Toxicological properties of Δ⁹-tetrahydrocannabinol and cannabidiol

The bioavailability of dronabinol is low (4–20 %) because of its high lipid solubility and extensive first-pass hepatic metabolism (15, 16). Its effects do not show clear dose dependence (17). Due to lipid solubility, the apparent volume of distribution is high (10 L/kg). Dronabinol is extensively metabolised in the liver, primarily by cytochrome P450 enzymes CYP2C9 and CYP3A4. CYP2C9 is probably responsible for the formation of the primary active metabolite hydroxy-Δ⁹-THC. Pharmacogenomics studies indicate two to three times higher plasma THC in individuals with a less active form of CYP2C9, so adverse drug reaction in these individuals may be more frequent and/or severe. The major route of excretion is faeces (65 %), and the minor is urine (20 %) (16). Urinary metabolites of dronabinol are identical to those of marijuana and may be excreted over long time (18).

Nabilone has better bioavailability (at least 60 %) than dronabinol and demonstrates dose linearity (15, 19). Multiple cytochrome P450 enzymes extensively metabolise nabilone to various metabolites, which have not been fully characterised yet. Two major metabolic pathways are probably involved in the biotransformation of nabilone: 1) enzymatic reduction of the 9-keto group to form carbinol metabolites; and 2) direct enzymatic oxidation of the aliphatic side-chain to produce carboxylic and hydroxylic analogues. The formation of carbinol metabolites is a major nabilone metabolic pathway in dog. Hydroxylic analogues appear to be more important in rhesus monkey and man. Carbinols are long-lived metabolites that accumulate in the plasma and concentrate in the brains of treated dogs over time (see chronic toxicity) (20). Nabilone and its metabolites are primarily eliminated in faeces (~65 %) and to a lesser extent in urine (~20 %) (14, 17). Although no accumulation of nabilone was observed after repeated doses, some accumulation was observed for its metabolites (21).

Acute oral toxicity of THC in rats is lower in males (LD₅₀=1910 mg/kg) than in females (LD₅₀=1040 mg/kg) (22). The LD₅₀ of oral nabilone is >1000 mg/kg in rats of both sexes (21). The signs of acute toxicity of THC and nabilone are similar and include lower respiratory rate, ataxia, decreased activity, catatonia, hypothermia, hypersensitivity to touch, and generalised body twitching. Death was reported to be due to respiratory arrest (21, 22).

Sub-chronic and chronic effects of THC (5, 15, 50, 150, and 500 mg/kg/day) administered by gavage were assessed in rats in a 13-week study followed by a 9-week recovery period and in a 2-year study (12.5, 25, and 50 mg/kg/day) (23). Briefly, THC-treated rats had lower body weight than controls and exhibited convulsions, hyperactivity, and changes in the reproductive organs of both male and female rats. Reduced body weight was notable even at low dose exposure and was attributed to metabolic changes caused by THC. Weight loss was not associated with lower feed consumption but with increased energy consumption (evidenced by higher plasma corticosterone levels) needed for hyperactivity, adaptation, and detoxification from THC. Convulsions and hyperactivity were observed at all doses. The onset and frequency of convulsions were also dose-related. However, Chan et al. (23) observed no histological changes in brain tissue of rats with a history of THC-related convulsion or seizures. Luthra et al. (24) reported generalised depression, followed by hyperactivity, irritability, aggressiveness, and convulsion in rats treated with THC for 119 days. The highest dose of THC in a sub-chronic study in rats induced testicular atrophy and uterine and ovarian hypoplasia (23). This study also found higher serum FSH and LH at all doses.

Nabilone was assessed in two chronic toxicity studies (21). The one in beagle dogs (0.5, 1.0, 2.0 mg/kg/day) was planned to last one year but was terminated after seven months due to high mortality. Most deaths were preceded by convulsions, and toxicity was attributed to accumulation of carbinol metabolites in the brain over time. In contrast to dogs, nabilone chronic toxicity was minimal in rhesus monkeys receiving doses of up to 2.0 mg/kg/day for one year. Transient periods of anorexia, emesis, and ataxia were observed only at the highest dose.

Chan et al. (23) also evaluated THC carcinogenicity in rats and mice and found no evidence in rats at doses of up to 50 mg/kg/day [~20 times the maximal human recommended dose (MHRD)]. In mice, THC produced thyroid follicular cell adenoma (a common benign neoplasm of the thyroid) in both sexes, but the effect was not dose-dependent, as the hyperplasia was increased compared to control at all doses and in both sexes. It is unclear what these findings mean. Carcinogenicity studies have not been performed with nabilone.

THC and nabilone have no mutagenic potential (11, 12, 13, 23). Positive Ames and skin test results in mice for THC in some in vitro tests are attributed to cytotoxic rather than mutagenic action (25).

THC was evaluated in an oral embryo-foetal developmental study in rats (at doses ranging from 12.5 to 50 mg/kg/day) (26) and in rabbits (0.5, 1.5, 5 and 15 mg/ kg/day) (27). No teratogenic effects were observed in rats. Increased foetal mortality and early resorption were associated with maternal toxicity, which manifested itself as lower weight gain. In rabbits, one third of the foetuses in the high-dose group had multiple anomalies (such as acrania and spina bifida). In a single-generation reproductive study (28), male and female rats received 0.5, 1.5, and 5 mg/ kg/day of THC by gavage. Offspring to mothers receiving 1.5 and 5 mg/kg/day showed a dose-related drop in survival at day 12 of lactation and at weaning.

A reproduction study of nabilone in rats (1.4, and 12 mg/ kg/day) and rabbits (0.7, 1.6, and 3.3 mg/kg/day) (29) showed no teratogenic effects. However, it did find dose-related developmental toxicity, such as embryo death, foetal resorption, decreased foetal weight, and disrupted pregnancy. Another study in rats (24) revealed postnatal developmental toxicity of nabilone at 1.4 and 12 mg/kg/ day), manifested by smaller litter size and lower survival as well as lower initial body weight and hypothermia in pups from the high-dose group.

There are no sufficient data on pregnancy outcomes in women exposed to dronabinol (THC) or nabilone.

Safety data on dronabinol come from 10 randomised, double-blind, placebo-controlled clinical trials. In one trial (30) patients with AIDS-related anorexia (N=139) were receiving dronabinol as appetite stimulant (5 mg/day), and in nine trials patients with cancer (N=454) were receiving dronabinol as antiemetic in the dose range of 2.5–40 mg/ day (31, 32, 33, 34, 35, 36, 37, 38, 39) for no longer than six weeks. The most frequently reported adverse events (33 %) in patients with AIDS were euphoria, dizziness, somnolence, and thinking abnormalities. The most common adverse events in patients receiving the antiemetic dronabinol were drowsiness, dizziness and transient impairment of sensory and perceptual functions. Patients from both studies (24% in antiemetic and 8% in appetite stimulant) reported dose-related “highs” (elation, laughter, and heightened awareness). The frequency of adverse effects on the central nervous system (CNS) increased with doses, and their severity greatly varied between patients. After oral administration, dronabinol had an action onset of approximately 30 min to one hour and a peak effect at two to four hours (40). Psychoactive effects lasted four to six hours. Other than those affecting the nervous system, the most frequent adverse effects were gastrointestinal (abdominal pain, nausea, and vomiting) and cardiovascular (palpitation, tachycardia, vasodilatation/facial flush) (30, 31, 32, 33, 34, 35, 36, 37, 38, 39). The following were the most serious adverse effects of dronabinol: neuropsychiatric, haemodynamic instability, seizure, paradoxical nausea, vomiting, and abdominal pain. Dronabinol should be discontinued in patients experiencing a psychotic reaction or showing cardiovascular effects (tachycardia, transient changes in blood pressure) and used with caution in patients with a history of epilepsy or recurrent seizures (13).

Nabilone has systematically been evaluated in controlled clinical trials that lasted up to nine weeks (41, 42, 43). The lowest nabilone dose (2 mg) had a few adverse effects, whereas a 3–5 mg dose closely mirrored dronabinol’s (25 mg) effects (18).

High levels of CB₁ receptors are found in the brain areas that are part of the mesocorticolimbic dopaminergic pathway and are implicated in motivational and reward processes (44). Being partial CB₁ receptor agonists, THC and its analogues should be tested for their addictive potential (45). Many abused drugs that can lead to addiction increase synaptic dopamine levels in the human limbic striatum. The same was reported for THC in human studies in healthy participants (46, 47, 48). Dopamine release was small compared to amphetamine, cocaine, alcohol (10–15 %), and nicotine (~10 %).

First studies in monkeys (49, 50) failed to show the rewarding effects of THC, but newer studies with intravenous dronabinol injection (1–6 μg/kg) confirmed it in squirrel monkeys (51, 52). Another widely used predictor of a reinforcing (and therefore addictive) effect is the conditional place preference (CCP) test, in which a compartment in a cage is associated (paired) with a tested substance. Lepore et. al. (53) reported that CCP depended on the dose and intervals between administration and that dronabinol doses of 2 or 4 mg/kg every 24 h produced a reliable shift in favour of the dronabinol-paired compartment.

Reinforcing effects have also been observed in humans (12). Nabilone (4–8 mg/day) and dronabinol (10–20 mg/ day) produced stronger marijuana-like subjective effects, such as feeling good, feeling “high”, and feeling “stoned” than placebo. Nabilone had a slower onset of the peak subjective effects.

Chronic therapy with dronabinol can lead to physical dependence. One human study (17) showed that dronabinol doses of 210 mg/day (~10 times higher than MHRD) administered for 12 to 16 consecutive days produced withdrawal syndrome within 12 h after discontinuation. Initial symptoms were irritability, insomnia, and restlessness. By hour 24 of discontinuation, withdrawal symptoms intensified to include “hot flashes”, sweating, rhinorrhoea, loose stool, hiccoughs, and anorexia. We still do not know whether nabilone can also lead to physical dependence. Patients that participated in clinical trials for up to five days showed no withdrawal symptoms after discontinuation of dosing (54).

Plasma CBD concentrations show a nonlinear increase with dose and 6.5 % bioavailability at a 3000-mg dose (60).

CBD absorption increases three times with a high-fat meal and six times with new oral delivery system for lipophilic active compounds (61, 62). Its high estimated volume of distribution (18,800—30,959 L) indicates accumulation of CBD in body fat (63). CBD is extensively metabolised in the liver and gut, mainly by the CYP2C19, CYP3A4, UGT1A7, UGT1A9, and UGT2B7 enzymes (64). Drug interaction trials to assess the effect of CBD on these enzymes in healthy volunteers will be conducted during the post-marketing period (Table 1) (55, 56). The metabolism of CBD is very complex, especially in hepatocytes. The main human metabolite is 7-carboxy-cannabidiol (7-COOH-CBD; ~90 % of all drug-related substances measured in the plasma) (64). Its toxicological profile has not been investigated because experimental animals for toxicological studies (mice, rats, and dogs) do not metabolise CBD to a comparable extent as humans (65). The major concern with 7-COOH-CBD could be its reactive acyl-glucuronide (66) The primary excretion route of CBD is through faeces (84 %), followed by urine (8 %) (63).

In a study of acute effects in rhesus monkeys (67), intravenous CBD caused death by respiratory arrest and cardiac failure at doses above 200 mg/kg (LD₅₀=212 mg/ kg). At the lower dose of 150 mg/kg, survivors recovered in one to three days, and liver weights increased from 19 to 142 %. In the part of the study investigating subchronic effects (after 90 days of oral administration), the authors reported inhibition of spermatogenesis at the highest oral dose of 300 mg/kg (67).

Animal studies of CBD alone described below make part of the Epidiolex® European Public Assessment Report (EPAR, EMA’s scientific monography) (56). To the best of my knowledge, they have not been published and therefore no further detail or original references are currently available. All these studies were conducted in accordance with medicinal product safety standards and protocols and reviewed by the EMA committee (9).

Two oral chronic toxicity studies (referred to in 56) have assessed CBD in Wistar rats (receiving 15, 50, or 150 mg/ kg/day for 6 months) and Beagle dogs (receiving 10, 50, 100 mg/kg/day for 9 months). In both species the primary target organ was the liver. Hepatocellular hypertrophy was detected at all doses, accompanied by an increase in alanine transferase (ALT) and alkaline phosphatase (ALP).

A 104-week oral carcinogenicity study in Wistar rats (referred to in 56) revealed no drug-related neoplastic findings. However, the study had several drawbacks, including impure active substance, excessive effect of body weight, and unknown exposure to the two major human metabolites.

The genotoxic potential of CBD was also investigated in a standard battery of tests, but their results were negative for mutagenicity and clastogenicity (referred to in 56).

A full battery of oral reproductive and developmental studies has been conducted with purified CBD. In an embryo-foetal development study in Wistar rats, litter loss was noted at the highest applied dose of 250 mg/kg. In a prenatal and postnatal development study (referred to in 56) rat exposure to the highest doses (150 and 200 mg/kg/ day) affected reproductive organs (smaller testes in males, reduced fertility index in females). A high dose of 125 mg/ kg also reduced foetal body weight in New Zealand white rabbit, which was related to maternal toxicity. The developmental toxicity in rabbits occurred at maternal plasma concentration similar to human at therapeutic doses (referred to in 56). In rats these concentrations were much higher. No adequate data are available on pregnancy outcome in women exposed to CBD.

A juvenile toxicity study in Wistar rats (referred to in 56) showed neurobehavioral deficits and delayed sexual maturation in males. A no observed effect level (NOAEL) was 150 mg/kg/day.

Safety data on Epidiolex® were obtained from four randomised, double-blind, placebo-controlled multicentre trials with exposure to CBD doses of 5, 10, and 20 mg/kg/day (68, 69, 70). These phase II studies were conducted in 2 to 55 year-old patients with LGS (N=235) and DS (N=88) for up to 14 weeks.

Additional non-controlled safety data have been obtained from an ongoing open-label Phase III study (Study 1415) in LGS and DS patients (N=644), which is being conducted at 38 sites in the USA and Australia. Since this trial is not finished, an interim analysis of long-term safety was conducted (71, 72).

The most common adverse events in CBD-treated patients affected the following systems: CNS (somnolence, sedation), gastrointestinal tract (lower appetite, diarrhoea), liver (higher transaminase), and the lungs (pneumonia). The severity of these events was generally mild to moderate. Diarrhoea, weight loss, higher ALT, and somnolence/ sedation/lethargy were all dose-related. There were two serious cases of transaminase elevation, two severe events with rash (one consistent with a hypersensitivity reaction) and three severe cases of appetite loss. The CBD-treated and the placebo group did not differ in the rate of respiratory failure. Children had lower weight, which was associated to a certain extent with appetite loss (68, 69, 70, 71).

Treatment with CBD is clearly associated with an increased risk of hepatotoxicity (68, 69, 70, 71). Higher doses of CBD and concomitant use of valproate increase the risk of transaminase elevation in patients. Two patients concomitantly treated with valproate experienced toxic hepatocellular injury, metabolic acidosis, and encephalopathy. There appears to be no pharmacokinetic interaction between CBD and valproate, although a pharmacodynamical interaction is currently being investigated. The potential of CBD to cause chronic liver injury should be evaluated in the post-marketing period (55, 56) (Table 1).