Current Drug Metabolism (v.12, #3)

The question to what extent acyl glucuronides, which are chemically reactive electrophiles formed from carboxylic acid-containing drugs, should be considered a potential hazard in drug development is an old bone of contention. On the one hand, a recent guideline issued by the FDA in 2008 states that and#x201C; - if a conjugate forms a toxic compound such as acylglucuronide, additional safety assessment may be needed - and#x201D; [1]. If one really considers acyl glucuronides as and#x201C;toxic compoundsand#x201D;, then the provocative question may arise of whether a drug should be stopped from further development if in human trials there is significant formation of acyl glucuronides. This view has certainly been influenced by the observation that a number of drugs that were withdrawn from the market due to idiosyncratic drug reactions actually did form acyl glucuronide metabolites - but the relationship is correlative, rather than causal. On the other hand, even endogenous compounds can form acyl glucuronides, and many drug acyl glucuronides exhibit very low reactivity and may not be a concern during therapy. Both views are probably gross oversimplifications. What one needs to take into account is, first, mechanistic aspects of acyl glucuronide protein acylation and its downstream consequences, and, second, pharmacokinetic aspects (e.g., disposition of the acyl glucuronides). Importantly, one also needs to see the hazard in the context of therapeutic dose and human exposure at the tissue level to a given drug acyl glucuronide. From a historical point of view, it was reported decades ago that acyl glucuronides formed from carboxylic acid-containing drugs by UDP-glucuronosyltransferase (UGT)-mediated glucuronidation are potentially reactive electrophiles that can interact with and covalently bind to nucleophilic targets [2]. Accordingly, it was proposed that glucuronidation may no longer be considered a harmless detoxication reaction but that it could be a bioactivation pathway leading to potential toxicity [3]. It was also recognized early on that the isomers of acyl glucuronides (formed as a result of intramolecular acyl migration) can be equally or even more potent electrophilic species than the parent acyl glucuronide, and that these iso-glucuronides covalently bind to proteins via another mechanism [4]. Furthermore, acyl glucuronides are not only able to directly acylate cellular proteins, but they can also transacylate the cysteine thiol of glutathione (GSH), leading to drug-Sacyl- GSH, which in turn is a highly reactive species [5]. However, we still know very little about the overall toxicological significance of acyl glucuronides or their derivatives. A discussion about the causal role of acyl glucuronides in drug toxicity must not only consider the differential reactivity of the drug acyl glucuronides (e.g., type of substitution at the alpha carbon, half life [6-8]) but also the nature of the nucleophilic targets. In fact, a number of molecular targets to which acyl glucuronides covalently bind have been identified in different organs including liver, small intestine, colon, and kidney. The traditional view holds that identification of the most prevalent protein targets would give a clue to possible mechanisms of toxicity (e.g., due to inactivation of a critical protein). In this respect, the discussion about the significance of acyl glucuronide formation very much resembles the speculations about the underlying mechanisms of acetaminophen hepatotoxicity mediated by its protein-reactive intermediate, NAPQI [9]. More recently, it has become clear that perhaps an electrophilic stress per se might be important in activating electrophile sensors and downstream stress response pathways, maybe even through the simultaneous activation of both proapoptotic and protective pathways [10]. Alternatively, haptenated proteins, in combination with and#x201C;danger signalsand#x201D;, have in some cases been associated with immune responses towards the drug-modified proteins [11]. On the other hand, although glucuronidation of carboxylic acid-containing drugs may cause delayed toxicity due to reactive acyl glucuronide intermediates, this process may at the same time protect from the acute toxicity of the aglycone or its oxidative metabolite(s). For example, the acute cytotoxicity of diclofenac was increased in the presence of (-) borneol, an inhibitor of UGT, both in rat hepatocytes [12] or HEK293 cells stably expressing human UGT1A3 [13], while, as expected, the formation of the acyl glucuronide was inhibited and covalent protein adduct formation was greatly diminished. Like for other signaling paradigms, it seems that the complex balance between bioactivating and protective pathways may ultimately determine the outcome in vivo, rather than one single factor (reactivity of an acyl glucuronide) alone. Thus, the exact role of acyl glucuronides in drug safety assessment is simply not known and cannot be generalized. While covalent protein binding in vitro and in vivo has been demonstrated and a number of target molecules have been identified, the biological consequences are largely unknown [14]. It is extremely difficult to dissociate acyl glucuronide-mediated covalent protein adduct formation from other mechanisms. In addition, many discussions about the formation of acyl glucuronides often ignore the fact that some of these glucuronoconjugates are equally rapidly degraded by enzymatic or non-enzymatic hydrolysis; for example, the half life of diclofenac acyl glucuronide in human plasma is only 6 minutes [8]. Finally, glucuronidation not only can generate a potentially reactive metabolite, but this process can also simply provide a transport form for many carboxylic acid-containing drugs, drastically changing the local concentrations of these drugs. For example, due to their uphill transport across the hepatic canalicular membrane into bile, some drug acyl glucuronides can reach high (millimolar) concentrations in the biliary tree, followed by transport to the more distal parts of the intestinal tract. Thus, although simple, plausible, and not new, the kinetic aspect of acyl glucuronide toxicity has been neglected; due to the conjugation to glucuronic acid, certain drugs are now delivered at high concentrations to remote target tissues, e.g., the small intestinal mucosa [15]....

Chemistry and Reactivity of Acyl Glucuronides by Andrew V. Stachulski (215-221).
This review presents a survey of acyl glucuronides (AGs) from the perspective of a synthetic medicinal chemist. After a brief introduction to the biogenesis of AGs and current attitudes of the pharmaceutical industry towards them, the importance of the availability of pure synthetic AGs as probe molecules is stressed. Current synthetic methods including the selective acylation method and enzymatic procedures are discussed with actual drug examples. The major reaction pathways of AGs, acyl migration and direct acyl transfer, are then examined with consideration of both in vitro and in vivo situations. Both the aglycone and the carbohydrate residue are shown to be important in determining the reactivity of AGs, especially the and#945;-substitution in aryl acetates and propionates and aryl substitution in benzoates. The significance of these effects for important drugs such as the NSAIDs diclofenac and ibuprofen is noted. Appropriate models of the reactivity of AGs with proteins are presented, from short model peptides to body proteins, and examples where AGs clearly interact with protein in vivo (e. g. mycophenolic acid) are presented. Recent results with AGs in plasma, where half-lives differ significantly from their values in aqueous buffer, show the importance of considering the external medium especially when other hydrolytic enzymes are present. Finally, the importance of considering the reactivity of acyl thioesters-which may be formed directly or via AGs as intermediates- is also stressed. This pathway may lead to different protein reactivity than shown for AGs themselves, including interference with the glutathione pathway as shown for NSAID-like carboxylic acids.

Iso-glucuronides by Ronald G. Dickinson (222-228).
This review on isomers or acyl glucuronides (iso-glucuronides) updates earlier reviews, and attempts to place in context the advances that have been made, especially over the last 15 years. The essential chemistry behind the intramolecular acyl migration and anomerization reactions of acyl glucuronides has been appreciated for 30 years. The great advances in the past 15 years have been in understanding the dynamics and kinetics of these processes in vitro, using highly sophisticated modern technology, e.g. LC-NMR, LCMS/ MS. In this way, earlier assumptions on kinetics and identification of migration isomers and anomers have come under intense review and update. Extensive structure-activity relationships, involving electronic and steric characteristics of an acyl glucuronide and its possible 7 isomers (excluding transient open-chain species) have been delineated. The covalent modification of endogenous proteins and other macromolecules has been further explored, though direct linkage between such modification and toxic sequelae remains elusive. An alternative view of acyl glucuronides and iso-glucuronides as just xenobiotics has perhaps added the dimension that acyl glucuronidation (and attendant formation of iso-glucuronides) does not necessarily mean that glucuronidation of the aglycone has ended metabolic sequences in vivo.

Carboxylic acid-containing drugs can be metabolized to chemically-reactive acyl glucuronide, S-acyl-CoA thioester, and/or intermediate acyl-adenylate metabolites that are capable of transacylating the cysteinyl-thiol of glutathione (GSH) resulting in the formation of drug-S-acyl-GSH thioesters detected in vivo in bile and in vitro in hepatocytes. Authentic S-acyl-GSH thioesters of carboxylic acids can be readily synthesized by modifying the cysteinyl-thiol group of GSH with an applicable acylating reagent. Bionanalytical characterization of S-acyl-GSH derivatives has demonstrated enhanced extraction efficiency from biological samples when formic acid is included in appropriate extraction solvents, and that tandem mass spectrometry of S-acyl-GSH conjugates results in fragmentation producing a common MH+-147 Da product ion. Chemical reactivity comparisons have shown that S-acyl-CoA thioester and acyl-adenylate conjugates are more reactive than their corresponding 1-and#946;-O-acyl glucuronides toward the transacylation of GSH forming S-acyl-GSH thioesters. S-Acyl-GSH thioester derivatives are also chemically-reactive electrophiles capable of transacylating biological nucleophiles. Glutathione S-transferases (GSTs) weakly catalyze S-acyl-GSH conjugate formation from S-acyl-CoA, acyl-adenylate, and 1-and#946;-O-acyl glucuronide substrates; however purified-GSTs have also been shown to hydrolyze S-acyl-GSH thioesters. Mechanistic in vitro studies in hepatocytes have revealed the primary importance of the S-acyl-CoA formation pathway leading to S-acyl-GSH-adduct formation. In addition to being hydrolytically-unstable in hepatocytes and plasma, S-acyl-GSH thioesters undergo and#947;-glutamyltranspeptidase-mediated cleavage of the and#947;-glutamyl-group leading to N-acyl-cysteinylglycine amide-linked products. In summary, S-acyl-GSH thioesters are indicators of reactive transacylating metabolite formation produced from the biotransformation of carboxylic acids, but since they are also chemically-reactive, perhaps these derivatives can contribute to covalent binding to tissue proteins and potential toxicity.

NSAID Acyl Glucuronides and Enteropathy by Urs A. Boelsterli, Veronica Ramirez-Alcantara (245-252).
Many nonsteroidal anti-inflammatory drugs (NSAIDs) are carboxylic acid-containing compounds that are conjugated in the liver to acyl glucuronides and excreted across the hepatocanalicular membrane into bile. Chronic and acute NSAID use has not only been associated with gastric injury but also increasingly recognized to cause small intestinal injury (enteropathy). The mechanisms of NSAID enteropathy are still unknown, but a combination of topical effects (including mitochondrial injury) combined with inhibition of COX1/2, followed by an inflammatory response triggered by LPS-mediated activation of TLR4 on macrophages, have been implicated in the pathogenesis. Some of the nucleophilic proteins that are targeted by the electrophilic NSAID acyl glucuronides or their iso-glucuronides have been identified both in bile canaliculi and on the apical membrane domain of enterocytes (e.g., aminopeptidase N); however, the mechanistic role of covalent adducts has remained enigmatic. In contrast, it has become increasingly clear that acyl glucuronide formation is a major toxicokinetic determinant, in that the drug conjugates are a transport form delivering the drug to the more distal parts of the jejunum/ileum, where the glucuronic acid moiety is cleaved off the aglycone due to higher local pH and the presence of bacterial and#946;- glucuronidase. Through this mechanism, high local concentrations of the parent NSAID can be attained, potentially leading to local tissue injury. Thus, even if one considers the formation of acyl glucuronides not as a potentially dangerous toxophore by virtue of their proteinreactivity, acyl glucuronides could still be a red flag in drug development if excreted at high rates into bile and delivered to more distal areas of the small intestine where high amounts of parent drug is released.

Analytical and Pharmacological Aspects of Therapeutic Drug Monitoring of mTOR Inhibitors by Maria Pieri, Nadia Miraglia, Giuliano Polichetti, Giovanni Tarantino, Antonio Acampora, Domenico Capone (253-267).
Mammalian Target Of Rapamycin (mTOR) inhibitors represent a new class of immunosuppressant drugs extensively used for the prevention and the treatment of graft rejection in organ transplant recipients. Their current use is due to referred low nephrotoxic effects, particularly important in kidney transplanted and/or patients with renal failure. The most representative drugs of such class are Sirolimus (Siro) and Everolimus (Rad). Both drugs show a narrow therapeutic window, therefore, monitoring of whole-blood drug levels is recommended in order to optimize the therapy. Among the available assays, Liquid Chromatography coupled with UltraViolet or Electrospray Tandem Mass Spectrometry methods (LC/UV or LC/ESI-MSMS) are the most accurate and specific ones. A reliable alternative is represented by immunoassays, which offer the opportunity to minimize sample pre-treatment, thus reducing the time between drawing blood sample and measuring the drug concentration, an important aspect in high-throughput analyses. Despite this, a limitation in the use of immunoassays for therapeutic drug monitoring is the lower specifity compared with the chromatographic methods when analysing structurally-related drugs. New insights to optimize mTOR inhibitors regimens seem to be offered by the evaluation of CYP450 3A activity by using the probe drug approach. To such purpose, there are a number of major probe drugs used for in vivo studies including: midazolam, cortisol, lidocaine, nifedipine, dextromethorphan, erythromycin, dapsone and alfentanil. The aim of the present paper is to report the most recent knowledge concerning this issue, supplying a critical and comprehensive review for whom are involved both in the clinical and analytical areas.

Multidrug Resistance ABC Transporter Structure Predictions by Homology Modeling Approaches by Mylene Honorat, Pierre Falson, Raphael Terreux, Attilio Di Pietro, Charles Dumontet, Lea Payen (268-277).
Human multidrug resistance ABC transporters are ubiquitous membrane proteins responsible for the efflux of multiple, endogenous or exogenous, compounds out of the cells, and therefore they are involved in multi-drug resistance phenotype (MDR). They thus deeply impact the pharmacokinetic parameters and toxicity properties of drugs. A great pressure to develop inhibitors of these pumps is carried out, by either ligand-based drug design or (more ideally) structure-based drug design. In that goal, many biochemical studies have been carried out to characterize their transport functions, and many efforts have been spent to get high-resolution structures. Currently, beside the 3D-structures of bacterial ABC transporters Sav1866 and MsbA, only the mouse ABCB1 complete structure has been published at high-resolution, illustrating the tremendous difficulty in getting such information, taking into account that the human genome accounts for 48 ABC transporters encoding genes. Homology modeling is consequently a reasonable approach to overcome this obstacle. The present review describes, in the first part, the different approaches which have been published to set up human ABC pump 3D-homology models allowing the localization of binding sites for drug candidates, and the identification of critical residues therein. In a second part, the review proposes a more accurate strategy and practical keys to use such biological tools for initiating structure-based drug design.

Clinical Pharmacogenetics of Methotrexate by Pasqualina Castaldo, Simona Magi, Annamaria Assunta Nasti, Sara Arcangeli, Vincenzo Lariccia, Nicola Alesi, Massimo Tocchini, Salvatore Amoroso (278-286).
It is well known that interindividual variability can affect the response to many drugs in relation to age, gender, diet, and organ function. Pharmacogenomic studies have also documented that genetic polymorphisms can exert clinically significant effects in terms of drug resistance, efficacy and toxicity by modifying the expression of critical gene products (drug-metabolizing enzymes, transporters, and target molecules) as well as pharmacokinetic and pharmacodynamic parameters. A growing body of in vitro and clinical evidence suggests that common polymorphisms in the folate gene pathway are associated with an altered response to methotrexate (MTX) in patients with malignancy and autoimmune disease. Such polymorphisms may also induce significant MTX toxicity requiring expensive monitoring and treatment. Although the available data are not conclusive, they suggest that in the future MTX pharmacogenetics could play a key role in clinical practice by improving and tailoring treatment. This review describes the genetic polymorphisms that significantly influence MTX resistance, efficacy, and toxicity.

Overview of the Metallometabolomic Methodology for Metal-Based Drug Metabolism by Ruiguang Ge, Xuesong Sun, Qing-Yu He (287-299).
Metallometabolomics is an emerging field integrating the research technologies related to the comprehensive analysis of metabolites of a metallodrug in a biologically relevant sample, requiring high-throughput and targeted analyses of the transformation, speciation, localization and structural characteristics of the metallometabolites. This review discusses the concept of metallometabolomics with a focus on analytical techniques and methods, particularly the hyphenated approaches that combine high resolution separation techniques (liquid chromatography or capillary electrophoresis) with highly sensitive detection methods such as mass spectrometry (elemental (ICP) or molecular (ESI)) or nuclear analytical methods (X-ray fluorescence/absorption/emission/diffraction and nuclear magnetic resonance). The application of these advanced analytical technologies in the speciation analysis, identity determination and structural elucidation of metallometabolites will be selectively outlined, along with their advantages and limitations.

Xenosensors CAR and PXR at Work: Impact on Statin Metabolism by Maria Marino, Alessandra di Masi, Viviana Trezza, Valentina Pallottini, Fabio Polticelli, Paolo Ascenzi (300-311).
The xenobiotic response represents a complex group of chemical reactions aimed to inactivate and eliminate foreign chemicals, to antagonize their toxic effects, and to repair eventually damaged tissues. Intriguingly, xenobiotic response is also active against endogenous products of metabolism. Members of the nuclear receptor superfamily play a key role in xenobiotic detection and response. In particular, the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are transcription factors activated by a variety of endogenous and exogenous ligands. However, a high affinity endogenous ligand for CAR or PXR is not known, therefore these NRs have been put in the class of and#x201C;orphan receptorsand#x201D;. Both CAR and PXR elevate the expression of the detoxification machinery in the presence of endogenous and exogenous ligands, regulating the expression of Phase I and II metabolizing enzymes and of phase III transporters. Unfortunately, prescription drugs are included in the list of xenobiotic recognized by CAR and PXR. Among others, statins represent a good example of CAR- and PXR-mediated drug metabolism. Indeed statins, the most effective and prescribed cholesterollowering drugs, are target of xenobiotic response mediated by CAR and PXR. Here, we review the structural and molecular bases of CAR- and PXR-mediated drug response highlighting how these mechanisms could impact on statin metabolism. Moreover, the alteration of statin pharmacodynamics and/or pharmacokinetics induced by concomitant drug treatment or dietary factors will be also examined.