Drug Metabolism and Disposition最新文献

Tigecycline (TIG) exhibits poor oral bioavailability and brain distribution. Because bacterial efflux pumps that expel tetracyclines are homologous to mammalian P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), we investigated whether these transporters, along with TIG metabolism, affect TIG pharmacokinetics in mice. BALB/c and wild-type mice received TIG (10 mg/kg) intraperitoneally or orally alone, or in combination with the selective P-gp/BCRP inhibitor elacridar (ELA, 100 mg/kg p.o.) and/or the metabolic inhibitor voriconazole (VORI, 40 mg/kg i.p. or p.o.). One group also received a double TIG dose (20 mg/kg i.p.). Plasma and brain TIG concentrations were quantified via ultraperformance liquid chromatography coupled with tandem mass spectrometry. Differences in area under the curve (AUC) and C_max were analyzed using Bayesian hierarchical models. In both mouse strains, oral ELA substantially increased the plasma AUC_tlast (>1.8-fold; lower 95% credible limit [LCL], >1.6-fold) and C_max (>1.8-fold; LCL, >1.2-fold) of intraperitoneal TIG. The TIG double dose produced somewhat larger increases. With oral TIG, oral ELA markedly increased plasma AUC_tlast (>1.8-fold; LCL, >1.6-fold) in both strains, though C_max increases were more modest (LCLs, ∼ 1). Coadministration with VORI transiently (0.25-0.5 hours) raised plasma TIG, with oral VORI producing greater effects than intraperitoneal VORI. Oral ELA markedly increases systemic exposure to TIG, suggesting that P-gp/BCRP efflux restricts gastrointestinal absorption and increases systemic elimination. Results with VORI suggest that intestinal metabolism further limits oral uptake. Thus, efflux pump inhibition may be a viable strategy to improve TIG therapy. SIGNIFICANCE STATEMENT: Blocking efflux pumps by elacridar in the gut, liver, and brain increases tigecycline absorption and systemic retention. Coadministration of voriconazole, an inhibitor of metabolism, also suggests a significant role of intestinal metabolism in restricting tigecycline's oral bioavailability.

A dual prodrug linking clopidogrel and indobufen-an established dual antiplatelet therapy combination-was designed to enhance the bioactivation of clopidogrel while enabling coordinated inhibition of the ADP and thromboxane A₂ pathways of platelet activation. Because these 2 agents differ markedly in mechanism and duration of action, conventional combination therapy necessitates asymmetrical dosing. The fixed 1:1 molar ratio imposed by covalent conjugation introduces an inherent constraint on achieving balanced dual-pathway inhibition, a key consideration for defining the conjugate's therapeutic positioning. Three conjugates-deuterated clopidogrel-indobufen (1a), clopidogrel-indobufen (1b), and clopidogrel-(S)-indobufen (1c)-were synthesized and evaluated in rats. A single dose of these conjugates produced a delayed time to maximum plasma concentration and a sustained-release profile for both active metabolites. Covalent conjugation enhanced systemic exposure to the clopidogrel active metabolite while reducing exposure to released indobufen. Because conjugates 1b and 1c exhibited pharmacokinetic profiles more comparable to equimolar coadministration, they were selected for pharmacodynamic assessment. ADP receptor P2Y₁₂ occupancy and plasma thromboxane B₂ served as pathway-specific biomarkers, each bridging the pharmacokinetics and pharmacodynamics of the irreversible inhibition by clopidogrel and the reversible inhibition by indobufen, respectively. Both biomarkers showed strong correlations with inhibition of the corresponding platelet activation pathways. A single dose of 1b or 1c yielded synchronized maximal inhibition of both pathways at 8 hours-4 hours later than conventional coadministration-while retaining comparable peak efficacy. In the repeated dosing study, assessments aligned with the maximal-effect time point of the coadministration reference demonstrated that both conjugates-when supplemented with an interdose of indobufen-achieved pathway inhibition equivalent to the clinical regimen. These findings support conjugates 1b and 1c as promising alternatives to standard clopidogrel therapy and as potential tools for controlled de-escalation of antiplatelet therapy. SIGNIFICANCE STATEMENT: The clopidogrel-indobufen dual prodrugs enable synchronous, sustained release of both antiplatelet species in rats. P2Y₁₂ receptor occupancy and plasma thromboxane B₂ effectively capture the pharmacokinetic-pharmacodynamic relationships of this irreversible/reversible dual-antagonist combination.

Cytochrome P450 2E1 (CYP2E1) plays an important role in inflammatory disease, but the role of CYP2E1 in gout remains elusive. The pharmacokinetics in rats of Q11, a CYP2E1 inhibitor synthesized by our group, were measured. The anti-inflammatory and uric acid (UA)-lowering levels mechanism of Q11 was investigated in hyperuricemic (HUA) mice and acute gouty arthritis (AGA) rats, and in macrophage cells stimulated by monosodium urate. The half-life (t_1/2) of Q11 in the ankle joint and kidney was approximately 9 hours, which was more than 3 times longer than that in plasma. The expression of CYP2E1 was increased in the kidneys of HUA mice and in the ankles of AGA rats. Q11 reduced plasma UA levels in HUA mice by decreasing xanthine oxidase activity. In addition, Q11 also decreased renal inflammatory injury in HUA mice. Moreover, Q11 decreased the levels of interleukin-1β and tumor necrosis factor-α in AGA rats, increased the levels of catalase and glutathione, and blocked the activation of the Nod-like receptor protein 3 inflammasome. A similar phenomenon was observed in J774A.1 and THP-1 cells treated with sodium urate.CYP2E1 may be a new therapeutic target for the treatment of gout and Q11 has the dual effect of being anti-inflammatory and lowering UA. SIGNIFICANCE STATEMENT: The specific CYP2E1 inhibitor Q11 reduced plasma uric acid levels by decreasing xanthine oxidase activity in a gout animal model, while attenuating joint and renal inflammatory injury through the inhibition of oxidative stress and Nod-like receptor protein 3 inflammasome activation.

Plasma levels of Coproporphyrin I (CP-I), an endogenous biomarker used to gauge hepatic organic anion transporting polypeptide (OATP)1B1 and OATP1B3 activities, are linked to covariates, namely ethnicity, sex, and hemoglobin level. We developed and verified a mechanistic physiologically based pharmacokinetic model for CP-I considering these covariates in basal conditions and with a range of OATP1B perpetrators in virtual healthy subjects of various ethnicities. Simulations recovered the observed steady-state baseline levels and concentrations with interaction (C_max and area under the curve ratios, n = 12 studies) within 2-fold. Published CP-I plasma data in hepatic impairment (HI) indicated a progressive reduction in OATP1B activity in vivo. We applied our verified CP-I model to simulate CP-I plasma levels reported in individuals with increasing severity of HI as classified based on the Child-Pugh classes (A, B, and C) to assess the hepatic OATP1B transporter activity in cirrhotic virtual populations. A biomarker-informed physiologically based pharmacokinetic (BI-PBPK) approach was applied to close the gap between known expression differences for human hepatic OATP1B and multidrug resistance-associated protein 2 (MRP2) in HI and observed activity differences in HI relative to healthy individuals. HI-associated relative activity factor scalars derived from BI-PBPK simulations were developed and verified using 9 OATP1B substrates (n = 7 studies), with an average fold error and absolute average fold error of 0.93 and 1.74 for C_max, and 1.29 and 1.47 for area under the plasma concentration-time curve ratios between HI and healthy. The BI-PBPK approach offers a powerful means to establish model system parameters to improve predictive performance, particularly in disease populations and to explore the mechanisms behind the changes in plasma level. SIGNIFICANCE STATEMENT: Biomarker-informed physiologically based pharmacokinetic approach was used to bridge abundance differences in transporter expression and observed activity differences between healthy volunteer and hepatically impaired patients. A PBPK model for the endogenous biomarker, Coproporphyrin I, was developed where its synthesis rate is linked to body weight, sex, ethnicity, and hemoglobin levels. The model was verified with an extensive set of weak-to-strong OATP1B perpetrator drugs and applied to recover plasma concentrations for multiple OATP1B substrates using associated drug-drug interactions.

(+)-Dihydromethysticin (DHM) is a major kavalactone isolated from kava plants. Previous studies have identified (+)-DHM as a CYP2B6 inhibitor, with intriguing structural specificity. It is the most potent CYP2B6 inhibitor identified to date, and CYP2B6 is the most sensitive of any CYP isoform to inhibition by DHM. This investigation evaluated the stereochemistry of DHM inhibition of CYP2B6 and the role of methylenedioxyphenyl group bioactivation in CYP2B6 inhibition, using expressed CYP2B6 and the probe substrates 7-ethoxy-4-trifluoromethyIcoumarin and S-ketamine. The unnatural enantiomer (-)-DHM and racemic (±)-DHM exhibited similar inhibitory activities. Both DHM enantiomers were noncompetitive inhibitors of CYP2B6, with Ki values of 0.2 μM. DHM analogs lacking a methylenedioxy group were devoid of inhibitory effects in both CYP2B6 metabolism assays. Difluoro substitution of the methylene hydrogens on DHM abolished DHM inhibitory activity, whereas dideuterio substitution had no effect on CYP2B6 inhibition. Both DHM enantiomers and the dideuterio analog, but not the difluorinated analog or methysticin, generated a difference spectrum consistent with a metabolite-inhibitor complex. Results suggest CYP2B6-catalyzed methylenedioxyphenyl bioactivation of DHM to a metabolite-inhibitor complex with subsequent enzyme inhibition. DHM may have potential clinical implications or application as a selective CYP2B6 index inhibitor probe. SIGNIFICANCE STATEMENT: Enantiomers of the kavalactone dihydromethysticin are among the most potent CYP2B6 inhibitors identified to date, undergo metabolite-inhibitor complex formation, and exhibit substrate-dependent competitive and noncompetitive inhibition, which may have potential clinical implications or application. Because there is presently no inhibitor of CYP2B6 recommended for in vitro studies and no strong index inhibitor available for CYP2B6 for clinical studies, due in part to specificity considerations, dihydromethysticin may be a candidate for this purpose.

Drug-induced kidney injury, often resulting from the intracellular accumulation of drugs in renal proximal tubule cells via uptake transporters such as organic anion transporters 1 and 3 (OAT1/3), remains a major obstacle in drug development. Conventional 2-dimensional cultures of human renal proximal tubule epithelial cells (RPTECs) hardly express OAT1/3, limiting their utility for toxicity assessment. In contrast, 3-dimensional (3D) cultures of RPTEC have been shown to markedly upregulate OAT1/3 expression, offering a more physiologically relevant in vitro model for evaluating the toxicity of anionic compounds. In this study, we investigated the mechanism underlying OAT1/3 upregulation in 3D-RPTEC and explored a strategy to mitigate transporter-mediated toxicity. We found that hepatocyte nuclear factor (HNF) 4α expression is also increased in 3D-RPTEC. Motif analysis and cleavage under targets and release using nuclease-quantitative polymerase chain reaction revealed that HNF4α directly binds to the promoters of SLC22A6 and SLC22A8, identifying it as a key transcriptional regulator of OAT1/3 expression. Activation of the farnesoid X receptor (FXR), which represses the binding of HNF4α to promoters through the upregulation of small heterodimer partner (SHP), decreased OAT1/3 expression. Treatment with FXR ligands reduced cellular uptake of OAT1/3 substrates (eg, tenofovir and adefovir) and decreased their cytotoxic effects in 3D-RPTEC. These findings elucidate a transcriptional mechanism by which HNF4α regulates OAT1/3 expression in 3D-RPTEC and demonstrate that FXR agonists can downregulate OAT1/3 expression via the HNF4α-SHP axis. The present study highlights the utility of 3D-RPTEC as a valuable platform for mechanistic studies of transporter regulation. SIGNIFICANCE STATEMENT: This study shows that farnesoid X receptor agonists suppress hepatocyte nuclear factor 4α-mediated OAT1/3 activity in 3-dimensional-cultured renal proximal tubular cells and reduce nucleotide analog-induced toxicity. These findings provide mechanistic insight into transporter regulation and suggest a potential strategy to prevent nephrotoxicity.

A novel workflow for quantification of a drug and its metabolites in in vivo studies has been developed in the context of a radiolabeled human mass balance study. Samples are analyzed with ultra-high-performance liquid chromatography, and fractions are collected in a 384-well plate, which is subjected to offline counting, providing improved detection limits over online radioactivity detection. We discuss an advanced strategy to account for signal suppression or quenching, which significantly affected results in the offline counting of feces and urine samples in the selected case example, to provide more accurate quantification. The new quench model fits 2 data sets from 384-well plates with the actual matrices present to perform counting efficiency correction. Improved results were obtained over the existing approach, where a generic quench curve is defined by only a limited number of points made from a dilution series of a quenching agent. To account for outliers, a robust quartic model was applied. The new model effectively describes matrix-induced quenching and corrects for this, resulting in correct profiles with improved overall recovery as corroborated by comparison with online radioactivity detection and liquid scintillation counting, and can generically be applied postacquisition. The strategy was applied to all 36 fecal extracts from a human absorption, distribution, metabolism, and excretion study, where half of the samples present less than 20,000 disintegrations per min/mL, increasing the average column recovery (sum of individually quantified peaks relative to the total injected radioactivity) to >85%. SIGNIFICANCE STATEMENT: To improve interpretation in radiolabeled absorption, distribution, metabolism, and excretion studies, a matrix-based quench correction model is developed. It compensates for matrix-induced signal suppression when analyzing in vivo samples via offline radioactivity counting. It greatly improves data quality and enables accurate assessment of the true significance of detected metabolites.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a significant public health concern. Accumulating evidence suggests that long noncoding RNAs are dysregulated in MASLD. However, the roles and underlying mechanism of long noncoding RNAs in MASLD progression have not been fully elucidated. Here, we investigated the liver-specific functions of hepatocyte nuclear factor 4 α antisense 1 (HNF4A-AS1) and its mouse homolog, HNF4A opposite strand (Hnf4aos), in the pathogenesis of MASLD. HNF4A-AS1 and Hnf4aos were significantly upregulated in MASLD and diet-induced obese mice, respectively. Functionally, liver-specific knockdown of Hnf4aos reduced blood glucose levels and improved insulin sensitivity in the MASLD mouse model. Similarly, HNF4A-AS1 knockdown suppressed lipid droplet formation, intracellular triglyceride accumulation, and total cholesterol production in free fatty acid-induced HepG2 cells. Conversely, HNF4A-AS1 overexpression produced the opposite effects. Mechanistically, HNF4A-AS1 bound to the HNF4A protein and recruited heterogeneous nuclear ribonucleoprotein C (HNRNPC), thereby promoting HNF4A protein degradation. Taken together, our findings highlight the critical role of HNF4A-AS1 in MASLD progression and indicate that repressing HNF4A-AS1/HNF4A axis might be a potential therapeutic strategy for MASLD. SIGNIFICANCE STATEMENT: Long noncoding RNA HNF4A-AS1 and its mouse homolog, Hnf4aos, are upregulated in metabolic dysfunction-associated steatotic liver disease (MASLD) progression. Knockdown of HNF4A-AS1 or Hnf4aos alleviates MASLD progression in vitro or in vivo. HNF4A-AS1 interacts with HNF4A and promotes its protein degradation via HNRNPC, therefore aggravating MASLD progression.

The bioavailability of a drug is influenced by its physicochemical properties, dissolution rate, solubility, gastrointestinal tract permeability, and its absorption, distribution, and metabolism. Zileuton, a poorly soluble biopharmaceutical classification system class II drug prescribed for the treatment of asthma, was selected as a candidate for nanocrystal drug formulation. Our previous study demonstrated that zileuton nanocrystal formulation (NfZ) efficiently suppressed proinflammatory cytokines associated with asthma pathogenesis. Hence, this study aims to evaluate whether sex-dependent differences in the expression of genes related to drug metabolism and intestinal permeability influence the activity of the nanocrystal-formulated drug. In this study, we used the same animal samples as those reported in our earlier publications. To the best of our knowledge, this is the first study to conduct a comparative analysis of the effects of pure active pharmaceutical ingredient (API) and its nanocrystal formulation on metabolic enzyme genes and intestinal permeability-related genes. Our results demonstrated a significant increase in several genes involved in the metabolism of API, physical mixture (PM), and NfZ in males compared with females. PM and NfZ-treated animals showed increased expression of metabolic enzyme-related genes (phase I and phase II) and reduced alanine aminotransaminase activity at an equivalent dose compared with API-treated animals. However, genes related to oxidative metabolism and detoxification pathways were highly expressed in PM-treated animals; comparatively, NfZ treatment showed modest induction. Genes related to intestinal permeability were most significantly altered in API-treated animals compared to those dosed with NfZ. SIGNIFICANCE STATEMENT: The significance of this study is the sex-dependent difference in the expression of phase I and phase II metabolic enzymes and intestinal permeability-related genes upon treatment with different formulations of zileuton. Approximately 5% of asthmatic patients develop hepatocytotoxicity due to zileuton treatment and show several fold elevated levels of ALT in plasma. In the present study, differences in the decrease of ALT levels between the formulations highlight the advantages of the nanocrystal formulation.