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Peter C. Preusch, Ph.D. Pharmacology, Physiology, and Biological Chemistry Division National Institute of General Me

Principles of Clinical Pharmacology January 9, 2003 Module 2: Drug Metabolism and Transport Unit 6: Concentrative and Equilibrative Drug Transport. Peter C. Preusch, Ph.D. Pharmacology, Physiology, and Biological Chemistry Division

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Peter C. Preusch, Ph.D. Pharmacology, Physiology, and Biological Chemistry Division National Institute of General Me

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  1. Principles of Clinical PharmacologyJanuary 9, 2003Module 2: Drug Metabolism and TransportUnit 6: Concentrative and Equilibrative Drug Transport Peter C. Preusch, Ph.D. Pharmacology, Physiology, and Biological Chemistry Division National Institute of General Medical Sciences

  2. Objectives • Vision, reality, and the path between. • Methods of measuring drug transport in vitro and in vivo. • Mechanisms of drug transport. • Recent advances in understanding the role of membrane transport proteins. • Clinical significance.

  3. Measurements of Drug Distribution Reflect Membrane Transport In Vivo • Blood/tissue Samples, Biopsies, and Assays • Autoradiography • Perfusion/Cannulation Methods • Note ability to biopsy lumen wall or collect shed cells in the same intestinal perfusionexperiments – Loc-I-Gut • In vivo P(eff) amoxicillin +/- amiloiride (Na/H exchange inhibitor) – no effect on low P(eff) drug • Radiology - X-ray, PET, SPECT • Magnetic Resonance Imaging • Microdialysis

  4. Measurements of MembraneTransport In Vitro • Ussing chamber - excised tissue samples • Everted gut sac - uptake from medium • Uptake/efflux by membrane vesicles, liposomes, BLM, PAMPA, cells in culture (CHO) - filtration, centrifugation, oil-stop separatory assays • Fluorescent (confocal) microscopy of cultured cells fluorescent drugs (mitoxantrone, rhodamine) • Electrophysiology in cells (e.g., oocytes) • Monolayer cell cultures on permeable supports • Caco-2 cells, MDCKII, brain MVECs

  5. Measurement of Transport inExcised Tissue Samples Modified Ussing-chamber allows perfusion of solutions on both sides of membrane holder, control of pressure differential, measurement of potential, conductivity, pH. Adapted from Ref. 7.

  6. Monolayer Epithelial Cell Culture I.J. Hidalgo, in ref. 6, Models for Assessing Drug Absorption and Metabolism (Borchardt, et al., Eds.) Plenum Press, NY, 1996, p. 38.

  7. Thermodynamics of Transport • Transport of neutral species • Ions & transmembrane potentials • Ionizable species & proton gradients • Metals and other titrants • Macromolecular and cellular binding sites • Coupled transport and ATP driven pumps • Chemical conversions

  8. Equilibrative Transport Compartment Model  + - pHo pHi Gtransp SHo+ So Si SHi+ Gpump KBo KBi S'i SoBo SiBi

  9. Equations for Membrane Transport Thermodynamics Gtransp = 2.303RT log[Si]/[So] + nF + Gpump R = 8.314 joules/molK = 1.987 cal/molK F = 96.5 Joules/mol-mV = 23.06 cal/mol-mV  [Si]/[So] = 10x @ 296K (23°C) 310°K (37°C) (G) = 5.67 kJ/mol 5.936 kJ/mol = 1.35 kcal/mol 1.41 kcal/mol () = 58.5 mV 61.5 mV pH = pKa + log[S]/[SH]

  10. Examples Driving Force/Drug/Compartment Diffusion caffeine total body water Ion trapping Tc-Sestamibi heart mitochondria pH trapping quinidine renal excretion Binding warfarin plasma/liver ratio Active captopril GI absorption

  11. Proposed Mechanisms of Tetracycline Uptake and Efflux

  12. Mechanisms of Transmembrane Drug Transport - Example Drugs • Paracellular diffusion - ions, mannitol, polymers • Passive diffusion across lipid bilayer • fluoroquinolones, tetracycline (hydrophobic) • Diffusion through OM channels and porins • B-lactams, tetracyclins (hydrophilic, charged) • Facilitated diffusion • imipenem, catechols, albomycin, albicin • Active Transport • aminoglycosides, cycloserine, phosphomycin, alaphosphin • Vesicle Trafficking Mediated Transport • polymers, peptide hormones, targeted delivery

  13. Transcellular vs Paracellular Pathways Transepithelial Resistance (Ω cm2) renal tubule 6-7 gallbladder 20-30 intestine 30-100 chroid plexus 80 colon 290-500 Caco-2 230-1000 Gastric mucosa >1700 urinary bladder >2000 N-trimethyl chitosan chloride coadmin increased permeation of nonopeptide buserelin in Caco-2 cells and enhanced bioavailability in rats from 0.8% to 6-13%

  14. Details of Tight Junction EM of Caco-2 zona occluden zona adherens desmosome Ca++, IP3, PKC, CamK, MLCK

  15. Apparatus for On-Line Fluorescence Measurement of Transport in Epithelial Cell Cultures MDCKII  MDR1  SDZ PSC 833 Daunorubicin ex = 480, em = 590 FITC-dextran ex = 480, em = 525 Trans Epithelial Resistance (TER) = 300 - 600 mm2 Ref. 8: Wielinga, et al., J. Pharm. Sci, 88(12), 1340, 1999.

  16. Paracellular versus Transcellular Transport Ref. 8: Wielinga, et al., J. Pharm. Sci, 88(12), 1340, 1999.

  17. Paracellular Permeability Enhancers • Examples: Ca++chelators, bile salts, anionic surfactants, medium chain FAs, alkyl glycerols, cationic polymers, cytochalsin D, hormones, TNF-α, enterotoxins, zonula occludens toxin (V. cholerae) • Substrates: Ions, mannitol, ceftoxin, dextrans, proteins • Advantages: • hydrophilic & macromolecular substrates • avoids intracellular degradation • Disadvantages: • toxicity due high mM concentrations needed • non-selectivity of substrate transport • Concern: systemic toxicity of lumenal contents, blood brain barrier effects (intended and/or not)

  18. Pinocytosis, Endocytosis, and Receptor Mediated Transcytosis • Pinocytosis (cell sipping - non-mediated) • non-specific, non-saturable, bulk fluid phase uptake, large particles, polymer-conjugates, obsolete term? • Endocytosis (receptor-mediated uptake) • specific, relevant to macromolecules, used to deliver small molecules as prodrugs, mediates clearance • insulin, growth hormone, erythropoetin, G-CSR, ILs • Transcytosis (receptor-mediated uptake and secretion on the translateral surface) • useful for macromolecules and small molecule prodrugs, GI, BBB, and pulmonary epithelia. • Protein translocation domain fusions (mechanism?) • Antennapedia homeodomain, HIV TAT protein, R7

  19. Transcytosis Delivery of Prodrug vesicular transport Brain Blood From: Bickel & Pardridge in Ref. 22, p. 30. Transferrin receptor-mediated transcytosis of an mAB-avidin-biotin-disulfide cross-linked vasoactive intestinal peptide. Endothelial Cell TfR, VitB12R, FcRn, PigR are under commercial development.

  20. Passive Diffusion • Characteristics of passive diffusion • kin = kout, net rate = k([So] - [Si]), non-selective • Model Membranes (experimental systems) • monolayers, bilayers, liposomes, BLMs, IAMs • Membrane Models (functional/mathematical) • structural, electrical, single/multiple barrier, partition adsorption/diffusive, unstirred layers • Simulation of bilayers and transport • molecular dynamics - diffusion within bilayer • QSAR - structure/transport correlations

  21. Molecular Dynamics Simulation of Membrane Diffusion From: Bassolino-Klimas, Alper and Stouch, ref. 16. See also ref. 17. Snapshot from 10 nsec MD simulation in 100 fs steps. Showed hopping motions of 8 Å over ca 5 psec vs RMS motions of 1.5 Å. Motions differ in center and near surface, both differ from bulk organic. Rotational isomerizations (gauche/trans) gate channels between voids. Differing motions available to adamantane, nifedipine.

  22. QSAR of Transport • Hansch Equation • log (1/C) = -k(logP)2 + k'(logP) + + k" • C = dose or [S] for effect (ED50, IC50, rate) • logP = partition coef or  = lipophilicity factor •  = Hammett electronic substituent effects • k, k', k",  = regression coefficients • Free-Wilson Model • BA = ajXj +  • BA = biological activity (e.g., log(1/c)) • aj = substituent constant, Xj = substituent presence,  = overall average activity

  23. QSAR of TransportSelected from: V.Austel & E. Kutter in Ref. 18. ABSORPTION - log (%abs), log Perm, log k Barbiturates Gastric log PCHCL3/w Sulfonamides Gastric log Pisoam-OAC/w Anilines Gastric pKa Xanthines Intestine Distribution Coef C-glycosides Intestine log Po/w, Rm Excretion - log (%excreted), log ClR, log k Penicillins Biliary logP, Rm Suflathiazoles Biliary logPo/w, pKa Sufapyridines Renal Rm, pKa Sulfonamides Renal , pKa Amphetamines Renal logPh/w

  24. QSAR Conclusions Passive Diffusion is a function of: • Lipophilicity (logPo/w or CLOGP) • GI (0.5-2.0), buccal (4-4.5), topical (>2.0) • Hydrogen bond donors/acceptors, polarity/charge • Water solubility (measured or calculated) • melting point, solvation energy, pH/buffers • pKa - fraction of neutral species available • mw - D  1/mw; mw < 500 Da • Confounding factors - inaccurate data, paracellular transport, mediated transport

  25. http://www.simulations-plus.com/pdf_files/aaps_2000_report.pdfhttp://www.simulations-plus.com/pdf_files/aaps_2000_report.pdf Neural Net models trained on up to 1337 compounds.

  26. Mediated Transport: Facilitated Diffusion and Active Transport • Rates > passive, solute specific, high Q10 • Non-symmetrical (kin kout at [Si] = [So]) • Saturable transport - Michaelis-Menten • Inhibitable - competitive, non-competitive • Regulated - inducibility & repression • Tissue specific- differential expression • Energy dependent - active transport • primary pumps - respiration, photosyn, ATPase • secondary transporters (coupled to H+, Na+ etc.)

  27. Membrane Transporter Models Circa 1991 Transporter Channel Pore

  28. Membrane Transporter Models Circa 2001 KscA OmpA GlpF FepA From: http://blanco.biomol.uci.edu/mptopo/

  29. Cell Culture and Molecular Biology Methods (I) • Isolation of MXR genes (Ref. 25). • Cells cultured from patients w/ resistant tumors. • mitoxantrone uptake measured microscopically • Cells grown under progressively selective conditions mitoxantrone, adriamycin, verapamil • Isolation of differentially expressed mRNA as cDNA clones and cDNA sequencing. • Northern analysis of mRNA expression levels. • Southern analysis of gene copy amplification. • Quantitative PCR analysis of expression levels in non-selected resistant cells.

  30. Cell Culture and Molecular Biology Methods (II) • Isolation of BCRP genes (Ref. 26-27) • cells same as from Ref. 25 • cultured under selective conditions w/ doxorubicin and verapamil • RNA fingerprinting used to isolate cDNAs. • transfection of non-selected cells confers resistance to mitoxantrone, doxorubicin, daunorubicin • reduced uptake (dauno), enhance efflux (rhodamine 123) • Northern/Southern analysis of various cell lines

  31. Cell Culture and Molecular Biology Methods (III) • Isolation of MOAT-B,C,D (Ref. 28) • cMOAT (cannicular multispecific organic anion transporter) = MRP previously isolated • MOAT-B isolated by PCR • Homology search against EST datase suggests MOAT-C & MOAT-D • EST probe isolation of cDNA from human • RNA blot analysis of tissue expression library • chromosomal location by FISH

  32. Cell Culture and Molecular Biology Methods (IV) • Other Cloning Methods • Expression cloning in oocytes • Homologous hybridization • Cloning by RT-PCR with degenerate primers • Cloning by functional complementation

  33. Cell Culture and Molecular Biology Methods (V) • What have you got? MXR, BCRP, MDR, MRP, ABC • Homology search against database - BLAST • Sequence alignments and phylogenetic trees • Hydropathy analysis and transmembrane topology predictions - Kyte-Dolittle • ATP binding and other consensus motifs • Homology modeling from known transporters • Inferences about possible substrates/functions

  34. Biochemistry and Biophysics • Functional characterization • Expression of Transport Activity in Vitro • Substrate structure/activity profiles and co-substrates (GSH, ATP, H+, Na+), uncouplers • Tissue distribution - EST database, RNA expression levels, antibodies, in situ methods • Phenotypes in Knock Out Rodents • Subcellular localization microscopy • Isolation, purification, reconstitution • Structural biology - EM, X-ray, NMR • Mechanism of substrate transport and energy coupling - enzymology, inhibition, drug design

  35. Structure of MsbA from E. coli: A Homolog of the Multidrug Resistance ATP Binding Cassette (ABC) TransportersGeoffrey Chang and Christopher B. Roth, Science Sep 7 2001: 1793-1800.

  36. Structure of bacterial oxalate transporter: a paradigm for the multifacilitator superfamily.T. Hirai, et al. (Subramaniam lab, NIH), Nature Structural Biology 9(8): 597-600. Low (6.5 Ǻ) resolution based on EM of 2D crystals.

  37. ABC Superfamily ABC peptide transporter family P-glycoprotein (MDR) family MDR1a,1b,2,3 - organic cations, lipids (PC) MRP1,2,3 - organic anions, GSX conjugates cMOAT - canalicular multispecific organic anion transporter = MRP2 cBAT - canalicular bile acid transporter Porins & Channels Major Facilitator Superfamily POT - proton coupled oligopeptide transporter NT - Na+ coupled nucleotide transporter NTCP - N+ coupled taurocholate protein OATP - polyspecific organic anion transport protein OAT-K1 - renal methotrexate transporter OCT - organic cation transporter - electrogenic RFC - reduced folate carrier sGSHT - glutathione conjugate transporter Membrane Transporter Families

  38. Organic Cation Substrates (MDR & OCT) (From Zhang, Brett, & Giacomini, Ref. 32)

  39. Substrates of cMOAT(canalicular multispecific organic anion transporter)Selected from Table IV in Chap. 14 in ref 42a. • glutathione disulfide • leukotrienes (C4, D4, E4, N-acetyl-E4) • glutathione conjugates (e.g., DNP, bromosulfophthalein, metals Sb, As, Bi, Cd, Cu, Ag, Zn) • glucuronide conjugates (bilirubin, T3, p-nitrophenol, grepafloxacin) • bile acid conjugates (glucuronides and sulfates) • organic anions (folates, methotrexate, ampicillin, ceftiaxone, cefadozime, grepafloxacin, prevastatin, temocaprilate)

  40. Transporter Amino Acid Organic Anion Nucleoside Oligopeptide Monocarboxylic Acid Organic Cation Substrates L-DOPA, gabapentin Captopril, acyclovir Didanosine, idoxuridine Β-Lactam antibiotics Valproic acid, pravastatin Cimetidine, verapamil Drug Uptake by Endogenous Transporters in the Small Intestine Lee, et al., Adv.Drug Delivery Reviews, 2001. Table 1.

  41. Nucleotide Transporters of Mammalian Cells (CNT1) Cloned from kidney. Apically expressed. N1 = SPNT or CNT2 N2 = CNT1 From: C.E. Cass, in Ref. 31, Fig. 3, p. 413. es,ei = sensitivity versus nitrobenzylthioinosine

  42. Nucleoside Drug TransportersAdapted from:C.E. Cass (Tables 1-4) in Ref 31. Cladribine (Cl-dAdo) Leukemia es, ei, N1, N5 Cytarabine (araC) Leukemia es, ei 2-Fludarabine (F-araA) Leukemia es, N1, N5 Pentostatin (dCF) Leukemia es Floxidine (F-dUrd) Colon Cancer es, ei Didanosine (ddI) HIV es, NB Zalcitabine (ddC) HIV es, N2 Zidovudine (AZT) HIV N2 Acyclovir (ACV) HSV NB Gancyclovir (GCV) HSV es, NB Vidarabine (araA) HSV es, ei, N1 Idoxuridine (IdUrd) HSV es Trifluridine (F3-dThd) HSV ND Ribavirin (RBV) RNA/DNA ND

  43. Tissue Uptake and Intracellular Drug Transport (subcellular PK) mito doxo AZT NT MXR Place Holder - Figure TBN MDR RFC OC+ MTP MTX AT MRP VATP PEPT GSX H+ valcyclo

  44. Exploiting Nutrient Transporters to Enhance Drug Bioavailability • Valacyclovir is an amino acid ester prodrug of the antiviral drug acyclovir. • Oral biovailability (AUC) is increased in humans 3-5x. • Intestinal permeability in a rat perfusion model is increased 3-10x. Effect is specific (SAR), stereospecific (L), saturable, and inhibitable by PEPT1 subsrates (cephalexin, dipeptides), and by gly-acyclovir, val-AZT. • Competitive with 3H-gly-sarc in CHO/hPEPT1 cells. • Enhanced, saturable, inhibitable mucosal to serosal transport demonstrated in CACO-2 cells and accompanied by hydrolysis. Serosal to mucosal transport is passive. • Rationale applied by Roche to design of valgancylcovir. • XenoPort, Inc. working on gabapeptin-XP

  45. Drug Interactions & Drug Transport • Digoxin - non-metabolized substrate for PgP • Verapamil, amiodarone, and quinidine increase plasma levels, reduce renal and non-renal clearance, increase blood/brain barrier transport. • Dose adjustment may be needed in 50% of cases. • St. John's wort (Hypericum perforatum) decreased digoxin AUC by 25% after 10 days treatment through induction of PgP. • HIV Protease Inhibitors • Amprenavir clearance reduced by nelfinavir (-41%) and by indinavir (-54%), but not saquinavir. • FDA warning against Hypericum supplements

  46. Drug Resistance & Reversal • MDR1 (P-glycoprotein) – drug efflux pump • Multiple trials of multiple agents – recent efforts at inhibiting transcription • Steady state digoxin therapy was established in normal healthy volunteers (1 mg then 0.125 mg/day). Initiation of valspodar (400 mg followed by 200 mg twice per day) caused immediate and progressive increases in digoxin AUC (+211%) and decreases in total body, renal, and non-renal clearance (-67%, -73%, -58%) after 5 days. • BCRP (breast cancer resistance protein or ABCG2) • Inhibited by fungal toxin fumitremorgin C, but neurotoxic side effects • Kol143 and other derived analogs developed inhibit BCRP, but not PgP or MRP • Non-toxic in mice, increased oral availability of topotecan in mice • RFC (reduced folate carrier) - antifolate drugs (methotrexate) • Resistant leukemia cell lines were selected by stepwise doses • Cross resistance (>2000x) to five novel hydrophilic antifolates shown • Intracellular folate levels reduced, increased requirement 42x • Hypersensitive to hydrophobic antifolates • Mutations clustered in exons 2 and 3, TMD1

  47. Microbial Drug Transport and Resistance Mechanisms • Mechanisms of Drug Uptake in Bacteria • OM porins, periplasmic binders, and IM pumps • B-lactam channels - imipenem resistance • nutrient uptake transporters - amino-glycosides • siderophore uptake is a drug delivery target • Mechanisms of Drug Efflux in Bacteria • Major facilitatory (MF) family • RND family (AcrAB, EmrAB, TolC) • SMR (small multidrugresistance pumps) • ABC (ATP binding cassette) family

  48. Structures of MDR substrates (From K. Lewis, Ref. 47).

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