biochemical and molecular toxicology and molecular toxicology envr 442/toxc 442/bioc 442 the role of...

K.L.R. Brouwer1

September 8, 2009TOXC 442

Xenobiotic Transporters

Biochemical and Molecular ToxicologyENVR 442/TOXC 442/BIOC 442

The Role of Transporters (Phase III)in Xenobiotic Disposition

Kim L.R. Brouwer, PharmD, PhD

William R. Kenan Distinguished Professor and Chair,Division of Pharmacotherapy & Experimental Therapeutics

UNC Eshelman School of [email protected]; 919-962-7030

PP

P

P

COOH

NH2

PPPP

PP

PP

COOH

NH2

Organic Anion TransportersOrganic Anion TransportersSolute Carrier Family 22Solute Carrier Family 22

PAH, OTA, DHEA-sulfate, estrone-sulfate, AZT, cimetidine, MTX

antiport (dicarboxylates)

kidney, placentaOAT4

PAH, OTA, salicylate, estrone-sulfate, cAMP, PGE2, cimetidine


kidney, liver, bone, brain, eye

OAT3

PAH, MTX, salicylate, acetylsalicylate, PGE2

?kidney, liverOAT2

PAH, PSP, methotrexate (MTX), cidofovir, ochratoxin A (OTA), cephaloridine, indoxyl sulfate, AZT, penicillins, cAMP, cGMP, PGE2, etc.


kidney, brain, choroid plexus

OAT1

SubstratesTransport mechanism

Tissue distributionTransporter

F. Russel

K.L.R. Brouwer2



Organic Anion TransportingOrganic Anion Transporting PolypeptidesPolypeptidesSolute Carrier Family 21/SLCOSolute Carrier Family 21/SLCO

MTX, folate, DHEA sulfate, E217βG ochratoxin A, digoxin, MTX, AZT

BSP, DHEA, estradiol glucuronide, estrone sulfate, thyroxine, T3, ochratoxin A, bile acids, fexofenadine, oubain, rocuronium, chlorambucil

digoxin, ouabain, thyroxine, T3, cAMP, MTX

?

antiport?(GSH)

?

kidney

brain, kidney, liver

kidney

Oat-k1/k2 (1a3_v1/v2)

OATP-A (1A2)

OATP-H (4C1)

??kidneyOatp5 (1a6)

taurocholate, thyroxine, T3?kidney, lung, retina, liver

Oatp3 (1a5)

BSP,ouabain, taurocholate, estradiol glucuronide (E217βG), estrone sulfate, DHEA sulfate, aldosterone, cortisol, enalapril, thyroxine, triiodo-L-thyronine (T3), leukotriene C4, PGE2, ochratoxin A

antiport(GSH)

kidney, liverOatp1 (1a1)



F. Russel

Organic Cation TransportersOrganic Cation TransportersSolute Carrier Family 22Solute Carrier Family 22

PP

P

P

COOH

NH2

PPPP

PP

PP

COOH

NH2

MPP+, guanidine, monoamines, cimetidine, tyramine

uniportliver, skeletal muscle, placenta, kidney,heart, brain

OCT3

TEA, MPP+, carnitine, choline, quinidine, verapamil, pyrilamine, valproate

uniport Na+-carnitine cotransport

skeletal muscle, kidney, placenta, liver, intestine, heart, etc.

OCTN2

TEA, MPP+, carnitine, quinidine, verapamil, pyrilamine

antiport (H+)

kidney, skeletal muscle, placenta, prostate, heart

OCTN1

TEA, MPP+, NMN, monoamines, amantadine

uniportkidney, brain, neurons

OCT2

MPP+, TEA, acyclovir, gancicloviruniportliverOCT1



F. Russel

K.L.R. Brouwer3



BQ123, glutathione conjugates

E217βG, calcein, rhodamine 123, digoxin, anticancer drugs, verapamil, anti-HIV drugs, steroid hormones

pump (ATP)

pump (ATP)

kidney, liver

kidney, liver, intestine, brain, placenta, lung

MRP6

MDR1

cidofovir, PMEA, AZTMP, MTX, PAH, cAMP, cGMP, prostaglandins

pump (ATP)kidney, liver, intestine, brain, prostate

MRP4

glutathione, glucuronide, and sulfate conjugates, PAH, GSH, GSSG, cisplatin, methotrexate, ochratoxin A

pump (ATP) kidney, liver, intestine MRP2

glutathione, glucuronide, and sulfate conjugates, anticancer agents, GSH, GSSG, PAH

pump (ATP)kidney, (liver), lung, intestine, brain

MRP1



Multidrug Resistance TransportersMultidrug Resistance TransportersATPATP--Binding Cassette subfamily (ABCB/ABCC)Binding Cassette subfamily (ABCB/ABCC)

out

in

NH2

COOHATP ATP

out

in

NH2 COOHATP ATP

out

in

out

in

NH2

COOHATP ATP

out

in

out

in

NH2 COOHATP ATP

MRP1MRP2MRP6

MRP4MRP5MDR1

F. Russel

Ayrton & Morgan, Xenobiotica 31:469, 2001

Organ Distribution of Transport Proteins

BSEP

K.L.R. Brouwer4



Mdr1a P-gp

Mdr1b P-gp

BBB

Gallbladder

Liver

Stomach

Intestine

Kidney

Adapted fromSchinkel, 1997

Organ Distribution of Multidrug Resistance Mdr1 P-glycoprotein

P-gp Expression in Murine Brain Capillary Endothelial Cells

Graff and Pollack, 2005

K.L.R. Brouwer5



Ivermectin Toxicity inMdr1a(-/-) and Mdr1a(+/+) Mice

Schinkel et al., Cell, 77:491, 1994

Maintenance of Barrier Function:Xenobiotic Transporters in the Brain

Ho and Kim, Clin Pharmacol Ther 78:260, 2005

K.L.R. Brouwer6



P-glycoprotein staining

Endothelial cell staining Co-localization

Maintenance of Barrier Function:Endothelial Cells Lining the Olfactory Bulb

Graff and Pollack, Pharm Res 22:86, 2005

Maintenance of Barrier Function in Sanctuary Site Tissues

Placenta

Leslie et al., Tox Appl Pharmacol 204:216, 2005

K.L.R. Brouwer7



Maintenance of Barrier Function:Xenobiotic Transporters in the Intestine


Role of Mrp1 in Intestinal Toxicity of MethotrexateIntestinal toxicity induced by methotrexate treatment in wild-type [Mrp1(+/+)] and

Mrp1 gene knockout [Mrp1(−/−)] mice in vivo. Mrp1 is localized primarily in proliferative cells in crypts where it is involved in active efflux of methotrexate as a

defensive mechanism to protect the small intestinal epithelial cells from toxicity.

Tissue sections from ‘lower’ part of the small intestine were analyzed for morphology with H&E staining (top panel) and for S-phase cells with immunostaining using anti-BrdU antibody (bottom panel).


K.L.R. Brouwer8



Xenobiotic Transporters in the Kidney


Effect of Probenecid on Renal Content and Urinary Excretion of Cadmium (Cd) in Mice

Mice were injected i.p. with saline or probenecid (1 mmol/kg), and injected i.v. 30 min later with either Cd alone (1 mg Cd/kg, 74 kBq 109Cd) or Cd with dithiocarbamate

chelating agents (1:30 molar ratio); urine samples were collected for 3 h and renal Cd content was determined from radioactivity.

Kamenosono et al., Comp Biochem Physiol C Toxicol Pharmacol 132:61, 2002

K.L.R. Brouwer9



Oatk1/2Oatk1/2

MRP1MRP1PTPT

TALTALCDCD

rOat2rOat2

MRP6MRP6

rOat3rOat3

Oatp1Oatp1OAT4OAT4

OAT1OAT1OAT3OAT3

cortexcortexmedullamedulla

Nephron Distribution of Organic Anion TransportersNephron Distribution of Organic Anion Transporters

MRP2MRP2MRP4MRP4

Oatv1Oatv1F. Russel

PTPT

TALTALCDCD

cortexcortexmedullamedulla

MDR1MDR1Mdr1a/1bMdr1a/1b

OCT2OCT2

rOct1rOct1rOct2rOct2

OATP4C1OATP4C1

rOatp4c1rOatp4c1

OCTN1OCTN1OCTN2OCTN2

Nephron Distribution Organic Cation TransportersNephron Distribution Organic Cation Transporters

rOct3rOct3??

F. Russel

K.L.R. Brouwer10



NPT1 pOatv1OAT1/3

MRP6

α-KGOat1/3

Mrp6

α-KG

OAT4

MRP2/4

α-KGOat4

Mrp2/4

α-KG

OCT2 Oct1/2

MDR1

OCTN1/2H+

Mdr1a/1b

Octn1/2H+

OATP4C1 Oatp4c1OATP1A2GSH

GSHOatp1a1

Oatp1a3(Oat-k1/k2)

Oatp1a4/a5

blood bloodurine

human rat

Species differences

F. Russel

Available Models To ExamineRenal Transport Processes

Intact kidney in vivoIsolated perfused kidneyIsolated perfused or nonperfused tubules Cultured renal cellsIsolated plasma membrane vesicles(basolateral or brush border)

K.L.R. Brouwer11



Hepatic Elimination: Phase I (P450s),Phase II (conjugation) & Phase III (transport)

bile

tight junction

sinusoidalmembrane

hepatocyte

canalicularmembrane

Metabolite

uptake

egress

blood flow

reabsorptionbile

blood flow

sinusoidalmembrane

Parentegress

IntracellularSequestration

protein binding

Bile and Urine as Complementary Pathways for Excretion of Foreign Compounds in Rats: Molecular Weight Threshold Hypothesis

Hirom et al., Xenobiotica 6:55-64, 1976

% of dose recovered inMW Bile Urine

Sulphathiazole 255 3.1 844.8 ---a

Succinylsulphacetamide 314 1.8 626.3 ---a

Glutarylsulphathiazole 369 42 4785 ---a

---b 831,2,3,6-Tetrahydrophthalyl- 407 45 34

sulphathiazole 81 ---a

---b 83Bromophenol blue 670 69 3.6

---b 19Indocyanine green 752 82 0

---b 0

aRenal pedicles were ligated before biliary cannulation to prevent urine formationbBile ducts were ligated; kidneys were left intact

K.L.R. Brouwer12



Ho and Kim, Clin Pharmacol Ther78:260, 2005

The Structureof the Liver

Basolateral Transporters: Organic Anions

bile bile

tight junction

sinusoidalmembrane

hepatocyte

NTCPTC Na+ 2 K+

3 Na+

ATP

sinusoidalmembrane

-40mV

blood flow

blood flow

K.L.R. Brouwer13



Bile acids [cholate; glycocholate taurochenodeoxycholate; tauroursodeoxycholate; taurocholate (TC)]BSP; estrone-3-sulfate;

SLC10A1NTCP

SubstratesGene Symbol

Trivial Names

Transport Protein

Hepatic Basolateral Uptake TransportersSolute Carrier (SLC) Family 10, Member A1

Na+-Taurocholate Cotransporting Polypeptide

• Liver-specific transporter• Mediates Na+-dependent uptake of bile acids• Driving force: secondary active transport, Na+ gradient• BQ123, indomethacin, various steroid conjugates, bumetanide,

furosemide and verapamil inhibit NTCP-mediated bile salt uptake but are not substrates.


bile bile

tight junction

sinusoidalmembrane

hepatocyte

NTCPTC Na+

OATPsOA-2 K+

3 Na+

ATP

sinusoidalmembrane

-40mV

Cl-+

blood flow

blood flow

K.L.R. Brouwer14



Bile acids; BQ-123; BSP; DHEAS; DPDPE; E217G; estrone-3-sulfate; n-methyl quinine; ouabain; T3; T4; fexofenadine

SLCO1A2(previously SLC21A3)

OATP-AOATP-1OATP

OATP1A2

Bile acids; BQ-123; BSP; DHEAS; DPDPE; E217G; estrone-3-sulfate; ouabain; T3; T4; bilirubin; bilirubin glucuronides; LTC4; prostaglandin E2; pravastatin; rifampin

SLCO1B1(previously SLC21A6)

OATP-CLST-1OATP2

OATP1B1

Bile acids; BQ-123; BSP; CCK-8; DHEAS; digoxin; DPDPE; E217G; estrone-3-sulfate; n-methyl quinine, ouabain; T3; T4; monoglucuronosyl bilirubin; rifampin

SLCO1B3(previously SLC21A8

OATP-8LST-2

OATP1B3

benzylpenicillin; BSP; DHEAS; estrone 3-sulfateSLCO2B1(previously SLC21A9)

OATP-BOATP2B1


Trivial Names

Transport Protein

Hepatic Basolateral Uptake TransportersSolute Carrier Family 21 (SLCO), Member #

Organic Anion Transporting Polypeptides

Estimated Cumulative Risk of Myopathy Associated with Taking 80 mg Simvastatin Daily,

According to SLCO1B1 rs4149056 Genotype

The SEARCH Collaborative Group, New Eng J Med 359:2008

K.L.R. Brouwer15



Rodent Hepatic Basolateral Uptake TransportersSolute Carrier Family 21 (SLCO), Member #

Nomenclature SpecificNew Old Substrate Homology Human

Oatp1a1 Oatp1 Deltorphin II 67% (OATP-A)Slco1a1 Slc21a1

OATP1A2 (OATP-A)SLCO1A2

Oatp1a4 Oatp2 Digoxin 77% (Oatp1)Slco1a4 Slc21a5

OATP1B1 (OATP-C)SLCO1B1

Oatp1b2 Oatp4 CCK-8 60% (OATP-C)Slco1b2 Slc21a10 66% (OATP-8)

OATP1B3 (OATP-8)SLCO1B3

OATP2B1 (OATP-B)SLCO2B1

Substrates for the Rat Organic Anion Transporting Polypeptides (Oatp)

Kullak-Ublick, J. Hepatology 31:563-573, 1999

Oatp1a1 (Oatp1)BromosulphophthaleinBile AcidsEstrone-3-sulfateEstradiol-17β-glucuronideLTC4DHEASOuabainPravastatinCRC 220BQ123Ochratoxin AAPD-ajmaliniumTemocaprilatGadoxetateFexofenadineDPDPEDexamethasone

Oatp1a4 (Oatp2)DigoxinTaurocholateEstrone-3-sulfateEstradiol-17β-glucuronideDHEASLTC4OuabainT3, T4APD-ajmaliniumBQ123DPDPEFexofenadine

Oatp1b2 (Oatp4)BromosulphophthaleinTaurocholateEstrone-3-sulfateEstradiol-17β

-glucuronide DHEASLTC4T3, T4BQ123DPDPEBilirubin/glucuronidesPGE2CCK-8

Faber et al., Adv. Drug Deliv. Rev. 55:107-124, 2003

K.L.R. Brouwer16




bile bile

tight junction

sinusoidalmembrane

hepatocyte

NTCPTC Na+

OATPsOA-2 K+

3 Na+

ATP

sinusoidalmembrane

-40mV

Cl-+

blood flow

blood flow

OA-

OATs

prostaglandin E2; prostaglandin F2α; salicylate; tetracycline; zidovudine

SLC22A7OAT2

bumetanide; estrone-3-sulfate; ketoprofen; salicylate; MTX; ochratoxin A; prostaglandin E2; prostaglandin F2α; tetracycline; zidovudine

SLC22A11OAT4


Trivial Names

Transport Protein

Hepatic Basolateral Uptake TransportersSolute Carrier (SLC) Family 22, Member #

Organic Anion Transporters

PAH, estrone sulfate, ochratoxin A, cimetidineOat3

p-Aminohippurate (PAH), dicarboxylates, PGE2, salicylate, methotrexate, indomethacin, nucleoside derivatives

Oat2

SubstratesTransport Protein

K.L.R. Brouwer17



Chemical Structure of Cationic Drugs Taken Up by Two Separate Hepatic

Transport Systems

Basolateral Transporters:Organic Anions and Cations

bile bile

tight junction

sinusoidalmembrane

hepatocyte

NTCPTC Na+

OATPs OA-2 K+

3 Na+

ATP

sinusoidalmembrane

OC+

Type IOA-

OCT1

-40mV

Cl-+

blood flow

blood flow

OATs

K.L.R. Brouwer18



azidoprocainamide methoiodide; n-methyl-quinidine; n-methyl-quinine; tributylmethylammonium; MPP+; tetraethylammonium

SLC22A1OCT1

adrenaline; noradrenaline; tyramine; agmatine; MPP+

SLC22A3EMTOCT3


Trivial Names

Transport Protein

Hepatic Basolateral Uptake TransportersSolute Carrier (SLC) Family 22, Member #

Organic Cation Transporters

bile bile

tight junction

sinusoidalmembrane

hepatocyte

sinusoidalmembrane

-40mV

blood flow

blood flow

MRP5 (ABCC5)

cAMP cGMP

ATP

MRP1,6(ABCC1,6)

ATP

OA-

ATP

cAMP,cGMPMTX,OA-

ATP

MRP3(ABCC3)

MRP4(ABCC4)

OA-

Hepatic Basolateral Export Transporters

2 K+

3 Na+

ATP

K.L.R. Brouwer19



cAMP; cGMP; PMEAABCC5MOAT-C ABC11

MRP5

BQ-123ABCC6MOAT-E MLP1

MRP6

E217G; LTC4ABCC10MRP7

daunorubicin; doxorubicin; etoposide; vincristine ABCC1MRP, GS-X MRP1

acetaminophen glucuronide; E217G; monovalent and sulfated bile salts; MTX

ABCC3MOAT-D MLP2 cMOAT2

MRP3

azidothymidine; cAMP; cGMP; PMEA; MTX ABCC4MOAT-B MRP4

cAMP; cGMP ABCC11MRP8


Trivial Names

Transport Protein

Hepatic Basolateral Export TransportersATP-Binding Cassette (ABC) Subfamily C, Member #

Multidrug Resistance-Associated Proteins

bile bile

tight junction

sinusoidalmembrane

hepatocyte

OATP1B1, 1B3, 2B1(SLCO1B1,1B3, 2B1)

OA-

OC+,Type II

sinusoidalmembrane

-40mV

blood flow

blood flow

OAT2(SLC22A7)

OA-

MRP5 (ABCC5)

cAMP cGMP

ATP

MRP1,6(ABCC1,6)

ATP

OA-

ATP

cAMP,cGMPMTX,OA-

ATP

MRP3(ABCC3)

MRP4(ABCC4)

OCT1(SLC22A1)

OC+Type I

OA-

Hepatic Basolateral Export Transporters

Chandra and Brouwer, Pharm Res, 21:719, 2004

K.L.R. Brouwer20



Hepatic Canalicular Transporters


sinusoidalmembrane

hepatocyte-40mV

blood flow

bile

BSEP(ABCB11)

TCATP

blood flow

sinusoidalmembrane

Canalicular Transporters

tight junction

K.L.R. Brouwer21



conjugated and unconjugated bile salts; TCABCB11Sister PgpBSEP


Trivial Names

Transport Protein

Canalicular TransportersATP-Binding Cassette (ABC) Subfamily B, Member 11

Bile Salt Export Pump

Numerous drugs (cyclosporin A, rifampicin, glibenclamide, bosentan, troglitazone) recently have been shown to inhibitBSEP function, but these compounds are not substrates for BSEP.

Fattinger et al., Clin Pharmacol Ther 69:223, 2001

Concentrations of Serum Bile Salts in Patients w/

Bosentan-Induced Liver Injury

Effect of Bosentan and Metabolites on ATP-Dependent

Taurocholate Transport in cLPMs and Sf9 Vesicles

Clinical Relevance of Drug Transport Interactions:Bosentan Inhibits BSEP

K.L.R. Brouwer22



sinusoidalmembrane

hepatocyte-40mV

blood flow

MRP2 (ABCC2)

bile

ATPOA-

blood flow

sinusoidalmembrane


tight junction

acetaminophen glucuronide; carboxydichlorofluorescein; camptothecin; doxorubicin; cisplatin; vincristine; etoposide; glibenclamide; indomethacin; rifampin; glucuronide, glutathione, and sulfate conjugates; LTC4; MTX; pravastatin

ABCC2CMOATcMRP

MRP2


Trivial Names

Transport Protein

Canalicular TransportersATP-Binding Cassette (ABC) Subfamily C, Member 2

Multidrug Resistance-Associated Protein

K.L.R. Brouwer23



Substrates for the Canalicular Multispecific Organic Anion Transporter (Mrp2; cMOAT)

Oude Elferink, R.P.J. et al., Biochim. Biophys. Acta 1241:215-268, 1995

# of NegativeEndogenous Compounds ChargesConjugated Bilirubin 2Glutathione GSH 2Glutathione GSSG 4Cysteinyl-leukotrienes 2/3Triiodothyronine-glucuronide 2Coproporphyrin I 2

Bile Salt ConjugatesCholate 3-O-glucuronide 2Lithocholate 3-O-glucuronide 2Nordeoxycholate 3-O-glucuronide 2 Tauro-glycolithocholate 3-sulfate 2Taurochenodeoxycholate 3-sulfate 2Nordeoxycholate-3-sulfate 2

# of NegativeExogenous Compounds ChargesCeftriaxone 2Ampicillin 2Carboxydichlorofluorescein 2Dibromosulfophthalein 2Bromosulfophthalein-glutathione 4Dinitrophenyl-glutathione 2Glutathionyl-bromoisovalerylurea 2Naphthol-1-glucuronide 1Indocyanine green 2Gadolinium-ethoxybenzyl-DTPA 2Acetaminophen glucuronide 1

MetalsZincCopperManganese

Hepatic Mrp3 Protein Levels inEHBR (Mrp2-deficient), Gunn, Bilirubin-

treated and Sprague-Dawley Rats

Ogawa et al., Am J Physiol 278:G438, 2000

K.L.R. Brouwer24



sinusoidalmembrane

hepatocyte-40mV

blood flow

ATP

bile MDR1 (ABCB1)OC+

PhospholipidsMDR3 (ABCB4)ATP

blood flow

sinusoidalmembrane


tight junction

amprenavir; indinavir; nelfinavir; ritonavir; saquinaviraldosterone; corticosterone; dexamethasone; digoxincyclosporin A; MX; debrisosoquine; erythromycin; lovastatin; terfenadine; digoxin; quinidine; doxorubicin; paclitaxel; rhodamine 123; etoposide; fexofenadine; losartan; vinblastine; tacrolimus; talinolol

ABCB1P-gpMDR1

phospholipidsABCB4PFIC3Phospholipid flippase

MDR3


Trivial Names

Transport Protein

Canalicular TransportersATP-Binding Cassette (ABC) Subfamily B, Member #

Multidrug Resistance Proteins

K.L.R. Brouwer25



Hepatic Export TransportersATP-Binding Cassette (ABC), Subfamily A-G, Member #

ABC Subfamily Rat Homology Human HomologyMDR/TAP Mdr1a 83% (MDR1) MDR1 (P-gp) 78% (Mdr1b)Subfamily B Abcb1a ABCB1

Mdr1b 79% (MDR1)Abcb1bMdr2 72.1% (Mdr1b) MDR2/3 74.9% (MDR1)Abcb4 ABCB4Bsep 48% (Mdr2) BSEP (SPGP) 88% (rat)Abcb11 ABCB11

MRP/CFTR Mrp1 MRP1 (GS-X) 19% (CFTR)Subfamily C Abcc1 ABCC1

Mrp2(cMoat) 48% (MRP1) MRP2 (cMOAT) 46% (MRP1)Abcc2 ABCC2Mrp3 MRP3 (MLP2) 58% (MRP1)Abcc3 ABCC3Mrp4 MRP4Abcc4 ABCC4Mrp5 MRP5Abcc5 ABCC5Mrp6 MRP6 (MLP1)Abcc6 ABCC6

sinusoidalmembrane

hepatocyte-40mV

blood flow

bile

ATP BCRP (ABCG2)MX

blood flow

sinusoidalmembrane


tight junction

K.L.R. Brouwer26



daunorubicin; doxorubicin; MX; sulfated conjugates

ABCG2MXR, ABCP

BCRP


Trivial Names

Transport Protein

Canalicular TransportersATP-Binding Cassette (ABC) Subfamily G, Member 2

Breast Cancer Resistance Protein

ATP-dependent half-transporterSubstrates include:– Estrone-3-sulfate– SN-38

Suzuki et al., Hepatology 36:218A (205), 2002

Hepatic Uptake and Export Transporters

-

B

OA-

m-

OA-

MITOCHONDRIA

GOLGI

OCT1 OAT2

ATPATP ATP

m-

m-

OATPs

OA-

X-E-

OST α/β

B

SPACE OF DISSE

NTCP

B Na+

B

ATP

ATP

m-X-

DrugsOther Xenobiotics/Endobiotics

X-m- OA-

m-

X+

OA-

X-E-

B

ATP

m-

MATE

BSEP

ATP

ATP

ATP

ATP

Fic1

Pgp

MDR3

ABCG5-8

MRP3 MRP4 MRP5,6MRP1

ATP

BCRP

MRP2

X-

X-

X-

X- m-B

X+

H+

OA-

A. Rizwan

K.L.R. Brouwer27



Model Systems to Investigate Hepatobiliary Disposition and Biliary Excretion

Intact Liver (in vivo)Isolated Perfused LiverLiver SlicesHepatocytes (suspension, couplets, cultures)– Sandwich-Cultured Hepatocytes

Plasma Membrane Vesicles (cLPM, bLPM)Transfected Transport Proteins

Species Differences in Canalicular Transport Proteins Primarily Responsible for Biliary

Excretion of Organic Anions

??Bcrp & Mrp2Mrp2APAP-Glucuronide??BcrpMrp2 & BcrpAPAP-Sulfate

P-gp and ??

Mrp2P-gpFexofenadine

??

????

Human

Mrp2Mrp2Carboxydichloro-fluorescein

BcrpMrp2 & Bcrp4MU-SulfateBcrp & Mrp2Mrp24MU-Glucuronide

MouseRatBiliary Excretion

Zamek-Gliszczynski et al., Mol Pharm, 70:2127, 2006

K.L.R. Brouwer28



Sandwich-Cultured HepatocytesUtility– Determine hepatic uptake and biliary clearance– Determine metabolic clearance

Advantages – Normal cell polarity re-established– Enzyme/transport activity may be modulated by culture

conditions– Applicable to hepatocytes from animals or humans– Enzyme/transport proteins may be inhibited/induced in

culture– Amenable to higher throughput

Limitations– Requires 3-4 days for proper localization of canalicular

transport proteins

Pre-isolation 0 hours 24 hours 48 – 96 hours

Percoll Gradient85 – 95% Viability

Liver Perfusion (~35 ml/min, 37oC):10 min Ca2+-free with chelator10 min collagenase digestionSingle pass or recirculating flow

Hepatocyte isolationLiver capsule gently torn

Sandwich-Cultured Hepatocytes: Experimental Procedures

Liu et al., Am J Physiol277: G12-G21, 1999

K.L.R. Brouwer29



Immunohistochemical Localization of Mrp2 (green)and Mrp3 (red) in Day 4 Sandwich-Cultured

Rat Hepatocytes

Zhang et al., AAPSPharmSci, 2001

Time course of Carboxydichlorofluorescein in Sandwich-Cultured Hepatocytes

4 hours

28 hours

48 hours

96 hours

Zhang et al., AAPSPharmSci, 2001

K.L.R. Brouwer30



Bosentan: Species-dependent differences in inhibition of bile acid uptake and excretion.Troglitazone: Hepatocyte accumulation of troglitazone sulfate

Can Hepatotoxicity be Predicted from In Vitro Systems?

Effect of Bosentan on 3H-Taurocholate Disposition in Sandwich-Cultured Rat Hepatocytes

Kemp et al., Toxicol Sci 83:207, 2005

Taurocholate accumulation in the absence of bosentan ( ) or presence of 1 μM ( ), 10 μM ( ), 20 μM ( ), 50 μM ( ), and100 μM ( ) bosentan. Taurocholate accumulation in cells + bile canaliculi (black) or cells (grey) was simulated in control (solid line) and 100 μM bosentan-treated (dashed line) sandwich-cultured rat hepatocytes.

K.L.R. Brouwer31



Effect of Bosentan on 3H-Taurocholate Accumulation in Sandwich-Cultured Human Hepatocytes

Leslie et al., J Pharmacol Exp Ther, 321:1170, 2007

[3H]-Taurocholate accumulation in cells + bile canaliculi (black) or cells (white) during 10-min exposure in sandwich-cultured human hepatocytes.

0 1 10 30 50 1000

50

100

bosentan (μM)

taur

ocho

late

accu

mul

atio

n(%

cont

rol)

0.01 0.1 1 10 1000

50

100

[bosentan] (μM)

taur

ocho

late

upta

ke(%

con

trol)

IC50 (µM)

Rat-Ntcp 9

NtcpNTCP Human-NTCP 140

Bosentan Inhibits 3H-Taurocholate Uptake by NTCP/Ntcp-Transfected HeLa Cells

Bosentan inhibits rat Ntcp ~15-fold more potently than human NTCP

Experiments performed in Dr. Richard Kim’s laboratory at Vanderbilt University


K.L.R. Brouwer32



0.01 0.1 1 10 100 10000

50

100

humanrat

[bosentan] (μM)

taur

ocho

late

upta

ke(%

con

trol)

Bosentan Inhibits Na+-Dependent3H-Taurocholate Uptake in Rat and Human

Suspended Hepatocytes

Bosentan inhibits Na+-dependent uptake of 3H-taurocholate in rathepatocytes ~6-fold more potently than human hepatocytes

Hepatocytes IC50(μM)

Rat 5 ± 1.7(n=4)

Human 30(n=2)


Taurocholate

Hepatocyte

Bosentan

Sinusoidal Blood

Bsep

NtcpNaNa++

OatpsOatps

OAOA--

Bile AcidsBile Acids


Current HypothesisBosentan inhibits rat Bsep but is not hepatotoxic in rats because inhibition of Ntcp protects the hepatocyte from

accumulation of toxic bile salts

Bile

K.L.R. Brouwer33



HepatocyteSinusoidal

Blood

Bsep

NtcpNaNa++

OatpsOatps

OAOA--



Rat Human

Current HypothesisBalance between inhibition of bile acid uptake

(NTCP/Ntcp) and excretion (BSEP/Bsep) may explain some species differences in drug-induced liver injury

Not Hepatotoxic Hepatotoxic

Taurocholate Drug with Hepatotoxic Potential

HepatocyteNTCP

Sinusoidal Blood

NaNa++


OATPsOATPsBile AcidsBile Acids

OAOA--

BSEP

Bile Bile

B

m-

MITOCHONDRIA

GOLGI

OCT1 OAT2

ATPATP ATP

OATPs

OA-

X-E-

OST α/β

B

NTCP

B Na+

B

ATP

ATP

m-

X-

X+

OA-

X-E-

ATP

MATEH+

BSEP

ATP

ATP

ATP

ATP

Fic1

Pgp

MDR3

ABCG5-8

MRP3 MRP4 MRP5,6MRP1

ATP

BCRP

MRP2

Troglitazone (IC50 = 3.9 µM) vs. TS (IC50= 0.4-0.6 µM)

B

B

B

B

B

B

BB

BB

Funk et al., 2001

Troglitazone Sulfate is a More Potent Inhibitorof BSEP than Troglitazone

K.L.R. Brouwer34



• A compartment model was developed to describe the hepatobiliary disposition of TGZ and its metabolites in medium, cell and bile of sandwich-cultured hepatocytes

• A first-order rate constant for bile leakage for TS and TG was incorporated in the model after 20 min

Medium Cell Bile

Disposition of Troglitazone and Metabolites in Sandwich-Cultured Rat Hepatocytes

Lee et al., 2009

• Cellular accumulation of TS was extensive when Kbile,TS was impaired

• Cellular accumulation measurements were 1.4- to 270-times more sensitive than medium accumulation measurements for TG, and 2.2- to 10-times more sensitive for TS

Impact of Kbile,TS Modulation onTroglitazone Sulfate (TS) Accumulationin Sandwich-Cultured Rat Hepatocytes

Lee et al., 2009

K.L.R. Brouwer35



Transport Systems:Implications for Xenobiotic Disposition

Does the parent compound and/or metabolite(s) undergo transport?– What transporters are involved?– What are relative affinities?– Potential for xenobiotic interactions?– Potential for disease state alterations in transport?

Does the xenobiotic and/or metabolite(s) alter the expression and/or function of transport systems?– Potential for xenobiotic interactions?– Potential for organ toxicity?

Xenobiotic Transport: “The More We Learn, The More We Realize

How Little We Know!”

Identification of Transport Proteins in Relevant OrgansStructure-Transport RelationshipsFactors that Regulate Transport Protein Expression, Localizationand Function

GeneticsAgeDietary InfluenceEnvironmental FactorsDisease StatesDrug Interactions

Effects of Altered Transport Function on Xenobiotic DispositionIn Vitro/In Vivo Correlations

biochemical and molecular toxicology and molecular toxicology envr 442/toxc 442/bioc 442 the role of...

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