Concentrative and Equilibrative Drug Transport

Concentrative and Equilibrative Drug Transport

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 National Institute of General Medical Sciences 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. 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 Measurements of Membrane Transport 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 Measurement of Transport in Excised 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. 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. 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 Equilibrative Transport Compartment Model + pHo SHo+ So Gtransp Gpump

KBo SoBo pHi Si SHi+ KBi SiBi S'i Equations for Membrane Transport Thermodynamics

Gtransp = 2.303RT log[Si]/[So] + nF + Gpump R = 8.314 joules/molK = 1.987 cal/molK F = 96.5 Joules/mol-mV = 23.06 cal/mol-mV [Si]/[So] = 10x @ 296K (23C) 310K (37C) (G) = = () = 5.67 kJ/mol 1.35 kcal/mol 58.5 mV

5.936 kJ/mol 1.41 kcal/mol 61.5 mV pH = pKa + log[S]/[SH] Examples Driving Force/Drug/Compartment Diffusion Ion trapping pH trapping Binding Active caffeine

Tc-Sestamibi quinidine warfarin captopril total body water heart mitochondria renal excretion plasma/liver ratio GI absorption Proposed Mechanisms of Tetracycline Uptake and Efflux 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 Transcellular vs Paracellular Pathways 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% Transepithelial Resistance ( 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 Details of Tight Junction EM of Caco-2 zona occluden zona adherens desmosome Ca++, IP3, PKC, CamK, MLCK 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. Paracellular versus Transcellular Transport

Ref. 8: Wielinga, et al., J. Pharm. Sci, 88(12), 1340, 1999. Paracellular Permeability Enhancers Examples: Ca++chelators, bile salts, anionic surfactants, medium chain FAs, alkyl glycerols, cationic polymers, cytochalsin D, hormones, TNF-, 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)

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 vesicular transport Blood Brain Transcytosis Delivery of Prodrug Endothelial Cell From: Bickel & Pardridge in Ref. 22, p. 30. Transferrin

receptor-mediated transcytosis of an mAB-avidin-biotindisulfide cross-linked vasoactive intestinal peptide. TfR, VitB12R, FcRn, PigR are under commercial development. 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 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 over ca 5 psec vs RMS motions of 1.5 . 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. 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, average activity = overall QSAR of Transport Selected 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 Amphetamines Renal , pKa Renal logPh/w 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 http://www.simulations-plus.com/pdf_files/aaps_2000_report.pdf Neural Net models trained on up to 1337 compounds. 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.) Membrane Transporter Models Circa 1991 Channel Pore Transporter Membrane Transporter Models Circa 2001 KscA OmpA

GlpF FepA From: http://blanco.biomol.uci.edu/mptopo/ 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. 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 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 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 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 Biochemistry and Biophysics Functional characterization Expression of Transport Activity in Vitro Substrate structure/activity profiles and cosubstrates (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

Structure of MsbA from E. coli: A Homolog of the Multidrug Resistance ATP Binding Cassette (ABC) Transporters Geoffrey Chang and Christopher B. Roth, Science Sep 7 2001: 1793-1800. 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 ) resolution based on EM of 2D crystals. Membrane Transporter Families 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 Organic Cation Substrates (MDR & OCT) (From

Zhang, Brett, & Giacomini, Ref. 32) 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) Drug Uptake by Endogenous Transporters in the Small Intestine Lee, et al., Adv.Drug Delivery Reviews, 2001. Table 1. Transporter Amino Acid Organic Anion Nucleoside Oligopeptide Monocarboxylic Acid Organic Cation

Substrates L-DOPA, gabapentin Captopril, acyclovir Didanosine, idoxuridine -Lactam antibiotics Valproic acid, pravastatin Cimetidine, verapamil Nucleotide Transporters of Mammalian Cells (CNT1) From: C.E. Cass, in Ref. 31, Fig. 3, p. 413. es,ei = sensitivity versus nitrobenzylthioinosine

Cloned from kidney. Apically expressed. N1 = SPNT or CNT2 N2 = CNT1 Nucleoside Drug Transporters Adapted 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 Tissue Uptake and Intracellular Drug Transport (subcellular PK) mito doxo

OC+ MXR MDR Place Holder - Figure TBN MTP AT MRP RFC VATP GSX AZT

NT H + MTX PEPT valcyclo 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 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 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 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 Structures of MDR substrates (From K. Lewis, Ref. 47).

Topology Models of Microbial Multidrug Resistance Pumps From: K. Lewis, Ref. 47, Fig. 2. Pharmacogenetics of Transport (I) P-glycoprotein Polymorphism in cell cultures Gly185Val - selected for colchicine sensitivity Ser893Ala - naturally occuring polymorphism RFLP predicts ivermectin neurotoxicity sensitive P-gpdeficient mice. Polymorphisms in 24 normal volunteers Function effects on digoxin uptake measured 7 intron, 3 wobble, 3' & 5' non-coding, 3 a.a. changes [C/T 3435 lowers 2-fold]

Polymorphisms now known in Pgp, MRP1 and MRP2 Pharmacogenetics of Transport (II) OATP-C (organic anion transporting polypeptide-C) liver specific uptake transporter multiple SNPs detected, including 14 non-synonymous, gene frequency depends on race 16 assessed in vitro, 8 result in reduced transport, esp. T521C (val174ala) occurs in 14% European- and G146C (gly488ala) in 9% of African-Americans OCT1 (organic cation transporter 1) electrogenic uptake, drugs neurotransmitters - 25 variations identified in 57 Caucasian samples - 3 (arg61cys, cys88arg, gly401ser) reduce transport and occur frequently (9/1%, 0.6%, 22%)

Pharmacogenetics of Transport (III) Pharmacogenetics Network - UCSF Project http://pharmacogenetics.ucsf.edu OCT2 Transporter - renal tubule basolateral Adverse Effects - procainamide, clonidine Chromosome locus 6q26 Aliases - Organic Cation Transporter 2 - Solute Carrier Family 22, Member 2 - SLC22A2 Links to NCBI Data OMIM On-line Mendelian Inheritance in Man LocusLink Data Reference Sequence: Homo Sapiens mRNA for OCT2 from kidney.

Gene Structure Introns/Exons Transmembrane Topology Prediction 1 2 3 4 Variants occur with frequency of 15% Coding regions and Exon/Intron boundaries For 247 DNA samples from Coriell Institute SNPs found at: Synonomous: 130, 223, 297, 401, 466, 502, 529

Non-Syn: 54, 161, 165 (2), 270 (2), 400, 432 Cellular phenotyping: Data to be gathered. Clinical studies: Data to be gathered 5 6 7 8 9 10

1 1 Pharmacogenomics of Transport (I) Classification by mechanism, origin, topology, domain structure, energetics, energy source, substrate specificity, sequences, 3D structures BLAST (Basic Local Alignment Search Tool) INCA (Integrative Neighborhood Cluster Analysis) T.C.# W.X.Y.Z (Saier et al)., e.g., MDR1 = 3.A.1.201 W = type and energy source (3 = primary transporter) Z = transporter family or superfamily(3.A = P-P cleavage) Y = transporter subfamily (3.A.1 = ABC family) Z = substrate transported (3.A.1.201 = multiple drugs) http://www.biology.ucsd.edu/~msaier/transport/titlepage.html Pharmacogenomics of Transport (II)

http://www.biology.ucsd.edu/~ipaulsen/transport/findings.html Pharmacogenomics of Transport (III) Database of Bacterial Transporters (Saier & Paulsen) http://www.biology.ucsd.edu/~ipaulsen/transport/index2.html TIGR (The Institute for Genome Research EGAD (Expressed Gene Anatomy Database) http://www.tigr.org/docs/tigr-scripts/egad Human Membrane Transporter Database Sadee et al (AAPS Pharm Sci) search by transporter, family, tissues/organ, substrates/drugs http://lab.digibench.net/transporter/ Human SNPs Consortium - search transporter => 58 hits 12/28/01 http://snp.cshl.org/ 175 hits 12/31/02

Other databases: http://wwwncbi.nlm.nih.gov/ Membrane Transporter Families H+/Dipeptide Symporters Proton-dept oligopeptide transporters (POT) 70 cloned from nature; 2 from humans (PEPT1 and PEPT2) Comparisons between worm, rat, mouse sequences identified two additional human POTs (hPHT1 and hPHT2) related to cloned rodent histidine/peptide transporters.

Facilitative Glucose Transporter Family Sodium/Glucose and Sodium/Nucleoside Amino Acid Transporters Sodium Neurotransmitter Symporters ABC Transporters Pharmacogenomics of Transport (IV) Expression Patterns using MicroArray Chips In vivo permeabilities measured in human duodenum using perfusion methods. In vitro permeabilities measured using Caco-2 cells. Expression patterns of 12,599 gene sequences analyzed using GeneChip (including 443 expected ADME genes). Sun, et al., 2002. Results: Functional Genomics 1) 37-47% of genes (26-44% of ADME genes) expressed in both cell types, but >1,000 sequences showed >5x variation between cell types. Variation >3x for >70 transporters detected.

2) In vivo/in vitro permeability correlated well (R2 = 85%) for passively absorbed drugs. 3) Variations (3-35x) above expected passive values were observed for mediated absorption and correlated with differences (2-595x) in gene expression. 4) Interhuman variability (3-294% of mean) for 31% of genes.

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