This study aimed to evaluate the interspecies difference in metabolism of mulberrin and examine the interaction between mulberrin and CYP enzymes or recombinant human uridine 5′-diphosphate (UDP)glucuronosyltransferase (UGT) enzymes. Liver microsomes from human (HLMs), Beagle dog (DLMs), minipig (PLMs), monkey (MLMs), rabbit (RLMs), rat (RAMs), and mouse (MIMs) were used to investigate metabolic diversity among different species. Additionally, recombinant human supersomes were used to confirm that metabolic enzymes are involved in the biotransformation of mulberrin. We also evaluated the influence of mulberrin on protein expression by Western blot analysis. Mulberrin metabolism showed significant interspecies differences. We found four and two metabolites in phase I and II reaction systems, respectively. In phase I metabolism profiles of mulberrin for HLMs, PLMs and MLMs conformed to the classic Michaelis-Menten kinetics, RAMs and MIMs followed biphasic kinetics; phase II reaction of mulberrin in HLMs, DLMs, PLMs, MLMs, RLMs, RAMs and MIMs followed biphasic kinetics. UGT1A1 were the major CYP isoforms responsible for the metabolism of mulberrin. Mulberrin showed potent inhibitory effects against CYP3A4, CYP2C9, CYP2E1, UGT1A1, UGT1A3 and UGT2B7 with IC 50 values of 54.21, 9.93, 39.12, 3.84, 2.01, 16.36 µM, respectively. According to Western blot analysis, mulberrin can upregulate the protein expression of CYP2C19, and downregulate the expression levels of CYP3A5 and CYP2C9 in HepG2 cells as concentration increased. The interspecies comparisons can help find other species with metabolic pathways similar to those in humans for future in vivo studies.

Context Toddalolactone, the main component of Toddalia asiatica (L.) Lam. (Rutaceae), has anticancer, antihypertension, anti-inflammatory, and antifungal activities. Objective This study investigated the metabolic characteristics of toddalolactone. Materials and methods Toddalolactone metabolic stabilities were investigated by incubating toddalolactone (20 μM) with liver microsomes from humans, rabbits, mice, rats, dogs, minipigs, and monkeys for 0, 30, 60, and 90 min. The CYP isoforms involved in toddalolactone metabolism were characterized based on chemical inhibition studies and screening assays. The effects of toddalolactone (0, 10, and 50 µM) on CYP1A1 and CYP3A5 protein expression were investigated by immunoblotting. After injecting toddalolactone (10 mg/kg), in vivo pharmacokinetic profiles using six Sprague–Dawley rats were investigated by taking 9-time points, including 0, 0.25, 0.5, 0.75, 1, 2, 4, 6 and 8 h. Results Monkeys showed the greatest metabolic capacity in CYP-mediated and UGT-mediated reaction systems with short half-lives ( T 1/2 ) of 245 and 66 min, respectively, while T 1/2 of humans in two reaction systems were 673 and 83 min, respectively. CYP1A1 and CYP3A5 were the major CYP isoforms involved in toddalolactone biotransformation. Induction of CYP1A1 protein expression by 50 μM toddalolactone was approximately 50% greater than that of the control (0 μM). Peak plasma concentration ( C max ) for toddalolactone was 0.42 μg/mL, and T max occurred at 0.25 h post-dosing. The elimination t 1/2 was 1.05 h, and the AUC 0–t was 0.46 μg/mL/h. Conclusions These findings demonstrated the significant species differences of toddalolactone metabolic profiles, which will promote appropriate species selection in further toddalolactone studies.

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