Fred J. Evans

Cannabinoids in Clinical Practice

Cannabis has a potential for clinical use often obscured by unreliable and purely anecdotal reports. The most important natural cannabinoid is the psychoactive tetrahydrocannabinol (Δ9-THC); others include cannabidiol (CBD) and cannabigerol (CBG). Not all the observed effects can be ascribed to THC, and the other constituents may also modulate its action; for example CBD reduces anxiety induced by THC. A standardised extract of the herb may be therefore be more beneficial in practice and clinical trial protocols have been drawn up to assess this. The mechanism of action is still not fully understood, although cannabinoid receptors have been cloned and natural ligands identified.Cannabis is frequently used by patients with multiple sclerosis (MS) for muscle spasm and pain, and in an experimental model of MS low doses of cannabinoids alleviated tremor. Most of the controlled studies have been carried out with THC rather than cannabis herb and so do not mimic the usual clincal situation. Small clinical studies have confirmed the usefulness of THC as an analgesic; CBD and CBG also have analgesic and antiinflammatory effects, indicating that there is scope for developing drugs which do not have the psychoactive properties ofTHC. Patients taking the synthetic derivative nabilone for neurogenic pain actually preferred cannabis herb and reported that it relieved not only pain but the associated depression and anxiety. Cannabinoids are effective in chemotherapy-induced emesis and nabilone has been licensed for this use for several years. Currently, the synthetic cannabinoid HU211 is undergoing trials as a protective agent after brain trauma. Anecdotal reports of cannabis use include case studies in migraine and Tourette’s syndrome, and as a treatment for asthma and glaucoma.Apart from the smoking aspect, the safety profile of cannabis is fairly good. However, adverse reactions include panic or anxiety attacks, which are worse in the elderly and in women, and less likely in children. Although psychosis has been cited as a consequence of cannabis use, an examination of psychiatric hospital admissions found no evidence of this, however, it may exacerbate existing symptoms. The relatively slow elimination from the body of the cannabinoids has safety implications for cognitive tasks, especially driving and operating machinery; although driving impairment with cannabis is only moderate, there is a significant interaction with alcohol.Natural materials are highly variable and multiple components need to be standardised to ensure reproducible effects. Pure natural and synthetic compounds do not have these disadvantages but may not have the overall therapeutic effect of the herb.

Activation of the PKC‐isotypes α, β1, γ, δ, and ε by phorbol esters of different biological activities

Phorbol esters, tetradecanoylphorbolacetate, sapintoxin‐A, 12‐deoxyphorbol‐phenylacetate, 12‐deoxyphorbol‐phenylacetate‐20‐acetate, thymeleatoxin and resiniferatoxin were investigated for their abilities to activate the PKC‐isotypes α, β1, γ, δ and ε. PKC‐isotypes were grouped into two classes on the basis of Ca2+ requirements for activation by phorbol esters; α, β1, and γ being Ca2+‐dependent forms and δ and ε being Ca2+‐independent. PKC‐isotype selective activation by phorbol esters was observed in that SAPA failed to activate PKC‐δ up to a concentration of 1000 ng ml−1 and DOPPA only activated PKC‐β1, over the same range of concentrations.

Polyphenolic compounds from Croton lechleri

The blood-red sap of Croton lechleri was found to contain proanthocyanidins as major constituents which accounted for up to 90% of the dried weight. In addition to (+)-catechin, (−)-epicatechin, (+)-gallocatechin, (−)-epigallocatechin and dimeric procyanidins B-1 and B-4, five novel dimers and trimers were isolated and characterized as catechin-(4α→8)- epigallocatechin, gallocatechin-(4α→8)-epicatechin, gallocatechin-(4α→6)-epigallocatechin, catechin-(4α→8)- gallocatechin-(4α→8)-gallocatechin and gallocatechin-(4α→8)-gallocatechin-(4α→8)-epigallocatechin. Higher oligomers were also obtained. A new procedure combining chemical degradation with 1H NMR spectroscopy has been developed for determination of the composition and molecular size of oligomeric/polymeric proanthocyanidins. The oligomers of the sap were shown to have the mean degree of polymerization of 4,5–6 and 6–7, respectively, and Mr up to 2130. The heterogeneity of the oligomers was clearly indicated by the presence of a variety of flavan-3-ols as extension and terminal units. An exceptionally high content of gallocatechin and epigallocatechin in the oligomers was observed.

Analgesic and antiinflammatory activity of constituents of Cannabis sativa L.

Two extacts ofCannabis sativa herb, one being cannabinoid-free (ethanol) and the other containing the cannabinoids (petroleum), were shown to inhibit PBQ-induced writhing in mouse when given orally and also to antagonize tetradecanoyl-phorbol acetate (TPA) -induced erythema of mouse skin when applied topically. With the exception of cannabinol (CBN) and δ1-tetrahydrocannabinol (δ1-THC), the cannabinoids and olivetol (their biosynthetic precursor) demonstrated activity in the PBQ test exhibiting their maximal effect at doses of about 100Μg/kg. δ1-THC only became maximally effective in doses of 10 mg/kg. This higher dose corresponded to that which induced catalepsy and is indicative of a central action. CBN demonstrated little activity and even at doses in excess of 10 mg/kg could only produce a 40% inhibition of PBQ-induced writhing. Cannabidiol (CBD) was the most effective of the cannabinoids at doses of 100Μg/kg. Doses of cannabinoids that were effective in the analgesic test orally were used topically to antagonize TPA-induced erythema of skin. The fact that δ1-THC and CBN were the least effective in this test suggests a structural relationship between analgesic activity and antiin-flammatory activity among the cannabinoids related to their peripheral actions and separate from the central effects of δ1-THC.

An assay procedure for the comparative irritancy testing of esters in the tigliane and daphnane series.

A method is described for testing of diterpene esters for irritancy. The technique involves the application of acetone solutions of the toxins to the inside ears of female LACA mice. The number of mice responding per group and the log10 dose data were evaluated using probit analysis with the assistance of a computer program. This evaluation has the advantage that approximations inherent with an arithmetical evaluation were eliminated, and limits may be placed upon the standard deviation of the irritant dose 50% (ID50). In addition, the use of a χ2 test automatically eliminated results which were not attributed to random biological variation. Observations of the time to onset and the persistence of the inflammation have led to the suggestion that daphnane orthoester diterpenes may elicit their effect by means of a direct action at a receptor site in skin, whereas the tiglianeO-acyl esters may in part act by causing more general tissue damage.

Cannflavin A and B, prenylated flavones from Cannabis sativa L.

Two novel prenylat flavones, termed Cannflavin A and B, were isolated from the cannabinoid free ethanolic extract ofCannabis sativa L. Both compounds inhibited prostaglandin E2 production by human rheumatoid synovial cells in culture.

Investigations into the skin-irritant properties of resiniferonol ortho esters.

A series of esters were produced by partial synthesis from 9,13,14-orthophenylacetyl-resiniferonol. These compounds were tested for irritant effects by means of a mouse ear assay. All of the derivatives, including the parent compound, produced short-term inflammation of mice ears within 1–2 h and the effects did not persist for 24 h. This is in contrast to esters of structurally related tigliane diterpenes which produce a longer-term effect on mice ears. Highly potent irritants were synthesized which exhibited irritant doses 50% in the 0.0012–0.00021 nmol range. These esters were all substituted phenylacetates of the C-20 position of 9,13,14-orthophenylacetyl-resiniferonol. The meta or para positions of the phenylacetate moiety were substituted with electronegative groups for maximum activity. Compounds exhibiting substituted phenyl propionates at C-20 were not irritants in the test used.

The Inhibitory Effects of Cannabinoids, The Active Constituents of Cannabis sativa L. on Human and Rabbit Platelet Aggregation

Olivetol, cannabigerol (CBG), cannabidiol (CBD), cannabinol (CBN) and tetrahydrocannabinol (δ1‐THC) were assessed for their ability to inhibit agonist‐induced platelet aggregation and [14C]5‐HT release. With the exception of olivetol, (40% maximal effectiveness), none of the compounds inhibited tetradecanoylphorbolacetate (TPA)‐induced aggregation of human or rabbit platelets. All of these cannabinoids partially inhibited primary aggregation and totally inhibited secondary aggregation of human platelets when adrenaline was used as the agonist. Inhibition was dose‐dependent over the range 10−3–10−5 M. Both rabbit and human platelet aggregation induced by adenosine diphosphate was inhibited in a dose‐dependent manner and the order of potency was CBG > CBD > olivetol > THC > CBN, the IC50 of CBG being 2·7 × 10−4M. PAF‐induced aggregation of rabbit platelets was also inhibited by these compounds in a dose‐dependent manner over the concentration range 10−3 to 10−4 M, however [14C]5‐HT release was only partially prevented by the cannabinoids in a manner which did not correlate with inhibition of aggregation.

Sapintoxin A, a new biologically active nitrogen containing phorbol ester

From the unripe fruits ofSapium indicum, an irritant compound, sapintoxin, was isolated. Spectroscopic data together with selective hydrolysis and partial synthesis confirmed sapintoxin as 12-O-[N-methylaminobenzoyl]-13-O-acetyl-4-deoxyphorbol.

Activation of phospholipase A2 by cannabinoids: Lack of correlation with CNS effects

Cannabinoids Δ1-tetrahydrocannabinol, cannabinol, cannabidiol and cannabigerol have been shown to affect directly the activity of phospholipase A2 in a cell-free assay. The compounds produced a biphasic activation of the enzyme, with EC50 values in the range 6.0–20.0 × 10−6 M and IC50 values in the range 50.0–150.0 × 10−6M. These results correlated well with the relative potencies reported for the stimulation of prostaglandin release from human synovial cells in vitro, confirming that activation of phospholipase A2 is the predominant action of cannabinoids on arachidonate metabolism in tissue culture. However, since Δ1-tetrahydrocannabinol is unique among these compounds in possessing cataleptic activity, it is unlikely that phospholipase A2 is the major receptor mediating the psychotropic effects of cannabis.Cannabinoids Δ1‐tetrahydrocannabinol, cannabinol, cannabidiol and cannabigerol have been shown to affect directly the activity of phospholipase A2 in a cell‐free assay. The compounds produced a biphasic activation of the enzyme, with EC50 values in the range 6.0–20.0 × 10−6 M and IC50 values in the range 50.0–150.0 × 10−6M. These results correlated well with the relative potencies reported for the stimulation of prostaglandin release from human synovial cells in vitro, confirming that activation of phospholipase A2 is the predominant action of cannabinoids on arachidonate metabolism in tissue culture. However, since Δ1‐tetrahydrocannabinol is unique among these compounds in possessing cataleptic activity, it is unlikely that phospholipase A2 is the major receptor mediating the psychotropic effects of cannabis.