Strategies and Chemical Design Approaches to Reduce the Potential for Formation of Reactive Metabolic Species

Metabolic activation of new chemical entities to reactive intermediates is routinely monitored in drug discovery and development. Reactive intermediates may bind to cellular macromolecules such as proteins, DNA and may eventually lead to cell death via necrosis, apoptosis or oxidative stress. The evidence that the ultimate outcome of metabolic activation is an adverse drug reaction manifested as in vivo toxicity, is at best circumstantial. However, understanding the process of bioactivation of structural alerts by trapping the reactive intermediates is critical to guide medicinal chemistry efforts in quest for safer and potent moelcues. This commentary provides a brief introduction to adverse drug reactions and mechanisms of reactive intermediate formation for various functional groups, followed by a review of chemical design approaches, examples of such strategies, possible isosteric replacements for structural alerts and rationalization of laboratory approaches to determine reactive intermediates, as a guide to today’s medicinal chemist

Identification of saturated and unsaturated fatty acids as microsomal degradation by-products by direct high resolution -ESI-MS/MS

In vitro clearance in liver microsomes is routinely measured in drug discovery and development for understanding the disposition properties of new chemical entities. Literature reports indicate that long chain fatty acids such as arachidonic, linoleic and oleic acids may be released over a period of time during a microsomal incubation. These fatty acids are known to be substrates for conjugating enzymes like Uridine diphosphoglucuronosyl transferases (UGTs), thus potentially inhibiting microsomal clearance of UGT substrates. The present study was aimed at deciphering the fatty acids that may be released as by-products of microsomal degradation. LC-MS analytical methods were developed to characterize and qualitatively assess the fatty acids without chemical derivatization in rat, monkey and human liver microsomes. Additionally, incubations with UDPGA were utilized to trap the released fatty acids in their glucuronate ester form, also characterized and confirmed by high resolution LC-MS/MS. Our results indicate for the first time that eicosapentanoic, trans- and cis- eicosenoic, docosanoic, and nervonic acid were released as microsomal by-products in incubation conditions. Our results corroborate that palmitic, stearic, linoleic, and arachidonic acid were also formed as previously reported. Additionally, α- and γ-linolenic, eicosapentaenoic, palmitoleic, linoleic, arachidonic, palmitic, oleic, and stearic acid were identified as their corresponding acyl-glucuronides in rat, monkey and human liver microsomes. Our investigation renders a deeper understanding about the fatty acids which may be released by degradation of liver microsomes

Identification of a novel N-carbamoyl glucuronide: In vitro, In vivo and mechanistic studies

1-[4-Aminomethyl-4-(3-chloro-phenyl )-cyclohexyl]-tetrahydro-pyrimidin-2-one, 1, was developed as an inhibitor of dipeptidyl peptidase-4 enzyme (DPP-4). Biotransformation studies with 1 revealed the presence of an N-carbamoyl glucuronide metabolite (M1) in rat bile and urine. N-carbamoyl glucuronides are rarely observed, and little is understood regarding the mechanism of N-carbamoyl glucuronidation. The objectives of the current investigation were to elucidate the structure of the novel N-carbamoyl glucuronide, to investigate the mechanism of N-carbamoyl glucuronide formation in vitro using stable labeled CO2, UGT reaction phenotyping, and to assess whether M1 was formed to the same extent in vitro across species – mouse, rat, hamster, dog, monkey and human. Structure elucidation was carried out on a Thermo LTQ-Orbitrap® with accurate mass measurement and MSn capabilities. 13C-labeled carbon dioxide (13CO2) was used for identification of the mechanism of N-carbamoyl glucuronidation. Mechanistic studies with 13C-labeled CO2 in rat liver microsomes revealed that CO2 from the bicarbonate buffer (in equilibrium with exogenous CO2) may be responsible for the formation of M1. M1 was formed in vitro in liver microsomes from multiple species – mainly rat and hamster, followed by similar formation in dog, monkey, mouse, human. M1 could be detected in UGT1A1, UGT1A3 and UGT2B7 Supersomes® in a CO2 rich environment. In conclusion our study demonstrates that formation of M1 was observed in microsomal incubations across various species and strongly suggests the incorporation of CO2 from the bicarbonate buffer, in equilibrium with exogenous CO2 into the carbamoyl moiety of the formed N-carbamoyl glucuronide

High-Throughput Analysis of In-Vitro Cytochrome P450 Inhibition Samples Using Mass Spectrometry Coupled with an Integrated Liquid Chromatography and Autosampler System

No Abstrac

Oxidative ipso Substitution of 2,4-Difluoro-benzylphthalazines: Identification of a Rare Stable Quinone Methide and Subsequent GSH Conjugate

ABSTRACT: In vitro metabolite identification and GSH trapping studies in human liver microsomes were conducted to understand the bioactivation potential of compound 1 [2-(6-(4-(4-(2,4-difluorobenzyl)phthalazin-1-yl)piperazin-1-yl)pyridin-3-yl)propan-2-ol], an inhibitor of the Hedgehog pathway. The results revealed the formation of a unique, stable quinone methide metabolite (M1) via ipso substitution of a fluorine atom and subsequent formation of a GSH adduct (M2). The stability of this metabolite arises from extensive resonance-stabilized conjugation of the substituted benzylphthalazine moiety. Cytochrome P450 (P450) phenotyping studies revealed that the formation of M1 and M2 were NADPH-dependent and primarily catalyzed by CYP3A4 among the studied P450 isoforms. In summary, an unusual and stable quinone methide metabolite of compound 1 was identified, and a mechanism was proposed for its formation via an oxidative ipso substitution

A Phase 1b Trial to Assess the Pharmacokinetics of Ezutromid in Pediatric Duchenne Muscular Dystrophy Patients on a Balanced Diet.

Ezutromid (SMT C1100) is a small-molecule utrophin modulator that was developed to treat Duchenne muscular dystrophy (DMD). Previous clinical trials of this agent revealed lower exposure in DMD patients compared with healthy volunteers, which may reflect differences in diet. This study evaluated the pharmacokinetics of ezutromid in patients with DMD who followed a balanced diet. This was a multicenter, double-blind, placebo-controlled, ascending single and multiple oral dose study. Twelve pediatric patients were randomly allocated to 1 of 3 treatment sequences within which were 3 treatment periods of 2 weeks each. Each patient received, in a dose-escalating fashion, 1250 mg and 2500 mg twice daily (BID) of ezutromid administered orally as a microfluidized suspension (F3) with placebo in the other treatment period. Throughout the study, patients followed a balanced diet including recommended proportions of major food groups and administration of drug accompanied with 100 mL of full-fat milk. This approach improved the absorption of ezutromid, resulting in higher systemic exposure, with considerable variability in exposure between patients at each dose level. Single and multiple oral doses of 1250 mg and 2500 mg BID were considered safe and well tolerated. No severe or serious adverse events and no study discontinuations due to adverse events were reported. This study provides assurance that, with the formulation tested (F3) and instructions regarding food (balanced diet and whole-fat milk), 2500 mg BID of ezutromid achieves plasma concentrations that, based on preclinical studies, should be able to modulate utrophin expression in future clinical trials

Comprehensive assessment of human pharmacokinetic prediction based on in vivo animal pharmacokinetic data, part 2: Clearance

A comprehensive analysis on the prediction of human clearance based on intravenous pharmacokinetic data from rat, dog, and monkey for approximately 400 compounds was undertaken. This data set has been carefully compiled from literature reports and expanded with some inhouse determinations for plasma protein binding and rat clearance. To the authors' knowledge, this is the largest publicly available data set. The present examination offers a comparison of 37 different methods for prediction of human clearance across compounds of diverse physicochemical properties. Furthermore, this work demonstrates the application of each prediction method to each charge class of the compounds, thus presenting an additional dimension to prediction of human pharmacokinetics. In general, the observations suggest that methods employing monkey clearance values and a method incorporating differences in plasma protein binding between rat and human yield the best overall predictions as suggested by approximately 60% compounds within 2-fold geometric mean-fold error. Other single-species scaling or proportionality methods incorporating the fraction unbound in the corresponding preclinical species for prediction of free clearance in human were generally unsuccessful. © The Author(s) 2012

Comprehensive assessment of human pharmacokinetic prediction based on in vivo animal pharmacokinetic data, part 1: Volume of distribution at steady state

The authors present a comprehensive analysis on the estimation of volume of distribution at steady state (VDss) in human based on rat, dog, and monkey data on nearly 400 compounds for which there are also associated human data. This data set, to the authors' knowledge, is the largest publicly available, has been carefully compiled from literature reports, and was expanded with some in-house determinations such as plasma protein binding data. This work offers a good statistical basis for the evaluation of applicable prediction methods, their accuracy, and some methods-dependent diagnostic tools. The authors also grouped the compounds according to their charge classes and show the applicability of each method considered to each class, offering further insight into the probability of a successful prediction. Furthermore, they found that the use of fraction unbound in plasma, to obtain unbound volume of distribution, is generally detrimental to accuracy of several methods, and they discuss possible reasons. Overall, the approach using dog and monkey data in the Oie-Tozer equation offers the highest probability of success, with an intrinsic diagnostic tool based on aberrant values (1) for the calculated fraction unbound in tissue. Alternatively, methods based on dog data (single-species scaling) and rat and dog data (Oie-Tozer equation with 2 species or multiple regression methods) may be considered reasonable approaches while not requiring data in nonhuman primates. © The Author(s) 2012