Eisai Hyperbilirubinemic Rat (EHBR) as an Animal Model Affording High Drug-Exposure in Toxicity Studies on Organic Anions

Drug Metab. Pharmacokin. 19 (5): 339–351 (2004).

Regular Article Eisai Hyperbilirubinemic Rat (EHBR) as an Animal Model AŠording High Drug-Exposure in Toxicity Studies on Organic Anions Hiroyasu NABA, Chitose KUWAYAMA, Chihaya KAKINUMA, Shuhei OHNISHI and Takuo OGIHARA Pharmaceutical Research Center, Mochida Pharmaceutical Co., Ltd, Shizuoka, Japan Full text of this paper is available at http://www.jssx.org

Summary: The Eisai hyperbilirubinemic rat (EHBR) should be a useful animal model for studies on the toxicity of organic anions which are substrates of multidrug resistance-associated protein 2 (Mrp2), since the systemic exposure to these compounds is expected to be increased in EHBR. In this study, we tested the value of EHBR for this purpose, using pravastatin (PV) and methotrexate (MTX) as model compounds. In the case of a single oral dose of PV (200 mg W kg), Cmax in plasma was 4.0-fold higher and AUC0-/ was 3.6-fold larger than those of normal Sprague-Dawley rats (SDR), respectively. When multiple doses of PV were given to EHBR without co-administration of any other compound, druginduced skeletal muscle toxicity (myopathy W rhabdomyolysis) and increased creatine phosphokinase (CPK) level were observed, whereas a control experiment using SDR did not show any toxic change. When a single dose of MTX (0.6 mg W kg) was given to EHBR orally, Cmax was 1.7-fold higher and AUC0-/ was 1.6-fold larger than those of SDR, respectively. When multiple doses of MTX were given to EHBR, the changes in bone marrow, spleen and intestines were more severe than those in SDR. These ˆndings support the view that EHBR would be a valuable animal model for toxicity studies on organic anion compounds which are substrates of Mrp2.

Key words: EHBR, MRP2 W Mrp2; organic anions; toxicity; animal model; pravastatin; myopathy W rhabdomyolysis; methotrexate and models that are suitable for toxicity evaluation at an early stage of drug development is a priority. The Eisai hyperbilirubinemic rat (EHBR) is mutant strain of inbred Sprague-Dawley rat (SDR) with autosomal recessive hyperbilirubinuria.1) The rat exhibits jaundice immediately after birth and hyperbilirubinemia throughout life. Plasma biochemistry in EHBR shows an increase in total cholesterol and total bile acid, as biliary excretion of bilirubin is dramatically decreased in the homozygotes compared with normal animals. Therefore, EHBR is used as a model for studying constitutive hyperbilirubinemia, bilirubin metabolism, cholestasis, and glomerulonephropathy subsequent to hepatic dysfunction.2) There have also been several reports of reduced biliary excretion and W or increased plasma concentration of not only endogenous materials, but also xenobiotic anionic drugs, including pravastatin (PV),3–6) methotrexate (MTX),7) irinotecan,8,9) temocaprilat,10) olam‰oxacin,11) grepa‰oxacin,12) and probenecid13) in EHBR.14–16) All of the above compounds are substrates of multidrug

Introduction Recently, new technologies for drug discovery, such as combinatorial chemistry, have yielded vast numbers of candidate compounds, and high throughput screening (HTS) for pharmacological evaluation has also made rapid progress. There is now a need for pharmacokinetic scientists and toxicologists to assess small amounts of many compounds in the early stage of drug discovery to eliminate compounds with unsuitable kinetic and toxicity proˆles before further pre-clinical and clinical studies. Studies on pharmacokinetics by using HTS with human liver microsomes or transgenic cells in vitro, and cassette dosing methods in vivo, and also by employing new analytical equipment such as liquid chromatography with tandem mass spectrometry (LC W MS W MS) are relatively well advanced. On the other hand, toxicological studies at the drug-discovery stage are still a bottleneck because of the lack of appropriate methods and animal models. Therefore, providing toxicologists with speedy, simple and reliable methods

Received; February 22, 2004, Accepted; July 14, 2004 To whom correspondence should be addressed : Takuo OGIHARA, Ph.D., Pharmaceutical Research Center, Mochida Pharmaceutical Co., Ltd., 722, Uenohara, Jimba, Gotemba, Shizuoka 412-8524, Japan. Tel. ＋81-550-89-7881, Fax. ＋81-550-89-8070, E-Mail: togihara＠mochida.co.jp

339

340

Hiroyasu NABA, et al.

resistance-associated protein 2 (rodents, Mrp2; humans, MRP2, ABCC2 or canalicular multispeciˆc organic anion transporter (cMOAT)), which plays an important role in the excretion of organic anions from the body. Since it was established that deˆciency in the expression and function of Mrp2 in the liver causes the hyperbilirubinemia in EHBR,17,18) this mutant rat would be an appropriate model of the Dubin-Johnson syndrome,19,20) which is a hereditary disease caused by mutation of MRP2 in humans.21) Further, EHBR could be a useful model animal for toxicity studies on organic anion compounds that are substrates of Mrp2, since systemic exposure to these drugs is considered to be increased in EHBR as compared with normal rats. The aim of this study was to evaluate the usefulness (simplicity and reliability) of EHBR as a model animal for toxicity evaluation of organic anion compounds, by dosing of PV, a 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase inhibitor, and MTX, a dihydrofolate reductase inhibitor, to EHBR and measuring the serum W plasma biochemical and histopathologic changes. We chose PV and MTX as model compounds for the present study, because ˆrstly, they are typical substrates of MRP2 W Mrp2,3–7) secondly, they are commercially available at high purity, and ˆnally, side eŠects of these compounds could be rarely traced in experimental toxicological studies. Materials and Methods Chemicals and animals: PV (sodium salt) was purchased from APIN Chemicals (Oxon, UK). MTX was obtained from Wako Pure Chemical Industries (Osaka, Japan). The other reagents and solvents used in this study were commercially available products of analytical grade or chromatographic grade. The animal study was performed according to the Guidelines for the Care and Use of Laboratory Animals in the Pharmaceutical Research Center, Mochida Pharmaceutical Co., Ltd. and approved by the Committee of Ethics of Animal Experimentation of Pharmaceutical Research Center, Mochida Pharmaceutical Co., Ltd. Male SDR and EHBR, 6 weeks of age were obtained from Charles River Japan, Inc. (Yokohama, Japan) and from Japan SLC (Hamamatsu, Japan), respectively. The animals were individually C) and humidity housed at constant temperature (23±29 (55±15z), with 12 h of light per day. They were allowed access to water and were given commercially available diet, CE-2 (Clea Japan Inc., Tokyo, Japan). Pharmacokinetic study: PV was dissolved at an appropriate concentration in 0.5z hydroxypropyl methyl cellulose (HPMC) and orally administered to kg. Blood rats at a single dose of 100, 200 or 400 mg W samples were withdrawn via the jugular vein with heparinized syringes at designated times and were

centrifuged (1700×g) for 15 min at 49C to obtain plasma samples. PV was extracted from rat plasma using ethyl acetate. The organic layer was collected and evaporated, then the dried residue was dissolved in acetonitrile mixed 20 mM phosphate buŠer (pH 4.5) W solution and an aliquot was injected into a high- performance liquid chromatography (HPLC) system (Millennium32TM; Waters, Milford, MS, USA). Analyses were performed on a DevelosilTM ODS HG-5 column (150×2.0 mm i.d.; Nomura Chemical, Seto, Japan) at 0.2 mL W min using gradient elution. Mobile phases used were 20 mM phosphate buŠer (pH 4.5) as 30z 20 mM phosphate solvent A and 70z acetonitrile W buŠer (pH 4.5) as solvent B, and gradient elution was done with 75z solvent A W 25z solvent B initially, followed by a linear gradient to 25z solvent A W 75z solvent B. The detection wavelength was set at 237 nm. MTX was dissolved in physiological saline containing 1 mM sodium hydroxide and orally administered to rats at a single dose of 0.2 or 0.6 mg W kg. Plasma samples were obtained from blood in the same manner as described above, and folinic acid (internal standard) and acetonitrile were added. The supernatant of the centrifuged solution was evaporated, and the dried residue was dissolved with aqueous methanol. Aliquots were analyzed in an HPLC system (HP-1050TM; Agilent Technologies, Palo Alto, CA, USA) equipped with a CAPCELL PAKTM MG column (150×2.0 mm i.d., Shiseido, Tokyo, Japan) using isocratic elution at 0.2 mL W min with 33z water W 67z methanol containing 5 mM di-n-hexylammonium, an ion-pair reagent. MTX was detected using a triple quadrupole mass spectrometer (TSQ700TM; Thermo Electron Corporation, San Jose, CA, USA) ˆtted with an electrospray ionization (ESI) source. Positive ion selected reaction monitoring (SRM) was done using the transitions 455 to 308 for MTX, and 474 to 327 for the internal standard. Calculation of pharmacokinetic parameters: Pharmacokinetic analysis of the plasma concentration timecurve of PV and MTX after a single oral administration was performed by use of the nonlinear estimation program WinNonlinTM (Pharsight, Mountainview, CA, USA). The elimination half-lives (T1 W2) and the area under the plasma concentration time-curve from time 0 to inˆnity (AUC0-/) were estimated by non-compartmental analysis, and the trapezoidal method with extrapolation to inˆnite time, respectively. The maximum plasma concentration (Cmax) and time to Cmax (Tmax) were determined directly from the observed data. The total body clearance corrected for absorption (CL W F) was calculated by dividing the dose by the AUC0-/ value. Each value is the mean±S.D. of three animals. Statistical analysis was performed by means of Student's two-tailed t test. A diŠerence between means was considered to be signiˆcant when the P-value was

EHBR as an Animal Model for Toxicity of Organic Anions

341

Fig. 1. Scoring system for the degree of muscular degeneration. A) Grade 0: no abnormalities are seen. B) Grade 1: ˆne vacuoles ( ) are seen in muscle ˆbers, but necrosis ( ) is very slight. C) Grade 2: necrosis is recognized. Edema (*) and in‰ammatory cell inˆltration can be seen. D) Grade 3: necrotic area up to one-third of the observed area. E) Grade 4: necrotic area exceeds one-third of the observed area. Bar＝100 mm.

less than 0.05. Plasma protein binding: Plasma protein binding ratio was measured with an ultraˆltration method. Rat plasma spiked with an appropriate concentration of PV or MTX was incubated at 379C for 20 min, and ultraˆltered (Centrifree}, Millipore Corporation, Billerica, MA, USA), and the ˆltrate was centrifuged (1400×g) for 15 min at room temperature. Plasma and ˆltrate concentrations of PV and MTX were determined by the method described above. Toxicity study: SDR and EHBR were orally given with PV at a dose of 200 mg W kg W day once a day for 7 or 14 days or with vehicle (0.5z HPMC) for 14 days.

Another group of EHBR was treated with PV at a dose of 400 mg W kg W day once a day for 7 days. The body weight of each animal was monitored several times throughout the study period. At 24 h after the last dosing, the animals were anesthetized with 2.5z pentobarbital sodium, (SomnopentylTM; ScheringPlough, Kenilworth, NJ, USA), and heparinized blood samples were collected from the abdominal aorta for analysis of creatine phosphokinase (CPK) level, which was performed by an autoanalyzer (COBAS MIRATM PLUS; Roche Diagnostics, Basel, Switzerland). Subsequently, each animal was killed and skeletal muscles were taken from each of three sites, musculus

342

Hiroyasu NABA, et al.

Fig. 2. Plasma concentration of unchanged drug after a single oral administration of PV to male SDR at a dose of 200 mg W kg ($) or to male EHBR at a dose of 100 (), 200 (#) or 400 mg W kg (). Each point represents the mean＋S.D. of three animals.

rectus femoris, musculus biceps femoris and musculus gastrocnemius, of the right and left hind limbs. All muscles were preserved in 10z phosphate-buŠered formaldehyde solution, routinely processed, embedded in para‹n, and sectioned at 3-mm thickness. These sections were stained with hematoxylin and eosin. In histopathological evaluation, the degree of muscular degeneration was evaluated according to the following scoring system:22) grade 0, within normal limits; grade 1, small amounts of muscle ˆbers showing a few vacuoles but no necrosis; grade 2, scattered necrosis of muscle ˆbers; grade 3, necrotic area reaching one-third of the observed area; grade 4, necrotic area exceeding onethird of the observed area (see Fig. 1). The mean of the scores of six specimens was taken as representative of the individual. Moreover, the average of these individual values was regarded as representative of the group. Body weight gain and plasma CPK level are shown as the mean±S.D. of three or ˆve animals. SDR and EHBR were treated orally with MTX at a dose of 0.2 mg W kg W day or with vehicle (physiological saline containing 1 mM sodium hydroxide) once a day for 14 days or at a dose of 0.6 mg W kg W day once a day for 7 days. Each individual was weighed several times during the study period. At 24 h after the end of the last dosing, the animals were anesthetized with 2.5z pentobarbital sodium, and blood samples were collected from the jugular vein using EDTA-2K as the anticoagulant, for hematological examinations. Heparinized blood samples were collected from the abdominal aorta for biochemical examinations. After each animal was killed, the liver, kidney, heart, lung, spleen and thymus were removed and weighed. The

submaxillary glands, adrenal glands, mesenteric lymph node, femoral bone marrow, stomach and intestines were also removed. All organs were prepared for histopathological study in the same manner as previously described. In hematological examinations, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), leucocytes and thrombocytes were determined using a fully automated hematology analyzer (SF3000TM; Sysmex, Kobe, Japan). Leukocytic diŠerential fraction, that is, basophils, eosinophils, band neutrophils, segmented neutrophils, lymphocytes and monocytes, was determined with an automatic diŠerential counter (MICROXTM HEG-120; OMRON Corporation, Tokyo, Japan). In biochemical examinations, aspartate aminotransferase (GOT), CPK, total cholesterol, triglyceride and phospholipid analyses were performed by COBAS MIRATM PLUS. In addition, alanine aminotransferase (GPT), alkaline phosphatase (ALP), glucose, total protein, albumin, total bilirubin, blood urea nitrogen (BUN), creatinine, calcium (Ca) and potassium (K) analyses were performed by an autoanalyzer (AbaxisTM EA; Abaxis, Inc., Union City, CA, UCA). Each value represents the mean±S.D. of four or ˆve animals. Statistical analysis was performed with Student's two-tailed t test and Dunnet's test. A diŠerence between means was considered to be signiˆcant when the P-value was less than 0.05. Results Pharmacokinetics of PV: The plasma concentration-time data are shown in Fig. 2, and the derived

EHBR as an Animal Model for Toxicity of Organic Anions Table 1.

343

Pharmacokinetic parameters of PV after a single oral administration to SDR or EHBR

Strain

Dose (mg W kg)

Cmax ( mg W mL)

Tmax (h)

T1 W2 (h)

AUC0-/ (mg･h W mL)

F CL W (L W kg W h)

SDR

200

0.56±0.16

0.8±0.3

6.9±3.8

2.29±0.38

89.11±15.00

EHBR

100 200 400

0.43±0.05 2.22±0.41* 7.56±1.66

0.8±0.3 0.8±0.3 0.7±0.3

14.8±1.9 14.5±4.6 16.4±13.7

3.96±1.05 8.30±0.36* 22.48±4.65

26.34±6.16 24.12±1.06* 18.26±3.41

Each value represents the mean±S.D. of three animals. *pº0.05: signiˆcantly diŠerent between SDR and EHBR at 200 mg W kg by Student's t-test.

pharmacokinetic parameters are summarized in Table 1. The Cmax and AUC0-/ of PV after a single oral kg were 0.56±0.16 administration at a dose of 200 mg W mg W mL and 2.29±0.38 mg･h W mL in SDR, and 2.22± 0.41 mg W mL and 8.30±0.36 mg･h W mL in EHBR, respectively, with Cmax being 4.0-fold higher and AUC0-/ 3.6-fold larger in EHBR than in SDR. The T1 W2 was 6.9±3.8 h in SDR and 14.5±4.6 h in EHBR. The CL W F value in EHBR, 24.12±1.06 L W kg W h, was onequarter of that in SDR, 89.11±15.00 L W kg W h. The Cmax and AUC0-/ values increased with dose escalation, and the value of CL W F at doses of 100, 200 and kg of PV in EHBR were similar. 400 mg W Plasma protein binding of PV: Plasma protein binding of PV amounted to 35z in SDR and 42z in EHBR, at 1.0 mg W mL. Toxicity of PV: Body weight gain, plasma CPK level and grade of skeletal muscle degeneration in male rats treated orally with PV for 7 or 14 days are shown in kg W day Table 2. In EHBR treated with PV at 200 mg W for 14 days, suppression of body weight gain was observed. The plasma CPK level, one of the indices of muscular damage, was also greatly increased. Histopathologically, the incidence and extent of muscular degeneration were prominent in EHBR treated with kg W day for 14 days (incidence: 5 W 5, PV at 200 mg W average grade: 3.5). The degree of muscular degeneration in these animals ranged from grade 2 to grade 4, and the individual averages were within grades 3.2–3.8. kg W day Moreover, EHBR treated with PV at 400 mg W for 7 days also showed muscular degeneration (incidence: 3 W 3), although the grade (average grade: 0.6) was less than that in the group given 200 mg W kg W day for 14 days. The Increase of CPK level was also less than in the 200 mg W kg W day group. Decrease of body weight was observed in only the 400 mg W kg W day group. On the other hand, SDR treated with PV at 200 mg W kg W day for kg W day for 7 days 7 or 14 days and EHBR at 200 mg W showed an increase in plasma CPK level, but the degree was very weak compared with that of EHBR at a dose of 200 mg W kg W day for 14 days. Moreover, histopathologically, these groups showed no abnormalities at all. Pharmacokinetics of MTX: The plasma concen-

tration-time data are shown in Fig. 3, and the derived pharmacokinetic parameters are summarized in Table 3. The Cmax and AUC0-/ of MTX after a single oral administration at a dose of 0.2 mg W kg were 0.039± 0.012 mg W mL and 0.166±0.036 mg･h W mL in SDR, and 0.033±0.011 mg W mL and 0.169±0.026 mg･h W mL in EHBR, respectively, being essentially the same in SDR kg of MTX, and EHBR. At the dose of 0.6 mg W however, Cmax and AUC0-/ were 0.087±0.030 mg W mL and 0.382±0.067 mg･h W mL in SDR, and 0.145±0.024 mg W mL and 0.607±0.103 mg･h W mL in EHBR, respectively, with Cmax being 1.7-fold higher and AUC0-/ 1.6kg, the fold larger in EHBR than in SDR. At 0.6 mg W CL W F value in EHBR was decreased to two-thirds of that in SDR, while T1 W2 was about 3 h in both. Plasma protein binding of MTX: Plasma protein binding of MTX was 44z in SDR, but only 20z in mL. EHBR at 0.1 mg W Toxicity of MTX: All groups given MTX showed suppression of body weight gain (78–93z of vehicle control at the same time point), and a slight weight reduction was observed in the EHBR group given kg W day for 7 days (96z of initial value). The 0.6 mg W groups given MTX showed a decrease in several hematological parameters (Table 4). For the most part, the degrees of decrease in EHBR were more prominent than those in SDR. Erythrocytes, short-lived blood kg W day for 14 days, cells, decreased in EHBR at 0.2 mg W and in contrast, leucocytes, long-lived ones, decreased in EHBR at 0.6 mg W kg W day for 7 days. Decreasing parameters for hemopoietic or immune systems: segmented neutrophilis, lymphocytes and monocytes in leukocytic fraction, and thrombocytes in this group were quite noticeable. Biochemically (Table 5), EHBR kg W day for 7 days showed treated with MTX at 0.6 mg W decreased GOT, GPT and ALP. Moreover, several parameters which re‰ect a decrease in food consumption and weight loss (glucose, total cholesterol, triglyceride, phospholipid, total protein and albumin) were decreased in this group. However, SDR and EHBR treated at 0.2 mg W kg W day for 14 days showed no obvious changes. Histopathologically, SDR and EHBR kg W day for 7 days and EHBR treated treated at 0.6 mg W

344

Hiroyasu NABA, et al.

Table 2. Body weight gain, plasma creatine phosphokinase (CPK) activity and incidence and grade of skeletal muscle degeneration in male rats treated orally with PV for 7 or 14 days

Strain

Dose kg) (mg W

Vehicle

Treatment period (day)

14

7

SDR

Body weight gaina (g) day 4

day 7

day 14

36.7 ±4.7

60.8 ±9.8

110.9 ±21.0

36.1 ±6.8

59.5 ±12.2

—

Grade of skeletal muscle degenerationb Plasma CPK level (U W L)

Left side

Average

Bc

Gc

Rc

Bc

Gc

individual

Group

1 2 3

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0.0 0.0 0.0

0.0

735±371

1 2 3 4 5

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0.0 0.0 0.0 0.0 0.0

0.0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0.0 0.0 0.0 0.0 0.0

0.0

14

35.9 ±7.6

67.2 ±7.8

116.1 ±13.6

655±235

14

30.2 ±4.3

51.1 ±7.1

93.2 ±15.2

582±153

1 2 3

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0.0 0.0 0.0

0.0

907±547

1 2 3 4 5

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0.0 0.0 0.0 0.0 0.0

0.0

29.5 ±32.8

9660±7665

1 2 3 4 5

4 3 4 4 4

3 4 4 4 4

2 3 3 2 3

4 3 4 4 4

4 4 4 4 4

2 2 3 3 4

3.2 3.2 3.7 3.5 3.8

3.5

—

822±391

1 2 3

1 1 0

1 0 0

0 1 0

1 1 0

1 0 1

1 1 0

0.8 0.7 0.2

0.6

23.8 ±1.7

31.6 ±9.7

—

200

400

Right side Rc

1 2 3 4 5

7 EHBR

Animal No.

238±52

200

Vehicle

a

14

22.6 ±2.5

7

－27.6 7.3 ±15.9 ±26.8

21.7 ±7.3

a

Each value of body weight and plasma CPK level represents the mean±S.D. of three or ˆve animals. The grade of skeletal muscle degeneration described in the method section and shown in Fig. 1: grade 0, within normal limits; grade 1, small amounts of muscle ˆbers showed a few vacuoles but no necrosis; grade 2, scattered necrosis of muscle ˆbers; grade 3, necrotic area up to one-third of observed area; grade 4, necrotic area exceeded one-third of observed area. c R, musculus rectus femoris; B, musculus biceps femoris; G, musculus gastrocnemius. b

at 0.2 mg W kg W day for 14 days showed a decrease in hematopoietic cells (erythroblasts and myeloblasts) of the bone marrow, extramedullary hematopoiesis and small-size lymphocytes of spleen, and lymphocytes of thymus. All of these changes were more severe in the EHBR at 0.6 mg W kg W day for 7 days than in other groups. Decrease of lymphocytes in lymph nodes was observed only in one individual in the EHBR group given 0.6 mg W kg W day (Fig. 4, Table 6). In addition, some changes in the stomach and liver were observed kg W day. Both strains at 0.6 only in EHBR given 0.6 mg W kg W day showed necrosis and atrophy of mucosal mg W epithelial cells in the small and large intestine; the number of animals with these symptoms was larger in the case of EHBR than in SDR (Fig. 5, Table 7). The

absolute and relative organ weights in the thymus and spleen were also reduced in EHBR given at 0.6 mg W kg W day (data not shown). Discussion In the present study, we investigated the value of EHBR as a model animal with a high exposure to anionic drugs, because of its deˆciency of the organic anion biliary eŒux transporter, Mrp2. In order to assess the utility of this model, compared with SDR, we chose PV and MTX as model drugs, and examined their pharmacokinetics and toxicity in the two strains. One of the most important clinical adverse eŠects in therapy with HMG-CoA reductase inhibitors (statins) to improve the lipid proˆle of hyperlipemia patients is a

345

EHBR as an Animal Model for Toxicity of Organic Anions

Fig. 3. Plasma concentration of unchanged drug after a single oral administration of MTX to male SDR at a dose of 0.2 ($) or 0.6 mg W kg (), or to male EHBR at a dose of 0.2 (#) and 0.6 mg W kg (). Each point represents the mean＋S.D. of three animals.

Table 3.

Pharmacokinetic parameters of MTX after a single oral administration to male SDR or EHBR

Dose (mg W kg)

Strain

Cmax ( mg W mL)

Tmax (h)

T1 W2 (h)

AUC0-/ (mg･h W mL)

F CL W (L W kg W h)

0.2

SDR EHBR

0.039±0.012 0.033±0.011

1.7±0.6 0.8±0.3

2.3±0.6 3.7±1.2

0.166±0.036 0.169±0.026

1.24±0.27 1.20±0.19

0.6

SDR EHBR

0.087±0.030 0.145±0.024

1.0±0.0 1.0±0.0

3.2±1.3 3.6±2.1

0.383±0.067 0.607±0.103*

1.60±0.26 1.01±0.16*

Each value represents the mean±S.D. of three animals. *pº0.05: signiˆcantly diŠerent between SDR and EHBR at the same dose.

Table 4.

Hematology in male rats treated orally with MTX at the dose of 0.2 mg W kg W day for 7 days or at the dose of 0.6 mg W kg W day for 14 days SDR Dose (mg W kg W day) Period (days)

Number of animals examined Erythrocytes (×104 W mm3) Hemoglobin (g W dL) Hematocrit (z) MCV (fL) MCH (pg) MCHC (g W dL) Leucocytes (×102 W mm3) Basophils Eosinophils Band neutrophils Segmented neutrophils Lymphocytes Monocytes Thrombocytes (×103 W mm3)

EHBR

Vehicle 14

0.2 14

0.6 7

Vehicle 14

0.2 14

5 688±47 15.5±0.7 44.7±2.4 65.1±2.7 22.6±1.0 34.7±0.4 75±19 0.0±0.0 1.2±1.2 0.0±0.0 9.3±8.6 56.2±12.0 8.7±3.4 102.8±5.8

5 566±113* 12.7±2.4* 36.1±7.2* 63.7±0.9 22.4±0.4 35.1±0.7 78±15 0.0±0.0 1.2±1.1 0.0±0.0 4.3±1.2 67.1±11.9 5.3±3.5 176.7±79.5

5 579±17* 12.8±0.6* 37.5±0.8* 64.8±1.6 22.1±0.9 34.1±0.8 56±17 0.0±0.0 0.3±0.2 0.0±0.1 4.0±1.1 48.9±15.0 2.9±1.8* 107.6±54.9

4 639±49 13.8±1.2† 38.9±3.4† 60.8±0.7† 21.6±0.4 35.5±0.2† 63±15 0.0±0.0 0.8±0.7 0.2±0.3 4.6±0.9 48.9±12.6 9.0±3.3 127.0±8.5†

5 389±46*,† 8.5±1.0*,† 23.2±2.7*,† 59.8±0.5*,† 21.8±0.2† 36.5±0.2*,† 41±7*,† 0.0±0.0 1.1±0.6 0.0±0.0 3.3±2.2 34.0±4.6*,† 3.1±1.1* 174.0±41.0*

Each value represents the mean±S.D. of four or ˆve animals. *pº0.05: signiˆcantly diŠerent from vehicle values by Dunnett's test. †pº 0.05: signiˆcantly diŠerent between SDR and EHBR at the same dose by Student's t-test.

0.6 7 5 714±71† 16.0±1.5*,† 43.3±4.2† 60.7±0.4† 22.4±0.2* 37.0±0.2*,† 20±9*,† 0.0±0.0 0.1±0.1 0.1±0.1 2.8±4.4 15.7±4.8*,† 1.4±0.7* 77.8±14.0*

346

Hiroyasu NABA, et al.

Table 5. Biochemical parameters in male rats treated orally with MTX at the dose of 0.2 mg W kg W day for 14 days or at the dose of 0.6 mg W kg W day for 7 days SDR Dose (mg W kg W day) Period (days) Number of animals examined L) GOT (U W GPT (U W L) L) ALP (U W CPK (U W L) dL) Glucose (mg W Total cholesterol (mg W dL) Triglyceride (mg W dL) Phospholipid (mg W dL) Total protein (g W dL) Albumin (g W dL) Total bilirubin (mg W dL) BUN (mg W dL) dL) Creatinine (mg W Ca (mg W dL) L) K (mEq W

EHBR

Vehicle 14

0.2 14

0.6 7

Vehicle 14

0.2 14

5 122±25 51±11 389±96 1437±707 241±38 70±5 120±35 150±7 6.9±1.3 4.3±0.8 0.2±0.0 17±3 0.5±0.2 12.2±2.3 4.7±0.8

5 86±18 30±6* 361±103 644±362 214±17 58±15 94±32 122±13* 5.8±0.2* 3.8±0.2 0.2±0.0 17±2 0.5±0.1 10.6±0.6 4.1±0.5

5 94±29 29±11* 333±53 887±741 190±19* 65±7 76±21 128±8* 5.6±0.3* 3.8±0.2 0.2±0.0 14±3 0.4±0.1 10.9±0.8* 4.0±0.3

4 114±8 49±6 233±21† 1029±418 280±77 122±17† 70±21† 232±33† 7.2±0.9 4.7±0.6 4.0±1.0† 19±1 0.6±0.1 12.4±1.2 4.8±0.3

5 95±20 35±4* 211±55† 689±297 250±50 113±32† 44±15*,† 210±49† 6.3±0.3† 4.3±0.2† 5.1±2.0† 17±3 0.6±0.1 10.9±0.5 4.3±0.6

0.6 7 5 62±13* 18±7* 77±18*,† 809±569 246±70 65±20* 36±10*,† 115±20* 4.6±0.8*,† 2.5±0.6*,† 2.5±0.7† 17±4 0.3±0.1* 9.9±2.3 3.9±0.7

Each value represents the mean±S.D. of four or ˆve animals. *pº0.05: signiˆcantly diŠerent from vehicle values by Dunnett's test. †pº 0.05: signiˆcantly diŠerent between SDR and EHBR at the same dose by Student's t-test.

drug-induced skeletal muscle toxicity (rhabdomyolysis),23) whose incidence is signiˆcant (e.g., 0.1–0.5z in patients treated with PV). Matsuyama et al.24,25) reported that single oral administration of statin (50–500 mg W kg) to rats under urethane anesthesia, dose-dependently elevated the serum CPK level in the case of simvastatin, but not PV. No signiˆcant elevation of serum CPK level was observed without urethane infusion. Moreover, Pierno et al.26,27) reported that PV treatment at 100 mg W kg W day for three months did not produce any alteration of excitation-contraction coupling of the rat skeletal muscle. Smith et al.22) reported that the incidence (rate) 11 (0z), 2 W 3 (33z) of induced myopathy in rats was 0 W and 2 W 2 (100z) with uncertain degeneration grades at 400, 1200 and 2400 mg W kg W day (dosed on a bid regimen kg) of PV for 2–4 weeks, at 200, 600 and 1200 mg W respectively. They also reported in the same paper that 13 (62z) and 3 W 3 (100z) at the incidence (rate) was 8 W 400 and 1200 mg W kg W day (dosed on a bid regimen at 200 and 600 mg W kg) of PV, respectively, with concomitant intravenous administration of cyclosporin A (10 mg W kg W day) at various doses for the same period. We found that at 200 mg W kg of PV, the Cmax and AUC values were signiˆcantly higher in EHBR than in SDR. Drug-induced rhabdomyolysis was observed with severe degeneration in all EHBR given at 200 mg W kg W day (once a day) of PV alone for 14 days. Compared with previously described methods, ours has several advantages: it was possible to reduce the drug administration by dosing once a day, to simplify the procedure by the dosing of PV alone, to shorten the study period

and to obtain a high rate of induction of rhabdomyolysis. We examined whether dosing period could be shortened with the same total exposure of PV as in the case of kg W day for 14 days, that is, PV treatment with 200 mg W was administered to EHBR at 400 mg W kg W day for 7 days. In this case, the incidence of degeneration was complete; however, the severity was low, and the increase of CPK level was much smaller. Moreover, this group showed not only rhabdomyolysis but also decreased body weight, which may re‰ect unexpected acute toxicity. When younger EHBR (5 weeks old) were kg W day for 14 days, both the treated with PV at 200 mg W incidence and degree of degeneration were less than those of older animals (7 weeks old) (data not shown), as reported previously.28) In the study on MTX, we found that Cmax and AUC of MTX were 1.7-fold higher and 1.6-fold larger in kg. Plasma EHBR than in SDR at the dose of 0.6 mg W protein binding of MTX was much smaller in EHBR than in SDR. When SDR and EHBR were treated kg W day for 7 days (4.2 mg W kg), with MTX at 0.6 mg W hemopoietic changes, lymphotoxicity, gastrointestinal eŠects and hepatotoxicity, which re‰ect MTX-induced clinical adverse eŠects, were more severe in EHBR than in SDR. The reason for the diŠerence was considered to be the diŠerences in not only systemic exposure but also protein binding of MTX between EHBR and SDR. Murakami et al.29) reported that MTX-induced toxicological changes were observed in normal and unilaterally nephrectomized rats which received dosages of up to 0.6 mg W kg W day for one month (18 mg W kg). Gao et al.30)

EHBR as an Animal Model for Toxicity of Organic Anions

347

Fig. 4. Histologic characterization of hematopoietic and immune-related organs from SDR (A, C and E) and from EHBR (B, D and F) treated with MTX (0.6 mg W kg W day) for 7 days. A and B) Bone marrow. Decrease in hematopoietic cells is seen in both strains, but the change in the bone marrow of EHBR is far greater than in that of SDR. C and D) Spleen. Decrease in small-sized lymphocytes is seen in the spleen from EHBR. E and F) Thymus. Decrease in lymphocytes is seen in both strains. In SDR, cortical atrophy (*) is apparent. In EHBR, the decrease in lymphocytes is very severe over the entire area. G and H) Lymph node. Decrease in lymphocytes is seen in lymph node from EHBR. Bar＝100 mm.

348

Hiroyasu NABA, et al.

Table 6. Histopathological changes of hematopoietic and immune-related organs from male rats treated orally with MTX at the dose of 0.2 mg W kg W day for 14 days or at the dose of 0.6 mg W kg W day for 7 days Severity of changes Number of animals examined Bone marrow Decrease in hematopoietic cells

Spleen Decrease in extramedullary hematopoiesis

Decrease in small sized lymphocytes Thymus Decrease in lymphocytes

Lymph nodes Decrease in lymphocytes

SDR Dose (mg W kg W day) Period (day)

EHBR

Vehicle 14

0.2 14

0.6 7

Vehicle 14

0.2 14

0.6 7

5

5

5

4

5

5

± ＋ ＋＋ ＋＋＋

0a 0 0 0

0 0 0 0

1 1 3 0

0 0 0 0

0 3 2 0

0 0 2 3

± ＋ ± ＋

0 0 0 0

0 0 0 0

3 0 1 0

0 0 0 0

3 2 5 0

3 2 1 4

± ＋ ＋＋ ＋＋＋

0 0 0 0

0 0 0 0

1 1 0 0

0 0 0 0

3 1 2 0

0 2 1 2

＋

0

0

0

0

0

1

Severity of changes is indicated as follows: ±, slight; ＋, moderate; ＋＋, severe; ＋＋＋, very severe a Number of animals with the indicated severity of changes

Fig. 5. Histologic characterization of liver and small intestine from SDR (A and C) and from EHBR (B and D) treated with MTX (0.6 mg W kg W day) for 7 days. A and B) Liver. Hypertrophy of nuclei and nucleolus, eosinophilic degeneration ( ) and single-cell necrosis ( ) are seen in the liver from EHBR. C and D) Small intestine. In SDR, some necrosis ( ) and atrophy of mucosal epithelial cells are observed. In EHBR, necrosis and atrophy of mucosal epithelial cells are very severe; moreover, hemorrhage (*) and in‰ammatory cell inˆltration are apparent. Bar＝100 mm.

349

EHBR as an Animal Model for Toxicity of Organic Anions

Table 7. Histopathological changes of digestive organs from male rats treated orally with MTX at the dose of 0.2 mg W kg W day for 14 days or at kg W day for 7 days the dose of 0.6 mg W SDR Dose (mg W kg W day) Period (days) Number of animals examined Liver Hypertrophy of nuclei and nucleolus Eosinophilic degeneration Single cell necrosis Stomach Ulcer in non-glandular stomach Submucosal edema in non-glandular stomach Degeneration and necrosis of smooth muscle ˆbers Small intestines Necrosis of mucosal epithelial cells Atrophy of mucosal epithelial cells Mucosal hemorrhage In‰ammatory cell inˆltration Large intestines Necrosis of mucosal epithelial cells Atrophy of mucosal epithelial cells In‰ammatory cell inˆltration a

EHBR

Vehicle 14

0.2 14

0.6 7

Vehicle 14

0.2 14

0.6 7

5

5

5

4

5

5

0a 0 0

0 0 0

0 0 0

0 0 0

0 0 1

5 2 1

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

2 2 2

0 0 0 0

0 0 0 0

1 1 0 0

0 0 0 0

0 0 0 0

5 5 5 5

0 0 0

0 0 0

1 1 0

0 0 0

0 0 0

3 3 3

Number of animals with the indicated severity of changes

employed repeated administration of MTX at a dose of 15 mg W kg W day for 5 days (75 mg W kg) to evaluate the toxicity. Our method using EHBR requires less time at the same dose, or a much lower dose at almost the same time compared with the previous methods. Several authors have suggested that operated animals, such as nephrectomized animals, and methods involving co-administration of other drugs are useful to increase systemic exposure. However, these models might not be appropriate for toxicity evaluation because of the injurious eŠects of operations and the possibility of drug-drug interaction. In contrast, natural or artiˆcial gene-deˆcient model animals lacking drug-eŒux transporter gene(s) may be better models for toxicity study. Some work has been done to survey polymorphisms of gene expression in patients with statin-induced rhabdomyolysis,31,32) and it was suggested that the rhabdomyolysis is associated with mutations of transporters such as OATP-C (SLC21A6, also known as OATP2, LCT-1). Since OATP-C is a human organic anion transporter, which is engaged in the hepatic uptake of endogenous materials and xenobiotic drugs including statins such as PV, the plasma PV level of patients with a mutated OATP-C gene might be higher than patients with normal gene. These considerations justify our use of EHBR, in that gene mutation and deˆciency of transporters both cause an increase of systemic exposure, and consequently toxicity. Our results support the idea that EHBR will be a useful model for toxicological evaluation to assess limited amount of the compounds at the drug discovery stage.

References 1)

2)

3)

4)

5)

6)

7)

Yamazaki, K., Mikami, T., Hosokawa, S., Tagaya, O., Nozaki, Y., Kawaguchi, A., Funami, H., Katoh, H., Yamamoto, N. and Wakabayashi, T.: A new mutant rat with hyperbilirubinuria (hyb). J. Hered., 86: 314–317 (1995). Kawaguchi, A., Nozaki, Y., Hosokawa, S., Tagaya, O., Mikami, T. and Wakabayashi, T.: Establishment of hyperbilirubinuria rat mutant-a new animal model for jaundice. Jikken Dobutsu, 43: 37–44 (1994). Yamazaki, M., Kobayashi, K. and Sugiyama, Y.: Primary active transport of pravastatin across the liver canalicular membrane in normal and mutant Eisai hyperbilirubinemic rats. Biopharm. Drug Dispos., 17: 645–659 (1996). Adachi, Y., Okuyama, Y., Miya, H., Matsusita, H., Kitano, M., Kamisako, T. and Yamamoto, T.: Pravastatin transport across the hepatocyte canalicular membrane requires both ATP and a transmembrane pH gradient. J. Gastroenterol Hepatol., 11: 580–585 (1996). Yamazaki, M., Akiyama, S., Ni'inuma, K., Nishigaki, R. and Sugiyama, Y.: Biliary excretion of pravastatin in rats: contribution of the excretion pathway mediated by canalicular multispeciˆc organic anion transporter. Drug Metab. Dispos., 25: 1123–1129 (1997). Fukumura, S., Takikawa, H. and Yamanaka, M.: EŠects of organic anions and bile acid conjugates on biliary excretion of pravastatin in the rat. Pharm. Res., 15: 72–76 (1998). Masuda, M., I'izuka, Y., Yamazaki, M., Nishigaki, R., Kato, Y., Ni'inuma, K., Suzuki, H. and Sugiyama Y.: Methotrexate is excreted into the bile by canalicular

350

8)

9)

10)

11)

12)

13)

14)

15)

16)

17)

18)

19)

20)

Hiroyasu NABA, et al.

multispeciˆc organic anion transporter in rats. Cancer Res., 57: 3506–3510 (1997). Chu, X. Y., Kato, Y., Niinuma, K., Sudo, K., Hakusui, H. and Sugiyama, Y.: Multispeciˆc organic anion transporter is responsible for the biliary excretion of the camptothecin derivative irinotecan and its metabolites in rats. J. Pharmacol. Exp. Ther., 281: 304–314 (1997). Chu, X. Y., Kato, Y. and Sugiyama, Y.: Multiplicity of biliary mechanisms for irinotecan, CPT-11 and its metabolites in rats. Cancer Res., 57: 1934–1938 (1997). Ishizuka, H., Konno, K., Naganuma, H., Sasahara, K., Kawahara, Y., Niinuma, K., Suzuki, H. and Sugiyama, Y.: Temocaprilat, a novel angiotensin-converting enzyme inhibitor, is excreted in bile via an ATP-dependent active transporter (cMOAT) that is deˆcient in Eisai hyperbilirubinemic mutant rats (EHBR). J. Pharmacol. Exp. Ther., 280: 1304–1311 (1997). Murata, M., Tamai, I., Sai, Y., Nagata, O., Kato, H., Sugiyama, Y. and Tsuji, A.: Hepatobiliary transport kinetics of HSR-903, a new quinolone antibacterial agent. Drug Metab. Dispos., 26: 1113–1119 (1998). Sasabe, H., Tsuji, A. and Sugiyama, Y.: Carriermediated mechanism for the biliary excretion of the quinolone antibiotic grepa‰oxacin and its glucuronide in rats. J. Pharmacol. Exp. Ther., 284: 1033–1039 (1998). Chen, C., Scott, D., Hanson, E., Franco, J., Berryman, E., Volberg, M. and Liu, X.: Impact of Mrp2 on the biliary excretion and intestinal absorption of furosemide, probenecid, and methotrexate using Eisai hyperbilirubinemic rats. Pharm. Res., 20: 31–37 (2003). Yamazaki, M., Suzuki, H. and Sugiyama, Y.: Recent advances in carrier-mediated hepatic uptake and biliary excretion of xenobiotics. Pharm. Res., 13: 497–513 (1996). Konig, J., Nies, A. T., Cui, Y., Leier, I. and Keppler, D.: Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate speciˆcity, and MRP2-mediated drug resistance. Biochim. Biophys. Acta., 1461: 377–394 (1999). Keppler, D. and Konig, J.: Hepatic secretion of conjugated drugs and endogenous substances. Semin. Liver Dis., 20: 265–272 (2000). Keppler, D., Konig, J. and Buchler, M.: The canalicular multidrug resistance protein, cMRP W MRP2, a novel conjugate export pump expressed in the apical membrane of hepatocytes. Adv. Enzyme. Regul., 37: 321–333 (1997). Ito, K., Suzuki, H., Hirohashi, T., Kume, K., Shimizu, T. and Sugiyama, Y.: Molecular cloning of canalicular multispeciˆc organic anion transporter defective in EHBR. Am. J. Physiol., 272: G16–22 (1997). Drummond, G. S. and Kappas, A.: Sn-mesoporphyrin suppression of hyperbilirubinemia in EHBR W Eis rats, an animal model of Dubin-Johnson syndrome. Pharmacology. 56:158–164 (1998). Hosokawa, S., Tagaya, O., Mikami, T., Nozaki, Y., Kawaguchi, A., Yamatsu, K. and Shamoto, M.: A new rat mutant with chronic conjugated hyperbilirubinemia and renal glomerular lesions. Lab. Anim. Sci., 42: 27–34 (1992).

21)

22)

23)

24)

25)

26)

27)

28)

29)

30)

31)

32)

Suzuki, H. and Sugiyama, Y.: Single nucleotide polymorphisms in multidrug resistance associated protein 2 (MRP2 W ABCC2): its impact on drug disposition. Adv. Drug Deliv. Rev., 54: 1311–1331 (2002). Smith, P. F., Eydelloth, R. S., Grossman, S. J., Stubbs, R. J., Schwartz, M. S., Germershausen, J. I., Vyas, K. P., Kari, P. H. and MacDonald, J. S.: HMG-CoA reductase inhibitor-induced myopathy in the rat: cyclosporine A interaction and mechanism studies. J. Pharmacol. Exp. Ther., 257: 1225–1235 (1991). Omar, M. A., Wilson, J. P. and Cox, T. S.: Rhabdomyolysis and HMG-CoA reductase inhibitors. Ann. Pharmacother., 35: 1096–1107 (2001). Nakai, A., Nishikata, M., Uchida, T., Ichikawa, M. and Matsuyama, K.: Enhanced myopathy following administration of hypolipidemic agents under urethane anesthesia. Biol. Pharm. Bull., 20: 104–106 (1997). Matsuyama, K., Nakagawa, K., Nakai, A., Konishi, Y., Nishikata, M., Tanaka, H. and Uchida, T.: Evaluation of myopathy risk for HMG-CoA reductase inhibitors by urethane infusion method. Biol. Pharm. Bull., 25: 346–350 (2002). Pierno, S., De Luca, A., Tricarico, D., Roselli, A., Natuzzi, F., Ferrannini, E., Laico, M. and Camerino, D. C.: Potential risk of myopathy by HMG-CoA reductase inhibitors: a comparison of pravastatin and simvastatin eŠects on membrane electrical properties of rat skeletal muscle ˆbers. J. Pharmacol. Exp. Ther., 275: 1490–1496 (1995). Pierno, S., De Luca, A., Liantonio, A., Camerino, C. and Conte Camerino, D.: EŠects of HMG-CoA reductase inhibitors on excitation-contraction coupling of rat skeletal muscle. Eur. J. Pharmacol., 364: 43–48 (1999). Reijneveld, J. C., Koot, R. W., Bredman, J. J., Joles, J. A. and Bar, P. R.: DiŠerential eŠects of 3-hydroxy-3methylglutaryl-coenzyme A reductase inhibitors on the development of myopathy in young rats. Pediatr. Res., 39: 1028–1035 (1996). Murakami, Y., Yamazaki, K., Sakauchi, N., Ogasawara, H., Yamashita, N., Masuda, T. and Tauchi, K.: A one-month repeated oral dose toxicity study of methotrexate in unilaterally nephrectomized rats. J. Toxicol. Sci., 23 Suppl 5: 681–699 (1998). Gao, F., Tomitori, H., Igarashi, K. and Horie, T.: Correlation between methotrexate-induced intestinal damage and decrease in polyamine content. Life Sci., 72: 669–676 (2002). Nishizato, Y., Ieiri I., Suzuki, H., Kimura, M., Kawabata, K., Hirota, T., Takane, H., Irie, S., Kusuhara, H., Urasaki, Y., Urae, A., Higuchi, S., Otsubo, K. and Sugiyama, Y.: Polymorphisms of OATP-C (SLC21A6) and OAT3 (SLC22A8) genes: consequences for pravastatin pharmacokinetics. Clin. Pharmacol. Ther., 73: 554–565 (2003). Morimoto, K., Ueda, S., Seki, N., Igawa, Y., Kameyama, Y., Shimizu, A., Oishi, T., Hosokawa, M. and Chiba, K.: Candidate gene approach for the study of genetic factors involved in HMG-CoA reductase inhibitor-induced rhabdomyolysis. Eighteenth JSSX Annual Meeting, 8PE-32 (2003).

EHBR as an Animal Model for Toxicity of Organic Anions

33)

Nakai, D., Nakagomi, R., Furuta, Y., Tokui, T., Abe, T., Ikeda, T. and Nishimura, K.: Human liver-speciˆc organic anion transporter, LST-1, mediates uptake of

351

pravastatin by human hepatocytes. J. Pharmacol. Exp. Ther., 297: 861–867 (2001).