b Gene Disruption on Tissue Distribution in Mice

Drug Metab. Pharmacokin. 17 (5): 449–456 (2002).

Regular Article Metabolic Fate of Pitavastatin, a New Inhibitor of HMG-CoA Reductase—EŠect of cMOAT Deˆciency on Hepatobiliary Excretion b Gene Disruption on Tissue Distribution in Mice in Rats and of mdr1a W Hideki FUJINO, Iwao YAMADA, Syunsuke SHIMADA and Junji KOJIMA Tokyo New Drug Research Laboratories I, Kowa Company Ltd. Summary: Pitavastatin is a potent competitive inhibitor of HMG-CoA reductase. In the current study, to elucidate the hepatobiliary excretion of pitavastatin, we investigated the plasma concentration and biliab ry excretion of 14C-pitavastatin in EHBR. We also evaluated the distribution of pitavastatin in mdr1a W knockout mice by whole body autoradiography and quantitative radioassay. In view of the widespread clinical use of pitavastatin and the importance of drug-drug interaction, the inhibitory eŠect on Pgpmediated activation of ATPase was also investigated. No marked diŠerence was observed in the plasma concentration and biliary excretion of radioactivity between SDR and EHBR after dosing of 14Cpitavastatin. Little radioactive transfer into the brain was detected in mdr1a W b knockout mice and the ATPase activity of human Pgp was negligible in the presence of pitavastatin. Moreover, no inhibitory eŠect on the Pgp-mediated activation of ATPase by verapamil was found in the presence of pitavastatin over a wide concentration range. These results indicated that a cMOAT and Pgp-mediated transport mechanism did not play a major role in the distribution of pitavastatin.

Key words: pitavastatin; cMOAT; P-glycoprotein; EHBR and mdr1 this area of research has been partly due to the discovery of mutant rats such as the Eisai hyperbilirubinemic rat (EHBR) strain, which has an inherited deˆciency in the biliary excretion of organic anions. With the use of such animals, several organic anions and their conjugates have been reported to be excreted into bile via a primary active transporter, which is deˆcient in mutant rats.7,8) On the other hand, mdr1a W b double knockout mice carry a functional deˆciency in the blood brain barrier through disruption of the endogenous Pgp genes.9) This knockout mouse is applicable to research on the central nerve system, drug transport and the pharmacokinetics of several drugs.10) As of today, no studies have been published regarding the mechanism of the distribution and biliary excretion of pitavastatin. In the current study, we investigated the biliary excretion of pitavastatin across the canalicular membrane in EHBR. We also evaluated the pitavastatin distribution in the mdr1a W b knockout mouse by whole body autoradiography and quantitative radioassay. Moreover, the inhibitory eŠect on Pgp-mediated ATPase was also investigated.

Introduction Pitavastatin (NK-104) is a highly potent inhibitor of HMG-CoA reductase and causes a signiˆcant reduction in serum total cholesterol, low-density lipoprotein cholesterol and triglyceride levels in animals.1,2) In humans, the persistent eŠect on serum lipids and the safety of pitavastatin have been conˆrmed in clinical practice.3) In our previous study in rats, 14C-pitavastatin was selectively distributed to liver, a target organ of this drug.4) The maximum radioactivity in liver was approximately 54 times higher than that in plasma. A little radioactivity was transferred into the brain, accounting for less than 6z of that in plasma. On the other hand, almost all the radioactivity was excreted into feces and the renal handling was negligible in rats. The role and importance of active carrier systems in the transport of drugs across biological membranes are well recognized, but it is only in the past several years that speciˆc transporter proteins have been identiˆed and begun to be characterized.5) In particular, canalicular multispeciˆc organic anion transporter (cMOAT) and P-glyocoprotein (Pgp) play an important role in the hepatobiliary excretion of drugs.6) Recent progress in

Received; June 21, 2002, Accepted; September 18, 2002 To whom correspondence should be addressed: Hideki FUJINO, Ph.D., Tokyo New Drug Research Laboratories I, Kowa Company Ltd., 2-17-43 Noguchicho, Higashimurayama, Tokyo 189-0022, Japan. Tel. +81-42-391-6211, Fax. +81-42-395-0312, E-mail: h-fujino＠kowa.co.jp

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Materials and Methods Chemicals and reagents: Pitavastatin (M.W. 440.5), monocalcium bis[(3R,5S,6E)-7-[2-cyclopropyl4-(4-‰uorophenyl)-3-quinolyl]3,5-dihydroxy-6-hepteonate] and its metabolites (pitavastatin lactone, M-3, M-6, M-8 and M-11) which were determined by NMR and LC-MS spectrometry in the bile of animals after an intravenous infusion11) were synthesized by Nissan Chemical Industries, (Chiba, Japan). The pKa values of pitavastatin were 5.36 (nitrogen of quinoline ring) and 4.40 (carboxyl moiety of side chain), respectively. [Fluorobenzene-U-14C] pitavastatin was synthesized by Amersham Co., (Little Chalfort, UK). The speciˆc radioactivity of the labeled compound was 981 kBq W mg, and the radiochemical purity was more than 99z during the experimental period. The recombinant membrane derived from baculovirus expressing human Pgp (MDR1) and control membrane were purchased from GENTEST Co. Ltd. (Woburn, MA, USA). All other chemicals and reagents used were commercially available and of guaranteed purity. Animals: Male Sprague-Dawley rats (SDR) and male EHBR rats, weighing about 250 g (7 weeks old), were purchased from SLC Co., Ltd. (Shizuoka, Japan). b (+ W +)] and knockout Male control mice [mdr1a W mice [mdr1a W b („ W „)] with a pure FVB background, weighting about 28 g, were obtained from Taconic Farms (Germantown, NY). The rats and mice were acclimatized for at least 4 days, and were kept in a conC and relative trolled room at a temperature of 23±39 humidity of 55±15z. All animals were maintained on laboratory chow and fasted overnight prior to the drug administration, but had free access to water. Three or four animals were assigned to each test group. The animal experiments were carried out with the approval of the Animal Ethics Committee of the Kowa Company. Pharmacokinetic study in SDR and EHBR: 1) Plasma concentration of radioactivity; 14C-pitavastatin was intravenously administered into a tail vein of SDR kg (n＝3). Heparinized and EHBR at a dose of 1 mg W blood samples were taken from the jugular vein of rats for 4 hr. Plasma was separated after centrifugation of the blood sample. An aliquot of plasma (50 mL) was used for measuring radioactivity and the remainder used for the analysis of unchanged pitavastatin. The pharmacokinetic parameters were calculated from the individual plasma concentration-time curve of unchanged pitavastatin in each animal. The observed data were ˆtted in a biexponetial equation using a nonlinear least squares program, Win Nonlin (version 1.1; Pharsight, Palo Alto, CA). 2) Biliary excretion; Rats were surgically prepared with common bile duct cannulae under ether anesthesia.

After they awakened, 14C-pitavastatin was intravenously administered at a dose of 1 mg W kg and each animal was housed individually in a Bollman's cage (Natsume Seisakusyo, Tokyo, Japan). The bile was continuously collected until 6 h after dosing. The collection of the bile sample was performed in darkness to prevent the degradation of biliary metabolites. An aliquot of bile sample (50 mL) was used to measure the total radioactivity and the biliary metabolites were also investigated. To conˆrm the presence of phase-two reaction-mediated metabolites, the biliary samples were treated with enarylsulfatase zymatic hydrolysis using b-glucuronidase W (5000 ˆshman units and 80 units, respectively) at 379C for 3 h. The amounts of conjugates were calculated by subtracting the concentration of enzyme-untreated samples. b (+ W +) and Distribution of radioactivity in mdr1a W mdr1a W b („ W „): 1) Whole body autoradiography; The mouse was sacriˆced by ether inhalation at 15 min and 1, 6 and 24 hr after an intravenous dosing of 14 C-pitavastatin (1 mg W kg) for whole body autoradiography. Each animal was immediately frozen in a dry ice-hexane mixture. The frozen carcass was then embedded in 3z carboxy methyl cellulose on a microtome stage, held in a JUNG CRYOMACROCUT (Leica Instruments GmbH, Nussioch, Germany) and cut into about 30 mm slices at „209 C. The obtained sections were caught on tape (No. 810, Sumitomo 3M) and freeze-dried. The surface of the slices was covered with protective ˆlm (4 mm, Dia foil) and then exposed for 24 hr to a phosphor imaging plate (IP) and measured using a bioimaging analyzer (BAS-2500, Fuji Photo Film, Tokyo, Japan) for whole body autoradiograms. The scanning conditions were as follows: gradation 1024, resolution 50, latitude 5, and sensitivity 10000. 2) Quantiˆcation of radioactivity in tissue; The mice were anesthetized with ether inhalation and killed by exsanguinated from the abdominal vein at 6 h after administration. The following tissues were examined: the plasma, brain, heart, liver, kidney, adrenal and testis. The liver was cut to half size to use for measuring tissue concentrations and the remainder used for the analysis of metabolites. A plasma sample was obtained by centrifugation of the remaining blood. An aliquot of plasma and tissues was dissolved in tissue solubilizer (Soluene-350, Packard) and the radioactivity was measured. Human P-glycoprotein ATPase assay: A human Pgp mediated ATPase assay was performed according to previous reports with some modiˆcations.12,13) ATP assays were conducted in 96-well microtiter plates using human Pgp membrane at a concentration range of 1–120 mM of pitavastatin. The initial assay volume was 0.06 mL and incubation time was 20 min. Incubations were conducted in duplicate with and without the

b Knockout Mice Studies of Pitavastatin on EHBR and mdr1a W

Fig. 1. Plasma concentration of radioactivity and unchanged pitavastatin after intravenous administration of EHBR (1): SDR, (2): EHBR (dose: 1 mg W kg). Each points represents the mean±S.D. of three rats.

Table 1. Pharmacokinetic parameters of unchanged pitavastatin after intravenous administration to SDR and EHBR (dose: 1 mg W kg) Parameters 2a T1 W T1 W 2b Clp Vss AUC

(min) (min) (L W min W kg) (L W kg) mL) (min・mg W

SDR

EHBR

3.2±0.3 65.9±15.2 0.012±0.003 0.774±0.031 86.2±21.1

3.7±0.7 111.8±16.5 0.012±0.001 1.563±0.257 85.8±6.4

Each value represents the mean±S.D. of three rats.

presence of sodium vanadate. Verapamil was used as a positive control of Pgp substrate. The assay was stopped by the addition of 0.2 mL of sodium dodecyl developing reagent. The response was sulfate (SDS) W measured by absorbance at 800 nm and quantitated by comparison to a standard curve for potassium phosphate. The inhibitory eŠect of ATPase on verapamil control (120 mM) was also investigated using pitavastatin at a concentration range of 3–300 mM in parallel. Analytical methods: Total radioactivity was measured with a liquid scintillation counter (Tri-carb 1500, Packard Instrument Co., Meriden, CT) for 10 min after addition of a scintillation cocktail (Hionic-Fluor, Packard). The measurement of the radioactivity of pitavastatin and its metabolite was made according to the HPLCradioluminography method.4) The amount of unchanged drug and its metabolites was determined using BAS-2500 equipment. The radioactive metabolites were positively identiˆed by a comparison of retention times with authentic unlabeled standard. Results Pharmacokinetic study in SDR and EHBR:

1)

451

14

C-pitavastatin to SDR and

Plasma concentration of radioactivity; The plasma concentration of radioactivity and unchanged pitavastatin after intravenous administration to SDR and EHBR are shown in Fig. 1 and the pharmacokinetic parameters are given in Table 1. The composition of the radioactivity in plasma consisted mostly of the unchanged pitavastatin in both strains. A biexponential elimination of unchanged pitavastatin was observed with a terminal halflife value of 66 min in SDR and 119 min in EHBR. The Vss in SDR and EHBR was 0.77 and 1.56 L W kg, respectively. On the other hand, AUC in SDR and EHBR was 86.2 and 85.8 mg・min W mL, respectively. 2) Biliary excretion of pitavastatin and its metabolites; Typical HPLC radio-chromatograms of bile at 2–3 h after dosing to both strains of rats are shown in Fig. 2. In bile of both strains, mostly unchanged pitavastatin was found though pitavastatin lactone, M-3 (5-keto pitavastatin), M-11 (taurine conjugate of pentenoic acid derivative) and M-6 (pentenoic acid derivative) were also detected. After the enzymatic hydrolysis of bile, the appearance of M-6 was observed in both strains. Table 2 shows the cumulative biliary excretion of radioactivity in SDR and EHBR. In the case of SDR, the biliary excretion of radioactivity was 31.4z of dose within 1 h after dosing, 76.6z within 4 h, and 80.3z within 6 h. The cumulative biliary excretion of radioactivity in EHBR was 45.8z within 1 h after dosing, 82.6z within 4 hr, and 89.5z within 6 h. The unchanged pitavastatin accounted in the bile of SDR and EHBR for 57.8z and 74.1z of the dose until 6 h, respectively. The metabolites of pitavastatin, M-3, M-11 and M-6 glucuronide, accounted for 3.2, 4.9 and 5.4z of the dose in SDR, and 0.9, 2.9 and 3.3z in EHBR, respectively.

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Fig. 2. HPLC-radiochromatograms of bile samples at 2–3 h after intravenous administration of 14C-pitavastatin to SDR and EHBR. (1) SDR bile, (2) Enzymatic hydrolysis of SDR bile, (3) EHBR bile, (4) Enzymatic hydrolysis of EHBR bile.

Table 2.

Cumulative biliary excretion of pitavastatin and its metabolites after intravenous administration of 14C-pitavastatin in SDR and EHBR

Strain

Time

SDR

EHBR

(z of dose) Total Radioactivity

Pitavastatin

Lactone

M-11

M-6

M-6 glu.*

M-3

0–1 h 1–2 h 2–3 h 3–4 h 4–6 h

31.43±4.21 53.18±6.86 66.13±6.70 76.55±4.33 80.25±3.64

26.70±4.82 42.12±7.09 50.06±7.34 55.65±5.97 57.59±5.60

0.91±0.35 2.11±0.79 2.83±1.01 3.31±1.06 3.47±1.07

0.86±0.39 2.26±1.02 3.35±1.52 4.53±2.05 4.94±2.21

0.04±0.03 0.13±0.04 0.21±0.06 0.36±0.08 0.40±0.10

0.51±0.26 2.15±0.60 3.25±0.97 4.59±0.96 5.37±1.08

0.61±0.26 1.43±0.62 2.11±0.83 2.82±1.01 3.17±1.02

0–1 h 1–2 h 2–3 h 3–4 h 4–6 h

45.83±8.24 67.6±9.79 76.7±13.83 82.63±13.03 89.45±10.99

41.42±8.10 59.19±11.34 66.68±15.62 70.60±15.39 74.12±14.62

0.01±0.02 0.01±0.02 0.01±0.02 0.01±0.02 0.01±0.02

0.97±0.98 1.70±0.84 1.96±0.95 2.31±0.99 2.88±1.06

0.71±1.28 1.44±1.46 2.21±1.51 2.21±1.53 2.82±1.61

0.30±0.36 1.04±0.74 1.91±1.23 2.42±1.47 3.28±1.66

0.26±0.37 0.47±0.41 0.58±0.44 0.70±0.45 0.89±0.47

Each value represents the mean±S.D. of four rats. *: Glucuronide conjugate of M-6.

Distribution of radioactivity in mdr1a W b (+ W +) and b („ W „) mice: 1) Whole body autoradiomdr1a W grams; Whole body autoradiograms of mdr1a W b (+ W

+) and („ W „) mice after intravenous administration of 14C-pitavastatin are shown in Fig. 3. High levels of radioactivity were observed in the liver, gall bladder and

b Knockout Mice Studies of Pitavastatin on EHBR and mdr1a W

453

Fig. 3. Autoradiograms showing the distribution of radioactivity at various times after intravenous administration of 14C-pitavastatin to mdr1a W b knockout mice. 1. Brain, 2. Heart, 3. Liver, 4. Kidney, 5. Intestinal contents, 6. Testis, 7. Submaximal gland, 8. Brown fat, 9. Gall bladder.

intestinal tracts at all times after dosing in both mice. On the other hand, only trace levels of radioactivity were observed in the heart and kidney. The radioactivity

in brain and testis was fairly weak at all stages. Almost all of the radioactivity had disappeared from the organs and tissues at 24 h. In particular, the extent of the

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Table 3. Tissue concentration of radioactivity after intravenous administration of 14C-pitavastatin to mdr1a W b knockout mice g) Tissue concentration ( mg eq. W Time after dosing Tissue

6 hr

Ratio („ W „):(+ W +)

mdr1a W b (+ W +)

Plasma Brain Testis Heart Kidney Adrenal Liver

0.160±0.123 0.024±0.007 0.043±0.014 0.393±0.240 0.127±0.082 0.160±0.122 1.264±0.676

1.14 1.32 1.42 1.92 1.50 0.76 0.88

b („ W „) mdr1a W

Plasma Brain Testis Heart Kidney Adrenal Liver

0.182±0.096 0.031±0.014 0.061±0.018 0.756±0.359 0.190±0.138 0.121±0.036 1.107±0.644

Fig. 4. Concentration response for the stimulation of Pgp-mediated ATPase activity by pitavastatin and verapamil.

Each value represents the mean±S.D. of three mice.

Table 4. Concentration of unchanged pitavastatin and its metabolites in liver after intravenous administration of 14C-pitavastatin to mdr1a W b knockout mice Concentration ( mg W g) Pitavastatin M-3 mdr1a W b (+ W +) mdr1a W b („ W „)

0.441 0.403

M-8

M-6

M-11 Unknown*

0.320 0.015 0.286 0.121 0.236 0.060 0.165 0.093

0.035 0.059

Each value represents the mean of 3 liver samples. *: Unknown metabolites.

radioactive transfer into brain was fairly small in knockout mice independent of time after dosing. No marked diŠerence was found in the tissue distribution of b gene-disrupted mice. radioactivity in mdr1a W 2) Radioactivity in tissue; Table 3 shows the tissue concentration of radioactivity in mdr1a W b (+ W +) and „) mice at 6 h after intravenous administration of („ W 14 C-pitavastatin. The highest concentration was observed in the liver, being 6–8 times higher than in plasma, followed by the heart; being 2–4 times higher than in plasma in both mice. The concentrations in other tissues and organs were similar to or lower than that in the plasma. In particular, the brain and testis showed very b („ W „) mice. The concentration low levels in mdr1a W ratios of tissues between mdr1a W b (+ W +) and („ W „) were almost the same in both mice and a remarkable diŠerence was not observed. These results were consistent with the observation of whole body autoradiograms. b 3) Liver metabolites of pitavastatin on mdr1a W (+ W +) and mdr1a W b („ W „) mice; Table 4 shows the

concentration of unchanged pitavastatin and its metabolites in liver at 6 h after intravenous administration to mdr1a W b (+ W +) and („ W „). Unchanged pitavastatin was the major component with some pitavastatin metabolites in both mice. M-3 and M-6 were found as major metabolites. M-11 and M-8 (Propenoic acid derivative) were also detected in the liver as minor metabolites. However, the concentrations of these metabolites were lower than the concentration of unchanged pitavastatin. Activation of ATPase in Pgp membranes: The concentration responses for the stimulation of Pgp-mediated ATPase activity by pitavastatin or verapamil are shown in Fig. 4. There were no responses until 120 mM of pitavastatin. In the case of verapamil as a positive control of the Pgp membrane, saturating and nonsaturating concentration ranges were identiˆed in the ATPase assay. The apparent Km value of verapamil, demonstrated by a Lineweaver-Burk plot, was 27 mM. No marked diŠerence was observed in the ATPase activity of verapamil demonstrating that there was no inhibition of the Pgp-mediated activation of ATPase by pitavastatin over a wide concentration range (data not shown). Discussion The processes involved in metabolic biotransformation, especially those mediated by P450s, are frequently critical determinants in the disposition of drugs. Recently, it has been recognized that additional processes such as membrane-bound transport systems may also be similarly important.14) Biliary excretion is an important pathway for the elimination of xenobiotics, including drugs and their

Studies of Pitavastatin on EHBR and mdr1a W b Knockout Mice

metabolites. In our previous study, biliary excretion was recognized as a major elimination pathway for pitavastatin and its metabolites in rats and dogs.15,16) To gain a better understanding of pitavastatin hepatobiliary excretion and defects there in EHBR, we applied several approaches. Unchanged pitavastatin was the major component of plasma radioactivity in both 2 b and Vss of unstrains of rats. Although, the T1 W changed pitavastatin increased about twice in EHBR, a remarkable diŠerence in AUC value was not found after intravenous administration. Moreover, the radioactivity was mainly excreted into the bile and mostly unchanged pitavastatin was found in the bile of EHBR. Yamazaki et al. reported that the steady-state biliary excretion of pravastatin was markedly impaired in EHBRs compared with normal rats in an in vitro single-pass liver perfusion study, suggesting that biliary excretion of pravastatin was mediated mainly by cMOAT.17,18) However, our study showed that cMOAT was not critically involved in the hepatobiliary excretion of pitavastatin. Takikawa et al. also reported that two or more excretory pathways for organic anions exist at the canalicular membrane of EHBR other than the ATPdependent one.19) Further study is required to clarify the nature of the transport mechanism for pitavastatin. Ohmori et al. reported that the activity of rat-CYP3A2 to mediate the function of testosterone 6 b-hydroxylase was signiˆcantly decreased in EHBR as compared to control rats.20) CYP2C9 was the major enzyme responsible for the hydroxylation of pitavastatin in humans and pitavastatin was scarcely metabolized at all in rat hepatic microsomes.21) This report can explain the slight diŠerence in biliary metabolites between EHBR and SDR. Pgp has been extensively investigated with regard to the multidrug resistance phenomenon in tumor cells.9) In the body, Pgp is widely localized on the luminal surface of endothelial cells in brain and testis, adrenal cortex, canalicular membrane of hepatocytes, mucosa of small and large intestine and placenta.10,22) Pgp functions as an ATP-dependent eŒux pump, transporting many structurally unrelated xenobiotics out of cells. In our previous reports, little radioactive transfer was found in the brain or testis of male rats or in the fetus of pregnant rats implying that there was a carrier-mediated transporting mechanism for the tissue distribution of pitavastatin.4,23) In the current study, we investigated the distribution of 14C-pitavastatin using Pgp knockout mice. 14C-pitavastatin was mainly observed in the liver and little radioactive transfer was evident into brain or testis. Concentration ratios for tissues and metabolic patterns were the same in both mice. No evidence was found of a Pgp-mediated tissue distribution of b pitavastatin in whole body autoradiograms of mdr1a W knockout mice. Furthermore, the ATPase activity of

455

pitavastatin was negligible in human Pgp membrane compared with verapamil. These results indicated that the Pgp-mediated transport mechanism did not play a role in the distribution of pitavastatin. In the current study, pitavastatin did not inhibit the Pgp-mediated activation of ATPase by verapamil. Recently, an inhibitory eŠect on Pgp-mediated transport was reported for simvastatin, lovastatin and atorvastatin.24–26) Kusus et al. demonstrated that avoiding the use of drugs that are Pgp or CYP3A4 substrates reduces the risk of rhabdomyolysis caused by statins.27) These results indicate that the Pgp-mediated inhibition may be an additional reason for the clinical interaction of HMG-CoA reductase inhibitors with other drugs. The mechanism for transporting pitavastatin was not completely elucidated in the present study. However, our result suggests that the plasma level of pitavastatin would not be increased by the concomitant administration of cMOAT or Pgp inhibitors. Acknowledgments: The authors are grateful to Ms. Keiko Maekawa for technical assistance and Prof. Kenichi Miyamoto, Kanazawa Univ. for invaluable suggestion. References 1)

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