Concentrative and Equilibrative Drug Transport

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Transcript 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
SoBo
SHi+
Si
Gpump
KBo
pHi
Gtransp
KBi
SiBi
S'i
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
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 (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
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-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)
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 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
• Free-Wilson Model
– BA = ajXj + 
– BA = biological activity (e.g., log(1/c))
– aj = substituent constant, Xj = substituent presence,
 = overall average activity
QSAR of Transport
Selected from: V.Austel & E. Kutter in Ref. 18.
ABSORPTION - log (%abs), log Perm, log k
Barbiturates
Sulfonamides
Anilines
Xanthines
C-glycosides
Gastric
Gastric
Gastric
Intestine
Intestine
log PCHCL3/w
log Pisoam-OAC/w
pKa
Distribution Coef
log Po/w, Rm
Excretion - log (%excreted), log ClR, log k
Penicillins
Suflathiazoles
Sufapyridines
Sulfonamides
Amphetamines
Biliary
Biliary
Renal
Renal
Renal
logP, Rm
logPo/w, pKa
Rm, pKa
, pKa
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 Ǻ) 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
Β-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)
Cytarabine (araC)
2-Fludarabine (F-araA)
Pentostatin (dCF)
Floxidine (F-dUrd)
Didanosine (ddI)
Zalcitabine (ddC)
Zidovudine (AZT)
Acyclovir (ACV)
Gancyclovir (GCV)
Vidarabine (araA)
Idoxuridine (IdUrd)
Trifluridine (F3-dThd)
Ribavirin (RBV)
Leukemia
Leukemia
Leukemia
Leukemia
Colon Cancer
HIV
HIV
HIV
HSV
HSV
HSV
HSV
HSV
RNA/DNA
es, ei, N1, N5
es, ei
es, N1, N5
es
es, ei
es, NB
es, N2
N2
NB
es, NB
es, ei, N1
es
ND
ND
Tissue Uptake and Intracellular
Drug Transport (subcellular PK)
mito
doxo
OC+
MXR
MDR Place Holder - Figure TBN
MTP
AT
MRP
VATP
GSX
AZT
NT
H+
RFC
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-gp-deficient 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.