Reactive Drug Metabolites
Gaining an understanding of the role of drug metabolism in the liver injury caused by APAP has been important to the field of drug-induced hepatotoxicity from several perspectives. Second, the p -aminophenol sub-structure contained in the APAP molecule is imbedded in many other therapeutic agents, or can be introduced or unmasked via metabolic reactions. Awareness of the potential for further metabolic activation of this element to yield an electrophilic quinone imine species has been invoked retrospectively to account for the hepatotoxic properties of drugs such as diclofenac 17 , nefazodone 18 , trazadone 19 , tacrine 20 , amodiaquine 21 , and lapatinib 22 Fig.
Drugs which serve as precursors of quinone imine formation. In one case amodiaquine , the p -aminophenol moiety is present in the parent compound, whereas in the other examples, metabolic oxidation at the indicated sites is required to introduce this structural feature. Examples include thiophenes and other sulfur-containing heterocycles via S -oxidation , furans via epoxidation , anilines via N- or C-oxidation , nitrobenzenes via nitro reduction , hydrazines via oxidation to free radical species , and some carboxylic acid derivatives via acyl glucuronide or acyl-coenzyme A thioester formation.
It should be emphasized, however, that in the context of drug discovery and lead optimization efforts, it is imperative to determine experimentally whether one of these functional groups, if present in a candidate of interest, actually is subject to metabolic activation. In addition, acyl glucuronide conjugates differ widely in terms of their intrinsic reactivity and propensity to rearrange through acyl migration, and the mere observation of such a conjugate being formed as a metabolite of a carboxylic acid-containing drug should not necessarily be taken as a portent of liver toxicity The foregoing discussion has focused on the role of reactive, electrophilic metabolites in drug-induced liver injury where the underlying mechanism is believed to involve cellular damage through either covalent modification of critical cellular macromolecules or oxidative stress, or both.
However, an additional mechanism of hepatotoxicity that has attracted considerable interest in the area of idiosyncratic drug reactions involves activation of the immune system through haptenization of proteins by reactive drug metabolites Classic examples of this phenomenon are the inhalation anesthetic agent halothane, which undergoes CYP-mediated oxidation to yield trifluoroacetyl chloride, and the anti-inflammatory drug tienilic acid, which is metabolized to a thiophene- S -oxide intermediate. These reactive metabolites covalently modify hepatocellular proteins which, in turn, are recognized as foreign by the immune system and induce an antibody response.
Trifluoroacetyl chloride selectively acylates free amino groups on proteins, and antibodies have been detected in the sera of patients exposed to halothane that recognize liver neoantigens containing the trifluoroacetyl moiety Drug-induced toxicities that are mediated by the immune system are especially difficult to predict from preclinical toxicology studies due to the lack of generally applicable animal models for the human immune system. While the protein targets of some reactive metabolites are now known, much remains to be learned on this subject before a knowledge of the chemistry of metabolic activation can be linked to the toxicological outcome.
Consequently, it would seem prudent, during the lead optimization phase of early drug development, to evaluate candidate molecules of interest for their propensity to generate chemically reactive metabolites, and to attempt through appropriate structural modification to minimize this potential liability in order to reduce the risk of downstream metabolism-dependent toxicities Whereas drug-induced liver injury has been, and remains, an area of significant concern during both drug development and post-marketing surveillance, other metabolism-based iatrogenic organ toxicities can also occur.
Indeed, extra-hepatic tissue-selective toxicities caused by a variety of environmental chemicals are well documented. The expression of lung-selective CYP2F and CYP4B enzymes has been associated with the metabolic activation of 3-methylindole to 3-methyleneindolenine 32 and 4-ipomeanol to its reactive ene-dial 33 , respectively.
These two agents are well recognized pulmonary toxins in several experimental animal species. However, extrapolation to humans of the lung-specific toxicities displayed by such model compounds is complicated by the much lower concentrations of cytochrome P isoforms that are present in human lung compared to rodents and other susceptible species The organ-specific nephrotoxic potential of halogenated alkenes, such as trichloroethylene, has been long recognized Cytochrome Pdependent metabolism also is implicated in the renal damage observed with numerous environmental chemicals For example, chloroform can be bioactivated to the reactive metabolite, phosgene, by CYP2E1.
Good evidence exists that the kidney toxicity of chloroform in mice is attributable to renal Cyp2e1 because Cyp2e1-null mice were resistant to chloroform-induced kidney toxicity 38 , whereas mice harboring a liver-specific P reductase knockout still displayed renal damage Kidney damage can also result from the physical deposition of poorly soluble metabolites, such as those derived from ethylene glycol.
Case examples illustrating species differences in metabolism-based renal toxicity are provided in a later section. In addition to the drug metabolism-dependent organ toxicities described above, where cellular injury is caused by one or more metabolites of a single parent compound, drug-drug interactions in polytherapy may lead to a serious adverse event.
In most cases, the underlying mechanism involves inhibition of cytochrome P activity by one of the interactants, resulting in decreased clearance of the second agent and expression of a toxicity that normally is observed only in overdose situations. Although a number of organ systems may be affected by this mechanism, much attention in recent years has focused on the cardiovascular system.
Numerous approved drugs, including terodiline, sertindole, terfenadine, astemizole, grepafloxacin, cisapride, droperidol, levacetylmethadol, thioridazine, and dofetilide were withdrawn from the market as a result of adverse QTc effects, in many cases precipitated by drug-drug interactions.
However, the biotransformation of terfenadine to fexofenadine which binds weakly to hERG is catalyzed primarily by hepatic and intestinal CYP3A4, and can be blocked by potent inhibitors of this enzyme, e. Under such conditions, blood levels of terfenadine rise to values that may exceed its IC 50 for hERG, with resulting prolongation of the QTc interval and, in severe cases, precipitation of life-threatening cardiotoxicity in the form of Torsades de Pointes Recognition of the mechanism of this toxic drug-drug interaction led to the effective replacement of terfenadine by its active metabolite, fexofenadine Allegra in , and terfenadine was withdrawn from the U.
In light of this experience, screening of new chemical entities for hERG binding has been implemented throughout the pharmaceutical industry 41 , as have assays for cytochrome P inhibition 42 that are designed to eliminate potent P inhibitors as development candidates, thereby minimizing the risk of failure due to an underlying drug interaction liability Oxidation of terfenadine to its active carboxylic acid metabolite fexofenadine, and inhibition of this pathway by inhibitors of CYP3A4.
Animal studies during preclinical drug development are used to evaluate the absorption, distribution, metabolism and excretion ADME profile and safety of new candidate drugs.
Description
Ideally, data obtained from such animal studies can be used to screen out severe toxicities and to estimate a safe, maximum starting dose in human Phase I clinical trials. Nonetheless, attempts have been made to to address this question, through compilation of available toxicity data in non-human primates, dogs, rats and mice 4. Dogs and rats are the commonest non-rodent and rodent species, respectively, that are used for pre-clinical toxicology testing. Further analysis demonstrated that dogs are much more useful than rodents in predicting up to different human toxicities. However, the dog was still not predictive for about one-third of drug toxicities reported ultimately in humans.
Perhaps surprisingly, non-human primates performed no better than dogs as a predictive species for drug-related toxicities in humans 4. Differences in drug response, including toxicity, across species and individuals will reflect both pharmacokinetic and pharmacodynamic variables. Drug pharmacodynamics often are highly individualized according to the pharmacological target involved, whereas pharmacokinetics are dictated by relatively conserved ADME processes. However, cross-species ADME comparisons also are complicated by a variety of factors, cogently summarized by Lin For example, when considering the oral absorption of a drug, it is well recognized that dogs are poor acid secretors, whereas humans and rats are good acid secretors.
Therefore, when the solubility of a drug is pH-dependent, species differences in drug absorption can be expected. In a similar vein, species differences exist in biliary excretion. Rodents and dogs are considered good biliary excretors compared to humans, and this can be reflected in species differences in the extent of fecal excretion and enterohepatic recycling. However, of more importance than biliary excretion to the overall elimination of drugs are renal and metabolic clearance. Predicting renal excretion across species is one area where allometric scaling of relative glomerular filtration rates works quite well, at least for high extraction ratio drugs.
On the other hand, scaling metabolism across species is much more problematic. Moreover, metabolism often is an important determinant of toxicity through the formation of reactive electrophilic intermediates that may react with tissue nucleophiles and DNA vide infra. As noted above, metabolism dominates clearance routes, with about two-thirds of all drugs cleared primarily by Phase I and Phase II enzymes. For example, species sensitivity to organophosphates OPs has been linked to the widely differing plasma levels of paraoxonase PON1 which hydrolyzes many OPs.
Organophosphates elicit neurotoxicity by irreversibly inactivating acetylcholinesterase in the brain, so PON1 serves a protective function against exposure to these toxins. The particularly high sensitivity of birds to OP poisoning has been attributed to their extremely low of plasma PON1 On a more domestic level, inadvertent facile poisoning of cats with APAP is due to a lack of the feline UGT1A6 enzyme that conjugates and eliminates the drug, thereby permitting its build-up to toxic levels Of high relevance to drug disposition studies in industry is the knowledge that dogs and other canids completely lack N -acetyltransferase NAT genes Therefore, these species have a greatly reduced capacity to metabolize primary aromatic amines by the N -acetylation pathway.
Table 2 lists all the important human liver drug metabolizing Ps and identifies homologs in other species. For example, rats possess at least six forms of CYP2D enzymes. However, none of these rat forms have the same substrate preferences as human CYP2D6, and should be described as homologs. Another way to highlight inter-species differences in P metabolism is to compare the activity of a substrate probe for a given human enzyme across different species.
Pelkonen and coworkers compared the rates of metabolism of eleven such P probes in liver microsomes from mouse, rat, rabbit, dog, rabbit, mini-pig, monkey and human 51 , with their data demonstrating very extensive species differences in liver microsomal probe activities. Extension of this type of study to a comparison of cat, dog, horse and human liver microsomal activities revealed a similarly high species variability 52 , again with no single species reflecting human metabolism of the probe substrates employed.
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Notably, these latter workers found extremely high dextromethorphan O -demethylase activity a CYP2D6 probe in humans in the horse compared to other species, and extremely low tolbutamide hydroxylase activity a CYP2C9 probe in humans in dogs and cats. These findings could have implications in the development of veterinary medicines.
Smith and co-workers have identified well over a dozen substrates for CYP2C9, that are hydroxylated very poorly in dog liver microsomes 7. Therefore, while CYP2C21 appears to be the canine ortholog of human CYP2C9, species differences in metabolism likely occur due to low intrinsic catalytic activity of canine CYP2C21, although further studies are needed for confirmation. In the case of non-human primates, it might be anticipated that liver Ps in humans and monkeys would be very closely related - and this does, in fact, turn out to be the case. There are two points of caution, however.
Second, a new monkey P, CYP2C76, has been identified recently, but this enzyme has no ortholog in humans These considerations provide a molecular basis for the species differences in Pdependent drug metabolism that can arise between humans and non-human primates. To summarize, highly variable microsomal P probe metabolism across species could be a result of: Species differences in drug metabolism take on an added significance when they are responsible for species differences in toxicity.
The first example is 4-ipomeanol, a furan-containing compound that was first found to cause lung toxicity after farm animals ingested it unintentionally in their feed It was established more than 25 years ago that lung CYP4B1 catalyzed the bioactivation of 4-ipomeanol in animals 57 , and it has been shown recently that CYP4B1 can convert ipomeanol in vitro to a reactive ene-dial Fig.
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In the late s, 4-ipomeanol was evaluated in humans by the National Cancer Institute NCI as a possible treatment for non-small cell lung carcinoma 58 , but the compound was found to be ineffective probably because lung CYP4B1 in humans has little or no catalytic activity In other human studies, a dose-limiting liver toxicity was observed What, then, is the basis for the species difference in organ toxicity? At least two possibilities can be considered; a alternative bioactivating liver P enzyme s in humans, or b a lack of detoxifying or conjugating enzyme s in human liver.
Further studies are needed to discriminate between these possibilities. The second example is renal toxicity of the non-nucleoside reverse transcriptase inhibitor, efavirenz.
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During development, efavirenz was found to cause renal tubule epithelial cell toxicity in rats, but not in monkeys or humans. The drug was metabolized extensively in all three species, but only the cysteinylglycine conjugate M10 Fig. Evidence in support of this hypothesis was obtained from studies in rats pretreated with the glutathione-S-transferase GST inhibitor, acivicin, which protected animals from the renal toxicity of efavirenz. It was concluded, therefore, that the basis for the species difference in efavirenz renal toxicity likely is related to species difference in GST activity between rats, monkeys and humans.
Proposed pathway for the metabolism of efavirenz to a nephrotoxic metabolite s , which is believed to result from activation of the cysteine conjugate, either through S -oxidation. Finally, a very recent example of renal toxicity in humans arose with the experimental kinase inhibitor, SGX The neutral, AO-derived lactam metabolite was very poorly soluble in patient urine, and so lactam precipitation was suggested to be the cause of the renal crystal deposits.
Notably, in vitro studies with liver microsomes would not have revealed this metabolite because AO is a cytosolic enzyme, and in vivo studies in dogs and rats failed to identify M11 as a possible metabolite because AO activity is low or absent in these preclinical animal species.
There then follows an in-depth analysis of the toxicological potential of the top prescription drugs, illustrating the power and the limits of the toxicophore concept, backed by numerous case studies. Finally, a risk-benefi t approach to managing the toxicity risk of reactive metabolite-prone drugs is presented. Since the authors carefully develop the knowledge needed, from fundamental considerations to current industry standards, no degree in pharmacology is required to read this book, making it perfect for medicinal chemists without in-depth pharmacology training.
Deepak Dalvie received his Ph. Scott Obach received his Ph. He is an author or coauthor on over research publications. Dennis Smith has worked in the pharmaceutical industry for 32 years after gaining his Ph. The first chapters trace the development of our understanding of drug metabolite toxicity, covering basic concepts and techniques in the process, while the second part details chemical toxicophores that are prone to reactive metabolite formation.
This section also reviews the various drug-metabolizing enzymes that can participate in catalyzing reactive metabolite formation, including a discussion of the structure-toxicity relationships for drugs.
Two chapters are dedicated to the currently hot topics of herbal constituents and IADRs. The next part covers current strategies and approaches to evaluate the reactive metabolite potential of new drug candidates, both by predictive and by bioanalytical methods. There then follows an in-depth analysis of the toxicological potential of the top prescription drugs, illustrating the power and the limits of the toxicophore concept, backed by numerous case studies. Finally, a risk-benefi t approach to managing the toxicity risk of reactive metabolite-prone drugs is presented.
Since the authors carefully develop the knowledge needed, from fundamental considerations to current industry standards, no degree in pharmacology is required to read this book, making it perfect for medicinal chemists without in-depth pharmacology training.