Pragmatic approaches to determine the exposures of drug metabolites in preclinical and clinical subjects in the MIST evaluation of the clinical development phase.

The recent stream of regulatory guidelines on the Safety Testing of Drug Metabolites by the FDA in 2008 and the ICH in 2009 and 2012 has cast light on the importance of qualifying metabolite exposure as part of the safety evaluation of new drugs and has provided a much needed framework for the drug safety researcher. Since then, numerous publications interpreting the practicalities of the guidelines have appeared in the literature focusing on strategic approaches and/or adaptation of modern analytical methodologies, e.g., NMR and AMS, for the identification and quantification of metabolites in the species used in preclinical safety assessments and in humans. Surprisingly, there are few literature accounts demonstrating how, in practice, a particular strategy or analytical method has been used to qualify drug metabolites during the safety evaluation of a drug during clinical development. At the same time as the initial FDA and ICH guideline releases, the neuroscience therapy area of AstraZeneca had a number of projects in clinical development, or approaching this phase, which gave the authors a scaffold upon which to build knowledge regarding the safety testing of drug metabolites. In this article, we present how the MIST strategy was developed to meet the guidelines. Pragmatic approaches have evolved from the experience learned in various projects in DMPK at AstraZeneca, Södertälje, Sweden. Our experience dictates that there is no single strategy for qualifying the safety of drug metabolites in humans; however, all activities should be tied to two unifying themes: first that the exposure to drug metabolites should be compared between species at repeated administration using the relative method or a similar one; and second that the internal regulatory documentation of the metabolite qualification should be agnostic to external criteria (guidelines), indication, dose given, and timing.

Effect of clozapine on neutrophil kinetics in rabbits.

Clozapine is an atypical antipsychotic drug effective in the treatment of refractory schizophrenia; however, its use is limited due to its propensity to cause agranulocytosis in some patients. Little is known about the mechanism of idiosyncratic drug-induced agranulocytosis, in part because of the lack of a valid animal model. Clozapine is oxidized by activated human neutrophils and bone marrow cells to a reactive nitrenium ion by the myeloperoxidase-hydrogen peroxide system of neutrophils. This reactive metabolite has been shown in vitro to induce the apoptosis of neutrophils and bone marrow cells. While in vitro studies demonstrated the toxic potential of clozapine upon oxidation, it is not clear if similar conditions occur in vivo. In response to the difficulties encountered with detecting apoptotic neutrophils in vivo, we conducted a series of studies in rabbits using two fluorescent cell-labeling techniques to study the effect of clozapine treatment on neutrophil kinetics, that is, their rates of production and removal from circulation. The fluorescein dye, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), was used as a general cell label to measure the half-life of neutrophils in blood. In addition, the thymidine analogue, 5-bromo-2-deoxyuridine (BrdU), was used to label dividing cells, thus enabling the measurement of the efflux of neutrophils from the bone marrow. Clozapine, indeed, increased the rate of both the release of neutrophils from the bone marrow and their subsequent disappearance from circulation. Failure of the bone marrow to compensate for a shorter neutrophil half-life could lead to agranulocytosis. Alternatively, the damage to neutrophils caused by clozapine could, in some patients, lead to an immune-mediated response against neutrophils resulting in agranulocytosis.

Introduction to early in vitro identification of metabolites of new chemical entities in drug discovery and development.

The introduction of combinatorial chemistry and robotics for high throughput screening has changed the way drugs are discovered today compared with 10-15 years ago when fewer compounds were tested in animal or organ models. The introduction of new analytical techniques, especially liquid chromatography/mass spectrometry (LC/MS) has made it possible to characterize the chemical properties, permeability, metabolic stability and metabolic fate of a large number of screening hits for further development in a funnel-like manner. The purpose of this contribution is to discuss principles and recent strategies for metabolite identification and to give an introduction to biotransformation studies. Metabolites are experimentally generated with the use of animal and human recombinant expressed enzymes, and different liver and other tissue fractions like microsomes and slices. For separation and identification of structurally diverse metabolites, LC/MS and tandem mass spectrometry (LC/MS/MS) techniques are commonly used. The LC/MS and LC/MS/MS techniques are rapid, sensitive, easy to automate and robust, and therefore, they are the methods of choice for these studies. The outcome of the metabolite identification studies is detection of metabolites that could be pharmacologically active and contribute to the efficacy of a new chemical entity (NCE), and elimination of compounds that form reactive intermediates and/or toxic metabolites that could cause adverse effects of NCE. If such information is available at an early stage during the drug discovery process, the chemical structure of the compound may be modified to reduce the risk of idiosyncratic and/or adverse drug reactions during clinical development.

Hemoglobin adduct levels in rat and mouse treated with 1,2:3,4-diepoxybutane.

For cancer risk assessment of 1,3-butadiene from rodent cancer test data, the in vivo doses of formed 1,2:3,4-diepoxybutane (DEB) should be known. In vivo doses of DEB were measured through a specific reaction product with hemoglobin (Hb), a ring-closed adduct, N,N-(2,3-dihydroxy-1,4-butadiyl)valine (Pyr-Val), to N-terminal valines. An analytical method based on tryptic digestion of Hb and quantification of Pyr-modified heptapeptides by LC-MS/MS has been further developed and applied in vivo to DEB-treated rats. Furthermore, N-(2,3,4-trihydroxybutyl)valine adducts (THB-Val) to the N-terminal valine in Hb were measured in rats and mice treated with DEB and in a complementary experiment with 1,2-epoxy-3,4-butanediol (EBdiol), using a modified Edman degradation method and GC-MS/MS. In vitro reactions of hemolysate with DEB and EBdiol were used to measure reaction rates for adduct formation needed for calculation of doses and rates elimination in vivo. The results showed that the level of the Pyr-Val adduct per administered dose of DEB was approximately the same in rats as had earlier been observed in mice [Kautiainen et al. (2000) Rapid Commun. Mass Spectrom. 14, 1848-1853]. Levels of the THB-Val adduct after DEB treatment were 3-4 times higher in rat than in mouse, probably reflecting an enhanced hydrolysis of DEB to EBdiol catalyzed by epoxide hydrolase. After EBdiol treatment, the THB-Val adduct levels were about the same in rat and mouse. Calculations from in vitro data show that the Pyr-Val adduct is a relevant monitor for the in vivo dose of DEB and that THB-Val primarily reflects doses to EBdiol. The calculated rates of formation of adducts and rates of elimination agree with expectations. Procedures for quantification of Hb adducts as modified peptides as well as preparation and characterization of peptide standards have been evaluated.

Attomole detection of in vivo protein targets of benzene in mice: evidence for a highly reactive metabolite.

Modified proteins were detected in liver and bone marrow of mice following treatment with [(14)C]benzene. Stained sections were excised from one-dimensional and two-dimensional gels and converted to graphite to enable (14)C/(13)C ratios to be measured by accelerator mass spectrometry. Protein adducts of benzene or its metabolites were indicated by elevated levels of (14)C. A number of proteins were identified by in-gel proteolysis and conventional mass spectrometric methods with the low molecular weight proteins identified including hemoglobin and several histones. The incorporation of (14)C was largely proportional to the density of gel staining, giving little evidence that these proteins were specific targets for selective labeling. This was also true for individual histones subfractionated with Triton-acid-urea gels. A representative histone, H4, was isolated and digested with endopeptidase Asp-N, and the resulting peptides were separated by high performance liquid chromatography. (14)C levels in collected fractions were determined, and the peptides were identified by conventional mass spectrometry. The modifications were distributed throughout the protein, and no particular amino acids or groups of amino acids were identified as selective targets. Thus chemical attack by one or more benzene metabolites upon histones was identified and confirmed, but the resulting modifications appeared to be largely nonspecific. This implies high reactivity toward proteins, enabling such attack to occur at multiple sites within multiple targets. It is not known to what extent, if any, the modification of the core histones may contribute to the carcinogenicity of benzene.