Drug Metabolism and Pharmacokinetics (DMPK)
Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE Received; July 1, 2013 Published online; December 24, 2013 Accepted; December 14, 2013 doi; 10.2133/dmpk.DMPK-13-RG-072
Role of P-Glycoprotein in Regulating Cilnidipine Distribution to Intact and Ischemic Brain
KENTARO YANO 1, SHINOBU TAKIMOTO 1, TOSHIMITSU MOTEGI 1, TAKUMI TOMONO 1,
MIHOKO HAGIWARA 2, YOKO IDOTA 1, KAORI MORIMOTO 3, AKIRA TAKAHARA 2 and
TAKUO OGIHARA 1,*
1Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Gunma 370 0033, Japan
2Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Toho
University, Funabashi, Chiba 274 8510, Japan
3Department of Drug Absorption and Pharmacokinetics, Tohoku Pharmaceutical University, Sendai,
Miyagi 981 8558, Japan
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Copyright C 2013 by the Japanese Society for the Study of Xenobiotics (JSSX) Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE
Running Title: Role of P Glycoprotein in Cilnidipine Distribution to Brain
* Corresponding Author: Takuo Ogihara, Faculty of Pharmacy, Takasaki University of Health and
Welfare, Takasaki, Gunma 370 0033, Japan (Telephone: +81 27 352 1180; Fax: +81 27 352 1118;
E mail: togihara@takasaki u.ac.jp)
Number of text pages: 24
Number of tables: 0
Number of figures: 4
Number of words in Summary: 189
Number of words in the text: 3084
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Summary
Cilnidipine is reported to show antihypertensive and neuroprotective actions in a rat brain ischemia model, but is barely distributed to normal brain, suggesting that its uptake into normal brain is inhibited by efflux transporter(s), such as P glycoprotein (P gp). Here, we investigated whether P gp regulates the brain distribution of cilnidipine. Intracellular accumulation of cilnidipine was decreased in P gp overexpressing porcine kidney epithelial cells (LLC GA5 COL150 cells) compared with control LLC PK1 cells and the decrease was markedly inhibited by verapamil, a P gp inhibitor. Further, cilnidipine concentration in the brain of P gp knockout mice was significantly increased after cilnidipine administration, compared with that in wild type mice. Moreover, when cilnidipine was administered to male spontaneously hypertensive rats (SHR) with tandem occlusion of the distal middle cerebral and ipsilateral common carotid artery, its concentration in the ischemic hemisphere was 1.6 fold higher than that in contralateral hemisphere. This result was supported by visualization of cilnidipine distribution using matrix assisted laser desorption/ionization time of flight/mass spectrometry (MALDI TOF/MS) imaging. Our results indicated that cilnidipine is normally excluded from the brain by P gp mediated efflux transport, but P gp function is impaired in ischemic brain and consequently cilnidipine is distributed to the ischemic region.
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Keywords: Cilnidipine, P gp, efflux transporters, ABC transporter, ischemia, MALDI TOF/MS
imaging, knockout mice, brain, distribution
Abbreviations: P gp, P glycoprotein; ATP, adenosine triphosphate; ABCB1, ATP binding cassette transporter B1; MALDI TOF/MS, matrix assisted laser desorption/ionization time of flight/mass spectrometry; C/M ratio, ratio of intracellular concentration to medium concentration
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Introduction
Influx of Ca2+ from blood into vascular smooth muscle cells induces vasoconstriction and blood pressure elevation, and is regulated by L type Ca2+ channels. On the other hand, N type Ca2+ channels in presynaptic cells regulate the extrusion of noradrenaline to the synaptic cleft. Cilnidipine is a dihydropyridine type antihypertensive drug that inhibits not only L type, but also N type Ca2+ channels.1 3) It reduces blood pressure via inhibition of the L type channels,4) and prevents development of tachycardia by suppressing the N type channels on peripheral sympathetic nerve terminals.5) Cilnidipine also has a cerebroprotective effect, because inhibition of N type channels reduces the release of glutamate, which causes neuronal damage, from presynaptic sites in focal brain ischemia.6 9) However, cilnidipine is not well distributed to the brain, despite its high lipophilicity. The blood brain barrier (BBB), which is formed from vascular endothelial cells linked via tight junctions,10) expresses several efflux transporters, such as P glycoprotein (P gp), multidrug resistance associated proteins (Mrps) and breast cancer resistance protein (Bcrp).11) Accordingly, even if cilnidipine is imported into vascular endothelial cells, it may be subject to ATP dependent efflux transport to the blood, mediated by these transporters.12)
Interestingly, hypotensive doses of cilnidipine have been reported to reduce the size of the infarcted region in a rat model of cerebral ischemia.13) This result indicates that this drug can penetrate BBB in ischemic regions. Therefore, we speculated that cilnidipine is indeed a substrate of
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Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE
P gp, but that the transport activity of P gp is impaired in ischemic brain, permitting the entry of cilnidipine.
To investigate this hypothesis, we examined the contribution of P glycoprotein to cilnidipine efflux transport and brain distribution by means of in vitro and in vivo experiments, using
P gp overexpressing cells, P gp knockout mice, and rats with unilateral brain ischemia.
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Materials and Methods
Chemicals and animals
Cilnidipine was provided by Ajinomoto Co. Inc. (Kawasaki, Japan). P gp knockout
(mdra/b / ) and FBV (wild type of mdra/b-/-) mice were purchased from CLEA (Tokyo, Japan). Male spontaneously hypertensive rats (SHR) rats were purchased from Japan SLC Inc. (Shizuoka, Japan).
P gp overexpressing porcine kidney epithelial cell line LLC GA5 COL150 (MDR1 gene transfected cells) and parental LLC PK1 cell line were obtained from RIKEN Cell Bank. Medium 199 (M199) was purchased from Life Technologies. Mice and rats were maintained with free access to food and water until 8 weeks of age (body weights of 15 23 g and 190 220 g, respectively). All animal experiments were performed according to the Guidelines for the Care and Use of Animals at
Takasaki University of Health and Welfare.
Cell culture
LLC PK1 cells were grown in M199 containing 10% fetal bovine serum, 100 U/mL
penicillin, and 100 µg/mL streptomycin at 37 in an atmosphere of 5% CO2 and 95% relative humidity. LLC GA5 COL150 cells were cultured under the same conditions in the presence of 150 ng/mL colchicine. For the uptake study, LLC PK1 cells and LLC GA5 COL150 cells were seeded on 12 well cell culture plate (Greiner bio one) at cell densities of 0.5×105 and 1.5×105 cells/mL,
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respectively.
General surgical preparation
Focal brain ischemia was induced in male spontaneously hypertensive rats (SHR), 8 weeks of age, by tandem occlusion of the distal middle cerebral and ipsilateral common carotid artery, as previously described.13, 14) Twenty minutes after cilnidipine or Evans blue15,16) administration, the left common artery was ligated with a thread, and the distal middle cerebral artery was permanently occluded just lateral to the olfactory tract by electrocautery.
Cilnidipine uptake in vitro
To investigate the involvement of P gp in cilnidipine transport, uptake studies with
LLC GA5 COL150 and LLC PK1 cells were performed at 37 in Hank’s balanced salt solution
(HBSS) with 4 (2 hydroxyethyl) 1 piperazineethanesulfonic acid (HEPES) (pH 7.4) buffer containing 100 µM cilnidipine. In the time dependence study, cells were incubated for 20, 40, 60,
120 and 300 seconds, then washed three times with HBSS HEPES buffer and lysed by adding 200
µL acetonitrile. To examine the effect of P gp inhibitor on the uptake of cilnidipine, cells were pre incubated with or without 100 µM verapamil or 1 mM dinitrophenol (DNP) for 20 min. After pre incubation, cilnidipine uptake experiments were performed in HBSS HEPES buffer containing
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100 µM cilnidipine in the presence of 100 µM verapamil or 1 mM DNP for 2 min, and then the cells were treated as above. Cell lysate was centrifuged at 3000 rpm for 5 min at 4 and the cilnidipine concentration was determined by means of high performance liquid chromatography (HPLC) as described below.
In vivo experiments in mice and rats
Cilnidipine (1.0 mg/ 10 mL/kg B.W.) was intraperitoneally administered to FVB mice and
P gp knockout mice (KO mice). Rats received intraperitoneal administration of cilnidipine (0.1 mg/
1 mL/kg B.W.) or intravenous administration of Evans blue (100 mg/ 1 mL/kg B.W.). Twenty minutes after cilnidipine or Evans blue administration, the left common artery and the distal middle cerebral artery were occluded as described above. The whole brain was excised and irrigated with saline, and blood was collected from the veins of both rodents with heparinized syringes 1 hr after administration. Cilnidipine or Evans blue concentrations of blood and brain samples were measured as described below.
Measurement of cilnidipine and Evans blue concentrations
In vitro and in vivo samples were treated as follows. Blood samples were centrifuged at
1800×g, 4 , 5 min. To measure cilnidipine concentration, 600 µL methyl tert butyl ether (MTBE)
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Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE
was added to the supernatant. The mixture was vortexed and further centrifuged at 1800×g, 4 for
5 min. To the organic layer was added 600 µL MTBE and the above procedure was repeated. Brain was divided into right and left hemispheres, and each was homogenized. The homogenates were then treated as described above for the blood supernatants. Cell lysates from the in vitro experiments were treated similarly. The collected organic layer from each sample was evaporated to dryness under a stream of nitrogen, and the residue was taken up in 200 µL methanol. Cilnidipine concentration was determined by means of LC MS/MS (high performance liquid chromatography tandem mass spectrometry) for brain, and HPLC for blood samples and cell homogenates.
For LC MS/MS measurement, a CN 3 column (5 µm, GL Science Inc., Tokyo, Japan) was used. The sample volume was 5 µL and the flow rate was 0.2 mL/min. The mobile phase was a mixture of 10 mM ammonium acetate (pH 5.0) and methanol. For HPLC measurement, a Mightysil RP 18 Aqua
250 4.6 column (5 µm, Kanto Chemical Co. Inc.) was used. The sample volume was 50 µL and the flow rate was 1.0 mL/min. The mobile phase was a mixture of 5 mM ammonium acetate (pH 5.0) and acetonitrile. To measure Evans blue concentration, blood samples were centrifuged at 1800×g for 10 min at 4 , and the supernatants were used as plasma samples. Brain samples were homogenized in two volumes of saline and centrifuged at 13000×g, 4 for 30 min. The supernatants in both plasma and brain samples were measured at 615nm for absorbance using a spectrophotometer (Sunrise rainbow RC; Tecan).
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Brain to plasma concentration ratios (Kpbrain values) were calculated using the formula: Kpbrain
(mL/g brain) = brain concentration (ng/g brain) / plasma concentration (ng/mL)
MALDI-TOF/MS imaging
Brain samples embedded in Tissue Tek O.C.T. compound (Sakura Fine Technical, Tokyo,
Japan) were snap frozen, and sliced at 9 µm using a cryostat (Leica CM1850). Tissue sections were sprayed with matrix solution (2,5 dihydroxybenzoic acid (DHB) 50 mg/mL in 70 % methanol and
0.1 % trifluoroacetic acid) using an ImagePrep matrix coater (Bruker Daltonics, Bremen, Germany), and dried for 1 hr under vacuum. Various concentrations (0, 20, 40 and 80 pmol) of cilnidipine were dropped on the tissue sections as positive controls. Mass spectra were obtained using a positive linear mode in the range of mass to charge ratio (m/z) 300 to 2,000. MS imaging analysis was performed using a MALDI TOF/MS, Ultraflextreme (Bruker Daltonics, Bremen, Germany).
Cilnidipine (m/z 492.52) was confirmed by the use of negative control tissue.
Statistical analysis
All data are presented as mean ± standard error of the mean (S.E.M.), except in Fig. 4.
Statistical analysis was done using the two tailed t test. Differences between means at the level of p<0.05 or 0.01 were considered to be significant.
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Results
Efflux transport of cilnidipine by P-gp in LLC cells
When LLC PK1 and LLC GA5 COL150 cells were exposed to 100 µM cilnidipine, the cell/medium (C/M ratio) of cilnidipine concentration linearly increased in LLC PK1 cells from 0 to
120 seconds. On the contrary, the ratio in P gp overexpressing LLC GA5 COL150 cells showed little increase and reached a steady state within 60 seconds (Fig. 1A). In addition, the C/M ratio of cilnidipine concentration in LLC PK1 cells was higher than that in P gp overexpressing
LLC GA5 COL150 cells at 2 min after the start of uptake. When LLC GA5 COL150 cells were incubated with verapamil or dinitrophenol (DNP), the C/M ratio of cilnidipine was significantly increased compared with the control (1.40 and 1.37 fold, respectively) (Fig. 1B).
Uptake of cilnidipine into the brain in wild-type and P-gp KO mice
When cilnidipine (1.0 mg/ 10 mL/kg B.W.) was administered intraperitoneally to mice, cilnidipine concentration in the brain of P gp KO mice (62.1 ng/g brain) was 6.3 fold higher than that of FVB (wild type) mice (9.89 ng/g brain) (Fig. 2A). Plasma concentration of cilnidipine in
P gp KO mice (20.8 ng/mL) was not significantly different from that in FVB mice (18.3 ng/mL) at 1 hr after intraperitoneal administration of 1 mg/ 10 mL/kg body weight (B.W.) cilnidipine (Fig. 2B).
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Concentration of cilnidipine and Evans blue in the brain of control and ischemic rats
Rats underwent ligation of the left common artery and occlusion of the distal middle cerebral artery after intraperitoneal administration of cilnidipine (0.1 mg/ 1 mL/kg B.W.). The
Left/Right ratio of cilnidipine Kpbrain value was 1.62 mL/g brain in ischemic rats at 1 hr after administration of cilnidipine (Fig. 3), but was 1.08 in the sham operated control rats. In addition, when Evans blue was intravenously administered to left brain ischemic rats and corresponding
control rats, there was no significant difference in the L/R ratio of Kpbrain value between the two groups (Fig. 3).
Imaging of cilnidipine distribution in rat brain
Cilnidipine concentration was increased in the ischemic penumbra site of the left hemisphere, as visualized with MALDI TOF/MS (Fig. 4). Cilnidipine was barely distributed in the intact right hemisphere.
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Discussion
The C/M ratio of cilnidipine was decreased in LLC GA5 COL150 cells, compared with
LLC PK1 cells, after 2 min incubation (Fig. 1A). In addition, verapamil (a P gp inhibitor) and DNP
(an ATP synthesis inhibitor) each significantly increased the C/M ratio of clnidipine in
LLC GA5 COL150 cells (Fig. 1B). These results indicate that cilnidipine is a substrate of P gp.
Moreover, our in vivo study showed that the concentration of cilnidipine in the brain of P gp knockout mice was significantly higher than that in FVB mice, whereas the plasma concentration of cilnidipine showed no significant difference between them (Fig. 2A, B). P gp restricts the permeability of many drugs across BBB into the brain in vivo.17,18) Accordingly, our results suggest that the intracerebral concentration of cilnidipine is regulated by P gp in mice in vivo.
Since cilnidipine was reported to show a neuroprotective effect in ischemic rat brain,13) we speculated that this drug is taken up into the brain in the state of ischemia. Accordingly, we next investigated whether cilnidipine was preferentially distributed in the ischemic region in a rat model of focal brain ischemia.
Surgical operation was conducted according to the reported method.13) It is considered that the white dotted circle in our MALDI TOF/MS imaging (Fig. 4) corresponds to the ischemic core site.13) Moreover, each hemisphere of the brains of sham operated rats and left brain ischemic rats was homogenized separately. Thus, the homogenate of the left hemisphere included the ischemic
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core and penumbra site. Indeed, the L/R ratio of cilnidipine Kpbrain values in the ischemic rats was significantly increased compared with that in sham operated control rats (Fig. 3). Although focal brain ischemia caused down regulation of P gp, we confirmed that BBB permeability through the paracellular route was not affected for at least 40 min after brain ischemia (Fig. 3). This result is also supported by previous findings in animals subjected to brief ischemia.19,20)
The involvement of P gp was also supported by MALDI TOF/MS imaging of the cilnidipine distribution (Fig. 4). We consider that P gp activity was impaired in the ischemic penumbra site owing to hypoxia induced decrease in ATP, which is required to drive the transport process.21 24) This may be the reason why cilnidipine concentration was increased in the ischemic penumbra site.
In conclusion, our results suggested that cilnidipine distribution to the brain is physiologically regulated by P gp mediated efflux transport, but P gp is downregulated in ischemic regions due to hypoxia induced decrease in ATP, thereby resulting in an increase of cilnidipine concentration in the ischemic regions. Therefore, cilnidipine might contribute to reduction of cerebral infarction size by inhibiting N type Ca2+ channels in ischemic regions of the brain.
ACKNOWLEDGMENTS
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This study was supported by a grant from the Research Foundation for Pharmaceutical
Sciences, and by government grants in aid for scientific research.
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References
1) Oike, M., Inoue, Y., Kitamura, K. & Kuriyama, H.: Dual action of FRC8653, a novel
dihydropyridine derivative, on the Ba2+ current recorded from the rabbit basilar artery. Circ.
Res., 67: 993 1006 (1990).
2) Takahara, A., Fujita, S., Moki, K., Ono, Y., Koganei, H., Iwayama, S. & Yamamoto, H.:
Neuronal Ca2+ channel blocking action of an antihypertensive drug, cilnidipine, in IMR 32
human neuroblastoma cells. Hypertens. Res., 26: 743 747 (2003).
3) Ogihara, T., Kano, T. & Kakinuma, C.: Evaluation of the inhibitory effect of dihydropyridines
on N type calcium channel by virtual three dimensional pharmacophore modeling.
Arzneimittelforschung., 59: 283 288 (2009).
4) Rose, G. W., Kanno, Y., Ikebukuro, H., Kaneko, M., Kaneko, K., Kanno, T., Ishida, Y. &
Suzuki, H.: Cilnidipine is as effective as benazepril for control of blood pressure and
proteinuria in hypertensive patients with benign nephrosclerosis. Hypertens. Res., 24: 377 383
(2001).
5) Uneyama, H., Uchida, H., Konda, T., Yoshimoto, R. & Akaike, N.: Selectivity of
dihydropyridines for cardiac L type and sympathetic N type Ca2+ channels. Eur. J.
Pharmacol., 373: 93 100 (1999).
6) Aota, Y., Morimoto, S., Sakuma, T., Morita, T., Jo, F., Takahashi, N., Maehara, M., Ikeda, K.,
17
Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE
Sawada, S. & Iwasaka, T.: Efficacy of an L and N type calcium channel blocker in
hypertensive patients with neurovascular compression of the rostral ventrolateral medulla.
Hypertens Res., 32: 700 705 (2009).
7) Takei, K., Araki, N., Ohkubo, T., Tamura, N., Yamamoto, T., Furuya, D., Yanagisawa, C. T. &
Shimazu, K.: Comparison of the anti hypertensive effects of the L/N type calcium channel
antagonist cilnidipine, and the L type calcium channel antagonist amlodipine in hypertensive
patients with cerebrovascular disease. Intern. Med., 48: 1357 1361 (2009).
8) Takizawa, S., Matsushima, K., Fujita, H., Nanri, K., Ogawa, S. & Shinohara, Y.: A selective
N type calcium channel antagonist reduces extracellular glutamate release and infarct volume
in focal cerebral ischemia. J. Cereb. Blood Flow Metab., 15: 611 618 (1995).
9) Valentino, K., Newcomb, R., Gadbois, T., Singh, T., Bowersox, S., Bitner, S., Justice, A.,
Yamashiro, D., Hoffman, B. B., Ciaranello, R. & et al.: A selective N type calcium channel
antagonist protects against neuronal loss after global cerebral ischemia. Proc. Natl. Acad. Sci.
U S A., 90: 7894 7897 (1993).
10) Luissint, A. C., Artus, C., Glacial, F., Ganeshamoorthy, K. & Couraud, P. O.: Tight junctions at
theblood brain barrier: physiological architecture and disease associated dysregulation. Fluids.
Barriers. CNS., 9: 23 34 (2012).
11) Nicolazzo, J. A. & Katneni, K..: Drug transport across the blood brain barrier and the impact of
18
Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE
breast cancer resistance protein (ABCG2). Curr. Top. Med. Chem., 9: 130 147 (2009).
12) Shukla, S., Robey, R. W., Bates, S. E. & Ambudkar, S. V.: Sunitinib (Sutent, SU11248), a
small molecule receptor tyrosine kinase inhibitor, blocks function of the ATP binding cassette
(ABC) transporters P glycoprotein (ABCB1) and ABCG2. Drug Metab. Dispos., 37: 359 365
(2009).
13) Takahara, A., Konda, T., Enomoto, A. & Kondo, N.: Neuroprotective effects of a dual L/N type
Ca(2+) channel blocker cilnidipine in the rat focal brain ischemia model. Biol. Pharm. Bull.,
27: 1388 1391 (2004).
14) Brint, S., Jacewicz, M., Kiessling, M., Tanabe, J. & Pulsinelli, W.: Focal brain ischemia in the
rat: methods for reproducible neocortical infarction using tandem occlusion of the distal
middle cerebral and ipsilateral common carotid arteries. J. Cereb. Blood Flow Metab., 8:
474 485 (1988).
15) Soejima, Y., Hu, Q., Krafft, P. R., Fujii, M., Tang, J. & Zhang, J. H.: Hyperbaric oxygen
preconditioning attenuates hyperglycemia enhanced hemorrhagic transformation by
inhibiting matrix metalloproteinases in focal cerebral ischemia in rats. Exp. Neurol., 247:
737 743 (2013).
16) Wang, Z., Leng, Y., Tsai, L. K., Leeds, P. & Chuang, D. M.: Valproic acid attenuates blood brain
barrier disruption in a rat model of transient focal cerebral ischemia: the roles of HDAC and
19
Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE
MMP 9 inhibition. J. Cereb. Blood Flow Metab., 31: 52 57 (2011).
17) Schinkel, A. H., Mayer, U., Wagenaar, E., Mol, C. A., van Deemter, L., Smit, J. J., van der Valk,
M. A., Voordouw, A. C., Spits, H., van Tellingen, O., Zijlmans, J. M., Fibbe, W. E. & Borst, P.:
Normal viability and altered pharmacokinetics in mice lacking mdr1 type (drug transporting)
P glycoproteins. Proc. Natl. Acad. Sci. U S A., 94: 4028 4033 (1997).
18) Schinkel, A. H., Wagenaar, E., Mol, C. A. & van Deemter, L.: P glycoprotein in the
blood brain barrier of mice influences the brain penetration and pharmacological activity of
many drugs. J. Clin. Invest., 97: 2517 2524 (1996).
19) Fang, W., Lv, P., Geng, X., Shang, E., Yang, Q., Sha, L. & Li, Y.: Penetration of verapamil
across blood brain barrier following cerebral ischemia depending on both paracellular
pathway and P glycoprotein transportation. Neurochem. Int., 62: 23 30 (2013).
20) Sakata, A., Tamai, I., Kawazu, K., Deguchi, Y., Ohnishi, T., Saheki, A. & Tsuji, A.: In vivo
evidence for ATP dependent and P glycoprotein mediated transport of cyclosporin A at the
blood brain barrier. Biochem. Pharmacol., 48: 1989 1992 (1994)
21) Chong, Y., Wang, T., Wang, W., Zhang, L., Li, C., Yu, P., Wang, H. & Fu, F.: Down regulation
ofP glycoprotein expression contributes to an increase in Danshensu accumulation in the
cerebral ischemia/reperfusion brain. Mol. Med. Rep., 5: 812 816 (2012).
22) Thews, O., Gassner, B., Kelleher, D. K. & Gekle, M.: Activity of drug efflux transporters in
20
Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE
tumor cells under hypoxic conditions. Adv. Exp. Med. Biol., 614: 157 164 (2008).
23) Dan, Y., Murakami, H., Koyabu, N., Ohtani, H. & Sawada, Y.: Distribution of domperidone
into therat brain is increased by brain ischaemia or treatment with the P glycoprotein inhibitor
verapamil. J. Pharm. Pharmacol., 54: 729 733 (2002).
24) Sakata, A., Tamai, I., Kawazu, K., Deguchi, Y., Ohnishi, T., Saheki, A. & Tsuji, A.: In vivo
evidence for ATP dependent and P glycoprotein mediated transport of cyclosporin A at the
blood brain barrier. Biochem. Pharmacol., 48: 1989 1992 (1994).
21
Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE
Figure legends
Fig. 1
C/M ratio of cilnidipine in LLC-PK1 and LLC-GA5-COL150 cells
LLC PK1 and LLC GA5 COL150 cells were incubated with 100 µM cilnidipine. (A) Time dependence of cilnidipine uptake. Open circles (○) and open squares (□) show LLC PK1 and
LLC GA5 COL150 cells, respectively. (B) The effects of P gp inhibitor (verapamil) and ATP synthesis inhibitor (dinitrophenol) on cilnidipine uptake in LLC GA5 COL150 cells. White, grey and black columns showed control (cilnidipine only), cilnidipine + verapamil and cilnidipine + DNP treatment, respectively. Each value is the mean ± S.E.M. of three experiments. *P<0.05 compared with control.
Fig. 2
Intracerebral and plasma concentrations of cilnidipine in wild-type and P-gp knockout mice
Mice were intraperitoneally administered cilnidipine (1.0 mg/ 10 mL/kg B.W.). Blood and brain were collected 1 hr after administration. White and black columns showed the results for FVB and P gp knockout mice, respectively. Each value is the mean ± S.E.M. of three mice. **P<0.01 compared with FVB mice.
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Fig. 3
Kpbrain values of cilnidipine and Evans blue in the brain of control and ischemic rats
Rats were intraperitoneally administered cilnidipine (0.1 mg/ 1mL/kg B.W.) (left panel) and intravenously administered Evans blue (100 mg/ 1 mL/kg B.W.) (right panel). Blood and brain were collected 1 hr after administration. White and black columns show cilnidipine and Evans blue concentrations of control and ischemic rats, respectively. Each value is the mean ± S.E.M. of three experiments. *P<0.05 compared with control rats.
Fig. 4
MALDI-TOF/MS imaging of cilnidipine distribution in the intact right hemisphere and ischemic left hemisphere of rat brain
The white dotted circle indicates the ischemic area. Blue and pink colors represent the absence and presence of cilnidipine in the brain, respectively. In the right panel, the images in the dotted circles illustrate the appearance of the indicated cilnidipine concentration.
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AB ＊＊＊
30 160 ＊＊＊ 140 25 120 20 100 15 80
10 60 40 5 C/M ratio (% ofcontrol) (% ratioC/M
C/M ratio(µL/mg protein) ratio(µL/mgC/M 20 0 0 0 1 2 3 4 5 Control Verapamil DNP Time(min) Fig.1 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE
AB ＊＊ 80 25
70 20 60
50 15 40
30 10
20 5
10 (ng/mL) concentration Plasma Brain concentration (ng/g brain) (ng/g concentration Brain
0 0 FVB P-gp KO FVB P-gp KO
Fig.2 Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE
Brain of Ischemic rat
Right hemisphere Left hemisphere
Normal Ischemia
2.0 ＊＊＊
1.5 value (mL/g brain) (mL/g value 1.0 brain
0.5
0.0 (brain to plasma concentration ratio) concentration to plasma (brain L/R ratio of Kp of ratioL/R Control Ischemia Control Ischemia
Fig.3 Cilnidipine Evans blue Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE
20pmol0pmol Right hemisphere Left hemisphere
20pmol
40pmol
80pmol
Fig.4