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. Due to broad substrate specificity of CYP enzymes, it is possible for more than one enzyme to be involved in the metabolism of a single compound. It is also possible for one CYP enzyme to catalyze two or more metabolic pathways for the same drug. In vitro methods have been established to determine which CYP isoform(s) is (are) involved in the metabolism of a specific drug. This process, also referred to as CYP reaction phenotyping, integrates data obtained from native human liver microsomes, intact cell models, recombinant CYPs, and inhibition studies with CYP selective chemical inhibitors and specific antibodies (Parkinson, 1996; Rodrigues, 1999 This was shown by Guengerich and colleagues (1987; 1988) who demonstrated the CYP catalyzed oxidative ester cleavage of 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester. The product, 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid monoethyl ester, was formed in the presence of substrate, enzymatically active microsomes and an NADPH generating system. The formation of these products was observed with five different CYP enzymes, however the extent of formation varied with the enzyme (Guengerich, 1987; Guengerich et al., 1988). CYP Chemical Inhibitor Incubations with Pooled Human Liver Microsomes. Of the CYP inhibitors studied at 100 µ M 13 are known to mediate dexamethasone induction, with the GR requiring nanomolar levels and PXR requiring micromolar levels for activation. In addition to the liver, the expression of hCE-1 and hCE-2 has been observed in small intestine, colon, testis, kidney, spleen, and heart classes of carboxylesterases, respectively. As well, more general esterase classification systems have been proposed, based on substrate and inhibitor specificity or interactions with organophosphorous insecticides The role of esterases in the metabolism of compounds is becoming increasingly important as, during the discovery of new drugs, candidate molecules are being synthesized which may contain an ester function. However, due to the non-selective substrate and inhibition specificity of esterases, phenotyping of specific esterases involved in the metabolism of a given compound is difficult. Esterases have been reported to exhibit clinically relevant polymorphisms which can lead to variable clearance of drugs. Cholinesterase, or butyrylcholinesterase, is abundant in human plasma and serum and hydrolyzes the muscle relaxant succinylcholine to succinylmonocholine and choline (Lockridge, 1990; Daly, 1993). Succinylcholine was introduced for use in 1951 having a quick onset of action resulting in complete paralysis, and a rapid recovery free of toxic side-effects. In some patients however, paralysis lasted for hours, instead of minutes, which often required life-saving intervention. For detailed discussion of the biochemistry and pharmacogenomics of cholinesterase, the authors refer the reader to Lockridge (1990); Darvesh et al. (2003); and Kalow (2004). Another esterase identified as having genetic variants is paraoxonase-1 (PON1). PON1 is a serum enzyme that catalyzes the hydrolysis of organophosphate esters, carbamates and aromatic carboxylic acid esters. For detailed discussion of the pharmacogenomics and catalytic efficiency of PON1, refer to Costa et al. (2003). Since it has been shown that CYP enzymes, in addition to esterases, catalyze the cleavage of ester-containing compounds, it becomes important to understand the involvement of these different enzyme families in the metabolism of a drug candidate. The determination of which enzyme family is involved may have an impact on the Using FD to measure human liver microsomal esterase activity, alphanaphthoflavone, clotrimazole ketoconazole, miconazole, nicardipine, and verapamil significantly inhibited the formation of the fluorescein product. The apparent IC 50 values of esterase activity for alpha-naphthoflavone and ketoconazole were 18 µ M and 6.5 µ M, respectively Much attention is directed towards the CYP metabolism of drugs and as a result, many other biotransformation enzymes, such as esterases, often get overlooked. Esterases however, are responsible for the metabolism of a number of endogenous and exogenous compounds. They can be induced, inhibited, and are subject to genetic polymorphisms, which can have clinical implications for the development of a drug. The results from the current study suggest that CYP chemical inhibitors should not be used to assess the role of CYP enzymes in the biotransformation of esters. Their potential to inhibit human liver microsomal esterase activity may result in an overestimation of the contribution of CYP enzymes in the metabolism of esters leading to a misinterpretation of potential drug-drug interactions. In contrast, CYP mAbs may be a useful tool to determine the contribution of CYP enzymes on the metabolism of esters as they were shown to have no effect on human liver microsomal esterase activity. Additional experiments to assess the contribution of oxidative enzymes in the metabolism of esters may include incubations in the presence and absence of β -nicotinamide adenine dinucleotide 2'-phosphate reduced (β-NADPH). DMD #9704 17 ACKNOWLEDGMENT

Structural studies on the muscarinic acetylcholine receptor

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