Current Drug Metabolism, Volume 4, Number 4, 2003
Metabolite Characterization in Drug Discovery
Utilizing Robotic Liquid- Handling, Quadruple Time-of-Flight Mass Spectrometry
and In-Silico Prediction Pp. 259-271
A-E.F. Nassar and P.E. Adams
Utility of Mdr1-Gene Deficient Mice in
Assessing the Impact of P-glycoprotein on Pharmacokinetics and Pharmacodynamics
in Drug Discovery and Development Pp. 272-291
Cuiping Chen , Xingrong Liu and Bill J. Smith
Human Hepatocytes as a Tool for Studying Toxicity
and Drug Metabolism Pp.
292-312
M.J. Gomez-Lechon , M.T. Donato, J.V. Castell
and R. Jover
An Update on the Extraneuronal Monoamine
Transporter (EMT): Characteristics, Distribution and Regulation Pp. 313-318
F. Martel and
The Metabolism of Diclofenac - Enzymology and
Toxicology Perspectives Pp.
319-329
Wei Tang
Abstracts
[Back to top] Metabolite Characterization in Drug Discovery
Utilizing Robotic Liquid- Handling, Quadruple Time-of-Flight Mass Spectrometry
and In-Silico Prediction
A-E.F. Nassar and P.E. Adams
An assay method for identification of metabolites from in vitro microsomal incubations was developed for use in the early stage of drug discovery. We have developed a practical approach which involves integrated sample generation, sample preparation, bioanalysis, and data handling to maximize sample throughput and speed up the process for identification of metabolites. The assay system consisted of a robotic liquid handler (Genesis workstation) to generate and process samples, PALLAS MetabolExpert software to predict possible metabolites, exact mass measurement via a tandem quadrupole time-of-flight mass spectrometer (QTOF-MS) coupled with liquid chromatography to analyze samples, MetaboLynx software to find potential metabolites and Advanced Chemistry Development/MS (ACD/MS) software to provide guidance to the most likely hypothetical metabolite chemical structures. For purposes of evaluating this new method, dextromethorphan, alprenolol, and propranolol were incubated separately for up to 60 minutes with rat and human hepatic microsomes. The incubation and sample preparation were carried out in 96-well plates using the Genesis workstation. The bioanalysis was performed by LC-MS/MS using QTOF with MetaboLynx software to find metabolites. Metabolic products formed in vitro by rat and human microsomes were separated using an analytical column C18 with gradient elution at flow rate of 250 ml/min. The internal mass calibration was performed by continuous postcolumn infusion of Haloperidol. The mass spectra from incubations containing NADPH were compared to those without NADPH (control) using the MetaboLynx software to find potential metabolites. Finally, the MS/MS spectra were processed by the ACD/MS software to predict the chemical structure. MetaboLynx software successfully identified metabolites for each of the drugs studied by automatically discerning expected metabolites. Exact differences in masses between each metabolite and parent drug were measured from five replicate sample injections. All measured values are accurate to less than 0.001Da or 3.8 ppm with the standard deviation within 0.0015 Da, which allowed good prediction/confirmation of empirical formulae. Hypothetical chemical structures were achieved by the ACD/MS software and provided a useful tool to assist in prediction of the metabolic pathways of the drugs. The metabolites identified were in good agreement with previously published results for all three compounds. This new method will greatly enhance throughput, which in turn will facilitate our ability to rapidly provide this guidance to the synthetic chemist.
[Back to top] Utility of Mdr1-Gene Deficient Mice in Assessing the Impact
of P-glycoprotein on Pharmacokinetics and Pharmacodynamics in Drug Discovery and Development
Cuiping Chen , Xingrong Liu and Bill J. Smith
Since the generation of the multi-drug resistance 1 (mdr1) gene knockout (KO) mice in the early 90’s, these animals have been instrumental to our understanding of the physiological roles of mdr1 gene product P-glycoprotein. Located in crucial organs such as brain, intestine, liver, and kidney, P-glycoprotein-mediated transport has been shown to affect both the pharmacokinetics and pharmacodynamics of endogenous compounds and xenobiotics. It appears that P-glycoprotein may not be essential for the maintenance of normal body function as suggested by the similarity in life span and serum chemistry values of mdr1 gene KO mice compared to their genetically competent littermates. However, numerous studies have demonstrated that P-glycoprotein limits the brain penetration of many drug substrates. The reduced central nervous system (CNS) access of these compounds has been linked to decreased pharmacological or toxicological effects. In contrast to the critical role that P-glycoprotein plays in the brain, the extent of P-glycoprotein involvement in oral absorption and hepatobiliary or renal excretion of xenobiotics appears more variable. In addition to the mdr1 gene KO model, in vitro cell lines that over-express P-glycoprotein, and clinical trials using P-glycoprotein modulators have allowed for the comparison of in vitro-in vivo and species related difference in P-glycoprotein activity. For the most part, studies have shown reasonable in vitro-in vivo correlations, modest species-related differences, and comparable human-mouse in vivo P-glycoprotein effects on systemic drug disposition. Therefore, the mdr1 gene KO mouse model, when used appropriately, may allow for prediction of CNS drug access and certain drug-drug interaction.
[Back to top] Human Hepatocytes as a Tool for Studying
Toxicity and Drug Metabolism
M.J. Gomez-Lechon , M.T. Donato, J.V. Castell
and R. Jover
Drugs are usually biotransformed into new chemical species that may have either toxic or therapeutic effects. Drug metabolism studies are routinely performed in laboratory animals but, due to metabolic interspecies differences when compared to man, they are not accurate enough to anticipate the metabolic profile of a drug in humans. Human hepatocytes in primary culture provide the closest in vitro model to human liver and the only model that can produce a metabolic profile of a given drug that is very similar to that found in vivo. However their availability is limited due to the restricted access to suitable tissue samples. The scarcity of human liver has led to optimising the cryopreservation of adult hepatocytes for long-term storage and regular supply. Human hepatocytes in primary culture express typical hepatic functions and express drug metabolizing enzymes. Moreover, qualitative and quantitative similarities between in vitro and in vivo metabolism of drugs were observed. Different strategies have been envisaged to prolong cell survival and delay the spontaneous decay of the differentiated phenotype during culture. Thus, hepatocytes represent the most appropriate model for the evaluation of integrated drug metabolism, toxicity/metabolism correlations, mechanisms of hepatotoxicity, and the interactions (inhibition and induction) of xenobiotics and drug-metabolising enzymes. However, in view of limitations of primary hepatocytes, efforts are made to develop alternative cellular models (i.e. metabolic competent CYP-engineered cells stably expressing individual CYPs and transient expression of CYPs by transduction of hepatoma cells with recombinant adenoviruses). In summary, several cellular tools are available to address key issues at the earliest stages of drug development for a better candidate selection and hepatotoxicity risk assessment.
[Back to top] An Update on the Extraneuronal Monoamine
Transporter (EMT): Characteristics, Distribution and Regulation
F. Martel and I. Azevedo
Biological membranes prevent transmembrane diffusion in the majority of organic molecules that bear net charges at physiological pH. Consequently, these compounds must use more or less specific membrane-bound transport systems to be imported into or exported from cells or organisms.
The extraneuronal monoamine transporter (EMT) is a transmembranar transport system involved in the transfer of monoamine compounds across cell membranes. It was identified more than 30 years ago [1], its functional characteristics being thereafter described [review by 2]. The recent cloning of this transporter in man and rat reopened investigation and interest in this entity. EMT is a Na+ and Cl--independent, potential-dependent carrier, known to have a broad tissue distribution (eg. myocardium, vascular and non-vascular smooth muscle cells, glandular cells, placenta and CNS glial cells). According to its transport function and primary structure, EMT is included in the amphiphilic solute facilitator (ASF) family of transporters. Physiological substrates for EMT include the monoamines serotonin, dopamine, noradrenaline, adrenaline and histamine. Moreover, several xenobiotics including the neurotoxin 1-methyl-4- phenylpyridinium, clonidine, cimetidine and the K+-channel blocker tetraethylammonium interact with this transporter.
The aim of this work is to review knowledge concerning EMT, making an update on its functional characteristics, physiological importance and regulation. A special emphasis will be given to very recent investigations concerning regulation of EMT by intracellular second messenger systems and the interaction of modulators of P-glycoprotein, the product of the multidrug resistance gene MDR1, with EMT.
[Back to top] The Metabolism of Diclofenac - Enzymology and
Toxicology Perspectives
Wei Tang
Diclofenac is a nonsteroidal anti-inflammatory drug
bearing a carboxylic acid functional group. As a result, the metabolism of
diclofenac in humans partitions between acyl glucuronidation and phenyl
hydroxylation, with the former reaction catalyzed primarily by uridine 5¢-diphosphoglucuronosyl transferase 2B7 while
the latter is catalyzed by cytochrome P450 (CYP)2C9 and 3A4. Further
hydroxylation of diclofenac glucuronide was shown to occur in vitro with
recombinant CYP2C8, which may be of clinical significance in terms of defining
major metabolic routes involved in the elimination of diclofenac in humans. The
4¢-hydroxylation of the drug appears to
represent a feature reaction for CYP2C9 catalysis, and this regioselective
oxidation is presumably dictated by interactions of the carboxylate moiety of
the substrate with a putative cationic residue of the enzyme. Several other
residues of CYP2C9 were identified in studies with site-directed mutants that
influence substrate binding affinity and specificity, including Arg97, Phe114,
Asn289 and Ser286. The 5-hydroxylation of diclofenac is subject to CYP3A4
cooperativity elicited by quinidine. In this case, enhancement by quinidine of
diclofenac metabolism in vitro was attributed to increases in the Vmax
with little contribution from changes in the Km value. These
cooperative interactions in recombinant systems, however, appeared to be
influenced by enzyme host membranes of various cDNA-directed expressing CYP3A4.
Nevertheless, the in vivo significance of CYP3A cooperativity was demonstrated
in a pharmacokinetic study in monkeys, wherein the hepatic clearance of
diclofenac increased 2-fold when quinidine was co-administered.
Therapeutic use of diclofenac is associated with rare but sometimes fatal hepatotoxicity characterized by delayed onset of symptoms and lack of a clear dose-response relationship. The toxicity has consequently been categorized as metabolic idiosyncrasy. In this regard, the acyl glucuronide of the drug was demonstrated to be reactive and capable of covalent modification of cellular proteins, with covalent binding to liver proteins in rats depending on the activity of multidrug resistance protein 2, a hepatic canalicular transporter. One of the modified proteins was identified as dipeptidyl peptidase IV. Formation of protein adducts also was evident following the oxidative metabolism of diclofenac catalyzed by CYP enzymes. The reactive intermediates in this case were presumably diclofenac 1¢,4¢- and 2,5-quinone imines, both of which were trapped by conjugation with gutathione and identified as glutathione adducts. These same glutathione adducts were detected in rats as well as in human hepatocytes treated with diclofenac, and a corresponding mercapturic acid derivative was identified in urine from patients administered the drug. It is conceivable that the acyl glucuronide and benzoquinone imines derived from diclofenac modify proteins covalently and thereby produce toxicity in susceptible patients via either direct disruption of critical cellular functions or elicitation of immunological responses.