Androgen regulation of renal uridine diphosphoglucuronosyltransferase 1A1 in rats.

Many phase I and II enzymes are under hormonal regulation, resulting in sex-related expression patterns. This sex-related enzyme expression can result in differential metabolism of physiologically active endogenous substances, altered xenobiotic clearance, and differences in susceptibility to drug toxicities. Treatment of female Sprague-Dawley (SD) rats with 5 mg testosterone propionate/kg/day, 2 ml/kg s.c. for 8 days resulted in induction of renal uridine diphosphoglucuronosyltransferase (UGT) 1A1, as determined by immunoblot and probe substrate activity. Glucuronidation activity for mycophenolic acid, a substrate for rat UGT1A1, 1A6, and 1A7, was significantly elevated approximately 2-fold in renal microsomes from testosterone propionate-treated animals. Protein expression of rat UGT1A1 was also dramatically increased, whereas 1A6 and 1A7 remained unchanged as a result of treatment. Male SD rats were determined to express greater renal UGT1A1 than age-matched female rats. These data support the androgen regulation of rat renal UGT1A1.

Effect of omeprazole on the plasma concentrations of indinavir when administered alone and in combination with ritonavir.

PURPOSE: The effects of omeprazole on indinavir when administered alone or in combination with ritonavir were evaluated. METHODS: Fourteen men and women age 18-55 years not infected with human immunodeficiency virus who met study qualifications were randomized to receive placebo, 20 mg of omeprazole, or 40 mg of omeprazole daily. After seven days, the single-dose pharmacokinetic profile of an 800-mg dose of indinavir alone or in combination with 200 mg of ritonavir was evaluated. Study participants received each of four study regimens in one of four randomly assigned orders. Blood samples were collected, and plasma indinavir and ritonavir concentrations were analyzed using high-performance liquid chromatography. RESULTS: The coadministration of 20 or 40 mg of omeprazole with indinavir significantly reduced the mean indinavir area under the concentration-versus-time curve (AUC) from 30.0 mg x hr/L (95% confidence interval [CI], 21.9-41.1 mg x hr/L) to 19.7 mg x hr/L (95% CI, 14.6-26.8 mg x hr/L) or 16.0 mg x hr/L (95% CI, 11.8-21.7 mg x hr/L), respectively (p < 0.002). The addition of 200 mg of ritonavir to 800 mg of indinavir in combination with 40 mg of omeprazole significantly increased the mean indinavir AUC from 30.0 mg x hr/L (95% CI, 21.9-41.1 mg x hr/L) to 46.6 mg x hr/L (95% CI, 34.0-63.8 mg x hr/L), but it did not significantly affect mean omeprazole concentrations (p < or = 0.02). CONCLUSION: The AUC of indinavir was substantially decreased in healthy volunteers who received omeprazole 20 or 40 mg daily for seven days before the administration of a single 800-mg dose of indinavir. Concomitant administration of ritonavir 200 mg with indinavir in participants receiving omeprazole led to a significant increase in the AUC of indinavir.

Lysosomes contribute to anomalous pharmacokinetic behavior of melanocortin-4 receptor agonists.

PURPOSE: A series of melanocortin-4 receptor (MC4R) agonists, developed for use as anti-obesity agents, were found to have unusual pharmacokinetic behavior arising from excessive retention in the liver, with nearly undetectable levels in plasma following oral administration in mice. This work investigates the molecular basis of the prolonged liver retention that provided a rational basis for the design of an analog with improved behavior. MATERIALS AND METHODS: The livers of mice were harvested and techniques were utilized to fractionate them into pools differentially enriched in organelles. The distribution of organelles in the fractions was determined using organelle-specific enzymatic assays. Livers from mice dosed with drug were fractionated and comparisons with organelle distributions assisted in determining the subcellular localization of the drug. Further analysis in cell culture systems was used to confirm results from liver fractionation studies and also allowed for more extensive evaluations to examine the mechanism for organelle compartmentalization RESULTS: Fractionation of livers following oral administration of the agonist showed sequestration in lysosomes. Subsequent evaluations in a cell culture system confirmed this finding. Agents used to disrupt acidification of lysosomes led to decreased lysosomal accumulation of the drug, which implicated a pH-partitioning type sequestration mechanism. These findings led to the rational synthesis of an analog of the parent compound with properties that reduced lysosomal sequestration. When this compound was examined in mice, the liver retention was found to be greatly reduced and plasma levels were significantly elevated relative to the parent compound. CONCLUSIONS: Weakly basic drugs with optimal physicochemical properties can be extensively sequestered into lysosomes according to a pH-partitioning type mechanism. When administered orally in animals, this particular sequestration event can manifest itself in long term retention in the liver and negligible levels in blood. This work revealed the mechanism for liver retention and provided a rational platform for the design of a new analog with decreased liver accumulation and better opportunity for pharmacokinetic analysis and therapeutic activity.

The contribution of intestinal UDP-glucuronosyltransferases in modulating 7-ethyl-10-hydroxy-camptothecin (SN-38)-induced gastrointestinal toxicity in rats.

Life-threatening diarrhea afflicts a considerable percentage of patients treated with irinotecan, an anticancer agent with effects elicited through its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38). The primary detoxification pathway for SN-38 is glucuronidation. The purpose of this study was to evaluate the role that intestinal UDP-glucuronosyltransferases (UGTs) have from hepatic UGTs in modulating this diarrhea. To investigate this, Gunn rats devoid of UGT1A activity were injected with recombinant adenoviral vectors expressing UGT1A1, 1A6, and 1A7, resulting in reconstituted hepatic UGT expression comparable to a heterozygote. Hepatic microsome studies indicated that 4 to 7 days after adenoviral injection, transfected Gunn rats (j/jAV) had SN-38 glucuronide (SN-38G) formation rates three times higher than control heterozygote rats (j+AV). The adenovirus did not impart any glucuronidating capacity to the intestine in j/jAV rats, whereas j+AV rats possessed intestinal UGT function. After the administration of 20 mg/kg/day irinotecan i.p. to j/jAV rats 4 days after adenovirus injection, diarrhea ensued before the fourth irinotecan dose. j+AV rats were spared the diarrhea, and the toxicity was mild compared with the j/jAV rats, as measured by diarrhea scores, weight loss, and histological assessments of the cecum and colon. The pharmacokinetics of irinotecan, SN-38, and SN-38G indicate that the systemic exposure of SN-38 and SN-38G was higher and lower, respectively, in j/jAV rats. Despite this, the biliary excretion of irinotecan and metabolites was similar. Because intestinal UGTs are the main discriminating factor between j/jAV and j+AV rats, their presence seems to be critical for the gastrointestinal protection observed in j+AV rats.

UDP-glucuronosyltransferase 1A1 is the principal enzyme responsible for etoposide glucuronidation in human liver and intestinal microsomes: structural characterization of phenolic and alcoholic glucuronides of etoposide and estimation of enzyme kinetics.

Etoposide, an important anticancer agent, undergoes glucuronidation both in vitro and in vivo. In this study, three isomeric glucuronides of etoposide, including one phenolic (EPG) and two alcoholic glucuronides (EAG1 and EAG2), were biosynthesized in vitro with human liver microsomes (HLMs), and identified by liquid chromatography-electrospray ionization-mass spectrometry and confirmed by beta-glucuronidase cleavage. In vitro UDP-glucuronosyltransferase (UGT) reaction screening with 12 recombinant human UGTs demonstrated that etoposide glucuronidation is mainly catalyzed by UGT1A1. Although UGT1A8 and 1A3 also catalyzed the glucuronidation of etoposide, their activities were approximately 10 and 1% of UGT1A1. Enzyme kinetic study indicated that the predominant form of etoposide glucuronide in HLMs and human intestinal microsomes (HIMs) was EPG, whereas EAG1 and EAG2 were the minor metabolites, with approximately an 8 to 10% glucuronidation rate of EPG. For the formation of EPG, the V(max) of HLMs (110 pmol/min/mg protein) was very similar to that of recombinant UGT1A1 (124 pmol/min/mg protein), whereas the V(max) of HIMs (54.4 pmol/min/mg protein) was 2-fold lower than those of HIMs and UGT1A1. The K(m) values of HLMs (530 microM) and HIMs (608 microM) were 2-fold higher than that of UGT1A1 (285 microM). The V(max)/K(m) values for the formation of EPG were 0.21 and 0.09 microl/min/mg protein for HLMs and HIMs, respectively. The data indicated that UGT1A1 is principally responsible for the formation of etoposide glucuronides, mainly in the form of phenolic glucuronide, suggesting that etoposide can be used as a highly selective probe substrate for human UGT1A1 in vitro.

Gender-related differences in mycophenolate mofetil-induced gastrointestinal toxicity in rats.

Mycophenolate mofetil (MMF), the prodrug of mycophenolic acid (MPA), is included in current combination immunosuppressive regimens following organ transplant. Treatment with MMF often results in dose-limiting gastrointestinal (GI) side effects. The underlying mechanisms responsible for these side effects are not fully understood, but exposure of the intestinal epithelia to MPA during enterohepatic recycling may be involved. The present study demonstrated that female rats are more susceptible to MMF-induced GI toxicity than male rats. Female Sprague-Dawley rats treated chronically with an oral dose of 50 mg of MPA equivalents/kg/day experienced greater GI toxicity than male rats, as measured by diarrhea grade and weight loss. Intestinal microsomes harvested from the upper jejunum of female rats had approximately 3-fold lower MPA glucuronidation rates compared with male rats. In the remaining areas of the small and large intestine, there was also a trend toward decreased glucuronidation in the female rats. The area under the plasma concentration-time curve (AUC) for MPA following an oral dose of 50 mg of MPA equivalents/kg was roughly similar between genders, whereas the AUC for mycophenolic acid phenolic glucuronide (MPAG) was significantly lower in female rats. Female rats also excreted half of the biliary MPAG as male rats. The greater susceptibility of female rats to MMF-induced gastrointestinal toxicity, despite diminished intestinal MPA exposure via reduced biliary excretion of MPAG, may result from reduced protection of enterocytes by in situ glucuronidation. Likewise, susceptibility to MMF-induced GI toxicity in humans may also result from variable intestinal glucuronidation due to UDP glucuronosyltransferase polymorphisms or differential expression.

Integration of hepatic drug transporters and phase II metabolizing enzymes: mechanisms of hepatic excretion of sulfate, glucuronide, and glutathione metabolites.

The liver is the primary site of drug metabolism in the body. Typically, metabolic conversion of a drug results in inactivation, detoxification, and enhanced likelihood for excretion in urine or feces. Sulfation, glucuronidation, and glutathione conjugation represent the three most prevalent classes of phase II metabolism, which may occur directly on the parent compounds that contain appropriate structural motifs, or, as is usually the case, on functional groups added or exposed by phase I oxidation. These three conjugation reactions increase the molecular weight and water solubility of the compound, in addition to adding a negative charge to the molecule. As a result of these changes in the physicochemical properties, phase II conjugates tend to have very poor membrane permeability, and necessitate carrier-mediated transport for biliary or hepatic basolateral excretion into sinusoidal blood for eventual excretion into urine. This review summarizes sulfation, glucuronidation, and glutathione conjugation reactions, as well as recent progress in elucidating the hepatic transport mechanisms responsible for the excretion of these conjugates from the liver. The discussion focuses on alterations of metabolism and transport by chemical modulators, and disease states, as well as pharmacodynamic and toxicological implications of hepatic metabolism and/or transport modulation for certain active phase II conjugates. A brief discussion of issues that must be considered in the design and interpretation of phase II metabolite transport studies follows.

Differential rates of glucuronidation for 7-ethyl-10-hydroxy-camptothecin (SN-38) lactone and carboxylate in human and rat microsomes and recombinant UDP-glucuronosyltransferase isoforms.

7-ethyl-10-hydroxy-camptothecin (SN-38), the active metabolite of the anti-cancer agent irinotecan, contains a lactone ring that equilibrates with a carboxylate form. Since SN-38 lactone is the active and toxic form, it is prudent to examine whether the more soluble carboxylate is a surrogate for SN-38 lactone conjugation. Therefore, relative rates of glucuronidation and isoform specificity of SN-38 lactone and carboxylate were characterized. The stability of SN-38 lactone and carboxylate in incubation mixtures of microsomes and UDP-glucuronosyltransferase (UGT) isoforms was used to determine optimal incubation times. Microsomal incubations were conducted using rat and human intestinal and hepatic microsomes and human and rat recombinant UGT1A isoforms. Where estimates of lactone and carboxylate glucuronidation rates could not be established due to short incubation times and detection limits, kinetic modeling was used to recover these rate constants. The stability experiments revealed that the lactone was stabilized by rat microsomes, however, the opposite was observed in human microsomes and recombinant isoforms. For all tissues and most UGT isoforms examined, the lactone consistently had catalytic rates up to 6-fold greater than the carboxylate. The rank order of glucuronidation for both SN-38 lactone and carboxylate was 1A7 > 1A1 > 1A9 > 1A8 and 1A7 > 1A8 > 1A1 for human and rat isoforms, respectively. This study provides further support that SN-38 lactone and carboxylate may be considered pharmacokinetically distinct agents. The in vivo impact of this conjugation difference is unknown, since variations in protein binding and transport proteins may affect intracellular concentrations of the lactone or carboxylate.

Altered pharmacokinetics of omeprazole in cystic fibrosis knockout mice relative to wild-type mice.

The cftr(tm1Unc)-knockout (CF-KO) mouse is being evaluated as a model of increased drug clearance noted clinically in patients with cystic fibrosis (CF). This study investigated whether CF-KO mice exhibited altered omeprazole pharmacokinetics compared with wild-type mice. Clinical observations have suggested reduced responses to omeprazole in CF children, which may reflect alterations in bioavailability or clearance. Omeprazole was dosed intravenously and orally in a crossover fashion to age-matched CF-KO and wild-type male and female mice. The mean terminal half-life of approximately 6 min was found across genotype and gender groups. Blood to plasma ratio estimates for omeprazole were similar across genders and genotypes with a mean value of 0.69. Omeprazole blood clearance (Cl(b)) was significantly higher in both male (190 ml/min/kg) and female (168 ml/min/kg) CF-KO mice compared with wild-type controls of the same gender (73 ml/min/kg for males and 100 ml/min/kg for females). The distributional volume of omeprazole in CF-KO mice was also statistically higher than in control genotypes. Bioavailability estimates were similar between CF-KO and wild-type females but were unavailable for male mice, due to the large variability in plasma concentrations after oral administration and the difficulty estimating the area under the plasma curve when the terminal half-life suggested absorption rate-limited disposition. Potential mechanisms for the pharmacokinetic differences observed with omeprazole in CF-KO mice may be increased hepatic blood flow or an up-regulation of hepatic transporters. These results may provide support for using the CF-KO mouse as a model for the altered disposition of drugs in CF.