Chem. Res. Toxicol. 2009, 22, 1217–1220
1217
ReViews Interpretation and Considerations on the Safety Evaluation of Human Drug Metabolites Aisar H. Atrakchi* Office of New Drugs, Center for Drug EValuation and Research, U.S. Food and Drug Administration, SilVer Spring, Maryland 20993 ReceiVed April 1, 2009
The final Food and Drug Administration guidance on the safety testing of drug metabolites was published in February 2008. Discussions of the role and applications of this guidance were addressed at several public scientific meetings over the past year. One of the main differences between the draft and the finalized guidance is that in the latter, the human metabolite level was correlated to the parent drug level in plasma, whereas this parameter was considered in relationship to administered dose or total exposure in the draft guidance. The parent drug concentration in plasma has traditionally been the parameter commonly measured in animals during drug development and the one used to estimate drug clinical levels and to assess human risk. Moreover, circulating parent drug in general is the molecule with the intended therapeutic and pharmacologic effect. Therefore, it is appropriate to compare metabolite concentration to that of the parent drug. This report elaborates on this issue and supports other alternative rational and scientific approaches on the design of nonclinical studies that may be needed to test a human drug metabolite.
1. 2. 3. 4. 5.
Contents Introduction Role of Metabolites in Drug Development Rationale for Drug Metabolite Testing Comparative Basis for Metabolite Testing Conclusions
1217 1218 1218 1218 1219
1. Introduction Nonclinical animal testing is often the first step in a development plan for evaluating the safety and efficacy of a new drug. A drug candidate is generally studied in a series of defined nonclinical tests that help characterize the drug’s therapeutic and toxicologic targets, thereby minimizing potential harm to humans during clinical development. Regulatory guidances provide important recommendations to the pharmaceutical industry and to regulatory review staff and present the current thinking and procedures on specific topics or drug-related issues (1). Drug disposition has always been an integral component investigated during drug development. Knowledge of how and where a drug is metabolized help in the understanding of the activity, the duration of action, the time to peak plasma concentration, and the potential formation of toxic metabolites. Ultimately, metabolite data can be incorporated into the nonclinical safety assessment of the drug candidate. The term detoxification historically referred to a process by which a compound is rendered by the body to a less toxic substance (2). However, this term is not entirely accurate since it is well-known that the body can at times convert a drug into * To whom correspondence should be addressed. E-mail: aisar.atrakchi@ fda.hhs.gov.
10.1021/tx900124j CCC: $40.75
a molecule more toxic than the parent compound (3, 4). Metabolism is the biotransformation of a molecule through chemical reactions that, in general, renders a compound more polar and water-soluble and, therefore, pharmacologically inert and readily excreted in urine or feces. The liver is the major organ responsible for drug metabolism, and most drugs are substrates for the hepatic P450 mixed function oxidase enzyme system. To a lesser extent, extrahepatic metabolism occurs in the kidneys, intestinal mucosa, skin, and lungs. Many factors play a role in determining where and how a drug is metabolized; these include but are not limited to genetic polymorphism, age, gender, concomitant medications, hormones, disease, diet, and the environment. Differences in metabolism across and within species have been known for many years (5, 6). Such differences can either be quantitative (same metabolic pathway but different rates and amounts) or qualitative (different pathways) (3-6). Classical examples of the former include hexobarbitone and caffeine, whereas amphetamine metabolism through conjugation exemplifies qualitative differences among species where no conjugation occurs in the feline model but is completely conjugated in the swine; another example is the glucuronidation of quaternary amines by human and rabbit UGT1A4 but not by the rat (5-9). Because of potential species differences in metabolism, it is important to know the metabolism of the drug and the enzymes responsible for its breakdown early during the drug development process. This would allow selection of relevant animal species with similar metabolic profiles to humans, thus providing more meaningful assessment of clinical safety through extrapolation of the data.
This article not subject to U.S. Copyright. Published 2009 by American Chemical Society. Published on Web 06/29/2009
1218
Chem. Res. Toxicol., Vol. 22, No. 7, 2009
2. Role of Metabolites in Drug Development In a manner similar to the parent drug molecule, metabolites can impose their own effects through pharmacological, toxicological, and/or physiological interactions. Metabolites may play a significant role in drug toxicity. This is exemplified by the withdrawal of several drugs from the market in recent years such as iproniazid, troglitazone, benoxaprofen, phenfluramine, and pemoline and by the inclusion of “black box” warnings to the drug label because of significant toxicity, such as in the cases of gatifloxacin and pergolide, or via potential adverse effects caused by toxic and/or reactive metabolites like felbamate, valproic acid, tolcapone, and methylenedioxymethamphetamine (MDMA) (10-13). On the other hand, metabolites may contribute to the efficacy or are the efficacious moiety as with prodrugs. Interest in metabolites has always existed. However, their contribution to the overall toxicity profile generally remained unknown due to lack of sensitive analytical methods that sometimes prevented quantification and characterization of metabolites. Analytical instrumentations such as the accelerated mass spectrometry and their sensitivities have improved the detection of metabolites formed at very low levels (pg range) (14, 15). Identification and measurement of metabolites provide more comprehensive knowledge of the drug and may provide insight into an unexplained toxicity in animals and its relevance to humans. Characterizing metabolic pathways and identification of metabolites early in the drug development process help avoid unnecessary delays in the clinical program and, if the need arises, where the safety of a human metabolite should be investigated. This approach should help identify the most relevant nonclinical studies that may need to be conducted.
3. Rationale for Drug Metabolite Testing Traditionally, circulating drug (parent) concentration and/or exposure were measured in animals, and the data were used to calculate and extrapolate to safe exposures in humans. This approach has been and remains adequate when metabolism in animals and humans is qualitatively similar (same metabolic pathway). This is generally the case, since most metabolic enzyme families are conserved across species, and it is, therefore, uncommon that a metabolite would form only in humans but not in any one animal test species. The more common occurrences are the quantitative differences in metabolism among species where the ratio of metabolite to parent may differ due to differences in enzyme content and/or activities (16-18). Although rare, a metabolite may be formed only in humans and not in any animal test species; therefore, the effects of this metabolite are unknown and should be investigated in animals. Similarly, in instances when the drug metabolite is present at disproportionately lower concentrations in animals than those measured in humans, the effects of such a human metabolite may also need to be investigated in animals. However, in this latter scenario, many parameters must be considered and assessed before the final decision is made to test the human metabolite in independent nonclinical studies. On the basis of the FDA guidance, a disproportionate metabolite is defined as one that is formed only in humans or formed at quantitatively higher levels, >10% of parent plasma exposure measured at steady state. Measurement and identification of drug metabolites have become more prevalent in recent years due to advancements in analytical methodologies (14, 15). Understanding drug metabolism and the potential contribution of metabolites to the
Atrakchi
therapeutic effects and/or toxicity of the drug are not new concepts and have been recognized and addressed in a number of regulatory guidances and pharmaceutical venues. The 21 Code of Federal Regulations 312.23(a)(8)(i) states that information on metabolism is needed and should be included in the drug application (19). The importance of metabolism and metabolites is recognized in several International Committees on Harmonization (ICH) and guidelines such as the ICH S7A, which recommends that in addition to the parent compound, major human metabolites may need to be tested in nonclinical safety pharmacology studies if they were absent or present at relatively low concentrations in animals (20). Similarly, the ICH S3A confirms the role of metabolites during drug development and recommends measurement of metabolite concentrations in plasma or other body fluids during the toxicokinetic assessments (21). The ICH M3 goes as far as recommending determination of exposure in animals prior to the first human clinical trial and that general information on ADME in animals is to be made available to compare human and animal metabolic pathways (22). The OECD TG417 Toxicokinetic guidelines state that reasonable efforts should be made to identify all metabolites present at 5% or greater of the administered dose and to provide a metabolic profile for the test substance (23). Baillie et al. (24, 25) summarized the deliberations of a joint FDA-pharmaceutical industry workshop and considered a drug metabolite that accounts for >25% of total drug-related material to constitute a major metabolite and that the plasma concentrations of such a metabolite should be monitored in the clinic. The FDA in a follow-up commentary corroborated the importance of measuring human metabolites and considered them to be of safety concern when they are present at >10% of total exposure or radioactive dose (26, 27). Very recently, Chemical Research in Toxicology devoted the February issue to the topic of metabolism with experts in the field addressing several aspects of this important parameter (28). The FDA publication of the Safety Testing of Drug Metabolite guidance provides a roadmap describing the type and duration of animal studies needed to assess human safety associated with drug metabolites (29). In this FDA guidance, a human metabolite constituting >10% of circulating drug levels and either could not be detected in animals or, was present at much higher levels than in animals (disproportionate exposure), may need further toxicological evaluation. Historically, the circulating parent drug concentration has been the moiety generally measured when conducting toxicokinetic analyses; therefore, comparison of the metabolite levels to parent drug concentrations seems to be a logical approach and, therefore, recommended in the FDA guidance. There are, however, circumstances when such comparison is inappropriate and other metrics should be used. It should emphasized that there is no straightforward or one standardized approach to the evaluation of the safety of drug metabolites. A case-by-case approach should be exercised when the information provided in the guidance does not apply.
4. Comparative Basis for Metabolite Testing When a disproportionate human metabolite is identified, it may not need to be tested in independent nonclinical safety studies. Several factors should be considered before the decision is made to synthesize and independently test a human metabolite. Generally, phase 1 reactions can generate metabolites that are likely to interact with target and/or key proteins, receptors, enzymes, ion channels, etc. and cause pharmacological and/or toxicological effects more so than phase 2 metabolites. However, acyl glucuronides and sulfate esters of allyl and benzyl alcohols
ReView
are examples of phase 2 metabolites that would be of special safety concerns due to their chemical reactivity (30-32, 1, 33, 34). Also of concern are metabolites with structural alerts identified during in silico evaluation, those with positive genetic toxicity based on testing in one or more genetic toxicity assays, or a metabolite that was absent in the activation systems used with the parent molecule in genetic toxicity tests. In general, circulating metabolites are more of a safety concern than those identified only in excreta. The relative amount of a human metabolite is important in determining the level of concern; generally, the larger the contribution of the metabolite to the overall metabolic profile, the greater the concern, that is, 9 vs 50%. On the other hand, a human metabolite with positive genetic toxicity results and/or with a structural alert, even if present at 10% when their levels are compared to the parent if the latter has low concentrations due to extensive metabolism or if it has a high volume of distribution (high tissue binding), or the metabolites constitute disproportionately high concentrations because it is highly bound to plasma proteins (low volume of distribution). In these cases, it is more appropriate to make the comparison relative to total exposure or total administered dose. In most cases, absolute concentrations (free or total) of metabolites should also be considered in addition to relative exposure (35). Therefore, the criterion that signals attention to a metabolite should first be its relative exposure, but if inappropriate (for any of the reasons mentioned above), the next approach would be to consider its absolute amount. Thus, the decision to conduct additional animal tests with a disproportionate human metabolite should be scientifically justified based on the collective information available on the parent pharmacology and pharmacokinetics.
Chem. Res. Toxicol., Vol. 22, No. 7, 2009 1219
This is in agreement with the FDA metabolite guidance recommendations that support and encourage a scientific, rational, and flexible approach as well as the case-by-case assessment in decision making. As to when metabolism data should be investigated during the course of drug development is determined by the individual pharmaceutical organization, the FDA guidance only makes a recommendation that encourages one to submit any metabolism data early. Many drug applications in recent years are already providing in vitro metabolism data across species with the phase 1 clinical protocol. In vivo metabolism studies follow at some point, and the data verify the in vitro results. The FDA guidance recommends that the in vivo metabolism be conducted not too late during the development process to avoid any potential delays. If or when nonclinical safety studies with a human metabolite are needed, some of the parameters to consider include physical/ chemical properties of the metabolite such as stability in the stomach, solubility, etc.; this information is useful in selecting the appropriate route of administration of the metabolite. Also, data from in vitro metabolism and knowledge of metabolic enzymes and pathways help in the selection of the most relevant animal test species. In some cases, a general toxicity/toxicokinetic study of 3 months in duration may be adequate to address the safety concerns with the metabolite, thus eliminating the need for additional studies of longer duration per the ICH M3 guidelines. This is applicable if data from such a study did not show new findings relative to those observed with the parent drug and the plasma exposure to the metabolite is at least equal to those measured in humans. Also, the metabolite should test negative for genetic mutation, and the clinical indication and patient population are taken into account.
5. Conclusions Drug metabolites are important because they may contribute to the pharmacology and/or toxicity of a drug. Therefore, their overall effects should be evaluated in appropriate animal species to ensure human safety. While it is unlikely that a metabolite forms only in humans and not in any routinely used animal species, it is more likely that differences in the ratio of metabolite to parent are observed in humans and animals, that is, the relative amounts. When this ratio is high and there is no other information to support the safety of the human metabolite, animal studies may be needed to ensure clinical safety. The decision to conduct safety studies with disproportionate human metabolite should always be data driven and scientifically justified. If nonclinical testing is required, a flexible and prudent approach should be pursued in determining the type, duration, and timing of animal studies to ensure clinical safety and avoid delays in the drug development program. The complexities associated with interpreting data obtained from administration of an endogenously formed metabolite are acknowledged (36). However, when the safety of a human metabolite is unknown or unclear, nonclinical studies should be performed to adequately evaluate human risk. The FDA guidance on the safety testing of drug metabolites provides information on when to conduct nonclinical studies with human metabolites but also recognizes those circumstances, as scientifically justified, when other approaches are more appropriate. An example to the latter may be comparison of the human metabolite to total exposure, absolute amount, or other scientifically justifiable parameters. Acknowledgment. I thank Dr. David Jacobson-Kram for critical review of the manuscript. The views and opinions expressed in this document are solely those of the author and
1220
Chem. Res. Toxicol., Vol. 22, No. 7, 2009
are not the official policy of the U.S. Food and Drug Administration.
References (1) U.S. Food and Drug Administration U.S. Food and Drug Administration Guidances, Center for Drug Evaluation and Research, Rockville, MD; www.fda.gov/cder/guidances. (2) Levine, R. R. (2004) Pharmacology: Drug Actions and Reactions, 6th ed., Parthenon Publishing Nashville, TN. (3) Gibson, G., and Skett, P. (2001) Introduction to Drug Metabolism, 3rd ed., Nelson Thornes Publishers, United Kingdom. (4) Smith, D. A., and Schmid, E. F. (2006) Drug withdrawals and the lessons within. Curr. Opin. Drug DiscoVery DeV. 9, 38–60. (5) Caldwell, J. (2006) Drug metabolism and pharmacogenetics: The British contribution to fields of international significance. Br. J. Pharmacol. 147, 89–99. (6) Caldwell, J. (2001) The pharmacogenetic basis of adverse drug reactions. Int. J. Pharm. Med. 15, 83–84. (7) Smith, D. A., and Obach, R. S. (2009) Metabolites in safety testing: Considerations of mechanisms of toxicity with dose, abundance, and duration of treatment. Chem. Res. Toxicol. 22, 267–279. (8) Bruck, M., Lamb, J. G., and Tukey, R. H. (1997) Characterization of rabbit UDP-glucuronosyltransferase UGT1A7: Tertiary amine glucuronidation is catalyzed by UGT1A7 and UGT1A4. Arch. Biochem. Biophys. 348, 357–364. (9) Kubota, T., Lweis, B. C., Elliot, D. J., Mackenzie, P. I., and Miners, J. O. (2007) critical roles of residues 36 and 40 in the phenol and tertiary amine aglycone substrate selectvities of UDP-glucuronosyltransferases 1A3 and 1A4. Crit. Mol. Pharmacol. 72, 1054–1062. (10) Hogan, V. (2000) Pemoline (Cylert): Market withdrawal. CMAJ 162, 106–110. (11) Desanty, K. P., and Amabile, C. M. (2007) Antidepressant-induced liver injury. Ann. Phamracother. 41, 1201–1211. (12) Shenouda, S. K., Varner, K. J., Carvalho, F., and Lucchesi, P. A. (2009) Metabolites of MDMA induce oxidative stress and contractile dysfunction in adult rat left ventricular myocytes. CardioVasc. Toxicol. 9, 30–38. (13) Caldwell, G. W., and Yan, Z. (2006) Screening for reactive intermediates and toxicity assessment in drug discovery. Curr. Opin. Drug DiscoVery DeV. 9, 47–60. (14) Barile, F. A. (2008) Principles of Toxicology Testing, CRC Press, Boca Raton, FL. (15) Lappin, G., and Garner, R. C. (2003) Big physics, small doses, the use of AMS and PET in human microdosing of development drugs. Nat. ReV. 2, 233–240. (16) Lin, J. H. (1995) Species similarities and differences in pharmacokinetics. Drug Metab. Dispos. 23, 1008–1021. (17) Dieckhaus, C. M., Miller, T. A., Sofia, R. D., and Macdonald, T. L. (2000) A mechanistic approach to understanding species differences in felbamate bioactivation: relevance to drug induced idiosyncratic reactions. Drug Metab. Dispos. 28, 814–822. (18) Green, M. D., and Tephly, T. R. (1998) glucuronidation of amine substrates by purified and expressed UDP-glucuronosyltransferase proteins. Drug Metab. Dispos. 26, 860–867.
Atrakchi (19) 21 Code of Federal Regulations (2009) section 312.23(a)(8)(i). (20) U.S. Food and Drug Administration (2001) Guidance for Industry: Safety Pharmacology Studies for Human Pharmaceuticals, International Committee on Harmonization ICH S7A, Rockville, MD. (21) U.S. Food and Drug Administration (1995) Guideline for Industry: Toxicokinetics: The Assessment of Systemic Exposure in Toxicity Studies ICH S3A, Rockville, MD. (22) U.S. Food and Drug Administration (1997) Guidance for Industry: Nonclinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals ICH M3, Rockville, MD. (23) (2008) Organization for Economic Co-operation and Development (OECD) Guideline for the Testing of Chemicals: TG417 Toxicokinetics. (24) Baillie, T. A., Cayen, M. N., Fouda, H., Gerson, R. J., Green, J. D., Grossman, S. J., Klunk, L. J., LeBlanc, B., Perkins, D. G., and Shipley, L. A. (2002) Drug metabolites in safety testing. Toxicol. Appl. Pharmacol. 182, 188–196. (25) Baillie, T. A., Cayen, M. N., Fouda, H., Gerson, R. J., et al. (2003) Reply to the Editor. Toxicol. Appl. Pharmacol. 190, 93–94. (26) Hastings, K. L., El Hage, J., Jacobs, A., Leighton, J., Morse, D., and Osterberg, R. (2003) Drug metabolites in safety testing. Toxicol. Appl. Pharmacol. 190, 91–92. (27) Davis-Bruno, K. L., and Atrakchi, A. (2006) A regulatory perspective on issues and approaches in characterizing human metabolites. Chem. Res. Toxicol. 19, 1561–1563. (28) (2009) Chemical Research in Toxicology, February issue. (29) U.S. Food and Drug Administration (2008) Guidance for Industry: Safety Testing of Drug Metabolites, U.S. Food and Drug Administration, Rockville, MD. (30) Ogilvie, B. W., Zhang, D., Li, W., Rodrigues, A. D., Gipson, A. E., Holsapple, J., Toren, P., and Parkinson, A. (2006) Glucuronidation converts gemfibrozil to a potent, metabolism-dependent inhibitor of CYP2C8: Implications for drug-drug interactions. Drug Metab. Dispos. 34, 191–197. (31) Shipkova, M., and Wieland, E. (2005) Glucuronidation in therapeutic drug monitoring. Clin. Chim. Acta 358, 2–23. (32) Kroemer, H. K., and Klotz, U. (1992) Glucuronidation of drugs: A re-evaluation of the pharmacological significance of the conjugates and modulating factors. Clin. Pharmacokinet. 23, 292–310. (33) Olson, J. A., Moon, R. C., Anders, M. W., Fenselau, C., and Shane, B. (1992) Enhancement of biological activity by conjugation reactions. J. Nutr. 122, 615–624. (34) Benet, L. Z., Spahn-Langguth, H., Iwakawa, S., Volland, C., Mizuma, T., Mayer, S., Mutschler, E., and Lin, E. T. (1993) Predictability of the covalent binding of acidic drugs in man. Life Sci. 53, 141–146. (35) Smith, D. A., and Obach, R. S. (2006) Metabolites and safety: what are the concern, and how should we address them? Chem. Res. Toxicol. 19, 1570–1579. (36) Prueksaritanont, T., Lin, J. H., and Baillie, T. A. (2006) Complicating factors in safety testing of drug metabolites: Kinetic differences between generated and preformed metabolites. Toxicol. Appl. Pharmacol. 217, 143–152.
TX900124J
