Current Drug Metabolism - Volume 6, Issue 2, 2005

Our previous animal study has suggested that the accelerated metabolism of warfarin enantiomers with concurrent coenzyme Q10 (CoQ10) treatment accounts for the reduced anticoagulant effect of warfarin in rats. The present study was to assess the effect of CoQ10 on individual hydroxylation pathways of the in vitro microsomal metabolism of warfarin enantiomers and to extrapolate in vitro data to in vivo situation. The effect of the antioxidant CoQ10 on the hydroxylation of warfarin enantiomers was examined using rat and human liver microsomes. Based on the in vitro kinetic data, together with the information retrieved from the literature, the magnitude of warfarin-CoQ10 interaction in man was quantitatively predicted. In rat liver microsomes, CoQ10 exhibited a selective activation effect on the 4'-hydroxylation of S-warfarin, with a KA value (i.e. dissociation constant of the enzyme-activator complex) being one third and one fifth of those for the 6- and 7- hydroxylation, respectively. The activation effect of CoQ10 was selective towards the 6- and 7-hydroxylation of Rwarfarin at low substrate concentrations, but towards the 4'-hydoxylation of the R-enantiomer at high substrate concentrations. In human liver microsomes, CoQ10 was a selective activator of the 7-hydroxylation of both R- and Senantiomers of warfarin, with KA values being half to one twelfth of those for the other pathways. A relatively accurate prediction was made for the increase in the total and hepatic clearance of both S- and R-warfarin in rats with concurrent CoQ10 treatment based on their respective overall hydroxylation, when the active transport of CoQ10 into the hepatocytes was taken into consideration. In man, one would expect about 32% and 17% increase in the total clearance of S- and Rwarfarin, respectively, with coadministration of 100 mg CoQ10. In both species, CoQ10 had enzyme activation effect, which appeared to be regioselective but not stereoselective, on the formation of the phenolic metabolites of warfarin enantiomers. A moderate increase in the total clearance of warfarin enantiomers could occur with coadministration of CoQ10 in humans.

This paper reviews the empirical methods of quantitative microdialysis that have been used to interpret the results obtained from pharmacokinetic studies. The concept of extraction efficiency or recovery and the properties of recovery in vivo (variation with flow rate, time dependency and influence of the mode of administration) are considered. The most frequently used methods for determining recovery in vivo are described and evaluated in the light of recent theoretical studies. Specifically, we review the variation of flow rate method, the very slow flow method, the no net flux method and the delivery and retrodialysis methods. Special emphasis is placed on the description of each method, demonstrating its applicability to pharmacokinetic studies conducted under steady-state or transient conditions, and also its limitations. Finally, the more relevant studies that have compared the suitability of these methods are reviewed.

UDP-glucuronosyltransferase is a group of catabolic enzymes involved in the detoxification and excretion of many xenobiotic and endogeneous substances in intrahepatic and extrahepatic tissues. The group consists of two subfamilies, UGT1 and UGT2. UGT1 consists of 5 exons and has a unique gene structure. There are thirteen exon 1s from UGT1A1 to UGT1A13P, and exon 2 to exon 5 are used in common for all mRNAs expressed from the gene. Each isoform of UGT1 results from differential splicing of exon1s to common exon 2-5, and has an unique spectrum of substrate specificity. In contrast, the genes of the UGT2 family consist of 6 exons, and all the enzymes have an individual set of exon 1 to exon 6. In UGT1 there are no reports of polymorphism in the common exons, although a number of polymorphisms have been reported for exon 1s. The mutations of UGT1A1 cause hereditary unconjugated hyperbilirubinemias: Crigler-Najjar syndrome type I, type II and Gilbert syndrome. UGT1A1 has two major polymorphisms - a missense mutation of G71R and an insertion mutation of TATA box. Prevalence of Gilbert syndrome is attributed to these polymorphisms. Since UGT1A1 metabolizes not only bilirubin but also hormones and drugs, the mutations could be involved in carcinogenesis and adverse drug reactions. Recent studies also revealed a widespread presence of diverse polymorphisms in other isoforms of UGT1 as well as the UGT2 family, including UGT1A6, UGTG1A7, UGT1A8, UGT1A10, UGT2B4, UGT2B7 and UGT2B15. The incidences and types of the polymorphisms for these enzymes are quite different in region and ethnic groups. Understanding of these polymorphisms is essential for the prevention of adverse effects of a considerable number of drugs and to predict cancer risks.

By assessing how drug/new chemical entity (NCE) cytotoxicity is affected when their metabolic pathways are inhibited or activated, the metabolic pathways that activate versus detoxify drugs/NCEs can be identified. Reactive metabolites contributing to cytotoxicity can also be identified. In the following, the drug metabolizing enzyme inhibitors and activators used in vitro with freshly isolated rat hepatocytes for the accelerated cytotoxicity mechanism screening (ACMS) of drugs/NCEs (a technique used in our laboratory) are reviewed and, this technique is useful for determining in vivo rat hepatotoxicity mechanisms. The enzyme inhibitors/activators have been chosen on the basis of their selectivity, modulator effectiveness, and their lack of toxicity. The use of these inhibitors/activators with human hepatocytes or subcellular fractions for assessing human hepatotoxicity mechanisms is also reviewed.

Understanding the hepatotoxicity of drugs and chemicals is essential for progress in the pharmaceutical industry, medical science and academic research. The study of hepatotoxicity in vitro is complicated by the difficulty of maintaining hepatocytes in culture due to a lack of understanding of the humoral and matrix requirements of these cells. A variety of in vitro models of the liver have been developed, such as perfused livers, liver slices and three-dimensional perfused bioreactors, but the static cell culture is the most commonly used system. In this review we present the advantages and disadvantages of each system and their roles in the study of hepatotoxicity. We will also discuss how the various culture conditions such as medium and matrix composition affect the systems. The technological advances, which started the fields of genomics, proteomics and metabonomics are playing a very important role in uncovering novel biochemical pathways and markers of toxicity. Several of these studies have focused on hepatotoxicity, particularly on the effects of acetaminophen, carbon tetrachloride and aflatoxin B1. Finally, we will discuss the new field of systems biology, which focuses on interpreting and integrating data from all of the other fields.

Epilepsy is the most common primary neurological disorder known. Epileptiform neurons undergo paroxysmal depolarization shifts (PDS), which result in the excessive sustained neuronal firing seen in epilepsy. These shifts are due to either an impairment of GABA mediated inhibition, or an enhancement of aspartate or glutamate mediated excitatory transmission. Recent research has focused on the cellular biology of seizures. 4-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter of mammalian central nervous system. In neural and nonneural tissues, GABA is metabolized by three enzymes-glutamic acid decarboxylase (GAD), which produces GABA from glutamic acid, and the catabolic enzymes GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). Production of succinic acid by SSADH allows entry of the GABA carbon skeleton into the tricarboxylic acid cycle. GABA-T is present in a variety of circulating cells, including platelets and lymphocytes. SSADH, the final enzyme of GABA catabolism, has been detected in some of the tissues in which GAD and GABA-T have been identified. This paper is aimed at elucidating the organization of the GABA shunt and covers a review on the antiepileptic drugs, both established and currently under development targeted to the GABA shunt in order to bring about effective seizure control.

UDP-Glucuronosyltransferases (UGTs) are actively involved in detoxification of xenobiotics and endogenous compounds and are a major source of drug inactivation and drug-drug interactions. UGTs are membrane-bound enzymes mostly localized in the endoplasmic reticulum (ER) and inner and outer nuclear membranes. UGT activities are totally dependent on the phospholipid content of the membrane and, as a result, are usually inactive when isolated from the ER in the presence of detergent. Several UGT expression systems have been described by different laboratories. They include expression in mammalian cells such as COS, V79 and HEK293. Also, baculovirus-infected insect cells systems have been developed and allow the expression of UGT isoforms with or without histidine molecule tags (His-tags). Moreover, as for CYP450, UGT isoforms have been expressed in E.coli. This review concentrates on a detailed description of all these expression systems in terms of their use for substrate specificity studies and the preparation of pure UGT proteins for active site identification and other structural studies. The effect of detergents and alamethicin on UGT catalytic activity in different expression systems will be discussed. Moreover, extensive comparative studies on the characterization of recombinant UGTs in terms of substrate specificity, evaluation of kinetic parameters, and the effect of inhibitors will be presented in this review. An overall picture of the use of different UGT expression systems will help in selecting the best one for identification of the individual UGT isoforms involved in the glucuronidation of drugs, environmental pollutants and physiologically important endogenous compounds. Especially important is an expression system where UGTs are biosynthesized with His-tags. UGTs expressed in this system can be easily purified to homogeneity, which will result in significant development of structure-function relationship studies, including the identification of substrate active sites and eventual crystallization. These are underdeveloped areas of UGT research and the availability of these recombinant UGTs will allow these gaps to be filled.