Henry K. Borys

The Influence of Cannabidiol and Δ9-Tetrahydrocannabinol on Cobalt Epilepsy in Rats

Summary: The mechanisms of the anticonvulsant activity of cannabidiol (CBD) and the central excitation of Δ9‐tetrahydrocannabinol (Δ9‐THC) were investigated electrophysiologically with conscious, unrestrained cobalt epileptic rats. The well‐known antiepileptics, trimethadione (TMO), ethosuximide (ESM), and phenytoin (PHT), were included as reference drugs. Direct measurements were made of spontaneously firing, epileptic potentials from a primary focus on the parietal cortex and convulsions were monitored visually. ESM and TMO decreased the frequency of focal potentials, but PHT and CBD exerted no such effect. Although CBD did not suppress the focal abnormality, it did abolish jaw and limb clonus; in contrast, Δ9‐THC markedly increased the frequency of focal potentials, evoked generalized bursts of polyspikes, and produced frank convulsions. 11‐OH‐Δ9‐THC, the major metabolite of Δ9‐THC, displayed only one of the excitatory properties of the parent compound: production of bursts of polyspikes. In contrast to Δ9‐THC and its 11‐OH metabolite, CBD, even in very high doses, did not induce any excitatory effects or convulsions. The present study provides the first evidence that CBD exerts anticonvulsant activity against the motor manifestations of a focal epilepsy, and that the mechanism of the effect may involve a depression of seizure generation or spread in the CNS.

An Electrophysiological Analysis of the Anticonvulsant Action of Cannabidiol on Limbic Seizures in Conscious Rats

Summary: The effects of cannabidiol (CBD) on electrically evoked kindled seizures were studied in conscious, unrestrained rats with chronically implanted cortical and limbic electrodes, and the results were compared with those of Δ9 ‐tetrahydrocannabinol (Δ9‐THC), phenytoin (PHT), and ethosuximide (ESM). All drugs were anticonvulsant, but there were marked differences in their effects on afterdischarge (AD) threshold, duration, and amplitude. CBD, like PHT and Δ9‐THC, elevated the AD threshold; in contrast, ESM decreased the threshold but suppressed AD spread. CBD, however, also resembled ESM inasmuch as both drugs decreased AD duration and amplitude. Electrophysiologically, the antiseizure effects of CBD were a combination of those of PHT and ESM. The combination of effects may account for the observation that CBD was the most efficacious of the drugs tested against limbic ADs and convulsions. Other properties of CBD were also noted: For example, compared with Δ9‐THC, it is a much more selective anticonvulsant vis‐à‐vis motor toxicity. CBD also lacks the CNS excitatory effects produced by Δ9‐THC, PHT, and ESM. These characteristics, combined with its apparently unique set of electrophysiological properties, support the suggestion that CBD has therapeutic potential as an antiepileptic.

Influence of ?9 and cannabidiol on photically evoked after-discharge potentials

Two cannabinoids, Δ9 and cannabidiol, and several reference drugs were compared relative to their effects in a recently developed anticonvulsant test system, the after-discharge potentials of the visually evoked response; the potentials were recorded electrophysiologically from electrodes permanently mounted over the visual cortices of conscious rats. In anticonvulsant doses, trimethadione and ethosuximide produced an extensive depression of after-discharge activity, whereas diphenylhydantoin and cannabidiol exerted no such effect. In contrast, anticonvulsant doses of Δ9 and subconvulsant doses of pentylenetetrazol markedly increased after-discharge activity, which may represent a manifestation of their central nervous system excitatory properties. The data from the present study support our previously published observations from several other anticonvulsant tests that indicate the anticonvulsant characteristics of cannabidiol resemble those of diphenylhydantoin rather than those of trimethadione and that the central excitatory properties of Δ9 distinguish it from cannabidiol. The results consistently suggest that the cannabinoids will be effective against grand mal but not absence seizures.

Effect of cannabidiol on cytochrome P-450 and hexobarbital sleep time

Abstract The influences of acute and subacute cannabidiol (CBD) treatment and of subsequent drug withdrawal were investigated on hexobarbital-induced sleep time, on hepatic cytochrome P-450 concentration, on the in vitro formation of carbon monoxide (CO) associated with CBD metabolism, and on the kinetics of aminopyrine N-demethylase metabolism. In acutely treated mice, CBD prolonged sleep time, decreased cytochrome P-450 concentration, decreased the endogenous formation of CO, and increased an apparent Km for aminopyrine N-demethylase activity. In subacutely treated animals, tolerance developed to the effect on sleep time but not to that on cytochrome P-450 concentration nor on the endogenous formation of CO in vitro nor on the Km for the N-demethylase activity. Upon withdrawal from subacute treatment, tolerance to the sleep-time effect was still evident on day 14, but, by day 28, the sensitivity to CBD had returned to normal. In contrast, the cytochrome P-450 concentration returned to normal on day 14 of withdrawal, as did the Km for the N-demethylase activity and the ability of CBD to induce CO synthesis in vitro. The comparative results lead us to conclude that the CBD effect on sleep time does not correlate with either the total amount of cytochrome P-450 or with the CBD depressant effect on the cytochrome.

The pharmacokinetic fate of cannabidiol and its relationship to barbiturate sleep time

Abstract Cannabidiol (CBD) is a known inhibitor of a number of hepatic drug metabolism reactions. Its pharmacokinetics in the liver were studied to determine the relationship between the amount of CBD in this organ and the effect on barbiturate sleep time, which has been shown by others to reflect an inhibition of drug metabolism. A methanol extract of liver was subjected to thin-layer chromatography which yielded three distinct fractions: CBD, the monohydroxylated metabolites and a relatively polar, unidentified fraction. The quantitative analysis of these fractions, as a function of time after drug administration, indicated that CBD is metabolized rapidly: the apparent half life is only about 52 min, which is approximately the same value obtained for the monohydroxylated metabolites; both of these fractions have virtually disappeared from the liver at 4 hr, at which time a significant effect on sleep time still persists. In contrast, the unidentified metabolite fraction contains a substantial amount of cannabinoid throughout the course of the effect on sleep time. These data suggest that the inhibition of hepatic drug metabolism is not caused by either CBD or one of its monohydroxylated metabolites; rather, the effect correlates with the persistence of more polar metabolites.

Post-column derivatization procedure for the colorimetric analysis of tissue cannabinoids separated by high-performance liquid chromatography.

A new method for quantitating cannabidiol (CBD) and delta 9-tetrahydrocannabinol (THC) in mouse plasma and brain involves (1) the separation of CBD and THC from their major metabolites by the use of isocratic, reversed-phase high-performance liquid chromatography (HPLC), and (2) the on-line reaction of the cannabinoids with Fast Blue Salt B (FBB) as a the former elute from the column; the colored cannabinoid-FBB derivatives are then detected at 490 nm in a spectrophotometer with a sensitivity of less than 50 ng. In addition to this HPLC--FBB analytical procedure, a method for extracting CBD and THC from brain and plasma is described, and selected examples illustrate the procedures application to the analysis of CBD and THC in mouse plasma and brain samples taken from animals injected with these two cannabinoids.

Cannabidiol and δ9-tetrahydrocannabinol metabolism: In vitro comparison of mouse and rat liver crude microsome preparations

Abstract The in vitro biotransformations of cannabidiol (CBD) and Δ 9 -tetrahydrocannabinol (Δ-THC) by hepatic microsomal preparations from mouse and rat were compared and it was found that both cannabinoids are metabolized approximately two to three times faster by a mouse preparation than by a similar preparation from the rat. In both species, however, CBD was metabolized more slowly than Δ 9 -THC. The metabolite patterns resulting from the biotransformations of radioactive CBD and Δ 9 -THC were examined by thin-layer chromatography and were found to be qualitatively identical in both the mouse and rat. In contrast, studies of the rate of cannabinoid metabolism in the presence of excess substrate revealed that CBD metabolism was only linear for about 10 min, whereas Δ 9 -THC metabolism was linear for about 60 min. This difference was noted in both species. The evidence suggests that CBD metabolism is inhibited by its own metabolites. Furthermore, the results of other experiments indicate that CBD metabolites can also inhibit Δ 9 -THC and aminopyrine metabolism. The established ability of CBD to inhibit hepatic microsomal drug metabolism in vivo may also be the consequence of the formation of inhibitory metabolites.

DEVELOPMENT OF TOLERANCE TO THE PROLONGATION OF HEXOBARBITONE SLEEPING TIME CAUSED BY CANNABIDIOL

1 The effects of acute and subacute cannabidiol (CBD) administration on hexobarbitone sleeping time and on some constituents of the hepatic microsomal drug‐metabolizing system were assessed in the mouse. 2 Acutely administered CBD prolonged sleeping time; but with subacute treatment, tolerance to the effect rapidly developed. 3 Brain hexobarbitone concentration upon awakening was unchanged by either acute or subacute CBD treatment, which suggests that neither the prolongation of sleeping time nor the tolerance is the result of a change in sensitivity of the central nervous system to the barbiturate. 4 Acute CBD treatment increased the half‐time of hexobarbitone in the brain, which returned toward normal with the development of tolerance. 5 Acutely, CBD caused a 30% decrease in hepatic cytochrome P‐450 level; with tolerance, the cytochrome concentration returned to normal. 6 The evidence suggests that the CBD‐induced prolongation of barbiturate sleeping time and the tolerance to this effect are the result of changes in the rate of drug metabolism, which are related to changes in the amount of cytochrome P‐450. 7 The effects of CBD on the hepatic microsomal drug‐metabolizing enzyme system are different from those attributed to SKF 525‐A and piperonyl butoxide because the cannabinoid does not decrease cytochrome P‐450 quantitatively by complex formation, it does not produce a recovery overshoot in the cytochrome concentration and, finally, it does not cause an induction of the hexobarbitone‐metabolizing enzymes.

Influence of 22-day treatment on the anticonvulsant properties of cannabinoids

SummaryMice were given delta-9-tetrahydrocannabinol (delta-9-THC), cannabidiol (CBD) or phenytoin (PHT) daily for 22 days. Drug activity was measured weekly in three different anticonvulsant tests: the maximal electroshock threshold, the 60-Hz-electroshock threshold and the 6-Hz-electroshock threshold. In order to correlate potential pharmacodynamic and pharmacokinetic changes resulting from repeated treatment, brain-drug concentrations were determined at each test time. The results from the delta-9-THC study indicate that, although tolerance developed in all three tests, there were no changes in the brain-drug concentration. For CBD the pharmacodynamics were strikingly different: an increase in sensitivity to the drug developed in two of the tests, tolerance in only one test. Here again, there were no changes in brain-drug concentrations. The results of the PHT study differed from both the cannabinoids, for tolerance developed in one test, an increase in sensitivity in one test, and the activity was unchanged in the third test. Again, the brain concentrations remained constant throughout. The results demonstrate that both tolerance and increased sensitivity can develop concomitantly with anticonvulsant effects of the cannabinoids and PHT, and that these modifications in drug activity appear to result from cellular or functional rather than dispositional changes.