`
`dmd.aspetjournals.org
`
` at ASPET Journals on January 29, 2016
`
`0090-9556/09/3709-1970–1977$20.00
`DRUG METABOLISM AND DISPOSITION
`Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics
`DMD 37:1970–1977, 2009
`
`Vol. 37, No. 9
`27797/3499396
`Printed in U.S.A.
`
`A Zone Classification System for Risk Assessment of Idiosyncratic
`Drug Toxicity Using Daily Dose and Covalent Binding
`
`Shintaro Nakayama, Ryo Atsumi, Hideo Takakusa, Yoshimasa Kobayashi, Atsushi Kurihara,
`Yoko Nagai, Daisuke Nakai, and Osamu Okazaki
`
`Drug Metabolism and Pharmacokinetics Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd. Hiromachi, Shinagawa-
`ku, Tokyo, Japan
`
`Received March 31, 2009; accepted May 27, 2009
`
`ABSTRACT:
`
`The risk of idiosyncratic drug toxicity (IDT) is of great concern to
`the pharmaceutical industry. Current hypotheses based on retro-
`spective studies suggest that the occurrence of IDT is related to
`covalent binding and daily dose. We determined the covalent bind-
`ing of 42 radiolabeled drugs in three test systems (human liver
`microsomes and hepatocytes in vitro and rat liver in vivo) to assess
`the risk of IDT. On the basis of safety profiles given in official
`documentation, tested drugs were classified into the safety cate-
`gories of safe, warning, black box warning, and withdrawn. The
`covalent binding in each of the three test systems did not distin-
`guish the safety categories clearly. However, when the log-normal-
`ized covalent binding was plotted against the log-normalized daily
`
`dose, the distribution of the plot in the safety categories became
`clear. An ordinal logistic regression analysis indicated that both
`covalent binding and daily dose were significantly correlated with
`safety category and that covalent binding in hepatocytes was the
`best predictor among the three systems. When two separation
`lines were drawn on the correlation graph between covalent bind-
`ing in human hepatocytes and daily dose by a regression analysis
`to create three zones, 30 of 37 tested drugs were located in zones
`corresponding to their respective classified safety categories. In
`conclusion, we established a zone classification system using co-
`valent binding in human hepatocytes and daily dose for the risk
`assessment of IDTs.
`
`Idiosyncratic drug toxicity (IDT) occurs rarely but is often very
`serious and appears as severe hepatotoxicity, agranulocytosis, neutro-
`penia, Stevens-Johnson syndrome (SJS), or other illnesses. Because of
`its low frequency of occurrence (1/1000 –1/100,000), IDT is often
`found late in drug development or in the postmarketing phase
`(Kaplowitz, 2005; Baillie, 2006; Uetrecht, 2007). In recent years,
`several drugs, including troglitazone, zomepirac, and tienilic acid,
`have been withdrawn from the market because of IDT, or the use of
`drugs has been limited by the addition of black box warnings to the
`label, as in the cases of flutamide, nevirapine, and valproic acid. For
`the pharmaceutical industry, it is important that drugs with the poten-
`tial risk of IDT be screened out in the early phase of discovery and/or
`the development process. Unfortunately, conventional animal models
`of toxicity are poor predictors for clinical situations and the mecha-
`nisms of IDT are not fully understood despite many efforts to clarify
`them (Evans et al., 2004; Walgren et al., 2005; Masubuchi et al., 2007;
`Takakusa et al., 2008).
`Current hypotheses based on retrospective studies suggest that the
`metabolic activation of a drug to a reactive metabolite and its covalent
`binding to cellular macromolecules are involved in the occurrence of
`IDT (Uetrecht, 2001; Zhou et al., 2007). Estimation of covalent
`
`Article, publication date, and citation information can be found at
`http://dmd.aspetjournals.org.
`doi:10.1124/dmd.109.027797.
`
`binding to cellular macromolecules by using radiolabeled drugs is a
`direct and reliable method. There are several examples of reactive
`metabolites forming covalent bonds with IDT-causing drugs, such as
`tienilic acid, acetaminophen, and clozapine (Lecoeur et al., 1994;
`Hinson et al., 1995; Gardner et al., 1998). Evans et al. (2004) pro-
`posed a threshold level of 50 pmol/mg protein as a screening criterion
`of covalent binding to human liver microsomes (HLMs) in vitro and
`rat liver in vivo. A previous study by our group determined the
`covalent binding of a variety of drugs to HLMs in vitro and rat liver
`in vivo; these included drugs withdrawn from the market, drugs with
`black box warnings in the United States labeling, and some safe drugs.
`It was found that most of the problematic drugs exhibited higher HLM
`in vitro covalent binding than “safe” drugs (Takakusa et al., 2008).
`Some reports suggest that the exposure to or daily dose of a drug
`may be related to the occurrence of IDT. Uetrecht (1999) reported that
`the occurrence of IDT is rare with drugs given at a daily dose of 10
`mg or less. Walgren et al. (2005) also pointed out the contribution of
`high daily dose to IDT risk. For example, in the case of antidiabetic
`“glitazone” drugs, troglitazone caused a high incidence of IDT in
`patients and had to be withdrawn from the market, whereas rosigli-
`tazone and pioglitazone do not show significant IDT risk even though
`they have similar chemical structures. The daily dose of troglitazone
`is 400 to 600 mg, whereas the daily doses of rosiglitazone and
`pioglitazone are 4 to 8 and 15 to 45 mg, respectively.
`
`ABBREVIATIONS: IDT, idiosyncratic drug toxicity; SJS, Stevens-Johnson syndrome; HLM, human liver microsome; WDN, drugs withdrawn from
`the market; BBW, drugs with a black box warning for IDT in the PDR; WNG, drugs without a black box warning but with a warning for IDT (severe
`hepatotoxicity, neutropenia, agranulocytosis, or SJS) in either the PDR or Japanese labeling; SAFE, drugs without any warning in either the PDR
`or Japanese labeling.
`
`1970
`
`AstraZeneca Exhibit 2180
`Mylan v. AstraZeneca
`IPR2015-01340
`
`Page 1 of 8
`
`
`
`ASSESSMENT OF IDT USING COVALENT BINDING AND DOSE
`
`1971
`
`Downloaded from
`
`dmd.aspetjournals.org
`
` at ASPET Journals on January 29, 2016
`
`Laboratory Animal Welfare (Institute of Laboratory Animal Resources, 1996).
`The Institutional Animal Care and Use Committee approved the protocols.
`In Vitro HLM Covalent Binding Study. The experimental procedure was
`based on that used in a study reported previously (Masubuchi et al., 2007). The
`incubation mixture consisted of the following: 10 M radiolabeled test drug
`(substrate), 2 mg/ml HLMs, 100 mM potassium phosphate buffer (pH 7.4), 25
`mM glucose 6-phosphate, 2 units/ml glucose-6-phosphate dehydrogenase, and
`10 mM MgCl2. The mixture was preincubated for 3 min at 37°C. A reaction
`was initiated by the addition of -NADP⫹ to reach a final concentration of 2.5
`mM, and the final incubation volume was 0.5 ml. Because the substrates were
`dissolved in acetonitrile, the final incubation mixture contained 1% (v/v)
`acetonitrile. Radiolabeled drugs of at least 95% purity were used. After
`incubation of the mixture for 1 h, the reaction was terminated by the addition
`of 0.5 ml of ice-cold acetonitrile. After vortexing, sonication was performed in
`an ultrasonic bath, and the mixture was centrifuged. The precipitated protein
`was serially washed twice with the following solvents: 80% (v/v) aqueous
`methanol containing 10% (w/v) trichloroacetic acid, diethyl ether-methanol
`(1:1, v/v), and 80% methanol. The resulting precipitated protein was dissolved
`in 0.5 ml 1.0 M NaOH, and aliquots were taken for a protein assay with a DC
`Protein Assay Kit (Bio-Rad, Hercules, CA) and also for the determination of
`radioactivity using a liquid scintillation counter after mixing of the aliquot with
`Hionic-Fluor scintillation cocktail (PerkinElmer Life and Analytical Sciences).
`The amount of the test drug-related material, as radioactivity covalently bound
`to the microsomal protein, was determined as the covalent binding (picomoles
`per milligram of protein). All of the experiments were performed in triplicate.
`In Vitro Human Hepatocyte Covalent Binding Study. Cryopreserved
`human hepatocytes were carefully thawed in a water bath set at 37°C and were
`suspended in Williams’ E medium at a final cell concentration of 1.0 ⫻ 106
`cells/ml. The total cell count and the number of viable cells were determined
`by the trypan blue exclusion method, and hepatocytes with more than 70%
`viability were used. The final incubation volume was 1.5 ml on a six-well
`plate. The hepatocytes were preincubated for 5 min in a humidified 37°C
`incubator (5% CO2). Because the radiolabeled drugs were dissolved in meth-
`anol, the final incubation mixture contained 1% v/v methanol. Radiolabeled
`drugs of more than 95% purity were used. Reactions were initiated by adding
`the radiolabeled drugs at the final concentration of 10 M. After 2 h of
`incubation in a humidified 37°C incubator, 0.45 ml of the suspension was
`sampled into 1 ml of 1 mM unlabeled solution in ice-cold methanol. After
`vortexing, sonication was applied in an ultrasonic bath, and then the mixture
`was centrifuged. The precipitated protein was immediately washed with 1 mM
`unlabeled solution in ice-cold methanol. After centrifugation, the precipitated
`protein was serially washed three times with each of the following solvents:
`dimethyl sulfoxide-methanol (1:4, v/v), methanol containing 25% (w/v) tri-
`chloroacetic acid, 100% methanol, and 80% methanol. The resulting precipi-
`tated protein was dissolved in 0.5 N NaOH and neutralized by adding 5 N HCl.
`Aliquots were taken, and the protein amount and radioactivity were determined
`as described above. All of the experiments were performed in triplicate.
`Rat Liver in Vivo Covalent Binding Study. The experimental procedure
`was based on a study reported previously (Masubuchi et al., 2007). Radiola-
`beled drugs of more than 95% purity were used. Radiolabeled and unlabeled
`drugs were dissolved or suspended in 0.5% methylcellulose (400 centipoise) to
`prepare a solution at a concentration of 2 mg/ml as a free base or acid form for
`oral administration to fasted rats. After a single oral administration of each test
`drug at a dose of 20 mg/kg, the rats were exsanguinated at 2, 6, or 24 h (n ⫽
`3 animals for each time point), and liver samples were collected and stored
`frozen until analysis. The liver samples were weighed and then homogenized
`with aqueous 1.15% (v/v) KCl. In the same way as in the in vitro covalent
`binding study using HLMs, the liver homogenate was washed with organic
`solvents, followed by protein assay and determination of radioactivity co-
`valently bound to the protein, as described above. The highest value of three
`time points was used for the further analysis of IDT risk assessment.
`Data Analysis. Ordinal logistic regression analysis was performed to assess
`the relationship between covalent binding, daily dose, and safety category by
`the following equation using JMP 5.0.1 statistical software (SAS Institute,
`Cary, NC),
`
`log冉 p
`
`1 ⫺ p
`
`冊 ⫽ 1 ⫻ log(dose) ⫹ 2 ⫻ log(CB) ⫹ 0
`
`These retrospective studies suggest that the occurrence of IDT may
`be related to both covalent binding and exposure. To date, however,
`only limited numbers of systematic investigations have been reported
`with regard to the relationship between covalent binding, daily dose,
`and IDT (Obach et al., 2008). In this study, to assess the risk of IDT,
`we determined the covalent binding of 42 radiolabeled drugs in three
`test systems. These systems were HLMs, which are most commonly
`used for oxidative metabolism; human hepatocytes, which have a full
`component of cellular enzyme systems; and rat liver in vivo, which
`includes many biological processes such as absorption or tissue dis-
`tribution and is realistic in the assessment of reactive metabolite
`formation in the body. From these data, we clarified the relationship
`between covalent binding, daily dose, and the safety profile. Finally,
`we established a zone classification system for the risk assessment of
`IDTs.
`
`Materials and Methods
`
`Materials. A total of 42 radiolabeled drugs were used. Amodiaquine,
`benzbromarone, carbamazepine, clozapine, clopidogrel, donepezil, flutamide,
`furosemide, imipramine, nevirapine, olanzapine, pioglitazone, rosiglitazone,
`sulfamethoxazole, tienilic acid, tacrine, valsartan, zafirlukast, and zomepirac
`(all 14C-labeled) were obtained from BlyChem Ltd. (Billingham, UK). Levo-
`floxacin, olmesartan, pravastatin, and ticlopidine (all 14C-labeled) were ob-
`tained from Sekisui Medical Co., Ltd. (Ibaraki, Japan). 14C-Labeled atorva-
`statin was obtained from MDS Pharma Services (Montreal, QC, Canada).
`Celecoxib and warfarin (both 14C-labeled) and propranolol and tamoxifen
`(both 3H-labeled) were purchased from GE Healthcare (Little Chalfont, Buck-
`inghamshire, UK). Acetaminophen, aminopyrine, caffeine, diclofenac, eryth-
`romycin, procainamide, and valproic acid (all 14C-labeled) and ethinylestradiol
`and fluoxetine (both 3H-labeled) were purchased from American Radiolabeled
`Chemicals, Inc. (St. Louis, MO). Indomethacin and phenytoin (both 14C-
`labeled) and 3H-labeled verapamil were purchased from PerkinElmer Life and
`Analytical Sciences (Waltham, MA). 14C-Labeled amlodipine and 3H-labeled
`ritonavir were purchased from Morvek Biochemicals (Brea, CA). The specific
`radio activities of the 14C-radiolabeled compounds were 13 to 58 mCi/mmol,
`and 3H-radiolabeled compounds were diluted with cold compounds at the final
`activity of 200 mCi/mmol. Unlabeled acetaminophen, amodiaquine, benzbro-
`marone, carbamazepine, clozapine, diclofenac, erythromycin, ethinylestradiol,
`fluoxetine, flutamide, furosemide, imipramine, indomethacin, phenytoin, pro-
`pranolol, sulfamethoxazole, tacrine, tamoxifen, ticlopidine, verapamil, warfa-
`rin, and zomepirac were obtained from Sigma-Aldrich (St. Louis, MO). Un-
`labeled aminopyrine, amlodipine, and valproic acid were purchased from
`Wako Pure Chemicals (Osaka, Japan). Unlabeled nevirapine and olanzapine
`were purchased from Toronto Research Chemicals Inc. (North York, ON,
`Canada). Unlabeled caffeine and zafirlukast were obtained from Fluka (Buchs,
`Switzerland) and Cayman Chemical (Ann Arbor, MI), respectively. Unlabeled
`atorvastatin, celecoxib, clopidogrel, donepezil, levofloxacin, olmesartan, pio-
`glitazone, pravastatin, procainamide, rosiglitazone, tienilic acid, ritonavir, and
`valsartan were synthesized by Daiichi Sankyo Co., Ltd. (Tokyo, Japan) Pooled
`human microsomes (n ⫽ 50, mixed gender) were purchased from XenoTech,
`LLC (Lenexa, KS). NADP and glucose-6-phosphate dehydrogenase were
`purchased from Oriental Yeast Co., Ltd.
`(Tokyo, Japan) and glucose
`6-phophate (G6P) was obtained from Sigma-Aldrich. Cryopreserved human
`hepatocytes (lots HH-286, HH-281, and HH-288) were purchased from BD
`Biosciences (San Jose, CA), and lot POP (pooled from five individuals) and lot
`SKI (pooled from 20 individuals) were purchased from In Vitro Technologies
`(Baltimore, MD). Williams’ E medium was purchased from Sigma-Aldrich.
`All other reagents and solvents were of the highest grade commercially
`available.
`Animals. Male Crj:CD(SD)IGS rats (4 – 8 weeks) were obtained from
`Charles River Laboratories Japan, Inc. (Kanagawa, Japan). The rats were
`acclimatized for 1 week with a 12-h light/dark cycle in a humidity- and
`temperature-controlled environment and allowed free access to food and tap
`water until experimental use, whereupon food was withdrawn for 16 to 18 h
`before administration of the radiolabeled drugs. The rats were cared for and
`treated in accordance with the National Institute of Health Guidelines for
`
`Page 2 of 8
`
`
`
`Downloaded from
`
`dmd.aspetjournals.org
`
` at ASPET Journals on January 29, 2016
`
`1972
`
`NAKAYAMA ET AL.
`
`TABLE 1
`Information on tested drugs, typical daily doses, and safety profiles in relation to IDT
`
`Drug No.
`
`Drug Name
`
`WDN
`1
`2
`3
`4
`BBW
`5
`6
`7
`8
`9
`10
`11
`12
`WNG
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`24
`25
`26
`27
`28
`29
`30
`SAFE
`31
`32
`33
`34
`35
`36
`37
`38
`39
`40
`41
`42
`
`Aminopyrine
`Amodiaquine
`Tienilic acid
`Zomepirac
`
`Benzbromarone
`Carbamazepine
`Clozapine
`Flutamide
`Nevirapine
`Ritonavir
`Ticlopidine
`Valproic acid
`
`Acetaminophen
`Atorvastatin
`Celecoxib
`Clopidogrel
`Diclofenac
`Erythromycin
`Fluoxetine
`Furosemide
`Imipramine
`Indomethacin
`Phenytoin
`Procainamide
`Propranolol
`Sulfamethoxazole
`Tacrine
`Tamoxifen
`Verapamil
`Zafirlukast
`
`Amlodipine
`Caffeine
`Donepezil
`Ethinylestradiol
`Levofloxacin
`Olanzapine
`Olmesartan
`Pioglitazone
`Pravastatin
`Rosiglitazone
`Valsartan
`Warfarin
`
`Daily Dose
`
`mg
`
`130–3000
`1750–2450
`250–500
`200–600
`
`50–150
`600–1200
`100–900
`750–750
`200–400
`1200–1200
`250–600
`400–4200
`
`900–4000
`10–80
`100–400
`75–75
`75–200
`1000–1000
`20–80
`40–80
`75–300
`100–200
`300–600
`1000–4000
`160–480
`800–1600
`40–160
`10–40
`240–480
`40–40
`
`5–10
`200–900
`5–10
`0.02–0.035
`250–750
`5–20
`20–40
`15–45
`20–80
`4–8
`80–320
`2–10
`
`Possible Relevant Toxicity
`
`Agranulocytosis
`Hepatotoxicity, agranulocytosis
`Hepatotoxicity
`Hepatotoxicity
`
`Hepatotoxicity
`Agranulocytosis, SJS, neutropenia
`Agranulocytosis
`Hepatotoxicity
`Hepatotoxicity
`Drug-drug interaction (MBI)
`Hepatotoxicity, agranulocytosis,
`Hepatotoxicity
`
`Hepatotoxicity
`Hepatotoxicity, SJS
`SJS, neutropenia
`SJS
`Hepatotoxicity
`Hepatotoxicity, SJS
`SJS, drug-drug interaction (MBI)
`Neutropenia
`Hypersensitivity
`SJS
`SJS
`Hypersensitivity, agranulocytosis
`Hypersensitivity, agranulocytosis
`Hepatotoxicity, SJS
`Hepatotoxicity
`Hepatotoxicity
`SJS, drug-drug interaction (MBI)
`Hepatotoxicity
`
`MBI, mechanism-based inhibition.
`
`where p is the probability of each category, and the left side of the equation is
`the logit value between two categories. Dose is the daily dose of the tested
`drug, CB is the covalent binding in each test system, and 
`
`
`0, 1, and 2 are
`coefficient values of the equation. When the odds were unity between the
`categories, lines separating the zone were drawn where the logit values were
`zero and the equation was rearranged to yield the following:
`
`log(CB) ⫽
`
`0
`2
`
`⫺
`
`1
`2
`
`⫻ log(dose)
`
`Results
`Classification of Tested Drugs. The tested drugs were classified
`into four categories on the basis of their safety profiles in the Physi-
`cian’s Desk Reference (1995, 2000, 2004, 2008) and Japanese drug
`labeling (Table 1). The first safety category, WDN, included drugs
`withdrawn from the market because of IDT in forms such as severe
`hepatotoxicity, agranulocytosis, neutropenia, and SJS. This category
`included four drugs: aminopyrine, amodiaquine, tienilic acid, and
`zomepirac. The second safety category, BBW, included drugs that had
`
`a black box warning for IDT in the Physicians’ Desk Reference (2000,
`2004, 2008). This category included eight drugs: benzbromarone,
`carbamazepine, clozapine, flutamide, nevirapine, ticlopidine, ritona-
`vir, and valproic acid. Ritonavir was placed in the BBW category
`because of its black box warning about serious drug-drug interactions
`based on mechanism-based inhibition related to covalent binding;
`however, its labeling does not carry any alert regarding IDT (Koudria-
`kova et al., 1998; Zhou et al., 2007). The third safety category, WNG,
`included drugs that did not have a black box warning but had a
`warning for IDT in the Physicians’ Desk Reference (1995, 2004,
`2008) or in Japanese labeling. This category included 18 drugs:
`acetaminophen, atorvastatin, celecoxib, clopidogrel, diclofenac, eryth-
`romycin, fluoxetine, furosemide, imipramine, indomethacin, phenyt-
`oin, procainamide, propranolol, sulfamethoxazole, tacrine, tamoxifen,
`verapamil, and zafirlukast. The last safety category, SAFE, included
`drugs with no warnings in the Physicians’ Desk Reference (2004,
`2008) or Japanese labeling. This category included 12 drugs: amlo-
`dipine, caffeine, donepezil, ethinylestradiol, levofloxacin, olanzapine,
`
`Page 3 of 8
`
`
`
`ASSESSMENT OF IDT USING COVALENT BINDING AND DOSE
`
`1973
`
`Downloaded from
`
`dmd.aspetjournals.org
`
` at ASPET Journals on January 29, 2016
`
`pmol/mg protein, that of WNG drugs ranged from 0.8 (sulfamethox-
`azole) to 209.2 (atorvastatin) pmol/mg protein, and that of BBW/
`WDN drugs ranged from 1.0 (aminopyrine) to 91.3 (amodiaquine)
`pmol/mg protein. We compared the covalent bindings in human
`hepatocytes of the drugs in all of the safety categories (Fig. 2B). As
`was the case in HLMs, comparison of covalent binding in human
`hepatocytes was not useful for distinguishing the safety categories.
`Covalent binding of 42 drugs in rat liver in vivo was determined
`after a single administration of a 20-mg/kg dose of radiolabeled drug.
`A relatively high dose was chosen to highlight
`the potential of
`metabolic bioactivation and to balance maximizing analytical sensi-
`tivity with standardizing protocol. The covalent binding of SAFE
`drugs ranged from 0.0 (levofloxacin and olmesartan) to 210.2 (ethi-
`nylestradiol) pmol/mg protein, that for WNG drugs ranged from 1.4
`(celecoxib) to 326.8 (imipramine) pmol/mg protein, and that for
`BBW/WDN drugs ranged from 13.6 (benzbromarone) to 555.7 (amin-
`opyrine) pmol/mg protein. We compared the covalent bindings in rat
`liver in vivo of the drugs in all of the safety categories (Fig. 2C).
`Covalent binding in rat liver in vivo was not useful for distinguishing
`the safety categories.
`Correlations among Covalent Bindings in the Three Test Sys-
`tems. To clarify the correlations among the three test systems, the
`covalent bindings from the different systems were plotted in pairs.
`Figure 3 shows representative results for HLMs and rat liver in vivo.
`Application of a log-linear regression analysis revealed that between
`HLM and human hepatocytes the correlation coefficient (r) was 0.25,
`between HLMs and rat liver in vivo r ⫽ 0.56, and between human
`hepatocytes and rat liver in vivo r ⫽ 0.20. Weak correlations were
`therefore observed.
`Relationships among Covalent Binding, Daily Dose, and Safety
`Category. The log-normalized covalent bindings in HLM, human
`hepatocytes, and rat liver in vivo were plotted against the log-normal-
`ized daily dose. Figure 4 is a representative result for human hepato-
`cytes. Drugs with lower daily doses and lower covalent binding were
`safer, whereas drugs with higher doses and higher covalent binding
`were relatively problematic. To investigate the correlations between
`covalent binding, daily dose, and safety categories statistically, an
`ordinal logistic regression analysis was performed (Table 3). The
`results indicated that both covalent binding and daily dose were
`statistically significant in each of the three test systems and that daily
`dose was the more important factor, because the value of 兩
`兩 (the
`daily dose coefficient) was higher than that of 兩
`兩 (the covalent
`2
`binding coefficient) in HLMs or human hepatocytes. Among the three
`test systems, classification using human hepatocytes showed the larg-
`est logit r2, with a value of 0.49, from the results of a whole-model
`test. From the results of the ordinal logistic regression analysis, two
`separation lines were drawn in each correlation figure for which the
`odds were unity between SAFE and WNG and between WNG and
`BBW/WDN (Fig. 4). We assigned these zones separated by lines as
`“acceptable,” “problematic,” and “unacceptable,” corresponding, re-
`spectively, to the safety categories SAFE, WNG, and BBW/WDN.
`Twelve of 14 SAFE drugs, 12 of 14 WNG drugs, and 5 of 8
`BBW/WDN drugs were located in the acceptable, problematic, and
`unacceptable sections, respectively (Fig. 4).
`Interlot Differences in Covalent Binding in Human Hepato-
`cytes. To investigate interlot differences in covalent binding, we used
`eight drugs to evaluate four lots of hepatocytes, including two lots
`from a single donor (HH-281 and HH-288) and two lots pooled from
`5 or 20 individual donors (POP and SKI) to add to the data shown in
`Fig. 2B from lot HH-286. The eight drugs were zomepirac, clozapine,
`acetaminophen, atorvastatin, diclofenac, ethinylestradiol, olanzapine,
`and warfarin. Although there were 3-fold interlot differences in the
`
`1
`
`FIG. 1. Daily doses of the test drugs categorized by safety profile. Numbers
`associated with symbols correspond to drug names as follows: 1, aminopyrine; 2,
`amodiaquine; 3, tienilic acid; 4, zomepirac; 5, benzbromarone; 6, carbamazepine; 7,
`clozapine; 8, flutamide; 9, nevirapine; 10, ritonavir; 11, ticlopidine; 12, valproic
`acid; 13, acetaminophen; 14, atorvastatin; 15, celecoxib; 16, clopidogrel; 17, di-
`clofenac; 18, erythromycin; 19, fluoxetine; 20, furosemide; 21, imipramine; 22,
`indomethacin; 23, phenytoin; 24, procainamide; 25, propranolol; 26, sulfamethox-
`azole; 27, tacrine; 28, tamoxifen; 29, verapamil; 30, zafirlukast; 31, amlodipine; 32,
`caffeine; 33, donepezil; 34, ethinylestradiol; 35, levofloxacin; 36, olanzapine; 37,
`olmesartan; 38, pioglitazone; 39, pravastatin; 40, rosiglitazone; 41, valsartan; 42,
`warfarin.
`
`olmesartan, pioglitazone, pravastatin, rosiglitazone, valsartan, and
`warfarin. For the analysis below, the WDN and BBW categories were
`combined as BBW/WDN, because the difference between BBW and
`WDN was considered to depend on the clinical risk-benefit balances
`or the safety profile of the other drugs in the same class.
`Daily Dose of Tested Drugs. The daily doses of the tested drugs
`are shown in Table 1. Almost all of the daily dose data were obtained
`from the Physicians’ Desk Reference (1995, 2000, 2004, 2008),
`except in the cases of amodiaquine and benzbromarone, because these
`two drugs have not been sold on the market in the United States. The
`data of daily dose for amodiaquine were obtained from a publication
`by Van den Broek et al. (2003) and for benzbromarone from Japanese
`drug labeling. For the analysis, the maximum dose in clinical use was
`used as the daily dose. Figure 1 shows the daily dose of the tested
`drugs in each safety category: SAFE, WNG, and BBW/WDN. Al-
`though the daily doses of WNG and BBW/WDN drugs tended to be
`higher than those of SAFE drugs, daily dose could not be used to
`distinguish the safety categories clearly.
`Covalent Binding Study with Three Test Systems. We deter-
`mined the covalent binding of as many as 42 radiolabeled drugs in
`HLMs, human hepatocytes in vitro, and rat liver in vivo (Table 2).
`Covalent binding of the 42 radiolabeled drugs in HLMs was deter-
`mined by incubation for 1 h with the HLMs. The covalent binding of
`SAFE drugs ranged from 0.1 (levofloxacin) to 937.5 (ethinylestradiol)
`pmol/mg protein, that of WNG drugs ranged from 3.2 (sulfamethox-
`azole) to 417.4 (clopidogrel) pmol/mg protein, and that of BBW/
`WDN drugs ranged from 3.7 (carbamazepine) to 858.0 (ticlopidine)
`pmol/mg protein. We compared the covalent binding in HLMs of the
`drugs in all of the safety categories (Fig. 2A). This comparison was
`unable to distinguish the safety categories.
`The covalent binding of 37 radiolabeled drugs in human hepato-
`cytes was determined by incubation for 2 h with human hepatocytes.
`Tienilic acid, carbamazepine, erythromycin, furosemide, and indo-
`methacin were not tested in the hepatocyte system because of insuf-
`ficient purity of the radiolabeled drugs. The covalent binding of SAFE
`drugs ranged from 0.0 (levofloxacin) to 80.6 (ethinylestradiol)
`
`Page 4 of 8
`
`
`
`Downloaded from
`
`dmd.aspetjournals.org
`
` at ASPET Journals on January 29, 2016
`
`1974
`
`NAKAYAMA ET AL.
`
`TABLE 2
`Covalent binding of tested drugs in HLMs and human hepatocytes in vitro and rat liver in vivo
`
`Data are the mean ⫾ S.D.
`
`Drug No.
`
`Drug
`
`WDN
`1
`2
`3
`4
`BBW
`5
`6
`7
`8
`9
`10
`11
`12
`WNG
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`24
`25
`26
`27
`28
`29
`30
`SAFE
`31
`32
`33
`34
`35
`36
`37
`38
`39
`40
`41
`42
`
`Aminopyrine
`Amodiaquine
`Tienilic acida
`Zomepirac
`
`Benzbromarone
`Carbamazepinea
`Clozapine
`Flutamide
`Nevirapine
`Ritonavir
`Ticlopidine
`Valproic acid
`
`Acetaminophen
`Atorvastatin
`Celecoxib
`Clopidogrel
`Diclofenac
`Erythromycina
`Fluoxetine
`Furosemidea
`Imipramine
`Indomethacina
`Phenytoin
`Procainamidea
`Propranolol
`Sulfamethoxazole
`Tacrine
`Tamoxifen
`Verapamil
`Zafirlukast
`
`Amlodipine
`Caffeine
`Donepezil
`Ethinylestradiol
`Levofloxacin
`Olanzapine
`Olmesartan
`Pioglitazone
`Pravastatin
`Rosiglitazone
`Valsartan
`Warfarin
`
`HLMs
`
`30.9 ⫾ 3.3
`208.1 ⫾ 13.4
`439.2 ⫾ 73.2
`6.4 ⫾ 0.5
`
`389.9 ⫾ 18.9
`3.7 ⫾ 0.2
`44.7 ⫾ 2.6
`178.0 ⫾ 10.9
`19.1 ⫾ 1.3
`253.3 ⫾ 24.8
`858.0 ⫾ 25.4
`6.3 ⫾ 3.3
`
`85.2 ⫾ 5.7
`352.3 ⫾ 60.4
`13.0 ⫾ 2.4
`417.4 ⫾ 83.1
`15.9 ⫾ 3.4
`57.1 ⫾ 6.7
`15.0 ⫾ 3.9
`78.6 ⫾ 1.4
`133.8 ⫾ 7.0
`16.7 ⫾ 3.2
`4.4 ⫾ 0.4
`5.1 ⫾ 0.5
`70.0 ⫾ 12.3
`3.2 ⫾ 0.7
`137.0 ⫾ 7.5
`11.5 ⫾ 2.1
`65.6 ⫾ 10.0
`36.4 ⫾ 2.0
`
`7.3 ⫾ 1.0
`9.9 ⫾ 1.6
`29.7 ⫾ 0.5
`937.5 ⫾ 94.0
`0.1 ⫾ 0.5
`138.9 ⫾ 47.8
`3.4 ⫾ 0.3
`353.0 ⫾ 34.4
`3.7 ⫾ 1.1
`516.1 ⫾ 40.3
`1.4 ⫾ 0.6
`15.9 ⫾ 2.6
`
`Covalent Binding
`
`Human
`Hepatocytes
`
`pmol/mg protein
`
`1.0 ⫾ 0.5
`91.3 ⫾ 6.1
`N.T.
`7.2 ⫾ 0.4
`
`12.1 ⫾ 2.7
`N.T.
`82.7 ⫾ 7.7
`9.7 ⫾ 0.3
`2.9 ⫾ 1.9
`47.7 ⫾ 3.6
`89.5 ⫾ 7.8
`9.3 ⫾ 0.7
`
`8.4 ⫾ 1.5
`209.2 ⫾ 17.1
`7.1 ⫾ 2.5
`75.0 ⫾ 75.0
`52.6 ⫾ 2.6
`N.T.
`9.0 ⫾ 2.4
`N.T.
`15.5 ⫾ 0.3
`N.T.
`3.7 ⫾ 2.5
`N.T.
`9.4 ⫾ 0.7
`0.8 ⫾ 0.1
`5.4 ⫾ 0.2
`64.9 ⫾ 1.5
`16.0 ⫾ 0.4
`19.1 ⫾ 0.9
`
`13.3 ⫾ 1.8
`0.2 ⫾ 0.5
`13.5 ⫾ 1.1
`80.6 ⫾ 8.3
`0.0
`38.5 ⫾ 0.9
`1.4 ⫾ 0.9
`40.5 ⫾ 14.2
`2.5 ⫾ 0.6
`42.5 ⫾ 1.8
`0.4 ⫾ 0.2
`8.0 ⫾ 1.8
`
`Rat Liver in
`Vivo
`
`555.7 ⫾ 58.1
`126.3 ⫾ 11.0
`46.1 ⫾ 17.5
`28.1 ⫾ 9.1
`
`13.6 ⫾ 1.1
`59.3 ⫾ 4.6
`156.6 ⫾ 9.6
`59.8 ⫾ 5.0
`79.5 ⫾ 11.4
`68.9 ⫾ 16.3
`252.0 ⫾ 38.7
`135.7 ⫾ 21.5
`
`10.6 ⫾ 1.6
`16.9 ⫾ 2.4
`1.4 ⫾ 0.5
`177.7 ⫾
`23.9 ⫾ 2.7
`54.5 ⫾ 21.6
`93.4 ⫾ 10.3
`14.9 ⫾ 3.3
`326.8 ⫾ 86.0
`26.0 ⫾ 5.6
`34.2 ⫾ 7.6
`26.5 ⫾ 8.3
`87.1 ⫾ 5.3
`13.2 ⫾ 0.2
`46.3 ⫾ 3.3
`60.8 ⫾ 11.3
`42.5 ⫾ 3.8
`14.2 ⫾ 7.2
`
`1.2 ⫾ 0.5
`21.0 ⫾ 3.6
`3.9 ⫾ 0.9
`210.2 ⫾ 70.5
`0.0
`93.6 ⫾ 6.4
`0.0
`6.3 ⫾ 1.7
`9.5 ⫾ 0.6
`8.7 ⫾ 0.1
`6.3 ⫾ 1.0
`17.1 ⫾ 4.5
`
`N.T., not tested.
`a These drugs were not tested in the hepatocyte system because of insufficient purity of the radiolabeled drugs.
`
`covalent bindings of both acetaminophen and olanzapine, the covalent
`bindings of drugs that showed high-level binding, such as atorvastatin,
`clozapine, and ethinylestradiol, were not variable among lots (Fig. 5).
`
`Discussion
`To assess the risk of IDT caused by reactive metabolites, we
`established a zone classification system using daily dose and covalent
`binding in human hepatocytes, which was indicated by an ordinal
`logistic regression analysis as the best predictor. The zones, each
`separated by a border line along which the logit value was zero, were
`defined acceptable, problematic, and unacceptable. The safety cate-
`gories were well separated by these zones. Although 7 of 37 tested
`drugs were located falsely, 4 of these 7 compounds were plotted near
`a border line. This good correlation means that this zone system can
`be used for IDT risk assessment. For example, a drug with a covalent
`
`binding of 50 pmol/mg protein may be acceptable at a dose less than
`25 mg, problematic at a dose between 25 and 250 mg, and unaccept-
`able at a dose greater than 250 mg (Fig. 4). Although this zone
`classification system is not an absolute criterion, because other factors
`such as therapeutic area, unmet medical needs, and the dosing period
`of the drug should also be taken into account, this assessment system
`should help in the screening of compounds at the drug discovery stage
`or in making a decision for further drug development using the
`covalent binding data and the range of the daily dose predicted from
`preclinical or clinical study data. The advantage of using this risk
`assessment system is the ability to estimate the risk at an earlier stage.
`This study was based on the hypothesis that metabolic activation of
`a drug to a reactive metabolite and its covalent binding to cellular
`macromolecules are involved in the occurrence of IDT. Evans et al.
`(2004) proposed a “threshold” level of 50 pmol/mg protein for the
`
`Page 5 of 8
`
`
`
`Downloaded from
`
`dmd.aspetjournals.org
`
` at ASPET Journals on January 29, 2016
`
`ASSESSMENT OF IDT USING COVALENT BINDING AND DOSE
`
`1975
`
`FIG. 3. Correlation of covalent binding in HLMs and rat liver in vivo. Safety
`profiles of tested drugs are as follows: BBW/WDN, red; WNG, yellow; and SAFE,
`blue. Numbers associated with the symbols correspond to the same drug names as
`in the legend to Fig. 1.
`
`FIG. 2. Covalent bindings in HLMs (A) and human hepatocytes (B) in vitro and rat
`liver in vivo (C), categorized by safety profile. Numbers associated with symbols
`correspond to the same drug names as in the legend to Fig. 1. Each point is the mean
`of triplicate analyses.
`
`screening criteria of covalent binding to HLMs in vitro and rat liver in
`vivo. This is the only previous study to have shown a targeted
`threshold for covalent binding. In our study, we determined the
`covalent bindings of as many as 42 radiolabeled drugs in three test
`systems: HLMs, human hepatocytes in vitro, and rat liver in vivo.
`Almost all of the tested BBW/WDN drugs (the exception being
`zomepirac) exceeded the 50 pmol/mg protein threshold in HLM and
`rat liver in vivo studies (Fig. 3). However, many of the WNG drugs
`did not exceed the threshold in each test system, and 4 of the 12 SAFE
`drugs were falsely classified as problematic. These results