# Presentation: 917363

## Principles of Clinical PharmacologyJanuary 10, 2007Module 2: Drug Metabolism and TransportUnit 6:	 Equilibrative and Concentrative                   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 and structure of membrane transport proteins
- Clinically important transporters
- Pharmacogenetics & pharmacogenomics of transporters

## Measurements of Drug Distribution Reflect Membrane Transport In Vivo

- Blood/tissue Samples, Biopsies, and Assays
- Autoradiography
- Perfusion/Cannulation Methods
- Radiology - x-ray, PET, SPECT
- Magnetic Resonance Imaging
- Microdialysis

## Apparatus for In Vivo GI Permeability StudiesPetri, et al. (Lennernäs) Drug Metab & Disposition 31(6), 805-813, 2003.

- Absorption of Phytochemicals from Onion and Broccoli Extract
- _Compound_       _Permeability (cm/sec)_  _Absorbed_   _Metabolite_           _Induction_
- Sulforaphane   18.7 ± 12.6 x 10-4         74 ± 29%   GSH-conjugate    2.0 ± 0.4x
- Quercetin           8.9 ±   7.1 x 10-4         60 ± 31%   -3’-glucoronide   2.4 ± 1.2x
-   -3,4-glucoside

- Six healthy volunteers
- Proximal jejunum segment
- Single pass Qi = 2 mL/min
- Peff = (Ci-Co)Qi/2LCo
- PEG 4000 non-absorbable
-   marker => net water flux
- Fabs = 1- (CoPEGi/CiPEGo)
- mRNA in shed enterocytes
-   GSTA1 and UGT1A1

## 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.

## Monolayer Epithelial Cell Culture

- I.J. Hidalgo in  Models for Assessing Drug Absorption and Metabolism (Borchardt, et al., Eds.) Plenum Press, NY, 1996, p. 38.

## Mechanisms of Transport Across Biological Membranes

- _Diffusion Mechanisms_:  Equilibrative
- Passive (self) diffusion across the lipid bilayer
- 	fluoroquinolones, tetracycline (hydrophobic)
- Diffusion through non-selective OM channels and porins
-  B-lactams, tetracyclins (hydrophilic, charged)
- Facilitated diffusion through selective channels and equilibrative transporters
- imipenem, catechols, albomycin, albicin
- Carrier-mediated transport
- Ion transport by valinomycin, host-guest delivery agents
- Paracellular transport – ions, mannitol, polymers
- _Energy-requiring Mechanisms_:  Concentrative

## Examples of Transport 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
- Group transfer	nucleosides	white cell uptake
- Cytoskeletal	gentamicin		renal resorption

## Thermodyanmics of Transport I: Equilibrative Diffusion of a Neutral Compound

**S****o**

**S****i**

******G****transp**

******Gtransp = 2.303RT log[Si]/[So]**
**                  ****= ********H - T********S**
******H ********0, ********S > 0, ********G < 0**

- Caffeine

- Thermodyanmics of Transport I: Equilibrative Diffusion of a Neutral Compound

## Thermodyanmics of Transport II: Equilibrative Diffusion of a Charged Compound

**S****o****+**

**S****i****+**

**+**

**-**

****

******G****transp**

- 99mTc-Sestamibi

******Gtransp = 2.303RT log[Si]/[So] + nF******
******cell**** = 60 mV = 1.38 kcal => Si/So = 10x**
******mito**** = 168 mV = 3.87 kcal => Si/So = 622x  **

## Thermodyanmics of Transport III: Equilibrative Diffusion – pH Trapping

- Thermodyanmics of Transport III: Equilibrative Diffusion – pH Trapping

**S****o**

**S****i**

**SH****o****+**

**SH****i****+**

**pH****o**

**pH****i**

**pKa**

**pKa**

**pH = pKa + log[S]/[SH]**
**For pKa = 7, pH****o**** = 8, pH****i**** = 6,**
**S****o****/SH****o****= 10   S****i****/SH****i ****= 1/10**** **
**SH****i**** = 100x SH****o**

- Quinidine sulfate
- pKa1 = 5.4, pKa2 = 10

******G****transp**

## Thermodyanmics of Transport III: Equilibrative Diffusion – Protein Binding

**S****o**

**S****i**

**S****o****B****o**

**K****B****o**

**S****i****B****i**

**K****B****i**

******G****transp**

- Warfarin

**K****B**** = [SB]/[S][B]**
**KB****o**** ********KB****i**

## Passive Diffusion

- Characteristics of passive diffusion
- kin = kout, net rate = k([So] - [Si]), non-selective
-  Model Membranes (experimental systems)
- liposomes, BLMs, IAMs, PAMPA
- 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..
- 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.

## 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

## 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

## Lipinski Rules of Five

- Based on analysis of human clinical data for
- 2245 compounds in the World Drug Index
- H-bond donors – no more than five
- H-bond acceptors – no more than five
- N plus O atoms – no more than ten
- MW no more than 500 dalton
- CLOGP no more than 5 (or log P > 4.15)

## http://www.simulations-plus.com/pdf_files/aaps_2000_report.pdf

- Neural Net models trained on up to 1337 compounds.

## Apparatus for On-Line Fluorescence Measurement of Transport in Epithelial Cell Cultures

- Wielinga, et al., J. Pharm. Sci, 88(12), 1340, 1999.

- MDCKII  MDR1
-  SDZ PSC 833
- Daunorubicin
- ex = 480, em = 590
- FITC-dextran
- ex = 480, em = 525
- Trans Epithelial
- Resistance (TER) =
-  300 - 600 mm2

## Paracellular versus Transcellular Transport

- 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-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)

## Mechanisms of Transmembrane Drug Transport

- _Diffusion Mechanisms_:
- _Energy-requiring Mechanisms_:
- Endocytosis - receptor mediated
- aminoglycosides (renal tubule), polymers, peptide hormones, targeted delivery, prodrugs, proteins
- Transcytosis
- Drug, macromolecule, particle delivery across GI, BBB, lung
- Protein tranduction
-  	– HIV TAT, HSV VP22, antennapedia, other amphipathic peptides
- Active transport via membrane transport proteins
- aminoglycosides (bacteria), cycloserine, phosphomycin, alaphosphin, others

## Endocytosis of AminoglycosidesGentamicin Elimination Phase Preceeds Its Distribution Phase (Flip/Flop)

*** From Schentag JJ, et al. JAMA 1977;238:327-9.**

**ECF**

**KIDNEY**

## Receptor-Mediated Endocytosis Aminoglycoside Nephrotoxicity

- Proximal Renal Tubule

- Aminoglycosides (+++) bind to anionic phospholipids
- Endocytosis via chlathrin-coated pits into lysozomes
- Reduced by 95% in megalin (gp330/LDL-receptor related protein-2) KO mice
- Uptake of proteins and Ca++
- Intracellular release leads to selective mitochondrial damage in kidney
- Epithelium of inner ear also sensitive (ototoxicity), but see also rRNA polymorphism.

## Transcytosis Delivery of Prodrug

**Brain**

**Blood**

**Endothelial Cell**

- From: Bickel & Pardridge.  Transferrin receptor-mediated transcytosis of an mAB-avidin-biotin-disulfide cross-linked vasoactive intestinal peptide.

- vesicular transport

- TfR, VitB12R, FcRn, PigR are under commercial development.

## Protein Transduction by Cell Penetrating Peptides

- Non-receptor mediated uptake (and subcellular targeting)
- Self-inserting amphipathic peptides
- Energy dependent (or not) internalization NOT via clathrin coated pits
- Mediated by charge interaction with glycosamino glycans on cell surface
- D-enantiomers and inverted sequences are active
- Cargos are synthetic or biosynthetically linked or fused peptides, proteins, small molecules, nucleic acids, vesicles, nanoparticles
- Delivery to cells, perfused tissues, organism, expression in situ-gene therapy
-  HIV transactivator of transcription (TAT)
- Nuclear localization sequence Tat48-60
- Drosophila antennapedia transcription factor homeodomain
- Penetratin = Antp43-58 homeodomain 3rd helix
- SynB vectors from protegrin-1 (18 a.a. peptide from porcine leukocytes)
- Transportan – synthetic 27 aa chimera of galanin and mastoparin-X
- Amphiphatic model peptides, signal sequence peptides, homo-arginine polyers
- Example:  Arg7 peptide-PKC-ε agonist protection of ischemic rat heart
- Example:  SynB-doxorubicin delivery across BBB bypasses PgP

## 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.)

## Biochemistry of Transporters

- Discovery and functional definition in vivo and in vitro
- Genetic definition by cloning and sequencing
- Confirmation by 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

## 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 (>1,000)
- 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 Models Circa 1991

- Channel

- Pore

- Transporter

## Membrane Transporter Models Circa 2001

- KscA

- OmpA

- FepA

- Membrane Protein Resources web site by Stephen White lab. http://blanco.biomol.uci.edu/MemPro_resources.html

## Topology Model for Multifactilitator SuperfamilyThe Kamikaze Approach to Membrane Transport.Kaback, et al., Nature Reviews Mol Cell Biol 2; 610-620 (2001).

-  400-600 residues
-  12 TM helices
-  N- and C-terminal halves
-    weakly homologous
-  Signature sequence RXXRR
-    in L2-3 and L8-9
-  Essential residues:

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

## Structure and Mechanism of the Glycerol-3-Phosophate Transporter from Eschericia coli.  [G-3-P/Pi exchange Pi driven]

- Y. Huang, et al. (Da-Neng Wang lab at NYU) Science 301, 616-620, Aug 1, 2003.  High resolution (3.3 Å) based on x-ray crystallography.

## Proposed transport mechanism:  i) translocation pathway between N- and C-terminal halves; ii) binding of G-3-P between R45(H1) and R269(H7); iii) binding lowers barrier for conformational exchange; iv) rocking motion exposes binding site to alternate membrane faces; v) Pi gradient drives conformational return.

- Images courtesy of Da-Neng Wang

- Proposed transport mechanism:  i) translocation pathway between N- and C-terminal halves; ii) binding of G-3-P between R45(H1) and R269(H7); iii) binding lowers barrier for conformational exchange; iv) rocking motion exposes binding site to alternate membrane faces; v) Pi gradient drives conformational return.

## Ci

- Ci

- Courtesy of Da-Neng Wang

**Notes:**
Oxlt and GlpT, C-terminal domains aligned.

## Co-S

- 6° rotation for each domain

- Co-S

- Courtesy of Da-Neng Wang

**Notes:**
GlpT aligned with OxlT by a 12-15 rotation of the N-terminal domain 

## Co

- 10° rotation for each domain

- Co

- Courtesy of Da-Neng Wang

**Notes:**
Outward facing

## 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
- Β-Lactam antibiotics
- Valproic acid, pravastatin
- Cimetidine, verapamil

## Hepatic transporters circa 2003C. Pauli-Magnus & P.J. Meier, Pharmacogenetics (2003) Apr;13(4):189-98. 

## Substrates of cMOAT (MRP2)(canalicular multispecific organic anion transporter)Selected from Table IV in Chap. 14 of Amidon & Sadee.	

- 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)

## Nucleotide Transporters of Mammalian Cells

- From: C.E. Cass
- es,ei = sensitivity versus nitrobenzylthioinosine

- ENT1   ENT2  CNT1 CNT2 CNT3
- SLC29A1 A2  SLC28A1-A3 Cloned Transporters
- Basolateral        Apical in kidney
- Mangravite (Giacomini) EJ Pharm 479 (2003), 269-281.

## Tissue Uptake and Intracellular Drug Transport (subcellular PK)

- Place Holder - Figure TBN

**OC****+**

**MDR**

**GSX**

**MRP**

**MXR**

**mito**
**doxo**

**VATP**

**PEPT**

- valcyclo

- MTX

**RFC**

**NT**

**AZT**

**H****+**

**MTP**

**AT**

## Transport and Intracellular Metabolism of FIAU

**FIAU**

**FIAUMP**

**FIAUDP**

**FIAUTP**

**mtDNA polymerase γ**

***TK-2***

***dTMPK***

***5’NDPK***

**FIAUMP**

**FIAUDP**

**FIAUTP**

***TK-1***

***dTMPK***

***5’NDPK***

***Mitochondria***

***Cytosol***

**?**

**   ****?**

**FIAU**

**FIAU**

**Transport and Intracellular Metabolism of FIAU**
**Courtesy of J. Unadkat**

**Inhibition**

## Mitochondrial toxicity depends on differences in intracellular transport

- Nucleoside drugs target DNA replication
- Inhibition of polγ leads to mtDNA loss
- AZT, ddC, ddI, d4t are known mito toxins
-  hepatoxicity, pancreatitis, neuropathy, myopathy
-  but rarely fatal
- Fialuridine trial for hepatitis B at NIH resulted in hepatic failure in 7/14 (5 died)
- Human ENT1 and ENT2 are expressed in mito
- Mouse ENT1 and ENT2 are NOT
- Drugs differ in rates of transport and activation

## 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. (www.xenoport.com) gabapeptin-XP Pfizer

## 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

## Pharmacogenetics of Transport (I)Estimating contribution of genes to variation in renal drug clearance.  Leabman & Giacomini, Pharmacogenetics 13(9), 581-4, 2003.. 

- Based on Repeated Drug Administration Literature Data
- Comparison of variation between individuals and variation in
- response for a given individual.
- rGC = (SDbetween2 – SDwithin2)/SDbetween2
- Drug             CLmean   SD2between   SD2within     Pbetweenwithin
- Metformin     450        5343            299          <0.01     Mediated*
- Amoxicillin   154          476              44          <0.01     Mediated*
- Ampicillin     165          919            334          <0.01     Mediated*
- Terodiline       11.3            6.4             4          >0.05    Passive
- Iohexol          115          110               88          >0.05    Passive
- Digoxin         150         1093            958          >0.05    Passive
- Mediated by variations in OAT1, OT2, PEPT2, Npt1.

## Pharmacogenetics of Transport (II)

- OATP-C (organic anion transporting polypeptide-C)
- liver specific uptake transport – bile salts, estrone sulfate, estradiol-glucuronide
- multiple SNPs detected, including 14 non-synonymous, gene frequency depends on race.  Tirona et al. JBC 276(38), 35669-75, 2001.
- 16 assessed in vitro, 8 result in reduced transport, esp. T521C (val174ala) occurs in 14% European- and G146C (gly488ala) in 9% of African-Americans
- Effects on pravastatin pharmacokinetics noted for OATP-C 15 allele (Asp130/Ala174) versus OATP-C 1b allele (Asp130/Val174).  Nishizato et al. Clin Pharm Ther. 73(6), 554-65, 2003.
-      Non-renal clearances (l/kg/hr):
-      1b/1b = 2.01± 0.42  n = 4  P<0.05
-      1b/15 = 1.11± 0.34  n = 9  P<0.05
-      15/15 = 0.29

## 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

- 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

|   |   |   |   |   |   |   |   |   |   |   |   |   |   |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |   |  |   |  |   |  |   |  |   |  |   |  |   |  |   |  |   |  |   |  |   |  |
|   | 1 |   | 2 |   | 3 |   | 4 |   | 5 |   | 6 |   | 7 |   | 8 |   | 9 |   | 10 |   | 11 |   |

## Pharmacogenomics of Transport (I)

- Classification by mechanism, origin, topology, domain structure, energetics, energy source, substrate specificity, sequences, 3D structures, organisms, tissue localization, etc.
- BLAST (Basic Local Alignment Search Tool – NLM)
- INCA (Integrative Neighborhood Cluster Analysis – W. Sadee)
- 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/  **
- Recent Review: The ABCs of Solute Carriers.  M. Hediger, Pflugers Archiv – EJ Physiol.  See also:  **http://www.bioparadigms.org/**

## Pharmacogenomics of Transport (II)Expression Patterns using MicroArray Chips

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

- 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.

## Conclusions

- We have been lucky in the past
- We have selected for drugs that are readily transported by passive diffusion – many of which act extracellularly
- We are just beginning to understand other transport processes and their consequences
- We are just beginning to understand the interindividual variations of transport
- We are just beginning to exploit that knowledge to design drugs for transport