oa Editorial [Hot Topic:Acyl Glucuronides: Mechanistic Role in Drug Toxicity? (Guest Editor: Urs A. Boelsterli)]

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 “…if a conjugate forms a toxic compound such as acylglucuronide, additional safety assessment may be needed…” [1]. If one really considers acyl glucuronides as “toxic compounds”, 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 This view has certainly been influenced by the observation that a number of drugs that were withdrawn from the market due to idiosyncratic drug reactions actually did form acyl glucuronide metabolites - but the relationship is correlative, rather than causal.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 “danger signals”, 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]....