Inhibition and induction of cytochrome P450 and the clinical implications.

The cytochrome P450s (CYPs) constitute a superfamily of isoforms that play an important role in the oxidative metabolism of drugs. Each CYP isoform possesses a characteristic broad spectrum of catalytic activities of substrates. Whenever 2 or more drugs are administered concurrently, the possibility of drug interactions exists. The ability of a single CYP to metabolise multiple substrates is responsible for a large number of documented drug interactions associated with CYP inhibition. In addition, drug interactions can also occur as a result of the induction of several human CYPs following long term drug treatment. The mechanisms of CYP inhibition can be divided into 3 categories: (a) reversible inhibition; (b) quasi-irreversible inhibition; and (c) irreversible inhibition. In mechanistic terms, reversible interactions arise as a result of competition at the CYP active site and probably involve only the first step of the CYP catalytic cycle. On the other hand, drugs that act during and subsequent to the oxygen transfer step are generally irreversible or quasi-irreversible inhibitors. Irreversible and quasi-irreversible inhibition require at least one cycle of the CYP catalytic process. Because human liver samples and recombinant human CYPs are now readily available, in vitro systems have been used as screening tools to predict the potential for in vivo drug interaction. Although it is easy to determine in vitro metabolic drug interactions, the proper interpretation and extrapolation of in vitro interaction data to in vivo situations require a good understanding of pharmacokinetic principles. From the viewpoint of drug therapy, to avoid potential drug-drug interactions, it is desirable to develop a new drug candidate that is not a potent CYP inhibitor or inducer and the metabolism of which is not readily inhibited by other drugs. In reality, drug interaction by mutual inhibition between drugs is almost inevitable, because CYP-mediated metabolism represents a major route of elimination of many drugs, which can compete for the same CYP enzyme. The clinical significance of a metabolic drug interaction depends on the magnitude of the change in the concentration of active species (parent drug and/or active metabolites) at the site of pharmacological action and the therapeutic index of the drug. The smaller the difference between toxic and effective concentration, the greater the likelihood that a drug interaction will have serious clinical consequences. Thus, careful evaluation of potential drug interactions of a new drug candidate during the early stage of drug development is essential.

Site-directed mutagenesis of rat liver NAD(P)H: quinone oxidoreductase: roles of lysine 76 and cysteine 179.

We previously reported the expression of a full-length cDNA complementary to a rat liver NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) mRNA in Escherichia coli (Q. Ma, R. Wang, C. S. Yang, and A. Y. H. Lu, 1990, Arch. Biochem. Biophys. 283, 311-317). Since cysteine residues have been suggested to be important for the catalysis of flavoproteins and a lysine residue at position 76 in NAD(P)H:quinone oxidoreductase has been proposed to be involved in electron transfer of the enzyme, we investigated the roles of lysine 76 and cysteine 179 of this enzyme in catalysis by site-directed mutagenesis. Mutant cDNA clones replacing lysine 76 with valine (K76V) and cysteine 179 with alanine (C179A) were generated by a procedure based on the polymerase chain reaction. The mutant enzymes were expressed in E. coli. The cytosolic activities of the K76V and C179A mutants were 50 and 25% of that of the wild type (DTD), due to lower levels of the mutant proteins as shown by immunoblot analysis. The mutant proteins were purified to apparent homogeneity. The purified K76V and C179A mutant enzymes maintained full activities of 2,6-dichlorophenolindophenol (DCIP) reduction compared with that of the wild type. The mutant enzymes exhibited kinetic parameters for DCIP, NADH, and NADPH similar to those of DTD except that, with K76V, the Km for NADPH was doubled. Both mutant proteins contained two molecules of FAD per enzyme molecule. Dicumarol inhibited K76V and C179A mutant activities to greater than 90% at a concentration of 10(-7) M. Heat stability studies showed that C179A was much more sensitive to inactivation at 37 degrees C than both the wild-type and K76V enzymes. It is concluded from this study that lysine 76 and cysteine 179 are not essential in catalysis and in the binding of FAD, DCIP, and dicumarol. However, lysine residue 76 appears to play a role in NADPH binding and cysteine residue 179 is important in maintaining the stability of the enzyme.

Rat liver NAD(P)H: quinone reductase nucleotide sequence analysis of a quinone reductase cDNA clone and prediction of the amino acid sequence of the corresponding protein.

We have determined the nucleotide sequence of a cDNA clone, pDTD55, complementary to rat liver quinone reductase mRNA (Williams, J.B., Lu, A.Y.H., Cameron, R.G., and Pickett, C.B. (1986) J. Biol. Chem. 261, 5524-5528). The cDNA clone contains an open reading frame of 759 nucleotides encoding a polypeptide comprised of 253 amino acids with a Mr = 28,564. To verify the predicted amino acid sequence of quinone reductase, we have been able to align the amino acid sequences of a cyanogen bromide digest of the purified enzyme to the sequence deduced from the cDNA clone. A comparison of the quinone reductase sequence with other known flavoenzymes did not reveal a significant degree of amino acid sequence homology. These data suggest that the quinone reductase gene has evolved independently from genes encoding other flavoenzymes.

Rat liver NAD(P)H:quinone reductase. Construction of a quinone reductase cDNA clone and regulation of quinone reductase mRNA by 3-methylcholanthrene and in persistent hepatocyte nodules induced by chemical carcinogens.

We have used polysomal immunoabsorption techniques to purify rat liver quinone reductase mRNA (NAD(P)H:quinone oxidoreductase, EC 1.6.99.2, formerly called DT-diaphorase). Using the purified mRNA as template, cDNA clones complementary to quinone reductase mRNA have been constructed. One cDNA clone, pDTD55, has a 1900-base pair insert which has been demonstrated by hybrid-select translation experiments to be complementary to quinone reductase mRNA. Clone pDTD55 has been used in RNA and DNA blot hybridizations to show that quinone reductase mRNA is approximately 1900 nucleotides in length and is encoded by a gene which spans approximately 7000-8000 base pairs. We have also shown that quinone reductase mRNA is markedly elevated by 3-methylcholanthrene administration and in persistent hepatocyte nodules induced by chemical carcinogens. The elevation of quinone reductase mRNA in persistent hepatocyte nodules is not due to either gene amplification of DNA rearrangement. Rather, the quinone reductase gene is hypomethylated in persistent hepatocyte nodules compared to the gene in either liver tissue surrounding the nodule or normal liver. These data suggest that hypomethylation of specific gene sequences occurs at early stages during chemical carcinogenesis.

Liver microsomal drug-metabolizing enzyme system: functional components and their properties.

The liver microsomal drug-metabolizing enzyme system consists of two protein components, cytochrome P-450 and NADPH-cytochrome c reductase, and a lipid, phosphatidylcholine. Cytochrome P-450 serves as the binding site for oxygen and substrate while the reductase acts as an electron carrier shuttling electrons from NADPH to cytochrome P-450. The phospholipid facilitates the transfer of electrons from NADPH-cytochrome c reductase to cytochrome P-450 but itself is not an electron carrier. Different cytochromes P-450 and P-448 have been purified; the spectral, catalytic, and immunological properties as well as the molecular weight (determined by SDS-gel electrophoresis) of all these hemeproteins differ from one another. The presence of multiple cytochrome P-450s may explain the species, strain, age, tissue, and sex differences as well as the effect of inducers and nutritional status in mammlian drug metabolism.