MX2007005626A - New use for cannabinoid - Google Patents

Abstract

The invention relates to the use of one or more cannabinolds in the manufacture of medicaments for use in the treatment of diseases and conditions benefiting from neutral antagonism of the CB, cannabinoid receptor. Preferably the cannabinoid is tetrahydrocannabivarin (THCV). Preferably the diseases and conditions to be treated are taken from the group:obesity, schizophrenia, epilepsy, cognitive disorders such as Alzheimer's, bone disorders, bulimia, obesity associated with type II diabetes (non-insulin dependant diabetes) and in the treatment of drug, alcohol and nicotine abuse or dependency.

Description

NEW USE FOR CANABINOID FIELD OF THE INVENTION The present invention relates to the use of one or more cannabinoids in the manufacture of medicaments for use in the treatment of diseases and conditions that benefit from neutral antagonism of the CBi cannabinoid receptor. Preferably, the cannabinoid is tetrahydrocannabivarin (THCV). Preferably the diseases and conditions to be treated are taken from the group: obesity, schizophrenia, epilepsy, cognitive disorders such as Alzheimer's disease, bone disorders, bulimia, obesity associated with type II diabetes (non-insulin dependent diabetes) and in the treatment of abuse or dependence on drugs, alcohol and nicotine. BACKGROUND DESCRIPTION The action of many known cannabinoids can be attributed to their interaction with cannabinoid receptors. The discovery that cannabinoid receptors are present in mammalian systems has led to further research. For example, a class of Protein G-coupled receptors that are mainly present in the central nervous system has been identified, these have been called CBi receptors. Another type of receptor coupled with Protein G is the CB2 receptors that are substantially in the immune system. Cannabinoids are generally cannabinoid receptor agonists, meaning they are attracted to a cannabinoid receptor and activated. Well-known cannabinoid receptor agonists include the classical plant derived from cannabinoid delta-9-tetrahydrocannabinol (THC), the non-classical cannabinoid receptor agonist R- (+) - IN55212 and the cannabinoid receptor-dependent eicosanoid or anandamide receptor agonist. animal. All of these compounds have been shown to bind to the CBi receptor. Agonism in a receptor will often lead to an active response by the cell. Many disease states result from the active or abundant effects of agonists in their receptors. Research has led to the discovery of compounds that prevent the activation of cannabinoid receptors and as such are known as cannabinoid receptor antagonists. A competitive cannabinoid receptor antagonist is one that will bind to the receptor but will not cause a response in the cell. An inverse agonist acts on a receptor to produce an effect opposite to the response that the agonist would produce. The compound SR141716A (described in EP0576357) has been shown to be antagonistic to the cannabinoid receptor CBi.

There is evidence, however, that SR141716A is an inverse agonist rather than a silent or neutral antagonist (Pertwee, R. G., 2003). Maruani and Soubrie in US 6,444,474 and EP0969835 have described the use of a reverse CBX receptor agonist such as SR141716A in the regulation of appetite disorders. In many assay systems containing CBi, SR141716A itself produces effects that are opposite in direction to those produced by CBi agonists such as THC. Therefore, leading to the inference that it is an inverse agonist of the CBx receptor. While in some cases this may reflect antagonism of an endogenous CBi agonist (a CBi agonist produced by the assay system itself) in other cases it is thought to occur because the CBX receptors are constitutively active. It is generally considered that constitutively active receptors trigger effects even in the absence of any agonist administered or endogenously produced. Agonists improve their activity whereas inverse agonists are opposed. In contrast, neutral antagonists leave the constitutive activity unchanged. Neutral antagonists are favored over inverse agonists since they only block the ability of the receptor to interact with an endogenously produced CBi agonist such as anandamide or one that has been administered. There is evidence that the endogenous CBi agonist, anandamide, can be released in the brain to mediate processes such as feeding and appetite (Di Marzo et al., 2001). This raises the possibility that an antagonist of this receptor could be effective in the clinic as an appetite suppressant. The compound SR141615A is coupled to the CBi cannabinoid receptors so that they can not be activated. It is possible that blocking the CBi receptor system may adversely affect aspects mediated by CBi such as mood, sleep and pain relief. Since endocannabinoids have neuroprotective and anti-oxidant properties, it is possible that users of SR141716A may be at an increased risk of cancer and stroke. Neutral CBi receptor antagonists are likely to have a less complex pharmacology than those of an inverse agonist. Thus, when administered per se, said antagonist will only have effects on regions of the cannabinoid system in which release of endogenous cannabinoids is occurring towards CBi receptors but will not affect the activity of the endogenous cannabinoid system that arises from the presence of parts of this system of constitutively active CB ° receptors. CB receptor antagonists! , particularly neutral CBi receptor antagonists, are as such, probably useful in the treatment of diseases and conditions that are caused by an interaction with the CBi receptor. These diseases and conditions include, for example, obesity, schizophrenia, epilepsy or cognitive disorders such as Alzheimer's, bone disorders, bulimia, obesity associated with type II diabetes (non-insulin dependent diabetes) and in the treatment of drug abuse or dependence, alcohol or nicotine (Pertwee, R. G., 2000). The use of a neutral antagonist in place of an inverse antagonist would be particularly beneficial, since it is likely that fewer side effects occur since it would not increase the consequences of constitutive CBi receptor activity. Currently, there are a few neutral CBi receptor antagonists identified. A psychotropic cannabinoid THC analogue has been produced that behaves as a neutral CBi antagonist in vitro (Martin, B. R., Et al., 2002). The compound, O-2050 is a sulfonamide analogue of delta-8-tetrahydrocannabinol, and has acetylene incorporated into its side chain.

This analog behaves as a neutral CBi receptor antagonist in the mouse vas deferens. However, O-2050 does not behave as a CBi receptor antagonist in mice in vivo and, like established CBi receptor agonists, depresses the spontaneous activity of the mouse. In addition, O-2050 analogs with R = ethyl or R = butyl behave as typical CBi receptor agonists in mice in vivo. Surprisingly, applicants have shown that cannabinoid tetrahydrocannabinol (THCV) is a neutral antagonist of cannabinoid receptors CBi and CB2. The cannabinoid THCV is a classic plant cannabinoid, which is structurally related to THC, in which instead of the 3-pentyl side chain of THC, the THCV molecule has a 3-propyl side chain. The structures of the two cannabinoids are shown in Figure 1. The discovery that THCV appears to act as a neutral antagonist of CBi receptors was particularly surprising since THC is known to be a CBi agonist and therefore must follow that a structurally related compound such as THCV would also be an agonist rather than an antagonist. SUMMARY OF THE INVENTION In accordance with the first aspect of the present invention, the use of tetrahydrocannabivarin (THCV) in the manufacture of a medicament for use in the treatment of diseases or conditions benefiting from neutral antagonism of the CBi receptor is provided. Preferably, THCV is used in the manufacture of a medicament for the treatment of obesity, schizophrenia, epilepsy or cognitive disorders such as Alzheimer's, bone disorders, bulimia, obesity associated with type II diabetes (diabetes not dependent on insulin) and in the treatment of drug, alcohol or nicotine abuse or dependence. More preferably, THCV is used in the manufacture of a medicament for use as an appetite suppressant. A neutral antagonist is likely to have fewer side effects than those of an inverse agonist. This is because it is expected to oppose the drug-induced activation of CBi receptors but does not attenuate effects produced by constitutively active CBi receptors. In contrast, an inverse agonist will attenuate the effects produced not only by drug-induced activation of CBi receptors but also by constitutively active CBi receptors and thus would be expected to result in a greater number of side effects than a neutral antagonist.

Therefore, in a preferred embodiment of the invention, THCV can be used in the substantial absence of any substance or compound that acts as an inverse agonist of CBi receptors. References to THCV, particularly with respect to therapeutic use, will be understood to also encompass pharmaceutically acceptable salts of said compounds. The term "pharmaceutically acceptable salts" refers to salts or esters prepared from pharmaceutically acceptable non-toxic bases or acids, including bases or inorganic acids and bases or organic acids, as will be well known to those skilled in the art. Many suitable inorganic and organic bases are known in the art. The scope of the invention also extends to THCV derivatives that retain the desired activity of neutral CBi receptor antagonism. Derivatives that retain substantially the same activity as the starting material, or more preferably exhibit improved activity, can be produced in accordance with conventional principles of medicinal chemistry, which are well known in the art. These derivatives may exhibit a lower degree of activity than the starting material, as long as they retain sufficient activity to be therapeutically effective. The derivatives may exhibit improvements in other properties that are desirable in pharmaceutically active agents, such as, for example, improved solubility, reduced toxicity, improved admission. Preferably THCV is an extract of at least one cannabis plant. More preferably, the THCV extract from at least one cannabis plant is a botanical drug substance. In one embodiment, the THCV extract from at least one cannabis plant is produced by extraction with supercritical or subcritical C02. Alternatively, the THCV extract from at least one cannabis plant is produced by contacting plant material with a gas heated to a temperature that is greater than 100 ° C, sufficient to volatilize one or more of the cannabinoids in the plant material. to form a vapor, and to condense the vapor to form an extract. Preferably, the THCV extract from at least one cannabis plant comprises all the cannabinoids that occur naturally in the plant. Alternatively, the THCV is in a substantially pure or isolated form. A "substantially pure" cannabinoid preparation is defined as a preparation having chromatographic purity (of the desired cannabinoid) greater than 90%, more preferably greater than 95%, more preferably greater than 96%, more preferably greater than 97%, more preferably greater than 98%, more preferably greater than 99% and more preferably greater than 99.5%, as determined by the normalization of area of an HPLC profile. Preferably, the substantially pure THCV used in the invention is substantially free of any other synthetic or naturally occurring cannabinoids, including cannabinoids that occur naturally in cannabis plants. In this context "substantially free" can be taken as meaning that no cannabinoids other than THCV are detectable by HPLC. In another aspect of the present invention, THCV is in a synthetic form. Preferably THCV is formulated as a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, excipients or diluents. The invention also encompasses pharmaceutical compositions comprising THCV, or pharmaceutically acceptable salts or derivatives thereof, formulated in pharmaceutical dosage forms, together with suitable pharmaceutically acceptable carriers, such as diluents, fillers, salts, buffers, stabilizers, solubilizers, etc. The dosage form may contain other pharmaceutically acceptable excipients to modify conditions such as pH, osmolarity, taste, viscosity, sterility, lipophilicity, solubility, etc. The selection of thinners, carriers or excipients will depend on the desired dosage form, which in turn may depend on the intended route of administration to a patient. Appropriate dosage forms include, but are not limited to, solid dosage forms, e.g., tablets, capsules, powders, dispersible granules, sachets and suppositories, including sustained deliberation and delayed release formulation. The powders and tablets will generally comprise from about 5% to about 70% active ingredient. Suitable solid carriers and excipients are generally known in the art and include, eg, magnesium carbonate, magnesium stearate, talc, sugar, lactose, etc. Tablets, powders, sachets and capsules are suitable dosage forms for oral administration. Liquid dosage forms include solutions, suspensions and emulsions. The liquid form preparations can be administered by intravenous, intracerebral, intraperitoneal, parenteral or intramuscular injection or infusion. Sterile injectable formulations may comprise a sterile solution or suspension of the active agent in a non-toxic, pharmaceutically acceptable diluent or solvent. Liquid dosage forms also include solutions or sprays for intranasal, buccal or sublingual administration. Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be combined with a pharmaceutically acceptable carrier, such as an inert compressed gas. Also included are dosage forms for transdermal administration, including creams, lotions, aerosols and / or emulsions. These dosage forms may be included in transdermal patches of the matrix or reservoir type, which are generally known in the art. The pharmaceutical preparations can be conveniently prepared in unit dosage form, in accordance with conventional pharmaceutical formulation procedures. The amount of active compound per unit dose can be varied according to the nature of the active compound and the intended dosage regimen. Generally, this will be within the range of 0.1 mg to 1000 mg. In accordance with a second aspect of the present invention, there is provided a method for the treatment of a disease or condition benefiting from neutral antagonism of the cannabinoid receptor CBi by THCV, which comprises administering to a subject in need thereof a Therapeutically effective THCV. The disease or condition to be treated is selected from the group consisting of obesity, schizophrenia, epilepsy or cognitive disorders such as Alzheimer's, bone disorders, bulimia, obesity associated with type II diabetes (non-insulin dependent diabetes) or drug abuse or dependence, alcohol or nicotine In accordance with a third aspect of the present invention, there is provided a method for cosmetically beneficial weight loss comprising suppressing appetite in a subject by administering to the subject an effective amount of THCV. In certain circumstances, the appetite suppressant can be used in order to achieve a cosmetically beneficial weight loss in a human subject, without necessarily producing medical or therapeutic benefit to that subject. In this context, the administration of the appetite suppressant may not be considered as a medical or therapeutic treatment of the subject. According to a fourth aspect of the present invention, the use of a neutral cannabinoid receptor antagonist is provided in the manufacture of a medicament for use in the treatment of diseases or conditions that benefit from neutral antagonism of one or more types of cannabinoid receptor. Preferably the neutral cannabinoid receptor antagonist is used in the manufacture of a medicament for use in the treatment of diseases or conditions that benefit from neutral antagonism of the CBN cannabinoid receptor., and wherein the constant dissociation of the cannabinoid receptor antagonist in the CBi receptor is approximately 75 nM. Preferably, the neutral cannabinoid receptor antagonist is used in the manufacture of a medicament for use in the treatment of diseases or conditions that benefit from neutral antagonism of the CB2 cannabinoid receptor, and wherein the constant dissociation of the receptor antagonist of cannabinoid in the CB2 receptor is approximately 62 n. The term "approximately" refers to within ± 10% of the aforementioned value. Certain aspects of this invention are further described, by way of example only, with reference to the accompanying drawings, in which: Figure 1 shows the 2-dimensional structure of tetrahydrocannabivarin (THCV) cannabinoid and tetrahydrocannabinol (THC) . SPECIFIC DESCRIPTION Example 1: Investigation into the effects that THCV has on CBX or CB2 cannabinoid receptors. Experiments were performed with membranes prepared from healthy brain tissue, which is densely populated with CBi but not CB2 receptors (reviewed in Howlett et al 2002). Additional experiments were undertaken with Chinese hamster ovary (CHO) cells transfected with hCB2 receptors. These membranes were used to investigate the ability of THCV to displace binding sites of. { JH } CP55940 CB2. These experiments were used to determine whether THCV behaves as a receptor agonist or antagonist of CBi or CB2. The experiments were also carried out with the mouse isolated vas deferens, a tissue in which cannabinoid receptor agonists such as R- (+) - WIN55212, CP55940, THC and 2-arachidonoyl ethanolamide (anandamide) can inhibit contractions electrically evoked 8Devane et al., 1992; Pertwee et al., 1995). Cannabinoid receptor agonists are thought to inhibit electrically evoked contractions by acting on preconfunctional neuronal cannabinoid CBi receptors to inhibit the release of contractile neurotransmitters, ATP, (acting in purine postjunctional P2X receptors), and norepinephrine (acting in cytotoxicity). post-adrenoceptors), (Trendelenberg et al., 2000).

Experiments were also conducted with (-) - 7-hydroxy-canabidiol-dimethylheptyl, a synthetic analogue of the plant cannabinoid, (-) -canabidiol, which inhibits electrically evoked contractions of the mouse vas deferens through a mechanism that seems to operate in a prejunta and be at least partially independent of CBi receptor. Methods Radioligand Displacement Assay Trials were carried out with. { 3H} CP55940, 1 mg ml 1 bovine serum albumin (BSA) and 50 mT Tris buffer, total assay volume 500 ul, using the filtration procedure previously described by Ross et al (1999b). addition of either brain membranes (33 ug of pin per tube) or transfected hCB2 cells (25 ug of pin per tube) All assays were performed at 37 ° C for 60 minutes before termination by addition of ice cold wash buffer (50 mM Tris buffer, 1 mg ml -1 bovine serum albumin, pH 7.4) and vacuum filtration using a 24-well sampling distributor and GF / B filters that had been soaked with wash buffer at 4 ° C for at least 24 hours.Each extraction tube was washed six times with a 1.2 ml aliquot of washing buffer.The filters were baked in the oven for 60 minutes and then placed in 5 ml of scintillation fluid. The radioactivity was quantified by means of spectrometry to scintillation liquid. The specific link was defined as the difference between the link that occurred in the presence and absence of 1 uM of unlabelled CP55940. THCV was stored as a solution of 10 m material in DMSO, the vehicle concentration in all test tubes containing 01% DMSO. The link parameters for. { 3J} CP55940, were 2336 fmol mg-1 pin (Bmax) and 2.31 nM (Kd) in mouse brain membranes (Thomas et al., 2004), and 72570 fmol / mg pin (Braax) and 1043 nM (Kd) in transfected cells of hCB2. Enmlacde trial of. { 35S} GTPüS The method to measure link of. { 35S} GTPyS stimulated by agonist to cannabinoid CBi receptors was adapted from the methods of Jurkinen et al. (1997) and Breivogel et al (2001). The conditions used to measure in link. { 35S} GTPyS stimulated by agonist to transfected cannabinoid CB2 receptors were adapted from those used by MacLennan et al. (1998) and Griffin et al. (1999). Tests were carried out with GTPyS binding buffer (50 mM Tris-HCl, 50 mM Tris-Base, 5 mM mgCl2, 1 mM EDTA, 100 mM NaCl, 1 mM DTT, 0.1% BSA) in the presence of. { 35S} GTPyS and GDP, in a final volume of 500 ul. The link was started by adding. { 35S} GTPyS to the tubes. The non-specific binding was measured in the presence of 30 uM of GTPyS. The drugs were incubated in the assay for 60 minutes at 30 ° C. The reaction was terminated by a rapid vacuum filtration method using Tris buffer (50 mM Tris-HC1, 50 mM Tris-Base, 0.1% BSA), and radioactivity was quantified by liquid scintillation spectrometry. The concentrations of. { 35S} GTPyS and GDP present in the assay varied depending on whether the assay was conducted with mouse brain or transfected cell membranes. When the assay was conducted with mouse brain membranes, 0.1 nM of. { 35S} GTPyS and 30 uM of GDP, while the corresponding concentrations present when the assay was conducted with transfected cell membranes were 1 nM and 320 uM respectively. Additionally, the mouse brain membranes were preincubated for 30 minutes at 30 ° C with 0.5 U ml of adenosine deaminase to remove the endogenous adenosine.Agonists and antagonists were stored as a solution of 1 or 10 mM material in DMSO. , vehicle concentration in all test tubes being 0.11% DMSO Experiments in vas deferens Vasa deferentia were obtained from albino MF1 mice weighing 31 to 59 g The tissues were mounted vertically in 4 ml organ baths. they underwent an electrical stimulation of progressively greater intensity followed by an equilibrium procedure in which they were exposed to alternating periods of stimulation (2 minutes) and rest (10 minutes) until contractions were obtained with consistent amplitudes (Thomas et al., 2004) These contractions were monophasic and isometric and were evoked by 0.5 s pulse trains of 110% maximum voltage (train frequency 0.1 Hz; pulse frequency 5 Hz, pulse duration 0.5 ms). Except in experiments with phenylephrine, all drug additions were made to the organ baths after the equilibrium period and there was no washing between these additions. In most of the experiments there was an initial application of a potential antagonist or its vehicle. ~ z '. . l. ÷ v. : ic 38 minutes later for a period of 2 min;! e electrical stimulus at the end of which the lowest of a | ·: ii ·. ": · '. ·? ·: -? ·, - ~? · G; t rae ions of the contraction inhibitors, R- (+) -WIN55212, CP55940, THC, anandamine, (-) -7-hydroxy-canabidiol-dimethylheptyl or clonidine, was applied.After a rest period, tissues were stimulated electrically for 2 minutes and then subjected to an extra addition of contraction inhibition This cycle of drug addition, rest and 2 minutes of stimulation was repeated in order to construct cumulative concentration-response curves.Only a concentration-response curve It was constructed by tissue, resting periods were 3 minutes for clonidine, 13 minutes for R- (+) -WIN55212, CP55940 and anandamide, 28 minutes for THCD and THCV, and 58 minutes for (-) -7-hydroxy-cannabidiol -dimethylheptyl, also experiments were performed with capsaicin.This drug was added at intervals those of 3 minutes and the tissues did not rest from electrical stimulation between these additions. In some experiments, cumulative concentration-response curves for. THCV were constructed without prior addition of any other compound, again using a drug addition cycle, 28 minutes of rest and 2 minutes of stimulation. In experiments with ß, y-methylene-ATP were cumulatively constructed without washing. THCV, WIN or drug vehicle were added 30 minutes before the first addition of β, β-methylene-ATP, each subsequent addition of which was done immediately after the effect that the previous dose had reached a plane (dose cycles from 1 to 2 min). Only one addition of phenylephrine was made to each tissue and this was carried out 30 minutes after the addition of THCV, WIN or drug vehicle. Data analysis The values are expressed as means and variability as s.e. medium or as 95% confidence limits. The concentration of THCVG that produced a 50% displacement of radioligand-specific binding sites (IC50 value) was calculated using Prism GraphPad 4. This dissociation constant (Kx value) was calculated using the equation of Chen and Prusoff 81973). The link values of. { 35S} GTPyS stimulated by agonist were calculated by subtracting the baseline binding values (obtained in the absence of agonist) from values stimulated by agonist (obtained in the presence of agonist) as detailed elsewhere (Ross et al., 1999a). The inhibition of the electrically evoked contraction response of vas deferens has been expressed in percentage terms and this has been calculated by comparing the amplitude of the contraction response after each addition of a contraction inhibitor with its amplitude immediately before the first addition. of the inhibitor. The contractile responses to phenylephine and β, β-methylene-ATP have been expressed as increases in tension (g). The values for EC50m for maximum effect (Emax) and for the s.e. Mean or 95% confidence limits of these values have been calculated by non-linear regression analysis using the equation for a sigmoid concentration-response curve (Graphpad Prism).

The values of apparent dissociation constant (KB) for THCV agonists in the vas deferens or in the binding assay of. { 35S} GTPyS have been calculated by Schild analysis of the concentration ratio defined as the concentration of an agonist that elucidates a response of a particular size in the presence of a competitive reversible antagonist at a concentration, B, divided by the concentration of the same agonist that produces an identical response in the absence of the antagonist. The methods used to determine the concentration ratio and apparent KB values and to establish whether to record concentration-response traces significantly deviated from parallelism are detailed elsewhere (Pertwee et al., 2002). The mean values were compared using the two-tailed Student's t-test for unpaired data or one-way analysis of variance (ANOVA) followed by Dunnett's test (GraphPad Prism). A value P < 0.05 was considered to be significant. Results: THCV radioligand experiments displaced. { 3H} CP55940 of specific binding sites in mouse brain and cell membranes CH09-hCB2 in a way that was significantly better fit to a site than a two site competition curve (_ <0.05; GraphPad Prism 4).

Their mean Ki values were 75.4 nM and 62.8 nM, respectively. THCV also displaced a. { 3H} R- (+) -WIN55212 and. { H.}. SR141716A of specific binding sites in mouse brain membranes, their mean EC5o values with 95% confidence limits shown in parentheses being 61.3 nM (48.6 and 77.3 nM, n = 4 to 7) and 86.8 nM (63.8 and 188.1 nM; n = 4 to 6) respectively. The corresponding EC50 value of THCV for displacement of. { H.}. cp55940 EN 98.2 Nm (69.6 and 138.6 nM; n = 4 to 8). The ability of CP55940 to improve link. { 3 S.}. GTPyS to mouse brain and membranes CHO-hCB2 was attenuated by THCV, which at 1 uM produced significant destral shifts in the response curves of concentration record of this cannabinoid receptor agonist that did not deviate significantly from parallelism. The apparent mean KB values for this antagonism are shown in Table 1, as are apparent mean KB values of SR141716A for antagonism of CP55940 in mouse brain membranes and of SR144528 for antagonism of CP55940 in the CHO-hCB2 cell membranes. At 1 uM, THCV also produced a significant parallel handover in the concentration response curve record of R- (+) - WIN55212 for enhancement of GTPyS binding to mouse brain membranes. Table 1 Antagonist Agonist Preparation Medium 95% of membrane n ap- limitites remte of con- KB bail (nM) (nM =) THCV CP55940 Brain 93.1 66.5, (1000 nM) 130.6 THCV R- (+) -WIN55212 Brain 85.4 29.3 , (1000 nM) 270.5 SR141716A CP55940 Brain 0.09 0.021, (10 nM) 0.41 THCV CP55940 CH0-hCB2 10-1 5.0, (1000 nM) 20.5 SR144528 CP55940 CHO-hCB2 0.49 0.26, (100 nM) 0.85 Experiments with vas deferens THCV produced an inhibition related to concentration of electrically evoked contractions of isolated mouse vas deferens with an EC50 of 12.7 um (6.9 and 23.2 uM). This effect is unlikely to be mediated by CBi receptor since it is not attenuated by SR141716A at 100 nM (n = 7, data not shown), a concentration that equals or exceeds the concentrations of this previously found CBi selective antagonist that antagonizes agonists of CBi receptor established in the same bioassay (Pertwee et al., 1995; Ross et al., 2001). At 31.6 uM, a concentration at which it produced a marked inhibition of electrically evoked contractions, THCV also attenuated contractile responses of the vas deferens to both the P2 receptor agonist, β, β-methyl-ATO and the adrenoceptor agonist ai, phenylephrine hydrochloride . In contrast, at 1 uM, a concentration at which there was no detectable inhibitory effect on the electrically evoked contractions, THCV did not induce any significant reduction in the amplitude of induced contractions either by β, β-methylene-ATP (n = 8, data not shown) or by phenylephrine. These findings suggest that THCV inhibited electrically evoked contractions of the vas deferens, at least in part, by acting postjunctionally to block contractile responses to endogenously released ATP and norepinephrine. At concentrations well below those at which electrically evoked contractions were inhibited, THCV opposed to inhibition induced by R- (+) - WIN55212 of the spasm response in a manner that was related to concentration and not accompanied by any significant change in the maximum effect (Emax) of R- (+) - IN55212 (PZ0.05; ANOVA followed by Dunnett's test; n = 6-9). The weight displacements produced by THCV in the concentration response curve record of R- (+) - WIN55212 do not deviate significantly from parallelism and provide a Schild trace with an inclination that is not significantly different from unity. The mean apparent KB value of THCV was calculated by the Tallarida method (Pertwee et al., 2002) which is 1.5 nM as shown in Table 2. At l uM, a concentration that markedly attenuated electrically evoked contractions, R- (+ ) - IN55212 did not decrease the capacity of β, β-methylene-ATP (n = 7 or 10, data not shown) or phenylephrine to induce contractions of vas deferens. Table 2: THCV Apparent KB inhibitor 95% limits of n (nM) mean contraction of the confidence THCV (nM) (nM) 10 - R- (+) - IN55212 1.5 1.1, 2.3 6-9 1000 100 anandamide 1.2 0.2, 6.2 7 100 methanandamide 4.6 1.5, 11.6 12 100 CP55940 10.3 3.8, 31.7 14 1000 THC 96.7 15.4, 978 10 100 clonidine > 100 - 8 100 capsaicin > 100-800 100 7-OH-CBD-DMH > 100-8 THCV was shown to antagonize anandamide at 10, 100 and 1000 nM, and methanandamide and CP55940 at 100 nM. The weight displacements produced by THCV in the record of concentration response curves of these contraction inhibitors did not deviate significantly from parallelism. The apparent mean KB value for antagonism of anandamide by 10 nM THCV with its 95% confidence limits shown in parentheses is 1.4 nM (0.36 and 7.50 nM). The apparent mean KB values for antagonism of anandamide, methanandamide and CP55940 per 100 nM THCV are listed in Table 2. At 100 nM, THCV did not reduce the ability of clonidine, capsaicin or (-) -7-hydroxy-cannabidiol- dimethylheptyl to inhibit electrically evoked contractions, indicating that it possesses at least some degree of selectivity as an antagonist of contraction inhibitors in vas deferens. Also, 100 nM of THCV do not antagonize the cannabinoid receptor agonist, THC (n = ll, data not shown). However, at 1 uM, THCV produced a significant downwash in the THC concentration response curve record that did not deviate significantly from parallelism (see Table 2 for its apparent KB value against THC). From this data it is possible that the co-administration of a low dose of THCV with THC could improve the effects of high doses of THC such as an increased heart rate and psychoactivity. The low dose of THCV would act as a competitive antagonist on passable CBi receptors and thus block some of the effects of high dose THC. It is well established in the art that partial agonist potency and efficiency increase with receptor density and that the potency of a surmountable competitive antagonist is not affected by receptor density. The dose of THCV will be one that is not sufficient to prevent the therapeutic effects of THC but would be sufficient to prevent the side effects of high dosage of THC. Conclusions · A9-tetrahydrocannabivarin (THCV) displaced. { 3H} CP55940 of specific binding sites in brain and CHO-hCB2 cell membranes (Ki = 75.4 and 62.8 nM respectively), indicating that THCV is both a receptor antagonist of CBi and CB2. or THCV (1 u) also antagonized the unbalanced improvement by CP55950 of. { 35S} GTPyS that binds to these membranes (apparent KB = 93.1 and 10.1 nM, respectively), indicating that it is a reasonably potent competitive antagonist. The KB values indicate that THCV is more potent as a receptor antagonist of CB2 than of CBi. o In the mouse vas deferens, the ability of A9-tetrahydrocannabinol (THC) to inhibit electrically evoked contractions was antagonized by THCV, its apparent KB value (96.7 nM) approaching the apparent KB values for its antagonism of CP55940- and R - (+) -WIN55212- induced improvement of. { S.}. GTPyS that binds to mouse brain membranes. or THCV also antagonized R- (+) - WIN55212, anandamide, methanandamia and CP55940 in the vas deferens, but with apparent lower KB values (1.5, 1.2, 4.6 and 10.3 nM respectively), indicating that THCV behaves in a competitive manner and surmountable. or THCV produced its antagonism of cannabinoids at concentrations that did not affect the amplitude of the electrically evoked contractions, or the capacity of. { 35S} GTPyS from binding to mouse brain membranes or CHO-hCB2 cell membranes, suggesting that THCV is a neutral cannabinoid receptor antagonist, or THCV (100 nM) did not object to inhibition induced by clonidine, capsaicin or (-) - 7-hydroxy-canabidiol-dimethylheptyl of electrically evoked contractions of vas deferens. This is an indication that THCV has selectivity. o The contractile responses of vas deferens to phenylephrine hydrochloride or ß,? -methylene-ATP were not reduced by 1 uM of THCV or R- (+) - WIN55212, suggesting that THCV interacts with R- (+) - WIN55212 at sites of prejunta. o At 31.6 uM, THCV did not reduce contractile responses to phenylephrine hydrochloride and β, β-methylene-ATP, and above 3 uM inhibited the electrically evoked contractions of the vas deferens in an independent manner from SR141716A. In conclusion, THCV behaves as a neutral competitive CBX and CB2 receptor antagonist. In vas deferens, it antagonized several cannabinoids more potently than THC and was also more potent against CP55940 and R- (+) - WIN55212 in this tissue than in brain membranes. REFERENCES: BREIVOGEL, C.S. and col. (2001). Evidence of a new cannabinoid receptor coupled to G protein in mouse brain. Mol. Pharmacol., 60, 155-163. CHEN, Y.-C. AND PRUSOFF, W.H. (1973). Relationship between the inhibition constant (Ki) and the inhibitor concentration that causes 50 percent inhibition (IC50) of an enzymatic reaction. Biochem. Pharmacol., 22, 3099-3108.

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Claims (17)

1. CLAIMS 1. - The use of tetrahydrocannabindivarin (THCV in the manufacture of a medicament for use in the treatment of diseases and conditions benefiting from neutral antagonism of the cannabinoid receptor CBi
2. 2. - The use of THCV in accordance with claim 1 , in the manufacture of a medicament for the treatment of obesity, schizophrenia, epilepsy or cognitive disorders such as Alzheimer's, bone disorders, bulimia, obesity associated with type II diabetes (non-insulin dependent diabetes) or in the treatment of abuse or dependence of drug, alcohol or nicotine
3. 3. - The use of THCV according to claim 2, in the manufacture of a medicament for use as an appetite suppressant
4. 4. - The use of THCV < in accordance with any of the claims above, wherein the THCV is in the form of an extract prepared from at least one cannabis plant
5. 5. - The use of THCV in accordance with claim 4, in do The prepared extract of at least one cannabis plant is in the form of a botanical drug substance.
6. 6. The use of THCV according to claims 4 or 5, wherein the extract prepared from at least one cannabis plant is produced by extraction with supercritical or subcritical C02.
7. 7. The use of THCV according to claims 4 or 5, wherein the extract prepared from at least one cannabis plant is produced by contacting the plant material with a gas heated to a temperature that is greater than 100 °. C, sufficient to volatilize one or more of the cannabinoids in the plant material to form a vapor, and condense the vapor to form an extract.
8. 8. The use of THCV according to any of claims 4 to 7, wherein the extract prepared from at least one cannabis plant comprises all naturally occurring cannabinoids in the at least one cannabis plant.
9. 9. The use of THCV according to claim 1, wherein the THCV is in a substantially pure or isolated form.
10. 10. The use of THCV according to claim 1, wherein the THCV is in a synthetic form.
11. 11. The use of THCV according to any of the preceding claims, wherein the THCV is formulated as a pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers, excipients or diluents.
12. 12. - A method for the treatment of a disease or condition that benefits from neutral antagonism of the cannabinoid receptor CBi by THCV, which comprises administering to a subject in need thereof a therapeutically effective amount of THCV.
13. 13. - A method according to claim 12, wherein the disease or condition is selected from the group consisting of obesity, schizophrenia, epilepsy, cognitive disorders such as Alzheimer's, bone disorders, bulimia, obesity associated with type II diabetes (diabetes not dependent on insulin), and abuse or dependence on drugs, alcohol or nicotine.
14. 14. A method for cosmetically beneficial weight loss comprising suppressing appetite in a subject by administering to the subject an effective amount of THCV
15. 15. - The use of a neutral cannabinoid receptor antagonist in the manufacture of a medicament for use in the treatment of diseases or conditions that benefit from neutral antagonism of one or more types of cannabinoid receptor.
16. 16. The use according to claim 15, of a neutral cannabinoid receptor antagonist in the manufacture of a medicament for use in the treatment of diseases or conditions that benefit from neutral antagonism of the cannabinoid receptor CBi wherein the constant The dissociation of the cannabinoid receptor antagonist in the CBi receptor is approximately 75 nM.
17. 17. The use according to claim 15 of a neutral cannabinoid receptor antagonist in the manufacture of a medicament for use in the treatment of diseases or conditions that benefit from neutral antagonism of the CB2 cannabinoid receptor where the constant of Dissociation of the cannabinoid receptor antagonist in the CB2 receptor is approximately 62 nM. SUMMARY OF THE INVENTION The invention relates to the use of one or more cannabinoids in the manufacture of medicaments for use in the treatment of diseases and conditions that benefit from neutral antagonism of the CBi cannabinoid receptor. Preferably the cannabinoid is tetrahydrocannabivarin (THCV). Preferably, the diseases and conditions to be treated are taken from the group: obesity, schizophrenia, epilepsy, cognitive disorders such as Alzheimer's disease, bone disorders, bulimia, obesity associated with type II diabetes (non-insulin dependent diabetes) and in the treatment of abuse or dependence on drugs, alcohol and nicotine.