Reactive metabolites assay

While covalent binding studies have an advantage over the reactive metabolite assay in that they provide a quantitative estimate of covalently bound drug to proteins and therefore an indirect measure of reactive metabolite formation, there are no studies to date which show a correlation between the extent of covalent binding and/or reactive metabolite formed and the probability that a drug is... 

Baughman, T.M. Wells-Knecht, M. Wells-Knecht, K. Zhao, Z. Method Validation for a Glutathione-trapping Reactive Metabolite Assay Using Drugs with Structural Moieties Known to Produce Reactive Species, Drug Metab. Rev. 35(S2), 112 (2003). 

From a purely pragmatic perspective, it is clear that reactive metabolites are linked with toxicity and that a circumstantial link can be made to idiosyncratic toxicides. Consequently, even though the mechanism of this toxicity is not fully understood, since assays are available to measure the potential for bioactivation in an ideal world one would not carry this liability forward. Conversely, it is not an ideal world, all drug molecules have challenges and the definition of therapeutic index (i.e., the ratio between the toxic exposure and the therapeutic exposure) is critical. Covalent binding of reactive metabolites to macromolecules is a crude measure and not a full predictor of toxicity and it is well known that toxicity can be ameliorated by a lower dose. Furthermore, the so-called definitive assays require radiolabeled drug material which is expensive and generally slow to produce. 

Liver injury is clinically defined as an increase of serum alanine amino transferase (ALT) levels of more than three times the upper limit of normal and a total bilirubin level of more than twice the upper limit of normal [4]. The clinical patterns of liver injury can be characterized as hepatocellular (with a predominant initial elevation of ALT), cholestatic (with an initial elevation of alkaline phosphatase) or mixed. The mechanisms of drug-induced hepatotoxicity include excessive generation of reactive metabolites, mitochondrial dysfunction, oxidative stress and inhibition of bile salt efflux protein [5]. Better understandings of these mechanisms in the past decades led to the development of assays and models suitable for studying such toxic mechanisms and for selecting better leads in the drug discovery stage. 

Kubicka-Muranyi M, Goebels R, Goebel C, Uetrecht J, Gleichmann E. T l5nnphocytes ignore procainamide, but respond to its reactive metabolites in peritoneal cells Demonstration by the adoptive transfer popliteal lymph node assay. Toxicol Appl Pharmacol 1993 122 88-94. 

In vitro systems combined with LC-MS/MS can also be used for the prediction of problematic or reactive metabolites. As shown in Figure 12.3, Bertrand et al. [16] have used an LC-MS/MS assay to look for glutathione (GSH) adducts of a test compound. As they point out this technique can be successful without the need for a radio-labeled compound and is therefore applicable in the early drug-discovery process. 

Lacoste, S., Castonguay, A., and Drouin, R. (2006). Formamidopyrimidine adducts are detected using the comet assay in human cells treated with reactive metabolites of 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK). Mutat Res 600, 138-149. 

Cy Reactive Metabolite Screening and Covalent-Binding Assays... 

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