Hepatic Transcript Profiles of Cytochrome P450 Genes Predict Sex Differences in Drug Metabolism

Abstract

Safety assessments of new drug candidates are an important part of the drug development and approval process. Often, possible sex-associated susceptibilities are not adequately addressed, and better assessment tools are needed. We hypothesized that hepatic transcript profiles of cytochrome P450 (P450) enzymes can be used to predict sex-associated differences in drug metabolism and possible adverse events. Comprehensive hepatic transcript profiles were generated for F344 rats of both sexes at nine ages, from 2 weeks (preweaning) to 104 weeks (elderly). Large differences in the transcript profiles of 29 drug metabolizing enzymes and transporters were found between adult males and females (8-52 weeks). Using the PharmaPendium data base, 41 drugs were found to be metabolized by one or two P450 enzymes encoded by sexually dimorphic mRNAs and thus were candidates for evaluation of possible sexually dimorphic metabolism and/or toxicities. Suspension cultures of primary hepatocytes from three male and three female adult rats (10-13 weeks old) were used to evaluate the metabolism of 11 drugs predicted to have sexually dimorphic metabolism. The pharmacokinetics of the drug or its metabolite was analyzed by liquid chromatography/tandem mass spectrometry using multiple reaction monitoring. Of those drugs with adequate metabolism, the predicted significant sex-different metabolism was found for six of seven drugs, with half-lives 37%-400% longer in female hepatocytes than in male hepatocytes. Thus, in this rat model, transcript profiles may allow identification of potential sex-related differences in drug metabolism. SIGNIFICANCE STATEMENT: The present study showed that sex-different expression of genes coding for drug metabolizing enzymes, specifically cytochrome P450s, could be used to predict sex-different drug metabolism and, thus, provide a new tool for protecting susceptible subpopulations from possible adverse drug events.

Fig. 1.

Principal component (PC) analysis of expression of 298 drug metabolism/transporter genes. Expression of hepatic drug metabolism genes varies with sex and age in untreated F344 rats. Each sphere represents the hepatic expression profile of a single rat, with females represented by black vertices and males without. Numbers and colors indicate age (week). Arrowed line indicates trajectory of profiles with age for males (blue line) and females (red line). Dotted circles show large expression profile difference between males and females at ages of 8–52 week.

Fig. 2.

Real-time qPCR measurement of P450 mRNAs in untreated suspension cultures of primary hepatocytes derived from male (solid line) and female (dashed line) F344 rats. (A) Cyp2c7, (B) Cyp3a62, (C) Cyp3a2, and (D) Cyp2c11 were measured at 0, 30, 60, 90, and 120 minutes of culture. Measurements were normalized to the lowest measured value for each P450 and expressed as relative expression. P450 mRNA levels were quantified in hepatocytes from three males and three females, and all measurements were made in duplicate. The average and S.E.M. are shown for each culture time. Glyceraldehyde-3-phosphate dehydrogenase was used as the internal endogenous control. Males and females expressed significantly different levels of mRNA coding for Cyp2c7 (P < 0.01), Cyp3a62 (P < 10⁻⁵), Cyp3a2 (P < 10⁻³⁰), and Cyp2c11 (P < 10⁻²⁸) by Student’s t test.

Fig. 3.

Western blot analysis of P450 proteins in untreated suspension cultures of primary hepatocytes derived from male and female F344 rats. (A) CYP2C11 and (B) CYP3A2, along with a control protein, actin, were measured at 0, 30, 60, 90, and 120 minutes of culture. P450 protein levels were measured in hepatocytes from three males and three females, and representative blots are shown. Replicate of the sample at time 0 are shown in (A).

Fig. 4.

Metabolism of selected drugs during incubation with suspension cultures of primary hepatocytes derived from male (solid line) and female (dashed line) F344 rats. (A) azelastine, (B) buspirone, (C) doxorubicin, (D) fentanyl, (E) glimepiride, (F) irinotecan, (G) phenytoin, (H) pravastatin, and (I) tamsulosin were measured at 5, 15, 30, 60, 90, and 120 minutes of culture. Drug levels were measured in triplicate at each time in hepatocyte cultures derived from three male and three female rats. The average drug remaining in the culture and S.E.M. are shown for both male and female at each culture time. Reliable measurements were not possible for buspirone at 120 minutes. For each drug, the average drug remaining at each time point was compared between the sexes using a one-tail t test (* and ** indicate significant difference with P value <0.05 and <0.01, respectively). The calculations of slope and testing for heterogeneity (or homogeneity) of the slopes of the regression lines between males and females were performed using a generalized linear model procedure (Proc GLM) as described in Materials and Methods. Males metabolized azelastine, buspirone, fentanyl, glimepiride, and tamsulosin significantly more rapidly than females (P < 0.01).

Fig. 5.

Metabolism of tazarotene and formation of tazarotenic acid from tazarotene in suspension cultures of primary rat hepatocytes derived from male (solid line) and female (dashed line) F344 rats. Drug levels were measured in triplicate at each time (5, 15, 30, 60, 90, and 120 minutes of culture) in hepatocyte cultures derived from three male and three female rats. The average relative drug amount in the culture and S.E.M. are shown for both male (filled square) and female (filled circle) at each culture time. The calculations of slope and testing for heterogeneity (or homogeneity) of the slopes of the regression lines between males and females were performed using a generalized linear model procedure (Proc GLM) as described in Materials and Methods. (A) No significant difference between males and females was seen in the metabolism of tazarotene. (B) No significant difference between males and females was seen in the metabolism of tazarotene to tazarotenic acid. Dotted line indicates the average of male and female relative tazarotenic acid levels.

Fig. 6.

Formation of fexofenadine from terfenadine in suspension cultures of primary rat hepatocytes derived from male (solid line) and female (dashed line) F344 rats. Fexofenadine levels were measured in triplicate at each time in hepatocyte cultures derived from three male and three female rats. The average drug remaining in the culture and S.E.M. are shown for both male (filled square) and female (filled circle) at each culture time. The average drug remaining at each time point was compared between the sexes using a one-tail t test (* and ** indicate significant difference with P value <0.05 and <0.01, respectively). The calculations of slope and testing for heterogeneity (or homogeneity) of the slopes of the regression lines between males and females were performed using a generalized linear model procedure (Proc GLM) as described in Materials and Methods. Males metabolized terfenadine to fexofenadine significantly more rapidly than females (P < 0.0001).

Fig. 7.

Cytotoxicity of doxorubicin (A) and terfenadine (B). LDH release from hepatocytes was measured 24 hours after addition of the tested drugs (1 times to 30 times C_max; C_max is 110 ng/ml for doxorubicin and 229 ng/ml for terfenadine; Supplemental Table 3) and cytotoxicity expressed as the percentage of total cellular LDH. Significant cytotoxicity compared with untreated control cultures is indicated by * (P < 0.05). Significant difference in cytotoxicity between male and female hepatocytes at corresponding times is indicated by # (P < 0.05). Shown are the average measurements and S.E.M. for hepatocytes derived from three male (solid line) and three female (dashed line) F344 rats.