Drug Metabolism and Disposition最新文献

Pressure overload induced cardiac hypertrophy is a major contributor to heart failure, and the arachidonic acid (AA) metabolism through cytochrome P450 enzymes is one of the metabolic pathways implicated in the hypertrophic response. The aryl hydrocarbon receptor (AhR) regulates CYP1A1 and CYP1B1 that generate protective 19-hydroxyeicosatetraenoic (HETE) and hypertrophic midchain-HETEs eicosanoids, respectively. The endogenous AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ) is a potent and selective inducer of CYP1A1, but its role in pressure overload induced cardiac hypertrophy has not been examined. This study investigated whether daily AhR activation by FICZ alters AA metabolites and attenuates cardiac hypertrophy in the abdominal aortic constriction (AAC) model. Male Sprague-Dawley rats underwent AAC or sham surgery and received FICZ (0.2 mg/kg per day) for 5 weeks. Echocardiography was performed at baseline and 5 weeks post-AAC, and gene, protein, and midchain-HETEs levels were assessed by real-time polymerase chain reaction, western blot, and liquid chromatography-tandem mass spectrometry, respectively. FICZ significantly reduced AAC-induced increases in left ventricular mass, ventricular wall thickness, heart weight-to-tibial length ratio, and hypertrophic gene expression. FICZ produced selective induction of CYP1A1 and significant rise in cardiac 19(S)-HETE. AAC significantly increased CYP1B1 and 12-LOX protein expressions and midchain-HETEs, whereas FICZ significantly attenuated 12-LOX and midchain-HETE. AAC also upregulated G protein coupled receptor 31, and FICZ reduced this increase at both mRNA and protein levels. This study provides the first evidence that endogenously generated 19(S)-HETE is cardioprotective in a pressure overload model and identifies FICZ as a modulator of the CYP1A1/19(S)-HETE that suppresses midchain-HETEs/G protein coupled receptor 31 pathways. SIGNIFICANT STATEMENT: This study demonstrates that 6-formylindolo[3,2-b]carbazole protect against cardiac hypertrophy induced by abdominal aortic constriction in rats. The significance of this research lies in its novel discovery, which elucidates for the first time the involvement of G protein coupled receptor 31 and the induction of CYP1A1 and 19-hydroxyeicosatetraenoic acid and suppressing midchain-hydroxyeicosatetraenoic acid/G protein coupled receptor 31 pathways by 6-formylindolo[3,2-b]carbazole in the protection against pressure overload cardiac hypertrophy in rats.

The therapeutic efficacy of 5-fluorouracil (5-FU), a cornerstone of gastric cancer chemotherapy, is predominantly limited by its catabolic inactivation in tumors. Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme responsible for 5-FU inactivation, and its tumor-specific overexpression constitutes a primary mechanism of 5-FU resistance. Here, we report a novel strategy to increase the sensitivity of 5-FU by targeting the post-translational regulation of DPD. We demonstrate that the neural precursor cell expressed, developmentally downregulated 8-activating enzyme (NAE) inhibitor MLN4924 significantly enhances the antitumor activity of 5-FU in both cellular and animal models of gastric cancer without augmenting systemic toxicity. Mechanistically, MLN4924 treatment inhibits the neddylation of DPD, which is dependent on the NAE1/ubiquitin-conjugating enzyme 12 axis. This inhibition triggers the ubiquitination and subsequent proteasomal degradation of DPD, thereby reducing intracellular 5-FU catabolism and augmenting its cytotoxic effects. Our findings identify neddylation as a previously unrecognized regulatory mechanism governing DPD protein stability and activity. This work identifies the neddylation-DPD axis as a novel therapeutic target and provides a strong rationale for combining NAE inhibition with 5-FU-based chemotherapy in gastric cancer. SIGNIFICANCE STATEMENT: This study establishes neddylation as a previously unrecognized regulatory mechanism that stabilizes the drug-metabolizing enzyme dihydropyrimidine dehydrogenase to drive 5-fluorouracil resistance in gastric cancer. It further unveils that inhibiting neddylation with MLN4924 selectively depletes tumor dihydropyrimidine dehydrogenase, enhancing chemotherapy efficacy without increasing toxicity, thereby proposing a targeted strategy to overcome chemoresistance.

The efficacy of inhaled drugs is significantly influenced by airway epithelial transporters that mediate their transport and distribution. Because inhaled drugs are used across diverse populations with inflammatory airway diseases, understanding factors that modulate transporter expression is crucial. Therefore, we investigated how inflammatory states, demographic factors, and airway anatomic location impact drug transporter abundance. Using quantitative targeted absolute proteomics, we measured transporter abundance in primary human bronchial epithelia (HBE) from 42 demographically diverse donors. To model distinct inflammatory environments, HBE were exposed to interleukin (IL)-1β (T-helper type [TH]1/neutrophilic inflammation) or IL-13 (TH2/eosinophilic inflammation) for 4 days, alongside baseline controls. We also measured transporter protein concentrations in small airway HBE from 5 donors to assess regional influence. In the primary set of HBE, multidrug resistance protein (MRP)1 (3.96 ± 1.64 pmol/mg protein) and peptide transporter 2 (3.12 ± 0.95 pmol/mg protein) were the most abundant transporters, and their concentrations were responsive to inflammatory signals. Specifically, IL-1β significantly reduced MRP1 (0.80-fold) and peptide transporter 2 (0.65-fold), whereas IL-13 modestly increased MRP1 (1.1-fold) but reduced peptide transporter 2 (0.73-fold) and novel organic cation transporter (OCTN)1 (0.44-fold). Several transporters present at lower abundance, including MRP4, MRP5, MRP6, and OCTN1, were also impacted by inflammation. Demographic factors also played a role, with MRP4 higher in females, and age positively correlated with MRP6 but negatively with OCTN1. Analysis of small airway-derived HBE did not show a significant impact of anatomic location on transporter abundance. Our findings demonstrate that airway inflammation and donor demographics significantly impact the protein expression of key drug transporters, highlighting dynamic factors crucial for optimizing inhaled drug delivery and efficacy. SIGNIFICANCE STATEMENT: Human airway epithelia express multiple transporters, with multidrug resistance protein 1 and peptide transporter 2 present at the highest levels. Transporter abundance is influenced by inflammation, age, and biological sex, but not by airway anatomic location. This information can guide the modeling of inhaled drug pharmacokinetics.

Ervogastat (PF-06865571) is a potent and selective small-molecule inhibitor of diacylglycerol-O-acyltransferase-2 currently in clinical development for treating metabolic dysfunction-associated steatohepatitis with liver fibrosis. A fixed sequence 2-period ¹⁴C-microtracer crossover study was used to determine a comprehensive and quantitative overview of the total disposition of ervogastat after oral and intravenous administration. From these studies, ervogastat was determined to have a systemic clearance of 23.9 L/h, a volume of distribution of 38.8 L, extent of oral absorption of ∼93%, time to maximum plasma concentration of ∼3.0 hours, and absolute oral bioavailability of ∼75%. The plasma half-lives of both ervogastat and total radioactivity were similar, with no indication of long-lived metabolites. The total recovery of [¹⁴C]ervogastat after oral administration was 79.0% ± 16.7%, with 48.9% ± 16.3% in the urine and 30.1% ± 2.9% in the feces. Ervogastat metabolites observed in human plasma, urine and feces generally can be assigned as originating from 1 of 4 primary metabolic pathways: amide bond hydrolysis (M2); tetrahydrofuran oxidation (M4/M5); O-de-ethylation (M1); and direct glucuronidation (584). The major circulating drug-related components were ervogastat (43.8%), M2 (36.6%), and M6 (11.3%). The major drug-related components detected in urine were M2 (24.9%) and coeluting M6 (des-ethyl sulfate), M7 (des-ethyl amide hydrolysis) and 584 (11.4%). In the feces, the predominant drug-related products were coeluting M4 and 426 (10.0%), M2 (7.0), M7 (6.3%), and M1 (4.5%). Reaction phenotyping was carried out using a subsequent qualitative followed by quantitative approach identifying CYP3A metabolism as the predominant route of in vitro metabolism. As assignment of metabolic pathways in the hADME study was complicated by chromatographically coeluting metabolites or metabolites arising from multiple possible primary pathways, fractional clearance ranges were derived by assigning metabolites to only one primary pathway at a time to represent a potential permutation followed by evaluation of all possible permutations. This novel approach allowed us to overcome the aforementioned confounding issues and, along with in vitro chemical inhibition results, permitted the assignment of the various metabolic pathways and enzymes involved in the metabolism of ervogastat. SIGNIFICANCE STATEMENT: This study provides a comprehensive and quantitative overview of the disposition, clearance pathways, and pharmacokinetics in humans of ervogastat, a potent and selective inhibitor of diacylglycerol-O-acyltransferase-2 for the treatment of metabolic dysfunction-associated steatohepatitis with liver fibrosis.

PM-4321 is a selective aryl hydrocarbon receptor (AhR) modulator profiled as part of an AhR antagonist program for cancer immunotherapy. The AhR regulates transcription of xenobiotic metabolizing enzymes, including cytochrome P450 (CYP) 1A1 and CYP1A2. Although initial pharmacokinetic studies in preclinical species demonstrated low systemic clearance and high oral bioavailability, repeat-dose pharmacokinetic studies revealed dose- and time-dependent reductions in systemic exposure indicative of metabolic autoinduction. This study sought to elucidate the mechanistic basis of this phenomenon. Despite high metabolic stability in hepatocytes and single dose pharmacokinetic studies, PM-4321 exhibited a decline in systemic exposure after repeat-dosing in mice and monkeys. This was accompanied by robust induction of CYP1A1 and CYP1A2, confirmed by both quantitative polymerase chain reaction and liver transcriptomic profiling. Conventional enzyme phenotyping failed to detect involvement of these enzymes due to low parent compound turnover. However, identification of M457-1, a CYP1A1-specific mono-oxidation metabolite, provided direct evidence of enzyme activity and enabled quantification of the induction response. These findings demonstrate that PM-4321 undergoes AhR-mediated autoinduction via selective upregulation of CYP1A1, a mechanism not readily captured by standard drug metabolism and pharmacokinetics assays. Integration of transcriptomic analysis and metabolite-centric phenotyping was essential to uncover this noncanonical pathway. This work underscores the importance of applying advanced molecular and analytical tools to characterize the disposition of low clearance compounds, particularly those targeting ligand-activated transcription factors such as AhR. SIGNIFICANCE STATEMENT: This study identifies cytochrome P450 1A1 induction as the mechanistic driver of PM-4321 autoinduction and the reduced systemic exposure in preclinical species. By integrating transcriptomic profiling with metabolite-centric phenotyping, we resolved a liability that standard drug metabolism and pharmacokinetics assays failed to capture. This framework offers a practical path for characterizing low-clearance compounds that engage ligand-activated transcription factors, with direct implications for translational pharmacokinetics and drug-drug interaction risk assessment.

Drug exposure, in terms of plasma concentrations, may be affected by individual differences in intrinsic drug-metabolizing enzymes as much as by exogenous drug interactions. However, baseline variations in drug-metabolizing enzyme activities in normal subjects and acceptable ranges for these variations remain unclear. To elucidate these baseline variations, CYP3A-dependent endogenous cortisol and/or deuterium-labeled cortisol and their 6β-hydroxylated metabolite concentrations were determined in vivo as biomarkers in 103 cynomolgus monkeys and 20 immunodeficient mice transplanted with human hepatocytes. After a single intravenous administration of cortisol-d₄ (0.50 mg/kg) in monkeys, the mean maximum plasma level of cortisol-d₄ was 1020 ± 160 ng/mL (n = 3) at 0.083 hours and was similar to the mean background cortisol level at the same time point, although background levels remained roughly constant throughout the day. Using spot plasma samples from an additional 100 monkeys, ratios of endogenous 6β-hydroxycortisol to cortisol concentration indicated variability: the median value was 1.4% (5% and 95% percentiles, 0.82% and 2.3%, respectively). The lowest and highest in vivo clearance values for cortisol-d₄ in humanized-liver mice with hepatocytes from 4 different donors had a 2.6-fold range across the 4 groups. The endogenous cortisol levels in mouse plasma samples were below the detection limit (<1 ng/mL). Currently, drug interaction study alerts are triggered at 1.25- to 2-fold increases in object drug clearance or metabolite/parent concentration ratio; however, evaluation should consider multiple factors, and these could be within the range of intrinsic baseline variations. SIGNIFICANCE STATEMENT: The mean 6β-hydroxycortisol plasma concentration in 100 cynomolgus monkeys was 4.4 ng/mL (coefficient of variation, 36%). The lowest and highest clearances of cortisol-d₄ in humanized-liver mice indicated a 2.6-fold range among the 4 sources of hepatocytes. This large baseline range may inform drug interaction guidance, which currently triggers alerts at a 1.25-fold increase in blood concentrations.

Accurately characterizing the transfer of apixaban and rivaroxaban into human milk is essential for evaluating their safety during lactation and for guiding anticoagulant therapy in breastfeeding patients. This study aimed to establish a mechanistic physiologically based pharmacokinetic (PBPK) modeling approach linking maternal exposure, mammary drug transport, and infant systemic levels for both anticoagulants. Passive permeability and efflux transport across the mammary epithelium were measured using an in vitro human mammary epithelial cell model, providing information on diffusion and transporter-mediated clearance, whereas milk protein binding was determined using rapid equilibrium dialysis to account for unbound drug available for transfer. These experimentally derived parameters were incorporated into an adult PBPK lactation model, which closely reproduced observed plasma and milk pharmacokinetics across dosing regimens, with simulated milk-to-plasma ratios deviating less than 10% from clinical measurements. The simulated maternal breast milk concentration-time profiles were subsequently used to drive a pediatric PBPK model, enabling an estimation of infant exposure with 2-hour feeding intervals. Simulations showed low infant plasma concentrations for both drugs, with rivaroxaban producing relative infant daily dose values below the commonly accepted 10% safety threshold, whereas apixaban exceeded this threshold with high absolute systemic exposure in infants. Overall, this work demonstrates that experimentally informed PBPK modeling provides a robust and quantitative framework to predict in vivo maternal-infant drug exposure. This approach enhances the ability to assess lactational safety for anticoagulants and establishes a generalizable strategy for evaluating other drugs used during breastfeeding. SIGNIFICANCE STATEMENT: This study establishes an integrated in vitro-in silico PBPK modeling framework to predict drug transfer into human milk and resulting infant exposure. By incorporating permeability, transporter activity, and milk protein binding into maternal and pediatric models, the approach accurately reproduces milk pharmacolinetics and enables mechanistic prediction of infant exposure. The model reveals substantially higher infant exposure to apixaban than rivaroxaban, supporting quantitative assessment of lactational drug safety.

Triclosan (TCS) is an antimicrobial toxicant found in a wide range of consumer products and has been detected in human tissues at concentrations ranging from 0.001 to 5 ppm. Hepatotoxic chemicals such as TCS can cause steatotic liver disease, a condition referred to as toxicant-associated steatotic liver disease. Once TCS enters the body, it induces liver UDP-glucuronosyltransferase (UGT) 1A proteins, leading to the formation of TCS glucuronides, which are biologically inactive metabolites. The induction of UGT1A proteins by TCS is regulated by the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). In this study, we evaluated the impact of TCS on the PPARα-UGT1 axis in steatohepatitis and hepatocellular carcinoma using humanized UGT1 (hUGT1) male mice. We have demonstrated that TCS exposure promotes liver tumorigenesis and induces human UGT1A1 and other human UGT1A proteins in the liver. Furthermore, our results demonstrate that UGT1A gene induction in the liver is dependent on PPARα. When PPARα-deficient hUGT1 mice (hUGT1/Pparα^-/-) were exposed to TCS for 5 months, steatohepatitis was exacerbated, accompanied by increased fibrosis, immune cell infiltration, lipid accumulation, and hepatocyte ballooning. Finally, we assessed whether disruption of the PPARα-UGT1 axis contributes to liver tumorigenesis. Our data indicate that deletion of PPARα and consequent impairment of UGT1A gene induction in the liver have no effect on tumor progression and malignancy. Our hUGT1/Pparα^-/- mouse model was a suitable model to study the TCS metabolism and how ablation of UGT1A proteins worsens TCS-induced metabolic dysfunction-associated steatohepatitis. SIGNIFICANCE STATEMENT: Triclosan strongly induces hepatic UGT1A gene expression through a PPARα-dependent mechanism in humanized UGT1 (hUGT1) mice. Loss of PPARα in hUGT1 mice augments triclosan-induced steatohepatitis, characterized by increased fibrosis, lipid accumulation, immune cell infiltration, and hepatocyte ballooning.

The interaction between the gut microbiome and drug metabolism is bidirectional and can influence the pharmacokinetics of certain drugs. In mice, the gut microbiome has been shown to influence Cyp3a11. However, evidence for microbial regulation of human cytochrome P450 3A4 (CYP3A4) is lacking. We aimed to bridge this gap by manipulating the microbiome of a humanized mouse model expressing CYP3A4, CYP3A7, pregnane X receptor and constitutive androstane receptor. Three groups of male and female humanized mice were studied: conventional (CV), germ-free (GF), and germ-free mice conventionalized (GFCV) using sex-matched pooled human fecal samples. The presence of microbiome upregulated CYP3A4 expression by 7.6-fold in male CV mice (P < .001) but downregulated CYP3A4 expression by 1.69-fold in female CV mice (P = .012) compared with GF mice. The human fecal microbiome transplant to sex-matched GF mice resulted in decreased microbial diversity (P < .05 in males and P < .01 in females) and was not effective in restoring CYP3A4 expression, suggesting complex underlying microbe-CYP3A4 interactions. We show that the hepatic CYP3A4 mRNA and protein expression were strongly correlated (R = 0.91; P = 2.6 × 10^-6). A total of 57 bacterial species from the mouse gut microbiome were identified to be significantly correlated with CYP3A4 protein expression (P < .05). Five bile acids and no short-chain fatty acids were correlated with CYP3A4 protein expression. In summary, alterations in the gut microbiome influenced hepatic CYP3A4 in humanized mice in a sex-dependent manner, with distinct microbes strongly correlating with this regulatory pattern. SIGNIFICANCE STATEMENT: To the best of our knowledge, this study is the first to evaluate the expression of cytochrome P450 3A4 under different microbial conditions in a humanized mouse model, including conventionalization of germ-free mice using pooled sex-matched human feces. Alterations in the gut microbiome influenced hepatic cytochrome P450 3A4 in a sex-dependent manner and were strongly correlated with microbial species.

GDC-8264 (flizasertib) is a small molecule inhibitor of receptor-interacting serine/threonine-protein kinase 1 currently in clinical development. From a phase I dose escalation study in healthy subjects, 2 major circulating metabolites, M5 and M3, were identified and M5 was estimated to be >10% of total drug-related material. Mass spectrometric analysis, characterized by a prominent neutral loss of water, indicated that M5 was a ketone reduction product (+2 Da), whereas M3 was identified as its glucuronide conjugate. Comparison with synthetic standards confirmed M5 as the (S)-alcohol, resulting from stereospecific reduction. The formation of M5 was found to be NADPH dependent and mainly produced from human liver cytosolic but not microsomal fractions. Using chemical inhibitors and recombinant enzymes, the reaction was attributed primarily to carbonyl reductase 1 (CBR1), with minimal contributions from aldo-keto reductases (AKR1Cs) and negligible involvement of 11β-hydroxysteroid dehydrogenase 1. Assessment of extrahepatic metabolism revealed a distinct rank order of specific activity: intestine ≫ kidney ≈ liver > lung. Furthermore, M5 underwent efficient reoxidation to the parent GDC-8264, mediated mainly by CYP2C19 and CYP1A2, with potential contribution from CBR1. Collectively, CBR1-mediated stereoselective ketone reduction is the primary determinant of GDC-8264's circulating metabolite profile, whereas the dynamic interconversion (reduction to M5 and reoxidation back to the parent) represents a critical factor for GDC-8264 pharmacokinetics. SIGNIFICANCE STATEMENT: This study demonstrated that a clinical drug candidate, GDC-8264, has a major human circulating metabolite M5 that was formed via stereoselective ketone reduction primarily mediated by carbonyl reductase 1. This highlights (1) the critical role of carbonyl reductase 1 for ketone-containing molecules as a potential major drug metabolizing enzyme that warrants attention in new chemical entity development and that (2) reversible reduction-reoxidation as well as enterohepatic recycling could play an important role to drive parent drug exposures.