1. Transporters 2015/11/04 Jun Min Jung

2. Overview Membrane transporters increase the influx and efflux of substrate compounds Transporters are found in many tissues in vivo P-glycoprotein efflux in the blood-brain barrier, cancer cells, and intestine is important for some compounds.

3. Transporters as Drug Targets Membrane transporters (T) play roles in pharmacokinetic pathways (drug absorption, distribution, metabolism, and excretion), thereby setting systemic drug levels.

4. Many important nutrients, including glucose and amino acids, are water soluble Transporter proteins enable water soluble nutrients to cross the lipid double membrane and enter the cell Transporters can be divided into pumps (ATP binding cassette) which consume energy by hydrolyzing ATP, and carriers which facilitate the diffusion of solutes without use of ATP

5. Transporter Fundamentals

6. Basic Mechanisms of Transporter Light blue circles depict the substrate. Arrows show the direction of flux. Dark blue ovals depict transport proteins

7. Transporter Effects Uptake transporters enhance the absorption of some drug molecules in the intestine. Transporters assist the uptake of some molecules into hepatocytes to enhance metabolic ad biliary clearance. Elimination of many drugs and metabolites is enhanced by active secretion in the nephrons of the kidney.

8. Examples of Drugs with Active Uptake Transport 혈압약 고혈압, 심부전 페니실린 파킨스병 강압제 ( 고혈압 ) 항암제 진통제, 해열제 고지혈증 항히스타민

9. Kidney Basolateral Transport: O CT1, OCT2, OAT1, OAT2, OAT3, MRP1 Kidney Apical Transport: Secretion: P-gp, OAT4 Re absorption: PEPT2 Blood-Brain Barrier P-gp (MDR1), OAT3, OATP-A, MRP1, MRP3 Liver Sinusoidal Transport Uptake: OCT1, OATP-C, OATP-B, OATP8, NTCP, OAT2 Secretion: MRP1, MRP3 Liver Canalicular (Biliary) Transport Secretion: P-gp, MRP2, BCRP, BSEP, MDR3 Intestinal Lumen Abs orption: PEPT1 Secreti on: P-gp, OATP3 Transporter Expression

10. GeneAliasesTissueDrug SubstrateInhibitor ABCB1P-gp, MDR1intestine, liver, kidney, brain, placenta, adrenal, testes digoxin, fexofenadine, indinavir, vincristine, colchicine, topotecan, paclitaxel ritonavir, cyclosporine, verapamil, erythromycin, ketocoanzole, itraconazole, quinidine, elacridar (GF 120918) LY335979, valspodar (PSC833) ABCB4MDR3liverdigoxin, paclitaxel, Vinblastine ABCB11BSEPlivervinblastine ABCC1MRP1intestine, liver, kidney, brainadefovir, indinavir ABCC2MRP2, CMOAT intestine, liver, kidney, brainindinavir, cisplatin,cyclosporine ABCC3MRP3, CMOAT2 intestine, liver, kidney, placenta, adrenal etoposide, methotrexate, tenoposide ABCC4MRP4 ABCC5MRP5 ABCC6MRP6liver, kidneycisplatin, daunorubicin ABCG2BCRPintestine, liver, breast, placenta daunorubicin, doxorubicin, topotecan, rosuvastatin, sulfasalazine elacridar (GF120918), gefitinib Major Human Transporters (ABC)

11. GeneAliasesTissueDrug SubstrateInhibitor SLCO1B1 OATP1B1, OATP-C OATP2 liverrifampin, rosuvastatin, methotrexate, pravastatin, thyroxine cyclosporine, rifampin SLCO1B3OATP1B3, OATP8,liverdigoxin, methotrexate, rifampin, SLCO2B1SLC21A9, OATP-Bintestine, liver, kidney, brainpravastatin SLC10A1NTCPliver, pancreasrosuvastatin SLC10A2ASBTileum, kidney, biliary tract SLC15A1PEPT1intestine, kidneyampicillin, amoxicillin, captopril, valacyclovir SLC15A2PEPT2kidneyampicillin, amoxicillin, captopril, Valacyclovir SLC22A1OCT1liveracyclovir, amantadine, desipramine, ganciclovir, metformin disopyramide, midazolam, phenformin, phenoxy-benzamine qu inidine, ritonavir, verapamil SLC22A2OCT2kidney, brainamantadine, cimetidine, memantine desipramine, phenoxy-benzamine, quinine SLC22A3OCT3skeletal muscle, liver, placenta, kidney, heart cimetidinedesipramine, prazosin, phenoxy- benzamine SLC22A4OCTN1kidney, skeletal muscle, placenta, prostate, heart quinidine, verapamil SLC22A5OCTN2kidney, skeletal muscle, prostate, lung, pancreas, heart, sm all intestine, liver quinidine, verapamil SLC22A6OAT1kidney, brainacyclovir, adefovir, methotrexate, zidovudine probenecid, cefadroxil, cefamandole, cefazolin, SLC22A7OAT2liver, kidneyzidovudine SLC22A8OAT3kidney, braincimetidine, methotrexate, zidovudineprobenecid, cefadroxil, cefamandole, cefazolin, Major Human Transporters (SLC)

12. Drug-Drug Interactions Due to Transporters Currently considered most important transporters for Drug- Drug Interactions (DDI): – ABC: P-gp, BCRP, BSEP – SLC: OATP1B1, OATP1B3, OATP2B1, OCT1, OCT3, OAT2 Currently considered less important: – MRP transporters Depending on the expression pattern of the affected transporter, DDI can result in changes to absorption, tissue distribution (CNS, tumors), or elimination of the victim drug.

13. Various Transporters

14. Intestinal epithelium Absorption direction = Drug Concentration Increase Secretion direction = Drug Concentration Decrease

15. Liver Hepatocytes Uptake (Excretion) Efflux(Retention recirculation) Clearance

16. Kidney Epithelial Cells

17. Blood-Brain Barrier Cells Efflux (from the BBB endothelial cells back into the blood) Uptake (from the blood, through the BBB endothelial cells and into the brain)

18. Efflux Transporters Facilitate the export of compounds from the cell Belong to the ATP-binding cassette (ABC) family

19. P-glycoprotein (Pgp) 170KD protein with 1280 amino acids & 12 trans-membrane segments Member of the ATP Binding Cassette family of transporters Gene known as MDR1 or ABCB1

20. 170KD protein with 1280 amino acids Member of the ATP Binding Cassette family of transporters Gene known as MDR1 or ABCB1 Positions of mutations that alter the substrate specificity of P-gp The N-linked glycosylation sites (N91, N94, and N99) Phosphorylation sites (S661, S667, S671, and S683)

21. Initially identified as a major cause of resistance by cancer cells to multiple drugs (e.g., paclitaxel, etoposide) having a variety of structures. It is present in many tissues of the body. Pgp is abundant in cell barriers that have a protective function blood-brain barrier, small and large intestine liver, kidney, adrenal gland, pregnant uterus. In the liver and kidney, Pgp enhances drug and metabolite clearance to the bile and urine, respectively. Pgp attenuates penetration of some compounds into the brain, uterus, testes and other tissues.

22. P-glycoprotein (Pgp) A drug molecule attaches to the binding domain of pgp 2 ATPs bound to the ATP binding regions, become hydrolyzed and induce conformational change to open pathway for the drug molecule to pass through into the extracellular fluid

23. Rules for Pgp Efflux Substrates More likely to be a Pgp substrateMore likely to be a Pgp non-substrate  Increasing numbers of hydrogen bond acceptors (N+O) appear to confer increasing likelihood of Pgp efflux.  This may be because binding to Pgp occurs in the lipophilic membrane region.

24. Models to Study Pgp Interactions Assay TypeTissuesParametersComments Bi-Directional Transport Caco-2 cells MDCK-MDR1 cells L LC-PK1-MDR1cells Net drug flux ratio of B to A and A to B Directly measure efflux across cell barrier Evaluation of P-gp transport and inh ibition Allow for localization/identification of t he transporters within the apical or basol ateral side of the membrane Uptake/effluxtumor cells cDNA transfected cells oocyte s injected with cRNA of transp orters Inhibition of uptake or efflux of fluorescent probe (Calcein-AM, rhodamine-123) Cannot distinguish substrate from inhibitor Tends to fail to identify substrate and/or inhibitor with low permeability ATPasemembrane vesicles from tissues or cells expressing P gp, Reconstituted P-gp ATPase stimulationSame comments as uptake/efflux assay

25. Cell monolayers grown on filters and placed in cluster plates Filters are typically PET or PC membranes with 0.4-1 μm pores Transport is measured in two directions: –Apical (A) to Basolateral (B), i.e. test compound added to apical side –Basolateral (B) to Apical (A), i.e. test compound added to basolateral side Drug = P-gp-mediated transport = Passive diffusion 1212 Filter membrane Cell monolayer ATP A (apical) B (basolateral) 1 2 Bi-directional Pgp Transport Assays

26. Cell Lines for Pgp Transport Assay Cells used for bi-directional transport studies should form a functionally polar ized cell monolayer, complete with tight junctions (verified by pre-experiment al TEER of 100-800 Ω·cm 2 ) At present, preferred cells lines include – Caco-2 cells – MDR1 transfected LLC-PK 1 cells – MDR1 transfected MDCK cells LLC-PK 1 and MDCK wild type cells should be used as negative controls Cells should be allowed to grow to confluence (typically 3-7 days for LLC-PK 1 or MDCK, 18-21 days for Caco-2, however accelerated 3-5 day Caco-2 models are available and produce similar results)

27. Cell Lines for Pgp Transport Assay Caco-2 – Human colon carcinoma cell line – Morphologically similar to small intestinal epithelial cells – Most extensively characterized human cell-based model for investigating permeability and Pgp transport of drugs – Various uptake and efflux transporters are expressed in Caco-2 cells, however, Pgp is functionally the most predominant – No wild-type cells to run alongside LLC-PK 1 -MDR1 – Transfected porcine kidney cell line – Low transporter background, especially for Pgp MDCK-MDR1 – Transfected canine kidney cell line – High background dog Pgp activity

28. Case Study of Pgp Efflux

29. Apparent Permeability (P app ) P app [cm/sec] = V r /C 0 x 1/S x dC/dt V r C 0 S dC/dt is the volume in the receiver chamber [cm 3 ] is the concentration in the donor chamber at t=0 is the filter surface area [cm 2 ] is the is the linear slope of the drug concentration in the receiver chamber with time after correcting for dilution [mM/sec] Efflux Ratio (RE) RE = P app (B to A) / P app (A to B) P app (B-A) is the P app value measured in the B to A direction P app (A-B) is the P app value measured in the A to B direction Calculation of P app and Efflux Ratios

30. Structure Modification Strategies to Reduce Pgp Efflux Introduce steric hindrance to the hydrogen bond donating atoms Attach a bulky group Methylate the nitrogen Decrease H-bond acceptor potential Add an adjacent electron withdrawing group Replace or remove the hydrogen bonding group Modify the overall structure’s Log P to reduce penetration into the lipid bilayer

31. Structure Modification Strategies to Reduce Pgp Efflux

33. Bidirectional transport assay In vivo drug interaction study with a P-gp inhibitor may be warranted In vivo drug interaction study with a Pgp inhibitor may be warranted Is efflux ratio ≥ 2 ? * Likely to be P-gp substrate Likely to be Pgp substrate Unlikely to be P-gp substrate Unlikely to be Pgp substrate YESNO Is efflux inhibited by P-gp inhibitors? ** Is efflux inhibited by Pgp inhibitors? ** Is efflux ratio < 2 ? * Transporters other than P-gp m ight be involved Transporters other than Pgp mi ght be involved Further in vitro studies to identify t ransporters may be warranted Decision Tree for Pgp Substrates FDA DRAFT Guidance for Industry (Sept 2006)

34. Bidirectional transport assay wit h P-gp probe substrate In vivo drug interaction study with a P-gp probe substrate (e.g. digoxin) is recommended In vivo drug interaction study with a Pgp probe substrate (e.g. digoxin) is r ecommended Net flux ratio of probe substrate de creases with increasing concentrati ons of test compound [I]/IC 50 > 0.1 Poor or non-inhibitor of P-gp Poor or non-inhibitor of Pgp Determine IC 50 (or K i ) Net flux ratio of probe substrate is n ot affected by increasing concentrati ons of test compound [I]/IC 50 < 0.1 In vivo drug interaction study w ith a P-gp probe substrate is not needed In vivo drug interaction study w ith a Pgp probe substrate is not needed P-gp inhibitor Pgp inhibitor Decision Tree for Pgp Inhibitors

35. Uptake Transporters Organic Anion Transporting Polypeptides (OATPs, SLCOs) OATP1B1 Expressed in liver Rifampin, Pravastatin, Rosuvastatin, Cerivastatin, Benzylpenicillin OATP1A2 Found in BBB, Hepatocytes, Renal epithelium Fexofenadine, Enalapril, Temocaprilat, N-methyl quinidine Di/Tri Peptide Transporters (PEPT1, PEPT2) Organic Anion Transporters (OATs) Organic Cation Transporters (OCT) Large Neutral Amino Acid Transporter (LAT1) Monocarboxylic Acid Transporter (MCT1)

36. Other Uptake Transporters Glucose Transporter (GLUT1) Bile Salt Export Pump (BSEP, ABCB11) Sodium Dependent Taurocholate Co-transporting Polypeptide (NCTP) Uptake transporters in the BBB CAT1 (Cationic amino acids) CNT2 (Nucleosides) CHT (Choline) NBT (Nucleobase)

37. Q&A A,B,C,D B,D A,B,C,D

38. Thank You