Louis Leung

Metabolic activation in drug-induced liver injury

It is generally believed that metabolic bioactivation of drug molecules to form reactive metabolites, followed by their covalent binding to endogenous macromolecules, is one of the mechanisms that can lead to hepatotoxicity or idiosyncratic adverse drug reactions (IADRs). Although the role of bioactivation in drug-induced liver injury has been reasonably well established and accepted, and methodologies (e.g., structural alerts, reactive metabolite trapping, and covalent binding) continue to emerge in an attempt to detect the occurrence of bioactivation, the challenge remains to accurately predict the likelihood for idiosyncratic liver toxicity. Recent advances in risk-assessment methodologies, such as by the estimate of total body burden of covalent binding or by zone classification, taking the clinical dose into consideration, are positive steps toward improving risk assessment. The ability to better predict the potential of a drug candidate to cause IADRs will further be dependent upon a better understanding of the pathophysiological mechanisms of such reactions. Until a thorough understanding of the relationship between liver toxicity and the formation of reactive metabolites is achieved, it appears, at present, that the most practical strategy in drug discovery and development to reduce the likelihood of idiosyncratic liver toxicity via metabolic activation is to minimize or eliminate the occurrence of bioactivation and, at the same time, to maximize the pharmacological potency (to minimze the clinical dose) of the drug of interest.

Cerebrospinal Fluid Amyloid-β (Aβ) as an Effect Biomarker for Brain Aβ Lowering Verified by Quantitative Preclinical Analyses

Reducing the generation of amyloid-β (Aβ) in the brain via inhibition of β-secretase or inhibition/modulation of γ-secretase has been pursued as a potential disease-modifying treatment for Alzheimers disease. For the discovery and development of β-secretase inhibitors (BACEi), γ-secretase inhibitors (GSI), and γ-secretase modulators (GSM), Aβ in cerebrospinal fluid (CSF) has been presumed to be an effect biomarker for Aβ lowering in the brain. However, this presumption is challenged by the lack of quantitative understanding of the relationship between brain and CSF Aβ lowering. In this study, we strived to elucidate how the intrinsic pharmacokinetic (PK)/pharmacodynamic (PD) relationship for CSF Aβ lowering is related to that for brain Aβ through quantitative modeling of preclinical data for numerous BACEi, GSI, and GSM across multiple species. Our results indicate that the intrinsic PK/PD relationship in CSF is predictive of that in brain, at least in the postulated pharmacologically relevant range, with excellent consistency across mechanisms and species. As such, the validity of CSF Aβ as an effect biomarker for brain Aβ lowering is confirmed preclinically. Meanwhile, we have been able to reproduce the dose-dependent separation between brain and CSF effect profiles using simulations. We further discuss the implications of our findings to drug discovery and development with regard to preclinical PK/PD characterization and clinical prediction of Aβ lowering in the brain.

Drug Metabolites as Cytochrome P450 Inhibitors: a Retrospective Analysis and Proposed Algorithm for Evaluation of the Pharmacokinetic Interaction Potential of Metabolites in Drug Discovery and Development

Understanding drug-drug interactions (DDIs) is a key component of clinical practice ensuring patient safety and efficacy of medicines. The role of drug metabolites in DDIs is a developing area of science, and has been recently highlighted in a draft regulatory guidance. The guidance states that metabolites representing ≥25% of the parent drug’s area under the plasma concentration/time curve and/or >10% of exposure of total drug-related material should trigger in vitro characterization of metabolites for cytochrome P450 inhibition and propensity for DDIs. The relationship between in vitro cytochrome P450 inhibitory potency, systemic exposure, and DDI potential of drug metabolites was examined using the Pfizer development database to identify compounds with pre-existing in vivo biotransformation data, where circulating metabolites were identified in humans. The database yielded 33 structurally diverse compounds with collectively 115 distinct circulating metabolites. Of these, 52% (60/115) achieved exposures >25% of parent drug levels as judged from mass balance/metabolite identification studies. It was noted that 14 metabolite standards for 12 parent drugs had been synthesized, monitored in clinical studies, and examined for cytochrome P450 inhibition. For the 14 metabolite/parent drug pairs, no clinically relevant DDIs were expected to occur against the major human cytochrome P450 isoforms. A review of the literature for parent/metabolite DDI information was also conducted to examine trends using a larger data set. Leveraging the analysis of both internal and literature-based data sets, an algorithm was devised for use in drug discovery/early development to assess cytochrome P450 inhibitory potential of drug metabolites and the propensity to cause a clinically relevant DDI.

Pharmacokinetics and metabolic disposition of sirolimus in healthy male volunteers after a single oral dose.

The pharmacokinetics and metabolic disposition of sirolimus (rapamycin, Rapamune), a macrocyclic immunosuppressive agent for the prevention of allograft rejection in organ transplantation, were investigated in 6 healthy male volunteers after a single nominal 40-mg oral dose of the 14C-radiolabeled drug, with the added aim of assessing the potential role of sirolimus metabolites in the clinical pharmacology of the parent drug. The absorption of parent drug and derived materials was rapid (tmax 1.3 ± 0.5 hours, mean ± SD), and the elimination of sirolimus was slow (t½ 60 ± 10 hours, mean ± SD) in whole blood. The high whole blood to plasma (B/P) concentration ratio of sirolimus (142 ± 39) was consistent with its extensive partitioning into formed blood elements. The markedly lower B/P value based on radioactivity (2.7 ± 0.4) suggested that drug-derived products partitioned into formed blood elements to a much lesser extent. Based on AUC0-144h values, unchanged sirolimus represented an average 35% of total radioactivity in whole blood. Drug-derived products in whole blood were characterized by HPLC, LC/MS, and LC/MS/MS as 41-O-demethyl, 7-O-demethyl, and several hydroxy, dihydroxy, hydroxy-demethyl and didemethyl sirolimus metabolites. The percentage distribution of sirolimus metabolites in whole blood ranged from 3%-10% at 1 hour to 6%-17% at 24 hours after drug administration. Based on their low immunosuppressive activities and relative abundance in whole blood of humans after sirolimus administration, metabolites of sirolimus do not appear to play a major role in the clinical pharmacology of the parent drug. A majority of the administered radioactivity (91.0 ± 8.0%) was recovered from feces, and only 2.2% ± 0.9% was renally excreted.

New insights into drug absorption: Studies with sirolimus

Sirolimus is a recently marketed immunosuppressant that, in common with cyclosporine and tacrolimus, exhibits a low average oral bioavailability (~20%). Likewise, sirolimus is a substrate for the major drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) and the efflux transporter P-glycoprotein (P-gp), both of which are expressed in close proximity in epithelial cells lining the small intestine. Using CYP3A4-expressing Caco-2 cell monolayers, we examined the interplay between metabolism and transport on the intestinal first-pass extraction of sirolimus. Modified Caco-2 cells metabolized [14C]sirolimus to the same CYP3A4-mediated metabolites as human small intestinal and liver microsomes. [14C]Sirolimus also degraded to the known ring-opened product, seco-sirolimus. A ring-opened dihydro species (M2) was, surprisingly, the major product detected in cells at all sirolimus concentrations examined (2–100 μmol/L) and in incubations with human liver and intestinal homogenates but not in corresponding microsomes. M2 formation was NADPH-dependent but unaffected by prototypical CYP3A4 inhibitors. Although M2 was formed from purified seco-sirolimus (20 μmol/L) in the homogenates, it was not detected in cells when seco-sirolimus was added to the apical compartment because seco-sirolimus was essentially impermeable to the apical membrane. Sirolimus, seco-sirolimus (basolaterally dosed), and M2 were all secreted across the apical membrane, and secretion of each was inhibited by the P-gp inhibitor LY335979 (zosuquidar trihydrochloride). Along with CYP3A4-mediated metabolism and P-gp-mediated efflux, a novel elimination pathway was identified that may also contribute to the first-pass extraction, and hence low oral bioavailability, of sirolimus. This new insight into the intestinal elimination of sirolimus, which was not identified using traditional drug metabolism/transport screening methods, may represent another source for the limited absorption of sirolimus.

Evaluation of a New Molecular Entity as a Victim of Metabolic Drug-Drug Interactions - an Industry Perspective

Under the guidance of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ), scientists from 20 pharmaceutical companies formed a Victim Drug-Drug Interactions Working Group. This working group has conducted a review of the literature and the practices of each company on the approaches to clearance pathway identification (fCL), estimation of fractional contribution of metabolizing enzyme toward metabolism (fm), along with modeling and simulation-aided strategy in predicting the victim drug-drug interaction (DDI) liability due to modulation of drug metabolizing enzymes. Presented in this perspective are the recommendations from this working group on: 1) strategic and experimental approaches to identify fCL and fm, 2) whether those assessments may be quantitative for certain enzymes (e.g., cytochrome P450, P450, and limited uridine diphosphoglucuronosyltransferase, UGT enzymes) or qualitative (for most of other drug metabolism enzymes), and the impact due to the lack of quantitative information on the latter. Multiple decision trees are presented with stepwise approaches to identify specific enzymes that are involved in the metabolism of a given drug and to aid the prediction and risk assessment of drug as a victim in DDI. Modeling and simulation approaches are also discussed to better predict DDI risk in humans. Variability and parameter sensitivity analysis were emphasized when applying modeling and simulation to capture the differences within the population used and to characterize the parameters that have the most influence on the prediction outcome.

Design of a Janus Kinase 3 (JAK3) Specific Inhibitor 1-((2S,5R)-5-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidin-1-yl)prop-2-en-1-one (PF-06651600) Allowing for the Interrogation of JAK3 Signaling in Humans

Significant work has been dedicated to the discovery of JAK kinase inhibitors resulting in several compounds entering clinical development and two FDA approved NMEs. However, despite significant effort during the past 2 decades, identification of highly selective JAK3 inhibitors has eluded the scientific community. A significant effort within our research organization has resulted in the identification of the first orally active JAK3 specific inhibitor, which achieves JAK isoform specificity through covalent interaction with a unique JAK3 residue Cys-909. The relatively rapid resynthesis rate of the JAK3 enzyme presented a unique challenge in the design of covalent inhibitors with appropriate pharmacodynamics properties coupled with limited unwanted off-target reactivity. This effort resulted in the identification of 11 (PF-06651600), a potent and low clearance compound with demonstrated in vivo efficacy. The favorable efficacy and safety profile of this JAK3-specific inhibitor 11 led to its evaluation in several human clinical studies.

Cerebrospinal fluid β-Amyloid turnover in the mouse, dog, monkey and human evaluated by systematic quantitative analyses.

Background: Reducing brain β-amyloid (Aβ) via inhibition of β-secretase, or inhibition/modulation of γ-secretase, has been widely pursued as a potential disease-modifying treatment for Alzheimers disease. Compounds that act through these mechanisms have been screened and characterized with Aβ lowering in the brain and/or cerebrospinal fluid (CSF) as the primary pharmacological end point. Interpretation and translation of the pharmacokinetic (PK)/pharmacodynamic (PD) relationship for these compounds is complicated by the relatively slow Aβ turnover process in these compartments. Objective: To understand Aβ turnover kinetics in preclinical species and humans. Methods: We collected CSF Aβ dynamic data after β- or γ-secretase inhibitor treatment from in-house experiments and the public domain, and analyzed the data using PK/PD modeling to obtain CSF Aβ turnover rates (kout) in the mouse, dog, monkey and human. Results: The kout for CSF Aβ40 follows allometry (kout = 0.395 × body weight-0.351). The kout for CSF Aβ40 is approximately 2-fold higher than the turnover of CSF in rodents, but in higher species, the two are comparable. Conclusion: The turnover of CSF Aβ40 was systematically examined, for the first time, in multiple species through quantitative modeling of multiple data sets. Our result suggests that the clearance mechanisms for CSF Aβ in rodents may be different from those in the higher species. The understanding of Aβ turnover has considerable implications for the discovery and development of Aβ-lowering therapeutics, as illustrated from the perspectives of preclinical PK/PD characterization and preclinical-to-clinical translation.

Disposition and Metabolic Profiling of [ 14 C]Cerlapirdine Using Accelerator Mass Spectrometry

Cerlapirdine (SAM-531, PF-05212365) is a selective, potent, full antagonist of the 5-hydroxytryptamine 6 (5-HT6) receptor. Cerlapirdine and other 5-HT6 receptor antagonists have been in clinical development for the symptomatic treatment of Alzheimer’s disease. A human absorption, distribution, metabolism, and excretion study was conducted to gain further understanding of the metabolism and disposition of cerlapirdine. Because of the low amount of radioactivity administered, total 14C content and metabolic profiles in plasma, urine, and feces were determined using accelerator mass spectrometry (AMS). After a single, oral 5-mg dose of [14C]cerlapirdine (177 nCi), recovery of total 14C was almost complete, with feces being the major route of elimination of the administered dose, whereas urinary excretion played a lesser role. The extent of absorption was estimated to be at least 70%. Metabolite profiling in pooled plasma samples showed that unchanged cerlapirdine was the major drug-related component in circulation, representing 51% of total 14C exposure in plasma. One metabolite (M1, desmethylcerlapirdine) was detected in plasma, and represented 9% of the total 14C exposure. In vitro cytochrome P450 reaction phenotyping studies showed that M1 was formed primarily by CYP2C8 and CYP3A4. In pooled urine samples, three major drug-related peaks were detected, corresponding to cerlapirdine-N-oxide (M3), cerlapirdine, and desmethylcerlapirdine. In feces, cerlapirdine was the major 14C component excreted, followed by desmethylcerlapirdine. The results of this study demonstrate that the use of the AMS technique enables comprehensive quantitative elucidation of the disposition and metabolic profiles of compounds administered at a low radioactive dose.

Clearance Prediction of Targeted Covalent Inhibitors by In Vitro-In Vivo Extrapolation of Hepatic and Extrahepatic Clearance Mechanisms

The concept of target-specific covalent enzyme inhibitors appears attractive from both an efficacy and a selectivity viewpoint considering the potential for enhanced biochemical efficiency associated with an irreversible mechanism. Aside from potential safety concerns, clearance prediction of covalent inhibitors represents a unique challenge due to the inclusion of nontraditional metabolic pathways of direct conjugation with glutathione (GSH) or via GSH S-transferase–mediated processes. In this article, a novel pharmacokinetic algorithm was developed using a series of Pfizer kinase selective acrylamide covalent inhibitors based on their in vitro-in vivo extrapolation of systemic clearance in rats. The algorithm encompasses the use of hepatocytes as an in vitro model for hepatic clearance due to oxidative metabolism and GSH conjugation, and the use of whole blood as an in vitro surrogate for GSH conjugation in extrahepatic tissues. Initial evaluations with clinical covalent inhibitors suggested that the scaling algorithm developed from rats may also be useful for human clearance prediction when species-specific parameters, such as hepatocyte and blood stability and blood binding, were considered. With careful consideration of clearance mechanisms, the described in vitro-in vivo extrapolation approach may be useful to facilitate candidate optimization, selection, and prediction of human pharmacokinetic clearance during the discovery and development of targeted covalent inhibitors.