Sex differences in drug metabolism have been reported in numerous species, including humans. In rats and mice, sexdependent differences in circulating growth hormone profiles are responsible for the differential expression of multiple sexdependent isoforms of cytochrome P450, which is the basis for the sexual dimorphism in drug metabolism. In contrast, very little is known about sex differences in human isoforms of cytochrome P450 and their regulation by growth hormone. In this study, we have examined the effects of physiologic-like exposure doses to dexamethasone and/or pulsatile (masculine) or constant (feminine) human growth hormone on expression levels of CYP3A4, 1A2, 2D6, and 2E1 and the glucocorticoid and growth hormone receptors in hepatocyte cultures obtained from men and women donors. We report that growth hormone can regulate expression of CYP3A4, 1A2, and 2D6. The masculine-like pulsatile growth hormone profile suppresses dexamethasone-induced CYP3A4, 1A2, and 2D6, whereas the feminine-like constant profile is permissive allowing isoform expression to be equal to or greater than glucocorticoid induction alone. There are intrinsic sexual differences in hepatocytes of men and women resulting in different levels of responsiveness of CYP3A4, 1A2, and hormone receptor expression to the same sexually dimorphic growth hormone profiles. Last, although real, the sexually dimorphic effects of growth hormone on human cytochrome P450 expression are not as dramatic as those observed in rats and could easily be overlooked by the heterogeneous backgrounds of human populations.

ABSTRACT:Although in vivo expression levels of the male-specific hepatic isoforms of cytochrome P450 (P450) (CYP2C11, CYP2C13, CYP2A2, and CYP3A2) are determined by the episodic growth hormone profile secreted by male rats, these isoforms have been completely refractory to growth hormone regulation in hepatocyte culture. By using species-specific rat growth hormone, at subphysiologic in vivo concentrations administered in two daily episodic pulses, we successfully induced CYP2C11 and CYP2A2 to near normal concentrations. Whereas inductive levels of CYP2C13 were subnormal, CYP3A2 was unresponsive to all hormonal treatments, quickly declining to undetectable concentrations. In agreement with in vivo findings, we observed that induction levels of the isoforms were always greatest when the male hepatocytes were exposed to the masculine-like episodic growth hormone profile and least stimulated by the continuous feminine-like hormone profile. When administered alone, dexamethasone consistently increased isoform levels. However, when administered with growth hormone, the glucocorticoid was always antagonistic, suppressing growth hormone induction of CYP2C11, CYP2C13, and CYP2A2. Finally, the P450 isoforms were completely unresponsive to all treatments when the hepatocytes were derived from female rats, supporting earlier findings that expression levels of sexually dimorphic P450 isoforms are inherently irreversible between sexes.

The lipid kinase phosphoinositide 3-kinase γ (PI3Kγ) has attracted attention as a potential target to treat a variety of autoimmune disorders, including multiple sclerosis, due to its role in immune modulation and microglial activation. By minimizing the number of hydrogen bond donors while targeting a previously uncovered selectivity pocket adjacent to the ATP binding site of PI3Kγ, we discovered a series of azaisoindolinones as selective, brain penetrant inhibitors of PI3Kγ. This ultimately led to the discovery of 16, an orally bioavailable compound that showed efficacy in murine experimental autoimmune encephalomyelitis (EAE), a preclinical model of multiple sclerosis.

This paper is available online at http://dmd.aspetjournals.org ABSTRACT:Rat liver, as well as other species, contains numerous sex-dependent isoforms of cytochrome P450 (P450) that are regulated by the sexually dimorphic profiles of circulating growth hormone. During puberty, young adulthood, and senescence, changes in the hormonal profiles appear to be responsible for alterations in ageassociated expression levels of selective P450 isoforms. In contrast, little is known about the growth hormone secretory profiles and their P450-dependent expression levels during middle age. In the present study, we observed subtle changes in the hormonal concentrations, and frequencies of peaks and interpulse periods in the sexually dimorphic growth hormone profiles of 1-year-old male and female rats correlated to suppression of male-specific isoforms CYP2C11 and CYP2C13 and female-predominant CYP2C7. To identify possible causes for the age-associated changes in the circulating growth hormone profiles, the responsiveness of the hypothalamic-pituitary axis to growth hormone secretagogues clonidine and growth hormone-releasing factor (GRF) were examined in middle-aged male and female rats. In spite of the same sexually dimorphic response in young adult and middle-aged rats to both secretogogues (males > females), the pituitary somatotrophs in the older animals exhibited a dramatic decrease in sensitivity to clonidine, characterized by subnormal growth hormone release levels and an inordinate delay in pituitary response to clonidine stimulation. Results from similar studies conducted on middle-aged arcuate nucleus-lesioned rats suggest that a decline in GRF secretion is a possible contributor to the age-associated alterations in plasma growth hormone profiles during middle age. These changes in GRF-induced, sexually dimorphic secretory growth hormone profiles and the accompanying decline in P450 expression levels may anticipate similar, but more profound, changes to occur during senescence.

The importance of photoperiod and ambient temperature on the accumulation of cadmium in the liver and kidneys of bank voles was determined in the present study. Males and females, aged 1 month, were given 3.0 micrograms Cd ml-1 drinking water and divided into four groups according to photoperiod (16 h light/8 h dark and 8 h light/16 h dark) and ambient temperature (20 or 5 degrees C); liver and kidneys were removed for cadmium as well as copper, iron and zinc analyses at the end of 6 weeks. Bank voles exposed to 5 degrees C in both photoperiods consumed approximately 30% less water containing cadmium than those kept at 20 degrees C. However, the total accumulation of cadmium in the liver and kidneys of males and females exposed to the low temperatures was 4.3-4.8 and 2.2-3.3 times less than that in animals maintained at room temperature in the long and short photoperiod, respectively. Simultaneously, the low temperature brought about an increase in the copper concentrations in the liver (12-43%) and kidneys (47-78%), giving rise to an inverse correlation between the cadmium accumulation and the tissue copper concentration. In contrast to cadmium and copper, the concentrations of iron and zinc were affected primarily by photoperiod. These findings indicate that ambient temperature is an important determinant of cadmium retention in the bank vole. It appears that low temperature decreases tissue cadmium accumulation not only by reducing cadmium intake but also through changes in copper metabolism.

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Adult male and female rat hepatocytes were individually transplanted into the spleens of adult male and female rats. The recipients were euthanized at either 8, 16, 30 or 45 weeks following transplantation at which time hepatic and splenic levels of liver-specific rat albumin mRNA as well as sex-dependent transcript levels of CYP2C11, -2C12, -2C7, -2A1 and -3A2, comprising >60% of the total concentration of hepatic constituent cytochrome P450, were determined. Whereas the pre-infused hepatocytes expressed their expected cytochrome P450 sexual dimorphisms (female-specific CYP2C12, male-specific CYP3A2 and female-predominant CYP2A1), their post transplantational competence now reflected the sexual dimorphisms of the recipient (as observed in the host’s liver) supporting the concept that the sex-dependent growth hormone circulating profiles are the determinants regulating the expression levels of hepatic cytochromes P450. Also expressed at normal concentrations in the pre-infused hepatocytes, male-specific CYP2C11 and female-predominant CYP2C7 were inexplicably undetectable in the spleens of both recipient males and females, irrespective of the sex of the donor hepatocytes, almost one year after transplantation.

The ability to predict human liver-to-plasma unbound partition coefficient (K puu ) is important to estimate unbound liver concentration for drugs that are substrates of hepatic organic anion-transporting peptide (OATP) transporters with asymmetric distribution into the liver relative to plasma. Herein, we explored the utility of PXB chimeric mice with humanized liver that are highly repopulated with human hepatocytes to predict human hepatic disposition of OATP substrates, including rosuvastatin, pravastatin, pitavastatin, valsartan, and repaglinide. In vitro total uptake clearance and transporter-mediated active uptake clearance in C57 mouse hepatocytes were greater than in PXB chimeric mouse hepatocytes for rosuvastatin, pravastatin, pitavastatin, and valsartan. Consistent with in vitro uptake data, enhanced hepatic uptake and resulting total systemic clearance were observed with the above four compounds in severely compromised immune-deficient (SCID) control mice compared with the PXB chimeric mice, which suggest that mouse has a stronger transporter-mediated hepatic uptake than human. In vivo liver-to-plasma K puu from PXB chimeric and SCID control mice were also compared, and rosuvastatin and pravastatin K puu in SCID mice were more than 10-fold higher than that in PXB chimeric mice, whereas pitavastatin, valsartan, and repaglinide K puu in SCID mice were comparable with K puu in PXB chimeric mice. Finally, PXB chimeric mouse liver-to-plasma K puu values were compared with the reported human K puu , and a good correlation was observed as the PXB K puu vales were within 3-fold of human K puu . Our results indicate that PXB mice could be a useful tool to delineate hepatic uptake and enable prediction of human liver-toplasma K puu of hepatic uptake transporter substrates. SIGNIFICANCE STATEMENTWe evaluated PXB mouse with humanized liver for its ability to predict human liver disposition of five organic anion-transporting polypeptide transporter substrates. Both in vitro and in vivo data suggest that mouse liver has a stronger transporter-mediated hepatic uptake than the humanized liver in PXB mouse. More importantly, PXB liver-to-plasma unbound partition coefficient (K puu ) values were compared with the reported human K puu , and a good correlation was observed. PXB mice could be a useful tool to project human liver-to-plasma K puu of hepatic uptake transporter substrates.The work was supported by Vertex Pharmaceutical Company Limited and Chimeric Mouse with Humanized Liver (CMHL) Consortium. Y.M. is an employee of PhoenixBio Co., Ltd., Hiroshima, Japan. He provided PXB mice but did not have any additional role in conducting the experiment, performing the data analysis, or preparing the manuscript. All other authors (B.