Current Drug Metabolism - Volume 6, Issue 5, 2005

Clinically observed warfarin-omeprazole interaction has been found to be associated with the inhibition of Rwarfarin hydroxylation by omeprazole. The present study was conducted in human liver microsomes and cDNAexpressed cytochrome P450s to assess the inhibitory potential of omeprazole on the hydroxylation of warfarin enantiomers, and to identify the cytochrome P450 isozymes involved in the inhibition of hydroxylation of warfarin enantiomers by omeprazole, and to evaluate the extent to which the in vitro data is predictive of the actual pharmacokinetic interaction between warfarin and omeprazole observed in vivo. Omeprazole inhibited the formation of R-6-, R-7- and S-7-hydroxywarfarin with the Ki values of 40, 22 and 116 μM, respectively. Its inhibitory effect was selective towards R-warfarin. Further study conducted in cDNA-expressed cytochrome P450s (CYPs) demonstrates that the inhibition of the in-vitro biotransformation of warfarin enantiomers by omeprazole is attributed to its inhibitory effect on the activities of CYP1A2, CYP3A4, CYP2C9 and CYP2C19. The extent of the in vivo warfarin-omeprazole interaction was underestimated as based on the Ki values obtained from the in-vitro inhibition study, suggesting an underestimation of the effective concentration of the inhibitor at the site of interaction or some other mechanisms involved in the drug interaction between warfarin and omeprazole.

The inhibition of human cytochrome P450s (CYPs) is one of the most common mechanisms which can lead to drug-drug interactions. The inhibition of CYPs can be reversible (competitive or non-competitive) or irreversible. Irreversible inhibition usually derives from activation of a drug by CYPs into a reactive metabolite, which tightly binds to the enzyme active site, leading to a long lasting inactivation. This process is called "mechanism based inhibition" or "suicide inhibition". The irreversible inactivation usually implies the formation of a covalent bond between the metabolite and the enzyme, which can lead to hapten formation and can in some cases trigger an autoimmune-response. For these reasons it is of utmost importance to study the mechanism of the CYP inhibition of new potential drugs as early as possible during the drug discovery process. The literature on CYPs is vast and covers numerous aspects of their biology and biochemistry, however to our knowledge there is no general and systematic review focusing on mechanism-based inhibitors; we have reviewed the literature and compiled all the available data on chemical entities, which are known to be CYP suicide inhibitors. Each compound is reported together with its chemical structure, the CYP isoform and the parameters describing the inactivation. Literature references are reported together with their PMID (PubMed ID number) to allow a fast retrieval of the papers. This review offers a quick reference to help predict liabilities of new chemical entities without carrying out extensive in vitro work, and will hopefully help in designing safer drugs.

Conjugating enzymes are traditionally recognized as one of the major biological barriers to the entry of xenobiotics/ drugs into systemic circulation and represent one of the main pathways for their elimination. Similar to drugs that undergo extensive phase I metabolism, drugs that undergo extensive conjugation have poor bioavailability and are more prone to metabolism-based drug interactions. Previously, enterohepatic recycling is used to explain why certain xenobiotics have half-lives that are much longer than expected from intravenous injection studies. In addition, changes in expression levels of metabolic enzymes due to chemical induction or suppression are often recognized as the source of drug interaction or toxicity of pollutants and carcinogens. These traditional approaches, whereas yielding highly valuable information, fail to recognize the fact that many conjugates (especially hydrophilic ones) cannot permeate the cell membrane. In the present review, we will focus on the coupling process that involves both conjugating enzymes and efflux transporters. We will briefly review conjugating enzymes capable of producing highly hydrophilic metabolic products. The other focus of this review is on various transporters capable of moving negatively charged hydrophilic conjugates across the cellular membrane. Evidence will support the hypothesis that efficient coupling of the conjugating enzymes and efflux transporters enables enterohepatic recycling and enteric recycling processes. Termed as a "revolving door" theory, the hypothesis focuses on the role played by efflux transporter capable of modulating the cellular excretion of hydrophilic metabolites. Coupling process in intestine, liver and kidney will be discussed with an emphasis on the intestinal coupling process, since we have just begun to understand it. Biological consequence and new insights into how coupling process can impact bioavailability of xenobiotics, biological functions of drugs and carcinogens, and drug interactions will be discussed.

Granisetron, a potent 5-HT3 receptor antagonist, has been reported to be mainly metabolized to 7- hydroxygranisetron and a lesser extent to 9'-desmethylgranisetron in humans. A previous study indicated that cytochrome P450 (CYP)3A4 is a major catalyst of 9'-demethylation, although the major CYP isoform(s) responsible for 7- hydroxylation are unknown. To clarify granisetron 7-hydroxylase, the in vitro metabolism of granisetron using expressed human CYPs and human liver microsomes was investigated. 7-Hydroxygranisetron was produced almost exclusively by CYP1A1, while, apparently, 9'-desmethylgranisetron was preferentially produced by CYP3A4. Marked inter-individual differences in the ratio of the formation of 7-hydroxygranisetron and 9'-desmethylgranisetron in human liver microsomes was observed. Granisetron 7-hydroxylase activity was strongly correlated with benzo[a]pyrene 3-hydroxylase activity (p<0.0001), but not with testosterone 6b-hydroxylase activity in human liver microsomes. Furthermore, an anti-human CYP1A1 antibody completely inhibited 7-hydroxylation in human liver microsomes, however, the reaction was not inhibited at all by an anti-CYP3A4 antibody. On the other hand, granisetron 9'-demethylase activity correlated significantly not only with testosterone 6b-hydroxylase activity (p<0.0001) but also with benzo[a]pyrene 3-hydroxylase activity (p<0.01). Consistent with this, both the anti-CYP1A1 and anti-human CYP3A4 antibodies inhibited the 9'-demethylase activity. These data indicate that CYP1A1 is a major enzyme responsible for the metabolism of granisetron via a main 7- hydroxylation pathway and an alternative 9'-demethylation route. This is the first report demonstrating the substantial contribution of CYP1A1 to the metabolism of a drug, although its role in the metabolism of environmental compounds is well established.

The effect on hepatic drug metabolising enzymes was evaluated for three representative structures (1, 2 and 3) that were selected from a series of substituted oxazine derivatives designed to possess particular pharmacological properties such as analgesic, antioxidant and hypolipidemic activity. In addition, since xenobiotic metabolism, reactive oxygen and nitrogen species, atherosclerosis and inflammation are interrelated and mutually affected, the effects of (2) and (3) on acute inflammation in vivo and lipoxygenase activity in vitro were also investigated. It was found that treatment of rats with (1) caused induction of cytochrome P450, enhancement of the metabolism of aminopyrine in vitro and of zoxazolamine and hexobarbital in vivo. Compound (2) appeared to induce particularly erythromycin N-demethylation, while (3), a nitric ester, reduced the catalytically active cytochrome P450, although it increased the metabolism of specific cytochrome P450 substrates, i.e. 4-nitrophenol and erythromycin. Compounds (2) and (3), with strong hypolipidemic and antioxidant properties, reduced acute inflammatory response in two inflammation models and inhibited lipoxygenase activity in vitro. These results are helpful in optimising the biological profile as well as the potential applications of substituted oxazines.

The present study was designed to investigate the hepatic disposition of the prodrug AM365 and the generated antiviral guanosine analogue, AM188 in the isolated perfused rat liver (IPL). The livers of rats (n = 12) were isolated and perfused with Krebs-Henseleit pH 7.4 buffer to which AM365 was added as a bolus to achieve an initial perfusate concentration of 22.4 μmol/L. During the 120-min period after administration of AM365, bile was collected in 10-min intervals and perfusate was collected at the mid-point of these intervals. Concentrations of AM365 and AM188 in perfusate and bile were quantified by HPLC. Following administration of AM365, its concentration in perfusate declined and the concentration of AM188 increased; the sum of the molar concentrations remained constant. The clearance and hepatic extraction ratio of AM365 were 3.3 ± 2.4 mL/min and 0.110 ± 0.079, respectively. The cumulative amount of AM365 excreted in bile during the 120-min perfusion period was approximately 0.21% of the bolus dose, and 0.36% of the total amount of AM365 cleared by the liver during the period. The cumulative amount of AM188 excreted in bile was about 0.48% of the total amount of AM188 formed during the perfusion period. In conclusion, AM365 was metabolised to AM188, which appeared to be the only metabolite and was not further biotransformed. The biliary excretion of AM365 and AM188 was negligible.

Defective sperm function is the most common cause of infertility, and until recently, was difficult to evaluate and treat. Mammalian spermatozoa membranes are rich in poly unsaturated fatty acids and are sensitive to oxygen induced damage mediated by lipid peroxidation. Hence, free radicals and reactive oxygen species [ROS] are associated with oxidative stress and are likely to play a number of significant and diverse roles in reproduction. The excessive generation of reactive oxygen species by abnormal spermatozoa and by contaminating leukocytes [leukocytospermia] has been identified as one of the few defined etiologies for male infertility. Moreover, environmental factors, such as pesticides, exogenous estrogens, and heavy metals may negatively impact spermatogenesis since male sperm counts were declined. In addition, aging is also likely to further induce oxidative stress. Limited endogenous mechanisms exist to reverse these damages. In a normal situation, the seminal plasma contains antioxidant mechanisms which are likely to quench these ROS and protect against any likely damage to spermatozoa. However, during genitourinary infection/inflammation these antioxidant mechanisms may downplay and create a situation called oxidative stress. Assessment of such oxidative stress status [OSS] may help in the medical treatment of male infertility by suitable antioxidants. The cellular damage in the semen is a result of an improper balance between ROS generation and scavenging activities. Therefore, numerous antioxidants such as vitamin C, vitamin E, glutathione, and coenzyme Q10, have proven beneficial effects in treating male infertility. A multi-faceted therapeutic approach to improve male fertility involves identifying harmful environmental and occupational risk factors, while correcting underlying nutritional imbalances to encourage optimal sperm production and function.

An important role of human cytochrome P450s (P450s) has been well recognized in the area of drug metabolism and pharmacokinetics. It has become possible in recent years to express catalytically active forms of these enzymes in various host systems. The resulting recombinant human P450s are either purified for studies of protein structure and the mechanism of catalysis or isolated in microsomal forms to serve the purposes of P450 phenotyping, metabolic stability screening and inhibitory potential evaluation. Intact mammalian cells expressing human enzymes may also be used to test the mutagenic and toxicity potential of drug candidates. The issue remains, however, that the data derived from recombinant P450s are not always consistent with those generated from human tissue preparations. The aim of this communication is to discuss applications of recombinant P450s in the drug discovery and development setting, with an emphasis on comparison of recombinant and human liver microsomal systems.