Cannabis Research A-Z

Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universit del Piemonte Orientale, Via Bovio 6, 28100 Novara, Italy, Consorzio per lo Studio dei Metaboliti Secondari (CSMS), Viale S. Ignazio 13, 09123 Cagliari, Italy, Centre for Pharmacognosy and Phytotherapy,

 

The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, U.K., and CRA-CIN Centro di Ricerca per le Colture Industriali, Sede distaccata di Rovigo, Via Amendola 82, 45100 Rovigo, Italy

* To whom correspondence should be addressed. Tel: +39 0321 373744(G.A.); +44 207 753 5913(S.G.). Fax: +39 0321 375621 (G.A.); +44 207 753 5909 (S.G.). E-mail: appendino@pharm.unipmn.it (G.A.); simon.gibbons@pharmacy.ac.uk (S.G.)., †

Universit del Piemonte Orientale.

Consorzio per lo Studio dei Metaboliti Secondari.

University of London.

Centro Ricerca Colture Industriali.

Abstract

Marijuana (Cannabis sativa) has long been known to contain antibacterial cannabinoids, whose potential to address antibiotic resistance has not yet been investigated.

All five major cannabinoids (cannabidiol, cannabichromene, cannabigerol, Δ⁹-tetrahydrocannabinol, and cannabinol showed potent activity against a variety of methicillin-resistant Staphylococcus aureus (MRSA) strains of current clinical relevance.

Activity was remarkably tolerant to the nature of the prenyl moiety, to its relative position compared to the n-pentyl moiety (abnormal cannabinoids), and to carboxylation of the resorcinyl moiety (pre-cannabinoids).

Conversely, methylation and acetylation of the phenolic hydroxyls, esterification of the carboxylic group of pre-cannabinoids, and introduction of a second prenyl moiety were all detrimental for antibacterial activity.

Taken together, these observations suggest that the prenyl moiety of cannabinoids serves mainly as a modulator of lipid affinity for the olivetol core, a per se poorly active antibacterial pharmacophore, while their high potency definitely suggests a specific, but yet elusive, mechanism of activity.

Several studies have associated the abuse of marijuana (Cannabis sativa L. Cannabinaceae) with an increase in opportunistic infections, and inhalation of marijuana has indeed been shown to interfere with the production of nitric oxide from pulmonary macrophages, impairing the respiratory defense mechanisms against pathogens and causing immunosuppression.

 

The association of C. sativa with a decreased protection against bacterial infections is paradoxical, since this plant has long been known to contain powerful antibacterial agents. Thus, preparations from C. sativa were investigated extensively in the 1950s as highly active topical antiseptic agents for the oral cavity and the skin and as antitubercular agents.

 

Unfortunately, most of these investigations were done at a time when the phytochemistry of Cannabis was still in its infancy, and the remarkable antibacterial profile of the plant could not be related to any single, structurally defined and specific constituent. Evidence that pre-cannabidiol  is a powerful plant antibiotic was, nevertheless, obtained, and more recent investigations have demonstrated, to various degrees, antibacterial activity for the nonpsychotropic cannabinoids cannabichromene  cannabigerol CBG,  and cannabidiol as well as for the psychotropic agent Δ⁹-tetrahydrocannabinol (THC,).

 

These observations, and the inactivity of several noncannabinoid constituents of C. sativa as antibacterial agents, suggest that cannabinoids and their precursors are the most likely antibacterial agents present in C. sativa preparations.(8) However, differences in bacterial strains and end-points make it difficult to compare the data reported in these scattered studies, and the overall value of C. sativa as an antibacterial agent is therefore not easy to assess.

 

There are currently considerable challenges with the treatment of infections caused by strains of clinically relevant bacteria that show multidrug-resistance (MDR), such as methicillin-resistant Staphylococcus aureus (MRSA) and the recently emerged and extremely drug-resistant Mycobacterium tuberculosis XDR-TB.

 

New antibacterials are therefore urgently needed, but only one new class of antibacterial has been introduced in the last 30 years.

 

Despite the excellent antibacterial activity of many plant secondary metabolites and the ability of some of them to modify the resistance associated with MDR strains and efflux pumps, plants are still a substantially untapped source of antimicrobial agents.

 

These considerations, as well as the observation that cross-resistance to microbial and plant antibacterial agents is rare, make C. sativa a potential source of compounds to address antibiotic resistance, one of the most urgent issues in antimicrobial therapy.

 

To obtain structure−activity data and define a possible microbiocidal cannabinoid pharmacophore, we investigated the antibacterial profile of the five major cannabinoids, of their alkylation and acylation products, and of a selection of their carboxylic precursors (pre-cannabinoids) and synthetic positional isomers (abnormal cannabinoids).

 

• • Top of Page
  • Results and Discussion
  • Experimental Section
  • References
  
  

Results and Discussion

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The antibacterial cannabinoid chemotype is poorly defined, as is the molecular mechanism of its activity.

 

Since many simple phenols show antimicrobial properties, it does not seem unreasonable to assume that the resorcinol moiety of cannabinoids serves as the antibacterial pharmacophore, with the alkyl, terpenoid, and carboxylic appendices modulating its activity.

 

To gain insight into the microbiocidal cannabinoid pharmacophore, we have investigated how the nature of the terpenoid moiety, its relative position compared to the n-pentyl group, and the effect of carboxylation of the resorcinyl moiety are translated biologically, assaying the major cannabinoids and a selection of their precursors and regioisomeric analogues against drug-resistant bacteria of clinical relevance.

 

Within these, we have selected a panel of clinically relevant Staphylococcus aureus strains that includes the (in)famous EMRSA-15, one of the main epidemic methicillin-resistant strains, and SA-1199B, a multidrug-resistant strain that overexpresses the NorA efflux mechanism, the best characterized antibiotic efflux pump in this species. SA-1199B also possesses a gyrase mutation that, in addition to NorA, confers a high level of resistance to certain fluoroquinolones.

 

A macrolide-resistant strain (RN4220),(15) a tetracycline-resistant line overexpressing the TetK efflux pump (XU212), and a standard laboratory strain (ATCC25923) completed the bacterial panel.

 

Δ⁹-Tetrahydrocannabinol (THC), cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), and cannabinol (CBN) are the five most common cannabinoids.

 

They could be obtained in high purity (>98%) by isolation from strains of C. sativa producing a single major cannabinoid (THC, CBD, CBG), by total synthesis (CBC), or by semisynthesis (CBN). Their antimicrobial properties are listed in Table 1.

 

All compounds showed potent antibacterial activity, with MIC values in the 0.5−2 μg/mL range. Activity was exceptional against some of these strains, in particular the multidrug-resistant (MDR) SA-1199B, which has a high level of resistance to certain fluoroquinolones. Also noteworthy is the potent activity demonstrated against EMRSA-15 and EMRSA-16, the major epidemic methicillin-resistant S. aureus strains occurring in U.K. hospitals.

 

These activities compare highly favorably with the standard antibiotics for these strains. The potent activity against strains possessing the NorA and TetK efflux transporters suggests that cannabinoids are not substrates for the most common resistance mechanisms to current antibacterial agents, making them attractive antibacterial leads.

 

Table 1. MIC (μg/mL) Values of Cannabinoids and Their Analogues toward Various Drug-Resistant Strains of Staphylococcus aureus

 

compound	SA-1199B	RN-4220	XU212	ATCC25923	EMRSA-15	EMRSA-16
1a	2	2	2	2	2	2
1b	1	1	1	0.5	1	1
2	2	2	1	2	2	2
3a	4	2	4	4	2	4
3b	1	1	1	1	2	1
3f	64	c	64	c	c	c
4a	8	4	8	4	8	4
4b	2	1	1	1	2	0.5
5	1	1	1	1	1	c
6	1	1	1	1	1	1
7	2	1	0.5	1	2	c
8	32	32	16	16	16	32
10	64	64	64	128	64	64
norfloxacin	32	1	4	1	0.5	128
erythromycin	0.25	64	>128	0.25	>128	>128
tetracycline	0.25	0.25	128	0.25	0.125	0.125
oxacillin	0.25	0.25	128	0.125	32	>128

a

Compounds 1c−g, 3c−e, 3g, and 9 exhibited MIC values of >128 μg/mL for all organisms in which they were evaluated.

b

Compound 11 exhibited MIC values of >256 μg/mL for all organisms in which they were evaluated.

c

Not tested.

Given their nonpsychotropic profiles, CBD (1b) and CBG (3b) seemed especially promising, and were selected for further structure−activity studies. Thus, acetylation and methylation of their phenolic hydroxyls (compounds 1c−e and 3c−e, respectively) were both detrimental for activity (MIC >100 μg/mL), in accordance with the essential role of the phenolic hydroxyls in the antibacterial properties. However, in light of the potent activity of the monophenols CBC, THC (4b), and CBN, it was surprising that monomethylation of the diphenols CBD (1b) and CBG (3b) was so poorly tolerated in terms of antibacterial activity.

 

Cannabinoids are the products of thermal degradation of their corresponding carboxylic acids (pre-cannabinoids).

 

Investigation of the antibacterial profile of the carboxylated versions of CBD, CBG, and THC (compounds 1a, 3a, and 4a, respectively) showed a substantial maintenance of activity. On the other hand, methylation of the carboxylic group (compounds 1f and 3f, respectively) caused a marked decrease of potency, as did esterification with phenethyl alcohol (compounds 1g and 3g, respectively).

 

This operation is associated with a potentiation of the antibacterial properties of phenolic acids, as exemplified by phenethyl caffeate (CAPE), the major antibacterial from propolis, compared to caffeic acid.(20) Remarkably, the synthetic abnormal cannabinoids abn-CBD and abn-CBG (7)(22) showed antibacterial activity comparable to, although slightly less potent than, their corresponding natural products, while olivetol  showed modest activity against all six strains, with MICs of 64−128 μg/mL, and resorcinol did not exhibit any activity even at 256 μg/mL.

 

Thus, the pentyl chain and the monoterpene moiety greatly enhance the activity of resorcinol.

 

Pharmacists and chemists have found another use for the multipurpose cannabis as a source of antibacterial chemicals for multidrug resistant bacteria. Ironically, inhaling cannabis is known to damage the lung's ability to fend off invading pathogens, but the ingredients in cannabis, particularly the cannabinoids, have antiseptic properties. Although scattered research has been conducted since the 1950s, no comprehensive study existed that relates the structure of cannabinoids with antibacterial activity. Giovanni Appendino, Simon Gibbons, and coworkers attempted to remedy that problem by examining the activity of five common cannabinoids and their synthetic derivatives.

Five of the most common cannabinoids. 

All five cannabinoids (THC, CBD, CBG, CBC, and CBN) were potent against bacteria. Notably, they performed well against bacteria that were known to be multidrug resistant, like the strains of MRSA that plagued U.K. hospitals. CBD and CBG have the most potential for consumer use because they are nonpsychotropic.

Besides identifying antibacterial capability, the researchers wanted to figure out why these cannabinoids are so good at killing bacteria. They obviously are very effective at specifically targeting some vital process in the bacteria. Unfortunately, even after extensive work at modifying the cannabinoids and comparing their activities, that targeting mechanism remains a mystery. The scientists were able to figure out that the position of the n-pentyl chain (orange) relative to the terpenoid moiety (blue) serves to control lipid affinity.

These cannabinoids are promising enough to warrant rigorous clinical trials. They are applicable as topical antiseptics, biodegradable antibacterial compounds for cosmetics, and systematic antibacterial agents.

Sept. 4, 2008 -- Chemicals in marijuana may be useful in fighting MRSA, a kind of staph bacterium that is resistant to certain antibiotics.

Researchers in Italy and the U.K. tested five major marijuana chemicals called cannabinoids on different strains of MRSA (methicillin-resistant Staphylococcus aureus). All five showed germ-killing activity against the MRSA strains in lab tests. Some synthetic cannabinoids also showed germ-killing capability. The scientists note the cannabinoids kill bacteria in a different way than traditional antibiotics, meaning they might be able to bypass bacterial resistance.

At least two of the cannabinoids don't have mood-altering effects, so there could be a way to use these substances without creating the high of marijuana.

MRSA, like other staph infections, can be spread through casual physical contact or through contaminated objects. It is commonly spread from the hands of someone who has it. This could be in a health care setting, though there have also been high-profile cases of community-acquired MRSA.

It is becoming more common for healthy people to get MRSA, which is often spread between people who have close contact with one another, such as members of a sports team. Symptoms often include skin infections, such as boils. MRSA can become serious, particularly for people who are weak or ill.

In the study, published in the Journal of Natural Products, researchers call for further study of the antibacterial uses of marijuana. There are "currently considerable challenges with the treatment of infections caused by strains of clinically relevant bacteria that show multi-drug resistance," the researchers write. New antibacterials are urgently needed, but only one new class of antibacterial has been introduced in the last 30 years. "Plants are still a substantially untapped source of antimicrobial agents," the researchers conclude.

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Cannabis Science, Inc. (OTCBB: GFON). Dr. Robert Melamede, PhD., Director and Chief Science Officer, reported to the Board on the current state of research into the use of natural plant cannabinoids to reduce the spread of drug-resistant bacteria, including methicillin-resistant Staphyloccus aureus (MRSA), and the prospects for development of topical whole-cannabis treatments.

According to studies published in the Journal of the American Medical Association and by the Center for Disease Control in 2007, MRSA is responsible for more than 18,500 hospital-stay related deaths each year, and increased direct healthcare costs of as much as $9.7 billion.

Dr. Melamede stated, "Research into use of whole cannabis extracts and multi-cannabinoid compounds has provided the scientific rationale for medical marijuana's efficacy in treating some of the most troubling diseases mankind now faces, including infectious diseases such as the flu and HIV, autoimmune diseases such as ALS (Lou Gehrig's Disease), multiple sclerosis, arthritis, and diabetes, neurological conditions such as Alzheimer's, stroke and brain injury, as well as numerous forms of cancer. One common element of these diseases is that patients often suffer extended hospital stays, risking development of various Staphyloccus infections including MRSA. A topical, whole-cannabis treatment for these infections is a functional complement to our cannabis extract-based lozenge."

Investigators at Italy's Universita del Piemonte Orientale and Britain's University of London, School of Pharmacy reported in the Journal of Natural Products that five cannabinoids - THC, CBD, CBG, CBC, and CBN - "showed potent antibacterial activity" and "exceptional" antibacterial activity against two epidemic MRSA occurring in UK hospitals. The authors concluded: "Although the use of cannabinoids as systemic antibacterial agents awaits rigorous clinical trials, … their topical application to reduce skin colonization by MRSA seems promising. … Cannabis sativa … represents an interesting source of antibacterial agents to address the problem of multidrug resistance in MRSA and other pathogenic bacteria."

About Cannabis Science, Inc.

Cannabis Science, Inc. is at the forefront of medical marijuana research and development. The Company works with world authorities on phytocannabinoid science targeting critical illnesses, and adheres to scientific methodologies to develop, produce, and commercialize phytocannabinoid-based pharmaceutical products. In sum, we are dedicated to the creation of cannabis-based medicines, both with and without psychoactive properties, to treat disease and the symptoms of disease, as well as for general health maintenance.

Forward-Looking Statements

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Nine new cannabinoids (1-9) were isolated from a high-potency variety of Cannabis sativa. Their structures were identified as (+/-)-4-acetoxycannabichromene (1), (+/-)-3''-hydroxy-Delta((4'',5''))-cannabichromene (2), (-)-7-hydroxycannabichromane (3), (-)-7R-cannabicoumarononic acid A (4), 5-acetyl-4-hydroxycannabigerol (5), 4-acetoxy-2-geranyl-5-hydroxy-3-n-pentylphenol (6), 8-hydroxycannabinol (7), 8-hydroxycannabinolic acid A (8), and 2-geranyl-5-hydroxy-3-n-pentyl-1,4-benzoquinone (9) through 1D and 2D NMR spectroscopy, GC-MS, and HRESIMS. The known sterol beta-sitosterol-3-O-beta-d-glucopyranosyl-6'-acetate was isolated for the first time from cannabis. Compounds 6 and 7 displayed significant antibacterial and antifungal activities, respectively, while 5 displayed strong antileishmanial activity.

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Ferenczy, L.; Gracza, L.; Jakobey, I.

Naturwissenschaften (1958), 45, 188 CODEN: NATWAY; ISSN: 0028-1042. English.

Exts. of resinous organs of the plant gives an active antibacterial compound (I). I has little activity against gram-neg. bacteria, yeasts, or molds, but is active against Streptomyces griseus and gram-pos. bacteria, especially in slightly acidic media. I shows an intensive hashish reaction which appears proportionate to its antibacterial activity.

Krejci, Z.

Pharmazie (1958), 13, 155-66 CODEN: PHARAT; ISSN: 0031-7144. Unavailable.

cf. C.A. 52, 4929d. In the course of a systematic investigation of Central European plants for antibacterial components, substances in hemp were found to have high activity.

Ascending paper chromatography was used to sep. the components of the extract, and strips of the chromatogram were laid on plate cultures of Staphylococcus aureus (in prelim. expts.) to demonstrate activity through inhibitory effects. Under conditions of low conch., chlorophyll so tested with the chromatogram was without antibiotic action (perhaps because of low concentration). The active principle was an amorphous resinous substance, soluble in EtOH, pert. ether, and C6H6, of acid nature, with phenolic and COOH groups, and could be acetylated. The Ac derivs., crystalline substances, showed slight reductions in antibacterial activity over the amorphous material, and were not studied as antibiotics.

Purified exts. of Cannabis representing antibiotic-active materials were prepared as follows (cf. Krejci and Santav.acte.y, C.A. 50, 12080d): the comminuted drug was extracted with petr. ether, light benzene, or C6H6; the extract was shaken with N NaOH to form a water-soluble salt; HCl added to precipitate the resin, and this extracted with Et2O, the latter evaporated to leave the antibacterial substance.

This could be crystallized by acetylation. This extract was antibacterial to Mycobacterium tuberculosis, even when diluted to 1:150,000. Gram-neg. organisms of the coli-typhus group, Pseudomonas aerogenes, and Proteus vulgaris were not affected. Blood, blood plasma, and Blood serum partially inactivated this substance, reducing the antibiotic effect. Thus, a diln. of 1:100,000, which inhibited S. aureus, was inactivated by adding 10% blood or blood plasma; there was lesser inactivation at 1:10,000 dilns.

Antibiotic activity was compared at different pH values (cf. Stoll, et al., Schweiz. Z. allgem. Pathol. Bakteriol. 14, 591(1952)) with penicillin and streptomycin. Na salts of the isolated amorphous substance in aqueous alkaline solution showed increasing activity with increase of pH from 5 to 7.5, whereas crystallized Ac derivs. (acids) showed increasing activity when the pH was decreased from 8 to 5.

The rapidity of antibacterial action varied with differing dilns., using S. aureus cultures for testing. Thus, 1: 100 dilns. produced immediate sterilization, 1:10,000 were sterile after 3 hrs., and 1:100,000 dilns. were sterile after 8 hrs. Clinical usage in stomatology, otorhinolaryngology, dermatology, and treatment of tuberculosis is reviewed. 44 references.

Nissen L, Zatta A, Stefanini I, Grandi S, Sgorbati B, Biavati B, Monti A.

Microbiology Area, DiSTA (Department of Agroenvironmental Sciences and Technologies), Italy. lorenzo.nissen@unibo.it

 

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