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Uptake Mechanism of Pitavastatin, a New Inhibitor of HMG-CoA Reductase, in Rat Hepatocytes
Drug Metab. Pharmacokin. 18 (4): 245–251 (2003).
Regular Article Uptake Mechanism of Pitavastatin, a New Inhibitor of HMG-CoA Reductase, in Rat Hepatocytes Syunsuke SHIMADA1, Hideki FUJINO1, Takashi MORIKAWA2, Matsuko MORIYASU2 and Junji KOJIMA1 1Tokyo
New Drug Research Laboratories, Kowa Company Ltd., Tokyo, Japan of Biochemistry & Immunochemistry Research Division, Panapharm Laboratories Co., Ltd., Kumamoto, Japan
2Department
Summary: To understand the mechanism underlying the highly liver-selective distribution of pitavastatin, uptake experiments were performed using rat hepatocytes. The uptake of pitavastatin into rat hepatocytes is carrier-mediated and involved nonspeciˆc diŠusion in the presence of Na＋. The michaelis constant (Km ) was 26.0 mmol W L, maximal uptake velocity (Vmax ) was 3124 pmol W min W mg protein, and non-speciˆc uptake (Pdif ) was 1.16 mL W min W mg protein. There were no remarkable diŠerences in these kinetic parameters between the presence and absence of Na＋. Experiments using metabolic inhibitors revealed that energy-dependent systems contribute to the uptake of pitavastatin in the liver. Some organic anions reduced the uptake into rat hepatocytes in a concentration-dependent manner. The observed rates of inhibition of pitavastatin uptake by BSP, TCA and pravastatin were compared with the predicted rates. The predicted values were calculated, assuming that BSP, TCA and pravastatin inhibit the uptake of pitavastatin in a competitive manner. The observed inhibition by BSP and TCA was similar to that predicted, but the observed inhibition by pravastatin was considerably less than that predicted. In conclusion, most of the pitavastatin taken up into the liver is transported by multiple carrier-mediated transporters such as Na＋-independent multispeciˆc anion transporters and energy-dependent transporters. In addition, these systems for pitavastatin may have features in common with the BSP and TCA transport system, and may partially involve the pravastatin transport system.
Key words: pitavastatin; HMG-CoA reductase inhibitor; rat hepatocyte; Na＋-independent multispeciˆc anion transporter; carrier-mediated uptake
these actions both in animals and in humans.5–7) In our previous study in rats, 14C-pitavastatin was selectivity distributed into the liver, a target organ of this drug.8) The maximum radioactivity in liver was approximately 54-times that in plasma and analysis of the metabolites revealed that most of the radioactivity in liver was from unchanged pitavastatin.8) Other statins such as pravastatin, lovastatin, simvastatin, ‰uvastatin, atorvastatin, and cerivastatin are also known to be distributed mainly in liver.9–12) Also, these statins were taken up into hepatocytes by not only simple diŠusion but also active transport systems, including Na＋-independent multispeciˆc transport systems and Na＋- and ATPdependent transport systems.13–17) To date, no studies have been published regarding the mechanism of the hepatic distribution of pitavastatin. In the present study, the potential active transport
Introduction In general, knowing the metabolic character of a drug can greatly assist in predicting its e‹cacy and interactions that may be clinically relevant.1) The importance of carrier-mediated transport systems to the distribution of a drug is well recognized2) and the mechanism of the distribution and the drug-drug interaction via transporters have been elucidated.3,4) These reports indicate that the transporter may provide additional information on clinical e‹cacy and interaction. Pitavastatin is a potent and selective inhibitor of HMG-CoA reductase (statin) which increases the uptake of blood low-density lipoprotein (LDL)-cholesterol into the liver by enhancing the activity of the LDL receptor. The drug powerfully reduces the serum levels of total cholesterol, LDL-cholesterol and triglyceride by
Received; April 26, 2003, Accepted; August 12, 2003 To whom correspondence should be addressed : Syunsuke SHIMADA, Tokyo New Drug Research Laboratories, Kowa Company Ltd., 2-17-43 Noguchicho, Higashimurayama, Tokyo 189-0022, Japan. Tel. ＋81-42-391-6211, Fax. ＋81-42-395-0312, E-mail: s-simada＠kowa.co.jp
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Fig. 1. Chemical structure of [ 14C]Pitavastatin. An asterisk denotes the 14C-labeled position.
mechanisms for the uptake of pitavastatin into liver were investigated using primary cultures of rat hepatocytes and some inhibitors for the carrier-mediated transporter. Materials and Methods Chemicals and Reagents: [ 14C]Pitavastatin (Fig. 1) was synthesized by Amersham Biosciences UK Ltd. (Bucks., UK). The speciˆc radioactivity of the labeled compound was 981 kBq W mg, and the radiochemical purity was more than 98z during the experimental period. Pitavastatin was synthesized by Nissan Chemical Industries, Ltd. (Chiba, Japan). Pravastatin was synthesized at Kowa Co. Ltd. (Tokyo, Japan). Taurocholic acid (TCA), carbonylcyanimide-4tri‰uoromethoxy-phenylhydrazone (FCCP), bromosulfophthalein (BSP) and 8-anilino-1-naphthalene sulfonic acid (ANS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Cholic acid (CA), 2,4-dinitrophenol (DNP), ethylene glycol-bis-( baminoethylether)-N,N,N?,N?-tetraacetic acid (EGTA) and N-2-hydroxyethylpiperazine-N?-2-ethanesulfonic acid (HEPES) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals were of reagent grade. Krebs-Henseleit buŠer was prepared as reported,18) with 142 mmol W L L NaHCO3, 4.83 mmol W L KCl, 0.96 NaCl, 23.8 mmol W mmol W L KH2 PO4, 1.20 mmol W L MgSO4, 12.5 mmol W L HEPES, 5.0 mmol W L glucose and 1.53 mmol W L CaCl2 (pH 7.3). The Na＋-free Krebs-Henseleit buŠer contains choline chloride and choline bicarbonate instead of NaCl and NaHCO3 in Krebs-Henseleit buŠer, respectively. [ 14C]pitavastatin and other reference compounds were dissolved in Krebs-Henseleit buŠer without HEPES. Cell preparation: Rat hepatocytes were prepared as described by Horiuti et al.18) Brie‰y, male SpragueDawley (SD) rats (6–8 weeks, 240¿320 g; Charles River Japan, Inc., Kanagawa, Japan) were anesthetized with an intraperitoneal injection of pentobarbitone sodium, and the portal vein was cannulated. The liver was perfused via the cannula with Ca＋, Mg＋-free Hanks'
solution containing 0.5 mmol W L EGTA (pH 7.2) and 0.05z collagenase. After perfusion, the liver was resected and the parenchymal cells were isolated. The cells were dispersed in Williams' medium E containing 10z fetal bovine serum. Approximately 1×106 cells W well were placed onto collagen-coated six-well plates (Greiner GmbH, Frickenhausen, Germany) and were cultured at 379C for 3–4 h. Cell viability was routinely checked using the trypan blue exclusion test, and the cells with viability of more than 90z were used for the experiments. Uptake-study of pitavastatin: Hepatocytes were preincubated in Krebs-Henseleit buŠer for 10 min at either 379C or 49 C, and [ 14C]pitavastatin (1 mmol W L) was added to the medium. After incubation for 1 to 60 min, the reaction medium was placed in a vial. Subsequently, the cells were washed four times with icecold phosphate buŠer. Thereafter, they were solubilized L NaOH, and the lysate was placed in 0.7 mL of 1 mol W in a vial. Both medium and cell lysate radioactivity were measured by a liquid scintillation counter (Packard Instrument Co., CT). In a parallel experiment, the protein concentration was determined by Bradford's method using bovine serum albumin as a standard.19) The concentration-dependence of pitavastatin uptake was investigated after incubation for 2 min in KrebsHenseleit buŠer at either 379C or 49 C. In addition, to estimate the Na＋-independence of pitavastatin uptake, the experiment was performed in the absence of external Na＋, using Na＋-free Krebs-Henseleit buŠer. EŠect of metabolic inhibitors and organic anions on pitavastatin uptake into hepatocytes: Various inhibitors such as FCCP, DNP, ANS, BSP, CA, TCA, and pravastatin were added to hepatocytes 5 min (3 min for FCCP) prior to the addition of [ 14C]pitavastatin (2 mmol W L). The concentrations of inhibitors were 10, L (2 mmol W L for FCCP). The extent 100 and 1000 mmol W of inhibition was shown as a ratio against the control uptake of pitavastatin for 2 min. Determination of kinetic parameters: The uptake of pitavastatin at 379 C or 49C for 2 min was used to determine the kinetic parameters because the initial velocity of the uptake is linear during this incubation period. The kinetic parameters were estimated according to the following equations; (Km＋[S])＋Pdif･[S] V0,379C＝(Vmax･[S]) W V0,49C＝Pdif･[S]
(Eq. 1) (Eq. 2)
where V0,379C and V0,49C represent the initial uptake min W mg protein) of pitavastatin at 379 C velocity (pmol W and 49C, respectively, [S] is the concentration ( mmol W L) of pitavastatin in the medium, Vmax is the maximum uptake velocity (pmol W min W mg protein), Km is the Michaelis constant ( mmol W L), and Pdif is the nonspeciˆc uptake clearance (mL W min W mg protein). The kinetic
Uptake Mechanism of Pitavastatin in Rat Hepatocytes
Fig. 2. Time course of [ 14C]pitavastatin (1 mmol W L) uptake by isolated rat hepatocytes in the presence of Na＋ . Closed and open circles represent uptake of pitavastatin at 379C and 49C, respectively. Each point and vertical bar give the mean±S.D. from 3 determinations.
parameters of pitavastatin at 379C and 49 C were estimated by curve ˆtting to Eq. 1 and Eq. 2, respectively, using a MULTI program.20) Input data was not weighted, and the algorithm used for the ˆtting was the Damping Gauss Newton Method. Analytical methods: The radioactivity of pitavastatin was measured by HPLC-radioluminography.8) The amount of pitavastatin was determined using a BAS-2500. The radioactive pitavastatin was positively identiˆed by a comparison of retention times with authentic unlabeled standard. Statistical analyses: Results are expressed as the mean±S.D. and statistical analyses were performed using Student's t test or Dunnet's test for multiple comparisons with pº0.05 as the minimum level of signiˆcance. Results Time course of pitavastatin uptake: Figure 2 shows the time-dependent uptake of [ 14C]pitavastatin in rat hepatocytes at 49 C and 379C. The uptake at 379C was almost linear up to 2 min, and reached equilibrium after 20 min of incubation. The uptake at 49C was almost linear up to 60 min, and considerably less than that at 379 C. Concentration-dependence of pitavastatin uptake in the presence or absence of Na＋: Figure 3 shows the uptake velocity of [ 14C]pitavastatin in the presence or absence of Na＋ at 49C and 379C. The uptake velocity at C in both conditions was saturated at a high concen379 trations. The Km, Vmax, and Pdif for the uptake of pitavastatin in the presence of Na＋ were found to be 26.0 mmol W L, 3124 pmol W min W mg protein and 1.16 mL W min W mg protein, respectively. The corresponding parameters for the uptake of pitavastatin in the absence
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Fig. 3. Concentration-dependency of [ 14C]pitavastatin uptake by isolated rat hepatocytes for 2 min in the presence and absence of Na＋ . , the uptake in the presence of Na＋ at 49 C; , the uptake in the presence of Na＋ at 379C; #, the uptake in the absence of Na＋ at 49C; $, the uptake in the absence of Na＋ at 379 C. Obtained kinetic L, maxparameters are as follows: Michaelis content (Km ) 26.0 mmol W imal uptake rate (Vmax ) 3124 pmol W min W mg protein, and Pdif 1.16 mL W min W mg protein when Na＋ was present. Km 21.0 mmol W L, Vmax 2297 min W mg protein, and Pdif 1.12 mL W min W mg protein for Na＋-free pmol W experiment.
Fig. 4. Inhibition by FCCP and DNP of [ 14C]pitavastatin (2 mmol W L) uptake into rat hepatocytes. Closed column, untreated inhibitor. Dotted and open columns, treated with FCCP (2 mmol W L) and DNP (10, 100, 1000 mmol W L), respectively. Each column and vertical bar gives the mean±S.D. from 3 determinations. Signiˆcant diŠerences were analyzed with Student's t-test (#: pº0.01 , ##: pº0.001).
of Na＋ were found to be 21.0 mmol W L, 2297 pmol W min W mg protein and 1.12 mL W min W mg protein, respectively. There were no practical diŠerences in the kinetic parameters between the presence and absence of Na＋. EŠect of metabolic inhibitors, organic anions and pravastatin on pitavastatin uptake: As shown in Fig. 4, in the presence of a metabolic inhibitor, FCCP at 2 mmol W L or DNP at 1000 mmol W L, the velocity of the uptake of pitavastatin into rat hepatocytes was inhibited by 19.4z and 38.6z, respectively. The velocity of the uptake was inhibited in a concentration-dependent manner by organic anions such as ANS, BSP, CA and TCA, and also by pravastatin (Fig. 5). In the presence of ANS (10, 100 and 1000 mmol
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Fig. 5. Inhibition by ANS, BSP, CA, TCA and pravastatin of [ 14C]Pitavastatin (2 mmol W L) uptake into rat hepatocytes. Each column and vertical bar gives the mean±S.D. from 3 determinations. Asterisk represents Na＋ -free experiment. Signiˆcant diŠerences were analyzed using Dunnet's multiple comparison (#: pº0.001).
L) and BSP (10, 100 and 1000 mmol W L), it was inhibitW ed 12.3, 45.7 and 81.2z and 38.1, 74.8 and 92.5z, respectively. Similarly, in the presence of bile acid, CA and TCA at 10, 100 and 1000 mmol W L, the velocity of the uptake was inhibited 12.0, 54.5 and 82.1z and 5.7, L, 47.2 and 80.0z, respectively. At 100 and 1000 mmol W pravastatin known as a substrate of the rat organic anion transporting polypeptides (roatps), also reduced the uptake velocity of pitavastatin by 27.0 and 64.9z, respectively. In the absence of Na＋, ANS, BSP, CA, L inhibited the TCA, and pravastatin at 1000 mmol W velocity of the uptake 90.0, 93.3, 85.0, 84.2, and 76.4z, respectively. Discussion It has been reported that pitavastatin is predominantly distributed in the liver and mostly excreted through the bile in rats.8) We have previously found that mdr-1 and mrp-2 transporters, which play a major role in the biliary excretion of some other statins,21,22) were scarcely involved in the biliary excretion and the brain eŒux of pitavastatin.23) As little information was available on the mechanisms for the uptake of pitavastatin into liver, we performed experiments using rat hepatocytes. In the present study, more than 70z of the radioactivity in rat hepatocytes was identiˆed as unchanged pitavastatin after incubation at 379C for 60 min (Fig. 6). Therefore, the majority of the radioactivity measured in this study was evaluated as unchanged pitavastatin rather than metabolites. Further, according to the concentration-dependent experiments in the presence of Na＋, the analysis of uptake kinetics provided Vmax W Km (120 mL W min W mg protein) as saturable component parameters and Pdif (1.16 mL W min W mg
Fig. 6. Representive radio-chromatogram of the medium fraction after 60 min incubation with rat hepatocytes at 379C.
protein) as a nonspeciˆc diŠusion parameter. The Vmax W Km of pitavastatin was approximately three to six times larger than that of other statins, cerivastatin (44.4 mL W min W mg protein),16) ‰uvastatin (23.1 mL W min W mg pro17) tein) and pravastatin (18.8 mL W min W mg protein).14) The large Vmax W Km support our previous ˆnding in vivo8) that pitavastatin is selectively distributed to rat liver. The contribution of carrier-mediated uptake (CLcarrier ) to the whole uptake (CLwhole ) at a low concentration was calculated to be 0.99. The maximum plasma concentration of pitavastatin in rats (1 mg W kg) was reported to be 0.5 mmol W L.8) Even without considering protein binding, it was lower than the Km. These results suggest that the carrier-mediated uptake is a major function at the pharmacological level. There have been reports of several mechanisms responsible for the transport of compounds into hepatocytes, including Na＋-dependent and independent trans-
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Uptake Mechanism of Pitavastatin in Rat Hepatocytes
porters such as oatp1, 2 and 4. It is known that Na＋ taurocholate co-transporting polypeptide (Ntcp) transports TCA in a Na＋-dependent manner in rat liver.24,25) To investigate the Na＋-dependent uptake of pitavastatin, experiments on the uptake were performed in the presence and absence of Na＋. There were no remarkable diŠerences in the uptake of pitavastatin between the presence and absence of Na＋. These ˆndings suggest that pitavastatin is little transported by any Na＋-dependent transporters or by Ntcp. ANS is one of the organic anion reagents, which was taken up into hepatocytes in an ATP-dependent manner.26) As shown in Fig. 5, the uptake of pitavastatin in the hepatocytes was inhibited in a concentration-dependent manner by ANS. Similarly, in the presence of metabolic inhibitors such as FCCP and DNP, which have been reported to reduce cellular ATP content,14,27) the uptake of pitavastatin was inhibited at a high concentration. This suppression of the uptake of pitavastaL FCCP (19.4z) is less than that for the tin by 2 mmol W uptake of pravastatin (85z).14) In addition, the suppression by 1000 mmol W L DNP (38.6z) was also less than that for cerivastatin (59.3z),16) and more than that for ‰uvastain (20z).17) These results indicate that energydependent systems contribute less to the uptake of pitavastatin than the uptake of pravastatin and cerivastatin. Inhibitors of rat organic anion transporting polypeptides (roatps) such as BSP, CA and TCA reduced the uptake of pitavastatin in a concentration-dependent manner.28–30) Further, a HMG-CoA reductase, pravastatin, also reduced the uptake. Pravastatin underwent rat organic anion transporting polypeptide 2 (roatp2)mediated transport in roatp2 cRNA-injected oocytes.15) However, kouzuki et al. reported that pravastatin did not undergo roatp1-mediated transport in COS-7.29) If pitavastatin is taken up through the same transporters as pravastatin, pravastatin would competitively inhibit the uptake of pitavastatin. The rate of inhibition [100× (1－Vi W V0 )] which suggests the extent of the reduction in pitavastatin uptake can be estimated using to the following equations, regarding the Km for pravastatin in the uptake experiment in isolated rat hepatocytes (29.1 mmol W L)14) as the inhibition constant (Ki ). (Km＋[S])＋Pdif･[S] V0＝Vmax･[S] W
(Eq. 3)
Vi＝Vmax･[S]〔 Ki )＋[S]〕＋Pdif･[S] WKm･(1＋ [i] W
(Eq. 4)
where [S] and [i] are the concentration of substrate and inhibitor, respectively. The predicted inhibition rates, when pravastatin was used at concentrations of 10, 100 and 1000 mmol W L, were 23.9z, 75.4z and 96.0z, respectively (Table 1). These predicted values were considerably higher than the observed rates and showed that pravastatin partially inhibits the uptake of
Table 1. Comparison of the predicted and observed inhibition for pitavastatin Inhibitor
L) Conc. ( mmol W
Inhibition rate Predicted (z)
Observed (z)
BSP
10 100 1000
59.3 92.8 98.3
38.1 74.8 92.5
TCA
10 100 1000
21.1 72.3 95.4
5.7 47.2 80.0
Pravastatin
10 100 1000
23.9 75.4 96.0
0.0 27.0 64.9
pitavastatin into hepatocytes. In the same way, the predicted inhibition of pitavastatin uptake by BSP and TCA was estimated (Table 1) using reported Km values L) and TCA (34.3 mmol W L) from of BSP (6.2 mmol W uptake experiments into isolated and cultured rat hepatocytes, respectively, instead of their Ki in Eq. 4.31,32) The predicted rates of inhibition by these inhibitors were similar to the observed rates. Recent reports showed that roatp1, 2, and 4 and moat1 were highly expressed in rat liver.33,34) BSP and TCA are taken up into cells through these multiple transporters.34,35) Therefore, these multiple transporters may be responsible for the active transport of pitavastatin into rat hepatocytes. In conclusion, the highly liver-selective distribution of pitavastatin is mainly due to carrier-mediated uptake via several Na＋-independent multispeciˆc anion transporters. The clearance of carrier-mediated hepatic uptake was greater than that for pravastatin, ‰uvastatin and cerivastatin. In addition, the systems involved in the uptake of pitavastatin may have features in common with the BSP and TCA transport system, and may partially include the pravastatin transport system. Acknowledgments: The authors are grateful to Dr. T. Kanke and Mr. M. Hirano for helpful suggestions. References 1)
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Uptake Mechanism of Pitavastatin in Rat Hepatocytes
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Li, N., Hartley, D. P., Cherrington, N. J. and Klaassen, C. D.: Tissue expression, ontogeny, and inducibility of rat organic anion transporting polypeptide 4. J. Pharmacol. Exp. Ther., 301: 551–560 (2002). Nishio, T., Adachi, H., Nakagomi, R., Tokui, T., Sato, E., Tanemoto, M., Fujiwara, K., Okabe, M., Onogawa, T., Suzuki, T., Nakai, D., Shiiba, K., Suzuki, M., Ohtani, H., Kondo, Y., Unno, M., Ito, S., Iinuma, K., Nunoki, K., Matsuno, S. and Abe, T.: Molecular identiˆcation of a rat novel organic anion transporter moat1, which transports prostaglandin D(2), leukotriene C(4), and taurocholate. Biochem. Biophys. Res. Commun., 275: 831–838 (2000). Cattori, V., van. Montfoort, J. E., Stieger, B., Landmann, L., Meijer, D. K., Winterhalter, K. H., Meier, P. J. and Hagenbuch, B.: Localization of organic anion transporting polypeptide 4 (Oatp4) in rat liver and comparison of its substrate speciˆcity with Oatp1, Oatp2 and Oatp3. P‰ugers Arch., 443: 188–195 (2001).
Regular Article Uptake Mechanism of Pitavastatin, a New Inhibitor of HMG-CoA Reductase, in Rat Hepatocytes Syunsuke SHIMADA1, Hideki FUJINO1, Takashi MORIKAWA2, Matsuko MORIYASU2 and Junji KOJIMA1 1Tokyo
New Drug Research Laboratories, Kowa Company Ltd., Tokyo, Japan of Biochemistry & Immunochemistry Research Division, Panapharm Laboratories Co., Ltd., Kumamoto, Japan
2Department
Summary: To understand the mechanism underlying the highly liver-selective distribution of pitavastatin, uptake experiments were performed using rat hepatocytes. The uptake of pitavastatin into rat hepatocytes is carrier-mediated and involved nonspeciˆc diŠusion in the presence of Na＋. The michaelis constant (Km ) was 26.0 mmol W L, maximal uptake velocity (Vmax ) was 3124 pmol W min W mg protein, and non-speciˆc uptake (Pdif ) was 1.16 mL W min W mg protein. There were no remarkable diŠerences in these kinetic parameters between the presence and absence of Na＋. Experiments using metabolic inhibitors revealed that energy-dependent systems contribute to the uptake of pitavastatin in the liver. Some organic anions reduced the uptake into rat hepatocytes in a concentration-dependent manner. The observed rates of inhibition of pitavastatin uptake by BSP, TCA and pravastatin were compared with the predicted rates. The predicted values were calculated, assuming that BSP, TCA and pravastatin inhibit the uptake of pitavastatin in a competitive manner. The observed inhibition by BSP and TCA was similar to that predicted, but the observed inhibition by pravastatin was considerably less than that predicted. In conclusion, most of the pitavastatin taken up into the liver is transported by multiple carrier-mediated transporters such as Na＋-independent multispeciˆc anion transporters and energy-dependent transporters. In addition, these systems for pitavastatin may have features in common with the BSP and TCA transport system, and may partially involve the pravastatin transport system.
Key words: pitavastatin; HMG-CoA reductase inhibitor; rat hepatocyte; Na＋-independent multispeciˆc anion transporter; carrier-mediated uptake
these actions both in animals and in humans.5–7) In our previous study in rats, 14C-pitavastatin was selectivity distributed into the liver, a target organ of this drug.8) The maximum radioactivity in liver was approximately 54-times that in plasma and analysis of the metabolites revealed that most of the radioactivity in liver was from unchanged pitavastatin.8) Other statins such as pravastatin, lovastatin, simvastatin, ‰uvastatin, atorvastatin, and cerivastatin are also known to be distributed mainly in liver.9–12) Also, these statins were taken up into hepatocytes by not only simple diŠusion but also active transport systems, including Na＋-independent multispeciˆc transport systems and Na＋- and ATPdependent transport systems.13–17) To date, no studies have been published regarding the mechanism of the hepatic distribution of pitavastatin. In the present study, the potential active transport
Introduction In general, knowing the metabolic character of a drug can greatly assist in predicting its e‹cacy and interactions that may be clinically relevant.1) The importance of carrier-mediated transport systems to the distribution of a drug is well recognized2) and the mechanism of the distribution and the drug-drug interaction via transporters have been elucidated.3,4) These reports indicate that the transporter may provide additional information on clinical e‹cacy and interaction. Pitavastatin is a potent and selective inhibitor of HMG-CoA reductase (statin) which increases the uptake of blood low-density lipoprotein (LDL)-cholesterol into the liver by enhancing the activity of the LDL receptor. The drug powerfully reduces the serum levels of total cholesterol, LDL-cholesterol and triglyceride by
Received; April 26, 2003, Accepted; August 12, 2003 To whom correspondence should be addressed : Syunsuke SHIMADA, Tokyo New Drug Research Laboratories, Kowa Company Ltd., 2-17-43 Noguchicho, Higashimurayama, Tokyo 189-0022, Japan. Tel. ＋81-42-391-6211, Fax. ＋81-42-395-0312, E-mail: s-simada＠kowa.co.jp
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Fig. 1. Chemical structure of [ 14C]Pitavastatin. An asterisk denotes the 14C-labeled position.
mechanisms for the uptake of pitavastatin into liver were investigated using primary cultures of rat hepatocytes and some inhibitors for the carrier-mediated transporter. Materials and Methods Chemicals and Reagents: [ 14C]Pitavastatin (Fig. 1) was synthesized by Amersham Biosciences UK Ltd. (Bucks., UK). The speciˆc radioactivity of the labeled compound was 981 kBq W mg, and the radiochemical purity was more than 98z during the experimental period. Pitavastatin was synthesized by Nissan Chemical Industries, Ltd. (Chiba, Japan). Pravastatin was synthesized at Kowa Co. Ltd. (Tokyo, Japan). Taurocholic acid (TCA), carbonylcyanimide-4tri‰uoromethoxy-phenylhydrazone (FCCP), bromosulfophthalein (BSP) and 8-anilino-1-naphthalene sulfonic acid (ANS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Cholic acid (CA), 2,4-dinitrophenol (DNP), ethylene glycol-bis-( baminoethylether)-N,N,N?,N?-tetraacetic acid (EGTA) and N-2-hydroxyethylpiperazine-N?-2-ethanesulfonic acid (HEPES) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals were of reagent grade. Krebs-Henseleit buŠer was prepared as reported,18) with 142 mmol W L L NaHCO3, 4.83 mmol W L KCl, 0.96 NaCl, 23.8 mmol W mmol W L KH2 PO4, 1.20 mmol W L MgSO4, 12.5 mmol W L HEPES, 5.0 mmol W L glucose and 1.53 mmol W L CaCl2 (pH 7.3). The Na＋-free Krebs-Henseleit buŠer contains choline chloride and choline bicarbonate instead of NaCl and NaHCO3 in Krebs-Henseleit buŠer, respectively. [ 14C]pitavastatin and other reference compounds were dissolved in Krebs-Henseleit buŠer without HEPES. Cell preparation: Rat hepatocytes were prepared as described by Horiuti et al.18) Brie‰y, male SpragueDawley (SD) rats (6–8 weeks, 240¿320 g; Charles River Japan, Inc., Kanagawa, Japan) were anesthetized with an intraperitoneal injection of pentobarbitone sodium, and the portal vein was cannulated. The liver was perfused via the cannula with Ca＋, Mg＋-free Hanks'
solution containing 0.5 mmol W L EGTA (pH 7.2) and 0.05z collagenase. After perfusion, the liver was resected and the parenchymal cells were isolated. The cells were dispersed in Williams' medium E containing 10z fetal bovine serum. Approximately 1×106 cells W well were placed onto collagen-coated six-well plates (Greiner GmbH, Frickenhausen, Germany) and were cultured at 379C for 3–4 h. Cell viability was routinely checked using the trypan blue exclusion test, and the cells with viability of more than 90z were used for the experiments. Uptake-study of pitavastatin: Hepatocytes were preincubated in Krebs-Henseleit buŠer for 10 min at either 379C or 49 C, and [ 14C]pitavastatin (1 mmol W L) was added to the medium. After incubation for 1 to 60 min, the reaction medium was placed in a vial. Subsequently, the cells were washed four times with icecold phosphate buŠer. Thereafter, they were solubilized L NaOH, and the lysate was placed in 0.7 mL of 1 mol W in a vial. Both medium and cell lysate radioactivity were measured by a liquid scintillation counter (Packard Instrument Co., CT). In a parallel experiment, the protein concentration was determined by Bradford's method using bovine serum albumin as a standard.19) The concentration-dependence of pitavastatin uptake was investigated after incubation for 2 min in KrebsHenseleit buŠer at either 379C or 49 C. In addition, to estimate the Na＋-independence of pitavastatin uptake, the experiment was performed in the absence of external Na＋, using Na＋-free Krebs-Henseleit buŠer. EŠect of metabolic inhibitors and organic anions on pitavastatin uptake into hepatocytes: Various inhibitors such as FCCP, DNP, ANS, BSP, CA, TCA, and pravastatin were added to hepatocytes 5 min (3 min for FCCP) prior to the addition of [ 14C]pitavastatin (2 mmol W L). The concentrations of inhibitors were 10, L (2 mmol W L for FCCP). The extent 100 and 1000 mmol W of inhibition was shown as a ratio against the control uptake of pitavastatin for 2 min. Determination of kinetic parameters: The uptake of pitavastatin at 379 C or 49C for 2 min was used to determine the kinetic parameters because the initial velocity of the uptake is linear during this incubation period. The kinetic parameters were estimated according to the following equations; (Km＋[S])＋Pdif･[S] V0,379C＝(Vmax･[S]) W V0,49C＝Pdif･[S]
(Eq. 1) (Eq. 2)
where V0,379C and V0,49C represent the initial uptake min W mg protein) of pitavastatin at 379 C velocity (pmol W and 49C, respectively, [S] is the concentration ( mmol W L) of pitavastatin in the medium, Vmax is the maximum uptake velocity (pmol W min W mg protein), Km is the Michaelis constant ( mmol W L), and Pdif is the nonspeciˆc uptake clearance (mL W min W mg protein). The kinetic
Uptake Mechanism of Pitavastatin in Rat Hepatocytes
Fig. 2. Time course of [ 14C]pitavastatin (1 mmol W L) uptake by isolated rat hepatocytes in the presence of Na＋ . Closed and open circles represent uptake of pitavastatin at 379C and 49C, respectively. Each point and vertical bar give the mean±S.D. from 3 determinations.
parameters of pitavastatin at 379C and 49 C were estimated by curve ˆtting to Eq. 1 and Eq. 2, respectively, using a MULTI program.20) Input data was not weighted, and the algorithm used for the ˆtting was the Damping Gauss Newton Method. Analytical methods: The radioactivity of pitavastatin was measured by HPLC-radioluminography.8) The amount of pitavastatin was determined using a BAS-2500. The radioactive pitavastatin was positively identiˆed by a comparison of retention times with authentic unlabeled standard. Statistical analyses: Results are expressed as the mean±S.D. and statistical analyses were performed using Student's t test or Dunnet's test for multiple comparisons with pº0.05 as the minimum level of signiˆcance. Results Time course of pitavastatin uptake: Figure 2 shows the time-dependent uptake of [ 14C]pitavastatin in rat hepatocytes at 49 C and 379C. The uptake at 379C was almost linear up to 2 min, and reached equilibrium after 20 min of incubation. The uptake at 49C was almost linear up to 60 min, and considerably less than that at 379 C. Concentration-dependence of pitavastatin uptake in the presence or absence of Na＋: Figure 3 shows the uptake velocity of [ 14C]pitavastatin in the presence or absence of Na＋ at 49C and 379C. The uptake velocity at C in both conditions was saturated at a high concen379 trations. The Km, Vmax, and Pdif for the uptake of pitavastatin in the presence of Na＋ were found to be 26.0 mmol W L, 3124 pmol W min W mg protein and 1.16 mL W min W mg protein, respectively. The corresponding parameters for the uptake of pitavastatin in the absence
247
Fig. 3. Concentration-dependency of [ 14C]pitavastatin uptake by isolated rat hepatocytes for 2 min in the presence and absence of Na＋ . , the uptake in the presence of Na＋ at 49 C; , the uptake in the presence of Na＋ at 379C; #, the uptake in the absence of Na＋ at 49C; $, the uptake in the absence of Na＋ at 379 C. Obtained kinetic L, maxparameters are as follows: Michaelis content (Km ) 26.0 mmol W imal uptake rate (Vmax ) 3124 pmol W min W mg protein, and Pdif 1.16 mL W min W mg protein when Na＋ was present. Km 21.0 mmol W L, Vmax 2297 min W mg protein, and Pdif 1.12 mL W min W mg protein for Na＋-free pmol W experiment.
Fig. 4. Inhibition by FCCP and DNP of [ 14C]pitavastatin (2 mmol W L) uptake into rat hepatocytes. Closed column, untreated inhibitor. Dotted and open columns, treated with FCCP (2 mmol W L) and DNP (10, 100, 1000 mmol W L), respectively. Each column and vertical bar gives the mean±S.D. from 3 determinations. Signiˆcant diŠerences were analyzed with Student's t-test (#: pº0.01 , ##: pº0.001).
of Na＋ were found to be 21.0 mmol W L, 2297 pmol W min W mg protein and 1.12 mL W min W mg protein, respectively. There were no practical diŠerences in the kinetic parameters between the presence and absence of Na＋. EŠect of metabolic inhibitors, organic anions and pravastatin on pitavastatin uptake: As shown in Fig. 4, in the presence of a metabolic inhibitor, FCCP at 2 mmol W L or DNP at 1000 mmol W L, the velocity of the uptake of pitavastatin into rat hepatocytes was inhibited by 19.4z and 38.6z, respectively. The velocity of the uptake was inhibited in a concentration-dependent manner by organic anions such as ANS, BSP, CA and TCA, and also by pravastatin (Fig. 5). In the presence of ANS (10, 100 and 1000 mmol
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Fig. 5. Inhibition by ANS, BSP, CA, TCA and pravastatin of [ 14C]Pitavastatin (2 mmol W L) uptake into rat hepatocytes. Each column and vertical bar gives the mean±S.D. from 3 determinations. Asterisk represents Na＋ -free experiment. Signiˆcant diŠerences were analyzed using Dunnet's multiple comparison (#: pº0.001).
L) and BSP (10, 100 and 1000 mmol W L), it was inhibitW ed 12.3, 45.7 and 81.2z and 38.1, 74.8 and 92.5z, respectively. Similarly, in the presence of bile acid, CA and TCA at 10, 100 and 1000 mmol W L, the velocity of the uptake was inhibited 12.0, 54.5 and 82.1z and 5.7, L, 47.2 and 80.0z, respectively. At 100 and 1000 mmol W pravastatin known as a substrate of the rat organic anion transporting polypeptides (roatps), also reduced the uptake velocity of pitavastatin by 27.0 and 64.9z, respectively. In the absence of Na＋, ANS, BSP, CA, L inhibited the TCA, and pravastatin at 1000 mmol W velocity of the uptake 90.0, 93.3, 85.0, 84.2, and 76.4z, respectively. Discussion It has been reported that pitavastatin is predominantly distributed in the liver and mostly excreted through the bile in rats.8) We have previously found that mdr-1 and mrp-2 transporters, which play a major role in the biliary excretion of some other statins,21,22) were scarcely involved in the biliary excretion and the brain eŒux of pitavastatin.23) As little information was available on the mechanisms for the uptake of pitavastatin into liver, we performed experiments using rat hepatocytes. In the present study, more than 70z of the radioactivity in rat hepatocytes was identiˆed as unchanged pitavastatin after incubation at 379C for 60 min (Fig. 6). Therefore, the majority of the radioactivity measured in this study was evaluated as unchanged pitavastatin rather than metabolites. Further, according to the concentration-dependent experiments in the presence of Na＋, the analysis of uptake kinetics provided Vmax W Km (120 mL W min W mg protein) as saturable component parameters and Pdif (1.16 mL W min W mg
Fig. 6. Representive radio-chromatogram of the medium fraction after 60 min incubation with rat hepatocytes at 379C.
protein) as a nonspeciˆc diŠusion parameter. The Vmax W Km of pitavastatin was approximately three to six times larger than that of other statins, cerivastatin (44.4 mL W min W mg protein),16) ‰uvastatin (23.1 mL W min W mg pro17) tein) and pravastatin (18.8 mL W min W mg protein).14) The large Vmax W Km support our previous ˆnding in vivo8) that pitavastatin is selectively distributed to rat liver. The contribution of carrier-mediated uptake (CLcarrier ) to the whole uptake (CLwhole ) at a low concentration was calculated to be 0.99. The maximum plasma concentration of pitavastatin in rats (1 mg W kg) was reported to be 0.5 mmol W L.8) Even without considering protein binding, it was lower than the Km. These results suggest that the carrier-mediated uptake is a major function at the pharmacological level. There have been reports of several mechanisms responsible for the transport of compounds into hepatocytes, including Na＋-dependent and independent trans-
249
Uptake Mechanism of Pitavastatin in Rat Hepatocytes
porters such as oatp1, 2 and 4. It is known that Na＋ taurocholate co-transporting polypeptide (Ntcp) transports TCA in a Na＋-dependent manner in rat liver.24,25) To investigate the Na＋-dependent uptake of pitavastatin, experiments on the uptake were performed in the presence and absence of Na＋. There were no remarkable diŠerences in the uptake of pitavastatin between the presence and absence of Na＋. These ˆndings suggest that pitavastatin is little transported by any Na＋-dependent transporters or by Ntcp. ANS is one of the organic anion reagents, which was taken up into hepatocytes in an ATP-dependent manner.26) As shown in Fig. 5, the uptake of pitavastatin in the hepatocytes was inhibited in a concentration-dependent manner by ANS. Similarly, in the presence of metabolic inhibitors such as FCCP and DNP, which have been reported to reduce cellular ATP content,14,27) the uptake of pitavastatin was inhibited at a high concentration. This suppression of the uptake of pitavastaL FCCP (19.4z) is less than that for the tin by 2 mmol W uptake of pravastatin (85z).14) In addition, the suppression by 1000 mmol W L DNP (38.6z) was also less than that for cerivastatin (59.3z),16) and more than that for ‰uvastain (20z).17) These results indicate that energydependent systems contribute less to the uptake of pitavastatin than the uptake of pravastatin and cerivastatin. Inhibitors of rat organic anion transporting polypeptides (roatps) such as BSP, CA and TCA reduced the uptake of pitavastatin in a concentration-dependent manner.28–30) Further, a HMG-CoA reductase, pravastatin, also reduced the uptake. Pravastatin underwent rat organic anion transporting polypeptide 2 (roatp2)mediated transport in roatp2 cRNA-injected oocytes.15) However, kouzuki et al. reported that pravastatin did not undergo roatp1-mediated transport in COS-7.29) If pitavastatin is taken up through the same transporters as pravastatin, pravastatin would competitively inhibit the uptake of pitavastatin. The rate of inhibition [100× (1－Vi W V0 )] which suggests the extent of the reduction in pitavastatin uptake can be estimated using to the following equations, regarding the Km for pravastatin in the uptake experiment in isolated rat hepatocytes (29.1 mmol W L)14) as the inhibition constant (Ki ). (Km＋[S])＋Pdif･[S] V0＝Vmax･[S] W
(Eq. 3)
Vi＝Vmax･[S]〔 Ki )＋[S]〕＋Pdif･[S] WKm･(1＋ [i] W
(Eq. 4)
where [S] and [i] are the concentration of substrate and inhibitor, respectively. The predicted inhibition rates, when pravastatin was used at concentrations of 10, 100 and 1000 mmol W L, were 23.9z, 75.4z and 96.0z, respectively (Table 1). These predicted values were considerably higher than the observed rates and showed that pravastatin partially inhibits the uptake of
Table 1. Comparison of the predicted and observed inhibition for pitavastatin Inhibitor
L) Conc. ( mmol W
Inhibition rate Predicted (z)
Observed (z)
BSP
10 100 1000
59.3 92.8 98.3
38.1 74.8 92.5
TCA
10 100 1000
21.1 72.3 95.4
5.7 47.2 80.0
Pravastatin
10 100 1000
23.9 75.4 96.0
0.0 27.0 64.9
pitavastatin into hepatocytes. In the same way, the predicted inhibition of pitavastatin uptake by BSP and TCA was estimated (Table 1) using reported Km values L) and TCA (34.3 mmol W L) from of BSP (6.2 mmol W uptake experiments into isolated and cultured rat hepatocytes, respectively, instead of their Ki in Eq. 4.31,32) The predicted rates of inhibition by these inhibitors were similar to the observed rates. Recent reports showed that roatp1, 2, and 4 and moat1 were highly expressed in rat liver.33,34) BSP and TCA are taken up into cells through these multiple transporters.34,35) Therefore, these multiple transporters may be responsible for the active transport of pitavastatin into rat hepatocytes. In conclusion, the highly liver-selective distribution of pitavastatin is mainly due to carrier-mediated uptake via several Na＋-independent multispeciˆc anion transporters. The clearance of carrier-mediated hepatic uptake was greater than that for pravastatin, ‰uvastatin and cerivastatin. In addition, the systems involved in the uptake of pitavastatin may have features in common with the BSP and TCA transport system, and may partially include the pravastatin transport system. Acknowledgments: The authors are grateful to Dr. T. Kanke and Mr. M. Hirano for helpful suggestions. References 1)
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Uptake Mechanism of Pitavastatin in Rat Hepatocytes
30)
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