Mixtures of cannabinoid compounds, and production and use thereof

Abstract

Specific compositions comprising one or multiple (cannabinoid) compound(s) of formula (A) and/or one or multiple salt(s) thereof are described as well as methods for their manufacture. ##STR00001## A compound of formula (A), a salt of formula (A) and a respective composition for use as medicine and for use in a method for the therapeutic treatment of the human or animal body, respectively, are also described. Furthermore, corresponding pharmaceutical formulations, cosmetic preparations and foodstuff and/or gourmet or snack preparations fit for consumption as well as a method for the manufacture of delta-9-tetrahydrocannabinol are described.

Claims

1. A compound selected from the group consisting of: ##STR00025## or a salt thereof.

2. A composition comprising one or more compounds and/or salts of claim 1.

3. A pharmaceutical composition comprising one or more compounds and/or salts of claim 1.

4. A method for manufacturing a compound of claim 1 comprising converting menthadienol with an olivetolic acid ester to derive the compound.

5. A method for manufacturing cannabidiol comprising: (a) obtaining a compound or salt of claim 1; and (b) subjecting the compound or salt to saponification and decarboxylation to generate cannabidiol.

6. A composition comprising: (a) one or more compounds selected from the group consisting of: ##STR00026## ##STR00027## or a salt thereof; (b) optionally, cannabidiol, wherein if cannabidiol is present, the ratio of the total amount of the one or more compounds of (a) and/or salts thereof to the total amount of cannabidiol is greater than 1:1; and (c) optionally, a compound of Formula (I) ##STR00028## wherein if a compound of Formula (I) is present, the ratio of the total amount of the one or more compounds of (a) and/or salts thereof to the total amount of the compound of Formula (I) is greater than 1:1.

7. A medicine comprising a composition of claim 6.

8. A method for the therapeutic treatment of a human or animal comprising administering a composition of claim 6 to the human or animal, wherein the therapeutic treatment provides: an appetite-stimulating effect, an anti-emetic effect to inhibit nausea and vomiting, reduction of muscular cramps and spasticity, alleviation of pain symptoms, alleviation of migraine symptoms, reduction of intraocular pressure related to glaucoma, mood enhancement, immunostimulation, and/or antiepileptic effect.

9. A method for manufacturing a composition of claim 6 comprising converting menthadienol with one or more olivetolic acid esters to derive the one or more compounds and/or salts of (a) in the composition.

10. A method for manufacturing cannabidiol comprising: (a) preparing a composition of claim 6; and (b) subjecting the one or more compounds of (a) and/or salts in the composition to saponification and decarboxylation to generate cannabidiol.

Description

A. STUDIES ON THE EFFECT OF COMPOUNDS ACCORDING TO THE INVENTION ON CANNABINOID RECEPTORS

(1) Binding Affinity:

(2) In particular, the following substances III-V and XI-XIII of the generic formula (A) were examined in own studies regarding their effect on cannabinoid receptors.

(3) ##STR00020##

(4) Substances III-V and XI-XIII were tested in competition studies regarding their binding affinity and their resulting binding profile for CB1 and CB2 receptors. Such studies enable the comparison of the affinity of each of the substances III-V and XI-XIII (K.sub.i values) with the affinity of another ligand for the cannabinoid receptors. The competition studies were carried out in cell membranes, which were transfected with CB1 and CB2 receptors.

(5) To this end, membranes of human cells were used, in which the CB1 and CB2 receptors (RBHCB1M400UA and RBXCB2M400UA) with a B.sub.max and K.sub.d value for CP55940 for CB1 or CB2 of, for example, 1.9 pmol/mg membrane protein and 0.18 nM for CB1 and 5.2 pmol/mg membrane protein and 0.18 nM for CB2 were transfected.

(6) In an exemplary experiment the protein concentration of the CB1 receptor-carrying membrane was 8.0 mg/mL and the one of the CB2 receptor-carrying membrane was 4.0 mg/mL. These and further values resulted from the specifications of the manufacturer of the membranes and can be easily followed by a knowledgeable person skilled in the art just as well as the techniques with which the studies were carried out. The membrane suspensions were diluted in a dilution of 1:20 with buffer solution (50 nM TrisCl, 5 nM MgCl.sub.2H.sub.2O, 2.5 nM EDTA, 0.5 mg/mL BSA and pH 7.4 for CB1 binding buffer; 50 nM TrisCl, 5 nM MgCl.sub.2H.sub.2O, 2.5 nM EGTA, 1 mg/mL BSA and pH 7.5 for CB2 binding buffer). [.sup.3H]-CP55940 (144 Ci/mmol) was used as radioligand. In this context, exemplary concentrations were 0.10 nM with a volume of 200 L for CB1 binding studies and 0.15 nM with a final volume of 600 L for CB2 binding studies. The membranes were resuspended in the buffer, incubated with the radioligand and with each substance for 90 min at 30 C. Nonspecific binding was determined with the aid of the classic ligand WIN55,212-2 and 100% binding of the radioligand was determined by incubating the membrane without any other substance. After filtration of the respective preparation, it was washed nine times with the respective binding buffer and subsequently dried. The radioactivity was determined with a suitable scintillation counter. Corresponding models are already known from the literature (Granja, A. G. et al. J. Neuroimmune Pharmacol. 2012, 7, 1002-1016; Cumella, J. et al. Chem Med Chem. 2012, 7, 452-463; Di Marzo, V. et al. 2000, J. Neurochem., 2000, 74, 1627-1635).

(7) An assessment of the components was carried out in two phases. The first phase consisted of a screening with a single high dose of each substance regarding their binding abilities. The following table shows the percentage values for the binding to CB1 and CB2:

(8) TABLE-US-00002 TABLE 1 Percentage binding of selected cannabinoids at cannabinoid receptors Substance CB1 (% binding) CB2 (% binding) III 77.3 6.5 90.3 2.4 IV 90.2 3.8 101.6 0.5 V 82.0 8.0 83.9 5.6 XI 92.2 1.7 97.2 3.4 XII 81.5 6.8 99.2 2.3 XIII 98.9 7.8 104.9 4.8

(9) Note: The measured values 101.60.5 and 104.94.8 are based on a usual, scientifically accepted measuring inaccuracy of the used model.

(10) Substances that display over 50% binding and therefore display displacement of [.sup.3H]-CP55940, were tested in a second phase for their competition for CB1 and CB2 by incubating different concentrations of the substances together with [.sup.3H]-CP55940 inside the receptor model. The resulting data was evaluated with the aid of a suitable statistics software (e.g. GraphPrism Version 5.01). The following table shows the dissociation constants (K.sub.i) for the substances as mean value+/standard error (SEM):

(11) TABLE-US-00003 TABLE 2 Dissociation constants of the cannabinoid compounds III-V and XI-XIII K.sub.i (CB1): K.sub.i (CB2) K.sub.i for CB1 K.sub.i for CB2 Selectivity for CB2 in Substance (nM) (nM) comparison to CB1 (rounded) III 3,923 1,547 374.5 47.7 10.4 IV 2,174 1,149 277.1 78.7 7.8 V 538.2 53.9 66.7 13.1 8.1 XI 538.2 53.9 510 29 1 XII 538.2 53.9 67 4 37 XIII 538.2 53.9 0.012 0.001 22.5

(12) In comparison, substance WIN55,212-2, which was used as classical nonspecific ligand as positive control for such an experiment, showed a dissociation constant of 28.8+/1 nM for CB1 and 3.7+/1 nM for CB2 and therefore corresponds to the literature values (e.g. Pertwee, R. G. et al. Pharmacol. Rev. 2010, 62, 588-631).

(13) Conclusion/Comparative Evaluation:

(14) The cannabinoid substances III-V and XI-XIII bind to cannabinoid receptors in nM concentrations and therefore at physiological doses. They are weak ligands for CB1 receptors and bind preferentially to CB2 receptors. Their selectivity for CB2 receptors particularly predestines them for the use as CB2 receptor modulators (as described above).

(15) Cannabinoids known in the literature and substances that do not number among the classical cannabinoids, are divided into groups on the basis of their affinity for CB1 and CB2 receptors (Pertwee, R. G. et al. Pharmacol. Rev. 2010, 62, 588-631). The group assignment and hence the pharmacodynamic mechanism determines the mode of the effect of the substances.

(16) While CBD exerts a very weak effect with low affinities (CB1: 4,350 to >10,000 nM; CB2: 2,399 to >10,000 nM), delta-9-THC is a strong ligand for both receptors with CB1: 5.05 to 80.3 nM and CB2: 3.13 to 75.3 nM, which also explains its strong effects on the central nervous system (also side effects) and simultaneous peripheral effects (and side effects). The psychotropic effects of delta-9-THC are attributed to its complex interaction with the CB1 receptor. The activation of the CB1 receptor causes undesired effects on the psyche (and the circulation), on the contrary the activation of the CB2 receptor does not seem to do this, which also is because of the localization of the CB2 receptors in the periphery (Atwood, B. K. Prog. Neuropsychopharmacol. Biol. Psychiatry 2012, 38, 16-20).

(17) The cannabinoids described herein display an advantageous and unique distribution of their binding affinity (see table 2). Their binding affinity towards an attenuated, but not completely abrogated activation of the CB1 receptor predestines the substances as pharmaceuticals. The evidence of an advantageous effect of CB2 modulators in pathological situations that have not been accessible to pharmacotherapy to date, has grown strongly over the past couple of years. The two most important indications for CB2 modulators are neuroinflammation and pain (Cheng, Y., Hitchcock, S. A. Expert Opin. Invest. Drugs 2007, 16, 951-965; Guindon, J., Hohmann, A. G. J. Pharmacol. 2008, 153, 319-334). Furthermore, substances according to the invention can also influence the following pathological situations via CB2 modulation: Systemic inflammation, osteoporosis, cancer, transplantation-induced pathological conditions, different pathological conditions of the central nervous system including drug addiction and anxiety states as well as liver conditions (Bab, I. et al. Ann. Med. 2009, 41, 560-567; Karsak, M. et al. Science 2007, 316, 1494-1497; Mallat, A., Lotersztajn, S. Dig. Dis. 2010, 28, 261-266; Nagarkatti, M. et al. Trends Pharmacol. Sci. 2010, 31, 345-350; Patel, K. D. et al. Curr. Med. Chem. 2010, 17, 1393-1410; Xi, Z. X. et al. Nat. Neurosci. 2011, 14, 1160-1166).

(18) Signal Transduction at CB1 and CB2 Transfected CHO Cells:

(19) Dermuth et al. (2006) describe the signal transduction via cannabinoid receptors. The mode of signal transduction has already been explained sufficiently.

(20) After the binding affinity of the substances according to the invention designated above was established, their intrinsic activity was examined on the basis of a functional assay of cannabinoid receptor transfected cells. To this end, CHO cells (immortalized Chinese Hamster Ovary cells) were transfected with CB1 and CB2 receptors via the transfer of cDNA. The hence obtained transfected cells (CHO-CB1 and CHO-CB2) were transiently transfected with plasmid CRE-luc, which contains several (e.g. 6) consensus cCAMP responsive elements (CRE) and firefly luciferase (luc). The techniques necessary for this purpose are accessible to the knowledgeable person skilled in the art via the relevant technical literature.

(21) In order to investigate the agonistic activity, the transfected cells (CHO-CB1-CREluc and CHO-CB2-CREluc) were treated either with increasing concentrations of the molecules according to the invention or with WIN55,212-2 (WIN), which is a classical nonspecific agonist for CB1 as positive control, incubated and subsequently tested for their activity through the addition of luciferin (a chemoluminescent substrate of firefly luciferase). Forskolin, an adenylate cyclase activator, was used as positive control, since its activation of the cAMP pathway takes place independently from the cannabinoid receptors. In order to investigate a possible antagonism at the CB1 receptors, the CHO-CB1-CREluc cells were pre-incubated with the test substances and then stimulated with WIN. In order to investigate the agonism at the CB2 receptors, CHO-CB2-CREluc cells were treated with either increasing concentrations of substances according to the invention or with WIN, also a classical nonspecific agonist for CB2 as positive control, for a short period of time (15 min). Then forskolin was added and the preparation was incubated. In order to confirm the agonistic effect at CB2 receptors, CHO-CB2-CREluc were furthermore incubated with the specific antagonist AM630 (Ross et al., 1999). After an adequate incubation time and subsequent lysis, the luciferase activity was measured. The background activity (buffer) was subtracted from the result, respectively. FIG. 1 (Analysis scheme of the signal transduction at CB1 and CB2 transfected CHO cells) depicts a possible analysis scheme for illustration of the activity of substances according to the invention.

(22) Substances according to the invention possess a particularly advantageous ratio of activation of CB1 receptors to CB2 receptors. Preferably, CB2 receptors are activated by substances according to the invention, whereas CB1 receptors are only activated to a negligible extent or not at all or are even inhibited.

(23) Glyceryl cannabidiolate shows a weak activation of CB1 receptors and a strong activation of CB2 receptors. 2-Hydroxyethyl cannabidiolate and 2-hydroxypentyl cannabidiolate show a strong activation of the CB2 receptor and inhibit the CB1 receptor. Hexyl cannabidiolate shows antagonism at both receptors, while cyclohexyl cannabidiolate has an antagonistic effect on the CB1 receptor. N-Methyl-sulfonyl cannabidiolate shows, besides a high binding affinity for CB1 and CB2, an antagonistic effect on CB1 and an agonistic effect on CB2.

(24) TABLE-US-00004 TABLE 3 Agonism and antagonism of cannabinoids according to the invention at cannabinoid receptors Substance CB1 CB2 2-Hxdroxyethyl cannabidiolate + (compound III) Glyceryl cannabidiolate + + (compound V) 2-Hydroxypentyl cannabidolate + (compound IV) Hexyl cannabidiolate + + (compound XI) Cyclohexyl cannabidiolate 0 (compound XII) N-Methyl-sulfonyl cannabidiolate + (compound XIII) Legend: : Antagonism +: Agonism 0: no activity
Endogenous Signal Transduction in Jurkat Cells:

(25) CB2 receptor agonists are particularly suitable for triggering immunomodulating effects. There is evidence for the inhibition of T cell activation by CB2 agonists. Particularly in Jurkat T cells CB2 agonists inhibit their activation according to Brner et al. (2009). Transferred to the physiological situation such functionality can be beneficial to the prophylaxis and therapy of immune diseases, e.g. autoimmune diseases such as multiple sclerosis.

(26) Substances according to the invention were investigated in an accepted Jurkat T cell model. The underlying mechanism is based on the fact that the transcriptional activity of lymphokines, such as e.g. the one of IL-2, is based on the coordinated activation of different transcription factors, such as e.g. NFAT and NF-B. The effect of the substances according to the invention on said factors was evaluated in-vitro with the aid of a luciferase-coupled construct (KBF-luc). Thereby, an activation of transiently transfected cells (Jurkat T cells) through PMA (plus ionomycin in case of NFAT activation), driven by a NF-B or NFAT dependent promoter, leads to a strong induction of luciferase gene expression. The inhibition of the luciferase activity was measured as a function of the dose rate of the substance according to the invention. A knowledgeable person skilled in the art can easily follow such procedure from the literature (e.g. in Yuan et al. (2002), Sancho et al. (2003), Do et al. (2004) and Cencioni et al. (2010)).

(27) A characteristic of substances according to the invention may be the inhibition of the NF-B or NFAT dependent activation of the T cells. Thus, for substance N-methyl-sulfonyl cannabidiolate according to the invention a strong inhibition of the activation of T cells via NF-B and NFAT is detectable. The substances 2-hydroxyethyl cannabidiolate, glyceryl cannabidiolate and 2-hydroxypentyl cannabidiolate according to the invention show an inhibition of the NFAT dependent activation of T cells.

B. SYNTHESIS OF DELTA-9-THC VIA 2-HYDROXYETHYL CANNABIDIOLATE (III)

(28) Step 1: Coupling Step (in the Continuous Process); Synthesis of Cannabidiolic Acid Methyl Ester (I)

(29) 300 g (2.0 mole) menthadienol and 476 g (2.0 mole) olivetolic acid ester are dissolved at ca. 22 C. in 1,370 g of chlorobenzene (2,000 mL solution A), likewise 94 g (0.66 mole) boron trifluoride*etherate are dissolved in 640 g of chlorobenzene at ca. 22 C. (666 mL solution B)., Solution A at a flow rate of 72 mL/min and solution B at a flow rate of 24 mL/min are pumped into a stirred reaction chamber via two separate dosing pumps, from the reaction chamber the reaction composition runs via a PTFE hose into a stirred solution of 1,000 g of sodium bicarbonate. The total reaction time is ca. 20 min. After termination of the metering the hydrolyzed reaction solution is stirred for a further 30 min.

(30) Then the hydrolyzed reaction solution is transferred into a 5 L jacket reaction vessel, the aqueous phase is separated and the solvent chlorobenzene is removed in vacuo. Ca. 2,000 g of toluene are added to the remaining 730 g of raw material and the unreacted olivetolic acid ester is extracted through the addition of 1,200 g 1% aqueous sodium hydroxide solution (four times). After acidifying with semi conc. sulfuric acid and re-extraction of this aqueous phase, ca. 30% (140 g) of non converted olivetolic acid ester are recovered.

(31) There are ca. 520 g of cannabidiolic acid methyl ester (I) in the toluene phase, which corresponds to a theoretical yield of ca. 70%. This first intermediate serves as starting material for the following transesterification.

(32) Step 2: Transesterification, Synthesis of 2-Hydroxyethyl Cannabidiolate (III):

(33) The toluene is removed by destillation and to the remaining first intermediate 600 g of ethylene glycol are added under stirring followed by a solution of 85 g of potassium hydroxide in 300 g ethylene glycol. A vacuum of ca. 0.5 bar is applied and it is heated to 120 C. for 2 h, whereby ca. 40 g of methanol distill off. The resulting product composition mainly comprises 2-hydroxyethyl cannabidiolate (III).

(34) Step 3: Saponification/Decarboxylation, Synthesis of Cannabidiol (X):

(35) Subsequently, the temperature is increased to 150 C. and it is stirred at this temperature for 2 h. The product composition resulting from the transesterification comprising mainly 2-hydroxyethyl cannabidiolate (III) is cooled down to ca. 40 C. and 500 g of water as well as 500 g of n-heptane are added and ca. 150 g of semi conc. sulfuric acid are added for neutralization. After phase separation, the solvent is removed using a rotary evaporator and the remainder is distilled over a thin-film evaporator using a vacuum of ca. 0.5 mbar and a jacket temperature of 230 C. 310 g of cannabidiol (X) are obtained in the form of a viscous, yellowish oil with a purity of 85%, which corresponds to a theoretical yield of 60% in relation to the used cannabidiolic acid ester.

(36) This viscous, yellowish oil is then recrystallized in ca. 200 g of n-heptane at ca. 5 C., after which 210 g of white crystallizate with a purity of 99% cannabidiol (X) are obtained.

(37) Step 4: Cyclization, Synthesis of Delta-9-THC:

(38) 50 g of pure cannabidiol are dissolved in 250 g methyl-tert-butylether and 40 g of boron trifluoride*acetic acid complex are added under stirring within 10 min at ca. 22 C. It is stirred for 3 h at said temperature and then 200 g of ice water are added, the organic phase is washed with sodium bicarbonate solution and the solvent is removed using a rotary evaporator. The remaining raw material of ca. 50 g contains 74% -9-tetrahydrocannabinol (delta-9-THC), 25% of side products as well as <1% cannabidiol. After purification by column chromatography, 30 g of pure delta-9-THC are obtained, which corresponds to a theoretical yield of 60%.

(39) The steps of the synthesis of delta-9-THC via 2-hydroxyethyl cannabidiolate (III) are depicted schematically below:

(40) Step 1: Coupling Step (in the Continuous Process); Synthesis of Cannabidiolic Acid Methyl Ester (I)

(41) ##STR00021##
Step 2: Transesterification, Synthesis of 2-Hydroxyethyl Cannabidiolate (III):

(42) ##STR00022##
Step 3: Saponification/Decarboxylation, Synthesis of Cannabidiol (X):

(43) ##STR00023##
Step 4: Cyclization, Synthesis of Delta-9-THC:

(44) ##STR00024##

C. APPLICATION EXAMPLES

(45) The use of compounds of formula (A) according to the invention is explained in greater detail by means of the following examples of preferred pharmaceutical formulations according to the invention. The use of compound (V) is preferred in this respect.

Application Example 1Capsules According to the Neuen Rezeptur Formularium, 18th Addition, 2001

(46) Preparation for 1 Capsule

(47) TABLE-US-00005 2.5 mg 5 mg 10 mg Compound of formula (A) 0.0025 g 0.005 g 0.010 g Hydrogenated fat (slip point: to 0.430 g to 0.430 g to 0.430 g 37-40 C.; OH-number: 7-17; sap-number: 245-260) Two-piece hard gelatin capsule 1 piece 1 piece 1 piece shell, size 1
Preparation for 30 Capsules Including 10% Excess of the Melt

(48) TABLE-US-00006 2.5 mg 5 mg 10 mg Compound of formula (A) 0.083 g 0.165 g 0.33 g Hydrogenated fat (slip point: to 14.2 g to 14.2 g to 14.2 g 37-40 C.; OH-number: 7-17; sap-number: 245-260) Two-piece hard gelatin capsule 30 pieces 30 pieces 30 pieces shell, size 1
Preparation for 60 Capsules Including 5% Excess of the Melt

(49) TABLE-US-00007 2.5 mg 5 mg 10 mg Compound of formula (A) 0.158 g 0.315 g 0.63 g Hydrogenated fat (slip point: to 27.1 g to 27.1 g to 27.1 g 37-40 C.; OH-number: 7-17; sap-number: 245-260) Two-piece hard gelatin capsule 60 pieces 60 pieces 60 pieces shell, size 1
1. In a horizontally adjusted capsule filling machine the inserted two-piece hard gelatin capsule shells are opened, the fixed lower parts of the capsules are exposed and made available for filling.
2. A little bit more hydrogenated fat than required for the preparation is melted in a beaker. In-process testing: The hydrogenated fat melting must be clear at visual inspection. It may be of a faintly yellow color.
3. In a second beaker molten hydrogenated fat is added to the compound of formula (A) according to the preparation amount specified above. The substance is dissolved under stirring with a glass rod. In-process testing: The fat melting must be clear at visual inspection. It may be of a faintly yellow color.
4. The fat melting is left inside the still warm, but no longer boiling water bath until the last capsule is filled, or it is removed from the water bath and reheated as needed. In-process testing (to be repeated from time to time): The temperature of the melting has to be between 35 and 45 C.
5. Ca. 1 mL of the fat melting is drawn up into a 1 mL disposable syringe preferably via a wide-lumen hollow needle (see under Pharmazeutische ErluterungenHerstellungstechnik und Abfllung). Two capsule lower parts are filled immediately. In-process testing: The upper rim of the lower part of the capsule has to be fully coated with fat melting from the inside. The surface of the liquid has to be planar or slightly concave.
6. The syringe is refilled and the filling of further capsules is continued until all of the capsules are filled. The empty space, which is generated in the capsule through the cooling of the melting may not be refilled. In-process testing: Only a small residue of fat melting of about 1 mL is supposed to remain in the beaker.
7. After solidification of the fat melting in the lower parts of the capsules, the capsules are closed tightly.

(50) In-process testing: The surface of the fat melting has to have the same opaque appearance in all of the capsule lower parts.

(51) End-product testing: The closed capsules have to have a uniform appearance. Only as needed: The single mass of all capsules has to be between 460 and 540 mg each.

Application Example 2Oily Solutions According to the Neuen Rezeptur Formularium, 19th Addition, 2002

(52) Ingredients

(53) TABLE-US-00008 20 g 100 parts by weight Compound of formula (A) 0.5 g 2.5 parts (see ,,Bezugsquellennachweis fr Rezepturbestandteile, chapter III.2.) Medium-chain triglycerides to 20.0 g to 100.0 parts
1. The compound of formula (A) is liquefied inside the storage vessel through gentle heating.
2. The compound of formula (A) is weighed into a beaker and dissolved in the medium-chain triglycerides under heating and stirring.

(54) End-product testing: The solution must be clear at visual inspection. It may be of a faintly yellow color.