1. Transporters 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-barrier, cancer cells, and intestine is a iabilitiy for some compounds. Membrane transporters are responsible for two important permeability mechanisms, active uptake and efflux.

3. Transporter Fundamentals Passive diffusion is the predominant mechanism for the permeation of drugs throughout the body. Compound must have favorable physicochemical properties (i.e., lipophilicity, hydrogen bonds, molecular weight)

4. Uptake (import) transporters provide necessary nutrients and other compounds. Often referred to as active transport Efflux transporters (export) assist the bulk movement of compounds.

5. Transporter Effects Uptake transporters enhance the absorption of some drug molecules in the intestine. Efflux transporters on the luminal surface of G.I epithelial cells oppose, the absorption of some molecules. Assist the uptake of some molecules into hepatocytes to enhance metabolic and biliary clearance. Elimination of many drugs and metabolites is enhanced by active secretion in the nephrons of the kidney.

7. Many of transporters that are known to affect drug ADME/Tox are listed.

8. Secretion vs Excretion Secretion - Active process, releasing chemicals from cell or gland. (Enzyme, Hormone, Saliva, etc) Movement of material one point to another Excretion – Passive process, process of waste products of metabolism. (Tears, urine, sweat, etc) Removal of material from living thing.

9. Involved in absorptive uptake, efflux, secretory efflux Uptake increases the concentration of drug in the blood While secretory efflux lowers the concentration in blood Involved in hepatic uptake, biliary clearance, and Hepatocyte efflux Involved in tubular secretion and reabsorption

10. Consequences of Chirality on Transporters Have enantiomeric selectivity which results effecting on any barriers in vivo where particular transporter has a significant role.

11. Efflux Transporters P-glycoprotein (Pgp) Breast Cancer Resistance Protein Multidrug Resistance Protein 2 Efflux transporters in the BBB ( Pgp, MRPs, BCRP, OATs, OATPs, EAATs), TAUT

12. P-glycoprotein 170KD protein with 1280 amino acids and 12 trans-membrane segments. Abundant in cell barriers that have a protective function such as BBB, small and large intestine, liver, kidney, adrenal gland, pregnant uterus.

13. Rules for Pgp Efflux Substrates Referred to as “Rule of 4”  More likely to be a Pgp substrate when N + O ≥ 8 MW ＞ 400 Acid with pKa ＞ 4  More likely to be a Pgp non-substrate when N + O ≤ 4 MW ＜ 400 Base with pKa ＜ 8

14. Substitution of moieties in reducing the Pgp efflux while maintaining potency

15. Structure Modification Strategies to Reduce Pgp Efflux 1. Introduce steric hindrance to the hydrogen bond donating atoms by ▫ A. Attach a bulky group ▫ B. Methylate the nitrogen 2. Decrease H-bond acceptor potential A. Add an adjacent electron withdrawing group B. Replace or remove the hydrogen bonding group 3. Modify other structural features so that they may interfere with Pgp binding 4. Modify the overall structure’s Log P to reduce penetration into the lipid bilayer where binding to Pgp occurs.

16. Steric hindrance can be increased to reduce Pgp efflux

17. Breast Cancer Resistance (BCRP, ABCG2) ▫ Expressed normally in many tissues ▫ Naturally involved in the efflux of porphyrins and metabolites Multidrug Resistance Protein 2 (MRP2, ABCC2) ▫ Contribute to cancer multidrug resistance ▫ Transports glutathione, glucuride, and sulfate conjugates of lipophilic compounds Efflux Transporters in the BBB ▫ Export substrates from the brain and BBB endothelial cells

18. Uptake Transporters Facilitate the permeation of compounds into cells ▫ Organic Anion Transporting Polypeptides ▫ Di/Tri Peptide Transporters ▫ Organic Anion Transporters ▫ Organic Cation Transporter ▫ Large Neutral Amino Acid Transporter ▫ Monocarboxylic Acid Transporter ▫ Glucose Transporter ▫ Bile Salt Export Pump ▫ Sodium Dependent Taurocholate Co-transporting Polypeptide

19. Organic Anion Transporting Polypeptides OATP1A2 ▫ Found in BBB, hepatocytes, renal epithelium ▫ Transport organic anions, ouabain, cortisol, large organic cations ▫ Transport drugs – fexofenadine, enalapril, temocaprilat, N-methyl quinidine, DPDPE, deltrophin II OATP1B1 ▫ Similar to OATP1A2 ▫ Transport – eicosanoid, benzylpenicillin, methotrexate, rifampin, pravastatin, rosuvastatin, cerivastatin

20. Di/Tri Peptide Transporters Enhance uptake of dipeptides and tripeptides (not individual amino aids or tetrapeptides) Hydrophobicity increases PEPT1 binding and aromatic residues are preferred.

21. Organic Anion Transporters (OATs) ▫ Enhance renal clearance of some drugs Organic Cation Transporter (OCT) ▫ Enhance transport into the urine in kidney Large Neutral Amino Acid Transporter (LAT1) ▫ Transports amino acids such as L and F, L-DOPA, methyl DOPA, daclofen, melphalan Monocarboxylic Acid Transporter (MCT1) ▫ Enhances uptake of salicyclic acid, pravastatin, lovastatin, simvastatin

22. Other Uptake Transporters Glucose Transporter Bile Salt Export Pump Sodium Dependent Taurocholate Co - transporting Polypeptide Uptake transporters in the BBB ( GLUT1, LAT1, MCT1, CAT1, CNT2, CHT, NBT)

23. Transporter Methods In Vivo In vitro In Silico

24. Overview In silico predictions are increasingly available for transporters In vitro cell layer assays use inhibitors and cell lines with transfected transporters High – throughput ATPase and calcein acetoxymethyl ester assays are used, but cell layer transport is most relevant Transporters are knocked out genetically or chemically in vivo for in – depth study

25. In Vivo Methods for Transporters Genetic Knockout Animal Experiments for Transporters ▫ Mdr1a, mdr1b, mrp1 genes are knocked out  Does the compound have a much higher BBB permeability in the knockout versus the wild- type?  For a compound that is poorly absorbed after oral dosing and has good solubility, passive diffusion permeability, and metabolic stability, does it have higher absorption in the knockout versus the wild – type?

26. In Vivo Methods for Transporters Chemical Knockout Experiments for Transporters ▫ Saturate the transporters with the test compound. ▫ Increasing permeability or absorption is observed, then efflux transporter likely is being saturated

27. In Silico Transporter Methods Commercial software for transporters is limited to the classification of Pgp substrates

28. In Vitro Transporter Methods Cell Layer Permeability Methods for Transporters Caco-2 Permeability Method for Transporters Transfected Cell Line Permeability Method for Transporters Uptake Method for Transporters Oocyte Uptake Method for Transporters Inverted Vesicle Assay for Transporters ATPase Assay for ATP Binding Cassette Transporters Calcein AM Assay for Pgp Inhibitor

29. Cell Layer Permeability Methods for Transporters

30. Caco-2 Permeability Method for Transporters Discussed in previous chapter Most common application of Caco-2 is to estimate intestinal absorption Two important points when it is used ▫ Expression levels of transporters can vary ▫ Test compound may be a substrate for more than one transporter  Pgp (MDR1), breast cancer resistance protein (BCRP), PepT1, PepT2, multidrug resistance protein 2 (MRP2)

31. Transfected Cell Line Permeability Method Cell lines naturally express only low levels of membrane transporters, so background signal is low Transporter expression levels are high, so high signal-to-noise ratios are obtained. Larger transport ratios are produced. Require fewer resources than Caco-2 to maintain. MDCK, LLC-PK1, HEK 293 are transfected.

32. Transfection Cultured eukaryotic cell Stable transfection & transient transfection Calcium phosphate precipitation Lipofectamin – mediated transfection Electroporation Microinjection

34. Transfected Cell Line Permeability Method Cells are seeded in transwell plate and cultured for 3days. Transepithelial electrical resistance values are checked to ensure minimal paracelluar permeation Atenolol (paracelluar marker) is used to check layer integrity Growth medium is removed and replaced with 37 ℃ reduced serum medium Small volumes from the apical and basolateral chambers are withdrawn at specific time points ( 30, 60, 120 mins) Quantitated using liquid chromatography/mass spectrometry (LC/MS) techniques

35. Uptake Method Measure the rate of test compound concentration cells that are living in standard solid-bottom culture plates. Convenient format for higher-throughput assays due to not requiring complexity and high cost of transwell system Beneficial for in vitro studies, in which the observed functional in vitro activity is correlated to the intracellular test compound concentration

36. Oocyte Uptake Method

37. Inverted Vesicle Assay Transporter genes have been cloned into insect cells Vesicles are produced that contain the transporter in their membranes. Quantitation uses scintillation counting or LC/MS

38. ATPase Assay for ATP Binding Cassette Transporters ATP binding cassette transporters bind and hydrolyze ATP molecules

39. Calcein - AM Assay Calcein acetoxymethyl ester is a Pgp substrate which limits its ability to enter cells. Calcein-AM is rapidly hydrolyzed inside the cells to form calcein If coincubated test compound is Pgp transporter inhibitor, the test compound will inhibit Pgp efflux and more calcein-AM will enter the cells

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