Drug metabolism letters最新文献

Background: Cytochrome P450 3A4 (CYP3A4) is an important drug-metabolizing enzyme that is expressed in the liver and small intestine of humans. Various factors influence the expression of CYP3A4, but gender difference in CYP3A4 expression remains debatable.

Objective: To clarify gender difference of hepatic and intestinal CYP3A4 in CYP3A-humanized mice generated by a human artificial chromosome (HAC) vector system. The CYP3A-humanized (CYP3AHAC) mice have essential regulatory regions, including promoters and enhancers, and unknown elements affecting the expression of CYP3A4.

Methods: We examined the expression and activity of hepatic and intestinal CYP3A4 in male and female CYP3A-HAC mice. CYP3A activity was determined as α- and 4-hydroxylation activity of triazolam in liver and intestinal microsomes. Expression level of CYP3A protein was determined by Western blot analysis. Expression level of CYP3A4 mRNA was measured by quantitative real-time PCR.

Results: The results showed that triazolam hydroxylation activities and protein levels of CYP3A in the liver were significantly higher in female than in male CYP3A-HAC mice, whereas those in the intestine were not significantly different between the genders. In addition, the expression of CYP3A4 mRNA showed a tendency similar to that found for the activity and expression of CYP3A protein in the liver and intestine of CYP3A-HAC mice.

Conclusion: These findings suggest that the expression and activity levels of CYP3A4 in the liver are higher in females than in males, whereas there is no gender difference in the levels in the intestine of CYP3A-HAC mice.

Background: The regulatory guidances on metabolites in safety testing (MIST) by US Food and Drug Administration (FDA) and International Conference on Harmonisation (ICH) describe the necessity to assess exposures of major circulating metabolites in humans at steady state relative to exposures achieved in nonclinical safety studies prior to the initiation of large scale clinical trials. This comparison can be accomplished by measuring metabolite concentrations in animals and humans with validated bioanalytical methods. However, bioanalysis of metabolites in multiple species and multiple studies is resource intensive and may impact the timelines of clinical studies.

Method: A simple, reliable and accurate method has been developed for quantitative assessment of metabolite coverage in preclinical safety species by mixing equal volume of human plasma with blank plasma of animal species and vice versa followed by an analysis using LC-SRM or LC-HRMS. Here, we explored the reliability and accuracy of this method in several development projects at Genentech and compared the results to those obtained from validated bioanalytical methods.

Results: The mixed-matrix method provided comparable accuracy (within ±20%) to those obtained from validated bioanalysis but does not require authentic standards or radiolabeled compounds, which could translate to time and resource savings in drug development.

Conclusion: Quantitative assessment of metabolite coverage in safety species can be made using mixed matrix method with similar accuracy and scientific rigor to those obtained from validated bioanalytical methods. Moving forward, we are encouraging the industry and regulators to consider accepting the mixed matrix method for assessing metabolite exposure comparisons between humans and animal species used in toxicology studies.

Background: Within the sulfotransferase (SULT) superfamily of metabolic enzymes, SULT1A1 and 1A3/4 isoforms are of particular interest, due to their abilities to catalyze the sulfation of phenolic endobiotics and xenobiotics. Although the difference in their substrate specificity is well documented, an isoform-specific quantification method is still not available.

Objective: To detect and quantify SULT1A1 and 1A3/4 in S9 fractions and cell lines using targeted mass spectrometry-based proteomics.

Method: Samples were tryptically digested, and signature peptides were quantified using liquid chromatography- multiple reaction monitoring mass spectrometry (LC-MRM/MS). Stable isotopelabeled (SIL) peptides were used as internal and calibration standards. SULT1A1 and SULT1A3/4 were quantified in various S9 fractions and cell line samples.

Results: Intraday and interday variabilities were low for relative quantification in S9 and cell line matrices (<8%). Expression profiles were validated using Western blot analysis of S9 fractions and lentiviral transduced SULT1A-overexpressing cell lines.

Conclusion: A reproducible method for simultaneous quantification of SULT1A1 and SULT1A3/4 in S9 fractions and cell line samples was established and validated.

Background: Non-selective chemical inhibitors of phase I and phase II enzymes are commonly used in in vitro metabolic studies to elucidate the biotransformation pathways of drugs. However, the inhibition of the inhibitors on efflux and uptake transporters is not well investigated, potentially leading to unexpected and ambiguous results in these studies.

Objective: The commonly used metabolizing enzyme inhibitors, 1-aminobenzotriazole (ABT), SKF- 525A, pargyline, allopurinol, menadione, methimazole, piperine and raloxifene, were examined for their potential inhibition of the major hepatic ABC (ATP binding cassette) and SLC (solute carrier) transporters.

Methods: Different concentrations of the metabolizing enzyme inhibitors were used to study their effects on ABC and SLC transporters expressed in MDR1-MDCKI, Bcrp1-MDCKII, OATP1B1-HEK, OATP1B3-HEK, OCT1-HEK, OCT3-HEK cells and MRP2 vesicles.

Results: ABT, allopurinol and methimazole had no inhibitory effects on MDR1, Bcrp1, MRP2 or on OATP1B1, OATP1B3, OCT1 or OCT3. Pargyline did not inhibit OATP1B1 or OATP1B3, but weakly inhibited OCT1 and OCT3. In contrast, SKF-525A showed inhibition of not only MDR1, Bcrp1 and MRP2 but also OATP1B1, OATP1B3 and OCT1. Menadione and raloxifene weakly inhibited Bcrp1, but the inhibition of raloxifene on MDR1 was as potent as on the xanthine oxidase pterin oxidation. Piperine showed inhibition of MDR1, Bcrp1, OATP1B1, OCT1 and OCT3.

Conclusion: ABT, pargyline, allopurinol and methimazole have no inhibitory effects on the studied ABC and SLC transporters, suggesting the inhibitors are unlikely to cause confounding inhibition of transporters when used in metabolism studies. However, SKF525A, menadione, raloxifene and piperine can inhibit the activities of ABC and/or SLC transporters.

Background: Epidermal Growth Factor (EGF) is a well-known mitogen that has importance in cell proliferation and differentiation. This property has led to the common use of EGF as an additive to some cell culture media. EGF has been previously shown to modulate constitutive Cytochrome P450 (CYP) expression in vitro.

Objectives: To assess the influence of EGF on the basal and induced expression of CYP3A4, CYP1A2 and CYP2B6 in plated human hepatocytes.

Methods: Human hepatocytes were treated with EGF with and without in the presence of positive control inducers. After treatment, CYP isoform mRNA expression and enzyme activity were measured.

Results: EGF at concentrations ranging from 0.001-500 ng/mL resulted in a concentration-dependent decrease in basal CYP3A4 catalytic activity by up to 92%. In contrast, rifampicin (RIF)-induced activity was decreased only slightly (up to 23%). CYP3A4 mRNA also decreased in an EGF concentrationdependent manner. In contrast to CYP3A4, CYP1A2 and CYP2B6 activity and mRNA were either not suppressed or suppressed to a lower extent. The preferential effect with CYP3A4 was confirmed in 4 additional donors using a single concentration of EGF (10 ng/mL) and time-dependence experiments revealed that suppression appeared after only 24h of treatment.

Conclusion: Because of the larger effect on the basal CYP3A4 compared to the induced response, EGF as a media additive enables a higher dynamic range in a CYP3A4 induction assay, potentially expanding the range of donor hepatocytes suitable for use in induction studies. These findings also suggest that EGF may be an important regulator of CYP3A4 expression in vivo.

Background: Nuclear Receptors (NRs), including PXR and CAR, are presumed to be ligand-dependent transcription factors, but ligand binding is not an absolute requirement for activation. Indeed, many compounds activate PXR and CAR by indirect mechanisms. Detecting these indirect activators of specific nuclear receptors in vitro has been difficult. As NR activation of either or both PXR and CAR can lead to drug-drug interactions and adverse drug effects, false negatives obtained with screening tools incapable of detecting indirect activators could present liabilities.

Objective: The aim of this study was to establish assays that identify indirect activators of human PXR and CAR.

Methods: Commercially available human PXR and CAR transactivation assays were used for analyses.

Results: We show that transactivation assays containing full-length nuclear receptors with native promoters can identify indirect activators of human CAR and PXRwhen compared to those of commercially available assays containing only the LBD of PXR and CAR. Of these two assay systems, only human PXR and CAR1 assays with full-length receptors and native promoters are capable of detecting indirect and ligand activators. With this capability, several kinase inhibitors were identified that activate PXR and CAR by indirect mechanisms. Furthermore by using both the LBD and full-length receptors, phenobarbital and midostaurin were found to be direct and indirect activators of PXR while human CAR activation by phenobarbital occurs by indirect mechanisms only.

Conclusion: Cell based transactivation assays employing the full-length receptors and native promoters identify both direct and indirect activators of either or both human PXR and CAR.

Background: Timolol is clinically administered topically (ocular) to reduce intraocular pressure and treat open-angle glaucoma. Ocular administration of timolol in low doses (0.5% w/v in the form of eye drops) has led to challenges for in vivo metabolite identification. An understanding of drug metabolism in the eye is important for clinical ocular therapeutics and potential drug candidates.

Methods: We aimed to investigate the metabolism of timolol in rat ocular and liver S9 fractions, as well as rat ocular tissue and plasma following a 0.5% topical (ocular) dose of timolol. We explored the potential in vitro metabolic bioactivation in the eye/liver by conducting trapping studies for putative aldehyde and iminium ion intermediates that may arise from the morpholine functionality.

Results: Oxidative metabolism of timolol to its major metabolite (M4) in ocular S9 and recombinant rat cytochrome P450 (CYP) isoforms supports the possible role of rat ocular CYP2D2, 2D4, and/or 2D18. Observation of N-acetyl-timolol (M5) is suggestive that the ocular N-acetyltransferases may also play a larger role in ocular disposition of timolol, a previously unreported finding. This research is the first comprehensive report of in vitro ocular metabolism of timolol in rat.

Conclusion: This study also indicates that in vitro hepatic metabolism is over-predictive of ocular metabolism following topically ocular dosed timolol. The research, herein, highlights the eye as an organ capable of first pass metabolism for topical drugs. Thus, new ophthalmologic considerations for studying and designing long term topical therapies in preclinical species are needed in drug discovery.