- Email: [email protected]
Overview: Evaluation of metabolism-based drug toxicity in drug development
Chemico-Biological Interactions 179 (2009) 1–3
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Editorial
Overview: Evaluation of metabolism-based drug toxicity in drug development a r t i c l e
i n f o
Keywords: Drug metabolism Drug toxicity Idiosyncratic drug toxicity P450 In vitro Human hepatocytes Metabolite identification Reactive metabolites
a b s t r a c t Drug metabolism can be a key determinant of drug toxicity. A nontoxic parent drug may be biotransformed by drug metabolizing enzymes to toxic metabolites (metabolic activation). Conversely, a toxic drug may be biotransformed to nontoxic metabolites (detoxification). The approaches to evaluate metabolism-based drug toxicity include the identification of toxic metabolites and the evaluation of toxicity in metabolically competent and metabolically compromised systems. A clear understanding of the role of drug metabolism in toxicity can aid the identification of risk factors that may potentiate drug toxicity, and may provide key information for the development of safe drugs. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
2. Drug metabolism and drug toxicity
Accurate prediction of drug toxicity remains a major challenge in drug development. It has been estimated that over 25% of drug candidates would fail in clinical trials due to unexpected adverse drug properties. Furthermore, there are examples of new drugs entering the market that are required to be withdrawn or to have their use limited due to severe adverse effects that are not detected in preclinical or Phase I, II and III clinical trials [1]. There are two likely reasons for the difficulty in the prediction of human drug toxicity:
Biotransformation of a xenobiotic by drug metabolizing enzymes can significantly alter its toxicity [2]. There have been decades of research with toxicants such as polycyclic hydrocarbons and nitrosamines demonstrating clearly that a xenobiotic can be “metabolic activated” from being rather inert molecules to extremely toxic metabolites. This area of research has led to the universal application of “exogenous activation” in genetic toxicity assays—the application of a liver homogenate/cofactors mixture for the metabolism of a “promutagen” to mutagenic metabolites. There also has been extensive research towards the identification of “reactive” metabolites. Formation of reactive metabolites and the subsequent covalent binding to macromolecules, leading to the formation of “neoantigens” and toxic immune reaction with the “neoantigens”, are steps postulated for a possible mechanism of idiosyncratic drug toxicity [3]. Another aspect of drug metabolism is detoxification: the metabolic transformation of a toxic molecule to less toxic metabolites. Phase II conjugation reactions are in general believed to be detoxifying, although some Phase II conjugates are also toxic. Glutathione conjugation of reactive metabolites is a well-established detoxification pathway. The major drug metabolizing enzyme pathway for metabolic activation is the cytochrome P450 monooxygenases (CYP). CYP is an enzyme family with multiple isoforms. It is now known that there are isoform-specific substrates, inhibitors, and inducers. UDPdependent glucuronosyl transferase (UGT), sulfotranferases (ST) and glutathione transferases (GST) are the major conjugative pathways known generally for detoxification. It is to be noted that while oxidative pathways such as P450 metabolism are generally known for metabolic activation, and conjugative pathways for detoxification, there is also evidence that both pathways can be involved in both activation and detoxification, depending on the chemicals involved. Species differences in drug metabolizing enzymes are a major factor for species differences in drug metabolism, and therefore a
1. Species differences in drug toxicity: Drugs with human-specific drug toxicity by definition cannot be predicted with nonhuman laboratory animals. The species difference may be due to drug metabolism. If a toxic metabolite is human specific, laboratory animals would not form the toxic metabolite and therefore would under predict toxicity (and vice versa). Species differences in response to toxicants can also be due to differences in target organ sensitivity, which may be related to species-specific xenobiotic distribution as well as toxic mechanisms. 2. Lack of mechanistic understanding of drug toxicity: Toxicology, especially when practiced for regulatory approval of a product, is often treated as an empirical discipline. Decisions on safety are often based on dose–response curves. The acceptability of the toxicity observed is often based on the level of exposure, regardless of toxic mechanisms. This lack of mechanistic understanding precludes logical deductions of human adverse effects based on human biology, especially in the identification of populations that, for mechanistic reasons, may have high sensitivity to drug toxicity that are deemed “nontoxic” based on preclinical data. In this overview, the importance of the understanding of the relationship between drug metabolism and drug toxicity to aid the accurate prediction of human drug toxicity will be discussed. 0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2008.11.013
2
Editorial / Chemico-Biological Interactions 179 (2009) 1–3
determinant for species differences in drug toxicity that involves drug metabolism. For instance, of the key P450 isoforms for drug metabolism: 1A, 2A, 2B, 2C, 2D, 2E and 3A, human and the four commonly used laboratory animals: mouse, rat, dog and monkey are different with the exception of CYP1A and CYP2E [2]. This difference is known to lead to species differences in drug metabolism, and may contribute to species differences in drug toxicity. 3. Approaches to evaluate metabolism-based drug toxicity As mentioned previously, exogenous activation is commonly used to evaluate genotoxicity. For drug toxicity evaluation, however, the role of metabolism in the observed toxicity is not routinely defined, at least not in a systematic manner. I have summarized here the promising approaches that can be used routinely for the evaluation of the role of drug metabolism in drug toxicity. As the objective is to evaluate human drug toxicity, in vitro experimental system for human drug metabolism such as human liver microsomes and human hepatocytes are useful tools for this purpose. 1. Identification of toxic metabolites: a. Identification of toxic moieties: It is difficult to assign toxicity to metabolites without actual experimentation. However, there are chemical moieties (e.g. quinones, epoxides) that are known to associate with toxicity. One assumption that can be used as an initial assessment of metabolite toxicity is to structurally identify the metabolites to evaluate if known toxic moieties are present. For the identification of human metabolites, the most effective preclinical approach is to perform in vitro studies (e.g. incubation with liver microsomes or hepatocytes). Toxic metabolites from laboratory animals can be identified from body fluids (e.g. plasma, bile, urine, feces) or in vitro studies. The identity of the metabolites usually is determined based on mass spectrometry (MS), especially tandem mass spectrometry (MS/MS). A comprehensive databases for toxic metabolites would be extremely helpful in this exercise [4]. b. Identification of reactive metabolites: Highly electrophilic metabolites are known to reactive with reduced GSH. Identification of GSH conjugates therefore can provide initial evidence of the formation of reactive metabolites. The studies are usually performed with an in vitro metabolism system such as liver microsomes or hepatocytes [5]. With liver microsomes, exogenous GSH is usually added to allow formation of GSH conjugates. With hepatocytes, the endogenous GSH are usually sufficient for the formation of GSH conjugates. c. Identification of unique human metabolites: While some chemical structures are consistently associated with toxicity, the state-of-the-art as of this writing does not allowed the toxicity of metabolites to be estimated with certainty. Metabolites unique to humans (not found in nonhuman animals) may have toxic consequences which cannot be studied by treating laboratory animals with the parent compound. Identification of unique human metabolites allows planning of studies to evaluate toxicity of such metabolites. It is to be noted that the final metabolite may not be the ultimate toxic structure. Evaluation of the toxicity of a compound which may be metabolized to unique metabolites in humans may be betterserved using a system with human-specific metabolism (e.g. human hepatocytes [6]; humanized chimeric mice [7]) than testing these metabolites directly. FDA currently is recommending direct safety testing of unique human metabolites
(metabolites in safety testing [8]) the virtual of which is being discussed among experts of the field [9,10]. d. Identification of the drug metabolizing enzyme pathways involved in the formation of toxic metabolites: This study, in general called Metabolic Pathway Identification, can be performed systematically. A common approach is to firstly use a non-specific P450 inhibitor (e.g. 1-aminobenzotriazole (ABT)) to evaluate whether P450 isoforms are involved. If ABT inhibits the formation of metabolites, isoform-specific inhibitors can be applied to pinpoint the exact isoforms involved. The results with P450 isoform-specific inhibitors can be further confirmed with individual cDNA-expressed human P450 isoforms [11]. 2. Evaluation of toxicity in the absence and presence of drug metabolism: a. Exogenous activation system: In genotoxicity studies such as Ames/Salmonella histidine reversion assay or in vitro mammalian gene mutation assay, liver homogenate and cofactors are added exogenously to provide hepatic metabolism for the evaluation of promutagens [12]. Evaluation of results with and without exogenous activation will provide information on the role of metabolism on the genotoxicity of the chemical being studied. For cytotoxicity studies, such an approach is more difficult as liver homogenates can be cytotoxic. Exogenous activating systems are generally not used in cytotoxicity studies, mainly because of the cytotoxicity of the liver homogenate. However, use of whole cells (e.g. hepatocytes) to provide exogenous activation is now being investigated by multiple laboratories as approach to understand the role of hepatic metabolism in drug toxicity (e.g. the Integrated Discrete Multiple Organ Co-culture (IdMOC) system [13,14]). b. Differential toxicity studies: This approach may represent the most efficient approach to evaluate if drug metabolism is involved in toxicity. The study involves the evaluation of toxicity or cytotoxicity in a metabolically competent system (e.g. hepatocytes) and a metabolically incompetent system (e.g. Chinese hamster ovary cells) [15]. Transgenic animals (e.g. “knock-out” animals deficient in 1 or more P450 isoforms [16]) have also been used successfully as in vivo system to evaluate the role of a specific drug metabolizing enzyme pathway in drug toxicity. A drug that is more toxic in a metabolically competent system than in an incompetent system can be initially classified as one that would require metabolism to be toxic. Conversely, a drug that is more toxic in a metabolically incompetent system would be one that is metabolically detoxified. Another differential toxicity studies is to compare cytotoxicity of a drug in hepatocytes or tissue slices from man to that from multiple laboratory animals which may aid to the understanding of species differences in toxicity (e.g. [17,18]). c. Metabolic inhibitors: Via the use of inhibitors of drug metabolizing enzymes, one can ascertain the role of the enzymes in drug toxicity. This approach is similar to that described above for Metabolite Pathway Identification, except that instead of metabolite identification, cytotoxicity is used as endpoint. The non-specific, mechanism-based P450 inhibitor ABT can be used to evaluate P450-dependent drug toxicity. P450 isoform-specific inhibitors can be used to evaluate the role of individual isoforms (e.g. [15]). Attenuation of cytotoxicity by metabolic inhibitors would infer that the pathways inhibited are involved in the generation of toxic metabolites. Similarly, enhancement of cytotoxicity by the inhibitors would suggest that the pathways inhibited are involved in detoxification. The cytotoxicity results can be correlated to the metabolite identification results to further understand the role of metabolic pathways and metabolites in the toxicity of the drug in question.
Editorial / Chemico-Biological Interactions 179 (2009) 1–3
4. Conclusions The field of toxicology, especially toxicology practices for regulatory purposes, has not changed in several decades. Preclinical safety testing is centered on in vivo laboratory animal studies. These in vivo studies have been valuable in the prevention of some toxic drug candidates from further development, as they are effective in the detection of toxicity that are common to both humans and nonhuman animals. However, toxicity that is unique to humans cannot be detected. Preclinical experimental systems to provide human-specific information should be incorporated in the preclinical safety evaluation program. The recent success in the application of human-based in vitro experimental system in the understanding of human drug metabolism, pharmacokinetics, and drug–drug interactions suggest that similar approaches can also aid the better prediction of human drug toxicity. Evaluation of the role of drug metabolism and toxicity is arguably a necessary activity for the evaluation of human drug toxicity. It allows a rationale design of a safer molecule (e.g. by blocking sites critical for toxic metabolite formation), assessment of sensitive human population (e.g. populations with high level of the drug metabolizing enzyme pathway for the formation of toxic metabolites; populations with low detoxifying activities; environmental factors leading to high levels of “activating” activities or low levels of “detoxifying” activities). The approaches suggest in this overview can be used as basis for a systematic approach for this important discipline of research during drug discovery and development. It is possible that via the understanding of the risk factors defined by drug metabolism, one can understanding the reasons behind the elusive idiosyncratic drug toxicity [19]. To accomplish the goal of a better prediction of human drug toxicity , a paradigm change is needed. Toxicology needs to be practiced not as an empirical discipline, but as an investigative discipline. Good Scientific Practice (GSP) is as important as Good Laboratory Practice (GLP). Adverse drug effects need to be studied under physiologically relevant conditions (e.g. avoid the testing of unacceptably high drug concentrations) [20]. Cross fertilization of the disciplines of pharmacology, drug metabolism, pharmacokinetics and toxicology are important for the understanding of adverse drug effects and the ultimate accurate prediction human drug toxicity [21]). References [1] A.P. Li, Accurate prediction of human drug toxicity: a major challenge in drug development, Chem. Biol. Interact. 150 (2008) 3–7. [2] F.P. Guengerich, Cytochrome P450s and other enzymes in drug metabolism and toxicity, AAPS J. 8 (1) (2006) E101–E111. [3] P.H. Beaune, S. Lecoeur, Immunotoxicology of the liver: adverse reactions to drugs, J. Hepatol. 26 (1997) 37–42.
3
[4] R.P. Hanzlik, J. Fang, Y.M. Koen, Filling and mining the reactive metabolite target protein database, Chem. Biol. Interact. 179 (2009) 38–44. [5] S. Ma, M. Zhu, Recent advances in applications of liquid chromatography–tandem mass spectrometry to the analysis of reactive drug metabolites, Chem. Biol. Interact. 179 (2009) 25–37. [6] A.P. Li, Human hepatocytes: isolation, cryopreservation and applications in drug development, Chem. Biol. Interact. 168 (2007) 16–29. [7] Y. Sato, H. Yamada, K. Iwasaki, C. Tateno, T. Yokoi, K. Yoshizato, I. Horii, Human hepatocytes can repopulate mouse liver: histopathology of the liver in human hepatocyte-transplanted chimeric mice and toxiologic responses to acetaminophen, Toxicol. Pathol. 36 (2008) 581–591. [8] FDA Guidance for Industry, Saftey testing of drug metabolites, Pharmcology and Toxicology, February 2008, http://www.fda.gov/cder/guidance/. [9] D.A. Smith, R. Scott Obach, D.P. Williams, B Kevin Park, Clearing the MIST (metabolites in safety testing) of time: the impact of duration of administration on drug metabolite toxicity, Chem. Biol. Interact. 179 (2009) 60–67. [10] K.S. Pang, Safety testing of metabolites: expectations and outcomes, Chem. Biol. Interact. 179 (2009) 45–59. [11] A.P. Li, Preclinical in vitro screening assays for drug-like properties, Drug Discov. Today 2 (2005) 79–85. [12] A.P. Li, Use of Aroclor 1254-induced rat liver homogenate in the assaying of promutagens in Chinese hamster ovary cells, Environ. Mutagen. 6 (1984) 539–544. [13] A.P. Li, A novel in vitro system, the integrated discrete multiple organ cell culture (IdMOC) system, for the evaluation of human drug toxicity: comparative cytotoxicity of tamoxifen towards normal human cells from five major organs and MCF-7 adenocarinoma breast cancer cells, Chemico-Biol. Interact. 150 (2004) 129–136. [14] A.P. Li, In vitro evaluation of human xenobiotic toxicity: scientific concepts and the novel integrated discrete multiple cell co-culture (IdMOC) technology, ALTEX 25 (2008) 43–49. [15] A.P. Li, Metabolism Comparative Cytotoxicity Assay (MCCA) and Cytotoxic Metabolic Pathway Identification Assay (CMPIA) with cryopreserved human hepatocytes for the evaluation of metabolism-based cytotoxicity in vitro: proofof-concept study with aflatoxin B1, Chemico-Biol. Interact. 179 (2009) 4–8. [16] F.J. Gonzalez, The use of gene knockout mice to unravel the mechanisms of toxicity and chemical carcinogenesis, Toxicol. Lett. 120 (2001) 199–208. [17] B. Lauer, G. Tuschl, M. Kling, S.O. Mueller, Species-specific toxicity of diclofenac, troglitazone in primary human and rat hepatocytes, Chemico-Biol. Interact. 179 (2009) 17–24. [18] A.E.M. Vickers, Tissue slices for the evaluation of metabolism-based toxicity with the example of diclofenac, Chemico-Biol. Interact. 179 (2009) 9–16. [19] A.P. Li, A review of the common properties of drugs with idiosyncratic hepatotoxicity and the “multiple determinant hypothesis” for the manifestation of idiosyncratic drug toxicity, Chem. Biol. Interact. 142 (2002) 7–23. [20] D.A. Smith, Commentary-Dogma driven science, the need to establish a common base line, Chemico-Biol. Interact. 179 (2009) 68–70. [21] A.P. Li, A comprehensive approach for drug safety assessment, Chemico-Biol. Interact. 150 (2004) 27–33.
Albert P. Li ∗ Advanced Pharmaceutical Sciences Inc. and In Vitro ADMET Laboratories LLC, 9221 Rumsey Road Suite 8, Columbia, MD 21044, USA ∗ Tel.:
+1 410 869 9037; fax: +1 410 869 9034. E-mail address: [email protected] Available online 25 November 2008
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Editorial
Overview: Evaluation of metabolism-based drug toxicity in drug development a r t i c l e
i n f o
Keywords: Drug metabolism Drug toxicity Idiosyncratic drug toxicity P450 In vitro Human hepatocytes Metabolite identification Reactive metabolites
a b s t r a c t Drug metabolism can be a key determinant of drug toxicity. A nontoxic parent drug may be biotransformed by drug metabolizing enzymes to toxic metabolites (metabolic activation). Conversely, a toxic drug may be biotransformed to nontoxic metabolites (detoxification). The approaches to evaluate metabolism-based drug toxicity include the identification of toxic metabolites and the evaluation of toxicity in metabolically competent and metabolically compromised systems. A clear understanding of the role of drug metabolism in toxicity can aid the identification of risk factors that may potentiate drug toxicity, and may provide key information for the development of safe drugs. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
2. Drug metabolism and drug toxicity
Accurate prediction of drug toxicity remains a major challenge in drug development. It has been estimated that over 25% of drug candidates would fail in clinical trials due to unexpected adverse drug properties. Furthermore, there are examples of new drugs entering the market that are required to be withdrawn or to have their use limited due to severe adverse effects that are not detected in preclinical or Phase I, II and III clinical trials [1]. There are two likely reasons for the difficulty in the prediction of human drug toxicity:
Biotransformation of a xenobiotic by drug metabolizing enzymes can significantly alter its toxicity [2]. There have been decades of research with toxicants such as polycyclic hydrocarbons and nitrosamines demonstrating clearly that a xenobiotic can be “metabolic activated” from being rather inert molecules to extremely toxic metabolites. This area of research has led to the universal application of “exogenous activation” in genetic toxicity assays—the application of a liver homogenate/cofactors mixture for the metabolism of a “promutagen” to mutagenic metabolites. There also has been extensive research towards the identification of “reactive” metabolites. Formation of reactive metabolites and the subsequent covalent binding to macromolecules, leading to the formation of “neoantigens” and toxic immune reaction with the “neoantigens”, are steps postulated for a possible mechanism of idiosyncratic drug toxicity [3]. Another aspect of drug metabolism is detoxification: the metabolic transformation of a toxic molecule to less toxic metabolites. Phase II conjugation reactions are in general believed to be detoxifying, although some Phase II conjugates are also toxic. Glutathione conjugation of reactive metabolites is a well-established detoxification pathway. The major drug metabolizing enzyme pathway for metabolic activation is the cytochrome P450 monooxygenases (CYP). CYP is an enzyme family with multiple isoforms. It is now known that there are isoform-specific substrates, inhibitors, and inducers. UDPdependent glucuronosyl transferase (UGT), sulfotranferases (ST) and glutathione transferases (GST) are the major conjugative pathways known generally for detoxification. It is to be noted that while oxidative pathways such as P450 metabolism are generally known for metabolic activation, and conjugative pathways for detoxification, there is also evidence that both pathways can be involved in both activation and detoxification, depending on the chemicals involved. Species differences in drug metabolizing enzymes are a major factor for species differences in drug metabolism, and therefore a
1. Species differences in drug toxicity: Drugs with human-specific drug toxicity by definition cannot be predicted with nonhuman laboratory animals. The species difference may be due to drug metabolism. If a toxic metabolite is human specific, laboratory animals would not form the toxic metabolite and therefore would under predict toxicity (and vice versa). Species differences in response to toxicants can also be due to differences in target organ sensitivity, which may be related to species-specific xenobiotic distribution as well as toxic mechanisms. 2. Lack of mechanistic understanding of drug toxicity: Toxicology, especially when practiced for regulatory approval of a product, is often treated as an empirical discipline. Decisions on safety are often based on dose–response curves. The acceptability of the toxicity observed is often based on the level of exposure, regardless of toxic mechanisms. This lack of mechanistic understanding precludes logical deductions of human adverse effects based on human biology, especially in the identification of populations that, for mechanistic reasons, may have high sensitivity to drug toxicity that are deemed “nontoxic” based on preclinical data. In this overview, the importance of the understanding of the relationship between drug metabolism and drug toxicity to aid the accurate prediction of human drug toxicity will be discussed. 0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2008.11.013
2
Editorial / Chemico-Biological Interactions 179 (2009) 1–3
determinant for species differences in drug toxicity that involves drug metabolism. For instance, of the key P450 isoforms for drug metabolism: 1A, 2A, 2B, 2C, 2D, 2E and 3A, human and the four commonly used laboratory animals: mouse, rat, dog and monkey are different with the exception of CYP1A and CYP2E [2]. This difference is known to lead to species differences in drug metabolism, and may contribute to species differences in drug toxicity. 3. Approaches to evaluate metabolism-based drug toxicity As mentioned previously, exogenous activation is commonly used to evaluate genotoxicity. For drug toxicity evaluation, however, the role of metabolism in the observed toxicity is not routinely defined, at least not in a systematic manner. I have summarized here the promising approaches that can be used routinely for the evaluation of the role of drug metabolism in drug toxicity. As the objective is to evaluate human drug toxicity, in vitro experimental system for human drug metabolism such as human liver microsomes and human hepatocytes are useful tools for this purpose. 1. Identification of toxic metabolites: a. Identification of toxic moieties: It is difficult to assign toxicity to metabolites without actual experimentation. However, there are chemical moieties (e.g. quinones, epoxides) that are known to associate with toxicity. One assumption that can be used as an initial assessment of metabolite toxicity is to structurally identify the metabolites to evaluate if known toxic moieties are present. For the identification of human metabolites, the most effective preclinical approach is to perform in vitro studies (e.g. incubation with liver microsomes or hepatocytes). Toxic metabolites from laboratory animals can be identified from body fluids (e.g. plasma, bile, urine, feces) or in vitro studies. The identity of the metabolites usually is determined based on mass spectrometry (MS), especially tandem mass spectrometry (MS/MS). A comprehensive databases for toxic metabolites would be extremely helpful in this exercise [4]. b. Identification of reactive metabolites: Highly electrophilic metabolites are known to reactive with reduced GSH. Identification of GSH conjugates therefore can provide initial evidence of the formation of reactive metabolites. The studies are usually performed with an in vitro metabolism system such as liver microsomes or hepatocytes [5]. With liver microsomes, exogenous GSH is usually added to allow formation of GSH conjugates. With hepatocytes, the endogenous GSH are usually sufficient for the formation of GSH conjugates. c. Identification of unique human metabolites: While some chemical structures are consistently associated with toxicity, the state-of-the-art as of this writing does not allowed the toxicity of metabolites to be estimated with certainty. Metabolites unique to humans (not found in nonhuman animals) may have toxic consequences which cannot be studied by treating laboratory animals with the parent compound. Identification of unique human metabolites allows planning of studies to evaluate toxicity of such metabolites. It is to be noted that the final metabolite may not be the ultimate toxic structure. Evaluation of the toxicity of a compound which may be metabolized to unique metabolites in humans may be betterserved using a system with human-specific metabolism (e.g. human hepatocytes [6]; humanized chimeric mice [7]) than testing these metabolites directly. FDA currently is recommending direct safety testing of unique human metabolites
(metabolites in safety testing [8]) the virtual of which is being discussed among experts of the field [9,10]. d. Identification of the drug metabolizing enzyme pathways involved in the formation of toxic metabolites: This study, in general called Metabolic Pathway Identification, can be performed systematically. A common approach is to firstly use a non-specific P450 inhibitor (e.g. 1-aminobenzotriazole (ABT)) to evaluate whether P450 isoforms are involved. If ABT inhibits the formation of metabolites, isoform-specific inhibitors can be applied to pinpoint the exact isoforms involved. The results with P450 isoform-specific inhibitors can be further confirmed with individual cDNA-expressed human P450 isoforms [11]. 2. Evaluation of toxicity in the absence and presence of drug metabolism: a. Exogenous activation system: In genotoxicity studies such as Ames/Salmonella histidine reversion assay or in vitro mammalian gene mutation assay, liver homogenate and cofactors are added exogenously to provide hepatic metabolism for the evaluation of promutagens [12]. Evaluation of results with and without exogenous activation will provide information on the role of metabolism on the genotoxicity of the chemical being studied. For cytotoxicity studies, such an approach is more difficult as liver homogenates can be cytotoxic. Exogenous activating systems are generally not used in cytotoxicity studies, mainly because of the cytotoxicity of the liver homogenate. However, use of whole cells (e.g. hepatocytes) to provide exogenous activation is now being investigated by multiple laboratories as approach to understand the role of hepatic metabolism in drug toxicity (e.g. the Integrated Discrete Multiple Organ Co-culture (IdMOC) system [13,14]). b. Differential toxicity studies: This approach may represent the most efficient approach to evaluate if drug metabolism is involved in toxicity. The study involves the evaluation of toxicity or cytotoxicity in a metabolically competent system (e.g. hepatocytes) and a metabolically incompetent system (e.g. Chinese hamster ovary cells) [15]. Transgenic animals (e.g. “knock-out” animals deficient in 1 or more P450 isoforms [16]) have also been used successfully as in vivo system to evaluate the role of a specific drug metabolizing enzyme pathway in drug toxicity. A drug that is more toxic in a metabolically competent system than in an incompetent system can be initially classified as one that would require metabolism to be toxic. Conversely, a drug that is more toxic in a metabolically incompetent system would be one that is metabolically detoxified. Another differential toxicity studies is to compare cytotoxicity of a drug in hepatocytes or tissue slices from man to that from multiple laboratory animals which may aid to the understanding of species differences in toxicity (e.g. [17,18]). c. Metabolic inhibitors: Via the use of inhibitors of drug metabolizing enzymes, one can ascertain the role of the enzymes in drug toxicity. This approach is similar to that described above for Metabolite Pathway Identification, except that instead of metabolite identification, cytotoxicity is used as endpoint. The non-specific, mechanism-based P450 inhibitor ABT can be used to evaluate P450-dependent drug toxicity. P450 isoform-specific inhibitors can be used to evaluate the role of individual isoforms (e.g. [15]). Attenuation of cytotoxicity by metabolic inhibitors would infer that the pathways inhibited are involved in the generation of toxic metabolites. Similarly, enhancement of cytotoxicity by the inhibitors would suggest that the pathways inhibited are involved in detoxification. The cytotoxicity results can be correlated to the metabolite identification results to further understand the role of metabolic pathways and metabolites in the toxicity of the drug in question.
Editorial / Chemico-Biological Interactions 179 (2009) 1–3
4. Conclusions The field of toxicology, especially toxicology practices for regulatory purposes, has not changed in several decades. Preclinical safety testing is centered on in vivo laboratory animal studies. These in vivo studies have been valuable in the prevention of some toxic drug candidates from further development, as they are effective in the detection of toxicity that are common to both humans and nonhuman animals. However, toxicity that is unique to humans cannot be detected. Preclinical experimental systems to provide human-specific information should be incorporated in the preclinical safety evaluation program. The recent success in the application of human-based in vitro experimental system in the understanding of human drug metabolism, pharmacokinetics, and drug–drug interactions suggest that similar approaches can also aid the better prediction of human drug toxicity. Evaluation of the role of drug metabolism and toxicity is arguably a necessary activity for the evaluation of human drug toxicity. It allows a rationale design of a safer molecule (e.g. by blocking sites critical for toxic metabolite formation), assessment of sensitive human population (e.g. populations with high level of the drug metabolizing enzyme pathway for the formation of toxic metabolites; populations with low detoxifying activities; environmental factors leading to high levels of “activating” activities or low levels of “detoxifying” activities). The approaches suggest in this overview can be used as basis for a systematic approach for this important discipline of research during drug discovery and development. It is possible that via the understanding of the risk factors defined by drug metabolism, one can understanding the reasons behind the elusive idiosyncratic drug toxicity [19]. To accomplish the goal of a better prediction of human drug toxicity , a paradigm change is needed. Toxicology needs to be practiced not as an empirical discipline, but as an investigative discipline. Good Scientific Practice (GSP) is as important as Good Laboratory Practice (GLP). Adverse drug effects need to be studied under physiologically relevant conditions (e.g. avoid the testing of unacceptably high drug concentrations) [20]. Cross fertilization of the disciplines of pharmacology, drug metabolism, pharmacokinetics and toxicology are important for the understanding of adverse drug effects and the ultimate accurate prediction human drug toxicity [21]). References [1] A.P. Li, Accurate prediction of human drug toxicity: a major challenge in drug development, Chem. Biol. Interact. 150 (2008) 3–7. [2] F.P. Guengerich, Cytochrome P450s and other enzymes in drug metabolism and toxicity, AAPS J. 8 (1) (2006) E101–E111. [3] P.H. Beaune, S. Lecoeur, Immunotoxicology of the liver: adverse reactions to drugs, J. Hepatol. 26 (1997) 37–42.
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[4] R.P. Hanzlik, J. Fang, Y.M. Koen, Filling and mining the reactive metabolite target protein database, Chem. Biol. Interact. 179 (2009) 38–44. [5] S. Ma, M. Zhu, Recent advances in applications of liquid chromatography–tandem mass spectrometry to the analysis of reactive drug metabolites, Chem. Biol. Interact. 179 (2009) 25–37. [6] A.P. Li, Human hepatocytes: isolation, cryopreservation and applications in drug development, Chem. Biol. Interact. 168 (2007) 16–29. [7] Y. Sato, H. Yamada, K. Iwasaki, C. Tateno, T. Yokoi, K. Yoshizato, I. Horii, Human hepatocytes can repopulate mouse liver: histopathology of the liver in human hepatocyte-transplanted chimeric mice and toxiologic responses to acetaminophen, Toxicol. Pathol. 36 (2008) 581–591. [8] FDA Guidance for Industry, Saftey testing of drug metabolites, Pharmcology and Toxicology, February 2008, http://www.fda.gov/cder/guidance/. [9] D.A. Smith, R. Scott Obach, D.P. Williams, B Kevin Park, Clearing the MIST (metabolites in safety testing) of time: the impact of duration of administration on drug metabolite toxicity, Chem. Biol. Interact. 179 (2009) 60–67. [10] K.S. Pang, Safety testing of metabolites: expectations and outcomes, Chem. Biol. Interact. 179 (2009) 45–59. [11] A.P. Li, Preclinical in vitro screening assays for drug-like properties, Drug Discov. Today 2 (2005) 79–85. [12] A.P. Li, Use of Aroclor 1254-induced rat liver homogenate in the assaying of promutagens in Chinese hamster ovary cells, Environ. Mutagen. 6 (1984) 539–544. [13] A.P. Li, A novel in vitro system, the integrated discrete multiple organ cell culture (IdMOC) system, for the evaluation of human drug toxicity: comparative cytotoxicity of tamoxifen towards normal human cells from five major organs and MCF-7 adenocarinoma breast cancer cells, Chemico-Biol. Interact. 150 (2004) 129–136. [14] A.P. Li, In vitro evaluation of human xenobiotic toxicity: scientific concepts and the novel integrated discrete multiple cell co-culture (IdMOC) technology, ALTEX 25 (2008) 43–49. [15] A.P. Li, Metabolism Comparative Cytotoxicity Assay (MCCA) and Cytotoxic Metabolic Pathway Identification Assay (CMPIA) with cryopreserved human hepatocytes for the evaluation of metabolism-based cytotoxicity in vitro: proofof-concept study with aflatoxin B1, Chemico-Biol. Interact. 179 (2009) 4–8. [16] F.J. Gonzalez, The use of gene knockout mice to unravel the mechanisms of toxicity and chemical carcinogenesis, Toxicol. Lett. 120 (2001) 199–208. [17] B. Lauer, G. Tuschl, M. Kling, S.O. Mueller, Species-specific toxicity of diclofenac, troglitazone in primary human and rat hepatocytes, Chemico-Biol. Interact. 179 (2009) 17–24. [18] A.E.M. Vickers, Tissue slices for the evaluation of metabolism-based toxicity with the example of diclofenac, Chemico-Biol. Interact. 179 (2009) 9–16. [19] A.P. Li, A review of the common properties of drugs with idiosyncratic hepatotoxicity and the “multiple determinant hypothesis” for the manifestation of idiosyncratic drug toxicity, Chem. Biol. Interact. 142 (2002) 7–23. [20] D.A. Smith, Commentary-Dogma driven science, the need to establish a common base line, Chemico-Biol. Interact. 179 (2009) 68–70. [21] A.P. Li, A comprehensive approach for drug safety assessment, Chemico-Biol. Interact. 150 (2004) 27–33.
Albert P. Li ∗ Advanced Pharmaceutical Sciences Inc. and In Vitro ADMET Laboratories LLC, 9221 Rumsey Road Suite 8, Columbia, MD 21044, USA ∗ Tel.:
+1 410 869 9037; fax: +1 410 869 9034. E-mail address: [email protected] Available online 25 November 2008