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Transporters 2015/11/04 Jun Min Jung 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. Transporters as Drug Targets • Membrane transporters (T) play roles in pharmacokinetic pathways (drug absorption, distribution, metabolism, and excretion), thereby setting systemic drug levels. • 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 Transporter Fundamentals Basic Mechanisms of Transporter • Light blue circles depict the substrate. Arrows show the direction of flux. • Dark blue ovals depict transport proteins 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. Examples of Drugs with Active Uptake Transport 혈압약 파킨스병 고혈압, 심부전 강압제(고혈압) 페니실린 페니실린 항암제 진통제, 해열제 고지혈증 항히스타민 Transporter Expression Liver Sinusoidal Transport Uptake: OCT1, OATP-C, OATP-B, OATP8, NTCP, OAT2 Secretion: MRP1, MRP3 Intestinal Lumen Abs orption: PEPT1 Secreti on: P-gp, OATP3 Blood-Brain Barrier P-gp (MDR1), OAT3, OATP-A, MRP1 , MRP3 Liver Canalicular (Biliary) Transport Secretion: P-gp, MRP2, BCRP, BSEP, MDR3 Kidney Basolateral Transport: O CT1, OCT2, OAT1, OAT2, OAT3, MRP1 Kidney Apical Transport: Secretion: P-gp, OAT4 Re absorption: PEPT2 Major Human Transporters (ABC) Gene ABCB1 Aliases P-gp, MDR1 ABCB4 MDR3 liver ABCB11 ABCC1 BSEP MRP1 liver intestine, liver, kidney, brain digoxin, paclitaxel, Vinblastine vinblastine adefovir, indinavir ABCC2 MRP2, CMOAT MRP3, CMOAT2 MRP4 MRP5 MRP6 BCRP intestine, liver, kidney, brain indinavir, cisplatin, intestine, liver, kidney, placenta, adrenal etoposide, methotrexate, tenoposide liver, kidney intestine, liver, breast, placenta cisplatin, daunorubicin daunorubicin, doxorubicin, topotecan, rosuvastatin, sulfasalazine ABCC3 ABCC4 ABCC5 ABCC6 ABCG2 Tissue Drug Substrate intestine, liver, kidney, brain, digoxin, fexofenadine, indinavir, vincristine, placenta, adrenal, testes colchicine, topotecan, paclitaxel Inhibitor ritonavir, cyclosporine, verapamil, erythromycin, ketocoanzole , itraconazole, quinidine, elacridar (GF 120918) LY335979, valspodar (PSC833) cyclosporine elacridar (GF120918), gefitinib Major Human Transporters (SLC) Gene SLCO1B1 Aliases OATP1B1, OATP-C OATP2 Tissue liver Drug Substrate rifampin, rosuvastatin, methotrexate, pravastatin, thyroxine SLCO1B3 SLCO2B1 SLC10A1 SLC10A2 SLC15A1 OATP1B3, OATP8, SLC21A9, OATP-B NTCP ASBT PEPT1 liver intestine, liver, kidney, brain liver, pancreas ileum, kidney, biliary tract intestine, kidney digoxin, methotrexate, rifampin, pravastatin rosuvastatin SLC15A2 PEPT2 kidney ampicillin, amoxicillin, captopril, Valacyclovir SLC22A1 OCT1 liver acyclovir, amantadine, desipramine, ganciclovir, metformin SLC22A2 OCT2 kidney, brain amantadine, cimetidine, memantine desipramine, phenoxy-benzamine, quinine SLC22A3 OCT3 skeletal muscle, liver, placenta, kidney, heart cimetidine desipramine, prazosin, phenoxybenzamine SLC22A4 OCTN1 kidney, skeletal muscle, placenta, prostate, heart quinidine, verapamil SLC22A5 OCTN2 SLC22A6 OAT1 kidney, brain acyclovir, adefovir, methotrexate, zidovudine SLC22A7 SLC22A8 OAT2 OAT3 liver, kidney kidney, brain zidovudine cimetidine, methotrexate, zidovudine kidney, skeletal muscle, prostate, lung, pancreas, heart, sm all intestine, liver Inhibitor cyclosporine, rifampin ampicillin, amoxicillin, captopril, valacyclovir disopyramide, midazolam, phenformin, phenoxy-benzamine qu inidine, ritonavir, verapamil quinidine, verapamil probenecid, cefadroxil, cefamandole, cefazolin, probenecid, cefadroxil, cefamandole, cefazolin, Drug-Drug Interactions Due to Transporters • Currently considered most important transporters for DrugDrug 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. Various Transporters Intestinal epithelium Absorption direction = Drug Concentration Increase Secretion direction = Drug Concentration Decrease Liver Hepatocytes Uptake (Excretion) Efflux(Retention recirculation) Clearance Kidney Epithelial Cells 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) Efflux Transporters • Facilitate the export of compounds from the cell • Belong to the ATP-binding cassette (ABC) family 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 The N-linked glycosylation sites (N91, N94, and N99) Positions of mutations that alter the substrate specificity of P-gp Phosphorylation sites (S661, S667, S671, and S683) • 170KD protein with 1280 amino acids • Member of the ATP Binding Cassette family of transporters • Gene known as MDR1 or ABCB1 • 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. 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 Rules for Pgp Efflux Substrates More likely to be a Pgp substrate More 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. Models to Study Pgp Interactions Assay Type Bi-Directional Transport Tissues Caco-2 cells MDCK-MDR1 cells L LC-PK1-MDR1cells Parameters Net drug flux ratio of B to A and A to B Comments 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/efflux tumor cells Inhibition of uptake or efflux of cDNA transfected cells oocyte fluorescent probe s injected with cRNA of transp (Calcein-AM, rhodamine-123) orters ATPase membrane vesicles from ATPase stimulation tissues or cells expressing P gp, Reconstituted P-gp Cannot distinguish substrate from inhibitor Tends to fail to identify substrate and/or inhibitor with low permeability Same comments as uptake/efflux assay Bi-directional Pgp Transport Assays • 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 1 = P-gp-mediated transport 2 = Passive diffusion A (apical) B (basolateral) 1 ATP 2 Cell monolayer Filter membrane 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·cm2) • At present, preferred cells lines include – Caco-2 cells – MDR1 transfected LLC-PK1 cells – MDR1 transfected MDCK cells • LLC-PK1 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-PK1 or MDCK, 18-21 days for Caco-2, however accelerated 3-5 day Caco-2 models are available and produce similar results) 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-PK1-MDR1 – Transfected porcine kidney cell line – Low transporter background, especially for Pgp • MDCK-MDR1 – Transfected canine kidney cell line – High background dog Pgp activity Case Study of Pgp Efflux Calculation of Papp and Efflux Ratios Apparent Permeability (Papp) Papp [cm/sec] = Vr/C0 x 1/S x dC/dt Vr C0 S dC/dt is the volume in the receiver chamber [cm3] is the concentration in the donor chamber at t=0 is the filter surface area [cm2] 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 = Papp (B to A) / Papp (A to B) Papp (B-A) is the Papp value measured in the B to A direction Papp (A-B) is the Papp value measured in the A to B direction 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 Structure Modification Strategies to Reduce Pgp Efflux Structure Modification Strategies to Reduce Pgp Efflux Decision Tree for Pgp Substrates Bidirectional Bidirectional transport transportassay assay IsIsefflux effluxratio ratio≥≥22??** IsIsefflux effluxratio ratio<<22??** IsIsefflux effluxinhibited inhibitedby byPgp P-gpinhibitors? inhibitors?**** YES Likely Likelyto tobe bePgp P-gpsubstrate substrate In Invivo vivodrug druginteraction interaction study studywith withaa Pgp P-gpinhibitor inhibitormay maybebewarranted warranted Unlikely Unlikelyto tobe bePgp P-gpsubstrate substrate NO Transporters Transporters other otherthan thanPgp P-gpmim ght be involved ight be involved Further Further in invitro vitrostudies studiesto toidentify identify tt ransporters ransportersmay may be bewarranted warranted FDA DRAFT Guidance for Industry (Sept 2006) Decision Tree for Pgp Inhibitors Bidirectional Bidirectional transport transport assay assay wit wit hh P-gp P-gp probe probesubstrate substrate Net Netflux fluxratio ratioof of probe probe substrate substrate de de creases with increasing concentrati creases with increasing concentrati ons ons of of test testcompound compound Pgp P-gpinhibitor inhibitor Net Net flux flux ratio ratio of of probe probe substrate substrate isis nn ot affected by increasing ot affected by increasing concentrati concentrati ons of test compound ons of test compound Poor Pooror ornon-inhibitor non-inhibitor of ofPgp P-gp Determine DetermineIC IC5050(or (or KKi)i) [I]/IC [I]/IC5050>> 0.1 0.1 [I]/IC [I]/IC5050<< 0.1 0.1 In In vivo vivodrug drug interaction interaction study studywith withaa Pgp P-gpprobe probesubstrate substrate(e.g. (e.g.digoxin) digoxin)isisr ecommended recommended In In vivo vivodrug drug interaction interaction study study w w ith ith aa Pgp P-gpprobe probesubstrate substrateisis not not needed needed FDA DRAFT Guidance for Industry (Sept 2006) 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) 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) Q&A A,B,C,D B,D A,B,C,D Thank You