Biological Activity of Cannabichromene, Its Homologs and Isomers

J C/in Pharmacol. 1981; 21:283S-291 S.

Biological Activity of Cannabichromene, its Homologs and Isomers

CARLTON E. TURNER, Ph.D., and MAHMOUD A. ELSOHLY, Ph.D. University, Miss.

Abstract: Cannabichromene (CBC) is one of four major cannabinoids in Csnnebb sativa L. and isthe second most abundant eannabinoid in drug-type cannabis.Cartna- bichromene and some of its homologs, analogs, and isomers were evaluated for- antiinflammatory, antibacterial, and antifungal activity. Antiinflammatory activity was evaluated by the carrageenan-induced rat paw edema andthe eqfhrocyte membrane- stabilization method. In both tests, CBC was superior to phenylbutazone. Antibacte- rial activity of CBC and its isomers and homologs was evaluated using gram-positive, gram-negative, and acid-fast-bacteria. Antifungal activity was evaluated using yeast- like and filamentous fungi and a dermatophyte. Antibacterial activity was strong, and the antifungal activity was mild to moderate.

RUDE drugs from the cannabis plant was carried out on mixtures of these com- C have been used by man for over 5000 pounds in the ratios in which they occur in years. Some of these crude drugs have been the crude drugs. Also, the “most active” in- associated with varying degrees of medical gredient, THC, was considered to be the applications in many countries. However, only significant compound in understand- as new and better medicinal agents were ing the pharmacologic action of mari- discovered, these crude drugs lost favor in huana. Actually, marihuana contains formal medical clinics. Interest in these many compounds that are active in biologic crude drugs has recently been renewed. The systems,’ U.S. Federal Government, since 1968, has Cannabis-is-a comprtix lplant which con- sponsored considerable research on can- tai’ns 421 known compoufids Fran 18 differ--~ nabis (mostly marihuana and hashish) and ent chemical classes. Sixty-one cam-&in-~ synthetic cannabinoids, notably (-Mans- oids and their hotnblogs, as defined by A’-tetrahydrocannabinol (THC), (-)-trans- Mechoula-m,2~~a-re known to occur in the A’-tetrahydrocannabinol (ad-THC), can- plarit.a These 61- cannabinoids can- be nabinol (CBN), and cannabidiol (CBD). Due further classified into at least 15 different to the problems associated with quantitat- typesA’en of these cannabinoids can be ing the quality of any crude drug, particu- quantitated routinely using -~methoda re- larly marihuana in the smoke form, much ported-Ii the literature.’ The chemical com- of the early laboratory work was done with position of crude drugs from cannabis is synthetic cannabinoids. Early researchers, much better understood today, and it is ob- using pure synthetic cannabinoids, found vioue that-the THC content of mari~huana~ leads to support some of the anecdotal re- cannot-be used to adequately describe the Ports about the crude drug marihuana. Al- pharmacologic activity of the drug. In an though these laboratory findings were attempt tom integrate ~analytic and biologic Primarily for the four previously mentioned data on the major, naturally occurring synthetic cannabinoids, very little work cannabinoids, it was discovered that cannabidiol~ was totally absent in many can- From the me search Institute of Pharmaceutical Scien- ce& The University of Mississippi, School of Pharmacy, nabis samples and present fin various University, Miss. 38677. amounts in othe~a.~ Cannambichromene was 283s

1 TURNER AND ELSOHLY I3 A CAFY-80 CAFY-80 6% ov lilB9' 2% OV r7;210°

AS-TN

I.% CBN CzqHC :BCI/CBC

A’-THC

A .RGM cao CBN CBC CBDV i

40 35 30 25 20 15 10 5 0 30 25 20 15 10 5 0 Fig. 1. Gas-chromatographic analysis of the cannabinoids in a chloroform extract of Afghanistan cannabie using 2% OV-17 (A) and 6% OV-1 (B) columns.,ii__I For details regarding, the, chromatographic, , conditions,, see, Refs. 4, and 5. identified 8s thre cannabinoid under the amounti in certain types; for example, chromatogrampeak~labeledcannabidiolin Afghanistani and Pakistani. In other sam- drug-type cannabis, for example, Mexican, ples, cannabidiol was th-e-major cannabin- Costa Rican, Brazilian, Jamaican, land oid under the peak, with a t%=ace of canna- South African. Cannabidiolm and cannabichromene; for example, Iranian, Leba- bichromene were present in nearly equal nese, and Turkish. See Fig. 1 for gas-

0 OY 0 %0 55, A'-tHt (i) . CBN (II)

CBD (III) Fig. 2. Structures of the major cannabirwids found in Cannabie sativa L. 284s BIOLOGICAL ACTIVITY OF CANNABICHROMENE chromatographic separation af sanna- All compounds were purified by column bidiol and cannabichromene on two dif- chromatography over silica gel and were ferent columns using Afghanistan can- characterized by spectral methods includ- nabis extracts. ing MS, ‘H-NMR, 13C-NMR, IR and UV, Cannabichromene (CBC), along with THC, carrnabidiol, and cannabinol (Fig. 2), Determination of Acute LDSO are thus the moat abundant naturally oc- of Cannabichromene curring cannabifioids. In freshly harvested drug-type cannabis- material, THC and ICR mice (Laboratory Supply Company, cannabichromene are the major sannabin- Inc., Indianapolis, Ind.) weighing 30 to 35 aids. Cannabinol is a product of the decom- Gm were used to determine the LIMO of can- position of THC.” nabichromene using two routes of admin- In view of these findings regarding the istration-subcutaneous and oral. Drug- abundance of cannabichromene in drug- dosed mice were observed for seven days. type cannabis -and after developing a syn- Cannabichromene was emulsified with theticlethod to produce large quantities of gum acacia (5%) and olive oil (10%) in water. this naturally occurring cannabinoid,7 an A vehicle made in the same manner showed extensive biolo@c study on cannabichro- no lethality over a two-week period. mene was initiated in- our laboratoties. In this connection, we have reported data in- Antiinflammatory Screening dicating~ that cannabichromene, in mon- keys, potentiates the mind-altering proper- Two methods were used to assess the anti- ties of THCs In this report, data on inflammatory activity ofcannabichromene: cannabichromen-e and its homologs and iso- the carrageenan-induced rat paw edema”’ mers are confined to the following three and erythrocyte membrane stabilization areas: (1) antiinflammatory, (2) antibacte- assay.ll Groups of eight Sprague-Hawley rial, and (3) antifungal. rats (Laboratory Supply Company, Inc., Indianapohs, Ind.) were used for the carrageenan-induced rat paw edema assay. , Methods Rats of both sexes were used in performing Preparation of Cannabichromsne (IV) initial assays, and results for both sexes and Its Horndogs and were identical. Thus, male rats were used Isomers (V-VIII) for the remainder of the study. Intraperito- Cannabichromene (IV), cannabichro- neal and oral routes were used. All animals mene-C1 (VI), and cannabichromene-Co received a 750 mgikg intraperitoneal injec- (VII) were prepared by condensation of cit- tion of ethylurethane 1 hour before testing ral with olivetol, orcinol, and resorcinol, re- to render them tractable. Negative control spectively, following our previously pub- groups received the vehicle, while positive lished method for synthesis of cannabi- control groups received a dose of phenyl- chromene.7 The yield for VI was 48.3 per butazone (PBZ) as shown in Table II. The cent while that of VII was only 10.8 per test compounds were prepared for dosing cent. by emulsification in 3% arlacel and 2% Isocannabichromene-Co (VIII) was sep- Tween-60 in water. arated from the same reaction mixture of The published procedure was followed for VII as a side product However isocanna- the erythrocyte membrane stabilization bichromene (V) was prepared by condens- method.” All compounds were prepared by ing olivitol and citral in pyridine at 110” for dissolving in physiologic saline or as a fine 7 hours,’ where both IV and V were formed suspension in 2% ethanol in saline and the along with other side products. pH adjusted to 7.4. The concentrations 2855 TURNER AND ELSlllHL Y TABLE I Antimicrobial Screening Using the Agar Well Diffusion Assay

Organism ATCC# Classiiication

Bacillus subtilis 6633 gram-positive bacterium Staphylococcus aureus 6538 gram-positive bacterium Escherichia coli 10536 gram-negative bacterium Pseudomonas aeruginosa 15442 gram-negative bacterium Mycobacterium smegmatis 607 acid-fast bacterium Candida albicans 10231 yeast-like fungus Saccharomyces cerevisiae 9763 yeast-like fungus Aspergillus niger 16888 filamentous fungus Trichophyton mentagrophytes 9972 dsrmatophyte tested are given in Table III. Apirin (ASA) Both qualitative and quantitative screens and phenylbutazone were used as positive were performed. Qualitative screening was controls for comparison of activity. accomplished using the agar well diffusion assay, and quantitative screening was accomplished by the two-fold (broth) serial A ntinicrobial Screening dilution method tv determine the minimum (antibacterial and antifungul) inhibitory concentration (M1Cj. Innocula- All compounds were screened for activity tion was accomplished with one loop-full of against the organisms listed in Table I. a 1:lO dilution of the 24-hour-old broth cul-

TABLE II Inhibition of Carrageenan-Induced Rat Paw Edema by Cannabichromene and its G-Homolog

Dose Route of Inhibition Compound (m&W administration (W)

Cannabichromene (IV) 120 intraperitoneal injection 70* 240 intraperitoneal injection 86* 480 intraperitoneal injection 100

Cannabichromene-Cl (V) 60 intraperitoneal injection 53* 120 intraperitoneal injection 63* 240 intraperitoneal injection 92*

Phenylbutazone 60 intraperitoneai injection 52* 120 intraperitoneal injection 76

Cannabichromene (IV) 120 oral, fasted rats 35 (N.S.) 240 oral, fasted rats 67* 480 oral, fasted rats 55**

Phenylbutazone 120 oral, fasted rats 36 (N.S.)

Cannabichromeae (IV) 120 oral, nonfasted rats 21 (N.S.) 240 oral, nonfasted rats 34** 480 oral, nonfasted rats 50*

Phenylbutazone 60 oral, nonfasted rats 37* 120 oral, nonfasted rats 22f

* P = 0.01. ** P = 0.05 286s BIOLOGICAL ACTIVITY OF CANNABICHROMElVE

TABLE III Inhibition of Heat-Induced Erythrocyte Hemolysis by Cannabichromene and its Homologs and Isomers

Concentration Inhibition Compound of test solution (MJ @)

Cannabichromene (IV) 1 x lo-’ 98 5x 10” 79 2.5 X lo5 67 1.25 X lo- 55

Isocannabichromene (VII) 1 x lo-” 75

Cannabichromene-C, (V) 1 x 10’ 86

Cannabichromene-C, (VI) 1 x 10’ 69

Isocannabichromene-C, (VIII) 1 x 10” 43

Phenylbutazone 1 x 10 16 2x 10” 10

Aspirin 2.5 x lo-’ 21

ture* of the test organisms in sterile water. chromene for possible therapeutic value. Streptomycin sulfate was used as a positive This report deal6 with the antiinflamma- control for antibacterial activity, while tory and antimicrobial (both antibacterial amphotericin B was used as an antifungal and antifungal) effects of cannabichro- positive control. mene. In addition, we investigated the same properties for some of its homologs Results and Discussion and isomers whose structures are shown in Although cannabichromene is now Fig. 3. The acute L&O of cannabichromene known to be one of the major cannabinoids , was determined in mice using two routes of in cannabis, little work has been done to administration, There were no d-t&a-re investigate its biologic activity. We devel- suiting from- oral admini&r&ion at doses oped a synthetic procedure to prepare can- up to 3009 nrg/kg,-while the-same-dose-level nabichromene in large quantities’ in order given su~cu~ne~usly~resulted in 10 to 20 to study the pharmacology of cannabi- per cent mortality. * Fungai cultures and M. smpgmatis often required 48 to The antiinflamm-atory a&iv&y of canna---- 72 hours of incubation. Eugon broth (50 ml) for bacteria, and Mycophil broth (50 ml) for fungi and yeasts were bichromene lIJ?1 and its G-homolog (VI) z used. was determined by both the carrageenan-

on I

I I I I 0 0 I m0 0 @# CBC, R = C5Hl, (IV) i5.0-CBc,aR = C5Hll (VII) CBC-Cl, R = CH3 (V) iso-CBC-C,, R = H (VIII) CBC-Co, R = t! (VI) Fig. 3. Cannabichromene (CBCI and its homologs and isomers. 287s Oral, non 500 I. P, faste: rats

400

m < F _ 300 Fz in x 200

100

50 75 1Oc) 125 Inhibition, Y Fig. 4. Dose-response curve for the inhibition of the carrageenan-induced rut paw edema using cannabichromene by both intraperitoneal and oral administration.

induced rat paw edema assay and the ery- the heat-induced hemolysis of erythrocytes. throcyte~ membrane stabilization method. Other homologs and isomers (VI-VIII) Table II shows the activity of both com- were tested using the same assay. Table III pounds at different dose levels given in- gives the per cent inhibition produced by traperitoneally and also the activity of solutions of the different compounds as cannabishromene given orally to both compared to PBZ and aspirin (ASA). All fasted and nonfasted rats. In each test, homologs and isomers were tested at 1 X comparison was made with phenylbuta- 10-“M dilution; however, cannabichro- zone (PBZ). It is clear ~that cannabichromene was tested at different concentra- mene was more active b-y the mtraperito- tions. The dose-response curve for the in- neal route than ar&lly and that the activity hibition of various concentrations of CBC was comparable to that -of its G-homolog is shown in Fig. 5. Results ntilizingmthie (V) and to PBZ (Table II). Cannabichro- assay-indicated that all compounds tested mene~was more active tit 240 or 480 mg/kg werem~signifidntly mar%? active than PBZ than wa3 PBZ~%tXN mg/kg, PBZ could~not and-aspirin. The activity of IV and V indi- be tested at higher doses because of its tox- cated by this screen was parallel to their ic&y. The dose-response curve for the an- activity in the rat paw edema assay. tiinflammatory activity of cannabichro- It sh-ould -be concluded that cannabi- mene when given intraperitoneally and chromene and its homologs and isomers do orally is given in Fig. 4. possess antiinfl2hrimatory activity as mea- In addition to the previous aesay, the an- sured by these two screens. The activity of tiinflamma~tory activity of compounds IV cannabichromene through the oral route, and V was confirmed by their inhibition of its safety and its lack of behavioral-type 288s BIOLOGICAL ACTIVITY OF CANNABICHROMENE

50 75 100 125 Inhibition, E

Fig. 5. Dose-response curve for the inhibition of heat-induced erythrocyte hemolysis using different concentrations of cannabichromene.

(psychotomimetic) activity characteristic tory concentrations (MC) for these com- of THQI) indicate its therapeutic potential pounds were determined using selected bac- for the treatment of inflammatory dkeasees. teria and fungi as recorded in Tables IV Further testing is under way to ascertain and V, respectively. The organisms se- the nature of this potential. lected for the NC determination were Compounds IV-VIII were subjected to based on the largest zone of inhibition re- antibacterial and antifungal activity sulting in the qualitative screen. It is evi- screens in addition to the antiinflamma- dent from TabkWthat cannahichromene tory activity screens. Organisms utilized in (IV) and cannabichromene-C, (VI), their the screens included gram-positive, gram- isoderivatives (VII and VIII}, and cannabi- negative, and acid-fast bacteria as well as chromeno (V) were more active against different types of fungi (see Table I). B. subtilis than streptomycin sulfate. Both The quahtative screen, the agar well dif- IVand V were more active against S. UU- fusion assay, showed that these com- rends than the reference compound. On the pounds possess strong antibacterial and other hand, the-only homolog with com- mild antifungal properties. These comparable activity to streptomycin against M. pounds exhibited large zones of inhibition sm~gntatis was CannabichromeneG. Al- when compared to positive standards at the though the other homologs and isomers same concentrations. The minimum inhibi- were moderately active, they showed 289s TURNER AND ELSOHL Y

TABLE IV Minimum Inhibitiry Concentration (MIC) of Cannabichrrzmene and Its Homologs and Isomers Against Different Organisms

MZC (pg/ml) B. subtilis S. aweus M. smegmatis Compound 24 hr 48 hr 24 hr 48 hr 24 hr 48 hr

Cannabichromene (IV) 0.39 0.78 1.56 1.56 12.3 25.0 Ieocannabichmmene (VII) 0.78 3.12 NT NT 25.0 25.0 Cannabichromene-C, (VI) 6.25 12.5 12.5 12.5 12.5 12.5 Streptomycin SOI* 6.25 25 3.12 12.3 1.56 1.56 Cannabichromene-Cl (VJ 3.12 3.12 3.12 3.12 3.12 6.25 Isocannabichromene-C. rv111\ 6.25 6.25 12.5 12.5 12.5 12.5 Streptomycin SOP* 3.12 6.25 6.25 6.25 6.25 6.25

* Streptomycin SO, was used as standard for teete performed on two different days. higher MfC values when compared to the compounds had mild to moderate activity standard. against all fungi tested. In the antifungal assay (Table V), canna- The combined antibacterial and antifun- bichromene-Ct and-- isocannabichromene- gal activities crf cannabichromene and its C, showed better activity against T. men- homslogs and isomers are quite encourag- tagroph ytes than amghstericin B. All other ing. Further testing is in-progress tu assess

TABLE V Minimum Inhibitory Concentration (&UC) of Cannabichromene and Its Homologs and Isomers Against Rifferent Fungi

C. albicans S. cerevisiee T. mentagrophytes Compound 48 hr 72 hr 48 hr 72 hr 48 hr 72 hr

Cannabichromene (IV) NT NT 25 50 25 50 Amphotericin B* NT NT 3.12 3.12 NT NT

Ieocannabichromene WI) 50 100 NT NT NT NT Cannabichromene-C, (VT) 50 50 25 25 25 25 Amphotericin B* 1.56 1.56 0.78 0.78 NT NT

Cannabichromene-Cl (v) NT NT 6.25 12.5 6.25 6.25 Isocannabichmmene-C. (VIII) 12.5 25 NT NT 6.25 6.25 Amphotericin B* 1.56 6.25 0.19 0.78 12.5 25

* Amphotericin B was used as standard for t.HtB performed on three different days.

290s