Drug-Induced Idiosyncratic Hepatotoxicity —Prevention Strategy Developed After the Troglitazone Case

Drug-Induced Idiosyncratic Hepatotoxicity: Prevention Strategy Developed after the Troglitazone Case

Toshihiko IKEDA* Yokohama College of Pharmacy, Yokohama, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: Troglitazone induced an idiosyncratic, hepatocellular injury-type hepatotoxicity in humans. Statistically, double null genotype of glutathione S-transferase isoforms, GSTT1 and GSTM1, was a risk factor, indicating a low activity of the susceptible patients in scavenging chemically reactive metabolites. CYP3A4 and CYP2C8 were involved in the metabolic activation and CYP3A4 was inducible by repeated administrations of troglitazone. The genotype analysis, however, indicated that the metabolic idiosyncrasy resides in the degradation of but not in the production of the toxic metabolites of troglitazone. Antibody against hepatic aldolase B was detected in the case patients, suggesting involvement of immune reaction in the toxic mechanism. Troglitazone induced apoptotic cell death in human hepatocytes at a high concentration, and this property may have served as the immunological danger signal, which is thought to play an important role in activating immune reactions. Hypothesis is proposed in analogy to the virus- induced hepatitis. After the troglitazone-case, pharmaceutical companies implemented screening systems for chemically reactive metabolites at early stage of drug development, taking both the amount of covalent binding to the proteins in vitro and the assumed clinical dose level into consideration. At the post-marketing stage, gene analyses of the case patients, if any, to ﬁnd pharmacogenetic biomarkers could be a powerful tool for contraindicating to the risky patients.

Keywords: troglitazone; chemically reactive metabolite; idiosyncratic drug reactions; drug-induced liver injury; prevention

aminase, GPT¥, and the initial sign of high GOT and GPT Idiosyncratic hepatotoxicity caused activities ¤more than threefold of the control level¥ was by troglitazone observed about 2 months after starting the troglitazone Troglitazone ¤Fig. 1 for the chemical structure and treatment. The peak in the number of patients showing the metabolic pathway¥ was introduced into the Japanese and abnormality was found in 17 to 20 months after starting the U.S. market in March, 1997 as an innovative new drug for treatment, indicating a slow onset of the hepatotoxicity. the treatment of type II diabetes mellitus. In December of Serum level of the total bilirubin also increased markedly, the same year, however, a doctor letter warning the drug- and was regarded as a marker of bad prognosis since the induced hepatotoxicity by troglitazone was issued. In 2000, patients with the total bilirubin not more than 5 mg/dL all troglitazone was withdrawn from the market voluntarily by recovered after discontinuation of the troglitazone treatment the pharmaceutical companies in both countries, Sankyo while the level of total bilirubin in 4 death cases was more Co., Ltd. and Warner-Lambert Co. Kuramoto et al., than 8 mg/dL. Increase in the activity of serum alkali investigated 35 Japanese cases of the troglitazone-induced phosphatase was not much, and was 1.29-fold the control hepatotoxicity including 4 death cases, which were reported level. All cases showed no allergic reactions such as fever, to the Ministry of Health and Welfare.1¥ The patients were rash, and eosinophilia. 19 females ¤age: 46®77 years¥ and 16 males ¤age: 34®75 The death cases included 3 females ¤age: 60®66 years¥ and years¥, and they were taking daily 400 mg-troglitazone 1 male ¤age: 58 years¥ and had no previous history of ¤200 mg b.i.d.¥. The hepatotoxicity was accompanied with hepatitis virus infection. History of drug allergy was found in increased activities of serum transaminases ¤glutamic-oxalo- 2 female death cases, and a positive reaction in the drug acetic transaminase, GOT, and glutamic-pyruvic trans- lymphocyte stimulation test ¤DLST, or lymphocyte trans-

Received; September 2, 2010, Accepted; November 29, 2010, J-STAGE Advance Published Date: December 17, 2010 *To whom correspondence should be addressed: Toshihiko IKEDA, Yokohama College of Pharmacy, 601 Matanocho, Totsuka-ku, Yokohama 245- 0066, Japan. Tel. 045-859-1300, E-mail: [email protected]

60 Prevention of Idiosyncratic Drug-Induced Hepatotoxicity 61

Fig. 1. Metabolic pathway of troglitazone formation test: LTT¥ was found in the male death case. in total are allocated to three therapeutic dose levels, high, Unfortunately, the levels of serum transaminases had not medium and low levels, the probability of the susceptible been frequently monitored in these patients as indicated by patient, just one patient at the most in this study scale, being the severe hepatic dysfunction already evident when checked allocated to the highest dose group is only 1/3 while the by the first measurements. The patients showed a hepatic probability of allocation of the susceptible patient to the encephalopathy as a consequence of a high total bilirubin lowest and medium dose groups is 2/3 leading to a greater level more than 20 mg/dL at the end stage, and died of a chance of the idiosyncratic toxicity manifesting in a dose- fulminant hepatitis. independent manner than in a dose-dependent manner. It is Preclinical safety assessments using experimental animals thought that, in groups made up only by susceptible patients, conducted in the pharmaceutical company before new toxicity would be detected at a very low dose level, and be drug application failed to predict the troglitazone-induced dose-dependent as well in a low-dose range. hepatotoxicity found clinically in humans. Any additional The idiosyncratic hepatotoxicity is further classified into 2 animal experiments conducted extensively after the drug subtypes, 1¥ immune idiosyncrasy, where the immune approval were also not predictive of the troglitazone-induced reaction seems to be deeply involved as judged by the hepatotoxicity in human.2,3¥ fever, rash and eosinophilia accompanied, and 2¥ metabolic It is accepted that there are 2 types of drug-induced idiosyncrasy, where the immune reaction is apparently less hepatotoxicity in humans, one is the intrinsic hepatotoxicity important or ignorable.5¥ ¤Type A hepatotoxicity¥ and the other is the idiosyncratic Based on these findings and the classification criteria hepatotoxicity ¤Type B hepatotoxicity¥ and that the former is described above, it was concluded that hepatotoxicity dose-dependent and reproducible in experimental animals caused by troglitazone is an idiosyncratic, hepatocellular while the latter is dose-independent, and is found only in injury-type liver toxicity, where the idiosyncrasy is believed humans. It should be noted, however, that the idiosyncratic to reside in the specific drug metabolism ¤metabolic hepatotoxicity being not dose-dependent would be only idiosyncrasy¥. The metabolic idiosyncrasy was true at least apparently true in a population containing an extremely in part in the troglitazone-case as described later in this small number of susceptible patients in a large number of review but, at the same time, the involvement of immune non-susceptible patients.4¥ This is because the susceptible reaction was also suggested since the antibody against hepatic patients likely show the toxicity even at pharmacologically aldolase B was detected in the serum samples collected effective dose levels, and generally clinical studies are from two patients with troglitazone-hepatotoxicity.6¥ The conducted at the therapeutic dose levels; hence, even the positive reaction of DLST found in one death case was also lowest dose level is toxic to the susceptible patient. In an suggestive of the involvement of immune reaction through T assumed clinical trial, where not more than 10000 patients lymphocytes.

Table 1. Comparison of intrinsic hepatotoxicity and idiosyncratic hepatotoxicity

Type A Type B Intrinsic hepatotoxicity Idiosyncratic hepatotoxicity Genetic factor %¦a Dose-dependency ¦%b Reproducibility in animals ¦% Onset Rapid ¤level of days¥ Slow ¤level of months¥ Chemically reactive metabolite ¦¦ Major immune system involved Innate immune system Adaptive immune systemc aGenes involved have not been identiﬁed in most cases. Multiple genes are believed to be involved. bIdiosyncratic hepatotoxicity would be dose-dependent in the susceptible patients. cThe innate immune system is also likely involved in idiosyncratic hepatotoxicity.

Chemically reactive metabolites produced from troglitazone Fig. 2. Chemically reactive metabolites produced in vitro from There are some differences in the causal factors for 2 troglitazone types of hepatotoxicity, intrinsic hepatotoxicity and idiosyncratic hepatotoxicity as summarized in Table 1 but, quite interestingly, chemically reactive metabolites are thought to semiquinone radical, quinone methide-form metabolite,10¥ play important roles commonly in both types of the drug- sulfenic acid-form metabolite,10¥ Æ-ketoisocynate-form induced hepatotoxicity. The factors separating 2 types of metabolite,10¥ epoxide-form metabolite,11¥ and thiazolidine the drug-induced hepatotoxicity are not known exactly dione radical ¤Fig. 2¥.12¥ The troglitazone semiquinone while the immune system mainly involved in each type of radical is not demonstrated directly for troglitazone but is hepatotoxicity seems different. It is likely that the innate thought quite likely produced in analogy to other quinone- immune system is involved in the intrinsic hepatotoxicity as form compounds. In addition to these observations, 14C- in the case of acetaminophen-induced hepatotoxicity, where labelled troglitazone was found to bind covalently to the inflammatory cytokines such as interferon-È¤IFN-È¥ and proteins in primary cultured human hepatocytes.13¥ All these tumor necrosis factor-Æ¤TNF-Æ¥ predominate over the findings clearly demonstrate that the chemically reactive anti-inflammatory cytokines such as interleukin 4 ¤IL-4¥ and metabolites are produced from troglitazone in the liver in IL-10.7¥ The adaptive immune system is involved in the addition to the production of the stable, major metabolites. idiosyncratic hepatotoxicity as in the case of halothane- CYP2C8 and CYP3A4 are likely the most important induced hepatotoxicity, where sensitization is needed, and isoforms of cytochrome P450 in bioactivating troglita- many autoimmune antibodies are produced.8¥ It could be zone.11,14¥ However, the involvement of CYP3A4, the most assumed that the reactive metabolites chemically modify major isoform of cytochrome P450 present in human liver, the hepatocellular proteins in varying manners in terms of in producing the chemically reactive metabolites is more or either different protein species modified, different sites of less puzzling when considering a low incidence rate of the modification in the same protein or the different rate troglitazone-induced idiosyncratic hepatotoxicity, which is and extent of covalent binding, leading to a stimulation about one among more than 30000 patients. Although of different immune system and finally to manifestation CYP3A4 shows great inter-individual variability and high of different type of hepatotoxicity, though the precise CYP3A4 activity would produce an increased amount of mechanism is still unclear. chemically reactive metabolites and hence the increased risk The major metabolites of troglitazone detected in both of hepatotoxicity in some individuals, it goes reversely with the experimental animals and humans were glucuronide and an extremely low incidence rate of idiosyncratic hepato- the sulfo-conjugate of troglitazone ¤Fig. 1¥, which were toxicity caused by troglitazone. Several papers demonstrate obviously not chemically reactive and not assumed to that troglitazone is a potent inducer of CYP3A4 in human cause the hepatotoxicity.9¥ After removal of troglitazone liver both in vitro and in vivo.15®20¥ The idea of metabolic from the market, many studies have been carried out in idiosyncrasy suggests that a limited number of patients have order to explore the chemically reactive metabolites of a specific metabolic pathway leading to a rare toxicity while troglitazone in vitro, and several reports described the CYP3A4 being the most major cytochrome P450 isoform in production of chemically reactive metabolites from this human liver and its activity being inducible by repeated drug. As shown in Figure 2, these are the troglitazone administration of troglitazone would make troglitazone to be

Copyright © 2011 by the Japanese Society for the Study of Xenobiotics (JSSX) Prevention of Idiosyncratic Drug-Induced Hepatotoxicity 63 hepatotoxic in a greater number of patients rather than in a Although the GST double null genotype is no doubt the limited number of patients. Therefore, genetic variability in risk factor statistically for drug-induced hepatotoxicity, the the oxidative metabolism producing the chemically reactive risk of the GST double null genotype carrier is only about metabolites would not be a causal factor for the idiosyncratic threefold compared with the non-carrier, and does not nature of troglitazone-induced hepatotoxicity. elucidate a low incidence rate of the troglitazone-induced idiosyncratic hepatotoxicity. In fact, as shown in Table 2,it Gene analysis of the case patients: should be noted that the double null genotype of GST is Decreased activity of glutathione found in 13 troglitazone-tolerant, control patients ¤15%¥ and conjugation as a risk factor the double wild genotype of GST is found in 3 case patients Watanabe et al. reported the results of a genetic ¤3%¥, clearly indicating that the GST double null genotype polymorphic analysis using blood samples collected from solely is not an overriding determinant of the troglitazone- 25 Japanese patients with type II diabetes mellitus who induced hepatotoxicity and that polymorphism of unknown experienced the troglitazone-induced hepatotoxicity showing other genes is also involved. more than fivefold increase of GOT and GPT ¤the case Genetic polymorphisms reported in relation patients¥ and 85 diabetic Japanese patients who were to idiosyncratic drug reactions: taking troglitazone without any sign of hepatotoxicity ¤the ¥ Importance of polymorphism of control patients¥.21 Genotype analysis was performed at human leukocyte antigen ¤HLA¥ genes 68 polymorphic sites of 51 candidate genes related to drug metabolism, apoptosis, production and elimination of The Stevens-Johnson syndrome is known as one of the reactive oxygen species, and signal transduction pathways severe, idiosyncratic adverse skin reactions caused by drugs. of peroxisome proliferator-activated receptor È 2 and A very strong correlation of the polymorphism in the major insulin. As a result, a statistically significant correlation histocompatibility complex class I gene ¤MHC-I or human with hepatotoxicity was observed in the glutathione S- leukocyte antigen, HLA¥ with the carbamazepine-induced transferase ¤GST¥ isoforms, GSTT1 and GSTM1, double null Stevens-Johnson syndrome has been reported for the Han genotype ¤Table 2, odds ratio, 3.69¥. GSTs are the group Chinese.24¥ The genotype, HLA-B*1502, was found in 100% of enzymes catalyzing the conjugation of a sulfhydryl group of patients susceptible to the carbamazepine-induced of reduced glutathione to electrophilic centers in a wide Stevens-Johnson syndrome while it was found only in 3% variety of chemically reactive substrates, and the double null of the carbamazepine-tolerant patients ¤odds ratio: 2504¥. genotype of GSTT1 and GSTM1 is accompanied with a low The same research group also reported a strong correlation activity in this detoxication reaction. Therefore, the results of HLA-B*5801 with allopurinol-induced skin toxicity for of gene analysis indicate that the metabolic idiosyncrasy in the Han Chinese ¤odd ratio: 580¥.25¥ With regard to the the troglitazone-induced hepatotoxicity resides in the process drug-induced hepatotoxicity, a strong correlation of HLA- of scavenging the chemically reactive metabolites but not in A*3303 with idiosyncratic, cholestatic hepatotoxicity in- the process of producing those metabolites. Later, the duced by ticlopidine in the Japanese patients ¤odds ratio: double null genotype of GSTT1 and GSTM1 was generalized 36.5¥ and of HLA-B*5701 with idiosyncratic hepatotoxicity as a risk factor of drug-induced liver injury regardless of the induced by flucloxacillin in the European patients ¤odds type of drug involved in the recent study where 154 patients ratio: 80.6¥ has been reported.26,27¥ The odds ratios are quite with drug-induced liver injury and 250 age-matched healthy high in these studies, and, therefore, by contraindicating subjects were analyzed ¤odds ratio 2.70¥.22¥ A rapid method the carriers of known HLA-genotypes correlated with the for analyzing the double null genotype of GST1 and GSTM1 specific drug-toxicity, it is possible to prevent clinically the within 40 min is now available.23¥ occurrence of idiosyncratic drug toxicity. In fact, FDA is recommended to screen the patients for HLA-B*1502 before 28¥ Table 2. Genotype analysis of glutathione S-transferase starting the treatment with carbamazepine. This strategy (GST) was successfully applied to avoid prospectively the abacavir- induced skin hypersensitivity, and the treatment of only the Control Genotype of GST Case patients ¤tolerant patients¥ HLA-B*5701-negative patients resulted in no occurrence of 29¥ GSTT1 GSTM1 n ¤%¥ n ¤%¥ the hypersensitivity reaction. It should be noted, however, that HLA genotype involved ¤ ¥ ¤ ¥ Wild type Wild type 25 29 3 12 in the drug toxicity seems ethnicity-specific, and, as an Wild type Null 27 ¤32¥ 7 ¤28¥ example, HLA-B*1502 is not predictive of the carbamaze- Null Wild type 20 ¤24¥ 5 ¤20¥ pine-induced Stevens-Johnson syndrome in the Caucasian 30,31¥ ¤ ¥ ¤ ¥ and Japanese patients. It should be also noted that the Null Null 13 15 10* 40 HLA genotype related to the drug-induced adverse reactions Total 85 ¤100¥ 25 ¤100¥ differs drug by drug, and must be determined in a case-by- *P g 0.01 case manner. By contraindicating the high risk patients

Copyright © 2011 by the Japanese Society for the Study of Xenobiotics (JSSX) 64 Toshihiko IKEDA according to the HLA genotype determined for each drug killed either by severe cell dysfunction as a direct action or and for each ethnicity, therefore, drug-induced idiosyncratic by the activation of innate immune reaction leading to the adverse reactions could be prevented. Not only HLA predominance of the inflammatory cytokines over the anti- genotypes serve as biomarkers of the drug-induced toxicity inflammatory cytokines ¤pathway 3¥. The dying cells or but also other genotypes, such as those of drug-metabolizing activated immune system could emit the immunological enzymes and transporters, do so, and the use of more than danger signal as described later. Chemically modified one pharmacogenetic biomarker, when available, would be proteins undergo break-down by proteasomal processing effective in preventing idiosyncratic drug-toxicity.32¥ to peptide fragments, one of which being modified by the reactive metabolite ¤pathway 4¥. The peptide fragments are Hypothesized mechanism: transported by TAP into the endoplasmic reticulum ¤path- Analogy of drug-induced hepatotoxicity way 5¥, and each peptide binds to MHC-I protein specificto to virus-induced hepatitis each fragment ¤pathway 6¥. Most probably, the peptide The fact that MHC-I gene polymorphism is highly fragment modified by chemically reactive metabolite may related to the idiosyncratic drug-induced adverse reactions not have the counterpart MHC-I protein but the MHC-I including hepatotoxicity strongly suggests that the immune protein with a genetic variation may bind to the modified system is somehow working in these reactions. MHC-I is peptide ¤pathway 6¥. MHC-I®peptide complexes are moved involved in self and non-self recognition as indicated by to Golgi apparatus, and subsequently to the cell surface of the fact that, in the organ transplantation, HLA type- the hepatocytes ¤pathway 7 and pathway 8¥. The peptides matching is crucially important to cut down the risk of with normal amino acid sequences would be immunogically rejection, which is the immune-mediated reaction to ignored due to their úself û nature. On the other hand, the eliminate non-self from self through the action of cytotoxic únon-self û peptide fragment modified by the chemically T lymphocytes. However, it is still not known precisely how reactive metabolite is foreign to the immune system, and the immune reaction is involved through MHC-I proteins in after receiving somehow the danger signal ¤pathway 9¥, idiosyncratic drug-induced hepatotoxicity, although it is cytotoxic T lymphocytes ¤CTL in Fig. 3¥ will increase in believed that hepatic proteins chemically modified by the number and start binding to the hepatocytes through reactive metabolites would trigger a series of immune interaction between MHC-I protein and T cell receptor reactions. ¤pathway 10¥. Cytotoxic T lymphocytes kill the hepatocytes As a hypothesis, the mechanism for viral hepatitis could be by 3 ways ¤pathway 11¥ all causing apoptotic cell death applied to idiosyncratic, drug-induced hepatotoxicity.33,34¥ ¤pathway 12¥,1¥ by expressing Fas lignd, which binds to Fas Many hepatitis virus carriers are free from the symptom, on the hepatocyte surface, 2¥ by expressing TNF-Æ, which and a portion of the carriers develops the hepatitis much binds to TNF-Æ receptor and 3¥ by releasing perforin, which delayed after viral infection, exhibiting a similar feature to polymerizes on the hepatocyte surface to form a channel, idiosyncratic drug-induced hepatotoxicity. In the viral and an apoptotic serine-protease, glanzyme, which enters hepatitis, it is thought that peptide fragments are produced the hepatocyte though the channel. from the viral proteins by a proteasomal processing, taken It is known that the immunological danger signal is needed up by endoplasmic reticulum of hepatocytes by the action of to activate the immune reactions ¤pathway 9¥, and is thought transporter associated with antigen processing ¤TAP¥, bound to play an important role in the idiosyncratic drug within a groove of MHC class I protein in the endoplasmic toxicity.4,35,36¥ It is not exactly known what the danger signal reticulum, and the virus peptide - MHC class I protein is but the dying cells are said to emit the danger signal.37¥ complex is transported to the cell surface through the Golgi With regard to this point, troglitazone causes apoptotic cell apparatus, exhibiting this foreign peptide to the immune death in rat and human hepatocytes and other cells at a high system. Cytotoxic T lymphocytes recognize the virus- concentration more than 50 µM,38®41¥ and this may have derived foreign peptide caught on the groove of MHC class I served as the danger signal in the mechanism of troglitazone- protein and kill the hepatocytes, regarding these cells to induced hepatotoxicity. The major metabolite, troglitazone be foreign as a whole. Figure 3 shows schematically the sulfate has been reported to inhibit competitively the bile acid hypothesized mechanism for the drug-induced hepatotoxicity exporting pump ¤Bsep¥ with a low Ki value of 0.23 µM, constructed in analogy to the viral hepatitis. The drug is possibly causing a hepatocellular accumulation of bile acids, oxidized by cytochrome P450 ¤CYP¥ to the chemically which are cytotoxic endogenous substances to hepatocytes.42¥ reactive metabolite ¤pathway 1¥, which is ordinarily This also may have assisted the apoptosis, and served as the detoxified by the action of glutathione S-transferase ¤GST¥. danger signal. Under the condition of induced cytochrome P450, null GST The number of the circulating cytotoxic T lymphocytes, genotype or glutathione depletion, the chemically reactive that recognize the drug-modified peptide as foreign metabolites increase, and bind covalently to hepatocellular substance, would be small, and the danger signal likely proteins ¤pathway 2¥. When covalent binding occurs on stimulates the proliferation of these cells. Cytotoxic T important proteins for cell function, the hepatocytes will be lymphocytes increased in number are thought to cause many

Fig. 3. Hypothesized mechanism for drug-induced hepatotoxicity in analogy to viral hepatitis CYP: cytochrome P450, GST: glutathione S-transferase, GSH: glutathione, TAP: transporter associated antigen processing, MHC-I: major histocompatibility complex class I, SNP: single nucleotide polymorphism, CTL: cytotoxic T lymphocyte, TNF-¡: tumor necrosis factor-¡,TNF- R: tumor necrosis factor receptor. The arrow indicates a genetic polymorphism found in a particular haplotype of MHC-I. cell deaths propagated to a whole liver and consequent In a therapeutic area where no effective drug is available, severe hepatotoxicity. This process is accompanied with cell the first-in-class drug could be used clinically even if the proliferation, and needs time and hence the delayed toxicity. drug shows idiosyncratic drug toxicity since it is better to None of these processes in this hypothetic mechanism in treat a majority of no risk patients who need the treatment Figure 3 has been demonstrated experimentally for the rather than to apprehend the drug toxicity occurring only in drug-induced hepatotoxicity but the pathways 1, 2 and 3 quite a limited number of patients. However, the first-in- to pathway 12 may constitute the intrinsic hepatotoxicity class drug showing the idiosyncratic toxicity will be replaced and the pathways 1, 2, 4, 5, 6, 7, 8, 9, 10, and 11 to immediately by a second generation drug free from such pathway 12 may constitute the idiosyncratic hepatotoxicity. toxicity, which usually comes into the market within a few We know that the mechanism for the drug-induced idi- years following the launch of the first drug. In other osyncratic hepatotoxicity is quite complicated, and the above therapeutic areas where existing drugs are available, the described hypothesis would cover only a portion of the drug accompanied with idiosyncratic toxicity loses the mechanisms for drug-induced idiosyncratic hepatotoxicity competitiveness in the market, and the development of such and future modification of or addition to this hypothesis drug does not provide the company with a profit. Therefore, should be awaited. the development of safer drugs is critically important for pharmaceutical companies. Drug development and strategy for preventing The troglitazone case occurred in 1997®2000, and at that idiosyncratic drug-induced hepatotoxicity time in late 1990s, screening systems, either conventional or Unlike the intrinsic hepatotoxicity ¤type A hepatotox- high-throughput, for improving the pharmacokinetic proper- icity¥, idiosyncratic drug-induced hepatotoxicity could occur ties of lead compounds began to be implemented in repeatedly in future since preclinical animal experiments pharmaceutical companies. Since the idea of chemically are not predictive of such toxicity. Drug withdrawal reactive metabolites as a risk factor of idiosyncratic drug caused by the drug toxicity greatly discredits the pharma- toxicity was disseminated to the research and development ceutical company leading subsequently to many litigations people in the pharmaceutical companies, several screening and financial damage. methods for chemically reactive metabolites have been

Copyright © 2011 by the Japanese Society for the Study of Xenobiotics (JSSX) 66 Toshihiko IKEDA introduced. The most straightforward method was to use based on the pharmacological potency of lead compounds or 14C- or 3H-labeled compound and measure the amount based on the early toxicological and pharmacokinetic data of covalent binding to the proteins by radioassay after but not based on a future risk of idiosyncratic hepatotoxicity incubation with human liver microsomes in the presence of or adverse reactions. However, we need the criteria for NADPH or with human hepatocytes. This method needs a production of the chemically reactive metabolites at this radiolabeled substance, and could not be employed at an stage, considering possible chance of a risky project still extremely early stage. However, quantitative measurement being forwarded to the next stage and the compounds with a of the drug-related substances covalently bound to the high risk of idiosyncratic toxicity being placed for further microsomal protein is rapid and easy by this method, and a development. As described before, the amount of covalent well considered criterion in vitro is available ¤not more than binding not more than 50 pmole/mg-protein has been 50 pmole/mg-protein¥.43,44¥ In addition to the criterion advocated as the criterion in vitro. The validity and allowance in vitro, Uetrecht reported that a dose level of not more of this criterion are still not known in avoiding the than 10 mg/body/day would not cause drug-induced idiosyncratic drug toxicity clinically, and chemists would be toxicity,45¥ and two judging criteria: 50 pmole/mg-protein hesitant to strictly comply with this criterion since doing so in vitro and 10 mg/body/day in vivo could be used con- may eliminate otherwise promising compounds from further veniently for decision making, although some modification of development. Nevertheless, it has been reported that known these criteria has been made as described later. hepatotoxic drugs tend to show a high covalent binding to Measurement of the activities of cytochrome P450 is the protein compared to safer drugs with some excep- carried out by a high-throughput screening method using a tions.59,60¥ Therefore, more improved quantitative criterion 96-well titer plate, fluorescent substrate specific to each is needed. Use of only the criterion in vitro for covalent isoform of cytochrome P450 and robotics device. When a binding is clearly incomplete for making appropriate chemically reactive metabolite is produced from the drug judgment of idiosyncratic drug toxicity in vivo and the substrate, the reactive metabolite inactivates the enzyme, assumed dose level in humans should be also taken together and the inhibitory effect increases as the time of incubation is into consideration.45,61¥ As a more quantitative method prolonged. This phenomenon is known as mechanism-based for decision making, Nakayama et al. reported a zone inhibition or time-dependent inhibition, and is utilized in the classification system based on data on covalent binding high-throughput screening to check the production of collected using the radiolabeled drugs together with the data chemically reactive metabolites.46¥ on daily dose level.62¥ As shown in Figure 4, when the log- Other screening methods for chemically reactive metab- normalized covalent binding in human hepatocytes is plotted olites employ trapping agents that react rapidly with against the log-normalized daily dose, the drugs withdrawn chemically reactive metabolites forming stable adducts. from the market or the drugs with the label of black box Reduced form of glutathione, either unlabeled, stable warning are mostly located in the unacceptable zone, the isotope-labeled, radioisotope-labeled or fluorescence- drugs with the label of warning are mostly located in the labeled, is used most frequently as a trapping agent but problematic zone, and the safe drugs were located in the N-acetyl cysteine, N-acetyl cysteinyl lysine, potassium cyanide, semicarbazide and synthetic peptide containing cysteine, lysine and histidine are also used.47®58¥ The adduct formation is detected by radioactivity detection combined with chromatography when the radiolabeled trapping agents are used, by fluorescence detection when the fluorescence labeled trapping agents are used or by liquid chromatography/mass spectrometry or liquid chromatography/tandem mass spectrometry when non-labeled or stable isotope- labeled trapping agents are used. Information on risky compounds producing chemically reactive metabolites at a level more than the criteria established in each company is to be provided to the chemists for further structural modification to reduce risk. As the situation demands, it would be necessary to isolate and structurally determine the adduct in order to locate the site of adduct formation on the molecule and to find Fig. 4. Zone classification for risk assessment of idiosyncratic potentially risky chemical structure. This information is drug toxicity Figure from Nakayama et al. with modification,62) Safe drug quite useful for the chemists to plan a synthetic strategy. without any warning (12 compounds), Drug with a label of warning At the early stage of drug development, a úGo/No-Goû (14 compounds), Drug withdrawn or with a label of black box decision for each research project would be made generally warning (10 compounds).

Copyright © 2011 by the Japanese Society for the Study of Xenobiotics (JSSX) Prevention of Idiosyncratic Drug-Induced Hepatotoxicity 67 acceptable zone. Of 36 drugs tested, 30 drugs were located decrease greatly the risk since more than 1®2 months are to their respective safety categories ¤acceptable zone: safe, normally needed to develop idiosyncratic hepatotoxicity. problematic zone: warning, and unacceptable zone: black Therefore, the signal for the project developing drugs to be box warning or withdrawal¥, and this zone classification used only for a short period will be úGoû, disregarding the method would be helpful in decision making at the early drug being or not being the first-in-class drug. Even in the stage of development. case of short clinical use, however, it should be kept in mind As another method using the covalent binding and that a high dose level may cause other types of idiosyncratic daily dose together, a comparator database on the covalent toxicity such as penicillin-induced anaphylactic reactions, binding in vitro ¤B¥, clinical dose level ¤D¥, and absorption which are believed to be due to previous sensitization. ratio ¤A¥ would be useful, although such a database has not Therefore, the zone classification system should be applied been constructed. The product of 3 items ¤B&D&A¥ serves as a to the compound to determine relative safety. Depending on delegate of bodily exposure to the chemically reactive the safety positioning, clinical study to discover the phar- metabolites, and by comparing this value of the compound in macogenetic biomarkers is better to be planned in future to question with those values of the drugs withdrawn from the establish the contraindication to a high-risk patient. market, the drugs with warning and the safe drugs, a relative Step 3-2¥ the case of risky and unacceptable compounds: position in safety of the compound could be known. Results If the compound to be developed is not the first-in-class could be presented that the compound A is much safer than drug, and the treatment period is to be long, evaluation troglitazone and in between amlodipine and rosiglitazone, of safety by the zone classification method should be hence safe, while compound B is risky in between flutamide carried out as early as possible using 14C-labeled compound. and diclofenac. Data for the database should be collected When the safety of the compound is judged problematic or according to standardized protocol using a large pool of unacceptable, the signal for the project will be úNo-Goû human liver microsomes in order to obtain quality because the compound has no competitiveness against the controlled data over a long period. Since the activity of existing low-risk drugs available in the market. drug-metabolizing enzymes varies from one lot of human Step 4¥ the case of risky but clinically useful compounds: liver microsomes to the other, use of the same lot of human On the premise that the compound to be developed is the liver microsomes is mandatory. first-in-class drug having an extremely high efficacy, and no Strategy for preventing drug-induced idiosyncratic hep- other existing drug is available in the targeted therapeutic atotoxicity would be as follows. area, the signal for the project would be úGoû. At the final Step 1¥ the lead compounds for optimization: At early stage stage of lead optimization or drug candidate profiling, of drug development, conduct a high-throughput screening determine quantitatively the covalent binding using 14C- for chemically reactive metabolites. Feed back the data to labeled compound, and evaluate the relative position in the chemists for chemical modification of a group of lead safety of the compound according to the zone classification compounds to reduce the risk. Repeat this cycle until risky system as described above in Step 2. When judged risky, it compounds are eliminated from the project, and go to is indeed recommended to carry out a clinical study on Step 2. When the elimination of risky compounds from the pharmacogenetic biomarkers in future to establish the project is unsuccessful, go to Step 3. contraindication of susceptible patients. Use of the Step 2¥ the case of likely safe compounds: At the stage biomarkers will prolong the life cycle of the drug after of drug candidate profiling, determine quantitatively the being launched into the market. covalent binding using 14C-labeled compounds. Estimate the Conclusion clinical dose level by an extensive in vitro-in vivo extrap- olation and pharmacokinetic/pharmacodynamic correlation After the case of troglitazone-induced idiosyncratic analyses. Confirm the safety of the drug candidate using the hepatotoxicity, various screening methods for the chemically potency of covalent binding, estimated clinical dose level and reactive metabolites at the early stage of drug development zone classification system. are implemented in many pharmaceutical companies. Drug Step 3¥ the case of risky compounds: Determine whether a candidate compounds that produce no chemically reactive period of treatment by the compounds in question is to metabolite certainly would have very low possibility of be short or long clinically. When the treatment period is inducing idiosyncratic drug reactions. How effective these short, go to Step 3-1. When the treatment period is long, screening methods at early stage in preventing the the compounds are accompanied with increased risk of idiosyncratic hepatotoxicity when the drug is given to a vast idiosyncratic hepatotoxicity, and determine whether or not number of patients is, however, still unknown and needs the project is going to develop the first-in-class drug. Go to more time for validation. At the stages of post-marketing Step 3-2 in the case of not being the first-in-class drug and to surveillance, gene analyses of the susceptible patients, if Step 4 in the case of the first-in-class drug. any, would be helpful to find out the pharmacogenetic Step 3-1¥ the case of risky but acceptable compounds: biomarkers, and contraindication of susceptible patients Very short treatment period in the clinical situation would would prolong the life cycle of the drug in question.

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