Chemical Synthesis, Pharmacokinetic Properties and Biological Effects of JM-00266, a Putative Non-Brain Penetrant Cannabinoid Receptor 1 Inverse Agonist

Abstract

Targeting cannabinoid 1 receptors (CB1R) with peripherally restricted antagonists (or inverse agonists) shows promise to improve metabolic disorders associated with obesity. In this context, we designed and synthetized JM-00266, a new CB1R blocker with limited blood-brain barrier (BBB) permeability. Pharmacokinetics were tested with SwissADME and in vivo in rodents after oral and intraperitoneal administration of JM-00266 in comparison with Rimonabant. In silico predictions indicated JM-00266 is a non-brain penetrant compound and this was confirmed by brain/plasma ratios and brain uptake index values. JM-00266 had no impact on food intake, anxiety-related behavior and body temperature suggesting an absence of central activity. cAMP assays performed in CB1R-transfected HEK293T/17 cells showed that the drug exhibited inverse agonist activity on CB1R. In addition, JM-00266 counteracted anandamide-induced gastroparesis indicating substantial peripheral activity. Acute administration of JM-00266 also improved glucose tolerance and insulin sensitivity in wild-type mice, but not in CB1R^-/- mice. Furthermore, the accumulation of JM-00266 in adipose tissue was associated with an increase in lipolysis. In conclusion, JM-00266 or derivatives can be predicted as a new candidate for modulating peripheral endocannabinoid activity and improving obesity-related metabolic disorders.

Keywords: CB1R antagonist; Rimonabant; SWISSADME prediction; drug discovery; endocannabinoid system; obesity; pharmacokinetics.

Figure 1

Structure and Swiss ADME analysis of JM-00266 in comparison with Rimonabant. (A): Chemical structures of JM-00266 and Rimonabant and results of their respective analysis using the Swiss ADME server. Boxes indicate topological polar surface area (TPSA) and potential ability to cross blood–brain barrier (BBB). (B): WLOGP (lipophilicity) vs. TPSA (apparent polarity) boiled-egg view of JM-00266 in comparison with Rimonabant.

Figure 2

Plasma kinetic profile, tissue distribution and brain penetrance of JM-00266. (A,B): JM-00266 and Rimonabant plasma appearance after intraperitoneal and oral administration (10 mg·kg⁻¹). Drugs were dissolved in DMSO/Tween 80 (4/1; v/v) and diluted with physiological saline before intraperitoneal injection. Results are expressed as mean ± SEM (n = 4 per group). (C): Plasma clearance of JM-00266 and Rimonabant after intravenous administration (D): JM-00266 and Rimonabant partitioning in plasma, brain, liver and visceral adipose tissue after oral administration (10 mg·kg⁻¹). Results expressed as ng·g⁻¹ of body weight were obtained multiplying concentration in sample by total tissue mass and normalizing by body weight (n = 4 per group) * p < 0.05. (E): Brain/plasma ratio after intraperitoneal or oral administration of JM-00266 and Rimonabant (10 mg·kg⁻¹) to mice. Results are expressed as mean ± SEM (n = 4 per group) * p < 0.05. (F): Brain uptake index was determined in rats after intravenous co-administration of JM-00266 and Rimonabant (10 mg·kg⁻¹). Results are expressed as mean ± SEM (n = 4 per group). * p < 0.05. In all experiments, drugs were dissolved in DMSO/Tween 80 (4/1; v/v) and diluted in a thermostated-buffered solution for intraperitoneal and intravenous injections or dissolved in oil for oral administration.

Figure 3

cAMP functional assay. Effect of increasing concentrations of JM-00266 (A) or Rimonabant (B) on cAMP Glosensor luminescence in the absence or presence of anandamide (AEA; 0–1 µM). When used, pertussis toxin (PTX; 0.1 µg·mL⁻¹) was added 24 h before the cAMP assay. Intracellular cAMP levels were expressed as % variation of relative luminescence units (RLU) obtained after stimulation by forskolin (1 µM) for 10 min (*, p < 0.05).

Figure 4

Effect of JM-00266 on behavior, body temperature and gastrointestinal transit. (A): Cumulative 3-h food intake in 24-h food-deprived mice subjected to an intraperitoneal injection of ACEA (1 mg/kg) alone, in combination with Rimonabant (10 mg·kg⁻¹) or with JM-00266 (20 mg·kg⁻¹). Results are expressed as mean ± SEM (n = 4 per group), * p < 0.05. (B): Effect of AEA (10 mg·kg⁻¹) on rectal temperature in mice treated with JM-00266 or vehicle (20 mg·kg⁻¹). Results are expressed as mean ± SEM (n = 4 per group) * p < 0.05. (C): Effect of ACEA on rectal temperature measured at t = 30 min after injection of ACEA (1 mg·kg⁻¹) in mice treated with JM-00266 (20 mg·kg⁻¹) 20 min earlier. Rimonabant (10 mg·kg⁻¹) or vehicle. Results are expressed as mean ± SEM (n = 4 per group) * p < 0.05. (D): Effect of JM-00266 and Rimonabant on open field exploration. Total ambulatory distance was determined calculating total distance travelled for 10 min. Anxiety was evaluated monitoring total entries in the center of the arena and total time spent in the center on a 10-min period. Results are expressed as mean ± SEM (n = 5 per group). (E): Effect of JM-00266 on gastrointestinal motility. Mice were treated with JM-00266 (10 mg·kg⁻¹) or vehicle 10 min before an injection of anandamide (10 mg·kg⁻¹). Results are expressed as mean ± SEM (n = 3 per group) * p < 0.05.

Figure 5

Effect of acute administration of JM-00266 on carbohydrate metabolism. (A): Oral glucose tolerance test (OGTT) and corresponding AUC_{0–2 h} calculations in mice injected with JM-00266 (10 mg·kg⁻¹) or vehicle 10 min before glucose gavage (n = 10 per group). (B): Insulinemia during OGTT. (C): Insulin tolerance test (ITT) and corresponding AUC_{0–2 h} calculations in mice treated with JM-00266 (10 mg·kg⁻¹) or vehicle 10 min before insulin administration (n = 15 per group). (D): OGTT in CB1R^−/− and corresponding AUC_{0–2 h} calculations mice injected with JM-00266 (10 mg·kg⁻¹) or vehicle 10 min before glucose gavage (n = 7 per group). Results are expressed as mean ± SEM, * p < 0.05.

Figure 6

Effect of JM-00266 on adipose tissue lipolysis. (A): Plasma glycerol appearance and corresponding AUC_{0–2 h} calculations in mice treated with JM-00266 or vehicle 4 h before i.p. injection of BRL37344 (ß3-adrenergic receptor agonist, 5 mg·kg⁻¹). Results are expressed as mean ± SEM (n = 5 per group), * p < 0.05. (B): Glycerol produced by visceral adipose tissue explants prepared from animals (n = 4 per group) was injected 4 h earlier with a dose of JM-00266 (10 mg·kg⁻¹) or vehicle and cultured in DMEM-HAM/F12 medium supplemented with 1% BSA for 1 h. Results are expressed as mean ± SEM, * p < 0.05.