Drug Metabolism Letters - Volume 11, Issue 1, 2017

Background: Clopidogrel is a key antiplatelet drug that has substantial interpatient variability in pharmacodynamic response. Patients with lesser degrees of platelet inhibition in response to clopidogrel appear to be at increased risk of cardiovascular events. Obesity is an independent risk factor for cardiovascular morbidity and mortality due to atherothrombotic events and represents a group of patients who are in need of optimized antithrombotic therapy. Central to the obesity-related risk of atherothrombosis is a pro-thrombotic state characterized by increased levels of coagulation factors, impaired fibrinolysis and platelet hyper-reactivity, which results from the interaction among the features clustering in obesity: insulin resistance, inflammation, oxidative stress, and endothelial dysfunction. Results: A number of reports have demonstrated that obesity is a risk factor for a reduced clopidogrel pharmacodynamic response. The inflammatory state associated with obesity, particularly a metabolic endotoxemia, may set in motion, a number of mechanisms that increase platelet reactivity, suppress cytochrome P450 enzyme activity, and increase platelet turnover, all contributing to a poor clopidogrel response. Conclusion: Comprehensive understanding of the mechanisms underlying obesity-related high onclopidogrel platelet reactivity will help in the optimization of antithrombotic therapy in this patient population.

Background: Flunitrazepam (FNZ) is a potent hypnotic, sedative, and amnestic drug used to treat insomnia and as a pre-anesthetic agent. The illicit practice in drug-facilitated sexual assault led to important clinical and forensic concerns. Objective: In this work the metabolism of FNZ, and pharmacological- and toxicological-related effects, were fully reviewed. Methods: FNZ and related known metabolizing enzymes and metabolites were searched in books and in PubMed (U.S. National Library of Medicine) without a limiting period. Results: Major metabolic pathways include N-demethylation, 3-hydroxylation, nitro-reduction, and further N-acetylation of the amino group, yielding N-desmethylflunitrazepam, 3-hydroxy-flunitrazepam, 7-aminoflunitrazepam, and 7-acetamidoflunitrazepam, respectively. A combination of these reactions may lead to the formation of 7-amino-N-desmethylflunitrazepam, 7-acetamido-N-desmethylflunitrazepam, 3- hydroxy-7-aminoflunitrazepam, 3-hydroxy-7-acetamidoflunitrazepam, 3-hydroxy-N-desmethylflunitrazepam and glucuronide conjugates. Genotypic variations in enzymes, interactions with other drugs or stability of FNZ during storage can result in large interindividual variability in the toxicological results. Conclusion: It is aimed that knowing the metabolism of FNZ may lead to the development of new analytical strategies for early detection, since this drug is typically present in very low concentrations in blood and urine when used to facilitate sexual assault.

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: Bupropion (BUP) has a potential to be an effective pharmacotherapy for smoking cessation during pregnancy. Smoking during pregnancy stimulates placental carbonyl reductases that catalyze the biotransformation of BUP. 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen of cigarette smoke. Carbonyl reduction of NNK into 4- methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) constitutes a major step in NNK detoxification. Thus, placentas of pregnant smokers on BUP therapy can become a site of drug-drug interaction. Therefore, we investigated the effect of continuous exposure to BUP and cigarette smoke on the activity of placental carbonyl reductases in the formation of NNAL from NNK. Methods: The reductive metabolism of NNK was determined using microsomal and cytosolic subcellular fractions of placentas obtained from non-smoking women treated with BUP for depression, and women not exposed to BUP: non-smokers (control) and smokers. The effect of BUP and its metabolites on the reductive metabolism of NNK was investigated using subcellular fractions of control placentas. Results: The formation of NNAL from NNK by placental cytosolic fractions of heavy smokers (≥20 cigarettes per day) was lower than that of control (12.1±3.5 nmol.mgP-1 vs 16.5±6.0 nmol.mgP-1, P<0.05). While being exposed to BUP, the activity of placental carbonyl reductases remained unaffected, the formation of NNAL in the placental cytosolic fraction decreased only in the presence of high concentrations of BUP metabolites. Conclusion: Smoking during pregnancy decreases the detoxifying capacity of soluble carbonyl reductases towards NNK. Given the experimental conditions, exposure to BUP and its metabolites should not impede the reductive metabolism of NNK by placenta in vivo.

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 and Objective: Doxorubicin, an anthracycline anti-cancer drug, is effective for breast cancer and childhood lymphoma. Chronic cardiotoxicity has been known as a critical adverse effect of doxorubicin and is attributed to its metabolite doxorubicinol produced by carbonyl reductase 1 (CBR1, SDR21C1). Some flavonoids have been reported to act as inhibitors for CBR1, therefore, commercially available juices containing flavonoids are likely to be applicable as a prophylactic treatment against doxorubicin-induced cardiotoxicity by suppression of doxorubicinol production. In the study, fruit juices containing flavonoids were investigated for inhibitory effects on CBR1. Method: Recombinant CBR1 protein was subjected to in vitro enzymatic assays with/without juices. An apple juice and a grapefruit juice were selected in the present study as candidates capable of inhib-iting CBR1. Results: The enzymatic assays revealed that both juices potently inhibit the CBR1-mediated metabolic conversion of doxorubicin to doxorubicinol in concertation-dependent manner. The concentrations required for obtaining 50% inhibition (IC50) were 0.0012% (v/v) and 0.0014% (v/v) for apple and grapefruit juices, respectively. Additionally, it is worth noting that these juices showed inhibitory effects on doxorubicin metabolism by CBR1 even at very low concentrations (0.0001% (v/v)). Conclusion: An apple juice and a grape fruit juice showed strong inhibitory effects on doxorubicin metabolism by CBR1 in vitro. These results suggest that the intake of flavonoid-containing juices can be a promising measure for protection against doxorubicin-induced cardiac toxicity, enabling patients to keep higher adherence with routine use in light of safety, economic performance and stable supply to market.

Background: CYP2D6 is one of the most significant polymorphic genes of drugmetabolizing enzymes due to its association with different metabolic activities and the pharmacokinetics of CYP2D6 substrates. Objective: The objective of this study was to explore for a novel haplotype of CYP2D6 in the Japanese population by using a large database of previous clinical studies. Methods: We analyzed CYP2D6 genotype data from a total of 723 Japanese individuals for 8 loci (100C>T, 1758G>A, 1846G>A, 2573 insertion of C, 2850C>T, 2988G>A, 4125 insertion of 9bp, and 4180G>C) and gene deletion. Genotypes were determined by the designated alleles CYP2D6*2, *4, *5, *10, *14A, *14B, *18, *21, and *41. Results: The frequencies of the common major haplotypes CYP2D6*1, *10, and *2 in the Japanese population were respectively 43.5%, 38.0%, and 11.3%. In 11 subjects, diplotypes of CYP2D6 were not identified and the genotypes at the 8 loci suggested that there were 2 minor haplotypes, one with only a variation at 4180G>C compared with the wild type CYP2D6*1 (Hap1, frequency: 0.4%) and one with only a variation at 100C>T (Hap2, frequency: 0.4%). The Hap1 haplotype is considered to have no effect on metabolic activity, while it is estimated that the Hap2 haplotype does have an effect on metabolic activity. By comparing with the allele nomenclature for CYP2D6, the Hap2 haplotype was considered to be a potentially novel haplotype involving 100C>T without 4180G>C. Conclusion: Using a large database of CYP2D6 genotypes in the Japanese population, we found a novel haplotype which involves 100C>T without 4180G>C. Although the haplotype will need to be confirmed by full sequencing, it may be a unique haplotype with an exception to the strong linkage disequilibrium between 100C>T and 4180G>C.

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: Cytochrome P450 (CYP) 2D6 is a major drug-metabolizing enzyme, responsible for eliminating 25% of marketed drugs. We recently identified SHP as a negative regulator of CYP2D6 expression and showed that factors that alter SHP expression influence CYP2D6 expression. Fenofibrate, an agonist of peroxisome proliferator-activated receptor α(PPARα), has been previously reported to upregulate SHP expression in the mouse liver. The objective of this study was to determine whether fenofibrate decreases CYP2D6 expression via upregulating SHP expression. Methods: CYP2D6-humanized transgenic mice were administered with fenofibrate (100 mg/kg/day intraperitoneally for 5 days) or vehicle control. Hepatic mRNA and protein expression levels of CYP2D6 and SHP were measured. Results: Results showed that while mRNA levels of SHP did not differ between the groups, protein levels of SHP increased by 2-fold in fenofibrate-treated mice. Despite the increased SHP protein levels, CYP2D6 expression did not decrease at the mRNA or protein levels. Similar results were observed in human hepatocytes treated with fenofibrate. Results from transient transfection and promoter reporter assays indicate that PPARα can transactivate CYP2D6 promoter, suggesting that the lack of CYP2D6 downregulation by fenofibrate may be in part due to the activation of CYP2D6 promoter by PPARα. Conclusion: These results indicate that fenofibrate has minimal effects on CYP2D6 expression despite increased SHP expression.