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 cannabidivarin (6-OH CBDV), is a metabolite of cannabidivarin (CBDV). The cannabinoid can be produced by synthetic means and a method for the production of 6-OH CBDV is described herein. In addition, disclosed herein are data which demonstrate the efficacy of 6-OH CBDV in a model of disease.

Claims

1. A method of treating epilepsy in a subject in need thereof, comprising administering a therapeutically effective dose of 6-hydroxy cannabidivarin (6-OH CBDV) to the subject.

2. The method of claim 1, wherein the 6-OH CBDV is in the form of a synthetic compound.

3. The method of claim 1, wherein the 6-OH CBDV is in the form of a pure or isolated compound.

4. The method of claim 1, wherein the dose of 6-OH CBDV is greater than 100 mg/kg/day.

5. The method of claim 1, wherein the dose of 6-OH CBDV is less than 100 mg/kg/day.

6. The method of claim 1, wherein the subject is a mammal.

7. The method of claim 6, wherein the mammal is a human.

8. The method of claim 6, wherein the mammal is a dog.

9. The method of claim 1, wherein the dose of 6-OH CBDV is less than 10 mg/kg/day.

10. The method of claim 1, wherein the dose of 6-OH CBDV ranges from 10 mg/kg/day to 100 mg/kg/day.

11. The method of claim 1, wherein the dose of 6-OH CBDV ranges from 20 mg/kg/day to 100 mg/kg/day.

12. The method of claim 1, wherein the dose of 6-OH CBDV ranges from 50 mg/kg/day to 100 mg/kg/day.

13. The method of claim 1, wherein the dose of 6-OH CBDV ranges from greater than 100 mg/kg/day to 1500 mg/kg/day.

14. The method of claim 1, wherein the dose of 6-OH CBDV ranges from greater than 100 mg/kg/day to 1000 mg/kg/day.

15. The method of claim 1, wherein the dose of 6-OH CBDV ranges from greater than 100 mg/kg/day to 750 mg/kg/day.

16. The method of claim 1, wherein the dose of 6-OH CBDV ranges from greater than 100 mg/kg/day to 500 mg/kg/day.

17. The method of claim 1, wherein the treatment comprises producing an anticonvulsive effect in the subject.

18. The method of claim 1, wherein the treatment reduces seizures in the subject or increases a threshold of seizures in the subject, compared to a control.

19. The method of claim 1, wherein the treatment comprises treating tonic seizures and/or tonic-clonic seizures.

20. The method of claim 1, wherein the 6-OH CBDV is at least 96.7% pure as measured by HPLC.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

(2) FIG. 1 shows the effect of 6-OH CBDV in the MEST test in mouse.

(3) The cannabinoids described in the present application are listed below along with their standard abbreviations.

(4) TABLE-US-00001 CBD Cannabidiol 6-OH CBDV Alpha 6-hydroxy cannabidivarin

DETAILED DESCRIPTION

Example 1

Synthetic Production Method for Alpha 6-Hydroxy Cannabidivarin (6-OH CBDV)

(5) As previously described the compound 6-OH CBDV is a metabolite of cannabidiol.

(6) The synthetic pathway described below details a methodology that can be used in order to produce the cannabinoid alpha 6-OH CBDV.

(7) On the scheme R═C.sub.3H.sub.7

(8) To CBDV (5.00 g, 17.5 mmol) in anhydrous pyridine (20 mL) was added acetic anhydride (5.63 g, 5.20 mL, 55.2 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 CBDV diacetate (6.84 g, quantitative), as a yellow oil which was used without further purification.

(9) To CBDV diacetate (4.00 g, 10.8 mmol) in glacial acetic acid (9 mL) and acetic anhydride (4.96 g, 4.59 mL, 48.6 mmol) was added sodium dichromate (3.86 g, 13.0 mmol) and the mixture was stirred at room temperature for 4 days. The resulting solution was diluted with water (150 mL) and extracted with diethyl ether (2×150 mL). The combined organic extracts were washed with saturated aqueous sodium bicarbonate (150 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.33% ethyl acetate in petrol) with detection at 254 nm to give 6-oxo-CBDV diacetate (1.40 g 33%), as a colourless oil.

(10) Rf=0.36 (ethyl acetate-petrol, 1:4 v/v)

(11) To lithium aluminium hydride (0.58 g, 15.3 mmol) in diethyl ether (50 mL) at 0° C. was added 6-oxo-CBDV diacetate (1.40 g, 3.64 mmol) in diethyl ether (23 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 iced water (100 mL). 1 M Hydrochloric acid (60 mL) was added and the mixture was extracted with diethyl ether (100 mL+50 mL). The combined organic layers were washed with saturated brine (100 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.53% ethyl acetate in petrol) with detection at 254 nm to give 6-oxo-CBDV (0.70 g, 64%), as a white glassy solid.

(12) Rf=0.29 (ethyl acetate-petrol, 3:7 v/v)

(13) ##STR00003##

(14) The resultant material was confirmed to be alpha 6-hydroxy-cannabidivarin (6-OH CBDV). The compound is a yellow glassy semi-solid material with the chemical formula C.sub.19H.sub.26O.sub.3 and a molecular weight of 302.4 g/mol.

(15) Purity of the compound was tested by HPLC which was shown to produce a 96.7% pure material.

(16) 6-OH CBDV was stored at −20° C. and protected from light until required for testing.

Example 2

Evaluation of 6-Hydroxy Cannabidivarin (6-OH CBDV) for Anticonvulsant Activity Using the Supramaximal Electroshock Seizure (Mes) Test in the Mouse

(17) The efficacy of 6-OH CBDV was tested in a mouse model of seizure, the supramaximal electroshock seizure (MES) test.

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

(19) 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).

(20) Methods

(21) Naïve mice were acclimatised to the procedure room in their home cages, with food and water available ad libitum.

(22) Animals were dosed i.p. according to treatment group.

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

(24) The test compound, alpha 6-OH CBDV was prepared according to the method described in Example 1.

(25) The test compound, 6-OH CBDV was administered at doses of 3, 10, 30, and 100 mg/kg given at 10 ml/kg i.p. 60 min pre-treatment time.

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

(27) 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.

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

(29) 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.

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

(31) 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).

(32) Results

(33) Table 1 below demonstrates the data produced in this experiment.

(34) 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.

(35) In the 6-OH CBDV treatment groups, administered i.p. 60 minutes before the test, the dose of 3 and 10 mg/kg 6-OH CBDV only produced a 20% change in seizures compared to the vehicle control which was non-significant.

(36) However, the doses of 30 and 100 mg/kg 6-OH CBDV enabled all mice to withstand seizures and produced a statistically significant effect compared to vehicle (p<0.001).

(37) TABLE-US-00002 TABLE 1 Evaluation of effect of 6-OH CBDV in the MES test Dose Pre-treatment % change from Treatment (mg/kg) N time (mins) vehicle Significance Vehicle — 10 60 — — Valproate 250 10 30 100% P < 0.001 6-OH  3 10 60  20% Non-significant CBDV 6-OH  10 10 60  20% Non-significant CBDV 6-OH  30 10 60 100% P < 0.001 CBDV 6-OH 100 10 60 100% P < 0.001 CBDV

(38) Conclusions

(39) These data demonstrate for the first time a therapeutic effect for the compound 6-OH CBDV.

(40) These data are significant as they provide heretofore unknown evidence that this cannabinoid may be of therapeutic value.

Example 3

Evaluation of 6-Hydroxy Cannabidivarin (6-OH CBDV) for Anticonvulsant Activity Using the Maximal Electroshock Seizure Threshold (Mest) Test in the Mouse

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

(42) 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).

(43) 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.

(44) Conversely, a reduction in seizure threshold is indicative of a pro-convulsant effect as observed with known convulsant agents such as picrotoxin.

(45) 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.

(46) Methods

(47) Study Details:

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

(49) 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, 6-OH CBDV at 3, 10 or 30 mg/kg, diazepam at 2.5 mg/kg or sodium valproate at 250 mg/kg.

(50) Animals were individually assessed for the production of a tonic hind limb extensor convulsion at 15 min post-dose for vehicle, at 15, 15 and 30 min post-dose for 6-OH CBDV at 3, 10 and 30 mg/kg respectively, and 30 min post-dose for diazepam and sodium valproate, from a single electroshock.

(51) 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.

(52) Data generated from each treatment group were used to calculate the CC.sub.50±SEM values for the treatment group.

(53) Test Compounds:

(54) 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).

(55) Positive controls: diazepam was used at 2.5 mg/kg and sodium valproate at 250 mg/kg.

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

(57) Sample Collection:

(58) 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.

(59) 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.

(60) Statistical Analysis

(61) 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.

(62) 6-OH CBDV effects were also calculated as percentage change in CC.sub.50 from the vehicle control group.

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

(64) Results

(65) Table 2 below demonstrates the data produced in this experiment, and FIG. 1 illustrates these results.

(66) In the vehicle group, the CC.sub.50 value was calculated to be 25.7 mA.

(67) 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 57.5 mA. In the sodium valproate (250 mg/kg) treated group, administered i.p. 30 minutes before the test, the CC.sub.50 value was 281.5 mA. These results were statistically significant (p<0.001) compared to the vehicle control.

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

(69) Such data are indicative that this compound will be of therapeutic benefit.

(70) TABLE-US-00003 TABLE 2 Evaluation of effect of 6-OH CBDV in the MEST test Dose Pre-treatment % change Treatment (mg/kg) N time (mins) CC.sub.50 ± SEM from vehicle Significance Vehicle — 12 15 25.7 ± 0.4 — — Diazepam 2.5 12 30 57.5 ± 0.3 124% P < 0.001 Sodium 250 12 30 281.5 ± 5.8  997% P < 0.001 Valproate 6-OH CBD 3 12 15 54.2 ± 1.4 111% P < 0.001 6-OH CBD 10 12 15 63.5 ± 3.4 147% P < 0.001 6-OH CBD 30 12 30 92.5 ± 0.4 260% P < 0.001

(71) Conclusions

(72) 6-OH CBDV 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.

(73) These data are significant as they provide heretofore unknown evidence that this cannabinoid may be of therapeutic value.