Current Drug Metabolism - Volume 7, Issue 8, 2006

This issue of Current Drug Metabolism is dedicated to Professor Ian A. Blair of the University of Pennsylvania in honor of his scientific and professional achievements in the field of mass spectrometry for the analysis of lipids, xenobiotics, proteomics, lipidomics, genomics and DNA adducts, on the occasion of his 60th birthday. Dr. Blair has made pioneering and monumental contributions to the field of drug metabolism, chemistry, pharmacology and mass spectrometry in the last 35 years. We are privileged to dedicate this issue for his contributions. Dr. Blair received his Bachelor of Science degree in Chemistry and his doctoral degree in Organic Chemistry from the Imperial College of Science and Technology, University of London, UK with Nobel Laureate Sir Derek HR Barton. After working for five years as a Research Fellow in Uganda and Australia, Dr. Blair joined Imperial College of Science and Technology, University of London as a lecturer of Analytical Chemistry and advanced to Senior Lecturer in 1982. Dr Blair moved to the Vanderbilt University, Nashville in 1983 as a Professor of Pharmacology and Chemistry, and Director of Mass Spectrometry Center. In 1997, Dr. Blair moved to Department of Pharmacology, University of Pennsylvania, Philadelphia as an A.N. Richards Professor of Pharmacology and Chemistry. Dr Blair is also a Scientific Director, Genomics Institute Proteomics Facility and Director, Systems Biology, Institute for Translational Medicine and Therapeutics at University of Pennsylvania. Dr. Blair has made numerous outstanding contributions to the field of drug metabolism and mass spectrometry, especially in developing of sensitive, selective and novel mass spectrometric techniques, for the analysis of endogenous and exogenous compounds from biological matrices. He has also made a significant contribution to the characterization of covalent modifications to macromolecules, and determination of the factors that control lipid hydroperoxide-mediated damage to DNA, RNA, and proteins. Dr Blair received the 2005 Beynon Prize from Rapid Communications in Mass Spectrometry for his contribution to this field. He was elected as to a fellowship in the American Association for the Advancement of Science in 2005. Dr. Blair was cited for his “distinguished contributions to the field of mass spectrometry and its applications to pharmaceutical medicine and for moving autacoid biology forward with sensitive bioanalytical techniques”. In 2006, he was elected to a fellowship in the American Association of Pharmaceutical Scientists for his distinguished contribution to the pharmaceutical sciences. Dr. Blair is not only an outstanding researcher, but also an admirable educator. Many of his former graduate students and postdoctoral fellows are making major contributions to the drug metabolism field in pharmaceutical industry and academic research. Dr Blair is considered by colleagues as “an excellent teacher, who has had very substantial impact on chemistry, pharmacology and mass spectrometry through his students”. His superb ability to encourage and develop people as well as his undergraduate and graduate teaching efforts have infected many talented students with an enthusiasm for mass spectrometry, chemistry and pharmacology. In 2006, Dr Blair was awarded the University of Pennsylvania Dean's Award for excellence in Graduate Student Training. He has helped all of us as students and as professionals and influenced our careers and lives. The lessons in scientific vision, persistence, independence, creativity and self-criticism he has practiced throughout his career can serve as an inspiration to any scientist. Dr. Blair has had very substantial impact in the field of drug metabolism and chemistry both as a researcher and as an extraordinary teacher with a positive, inspiring, and long-lasting effect on his colleagues, postdoctoral fellows and graduate students. Few scientists can claim such a fruitful career. We would like to thank all the authors for their great contributions to this issue.

Studies of the metabolic fate of drugs and other xenobiotics in living systems may be divided into three broad areas: (1) elucidation of biotransformation pathways through identification of circulatory and excretory metabolites (qualitative studies); (2) determination of pharmacokinetics of the parent drug and/or its primary metabolites (quantitative studies); and (3) identification of chemically-reactive metabolites, which play a key role as mediators of drug-induced toxicities (mechanistic studies). Mass spectrometry has been regarded as one of the most important analytical tools in studies of drug metabolism, pharmacokinetics and biochemical toxicology. With the commercial introduction of new ionization methods such as those based on atmospheric pressure ionization (API) techniques and the combination of liquid chromatography- mass spectrometry (LC-MS), it has now become a truly indispensable technique in pharmaceutical research. Triple stage quadrupole and ion trap mass spectrometers are presently used for this purpose, because of their sensitivity and selectivity. API-TOF mass spectrometry has also been very attractive due to its enhanced full-scan sensitivity, scan speed, improved resolution and ability to measure the accurate masses for protonated molecules and fragment ions. This review aims to survey the utility of mass spectrometry in drug metabolism and toxicology and to highlight novel applications and future trends in this field.

This review provides an overview of the formation, pharmacology, and toxicology of endogenous glutathione (GSH)-adducts with particular emphasis on GSH-adducts that arise from lipid peroxidation. GSH is the major lowmolecular- weight thiol in mammalian cells. It is involved in the formation of endogenous bioactive eicosanoids and is a source of reducing equivalents in a number of biosynthetic reactions. GSH has long been recognized to act as a co-factor in the reduction of reactive oxygen species and lipid hydroperoxides by glutathione peroxidases and glutathione-Stransferases (GSTs). It also plays an important role in the reduction of reactive intermediates derived from arylamines and in the conjugation of reactive intermediates to form S-substituted endogenous GSH-adducts through its nucleophilic cysteine sulfhydryl group. Although some reactive intermediates can form adducts directly, GST-mediated reactions generally predominate. This results in the formation of bioactive endogenous GSH-adducts derived from eicosanoids, isoprostanes, estrogens, catecholamines, and 4-hydroxy-2(E)-nonenal (HNE). Cellular oxidative stress causes increased lipid peroxidation with the concomitant formation of DNA- and protein-reactive bifunctional electrophiles. It has generally been considered that HNE is the most abundant bifunctional electrophile that is formed. Several years ago we discovered that 4-oxo-2(E)-nonenal (ONE) was also a major lipid hydroperoxide-derived bifunctional electrophile. From in vitro studies, we showed that ONE and HNE arose from the common intermediate, 4-hydroperoxy-2(E)-nonenal and also showed that ONE was formed in greater amounts than HNE. We have recently made the unexpected discovery that GSH addition to ONE leads to the formation of an unusual thiadiazabicyclo-ONE-GSH-adduct (TOG), which was characterized as (2S,7R) - 7 - [N - (carboxymethyl)carbamoyl] - 5 - oxo - 12 - pentyl - 9 - thia - 1,6 - diazabicyclo[8.2.1]trideca - 10(13), 11-diene-2- carboxylic acid. TOG is one of the most abundant GSH-adducts formed during peroxide/FeII- or FeII-mediated oxidative stress in EA.hy 926 endothelial cells. As TOG is formed from ONE, these experiments have confirmed that ONE is a major lipid hydroperoxide-derived bifunctional electrophile formed during intracellular oxidative stress. TOG represents the first member of a new class of endogenous GSH-adduct biomarkers that can be used to quantify intracellular oxidative stress. Two other members of the TOG family arise from GST-mediated GSH-adduct formation with dioxododecenoic acid and dioxooctenoic acid, bifunctional electrophiles derived from the carboxy terminus of lipid hydroperoxides. The formation of TOG and TOG-related endogenous GSH-adducts can result from free radical- as well as cyclooxygenase- and lipoxygenase-mediated pathways. Analysis of the GSH-adducts by stable isotope dilution mass spectrometry-based methodology will provide a quantitative measure of enzymatic and non-enzymatic cellular oxidative stress to complement isoprostane measurements. In future studies, it will also be important to establish the biological activity of TOG and its analogs in view of the potent activity of many other endogenous GSH-adducts such as the leukotrienes.

Glucuronidation is an important mechanism used by mammalian systems to clear and eliminate both endogenous and foreign chemicals. Many functional groups are susceptible to conjugation with glucuronic acid, including hydroxyls, phenols, carboxyls, activated carbons, thiols, amines, and selenium. Primary and secondary amines can also react with carbon dioxide (CO2) via a reversible reaction to form a carbamic acid. The carbamic acid is also a substrate for glucuronidation and results in a stable carbamate glucuronide metabolite. The detection and characterization of these products has been facilitated greatly by the advent of soft ionization mass spectrometry techniques and high field NMR instrumentation. The formation of carbamate glucuronide metabolites has been described for numerous pharmaceuticals and they have been identified in all of the species commonly used in drug metabolism studies (rat, dog, mouse, rabbit, guinea pig, and human). There has been no obvious species specificity for their formation and no preference for 1° or 2° amines. Many biological reactions have also been described in the literature that involve the reaction of CO2 with amino groups of biomolecules. For example, CO2 generated from cellular respiration is expired in part through the reversible formation of a carbamate between CO2 and the -amino groups of the α and β-chains of hemoglobin. Also, carbamic acid products of several amines, such as β-N-methylamino-L-alanine (BMAA), ethylenediamine, and L-cysteine have been implicated in toxicity. Studies suggested that a significant portion of amino-compounds in biological samples (that naturally contain CO2/bicarbonate) can be present as a carbamic acid.

BMS-299897 is a γ-secretase inhibitor that has the potential for treatment of Alzheimer’s disease. The metabolism of [14C]BMS-299897 was investigated in human liver microsomes, in rat, dog, monkey and human hepatocytes and in bile duct cannulated rats. Seven metabolites (M1-M7) were identified from in vitro and in vivo studies. LC-MS/MS analysis showed that M1 and M2 were regioisomeric acylglucuronide conjugates of BMS-299897. Metabolites M3, M4 and M6 were identified as monohydroxylated metabolites of BMS-299897 and M5 was identified as the dehydrogenated product of monooxygenated BMS-299897. In vivo, 52% of the radioactive dose was excreted in bile within 0-6 h from bile duct cannulated rats following a single oral dose of 15 mg/kg of [14C]BMS-299897. Glucuronide conjugates, M1 and M2 accounted for 80% of the total radioactivity in rat bile. In addition to M1 and M2, M7 was observed in rat bile which was identified as a glucuronide conjugate of an oxidative metabolite M5. For structure elucidation and pharmacological activity testing of the metabolites, ten microbial cultures were screened for their ability to metabolize BMS-299897 to form these metabolites. Among them, the fungus Cunninghamella elegans produced two major oxidative metabolites M3 and M4 that had the same HPLC retention time and mass spectral properties as those found in in vitro incubations. NMR analysis indicated that M3 and M4 were stereoisomers, with the hydroxyl group on the benzylic position. However, M3 and M4 were unstable and converted to their corresponding lactones readily. Based on x-ray analysis of the synthetically prepared lactone of M3, the stereochemistry of benzylic hydroxyl group was assigned as the R configuration. Both the hydroxy metabolites (M3 and M4) and the lactone of M3 showed γ-secretase inhibition with IC50 values similar to that of the parent compound. This study demonstrates the usefulness of microbial systems as bioreactors to generate metabolites of BMS-299897 in large quantities for structure elucidation and activity testing. This study also demonstrates the biotransformation profile of BMS-299897 is qualitatively similar across the species including rat, dog, monkey and human which provides a basis to support rat, dog and monkey as preclinical models for toxicological testing.

Etoposide (VP-16), a DNA topoisomerase II poison widely used as an antineoplastic agent is also known to cause leukemia. One of its major metabolic pathways involves O-demethylation to etoposide catechol (etoposide-OH) by cytochrome P450 3A4 (CYP3A4). The catechol metabolite can undergo sequential one- and two-electron oxidations to form etoposide semi-quinone (etoposide-SQ) and etoposide quinone (etoposide-Q), respectively, which have both been implicated as cytotoxic metabolites. However, etoposide-Q is known to react with glutathione (GSH), which can protect DNA from oxidative damage by this reactive metabolite. In this study, etoposide-Q was reacted with GSH and the two etoposide-GSH conjugates were characterized. The major conjugate was etoposide-OH-6'-SG and the minor product was etoposide-OH-2'-SG. Etoposide-OH-6'-SG, which arose from Michael addition of GSH to etoposide-Q, was characterized by mass spectrometry and 2-D NMR. It was identified as the sole product from in vitro metabolism experiments using recombinant human CYP3A4 or liver microsomes incubated with etoposide in the presence of GSH. Etoposide-OH- 6'-SG was also detected from incubations of etoposide-OH and GSH alone. Therefore, the presence of etoposide-OH, which can be formed from etoposide metabolism by CYP3A4, is essential for formation of the GSH conjugate. The oxidation of etoposide-OH to a quinone intermediate is likely the precursor in the formation of etoposide-OH-6'-SG.

Although traditionally reserved for proteomic analysis, nanoESI has found increased use for small molecule applications related to drug metabolism/pharmacokinetics (DMPK). NanoESI, which refers to ESI performed at flow rates in the range of 200 to 1000 nL/min using smaller diameter emitters (10 to 100 μm id), produces smaller droplets than conventional ESI resulting in more efficient ionization. Benefits include greater sensitivity, enhanced dynamic range, and a reduced competition for ionization. These advantages may now be harnessed largely due to the introduction of a commercial system for automated nanoESI infusion. This development in turn has allowed ADME (absorption, distribution, metabolism, and excretion) scientists to consider novel approaches to mass spectrometric analysis without direct LC interfacing. While it is freely acknowledged that nanoESI infusion is not likely to supplant LC-MS as the primary analytical platform for ADME, nanoESI infusion has been successfully applied to both quantitative (bioanalysis) and qualitative (metabolite identification) applications. This review summarizes published applications of this technology and offers a perspective on where it fits best into the DMPK laboratory.

Reactive oxygen species (ROS) can mediate damage to cellular macromolecules and lipids. Lipid peroxidation is considered to be a major pathway by which ROS can cause tissue damage and alterations in cell membranes. Other factors affecting oxidative damage include the target molecules such as fatty acids, which are readily oxidized by ROS. Thus, lipid peroxidation may depend upon the cellular fatty acid composition. Analysis of saturated fatty acids that are present by liquid chromatography/mass spectrometry (LC/MS) is difficult because they are poorly ionized under electrospray ionization (ESI) conditions. The separation of short to very long chain saturated and unsaturated fatty acids is also very challenging when LC is employed instead of gas chromatography. The use of trimethylaminoethyl (TMAE) ester iodide derivatization has been shown previously to improve the sensitivities of saturated fatty acids in the ESI mode. A reversed- phase LC method using a diphenyl column was employed to separate 14 fatty acids as their TMAE derivatives. Stable isotope dilution LC/ESI/multiple reaction monitoring/MS methodology was then developed for the quantitative analysis of seven saturated and seven unsaturated forms of short (C14) to very long (C26) chain fatty acids as their TMAE ester iodide derivatives. This methodology has allowed the analysis of fatty acid composition from parental rat intestinal epithelial cell and rat intestinal epithelial cells transfected with cyclooxygenase-2, a model system of oxidative stress.

Human lung is a major target organ for all inhaled drugs, environmental toxicants and carcinogens. Recent hypotheses suggesting a role for environmental toxicants in the pathogenesis of lung diseases, such as lung cancer and chronic obstructive pulmonary disease have stimulated interest in research on the xenobiotic metabolizing capability of the lung. Many of the compounds associated with these diseases require enzymatic activation to exert their deleterious effects on pulmonary cells. Interindividual differences in in situ activation and inactivation of xenobiotics may contribute to the risk of developing of lung diseases associated with these compounds. The major xenobiotic metabolizing enzymes, including both phase I and phase II enzymes, have been detected in animal and human lung tissues. Although the lung cytochrome P450 (CYP) and other xenobiotic metabolizing enzymes share many common features with those present in other tissues such as liver, kidney and gut, there are some distinctive differences. It is evident from the studies carried out to date CYP1A1, 1B1, 2A13, 2F1, 2S1 and 4B1 are preferentially expressed in the lung together with CYP2E1 and 3A5. This review provides a detailed picture of major xenobiotic-metabolizing phase I (CYPs, epoxide hydrolases, flavin monooxygenases, etc.) and phase II enzymes (conjugation enzymes, including several transferases) expressed in human lung. The roles of individual metabolizing enzymes and their genetic polymorphisms are also discussed.