Raloxifene glucuronidation in liver and intestinal microsomes of humans and monkeys: contribution of UGT1A1, UGT1A8 and UGT1A9.

1. Raloxifene is an antiestrogen that has been marketed for the treatment of osteoporosis, and is metabolized into 6- and 4'-glucuronides by UDP-glucuronosyltransferase (UGT) enzymes. In this study, the in vitro glucuronidation of raloxifene in humans and monkeys was examined using liver and intestinal microsomes and recombinant UGT enzymes (UGT1A1, UGT1A8 and UGT1A9). 2. Although the K(m) and CL(int) values for the 6-glucuronidation of liver and intestinal microsomes were similar between humans and monkeys, and species differences in Vmax values (liver microsomes, humans > monkeys; intestinal microsomes, humans < monkeys) were observed, no significant differences were noted in the K(m) or S50, Vmax and CL(int) or CLmax values for the 4'-glucuronidation of liver and intestinal microsomes between humans and monkeys. 3. The activities of 6-glucuronidation in recombinant UGT enzymes were UGT1A1 > UGT1A8 >UGT1A9 for humans, and UGT1A8 > UGT1A1 > UGT1A9 for monkeys. The activities of 4'-glucuronidation were UGT1A8 > UGT1A1 > UGT1A9 in humans and monkeys. 4. These results demonstrated that the profiles for the hepatic and intestinal glucuronidation of raloxifene by microsomes were moderately different between humans and monkeys.

Effect of UDP-glucuronosyltransferase 1A8 polymorphism on raloxifene glucuronidation.

Raloxifene is an antiestrogen marketed for the treatment of osteoporosis. The major metabolic pathway of raloxifene is glucuronidation at 6- and/or 4'-positions, which is mainly catalyzed by UDP-glucuronosyltransferase 1A8 (UGT1A8) expressed in extrahepatic tissues such as the small intestine and colon. Two non-synonymous allelic variants, termed UGT1A8*2 (518C>G, A173G) and UGT1A8*3 (830G>A, C277Y), have been found in Caucasian, African-American and Asian populations. In this study, the effect of amino acid substitutions in UGT1A8 on raloxifene glucuronidation was studied using recombinant UGT1A8 enzymes of wild-type (UGT1A8.1) and variant UGT1A8 (UGT1A8.2 and UGT1A8.3) expressed in Sf9 cells. Raloxifene 6- and 4'-glucuronidation by UGT1A8.1 exhibited negative allosteric kinetics. The Km and Vmax values of UGT1A8.1 were 15.0 µM and 111 pmol/min/mg protein for 6-glucuronidation, and 9.35 µM and 232 pmol/min/mg protein for 4'-glucuronidation, respectively. The kinetics of raloxifene 6-glucuronidation by UGT1A8.2 was positive allosteric, whereas the kinetics of raloxifene 4'-glucuronidation was negative allosteric. The S50 value of raloxifene 6-glucuronidation was markedly low (1.2%) compared with the Km value of UGT1A8.1, and the Km value for raloxifene 4'-glucuronidation was 29% that of UGT1A8.1. The Vmax value for raloxifene 6-glucuronidation by UGT1A8.2 was comparable to that of UGT1A8.1, whereas the Vmax value for raloxifene 4'-glucuronidation was significantly lower (54%) than that of UGT1A8.1. The activities of raloxifene 6- and 4'-glucuronidation in UGT1A8.3 were markedly lower than those of UGT1A8.1. In mycophenolic acid glucuronidation, the kinetics by wild-type and variant UGT1A8s fitted the Michaelis-Menten model. The Km and Vmax values of UGT1A8.1 were 123 µM and 4820 pmol/min/mg protein, respectively. The Km and Vmax values of UGT1A8.2 were comparable to those of UGT1A8.1. The Km value of UGT1A8.3 was similar to that of UGT1A8.1, whereas the Vmax value was reduced to 2.4% of UGT1A8.1. These findings suggest that A173G and C277Y substitutions of UGT1A8 change the metabolic ability toward raloxifene, and that the polymorphic alleles of UGT1A8 may influence the clinical response and bioavailability of medicines metabolized mainly by UGT1A8.

Fish immunization using a synthetic double-stranded RNA Poly(I:C), an interferon inducer, offers protection against RGNNV, a fish nodavirus.

Viral nervous necrosis (VNN), caused by a fish nodavirus, is one of the most serious fish diseases worldwide. Here we report a unique vaccination method in sevenband grouper Epinephelus septemfasciatus using a synthetic double-stranded RNA polyinosinic polycytidylic acid (Poly(I:C)), an interferon inducer, followed by challenge with a live fish nodavirus. Fish injected with Poly(I:C) at 200 microg fish(-1) were highly protected from artificial challenge with red-spotted grouper nervous necrosis virus (RGNNV) (relative percentage survival, RPS: 100%), and specific antibodies against RGNNV were detected in sera from survivors. Moreover, the surviving fish were protected from rechallenge with RGNNV (relative percent survival RPS: 100%). Thus, it was confirmed that specific immunity against RGNNV was established in sevenband grouper by injection with live RGNNV following Poly(I:C) administration. Antiviral state was induced in fish by injection with Poly(I:C) at > or = 50 microg fish(-1), but no toxic response was observed in the fish even if Poly(I:C) was injected at a dose of 200 microg fish . In fish injected with Poly(I:C) at 200 pg fish(-1), a high level of antiviral state of > 90% RPS against RGNNV challenge lasted for at least 4 d after Poly(I:C) injection. However, no curative effect by Poly(I:C) injection was observed in fish already infected with RGNNV. It is considered that the present immunization method using Poly(I:C) followed by a live virus injection could offer protection against various viral infections in a broader range of fish species.

Enhanced propagation of fish nodaviruses in BF-2 cells persistently infected with snakehead retrovirus (SnRV).

Fish nodaviruses are causative agents of viral nervous necrosis causing high mortality in cultured marine fishes around the world. The first successful isolation of fish nodavirus was made with SSN-1 cells, which are persistently infected with snakehead retrovirus (SnRV). In the present study, a BF-2 cell line persistently infected with SnRV (PI-BF-2) was established to evaluate the influence of SnRV on the production of fish nodavirus. The PI-BF-2 cells were slightly more slender than BF-2 cells, but no difference was observed in propagation rate between both cell lines. No difference was observed in production of SnRV between PI-BF-2 and SSN-1 cell lines. Although both PI-BF-2 and BF-2 cell lines showed no cytopathic effect (CPE) after inoculation of striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), these fish nodaviruses could be amplified in BF-2 cells, and moreover, production of fish nodaviruses in the PI-BF-2 cell line was more than 40 times higher than in BF-2 cells. Thus, it was concluded that BF-2 cell permissiveness to fish nodaviruses was enhanced by persistent infection with SnRV. Furthermore, homologous cDNA to genomic RNA of SJNNV was detected from both PI-BF-2 and SSN-1 cell lines persistently infected with SnRV. The amount of nodavirus cDNA in SJNNV-inoculated PI-BF-2 cells was clearly lower than that in SJNNV-inoculated SSN-1 cells.