Drug Metabolism and Disposition最新文献

Camizestrant (AZD9833) is a next-generation oral selective estrogen receptor (ER) degrader and complete ER antagonist in phase 3 clinical development for the treatment of ER-positive breast cancer. Metabolite M14, an N-glucuronide of the acid metabolite M46 of camizestrant, is one of the major circulating metabolites in humans, in addition to a direct N-glucuronide M4. In light of the fact that the existence of the acidic functional group of the acid metabolite M46 prevented an efficient in vitro glucuronidation in human liver microsomes, a novel hybrid chemical and biological synthesis of AZ4678 (M14) was successfully achieved. The acidic functional group of AZ8713 (M46) was esterified with methanol to give the M46 methyl ester, which was then glucuronidated in human liver microsomes in the presence of UPDGA to form M14 methyl ester. Selective hydrolysis of M14 methyl ester by aqueous lithium hydroxide in tetrahydrofuran successfully generated AZ4678 (M14). The development of this synthesis route has introduced a new approach to synthesizing glucuronides of compounds with acidic functional groups that otherwise interfere with their susceptibility to glucuronidation. NMR analysis confirmed the structure of AZ4678 (M14) with the glucuronic acid attached to the position N45 of the azole nitrogen atom while retaining the original stereochemistry of camizestrant. Chromatography of AZ4678 (M14) was sensitive to 67% of organic content in samples as well as sensitive to solvents at a basic pH containing ammonium hydroxide, resulting in split peaks in liquid chromatography-mass spectrometry analysis. AZ4678 (M14) was stable in human feces and in aqueous solutions at a pH range of 1.5-12.0. SIGNIFICANCE STATEMENT: Metabolite M14 of camizestrant (AZD9833), a major circulating metabolite in humans, was successfully identified as an N-glucuronide of an acid metabolite of camizestrant, and its unusual chromatographic and stability properties were characterized. The development of a novel hybrid chemical and biological synthesis for M14 has introduced a new approach to synthesizing glucuronides of compounds with acidic functional groups that otherwise interfere with their own in vitro glucuronidation.

Interindividual variability in drug response stems largely from metabolic enzyme activity differences, with cytochrome P450 3A (CYP3A) being the predominant enzyme whose fluctuations critically impact exposure to narrow-therapeutic-index drugs (eg, tacrolimus) and anticancer agents. Current clinical probes such as midazolam (suboptimal hepatic uptake, sedation) and the erythromycin breath test (poor selectivity) remain inadequate. This study proposes deoxyschizandrin (DS), derived from Schisandra chinensis, as a novel in vivo CYP3A probe substrate. Using integrated rat in situ liver perfusion, whole-animal studies, physiologically based pharmacokinetic modeling, and proteomics, we demonstrated DS's advantageous properties: ideal biliary disposition (>80% hepatic accumulation, negligible parent/metabolite biliary excretion <0.02%); superior hepatic influx/uptake rate constants (0.24 s^-1 vs midazolam's 0.13 s^-1); robust metabolic kinetics (consistent in situ maximum metabolic rate [V_max] vs in vitro V_max: 3.30 vs 3.40 nmol/min/g liver); achievable in vivo enzyme saturation (physiologically based pharmacokinetic-predicted hepatic unbound concentrations > 5 × K_m) for ∼20 minutes after a bolus dose of 25 mg/kg, enabling linear metabolite generation and apparent V_max determination; and precise activity quantification (in vivo V_max strongly correlated with rat hepatic CYP3A2 protein content, R² = 0.89, P = .0047). Collectively, these findings demonstrate that DS enables accurate quantification of CYP3A activity through the V_max approach, thereby providing a promising candidate for the development of a clinically viable in vivo CYP3A probe. SIGNIFICANCE STATEMENT: This study identifies deoxyschizandrin as a novel, safe, and liver-specific in vivo probe substrate for cytochrome P450 3A (CYP3A). It enables the quantitative determination of hepatic CYP3A maximum metabolic capacity through a bolus dosing protocol, offering a promising candidate for the development of a clinically viable in vivo CYP3A probe.

Cytochrome P450s (CYPs) 2C subfamily (eg, 2C8, 2C9, and 2C19) and phase II enzymes such as uridine 5'-diphospho-glucuronosyltransferases (UGTs) are increasingly relevant in drug development and key targets for enzymatic induction. However, for these enzymes, weak induction signals in standard in vitro tools, such as sandwich-cultured human hepatocytes, challenge drug-drug interaction (DDI) risk assessment. This study evaluated an all-human hepatocyte coculture system (TruVivo) as a more sensitive model for CYP2Cs and UGT1A1 induction. After treatment of cells with rifampicin, carbamazepine, and phenytoin, we demonstrated robust mRNA and activity-fold-induction exceeding or meeting the 2-fold threshold in the coculture system, allowing for estimation of CYP2Cs and UGT1A1 induction parameters (Ind_C50, Ind_max), unlike sandwich culture. Using TruVivo Ind_C50, Ind_max of these precipitants in physiologically based pharmacokinetic (PBPK) modeling resulted in high predictive accuracy. In rifampicin studies, using TruVivo mRNA-derived data from the most sensitive donor 1 and average parameters across donors, was essential to properly predict in vivo DDI, particularly for object drugs mainly metabolized by CYP2Cs and UGT1A1, or with moderate to low CYP3A4 contribution (fm ≤ 0.5) in multipathway metabolism. For object drugs metabolized by CYP3A4 beyond 2Cs and UGTs, carbamazepine and phenytoin PBPK predictions highlighted the applicability of TruVivo uncalibrated CYP3A4 data for accurate assessment, whereas parameters calibrated against rifampicin showed a conservative trend in estimating DDI. Overall, the all-human coculture system, paired with PBPK, offers a breakthrough for CYP2Cs and UGT1A1 preclinical DDI induction risk assessment. SIGNIFICANCE STATEMENT: Cryopreserved human hepatocytes in sandwich culture show limited sensitivity toward cytochrome P450s 2C and uridine 5' -diphospho-glucuronosyltransferases induction, challenging in vitro-in vivo translation of the drug-drug interaction risk. This study confirms that TruVivo is a more sensitive in vitro model. By using physiologically based pharmacokinetic modeling, we investigated the impact of the measured induction parameters on predictive accuracy, showing TruVivo as a useful tool for cytochrome P450s 2C and uridine 5' -diphospho-glucuronosyltransferases risk assessment.

The pregnane X receptor (PXR), a ligand-activated transcription factor, regulates the expression of genes involved in endobiotic and xenobiotic metabolism, inflammation, and fibrosis. Disruption of PXR functions can affect processes critical to metabolic dysfunction-associated steatohepatitis (MASH) progression. Although ligand-dependent PXR functions are well studied, its regulation by post-translational modification, particularly phosphorylation, remains unclear. PXR has a conserved phosphorylation motif within its ligand binding domain (Ser347 in mice; Ser350 in humans). In vitro studies showed that this site mutation impairs human PXR transcriptional activity; however, the mechanism remains elusive. To investigate this phosphorylation site role in MASH development, wild-type and PXR Ser347Ala knock-in mutation (PXR-KI) mice were fed either a high-fat diet or a control chow diet for 16 weeks. On control chow diet, PXR-KI mice exhibited decreased expression of alternative bile acid (BA) synthesis genes compared with wild-type mice. On a high-fat diet, PXR-KI mice manifested more severe hepatic steatosis, revealed by elevated serum total cholesterol, and increased expression of genes involved in lipid metabolism. In addition, changes in BA metabolism and transporter genes suggested a cholestatic pattern in this group of mice. BA profiling showed higher levels of conjugated, hydrophilic, primary BA in the serum and liver, and increased unconjugated BA in the intestine. The data suggest that PXR Ser347 phosphorylation motif is essential for regulating PXR functions to maintain endobiotic metabolism and alleviate hepatotoxicity during MASH progression. SIGNIFICANT STATEMENT: The ligand-independent role of pregnane X receptor (PXR) is unclear. In phosphodeficient PXR knock-in mice, loss of Ser347 phosphorylation worsened hepatic steatosis and altered bile acid homeostasis under high-fat diet feeding, uncovering a novel role and therapeutic potential of PXR phosphorylation in fatty liver diseases.

This study examined the absorption, distribution, metabolism, and excretion of branaplam in healthy adult male volunteers and, additionally, compared the adult metabolite profiles obtained in plasma and urine to those obtained in infants with type 1 spinal muscular atrophy. Six volunteers received a single oral dose of 140 mg ¹⁴C-branaplam. Blood, plasma, urine, and fecal samples were analyzed using liquid scintillation counting, accelerator mass spectrometry, and liquid chromatography coupled with high-resolution or tandem mass spectrometry to assess radioactivity, generate metabolite profiles, and structurally characterize branaplam metabolites. Pediatric samples from various age groups were also evaluated. Mechanistic in vitro experiments enabled direct comparison between adult and pediatric results. The ¹⁴C-branaplam dose was well tolerated. Pharmacokinetic analysis showed that branaplam and metabolite UFB112 were the main circulating species, exhibiting delayed T_max (10 and 28 hours, respectively) and prolonged half-lives (218 and 199 hours, respectively). UFB112 formation was exclusively catalyzed by CYP3A4, and its plasma levels increased with age, reflecting hepatic enzyme maturation. Branaplam was primarily eliminated through metabolism. Renally excreted metabolites were formed via oxygenation, glucuronidation, glucosidation, or ribose conjugation, whereas metabolites in feces included glucosidation and oxidative products. Mass balance was almost complete, with 86.6% of the administered radioactivity recovered in urine and feces over 47 days. These findings highlight the pharmacokinetic behavior of branaplam and UFB112, including the role of the 2,2,6,6,-tetramethylpiperidine-1-oxyl moiety, in how metabolism of branaplam changes during physiological development. Mechanistic insights confirm that CYP enzyme ontogeny significantly influences metabolic profiles. SIGNIFICANCE STATEMENT: This study provides a comprehensive overview of the metabolism of the tetramethyl piperidine moiety, contextualizing enzyme maturation by comparing metabolic fates in infants and adults. It also clearly explains human metabolism of branaplam and summarizes a rare Adenosine Triphosphate pathway observed in these studies.

In our previous study, long-term cocultured hepatocytes were used to estimate the fraction of a drug metabolized by CYP3A4 (f_m,CYP3A4). Metabolic turnover was measured with and without a CYP3A4 selective inhibitor, and the results were verified against in vivo reference data. The current study followed a similar approach using direct or time-dependent inhibitors to evaluate f_m,CYP1A2, f_m,CYP2C8, f_m,CYP2C9, f_m,CYP2C19, and f_m,CYP2D6 for a set of marketed drugs. The used inhibitors were for CYP1A2 (20 μM furafylline), CYP2C8 (40 μM montelukast), CYP2C9 (40 μM sulfaphenazole), CYP2C19 (3 μM (-)N-3-benzyl-phenobarbital), and CYP2D6 (5 μM quinidine). We found that in vitro f_m values above 0.5 were comparable to in vivo values, falling within a 0.5 to 2-fold error in 9 of 11 CYP1A2 substrates, 5 of 8 CYP2C8 substrates, 5 of 8 CYP2C9 substrates, 2 of 3 CYP2C19 substrates, and 11 of 20 CYP2D6 substrates. The study also showed how uncertainty in measured metabolic turnover affects the estimated f_m,CYPs, revealing that when estimated f_m errors are <25%, 89% of predictions are within 2-fold of in vivo f_m, but this drops to 40% when there is higher uncertainty in measured turnover. Although some f_m values were poorly predicted and clinical studies revealed off-target inhibition by certain inhibitors, the chemical inhibition approach using human long-term cocultured hepatocytes showed useful prediction performance for early drug discovery enabling moderate-to-sensitive drug-drug interaction risk assessments, when metabolic turnover is adequate, and inhibitor selectivity is well defined. SIGNIFICANCE STATEMENT: Calculating in vitro fraction metabolized by cytochrome P450 enzymes in liver is vital in drug discovery for assessing the object drug-drug interaction risk of new chemical entities metabolized by cytochrome P450 enzymes before clinical data are available. Despite some limitations, the current study demonstrated that using long-term cocultured hepatocytes with chemical inhibitors is a reliable method for estimating fraction metabolized by cytochrome P450 enzymes in liver, complementing the drug interaction risk assessment.

The severe acute respiratory syndrome coronavirus-2 main protease inhibitor PF-07321332 (nirmatrelvir), in combination with ritonavir (Paxlovid), has been approved by the US Food and Drug Administration as an oral treatment option for coronavirus disease 2019 patients. In this perspective, we share the expediated absorption, distribution, metabolism, and excretion strategies, which were incorporated as part of discovery efforts, to design orally active severe acute respiratory syndrome coronavirus-2 main protease inhibitors. PF-07321332 (nirmatrelvir) emerged as a potential oral clinical candidate within ∼ 6 months from the time discovery efforts were first initiated. The review also delves into a discussion around the successful use of quantitative fluorine-19 nuclear magnetic resonance spectroscopy in the characterization of the human mass balance and excretion pathways of nirmatrelvir. Human absorption, distribution, metabolism, and excretion data that emerged from the fluorine-19 nuclear magnetic resonance study were used to support the Emergency Use Authorization and new drug application filing, which was accepted by regulatory agencies worldwide. Efficient operational and technical strategies, incorporating the elements of speed without sacrificing data quality, which were crucial to the success of the program, are highlighted. SIGNIFICANCE STATEMENT: This perspective discusses the expedited absorption, distribution, metabolism, and excretion efforts utilized in the discovery and development of the orally active severe acute respiratory syndrome coronavirus-2 main protease inhibitor nirmatrelvir, which in combination with the cytochrome P450 3A inhibitor ritonavir (Paxlovid), is used in the oral treatment of COVID-19. Paxlovid was granted an Emergency Use Authorization by global regulatory agencies in less than 2 years from the initiation of the discovery program and has since been fully approved by the US Food and Drug Administration.

Polychlorinated biphenyls (PCBs) are persistent environmental toxicants that bioaccumulate in the food chain and readily cross the placenta, raising concerns for developmental toxicity. Although PCB exposure has been associated with metabolic and neurodevelopmental disorders, its cell type-specific effects on liver development remain poorly understood. This study aimed to investigate how maternal exposure to an environmentally relevant Fox River PCB mixture affects liver development in female offspring at single-cell resolution. We hypothesized that developmental PCB exposure disrupts hepatic metabolic and immune function in a cell-type-specific manner. Using single-cell RNA sequencing on liver tissue from postnatal day 28 female mice exposed to PCBs throughout gestation and lactation, we identified major hepatic and immune cell populations and assessed cell-specific transcriptional responses. PCB exposure significantly altered the proportions of endothelial cells and Kupffer cells and reduced neutrophil abundance in the liver. Transcriptomic analysis revealed that PCBs dysregulated key functional pathways in hepatocytes and nonparenchymal cells, including endoplasmic reticulum stress responses, drug metabolism, and glucose/insulin signaling. Notably, hepatocytes exhibited upregulation of phase I drug-metabolizing enzymes and uptake transporters, but downregulation of phase II enzymes and efflux transporters. Kupffer cells and endothelial cells exhibited altered immune and metabolic gene expression, and intercellular communication analysis predicted that PCB exposure disrupted fibronectin, collagen, and chemokine signaling. Reverse transcription-quantitative polymerase chain reaction validation confirmed increased expression of hepatic endoplasmic reticulum stress markers. Together, these findings demonstrate that developmental PCB exposure induces persistent, cell-type-specific transcriptomic reprogramming in the liver, impairing metabolic and immune functions. This study highlights the utility of single-cell transcriptomics for revealing toxicant effects with cellular precision during critical windows of development. SIGNIFICANCE STATEMENT: Our findings showed that developmental exposure to a persistent organic pollutant led to transcriptomic changes that differed across cell types that form the liver. Our study highlights the use of single cell technology to investigate toxic effects for mechanistic insights with greater precision.

Accurately predicting human exposure to food-related compounds is crucial for evaluating their health effects without conducting animal testing. We previously reported an in vitro method using human induced pluripotent stem cell-derived small intestinal epithelial cells (hiSIECs) for predicting human maximum plasma concentrations (C_max) of food-related compounds. However, the C_max predictivity of flavonoids and their glycosides is relatively low, potentially due to complex gastrointestinal absorption processes. This study aimed to assess whether hiSIECs cultured using with a modified culture method (mod-hiSIECs) can mimic human intestinal absorption and enhance C_max predictivity. Consistent with observations in the human intestine, the mod-hiSIECs formed a column-like morphology. The expression levels of key saccharide-related genes, such as sucrase-isomaltase, lactase, and cytosolic β-glucosidase, and disaccharidase and glycosidase activities similar to those of human primary enterocytes were obtained using mod-hiSIECs when compared with hiSIECs cultured using the conventional hiSIEC culture method and Caco-2 cells. In addition, the mod-hiSIECs accurately mimicked the intestinal glucosidase, glucuronidase, and sulfatase activities against daidzein, genistein, and their corresponding glucosides. The C_max predictability for flavonoid aglycones and their glucosides was significantly improved with mod-hiSIECs compared with conventional hiSIEC culture method, reducing the fold-difference from 2.3-9.0 to 0.46-5.5. The mod-hiSIECs could be a useful tool for predicting gastrointestinal absorption of flavonoids in humans. SIGNIFICANT STATEMENT: Human induced pluripotent stem cell-derived small intestinal epithelial cells via a modified method, exhibited gene expression and metabolic profiles akin to primary enterocytes, and their permeability data predicted human plasma concentrations of isoflavones and glucosides more accurately than Caco-2 cells or conventional human induced pluripotent stem cell-derived small intestinal epithelial cells.