Current Drug Metabolism - Volume 12, Issue 4, 2011

Drug metabolism studies offer significant information on the clarification of the pharmacological and toxicological action of drugs and typically include isolation and identification of metabolites generated in vivo and in vitro. On the other hand, analytical chemistry and chemical analysis are useful tools that are ad nausea used in drug metabolism studies since they provide invaluable information. Among others, separation techniques (liquid chromatography, mass spectrometry, etc) are predominant when it comes to the analysis of complex matrices where enhanced selectivity and sensitivity is required. For instance, mass spectrometry could be considered to be the technique of choice that used in the elucidation of metabolic pathways of drugs. The aim of the current Hot Topic Issue of the international journal Current Drug Metabolism intends to provide to the scientific community comprehensive reviews covering the new trends and applications in the area of Analytical Chemistry towards Drug Metabolism and Biological Systems. The introductory paper by the research group of Prof. D.S. Hage aims towards to the characterization of drug interactions with serum proteins by high performance affinity chromatography. It covers theoretical and practical aspects on the studies of drug-protein interactions by affinity chromatography illustrating recent applications of the use of serum binding agents like human serum albumin, α1-acid glycoprotein and lipoproteins. Prof. Longsheng Sheng and his colleagues focused on a review dedicated to recent developments of LC-MS in drug metabolite identification. They describe a variety of experimental strategies and post acquisition data processing modes; tools that are needed of identification and characterization of drug metabolites. Finally, the authors comprehensively discussed aspects on mass spectrometric techniques including low resolution (quadrupole, ion trap, etc), high resolution MS analyzers (time-of-flight, Orbitrap, Fourier transform ion cyclotron resonance MS, etc) and novel MS technologies (ion mobility MS, imaging MS, etc). During the last years the -omics sciences (proteomics, genomics, metabolomics) are growing rapidly and on this basis the contribution of Dr. R. Wei, offers a complete overview of metabolomics and its practical value in pharmaceutical industries. Except for the brief historical overview of metabolomics, this article illustrates its practical role in pharmaceutical industries' issues dealing with validation of novel therapeutic targets, decision-making in the drug development processes and clinical trials. A selection of examples in metabolomics studies were also reviewed focusing on biochemical changes associates with pharmaceutical interventions. The research group of Dr. H.P. Permentier focused on the utilization of electrochemistry as a versatile analytical and preparative technique in the mimicry of oxidative drug metabolism by Cytochrome450s. This review also highlights oxidation methodologies that imitate the in vivo oxidation reactions including direct oxidation approaches and indirect electrochemical oxidation reactions by reactive oxygen species. Dr. J.M.P.J. Garrido briefly discussed the electrochemical outlook of tamoxifen biotransformation. This article includes the most important biotransformation steps of tamoxifen (a non-steroidal antiestrogen) based on recent pharmacological data and some applications of electrochemical methods for the determination of the drug and its metabolites. Prof. H. Orhan provides an important review on approaches in generating and characterization of reactive intermediates from drugs and drug candidates. They draw our attention to main criteria which a drug candidate should meet during its development and on reactive intermediates generated by biotransformation of the parent drug. Basic aspects on the screening and characterization of reactive intermediates using mass spectrometric techniques are also discussed. Finally, the research group of Prof. A. van Schepdael contributed an article orientated towards matrix metalloproteinase inhibitors. They focused on bioanalytical methodologies for the determination of matrix metalloproteinase inhibitors and their metabolites underlining aspects on sample preparation and detection techniques. In particular they describe features on pharmacokinetics and metabolism of matrix metalloproteinase inhibitors and their metabolites. From the position of the Guest Editor of this special issue, I would like to thank the Editor-in-Chief of Current Drug Metabolism Dr. Chandra Prakash for accepting my proposal to organize this special issue. Finally, I would like to thank all colleagues from all over the world that accepted my invitation and contributed with their high quality articles to this effort.

The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding.

Metabolism studies play a pivotal role in drug discovery and development since the active metabolites is critical to toxicological profile, efficacy and designing new drug candidates. From the instrumentation standpoint, liquid chromatography/mass spectrometry (LC/MS) has secured a central analytical technique for metabolite identification with the continuous developments and improvements in LC and MS technologies. Recently, a wide range of experimental strategies and post acquisition data processing and mining modes have emerged driven by the need to identify and characterize metabolites at ever increasing sensitivity and in ever more complex samples. In this article, the classical and practical mass spectrometry-based techniques, such as low resolution MS (quadruple, ion trap, linear ion trap, etc), high resolution MS (time-of-flight, hybrid time-of-flight instruments, Qrbitrap, Fourier transform ion cyclotron resonance MS, etc) and corresponding post acquisition data processing and mining modes (precursor ion filtering, neutral loss filtering, mass defect filter, isotope-pattern-filtering, etc) are described comprehensively. In addition, this review is also devote to discuss several novel MS technologies (ambient ionization techniques, ion mobility MS, imaging MS, LC/NMR/MS, etc) that hold additional promise for the advancement of metabolism studies.

Metabolomics is emerging as a promising systems biology approach for many research fields including functional genomics, disease diagnosis, nutrition science, and drug discovery. Following rapid development in academic and research institutes, metabolomics is drawing an attention in the pharmaceutical industry. This review aims to highlight the practical value of metabolomics in (a) potentially validating more novel therapeutic targets and indications, (b) facilitating decision-making on the advancement of therapeutics in the drug development process, and (c) leading to better patient stratification and cost-effective clinical trials, through the application of LC/MS/MRM-based targeted metabolomics studies at a relatively low cost in pharmaceutical companies. This paper provides a brief history of the development of metabolomics and common strategies for conducting metabolomics studies. The pros and cons of the most important technologies and the major components of metabolomics studies are reviewed and discussed. Finally, selected metabolomics study examples are reviewed to illustrate how metabolomics can be used to simultaneously capture underlying biochemical changes associated with pharmaceutical interventions, and effectively produce more accurate and/or alternative efficacy and ADR biomarkers, thereby, greatly extending our knowledge of disease, protein function and drug action and maximally benefiting the pharmaceutical industry.

Prediction of oxidative drug metabolism at the early stages of drug discovery and development requires fast and accurate analytical techniques to mimic the in vivo oxidation reactions by cytochrome P450s (CYP). Direct electrochemical oxidation combined with mass spectrometry, although limited to the oxidation reactions initiated by charge transfer, has shown promise in the mimicry of certain CYP-mediated metabolic reactions. The electrochemical approach may further be utilized in an automated manner in microfluidics devices facilitating fast screening of oxidative drug metabolism. A wide range of in vivo oxidation reactions, particularly those initiated by hydrogen atom transfer, can be imitated through the electrochemically-assisted Fenton reaction. This reaction is based on O-O bond activation in hydrogen peroxide and oxidation by hydroxyl radicals, wherein electrochemistry is used for the reduction of molecular oxygen to hydrogen peroxide, as well as the reduction of Fe3+ to Fe2+. Metalloporphyrins, as surrogates for the prosthetic group in CYP, utilizing metallo-oxo reactive species, can also be used in combination with electrochemistry. Electrochemical reduction of metalloporphyrins in solution or immobilized on the electrode surface activates molecular oxygen in a manner analogous to the catalytical cycle of CYP and different metalloporphyrins can mimic selective oxidation reactions. Chemoselective, stereoselective, and regioselective oxidation reactions may be mimicked using electrodes that have been modified with immobilized enzymes, especially CYP itself. This review summarizes the recent attempts in utilizing electrochemistry as a versatile analytical and preparative technique in the mimicry of oxidative drug metabolism by CYP.

Tamoxifen is a nonsteroidal antiestrogen that is currently and widely used in the treatment of breast cancer in all of its stages, in adjuvant therapy as a long-term suppressant of tumor recurrence and also as a chemopreventive agent in women that are in high risk of developing this type of estrogen-dependent cancer. From a toxicological and (bio)analytical point of view the knowledge of the metabolic pathways of a drug is found to be extremely important. So, in the present work the most important tamoxifen biotransformation steps were reviewed in the light of recent pharmacological data. This overview also includes the current controversy concerning tamoxifen DNA-damaging (genotoxic) versus non-genotoxic mechanisms. A special focus will be given to the putative application of electrochemical methods as a modern and reliable analytical tool for determination of tamoxifen and its metabolites. Moreover, the potential of DNA electrochemical sensors for detection of structural damage to DNA as a basis for toxicity screening is highlighted. Future prospects looking for the importance of developing new analytical methodologies are also discussed.

Despite several thousands of drugs are in use currently, research on new drug molecules is continuing. Because, there are diseases still without medication, successor/better drugs make the predecessor ones obsolete, and advancement in both life sciences and analytical technologies provide identification of previously unknown mechanisms of diseases, and discovery of novel drug targets. The two main criteria which a drug candidate should meet are high affinity for the target, and no or acceptable/tolerable toxicity in humans. Among these two, toxicity is the limiting one; developing a drug candidate with unacceptable toxicity has to be discontinued, even if it has an extremely high pharmacological activity. Drug would be withdrawn, if serious toxicity arises after marketing. Since drug development is a long (approximately 10 years), expensive, and infertile (one lead in 10.000 molecules) process, it is extremely important to detect the potential toxicity of drug candidate as early as possible. Today, it is believed that a great majority of toxic effects are caused by reactive intermediates generated by biotransformation of the parent drug. However, there are experimental difficulties in identifying such metabolite(s) in vivo. Their formation is affected by multi-factorial events; they can further be metabolized to structurally different products, and/or they may bind to a huge variety of biological sites or macromolecules. Hence, some reactive intermediates and their corresponding stable derivatives are generated in trace amounts, which make their determination more difficult. The ability of cytochrome P450s (CYP450) and other biotransformation enzymes to function in vitro offers a great flexibility to researchers, biotransformation of any compound can be simulated in a test tube, and metabolites/reactive intermediates are generated in an environment which has relatively much less background and less interfering multi-factorial events compared to in vivo. To simulate biotransformation, microsomal fraction is used most frequently from human and non-human sources. Purified or recombinant enzymes are used in determining the individual isoenzymes responsible for certain metabolites. Because of the chemical reactivity of intermediates, relevant, usually nucleophilic trapping agent(s) such as glutathione (GSH), N-acetylcysteine (NAC) and cyanide (CN-) are used to stabilize the metabolite. Trapped metabolites are subjected to spectrometric and/or nuclear magnetic resonance spectroscopic analyses for structural identification. Vertiginous advances especially in mass spectrometric technologies offer researchers new challenges in this area. This review is aimed at briefly summarizing the state of the art and compiling the highlighted studies in characterization of the reactive metabolites from drug molecules.

Enzymes are major drug targets in drug discovery and development processes in the pharmaceutical and biotechnology industry. A recent survey found that nearly half of all the marketed small-molecule drugs are inhibitors of enzymes. Matrix metalloproteinases (MMPs) are a family of 28 enzymes capable of degrading the constituents of the extracellular matrix (ECM) and the basementmembrane. MMPs play an essential role in several normal physiological processes including growth, wound healing and tissue repair. Over-expression and activation of MMPs has been linked to a range of diseases which include osteoarthritis, tumor metastasis, angiogenesis and cardiovascular diseases. The development of MMP inhibitors as therapeutic agents has kept an important place in drug discovery. Therefore, there is also an increasing need for robust analytical methods for evaluation of inhibitory potency and for the analysis of MMP inhibitors and their metabolites which can even play a more significant role than the parent drug. Modern analytical techniques and hyphenated instrumentations such as liquid chromatography-mass spectrometry with a function of structure elucidation can provide a profound insight into the research of MMP inhibitors and also serve as a complementary method to zymographic techniques for the analysis of biological samples. This review mainly summarizes bioanalytical methods, pharmacokinetics and related metabolites of MMP inhibitors over the last 12 years.