Drug Metabolism and Disposition最新文献

Acetaminophen (APAP) is an analgesic and antipyretic drug used worldwide, which is safe at therapeutic doses. However, an overdose can induce liver injury and even liver failure. Mechanistic studies in mice beginning with the seminal papers published by B.B. Brodie's group in the 1970s have resulted in important insight into the pathophysiology. Although the metabolic activation of APAP with generation of a reactive metabolite, glutathione depletion, and protein adduct formation are critical initiating events, more recently, mitochondria have come into focus as an important target and decision point of cell death. This review provides a comprehensive overview of the induction of mitochondrial superoxide and peroxynitrite formation and its propagation through a mitogen-activated protein kinase cascade, the mitochondrial permeability transition pore opening caused by iron-catalyzed protein nitration, and the mitochondria-dependent nuclear DNA fragmentation. In addition, the role of adaptive mechanisms that can modulate the pathophysiology, including autophagy, mitophagy, nuclear erythroid 2 p45-related factor 2 activation, and mitochondrial biogenesis, are discussed. Importantly, it is outlined how the mechanisms elucidated in mice translate to human hepatocytes and APAP overdose patients, and how this mechanistic insight explains the mechanism of action of the clinically approved antidote N-acetylcysteine and led to the recent discovery of a novel compound, fomepizole, which is currently under clinical development. SIGNIFICANCE STATEMENT: Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in western countries. Extensive mechanistic research over the last several decades has revealed a central role of mitochondria in the pathophysiology of APAP hepatotoxicity. This review article provides a comprehensive discussion of a) mitochondrial protein adducts and oxidative/nitrosative stress, b) mitochondria-regulated nuclear DNA fragmentation, c) adaptive mechanisms to APAP-induced cellular stress, d) translation of cell death mechanisms to overdose patients, and e) mechanism-based antidotes against APAP-induced liver injury.

Physiologically based pharmacokinetic (PBPK) modeling was used to predict the human pharmacokinetics and drug-drug interaction (DDI) of GDC-2394. PBPK models were developed using in vitro and in vivo data to reflect the oral and intravenous PK profiles of mouse, rat, dog, and monkey. The learnings from preclinical PBPK models were applied to a human PBPK model for prospective human PK predictions. The prospective human PK predictions were within 3-fold of the clinical data from the first-in-human study, which was used to optimize and validate the PBPK model and subsequently used for DDI prediction. Based on the majority of PBPK modeling scenarios using the in vitro CYP3A induction data (mRNA and activity), GDC-2394 was predicted to have no-to-weak induction potential at 900 mg twice daily (BID). Calibration of the induction mRNA and activity data allowed for the convergence of DDI predictions to a narrower range. The plasma concentrations of the 4β-hydroxycholesterol (4β-HC) were measured in the multiple ascending dose study to assess the hepatic CYP3A induction risk. There was no change in plasma 4β-HC concentrations after 7 days of GDC-2394 at 900 mg BID. A dedicated DDI study found that GDC-2394 has no induction effect on midazolam in humans, which was reflected by the totality of predicted DDI scenarios. This work demonstrates the prospective utilization of PBPK for human PK and DDI prediction in early drug development of GDC-2394. PBPK modeling accompanied with CYP3A biomarkers can serve as a strategy to support clinical pharmacology development plans. SIGNIFICANCE STATEMENT: This work presents the application of physiologically based pharmacokinetic modeling for prospective human pharmacokinetic (PK) and drug-drug interaction (DDI) prediction in early drug development. The strategy taken in this report represents a framework to incorporate various approaches including calibration of in vitro induction data and consideration of CYP3A biomarkers to inform on the overall CYP3A-related DDI risk of GDC-2394.

Heart failure (HF) is a chronic disease affecting 1%-2% of the global population.¹²³I-labeled meta-iodobenzylguanidine (mIBG) is US Food and Drug Administration-approved for cardiac imaging and prognosis risk assessment in patients with HF. As a norepinephrine analog, mIBG is believed to be transported into adrenergic nerve terminals by the neuronal norepinephrine transporter (NET) and hence image sympathetic innervation of the myocardium. We previously showed that mIBG is an excellent substrate of organic cation transporter 3 (OCT3), an extraneuronal transporter expressed in cardiomyocytes. Here, we evaluated the in vivo impact of Oct3 on mIBG disposition and tissue distribution using Oct3 knockout mice. Oct3 ^+/+ and Oct3 ^-/- mice were administered with mIBG intravenously, and mIBG plasma pharmacokinetics and tissue exposures were determined. In Oct3 ^+/+ mice, mIBG exhibited extensive accumulation in multiple tissues (heart, salivary gland, liver, and adrenal gland). No difference was observed in overall plasma exposure between Oct3 ^+/+ and Oct3 ^-/- mice. Strikingly, cardiac mIBG was depleted in Oct3 ^-/- mice, resulting in 83% reduction in overall cardiac exposure (AUC_{0-24 h}: 12.7 vs. 2.1 μg × h/g). mIBG tissue exposure (AUC_{0-24 h}) was also reduced by 66%, 36%, and 31% in skeletal muscle, salivary gland, and lung, respectively, in Oct3 ^-/- mice. Our data demonstrated that Oct3 is the primary transporter responsible for cardiac mIBG uptake in vivo and suggested that cardiac mIBG imaging mainly measures OCT3 activity in cardiomyocytes but not NET-mediated uptake in adrenergic nerve endings. Our findings challenge the current paradigm in interpreting cardiac mIBG imaging results and suggest OCT3 as a potential genetic risk marker for HF prognosis. SIGNIFICANCE STATEMENT: ¹²³I-labeled meta-iodobenzylguanidine is used for cardiac imaging and risk assessment in heart failure patients. Contrary to the current belief that meta-iodobenzylguanidine (mIBG) tracks cardiac sympathetic innervation due to its uptake by the neuronal norepinephrine transporter, the authors demonstrated that cardiac mIBG uptake is mediated by the extraneuronal transporter Oct3. Their findings warrant a re-evaluation of the scientific rationale behind cardiac mIBG scan and further suggest organic cation transporter 3 as a risk factor for disease progression in heart failure patients.

Asphyxiated neonates often undergo therapeutic hypothermia (TH) to reduce morbidity and mortality. Since both perinatal asphyxia (PA) and TH influence physiology, altered pharmacokinetics (PK) and pharmacodynamics (PD) are expected. Given that TH is the standard of care for PA with moderate to severe hypoxic-ischemic encephalopathy, disentangling the effect of PA versus TH on PK/PD is not possible in clinical settings. However, animal models can provide insights into this matter. The (neonatal) Göttingen Minipig, the recommended strain for nonclinical drug development, was selected as translational model. Four drugs-midazolam (MDZ), fentanyl (FNT), phenobarbital (PHB), and topiramate (TPM)-were intravenously administered under four conditions: control (C), therapeutic hypothermia (TH), hypoxia (H), and hypoxia plus TH (H+TH). Each group included six healthy male neonatal Göttingen Minipigs anesthetized for 24 hours. Blood samples were drawn at 0 (predose) and 0.5, 2, 2.5, 3, 4, 4.5, 6, 8, 12, and 24 hours post drug administration. Drug plasma concentrations were determined using validated bioanalytical assays. The PK parameters were estimated through compartmental and noncompartmental PK analysis. The study showed a statistically significant decrease in FNT clearance (CL; 66% decrease), with an approximately threefold longer half-life (t_1/2) in the TH group. The H+TH group showed a 17% reduction in FNT CL, with a 62% longer t_1/2 compared with the C group; however, it was not statistically significant. Although not statistically significant, trends toward lower CL and longer t_1/2 were observed in the TH and H+TH groups for MDZ and PHB. Additionally, TPM demonstrated a 28% decrease in CL in the H group compared with controls. SIGNIFICANCE STATEMENT: The overarching goal of this study using the neonatal Göttingen Minipig model was to disentangle the effects of systemic hypoxia and TH on PK using four model drugs. Such insights can subsequently be used to inform and develop a physiologically based pharmacokinetic model, which is useful for drug exposure prediction in human neonates.

Our understanding of the fundamental molecular mechanisms of acetaminophen (APAP) hepatotoxicity began in 1973 to 1974, when investigators at the US National Institutes of Health published seminal studies demonstrating conversion of APAP to a reactive metabolite that depletes glutathione and binds to proteins in the liver in mice after overdose. Since then, additional groundbreaking experiments have demonstrated critical roles for mitochondrial damage, oxidative stress, nuclear DNA fragmentation, and necrotic cell death as well. Over the years, some investigators have also attempted to translate these mechanisms to humans using human specimens from APAP overdose patients. This review presents those studies and summarizes what we have learned about APAP hepatotoxicity in humans so far. Overall, the mechanisms of APAP hepatotoxicity in humans strongly resemble those discovered in experimental mouse and cultured hepatocyte models, and emerging biomarkers also suggest similarities in liver repair. The data not only validate the first mechanistic studies of APAP-induced liver injury performed 50 years ago but also demonstrate the human relevance of numerous studies conducted since then. SIGNIFICANCE STATEMENT: Human studies using novel translational, mechanistic biomarkers have confirmed that the fundamental mechanisms of acetaminophen (APAP) hepatotoxicity discovered in rodent models since 1973 are the same in humans. Importantly, these findings have guided the development and understanding of treatments such as N-acetyl-l-cysteine and 4-methylpyrazole over the years. Additional research may improve not only our understanding of APAP overdose pathophysiology in humans but also our ability to predict and treat serious liver injury in patients.

The role of inflammatory cells and other components of the immune system in acetaminophen (APAP)-induced liver injury and repair has been extensively investigated. Although this has resulted in a wealth of information regarding the function and regulation of immune cells in the liver after injury, apparent contradictions have fueled controversy around the central question of whether the immune system is beneficial or detrimental after APAP overdose. Ultimately, this may not be a simple assignment of "good" or "bad." Clinical studies have clearly demonstrated an association between immune dysregulation and a poor outcome in patients with severe liver damage/liver failure induced by APAP overdose. To date, studies in mice have not uniformly replicated this connection. The apparent disconnect between clinical and experimental studies has perhaps stymied progress and further complicated investigation of the immune system in APAP-induced liver injury. Mouse models are often dismissed as not recapitulating the clinical scenario. Moreover, clinical investigation is most often focused on the most severe APAP overdose patients, those with liver failure. Notably, recent studies have made it apparent that the functional role of the immune system in the pathogenesis of APAP-induced liver injury is highly context dependent and greatly influenced by the experimental conditions. In this review, we highlight some of these recent findings and suggest strategies seeking to resolve and build on existing disconnects in the literature. SIGNIFICANCE STATEMENT: Acetaminophen overdose is the most frequent cause of acute liver failure in the United States. Studies indicate that dysregulated innate immunity contributes to the transition from acute liver injury to acute liver failure. In this review, we discuss the evidence for this and the potential underlying causes.

The role of transporters in drug clearance is widely acknowledged, directly and indirectly by facilitating tissue/enzyme exposure. Through the latter, transporters also affect volume of distribution. Drug-drug interactions (DDIs) involving organic anion transporting polypeptides (OATPs) 1B1/1B3 and SLCO1B1 pharmacogenetics lead to altered pharmacokinetics of OATP1B substrates; however, several factors may confound direct interpretation of pharmacokinetic parameters from these clinical studies using noncompartmental analysis (NCA). A review of clinical data herein indicates a single dose of OATP1B inhibitor rifampin almost never leads to increased substrate half-life but often a decrease and that most clinical OATP1B substrates are CYP3A4 substrates and/or undergo enterohepatic cycling (EHC). Using hypothetically simple OATP1B substrate physiologically based pharmacokinetic (PBPK) models, simulated effect of rifampin differed from specific OATP1B inhibition due to short rifampin half-life causing dissipation of OATP1B inhibition over time combined with CYP3A4 induction. Calculated using simulated tissue data, volume of distribution indeed decreased with OATP1B inhibition and was expectedly limited to the contribution of liver volume. However, an apparent and counterintuitive effect of rifampin on volume greater than that on clearance resulted for CYP3A4 substrates using NCA. The effect of OATP1B inhibition and rifampin on OATP1B substrate models incorporating EHC plus or minus renal clearance was distinct compared with simpler models. Using PBPK models incorporating reversible lactone metabolism for clinical OATP1B substrates atorvastatin and pitavastatin, DDIs reporting decreased half-life with rifampin were reproduced. These simulations provide an explanation for the distinct change in OATP1B substrate pharmacokinetics observed in clinical studies, including changes in volume of distribution and additional mechanisms. SIGNIFICANCE STATEMENT: Transporters are involved in drug clearance and volume of distribution, and distinct changes in OATP1B substrate pharmacokinetics are observed with OATP1B inhibitor rifampin. Using hypothetical and validated PBPK models and simulations, this study addresses the limitations of single-dose rifampin and complicated clinical OATP1B substrate disposition in evaluating the pharmacokinetic parameters of OATP1B substrates during rifampin drug-drug interactions (DDIs). These models account for change in volume of distribution and identify additional mechanisms underlying apparent pharmacokinetic changes in OATP1B DDIs.

Arsenite is an important heavy metal. Some Chinese traditional medicines contain significant amounts of arsenite. The aim of this study was to investigate subacute exposure of arsenite on activities of cytochrome P450 enzymes and pharmacokinetic behaviors of drugs in rats. Midazolam, tolbutamide, metoprolol, omeprazole, caffeine, and chlorzoxazone, the probe substrates for cytochrome P450 (CYP) s3A, 2C6, 2D, 2C11, 1A, and 2E, were selected as probe drugs for the pharmacokinetic study. Significant decreases in areas under the curves of probe substrates were observed in rats after consecutive 30-day exposure to As at 12 mg/kg. Microsomal incubation study showed that the subacute exposure to arsenite resulted in little change in effects on the activities of P450 enzymes examined. However, everted gut sac study demonstrated that such exposure induced significant decreases in intestinal absorption of these drugs by both passive diffusion and carrier-mediated transport. In addition, in vivo study showed that the arsenite exposure decreased the rate of peristaltic propulsion. The decreases in intestinal permeability of the probe drugs and peristaltic propulsion rate most likely resulted in the observed decreases in the internal exposure of the probe drugs. Exposure to arsenite may lead to the reduction of the efficiencies of pharmaceutical agents coadministered resulting from the observed drug-drug interactions. SIGNIFICANCE STATEMENT: Exposure to arsenite may lead to the reduction of the efficiencies of pharmaceutical agents coadministered resulting from the observed drug-drug interactions. The present study, we found that P450 enzyme probe drug exposure was reduced in arsenic-exposed animals (areas under the curve) and the intestinal absorption of the drug was reduced in the animals. Subacute arsenic exposure tends to cause damage to intestinal function, which leads to reduced drug absorption.

In vitro clearance assays are routinely conducted in drug discovery to predict in vivo clearance, but low metabolic turnover compounds are often difficult to evaluate. Hepatocyte spheroids can be cultured for days, achieving higher drug turnover, but have been hindered by limitations on cell number per well. Corning Elplasia microcavity 96-well microplates enable the culture of 79 hepatocyte spheroids per well. In this study, microcavity spheroid properties (size, hepatocyte function, longevity, culturing techniques) were assessed and optimized for clearance assays, which were then compared with microsomes, hepatocyte suspensions, two-dimensional-plated hepatocytes, and macrowell spheroids cultured as one per well. Higher enzyme activity coupled with greater hepatocyte concentrations in microcavity spheroids enabled measurable turnover of all 17 test compounds, unlike the other models that exhibited less drug turnover. Microcavity spheroids also predicted intrinsic clearance (CL_int) and blood clearance (CL_b) within threefold for 53% [9/17; average absolute fold error (AAFE), 3.9] and 82% (14/17; AAFE, 2.6) of compounds using a linear regression correction model, respectively. An alternate method incorporating mechanistic modeling that accounts for mass transport (permeability and diffusion) within spheroids demonstrated improved predictivity for CL_int (12/17; AAFE, 4.0) and CL_b (14/17; AAFE, 2.1) without the need for empirical scaling factors. The estimated fraction of drug metabolized by cytochrome P450 3A4 (fm,CYP3A4) using 3 μM itraconazole was within 25% of observed values for 6 of 8 compounds, with 5 of 8 compounds within 10%. In sum, spheroid cultures in microcavity plates permit the ability to test and predict clearance as well as fm,CYP3A4 of low metabolic turnover compounds and represent a valuable complement to conventional in vitro clearance assays. SIGNIFICANCE STATEMENT: Culturing multiple spheroids in ultralow attachment microcavities permits accurate quantitation of metabolically stable compounds in substrate depletion assays, overcoming limitations with singly cultured spheroids. In turn, this permits robust estimates of intrinsic clearance, which is improved with the consideration of mass transport within the spheroid. Incubations with 3 μM itraconazole enabled assessments of CYP3A4 involvement in hepatic clearance.