the role of transporters (phase iii) in xenobiotic...

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

mailto:[email protected]

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 PolypeptidesSolute Carrier Family 21/SLCO


mechanism

Substrates

Oatp1 (1a1) kidney, liver antiport

(GSH)

BSP,ouabain, taurocholate, estradiol

glucuronide (E217βG), 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, 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 F. Russel

Organic Cation TransportersSolute Carrier Family 22


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


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

E217βG, calcein, rhodamine 123,

digoxin, anticancer drugs, verapamil,

anti-HIV drugs, steroid hormones

Multidrug Resistance TransportersATP-Binding Cassette subfamily (ABCB/ABCC)

out

in

NH2

COOHATP ATP

out

in

NH2COOH

ATP ATP

out

in

out

in

NH2

COOHATP ATP

out

in

out

in

NH2COOH

ATP 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

bladderLiver

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


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


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).


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

NPT1 pOatv1

OAT1/3

MRP6

α-KG

Oat1/3

Mrp6

α-KG

OAT4

MRP2/4

α-KG

Oat4

Mrp2/4

α-KG

OCT2 Oct1/2

MDR1

OCTN1/2

H+

Mdr1a/1b

Octn1/2

H+

OATP4C1 Oatp4c1OATP1A2

GSH

GSH

Oatp1a1

Oatp1a3(Oat-k1/k2)

Oatp1a4/a5

blood bloodurine

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

reabsorptionbile

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 formationbBile ducts were ligated; kidneys were left intact

Ho and Kim, Clin Pharmacol Ther

78:260, 2005

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.


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 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

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

Courtesy of K. Hillgren

Lilly Research Laboratories

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)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


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 TransportersSolute 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 IOA-

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 TransportersSolute 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,cGMP

MTX,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 TransportersATP-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,cGMP

MTX,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


sinusoidal

membrane

hepatocyte

-40mV

blood flow

bile

BSEP(ABCB11)

TCATP

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


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

ATPOA-

blood flow

sinusoidal

membrane


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 TransportersATP-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+

PhospholipidsMDR3 (ABCB4)ATP

blood flow

sinusoidal

membrane


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

Multidrug Resistance Proteins

sinusoidal

membrane

hepatocyte

-40mV

blood flow

bile

ATP BCRP (ABCG2)

MX

blood flow

sinusoidal

membrane


tight junction

Transport

Protein

Trivial

Names

Gene

Symbol

Substrates

BCRP MXR,

ABCP

ABCG2 daunorubicin; doxorubicin; MX; sulfated

conjugates

Canalicular TransportersATP-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 α/β

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

BufferOutflow

PerfusateBile

Portal Vein Inflow

30 ml/min rat

5 ml/min mouse

Bile

Bile AUC

XCl =Perfusate

Basolateral AUC

XCl =

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 DuctGall 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% ViabilityLiver 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 mM ( ),

10 mM (), 20 mM (), 50 mM (), and

100 mM ( ) bosentan. Taurocholate accumulation

in cells + bile canaliculi (black) or cells (grey)

was simulated in control (solid line) and

100 mM bosentan-treated (dashed line)

sandwich-cultured rat hepatocytes.

Bosentan Inhibits Taurocholate Uptake by

NTCP/Ntcp-Transfected HeLa Cells

0.01 0.1 1 10 1000

50

100

[bosentan] (mM)

tau

roch

ola

teu

pta

ke

(% c

on

tro

l)

IC50

(µM)

Rat-Ntcp 9

Bosentan inhibits rat Ntcp ~15-fold more potently

than human NTCP

Ntcp

NTCP 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

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

Bosentan Inhibits Na+-Dependent

Taurocholate Uptake in Rat and Human

Suspended Hepatocytes

IC50

(µM)

5 ± 1.7

(n=4)

35

(n=1)

Hepatocyte

Rat

Human

Bosentan inhibits Na+ dependent uptake of taurocholate in rat

hepatocytes ~7-fold more potently than human

0.01 0.1 1 10 100 10000

50

100

human

rat

[bosentan] (mM)

tau

roch

ola

teu

pta

ke

(% c

on

tro

l)

Bosentan Inhibits Na+-Dependent3H-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(mM)

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

ATPATP ATP

OATPs

OA-

X-E-

OST α/β

B

NTCP

B Na+

B

ATP

m-

X-

X+

OA-

X-E-

ATP

ATP

ATP

ATP

ATP

Pgp

MDR3


MRP2

Rat cLPM: 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 Inhibitor

of Bsep than Troglitazone

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

mu

latio

n in

Ce

ll (p

mo

l)

0

200

400

600

800

1000

1200

TGZ

TS

TG

BSEP-expressing membrane vesicles: Troglitazone (IC50 =

20 µM)

vs.

Time (min)

0 20 40 60 80 100 120 140

Accu

mu

latio

n in

Ce

ll (p

mo

l)

0

200

400

600

800

1000

1200

1400

1600

TGZ

TS

TG

Rat cLPM: (IC50 = 3.9 µM)

vs.

Rat cLPM: Troglitazone Sulfate (IC50 = 0.4-0.6

µM)

vs.

Human

Rat

(Incubation with 10 mM Troglitazone)

µM8.44µl/well 6.83

pmol/well 306

Volumelar Intracellu

Cellin neTroglitazo ofon Accumulati ion Concentratular Hepatocell

µM5.6µl/well 6.83

pmol/well 7.44 Conc. neTroglitazo

µM173µl/well 6.83

pmol/well 1182 Conc. Sulfate neTroglitazo


• 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


OA-

B

m-

MITOCHONDRIA

GOLGI

OCT1 OAT2

ATPATP ATP

OATPs

OA-

X-E-

OST α/β

B

NTCP

B Na+

B

ATP

TS

TS

X+

OA-

X-E-

ATP

ATP

ATP

ATP

ATP

Pgp

MDR3


MRP2

B

B

B

B

BB

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

the role of transporters (phase iii) in xenobiotic...

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