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DMPK, Aventis Pharma, Frankfurt Paris, February 4th 2004

Transporters and Pharmacokinetics: Optimization of PK Parameters Using Double Transfected MDCK Cells (OATP8/MRP2 and OATP2/MRP2). DMPK, Aventis Pharma, Frankfurt Paris, February 4th 2004. Why should we worry about Transporters?. Decrease fraction absorbed due to efflux- to which degree?

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DMPK, Aventis Pharma, Frankfurt Paris, February 4th 2004

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  1. Transporters and Pharmacokinetics:Optimization of PK Parameters Using Double Transfected MDCK Cells (OATP8/MRP2 and OATP2/MRP2) DMPK, Aventis Pharma, Frankfurt Paris, February 4th 2004

  2. Why should we worry about Transporters? • Decrease fraction absorbed due to efflux- to which degree? • increase fraction absorbed in drug-drug-interactions (p.o.) • Cause dose-dependent absorption • Induction, inhibition, NHR regulation • patient variability (polymorphisms) • drug-drug interactions • Transporter-excipient interaction possible • kinetics not zero or first order

  3. Systemic Availability: Identification of PK Problems Central Compartment Intestinal Wall Systemic Circulation Intestinal Lumen Portal Vein Liver Intestinal Metabolism Efflux Liver Metabolism Enterohepatic Circulation Renal Elimination Hepatobiliary Elimination Faeces

  4. PK: Transport Processes • GI: lumenal uptake/efflux- enterocytes (apical) • GI: release/partition to blood stream (basolat) • Liver: sinusoidal uptake (basolat) • Liver: secretion into bile canaliculi (apical) • Liver: secretion of metabolites into bile and blood • Kidney: tubular uptake/ partition from blood (basolat) • Kidney: tubular release/resorption to lumen (apical) • Tissue: uptake to site of action (membrane uptake/secretion; uptake into subcellular compartments) • BBB: uptake from blood (apical) • BBB: secretion into brain interstitial space (basolateral) • BCF: uptake from extravascular fluid into epithelia (basolateral) • BCF: release to ventricle (apical)

  5. Solute Carrier Family (Uptake transporters) • Organic Anion Transporters (OAT) (SLC22A) • Organic Anion Transporting Polypeptides (OATP) (SLC21A) • Na+/Taurocholate Cotransporting Polypeptides (NTCP) (SLC10A) • Organic Cation Transporters (OCT) (SLC22A) • Na+/Phosphate Cotransproters (NPT) (SLC17A) • Peptide Transporters (PEPT) (SLC15A) • And others

  6. ATP Binding Cassette Transporter Family (Exporter) • Bile Salt Excreting Protein (BSEP) (ABCB) • P-glycoprotein (Pgp = MDR1) (ABCB) • Multidrug Resistance associated Proteins (MRP) (ABCC) • Breast Cancer Resistance Protein (BCRP) (ABCG)

  7. Intestinal Barrier Sucr IsoM DS IBAT Oatp3 ATPase LNAAT MCT SGLT1 MDR1 Glut2  GT MRP2 PhT ALP OCT1 MXR OAT TauT PEPT1 2J 2D 3A Gluc Sulf 1A Glut5 MRP1 Na/K- ATPase MRP5 MRP3 AdCycl PhT- phosphate tr. LNAAT- amino acid tr. 3A, 1A, 2D, 2J- CYP‘s/ Phase I Sulf- sulfatation/ Phase II Gluc- glucuronidation/ Phase II PEPT1- di/tripeptides OCT- org. cation tr. IBAT-int.bile acid tr. AdCycl- adenylat- cyclase MDR, MRP- ABC transporter ALP- alk. phosph. GT-glut.transpept. OAT- org. anion tr. Sucr- sucrase IsoM- isomaltase DS- disaccharidase TauT- taurocholic acid tr. MCT- monocarb. acid tr.

  8. Kidney Proximal Tubular Cell Apical side human Lumen URAT1 OATP-A Glut1/2 PEPT2 PEPT1 MRP2 OAT4 SGLT1/2 MDR1 NPT1 BCRP OCTN1/2 MRP4? OCT? OAT-K1 OAT-K2? Na/K- ATPase SDCT2 OCTN1/2 MCT1/2 OAT1 MRP5? OCT2 OAT2 ASBT? rat Oatp3? Oatp1 MRP6? OCT1 OAT3 Glut1 Basolateral side Interstitium Oat-k 1 OAT2 OAT4 Glut3/5 MRP1: collecting duct MRP3-distal cells OATP-D OATP-E

  9. Blood-Brain Barrier Transporters Sero- tonine Choline / thiamine: OCT? Blood OAT1 L ALP GGT GLUT1 SGLT1 N2 Oatp2/3 MCT1 TR MRP2 Na / H exchanger GLUT3 NT(es) NT(ei) OATP-A MCT2 Pgp L-carnitine: OCT3? MAO Brain endothelial cell 2B1/2 NOS AAD ACE ALD 2E1 GSH-T GLUT1 oatp2 MCT1 L A ALP GGT Na / K ATPase Na / H exchanger K+pump L-carnitine: OCT3? TNF, IL6 Pgp Pericyte MRP ABCA1 ABCA2 SUR1 ABCG2 Pgp OATP-F Glut-8 PEPT2? Brain Microglia Astrocytic endfeet Pgp Mertsch and Maas, Curr Med Chem, 2002

  10. Transporter regulation via networks of nuclear hormone receptors Antagonists Agonists PXR CYP 3A MRP2 Pgp Oatp2 PPAR a ASBT Triglycerids Fatty acids MDR3 CYP 7A1 FXR OATP8 CYP 7A1 BSEP IBABP MRP2 NTCP CAR CYP 2B MRP2 MRP3 Oatp2 LXR CYP 7A1 Cholesterol and Bile salt Transport IBABP

  11. Transporter Studies • In vivo only indications via tissue distribution, expression levels, in situ methods, K.O. mice, bile fistula studies • In vitro: biochemical and physiological • ATPase assay, BBMV, inverted out vesicles • single transfectants (Sf9, mammalian cells): uptake, binding • double transfectants: transport

  12. Case Study: Hepatobiliary Elimination

  13. Several drug targets in diabetes research are located in the liver Problem: fast elimination of drug candidates via liver Drug candidate with in vitro potency Solubility, S9 metabolic Stability, CACO-2, Protein Binding, Metabolic profile, in vivo PK (i.v., p.o.: T1/2, Cl, Cmax, Vss), Portal vein study, Bile fistula study Rapid hepatobiliary elimination Insufficient PD and PK profile

  14. Hepatobiliary Elimination SMALL ORGANIC CATIONS SMALL ORGANIC ANIONS BULKY ORGANIC COMPOUNDS MONOVALENT BILE SALTS OATP2 (OATP-C) OATP8 OATP-B BLOOD OAT2 (OAT4) (OAT5) Na+ OCT1 NTCP GSH MDR1/ Pgp MRP2 BILE BILE BILE BCRP BSEP/ SPGP MDR3 PHOSPHATIDYL- CHOLINE MRP 3 MRP 6 modified from Muller M and Jansen PL (1997) Am J Physiol Gastrointest Liver Physiol 272: G1285-G1303

  15. Substrate Specificity of Liver Transporters OATP2 bulky organic compounds estradiol-17-b-glucuronide, pravastatin, leukotriene C4, estrone-3-sulfate, ouabain, digoxin, taurocholate, bromosulfophthalein (BSP) OATP8 bulky organic compounds estradiol-17-b-glucuronide, leukotriene C4, bromosulfophthalein (BSP), DHEAS, ouabain, digoxin, MRP2 neutral and anionic compounds bromosulfophthalein (BSP), leukotriene C4, estradiol-17- b-glucuronide, GSH, folates, methotrexate, pravastatin,

  16. In vitro Approaches to Study Hepatobiliary Elimination • Oocytes (Becton Dickinson) for uptake transporters: different membranes, different glycosylation motif, different Km values expected • HEK, CHO cells for uptake transporters: background, lower protein expression levels • Sf9 (Solvo) cells for efflux transporters: different membranes, different glycosylation motif, different Km values expected • MDCK cells transfected with uptake and efflux transporters and double transfected cells (collaboration with Prof. Keppler, University of Heidelberg)

  17. MDCKII CELL MONOLAYER APICAL COMPARTMENT POROUS FILTER BASOLATERAL COMPARTMENT Assay Design (B) (C) (A) EFFLUX Apical Tight junction Basolateral UPTAKE UPTAKE MDCKII MDCKII-OATP8 or MDCKII-OATP2 MDCKII-OATP8/MRP2 or MDCKII-OATP2/MRP2

  18. Expression of Transporters (Western Blot Analysis) * * * * * * * g g g: glycosylated protein; MRP2 (190 kDa); OATP2 (76 kDa); OATP8 (77 kDa)

  19. Localization of Transporters (CLSM) top view middle view bottom view Immunostaining of MDCK-hMRP2/hOATP2 cells OATP2 lateral (blood) MRP2 apical (bile)

  20. MRP2 OATP8 DNA Immunoblot analysis of MRP2 and OATP8 a-MRP2 a-OATP8 X/Y MDCK- OATP8 MDCK- M2/O8 MDCKII 250 250 0 4 24 0 4 24 0 4 24 h 150 150 MDCK- OATP8 MDCK- M2/O8 MDCKII 100 100 0 4 24 0 4 24 0 4 24 h 75 75 X/Z Localization of Transporters (CLSM)

  21. MDCK M2/O8 # 8 # 3 # 11 # 23 + - + - + - + - + - + - OATP8 MDCK M2/O8 # 8 # 3 # 11 # 23 + - + - + - + - + - + - MRP2 Western blots of cloned cells Klon #8 und #23

  22. Transport Assays with BSP Clone #8 and #23

  23. Physiological Transport Experiments MRP2/OATP2 MRP2/OATP8 * * Leukotriene C4

  24. Physiological Transport Experiments MRP2/OATP8 MRP2/OATP2 Estradiol-17-b -Glucuronide * *

  25. Selection of the Standard: BSP

  26. Quality Assurance

  27. GLUCOSE-6-PHOSPHATASE SYSTEM • Glucose-6-phosphatase system catalyses the final step of hepatic glucose production from gluconeogenesis and glycogenolysis •  Key regulatory factor of blood glucose homeostasis • HEPATIC GLUCOSE PRODUCTION IS INCREASED IN TYPE 2 DIABETIC PATIENTS Chlorogenic acid derivatives Cytosol ER lumen • Chlorogenic acid derivatives are potent and specific inhibitors of the glucose-6-phosphate translocase • Inhibition of glucose-6-phosphate translocase results in a decrease in hepatic glucose production and plasma glucose levels Image modified from Van Schaftingen and Gerin I, Biochem. J. (2002) 362, 513-532 CHLOROGENIC ACID DERIVATIVES ARE POTENTIAL NOVEL THERAPEUTICS IN PATIENTS WITH TYPE 2 DIABETES

  28. S 4048 Pharmacodynamic profile of S 4048 Herling AW et al., Biochim.Biophys.Acta 1569 (2002), 105-110

  29. S 4048 Hepatobiliary elimination and PK of S 4048 in Wistar and GY/TR– rats 10 mg/kg i.v. bolus injection Herling AW et al., Biochim.Biophys.Acta 1569 (2002), 105-110

  30. S 4048 S 3025 Hepatobiliary elimination and plasma kinetics of S 3025 in Wistar and GY/TR– rats 10 mg/kg i.v. bolus injection Herling AW et al., Biochim.Biophys.Acta 1569 (2002), 105-110

  31. Project: Glucose-6-phosphate Translocase Inhibitors Results: In vitro identification of transporters involved in hepatobiliary elimination of drug candidates Control Active transport  by OATP8/MRP2 No transport

  32. Project: Glucose-6-Phosphate Translocase Inhibitors control positive transport (MRP2/OATP8) no transport no statement possible (high background) * * * *

  33. Project: Glucose-6-Phosphate Translocase Inhibitors Correlation of in vitro and in vivo data Transported by MRP2/OATP8 in vitro in vivo NOT transported by MRP2/OATP8/OATP2 in vitro in vivo + - 40% 76% A000251691 (S 4048) A000254060 (S 3483) + - 5% 62% A000248238 (S 0582) A000247923 (S 1160) + 58% Additional studies are needed for three compounds. A000297170 (S 3025) + 40% A000249425 (S 3994)

  34. Project: Glucose-6-Phosphate Translocase Inhibitors Summary: correlation between in vitro and in vivo data • More than 20 cpds tested so far • Identification of transporter combination responsible for drug elimination possible • Evidence for participation of azabenzimidazole ring system in MRP2/OATP8/OATP2 mediated drug transport in vitro (G6PT) • For one compound no transport was measured despite of its high hepatobiliary elimination rate in rat: • possibly other transporters than MRP2/OATP8/OATP2 are involved or differences between rat and human transporters are responsible • Additional methods are needed (for three compounds) • Additional methods: vesicles (for efflux studies), uptake (new cell lines) • Further cell lines necessary (Molecular Liver) including rat transporters

  35. Reports at Aventis • Standard Operating Procedure • Standard Validation Report • Project Reports

  36. Summary of Results from Double-Transfectants Model • More than 30 cpds tested so far in both double transfectants and single transfectants • For some compounds high background due to endogenous canine transporters like MDR1 and MRP2: alternative methods needed • Compounds with high permeability rates (Papp values higher than 10-6 cm/sec): • Partitioning is faster than active transport processes (PAMPA will be used) • One cpd identified as OATP2 and MDR1 substrate, less MRP2 • One cpd + metabolite characterized for approval in Japan • Cpds from one project are inhibitors of OATP2/MRP2 or/and OATP8/MRP2: additional studies needed!!! • Cpds from Aventis being on the market will be characterized

  37. Discussion and Limitations of the Double-Transfectants Model • For some compounds high background due to endogenous canine transporters like MDR1 and MRP2 • Compounds with high permeability rates (Papp values higher than 10-6 cm/sec): • Partitioning is faster than active transport processes (PAMPA useful ?) • Metabolites are not formed: advantage or disadvantage? • For inhibitors of OATP2/MRP2 or/and OATP8/MRP2: additional studies needed!!! • No Km value determination possible: alternative is inverted out vesicles, uptake assays • ATPase assay, binding sides • Cell lines without background? • Tight cell lines for transfection of efflux transporters-alternatives for MDCK? • In vivo experiments? How to interprete K.O. mice results in terms of PK? • DDI experiments? • Throughput of the filter experiment: low to medium (controls, standards)- for comparison CACO-2 throughput 1700-2500 cpds/year in FFM • Throughput of the LC-MS analytics: medium

  38. Joined Initiative DMPK Frankfurt DG Metabolic Diseases Stefan Theis Tanja Eisenblätter Reinhard Liebe Wolfgang Schmider Katharina Mertsch Jochen Maas Werner Kramer

  39. Efflux Studies: do we have a Strategy?

  40. How to Study MDR1: Complications Regarding MDR1 • Multiple binding sides • distinct substrate specificity • binding sites can interact in a cooperative manner • Competitive or cooperative inhibition possible • Interaction at a third side: difficult to identify • ATP dependent action • Compounds with high partitioning are no substrates but can be inhibitors Shapiro & Ling, 1997

  41. Caco-2 Cell Line: Expression of Transporters P-Glycoprotein Expression and Functionality Caco-2 #51 #52 #53 A B C D E F A B C D E F B A 250 kD MDR1 (170 kD) 150 kD 100 kD 75 kD * Digoxin-Ratio: ? 6,6 3,9 4,3 2,0 4,0 2,6 1,2 1,2 6,4 3,8 3,2 2,9 2,4 * R. Liebe

  42. Caco-2 Cell Line: Localization of Transporters Immunofluorescence (CLSM) hMDR1 (Pgp) Apical Expression of human MDR1 Nucleus Staining Filter Membrane with Pores

  43. CACO-2 Permeability Screen: Flux and Efflux Digoxin Ratio: 5-9 BSP Ratio: 3.5-5

  44. Substrate Efflux ratios normalized to digoxin (=100%) Digoxin 100% Doxorubicin 118% Etoposide 123% Rhodamine 123 109% Verapamil 105% Colchicine 200% Vincristine 423% Ketoconazole No efflux (inhibitor!) Vinblastine 250% Known MDR1 Substrates and Inhibitors in the CACO-2 Model in FFM

  45. No effect on A... Inhibition of A... efflux Increase in A... efflux Other effects etoposide Cyclosporin A (at 10µM) by 39% Cyclospsorin A (1µM) by 61% inhibition of BSP efflux by 9% vinblastine probenecid by 19% colchicine by 29% inhibition of digoxin efflux by 8% verapamil by 56% daunorubicine by 18% inhibition of etoposide efflux by 72% verapamil+probenecid by 43% (ritonavir) Summary For A...

  46. Strategy for MDR1 and Open Questions: • Calcein assay, CACO-2 efflux • Additional tests necessary: inverted out vesicles, ATPase assay, inhibition tests • Alternative assays for other binding sites? • How to handle false positives and negatives? • Cell lines without background? • Tight cell lines for transfection of efflux transporters-alternatives for MDCK? • In vivo experiments? How to interprete K.O. mice results in terms of PK? • DDI experiments?

  47. Joined Initiative DMPK Frankfurt DG Metabolic Diseases Stefan Theis Tanja Eisenblätter Reinhard Liebe Wolfgang Schmider Katharina Mertsch Jochen Maas Werner Kramer

  48. Back ups

  49. Caco-2 Cell Line: Expression of Transporters Western Blot Analysis + + -

  50. Caco-2 Cell Line: Expression of Transporters Western Blot Analysis + - + +

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