Overview of Drug Transporter Families and Their Importance in ADME

1
Overview of Drug Transporter Families and Their
Importance in ADME
AAPS Workshop on Drug Transporters in ADME From
the Bench to the Bedside
Jeffrey A. Silverman Sunesis Pharmaceuticals, Inc

• March, 2005

2
Outline

• Objective To review the major drug transporters
  in key elimination/barrier organs and provide a
  framework for subsequent talks
• Impact of Drug Transporters in ADME
• Overview of Major Drug Transporters

3
Transporters Have a Important Role in ADME

• Barrier to Absorption
• Determinant of Tissue Distribution
• Modulation of Metabolism
• Role in Elimination

4
P-glycoprotein Mediates Bioavailability in Humans
Paclitaxel
CsA increases the oral absorption of paclitaxel 9
fold. Plasma paclitaxel reached above active
threshold concentration (0.1 µM) for gt 4
hr Terwogt et al. Lancet 352285, 1998

• Docetaxel
• Co-administration with CsA increased
  bioavailability from ?6 to 90 (JCO 191160,
  2001)

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Impact of P-glycoprotein on CNS Exposure to
Loperamide in Humans
Change of ventilatory response with increasing
end-tidal carbon dioxide after administration of
loperamide with placebo and quinidine. (Representa
tive measurement from a single timepoint in one
subject)
Sadeque et al. Clin Pharmacol Ther 68231-7,2000
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Transport Proteins

• Few molecules enter or leave cells, or
  organelles, unaided by proteins
• Channels
• Facilitative transporters
• Active transporters
• Estimates range up to 5 of human genome (gt2000
  proteins) are transporter related

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Active vs Passive Transport
Electrochemical gradient
Lipid Bilayer
Energy
Simple Diffusion
Channel- mediated Diffusion
Carrier- mediated Diffusion
Passive Transport
Active Transport
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Carrier-Mediated (Facilitated) Diffusion

• Movement mediated by proteins
• uniporter
• Transport is highly specific
• No external energy source
• Down concentration/electrochemical gradient
• Transport is mediated by a limited number of
  proteins
• Saturable
• Polar and charged molecules
• Carbohydrates, amino acids, nucleosides and ions

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Active Transport

• Primary Active Transport
• Directly coupled to energy source (ATPase)
• Secondary Active Transport
• Uphill transport of one solute is coupled to the
  downhill transport of a second solute
• Co-transporter
• Same direction symporter
• Opposite direction antiporter

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Examples

• Passive Transporters
• SLC transporters
• Glucose transporters
• GLUT1-5
• Active Transporters
• ABC transporters
• P-gp TAP CFTR MRP
• SLC transporters
• OATPs

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ATP Binding Cassette Proteins

• Large gene family
• Defined by sequence homology in several key areas
• Share a conserved Walker A and B nucleotide
  binding motifs
• Also share a consensus C- region (aka signature
  sequence)
• Critical for moving a wide range of substances
• nutrients, amino acids, sugars, ions and
  metabolites
• Approximately 1000 ABC proteins have been
  identified, 48 in humans
• http//nutrigene.4t.com/humanabc.htm

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P-glycoprotein

• P-glycoprotein is a cellular efflux pump encoded
  by the MDR1 gene.
• MDR1 is a member of the ATP-Binding Cassette
  (ABC) family of transporters.
• P-glycoprotein is responsible for the resistance
  of tumor cells to multiple chemotherapeutic
  agents.
• P-glycoprotein is expressed on the apical
  membrane of epithelial cells in the intestine,
  liver, kidney, testes, blood-brain barrier and
  adrenals.
• P-glycoprotein plays a role in the absorption,
  distribution and elimination of numerous drugs.

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P-glycoproteinSummary of Characteristics

• Integral Membrane Protein
• 1280 Amino Acids Calculated MW 140 kDa Observed
  170 kDa
• N-Glycosylated

• 12 Membrane Spanning Domains
• 2 ATP-Binding Sites
• Highly Conserved
• Member of ABC Transporter Family

Gottesman, MM
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MDR Gene Family
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Paclitaxel Absorption in Rats
6 fold increase in Cmax 10 fold increase in AUC
16
P-gp effect on Vinblastine Distribution
4 hours following 1 mg/kg I.V. dose
Schinkel et al Cell 77491-502, 1994
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P-gp Affects Metabolism
Apical Dose
Basolateral Dose

• Substrates diffusing into cells
• will be pumped out by P-gp
• but not diffuse back in because it is
• against the concentration gradient
• less metabolites formed
• more parent traversing membrane

• Substrates diffusing into cells
• will be pumped out by P-gp
• and have another opportunity
• to diffuse in
• more metabolites formed
• less parent traversing membrane

Les Benet, UCSF
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MRPs Multidrug Resistance-associated Proteins

• Multigene family of proteins associated with
  resistance to drugs
• MRP1 isolated from non-P-gp expressing,
  multidrug-resistant, small cell lung carcinoma
  cells
• Currently there are 8 MRP genes identified

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Human MRP Gene Family
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MRP2 Impact on Blood Brain Barrier
Probenecid, an MRP1/2 inhibitor, increased BBB
penetration of phenytoin but not phenobarbital
Potschka et al. JPET 306124-131, 2003
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Breast Cancer Resistance ProteinBCRP MXR ABCP

• Novel ABC transporter
• Related to MRP family
• Expressed in liver, small intestine, colon,
  placenta
• Export pump (apical membrane localization
• Inhibition may increase oral absorption of
  substrates e.g. topotecan Other substrates
  mitoxantrone, anthracylines
• GG918 (GF120918) inhibits BCRP/MXR as well as P-gp

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Drugs Effluxed by BCRP

• Mitoxantrone
• Topo I inhibitors
• Topotecan, Ininotecan (SN-38) 9-amino camptotecan
• Anthracylines
• Flavopiridol
• Qinazolines
• HER tyrosine kinase inhibitor (CI1033)
• Prazosin

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BCRP/MXR in Oral Bioavailability
MDR1a/1b -/- mice
Jonker et al JNCI 921651-1656, 2000
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SLC Transporters

• Solute carriers
• Currently 43 gene families with approximately 298
  transporter genes

Antiporters/ Exchangers
Symporters/ Coupled transporter
Passive Transporters
http//www.bioparadigms.org/slc/menu.asp
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SLC Transporters 43 Families, 298 Genes
SLC22 The organic cation/anion/zwitterion
transporters SLC23 The Na-dependent ascorbic
acid transporters SLC24 The Na/(Ca2-K)
exchangers SLC25 The mitochondrial carriers
SLC26 The multifunctional anion exchangers
SLC27 The fatty acid transport proteins
SLC28 The Na-coupled nucleoside transporters
SLC29 The facilitative nucleoside transporters
SLC30 The zinc efflux proteins SLC31 The
copper transporters SLC32 The vesicular
inhibitory amino acid transporter SLC33 The
Acety-CoA transporter SLC34 The type II
Na-phosphate cotransporters SLC35 The
nucleoside-sugar transporters SLC36 The
proton-coupled amino acid transporters SLC37
The sugar-phosphate/phosphate exchangers
SLC38 The System A N, sodium-coupled neutral
amino acid transporters SLC39 The metal ion
transporters SLC40 The basolateral iron
transporter SLC41 The MgtE-like magnesium
transporters SLC42 The Rh ammonium
transporters (pending) 3 SLC43
Na-independent, system-L like amino acid
transporters 3
SLC1 The high affinity glutamate and neutral
amino acid transporters SLC2 The facilitative
GLUT transporters SLC3 The heavy subunits of
the heteromeric amino acid transporters SLC4
The bicarbonate transporters SLC5 The sodium
glucose cotransporters SLC6 The sodium- and
chloride- dependent neurotransmitter transporters
SLC7 The cationic amino acid
transporter/glycoprotein-associated SLC8 The
Na/Ca2 exchangers SLC9 The Na/H
exchangers SLC10 The sodium bile salt
cotransporters SLC11 The proton coupled metal
ion transporters SLC12 The electroneutral
cation-Cl cotransporters SLC13 The human
Na-sulfate/carboxylate cotransporters SLC14
The urea transporters SLC15 The proton
oligopeptide cotransporters SLC16 The
monocarboxylate transporters SLC17 The
vesicular glutamate transporters SLC18 The
vesicular amine transporters SLC19 The
folate/thiamine transporters SLC20 The type
III Na-phosphate cotransporters SLC21/SLCO
The organic anion transportings
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OATP/SLC21 Family

• Organic anion transporting polypeptides
• 36 human, rat and mouse OATP/Oatps
• Substrates
• Bile salts, organic anions, Eicosanoids,
  Taurocholate, T3, T4
• Tissue Distribution
• Many OATPs are expressed in multiple tissues
• Liver, kidney, lung, heart, intestine, BBB,
  choroid plexus, testis and placenta
• Driving mechanism anion exchange
• Bicarbonate, GSH, GSH-conjugates

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OATP/SLC21 Family
Hagenbuch Meier, Eur J Physiol (2004)
447653-665
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OATP Substrates, Inhibitors and Inducers
Substrates Taurocholate cholate glycocholate estra
diol glucuronide estrone sulphate prostaglandin
E2 thromboxane B2 dihydroepiandosterone
sulphate LTC4 BQ-123 enkephalin benzylpenicillin m
ethotrexate pravastatin cerivastatin
Substrates bilirubin mono/bi-glucuronosyl
bilirubin bromosulfophthalein microcystin phalloid
in CCK-8 deltorphin II digoxin ouabain rifampicin
fexofenadine thyroid hormones celiprolol
Inhibitors quinidine rifampicin rifamycin digoxin
indocyanine green amiodarone quinine ibuprofen ind
omethacin verapamil cyclosporin
A deoxycorticosterone glibenclamide pravastatin ri
tonavir
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SLC22 Family OCT, OAT Zwitterion Transporters

• Organic cation/anion transporters
• 19 human OCT OATs
• Substrates
• Organic cations, Organic anions
• Tissue Distribution
• OCTs and OATPs are expressed in multiple tissues
• Kidney, liver, lung, heart, skeletal muscle,
  intestine, brain, choroid plexus, adrenal gland,
  thyroid, testis and placenta
• Uniporters, Symporters and Antiporters
• Driving mechanism cation or anion exchange

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SLC22 Family
Koepell and Endou, Eur J Physiol (2004)
447666-676
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Peptide Transporters

• Transport di or tri peptides
• Critical role in the absorption of protein
  digestion products
• Driving force proton gradient (secondary active
  transporter)
• Saturable and concentration dependent
• Pharmacological role mechanism for absorption of
  a variety of drugs
• peptidomimetics (ß lactam antibiotics) ACE
  inhibitors, renin inhibitors, thrombin
  inhibitors, cephalosporins, bestatin

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Peptide Transporters 2

• PEPT1
• Small intestine, kidney, bile duct, pancreas
• Low affinity, Km in mM
• PEPT2
• Kidney, CNS, PNS, lung, mammary gland, spleen,
  colon, pancreas
• High affinity, Km in µM

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Model of Peptide Transport
Di/tri peptides ß lactams ACE inhibitors
H
Na
Apical Membrane
PEPT1(2)
Na
H
3Na
2K
Basolateral Membrane
Basolateral Peptide Transporter
Drug Transporters
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Peptide Transporters 3

• Drug Substrates
• ß lactam antibiotics (penicillins,
  cephalosporins)
• Ampicillin, Amoxicillin, Cefadroxil, Cefadrine
  Cefixime, Ceftibuten Cefalexin
• Angiotensin Converting Enzyme Inhibitors
• Captopril, Enalapril, Fosinopril
• Anticancer
• Bestatin
• Prodrugs
• Valacylovir, Valganciclovir, DOPA-amino-acids

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Summary

• Many human transporters have been identified and
  a critical role in the absorption, distribution,
  metabolism and elimination of drugs
• ABC Transporters
• 48 human proteins
• SLC Transporters
• 43 Families, 300 proteins