Nicotine is of importance as the addictive chemical in tobacco, pharmacotherapy for smoking cessation, a potential medication for several diseases, and a useful probe drug for phenotyping cytochrome P450 2A6 (CYP2A6). We review current knowledge about the metabolism and disposition kinetics of nicotine, some other naturally occurring tobacco alkaloids, and nicotine analogs that are under development as potential therapeutic agents. The focus is on studies in humans, but animal data are mentioned when relevant to the interpretation of human data. The pathways of nicotine metabolism are described in detail. Absorption, distribution, metabolism, and excretion of nicotine and related compounds are reviewed. Enzymes involved in nicotine metabolism including cytochrome P450 enzymes, aldehyde oxidase, flavin-containing monooxygenase 3, amine N-methyltransferase, and UDP-glucuronosyltransferases are represented, as well as factors affecting metabolism, such as genetic variations in metabolic enzymes, effects of diet, age, gender, pregnancy, liver and kidney diseases, and racial and ethnic differences. Also effects of smoking and various inhibitors and inducers, including oral contraceptives, on nicotine metabolism are discussed. Due to the significance of the CYP2A6 enzyme in nicotine clearance, special emphasis is given to the effects and population distributions of CYP2A6 alleles and the regulation of CYP2A6 enzyme.

Metabolism and Disposition Kinetics of Nicotine

Nicotine underlies tobacco addiction, influences tobacco use patterns, and is used as a pharmacological aid to smoking cessation. The absorption, distribution and disposition characteristics of nicotine from tobacco and medicinal products are reviewed. Nicotine is metabolized primarily by the liver enzymes CYP2A6, UDPglucuronosyltransfease (UGT), and flavin-containing monooxygenase (FMO). In addition to genetic factors, nicotine metabolism is influenced by diet and meals, age, sex, use of estrogen-containing hormone preparations, pregnancy and kidney disease, other medications, and smoking itself. Substantial racial/ethnic differences are observed in nicotine metabolism, which are likely influenced by both genetic and environmental factors. The most widely used biomarker of nicotine intake is cotinine, which may be measured in blood, urine, saliva, hair, or nails. The current optimal plasma cotinine cut-point to distinguish smokers from non-smokers in the general US population is 3 ng ml−1. This cut-point is much lower than that established 20 years ago, reflecting less secondhand smoke exposure due to clear air policies and more light or occasional smoking.

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Nicotine Chemistry, Metabolism, Kinetics and Biomarkers

Currently available HIV-1 protease inhibitors are potent agents in the therapy of HIV-1 infection. However, limited oral absorption and variable tissue distribution, both of which are largely unexplained, complicate their use. We tested the hypothesis that P-glycoprotein is an important transporter for these agents. We studied the vectorial transport characteristics of indinavir, nelfinavir, and saquinavir in vitro using the model P-glycoprotein expressing cell lines L-MDR1 and Caco-2 cells, and in vivo after intravenous and oral administration of these agents to mice with a disrupted mdr1a gene. All three compounds were found to be transported by P-glycoprotein in vitro. After oral administration, plasma concentrations were elevated 2-5-fold in mdr1a (-/-) mice and with intravenous administration, brain concentrations were elevated 7-36-fold. These data demonstrate that P-glycoprotein limits the oral bioavailability and penetration of these agents into the brain. This raises the possibility that higher HIV-1 protease inhibitor concentrations may be obtained by targeted pharmacologic inhibition of P-glycoprotein transport activity.

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The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors.

Literature data on compounds both well- and poorly-absorbed in humans were used to build a statistical pattern recognition model of passive intestinal absorption. Robust outlier detection was utilized to analyze the well-absorbed compounds, some of which were intermingled with the poorly-absorbed compounds in the model space. Outliers were identified as being actively transported. The descriptors chosen for inclusion in the model were PSA and AlogP98, based on consideration of the physical processes involved in membrane permeability and the interrelationships and redundancies between available descriptors. These descriptors are quite straightforward for a medicinal chemist to interpret, enhancing the utility of the model. Molecular weight, while often used in passive absorption models, was shown to be superfluous, as it is already a component of both PSA and AlogP98. Extensive validation of the model on hundreds of known orally delivered drugs, “drug-like” molecules, and Pharmacopeia, Inc. compounds, whic...

Prediction of drug absorption using multivariate statistics.

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.

Inhibition and Induction of Cytochrome P450 and the Clinical Implications

The gastrointestinal tract remains the most popular and acceptable route of administration for drugs. It offers the great advantage of convenience and many compounds are well absorbed and thereby provide acceptable plasma concentration-time profiles. Currently there is considerable interest from the pharmaceutical industry in development of cell culture systems that would mimic the intestinal mucosa in order to evaluate strategies for investigating and/or enhancing drug absorption. The intestinal epithelial cells of primary interest, from the standpoint of drug absorption and metabolism, are the villus cells, which are fully differentiated cells. An in vitro cell culture system consisting of a monolayer of viable, polarized and fully differentiated villus cells, similar to that found in the small intestine, would be a valuable tool in the study of drug and nutrient transport and metabolism. The Caco-2 cell line, which exhibits a well-differentiated brush border on the apical surface and tight junctions, and expresses typical small-intestinal microvillus hydrolases and nutrient transporters, has proven to be the most popular in vitro model (a) to rapidly assess the cellular permeability of potential drug candidates, (b) to elucidate pathways of drug transport (e.g., passive versus carrier mediated), (c) to assess formulation strategies designed to enhance membrane permeability, (d) to determine the optimal physicochemical characteristics for passive diffusion of drugs, and (e) to assess potential toxic effects of drug candidates or formulation components on this biological barrier. Since differentiated Caco-2 cells express various cytochrome P450 isoforms and phase II enzymes such as UDP-glucuronosyltransferases, sulfotransferases and glutathione-S-transferases, this model could also allow the study of presystemic drug metabolism.

The human intestinal epithelial cell line Caco-2; pharmacological and pharmacokinetic applications.

Cytochrome P450 chemical inhibitors are widely used to define the role of individual cytochrome P450 isozyme(s) in a metabolism process. In this study, cytochrome P450 isoform-dependent reactions were investigated on our human liver microsomes bank (n = 34) and characterized for both KM and VMAX values (n > or = 3). These metabolic reactions were: 7-ethoxyresorufin O-deethylation (CYP1A1), phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), tolbutamide 4-methylhydroxylation (CYP2C9), dextromethorphan O-demethylation (CYP2D6), aniline 4-hydroxylation (CYP2E1) and nifedipine dehydrogenation (CYP3A4). Literature data-based specific inhibitors were selected and characterized for both their inhibitory constant (Ki) and the inhibition-type toward their specific substrate. Results were as follows: alpha-naphthoflavone (CYP1A1; mixed-type interaction with a Ki = 0.01 microM), furafylline (CYP1A2; competitive-type interaction with a Ki = 3 microM when microsomes were incubated with both furafylline and phenacetin; noncompetitive-type interaction with a Ki = 0.6 microM when microsomes were preincubated with furafylline and NADPH), pilocarpine (CYP2A6; competitive-type interaction with a Ki = 4 microM), sulfaphenazole (CYP2C9; competitive-type interaction with a Ki = 0.3 microM), quinidine (CYP2D6; competitive-type interaction with a Ki = 0.4 microM, diallyldisulfide (CYP2E1; noncompetitive-type interaction with a Ki = 150 microM on an aniline concentration range of 10-60 microM; competitive-type interaction with a Ki = 100 microM on an aniline concentration range of 80-2000 microM) and ketoconazole (CYP3A4; mixed-type interaction with a Ki = 0.015 microM). Once the inhibitors' potency was determined, the selective effects of these inhibitors were evaluated after incubation of human hepatic microsomes with isoform-selective substrates in the presence of the different chemical inhibitors. Up to 10 times the Ki value toward the isoform-selective probe, pilocarpine, sulfaphenazole, quinidine and ketoconazole exhibited potent inhibitory and specific effects. alpha-Naphthoflavone and furafylline both inhibited phenacetin and 7-ethoxyresorufin O-deethylation processes, a consequence of the absence of CYP1A1 in noninduced human liver. Diallyldisulfide exhibited broad and nonspecific inhibitory effects. When used in their "window of selectivity," ie., up to 10-fold the Ki value, most chemical inhibitors powerfully and specifically inhibited cytochrome P450 isoform-specific reactions when analyzed at their KM values.

Cytochrome P450 isoform inhibitors as a tool for the investigation of metabolic reactions catalyzed by human liver microsomes.

1. The aims were to refine experimental conditions (using 76 human hepatocyte preparations) in terms of the selection of enzyme inducers and their optimal concentration, the treatment duration with inducers and the choice of specific cytochrome P450 isoform(s) probes to optimize the use of primary hepatocytes for predicting the potential induction by new chemical entities of cytochrome P450 isoforms in vivo in man. 2. In the absence of any inducer, basal cytochrome P450 isoform(s)-mediated activities decreased to 20% of their initial activity (end of the seeding period) by 72-96 h. In contrast, UGT-dependent enzyme activities remained at a constant level (+/- 20%) up to the fifth day of culture. 3. Beta-naphthoflavone, at an optimal concentration of 50 microM and after a 3-day treatment, specifically and potently induced 7-ethoxyresorufin (10.4 +/- 10.4-fold, n = 74) and phenacetin (6.6 +/- 6.4-fold, n = 60) O-deethylation processes, markers for CYP1A1 and CYP1A2 isoforms respectively. Only a 2-fold increase was noted following treatment with 2 mM phenobarbitone, whereas dexamethasone and rifampicin had no effect at all. 4. A 3-day treatment of human hepatocytes with 50 microM dexamethasone was associated with a major induction of both coumarin 7-hydroxylation (9.4 +/- 11.4-fold, n = 49) and nifedipine dehydrogenation (4.7 +/- 3.8-fold, n = 61), markers for CYP2A6 and CYP3A4 respectively. Phenobarbitone, however, exhibited a broad but moderate inducing effect on 7-ethoxyresorufin (2.2 +/- 1.5-fold, n = 55) and phenacetin (1.7 +/- 0.9-fold, n = 54) O-deethylation, coumarin 7-hydroxylation (3.9 +/- 9.2-fold, n = 50) and nifedipine dehydrogenation (2.1 +/- 2.0-fold, n = 47). 5. Km obtained for the different cytochrome P450 isoform substrates in untreated hepatocytes were in the same range of magnitude that those determined on human hepatic microsomal fractions. Enzyme induction processes were characterized by a large increase in apparent Vmax whereas apparent Km were not affected. 6. These studies demonstrate that human hepatocytes in primary culture can respond specifically and quantitatively to model inducers. This in vitro system offers a useful approach to study the regulation of human hepatic biotransformation activities and should facilitate the demand for a reproducible method for addressing cytochrome P450 induction.

Expression and induction of CYP1A1/1A2, CYP2A6 and CYP3A4 in primary cultures of human hepatocytes : a 10-year follow-up

The expression and inducibility of cytochrome P450IA1 isozyme was investigated in the human carcinoma cell line Caco-2 cultured between days 7 and 35 in the absence or the presence of various enzyme inducers such as 3-methylcholanthrene, beta-naphthoflavone (beta NF), dioxin, isosafrole, rifampycin, dexamethasone or phenobarbital. 7-Ethoxyresorufin O-deethylase activity (EROD) was maximal at day 25 when the differentiation of Caco-2 cells, characterized by the level of the brush border associated enzymes such as sucrase isomaltase and alkaline phosphatase, was higher. The inducibility of this enzyme activity was found to be maximal when cells were treated between days 7 and 10. After a 3-day treatment of Caco-2 cells with 50 microM beta NF, EROD achieved 36.6 +/- 14.6 pmol/min/mg compared to 2.5 +/- 1.1 pmol/min/mg in untreated cells. This enzyme activity appeared to be supported only by P450IA1 isozyme because: 1) EROD was quantitatively inhibited by alpha-naphthoflavone, a P450IA1-specific inhibitor; otherwise, phenacetin O-deethylation was completely abolished in the presence of alpha-naphthoflavone and not by furafylline, a P450IA2-specific inhibitor; 2) EROD was induced after treatment with 3-methylcholanthrene, beta NF and dioxin, which are P450IA1 inducers, but not by isosafrole, a P450IA2-specific inducer; 3) cytochrome P450IA1 apoprotein could be immunodetected by antibodies directed against rabbit cytochrome P450-LM6, orthologous to P450IA1, in polycyclic hydrocarbon-treated cells; 4) under the latter conditions, P450IA1 mRNA accumulation was specifically detected, but not P450IA2 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of cytochrome P450IA1 gene expression in a human intestinal cell line, Caco-2.

The glucuronidation of the AT1 nonpeptide angiotensin II receptor antagonist, SR 47436 (BMS 186295), was investigated in hepatic microsomes prepared from various species, i.e., Sprague-Dawley rat, Cynomolgus monkey and Caucasian humans. The drug was found to undergo N-glucuronidation on the tetrazole moiety as confirmed by its hydrolysis by beta-glucuronidase, its associated radioactivity when UDP-[U-14C]glucuronic acid was used as substrate and by different techniques such as high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. Glucuronide formation was optimal at pH 5.0 along with a "0.2 mg of Brij 58 per mg of protein" ratio, regardless of the investigated species. Cynomolgus monkey microsomes glucuronidated SR 47436 (BMS 186295) to the greatest extent, with a relative catalytic efficiency 11.0- and 2.6-fold higher than that observed in rat and human, respectively. SR 47436 (BMS 186295) glucuronidation followed Michaelis-Menten kinetics. Bilirubin:UDP-glucuronosyltransferase isoform was not involved, inasmuch as bilirubin did not affect its glucuronidation, 7,7,7-triphenylheptanoic acid was a noncompetitive inhibitor and glucuronidation was only decreased 2-fold in Gunn rats. SR 47436 (BMS 186295) glucuronidation was enhanced markedly after treatment of rats with dexamethasone (Vmax/Km = 71.5 vs. 2.6 in untreated animals). Among the drugs used which undergo phenolic, carboxylic acid, alcohol or tertiary amine glucuronidation, only monodigitoxigenin-monodigitoxoside, flurbiprofen, naproxen, testosterone and estrone inhibited SR 47436 (BMS 186295) glucoronidation in a noncompetitive manner. These data suggest that SR 47436 (BMS 186295) was glucuronidated by a highly dexamethasone-inducible UDP-glucuronosyltransferase isoform(s), different from that involved in the glucuronidation of monodigitoxigenin-monodigitoxoside.

In vitro N-glucuronidation of SB 47436 (BMS 186295), a new AT1 nonpeptide angiotensin II receptor antagonist, by rat, monkey and human hepatic microsomal fractions.

Y Berger

Papers

The human intestinal epithelial cell line Caco-2; pharmacological and pharmacokinetic applications.

Cytochrome P450 isoform inhibitors as a tool for the investigation of metabolic reactions catalyzed by human liver microsomes.

Expression and induction of CYP1A1/1A2, CYP2A6 and CYP3A4 in primary cultures of human hepatocytes : a 10-year follow-up

Regulation of cytochrome P450IA1 gene expression in a human intestinal cell line, Caco-2.

In vitro N-glucuronidation of SB 47436 (BMS 186295), a new AT1 nonpeptide angiotensin II receptor antagonist, by rat, monkey and human hepatic microsomal fractions.