Molecular Pharmacology & Therapeutics

Molecular Pharmacology & Therapeutics
The Pharmacology of Drug Transporters Neil A. Clipstone, Ph.D. Molecular Pharmacology & Therapeutics

How drugs cross cellular membranes
Passive diffusion Drug Carrier-mediated Influx/Uptake transporter Efflux Drug- H+ Drug Extracellular Intracellular

Significance of drug transporters in the drug response
Influx/Uptake transporter Efflux Influx Efflux Tissue expression Expression levels Activity Polymorphisms Inhibitors Plasma/Tissue drug concentration Drug distribution Drug Toxicity Drug Efficacy

Dietary and Evironmental
Normal endogenous function of transporter proteins Nutrients e.g. AA/sugars Influx/Uptake transporter Essential Metabolites e.g. vitamins Signaling Molecules e.g. steroids/hormones Normal Cell Function And Metabolism Dietary and Evironmental Toxins Efflux transporter EXCRETION/DETOXIFICATION Bile/Urine/Gut

conformational change
Carrier-mediated drug transport Cargo forms complex with transporter Transport is subject to saturability Transport can be reversible (depending on concentration gradient) Transport is similar to enzyme kinetics Rate of transport determined by Michaelis-Menten kinetics Transport can be inhibited by other compounds Transporter: Open conformation Cargo binds to transporter Transporter undergoes conformational change Cargo is released on other side of membrane Drug

Different mechanisms of carrier-mediated drug transport
Passive Transport Active Transport High Electrochemical potential gradient of substrate Low Low Electrochemical potential gradient of substrate High membrane membrane ATP Primary Active Transport ADP Facilitated Diffusion Antiporter Co-transported substrate Secondary Active Transport Transported substrate Symporter

Influx and Efflux transporters co-operate to promote
vectorial transport of drugs across epithelial/endothelial barriers - absorption/cellular uptake/excretion Epithelial/Endothelial barrier Drug 1 Influx Transporter 1 Efflux Transporter 1 Drug 2 Efflux Transporter 2 Influx Transporter 2 Gut lumen/Blood Blood/Bile Blood/Urine Blood/CNS

Selective expression of transporters promotes tissue-specific
drug uptake and barrier functions Tissue 1 Tissue 2 Tissue 3 Drug efficiently taken up into Tissue 1 Drug not a substrate for influx transporter expressed in Tissue 2 - No drug uptake Efflux transporters form a “drug-barrier” by exporting any drug taken up by influx transporters Drug Response Drug Response Drug Response

Role of drug transporters in Pharmacokinetics
Intestinal epithelia - ABSORPTION/EXCRETION Target tissues - SELECTIVE DRUG UPTAKE/DISTRIBUTION Liver epithelia - HEAPTIC UPTAKE/METABOLISM/ELIMINATION Kidney epithelia - CLEARANCE/ELIMINATION CNS endothelium - BLOOD BRAIN BARRIER Efflux transporters & Blood Brain Barrier

Role of drug transporters in mediating adverse drug reactions
Example 1 Uptake and/or Efflux in LIVER/KIDNEY Clearance/Plasma Conc Target Organ Conc TOXICITY Example 2 Uptake and/or Efflux in toxicological target organ e.g. Brain Cellular Conc/Toxicity e.g. Drugs can inhibit either the Hepatic uptake of bilirubin or the excretion of bilirubin-conjugates into the bile resulting in either Hyperbilirubinemia (serum bile) or cholestasis ( bile flow to gut) Example 3 Drug inhibits transport of endogenous transporter substrates Plasma OR Cell conc of substrates/Toxicity

Drug Transporters as mediators of Drug-Drug interactions
binds transporter and prevents transport of Drug #1 Drug #1 is specifically taken up into tissue via a specific drug transporter Drug #2: Substrate or Inhibitor of Drug transporter Toxicity Plasma concentration Reduced efficacy of Drug #1 due to decreased uptake

Major classes of transporter proteins implicated in drug transport
Drug Transporters 48 sub-families >380 unique proteins 11 sub-families >49 unique proteins Solute Carrier (SLC) Superfamily ATP-binding Cassette (ABC) Superfamily Predominantly Influx/Uptake transporters (* - efflux, non ATP-dependent) ATP-dependent Efflux transporters 4 most important sub-families 3 most important sub-families OAT Organic Anion Transporters P-gp/MDR1 P-glycoprotein/Multidrug resistance 1 OATP Organic Anion Transporting Polypeptides BCRP Breast Cancer Resistance Protein OCT Organic Cation Transporters MRPs Multidrug resistance proteins *MATE Multi-drug and Toxin Extrusion transporters

A. OAT: Organic Anion Transporters
Uptake/Influx transporters: - Transport organic anions against a negative membrane potential by linking to the facilitated efflux of the counter ion a-ketoglutarate, whose intracellular concentration is maintained by the Na/dicarboxylate co-transporter acting in concert with the Na+/K+ ATPase Expressed in liver and kidney proximal tubules (+other tissues) Substrate specificity: Broad range of low Mr substrates; Endogenous: cGMP; Bile salts; Citric acid cycle intermediates; Hormones Drugs: Methotrexate (anti-cancer); NSAIDs; Captopril (ACE Inhib); furosemide (diuretic); antibiotics; ganciclovir & cidefovir (anti-viral); OAT OA- aKG Na/aKG Na+ Na+/K+ K+ ATP ADP Tertiary active transport OAT1-3 OAT4 OAT2 Kidney Liver Blood basolateral Apical/luminal Uptake from blood Renal reabsoprtion

+ Clinical significance of OAT Transporters: Drug Interactions I
Methotrexate and NSAIDs drug interactions Methotrexate: - cancer/Rheumatoid Arthritis - narrow therapeutic index - eliminated via renal tubular elimination - transported from blood to kidney via OAT1 NSAIDS: - competitive inhibitors of OAT1 transporter activity Methotrexate Toxicity OAT1 Kidney Blood Apical/luminal OAT1-dependent uptake from blood Methotrexate URINE Elimination Methotrexate OAT1 Methotrexate elimination Plasma Methotrexate + NSAIDs NSAIDs NSAIDs inhibit OAT1-dependent Methotrexate uptake into the kidney

Clinical significance of OAT Transporters: Drug Interactions II
Using Probenecid to prevent Cidefovir-induced nephoroxicity Probenecid: - a potent inhibitor of OAT1 Cidefovir: - anti-viral used to treat CMV retinitis - transported into proximal tubules by OAT1 - treatment limited by severe renal toxicity - Cidefovir always co-administered with probenecid Cidefovir elimination (glomerular filtration) Cidefovir Cidefovir Blood OAT1-dependent uptake from blood OAT1 OAT1 Kidney Drug-induced nephrotoxicity Apical/luminal + Probenecid Probenecid prevents Cidefovir-induced nephrotoxicity by blocking OAT1-dependent Cidefovir uptake into the proximal tubules allowing for Cidefovir to be eliminated via Glomerular filtration Probenecid

B. OATP: Organic Anion Transporting Polypeptides
Uptake/Influx transporters: Electroneutral exchangers Transports substrates in exchange for HCO3- Broadly expressed in many tissues including gut, liver and kidney proximal tubules Substrate specificity: Broad range of substrates; Amphipathic Anions, Mr > 350 Da Endogenous: Bile acids, steroids, thyroid hormones Drugs: Statins, Antibiotics, Anti-cancer drugs, Anti-diabetes drugs Inhibitors: Cyclosporin, Macrolide antibiotics, Flavanoids OATP OA- HCO3- OATP4C1 OATP 1B1/1B3/2B1 Kidney Liver basolateral 1A2/2B1 Intestine Blood Apical/luminal OATP1A2 Intestinal uptake Renal reabsorption Hepatic reabsorption from bile Uptake from blood

Clinical significance of OATP transporters
OATP1B1: - important in response to STATIN drugs (anti-cholesterol) - responsible for STATINs principle effects in the liver (First Pass Effect) - multiple SNP in OATP1B1 influence STATIN efficacy & systemic exposure - OATP1B1*5 and OATP1B1*15 have  transporter activity  STATIN uptake/ STATIN efficacy   systemic STATIN exposure/ STATIN TOXICITY - Cyclosporin is potent inhibitor of OATP1B1 and blocks STATIN uptake Toxicity STATINs STATINs OATP1B1 basolateral Blood Liver STATINs OATP1B1 Liver STATINs OATP1B1*15 Liver OATP1B1 polymorphism Cyclosporin STATINs STATINs effectively taken up into liver by OAT1B1 STATIN uptake by OAT1B1*15 Efficacy/Systemic [STATIN] Cyclosporin inhibits OATP1B1  STATIN Uptake Efficacy/Risk of toxicity

C. OCT: Organic Cation Transporters
(OCT1-4 & OCTN1 and OCTN2) Uptake/Influx transporters - mediate simple passive facilitated diffusion of substrates - (Na+/H+-independent) Expressed in Gut, Kidney and Liver and other tissues Substrate specificity: Small positively charged compounds Endogenous: monoamine neurotransmitters, creatine, catecholamines Drugs: Cisplatin (chemotherapy); metformin (anti-diabetes); imatinib (anti-cancer); cimetidine (H2 receptor antagonist); procainamide (antiarrhythmic -narrow therapeutic window) OCT2/OCT3 OCT1/OCT3 Kidney Liver basolateral OCT3/N1/N2 Intestine Blood Apical/luminal OCTN1 Uptake from blood Intestinal Absorption

D. MATE: Multidrug and Toxin Extrusion transporters
(MATE-1/MATE-2) Organic Cation/Proton Antiporter Efflux transporters - Transport organic cations - Secondary active transport- driven by H+ antiport - Play a major role in excretion of +ve charged drugs - Overlapping substrate specificity with OCTs - Primarily responsible for secretion of OCT-transported substrates - Expressed in Liver and Kidney- luminal brush border surfaces - Responsible for: - renal tubular secretion of cationic drugs into urine - hepatic elimination of cationic drugs into bile MATE1 OC+ H+ extracellular intracellular OCT2/OCT3 OCT1/OCT3 Kidney Liver basolateral Blood Apical/luminal MATE1 Bile MATE2 Urine

Clinical significance of OCT/MATE transporters
A. Transporter polymorphisms Multiple SNPs affect OCT and MATE activity Influence the PK of multiple Organic Cation drugs (especially metformin) Metformin is a very basic anti-diabetic drug that acts in the liver and is eliminated unchanged by renal tubular excretion OCT/MATE SNPs loss of transporter activity kidney uptake/excretion  systemic drug availability B. Drug Interactions Most drug interactions mediated by OCT/MATE are caused by CIMETIDINE - an histamine H2 receptor antagonist used in the treatment acid peptic disorder - extensively eliminated via the kidney - potent competitive inhibitor of OCT transporters and prevents renal elimination of other OCT-dependent drugs e.g. PROCAINAMIDE- basic anti-arrhythmic drug extensively eliminated via renal tubular secretion CIMETIDINE  blocks PROCAINAMIDE renal elimination plasma concentration of PROCAINAMIDE (narrow therapeutic window, TOXICITY) C. Prevention of Cisplatin-induced nephrotoxicity Cisplatin is a chemotherapeutic agent used in the treatment of certain cancers - primarily eliminated via renal tubular excretion - use is limited by nephrotoxicity - Co-administration of CIMETIDINE blocks Cisplatin uptake into the kidney and prevents Cisplatin-induced nephrotoxicity

ABC: ATP-binding Cassette family of Efflux transporters
A. P-glycoprotein (P-gp)/MultiDrug Resistant protein-1 (MDR1) B. Breast Cancer Resistant Protein (BCRP) C. Multidrug resistant proteins (MRP) Active transport Efflux transporters - Use hydrolysis of ATP to generate the energy needed to move substrates across membranes against their concentration gradient Present on the apical luminal brush border membranes of gut, liver and kidney epithelia - Involved in the active secretion of drugs across epithelial surfaces into the gut lumen, urine and bile - major role in systemic drug elimination Expressed on endothelial cells of the Blood Brain Barrier (BBB) - prevent access of xenobiotic compounds (drugs) to the CNS Upregulated in certain cancer cells - implicated in resistance of cancer cells to chemotherapeutic drugs ATP ADP ABC Transporter Drug

P-gp pumps drug out of the cell
ABC: ATP-binding Cassette family of transporters A. P-glycoprotein (P-gp)/MultiDrug Resistant protein-1 (MDR1) Kidney Liver Intestine Blood Apical/luminal Pgp/MDR1 Urine ATP ADP Gut lumen Bile Drug P-gp pumps drug out of the cell across the membrane and into lumen Drug elimination Substrate Specificity: - Broad substrate specificity - Typically bulky hydrophobic structures with neutral/positive charge - Specificity overlaps with CYP3A4 Drugs: Digoxin (narrow therapeutic range); Ca2+ channel blockers (e.g. verapamil), HIV protease inhibitors; anti-cancer drugs (e.g. etoposide); erythromycin; ketoconazole; Cyclosporin; Loperamide Inhibitors: Cyclosporin; verapamil; Clarithromycin; ritonovir Inducers: Rifampin & St. John’s wort- both induce  expression of P-gp resulting in  drug efflux & drug plasma concentration Similar to CYP3A4

B. Breast Cancer Resistant Protein (BCRP)
Expression- gut enterocytes & liver; blood brain barrier; mammary epithelium Substrates: Neutral/negatively charged compounds Endogenous: Riboflavin (Vit B12)- BCRP responsible for concentrating in breast milk Drugs: STATINS; antibiotics; etoposide, imatinib & gefitinib (anti-cancer drugs) C. Multidrug resistant proteins (MRP) Expression: - broadly expressed in many tissues Substrates: - amphipathic molecules with at least one –ve charge Endogenous substrates: - glutathione-, glucuronide-, and sulfate-conjugates Drugs: anthracyclines, vinca alkaloids, etoposide, vincristine, methotrexate, antivirals

The Blood Brain Barrier
Partridge, W.M. NeuroRx 2: 3-14 (2005) Whole body imaging of radioactive histamine distribution in a mouse 30 min post-injection Main function is to protect the brain from xenobiotics and toxins Formed by specialized brain endothelial cells - Tight junctions prevent ions and large molecules passing between endothelial cells - P-gp/MRP/BCRP ABC family efflux pumps form a barrier to a large range of drugs and other compounds - actively transport these compounds back into the blood and thereby prevent their entry into the CNS Excludes most drugs other than those small (< 400Da) and lipophilic - consequently only ~1% of all drugs gain access and are active in the CNS Pgp/MDR ATP ADP MRP 1/2/4/5 BCRP Blood OATP1A2 OATP2B1 CNS Brain endothelial cell Astrocyte Brain Barrier Tight Junctions Drug P-gp/MRP/BCRP family of efflux pumps Pumps drugs from endothelium back into into blood - Prevents drugs from entering the CNS

Clinical significance of the P-glycoprotein/MDR1 transporter I
A. Effects of P-gp/MDR1 inhibitors on drug pharmacokinetics - P-gp/MDR1 inhibitors (e.g. cyclosporin) inhibit P-gp/MDR1 efflux activity in the gut, kidney and liver this leads to decreased drug elimination of P-gp/MDR1 subtrates such as DIGOXIN (narrow therapeutic window) - leads to increased systemic drug bioavailability and increased risk of drug toxicity Kidney Liver Intestine Blood Pgp/MDR1 Renal/Hepatic elimination ATP ADP Bioavailability Drug P-gp inhibitor e.g. cyclosporin [DRUG] B. Inducers of P-gp e.g. RIFAMPICIN and St. John’s wort (popular herbal medication) - induce increased expression of P-gp (and other ABC Cassette transporters) -   drug efflux in gut/kidney/liver  plasma concentration  drug efficacy

Clinical significance of the P-glycoprotein/MDR1 transporter II
C. Effect of P-gp/MDR1 inhibitors on the Blood Brain Barrier P-gp inhibitors can decrease the efficacy of the BBB EXAMPLE: - LOPERAMIDE is an opioid receptor agonist used in the treatment of diarrhea - Potent substrate for P-gp therefore does not cross the BBB - Co-administration with P-gp inhibitors (e.g. CYCLOSPORIN) inhibits P-gp in BBB This allows Loperamide to cross BBB and enter the CNS where it can cause respiratory depression (adverse effect) D. Effects of increased expression of P-gp/MDR1 in tumor cells - tumor cells often “upregulate” expression of P-gp/MDR1 - increased expression of P-gp/MDR1 in tumor cells promotes efflux of anti-cancer drugs - increased expression of P-gp/MDR1 in tumor cells is associated with more aggressive phenotype, poorer prognosis and decreased sensitivity to chemotherapeutic drugs

KIDNEY PROXIMAL TUBULE
Overview of the role of drug transporters in drug disposition Gut Absorption Pgp/MDR ATP ADP MRP2 BCRP MATE MRP3 OATP OCT3 OCTN1 OCTN2 Gut Elimination Intestinal Epithelium BLOOD Enterocytes GUT LUMEN Luminal Brush border membrane Pgp/MDR ATP ADP MRP BCRP MATE OAT2 OCT1 OATP1B1 OATP1B3 OATP2B1 MRP3 OCT3 BILE OATP1A2 BLOOD LIVER Hepatic Elimination Bile Canaliculus OAT4 Pgp/MDR ATP ADP MRP2 BCRP MATE OAT3 OAT2 OAT1 OCT3 OCT2 OATP4C1 OATP1A2 Blood Kidney Epithelium: Renal Proximal Tubule reabsorption Renal excretion KIDNEY PROXIMAL TUBULE OCTN1 Urine

Summary: Clinical significance of drug transporters
Pharmacokinetics Drug #2 binds transporter and prevents transport of Drug #1 Drug #1 is specifically taken up into tissue via a specific drug transporter Drug #2: Substrate or Inhibitor of Drug transporter Toxicity Plasma concentration Reduced efficacy of Drug #1 due to decreased uptake Drug Interactions

SUMMARY OAT OATP OCT MATE P-gp Type Substrate Prototypical Drug
Inhibitors Clin. significance/ Drug Interactions Uptake -linked to aKG antiport Bicarb exchanger Passive facilitated diffusion Efflux Proton exchanger Not dependent on ATP Active transport ATP-dependent Organic anions Broad range Low Mr Amphipathic anions Mr>350 Da Small cations Broad specificity Bulky hydrophobic Neutral/+ve charge Methotrexate NSAIDs Cidefovir STATINS Metformin Cisplatin Cimetidine Procainamide Digoxin Loperamide Etoposide Probenecid Cyclosporin Macrolides Verapamil NSAIDs block methotrexate elimination methotrexate toxicity Probenecid prevents Cidefovir renal uptake blocks cidefovir-induced nephrotoxicity OATP1B1 polymoprphisms Hepatic STATIN uptake STATIN toxicity Cyclosporin blocks STATIN uptake Main role: Renal tubular secretion of OCT substrates Cimetidine blocks OCT-mediated renal uptake of many drugs thereby blocking their elimination plasma concn Cimetidine blocks renal uptake of cisplatin prevents cisplatin-induced nephrotoxicity Cyclosporin inhibits P-gp-mediated elimination of digoxin Systemic availability Rifampicin/St. John’s wort induce P-gp expression  drug efflux  systemic availability Inhibitors enhance CNS accessibility by overcoming BBB Cimetidine

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