Biotransformation and Rearrangement of Laromustine.

This review highlights the recent research into the biotransformations and rearrangement of the sulfonylhydrazine-alkylating agent laromustine. Incubation of [(14)C]laromustine with rat, dog, monkey, and human liver microsomes produced eight radioactive components (C-1 to C-8). There was little difference in the metabolite profile among the species examined, partly because NADPH was not required for the formation of most components, which instead involved decomposition and/or hydrolysis. The exception was C-7, a hydroxylated metabolite, largely formed by CYP2B6 and CYP3A4/5. Liquid chromatography-multistage mass spectrometry (LC-MS(n)) studies determined that collision-induced dissociation, and not biotransformation or enzyme catalysis, produced the unique mass spectral rearrangement. Accurate mass measurements performed with a Fourier-transform ion cyclotron resonance mass spectrometer (FTICR-MS) significantly aided determination of the elemental compositions of the fragments and in the case of laromustine revealed the possibility of rearrangement. Further, collision-induced dissociation produced the loss of nitrogen (N2) and methylsulfonyl and methyl isocyanate moieties. The rearrangement, metabolite/decomposition products, and conjugation reactions were analyzed utilizing hydrogen-deuterium exchange, exact mass, (13)C-labeled laromustine, nuclear magnetic resonance spectroscopy (NMR), and LC-MS(n) experiments to assist with the assignments of these fragments and possible mechanistic rearrangement. Such techniques produced valuable insights into these functions: 1) Cytochrome P450 is involved in C-7 formation but plays little or no role in the conversion of [(14)C]laromustine to C-1 through C-6 and C-8; 2) the relative abundance of individual degradation/metabolite products was not species-dependent; and 3) laromustine produces several reactive intermediates that may produce the toxicities seen in the clinical trials.

An in vitro evaluation of the victim and perpetrator potential of the anticancer agent laromustine (VNP40101M), based on reaction phenotyping and inhibition and induction of cytochrome P450 enzymes.

Laromustine (VNP40101M, also known as Cloretazine) is a novel sulfonylhydrazine alkylating (anticancer) agent. Laromustine generates two types of reactive intermediates: 90CE and methylisocyanate. When incubated with rat, dog, monkey, and human liver microsomes, [(14)C]laromustine was converted to 90CE (C-8) and seven other radioactive components (C-1-C-7). There was little difference in the metabolite profile among the species examined, in part because the formation of most components (C-1-C-6 and 90CE) did not require NADPH but involved decomposition and/or hydrolysis. The exception was C-7, a hydroxylated metabolite, largely formed by CYP2B6 and CYP3A4/5. Laromustine caused direct inhibition of CYP2B6 and CYP3A4/5 (the two enzymes involved in C-7 formation) as well as of CYP2C19. K(i) values were 125 microM for CYP2B6, 297 muM for CYP3A4/5, and 349 microM for CYP2C19 and were greater than the average clinical plasma C(max) of laromustine (25 microM). There was evidence of time-dependent inhibition of CYP1A2, CYP2B6, and CYP3A4/5. Treatment of primary cultures of human hepatocytes with up to 100 microM laromustine did not induce CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, or CYP3A4/5, but the highest concentration of laromustine decreased the activity and levels of immunoreactive CYP3A4. The results of this study suggest the laromustine has 1) negligible victim potential with respect to metabolism by cytochrome P450 enzymes, 2) negligible enzyme-inducing potential, and 3) the potential in some cases to cause inhibition of CYP2B6, CYP3A4, and possibly CYP2C19 during and shortly after the duration of intravenous administration of this anticancer drug, but the clinical effects of such interactions are likely to be insignificant.

Drug interactions: concerns and current approaches.

Following the recent withdrawal of several prominent drugs from US and European markets because of detrimental drug-drug interactions, metabolic drug interactions have received considerable attention in the pharmaceutical industry. In turn, the question of drug safety has received significant legal, regulatory and commercial emphasis, bringing this issue to the forefront of both industry and government drug agendas. The value of predicting the drug interactions of compounds as early as possible in the drug discovery process for all therapeutic areas cannot be underestimated. From 1964 to 1999, approximately 8% of the drugs approved by the FDA were later withdrawn from the US market. Pharmaceutical companies are facing increasing pressure to prove the long-term safety of their products, and this is complicated by the fact that animal models are not perfectly predictive of human responses, and may provide contradictory information. The failure to address safety concerns successfully during the drug optimization process may result in companies withdrawing any approved drugs from the market; drug safety issues not only present human health consequences, but also have a negative economic and public relations impact on the pharmaceutical industry. This paper discusses the significance of drug interactions, and addresses strategies to evaluate the potential of a drug candidate for drug interactions.

The impact of recent innovations in the use of liquid chromatography-mass spectrometry in support of drug metabolism studies: are we all the way there yet?

Absorption, distribution, metabolism, excretion and toxicology (ADMET) studies are widely used in drug discovery and development to help obtain the optimal balance of properties necessary to convert lead compounds into drugs that are safe and effective for human use. Drug discovery efforts have been aimed at identifying and addressing metabolism issues at the earliest possible stage, by developing and applying innovative liquid chromatography-mass spectrometry (LC-MS)-based techniques and instrumentation, which are both faster and more accurate than prior techniques. Such new approaches are demonstrating considerable potential to improve the overall safety profile of drug candidates throughout the drug discovery and development process. These emerging techniques streamline and accelerate the process by eliminating potentially harmful candidates earlier and improving the safety of new drugs. In the area of drug metabolism, for example, revolutionary changes have been achieved by the combination of LC-MS with innovative instrumentation such as triple quadrupoles, ion traps and time-of-flight mass spectrometry. In turn, most ADMET studies have come to rely on LC-MS for the analysis of an ever-increasing workload of potential candidates. This article provides a discussion on the importance of LC-MS in supporting drug metabolism studies, and highlights the relative merits of current applications for LC-MS in drug metabolism testing and analysis. These applications include in vitro and in vivo testing, pharmacokinetic profiling, chiral separations, stable isotope labeling, metabolic activation testing, metabolite characterization and radiolabeled-drug testing.

Improving the decision-making process in structural modification of drug candidates: reducing toxicity.

The rule of three, relating to activity-exposure-toxicity, presents the single most difficult challenge in the design and advancement of drug candidates to the development stage. Absorption, distribution, metabolism and excretion (ADME) studies are widely used in drug discovery to optimize this balance of properties necessary to convert lead compounds into drugs that are both safe and effective for human patients. Idiosyncratic drug reactions (IDRs; referred to as type B reactions, which are mainly caused by reactive metabolites) are one type of adverse drug reaction that is important to human health and safety. This review highlights the strategies for the decision-making process involving substructures that, when found in drugs, can form reactive metabolites and are involved in toxicities in humans; the tools used to reduce IDRs are also discussed. Several examples are included to show how toxicity studies have influenced and guided drug design. Investigations of reactive intermediate formation in subcellular fractions with the use of radiolabeled reagents are also discussed.

Improving the decision-making process in the structural modification of drug candidates: enhancing metabolic stability.

The activity-exposure-toxicity relationship, which can be described as "the rule of three", presents the single most difficult challenge in the design of drug candidates and their subsequent advancement to the development stage. ADME studies are widely used in drug discovery to optimize the balance of properties necessary to convert lead candidates into drugs that are safe and effective for humans. Metabolite characterization has become one of the key drivers of the drug discovery process, helping to optimize ADME properties and increase the success rate for drugs. Various strategies can influence drug design in the decision-making process in the structural modification of drug candidates to reduce metabolic instability.

Strategies for dealing with metabolite elucidation in drug discovery and development.

Structural information on metabolites can be a considerable asset for enhancing and streamlining the process of developing new drug candidates. Modern approaches that generate and use metabolite structural information can accelerate the drug discovery and development process by eliminating potentially harmful candidates earlier in the process and improving the safety of new drugs. This review examines the relative merits of current and potential strategies for dealing with metabolite characterization.

Strategies for dealing with reactive intermediates in drug discovery and development.

Idiosyncratic drug reactions (IDRs; a specific type of drug toxicity characterized by delayed onset) are a major complication of drug therapy that need to be addressed during drug discovery and development. Efforts to improve drug safety are hampered by the lack of an accepted approach to predict IDRs, which in turn is due to the low incidence of occurrence of IDRs and the various potential mechanisms involved in these reactions. The concept of the relative rarity and formation of reactive metabolite of IDRs is briefly described. Hypothetical chemical mechanisms for the formation of reactive metabolites are summarized, including a classification of adverse drug reactions and types of reactive metabolites. The relative merits of current and potential strategies for dealing with reactive intermediates in drug discovery and development are examined, and the significance of covalent binding in drug discovery/development in vitro and in vivo systems is considered. Also discussed are the merits of tools (screening methods to trap reactive intermediates, enzyme inhibition and covalent binding) and strategies for predicting which new drugs have the potential to produce reactive intermediates and IDRs; these approaches may be considered to have the potential to improve the overall safety profile of drug candidates at various stages of the drug discovery and development process.