Anthony Y.H. Lu

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.

The environmental impact of the use of ivermectin: environmental effects and fate

Abstract The environmental effects and fate of ivermectin were studied either as the pure compound or in feces. Toxicities to microbes, earthworms, algae, fish, and Daphnia, soil-binding parameters and degradation half-lives were determined.

Acetone-inducible cytochrome P-450: Purification, catalytic activity, and interaction with cytochrome b5

A procedure was developed for the purification of an acetone-inducible form of cytochrome P-450 (P-450ac) to electrophoretical homogeneity from liver microsomes of acetone-treated rats. The P-450ac preparation containing 16.0 to 16.5 nmol P-450/mg protein moved as a single protein band with an estimated molecular weight of 52,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate. The ferric P-450ac showed an absorption maximum at 394 nm at 25 degrees C, suggesting that it exists mainly in the high-spin form. It also existed in the low-spin form, especially at lower temperatures, as indicated by the absorption maximum in the 412-nm region. Upon reconstitution with NADPH: cytochrome P-450 reductase and phospholipid, P-450ac efficiently catalyzed both the demethylation and denitrosation of N-nitrosodimethylamine (NDMA) showing Vmax values of 23.8 and 2.3 nmol min-1 nmol P-450-1, respectively. The catalytic activity of P-450ac was greatly affected by cytochrome b5 which decreased the Km values of these reactions by a factor of 10 and increased the Vmax values. Cytochrome b5 appeared to interact with P-450 at a molar ratio of 1:1 and an intact cytochrome b5 structure was required for such interaction. Among the substrates studied, the demethylation of NDMA was affected the most by cytochrome b5 and showed the highest rate. P-450ac also catalyzed the oxygenation of N-nitrosomethylethylamine and aniline and the activity was enhanced slightly by cytochrome b5. Cytochrome b5 did not enhance the P-450ac-catalyzed metabolism of other drug substrates such as benzphetamine, aminopyrine, and ethylmorphine. P-450ac appeared to be similar in property to the previously studied rat P-450et (ethanol-inducible), rat P-450j (isoniazid-inducible), and rabbit P-450LM3a (ethanol-inducible). These P-450 species represent a new class of P-450 isozymes that are important in the metabolism of many endobiotics and xenobiotics.

Selective inactivation of cytochrome P-450 isozymes by suicide substrates

Abstract The autocatalytic destruction of cytochrome P-450 by the following six substrates has been investigated in vivo and in vitro with microsomal and purified, reconstituted rat liver enzymes: 2-isopropyl-4-pentenamide (AIA), 1-ethinylcyclopentanol, 17α-propadienyl-19-nortestosterone, fluroxene, 5,6-dichloro-1,2,3-benzothiadiazole (DCBT), and 1-aminobenzotriazole (ABT). Administration of the first three substrates to rats pretreated with either phenobarbital (Pb) or 3-methylcholanthrene (3-MC), or their incubation with hepatic microsomes from such rats, produced a larger decrease in cytochrome P-450 levels in the membranes from Pb- than 3-MC-treated rats. Comparable losses, however, were observed in microsomes from rats pretreated with both Pb and 3-MC when the last three agents were used. Similar experiments were carried out using the major cytochrome P-450 isozymes purified from liver microsomes of Pb- or 3-MC-treated rats. The Pb isozyme was inactivated during catalytic turnover of all six substrates while only three substrates (DCBT, ABT, and fluroxene) were found to inactivate the 3-MC isozyme. Oxygen consumption studies with purified enzymes have shown that AIA is not a measurable substrate for the 3-MC isozyme, a fact which explains its failure to inactivate this isozyme. Similar studies with the Pb isozyme establish that one enzyme molecule is inactivated for approximately every 230–320 AIA molecules processed by the enzyme.

Environmental Aspects of Ivermectin Usage in Livestock: General Considerations

A detailed analysis of ivermectin’s effect on the environment was an integral component of the overall program to develop ivermectin as an antiparasitic drug for food-producing animals. The analytical studies were designed to determine whether using ivermectin in animals would result in any harmful or undesirable effects on the environment. These studies measured ivermectin’s physical properties and its mobility, distribution, and stability in soil and water. Additional studies investigated the drug’s effect on a variety of environmentally important organisms. These studies, combined with the clinical use pattern of ivermectin in cattle, sheep, and swine, provided the means to assess ivermectin’s environmental impact.

The association of cytochrome P-450 and NADPH-cytochrome P-450 reductase in phospholipid membranes

NADPH-cytochrome P-450 reductase and two purified isozymes of cytochrome P-450 have been incorporated into phospholipid vesicles by a cholate dialysis technique. The enzyme system reconstituted in this manner was catalytically active. The observed kinetics for substrate oxidation indicated that both enzymes were associated with the liposomal membranes, and were not simply entrapped in the interior of the vesicle. The N-demethylation of benzphetamine was measured in order to determine the effect of variations in the mole ratio between the two enzymes and between the lipid and the total enzyme on the observed steady-state kinetics. In addition, the kinetic isotope effects for the O-deethylation of 7-ethoxycoumarin were measured in order to compare these parameters to those previously observed in a reconstituted system [G. T. Miwa, and A. Y. H. Lu (1981) Arch. Biochem. Biophys. 211, 454-458]. The results were all consistent with the association of the two proteins by lateral diffusion in the vesicle membrane. Moreover, the observed reduction in catalytic activity, as the enzymes were diluted in the vesicle membrane, can only be explained by the formation of a transient P-450-reductase complex, and not by the existence of a stable complex between the two proteins. These results provide compelling evidence for a mass action model for the interaction of these two enzymes in liposomal membranes.

Comparative in vivo and in vitro metabolism of ivermectin in steers, sheep, swine, and rat.

(1987). Comparative in Vivoand in Vitro Metabolism of Ivermectin in Steers, Sheep, Swine, and Rat. Drug Metabolism Reviews: Vol. 18, No. 2-3, pp. 289-302.

Metabolism and tissue residues.

Ivermectin is used widely as an antiparasitic agent in food-producing animals. As in the case of any such drug, the residual tissue concentration of the therapeutic agent, or tissue residue, is a safety concern to the meat-consuming public. To evaluate the toxic potential of the residual tissue concentration of ivermectin and its metabolites, metabolism studies have been carried out in target species (cattle, sheep, swine) using the radiolabeled drug. Comparative metabolic studies were done in a laboratory animal, the rat, and in liver microsomes from various species.

Differential induction of rat hepatic cytochrome P-448 and glutathione S-transferase B messenger RNAs by 3-methylcholanthrene.

Abstract Liver poly(A + )-RNA isolated from untreated and 3-methylcholanthrene treated rats has been translated in the rabbit reticulocyte cell-free system in order to determine the level of translationally active cytochrome P-448, glutathione S-transferase B and serum albumin mRNAs. Translatable cytochrome P-448 mRNA was not detected in untreated rats; however in animals treated with 3-methylcholanthrene cytochrome P-448 mRNA was elevated markedly. Functional rat liver glutathione S-transferase B mRNA was elevated 2-fold by 3-methylcholanthrene administration, whereas the serum albumin mRNA level was decreased by 50%. Our results indicate that 3-methylcholanthrene is not just a specific inducer of drug metabolizing enzymes but can alter the mRNA level encoding other polypeptides and thus affect cellular homeostasis.

Drug residue formation from ronidazole, a 5-nitroimidazole. I. Characterization of in vitro protein alkylation

The metabolic activation of [14C]ronidazole by rat liver enzymes to metabolite(s) bound to macromolecules was investigated. The alkylation of protein by [14C]ronidazole metabolite(s) was catalyzed most efficiently by rat liver microsomes, in the absence of oxygen utilizing NADPH as a source of reducing equivalents. Based on a comparison of total ronidazole metabolized versus the amount bound to microsomal protein, approximately one molecule alkylates microsomal protein for every 20 molecules of ronidazole metabolized. Protein alkylation was strongly inhibited by sulfhydryl-containing compounds such as cysteine and glutathione whereas methionine had no effect. Based on HPLC analysis of ronidazole, cysteine was found not to inhibit microsomal metabolism of ronidazole ruling out a decrease in the rate of production of the reactive metabolite(s) as the mechanism of cysteine inhibition.