Assessment of inhibitory effect of many therapeutically important drugs on bile acid transport by NTCP, BSEP and other transporters

Transcription

1 ct 6, 26 Falk Symposium 155; XIX International Bile Acid Meeting Assessment of inhibitory effect of many therapeutically important drugs on bile acid transport by NTCP, BSEP and other transporters Kazuya Maeda and Yuichi Sugiyama Graduate School of Pharmaceutical Sciences, The University of Tokyo

2 Topics How do we predict the inhibitory effects of drugs on the bile acid transport in advance? 1) Inhibitory effects of PPARα, PPARγ and PPARα/γ agonists on the function of transporters for bile acids and drugs 2) In vitro assay system for the assessment of the inhibitory effects of drugs on the bile acid transport (NTCP/BSEP double transfectant)

3 Topics How do we predict the inhibitory effects of drugs on the bile acid transport in advance? 1) Inhibitory effects of PPARα, PPARγ and PPARα/γ agonists on the function of transporters for bile acids and drugs 2) In vitro assay system for the assessment of the inhibitory effects of drugs on the bile acid transport (NTCP/BSEP double transfectant)

4 PPAR (peroxisome proliferator-activated receptor) fatty acids PPARα nuclei Prostaglandines Leukotrienes PPARγ PPARα RXR PPRE Transcription Fatty Acid Metabolism Lipid Homeostasis PPARγ RXR PPRE Transcription Adipocyte Differentiation Glucose + Insulin Homeostasis Drugs targeting at PPARα and PPARγ PPARα agonist for hyperdyslipidemia PPARγ agonist for diabetes PPARα/γ dual agonist PPARγ agonist with avoidance of obesity (PPARα) (under clinical trial)

5 Chemical structures of PPARα, PPARγ and PPARα/γ agonists H Cl PPARα agonists CH 3 Gemfibrozil Bezafibrate Fenofibrate Clofibrate CH 3 Cl H 3 C CH 3 H 2 C H S H 3 C CH 3 H 2 C H S PPARγ agonists H CH 3 N Troglitazone CH 3 N S NH Rosiglitazone NH H 3 S Troglitazone Sulfate H 3 C CH 3 N Pioglitazone S NH NH PPARα/γ dual agonists H 3 C H 3 C LM 4156 H CH 3 CH 3 S Tesaglitazar H N N CH 3 Muraglitazar H 3 C H

6 Examples for interaction between PPAR agonist and transporters Case 1: possible mechanism of drug-drug interaction between cerivastatin and gemfibrozil Case 2: possible mechanism of drug-induced hepatotoxicity Liver BCRP? Bile Canaliculus Tro-S BSEP SULT Tro bile acids Troglitazone(Tro) Tro-Sulfate(Tro-S) ATPs? Portal vein

7 Purpose Troglitazone was withdrawn from the market because of idiosyncratic hepatotoxicity in 2. Gemfibrozil-cerivastatin combination therapy, caused rhabdomyolysis (transporter-mediated drug interaction). Strategies in vitro --- To clarify the inhibitory effects of PPAR agonists on the transporter-mediated uptake and efflux (Ki values) in vivo --- To investigate the relationship between the hepatic concentration of PPARγ agonists (-glitazones) and increase in the plasma bile acids level

8 Target transporters in liver rganic anions Bile acids Na + Blood ATP1B1 ATP1B3 NTCP NTCP Na + -taurocholate transporting polypeptide ADP rganic anions ADP ATP MRP2 ATP BCRP BSEP ATP Bile acids ADP ATP rganic anion transporting polypeptide BSEP Bile salt export pump MRP2 Multidrug resistanceassociated protein 2 Bile BCRP Breast cancer resistance protein

9 Experimental methods In vitro study Vesicle assay for efflux transporters (BSEP,MRP2,BCRP) expressing cells Uptake assay for uptake transporters (NTCP,ATP1B1&1B3) Construction of membrane vesicles & rapid filtration technique Method Method for for transport transport study study in transporter in mkatp1b1- expressing - cells infection Adeno Virus HEK293 cells ADP+Pi ATP Substrate rightside-out vesicle ATP ADP+Pi Substrate inside-out vesicle Membrane vesicles 37 +ATP incubation filtration filter Vesicle Assay count Cells 24-well 12-well plate insert Inhibitor + Substrate Counting of radioactivity after 72 hr Incubation Incubate for 55 min Wash Counting of radioactivity

10 Inhibitory effects of PPAR agonists on the transportermediated transport (1) Ki value (μm ) hntcp ATP1B1 ATP1B3 hbsep gemfibrozil 74.7 ±11.8% 51.4 ± ±8.7% 12%±6% α clofibrate bezafibrate 99.3 ±13.8% 58.4 ± 7.6% 29. ±1.9%(1 mm) 94.5 ± %±4% 67.3 ±4.6% 19 ±7% 17 ±6% fenofibrate 17 ±9% 7.4 ±8.8%(.5 mm) 15 ±4% 72.9%±3.2% Dual tesaglitazar 57.2 ±3.3% 21.5±6. >1μM 72.2%±4.8% α/γ muraglitazar LM ± ± ± ± ± ± ± ±3.2% (3μM) pioglitazone 3.14 ± ± ±5.%(3μM).183 ±.44 troglitazone.886± ± ± ±1.56 γ troglitazone sulfate troglitazone.739± ± ± ± ± ± ± ±1.5 glucuronide rosiglitazone 3.52± ± ± ±.27 Blue letters represent the percentage of the transport clearance in the presence of 1 μm (or designated conc.) inhibitors to that in the absence of inhibitors.

11 Inhibitory effects of PPAR agonists on the transportermediated transport (2) Ki value (μm ) hmrp2 hbcrp gemfibrozil 93. ±2.% 77.8 ±11.5% α clofibrate bezafibrate 87.8 ±17.4% 12 ± 15% 14 ±15% 6.6 ±1.97 Fenofibrate 122 ±9% 83.1 ±13.2% Dual tesaglitazar N.D ±2.7% α/γ muraglitazar LM4156 N.D. 18 ±12% 2.68 ±11.7% 2.76±1.63 pioglitazone 136 ±13% 89.1 ±13.7% troglitazone 11.9 ±18.1% 5.38 ±1.36 γ troglitazone sulfate troglitazone Glucuronide rosiglitazone ±5.77% 7.5 ±3.45% 19 ±18% 6.79± ±15.5% 47.5 ±7.1% N.D. = not determined Blue letters represent the percentage of the transport clearance in the presence of 1 μm (or designated conc.) inhibitors to that in the absence of inhibitors.

12 Inhibitory effects of PPARγ agonists on the transport of TCA in membrane vesicles expressing rat or human BSEP, and rat CMVs Troglitazone human BSEP 15 rat Bsep rat CMVs 125 TGZ-sul human BSEP rat Bsep 15 rat CMVs 125 Pioglitazone Human BSEP rat Bsep rat CMVs % of control % of control % of control % of control Troglitazone(μM) Rosiglitazone Human BSEP rat Bsep rat CMVs Rosiglitazone (μm) Troglitazone sulfate (μm) Ki value (μm) troglitazone TGZ-sul pioglitazone rosiglitazone human BSEP 5.5 ± ± ± ± rat Bsep 12 ± ±.48 >1 >1 rat CMVs 6.6 ± ±.211 > 1 > 1 Species difference in the inhibitory effects of PPARγ agonists on the BSEP-mediated transport was observed. Pioglitazone (μm)

13 Experimental methods In vivo study expressing cells Animal model: SD rats (male, 8 weeks old, n=5~6) Administration: 1mg/kg TGZ 2.5,1mg/kg pioglitazone.5,2mg/kg rosiglitazone (dissolved in glycofurol, i.v. bolus) Drug administration min Blood sampling total bile acids, drug conc. Liver sampling drug conc.

14 Plasma and hepatic concentration of PPARγ agonist after i.v. administration in rats 2 Plasma concentration Hepatic concentration (at 6 min) plasma conc. (μm) TGZ TGZ-sulfate Rosi_ time after administration (min) Troglitazone (1mg/kg) Troglitazone sulfate Rosiglitazone (.5mg/kg) Rosiglitazone (2mg/kg) Pioglitazone (2.5mg/kg) Pioglitazone (1mg/kg) Pio_1 Pio_2.5 liver conc. (μm) Rosi_2 4.9 TGZ 111 TGZsulfate Rosi_.5 Rosi_2 Pio_2.5 Pio_1 Intrahepatic conc. of TGZsulfate was much higher than that of parent troglitazone.

15 PPARγ agonist-induced increase in the concentration of total bile acids in plasma Absolute change in TBA level (μm) Control (n=11) Troglitazone, 1mg/kg (n=5) Rosiglitazone,.5mg/kg (n=4) Rosiglitazone, 2mg/kg (n=4) Pioglitazone, 2.5mg/kg (n=6) Pioglitazone, 1mg/kg (n=4) Time after administration (min) (Male SD rats, 2-25 g, iv)

16 PPARγ agonist-induced increase in the concentration of total bile acids in plasma (Individual data) control 1mg TGZ.5mg Rosi 2mg Rosi 2.5 mg Pio 1mg Pio absolute change in plasma TBA level (μm) -1

17 Summary table for the results of in vivo and in vitro analyses in humans and rats in vivo in vitro Rat Troglitazone (1mg/kg) Pioglitazone (1 mg/kg) Rosiglitazone (2 mg/kg) TGZ TGZ-S Max. Plasma conc. (μm) 28.2 (1min) 28.5 (3min) 152 (1min) 3.8 (1min) Liver conc. at 6min (μm) Ki value for rbsep (μm) 12± ±.48 >1 >1 Ki value for rcmvs* (μm) 6.6± ±.211 >1 95.3±21.2 Human Troglitazone (4-6mg, p.o.) Pioglitazone (3mg, p.o.) Rosiglitazone (4-8mg, p.o.) clinical pharmacokinetics t C 1/2 max (μm) (hr) TGZ TGZ-S Protein unbound fraction.2 % 1% < 1 %.2 % Ki value for hntcp (μm).886± ± ± ±.85 in vitro Ki value for hbsep (μm) 5.5± ± ± ±.27 *CMVs=canalicular membrane vesicles

18 Conclusions -1 # Some PPAR agonists, especially PPARγ agonists, can inhibit the transport function of transporters for organic anions (ATPs) and bile acids (NTCP, BSEP) # In vivo analyses supported that inhibition of BSEPmediated transport by troglitazone sulfate possibly results in the increase in plasma bile acid concentration in rats. # Species difference in the inhibition potency of PPARγ agonists for BSEP-meditated transport was clearly observed between rats and humans. Inhibition of transporters by drugs Drug-drug interaction, Cholestasis

19 Topics How do we predict the inhibitory effects of drugs on the bile acid transport in advance? 1) Inhibitory effects of PPARα, PPARγ and PPARα/γ agonists on the function of transporters for bile acids and drugs 2) In vitro assay system for the assessment of the inhibitory effects of drugs on the bile acid transport (NTCP/BSEP double transfectant)

20 Efflux transporter (BSEP) apical TRANSWELL Basal to Apical LLC-PK1 cell Uptake transporter (NTCP) basal transport Basal to Apical Apical to Basal Apply Apply If a compound is bisubstrate of uptake and efflux transporters. Apical to Basal Time Experiments using double transfectants (Mita S et al., Am J Physiol, 29, (26))

21 B A A B B A A B cholate H H TC CH chenodeoxy cholate TCDC CH ursodeoxy cholate TUDC CH Apical Transcellular transport (pmol/min/mg protein) H GC CA CDCA GCDC NTCP H BSEP conjugation NTCP/BSEP H H NTCP H BSEP conjugation NTCP/BSEP H TC TCDC TUDC Mock GC GCDC GUDC CA CDCA UDCA Mock NTCP NTCP/BSEP BSEP Mock 15 Mock GUDC UDCA NTCP H BSEP conjugation NTCP/BSEP Transcellular transport of primary bile acids Taurine conjugates Glycine conjugates Unconjugated Substrate specificity of NTCP/BSEP CA=CDCA=UDCA Conjugated>Unconjugated Basal

22 deoxycholate TDC H CH GDC lithocholate LCA CH Transcellular transport (pmol/min/mg protein) Transcellular transport (pmol/min/mg protein) H cholylsarcosine Cholylsarcosine 1 TDC 1 GDC 1 LC Mock NTCP BSEP NTCP/BSEP H conjugation Cholylsarcosine H H H CH Transcellular transport of secondary bile acids and bile acid analogue cholylsarcosine 1 H Sarcosine (N-methylglysine) conjugation Apical Basal Substrate specificity of NTCP/BSEP Primary bile acid > Secondary bile acid

23 Comparison of the rank order of the clearance of a series of bile acids PS1 PS2 PS ntcp PS ntcp PS bsep PS3 rat CDCA PS ntcp (TC=1) LCA UDCA CA GUDC human TC GC TUDC TCDC GCDC rat 2 1 PS bsep (TC=1) TUDC TCDC PS net 1 2 human No significant species difference in the rank order of clearance was observed between humans and rats TC GCDC UDCA LCA CDCA CA GC GUDC (Mita S et al., Am J Physiol, 29, (26))

24 PS b-a BSEP NTCP C cell PS apical BSEP NTCP % of control % of control % of control LLC-PK1 NTCP NTCP/BSEP 1 1 Rifampicin Inhibition of NTCP Inhibition of BSEP 1 1 Rifamycin SV 1 1 Glibenclamide Inhibitory effects of drugs on the transport of taurocholate (Mita S et al., Drug Metab Dispos, in press) Captopril (1) Cimetidine (1)

25 control NTCP NTCP/BSEP taurocholate H H H H N H N CGamF CH (Z) (Z) H (E) H 2 1 H H N H N CDCGamF CH (Z) (Z) H (E) basal-to-apical transport (μl/min/mg protein) N H H N H H H N N CamF basal-to-apical transport (μl/min/mg protein) basal-to-apical transport (μl/min/mg protein) Rapid Screening of the inhibitors H (Z) CH N of H NH CH 2 (Z) (E) CH bile acid transport 2 NH H S (R) NH (S) H NH H N 2 1 UDC-L-NBD basal-to-apical transport (μl/min/mg protein) 2 1 N 2 7β-NBD-NCT basal-to-apical transport (μl/min/mg protein) basal-to-apical transport (μl/min/mg protein) Transport properties of fluorescent bile acids in NTCP/BSEP double transfected cells (Mita S et al., Drug Metab Dispos, in press)

26 Conclusions -2 We characterized the transport properties of bile acids in rat and human versions of NTCP/BSEP double transfectant. This system will be useful for the rapid identification of the inhibitors of bile acid transport with the use of fluorescent bile acids.

27 Acknowledgements verall Dr. Yuichi Sugiyama, Dr. Hiroyuki Kusuhara Part I Ms. Tian Ying Sankyo Co. (providing us troglitazone and its metabolites and other glitazones) Merck KGaA (providing us LM 4156) Part II Dr. Alan F. Hofmann Ms. Sachiko Mita Mr. Hisamitsu Hayashi Dr. Hidetaka Akita Dr. Reiko nuki Dr. Hiroshi Suzuki Members of the Dept. of Molecular Pharmacokinetics