Metabolite-intermediate complex

Ma, B., Prueksaritanont, T. and Lin, J.H. (2000) Drug interactions with calcium channel blockers possible involvement of metabolite-intermediate complexation with CYP3A. Drug Metabolism and Disposition, 28 (2), 125-130. 

There are three biochemical mechanisms of CYP inhibition competitive, mechanism-based, and metabolite-intermediate-complex (Fig. 5.3). Each type of inhibitor differs in the nature of CYP binding. Competitive inhibitors are reversibly bound and can be competed off of the docking site if another substrate of higher affinity is present at a higher concentration. Therefore,... 

METABOLITE INTERMEDIATE COMPLEX Drug P cannot dock on CYP because Drug E has formed a quasi-irreversFble complex, glue," that inactivates CYP... 

Jones DR, Gorski JC, Hamman MA, et al. Diltiazem inhibition of cytochrome P-450 3A activity is due to metabolite intermediate complex formation. J Pharmacol Exp Ther 1999 290(3) 1116-1125. 

Metabolite intermediate complexation of CYP450. In this case the drug is acted upon by the enzyme to form an oxidised derivative with a high affinity for the iron at the active site. Examples of this type of inhibition include alkylamine drugs that undergo oxidation to nitrosoalkane derivatives. Inhibition of this type renders the enzyme unavailable for further oxidation and synthesis of new enzyme is required to restore CYP450 activity. 

Levine, M. and G.D. Bellward (1995). Effect of cimetidine on hepatic cytochrome P450 Evidence for formation of a metabolite-intermediate complex. Drug Metab. Dispos. 23, 1407-1411. 

Irreversible inhibition (mechanism-based inhibition, MBI) is among the most specific enzyme inhibitions, which includes CYP suicide inactivation process (the more widely studied process) and metabolite-intermediate complex (MI) formation (Silverman, 1995 Waley, 1980). The former involves metabolism of drugs to products that denature the CYP. In this case, the inactivator for the... 

Metabolite Intermediates Metabolite intermediate complexes are formed by the oxidation of amines to C-nitroso compounds or from oxidation of methylene dioxy-phenyl compounds to carbenes [195]. These bind extremely tightly to ferrous P450 iron. Both of the bound forms are characterized by their 455-nm absorption bands, which can be produced in in vitro experiments. A classic example is seen with troleandomycin (TAO) andP450 3A4 [196]. These complexes can be disrupted by KjFelCNlg oxidation of the iron (in vitro). 

These studies demonstrated that DNA-binding can be a reliable probe of metabolic activation. In contrast to studies of metabolites per se, which usually involve large numbers of metabolite intermediates, DNA-binding monitors only chemically reactive metabolites. Also, if there is no selective repair of specific adducts, DNA-binding monitors the cumulative production of metabolites over time, while direct measurement of metabolites can show the metabolite spectrum only at the time observed. This can be particularly critical for studies of activation of complex chemicals such as polycyclic aromatic hydrocarbons whose primary metabolites are subject to secondary and tertiary metabolism (8). 

CYP3A4 is mainly responsible for catalyzing the hydro-xylation of miocamycin metabolites, which can alter the metabolism of concomitantly administered drugs by the formation of a metabolic intermediate complex with CYP450 or by competitive inhibition of CYP450 (25). This can cause excessive sedation with benzodiazepines such as triazolam. 

Quasi-irreversible inhibition is observed when CYP metabohsm produces an intermediate that can form a stable metabolite-intermediate MI) complex. This is another example of mechanism-based inhibition. Erythromycin is one such quasi-irreversible CYP3A4 inhibitor. Upon didemethylation of its tertiary amine group and subsequent oxidation, the resulting nitroso species forms a tight complex with the Fe(II) atom of the CYP s heme unit. Unhke truly irreversible adducts, such complexes can be broken up, say by oxidation with potassium ferricyanide, but under normal physiological conditions this obviously doesn t happen. 

Abbreviations V, velocity at a given substrate concentration V nax, maximum velocity the binding affinity between substrate and enzyme Kg, dissociation constant of substrate-enzyme complex Ki, dissociation constant of inhibitor-enzyme complex fobs> rate of inactivation at a given inhibitor concentration krmot maximal rate of inactivation Ki, half maximal rate of inactivation (exact physical meaning is not defined) MI, metabolite-intermediate Ki, dissociation constant of inhibitor-enzyme complex in the presence of substrate S, substrate concentration IC50, concentration of inhibitor that gives rise to a 50% decrease in activity. 

Because of the experimental manifestation of time-dependency and requirement for CYP catalysis, mechanism-based CYP inactivation is often referred to as time-dependent, metabolism-dependent, or preincubation-dependent inhibition. A detailed description of the kinetic characteristics of this type of inhibition has been published (Silverman, 1988) and a simplified kinetic equation is presented in Table 5.1. In cases where CYP activity can be recovered by dialysis, the term quasi-irreversible inhibition has been proposed (Ma et al., 2000). In addition, a time-dependency of CYP inhibition can result from the formation of potent yet reversible metabolites (Ma et al., 2000 Zhao et al., 2002 Zhout et al., 2005). Formation of a metabolite-intermediate (MI) complex has been described as another cause for time-dependent CYP inhibition by many quasi-irreversible inhibitors in this situation the metabolite or intermediate coordinates with the heme-ion thus decreasing the rate of catalysis. 

Eirst the drug interacts with the enzyme to produce a drug-enzyme intermediate. Then the intermediate complex is further processed to produce a metabolite, with release of the enzyme. The released enzyme is recycled back to react with more drug molecules. 

Time-dependent inhibition defined mainly by mechanism-based inhibition (MBI), which includes CYP suicide inactivation (irreversible inhibition, the more widely studied process) and metabolite-intermediate (MI) complex formation (quasi-irreversible inhibition), is responsible for most clinically significant DDIs (Silverman, 1995 Waley, 1980 Zhou et al., 2005). Suicide inactivation involves the formation of a reactive intermediate that irreversibly inactivates the CYP in the process of catalytic turnover. Quasi-irreversible inhibition occurs when the CYP produces a metabolite (e.g., nitroso intermediate) with the capacity to bind tightly to the CYP heme. TDI (time-dependent inhibition) can be characterized (1) to be dose dependent, (2) to be preincubation time dependent, (3) to have bioactivation of the inhibitor that is required for inactivation of the target enzyme, (4) to have de novo protein synthesis that is required to recover metabolic capacity, and (5) to have potentially slow onset of the effects but be more profound than reversible inhibition. If present, then TDI is the major component of overall enzyme inhibition and frequently leads to clinically relevant DDIs. Table 4.5 contains a list of inhibitors of TDI observed in vitro and in vivo. 

The glycolytic pathway described in this chapter begins with the breakdown of glucose, but other sugars, both simple and complex, can enter the cycle if they can be converted by appropriate enzymes to one of the intermediates of glycolysis. Figure 19.32 shows the mechanisms by which several simple metabolites can enter the glycolytic pathway. Fructose, for example, which is pro-... 

The degradation of CCl4 by Pseudomonas sp. strain KC involved formation of intermediate COCI2 that was trapped as a HEPES complex, and by reaction with cysteine (Lewis and Crawford 1995). Further details of the pathway that is mediated by the metabolite pyridine-dithiocarboxylic acid have been elucidated (Lewis et al. 2001). 

The degradation of tetrachloromethane by a strain of Pseudomonas sp. presents a number of exceptional features. Although was a major product from the metabolism of CCI4, a substantial part of the label was retained in nonvolatile water-soluble residues (Lewis and Crawford 1995). The nature of these was revealed by the isolation of adducts with cysteine and A,A -dimethylethylenediamine, when the intermediates that are formally equivalent to COClj and CSClj were trapped—presumably formed by reaction of the substrate with water and a thiol, respectively. Further examination of this strain classified as Pseudomonas stutzeri strain KC has illuminated novel details of the mechanism. The metabolite pyridine-2,6-dithiocarboxylic acid (Lee et al. 1999) plays a key role in the degradation. Its copper complex produces trichloromethyl and thiyl radicals, and thence the formation of CO2, CS2, and COS (Figure 7.64) (Lewis et al. 2001). 

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