MRPs IMPACT EN PHARMACOCINETIQUE ET PHARMACODYNAMIQUE DES MEDICAMENTS

1. MRPs IMPACT EN PHARMACOCINETIQUE ET PHARMACODYNAMIQUE DES MEDICAMENTS Pascal Espié UCB Pharma, Belgium Journées GMP, 4-5 Février 2004 - Distribution et Transporteurs en Pharmacocinetique

2. Multidrug Resistance-associated Proteins are Members of the ABC Transporter Superfamily • Three major drug efflux pumps involved in intrinsic or acquired defense • of cells against drugs • MultiDrug Resistance 1-encoded Pgp (ABCB1) ... 70’s • MDR1 and mMdr1a,b confer MDR phenotype to tumor cells • (MDR2 and mMdr2 are flippases for PC at the CM of the liver - Do not confer MDR) • Multidrug Resistance-associated Proteins (ABCC family) • First MRP cloned in 1992 in cancer cells (Cole et al.) ... MRP1 • Breast Cancer Resistance Protein (ABCG2)

3. The BBB & BCSFB Isolate the Brain From Circulating Xenobiotics • Interfaces between the CNS and the peripheral circulation • Brain Capillary Endothelial Cells form the Blood Brain Barrier • Interface brain parenchyma / systemic blood • Epithelium of the Choroid Plexus forms the Blood CerebroSpinal Fluid Barrier • Interface CSF / systemic blood • Relative importance, • Keep & Jones, Dev. Brain Res., 1990 • CP shows an apical surface area in the same size range as the luminal • surface area of the ECs of the BBB • Lee et al., Pharmacol Rev , 2001 • BBB surface area ca. 500/1000-fold larger than that of the CP • Restrict penetration of CNS drugs (vincristine, doxorubicin ... despite high lipophilicity) • Transporter-mediated efflux : Saturable , Non-Linearity , Interactions

4. c Efflux Transport Systems at the BBB - MDR1, MRPs, BCRP d MRP1 in astrocytes (Declèves et al., 2000) e MRP1 in microglia (Lee et al., 2001) MRP1 a Pgp in astrocytes & pericytes (Pardridge et al., 1997; Golden et al., 1999; Declèves et al., 2000 ; Sisodiya et al., 2002) b Pgp basal (Miller et al., 2000; Golden & Partridge, 1999, 2000) c Pgp in microglia (Lee et al., 2001) d ATP MRP5 ATP MRP2 Blood Astrocyte foot process e Passive Diffusion MDR1 Pericyte b ATP a ATP BCRP CE Cell & TJ a

5. Pericytes Blood cells Expression of MRPs on the BBB - Isolated Rat Brain Capillaries (1) A. Transmitted light image B. Single confocal section C. Image constructed from the stack • Reconstructed • z-section • 1 et 3 • No pericyte • No blood cell • Lumen with high • Fluorescence • vs. medium • 2 • Contain blood cells • Luminal fluo. lower • vs. endothelium • (ca. medium) Incubation : 30 min. w. 5µM daunomycin Miller et al., Mol Pharmacol, 2000

6. Expression of MRPs on the BBB - Isolated Rat Brain Capillaries (2) At 20’ : 8-times vs. medium (uphill transport) Luminal fluorescence • Luminal accumulation of daunomycin • PSC 833, ivermectin, verapamil, • CSA & vanadate sensitive • but not to leukotriene C4 (LTC4) Involvement of P-glycoprotein. Miller et al., Mol Pharmacol, 2000

7. P BC BC Expression of MRPs on the BBB - Isolated Rat Brain Capillaries (3) • Luminal accumulation • of S-Rho 101 • LTC4, CSA & vanadate • sensitive • but not to PSC 833 • & verapamil Accumulation of FL-Methotrexate Accumulation of sulforhodamine 101 (1µM in medium - 30’) - Stacked image Involvement of Mrp(s) Miller et al., Mol Pharmacol, 2000

8. Confocal Immunolocalization of Pgp & Mrp2 Isolated Rat Brain Capillaries pAb-anti Pgp Stacked images mAb-anti Mrp2 (... Mrp3, -5) Capillary lumens are partially collapsed Both antibodies labeled structures that line the capillary lumens, indicating localization to the luminal surface of the ECs [No staining in Mrp2-deficientTR- rats, not shown] “Pgp also localized to punctate sites at or near the basal surface of the ECs” Miller et al., Mol Pharmacol, 2000

9. Dialysate Role of MRP2 in the BBB - In Vivo Microdialysis • Perfusion mock CSF : 2µL/min • 0.5 h : local admin. PROBENECID (PB 10mM i.e. CSF +/- inh.) • 1 h : i.p. AED, PHENYTOIN (PH 50mg/kg) • Sampling : 40µL outflow (w. concomit. blood) Increased extracellular brain level of PH Increased D-to-P ratio of PH Potschka et al., JPET, 2003

10. Mrp2-deficient Mrp2-deficient Dialysate BBB Permeability in Mrp2-deficient TR- Wistar - In Vivo Microdialysis Higher dialysate-to-plasma ratio in MRP2-deficient rats. Higher extracellular brain levels in Mrp2-deficientrats Potschka et al., JPET, 2003

11. Effect of Mrp2 Inhibition on AED Efficacy in Amygdala Kindled Rats Anticonvulsant activity is illustrated as alteration of the GSTs relative to control* 1 2 3 4 1 2 3 Generalized Seizure Threshold (%) * 64.4 ± 8.2 µA * 41.1 ± 4.8 µA TR- * 70.1 ± 4.9 µA [+] Normal rats +/- PB Mrp2 deficient TR- rats 1 - Control 2 - 6.25 mg/kg PH i.p. 3 - 50 mg/kg PB i.p. (15 min before PH) 4 - PH + PB 1 - Control 2 - PH i.p. in [+] 3 - PH in wistar TR- Enhanced penetration of PH into brain (after MRP2 inhibition or lack of MRP2 in the BBB) Seizure threshold increased by 90% Potschka et al., JPET, 2003

12. CP 3rd V CP LV (2) Ependyma CSF CP 4th V EC TJ AM BLM Fenestrated Capillaries (no TJ) The Blood-Cerebrospinal-Fluid Barrier • The exchanges between blood & CSF • are regulated by : • The arachnoid membrane • The epithelial cells (EC) of the choroid • plexus CSF

13. BCSF Permeability Barrier for 99mTc-sestamibi In Vivo in Human MRI image CP within the lat. ventricles Coregistered image SPECT image 90 min after iv inj. of 99mTc-sestaMIBI (Pgp & MRP subst.) “Coupes coronales” • Radioactivity confined to the CP within each lateral ventricle • No detectable activity in adj. CSF or surrounding parenchyma (high in blood) Evidence for a carrier-mediated permeability barrier for 99mTc-sestaMIBI (freely diffusible) in CP preventing entry into CSF.... MRP1 ? Single-Photon-Emission Computed Tomography (g-activity) Rao et al., PNAS, 1999

14. Localization of Pgp in Epithelial Cells of CP by Immunohistochemistry • Frozen rat brain tissue • mAb C219 (x 100) • Intra. cell. epitope, all Pgps • cp, CP within a lateral ventricle • Arrowhead, Pgp in the CEC of • brain parenchyma • Frozen human CP • mAb MRK16 (x 400) • Extra cell. epitope, spec. hMDR1 In CP, MDR1/Pgp confined in ECs Rao et al., PNAS, 1999

15. mAb MRPr1 (x 400) • mAb QCRL-1 (x 400) - h epitope Localization of MRP in Epithelial Cells of CP by Immunohistochemistry Human CP tissue • Rat-brain section • mAb MRPr1 (x100) • r, h epitope In all cases, in CP, MRP confined in ECs Rao et al., PNAS, 1999

16. Localization of MRP in Polarized Primary Cultures of CP Epithelial Cells • Cultured neonatal rat CPECs • (confocal microscopy - mAb MRPr1) • Top view • Optical sections (perpendicular / plane of • the layer) • Plasma membrane expression pattern obvious 10 µM Basolateral surface localization Rao et al., PNAS, 1999

17. Localization of Pgp in Polarized Primary Cultures of CP Epithelial Cells • Cultured neonatal rat CPECs • (confocal microscopy - mAb C219) • Top view : punctuate or granular staining • pattern throughout the cytoplasm • Plasma membrane expression pattern • occasional • Optical sections (perpendicular / plane of the • layer) • Scattered intracellular punctuate foci • Occasional basolateral foci Vesicular localization for Pgp 10 µM Predominantly subapical distribution Rao et al., PNAS, 1999

18. Transepithelial Transport of 99mTc-sestamibi and 3H-Taxol Across Monolayers of Rat CP Epithelial Cells - Opposing Efflux Functions for Pgp & Mrp1 B > A B > A B > A + I + I + I A > B A > B A > B + I + I + I 99mTc-sestaMIBI 99mTc-sestaMIBI [3H]Taxol +/- GF120918 (Pgp Inhib) +/- MK571 (MRP Inhib) +/- GF120918 Taxol : Pgp substrate SestaMIBI : Pgp & MRP substr. • Apical directed efflux function for Pgp in the CP • Basolateraldirected efflux function for Mrp1 in the CP Rao et al., PNAS, 1999

19. MRP1 Limits the Passage of Etoposide from the Blood Towards the CSF CSF Mdr1a/Mdr1b IV admin. (60mg/kg) 1h - CSF, brain & blood “sampling” DKO TKO Mrp1 Blood 10-fold accumulation into CSF No significant difference in brain and plasma concentrations Minor role for Mrp1 at the BBB Major role at the BCSFB Wijnholds et al., J Clin Invest, 2000

20. ATP Transport Systems at the Blood-CSF Barrier Oatp3 CSF Oat1 Estradiol 17b-G 1-Naphthyl 17b-G Estrone Sulfate Leukotriene C4 Pravastatine Oatp1 MDR1 MDR1 Subapical Vesicules Rao et al., 1999 ? X Concerted action of members of the OAT family and ABC family OA- X-Glu/SG cAMP, cGMP SG conjugates Oatp2 MRP1 ATP MRP5 Blood N. Joan Abbott et al., 2002 ; Spector, Pharmacology, 2000 ; Angeletti et al., PNAS, 1997 ; Rao et al., PNAS, 1999

21. Pharmacoresistance in Epilepsy • In ca. 30% of patients with epilepsy, seizures persist despite carefully monitoring • Multifactorial A consequence of epilepsy • Uncontrolled seizures - ineffective mechanisms of action • Polymorphism • Chronic treatment with AEDs - loss of efficacy (tolerance) • Most patients with refractory epilepsy are resistant to most (often all) AEDs • Patients not controlled on monotherapy with the first AED have a chance of ca. 10% • to be controlled by other AEDs • In refractory epilepsy AED blood concentrations are near normal therapeutic levels • This argues against epilepsy-induced alterations in specific targets • Nonspecific and adaptive mechanisms (decreased drug uptake into the brain) • Overexpression of multidrug transporters in the BBB

22. Over-Expression of MDR-Related Transporters in Brain Tissue of Pharmacoresistant Patients With Epilepsy Resected epileptogenic human brain tissues Which is(are) the mechanism(s) triggering overexpression of transporters in the BBB ? See also Regesta and Tanganelli, 1999

23. Over-Expression of MDR-Related Transporters in ECs from Patients with Refractory Epilepsy (TLE) n.s n.s n.s + 134 % + 225 % + 179 % + 321 % Gene expression in isolated ECs (cDNA array) Immunohistochemistry of MDR1 in Cap. Endothelium Pial surface Capillaries Pial vessel GFAP (red) Nuclei (blue) MDR1 (green) Gene-expression changes - MDR1, MRP2, -5, hCRA-a Regional differences of MDR1 protein expression Dombrowski et al., Epilepsia, 2001

24. Seizure (not repetitive AED treatment) Induced MDR1 Expression In the Brain Capillary Endothelium Acute seizure model Mdr1 mRNA in hippocampus after limbic seizures induced by kainic acid i.p. in mice Mdr1 mRNA levels of rats with self-sustained status epilepticus (electrical stimulation of HP) Chronic epileptic model Stimulated HP EC : contralateral enthorinal cortex (area recruited in seizure generalization) Agarose gel at 24h (lane 1: saline) Upregulation of mdr1 in the brain after seizures contribute to MDR in epilepsy Rizzi et al., J. Neurosci., 2002

25. Major MRPs in the Liver Rat : Ampicilin, bilirubin G, cholate-3-G, E2-17b-G, lithocholate-3-G, 1-naphtol G, nordeoxycholate G, triiodothyronin G, GSH, BSP-SG, GSSG, LKT-SG, DNP-SG, Methotrexate, irinotecan, pravastatin, temocaprilat Hum : LTC4, bilirubin(mono/bis)-G r,h MRP2 (ATP) rMdr 1 hMDR 1 (ATP) Blood Calcein, E2-17bG, etoposide-G, aflatoxin B1-G, GSSG, LKT-SG, estrone sulfate (with GSH), vincristine (with GSH), PSC 833 (inhibitor) Resistance to : Doxo-, dauno-, epirubicin, vinblastine, vincristine, etoposide rMdr 2 hMDR 2 hMDR 3 (ATP) Bile canaliculus BSEP / SPgp Rat : Bile acid (TC, GC), benzothiazole-G, E2-17b-G, taurolithocholate-3-sulfate Resist. : etoposide, teniposide (w/o GSH) r,h MRP1 (ATP) r,h MRP3 (ATP) Blood

26. BLOOD Normal Mutant 0.4 LIVER BILE 0.6 0.3 0.4 LIVER MEAN ACTIVITY CONCENTRATION (MBq/cm3) BLOOD 0.2 URINE 0.2 0.1 URINE BILE 0 0 0 10 20 30 40 50 0 10 20 30 40 50 TIME AFTER N-[11C]ACETYL-LTE4 (min) Hepatobiliary and Renal Elimination of Positon-Emitting Cysteinyl Leukotriene in Normal and Mrp2-deficient Rats From Dietrich Keppler, AAPS Workshop, Feb. 2003

27. Transport Mechanism of Organic Anions : MRP & OATP Working Jointly Transcellular transport of [3H] Pravastatin (1µM) across an MDCK II Monolayer OATP2 MRP2 B > A A > B MDCK II - OATP2 / MRP2 may be used to analyze the vectorial transport of OA- and to screen the transport profiles of NCEs Sasaki et al., JBC, 2002

28. Concentration Dependence of Transcellular Transport Across MDCKII Double Transfectants (MRP2/OATP2) DT OATP2 only OATP2 Km 23.8 ± 6.1 µM Vm 249 ± 60 pmol/min/mg Pdiff 0.81 ± 0.41 µL/min/mg DT Km 27.9 ± 4.1 µM Vm 560 ± 89 pmol/min/mg Pdiff 6.01 ± 0.61 µL/min/mg 3H-Pravastatin 3H-Estradiol17b G Secretory efflux (B > A) DT Km 24.3 ± 10.4 µM Vm 149 ± 56 pmol/min/mg Pdiff 0.99 ± 0.32 µL/min/mg The uptake by OATP2 is the rate determining step for E17b G transcellular transport Saturable transport Sasaki et al., JBC, 2002

29. The Role of Transporters in Governing Drug Trafficking in the Brain • Multiples sites of entry and exit(brain capillary endothelium, choroidal epithelium • and circumventricular organs) • Multiple influx or efflux transporters operate in parallel at a given membrane site • At each barrier influx and efflux transporters are working in concert or in opposition • Physiological relevance of Pgp in CP remains unclear, • Apical/Subapical localization of Pgp paradoxically seems to prevent trafficking of • certain substrates out of the CSF. • Pgp at the BCSF barrier opposes the action of Pgp at the BBB • Novel localization in brain parenchyma cells ... Second “barrier” • Reconsideration of the present conceptualization of brain barriers : “Parenchymal Barrier”

30. The Role of Transporters in Governing Drug Trafficking in the Brain • Transporters mediate drug exchange between various cells types in the brain • parenchyma (neurons, neuroglial cells) and brain interstitium (think beyond BBB !) • Varying density of transporters across brain regions or structures • The transcellular transport rate of ligands is governed by the cellular uptake rate • Inhibition or induction of MDR proteins at the BBB/BCSFB can be a cause of • drug interaction (toxicity, therapeutic failure) - demonstratedclinically ? • Up-regulation of MDR proteinsat the BBB : pharmacoresistance to CNS drugs (AEDs)

31. Many thanks to ... In Vitro ADME team ... Rhys Whomsley Anne Brochot Brigitte Gérin Lydia Wouters Maria Rosa Florence Toubeau Vincent Scohy Arlette Maes In Vivo ADME team ... Pascale Jacques Didier Houyoux Delphine Viot Mohamed Amghar Metabolism team ... Valérie Mancel Sylvie Dell’Aiera Xavier Delepine Olivier Lurkin ... et on embauche ;-) !