Drug Metabolism and Disposition最新文献

Exemestane (EXE) is an aromatase inhibitor used for the treatment of estrogen receptor-positive breast cancer. The metabolism of EXE includes reduction to form 17-β-hydroxy-EXE (17β-DHE) and subsequent UGT2B17-mediated glucuronidation to form 17-β-hydroxy-EXE-17-O-β-D-glucuronide (17β-DHE-Gluc), and GSTA1-mediated glutathione conjugation of EXE and 17β-DHE and subsequent sequential metabolism by γ-glutamyl transferases and dipeptidases to form 6-methylcysteinylandrosta-1,4-diene-3,17-dione (EXE-Cys) and 6-methylcysteinylandrosta-1,4-diene-17-β-hydroxy-3-one (17β-DHE-Cys). The aim of the present study was to determine the effects of UGT2B17 and GSTA1 genotype on the serum levels of EXE and its metabolites among subjects taking EXE. Genotypes of UGT2B17 and GSTA1 were determined by real-time polymerase chain reaction and serum EXE, 17β-DHE, 17β-DHE-Gluc, EXE-Cys, and 17β-DHE-Cys were quantified by ultra performance liquid chromatography-mass spectrometry. Shunting was observed between the 2 metabolic pathways of EXE, with serum EXE levels increased with increasing numbers of either the UGT2B17∗2 or GSTA1∗B alleles (P_trend < .0001). 17β-DHE-Gluc levels decreased (P_trend < .0001) and EXE-Cys levels increased (P_trend < .0001) with combined increasing numbers of the UGT2B17∗2 allele and decreasing numbers of the GSTA1∗B allele. Although GSTA1 genotype alone showed no effect on serum 17β-DHE-Gluc levels, the UGT2B17 (∗2/∗2) genotype was associated with a 10.4-fold decrease (P < .0001) in serum 17β-DHE-Gluc levels as compared with wild-type UGT2B17. The GSTA1 (∗B/∗B) genotype was associated with 1.4- (P < .0001) and 1.3-fold (P = .0005) decreases, whereas UGT2B17 (∗2/∗2) genotype was associated with 2.1- (P < .0001) and 2.3-fold (P < .0001) increases in EXE-Cys and 17β-DHE-Cys formation, respectively, as compared with their respective wild-type genotypes. These results suggest that GSTA1 and UGT2B17 genotypes play an important role in EXE metabolism variability and potentially in patient response to EXE. SIGNIFICANCE STATEMENT: To our knowledge, the present pharmacogenetic study is the first to examine interindividual variability in exemestane (EXE) metabolism for the 2 major phase II metabolism pathways of EXE. The UGT2B17 genotype was found to contribute to substantial interindividual variability in the metabolism of EXE, however, GSTA1 genotype was also significantly associated with altered EXE metabolism. Given their high polymorphic allele frequency, genotypes of UGT2B17 and GSTA1 potentially play important roles in interindividual variability in patient response including EXE efficacy and toxicity.

It is important to identify diagnostic biomarkers and demographic factors that characterize patients with COVID-19-drug interactions to mitigate safety and efficacy issues. This study investigated the impact of 6 different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on the mRNA expression of 36 genes associated with inflammation, anti-inflammation, transcriptional regulation, drug metabolism, and membrane transport in the lung and nasal turbinate tissues of 21 male and 21 female Syrian golden hamsters. Furthermore, the study investigated how individual and combined treatments with angiotensin II and D614G variant influence the lung expression of these genes in 7- and 4-month-old 23 male and 23 female Syrian golden hamsters. This study showed for the first time that SARS-CoV-2 variants cause greater heterogeneous dysregulation of drug processing genes in hamster lung tissue compared with nasal turbinate, because of an imbalance between inflammatory and anti-inflammatory responses, with P.1, D614G, and Delta variants playing a major role in this dysregulation. The study discovered a sex-dependent dysregulation of lung NAT2 expression by the D614G variant, and a more severe age-dependent dysregulation of genes associated with inflammation, anti-inflammation, transcriptional regulation, drug metabolism, and membrane transport in D614G variant-infected Syrian golden hamster lung tissues. On the contrary, angiotensin II administration did not contribute to the dysregulation of any of these genes in hamster lung tissues. Finally, potential biomarkers were identified for diagnosing dysregulation of drug processing genes based on SARS-CoV-2 variants, infection site, age, and sex. SARS-CoV-2 variants, infection site, age, and sex should be considered when treating patients with COVID-19-drug interactions. SIGNIFICANCE STATEMENT: COVID-19-drug interactions have been observed in several hospitalized patients; however, the clinicopathologic and demographic factors that define at risk patient population are poorly understood. This study shows for the first time that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, infection site, age, and sex impact SARS-CoV-2-mediated dysregulation of drug processing genes in Syrian golden hamsters. Potential biomarkers for SARS-CoV-2-drug processing gene interactions based on these factors were discovered and may be useful for the diagnosis and management of patients at risk of COVID-19-drug interactions.

Mercaptopurine (MP)-based maintenance therapy is essential to cure acute lymphoblastic leukemia (ALL). However, insufficient myelosuppression and MP-related hepatotoxicity occasionally occur. The investigation of the strategies to improve the efficacy and toxicities is warranted. The role of allopurinol combination therapy has been reported, but its effects need further verification, and the underlying mechanism remains unclear. Two hundred and eight pediatric patients requiring maintenance therapy were enrolled to investigate the effect of allopurinol combination and the potential mechanisms underlined. Blood samples were collected to determine drug metabolite levels [6-thioguanine nucleotides (6-TGN), 6-methyl mercaptopurine (6-MMP), DNA-thioguanine (DNA-TG)], thiopurine methyltransferase (TPMT) activity, and NUDT15/TPMT genotypes. Hematological values and hepatic function were measured as well. The results indicated that TPMT∗1/∗1/NUDT15∗1/∗1 patients with inadequate myelosuppression exhibited significantly higher doses of MP, increased 6-MMP concentrations, and elevated 6-MMP:6-TGN/6-MMP: DNA-TG ratios (P < .001), indicating that MP-methylated pathway was overactivated and intensifying MP dosing could not lead to adequate myelosuppression effect, but instead primarily lead to higher risk of hepatotoxicity. In contrast, after allopurinol combination with dose reduction in MP, TPMT activity decreased significantly leading to a significant reduction in the 6-MMP:6-TGN and 6-MMP: DNA-TG ratios and improvement in alanine aminotransferase and aspartate aminotransferase levels. Meanwhile, DNA-TG levels increased by an average of 257.5 fmol/μg DNA, resulting in better control of white blood cell and absolute neutrophil count. In conclusion, low-dose MP combined with allopurinol, rather than intensifying MP dose, could be an alternative strategy for better efficacy and lower risk of hepatotoxicity. SIGNIFICANCE STATEMENT: Patients requiring higher mercaptopurine doses may indicate the overactivation of methylation metabolism. Dose escalation is unlikely to improve myelosuppression but will increase the risk of hepatotoxicity. Low-dose mercaptopurine combined with allopurinol can improve efficacy and reduce the risk of hepatotoxicity.

Hydroxamic acid is an excellent metal ion chelating group and is commonly used in the design of histone deacetylase inhibitors. Despite its utility, the hydroxamic acid group suffers from poor metabolic stability and undergoes rapid conversion to carboxylic acid, a process that has generally been described as hydrolytic metabolism. By using the histone deacetylase inhibitor, bisthianostat as a model drug, this study provided novel insights into the role of cytochrome P450 (P450) enzymes in the metabolism of the hydroxamic acid group into the carboxylic acid metabolite (M351). The primary formation of M351 was observed in liver microsomes after incubating bisthianostat with different human liver, intestinal, and plasma fractions. Chemical inhibition experiments further indicated that P450 enzyme-mediated oxidation was the main pathway for the generation of M351, with hydrolysis making a minor contribution. ¹⁸O-labeling isotope experiments and Griess assays demonstrated that the peroxoferric species of P450 enzymes acted as a nucleophile, attacking the carbonyl carbon of hydroxamic acid, leading to the formation of carboxylic acid and reactive nitrogen species. The metabolism of 8 hydroxamic acid derivatives in human liver microsomes indicated that P450 enzymes exhibited substrate specificity in catalyzing the conversion of hydroxamic acids to carboxylic acids. Molecular docking results further revealed that oxidative metabolism occurred when the hydroxamic acid group was in appropriate proximity to the P450 catalytic center. Overall, this study demonstrated the important role of P450 enzymes in hydroxamic acid metabolism and provided valuable insights for future rational design and clinical applications of hydroxamic acid-based drugs. SIGNIFICANCE STATEMENT: Hydroxamic acid is a functional group widely used in histone deacetylase inhibitors. It has been traditionally held that the primary metabolic pathway of hydroxamic acids involves hydrolytic metabolism, resulting in the formation of carboxylic acid metabolites. However, this study revealed a previously unrecognized metabolic pathway: cytochrome P450-mediated oxidative cleavage. The results of this study provided novel mechanistic insights into the metabolism of hydroxamic acids, with significant implications for rational drug design, metabolic prediction, and safety evaluation of this important pharmacophore.

Interindividual differences in drug efficacy and safety are driven by variability in the expression of absorption, distribution, metabolism, and excretion (ADME) genes. A comprehensive analysis of 400 ADME-related genes across all available tissues from the Genotype-Tissue Expression v10 RNA-seq data set was conducted to characterize sex- and age-related expression variations. Principal component analysis revealed distinct tissue-specific ADME expression signatures, with the liver showing the highest ADME functional capacity, followed by the small intestine and kidney cortex. Sex-stratified analysis identified 117 ADME genes with significant expression differences (adjusted P < .05), with the digestive system showing numerous differentially expressed genes, predominantly female-biased. Notable sex differences included CYP3A4, CES1, CYP2C19, GSTM1, and various UDP-glucuronosyltransferases (UGTs), which showed a substantial female-biased expression. Age-related analysis across 6 age brackets (20-29 through 70+ years) revealed divergent pathway remodeling: cytochrome P450 enzymes showed consistent age-related declines across tissues, whereas UGT enzymes exhibited substantial increases in the digestive, immune, and urinary systems. Combined sex and age analysis demonstrated that elderly females showed the most pronounced changes, with UGT and transporter expression changing in most tissues relative to young males. This atlas reveals the complex demographic influences on drug metabolism, with female-biased and age-dependent expression changes in clinically important ADME genes, supporting the need for sex- and age-specific dosing strategies in precision medicine. SIGNIFICANT STATEMENT: This study provides a systematic analysis of the sex and age transcriptional differences in drug-metabolizing enzyme and transporter expression across human tissues. These data identify clinically significant demographic variations that may directly inform precision dosing strategies.

The drug candidate, 2-methoxy-N-[3-[4-[3-methyl-4-[(6-methyl-3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714), was discontinued because of hepatotoxicity observed in clinical studies. CP-724,714 is a substrate of aldehyde oxidase (AO) found in the human liver cytosol. CP-724,714 metabolization by AO in cryopreserved human hepatocytes generates several oxidative metabolites, including (E)-N-(3-(2-hydroxy-4-(3-methyl-4-(6-methylpyridin-3-yloxy)phenylamino)quinazolin-6-yl)allyl)-2-methoxyacetamide (CP-724,714-AOM). However, the structure of CP-724,714-AOM has not been identified. Therefore, we aimed to identify the structure of CP-724,714-AOM, determine the propensity of CP-724,714 and CP-724,714-AOM for toxic effects, and understand the underlying hepatotoxic mechanism in humans. A synthesized oxidized CP-724,714, identified as CP-724,714-AOM, was consistent with the AO metabolite of CP-724,714 generated in the human liver cytosol. The enzymatic kinetic parameters of CP-724,714 were calculated as a K_m value of 9.17 ± 0.70 μM and a V_max value of 3.57 ± 0.10 pmol/min/mg of human liver cytosol proteins, assessed by the production of CP-724,714-AOM. CP-724,714 showed a weak inhibitory effect on the bile salt export pump without inhibiting multidrug resistance protein 2, whereas CP-724,714-AOM showed no inhibitory effect. The trapping assay showed that both compounds formed reactive metabolites in the human liver microsomes. Moreover, the inflammasome activation potentials of the compounds were observed in HepaRG cells, which can also generate CP-724,714-AOM from CP-724,714. Thus, our findings show that confirming AO susceptibility at an early stage of drug development is crucial for understanding the potential risks of AO metabolism in terms of pharmacokinetics and toxicity. SIGNIFICANCE STATEMENT: The structure of the aldehyde oxidase metabolite of 2-methoxy-N-[3-[4-[3-methyl-4-[(6-methyl-3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) was identified using a synthetic standard, and its kinetic parameters in the human liver cytosol were determined. Reactive metabolite formation and inflammasome activation by CP-724,714 and (E)-N-(3-(2-hydroxy-4-(3-methyl-4-(6-methylpyridin-3-yloxy)phenylamino)quinazolin-6-yl)allyl)-2-methoxyacetamide were observed.

This study investigated the effects of indoxyl sulfate (IS), an endogenous metabolite and uremic toxin, on arsenic trioxide pharmacokinetics in acute promyelocytic leukemia patients with varying renal function. Plasma IS levels demonstrated a significant positive correlation with monomethylarsonic acid and dimethylarsinic acid concentrations in patients (P < .0001). In adenine-induced renally impaired rats, IS similarly correlated with elevated plasma inorganic arsenic (iAs), monomethylarsonic acid, and dimethylarsinic acid levels. Protein expression analysis indicated a downregulation of renal aquaporin (AQP) 7 and AQP3. In vitro studies confirmed that IS selectively inhibits AQP7 expression (62.1% reduction at 100 μM) through aryl hydrocarbon receptor activation in human embryonic kidney 293T cells, while AQP3 remained unaffected. Collectively, IS increases plasma arsenic concentration in renally impaired acute promyelocytic leukemia patients via aryl hydrocarbon receptor-mediated suppression of renal AQP7. SIGNIFICANCE STATEMENT: This study reveals that indoxyl sulfate inhibits renal aquaporin 7 via aryl hydrocarbon receptor activation, increasing plasma arsenic in arsenic trioxide-treated acute promyelocytic leukemia patients with renal impairment. As the first demonstration of this mechanism, to our knowledge, it provides crucial insights for optimizing therapy and reducing toxicity risks.

COVID-19 remains a significant health threat, particularly to people over the age of 65 with existing comorbidities like diabetes, hypertension, cancer, and viral infections. Despite expedited drug approvals, drug-drug interaction profiles for COVID-19 antiviral drugs have not yet been completely defined. The antiviral drugs remdesivir and molnupiravir are ester prodrugs with carboxylesterases (CES) playing a critical role in their bioactivation. In this study, we investigated the effect of COVID-19 antiviral drugs on CES hydrolysis activity. Of the 3 drugs tested, remdesivir inhibited 50% of CES2 activity in a nanomolar concentration range. Furthermore, time-dependent inhibition of CES2 activity by remdesivir was identified with an IC₅₀ shift of nearly 3-fold from 0.188 μM after 5-minute preincubation to 0.068 μM following 40-minute preincubation. Remdesivir inactivation of CES2 was characterized by a k_inact/K_I value of 1.6 × 10³ M^-1.s^-1. Through equilibrium dialysis and substrate protection experiments, we were able to further substantiate previous findings of the irreversible CES2 inhibition by remdesivir. Finally, in silico docking analysis of remdesivir to CES2 supported the proposed mechanism for covalent modification of the CES2 active site involving the catalytic triad serine via the phosphate group of remdesivir. SIGNIFICANCE STATEMENT: This study provides insights into human carboxylesterase inhibition by COVID-19 drugs and these findings demonstrate the underlying inhibition mechanism by remdesivir of recombinant human carboxylesterase 2.

Three-dimensional (3D) cultures of primary human hepatocytes (3D PHH) are successfully used to reduce and replace the use of animal experiments in biomedical research. Yet, the initial formation of 3D PHH is highly dependent on the supplementation with FBS. However, the molecular composition of FBS and its effects on cultured cells are poorly understood. Moreover, FBS is prone to batch-to-batch variation, immunogenic risk, and lack of adherence to the replacement, refinement, and reduction of animal experiments. Here, we demonstrate that FBS can be fully replaced by animal-free substitutes, thus facilitating fully chemically defined and animal serum-free 3D PHH cultures. Specifically, we combined a previously developed animal-free substitute cocktail with a normoglycemic (5.5 mM glucose and 0.58 ng/mL insulin) chemically defined culture medium. Morphological and viability evaluations, along with global proteomics data, demonstrated that serum-free cultured 3D PHH have comparable viability and functional performance of cytochrome P450s, rendering this medium useful for long-term studies and in vitro absorption, distribution, metabolism, excretion, and toxicity applications. This study marks a significant advancement in the development of animal serum-free culture conditions for primary human cell cultures, paving the way for more reliable and ethical in vitro studies. SIGNIFICANCE STATEMENT: Most in vitro cell models rely on FBS. However, the use of FBS leads to inconsistent experimental results and raises serious ethical concerns. In this study, a chemically defined animal product-free cell culture medium with physiologically relevant levels of key hormones and nutrients for liver spheroid cultures was developed and evaluated. This study marks a significant advancement in the development of animal serum-free culture conditions for primary human cell cultures used in drug disposition studies.