Measurement of Reactive Metabolites

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... 

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. 

Most P450 inhibitors act via reversible (competitive or noncompetitive mechanisms) with which their inhibitory potential can be estimated from their ICS0 or Ki values. Some inhibitors are "mechanism-based" or "time-dependent" inhibitors, which can cause irreversible inhibition due to the formation of reactive metabolites by the CYP isoform, leading to covalent binding to the active site and thereby causing irreversible inhibition of the affected enzyme molecule.31 Irreversible inhibitors therefore will have prolonged inhibition of the enzyme even after clearance of the drug in question. fCinact is a measurement of the potency of such "mechanism-based" inhibitors. 

The amino acid taurine has been suggested as a marker of hepatotoxicity because it is involved in the conjugation of bile acids to bile acids, the conjugation of xenobiot-ics, and the detoxification of reactive metabolites. Taurine is a product of sulfur acid metabolism and may reflect protein synthesis. The measurement of urinary taurine in models of hepatotoxicity has been described (Sanins et al. 1990 Waterfield et al. 1993a, 1993b, 1993c Timbrell, Seabra, and Waterfield 1995 Waterfield, Asker, and Timbrell 1996), but the measurement is reported to show diurnal variation, wide intraanimal variability, and less success at minor levels of hepatic injury (Maxuitenko, North, and Roebuck 1997). 

Because different chemically reactive metabolites react with various tissue nucleophiles at relatively different rates, it seemed likely to us that measuring the total covalent binding of reactive metabolites to proteins would not provide a reliable estimate of the relative toxicity of the chemically reactive metabolites. Indeed it seemed entirely possible that a chemically reactive metabolite could react extensively with protein and still be nontoxic. Moreover, it also seemed possible that a toxicant might be converted to a chemically reactive metabolite which combined with protein even though the toxicity is caused directly by the parent substance. 

In order to obtain some insight concerning the effect of fluorine on metabolic activation of fluorinated PAHs, isodesmic reactions (Figure 12) were calculated as a measure of the relative ease of formation of the fluorinated epoxides. Almost no energy differences were observed, consequently formation of these intermediates could be assumed not to be as relevant as carbocation formation in determining the relative reactivity of the ultimate metabolites. 

When reactive metabolites are formed by metabolic activation, some of them can escape from the active site and bind to external protein residues or be trapped by reduced glutathione (GSH) or other nucleophiles. The remaining molecules that are not released from the active site will cause the suicide inhibition [7]. The ratio of the number of reactive molecules remaining in the active site and those escaping is a measure of the reactivity of the intermediates formed. The addition of scavengers or GSH to the incubation mixture does not affect and cannot prevent the CYP mechanism-based inhibition. However, GSH can reduce the extent of the nonspecific covalent binding to proteins by those reactive molecules that escape from the active site. In contrast, addition of substrates or inhibitors that compete for the same catalytic center usually results in reduction of the extent of inhibition. 

Where related molecules are recognized by an antibody, they are said to cross-react and such antibodies are likely to be unsuitable for some assays. For example, where measurements of a particular drug are made, metabolites of the drug having similar structures may also react with the antibody. The extent of cross-reactivity may be assessed by comparing the concentration at which a 50% displacement of the related compound is obtained with that required for the antigen (Figure 7.11). 

Metabolites formed during the decolourization of the azo dye Reactive red 22 by Pseudomonas luteola were separated and identified by HPLC-DAD and HPLC-MS. The chemical structures of Reactive red 22 (3-amino-4-methoxyphcnyl-/fhydroxyl-sulphonc sulphonic acid ester) and its decomposition products are shown in Fig. 3.92. RP-HPLC measurements were carried out in an ODS column using an isocratic elution of 50 per cent methanol, 0.4 per cent Na2HP04 and 49.6 per cent water. The flow rate was 0.5 ml/min, and intermediates were detected at 254 nm. The analytes of interest were collected and submitted to MS. RP-HPLC profiles of metabolites after various incubation periods are shown in Fig. 3.93. It was concluded from the chromatographic data that the decomposition process involves the breakdown of the azo bond resulting in two aromatic amines [154],... 

Aging is of clinical interest in the treatment of poisoning because cholinesterase reactivators such as pralidoxime (2-PAM, Protopam) chloride are ineffective after aging has occurred. Measurement of metabolites of methyl parathion, p ra-nitrophenol, and dimethylphosphate in the urine has been used to monitor exposure to workers. ... 

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