| Nicousamide [3-(3'-carboxy-4'-hydroxy-anilino-carbo-)-6nitro-7-hydroxy-8-me- thyl-coumarin] is an inhibitor of rennin and TGF-β1 typeⅡreceptor for the treatment of renal inadequacy. Recent studies have shown that nicousamide can attenuate albuminuria, glomerular sclerosis, tubulointerstitial fibrosis and renal tubule vacuolar degenerationand in streptozotocin-induced diabetic rats. Interestingly, nicousamide also can reduce the incidence of diabetic cataract. The protective mechanism of nicousamide was believed to reduce the AⅡby rennin inhibition resulted the decrease of TGF-βsynthesis and prevent tubulointerstitial fibrosis by blocking the phosphorylation of TGF-β1 typeⅡreceptor.The present study was conducted to characterize the pharmacokinetics, distribution and excretion of nicousamide in rats and beagle dogs, identify the metabolites and the enzymes participated in the biotransformation of nicousamide in in vivo and in vitro, and evaluate the modulation on drug metabolic enzymes.After single oral doses of 30, 100 and 300mg/kg to rats, nicousamide appeared in male plasma at 5 min after administration. The peak plasma concentrations occurred at 20min-2h with the Cmax 1011.4, 1908.1 and 4055.0 ng/ml. Nicousamide appeared in female plasma at lmin after administration. The peak plasma concentrations occurred at 3-20min and the Cmax was 270.4, 728.4 and 1295.7 ng/ml, respectively.Pharmacokinetic parameters were determined using noncompartmental methods. The AUC was 7380.9, 13370.8, 32803.1ng/ml·h in the male and 695.6, 2326.4, 3940. lng/ml·h in the female with a dose dependent manner after single oral dosing of nicousamide while the MRT was all 5.8h in the male, and was 3.0, 3.7 and 3.8h in the female, respectively.Nicousamide was absorbed and eliminated more rapid in female than male rats. The AUC and MRT values demonstrated a linear pharmacokinetics after oral administration from 30 to 300mg/kg of nicousamide in rats. The oral bioavaiability of nicousamide was 1.34% in the male and 0.67% in female rats. Nicousamide (30, 100, and 300mg/kg) can be detected at 3-20 min after oral administration to male beagle dogs, with Tmax 6min-3h, and the Cmax 123.5, 153.6, and 136.7ng/ml, respectively. Similar absorption process also can be seen in female dogs, with Tmax 20min-1h, and the Cmax 33.5, 158.3, 110.2ng/ml, respectively. Pharmacokinetic parameters were determined using noncompartmental methods. After a single oral low-, mid- and high-dosing, the AUC was 109.5, 448.4, 1846.8ng/ml·h in the male and was 110.7, 468.6, 1907.8ng/ml·h in female dogs. The MRT was 1.4, 4.3, 12.1h in the male, and was 1.6, 3.2, 12.8h in female dogs, respectively. The Cmax of nicousamide was dose-independent suggesting the saturation of nicousamide absorption in vivo, while MRT significantly extended with the increaseing of dose, indicating the saturation in the nicousamide elimination. The above data demonstrated a nonlinear pharmacokinetics after single oral administration from 30 to 300mg/kg ofnicousamide in beagle dogs.After seven daily doses of nicousamide (100mg/kg), the Cmax of female and male dogs increased by 30% and 21%, AUCO-∞values raised 5.4 and 4.3 times, and MRT extended 3.2 and 2.4 times compared with single dosing, suggesting there was a potential accumulation in dogs following multiple doses.The results of tissues distribution showed that the concentration order of nicousamide in tissues measured at 10min was stomach＞fat＞epididymis＞muscle＞intestine＞adrenal gland＞testicle＞lung＞kidney＞liver＞heart＞spleen＞brain after a single oral dose of 30mg/kg to male rats. The distribution order at 120 and 480 min was similar with 10min. In female rats, the concentration order of nicousamide in tissues measured at 3min was stomach＞adrenal gland＞intestine＞ovaries＞lung＞fat＞hystera＞spleen＞muscle＞brain＞kidney＞heart＞liver. The brain, lung, kidney and adrenal gland concentration decreased under the limit of quantitation at 360min. Fat, ovaries, epididymis and adrenal gland had the highest concentration of nicousamide of all tissues examined, excluding the gastrointestinal tract. The brain had the lowest concentration of nicousamide of the tissues measured. The concentration of nicousamide in kidney, a reported target tissue, was lower.Nicousamide was excreted approximately 0.5% and 2% into the bile fluid in male and female rats through 72h postdose (30mg/kg). In feces, 18.2% was recovered in male rats and 21.4% in female rats. Less than 0.05% was apparent in the urine of both male and female rats. The total recovery after oral administration was 18.7% in male rats and 23.4% in female rats through 72h postdose.The plasma proteins binding of nicousamide to rats and human was approximately 100%.The major metabolic pathway of nicousamide was believed to be the nitro reduction. XLF-1[3-(3'-carboxy-4'-hydroxy-anilino-carbo-)-6amino-7-hydroxy-8-me- thyl-coumarin] was the main metabolite of nicousamide found in rat and human liver microsomes. The metabolite of nicousamide in rat plasma, urine, bile and liver was identified to be consistent with XLF-1. No changes of nicousamide and its metabolite levels in rat plasma, urine and bile were observed after hydrolysis withβ-glucuronidase and sulfatase which indicated that little of nicousamide was existed as glucuronate or sulfate conjugates.The formation of nicousamide metabolite can be catalized by certain reductases in both rat liver microsomes and cytosol. The major enzymes involved in the nicousamide reduction in liver microsomes were NADPH-cytochrome c reductases, cytochrome b5 reductases and CYP2C6. The reduction metabolism of nicousamide observed in cytosol was mediated by xanthine oxidase and DT-diaphorase.The highest metabolic rate of nicousamide was found in intestine, followed by fat and liver. Intestinal bacteria were also involved in the reduction of nicousamide with 50% decreasing after 2h incubation under anaerobic conditions. It was conclude that the poor oral bioavailability of nicousamide was mainly due to the reduction of nicousamide in intestine, liver and intestinal bacteria.The effect of nicousamide on liver mocrosomal CYP450s (CYP3A4, CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP2C19) was evaluated in both in vivo and in vitro studies. Nicousamide showed no effect on rat liver microsomal protein and CYP450 content. It was also found that nicousamide had no significant induction or inhibition on the activity of CYP450s, GST and UDPGT.
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