sub.0-24 for a 10-fold increase in dose level.

[0118] The absolute bioavailability of 6-OH CBD following IP administration was 52.1%, 75.8% and 173%, at 3, 10 and 30 mg/kg respectively.

[0119] After systemic administration, 6-OH CBD appeared in brain, with T.sub.max values of 0.117 hours after IV dosing and ranging from 0.250 to 1 hours. After reaching C.sub.max, 6-OH CBD concentrations declined, with half-life (t.sub.1/2) values of 0.312 hours following IV administration and ranging between 0.569 and 0.919 hours, with the trend to increase with increasing dose level, following IP administration.

[0120] As can be seen from Table 5, following IP administration, 6-OH CBD exposure in brain (as assessed by C.sub.max and AUC.sub.0-24 values) increased with each increase in dose level between 3 and 30 mg/kg. The observed increases in exposure were greater than dose-proportional, with increases of 15.8-fold for C.sub.max and 32.6-fold for AUC.sub.0-24 for a 10-fold increase in dose level.

[0121] Brain: plasma ratios for 6-OH CBD C.sub.max and AUC.sub.0-24 ranged from 1.23 to 2.03 for C.sub.max and 1.41 to 2.01 for AUC.sub.0-24. Following IP administration, the ratios for both parameters were similar across each of the dose levels assessed. However, the ratios for both parameters were higher following IV compared with IP administration.

CONCLUSION

[0122] After intraperitoneal administration, 6-OH CBD was absorbed with T.sub.max values of 15 min for all dosed IP; and 7 min for 1 mg/kg IV.

[0123] The results of this study provide evidence of good pharmacokinetic parameters (bioavailability, clearance etc.) for the compound 6-OH CBD.