Biochemical and Molecular Toxicology

ENVR 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 Pharmacy kbrouwer@unc.edu; 919-962-7030

mailto:kbrouwer@unc.edu

Organic Anion Transporters Solute Carrier Family 22

Transporter Tissue distribution

Transport mechanism

Substrates

OAT1 kidney, brain, choroid plexus

antiport (dicarboxylates)

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

OAT2 kidney, liver ? PAH, MTX, salicylate, acetylsalicylate, PGE2

OAT3 kidney, liver, bone, brain, eye

antiport (dicarboxylates)

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

OAT4 kidney, placenta antiport (dicarboxylates)

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

F. Russel

Organic Anion Transporting Polypeptides Solute Carrier Family 21/SLCO

Transporter Tissue distribution

Transport mechanism

Substrates

Oatp1 (1a1) kidney, liver antiport (GSH)

BSP,ouabain, taurocholate, estradiol glucuronide (E217G), estrone sulfate, DHEA sulfate, aldosterone, cortisol, enalapril, thyroxine, triiodo-L-thyronine (T3), leukotriene C4, PGE2, ochratoxin A

Oatp3 (1a5) kidney, lung, retina, liver

? taurocholate, thyroxine, T3

Oatp5 (1a6) kidney ? ?

Oat-k1/k2 (1a3_v1/v2) OATP-A (1A2) OATP-H (4C1)

kidney brain, kidney, liver kidney

? antiport? (GSH) ?

MTX, folate, DHEA sulfate, E217G 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 F. Russel

Organic Cation Transporters Solute Carrier Family 22

Transporter Tissue distribution

Transport mechanism

Substrates

OCT1 liver uniport MPP+, TEA, acyclovir, ganciclovir

OCT2 kidney, brain, neurons uniport TEA, MPP+, NMN, monoamines, amantadine

OCT3 liver, skeletal muscle, placenta, kidney, heart, brain

uniport MPP+, guanidine, monoamines, cimetidine, tyramine

OCTN1 kidney, skeletal muscle, placenta, prostate, heart

antiport (H+)

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

OCTN2 skeletal muscle, kidney, placenta, liver, intestine, heart, etc.

uniport Na+-carnitine cotransport

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

F. Russel

Transporter Tissue distribution

Transport mechanism

Substrates

MRP1 kidney, (liver), lung, intestine, brain

pump (ATP) glutathione, glucuronide, and sulfate conjugates, anticancer agents, GSH, GSSG, PAH

MRP2 kidney, liver, intestine pump (ATP) glutathione, glucuronide, and sulfate conjugates, PAH, GSH, GSSG, cisplatin, methotrexate, ochratoxin A

MRP4 kidney, liver, intestine, brain, prostate

pump (ATP) cidofovir, PMEA, AZTMP, MTX, PAH, cAMP, cGMP, prostaglandins

MRP6 MDR1

kidney, liver kidney, liver, intestine, brain, placenta, lung

pump (ATP) pump (ATP)

BQ123, glutathione conjugates E217G, calcein, rhodamine 123, digoxin, anticancer drugs, verapamil, anti-HIV drugs, steroid hormones

Multidrug Resistance Transporters ATP-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

MRP1 MRP2 MRP6

MRP4 MRP5 MDR1

F. Russel

Ayrton & Morgan, Xenobiotica 31:469, 2001

Organ Distribution of Transport Proteins

BSEP

Mdr1a P-gp Mdr1b P-gp

BBB

Gall bladder

Liver

Stomach

Intestine

Kidney

Adapted from Schinkel, 1997

Organ Distribution of Multidrug Resistance Mdr1 P-glycoprotein

P-gp Expression in Murine Brain Capillary Endothelial Cells

Graff and Pollack, 2005

Ivermectin Toxicity in Mdr1a(-/-) 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

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

Maintenance of Barrier Function: Xenobiotic Transporters in the Intestine

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

Role of Mrp1 in Intestinal Toxicity of Methotrexate Intestinal 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).

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

Xenobiotic Transporters in the Kidney

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

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

NPT1 pOatv1 OAT1/3

MRP6

-KG Oat1/3

Mrp6

-KG

OAT4

MRP2/4

-KG Oat4

Mrp2/4

-KG

OCT2 Oct1/2

MDR1

OCTN1/2 H+

Mdr1a/1b

Octn1/2 H+

OATP4C1 Oatp4c1 OATP1A2 GSH

GSH Oatp1a1

Oatp1a3 (Oat-k1/k2)

Oatp1a4/a5

blood blood urine

human rat

Species differences

F. Russel

Available Models To Examine Renal Transport Processes

Intact kidney in vivo Isolated perfused kidney Isolated perfused or nonperfused tubules Cultured renal cells Isolated plasma membrane vesicles (basolateral or brush border)

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

bile

tight junction

sinusoidal membrane

hepatocyte

canalicular membrane

Metabolite

uptake

egress

blood flow

reabsorption bile

blood flow

sinusoidal membrane

Parent egress

Intracellular Sequestration

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 in MW Bile Urine Sulphathiazole 255 3.1 84 4.8 ---a Succinylsulphacetamide 314 1.8 62 6.3 ---a Glutarylsulphathiazole 369 42 47 85 ---a ---b 83 1,2,3,6-Tetrahydrophthalyl- 407 45 34 sulphathiazole 81 ---a ---b 83 Bromophenol blue 670 69 3.6 ---b 19 Indocyanine green 752 82 0 ---b 0 aRenal pedicles were ligated before biliary cannulation to prevent urine formation bBile ducts were ligated; kidneys were left intact

Kck and Brouwer, Clin Pharmacol Ther

in press, 2012

The Structure of the Liver

Basolateral Transporters: Organic Anions

bile bile

tight junction

sinusoidal membrane

hepatocyte

NTCP TC Na+ 2 K+

3 Na+

ATP

sinusoidal membrane

-40mV

blood flow

blood flow

Transport Protein

Trivial Names

Gene Symbol

Substrates

NTCP SLC10A1 Bile acids [cholate; glycocholate taurochenodeoxycholate; tauroursodeoxycholate; taurocholate (TC)] BSP; estrone-3-sulfate;

Hepatic Basolateral Uptake Transporters Solute 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.

Basolateral Transporters: Organic Anions

bile bile

tight junction

sinusoidal membrane

hepatocyte

NTCP TC Na+

OATPs OA- 2 K+

3 Na+

ATP

sinusoidal membrane

-40mV

Cl- +

blood flow

blood flow

Transport Protein

Trivial Names

Gene Symbol

Substrates

OATP1A2 OATP-A OATP-1 OATP

SLCO1A2 (previously SLC21A3)

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

OATP1B1 OATP-C LST-1 OATP2

SLCO1B1 (previously SLC21A6)

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

OATP1B3

OATP-8 LST-2

SLCO1B3 (previously SLC21A8

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

OATP2B1 OATP-B SLCO2B1 (previously SLC21A9)

benzylpenicillin; BSP; DHEAS; estrone 3-sulfate

Hepatic Basolateral Uptake Transporters Solute 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

Mean serum concentration-time profiles after single oral pravastatin dose (40mg) in three OATP1B1 genotypic groups

Adapted from Nishizato, et al. Clin. Pharmacol. Ther. 73:554 (2003)

Effect of SLCO1B1 Genotype on

Oral Pravastatin Pharmacokinetics

SLCO1B1 *15/*15, n=1 SLCO1B1 *1b/*15, n=9 SLCO1B1 *1b/*1b, n=4

Courtesy of K. Hillgren Lilly Research Laboratories

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

Nomenclature Specific New 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) Bromosulphophthalein Bile Acids Estrone-3-sulfate Estradiol-17-glucuronide LTC4 DHEAS Ouabain Pravastatin CRC 220 BQ123 Ochratoxin A APD-ajmalinium Temocaprilat Gadoxetate Fexofenadine DPDPE Dexamethasone

Oatp1a4 (Oatp2) Digoxin Taurocholate Estrone-3-sulfate Estradiol-17-glucuronide DHEAS LTC4 Ouabain T3, T4 APD-ajmalinium BQ123 DPDPE Fexofenadine

Oatp1b2 (Oatp4) Bromosulphophthalein Taurocholate Estrone-3-sulfate Estradiol-17 -glucuronide DHEAS LTC4 T3, T4 BQ123 DPDPE Bilirubin/glucuronides PGE2 CCK-8

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

Basolateral Transporters: Organic Anions

bile bile

tight junction

sinusoidal membrane

hepatocyte

NTCP TC Na+

OATPs OA- 2 K+

3 Na+

ATP

sinusoidal membrane

-40mV

Cl- +

blood flow

blood flow

OA-

OATs

Transport Protein

Trivial Names

Gene Symbol

Substrates

OAT2 SLC22A7 prostaglandin E2; prostaglandin F2; salicylate; tetracycline; zidovudine

OAT4 SLC22A11 bumetanide; estrone-3-sulfate; ketoprofen; salicylate; MTX; ochratoxin A; prostaglandin E2; prostaglandin F2; tetracycline; zidovudine

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

Organic Anion Transporters

Transport Protein

Substrates

Oat2 p-Aminohippurate (PAH), dicarboxylates, PGE2, salicylate, methotrexate, indomethacin, nucleoside derivatives

Oat3 PAH, estrone sulfate, ochratoxin A, cimetidine

Chemical Structure of Cationic Drugs Taken Up by Two Separate Hepatic

Transport Systems

Basolateral Transporters: Organic Anions and Cations

bile bile

tight junction

sinusoidal membrane

hepatocyte

NTCP TC Na+

OATPs OA- 2 K+

3 Na+

ATP

sinusoidal membrane

OC+ Type I

OA-

OCT1

-40mV

Cl- +

blood flow

blood flow

OATs

Transport Protein

Trivial Names

Gene Symbol

Substrates

OCT1 SLC22A1 azidoprocainamide methoiodide; n-methyl-quinidine; n-methyl-quinine; tributylmethylammonium; MPP+; tetraethylammonium

OCT3 EMT SLC22A3 adrenaline; noradrenaline; tyramine; agmatine; MPP+

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

Organic Cation Transporters

bile bile

tight junction

sinusoidal membrane

hepatocyte

sinusoidal membrane

-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

Transport Protein

Trivial Names

Gene Symbol

Substrates

MRP1 MRP, GS-X ABCC1 daunorubicin; doxorubicin; etoposide; vincristine

MRP3 MOAT-D MLP2 cMOAT2

ABCC3 acetaminophen glucuronide; E217G; monovalent and sulfated bile salts; MTX

MRP4 MOAT-B ABCC4 azidothymidine; cAMP; cGMP; PMEA; MTX MRP5 MOAT-C

ABC11 ABCC5 cAMP; cGMP; PMEA

MRP6 MOAT-E MLP1

ABCC6 BQ-123

MRP7 ABCC10 E217G; LTC4

MRP8 ABCC11 cAMP; cGMP

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

Multidrug Resistance-Associated Proteins

bile bile

tight junction

sinusoidal membrane

hepatocyte

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

OA- OC+,Type II

sinusoidal membrane

-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

Hepatic Canalicular Transporters

Kck and Brouwer, Clin Pharmacol Ther, in press, 2012

sinusoidal membrane

hepatocyte -40mV

blood flow

bile

BSEP (ABCB11)

TC ATP

blood flow

sinusoidal membrane

Canalicular Transporters

tight junction

Transport Protein

Trivial Names

Gene Symbol

Substrates

BSEP Sister Pgp ABCB11 conjugated and unconjugated bile salts; TC

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

Bile Salt Export Pump

Numerous drugs (cyclosporin A, rifampicin, glibenclamide, bosentan, troglitazone) recently have been shown to inhibit BSEP 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

sinusoidal membrane

hepatocyte -40mV

blood flow

MRP2 (ABCC2)

bile

ATP OA-

blood flow

sinusoidal membrane

Canalicular Transporters

tight junction

Transport Protein

Trivial Names

Gene Symbol

Substrates

MRP2 CMOAT cMRP

ABCC2 acetaminophen glucuronide; carboxydichlorofluorescein; camptothecin; doxorubicin; cisplatin; vincristine; etoposide; glibenclamide; indomethacin; rifampin; glucuronide, glutathione, and sulfate conjugates; LTC4; MTX; pravastatin

Canalicular Transporters ATP-Binding Cassette (ABC) Subfamily C,

Member 2 Multidrug Resistance-Associated Protein

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 Negative Endogenous Compounds Charges Conjugated Bilirubin 2 Glutathione GSH 2 Glutathione GSSG 4 Cysteinyl-leukotrienes 2/3 Triiodothyronine-glucuronide 2 Coproporphyrin I 2 Bile Salt Conjugates Cholate 3-O-glucuronide 2 Lithocholate 3-O-glucuronide 2 Nordeoxycholate 3-O-glucuronide 2 Tauro-glycolithocholate 3-sulfate 2 Taurochenodeoxycholate 3-sulfate 2 Nordeoxycholate-3-sulfate 2

# of Negative Exogenous Compounds Charges Ceftriaxone 2 Ampicillin 2 Carboxydichlorofluorescein 2 Dibromosulfophthalein 2 Bromosulfophthalein-glutathione 4 Dinitrophenyl-glutathione 2 Glutathionyl-bromoisovalerylurea 2 Naphthol-1-glucuronide 1 Indocyanine green 2 Gadolinium-ethoxybenzyl-DTPA 2 Acetaminophen glucuronide 1 Metals Zinc Copper Manganese

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

treated and Sprague-Dawley Rats

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

Hepatic Protein Levels in Wistar and TR- Rats

Johnson et al., Drug Metab Dispos 34:556, 2006

sinusoidal membrane

hepatocyte -40mV

blood flow

ATP

bile MDR1 (ABCB1) OC

+

Phospholipids MDR3 (ABCB4) ATP

blood flow

sinusoidal membrane

Canalicular Transporters

tight junction

Transport Protein

Trivial Names

Gene Symbol

Substrates

MDR1 P-gp ABCB1 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

MDR3 PFIC3 Phospholipid flippase

ABCB4 phospholipids

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

Multidrug Resistance Proteins

sinusoidal membrane

hepatocyte -40mV

blood flow

bile

ATP BCRP (ABCG2) MX

blood flow

sinusoidal membrane

Canalicular Transporters

tight junction

Transport Protein

Trivial Names

Gene Symbol

Substrates

BCRP MXR, ABCP

ABCG2 daunorubicin; doxorubicin; MX; sulfated conjugates

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

Breast Cancer Resistance Protein

ATP-dependent half-transporter Substrates include:

Estrone-3-sulfate SN-38

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

B

OA-

m-

OA-

MITOCHONDRIA

GOLGI

OCT1 OAT2

ATPATP ATP

m-

m-

OATPs

OA-

X-E-

OST /

B

SPACE OF DISSE

NTCP

B Na+

B

ATP

m-X-

Drugs

Other Xenobiotics/EndobioticsX- m-

OA-

m-

X+

OA-

X-E-

B

ATP

m-ATP

ATP

ATP

ATP

Pgp

MDR3

MRP3 MRP4 MRP5,6MRP1

MRP2

X-

X-

X-

X- m-B

X+

OA-

Hepatic Uptake and Export Transporters

A. Rizwan

Sinusoid

Model Systems to Investigate Hepatobiliary Disposition and Biliary Excretion

Intact Liver (in vivo) Isolated Perfused Liver Liver Slices Hepatocytes (suspension, couplets, cultures)

Sandwich-Cultured Hepatocytes Plasma Membrane Vesicles (cLPM, bLPM) Transfected Transport Proteins

Isolated or In Situ Perfused Liver (IPL): Experimental Procedures

Buffer Outflow Perfusate Bile

Portal Vein Inflow 30 ml/min rat

5 ml/min mouse

Bile Bile AUC

X Cl = Perfusate Basolateral AUC

X Cl = Liver Liver

T1-T2

T1-T2

T1-T2

T1-T2

Chandra et al., Am J Physiol 288:G1252, 2005; Nezasa et al., Drug Metab Dispos 34:718, 2006

Inferior Vena Cava Outflow

Bile Duct Gall Bladder

CDF Biliary Excretion and Biliary Excretion Rate Constants (mean SD; min -1) in C57BL/6 WT(O),

Mrp2 KO () and Bcrp KO () Mouse IPLs

Nezasa et al., Drug Metab Dispos 34:718, 2006

Kbile (min -1) 0.061 0.005

0.039 0.011

N.D.

Species Differences in Canalicular Transport Proteins Primarily Responsible for Biliary

Excretion of Organic Anions

Biliary Excretion Rat Mouse Human APAP-Glucuronide Mrp2 Bcrp & Mrp2 ??

APAP-Sulfate Mrp2 & Bcrp Bcrp ?? 4MU-Glucuronide Mrp2 Bcrp & Mrp2 ??

4MU-Sulfate Mrp2 & Bcrp Bcrp ??

Carboxydichloro-fluorescein

Mrp2 Mrp2 ??

Fexofenadine P-gp Mrp2 P-gp and ??

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

Sandwich-Cultured Hepatocytes

Utility 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 Gradient 85 95% Viability

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

Hepatocyte isolation Liver capsule gently torn

Sandwich-Cultured Hepatocytes: Experimental Procedures

Liu et al., Am J Physiol 277: G12-G21, 1999

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

Fluorescence of 5 (and 6)-Carboxy -2,7-dichlorofluorescein in Canalicular Networks of

Day 4 Sandwich-Cultured Hepatocytes

Wistar Control TR-

Bow et al., in preparation, 2010

Mrp2 KO

WT

TR-

WT

Mouse Rat

Swift et al., Drug Metab Rev, in press, 2010

Fluorescence of 5 (and 6)-Carboxy -2,7-dichloro-fluorescein in Canalicular Networks of Day 4

Sandwich-Cultured Rat Hepatocytes

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

0.01 0.1 1 10 1000

50

100

[bosentan] (M)

taur

ocho

late

upta

ke(%

con

trol)

IC50 (M)

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 Kims laboratory at Vanderbilt University

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

0.01 0.1 1 10 100 10000

50

100

humanrat

[bosentan] (M)

taur

ocho

late

upta

ke(%

con

trol)

Bosentan Inhibits Na+-Dependent 3H-Taurocholate Uptake in Rat and Human

Suspended Hepatocytes

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

Hepatocytes IC50(M) Rat 5 1.7 (n=4) Human 30 (n=2)

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

Taurocholate

Hepatocyte

Bosentan

Sinusoidal Blood

Bsep

Ntcp Na+

Oatps

OA-

Bile Acids

Bile Acids

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

accumulation of toxic bile salts

Bile

Rat Human

Current Hypothesis Balance 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

Bile Bile

OA-

B

m-

MITOCHONDRIA

GOLGI

OCT1 OAT2

ATP ATP ATP

OATPs

OA-

X- E-

OST /

B

NTCP

B Na+

B

ATP

m-

X-

X+

OA-

X- E-

ATP

ATP

ATP

ATP

ATP

Pgp

MDR3

MRP3 MRP4

MRP5,6

MRP1

MRP2

Rat cLPM: Troglitazone (IC50 = 3.9 M) vs. TS (IC50= 0.4-0.6 M)

B

B

B

B

B

B

B B

B B

Funk et al.,2001

Troglitazone Sulfate is a More Potent Inhibitor of Bsep than Troglitazone

BSEP-expressing membrane vesicles: Troglitazone (IC50 = 20 M) Yabuuchi et al., 2008

Medium Cell Bile

Disposition of Troglitazone and Metabolites in Day 4 Sandwich-Cultured Rat Hepatocytes

Lee et al. J Pharmacol Exp Ther, 332:26, 2010

Estimated Hepatocellular Concentrations of Troglitazone and Troglitazone Sulfate (TS) in Human

and Rat Sandwich-Cultured Hepatocytes

Time (min)

0 20 40 60 80 100 120 140

Accu

mul

atio

n in

Cel

l (pm

ol)

0

200

400

600

800

1000

1200TGZTSTG

BSEP-expressing membrane vesicles: Troglitazone (IC50 = 20 M)

vs.

Time (min)

0 20 40 60 80 100 120 140

Acc

umul

atio

n in

Cel

l (pm

ol)

0

200

400

600

800

1000

1200

1400

1600TGZTSTG

Rat cLPM: (IC50 = 3.9 M)

vs. Rat cLPM: Troglitazone Sulfate (IC50 = 0.4-0.6 M)

vs.

Human

Rat

(Incubation with 10 M Troglitazone)

M8.44l/well 6.83

pmol/well 306 Volumelar Intracellu

Cellin neTroglitazo ofon Accumulati ion Concentratular Hepatocell

==

=

M5.6l/well 6.83

pmol/well 7.44 Conc. neTroglitazo ==

M173l/well 6.83

pmol/well 1182 Conc. Sulfate neTroglitazo ==

Lee et al. J Pharmacol Exp Ther, 332:26, 2010

Cellular accumulation of TS was extensive when Kbile,TS was impaired; intracellular TS concentrations increased 3- to 6-fold when biliary excretion of TS was decreased 2- and 10-fold, respectively.

Altered hepatobiliary transport and the extent of hepatocyte exposure may not be evident based on medium concentrations (analogous to systemic exposure in vivo).

Impact of Kbile,TS Modulation on TS Accumulation in Sandwich-Cultured Rat Hepatocytes

Lee et al. J Pharmacol Exp Ther, 332:26, 2010

OA-

B

m-

MITOCHONDRIA

GOLGI

OCT1 OAT2

ATP ATP ATP

OATPs

OA-

X- E-

OST /

B

NTCP

B Na+

B

ATP

TS

TS

X+

OA-

X- E-

ATP

ATP

ATP

ATP

ATP

Pgp

MDR3

MRP3 MRP4

MRP5,6

MRP1

MRP2

B

B

B

B

B B

B

B

Troglitazone Sulfate Inhibits BSEP- and MRP4-Mediated Hepatic Excretion of Bile Acids

TS TS TS

B

B

B

B

Marion et al. in preparation, 2010

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 Organs Structure-Transport Relationships Factors that Regulate Transport Protein Expression,

Localization and Function Genetics Age Dietary Influence Environmental Factors Disease States Drug Interactions

Effects of Altered Transport Function on Xenobiotic Disposition

In Vitro/In Vivo Correlations

Biochemical and Molecular Toxicology ENVR 442/TOXC 442/BIOC 442The Role of Transporters (Phase III)in Xenobiotic Disposition Kim L.R. Brouwer, PharmD, PhDWilliam R. Kenan Distinguished Professor and Chair,Division of Pharmacotherapy & Experimental TherapeuticsUNC Eshelman School of Pharmacykbrouwer@unc.edu; 919-962-7030Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Organ Distribution of Multidrug Resistance Mdr1 P-glycoprotein Slide Number 8Ivermectin Toxicity in Mdr1a(-/-) and Mdr1a(+/+) MiceSlide Number 10Maintenance of Barrier Function:Endothelial Cells Lining the Olfactory BulbMaintenance of Barrier Function in Sanctuary Site TissuesSlide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Available Models To ExamineRenal Transport ProcessesHepatic Elimination: Phase I (P450s),Phase II (conjugation) & Phase III (transport)Bile and Urine as Complementary Pathways for Excretion of Foreign Compounds in Rats: Molecular Weight Threshold Hypothesis Slide Number 21Basolateral Transporters: Organic AnionsSlide Number 23Basolateral Transporters: Organic AnionsSlide Number 25Slide Number 26Slide Number 27Rodent Hepatic Basolateral Uptake TransportersSolute Carrier Family 21 (SLCO), Member #Substrates for the Rat Organic Anion Transporting Polypeptides (Oatp)Basolateral Transporters: Organic AnionsSlide Number 31Slide Number 32Basolateral Transporters: Organic Anions and CationsSlide Number 34Hepatic Basolateral Export TransportersSlide Number 36Hepatic Basolateral Export TransportersSlide Number 38Canalicular TransportersSlide Number 40Concentrations of Serum Bile Salts in Patients w/ Bosentan-Induced Liver Injury Canalicular TransportersSlide Number 43Substrates for the Canalicular Multispecific Organic Anion Transporter (Mrp2; cMOAT)Hepatic Mrp3 Protein Levels in EHBR (Mrp2-deficient), Gunn, Bilirubin-treated and Sprague-Dawley RatsHepatic Protein Levels in Wistar and TR- RatsCanalicular TransportersSlide Number 48Canalicular TransportersSlide Number 50Hepatic Uptake and Export TransportersModel Systems to Investigate Hepatobiliary Disposition and Biliary ExcretionIsolated or In Situ Perfused Liver (IPL):Experimental ProceduresCDF Biliary Excretion and Biliary Excretion Rate Constants (mean SD; min -1) in C57BL/6 WT(O), Mrp2 KO () and Bcrp KO () Mouse IPLsSpecies Differences in Canalicular Transport Proteins Primarily Responsible for Biliary Excretion of Organic Anions Sandwich-Cultured HepatocytesSlide Number 57Immunohistochemical Localization of Mrp2 (green) and Mrp3 (red) in Day 4 Sandwich-Cultured Rat HepatocytesTime course of Carboxydichlorofluorescein in Sandwich-Cultured HepatocytesFluorescence of 5 (and 6)-Carboxy -2,7-dichlorofluorescein in Canalicular Networks of Day 4 Sandwich-Cultured HepatocytesFluorescence of 5 (and 6)-Carboxy -2,7-dichloro-fluorescein in Canalicular Networks of Day 4 Sandwich-Cultured Rat HepatocytesCan Hepatotoxicity be Predicted from In Vitro Systems?Effect of Bosentan on 3H-Taurocholate Disposition in Sandwich-Cultured Rat HepatocytesBosentan Inhibits 3H-Taurocholate Uptake by NTCP/Ntcp-Transfected HeLa CellsSlide Number 65Slide Number 66Slide Number 67Slide Number 68Slide Number 69Slide Number 70Slide Number 71Slide Number 72Transport Systems:Implications for Xenobiotic DispositionXenobiotic Transport: The More We Learn, The More We Realize How Little We Know!