The Preclinical Toxicology And Toxicity Mechanisms Of MJ15, A New Cannabinoid Receptor Antagonist Weight-Reducing Drug

Cannabinoid receptors and the endogenous cannabinoid system have played an important role in the regulations of food intake and consumptions, and the etiology of the obesity. The first cannabinoid receptor antagonist weight-reducing drug, rimonabant, have been successfully developed and marketed in 2006, with cannabinoid receptor 1 as a target. Some severe psychological adverse effects have been observed among the clinical users of rimonabant, however,a series of new cannabinoid receptor antagonists are in the development pipelines. MJ15 is the first cannabinoid receptor antagonist developed in China, and the current nonclinical pharmacological, pharmacodynamic and pharmaceutical studies demonstrated that it boosts a bright development prospective. In the present study, a comprehensive preclinical safety evaluation on MJ15 was undertaken, to explore its acute toxicity, repeated dose toxicity, the genetic, reproductive and the developmental toxicity, and the safety pharmacology, in compliance with the national or international nonclinical testing guidelines. After then, the relationship between the weight reducing effects of MJ15 and the lipid metabolisms in the liver has been studied, with the metabonomical technology.The single dose acute oral (by gavage) toxicity study in SD rats at a maximum feasible dose of 5000mg/kg MJ15 demonstrated that the main clinical finding in day1 were body weight decreases, hypoactivity, salivation, stagger, ataxia; and anorexia, with females were more severely affected than males. On the next day after dosing, all these clinical signs were disappeared. There were no major findings upon gross necropsy at day 15. LD50 for the single oral dose of MJ15 in SD rats is greater than 5000mg/kg. MJ15 formulated as capsules was administered once, by oral, at 250, 312.5, 375, 500 and 1000mg/kg, to Beagle dogs. This acute toxicity study demonstrated that the main finding was vomiting, salivation, limb tremor, stagger, loose stools and anorexia,and all these abnormal were recovered 2 days after dosing. There were no findings upon gross necropsy. The maximum tolerant dose was 250mg/kg.MJ15 was administered to SD rats once daily, 6 times a week, by oral gavage, for 6 months. One control group and 3 treated groups were given, 0 40, 160 and640 mg/kg/day. The results showed that there were a dose dependent clinical finding such as mild hypoactivity, emaciation, toe gait, reflex, startling and piloerection in some of the MJ15 dose rats, and the females were more severely affected than males. All these adverse effects recovered after 2 weeks of dosing. The body weights were decreased from day1, and persisted to day 91 in the high dose group. The multitudes and the persistence period of the body weight decreases were dose dependent, and the females were more severely affected than males. The food consumption of the animals showed the same change trend. There were no abnormal findings upon the hematological, clinical chemistry examination. The histopathological examination revealed no definitive toxic target. The toxicokinetic analysis demonstrated that after repeated dosing of MJ15, there were great individual variation on the toxicokinetic parameters of the prodrug and its N-oxides. The mean Tmaxfor the prodrug and the N-oxides of MJ15 are 1.5-5.2 h and 10.8-20.0h, respectively. The plasma exposures to the metabolites were about 24-29 folds and 42-53 folds of the exposures to the prodrug MJ15 after single and multiple dosing, respectively. After single and multiple dosing, there were no obvious dose dependent changes on the Cmax and AUC0-t of the prodrug and the N-oxides of MJ15 in the dose range of 40-640 mg/kg. The exposures after multiple dosing were lower than that after single dosing, suggesting that there were no dose accumulation for MJ15.The No Observed Adverse Effect Level (NOAEL)was 40.0mg/kg, and the main affected system was the nervous system.MJ15 formulated in capsules was administered to Beagle dogs once daily, 6 times a week, by oral gavage, for 9 months. One control group and 3 treated groups were given 0, 12, 28 and 66mg/kg/ day MJ15.Regular observations were made for clinical signs, body weight and food consumption, together with ophthalmoscopic examination, hematology, and blood chemistry at termination. Blood samples were collected for concomitant toxicokinetic analysis. Animals in the high and mid dose groups exhibited emesis, myasthenia of limbs, tremor, and salivation, and some other more severe symptoms such as twitch, dystasia, recumbence ( high dose only), with most of these effects recovered within 24 hour. The body weights were decreased from week 2, and persisted to dosing termination in the high dose group. The multitudes and the persistence period of the body weight decreases were dose dependent, and the females were more severely affected than males. The food consumption of the animals showed the same change trend. The food consumptions were dose dependently decreased from week 1, and persisted to dosing termination in the high and mid dose groups. There were no abnormal findings upon the hematological, clinical chemistry examination. The histopathological examination revealed no treatment related findings. The toxicokinetic analysis demonstrated that after repeated dosing of MJ15, there were great individual variation on the toxicokinetic parameters of the prodrug and its N-oxides. The mean Tmaxfor the prodrug and the N-oxides of MJ15 are 1.0-1.5 h and 1.9-3.4h, respectively. The plasma exposures to the metabolites were about 20 folds of the exposures to the prodrug MJ15 at all dose levels. In the dose range of 12-66 mg/kg, the proportional increases in the AUC of the prodrug and the N-oxides were lower than that of the dose of MJ15. There were no obvious dose accumulation for MJ15 at dose levels of 12 andå’Œ66mg/kg, but there was some dose accumulation at 12.0 mg/kg. The NOAEL was 12.0mg/kg, and the main affected system was the nervous system.The in vivo bone marrow micronucleus test in Kunming mice showed that no increase in the frequency of micronucleated cells was observed in the polychromatic erythrocytes of the bone marrow of animals treated with MJ15 at a dose up to 2000mg/kg. And there were no bone marrow toxicity observed. Ames test in Salmonella typhimurium demonstrated that MJ15 exhibited no mutagenic effects to the test strains of TA1535,TA97,TA98,TA100 and TA102 in the absence or presence of rat liver S9 metabolic activation at a dose range of 0.8～1000.0μg/plate. In the in vitro clastogenicity assay in CHL cells, the results showed that MJ15 did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations in the absence or presence of S9 mix, at a dose up to 4.08μg/ml.The general reproductive toxicity of MJ 15 were evaluated in Wistar rats at the doses of 0, 40, 160 and 640mg/kg/day by gavage. The results showed that there were no relevant effect on the copulation index, precoital interval, postcoitus body weight, and the pregnancy index at all dose levels. But the absolute weights of seminal vesicle were mild decreased, some motility parameters of sperms in the epididymis cauda were mild changed, the mean number of corpora lutea of pregnancy, implantations, and the ovary weight of females were decreased. The number of resorptions was increased. The Lowest Observed Adverse Effect Level (LOAEL) was considered to be 40 mg/kg/day. The developmental toxicity of MJ 15 were evaluated in Wistar rats at the doses of 0, 40, 160 and 640mg/kg/day by gavage. The results showed that the gestational body weight, food consumption, and the gestational weight gain were decreased in the high and mid dose groups. The main adverse signs of dams were prostration and convulsion. The number of pre-implantation loss and resorption were increased, and the number of live fetuses and the placenta weight were decreased. There was no effect on fetal body weight, sex ratio or fetal abnormalities. The maternal and embryo fetal NOAEL was considered to be 40 mg/kg/day. The perinatal toxicity of MJ 15 were evaluated in Wistar rats at the doses of 0, 40, 160 and 640mg/kg/day by gavage. The results showed that the body weight and food consumption in the gestation and lactation periods were decreased in the high and mid dose groups. The pup caring behaviors of some dams were abnormal, resulting in the mortality of F1 pups, a decreased body weight at lactational and post lactation periods, increased locomotive activity in male pups, and the abnormal learning and memory in pups. The perinatal toxicity NOAEL was considered to be 40 mg/kg/day.The safety pharmacology studies demonstrated that MJ15 could induce muscle tremor, unsteadiness, piloerection, alertness to the acoustic and tactile stimulation, convulsion, and urinary incontinence in mice at a dose level of 640.0mg/kg. Furthermore, it could induce the hypoalgesia in mice and enhance the hypnotic effects of pentobarbital, suggested that MJ15 could affect the motor, sensation and the coordination of the mice. There were no relevant effects on the coordination, balance, body temperature and locomotive activity in rats. There were no effects on the heart rate, ECG parameters, blood pressure, and respiration function of the anesthetized Beagle dogs at a dose up to 80mg/kg.The metabolites changes in the plasma and liver extracts were analyzed with NMR metabolic procedures for the rats after 14 days repeated dosing of different dosage of MJ15.The results showed that the metabolites phenotype in the plasma and liver extracts of high dose group (640mg/kg) was more representative for the effects of MJ15 on the organisms. The main metabolite changes included that the increases in the plasma levels of glutamine,pyruvate,choline,phenylalanine,and TMAO;the decreases in the plasma levels of 2-oxy-3-methyl-N-valerate, dodecanoate, trans-aconitic acid, lactic acid, sucrose, 2-hydroxy- glutaric acid; the increases in the aqueous liver extract levels of 3-hydroxy-butyrate, lactic acid, 2-hydroxy- glutaric acid; creatinine, ethanol, mannitol, 2-hydroxy-isovalerianic acid; the decrease in the aqueous liver extract levels of methylamine, taurine, trans-aconitic acid, isoleucine, and alanine. In the liver lipid-solvent extract, the levels of triglyceride terminal methyls, methylene (CH₂)n, CH₂CO, N+(CH3)3, CH₂OPO2, and CH₂OCOR were also increased. All these metabolite changes suggested there were some increases in the oxidative phosphorylation of mitochondria andβoxidation of peroxisome, in the liver of MJ15 treated rats.In summary, the comprehensive preclinical safety evaluation of MJ15, a cannabinoid receptor antagonist, has shown that its main preclinical toxicological characteristics were similar with its congener Rimonabant, with the main toxic target being the central nervous system. However, compared with Rimonabant, MJ15 has a more broad margin of safety. The metabonomic study demonstrated that MJ15 could affect the mitochondrial oxidative phosphorylation and peroxisomalβoxidation of liver, a finding consistent with its pharmacological effects on the lipid metabolism. Considering its preclinical pharmacology and pharmacodynamics, the present study on its preclinical toxicology has exhibited a excellent developmental perspectives for MJ15.