Download ROLE OF HEPATIC TRANSPORT PROTEINS IN DRUG

ROLE OF HEPATIC TRANSPORT PROTEINS
IN DRUG DISPOSITION AND DRUG-INDUCED LIVER INJURY
by
Jin Kyung Lee
A dissertation submitted to the faculty of the University of North Carolina at Chapel
Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in
the School of Pharmacy.
Chapel Hill
2009
Approved by:
Kim L. R. Brouwer, Pharm.D., Ph.D.
Gary M. Pollack, Ph.D.
David Miller, Ph.D.
Nita Patel, Ph.D.
Mary F. Paine, Ph.D.
Elaine M. Leslie, Ph.D.
© 2009
Jin Kyung Lee
ALL RIGHTS RESERVED
ii
ABSTRACT
Jin Kyung Lee: Role of Hepatic Transport Proteins in Drug Disposition and
Drug-induced Liver Injury
(Under the direction of Kim L. R. Brouwer)
Transport proteins in the liver play a crucial role in hepatic uptake and biliary
excretion of drugs/metabolites. Understanding how altered hepatic transport (due to
sex, genetic polymorphisms, disease states, drug interactions) influences systemic
and hepatic exposure of drugs/metabolites is important from both a therapeutic and
toxicologic standpoint. The objective of this dissertation research was to investigate
the role of hepatic transport proteins in the hepatobiliary disposition of
drugs/generated metabolites and drug-induced liver injury.
Initial studies in perfused livers from male and female wild-type (WT) and
breast cancer resistance protein (Bcrp)-deficient mice revealed that acetaminophen
sulfate and glucuronide hepatobiliary disposition was influenced by sex-dependent
Bcrp expression as well as other factors relating to conjugate formation and
basolateral transport. In a second set of studies, sandwich-cultured hepatocytes
(SCH), coupled with pharmacokinetic modeling/Monte Carlo simulations, were used
to examine the impact of impaired biliary excretion on the hepatobiliary disposition of
troglitazone (TGZ) and metabolites. Consistent with in vivo data, TGZ was
extensively metabolized to TGZ-sulfate, and to a lesser extent to TGZ-glucuronide
and TGZ-quinone, in both rat and human SCH. Modeling/simulation revealed that
hepatocyte accumulation of metabolites was extensive when biliary excretion
iii
pathways
were
impaired;
medium
concentrations
(analogous
to
systemic
concentrations in vivo) may not reflect changes in hepatic exposure. Subsequent
studies focused on two hepatotoxic drugs (trabectedin and sulindac) and the
potential role of altered hepatic transport in drug-induced liver injury. Trabectedinmediated cytotoxicity was significantly decreased in dexamethasone-pretreated WT
rat SCH and in Mrp2-deficient TR- rat SCH, compared to control WT rat SCH. These
data emphasize the importance of Mrp2 and Mrp3 in hepato-protection from
trabectedin-mediated cytotoxicity. Sulindac and metabolites inhibited multiple
hepatic transport proteins in rat and human hepatocytes; potent inhibition of the
hepatic transport of bile acids and/or drugs by sulindac sulfide and sulfone may play
a previously unrecognized role in sulindac-mediated hepatotoxicity. Collectively, this
dissertation research demonstrated the impact of sex-dependent transporter
expression on the hepatobiliary disposition of phase II conjugates, illustrated use of
an in vitro approach coupled with modeling/simulation to predict altered
drug/metabolite hepatobiliary disposition, and defined the involvement of multiple
transport proteins in drug-induced liver injury.
iv
ACKNOWLEDGEMENT
First of all, I would like to thank God for His constant help and guidance. To
Him be all the glory.
I would like to express my sincere thanks to my advisor, Dr. Kim L. R.
Brouwer, for her valuable support and guidance, but more importantly her patience
druing my graduate study. I am very grateful to my dissertation committee chair, Dr.
Gray M. Pollack for his outstanding teaching in pharmacokinetics, and his insightful
advice for conducting pharmacokinetic modeling. I also would like to give a special
thanks to my dissertation committee members, Drs. David Miller, Nita Patel, Elaine
M. Leslie, and Mary F. Paine, for their scientific advice and encouragement.
I am very grateful to my friends and colleagues. I want to thank Dr. Maciej J.
Zamek-Gliszczynski and Dr. Xianbin Tian for their support, advice, and
encouragement, as well as their training of isolated perfused liver technique. I want
to thank to Dr. Koji Abe for his excellent mentorship, professional advice, but most
importantly, his genuine kindness. I would like to thank to Drs. Wei Yue and Ahsan
Rizwan for their unwavering friendship and enthusiastic scientific discussion.
I
would like to extend my thanks to my sincere friends, Tracy Marion, Brandon Swift,
Dr. Kristina Wolf, Grace Yan, LaToya Griffin, Christina Won, and TaeEun Kim for
their unwavering friendship and continuous support.
v
Most importantly of all, I would like to express my sincere thanks to my family
for their unwavering love and continuous prayers over the years. Finally, I would like
to express my deepest gratefulness for my husband, Changwon Lim for his endless
love, friendship and support during the years of my doctoral program.
vi
TABLE OF CONTENTS
LIST OF TABLES…………………………………………………………………….……viii
LIST OF FIGURES………………………………………………………………….……..ix
LIST OF ABBREVIATIONS………………………………………………………….……xii
CHAPTER
1.
Introduction…………………………………………………………………………..1
2.
Sex-dependent Disposition of Acetaminophen Sulfate and
Glucuronide in the In Situ Perfused Mouse Liver……………………………..49
3.
Hepatobiliary Disposition of Troglitazone (TGZ) and Metabolites
in Rat and Human Sandwich-Cultured Hepatocytes:
Use of Monte Carlo Simulations to Assess Impact of
Changes in Biliary Excretion on Substrate Accumulation
in Cells versus Culture Medium………………………………………………….78
4.
Modulation of Trabectedin (ET-743) Hepatobiliary Disposition
by Multidrug Resistance-Associated Proteins (Mrps) May
Prevent Hepatotoxicity…………………………………………………………..113
5.
Interactions Between Sulindac, Sulindac Sulfone, Sulindac Sulfide
and Hepatic Transport Proteins………………………………………………...141
6.
Conclusion………………………………………………………………………...173
APPENDIX
1.
GF120918 (Elacridar) Inhibits Multidrug Resistance-Associated
Protein 3 (MRP3)- and MRP4-Mediated Transport………………………..…200
2.
Data Summary……………………………………………………………………220
vii
LIST OF TABLES
Table 1.1
Human hepatic uptake transport proteins………………………………..9
Table 1.2
Human hepatic efflux transport proteins…………………..…………....10
Table 2.1
Mouse body weights, liver weights normalized
to body weight, acetaminophen extraction
ratio and total recovery at 70 min in in situ single-pass
perfused livers from male and female wild-type
and Bcrp-deficient mice………………………………………………...…70
Table 2.2
Mean pharmacokinetic parameter estimates governing
APAP, AG and AS disposition in in situ single-pass
perfused livers from wild-type and Bcrp-deficient male
and female mice…………………………………………………….……..71
Table 3.1
Demographics of human liver donors……………………………….…102
Table 3.2
Disposition of TGZ and generated metabolites
in human SCH…………………………………………………………….103
Table 3.3
Disposition of TGZ and its generated metabolites
in rat SCH…………………………………………………………………104
Table 3.4
Kinetic parameters (min-1) estimated from compartmental
pharmacokinetic modeling analysis ……………………………………105
Table 3.5
Evaluation of the sensitivity of cell and medium
accumulation to detect changes in biliary excretion
of TS or TG utilizing data from Monte Carlo simulations…………….106
viii
LIST OF FIGURES
Figure 2.1
Model scheme depicting the hepatic disposition of
APAP, AG and AS in the single-pass perfused mouse liver………….72
Figure 2.2
APAP concentrations (nM) in outflow perfusate from
male wild-type (●) and Bcrp-deficient (○), and female
wild-type (▼) and Bcrp-deficient (∆) mouse liver perfusions………….73
Figure 2.3
Cumulative biliary excretion and basolateral excretion
of AG (A and B, respectively) in male and
female wild-type and Bcrp-deficient perfused mouse livers…………..74
Figure 2.4
Cumulative biliary excretion and basolateral excretion
of AS (A and B, respectively) in male and
female wild-type and Bcrp-deficient perfused mouse livers…………..75
Figure 2.5
Representative immunoblot of Bcrp, Mrp4 and actin in
crude membrane fractions from male and female wild-type
and Bcrp-deficient mouse liver homogenates…..………………………76
Figure 2.6
Representative outflow perfusate rate of AG (●) and AS (■),
and biliary excretion rate of AG (○) and AS (□) in male and
female wild-type and Bcrp-deficient mouse livers……………………...77
Figure 3.1
Model scheme depicting the mass of TGZ, TGZ sulfate (TS),
TGZ glucuronide (TG), and TGZ quinone (TQ)
in the medium, hepatocytes, and bile of rat SCH……………………..107
Figure 3.2
Accumulation of TGZ, TS, and TG in rat SCH…………………..……108
Figure 3.3
Mass vs. time profiles of TGZ (●), TS (○),
TG (▼) and TQ (∆) in the medium……………………………………..109
Figure 3.4
Mass vs. time profiles of TGZ (●), TS (○)
and TG (▼) in the hepatocytes in day 4 rat SCH…………………….110
Figure 3.5
Mass vs. time profiles of TS (○) and TG (▼)
in the bile canaliculi of day 4 rat SCH………………………………….111
Figure 3.6
Monte Carlo simulations with modulation of TS or TG
biliary excretion………….………………………………………………..112
Figure 4.1
Effect of DEX on trabectedin-mediated cytotoxicity
in SCRH isolated from wild-type Wistar rats…………………………..136
ix
Figure 4.2
Trabectedin-mediated cytotoxicity in SCRH
from wild-type and TR- rats……………………………………………...137
Figure 4.3
Effect of BSO on trabectedin-mediated cytotoxicity…………………..138
Figure 4.4
Representative Western blot from whole cell lysates for
Mrp2, 3, 4 and CYP3A1/2 in SCRH pretreated with vehicle
(0.1 µM DEX), additional DEX (1 µM) or BSO (500 µM)..…………...139
Figure 4.5
Trabectedin perfusate concentrations (A) and cumulative
biliary excretion (B) in isolated perfused livers from
wild-type (solid circle) and TR- (white triangle) rats…………..…...….140
Figure 5.1
Chemical structures of sulindac, sulindac
sulfone (S-sulfone) and sulindac sulfide (S-sulfide)…………….......165
Figure 5.2
Effect of sulindac and metabolites on
hepatobiliary disposition of TC in rat SCH………………………......166
Figure 5.3
Effect of sulindac and metabolites on
hepatobiliary disposition of E217G in rat SCH………………………167
Figure 5.4
Effect of sulindac and metabolites on
hepatobiliary disposition of NF in rat SCH…………………………..168
Figure 5.5
Inhibition of Na+-dependent TC initial uptake in suspended
rat hepatocytes by S-sulfone and S-sulfide ………...……………….169
Figure 5.6
Inhibition of Na+-independent E217G initial uptake in
suspended rat hepatocytes by S-sulfone and S-sulfide……….…...170
Figure 5.7
Effect of sulindac and metabolites on
hepatobiliary disposition of TC in human SCH………………………171
Figure 5.8
Inhibition of Na+-dependent TC initial uptake in
suspended human hepatocytes by S-sulfone and S-sulfide ....…...172
Figure A.1
Representative Western blot of MRP3 and MRP4 in MRP3and MRP4- overexpressing membrane vesicle preparations….……216
Figure A.2
Functional activity of MRP3- or MRP4-overexpressing
membrane vesicles………………………………………………………217
x
Figure A.3
Inhibitory effect of GW918 and MK571 on MRP3and MRP4-mediated [3H]E217G and [3H]MTX uptake
in the absence of GSH.......................................................................228
Figure A.4
Inhibitory effect of GW918 and MK571 on MRP3and MRP4-mediated [3H]E217G and [3H]MTX uptake
in the presence of GSH (3 mM)………………………………….....…..229
xi
LIST OF ABBREVIATIONS
ABC
ATP binding cassette
AG
acetaminophen glucuronide
ALP
alkaline phosphatase
ALT
alanine aminotransferase
APAP
acetaminophen
AS
acetaminophen sulfate
BCRP
breast cancer resistance protein
BEI
biliary excretion index
BSEP
bile salt export pump
BSO
buthionine sulfoximine
CDF
5 (and 6)-carboxy-2',7'-dichlorofluorescein
CYP
cytochrome P450
DEX
dexamethasone
DHEAS
dehydroepiandrosterone sulfate
DILI
drug-induced liver injury
DMEM
dulbecco’s modified eagle’s medium
DPDPE
[D-Pen2, D-Pen5]-enkephalin
E217G
estradiol-17-β-glucuronide
EHBR
eisai hyperbilirubinemic rats
GSH
reduced glutathione
GST
glutathione-S-transferase
xii
HBSS
hanks’ balanced salts solution
LDH
lactate dehydrogenase
MRP
multidrug resistance-associated protein
MTX
methotrexate
NF
nitrofurantoin
NSAID
nonsteroidal anti-inflammatory drug
NTCP
sodium/taurocholate cotransporting polypeptide
OATP
organic anion transporting polypeptide
OST
organic solute transporter
P-gp
P-glycoprotein
SCH
sandwich-cultured primary hepatocytes
S-sulfide
sulindac sulfide
S-sulfone
sulindac sulfone
SULT
sulfotransferase
TC
taurocholate
TGZ
troglitazone
TG
troglitazone glucuronide
TS
troglitazone sulfate
TQ
trolitazone quinone
UGT
uridine diphosphoglucuronosyl transferase.
xiii
CHAPTER 1
Introduction
This work has been published in part in Drug Transport in Liver. You, G., and Morris,
M.E. (eds), In: Drug Transporters. Wiley Publishers, 2007: 359-410.
Transport of xenobiotics by the liver is supported by the polarized nature of
hepatocytes; the basolateral (sinusoidal and lateral) membrane domain represents
the interface with sinusoidal blood, while the canalicular (apical) membrane domain
faces the bile duct lumen. Basolateral transport proteins mediate the movement of
compounds to and from the blood, while canalicular transport proteins mediate flux
of compounds from hepatocytes to bile.
Nomenclature for the human hepatic
transport proteins, example substrates and relevant references are provided in Table
1.1 (uptake transport proteins) and Table 1.2 (efflux transport proteins).
Hepatic Uptake Transport Proteins
The
Na+-taurocholate
cotransporting
polypeptide
[NTCP,
(SLC10A1)]
mediates the basolateral uptake of both conjugated and unconjugated bile acids in a
sodium-dependent manner with a 2:1 sodium-to-taurocholate stoichiometry. An
inwardly directed Na+ gradient maintained by the activity of Na+/K+ ATPase serves
as the driving force for NTCP (Meier and Stieger, 2002). Besides serving as the
primary uptake mechanism for conjugated and unconjugated bile acids, NTCP also
transports sulfated bile acids (e.g. chenodeoxycholate-3-sulfate, taurolithocholate-3sulfate) and steroid sulfates, as well as certain drugs (e.g. rosuvastatin,
cholorambucil-taurocholate) (Trauner and Boyer, 2003; Ho et al., 2006). Ho et al.
recently demonstrated ethnicity-dependent polymorphisms in the human NTCP gene
resulting in decreased transport function of bile acid (Ho et al., 2004).
The family of organic anion transporting polypeptides [OATPs, (SLCO)] is
responsible for the sodium independent uptake of a large variety of amphipathic
2
compounds including bile acids, hormones, eicosanoids, and various drugs
(Hagenbuch and Meier, 2004). Although a number of studies have revealed the
substrate specificity of OATPs, the driving force behind OATP-mediated transport
remains controversial. Bi-directional transport of substrates and anions (e.g. GSH
and HCO3-) has been demonstrated for Oatp1a1 (Li et al., 1998; Li et al., 2000). In
contrast, Briz et al. demonstrated cis-stimulation of taurocholate uptake by GSH in
oocytes expressing OATP1B3, suggesting co-transport of GSH and bile acids (Briz
et al., 2006). Mahagita et al. proposed that OATP1B3 functions as a bidirectional
facilitated diffusion transporter rather than a bidirectional exchanger or cotransporter
(Mahagita et al., 2007). Because some rodent Oatps are not orthologs of human
OATP proteins, the prediction of human OATP-mediated transport from animal
models is challenging (Hagenbuch and Meier, 2003). Among 11 human OATP
isoforms identified, the human liver expresses OATP1A2 (previously named OATPA), OATP1B1 (previously named OATP-C), OATP1B3 (previously named OATP-8),
and OATP2B1 (previously named OATP-B). OATP1A2 and OATP2B1 are widely
expressed in several tissues, whereas OATP1B1 and OATP1B3 are selectively
expressed in the liver (Konig et al., 2000a; Konig et al., 2000b). Despite distinct
substrate affinity profiles, there are overlapping substrates for OATP1A2, 1B1 and
1B3, such as bile acids, bromosulfophthalein (BSP), dehydroepiandrosterone sulfate
(DHEAS) and estrone-3-sulfate. As detailed in Table 1.1, the substrate specificity for
OATP2B1 is selective, while the other three liver-expressed OATPs transport
diverse compounds including organic anions, bulky cations, and neutral steroids.
3
In addition to NTCP and OATPs, organic anion transporter 2 [OAT2,
(SLC22A7)] and organic cation transporter 1 and 3 [OCT, (SLC22A1 and SLC22A3,
respectively)] also are expressed in the human liver and function as ion exchangers
(Sun et al., 2001; Choi and Song, 2008). OAT2 is responsible for the hepatic uptake
of negatively charged compounds such as methotrexate, zidovudine and taxol.
While OATPs mediate transport of bulky type II cations, small cations (previously
type I) are transported by OCTs. OCT1, which is expressed exclusively in liver,
mediates transport of several therapeutic drugs including imatinib, metformin, and
famotidine, whereas OCT3 is selective towards endogenous substrates (Table 1.1).
Canalicular Efflux Transport Proteins
All canalicular xenobiotic transport proteins, except the recently-identified
MATE1 organic cation-H+ exchanger (Terada and Inui, 2008), belong to the ATPbinding cassette (ABC) family of proteins which mediate ATP-dependent transfer of
solutes. The canalicular efflux transport system includes the multidrug resistance
protein 1 [MDR1, (ABCB1)], MDR 3 (ABCB4), the bile salt export pump [BSEP,
(ABCB11)], the multidrug resistance associated protein 2 [MRP2, (ABCC2)] and the
ABC half-transporter protein breast cancer resistance protein [BCRP, (ABCG2)].
MDR1 (ABCB 1), generally referred to as P-glycoprotein, was first identified
over two decades ago in multidrug resistant (MDR) tumor cells (Juliano and Ling,
1976); MDR1 represents the most widely studied ABC transport protein. MDR1 is
responsible for biliary excretion of bulky hydrophobic and cationic substrates such as
chemotherapeutic agents (e.g., daunorubicin, doxorubicin, etoposide, paclitaxel,
4
vinblastine, vincristine), cardiac glycosides (e.g., digoxin), rhodamine 123,
cyclosporine A, and HIV-1 protease inhibitors (e.g., amprenavir, inidnavir, nelfinavir,
ritonavir, saquinavir). MDR1 modulators have been evaluated for their ability to
increase drug sensitivity during chemotherapy because overexpression of MDR1 on
the surface of tumor cells causes multidrug resistance (Honma et al., 2002). MDR3
(ABCB4) is a phospholipid flippase and is involved in the biliary secretion of
phospholipids and cholephilic compounds. Thus, MDR3 plays a crucial role in basic
liver physiology in human and rats because phospholipids and cholesterol are
responsible for the formation of micelles that solubilize bile acids in the lumen of the
bile canaliculus (Carrella and Roda, 1999). Mutations in the MDR3 gene lead to
progressive familial intrahepatic cholestasis type 3 (PFIC3), a disease that is
characterized by increased γ-glutamyltranspeptidase levels, ductular proliferation,
and inflammatory infiltrate which can progress to biliary cirrhosis. MDR3 is reported
to have substrate specificity similar to MDR1, but the rate of transport of substrate is
lower than for MDR1 (Evers et al., 2000).
The bile salt export pump [BSEP, (ABCB11)] is the predominant bile acid
efflux transport protein, and mediates excretion of conjugated and unconjugated bile
acids into bile. It was reported that BSEP might also participate in transport of nonbile acid substrates such as pravastatin (Kullak-Ublick et al., 2000; Hirano et al.,
2005b). Patients with progressive familial intrahepatic cholestasis type 2 (PFIC2)
have a mutation in the ABCB11 gene and absence of BSEP protein expression,
which leads to hepatocellular injury and necrosis caused by the increased
intracellular concentration of detergent-like bile acids (Kullak-Ublick et al., 2004).
5
Inhibition of BSEP by several drugs such as troglitazone, bosentan, cyclosporine A
and rifampicin has been suggested as one mechanism of drug-induced liver injury
(Fattinger et al., 2001; Funk et al., 2001a; Mita et al., 2006).
MRP2 (ABCC2) is a major xenobiotic efflux pump at the canalicular
membrane.
MRP2 plays a key role in the biliary excretion of organic anions
including bilirubin-diglucuronide, glutathione conjugates, sulfated bile acids, and
divalent bile acid conjugates as well as numerous drugs such as sulfopyrazone,
indomethacin, penicillin, vinblastine, methotrexate and telmisartan (Table 1.2).
Patients with Dubin-Jonson syndrome suffer from defective hepatic biliary excretion
due to absence of MRP2. Animals with hereditary conjugated hyperbilirubinemia
(Mrp2-deficient rats (GY/TR(-)) and Eisai hyperbilirubinemic rats (EHBR)) exhibit
elevated MRP3 expression. This phenomenon has been explained as a
compensatory mechanism to extrude MRP2 substrates into the systemic circulation
under cholestatic conditions in order to reduce the burden on the liver (Konig et al.,
1999b; Kuroda et al., 2004).
Breast cancer resistance protein [BCRP, (ABCG2)] is highly expressed in the
canalicular membrane of the liver as well as in the intestine, breast, and placenta.
BCRP is a member of the ABC half-transporter protein family and forms a functional
homodimer. It mediates transport of estrone-3-sulfate and various sulfated steroid
compounds. It is also known to be involved in development of resistance to a variety
of anticancer agents such as SN-38, mitoxantrone, topotecan and doxorubicin.
Coadministration of topotecan in combination with GF120918, a potent BCRP and
MDR1 inhibitor, significantly increased topotecan oral bioavailability in mice,
6
probably due to the inhibition of biliary clearance and enhanced intestinal absorption
(Kruijtzer et al., 2002).
Recently, several mammalian orthologues of the bacterial multidrug and
toxin extrusion (MATE)-type transporters have been identified. In humans, the
isoform MATE1 is localized to the bile canalicular membrane in the liver (Otsuka et
al., 2005b). The driving force for organic cation extrusion by MATE1 is the inwardly
directed H+ gradient (Otsuka et al., 2005b). Several organic cations (e.g. metformin,
cimetidine and tetraethyl ammonium) have been identified as substrates for MATE1
(Table 1.2). The role of MATE1 in hepatobiliary disposition of drugs remains to be
elucidated.
Basolateral Efflux Transport Proteins
The organic anion transporting polypeptides [OATPs, (SLCO)] represent the
major family of basolateral uptake transport proteins for organic anions and have
been hypothesized to also function as basolateral efflux transport proteins under
certain conditions.
However, the in vivo role of these transport proteins for
basolateral egress of drugs from the hepatocyte warrants further studies (Durr et al.,
2000).
The ATP-dependent multidrug resistance-associated protein [MRP, (ABCC)]
subfamily is a major class of efflux transport proteins on the hepatic basolateral
membrane. MRP1, 3, 5, and 6 are involved in cellular transport of both hydrophobic
uncharged molecules and hydrophilic anionic compounds.
MRP1 (ABCC1) is
responsible for efflux of various organic anions including glucuronide-, glutathione-,
and sulfate-conjugated drugs. The expression of MRP1 (ABCC1) is very low on the
7
basolateral membrane in healthy liver, but MRP1 is highly inducible during severe
liver injury and this induction is believed to play a role in liver protection (Wilson et al.,
2003).
MRP3 (ABCC3) is involved in the hepatic excretion of glucuronide
conjugates (e.g., acetaminophen glucuronide), methotrexate, and estradiol-17βglucuronide (E217G). The expression level of MRP3 is markedly increased under
cholestatic conditions (Hirohashi et al., 1999). MRP4 (ABCC4) and MRP5 (ABCC5)
transport cyclic nucleotides such as cAMP and cGMP, as well as the purine analogs
6-mercaptopurine and 6-thioguanine (Zhou et al., 2001). MRP4 has been implicated
in the transport of nucleoside-like drugs such as zidovudine, lamivudine, and
stavudine, as well as non-nucleotide substrates (e.g., methotrexate), and the reverse
transcriptase inhibitor azidothymidine. MRP4 also is involved in the transport of
sulfate conjugates of bile acids and steroids (Zelcer et al., 2003).
While the
expression level of MRP5 in healthy liver is relatively low, treatment with
lipopolysaccharide (LPS) results in down-regulation of MRP2 and induction of MRP5,
suggesting that MRP5 may participate in the hepatic response to cholestasis
(Donner et al., 2004).
MRP6 is localized at the lateral side and canalicular
membrane. MRP6 transports glutathione conjugates and the endothelin receptor
antagonist, BQ-123 (Madon et al., 2000).
Mutation of the MRP6 gene causes
pseudoxanthoma elasticum (PXE), an inheritable systemic connective tissue
disorder affecting the skin, eyes and blood vessels (Perdu and Germain, 2001).
Clarification of the role of MRP6 in canalicular membrane transport awaits further
investigation.
8
Table 1.1: Human Hepatic Uptake Transport Proteins
Protein/
Gene symbol
NTCP/
SLC10A1
OATP1A2/
SLCO1A2
OATP1B1/
SLCO1B1
Substrates
References
BSP;
cholate;
estrone-3-sulfate;
glycocholate;
taurochenodeoxycholate;
tauroursodeoxycholate;
taurocholate
Rosuvastatin
Bile acids; BQ-123; Bromosulfophthalein (BSP);DHEAS;
2,5
[D-penicillamine ]enkephalin (DPDPE); Estradiol-17βglucuronide (E217G); estrone-3-sulfate; n-methyl quinine;
ouabain; prostaglandin E2; thyroid hormones (T3, T4)
Deltorphin II
Fexofenadine
Levofloxacin
Methotrexate
Microcystin-LR
Pitavastatin
Saquinavir
Arsenic (arsenite, arsenate)
Atrasentan
Atorvastatin; Simvastatin
(Meier et al., 1997)
Benzylpenicillin
Bile acids; BQ-123; BSP; DHEAS; DPDPE; E217G;
estrone-3-sulfate;
T3,
T4;leukotriene
C4(LTC4);
prostaglandin E2
Bilirubin, bilirubin glucuronides
Bosentan
Caspofungin
Cerivastatin; Pravastatin
Fluvastatin
Irinotecan metabolite (SN-38)
Methotrexate
Microcystin-LR
Olmesartan
OATP1B3/
SLCO1B3
Pitavastatin
Pravastatin
Repaglinide
Rifampin
Rosuvastatin
Troglitazone-sulfate
Valsartan
Amanitin
Bile acids; BQ-123; BSP; Deltorphin-II; DHEAS; digoxin;
DPDPE; E217G; estrone-3-sulfate; LTC4; ouabain; T3;
T4
Bosentan
CCK-8
Digoxin
Docetaxel
Enalapril
Fexofenadine
9
(Ho et al., 2006)
(Kullak-Ublick et al., 2001)
(Gao et al., 2000)
(Cvetkovic et al., 1999)
(Maeda et al., 2007)
(Badagnani et al., 2006)
(Fischer et al., 2005)
(Fujino et al., 2005)
(Su et al., 2004)
(Lu et al., 2006)
(Katz et al., 2006)
(Kameyama et al., 2005;
Lau et al., 2006)
(Tamai et al., 2000)
(Kullak-Ublick et al., 2001)
(Cui et al., 2001)
(Treiber et al., 2007)
(Sandhu et al., 2005)
(Kameyama et al., 2005)
(Kopplow et al., 2005)
(Nozawa et al., 2004b)
(Abe et al., 1999)
(Fischer et al., 2005)
(Nakagomi-Hagihara et al.,
2006)
(Fujino et al., 2004)
(Hsiang et al., 1999)
(Kajosaari et al., 2005)
(Vavricka et al., 2002)
(Ho et al., 2006)
(Nozawa et al., 2004b)
(Yamashiro et al., 2006)
(Letschert et al., 2006)
(Kullak-Ublick et al., 2001)
(Treiber et al., 2007)
(Ismair et al., 2001)
(Kullak-Ublick et al., 2001)
(Smith et al., 2005a)
(Liu et al., 2006)
(Shimizu et al., 2006)
(Kopplow et al., 2005)
Fluvastatin
Methotrexate
Microcystin-LR
Monoglucuronosyl bilirubin
Olmesartan
OATP2B1/
SLCO2B1
OAT2/
SLC22A7
OCT1/
SLC22A1
OCT3/
SLC22A3
Paclitaxel
Pitavastatin
Rifampin
Rosuvastatin
Telmisartan
Valsartan
Atorvastatin
Benzylpenicillin
Bosentan
BSP; DHEAS; estrone-3-sulfate
Glibenclamide
Fluvastatin
Pravastatin
Pitavastatin
Rosuvastatin
5-Fluorouracil; Allopurinol; L-ascorbic acid; Bumetanide;
DHEAS; estrone-3-sulfate; glutarate; prostaglandin E2
Erythromycin; theophylline
Methotrexate
Prostaglandin F2α
Ranitidine
Tetracycline
Zidovudine
Acyclovir, ganciclovir
Azidoprocainamide methoiodide; n-methyl-quinidine; nmethyl-quinine; tributylmethylammonium
Choline
Imatinib
Metformin
Famotidine, ranitidine
Prostaglandin E2, Prostaglandin F2α
Adrenaline, noradrenaline, tyramine
+
Agmatine, MPP ; tetraethylammonium
Atropine, etilefrine
Cimetidine
(Abe et al., 2001)
(Fischer et al., 2005)
(Cui et al., 2001)
(Nakagomi-Hagihara et al.,
2006)
(Smith et al., 2005a)
(Fujino et al., 2004)
(Vavricka et al., 2002)
(Ho et al., 2006)
(Ishiguro et al., 2006)
(Yamashiro et al., 2006)
(Grube et al., 2006)
(Tamai et al., 2000)
(Treiber et al., 2007)
(Kullak-Ublick et al., 2001)
(Satoh et al., 2005)
(Kopplow et al., 2005)
(Nozawa et al., 2004a)
(Hirano et al., 2006)
(Ho et al., 2006)
(Kobayashi et al., 2005a)
(Kobayashi et al., 2005b)
(Sun et al., 2001)
(Enomoto et al., 2002)
(Tahara et al., 2005)
(Babu et al., 2002)
(Takeda et al., 2002)
(Takeda et al., 2002)
(van Montfoort et al., 2001)
(Grundemann et al., 1999)
(Thomas et al., 2004)
(Kimura et al., 2005)
(Bourdet et al., 2005)
(Kimura et al., 2002)
(Grundemann et al., 1998)
(Hayer-Zillgen et al., 2002)
(Muller et al., 2005)
(Grundemann et al., 1999)
Table 1.2: Human Hepatic Efflux Transport Proteins
Protein/
Gene symbol
MRP1/
ABCC1
MRP3/
ABCC3
Substrates
References
Daunorubicin; Doxorubicin; etoposide; vindcristine
Glutathione
Methotrexate (MTX)
Acetaminophen glucuronide
E217G; monovalent and sulfated bile salts; MTX
(Cole et al., 1994)
(Madon et al., 2000)
(Zeng et al., 2001)
(Xiong et al., 2000)
(Hirohashi et al., 1999; Zeng
et al., 2001)
(Madon et al., 2000)
Etoposide
10
MRP4/
ABCC4
MRP5/
ABCC5
MRP6/
ABCC6
BSEP/
ABCB11
MRP2/
ABCC2
MDR1/
ABCB1
Fexofenadine
(Matsushima et al., 2008)
Azidothymidine
cAMP; cGMP; PMEA
(Schuetz et al., 1999)
(Jedlitschky et al., 2000;
Chen et al., 2001; Reid et
al., 2003a)
(Chen et al., 2002)
(Reid et al., 2003a)
(Jedlitschky et al., 2000)
(Reid et al., 2003a)
(Madon et al., 2000)
MTX
Prostaglandin E1; Prostaglandin E2
cAMP; cGMP
Adefovir
BQ-123
Verapamil; Norverapamil
Phospholipids
(Gerloff et al., 1998)
(Matsushima et al., 2008)
(Hirano et al., 2005a)
(Xiong et al., 2000)
(Koike et al., 1997)
(Hirano et al., 2005b)
(Kawabe et al., 1999)
(Konig et al., 1999b)
(Payen et al., 2000)
(Konig et al., 1999b)
(Hooijberg et al., 1999)
(Hirano et al., 2005b)
(Sasaki et al., 2002)
(Kitamura et al., 2008)
(Kim et al., 1998; Polli et al.,
1999)
(Bogman et al., 2001)
(Ueda et al., 1992)
(Marie et al., 1992)
(Kim et al., 1999)
(Fromm et al., 1999)
(van der Sandt et al., 2000;
Advani et al., 2001)
(Lum et al., 1993)
(Cvetkovic et al., 1999)
(Yamaguchi et al., 2000)
(Soldner et al., 1999)
(Hirano et al., 2005b)
(Kitamura et al., 2008)
(Floren et al., 1997)
(Spahn-Langguth et al.,
1998)
(Pauli-Magnus et al., 2000)
(Evers et al., 2000)
Albendazole sulfoxide; Oxfendazole
Daunorubicin; doxorubicin; MX; sulfated conjugates
Ciprofloxacin; Ofloxacin; Norfloxacin
Cerivastatin
Dirithromycin; Erythromycin; Rifampicin
Genistein
Epirubicin
Irinotecan; Topotecan
Imatinib
(Merino et al., 2005a)
(Doyle et al., 1998)
(Merino et al., 2006)
(Matsushima et al., 2005)
(Janvilisri et al., 2005)
(Imai et al., 2004)
(Robey et al., 2003)
(Maliepaard et al., 2001)
(Burger et al., 2004)
Conjugated and unconjugated bile salts; TC
Fexofenadine
Pravastatin
Acetaminophen glucuronide; carboxydichlorofluorescein
Camptothecin; doxorubicin
Cerivastatin; estron-3-sulfate
Cisplatin; vincristine
Etoposide
Glibenclamide; indomethacin; rifampin
Glucuronide, glutathione, and sulfate conjugates; LTC4
MTX
Pitavastatin
Pravastatin
Rosuvastatin
Amprenavir; indinavir; nelfinavir; ritonavir; saquinavir
Atorvastatin
Aldosterone; corticosterone; dexamethasone; digoxin
Cyclosporin A; mitoxantrone (MX)
Debrisoquine; erythromycin; lovastatin; terfenadine
Digoxin; quinidine
Doxorubicin; paclitaxel; rhodamine 123
Etoposide
Fexofenadine
Levofloxacin; Grapafloxacin
Losartan; vinblastine
Pitavastatin
Rosuvastatin
Tacrolimus
Talinolol
MDR3/
ABCB4
BCRP/
ABCG2
11
Lamivudine
Methotrexate
Nitrofurantoin
Pitavastatin
Prazosin; rhodamine 123
Rosuvastatin
Sulfasalazine
MATE1/
SLC47A1
SN-38; SN-38 glucuronide
Testosterone; tamoxifen; estradiol
Zidovudine
Tetraethylammonium (TEA)
Cimetidine; metformin; procainamide; topotecan;
(Wang et al., 2003)
(Volk et al., 2002)
(Wang and Morris, 2007)
(Hirano et al., 2005b)
(Ozvegy et al., 2001)
(Kitamura et al., 2008)
(van der Heijden et al.,
2004)
(Yoshikawa et al., 2004)
(Janvilisri et al., 2003)
(Wang et al., 2004)
(Otsuka et al., 2005a)
(Tanihara et al., 2007)
Potential Role of Hepatic Transport Proteins in Drug-Induced Liver Injury
Drug-induced liver injury has emerged as the most common reason for the
withdrawal of FDA-approved drugs from the market (FDA, 2000).
Drug-induced
hepatotoxicity may be classified as intrinsic or idiosyncratic. Intrinsic hepatotoxicity
is generally predictable, dose-dependent, and characteristic for a particular drug. In
contrast, idiosyncratic toxicity is unpredictable, not dose-related, and displays no
clear underlying mechanism.
Drug-induced liver injury can be evaluated by
measuring the activity of alanine aminotransferase (ALT) and alkaline phosphatase
(ALP) in the plasma (Benichou, 1990); liver injury is designated hepatocellular injury
if the ALT level is greater than 2 times the upper limit of normal (ULN), or if a ratio of
ALT/ULN and ALP/ULN is greater than 5; cholestatic injury can be characterized by
elevations in ALP greater than 2 times the ULN or by a ratio of ALT/ULN and
ALP/ULN that is less than 2; mixed patterns of liver injury are defined as a ratio of
ALT/ULN and ALP/ULN that is greater than 2 but less than 5.
Age, gender, genetic and environmental factors may affect susceptibility to
drug-induced liver injury. Early studies focused on bioactivation, covalent adduct
formation, immunotoxicity, and disruptions in cellular bioenergetics as mechanisms
12
underlying hepatotoxicity, but more recent data suggest that alterations in hepatic
transport may be an important mechanism of toxic endpoints. Inhibition of
canalicular Bsep has been reported as one mechanism of drug-induced liver injury
because impaired Bsep function leads to elevated hepatic concentrations of
detergent-like bile acids, which can disrupt key membrane-associated cellular
functions (Spivey et al., 1993; Funk et al., 2001a). Troglitazone (Rezulin) was the
first thiazolidinedione approved for the treatment of type 2 diabetes prior to its
withdrawal from the market in 2000 due to idiosyncratic liver injury (Herrine and
Choudhary,
1999).
Several
mechanisms
underlying
troglitazone-induced
hepatotoxicity have been proposed (Smith, 2003), including Bsep inhibition by both
troglitazone (KI, apparent = 1.3 µM) and its primary metabolite, troglitazone sulfate (KI,
apparent
= 0.23 µM) (Funk et al., 2001b).
Troglitazone causes intrahepatic
accumulation of bile acids, which can lead to hepatocellular necrosis and severe
liver damage (Gores et al., 1998).
Following troglitazone administration, Mrp2-
deficient rats exhibited a marked delay in the biliary excretion and enhanced urinary
excretion of troglitazone sulfate leading to elevated serum bile acid concentrations
relative to wild-type rats (Kostrubsky et al., 2001). Although troglitazone has been
one of the most studied examples of hepatotoxicity resulting from Bsep inhibition,
other drugs, including bosentan, glibenclamide, cyclosporin, and rifampin also impair
Bsep function (Stieger et al., 2000; Fattinger et al., 2001). In addition to cis-inhibition
of Bsep, estradiol-17-β-glucuronide trans-inhibits Bsep following biliary excretion by
Mrp2 (Stieger et al., 2000). In the absence of Mrp2, estradiol-17-β-glucuronideinduced cholestasis is not observed (Sano et al., 1993; Stieger et al., 2000).
13
Hepatic transport proteins may provide a protective role against drug-induced
hepatotoxicity through changes in transporter expression (Ananthanarayanan et al.,
1994). Intrahepatic and obstructive cholestasis in humans and rats result in downregulation of Oatp/Ntcp isoforms and up-regulation of Mrp isoforms (Gartung et al.,
1996; Donner and Keppler, 2001; Wagner et al., 2003). In patients with hepatitis or
chronic cholestasis, up-regulation of major hepatic efflux transporters such as MDR1,
MRP1 and MRP3 have been reported (Wilson et al., 2003). Similarly, during liver
injury caused by acetaminophen and carbon tetrachloride which are both well-known
hepatic toxicants, expression of hepatic transport proteins in mice was modulated
(Aleksunes et al., 2005; Moffit et al., 2006): Mrp2 protein expression was slightly
increased; Mrp3 and Mrp4 expression was increased by 3.6-fold and 16-fold,
respectively; Oatps and Ntcp were down-regulated. Collectively, hepatocytes may
prevent accumulation of potentially toxic chemicals and protect against subsequent
liver injury by limiting influx and enabling efficient efflux of harmful toxicants following
down-regulation of uptake transporters and up-regulation of efflux transporters.
In certain cases, impaired hepatic transport may provide protection against
drug-induced liver injury.
Many electrophiles that form covalent adducts with
intracellular macromolecules are detoxified by conjugation with glutathione. Mrp2deficient rats do not excrete glutathione into bile, resulting in a ~2-3-fold increase in
hepatic glutathione concentrations (Dietrich et al., 2001).
Acetaminophen, a
commonly used antipyretic and analgesic agent, induces severe liver injury at high
doses when acetaminophen sulfation and glucuronidation become saturated and
oxidation to N-acetyl-p-benzoquinoneimine, a potent electrophile, becomes a
14
significant
metabolic
pathway
(Galinsky
and
Levy,
1981).
N-acetyl-p-
benzoquinoneimine may be detoxified by conjugation with glutathione, but as this
co-factor becomes depleted, the electrophilic metabolite forms covalent adducts,
which can lead to cellular necrosis (Dahlin et al., 1984). Administration of toxic
acetaminophen doses (1 g/kg) induced the expected hepatotoxicity in wild-type rats,
but had no apparent adverse effects in Mrp2-deficient rat livers (Silva et al., 2005).
Elevated glutathione concentrations in Mrp2-deficient rats appear to play a
protective role in acetaminophen-induced hepatotoxicity; a similar effect also would
be expected for other electrophiles in Mrp2-deficient rats (Silva et al., 2005).
In this dissertation project, the role of hepatic transport proteins in druginduced liver injury was assessed using troglitazone, trabectedin, and sulindac as
model hepatotoxic compounds. As mentioned above, Bsep inhibition by troglitazone
and troglitazone sulfate has been proposed as one mechanism for troglitazonemediated liver injury. In rats and humans, troglitazone is extensively metabolized to
troglitazone sulfate (TS) which is transported by BCRP (Enokizono et al., 2007).
Patients who have impaired functional activity of BCRP (e.g. due to a polymorphism
or drug-drug interaction) may have increased potential for troglitazone-mediated
hepatotoxicity due to higher accumulation of TS in the liver. Trabectedin is a
promising anticancer agent which is currently under phase II evaluation (Schoffski et
al., 2008). During phase I/II clinical trials, the most prevalent adverse effect was
hepatotoxicity with elevated ALP and/or bilirubin levels (Beumer et al., 2005b). Little
is known about the metabolic profiles of trabectedin in rats and humans, but multiple
cytochrome P450 enzymes (3A4, 2C9, 2C19, 2D6, and 2E1) appear to be involved
15
in trabectedin metabolism. Sulindac, a nonsteroidal anti-inflammatory drug (NSAID),
has been consistently associated with liver injury among NSAIDs (Aithal and Day,
2007). Sulindac is metabolized to sulindac sulfide, which has anti-inflammatory
properties, and is also transformed to sulindac sulfone, which has antiproliferative
effects against colon cancer (Davies and Watson, 1997). Several investigators have
demonstrated a linear correlation between sulindac sulfide concentrations and
hepatotoxicity (Duggan et al., 1978; Laffi et al., 1986). Although hepatocellular and
mixed patterns of sulindac-mediated hepatotoxicity have been reported, cholestatic
patterns of liver injury were most commonly observed (Tarazi et al., 1993).
Modulation of Hepatic Transport Proteins by Drugs and Patient-Specific Factors
As discussed above, hepatic transport proteins play an important role in the
uptake and excretion of endogenous and exogenous compounds. Altered hepatic
transport function (e.g., inhibition, induction, or altered translocation) may have a
significant impact on the hepatobiliary disposition of drugs.
Inhibition of cytochrome P450 (CYP) enzymes is an important mechanism of
many toxic drug interactions, but accumulating data suggest that some interactions
may be mediated, at least in part, by transport proteins. Numerous transport
interactions occur at the level of hepatic uptake. Cyclosporine A increased the AUC
of atovastatin, lovastatin and simvastatin ~7.4-fold, 5-fold and ~2.5-fold, respectively;
these statins are transported into the hepatocyte by OATPs, and are cleared by
CYP3A4-mediated metabolism and P-gp-mediated biliary excretion (Shitara and
Sugiyama, 2006). The observed interaction between some statins (atorvastatin,
16
simvastatin, and lovastatin) and cyclosporine A may be caused by the combined
effects on drug metabolizing enzymes and transport proteins; cyclosporine A not
only inhibits CYP3A4-mediated metabolism, but also affects OATP-mediated hepatic
uptake and P-gp-mediated biliary excretion. Cyclosporine A also increased the AUC
of rosuvastatin ~7.1-fold (Simonson et al., 2004). This interaction may be due to
impaired OATP-mediated hepatic uptake of rosuvastatin, since rosuvastain is neither
a P-gp nor a CYP3A4 substrate or inhibitor (Martin et al., 2002; Cooper et al., 2003),.
The interaction between atorvastatin or rosuvastatin and cyclosporine A is consistent
with pharmacokinetic alterations of these statins observed in subjects with genetic
polymorphisms in OATP1B1; the AUC of atorvastatin and rosuvastatin in subjects
with the SLCO1B1 c.521CC genotype was 144% and 65% higher than in subjects
with the SLCO1B1 c.521TT genotype (Pasanen et al., 2007).
Undoubtedly, interactions in biliary excretion or basolateral efflux also occur.
Although there is accumulating evidence of potential drug-drug interactions
mediated by inhibition of these hepatic efflux transport proteins based on in vitro
screening, clinical trials examining hepatic efflux transporter-mediated drug
interactions often demonstrate an undetectable or weak interaction in humans.
Impaired function of hepatic efflux transport proteins may increase hepatocelluar
exposure without affecting the plasma pharmacokinetics. Drug interactions at the
level of hepatic efflux can be assessed in human only via imaging techniques or
quantifying biliary clearance. Alternatively, the application of a modeling/simulation
approach coupled with in vitro data may address these issues as demonstrated in
Chapter 3 based on Monte Carlo simulations.
17
In addition to acute inhibition, long term administration of xenobiotics can
modulate
transport
function
by
affecting
transcriptional,
translational
and
posttranslational events (Hong and You, 2007). The transcriptional regulation of
transporter gene expression in hepatocytes is mediated by a large variety of orphan
nuclear receptors [e.g., pregnane X receptor (PXR), liver X receptor (LXR),
constitutive androstane receptor (CAR) and farnesoid X receptor (FXR)] coupled
with a heterodimeric partner (e.g., retinoic acid X receptor).
Although many
investigations have focused on the identification of regulatory mechanism(s)
responsible for the induction of transport proteins (Rizzo et al., 2005; Urquhart et al.,
2007), the overlapping gene targets and ligand specificity for orphan nuclear
receptors make it difficult to determine how regulatory processes ultimately affect the
functional pharmacokinetic outcomes of transport protein induction. Other than
orphan nuclear receptors, some signaling events also can modulate transport
protein expression. For example, an estrogen response element has been identified
in the BCRP promoter region, and BCRP expression appears to be influenced by
estrogen (E1) and/or estradiol (E2) in cancer cell lines when the E1 receptor (ER) is
present (Ee et al., 2004; Imai et al., 2005). However, whether these hormones
increase or inhibit BCRP expression is controversial; BCRP expression was
increased by E2-mediated transcriptional up-regulation in ER-α-positive MCF-7
breast cancer cells (Zhang et al., 2006b), while another group demonstrated
posttranscriptional BCRP down-regulation by E2 in the same cell line (Imai et al.,
2005). In addition, BCRP expression is regulated by the phophoinositol-3 kinase
(PI3K)-Akt signaling pathway via posttranscriptional/translational mechanisms in
18
BCRP-overexpressing K562 cells (Nakanishi et al., 2006). Interestingly, imatinib, a
potent tyrosine kinase inhibitor, decreased BCRP expression by modulating PI3KAkt signaling pathway in the same cell line (Nakanishi et al., 2006). However, the
effect of these signaling pathways on BCRP regulation in BCRP-expressing cancer
or non-polarized cell lines may not be reflected in hepatocytes, or in vivo.
Hepatic
transport
protein
function
can
be
modulated
by
altered
translocation of transport protein(s) between the plasma membrane and
intracellular vesicles. The mechanisms of intracellular trafficking of some hepatic
proteins to the correct plasma membrane domain have been examined previously
(Wakabayashi et al., 2006). Newly synthesized canalicular transport proteins in
the Golgi apparatus may be directly trafficked to the apical membrane (e.g., P-gp),
or may be stored transiently in intracellular compartments before apical delivery to
the membrane (e.g., Bsep) (Kipp et al., 2001; Kipp et al., 2003); some stimuli such
as taurocholate or dibutyryl (DB) cAMP are involved in Bsep and P-gp trafficking.
Trafficking of Mrp2 to the apical membrane is mediated by a protein kinase Cdependent mechanism (Beuers et al., 2001). Some xenobiotics interacting with
these stimuli may affect the intracellular trafficking of canalicular transport proteins,
thus resulting in changes in biliary excretion. Although limited information is
available for the mechanism(s) of basolateral transport protein trafficking, the
cysteine residues of extracellular loop IX-X, responsible for disulfide linkages,
appear to be involved in the targeting of OATP to the basolateral membrane
(Hanggi et al., 2006). The current understanding of the mechanism(s) of
intracellular translocation of xenobiotics is rudimentary. Interestingly, a recent
19
investigation by Munteanu et al. demonstrated the presence of functional P-gp in
the Golgi and the mitochondrial membrane of doxorubicin-resistant K562 leukemia
cells; doxorubicin was transported by P-gp into the mitochondria (Munteanu et al.,
2006). This finding suggests that xenobiotics may accumulate within intracellular
organelles via transport proteins prior to hepatic basolateral and canalicular
excretion.
Sex-related variations in the expression and activity of drug transport
proteins may contribute to sex-related differences in therapeutic response or
toxicity. P-glycoprotein has been reported to be higher in livers of men compared
with women, but phenotyping with fexofenadine did not demonstrate sex
differences in clearance (Schuetz et al., 1995; Kim et al., 2001). Sex-dependent
expression of Bcrp/BCRP have been demonstrated in mice and humans; men
have 2-3 fold higher BCRP expression than women (Merino et al., 2005b).
Experimental Tools for Studying Hepatic Transport
1) Membrane Vesicles Prepared From Transfected Systems: Uptake of substrates
into membrane vesicles prepared from purified basolateral and canalicular liver
plasma membranes is an established technique for elucidating the kinetics of
membrane transport proteins (Moseley et al., 1990; Wolters et al., 1991).
Theoretically, liver plasma membrane vesicles include every transport protein that
resides on the hepatocyte plasma membrane, and these can be used to assess the
overall contribution of all transport proteins to the uptake and efflux of substrates.
Utilization of recombinant expression systems enables the preparation of membrane
20
vesicles from cells over-expressing a single transport protein; this system has been
used to determine whether a compound is an inhibitor and/or substrate for a specific
transport protein.
Membrane vesicles prepared from transfected cells that over-
express the target transport protein have been used with either a radio-labeled
compound to directly measure the uptake, or unlabeled compound to evaluate the
ability of the compound to inhibit the transport of a radio-labeled probe substrate for
the transport protein (Leslie et al., 2003; El-Sheikh et al., 2007). Active transport in
membrane vesicles should be energy-dependent, temperature-dependent and
saturable (Leier et al., 1994; Allen et al., 2002). Membrane vesicles also are a
useful model system to characterize mechanisms of transport. For example, the
dependence of MRP1-mediated vincristine transport on reduced glutathione (GSH)
has been characterized using membrane vesicles (Allen et al., 2002). However, the
utility of membrane vesicles is somewhat limited because of labor intensive, timeconsuming processes of vesicle preparation.
2) Freshly-Isolated Suspended Hepatocytes: Freshly-isolated hepatocytes in
suspension are preferable to cultured hepatocytes for characterization of the
mechanisms of hepatic uptake due to the technical ease of determining initial rates
of uptake. To characterize uptake mechanism(s), various conditions have been
applied in experiments using suspended hepatocytes, such as addition of inhibitor(s)
for uptake pathways, substitution of sodium with choline, depletion of ATP and low
temperature (Brock and Vore, 1984). One major disadvantage of suspended
hepatocytes is that cells should be used within 6-8 h due to a rapid decrease in
activity of both metabolic and transport proteins. In addition, during cell isolation,
21
hepatocytes lose polarity, and canalicular transport proteins such as P-gp and Mrp2
are internalized. Thus, use of suspended hepatocytes to determine biliary clearance
is limited (Roelofsen et al., 1998; Bow et al., 2008).
3) Sandwich Cultured Hepatocytes: Hepatocytes cultured between two layers of
gelled collagen in a sandwich configuration re-create the three-dimensional in vivo
cellular environment and re-establish cell polarity forming canalicular membrane
domains bounded by junctional complexes and bile canalicular networks (Liu et al.,
1998; Liu et al., 1999c). In sandwich-cultured hepatocytes (SCH), transport proteins
are expressed on the appropriate membrane domains (Hoffmaster et al., 2004a).
For example, confocal microscopy revealed the co-expression of P-gp and Mrp2 on
the canalicular domain, and biliary excretion of [D-penicillamine2,5]enkephalin (3HDPDPE) and 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDF) confirmed P-gp and
Mrp2 function, respectively.
Expression and appropriate localization of transport
proteins in SCH enables the utilization of this model to investigate drug-drug
interactions mediated by inhibition of hepatobiliary excretion, and to elucidate the
consequences of protein induction (Annaert et al., 2001; Zamek-Gliszczynski et al.,
2003; Turncliff et al., 2004).
SCH exhibit down-regulation of NTCP and up-
regulation of MRP3; these changes may be due to the somewhat cholestatic nature
of the canalicular networks that lack normal bile drainage via the common bile duct
in vivo.
4) Isolated Perfused Liver Model: The isolated perfused rat liver system was first
reported by Claude Bernard in 1855 and has been used to investigate the
physiology and pathophysiology of the rat liver for many years (Wolkoff et al., 1979).
22
Although it is labor intensive and time- and animal-consuming, the isolated perfused
liver system has been used widely to investigate hepatobiliary transport of drugs
since it maintains intact hepatic architecture, remains viable for ~ 3 hr and allows for
collection of perfusate and bile. This system is applicable for use with chemical
modulators (e.g., inducers or inhibitors), and livers from rodents with transport
protein(s) knocked out may be perfused to determine the contribution of specific
proteins to hepatobiliary disposition. For example, in situ isolated perfused mouse
livers have been used to identify the role of P-gp and Bcrp in the biliary excretion of
4-methylumbelliferyl sulfate (4-MUS) using P-gp- and Bcrp-deficient mice (ZamekGliszczynski et al., 2006a); the biliary excretion of 4-MUS was obliterated in Bcrpdeficient mice while 4-MUS biliary excretion was comparable between wild-type and
P-gp-deficient mice, showing the exclusive role of Bcrp in 4-MUS biliary excretion.
The use of 10 µM GF120918 in isolated perfused livers from wild-type mice showed
significantly reduced biliary excretion of 4-MUS, which was comparable to that in
livers from Bcrp-deficient mice.
5) Animal Knockout Models: Genetic models of transport protein deficiencies also
have been used in in vivo pharmacokinetic and pharmacodynamic studies to assess
the role of a particular transport protein in drug disposition. Animal models with
transport protein deficiencies are a valuable tool to study hepatobiliary drug transport
due to intact liver physiology and preserved hepatic metabolic activity and transport
processes.
However, the potential disadvantage of the knockout animal is the
compensatory up-regulation of other metabolic enzymes and/or transport proteins as
a result of the loss of a transport system. For example, the mutational disruption of
23
mdr1 genes led to over-expression of CYP3A, 2B, and 1A proteins and increased
the catalytic activity of CYP enzymes (Schuetz et al., 2000). Interestingly, this upregulation of CYP enzymes was observed only in P-gp deficient mice housed in
Amsterdam, but not in P-gp deficient mice housed in the United States, suggesting
that both the genetic deficiency and environmental factors regulated P450
expression in P-gp-deficient mice. Another example is the increased expression of
basolateral Mrp3 in rodents with hereditary conjugated hyperbilirubinemia [Mrp2deficient rats (GY/TR-) and Eisai hyperbilirubinemic rats (EHBR)] (Kuroda et al.,
2004). This compensatory up-regulation of MRP3 also is observed in patients with
Dubin-Johnson syndrome who suffer from impaired biliary excretion of organic anion
due to the absence of MRP2 (Tsujii et al., 1999).
Another limitation of using
knockout animal models is the lack of predictability of these models to humans. For
example, it has been documented previously that substrate specificity between the
human MDR1 and mouse Mdr1a was markedly different, suggesting species
differences in P-gp-mediated transport (Yamazaki et al., 2001). Species differences
in the biliary excretion of phase II conjugates between rats and mice have been
demonstrated recently (Zamek-Gliszczynski et al., 2008); Bcrp1 was involved
exclusively in the biliary excretion of sulfate metabolites in mice, whereas both Mrp2
and Bcrp1 were responsible for canalicular transport of sulfate metabolites in rats.
24
Specific Aims
The goal of this dissertation project was to investigate the role of hepatic
transport proteins in drug disposition and drug-induced liver injury. Transport
proteins, individually and/or in concert, play a crucial role in hepatic uptake and
excretion of drugs and metabolites. Thus, altered hepatic transport protein
function (due to disease state, genetic polymorphism or drug-drug interactions)
may influence systemic and/or hepatic concentrations, thereby altering therapeutic
efficacy and/or toxicity. A multiexperimental approach was employed to address
these key issues regarding hepatic transport mechanisms: 1) sex-related
differences in the hepatobiliary disposition of phase II conjugates; 2) in vitro tools
to assess altered transport function; 3) the role of transport proteins in druginduced liver injury.
AIM #1. Elucidate the Mechanism of Differences in the Hepatobiliary
Disposition of Acetaminophen and Metabolites in Male and Female Mice.
Hypothesis: Sex-dependent hepatic Bcrp expression (male > female) results in sexdependent hepatobiliary disposition of phase II conjugates. This hypothesis was
tested using an in situ mouse liver perfusion approach to compare the disposition of
acetaminophen and metabolites in male and female wild-type and Bcrp-deficient
mice.
1.a. Characterize the impact of sex-dependent expression of Bcrp on the
hepatobiliary disposition of acetaminophen and generated metabolites
[glucuronide (AG); sulfate (AS)] in male and female wild-type and Bcrpdeficient mice.
25
1.b. Use a pharmacokinetic modeling approach to estimate pharmacokinetic
parameters and evaluate mechanisms by which the hepatobiliary disposition
of Bcrp substrates was altered.
1.c. Compare protein expression levels of the major biliary and basolateral
transport proteins among male and female wild-type and Bcrp-deficient mice.
AIM #2. Assess the Utility of an Integrated in vitro Approach [Sandwichcultured
Hepatocytes
(SCH)
and
Modeling/Simulation]
in
Predicting
Alterations in the Hepatobiliary Disposition of Troglitazone (TGZ) and
Metabolites.
Hypothesis: Rat and Human SCH can be used to mimic the in vivo disposition of
TGZ
and
generated
metabolites
in
rats
and
humans.
Compartmental
pharmacokinetic modeling and Monte Carlo simulation approaches can be applied to
data from SCH to examine the consequences of canalicular transport protein
modulation resulting from drug interactions or disease states, on hepatic exposure in
hypothetical “populations” of hepatocytes.
2.a. Assess the metabolism of TGZ, which is governed primarily by phase II
conjugation, and the hepatobiliary disposition of TGZ and generated metabolites
in rat and human SCH.
2.b. Use pharmacokinetic modeling and Monte Carlo simulation approaches to
predict alterations in
hepatic exposure and routes of excretion of TGZ and
generated metabolites.
26
AIM #3. Examine the Potential Role of Hepatic Transport Proteins in Liver
Injury Mediated by Trabectedin and Sulindac.
Hypothesis 1: Mrp2 as well as CYP3A1/2 play a role in the previously reported
hepato-protective effect of DEX against trabectedin-mediated hepatotoxicity in rats
and humans. SCH, which closely mimic the metabolic and transport capabilities of
the intact liver, can predict this DEX-mediated hepato-protection.
3.a. Assess the effect of DEX and BSO on trabectedin-mediated hepatotoxicity
in SCH from WT and Mrp2-deficient TR- rats.
3.b. Examine the effect of DEX and BSO on the expression levels of transport
proteins in WT SCH.
3.c. Determine the role of Mrp2 in the hepatobiliary disposition of trabectedin in
the rat isolated perfused liver (IPL) using WT and Mrp2-deficient TR- rats.
Hypothesis 2: Interaction of sulindac and metabolites with hepatic transport
proteins disrupts the hepatobiliary disposition of bile acids, bile acid conjugates
and/or therapeutic drugs, thereby resulting in sulindac-mediated liver injury.
4.a. Examine the impact of acute exposure of sulindac, sulindac sulfone and
sulindac sulfide on hepatobiliary disposition of nitrofurantoin (NF), taurocholate
(TC) and estradiol-17-β-glucuronide (E217G) in rat SCH.
4.b. Determine the inhibitory potency of sulindac sulfone and sulindac sulfide on
TC and E217G initial uptake in freshly-isolated rat hepatocytes.
4.c. Elucidate altered hepatobiliary disposition of TC by sulindac, sulindac
sulfone and sulindac sulfide in human SCH, and subsequently determine the
27
inhibitory potency of sulindac metabolites on TC initial uptake in freshly-isolated
human hepatocytes.
28
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48
CHAPTER 2
Sex-dependent Disposition of Acetaminophen Sulfate and Glucuronide
in the In Situ Perfused Mouse Liver
This work has been presented in part at the AAPS Workshop on Drug Transporters
in ADME: From the Bench to the Bedside, North Bethesda, MD, March 5-7, 2007
and has been submitted to Drug Metabolism and Disposition.
ABSTRACT
Breast cancer resistance protein (BCRP; ABCG2) is expressed in the hepatic
canalicular membrane and mediates biliary excretion of xenobiotics including sulfate
and glucuronide metabolites of some compounds. Hepatic Bcrp expression is sexdependent, with higher expression in male mice. The hypothesis that sex-dependent
Bcrp expression influences the hepatobiliary disposition of phase II metabolites was
tested in the present study using acetaminophen (APAP), and the generated APAP
glucuronide (AG) and sulfate (AS) metabolites in single-pass in situ perfused livers
from
male
and
female
wild-type
and
Abcg2(-/-)
(Bcrp-deficient)
mice.
Pharmacokinetic modeling was utilized to estimate parameters governing the
hepatobiliary disposition of APAP, AG and AS. In wild-type mice, the biliary excretion
rate constant was 2.5- and 7-fold higher in males compared to females for AS and
AG, respectively, reflecting male-predominant Bcrp expression. Sex-dependent
differences in AG biliary excretion were not observed in Bcrp-deficient mice and AS
biliary excretion was negligible. Interestingly, sex-dependent basolateral excretion of
AG (higher in males) and AS (higher in females) was noted in wild-type mice with a
similar trend in Bcrp-deficient mouse livers, reflecting an increased rate constant for
AG formation in male and AS formation in female mouse livers. In addition, the rate
constant for AS basolateral excretion was increased significantly in female
compared to male mouse livers. Interestingly, Mrp4 protein was higher in female
compared to male mouse livers. In conclusion, sex-dependent differences in
conjugation and transporter expression result in profound differences in the
hepatobiliary disposition of AG and AS in male and female mouse livers.
50
INTRODUCTION
The breast cancer resistance protein (BCRP) belongs to the ATP-binding
cassette transporter G family and was first cloned in 1998 from mitoxantroneresistant cell lines (Doyle et al., 1998). BCRP is highly expressed in the canalicular
membrane of the hepatocyte as well as in the intestinal epithelia, blood-brain barrier,
and placenta (Robey et al., 2008). BCRP has wide substrate specificity, including
organic anions, cations, and zwitterions. BCRP mediates the transport of sulfated
hormone metabolites, SN-38, mitoxantrone, topotecan, doxorubicin, flavopiridol,
nitrofurantoin, various flavonoids and the cholorophyll metabolite pheophorbide A
(Robey et al., 2001; Jonker et al., 2002; Nakagawa et al., 2002; Bates et al., 2004;
Imai et al., 2004; Jonker et al., 2005; Merino et al., 2005a).
Male-predominant expression of Bcrp has been reported in mouse liver
(Merino et al., 2005b; Tanaka et al., 2005). Bcrp is influenced by sex hormones;
estradiol suppresses and testosterone induces Bcrp expression (Tanaka et al.,
2005). Sex-dependent differences in Bcrp expression have been shown to influence
the pharmacokinetics of drugs (Merino et al., 2005b). For example, the area under
the plasma concentration-time curve of nitrofurantoin in female wild-type mice was
~2-fold higher than in male mice after oral administration; the cumulative biliary
excretion of nitrofurantoin was 9-fold higher in male compared to female mice. In
contrast, no significant sex differences in plasma concentrations or the hepatobiliary
excretion of nitrofurantoin were observed in Bcrp-deficient mice, indicating that
hepatic Bcrp protein contributes to sex differences in wild-type mice (Merino et al.,
2005b).
51
The role of various transport proteins in the biliary excretion of phase II
metabolites has been examined using several compounds including acetaminophen,
4-methylumbelliferone and harmol, which are metabolized primarily to glucuronide
and sulfate conjugates in mice (Zamek-Gliszczynski et al., 2006c). In situ singlepass liver perfusion in male mice demonstrated that Bcrp plays a predominant role in
the biliary excretion of sulfate conjugates, whereas the biliary excretion of
glucuronide conjugates was mediated by both Bcrp and, to a lesser extent, Mrp2.
Glucuronide and sulfate conjugates of acetaminophen (AG and AS, respectively) are
transported from the hepatocytes into sinusoidal blood by basolateral transport
proteins; Mrp3 mediates basolateral excretion of AG whereas Mrp3, Mrp4, and other
transport proteins may be involved in the basolateral excretion of AS in mice (Xiong
et al., 2002; Manautou et al., 2005; Zamek-Gliszczynski et al., 2006b). In the liver,
mRNA levels of Mrp4 were higher in female than in male C57BL/6 mice, but Mrp2
and Mrp3 mRNA levels did not differ based upon gender (Maher et al., 2005). Sexdependent differences in the disposition of some phase II conjugates might be
anticipated, considering the important role of transport proteins in the biliary and
basolateral excretion of phase II conjugates.
In addition, these sex-dependent
differences in hepatobiliary disposition may be of potential clinical relevance, as
some
sulfate
conjugates
are
important
from
a
physiologic
[e.g.,
dehydroepiandrosterone sulfate (DHEAS) and estrone-sulfate] or pharmacologic
(e.g., minoxidil sulfate) standpoint.
The purpose of this study was to examine the influence of Bcrp and sex on
the hepatobiliary disposition of phase II metabolites, using APAP as a model
52
substrate. In situ single-pass liver perfusion in male and female wild-type and Bcrpdeficient mice was utilized to examine the hepatobiliary disposition of AG and AS.
Compartmental pharmacokinetic modeling was performed to recover relevant
parameters governing hepatobiliary disposition of AG and AS.
In light of the
physiologic and pharmacologic roles that some conjugates play in vivo, it is
important to investigate the potential for significant sex-dependent differences in
hepatobiliary disposition of sulfate and glucuronide metabolites.
53
METHODS
Materials. APAP, AG, AS, cimetidine, taurocholate and Krebs-Henseleit buffer
packets were purchased from Sigma-Aldrich (St. Louis, MO). All other reagents
were of analytical grade and were available commercially.
Mice. Male and female C57BL/6 (wild-type) and Abcg2(-/-) (Bcrp-deficient) (created
by Deltagen Inc., San Mateo, CA) mice (23–30 g) were a gift from Eli Lilly and
Company. Further information regarding background and breeding of these mice
has been detailed previously (Nezasa et al., 2006). All animal procedures were
approved by The Institutional Animal Care and Use Committee at the University of
North Carolina at Chapel Hill.
In Situ Single-pass Liver Perfusion. The abdominal cavity of each mouse was
opened to expose the intestines, liver, and gallbladder. The common bile duct above
the duodenum was ligated and the gallbladder was cannulated with PE-10 tubing
(BD Biosciences, Franklin Lakes, NJ). A loose suture was placed around the inferior
vena cava below the liver. The portal vein was cannulated with a 20-gauge catheter
(B. Braun Medical, Inc., Bethlehem, PA) and the liver was perfused with oxygenated
drug-free Krebs-Henseleit buffer containing 5 µM taurocholate at a flow rate of 5
ml/min. The inferior vena cava below the liver was severed immediately by an
incision below the loose suture. Thereafter, the inferior vena cava above the liver
was cannulated with a 20-gauge catheter. The inferior vena cava below the liver
was ligated to direct all perfusate outflow to the inferior vena cava above the liver.
After a 15-min pre-perfusion period to equilibrate liver temperature and stabilize bile
flow, the liver was perfused with buffer containing 500 nM APAP for 40 min. Liver
54
perfusion was continued for an additional 30 min after APAP infusion with drug-free
buffer. Perfusate outflow was collected at designated time points throughout the 70min experiment. Bile was collected in toto at 10-min intervals. At the end of the
perfusion, livers were isolated and snap frozen.
Analytical Methods. Perfusate, bile and liver homogenate samples were diluted 1:5,
1:250 and 1:10, respectively, to obtain a final sample aliquot of 500 µl. Samples
were protein-precipitated by the addition of 390 µl ice-cold methanol containing the
internal standard cimetidine. After the removal of protein by centrifugation at 3,000g
for 15 min at 4ºC, the supernatants were analyzed by liquid chromatography with
detection by tandem mass spectrometry (Applied Biosystems API 4000 triple
quadrupole with TurboIonSpray interface; Foster City, CA). AS, AG and the internal
standard (cimetidine) were eluted from an Aquasil C18 column (2.1 x 50 mm, dp = 5
µm; Thermo Electron Corporation, San Jose, CA) using a mobile phase gradient (A,
0.1% formic acid in water; B, 0.1% formic acid in methanol; 0–0.75 min hold at 5% B;
0.75–2.5 min linear gradient to 70% B; 2.5–3.5 min hold at 70% B, 3.5–3.6 min linear
gradient to 5% B, 3.6–4 min hold at 5% B; flow rate, 0.75 ml/min; 0.8–4 min directed
to mass spectrometer) and were detected in negative ion mode using multiple
reaction monitoring: AS, 230
150 m/z; AG, 326
150 m/z; cimetidine, 251
157
m/z. Concentrations of APAP and the internal standard (cimetidine) were quantified
using the chromatographic conditions detailed above, but were detected in positive
ion mode using multiple reaction monitoring: acetaminophen, 152
cimetidine, 253
110 m/z;
117 m/z. Care was taken to confirm that no in-source
fragmentation of the conjugates back to APAP occurred. Standard curves of each
55
analyte (0.5-1000 nM) were prepared in the appropriate matrices; inter- and intraday CVs were <15%.
Western Blot Analysis. Mouse livers, perfused with phosphate-buffered saline
solution, were removed promptly and frozen. Livers were homogenized with 3
volumes of Tris-HCl solution (pH 7.4) containing complete protease inhibitor cocktail
(Roche Diagnostics, Mannheim, Germany). The homogenates were centrifuged at
1500×g for 10 min, and the supernatant was ultracentrifuged at 100,000×g for 30
min. The resulting pellet was suspended in Tris-HCl solution (pH 7.4) containing
complete protease inhibitor cocktail, 1% sodium dodecyl sulfate, and 1 mM ethylene
diamine tetraacetic acid. The protein concentration of the solution was determined
with the BCA protein assay reagent kit (Pierce Biotechnology, Inc., Rockford, IL).
Bcrp expression was determined using the anti-BCRP primary monoclonal antibody
(BXP-53; Alexis Biochemicals, Carlsbad, CA) at a 1:1000 dilution. Anti-MRP4 (M4I10; Alexis Biochemicals) was used at a 1:1000 dilution to determine the expression
of Mrp4. Protein loading was normalized to β-actin, which was detected using a βactin antibody at a dilution of 1:5000 (C4; Chemicon, San Fransisco, CA).
Immunoreactive protein bands were detected by chemilumiscence using a Bio-Rad
VersaDoc imaging system and densitometry analysis was achieved using the
Quantity One software package v.4.1 (Bio-Rad Laboratories, Hercules, CA).
Pharmacokinetic Modeling. A compartmental modeling approach was utilized to
describe the hepatobiliary disposition of APAP and metabolites in perfused livers
from male and female wild-type and Bcrp-deficient mice, and to recover estimates of
key pharmacokinetic parameters. AG and AS outflow perfusate and biliary excretion
56
rates were calculated as the product of flow (perfusate or bile) and outflow perfusate
or biliary concentrations, respectively, during each collection period, and then were
normalized to liver weight. Several structurally-distinct models were evaluated;
standard model selection criteria including residual distribution and Akaike’s
information criterion were used to identify the optimal structure. The compartmental
model that best described the data is shown in Figure 2.1. Differential equations
derived from the model scheme in Figure 2.1 were as follows:
dX APAP
= CLup × Cin − X APAP × (K perf,APAP + K gluc + K sulf ); X APAP 0 = 0; t ≤ 40min
dt
dX APAP
= − X APAP × (K perf,APAP + K gluc + K sulf ); t > 40min
dt
dX AG −1
= X APAP × K gluc − X AG−1 × (K perf,AG + K g1); X AG −10 = 0
dt
dX AG − 2
= X AG −1 × K g1 − X AG − 2 × K bile,AG ; X AG − 20 = 0
dt
dX AS −1
= X APAP × K sulf − X AS −1 × (K perf,AS + K s1); X AS −10 = 0
dt
dX AS − 2
= X AS −1 × K s1 − X AS −2 × K bile, AS ; X AS − 20 = 0
dt
dXperfusate, APAP
= Q × Cin − CLup × Cin + X APAP × K perf,APAP ; t ≤ 40min
dt
dXperfusate,
APAP = X
APAP × K perf,APAP ; t > 40min
dt
dXperfusate, AG
= X AG −1 × K perf,AG
dt
dXperfusate, AS
= X AS −1 × K perf,AS
dt
dXbile,AG
= X AG− 2 × K bile,AG
dt
dXbile,AS
= X AS − 2 × K bile,AS
dt
The final model included compartments representing hepatocellular spaces for
APAP, AG and AS. To describe the prolonged biliary excretion of AG and AS after
57
APAP washout relative to basolateral excretion of AG and AS, an intervening
compartment between the site of conjugate formation and the bile compartment was
included in the model.
This intervening compartment was required in order to
describe disposition of AG in all livers, and of AS in wild-type but not in Bcrpdeficient mouse livers (AS biliary concentrations measured in Bcrp-deficient mouse
livers were below the limit of quantitation of the assay).
Differential equations
describing substrate flux in each compartment were fit simultaneously to the
perfusate and biliary excretion rate data by nonlinear least-squares regression
(WinNonlin Pro version 4.2; Pharsight, Mountain View, CA) with a weighting scheme
of 1/(Y predicted).
The precision of parameter estimates was maximized with
stepwise nonlinear least-squares regression. AG perfusate and biliary excretion rate
data from male and female wild-type and Bcrp-deficient mouse livers were used to
estimate Kperf,
AG,
Kg1 and Kbile,AG, while Clup, Kperf,
APAP,
Kgluc and Ksulf were held
constant at the estimated values. Likewise, AS perfusate and biliary excretion rate
data were used to estimate Kperf,AS, Ks1 and Kbile,AS while Clup, Kperf, APAP, Kgluc and Ksulf
were held constant at the estimated values.
Data Analysis. All data are expressed as mean ± SD unless otherwise indicated.
The effects of Bcrp expression (wild-type or Bcrp-deficient) and sex (male or female)
were assessed using a two-way analysis of variance. Data shown in Table 2.1,
Figure 2.3 and 2.4 were analyzed further with Bonferroni’s post-hoc test, but no
further analysis was performed for pharmacokinetic parameter estimates shown in
Table 2.2, as an interaction between Bcrp expression and sex was observed for one
of the estimates in the two-way analysis of variance. An unpaired Student’s t-test
58
was used to determine the statistical significance of differences in AS biliary
excretion from male and female mouse livers because AS biliary excretion was
negligible in Bcrp-deficient mice.
Differences were considered to be statistically
significant when p < 05.
59
RESULTS
Statistically significant differences were observed in body weights among
male and female wild-type mice, but mouse liver weights normalized for body weight
were comparable between wild-type and Bcrp-deficient male and female mice (Table
2.1). Outflow perfusate concentrations of APAP reached a plateau within 20 min of
continuous perfusion (Figure 2.2). The hepatic extraction ratios of APAP, calculated
from inflow and outflow APAP perfusate concentrations between 30 and 40 min of
perfusion, ranged from 24.8 to 35.4% among mouse groups (Table 2.1). Since
APAP, AG and AS concentrations in liver homogenates after the 30-min washout
phase were negligible, total recovery (% of dose) was calculated by adding
recovered APAP, AG and AS in outflow perfusate and bile; in all cases, total
recovery was comparable among all mouse groups (Table 2.1).
Biliary excretion of AG in livers from male wild-type mice was ~2.5-fold higher
than in female wild-type mouse livers, but AG biliary excretion between male and
female Bcrp-deficient mice was comparable (Figure 2.3A).
Relative to the
corresponding wild-type mouse livers, biliary excretion of AG was decreased ~73%
and ~54% in male and female Bcrp-deficient mouse livers, respectively. Cumulative
AG excretion in outflow perfusate from male wild-type and Bcrp-deficient livers was
~1.6-fold higher compared to female wild-type and Bcrp-deficient livers (Figure 2.3B).
Biliary excretion of AS in livers from male wild-type mice was ~2.2-fold higher
than in female wild-type livers (Figure 2.4A).
In Bcrp-deficient mice, AS biliary
recovery was negligible. AS basolateral excretion in female wild-type and Bcrp-
60
deficient mouse livers was ~3.1-fold and ~2.4-fold higher, respectively, than in livers
from their male counterparts (Figure 2.4B).
Immunoblot analysis was performed with crude membrane fractions of liver
homogenates from male and female wild-type and Bcrp-deficient mice to compare
the levels of Bcrp and Mrp4 protein (Figure 2.5). As previously reported (Tanaka et
al., 2005), densitometry analysis revealed that hepatic Bcrp protein, normalized to
the β-actin signal, was 3-fold higher in male wild-type mice than in female wild-type
mice. Interestingly, there was a trend toward increased hepatic Mrp4 protein in
females; Mrp4 protein, normalized to β-actin signal, in female wild-type and Bcrpdeficient mouse livers was 3.5- and 1.6-fold higher than male mouse livers from wildtype and Bcrp-deficient mice, respectively.
The model scheme presented in Figure 2.1 best described the current
experimental data. Representative fits of the model to APAP, AG and AS biliary and
basolateral excretion rate vs. time data are shown in Figure 2.6. Pharmacokinetic
parameter estimates obtained by resolving the differential equations representing
biliary and basolateral excretion rate vs. time data for APAP, AG and AS are
presented in Table 2.2.
The uptake clearance (CLup) was comparable to the
perfusate flow rate (Q; 5 ml/min). The rate constant for APAP basolateral excretion
(Kperf,
APAP)
was comparable among male and female wild-type and Bcrp-deficient
mouse livers.
Sex-dependent differences in metabolite formation rate constants
were observed; the rate constant for AG formation (Kgluc) was ~2-fold higher in male
mouse livers, and the rate constant for AS formation (Ksulf) was ~2-4-fold higher in
female mouse livers. The basolateral excretion rate constant of AS (Kperf,AS), but not
61
AG (Kperf,AG), differed based on sex; the AS basolateral excretion rate constant was
~2-fold higher in female vs. male mouse livers.
The AG biliary excretion rate
constant (Kbile,AG) was higher in male than in female wild-type mouse livers (0.014
min-1 vs. 0.002 min-1), but was similar between male and female Bcrp-deficient
mouse livers. The rate constant for movement of AS from hepatic compartment 1 to
2 (Ks1) was higher in male compared to female wild-type mouse livers. Likewise, the
rate constant for AS biliary excretion (Kbile,AS) in male vs. female wild-type mouse
livers was higher (0.107 min-1 vs. 0.043 min-1).
62
DISCUSSION
The current studies support the hypothesis that sex-dependent hepatic Bcrp
expression influences the hepatobiliary disposition of sulfate and glucuronide
conjugates of the model substrate APAP during in situ single-pass mouse liver
perfusion. Male-predominant Bcrp protein expression in wild-type mouse livers is
consistent with ~2.5-fold higher biliary excretion of AG and AS in male compared to
female wild-type mouse livers. Sex-dependent differences in biliary excretion of AG
were not observed in Bcrp-deficient mice. Pharmacokinetic modeling revealed that
the rate constants governing AG and AS biliary excretion were significantly higher in
male compared with female wild-type mouse livers.
AS biliary excretion was
negligible and AG biliary excretion rates were markedly reduced in Bcrp-deficient
mice, consistent with recent findings demonstrating an important role of Bcrp in AG
and AS biliary excretion in male mice (Zamek-Gliszczynski et al., 2006c). Both Bcrp
and Mrp2 appear to be involved in AS biliary excretion in rats (Zamek-Gliszczynski
et al., 2005), however, AS biliary excretion was not decreased in Mrp2-deficient mice
(Zamek-Gliszczynski et al., 2006c). This apparent discrepancy in the mechanisms
of AS biliary excretion may be due to species-specific differences in the affinity of
multiple hepatic efflux proteins for AS.
Interestingly, sex differences were observed for basolateral excretion of AG
and AS, with more extensive basolateral excretion of AG in male and more
extensive AS basolateral excretion in female mouse livers (Figure 2.3B, 2.4B). One
explanation for these findings may be that the rate constant governing AG formation
(Kgluc) was greater in male mouse livers, whereas the rate constant for AS formation
63
(Ksulf) was greater in female mouse livers, suggesting sex-dependent differences in
formation
of
AG
and
AS.
Although
the
primary
isoforms
of
UDP-
glucuronosyltransferases (Ugt) and sulfotransferases (Sult) responsible for AG and
AS formation in mice remain to be elucidated, previously published data
demonstrate
male-predominant
mRNA
expression
of
Ugt1a5
and
female-
predominant mRNA expression of Sult1a1 and Sult2a1/2 in mouse livers (Alnouti
and Klaassen, 2006; Buckley and Klaassen, 2007). In human liver microsomes,
protein expression of UGT1A6, a major isoform responsible for AG formation, was
50% higher in males compared to females (Court et al., 2001).
Sex-dependent basolateral excretion of AG and AS also may be due to
differences in the expression of transport protein(s) responsible for AG and AS
basolateral excretion.
As previously reported, Mrp3 plays a significant role in
basolateral excretion of AG, whereas both Mrp3 and Mrp4, as well as other
unidentified mechanisms, may be involved in AS basolateral excretion (Xiong et al.,
2002; Manautou et al., 2005; Zamek-Gliszczynski et al., 2006b). Previous studies
showed moderate expression of Mrp3 mRNA in mouse livers, but no differences in
Mrp3 mRNA were noted between male and female mouse livers (Maher et al., 2005).
In the current study, pharmacokinetic modeling revealed that the rate constants for
AG basolateral excretion between male and female mouse livers were not different.
Thus, greater AG basolateral excretion in male vs. female mouse livers appears to
be due primarily to more extensive glucuronidation in male mouse livers. In contrast,
the rate constant governing AS basolateral excretion (Kperf,AS) was significantly
higher in female vs. male mouse livers; this finding cannot be explained by
64
increased AS formation in female mice.
Interestingly, Western blot analysis
demonstrated greater Mrp4 protein in crude membrane fractions from female
compared to male mouse livers, consistent with previous findings that hepatic Mrp4
mRNA was increased in female compared to male wild-type mice (Maher et al.,
2005). This sex-dependent increase in Mrp4 protein levels, coupled with more
extensive sulfation in female mouse livers, may explain, in part, the higher AS
basolateral excretion in female mouse livers since Mrp4 has been implicated in the
basolateral excretion of AS (Lickteig et al., 2007).
The pharmacokinetic model included an intervening compartment between
the sites of conjugate formation and excretion into bile. This additional compartment
was required to describe the prolonged biliary excretion of AG and AS during the
washout phase (Figure 2.1). Movement of AG and AS from the site of conjugate
formation to the site of biliary excretion was best described as a unidirectional rather
than bidirectional process, based on markedly lower coefficients of variation (CV%)
associated with this model fit. Similarly, pharmacokinetic modeling of the in vivo
disposition of APAP and AG in rats has suggested the existence of a discrete
cytosolic compartment prior to AG biliary excretion (Studenberg and Brouwer, 1993).
In a study of hepatic intracellular distribution of several compounds, the presence of
a cytosolic link between metabolite formation and subsequent biliary excretion also
was proposed (Levine and Singer, 1972). Arias and coworkers demonstrated that
newly synthesized canalicular transport proteins (e.g., P-gp) may be directly
trafficked from the Golgi apparatus to the apical membrane, or may be stored
transiently in intracellular compartments before apical delivery to the membrane (e.g.,
65
Bsep)(Kipp et al., 2001); taurocholate or dibutyryl cAMP can stimulate trafficking of
Bsep and P-gp. Immunohistochemical studies clearly revealed BCRP subcellular
localization in folate-deprived MCF cells. In contrast, intense BCRP localization was
noted at cell-to-cell contacts in parental MCF cells cultured in medium containing
high concentrations of folic acid (Ifergan et al., 2005).
Although subcellular
localization of BCRP in hepatocytes remains to be elucidated, substrate binding or
association with subcellularly-localized Bcrp protein prior to substrate biliary
excretion may explain the observation of unidirectional flux of AG and AS (Kg1 and
Ks1) suggested by the current pharmacokinetic model.
In conclusion, the present study demonstrated sex-dependent hepatobiliary
disposition of glucuronide and sulfate conjugates of APAP in mice due to differences
in hepatic transport protein expression and conjugation. In light of the extensive use
of the mouse as a model species to examine hepatobiliary drug disposition and
drug-induced liver injury, such sex-dependent differences in transport proteins and
phase II conjugation may explain different outcomes between male and female mice
during preclinical drug development.
66
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69
Table 2.1. Mouse body weights, liver weights normalized to body weight,
acetaminophen (APAP) extraction ratio and total recovery at 70 min in in situ singlepass perfused livers from male and female wild-type and Bcrp-deficient mice.a
Wild-type
Bcrp-deficient
Male
Female
Male
Female
Body weight (g)
28.4 ± 0.8b
25.8 ± 1.8b
25.9 ± 1.7
24.5 ± 1.7
Liver weight/Body
weight (%)
4.7 ± 0.2
4.8 ± 0.5
4.3 ± 0.2
4.4 ± 0.7
Extraction ratio (%)
35.4 ± 4.8
29.6 ± 7.1
30.8 ± 6.1
24.8 ± 1.2
Total recovery
98.4 ± 5.2
91.7 ± 8.5
93.6 ± 3.7
98.2 ± 4.8
(% of dose)
Livers were perfused with Krebs-Henseleit buffer containing 500 nM APAP for 40
min followed by an additional 30-min infusion with drug-free buffer.
a
mean ± SD (n = 3-4 per group)
b
p < 0.05
70
Table 2.2. Mean pharmacokinetic parameter estimates governing APAP, AG and AS
disposition in in situ single-pass perfused livers from wild-type and Bcrp-deficient
male and female mice.a
Parameter
Wild-type
Bcrp-deficient
Mean estimates (CV%)
Clup (ml/min)
Male
5.63 (12.1)
5.60 (13.2)
Female
5.79 (4.9)
5.55 (8.8)
-1
Kperf,APAP (min )
Male
0.468 (16.5)
0.588 (44.7)
Female
0.525 (31.1)
0.564 (36.3)
Kgluc (min-1)c
Male
0.183 (36.9)
0.199 (17.3)
Female
0.121 (16.5)
0.119 (8.2)
Ksulf (min-1)c
Male
0.007 (13.1)
0.009 (50.6)
Female
0.026 (8.8)
0.018 (44.6)
-1
Kperf, AG (min )
Male
0.168 (45.4)
0.247 (22.5)
Female
0.217 (49.3)
0.214 (77.3)
Kg1 (min-1)
Male
0.044 (89.3)
0.104 (36.7)
Female
0.093 (49.4)
0.092 (55.3)
Kbile, AG (min-1)b,c
Male
0.014 (45.4)
0.001 (66.4)
Female
0.002 (68.1)
0.001 (72.1)
Kperf, AS (min-1)c
Male
0.379 (38.6)
0.271 (11.7)
Female
0.663 (17.6)
0.634 (9.6)
Ks1 (min-1)c
Male
0.048 (25.3)
N/A
Female
0.017 (39.5)
N/A
Kbile, AS (min-1)c
Male
0.107 (8.0)
N/A
Female
0.043 (72.6)
N/A
a
n = 3-4 per group; N/A, not available; CV, coefficients of variation
b
p < 0.05, Wild-type vs. Bcrp-deficient mice
c
p < 0.05, Male vs. Female
71
Q×
×Cin-CLup×Cin
Perfusate
Cin
K perf, AG
XAG-1
CLup
K gluc
K perf, APAP
XAPAP
Kg1
K sulf
Perfusate
Cout
K perf, AS
XAS-1
Ks1
XAG-2
XAS-2
K bile, AG
K bile, AS
X bile, AG
X bile, AS
Figure 2.1. Model scheme depicting the hepatic disposition of APAP, AG and AS in
the single-pass perfused mouse liver. Q, flow rate; Cin, concentration of APAP in
inflow perfusate; Cout, concentration of APAP in outflow perfusate; XAPAP, amount of
APAP in liver compartment; XAG-1 and XAG-2, amount of AG in liver compartments 1
and 2, respectively; XAS-1 and XAS-2, amount of AS in liver compartments 1 and 2,
respectively; CLup, uptake clearance into the liver; Kperf,APAP, first-order rate constant
for basolateral excretion of APAP; Kperf,AG, first-order rate constant for basolateral
excretion of AG; Kperf,AS, first-order rate constant for basolateral excretion of AS; Kgluc
and Ksulf, first-order rate constant for formation of AG and AS, respectively; Kg1, firstorder rate constant for movement of AG from hepatic compartment 1 to 2; Ks1, firstorder rate constant for movement of AS from hepatic compartment 1 to 2; Kbile,AG
and Kbile,AS, first-order rate constants for biliary excretion of AG and AS, respectively.
AS biliary excretion was lower than the limit of quantitation in Bcrp-deficient mouse
livers.
72
APAP Concentration (nM)
500
400
300
200
100
0
0
20
40
60
80
Time (min)
Figure 2.2. APAP concentrations (nM) in outflow perfusate from male wild-type (●)
and Bcrp-deficient (○), and female wild-type (▼) and Bcrp-deficient (∆) mouse liver
perfusions. Livers were perfused with Krebs-Henseleit buffer containing 500 nM
APAP for 40 min followed by an additional 30-min infusion with drug-free buffer (n =
3-4/group).
73
A
AG Biliary Excretion
(nmol/g liver)
2.0
1.5
*
AG Basolateral Excretion
(nmol/g liver)
B
*
1.0
0.5
0.0
Male Female Male Female
Wild-type
25
*
*
20
15
10
5
0
Male Female Male Female
Bcrp-deficient
Wild-type
Bcrp-deficient
Figure 2.3. Cumulative biliary excretion and basolateral excretion of AG (A and B,
respectively; nmol/g liver) in male and female wild-type and Bcrp-deficient perfused
mouse livers through 70 min as described in Figure 2.2 legend (n = 3-4/group; p <
0.05).
74
A
AS Biliary Excretion
(nmol/g liver)
0.25
*
0.20
0.15
0.10
0.05
N.D.
N.D.
0.00
AS Basolateral Excretion
(nmol/g liver)
B
Male Female Male Female
Wild-type
5
*
*
4
3
2
1
0
Male Female Male Female
Bcrp-deficient
Wild-type
Bcrp-deficient
Figure 2.4. Cumulative biliary excretion and basolateral excretion of AS (A and B,
respectively; nmol/g liver) in male and female wild-type and Bcrp-deficient perfused
mouse livers through 70 min as described in Figure 2.2 legend (n = 3-4/group;
N.D.:not detected; p < 0.05).
75
Wild-type
Male
Female
Bcrp-deficient
Male
Female
Bcrp
Actin
Mrp4
Actin
Figure. 2.5. Representative immunoblot of Bcrp, Mrp4 and actin in crude membrane
fractions from male and female wild-type and Bcrp-deficient mouse liver
homogenates from three independent experiments (30 µg protein/well).
76
Female Wild-type
Rate (pmol/min/g liver)
Rate (pmol/min/g liver)
Male Wild-type
10000
1000
100
10
1
0.1
0.01
0.001
0
20
40
60
10000
1000
100
10
1
0.1
0.01
0.001
80
0
20
Time (min)
Rate (pmol/min/g liver)
Rate (pmol/min/g liver)
10000
1000
100
10
1
0.1
0.01
0.001
20
40
60
80
Female Bcrp-deficient
Male Bcrp-deficient
0
40
Time (min)
60
80
10000
1000
100
10
1
0.1
0.01
0.001
0
Time (min)
20
40
60
80
Time (min)
Figure. 2.6. Representative outflow perfusate rate (pmol/min/g liver) of AG (●) and
AS (■), and biliary excretion rate (pmol/min/g liver) of AG (○) and AS (□) in male and
female wild-type (solid symbols) and Bcrp-deficient (open symbols) mouse livers.
Livers were perfused with Krebs-Henseleit buffer containing 500 nM APAP for 40
min followed by an additional 30-min infusion with drug-free buffer. Curves represent
the best fit of the model scheme depicted in Figure 2.1 to the data.
77
CHAPTER 3
Hepatobiliary Disposition of Troglitazone (TGZ) and Metabolites in Rat and
Human Sandwich-Cultured Hepatocytes: Use of Monte Carlo Simulations to
Assess Impact of Changes in Biliary Excretion on Substrate Accumulation
in Cells versus Culture Medium
This Chapter will be submitted to Pharmaceutical Research.
ABSTRACT
This study examined the hepatobiliary disposition of troglitazone (TGZ) and
its derived metabolites [TGZ sulfate (TS), TGZ glucuronide (TG), and TGZ quinone
(TQ)] over time in rat and human sandwich-cultured hepatocytes (SCH).
To
determine the disposition of TGZ and its metabolites generated in rat and human
SCH, cells were incubated with 10 µM TGZ for 120 min. Rat and human SCH
mimicked the in vivo disposition of TGZ/metabolites; TGZ was metabolized primarily
to TS, and to a lesser extent to TG and TQ, in both human and rat SCH. In human
SCH, the biliary excretion index (BEI) was negligible for TGZ and TQ, ~16% for TS,
and ~43% for TG over the duration of the 120 min incubation; in rat SCH, BEI for TS
and TG was 13% and 41%, respectively. Pharmacokinetic modeling and Monte
Carlo simulations were employed to evaluate the impact of modulating the biliary
excretion rate constant (Kbile) for TS or TG on metabolite accumulation in medium
and hepatocytes. The required number of observations to discern statistically
significant alterations in response to changes in Kbile was greater in medium than in
hepatocytes,
suggesting
that
cellular
concentrations
(analogous
to
tissue
accumulation in vivo) is a more sensitive metric than medium concentrations
(analogous to systemic concentrations in vivo). Simulations supported the
hypothesis that altered hepatobiliary transport may not always be evident based on
medium concentrations in rat SCH. Pharmacokinetic modeling/simulation coupled
with data from SCH is a useful in vitro approach to predict alterations in
drug/metabolite hepatobiliary disposition in humans.
79
INTRODUCTION
Troglitazone (TGZ), a peroxisome proliferator-activated receptor gamma
(PPARγ) agonist, was withdrawn from the market in 2000 due to severe idiosyncratic
liver injury (Graham et al., 2003; Smith, 2003). Hepatotoxicity associated with TGZ
has been classified as hepatocellular liver injury (Chan et al., 2002). Several
mechanisms for TGZ’s hepatotoxicity have been proposed (Smith, 2003; Masubuchi,
2006), including inhibition of the bile salt export pump (BSEP, ABCB11), the hepatic
transport protein primarily responsible for biliary excretion of bile acids, by TGZ and
TGZ sulfate (TS) (Funk et al., 2001a). BSEP inhibition may cause hepatic
accumulation of detergent-like bile acids, thereby resulting in hepatocellular injury.
Interestingly, TS is a more potent inhibitor of BSEP (IC50 = 0.4-0.6 µM) than TGZ
(IC50 = 3.9 µM)(Funk et al., 2001a), emphasizing the importance of metabolitemediated hepatotoxicity, in addition to that of the parent drug.
TGZ is metabolized extensively by the liver in humans as well as in rats; the
in vivo metabolic profile and disposition of TGZ in rats are similar to that in humans.
In humans, TGZ is metabolized extensively in the liver primarily to a sulfate
conjugate mostly via sulfotransferase (SULT) 1A3 (Honma et al., 2002), and also to
a glucuronide conjugate mostly via UDP-glucuronosyltransferase (UGT) 1A1
(Watanabe et al., 2002; Nakagomi-Hagihara et al., 2006); TGZ also is oxidized to a
quinone derivative by cytochrome P450 isozymes CYP3A4 and CYP2C8 (Yamazaki
et al., 1999). After oral administration of TGZ for 7 days in humans, the area under
the plasma concentration vs. time curve at steady state (AUCss) for TS was ~7-10fold higher than that of TGZ; the plasma AUCss for TQ was comparable to that of
80
TGZ, but the AUCss for TG was only 20-40% of that for TGZ (Loi et al., 1999).
Similarly, after oral administration of [14C]TGZ to male rats, the AUC of TS
accounted for 89% of the AUC of total plasma radioactivity, while the AUC of TGZ
and TG was only 5% and 1% of the AUC of total plasma radioactivity, respectively
(Kawai et al., 1997). After intravenous administration of [14C]TGZ to male rats, the
biliary excretion of TS and TG was 61% and 2% of the dose, respectively; TGZ and
TQ recovery in bile accounted for less than 0.1% of the dose (Kawai et al., 1997).
Since TGZ is extensively metabolized and little parent compound is excreted
unchanged into urine or bile (Kawai et al., 1997), the total body clearance of TGZ is
governed by its metabolic clearance (Izumi et al., 1996).
Sensitive and rapid in vitro screening tools to predict hepatotoxicity are
needed in the pharmaceutical industry. Primary hepatocytes remain an important
tool for studying the metabolism and hepatotoxic potential of xenobiotics.
Hepatocytes cultured in a single layer of collagen are suitable for cytotoxicity assays,
but the use of this tool is limited due to loss of metabolic activity and short survival
periods (Hewitt et al., 2007).
Hepatocytes cultured in sandwich configuration
between layers of extracellular matrix have overcome many of the limitations of
conventional hepatocyte culture because the cells repolarize, regain their proper
morphology, and maintain liver-specific metabolic activity, including stable
expression of cytochrome P450 enzymes over longer times (Liu et al., 1998; Liu et
al., 1999b; Liu et al., 1999d; Liu et al., 1999e; Hamilton et al., 2001; Hewitt et al.,
2007; Mingoia et al., 2007). Furthermore, sandwich-cultured hepatocytes develop
functional networks of bile canalicular structures between cells that are sealed by
81
tight junctions (LeCluyse et al., 1994). Measurements of biliary excretion of drugs
and metabolites in humans are often indirect or require invasive procedures, but
cells and canalicular networks of sandwich-cultured hepatocytes are directly
accessible, allowing the quantitation of substrates and metabolites excreted into the
medium, retained within the cell, or excreted into the bile (Hoffmaster et al., 2004b;
Turncliff et al., 2006).
The present study utilized rat and human SCH to examine the metabolism of
TGZ, governed primarily by phase II conjugation, and the hepatobiliary disposition of
TGZ and its generated metabolites. Moreover, the ability of this in vitro system to
estimate the in vivo disposition of TGZ and metabolites previously reported in rats
and humans was assessed. A pharmacokinetic model was developed to estimate
key parameters governing the hepatic metabolism and disposition of TGZ and
metabolites in rat SCH. Monte Carlo simulations were utilized to predict the influence
of hepatic transport modulation on the hepatobiliary disposition of TGZ and
metabolites in a hypothetical “population” of hepatocytes.
82
MATERIALS AND METHODS
Chemicals.
Penicillin-streptomycin
solution,
dexamethasone
(DEX),
Hanks’
balanced salts (HBSS), HBSS modified (HBSS without Ca2+ and Mg+), collagenase
(type IV), and Triton X-100 were purchased from Sigma-Aldrich, Inc (St. Louis, MO).
Dulbecco’s modified Eagle’s medium (DMEM) and MEM non-essential amino acids
were purchased from Invitrogen (Carlsbad, CA). Insulin/transferrin/selenium culture
supplement (ITSTM), BioCoatTM culture plates and MatrigelTM extracellular matrix
were purchased from BD Biosciences Discovery Labware (Bedford, MA). TGZ was
purchased from Toronto Research Chemicals Inc. (Ontario, Canada). TS, TG, and
TQ were kindly provided by Daiichi-Sankyo Co., Ltd (Tokyo, Japan). All other
chemicals and reagents were of analytical grade and were readily available from
commercial sources.
Hepatocyte Culture. Male Wistar rats (220-300 g; Charles River Laboratories, Inc.,
Raleigh, NC) were maintained on a 12-hr light/dark cycle with free access to water
and rodent chow. The Institutional Animal Care and Use Committee of the University
of North Carolina at Chapel Hill approved all procedures. Rat hepatocytes were
isolated using a two-step collagenase perfusion method as described previously (Liu
et al., 1999c). Hepatocytes were plated at a density of 1.75 x 106 cells/well on 6-well
BioCoatTM plates in Dulbecco’s modified Eagle’s medium (DMEM; 1.5 ml) containing
5% fetal bovine serum, 10 µM insulin, 1 µM DEX, 2 mM L-glutamine, 1% MEM nonessential amino acids, 100 units penicillin G sodium and 100 µg streptomycin sulfate.
Hepatocytes were allowed to attach ~2 hr, and the medium was changed to remove
unattached cells. Cells were overlaid ~24 hr later with BD MatrigelTM at a
83
concentration of 0.25 mg/ml in 2 ml/well ice-cold DMEM supplemented with 0.1 mM
MEM non-essential amino acids, 2 mM L-glutamine, 100 units penicillin G sodium,
100 µg streptomycin, 1% ITSTM and 0.1 µM DEX. CellzDirect (Durham, NC) kindly
provided human hepatocytes cultured in 6-well plates, seeded at a density of 1.5 x
106 cells/well, and overlaid with MatrigelTM. Table 3.1 shows demographics of human
liver donors. Human cells were cultured in the same medium as rat hepatocytes.
Both rat and human hepatocytes were cultured at 37°C i n a humidified incubator
with 95% O2/5% CO2. Medium was changed daily until the experiments were
conducted.
Metabolism of TGZ and Accumulation of Parent and Metabolites in Medium,
Cells, and Bile in Rat and Human SCH. Modulation of Ca2+ in the incubation buffer
was used to manipulate the tight junctions sealing the canalicular networks between
cells. In Ca2+-containing standard buffer (HBSS with 0.35 g/L sodium bicarbonate),
tight junction integrity is preserved and substrates are excreted into the bile
canaliculi; incubation of cells in Ca2+-free buffer (HBSS modified, with 0.35 g/L
sodium bicarbonate and 0.38 g/L EGTA) disrupts the tight junctions, opens the
canalicular spaces, and canalicular contents are released into the incubation
medium (Liu et al., 1999e). Subtracting the amount of substrate accumulated in cells
after a pre-incubation in Ca2+-free buffer from the accumulated amount in cells + bile
after pre-incubation with Ca2+-containing buffer yields the amount of substrate
excreted into the canalicular networks.
To evaluate metabolism of TGZ, day-4 cells (rat; n=3 livers) and day-8 or day-10
cells (human; n=2 livers) were incubated at 37 °C for 10, 20, 30, 60, 90, and 120 min
84
for rat SCH, and for 30, 60, and 120 min for human SCH, with 10 µM TGZ in culture
medium. Aliquots of medium were removed at each timepoint, and samples were
diluted with methanol (1:2 v/v). At the designated time, cells were washed twice and
incubated at 37 °C for 5 min with HBSS with Ca 2+ (cells + bile) or without Ca2+ (cells).
After incubation, buffer was aspirated and cells were lysed with 1 ml of 70%:30%
(v/v) methanol:water, and sonicated for 20 sec with a sonic dismembrator (model
100, Fisher Scientific, Pittsburgh, PA). Medium, and cells or cells + bile lysate
samples were stored at <-70 °C until analysis.
Transport of TGZ, and Preformed TS and TG Metabolites in Rat SCH. To
confirm transport of TGZ and its preformed metabolites in rat SCH, media was
aspirated from cells on culture day 4 and cells were rinsed twice and then preincubated for 10 min at 37°C with 2 ml of warmed HBS S+Ca2+ buffer (cells + bile) or
Ca2+-free HBSS buffer (cells). Medium was aspirated and cells were treated with 5
µM TGZ, TS, or TG in 1.5 ml HBSS +Ca2+ buffer for 10 min. After incubation, the
dosing medium was removed, cells were washed 3x with 2 ml of ice-cold HBSS
+Ca2+ buffer. The wash buffer was aspirated, cells were lysed by adding 1 ml of
70%:30% (v/v) methanol:water, and samples were sonicated for 20 sec with a sonic
dismembrator (model 100, Fisher Scientific, Pittsburgh, PA). Cells and cells + bile
lysate samples were stored at <-70°C until analysis. Tre atments were performed in
triplicate in n=1 experiment (TGZ) or n=3 experiments (TS and TG).
Sample Analysis. Substrate accumulation was corrected for nonspecific binding to
the extracellular matrix by subtracting accumulation in MatrigelTM-coated BioCoatTM
plates without cells. The BCA protein assay (Pierce Biotechnology, Inc., Rockford,
85
IL) was used to quantify total protein concentration in cell lysate samples using
bovine serum albumin as the reference standard, and accumulation was normalized
to protein concentration. To account for the incompatibility of the protein assay with
methanol, the average protein concentration for standard HBSS or Ca2+-free HBSS
incubations in a representative plate from the same liver preparation was used to
normalize accumulation. The medium and cell or cell + bile lysate samples were
centrifuged at 12,000×g for 2 min at 4 °C, and the su pernatant was diluted 1:6 (v/v)
with 79%:21% (v/v) methanol:water containing an internal standard (ethyl warfarin).
A Shimadzu solvent delivery system (Columbia, MD) and a Leap HTC Pal
thermostated autosampler (Carrboro, NC) connected to an Applied Biosystems API
4000 triple quadruple mass spectrometer with a TurboSpray ion source (Applied
Biosystems, Foster City, CA) were used for analysis. Tuning, operation, integration
and data analysis were performed in negative ionization mode using multiple
reaction monitoring (Analyst software v.1.4.1, Applied Biosystems). Separation was
accomplished using an Aquasil C18, 50×2.1 mm column, with a 5 µm particle size
(ThermoElectron, San Jose, CA). Analysis required 15 µL of sample and a solvent
flow of 0.75 mL/min. Initial gradient conditions (80% 10 mM ammonium acetate
aqueous solution and 20% methanol) were held for 0.8 min. From 0.8 to 1.5 min, the
mobile phase composition increased linearly to 60% methanol, and the eluent was
directed to the mass spectrometer. From 1.5 to 4 min, the mobile phase composition
increased linearly to 65% methanol. At 4.1 min, the methanol composition was
increased to 90%. The flow was held at 90% methanol until 4.3 min. At 4.4 min, the
column was equilibrated with 80% 10 mM ammonium acetate aqueous solution. The
86
total run time, including equilibration, was 5 min per injection. Eight point calibration
curves (5-5000 nM) were constructed as composites of TGZ (440.0→397.1), TS
(520.2→440.1), TG (616.2→440.1), and TQ (456.1→413.1) using peak area ratios
of analyte and ethyl warfarin (320.8→160.9). All points on the curves backcalculated to within ± 15% of the nominal value.
B-CLEAR® technology (Qualyst, Inc., Raleigh, NC) was used to calculate the
biliary excretion index (BEI), which represents the percentage of accumulated
substrate that is excreted into bile, using the following equation (Liu et al., 1999c):
BEI (%) =
Accumulationcells +bile − Accumulationcells
× 100
Accumulationcells +bile
Pharmacokinetic Modeling. The average rat SCH accumulation data for TGZ and
metabolites, including TS, TG and TQ, were utilized in pharmacokinetic modeling
analysis. Several structurally-distinct models were evaluated, and standard model
selection criteria, including Akaike’s information criterion, residual distribution, and
the distribution of the residual error, were used to identify the optimal structure. The
model scheme shown in Figure 3.1 best described the disposition of TGZ and
generated metabolites in rat SCH over 120 min in the medium (M), hepatocytes (C)
and bile canaliculi (B). Differential equations derived from the model scheme in
Figure 3.1 (see legend for definition of parameters) were as follows:
87
dXTGZ,M
dt
dXTS,M
dt
dXTS,M
dt
dXTG,M
dt
dXTG,M
dt
dXTQ,M
dt
= K efflux,TS × XTS,C − K uptake,TS × XTS,M; XTS,M0 = 0; t ≤ 20min
= K efflux,TS × XTS,C − K uptake,TS × XTS,M + K flux,TS × XTS,B ; t > 20min
= K efflux,TG × XTG,C − K uptake,TG × XTG,M; XTG,M0 = 0; t ≤ 20min
= K efflux,TG × XTG,C − K uptake,TG × XTG,M + K flux,TG × XTG,B ; t > 20min
= K f,TQ × XTGZ,M −K other × XTQ,M; XTQ,M0 = 0
dXTGZ,C
dt
dXTS,C
dt
dXTG,C
dt
dXTS,B
dt
dXTS,B
dt
dXTG,B
dt
dXTG,B
dt
= −(K uptake,TGZ + K f,TQ ) × XTGZ,M; XTGZ,M0 = 0
= K uptake,TGZ × XTGZ,M −(K f,TS + K f,TG ) × XTGZ,C; XTGZ,C0 = 0
= K f,TS × XTGZ,C + K uptake,TS × XTS,M − (K efflux,TS + K bile,TS ) × XTS,C; XTS,C0 = 0
= K f,TG × XTGZ,C + K uptake,TG × XTG,M − (K efflux,TG + K bile,TG ) × XTG,C; XTG,C0 = 0
= K bile,TS × XTS,C; XTS,B0 = 0; t ≤ 20min
= K bile,TS × XTS,C − K flux,TS × XTS,B ; t > 20min
= K bile,TG × XTG,C; XTG,B0 = 0; t ≤ 20min
= K bile,TG × XTG,C − K flux,TG × XTG,B ; t > 20min
Differential equations describing the accumulation of TGZ and generated
metabolites in medium, hepatocytes and bile canaliculi were fit simultaneously by
nonlinear least-squares regression (WinNonlin Pro version 5.0.1; Pharsight,
Mountain View,CA) using a weighting scheme of 1/(Y predicted).
Monte Carlo Simulations. Parameter estimates with associated variance
(recovered by nonlinear least-squares regression of the flux data shown above) and
uncontrolled error (experimental or analytical error; 15%) served as input for Monte
88
Carlo simulations. To explore hepatic TS and TG disposition in response to impaired
transport in a hypothetical “population” of hepatocytes, simulations were performed
by modulating the rate constant for TS or TG biliary excretion. In order to estimate
the number of observations (sample size) necessary to detect a statistically
significant difference in TS or TG accumulation in cells or medium due to changes in
the TS or TG biliary excretion rate constant (Kbile, TS or Kbile, TG), data from Monte
Carlo simulations at designated time points (10, 20, 30, 60, 90 and 120 min) were
analyzed using one-way ANOVA. Based on the effect size (the square root of the
ratio of the between-group to within-group variance) calculated by Cohen’s f2 (Cohen,
1988), a sample size for each time point was calculated to achieve a power of 0.80
(p<0.05)(2008). The relative power to detect a statistically significant difference in
medium vs. cell at each time point was calculated as a ratio of the number of
observations (sample size) necessary to detect a statistically significant difference in
accumulation in medium to the number of observations necessary for accumulation
in cells.
89
RESULTS
Hepatobiliary disposition of TGZ and generated metabolites in human SCH.
The disposition of TGZ and the generated metabolites in the medium and
hepatocytes at 30, 60, and 120 min is shown in Table 3.2; cumulative recovery of
TGZ and generated metabolites (medium + hepatocytes + bile) was 109-117%. At
120 min, TGZ in the medium accounted for 64% of the dose. TS was the
predominant metabolite of TGZ in the medium (30% of dose) as well as hepatocytes
(5.6% of dose) during the 120-min incubation; TG and TQ were recovered
predominantly in the medium (3% and 2.4% of dose, respectively) with negligible
cellular accumulation (less than 1% of dose). Accumulation of TGZ and TQ in bile
canaliculi was negligible. The BEI values of TS (15-17 %) and TG (41-46 %) were
comparable at 30, 60, and 120 min in human SCH (Table 3.2).
Hepatobiliary disposition of TGZ and metabolites in rat SCH.
Initial experiments confirmed that TGZ and preformed metabolites of TGZ, TS
and TG, were taken up into rat SCH (Figure 3.2). Following a 10-min accumulation,
average cell + bile accumulation of TGZ was only slightly greater than cellular
accumulation (1600s. 1560 pmol/mg protein), consistent with a negligible BEI for
TGZ of 2.3%. Average cell + bile accumulation was slightly greater than cellular
accumulation for TS (2280 vs. 1940 pmol/mg protein) and TG (908 vs. 605 pmol/mg
protein), consistent with more extensive BEI values of 15% and 34% for TS and TG,
respectively.
The disposition of TGZ and its generated metabolites in medium, hepatocytes,
and bile are plotted in Figure 3.3, 3.4, and 3.5, respectively. Cumulative recovery of
90
TGZ and its metabolites from medium, hepatocytes and bile was comparable
throughout the 120-min incubation period (90-104%) (Table 3.3). At 120 min, 35% of
the dose remained in the medium as TGZ (Table 3.3). Among metabolites present in
the medium, TS was predominant (38% of dose), followed by TG (7% of dose) and
TQ (1% of dose) at 120 min. Figure 3.4. depicts the hepatocyte accumulation of TGZ
and its generated metabolites over the 120-min incubation period. TS formation was
rapid and extensive; cellular accumulation of TS exceeded TGZ even at 10 min
(5.4% vs. 1.5% of the dose, respectively; Table 3.3). The cellular accumulation of
TG was <0.4% of the dose over the 120-min incubation. TQ in the hepatocytes was
negligible. The average BEI of TS and TG was 13% and 41% over the 120-min
incubation time; accumulation of TGZ and TQ in bile was negligible (Figure 3.5;
Table 3.3).
Pharmacokinetic modeling of the hepatobiliary disposition of TGZ and its
generated metabolites in rat SCH.
Because data were collected at more time points in rat SCH, a compartmental
pharmacokinetic model was developed using data from rat SCH. The model scheme
depicted in Figure 3.1 best described the disposition of TGZ, TS, TG and TQ in rat
SCH during the 120-min incubation. Representative descriptions of the best fit of the
pharmacokinetic model to the TGZ/generated metabolite vs. time data are shown in
Figures 3.3-3.5. Pharmacokinetic parameter estimates obtained by resolving the
differential equations describing hepatocellular disposition of TGZ, TS, TG and TQ
vs. time data are included in Table 3.4. The first-order rate constant for TS formation
(0.382 min-1) was ~6-fold greater than that for TG formation (0.061 min-1). The
91
pharmacokinetic model incorporated a formation process for TQ from TGZ in the
incubation medium, instead of cellular TQ formation, because accumulation of TQ in
hepatocytes was negligible while TQ in the medium represented ~1% of the dose at
120 min. In order to explain the decreased accumulation of TS and TG in bile
canaliculi after 20 min, first-order rate constants for TS and TG flux from the bile
canaliculi to the incubation medium were incorporated into the pharmacokinetic
model. Consistent with data from the uptake study with preformed TGZ metabolites,
TS and TG (Figure 3.2), the first-order rate constant for TS uptake (Kuptake, TS; 0.005
min-1) was greater than for the TG uptake rate constant (Kuptake, TG; 0.001 min-1). The
first-order rate constant for biliary excretion of TG (0.061 min-1) was ~3-fold higher
for TS (0.023 min-1). The first-order rate constant for basolateral efflux of TG (0.160
min-1) was ~5-fold greater than for TS (0.031 min-1).
Monte Carlo simulations
Monte Carlo simulations were performed to assess the impact of modulation
of the first-order rate constant for biliary excretion of TS (Kbile, TS) and TG (Kbile, TG) on
the hepatobiliary disposition of TS and TG, respectively (Figure 3.6). Overall,
measurements of cellular accumulation were more sensitive to changes in kinetic
parameters than were measurements of substrate concentrations in medium.
Cellular accumulation measurements were 1.4- to 270-times more sensitive than
medium accumulation measurements for TG; cellular accumulation measurements
were 2.2- to 10.2-times more sensitive than medium accumulation measurements
for TS (Table 3.5).
92
DISCUSSION
The present study examined the hepatobiliary disposition of TGZ and its
metabolites in the SCH system; results confirmed that functional activity of phase II
conjugation enzymes is preserved in rat and human SCH. In addition,
pharmacokinetic modeling and simulation studies were employed to examine the
impact of impaired biliary excretion on hepatobiliary disposition of TS and TG in rat
SCH.
In human SCH, TS was most abundant, and TQ was more abundant than TG,
in the medium at 30 min; amounts of both TQ and TG were similar at 60 and 120
min in the medium. Amounts of TQ and TG in cells were also similar at all time
points, but no TGZ and TQ was found in the bile at any time. Kostrubsky and
colleagues treated conventionally-cultured human hepatocytes with 10 µM TGZ; at 1
hour, concentrations of TS and TQ were the highest in medium (depending on
donor), followed by parent TGZ, and TG (Kostrubsky et al., 2000). Our data show
that in human SCH at 1 hour, unconjugated TGZ is most abundant in the medium
(88.84% of dose), followed by TS (13.37% of dose), TQ (2.23% of dose), and lastly
TG (1.56% of dose).
Transport of TGZ and its preformed metabolites was confirmed in rat SCH.
While TGZ, TS, and TG were taken up into rat hepatocytes, it appears that TS was
taken up into cells more efficiently compared to TGZ even though TS is more
hydrophilic than TGZ. These data are consistent with the observation that TS is a
higher affinity substrate than TGZ or TG for the uptake transporter OATP1B1,
(Nozawa et al., 2004b). Further experiments revealed that in rat SCH, TS was the
93
major metabolite found in the medium, cells, and bile at all timepoints. TG was the
next most abundant metabolite found in the medium and bile at all time points, while
TQ was only detected in lesser amounts in the medium, but not in cells or bile. This
observation may be explained by photooxidation of TGZ to TQ; thus, subsequent
pharmacokinetic modeling incorporated conversion of TGZ to TQ in medium (Fu et
al., 1996).
Additionally, in rat and human SCH, TS and TG were transported into the bile
canaliculi, while transport of TGZ into the bile canaliculi was negligible. These
findings in SCH are consistent with published in vivo reports showing that little
parent TGZ is recovered in rat bile, while TS and TG (68.0% and 11.6% of dose,
respectively, after oral dosing of TGZ) are excreted into bile in rats (Izumi et al.,
1996; Kawai et al., 1997). These medium and biliary excretion profiles resembled
the plasma profiles (TS > TG) and biliary excretion (TS > TG), respectively, after oral
dosing of TGZ to rats in vivo (Izumi et al., 1997; Kawai et al., 1997). The greater
amount of TS compared to TG in rat SCH, and the greater value of Kf, TS compared
to Kf,TG correspond to data by Izumi et al. showing that the sulfate formation
clearance was 6-fold higher than glucuronide formation clearance in rats (Izumi et al.,
1997).
The accumulation of TS and TG in bile remained constant or decreased
during the incubation time after 20 min in both human and rat SCH (Table 3.2 and
Table 3.3). To account for this decrease, the pharmacokinetic model incorporated a
rate constant for flux of TS and TG from the bile into the medium (Figure 3.1). The
flux of other compounds from bile into the medium of SCH has been noted
94
previously. The first-order rate constant for flux of taurocholate (TC) and [Dpenicillamine2,5] enkephalin (DPDPE) from bile was reported to be 0.85 min-1 (Liu et
al., 1999e) and 1.6 min-1 (Hoffmaster et al., 2005), respectively, but only 0.05 min-1
for 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDF) [Abe, unpublished data]. The
greater rate constant for flux of TC and DPDPE compared to CDF, TS (0.17 min-1;
Table 3.4) and TG (0.094 min-1; Table 3.4) may be attributed to different culture
conditions: 2×106cells/60-mm dish overlaid with rat tail type 1 collagen for studies
conducted with TC and DPDPE vs. 1.75×106 cells/6-well plates overlaid with
MatrigelTM for studies conducted with CDF and TGZ.
The accumulation of TS and TG in the medium in rat SCH (38% and 7% of
the dose, respectively) was comparable to values obtained in human SCH (30% and
3% of the dose, respectively). Transport proteins responsible for the basolateral
excretion of TS and TG remain to be identified. It has been documented previously
that Mrp3 plays a predominant role in the basolateral excretion of glucuronide
conjugates, while both Mrp3 and Mrp4 are involved in the basolateral excretion of
sulfate conjugates along with additional yet-to-be-identified mechanism(s) (ZamekGliszczynski et al., 2006b). Pharmacokinetic modeling revealed a ~5-fold higher firstorder rate constant for TG than for TS basolateral efflux. This observation may
reflect more efficient basolateral efflux of TG, presumably by Mrp3; Mrp3 expression
was increased over time in culture in rat SCH (unpublished observation).
Monte Carlo simulations revealed that cellular accumulation is a more
sensitive measure of changes in the rate constant for biliary excretion of TS or TG
(Kbile,TS or Kbile,TG) than medium accumulation. In practice, the number of
95
observations necessary to observe a statistically significant difference in the medium
was too high (19-7271 for TG; 13-144 for TS), while the number of observations
necessary to observe a statistically significant difference in hepatocytes was more
reasonable (14-27 for TG; 4-27 for TS) (Table 3.5). SCH data coupled with modeling
and simulation are capable of estimating the impact of altered transport function on
hepatic (reflected in hepatocyte accumulation) and systemic (reflected in medium
accumulation) exposure. Peak plasma concentrations or AUCplasma often have been
used to examine the effect of modulation of canalicular transport proteins on drug
disposition, but results of this simulation suggest that those parameters may not
always reflect changes in hepatic exposure due to modulation of biliary excretory
pathways.
Physiologically-based
pharmacokinetic
modeling
of
pravastatin
distribution also suggested that plasma concentrations are not sensitive enough to
detect changes in canalicular efflux; altered canalicular efflux of pravastatin changed
liver concentrations markedly, but had minimal effect on plasma concentrations
(Watanabe et al., 2009).
The failure of preclinical testing to predict the hepatotoxicity of TGZ
emphasizes the importance of understanding the metabolism and hepatobiliary
disposition of drugs in development, in both humans and in species commonly used
for animal testing. One of the most efficient ways to study species differences in
metabolism and hepatobiliary disposition is with the use of in vitro models. In vitro
models are also an important tool for the prediction of the pharmacokinetics and
pharmacodynamics of drugs in humans. One benefit of using sandwich-cultured
hepatocytes is that concentrations of parent drug and metabolites in medium can be
96
measured, in addition to discriminating between accumulation within hepatocytes
and bile. Efflux into medium may be analogous to efflux from the hepatocytes into
plasma in vivo, while efflux into the bile may approximates biliary excretion in vivo.
Because data in humans regarding intracellular concentrations in the liver of TGZ
and its metabolites is lacking, as is data about efflux of parent or metabolites into
bile, the sandwich-cultured model may be a useful in vitro tool to predict human in
vivo hepatobiliary disposition. Herein we have shown that the sandwich-cultured
hepatocyte model can be used to elucidate the hepatobiliary disposition of TGZ and
metabolites in both humans and rats.
In conclusion, phase II metabolites of TGZ were detected in both rat and
human SCH, indicating the preservation of functional activity of sulfotransferase and
UDP-glucuronosyltransferase in the SCH system. The remarkable similarity in TGZ
metabolism and hepatobiliary disposition between rats and humans in vivo also was
observed in rat and human SCH, respectively, suggesting that the SCH model is a
useful in vitro tool to predict in vivo hepatobiliary disposition in rats and humans.
Pharmacokinetic modeling and simulation, coupled with data obtained from the SCH
model, may be used as an in vitro approach to determine likely alterations in
drug/metabolite hepatobiliary disposition in humans.
97
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101
Table 3.1. Demographics of human liver donors
Donor
Age
Gender
Ethnicity
Co-medications
1
36
Female
Caucasian
Oral contraceptive, HCTZ
2
64
Female
Caucasian
Atorvastatin, Amlodipine,
Benazepril, Alendronate
102
Table 3.2. Disposition of TGZ and generated metabolites in human SCH
TGZ, TS, TG and TQ (expressed as % of dose) in medium, cells and bile at 30, 60
and 120 min after incubation in medium initially containing 10 µM TGZ. BEI of TS
and TG was calculated as described in Materials and Methods Data represent mean
of a single determination in n=2 livers (N.D, not-detectable).
Time
(min)
30
60
120
Time
(min)
30
60
120
Time
(min)
30
60
120
TGZ
Medium
Cells
(% of dose)
93.94
3.5
88.84
2.8
64.24
2.04
TQ
Medium
Cells
(% of dose)
2.28
0.07
2.23
0.07
2.41
0.07
Recovery
(%)
112
117
109
Bile
N.D
N.D
N.D
Bile
N.D
N.D
N.D
103
TS
Medium
Cells
(% of dose)
3.69
6.27
13.37
6.61
30.29
5.64
TG
Medium
Cells
(% of dose)
0.63
0.06
1.56
0.06
2.98
0.05
Bile
1.29
1.22
0.99
Bile
0.05
0.04
0.04
BEI
(%)
17
17
15
BEI
(%)
46
41
43
Table 3.3. Disposition of TGZ and its generated metabolites in rat SCH
TGZ, TS, TG and TQ (expressed as % of dose) in medium, cells and bile at 10, 20,
30, 60, 90 and 120 min after incubation in medium initially containing 10 µM TGZ.
BEI of TS and TG was calculated as described in Materials and Methods after
incubation of TGZ (10 µM) in rat SCH. Data represent mean ± S.E.M (n=3 livers;
N.D, non-detectable).
TGZ
Cells
(% of dose)
Time
(min)
Medium
10
20
30
60
90
120
94.28 ± 9.29
86.38 ± 3.42
81.10 ± 3.68
63.55 ± 4.98
46.90 ± 3.07
35.30 ± 2.87
Time
(min)
1.50 ± 0.31
1.97 ± 0.21
1.57 ± 0.06
1.38 ± 0.45
0.73 ± 0.24
0.30 ± 0.13
TQ
Medium
Cells
(% of dose)
10
20
30
60
90
120
Time
(min)
1.00 ± 0.07
1.36 ± 0.23
1.27 ± 0.09
1.01 ± 0.13
0.85 ± 0.13
0.65 ± 0.14
Recovery
(%)
10
20
30
60
90
120
104
103
103
99
95
90
N.D
N.D
N.D
N.D
N.D
N.D
Bile
Medium
N.D
N.D
N.D
N.D
N.D
N.D
0.48 ± 0.06
2.97 ± 0.58
6.14 ± 0.57
18.61 ± 2.02
29.93 ± 2.64
37.80 ± 2.70
Bile
Medium
N.D
N.D
N.D
N.D
N.D
N.D
0.19 ± 0.02
1.18 ± 0.10
2.41 ± 0.16
3.86 ± 0.78
5.87 ± 1.15
6.89 ± 1.47
104
TS
Cells
(% of dose)
Bile
BEI
(%)
5.46 ± 0.72
7.15 ± 1.50
7.92 ± 2.17
9.20 ± 1.73
8.64 ± 1.74
7.88 ± 1.67
TG
Cells
(% of dose)
0.92 ± 0.47
1.56 ± 0.15
1.35 ± 0.26
1.16 ± 0.45
1.27 ± 0.34
0.75 ± 0.13
12 ± 6
19 ± 2
17 ± 5
12 ± 4
14 ± 4
10 ± 4
Bile
BEI
(%)
0.39 ± 0.01
0.51 ± 0.02
0.43 ± 0.04
0.31 ± 0.04
0.24 ± 0.01
0.16 ± 0.02
0.19 ± 0.02
0.33 ± 0.01
0.33 ± 0.07
0.24 ± 0.03
0.17 ± 0.09
0.13 ± 0.03
33 ± 2
40 ± 1
42 ± 5
44 ± 4
37 ± 15
45 ± 10
Table
3.4.
Kinetic
parameters
(min-1)
estimated
from
compartmental
pharmacokinetic modeling analysis
TGZ (10 µM) was incubated in day 4 rat SCH as described under Materials and
Methods. Parameter estimates were generated by nonlinear regression of the mean
TGZ, TS, TG and TQ data in the medium, hepatocytes and bile based on the
equations described in Materials and Methods according to the model scheme
depicted in Figure 3.1.
Parameters
Estimates (min-1)
CV%
Kuptake, TGZ
0.007
2
Kuptake, TS
0.005
39
Kuptake, TG
0.001
384
Kf, TS
0.382
11
Kf, TG
0.061
12
Kf, TQ
0.001
19
Kother
0.064
25
Kefflux, TS
0.031
15
Kefflux, TG
0.16
29
Kbile, TS
0.023
17
Kbile, TG
0.061
44
Kflux, TS
0.17
22
Kflux, TG
0.094
55
105
Table 3.5. Evaluation of the sensitivity of cell and medium accumulation to detect
changes in biliary excretion of TS or TG utilizing data from Monte Carlo simulations.
Monte Carlo simulations were performed by modulating the rate constant for biliary
excretion of TS or TG by 3 different scenarios [experimentally-determined parameter
estimates; 2-fold increase; 2-fold decrease]. The relative power to detect a
statistically significant difference in medium vs. cell was calculated as the ratio of the
number of observations (sample size) necessary to detect a statistically significant
difference in TS or TG accumulation in medium to the number of observations
necessary for accumulation in hepatocytes.
Time
TG
TS
Number of Observations to
Detect a Statistically
Significant Difference
Relative
Power
(min)
(Ncell)
(Nmedium)
(NMedium/NCell)
10
20
47
2.4
20
14
19
1.4
30
14
108
7.7
60
15
662
44.1
90
15
1265
84.3
120
27
7271
269.3
10
27
144
5.3
20
9
28
3.1
30
6
13
2.2
60
4
18
4.5
90
4
33
8.3
120
5
51
10.2
106
Kflux,TS
Kefflux,TS
XTS,M
Kbile,TS
XTS,C
XTS,B
Kuptake,TS
Kf,TS
Kuptake, TGZ
XTGZ,M
XTGZ,C
Kf,TQ
Kf,TG
XTQ,M
Kother
XTG,M
Kefflux,TG
XTG,C
Kbile,TG
XTG,B
Kuptake,TG
Kflux,TG
Figure 3.1. Model scheme depicting the mass (X) of TGZ, TGZ sulfate (TS), TGZ
glucuronide (TG), and TGZ quinone (TQ) in the medium (M), hepatocytes (C), and
bile (B) of rat SCH. Kuptake, Kefflux and Kbile represent first-order rate constants for
uptake, basolateral efflux and biliary excretion, respectively. Kf, TS, Kf, TG and Kf, TQ
represent first-order rate constants for formation of TS, TG and TQ, respectively;
Kflux, TS and Kflux, TG represent first-order rate constant for flux of TS and TG from the
bile canaliculi into the incubation medium, respectively. Dotted line represents flux
from bile canaliculi to medium that occurs during incubation after the 20-min
timepoint.
107
BEI (%)
2.3
15 ± 4
34 ± 2
Accumulation (pmol/mg protein)
3000
2500
2000
1500
1000
500
0
TGZ
TS
TG
Figure 3.2. Accumulation of TGZ, TS, and TG in rat SCH. Rat SCH were treated for
10 min with 5 µM TGZ, TS, or TG and accumulation (pmol/mg protein) was
measured in cells + bile (solid bars) and in cells (open bars). Values represent the
mean of triplicate determinations from n=1 experiment (TGZ) or mean ± SEM of
triplicate determinations in n=3 experiments (TS and TG).
108
Accumulation in Medium
(pmol/well)
10000
1000
100
10
0
20
40
60
80
100
120
Time (min)
Figure 3.3. Mass vs. time profiles of TGZ (●), TS (○), TG (▼) and TQ (∆) in the
medium initially containing 10 µM TGZ in day 4 rat SCH (Mean ± S.E.M.; n=3 livers
in triplicate). Dashed lines represent the best fit of the pharmacokinetic model
derived from the scheme depicted in Figure 3.1 to the data.
109
Accumulation in Hepatocytes
(pmol/well)
10000
1000
100
10
1
0
20
40
60
80
100
120
Time (min)
Figure 3.4. Mass vs. time profiles of TGZ (●), TS (○) and TG (▼) in the hepatocytes
in day 4 rat SCH; incubation medium initially contained 10 µM TGZ (Mean ± S.E.M.;
n=3 livers in triplicate). Dashed lines represent the best fit of the pharmacokinetic
model derived from the scheme depicted in Figure 3.1 to the data.
110
Accumulation in Bile
(pmol/well)
1000
100
10
1
0
20
40
60
80
100
120
Time (min)
Figure 3.5. Mass vs. time profiles of TS (○) and TG (▼) in the bile canaliculi of day 4
rat SCH; incubation medium initially contained 10 µM TGZ (Mean ± S.E.M.; n=3
livers in triplicate). Dashed lines represent the best fit of the pharmacokinetic model
derived from the scheme depicted in Figure 3.1 to the data.
111
A
B
C
D
Figure 3.6. Monte Carlo simulations were performed to examine the impact of
modulation of TS or TG biliary excretion on the disposition of TS or TG in
hepatocytes (A and C, respectively) and medium (B and D, respectively). The rate
constant for biliary excretion of TS (Kbile,TS) or TG (Kbile,TG) was modulated by 5
different scenarios [experimentally-determined parameter estimates (black); 2-fold
increase (red); 10-fold increase (green); 2-fold decrease (yellow); 10-fold decrease
(blue)].
112
CHAPTER 4
Modulation of Trabectedin (ET-743) Hepatobiliary Disposition by Multidrug
Resistance-Associated Proteins (Mrps) May Prevent Hepatotoxicity
This Chapter has been published in Toxicology and Applied Pharmacology
(228(1):17-23, 2008) and is presented in the style of that journal.
ABSTRACT
Trabectedin is a promising anticancer agent, but dose-limiting hepatotoxicity
was observed during phase I/II clinical trials. Dexamethasone (DEX) has been
shown to significantly reduce trabectedin-mediated hepatotoxicity. The current study
was designed to assess the capability of sandwich-cultured primary rat hepatocytes
(SCRH) to predict the hepato-protective effect of DEX against trabectedin-mediated
cytotoxicity. The role of multidrug resistance-associated protein 2 (Mrp2; Abcc2) in
trabectedin hepatic disposition also was examined. In SCRH from wild-type Wistar
rats, cytotoxicity was observed after 24-hr continuous exposure to trabectedin.
SCRH pretreated with additional DEX (1 µM) exhibited a 2-3-fold decrease in toxicity
at 100 nM and 1000 nM trabectedin. Unexpectedly, toxicity in SCRH from Mrp2deficient (TR-) compared to wild-type Wistar rats was markedly reduced. Depletion of
glutathione from SCRH using buthionine sulfoximine (BSO) mitigated trabectedin
toxicity associated with 100 nM and 1000 nM trabectedin. Western blot analysis
demonstrated increased levels of CYP3A1/2 and Mrp2 in SCRH pretreated with
DEX; interestingly, Mrp4 expression was increased in SCRH after BSO exposure.
Trabectedin biliary recovery in isolated perfused livers from TR- rats was decreased
by ~75% compared to wild-type livers. In conclusion, SCRH represent a useful in
vitro model to predict the hepatotoxicity of trabectedin observed in vivo. The
protection by DEX against trabectedin-mediated cytotoxicity may be attributed, in
part, to enhanced Mrp2 biliary excretion and increased metabolism by CYP3A1/2.
Decreased trabectedin toxicity in SCRH from TR- rats, and in SCRH pretreated with
BSO, may be due to increased basolateral excretion of trabectedin by Mrp3 and/or
114
Mrp4.
115
INTRODUCTION
Trabectedin (Ecteinascidin 743, YondelisTM) is a DNA alkylating agent
extracted from the marine tunicate Ecteinascidia turbinate that exhibits potent
antineoplastic activity against a variety of tumor cells at nanomolar concentrations
(Izbicka et al., 1998).
Trabectedin is currently under phase II/III evaluation in
advanced gastrointestinal stromal tumors, pretreated soft tissue sarcoma, ovarian
cancer and breast cancer (Laverdiere et al., 2003; Robey et al., 2004; Yovine et al.,
2004; Zelek et al., 2006).
Trabectedin is metabolized primarily by CYP 3A4; 2C9, 2C19, 2D6 and 2E1,
and
to
a
lesser
extent
conjugated
by
the
phase
II
enzymes
uridine
diphosphoglucuronosyl transferase (UGT) and glutathione-S-transferase (GST)
(Allen et al., 2002; Brandon et al., 2006). Extensive metabolism of trabectedin was
demonstrated in a mass balance study conducted in patients with advanced cancer
(Beumer et al., 2007b). Following i.v. [14C]trabectedin administration to patients,
~55.5% and ~5.9% of the dose was recovered in feces and urine, respectively,
suggesting that trabectedin and its metabolites undergo biliary excretion (Beumer et
al., 2005a).
However, the involvement of transport proteins in hepatobiliary
disposition of trabectedin remains to be elucidated.
During phase I/II clinical trials, the most prevalent adverse effect associated
with trabectedin treatment was dose-limiting hepatotoxicity secondary to increases
in blood alkaline phosphatase and/or bilirubin levels (Delaloge et al., 2001;
Taamma et al., 2001; Lee et al., 2005a). An in vitro cytotoxicity study using HepG2
cells showed increased trabectedin-mediated toxicity after 1-hr preincubation with
116
inhibitors of cytochrome P450 (CYP) enzymes involved in trabectedin metabolism;
CYP inducers and phase II enzyme inhibitors did not significantly affect
trabectedin-mediated toxicity, suggesting that trabectedin metabolites may be less
toxic compared to the parent compound (Brandon et al., 2005). Pretreatment of
female Fisher rats with DEX has been shown to ameliorate the hepatotoxic effects
of trabectedin while coadministration of DEX with trabectedin did not reduce
toxicity (Donald et al., 2003). Consistent with the protective effect of DEX on
trabectedin-mediated toxicity in rats, DEX premedication markedly reduced
trabectedin-mediated hepatotoxicity in patients with advanced sarcoma (Grosso et
al., 2006). However, this hepato-protective effect of DEX could not be mimicked in
vitro using primary rat hepatocytes cultured on a fibronectin matrix (Donald et al.,
2004).
Significant induction of Mrp2 mRNA and protein by DEX treatment has been
reported in primary rat and mouse hepatocytes (Kast et al., 2002; Luttringer et al.,
2002; Turncliff et al., 2004). Mrp2, a transport protein localized on the hepatic
canalicular plasma membrane, is responsible for the biliary excretion of organic
anions as well as numerous drugs including pravastatin, vincristine and etoposide
(Cui et al., 1999; Kawabe et al., 1999; Konig et al., 1999a; Sasaki et al., 2002).
Animals with hereditary conjugated hyperbilirubinemia [Mrp2-deficient rats (TR-)
and Eisai hyperbilirubinemic rats (EHBR)] have been used extensively to examine
the substrate specificity and in vivo function of Mrp2 (Westley et al., 2006; ZamekGliszczynski et al., 2006a). Patients with Dubin-Johnson syndrome lack MRP2
and hence suffer from hereditary conjugated hyperbilirubinemia (Paulusma et al.,
117
1997).
In the absence of Mrp2/MRP2, rats and humans exhibit increased
expression of basolateral Mrp3/MRP3 as a compensatory mechanism that enables
the excretion of Mrp2/MRP2 substrates into the systemic circulation, thus avoiding
excessive accumulation of organic anions in the hepatocyte (Kiuchi et al., 1998;
Hirohashi et al., 1999; Johnson et al., 2006; Nies and Keppler, 2007).
Drug-induced liver injury has emerged as the most common reason for the
withdrawal of Food and Drug Administration-approved drugs from the market
(Abboud and Kaplowitz, 2007; FDA, 2007).
Covalent adduct formation,
immunotoxicity, and disruption of cellular bioenergetics have been considered
previously as mechanisms underlying hepatotoxicity, but more recent data suggest
that hepatic transport proteins may be an important site of toxic interactions
(Fattinger et al., 2001; Funk et al., 2001a; Leslie et al., 2007). The objective of this
study was to evaluate the capability of sandwich-cultured primary rat hepatocytes
(SCRH), which more closely mimic the metabolic and transport capabilities of the
intact liver, to predict the hepato-protective effect of DEX against trabectedinmediated hepatotoxicity. An additional objective was to examine the role of Mrp2 in
trabectedin hepatobiliary disposition.
118
MATERIALS AND METHODS
Materials. Trabectedin (Ecteinascidin 743, YondelisTM, NSC 648766) was supplied
by the National Cancer Institute, Developmental Therapeutics Program Repository
(http://dtp.nci.nih.gov/branches/dscb/repo_open.html) and was prepared as a 10 mM
stock solution in DMSO. DEX and BSO were purchased from Sigma Chemical Co.
(St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), human recombinant
insulin, modified essential media non-essential amino acids, L-glutamine, penicillin
and streptomycin were purchased from GIBCO (Invitrogen Corporation, Grand
Island, NY).
ITS+ (insulin, transferin, and selenium) was purchased from BD
Biosciences (Bedford, MA).
Animals. Male wild-type Wistar rats (220-300 g; Charles River Laboratories, Raleigh,
NC) and male TR- Wistar rats (220-300 g; in-house breeding colony originally
obtained from Dr. Mary Vore, University of Kentucky, Lexington, KY) were used for
hepatocyte isolation and as liver donors for isolated perfused liver experiments. Male
wild-type Wistar rats (>400 g) were used as blood donors for isolated perfused liver
experiments.
Rats were anesthetized with ketamine/xylazine (60/12 mg/kg
intraperitoneally) before surgical manipulation. The Institutional Animal Care and
Use Committee of the University of North Carolina at Chapel Hill approved all
procedures.
Hepatocyte Isolation and Culture. Hepatocytes were isolated from male Wistar or
TR- rats (220-300 g) by a modification of the two-step collagenase digestion method
as described previously (Liu et al., 1998).
determined by trypan blue exclusion.
Hepatocyte viability was >85% as
Hepatocytes were plated at a density of
119
~1.5×106 cells/well in 6-well plates coated with 0.1 ml of rat tail collagen Type I
solution (1.5 mg/ml, pH 7.4) in 1.5 ml of plating medium (phenol red-free DMEM
containing 5% fetal bovine serum, 0.1 mM MEM nonessential amino acids, 2 mM Lglutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 4 µg/ml human recombinant
insulin and 1 µM DEX), and allowed to attach for 2-3 hr at 37 ˚C in a humidified
incubator with 95%O2/5%CO2. After attachment, medium was replaced to remove
dead/unattached cells. To achieve a sandwich configuration, medium was aspirated
and cells were overlaid with 0.1 ml of rat tail collagen Type I solution (1.5 mg/ml, pH
7.4) at 24 hr (day 1). Collagen was allowed to gel for 1 hr in a 37 ˚C humidified
incubator 95%O2/5% CO2, and then culture medium was added (1.5 ml/well; phenol
red-free DMEM supplemented with 0.1 mM MEM nonessential amino acids, 2 mM Lglutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1% ITS+ and 0.1 µM DEX).
Subsequently, the culture medium was changed daily until the conduct of
experiments.
DEX and BSO Pretreatment in SCRH. From day 2 to day4, additional DEX (1 µM)
or BSO (500 µM) was added to the culture medium containing 0.1 µM DEX. On day
3, SCRH were incubated for 24 hr in culture medium containing a range of
trabectedin
concentrations
(1-1000
nM)
to
compare
trabectedin-mediated
cytotoxicity between SCRH pretreated with vehicle (0.1 µM DEX) and additional
DEX (1 µM) or BSO (500 µM). DEX and trabectedin were prepared in DMSO, and
BSO was solubilized in water. Stock solutions were diluted in culture medium for
treatment.
The final concentration of DMSO was <0.2% and had no effect on
untreated controls.
120
Determination of Cell Viability Using LDH Release. LDH leakage into the culture
medium was measured at the end of the 24 hr exposure using the Cytotoxicity
Detection kit (Roche, Indianapolis, IN) according to the manufacturer’s instructions.
The degree of LDH release was expressed as a percentage of the maximum cellular
LDH release, which was measured by adding 2% Triton X-100 to SCRH.
Cytotoxici ty (% of control) =
LDH sample − LDHblank
LDH Triton X -100 − LDHblank
Western Blot Analysis of Mrp2, 3, 4 and CYP3A1/2 Proteins. On day 4, the
medium was removed and cells were washed twice with ice-cold phosphate-buffered
saline (PBS). Subsequently, cells were lysed with 400 µl of lysis buffer [1 mM EDTA,
1% sodium dodecyl sulfate (SDS), pH 8, with Complete protease inhibitor cocktail
tablets (Roche Diagnostics, Mannheim, Germany)] in each well of a 6-well plate.
Lysates were stored at -20 ˚C until western blot analysis. The whole cell lysates
were thawed on ice and protein concentrations were determined using the BCA
protein assay (Pierce, Rockford, IL). Protein samples (25 µg/well) were separated
by SDS-polyacrylamide gel electrophoresis, and electrotransferred to polyvinylidene
difluoride (PVDF) membranes (Millipore, Billerica, MA). Blots were blocked with Trisbuffered saline with 0.1% Tween 20 (TBS-T) containing 5% nonfat dry milk for 1 hr at
room temperature. Subsequently, the membrane was incubated with appropriate
primary antibodies for 2 hr at room temperature or over-night at 4 ˚C and then rinsed
three times at 10-min intervals with TBS-T. Mrp2, 3 and 4 were detected using
monoclonal antibodies M2III-6, M3II-9 and M4I-10, respectively, at a 1:1000 dilution
(Alexis Biochemicals, San Diego, CA).
CYP3A1/2 protein expression was
determined with a polyclonal antibody against rat CYP3A1/2 at a 1:1500 dilution
121
(Xenotech, Kansas City, KS). Protein loading was normalized to β-actin expression
using a β-actin antibody at a dilution of 1:5000 (C4; Chemicon, San Fransisco, CA).
Immunoreactive protein bands were detected by chemilumiscence using a Bio-Rad
VersaDoc imaging system and densitometry analysis was achieved using the
Quantity One software package v.4.1 (Bio-Rad Laboratories, Hercules, CA).
Isolated Perfused Liver Study. Recirculating isolated perfused liver (IPL) studies
were performed with male Wistar wild-type and TR- rat livers as described previously
(Brouwer and Thurman, 1996). Briefly, livers were perfused in situ with oxygenated
Krebs-Henseleit bicarbonate buffer (pH 7.4) after cannulation of the bile duct and
portal vein. Livers were removed from the body cavity and placed into a humidified
perfusion chamber at 37 ˚C.
Perfusion was continued with oxygenated buffer
containing 20% (v/v) heparinized male rat blood at a flow rate of 20 ml/min. Livers
were allowed to equilibrate for 10-15 min prior to bolus administration of 24 nmol of
trabectedin (preliminary recovery studies in the perfusion system without the liver
indicated that the actual dose delivered to the IPL was decreased by 50% due to
nonspecific binding/degradation in blood-containing perfusate). Liver viability was
assessed by monitoring portal pressure (<15 cm of H2O), observing gross
morphology, and measuring bile flow in perfused livers from control and TR- rats
(>0.8 and >0.3 µl/min/g liver, respectively). Taurocholate (0.5 µmol/min, in saline)
was infused to maintain bile flow. Bile was collected in toto every 10 min, and the
volume was determined gravimetrically (specific gravity 1.0).
Perfusate samples
(0.5 ml) were immediately centrifuged following collection to obtain plasma for
analysis.
122
Analytical Methods. Bile and perfusate samples were analyzed by liquid
chromatography with detection by tandem mass spectrometry (Applied Biosystems
API 4000 triple quadrupole with TurboIonSpray interface, MDS Sciex, Concord, ON,
Canada).
Trabectedin and cimetidine (internal standard) were eluted from an
Aquasil C18 column (dp=5µm, 2.1 x 50 mm, Thermo-Electron, Waltham, MA) using a
mobile phase gradient (A: water with 0.1% formic acid, B: methanol with 0.1% formic
acid); 0-2.5 min hold at 95% B, a linear gradient to 10% B from 2.5-3 min, 3-4 min
hold at 10% B; flow rate was 0.75 mL/min (Shimadzu solvent delivery system,
Columbia, MD). Trabectedin and cimetidine were detected in positive ion mode
using multiple reaction monitoring; trabectedin: 744.2 495.2 m/z, cimetidine: 250.9
156.9 m/z. Trabectedin was quantified with calibration curves prepared in the
appropriate matrix using analyte:internal standard (trabectedin:cimetidine) peak area
ratios. The lower limit of detection was 1 nM for all analytes and standard curves
ranged from 1 to 1000 nM.
Statistical Analysis. Statistical significance was assessed using two-way analysis of
variance with Bonferroni’s post hoc test, except where the groups being compared
had unequal variances, in which case log-transformed data were assessed. SCRH
data were presented as the mean ± S.E.M. when at least three sets of independent
experiments were performed in triplicate. In all cases, p < 0.05 was considered to
be statistically significant.
123
RESULTS
DEX effect on trabectedin-mediated cytotoxicity in SCRH from wild-type rats
Trabectedin-mediated cytotoxicity was defined as LDH release, relative to a
Triton X-100 treated control, from hepatocytes into the culture medium. Following 2
hr of trabectedin exposure, LDH release was not increased relative to untreated
SCRH (data not shown).
However, cytotoxicity was observed after 24 hr of
continuous trabectedin exposure to SCRH (Figure 4.1).
From day 2 to day 4,
additional DEX (1 µM) was added to the culture medium containing 0.1 µM DEX to
examine the effect of DEX on trabectedin-mediated cytotoxicity; SCRH were
incubated with trabectedin for 24 hr starting on day 3. This pretreatment of SCRH
with additional DEX (1 µM) decreased toxicity of trabectedin compared to vehiclepretreated SCRH (Figure 4.1).
Trabectedin-mediated cytotoxicity in SCRH from TR- rats
Primary hepatocytes from Mrp2-deficient TR- rats were utilized to examine the
involvement of Mrp2 in trabectedin-mediated cytotoxicity at 0.1 µM DEX.
Unexpectedly, cytotoxicity caused by exposure to 50 nM and 100 nM trabectedin in
TR- SCRH was reduced compared to that in wild-type SCRH (Figure 4.2).
BSO effect on trabectedin-mediated cytotoxicity in wild-type and TR- SCRH
Elevated levels of glutathione in TR- compared to wild-type rat hepatocytes
has been proposed as one mechanism that can protect the hepatocyte from druginduced hepatotoxicity (Lu et al., 1996). To determine the effect of GSH depletion on
trabectedin cytotoxicity, BSO (500 µM), an inhibitor of γ-glutamylcysteine synthetase
which is the rate-liming step in GSH synthesis, was incubated for 24 hr prior to
124
trabectedin exposure in SCRH from wild-type and TR- rats (Griffith and Meister,
1979). Interestingly, toxicity in wild-type SCRH pretreated with BSO was reduced
compared to vehicle-pretreated wild-type SCRH (Figure 4.3). Trabectedin-mediated
cytotoxicity in BSO-pretreated TR- SCRH was not different from vehicle-pretreated
TR- SCRH (data not shown).
Alteration of Mrp2, 3, 4 and CYP3A1/2 protein expression by DEX and BSO
When SCRH were cultured with additional DEX (1 µM), the fold increase in
expression of CYP3A1/2 and Mrp2 was 5.0 ± 2.5 and 2.5 ± 0.6, respectively,
compared to vehicle, whereas the fold increase in protein expression of Mrp3 and
Mrp4 was 1.2 ± 0.7 and 1.4 ± 0.4, respectively (Figure 4.4). Interestingly, in BSOpretreated SCRH, the fold increase in Mrp4 expression was 3.1 ± 0.7 whereas the
fold increase in Mrp2, Mrp3 and CYP3A1/2 protein expression was 1.5 ± 0.7, 1.9 ±
0.4 and 1.2 ± 0.9, respectively.
Hepatobiliary disposition of trabectedin in IPLs
IPLs from wild-type and TR- rats were utilized to examine the involvement of
Mrp2 in trabectedin hepatobiliary disposition.
Trabectedin concentrations in
perfusate were not different between livers from wild-type and TR- rats; trabectedin
was rapidly extracted by the liver and concentrations were negligible after 20 min
(Figure 4.5.a). Biliary recovery of trabectedin represented less than 3% of the dose
in wild-type rat livers, consistent with extensive metabolism of trabectedin in rats as
reported previously (Figure 4.5.b) (Allen et al., 2002). Biliary recovery of trabectedin
in IPLs from TR- rats was decreased by ~75% compared to wild-type IPLs.
125
DISCUSSION
SCRH were selected to investigate the hepato-protective effect of DEX
against trabectedin-mediated cytotoxicity because they maintain metabolic enzyme
activity and develop functional bile canalicular networks with proper localization of
hepatic transport proteins (Liu et al., 1998; LeCluyse et al., 1999; Liu et al., 1999a;
Hoffmaster et al., 2004a). Previous experiments in freshly isolated rat hepatocytes
cultured in a conventional configuration were unable to replicate the hepatoprotective effect of DEX against trabectedin-mediated cytotoxicity when rats were
pretreated orally with 20 mg/kg/day of DEX 24 hr prior to hepatocyte isolation
(Donald et al., 2004). When freshly isolated hepatocytes are cultured on a single
collagen substratum (conventional configuration), hepatocytes do not form bile
canalicular networks, do not re-establish cell polarity, and gradually lose expression
of CYP enzymes (LeCluyse et al., 1996; George et al., 1997). The loss of CYP
expression over time in culture of human hepatocytes varies for each isoform
(George et al., 1997); mRNA levels of some P450s such as CYP1A2 and CYP2C9
did not change over time in culture whereas CYP2E1 and CYP3A4 expression
declined gradually over time in culture. Therefore, induction of CYP3A protein by
DEX prior to hepatocyte isolation may be lost during culture in a conventional
configuration. In contrast, incubation of SCRH with additional DEX (1 µM) increased
CYP3A1/2 protein levels compared to vehicle-treated SCRH (Figure 4.4) and may
provide hepato-protection from trabectedin-mediated cytotoxicity.
Vectorial transport studies in polarized cell monolayers examining cellular
accumulation of [14C] trabectedin in the absence and presence of P-gp inhibitors
126
indicated that trabectedin is a substrate for P-gp (Beumer et al., 2007a). However,
P-gp was only able to confer resistance to trabectedin when expressed at very high
levels, suggesting that the intrinsic clearance of P-gp for trabectedin is low. Even
though trabectedin seems to be more toxic than its metabolites based on previous
cytotoxicity studies (Brandon et al., 2005), limited information is available on the
interaction of other hepatic transport proteins with trabectedin.
The present studies are the first to demonstrate the involvement of Mrp2 in
the biliary excretion of trabectedin in whole rat livers. Although the biliary excretion of
unchanged trabectedin was low as a percentage of the total dose, the biliary
excretion of trabectedin in IPLs from TR- rats was decreased ~75% compared to
wild-type IPLs (Figure 4.5.b). As shown in Figure 4.4, induction of Mrp2 protein
expression by additional DEX (1µM) in SCRH may provide hepato-protection from
trabectedin-mediated cytotoxicity by increasing the biliary excretion of parent
compound and/or derived metabolites.
Interestingly, TR- SCRH were more resistant to trabectedin-mediated
cytotoxicity relative to wild-type controls, despite the increased potential for
trabectedin accumulation in TR- hepatocytes due to impaired biliary excretion. This
observation could be explained by modulation of the expression/activity of hepatic
basolateral efflux transport proteins and metabolic enzymes in TR- rats.
Mrp2-
deficient rats are well-known to exhibit higher Mrp3 expression (Hirohashi et al.,
1998; Ogawa et al., 2000; Johnson et al., 2006). Mrp3 mediates hepatic basolateral
efflux of substrates from hepatocytes to the blood, and is thought to compensate for
impaired biliary excretion of organic anions during Mrp2 deficiency (Hirohashi et al.,
127
2000; Xiong et al., 2000). In addition, the expression and activity of several phase I
and II metabolic enzymes that may be involved in trabectedin detoxification are
modulated in TR- rats. For example, the total P450 content and activity of CYP1A1/2
and CYP2B1/2 were significantly higher in TR- compared to wild-type rats, although
testosterone 6β-hydroxylation mediated by CYP3A1/2 was lower in TR- compared to
wild-type rats (Newton et al., 2005; Silva et al., 2005). TR- rats exhibit up-regulated
UGT1a (Johnson et al., 2006). Since Mrp2 plays an important role in GSH biliary
excretion, impaired function of Mrp2 in TR- rats has been attributed to higher
hepatocellular concentrations of GSH relative to wild-type rats (Lu et al., 1996). The
important role of GSH in detoxification of the reactive metabolite of acetaminophen,
N-acetyl-p-benzoquinoneimine, was demonstrated recently in TR- rats (Silva et al.,
2005); TR- rats were more resistant to acetaminophen hepatotoxicity compared to
wild-type rats, but this protection against acetaminophen hepatotoxicity in TR- rats
was completely reversed by BSO treatment.
Previous studies in cultured rat
hepatocytes have documented that 200 µM BSO reduced GSH levels by 50% after 5
hr (Sinbandhit-Tricot et al., 2003). Interestingly, in the present study, pretreatment of
SCRH with BSO (500 µM) for 48 hr decreased trabectedin–mediated cytotoxicity
relative to vehicle-pretreated SCRH.
A novel finding in the current study was induction of Mrp4 in SCRH
associated with BSO treatment.
MRP4 is a basolateral protein involved in the
transport of xenobiotics and endogenous substrates such as cyclic nucleotides
(cAMP and cGMP) and bile acids (van Aubel et al., 2002; Reid et al., 2003b; Rius et
al., 2003; Tian et al., 2006).
MRP4 mediates the efflux of GSH across the
128
basolateral membrane of hepatocytes into blood by co-transport with monoanionic
bile acids (Rius et al., 2003).
BSO may play a role as a GSH-depleting agent not
only by inhibiting synthesis, but also by enhancing hepatic efflux of GSH out of the
hepatocyte by increasing the expression of Mrp4. Whether upregulation of Mrp4 by
BSO is a direct effect, or an indirect response associated with oxidative stress due to
GSH depletion, requires further investigation. MRP4 mRNA and protein expression
were significantly up-regulated in liver samples from patients with cholestasis,
indicating a potential role of MRP4 in protecting the liver by decreasing the
intracellular accumulation of detergent-like bile acids (Gradhand et al., 2007).
Hence, the protective role of BSO in trabectedin-mediated cytotoxicity may be due to
increased efflux of trabectedin or toxic metabolites by Mrp4, although the affinity of
trabectedin for Mrp4 is unknown.
In conclusion, the hepato-protective effect of DEX on trabectedin toxicity may
be due to increased metabolism by CYP3A1/2 as well as enhanced biliary excretion
by Mrp2. Although the interaction of trabectedin and/or derived metabolites with
basolateral transport proteins such as Mrp3 and 4 remains to be elucidated, the
unexpected attenuation of trabectedin toxicity in SCRH from TR- rats, and in SCRH
pretreated with BSO, may be associated with increased basolateral excretion of
trabectedin /metabolites by Mrp3 and/or Mrp4.
129
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135
Cytotoxicity (% of control)
35
30
25
20
*
15
10
*
5
*
*
0
1
10
50
100
1000
Trabectedin (nM)
Figure 4.1. Effect of DEX on trabectedin-mediated cytotoxicity in SCRH isolated
from wild-type Wistar rats. Cytotoxicity was determined by measuring LDH release
into the culture medium after 24 hr trabectedin (1-1000 nM) exposure. Each bar
represents the mean ± S.E.M. (n=3) for samples from vehicle- (0.1 µM DEX; solid
bars) or additional DEX (1 µM; white bars); *, p < 0.05.
136
Cytotoxicity (% of control)
35
30
25
20
15
10
*
*
50
100
5
0
1
10
Trabectedin (nM)
Figure 4.2. Trabectedin-mediated cytotoxicity in SCRH from wild-type and TR- rats.
Cytotoxicity was determined by measuring LDH release into the culture medium
after 24 hr trabectedin (1 - 100 nM) exposure. Each bar represents the mean ±
S.E.M. (n=3) for samples from wild-type (solid bars) or TR- (white bars) SCRH; *, p <
0.05.
137
Cytotoxicity (% of control)
35
30
25
20
*
15
10
5
*
*
0
50
100
1000
Trabectedin (nM)
Figure 4.3. Effect of BSO on trabectedin-mediated cytotoxicity. Cytotoxicity was
determined by measuring LDH release into the culture medium after 24 hr
trabectedin (50, 100 and 1000 nM) exposure. BSO (500 µM) was added to the
culture medium 24 h prior to trabectedin exposure. Each bar represents the mean ±
S.E.M. (n=3) for samples from vehicle- (solid bars) or BSO-pretreated (white bars)
SCRH; *, p < 0.05.
138
Mrp2
Mrp3
Actin
CYP3A1/2
Mrp4
Actin
Figure 4.4. Representative Western blot from whole cell lysates for Mrp2, 3, 4 and
CYP3A1/2 in SCRH pretreated with vehicle (0.1 µM DEX), additional DEX (1 µM) or
BSO (500 µM) (25 µg protein/well; n=3).
139
Trabectedin in Perfusate (nM)
A
60
50
40
30
20
10
0
0
5
10 20 30 40 50 60 70 80 90
B
Trabectedin Biliary Excretion
(% of dose)
Time (min)
4
3
2
1
0
0
5 10 20 30 40 50 60 70 80 90
Time (min)
Figure 4.5. Trabectedin perfusate concentrations (A) and cumulative biliary
excretion (B) in isolated perfused livers from wild-type (solid circle) and TR- (white
triangle) rats. Data are presented as mean ± S.D., n= 3/group.
140
CHAPTER 5
Interactions Between Sulindac, Sulindac Sulfone, Sulindac Sulfide and
Hepatic Transport Proteins
This Chapter will be submitted to the Journal of Pharmacology and Experimental
Therapeutics
ABSTRACT
Sulindac, a nonsteroidal anti-inflammatory drug (NSAID) used clinically to
relieve symptoms of rheumatoid arthritis and osteoarthritis, is associated with a
higher incidence of hepatotoxicity compared to other NSAIDS. The role of hepatic
transport proteins in sulindac-mediated hepatotoxicity remains to be evaluated.
Accordingly, model substrates for hepatic transport proteins [[3H]taurocholate (TC),
[3H]estradiol 17-β-glucuronide (E217G), nitrofurantoin (NF)] were utilized in rat and
human sandwich-cultured hepatocytes (SCH) and suspended hepatocytes to
determine whether sulindac and/or sulindac metabolites (S-sulfone and S-sulfide)
interfere with hepatic transport protein activity. In rat SCH, compared to control, Ssulfone and S-sulfide (100 µM) decreased TC biliary excretion index (BEI) by 42%
and 55%, respectively, but had no effect on E217G BEI; in contrast,
sulindac/metabolites (100 µM) decreased both TC and E217G in vitro biliary
clearance (Clbiliary) by 38-83% and 81-97%, respectively, consistent with impaired
uptake of TC and E217G and impaired excretion of TC. In suspended rat
hepatocytes, S-sulfone and S-sulfide inhibited Na+-dependent TC initial uptake (IC50
of 25.6±10.7 and 12.7±5.3 µM, respectively) and Na+-independent E217G initial
uptake (IC50 of 12.6±5.1 and 6.5±1.2 µM, respectively). In rat SCH, compared to
control, sulindac, S-sulfone and S-sulfide (100 µM) decreased NF BEI and Clbiliary by
30-57%. In suspended human hepatocytes, S-sulfone and S-sulfide inhibited Na+dependent TC initial uptake (IC50 of 42.2 and 3.1 µM, respectively). In conclusion,
inhibition of multiple hepatic transport proteins (Na+/taurocholate cotransporting
protein, organic anion transporting polypeptides, bile salt export pump, breast cancer
142
resistance protein) by sulindac/metabolites may play a role in clinically significant
drug interactions and/or liver injury.
143
INTRODUCTION
Sulindac is a nonsteroidal anti-inflammatory drug (NSAID) indicated for the
relief of signs and symptoms of arthritic conditions, including osteoarthritis and
rheumatoid arthritis. Sulindac is reduced by the aldehyde oxidase system to sulindac
sulfide (S-sulfide) (Figure 5.1), which has analgesic and anti-inflammatory properties.
S-sulfide is converted by flavin-containing monooxygenase 3 back to sulindac and to
sulindac sulfone (S-sulfone), which has been recognized as a promising
antiproliferative agent in colon cancer (Tatsumi et al., 1983; Davies and Watson,
1997; Hamman et al., 2000). Among NSAIDs, sulindac has been associated most
consistently with hepatotoxicity (Aithal and Day, 2007), particularly with cholestatic
hepatitis, and to a lesser extent with hepatocellular damage (Tarazi et al., 1993).
Despite the high incidence of liver injury, the mechanism(s) underlying sulindacmediated hepatotoxicity remain(s) to be elucidated.
Inhibition of hepatic transport proteins has been proposed as one mechanism
of drug-induced liver injury (DILI). Inhibition of the canalicular bile salt export pump in
rats (Bsep; ABCB11) by hepatotoxic drugs such as troglitazone and bosentan may
lead to elevated hepatic concentrations of detergent-like bile acids, which can
disrupt cellular function (Fattinger et al., 2001; Funk et al., 2001a). Several hepatic
transport proteins other than Bsep also are involved in bile acid transport, acting in
concert to maintain homeostasis of bile acids. In humans, conjugated and
unconjugated bile acids are taken up primarily by Na+-dependent taurocholate cotransporting polypeptide (NTCP; SLC10A1) with high affinity, as well as by Na+independent organic anion transporting polypeptides (OATPs; SLCOs). Once taken
144
up by the hepatocytes, bile acids are excreted into the canalicular lumen primarily by
the bile salt export pump (BSEP). Alternatively, bile acids can be excreted into
sinusoidal blood by basolateral hepatic transport proteins such as the multidrug
resistance-associated protein (MRP; ABCC) 3, MRP4 and the organic solute
transporter (OST) α/β (Kosters and Karpen, 2008).
The involvement of hepatic transport proteins in DILI and some drug
interactions has been established (Smith et al., 2005b; Pauli-Magnus and Meier,
2006). Furthermore, interactions between sulindac and hepatic transport proteins
have been demonstrated. Sulindac inhibited organic anion transporter (OAT) 1mediated [14C]para-aminohippurate uptake (IC50 ~36 µM) and OAT3-mediated
[3H]estrone sulfate uptake (IC50 ~3 µM) in stably transfected cells; sulindac also was
the most potent inhibitor among tested NSAIDs of organic cation transporter (OCT) 1
and 2 in stably transfected cells (Khamdang et al., 2002). Sulindac also inhibited
[3H]methotrexate transport in MRP2- and MRP4-overexpressing membrane vesicles,
with IC50 values of ~38 µM and ~2 µM, respectively (El-Sheikh et al., 2006); MRP2
functions to excrete compounds from the hepatocyte into bile. Finally, S-sulfide was
shown to be a potent inhibitor of MRP4-mediated leukotriene B4 and C4 transport in
MRP4-overexpressing membrane vesicles (Rius et al., 2008).
Sulindac is metabolized extensively by the liver in rats as well as in humans,
and plasma concentrations of sulindac metabolites are sustained due to extensive
enterohepatic recycling (Dujovne et al., 1983). In rats, S-sulfone is the major
metabolite in the plasma. After intravenous administration of sulindac (10 mg/kg) in
rats, the plasma area under the concentration-time curve (AUC) for S-sulfone was
145
~2.5-fold higher than that of sulindac, whereas the AUC for S-sulfide was only ~40%
that of sulindac (Duggan et al., 1978); Clbiliary of sulindac and S-sulfone were
comparable, whereas S-sulfide Clbiliary was only 40% of that of sulindac. In humans,
after oral administration of sulindac, peak plasma total concentrations (Cmax) were
achieved after 1 h for sulindac (10-34 µM), and after 2-4 h for S-sulfone (4-19 µM)
and S-sulfide (3-33 µM) (Davies and Watson, 1997); the apparent Clbiliary of sulindac
and S-sulfone, measured by an occludable T-tube that was placed in the common
bile duct of patients following elective gallbladder surgery, was ~25-fold and ~18-fold
greater, respectively, than that of S-sulfide (Dobrinska et al., 1983). Several groups
have reported a proportional relationship between S-sulfide concentrations and the
incidence of hepatotoxicity, emphasizing the potential for metabolites, in addition to
the parent drug to cause hepatotoxicity (Duggan et al., 1978; Laffi et al., 1986).
The aim of the present study was to examine the interactions between
hepatic transport proteins and sulindac, S-sulfone and S-sulfide. First, interactions
between sulindac/metabolites and the model substrates [3H]taurocholate (TC;
substrate for Ntcp/Oatp/Bsep), [3H]estradiol 17-β-glucuronide (E217G; substrate for
Oatp/Mrp2) and nitrofurantoin (NF; substrate for breast cancer resistance protein
(Bcrp, a canalicular efflux transport protein) were examined in rat sandwich-cultured
hepatocytes (SCH). Second, the inhibitory potency (IC50) of sulindac metabolites on
Na+-dependent TC initial uptake and Na+-independent E217G initial uptake was
examined
in
freshly-isolated
rat
hepatocytes.
Third,
the
impact
of
sulindac/metabolites on the hepatobiliary disposition of TC was examined in human
SCH, and suspended human hepatocytes were used to determine the IC50 of
146
sulindac metabolites on Na+-dependent TC initial uptake. Results suggested that
inhibition of multiple transport proteins by sulindac/metabolites could play a role in
sulindac-mediated liver injury.
147
MATERIALS AND METHODS
Chemicals and Reagents. [3H]TC, [3H]E217G and [14C]inulin were purchased from
PerkinElmer Life and Analytical Sciences (Boston, MA). Dulbecco’s modified Eagle’s
medium (DMEM) and MEM non-essential amino acids were purchased from
Invitrogen (Carlsbad, CA). BioCoatTM culture plates, MatrigelTM extracellular matrix,
and insulin/transferrin/selenium culture supplement (ITSTM) were purchased from BD
Biosciences Discovery Labware (Bedford, MA). NF, TC, E217G, sulindac, S-sulfone,
S-sulfide, penicillin-streptomycin solution, dexamethasone, Hanks’ Balanced Salt
Solutions (HBSS), HBSS modified (HBSS without Ca2+ and Mg+) and Triton X-100
were purchased from Sigma-Aldrich, Inc (St. Louis, MO). All other chemicals and
reagents were of analytical grade and were readily available from commercial
sources.
Culture of Primary Rat and Human Hepatocytes in a Sandwich Configuration.
Primary rat hepatocytes were cultured as described previously (Marion et al., 2007).
Briefly, hepatocytes were seeded at a density of 1.75×106 cells/well onto 6-well
BioCoatTM plates. Approximately 24 hours later, hepatocytes were overlaid with 2 mL
of 0.25 mg/mL BD MatrigelTM basement membrane matrix in DMEM containing 1%
(v/v) ITS+TM, 0.1 µM dexamethasone, 2 mM L-glutamine, 1% (v/v) MEM nonessential amino acids, 100 units penicillin G sodium/mL and 100 µg streptomycin
sulfate/mL. CellzDirect (Durham, NC) kindly donated human hepatocytes cultured in
6-well plates, seeded at a density of 1.5×106 cells/well, and overlaid with Matrigel™.
Hepatocytes were cultured for 4 days (rat) or 7-8 days (human) to allow extensive
148
formation of canalicular networks between cells before experimentation; medium
was changed daily.
Transport Studies using Rat and Human Sandwich-Cultured Hepatocytes
(SCH). The method to determine substrate accumulation in SCH has been
described previously (Liu et al., 1999e). Briefly, SCH were washed twice with 2 ml of
warm HBSS containing Ca2+ (standard; cells+bile) or Ca2+-free (cells), followed by
incubation in the same buffer for 10 min at 37˚C. Subsequently, cells were incubated
at 37˚C for 10 min with 1.5 mL standard HBSS containing NF (5 µM), [3H]TC (1 µM;
100 nCi/mL), or [3H]E217G (1 µM; 100 nCi/mL) in the presence of sulindac or its
metabolites (0-100 µM). After 10 min, cells were washed 3 times with ice-cold
standard HBSS. Cells were lysed either with 1 mL of methanol/water (70/30; v/v)
(NF studies) or 0.5% (v/v) Triton X-100 in phosphate-buffered saline (TC and E217G
studies), and plates were shaken for at least 20 min. Samples of cells+bile and cells
were quantified by HPLC-MS/MS for NF, and by liquid scintillation spectroscopy
(Packard Tricarb, Packard Corp., Meriden, CT) for [3H]TC or [3H]E217G, and
transport function was normalized to the protein content of the hepatocytes using a
BCA protein assay (Pierce, Rockford, IL). The biliary excretion index (BEI; %) and in
vitro biliary clearance (Clbiliary; mL/min/kg) were calculated using B-CLEAR®
technology (Qualyst, Inc., Raleigh, NC) based on the following equations:
BEI(%) =
Accumulation cells +bile − Accumulation cells
× 100
Accumulation cells +bile
In Vitro Biliary Clearance (mL/min/kg ) =
Accumulation cells +bile − Accumulation cells
AUC medium
149
where AUCmedium represents the area under the substrate concentration-time curve,
which was determined by multiplying the initial substrate concentration in the
incubation medium by the incubation time (10 min). Clbiliary values were converted to
milliliter per minute per kilogram based on previously reported values for liver weight
(200 mg/g liver weight for rats; 160 mg/g liver weight for humans) and protein
content in liver tissue (40 g/kg body weight for rats; 25.7 g/kg body weight for
humans) (Davies and Morris, 1993; Wilson et al., 2003).
HPLC/MS/MS Analysis of NF. NF accumulation in cells+bile and cells were
quantified by HPLC/MS/MS as described previously (Yue et al., 2008). Briefly,
samples obtained from rat SCH were deproteinated with 1 mL of methanol/water
(70/30, v/v), and sonicated for 20 sec. After the removal of protein by centrifugation
at 4°C (12000 g) for 3 min, the supernatant (20 µL) was mixed with 100 µL methanol
and water (3.8:1) containing internal standard (ethyl warfarin, 10 mM). A Shimadzu
solvent delivery system (Columbia, MD) and a Leap HTC Pal thermostatted
autosampler (Carrboro, NC) connected to an Applied Biosystems API 4000 triple
quadruple mass spectrometer with a Turbo Spray ion source (Applied Biosystems,
Foster City, CA) were used for analysis. Tuning, operation, integration and data
analysis were performed in negative ionization mode using multiple reaction
monitoring (Analyst software v.1.4.1, Applied Biosystems). NF and internal standard
were eluted from an Aquasil C18 column (50 × 2.1 mm, dp = 5 µm; Thermo Electron
Corporation, San Jose, CA) with 20 µL injection volume at a flow rate of 0.75 mL/min.
Initial gradient conditions (100% 10 mM ammonium acetate aqueous solution) were
held for 0.75 min. From 0.75 to 1.39 min, the mobile phase composition increased
150
linearly to 40% methanol, and the eluent was directed to the mass spectrometer. At
3.3 min, the methanol composition was increased to 90%. The flow was held at 90%
methanol until 4 min. At 4 min, the column was equilibrated with 100% 10 mM
ammonium acetate aqueous solution. The total run time, including equilibration, was
5 min per injection. Eight-point calibration curves (2-1000 nM) were constructed
based on peak area ratios of analyte to internal standard (ethyl warfarin) using the
following transitions: 236.8 151.8 (NF) and 320.8 160.9 (ethyl warfarin). All
points on the curves back-calculated to within ± 15% of the nominal value.
Transport Studies using Suspended Primary Rat and Human Hepatocytes.
[3H]TC and [3H]E217G initial uptake was determined in freshly-isolated hepatocytes
as described previously (Leslie et al., 2007). Briefly, hepatocytes were washed twice
with ice-cold standard HBSS modified with 10 mM Tris and 5 mM glucose (Na+containing condition) or Na+-free choline buffer (10 mM Tris, 5 mM glucose, 5.4 mM
KCl, 1.8 mM CaCl2, 0.9 mM MgSO4, 10 mM HEPES, and 137 mM choline).
Hepatocytes were suspended at 1×106 cells/mL in the same buffer, placed on ice,
and used immediately in experiments. Hepatocytes (2 mL) in 16 × 100 mm test
tubes were incubated at 37˚C in an orbital shaking water bath for 3 min. Vehicle
control (0.1 % DMSO), S-sulfone or S-sulfide (0.5–100 µM) was added, followed by
[3H]TC (1 µM; 60 nCi/mL) or [3H]E217G (1 µM; 60 nCi/mL). At 30 sec (TC) or 90 sec
(E217G), samples (200 µL) were placed into 0.4-mL polyethylene tubes on top of a
layer of silicone oil:mineral oil (82:18, v/v; 100 µL) and a bottom layer of 3 M KOH
(50 µL), and immediately centrifuged. Radioactivity in the cell pellet and supernatant
was quantified by liquid scintillation counting. The adherent fluid volume was
151
determined by the incubation of cells with [14C]inulin. At the end of each experiment,
an aliquot (10 µl) of suspended cells was used to determine protein concentrations
by the BCA protein assay.
Determination of Inhibitory Potency of Sulindac Metabolites on Na+-dependent
[3H]TC Uptake or Na+-independent [3H]E217G Uptake. The data obtained from
substrate transport studies ([3H]TC and [3H]E217G) in suspended hepatocytes were
plotted against the inhibitor concentration (S-sulfone and S-sulfide). Based on
standard model selection criteria including residual distribution, Akaike’s information
criterion and visual inspection, the sigmoidal inhibitory Emax model was selected to
estimate the concentration for 50% inhibition of uptake (IC50) using WinNonlin 5.0.1
(Pharsight, Mountain View, CA).
E = Emax × (1−
Cr
Cr + IC50r
)
where E is the rate of substrate uptake, Emax is maximum rate of substrate uptake, C
is the inhibitor concentration, and r is the curve shape factor.
Statistical Analysis. Data are expressed as mean and the associated S.E.M.
Statistical differences were determined using one-way analysis of variance with
Bonferroni’s post hoc test using SigmaStat 2.03 (Aspire Software International
Ashburn, VA). Differences were considered significant at p < 0.05.
152
RESULTS
Effect of Sulindac and Metabolites on TC Hepatobiliary Disposition in Rat SCH.
[3H]TC was used as a model substrate to examine the impact of sulindac and
metabolites on hepatic uptake and biliary excretion of bile acids in rat SCH (Figure
5.2A-C). The accumulation of TC (1 µM, 100 nCi/mL, 10 min) in cells+bile in the
presence of 100 µM S-sulfide was significantly decreased compared to vehicle
control. TC BEI in the presence of 1 and 10 µM sulindac, S-sulfone and S-sulfide
was not statistically different from those values in vehicle control. TC BEI was
decreased significantly by 100 µM S-sulfone (42% decrease) and S-sulfide (55%
decrease), but TC BEI in the presence of 100 µM sulindac was not different from
those values in vehicle control. Sulindac (10 µM; 53% decrease), S-sulfone (100 µM;
76% decrease) and S-sulfide (100 µM; 84% decrease) significantly inhibited TC
Clbiliary.
Effect of Sulindac and Metabolites on E217G Hepatobiliary Disposition in Rat
SCH.
[3H]E217G was used as a model substrate to examine the impact of sulindac
and metabolites on Oatp-mediated uptake and Mrp2-mediated biliary excretion in rat
SCH (Figure 5.3A-C). Accumulation of E217G (1 µM, 100 nCi/mL, 10 min) in
cells+bile and in cells was decreased significantly in the presence of sulindac (100
µM), S-sulfone (10 and 100 µM) and S-sulfide (10 and 100 µM). E217G BEI was not
affected by sulindac, S-sulfone or S-sulfide (1-100 µM), whereas E217G Clbiliary was
decreased significantly by S-sulfone (1, 10 and 100 µM) and S-sulfide (100 µM), but
not by sulindac.
153
Effect of Sulindac and Metabolites on NF Hepatobiliary Disposition in Rat SCH.
NF, a specific Bcrp substrate in rat SCH (Yue, 2008), was used as a model
substrate to examine the impact of sulindac and metabolites on Bcrp-mediated
biliary excretion in rat SCH (Figure 5.4). NF accumulation (5 µM, 10 min) in
cells+bile was not significantly different in the presence of 100 µM sulindac, Ssulfone or S-sulfide, but cellular accumulation was increased significantly in the
presence of S-sulfide. NF BEI was decreased by 30%, 39% and 57% in the
presence of 100 µM sulindac, S-sulfone and S-sulfide, respectively, compared to
vehicle control. Clbiliary of NF was decreased, with more potent inhibition by S-sulfide
(50% decrease), followed by sulindac (33% decrease) and S-sulfone (33%
decrease).
Influence of S-sulfone and S-sulfide on Na+-dependent Initial Uptake of TC and
Na+-independent Initial Uptake of E217G in Suspended Rat Hepatocytes.
To further examine the mechanisms of inhibition of hepatic transport proteins
by sulindac metabolites, the inhibitory potency of S-sulfone and S-sulfide on the
initial uptake of TC and E217G in the absence or presence of Na+ was determined in
suspended rat hepatocytes. Initial uptake was determined at 30 sec for TC (1 µM, 60
nCi/mL) based on preliminary studies (data not shown). E217G initial uptake (1 µM,
60 nCi/mL, 90 sec) was determined under Na+-free conditions to exclude Na+dependent E217G uptake, based on previously published data demonstrating that
E217G uptake was partially dependent on sodium (Brock and Vore, 1984). The IC50
values for inhibition of Na+-dependent TC initial uptake by S-sulfone and S-sulfide
were 25.6±10.7 µM (Figure 5.5A) and 12.7±5.3 µM (Figure 5.5B), respectively. The
154
IC50 values for inhibition of Na+-independent E217G initial uptake by S-sulfone and
S-sulfide were 12.6±5.1 µM (Figure 5.6A) and 6.5±1.2 µM (Figure 5.6B), respectively.
Effect of Sulindac and Metabolites on TC Hepatobiliary Disposition in Human
SCH.
In human SCH, [3H]TC accumulation in cells+bile and cells (1 µM, 100 nCi/mL,
10 min) in the presence of 10 and 100 µM sulindac and S-sulfone was comparable
to vehicle control, but S-sulfide at 10 and 100 µM inhibited TC accumulation by 52%
and 87%, respectively (Figure 5.7). TC BEI was not decreased by sulindac, Ssulfone or S-sulfide, but the Clbiliary of TC was decreased by 50% and 88% with 10
and 100 µM S-sulfide, respectively.
Influence of S-sulfone and S-sulfide on Na+-dependent TC Initial Uptake in
Suspended Human Hepatocytes.
The initial uptake of [3H]TC (1 µM, 60 nCi/mL) in suspended human
hepatocytes was determined at 30 sec based on previously published data
demonstrating that TC uptake was linear for at least 2 min in freshly-isolated human
hepatocytes (Shitara et al., 2003). The IC50 for inhibition of Na+-dependent TC initial
uptake by S-sulfone and S-sulfide were 42.2 µM (Figure 5.8A) and 3.1 µM (Figure
5.8B), respectively.
155
DISCUSSION
Among the NSAIDs, sulindac has been associated with the highest incidence
of DILI, which may involve interference with hepatic transport protein function. As
such, the present study was designed to examine interactions between
sulindac/metabolites and hepatic transport proteins using TC, E217G and NF as
model substrates for Ntcp-/Bsep-, Oatp-/Mrp2- and Bcrp-mediated transport,
respectively, in primary rat and human hepatocytes. BEI and Clbiliary from SCH were
used as indices of hepatobiliary disposition of each model substrate; BEI is a
measure of net canalicular efflux, whereas Clbiliary encompasses both uptake and
efflux.
Both the BEI and Clbiliary of TC in rat SCH were decreased significantly by Ssulfone and S-sulfide (100 µM) (Figure 5.2A-C). Moreover, both metabolites inhibited
Na+-dependent TC initial uptake in suspended rat hepatocytes (IC50 of 26 and 13 µM,
respectively). Collectively, these data indicate that S-sulfone and S-sulfide inhibit
Ntcp and Bsep. The balance between bile acid uptake and excretion in hepatocytes
may be influence the occurrence of DILI. Indeed, the preferential inhibition of rat
Ntcp rather than human NTCP by bosentan has been proposed as a plausible
mechanism for protection of the rat against bosentan-mediated hepatotoxicity by
preventing the further uptake of bile acids from blood when hepatocyte bile acid
concentrations are increased due to bosentan-mediated Bsep/BSEP inhibition
(Leslie et al., 2007).
In rats, E217G is taken up by multiple Oatps, whereas biliary excretion of
E217G is governed by Mrp2. The decreased hepatocellular accumulation of E217G
156
in the presence of S-sulfone and S-sulfide in rat SCH, combined with the inhibition of
E217G Clbiliary but not BEI, suggested that sulindac metabolites inhibited Oatpmediated uptake but not Mrp2-mediated biliary excretion of E217G (Figure 5.3A-C).
Subsequent investigations in suspended rat hepatocytes revealed that S-sulfone
and S-sulfide were potent inhibitors (IC50 of 13 and 6.5 µM, respectively) of E217G
initial uptake, consistent with inhibition of Oatp 1a1, 1a3, 1a4, 1a5 and 1b2.
NF was chosen as a model substrate for Bcrp-mediated transport based on
recent data demonstrating that NF is a specific Bcrp substrate in rat SCH (Yue et al.,
2008). Several clinical studies have reported that genetic polymorphisms in BCRP
(ABCG2) can influence drug disposition. For example, the ABCG2 C421A
polymorphism, which is associated with decreased protein expression and function
in vitro (Imai et al., 2002), increased plasma concentrations of several BCRP
substrates, including rosuvastatin, diflomotecan, and sulfasalazine (Morisaki et al.,
2005; Zhang et al., 2006a; Yamasaki et al., 2008). In the present study,
sulindac/metabolites significantly decreased NF BEI and Clbiliary in rat SCH,
consistent with inhibition of Bcrp (Figure 5.4). NF has been associated with
hepatotoxicity;
although
cholestatic
and
mixed
patterns
of
NF-mediated
hepatotoxicity have been reported, hypersensitivity or immunologic injury were most
common (Thiim and Friedman, 2003). NF-mediated oxidative stress via depletion of
GSH or NADPH has been postulated as one mechanism for NF-mediated
hepatotoxicity (Thiim and Friedman, 2003). Increased accumulation of NF in
hepatocytes may occur in patients who have impaired BCRP activity (due to genetic
variations or inhibitors), thus increasing the likelihood of NF-mediated hepatotoxicity.
157
Investigations are underway to determine whether sulindac and metabolites alter
human BCRP-mediated transport.
Due to the limited availability of human liver tissue combined with prominent
roles of bile acid toxicity, only TC was tested further in human SCH to examine the
effect of sulindac/metabolites on the hepatobiliary disposition of TC. In human SCH,
TC accumulation in cells+bile, and the TC Clbiliary, were markedly decreased by Ssulfide without affecting TC BEI. These findings may reflect, at least in part, Ssulfide-mediated inhibition of NTCP. The IC50 (3.1 µM) of Na+-dependent TC initial
uptake by S-sulfide determined in human hepatocyte suspensions, which do not
contain serum, was near the unbound plasma concentration range of S-sulfide
(~0.14-1.5 µM) based on reported Cmax values of 3-33 µM and an unbound fraction
(fu) of 4.6% after oral administration of a therapeutic dose of sulindac (Davies and
Watson, 1997). However, the IC50 (42.2 µM) of Na+-dependent TC initial uptake by
S-sulfone was ~100-fold greater than the unbound plasma concentration range
(~0.1-0.4 µM) of S-sulfone (Cmax, 4-19 µM; fu, 2.1%). Although the IC50 for inhibition
of Na+-dependent TC initial uptake by S-sulfide and S-sulfone was higher than
unbound plasma concentrations, it is probable that initial inhibitor concentrations are
higher than unbound plasma concentrations in the portal vein when sulindac is
administered orally, since hepatic blood flow consists of ~85% the portal venous
blood, which contains drug absorbed directly from the gastrointestinal tract (Ito et al.,
1998). In addition, it is plausible that the extensive enterohepatic recycling of
sulindac and metabolites may result in prolonged inhibition of hepatic transport
158
proteins due to much higher concentrations in the portal circulation (Dujovne et al.,
1983).
The FDA has recommended an in vivo evaluation of drug interactions when
the estimated [I]/Ki ratio is greater than 0.1, where [I] represents the mean steadystate Cmax value for total drug (bound plus unbound) following administration of the
highest proposed clinical dose (Huang et al., 2008). In theory, the IC50 values
determined in the present study would be identical to Ki values, regardless of the
mechanism of inhibition, based on previous data demonstrating the relationships
between the Ki and IC50 values (Cheng and Prusoff, 1973); that is, the Ki value is
equal to the IC50 value under conditions of either noncompetitive or uncompetitive
inhibition, whereas under competitive inhibition conditions, the IC50 value will be
identical to Ki(1+S/Km), where S is the substrate concentration and Km is an
approximate measure of the affinity of the substrate for the enzyme. Because the TC
concentration (1 µM) used for the IC50 determination in the present study was lower
than the previously reported Km for the uptake of TC in rat (18 µM) and human
hepatocytes (4.8 µM), the Ki values would be equal to or less than the IC50 value
(Kouzuki et al., 1998; Shitara et al., 2003). With this assumption, the [I]/Ki ratio
determined using data from human hepatocytes ranged from 0.9-10.6 for S-sulfide,
and from 0.1-0.4 for S-sulfone, based on Cmax values of 3-33 µM and 4-19 µM,
respectively. These findings suggest that S-sulfide and S-sulfone may interact with
NTCP-mediated transport of drugs and/or bile acids in humans (Davies and Watson,
1997).
159
In conclusion, the present studies demonstrated that hepatic transport
proteins (Ntcp/NTCP, Oatps, Bsep, Bcrp) are inhibited by sulindac and metabolites.
Considering the important role of Ntcp/NTCP, Oatp and Bsep in hepatic uptake and
excretion of bile acids in the liver, impaired hepatic transport of bile acids by
sulindac/metabolites may disturb bile acid homeostasis, resulting in sulindacmediated liver injury. Based on the present study demonstrating inhibition of multiple
transport proteins by sulindac and metabolites, the possibility of drug-xenobiotic
interactions should be considered when sulindac is coadministered with other
drugs/xenobiotics, warranting careful monitoring for liver injury.
160
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characterization of sulfasalazine disposition based on NAT2 and ABCG2
(BCRP) gene polymorphisms in humans. Clin Pharmacol Ther 84:95-103.
Yue W, Abe K and Brouwer KL (2008) Knocking Down Breast Cancer Resistance
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373:99-103.
164
Sulindac
H3CS
H
CH3
F
CH2COOH
Sulindac sulfide
Sulindac sulfone
Figure 5.1. Chemical Structures of Sulindac, Sulindac Sulfone (S-sulfone) and
Sulindac Sulfide (S-sulfide).
165
60
TC Accumulation
(pmol/mg protein)
A
50
40
30
20
10
0
CTL
1
10
100
Sulindac (µM)
BEI (%)
78 ± 5
79 ± 4
61 ± 7
in vitro Clbiliary
(ml/min/kg)
32 ± 5
26 ± 1a
15 ± 1
*
69 ± 13
20 ± 3a
100
TC Accumulation
(pmol/mg protein)
B
80
60
40
20
0
CTL
1
10
100
S-sulfone (µM)
*
BEI (%)
82 ± 2
84 ± 2a
76 ± 3a
47 ± 12
in vitro Clbiliary
(ml/min/kg)
45 ± 10
31 ± 7b
20 ± 4b
11 ± 5
100
TC Accumulation
(pmol/mg protein)
C
*
*
80
60
40
20
0
CTL
1
10
100
S-sulfide (µM)
*
BEI (%)
83 ± 2
79 ± 2a
79 ± 2a
37 ± 17
in vitro Clbiliary
(ml/min/kg)
49 ± 8
32 ± 6b
31± 9b
7.5 ± 5.0
*
Figure 5.2. Effect of Sulindac and Metabolites on Hepatobiliary Disposition of TC in
Rat SCH. Accumulation of [3H]TC (1 µM, 100 nCi/mL, 10 min) in cells+bile (solid
bars) and cells (white bars) was measured in the presence of increasing
concentrations (1-100 µM) of sulindac (A), S-sulfone (B) or S-sulfide (C) in rat SCH.
BEI and in vitro Clbiliary of TC were calculated as described in Materials and Methods.
Data are presented as the mean ± S.E.M (n=3-4 livers in triplicate); *, statistically
different from vehicle control (CTL) by one-way ANOVA with Bonferroni’s post-hoc
test.
166
A
*
E217G Accumulation
(pmol/mg protein)
180
*
160
140
120
100
80
60
40
20
0
CTL
1
10
100
Sulindac (µM)
BEI (%)
18 ± 3
15 ± 6
8±3
12 ± 6
in vitro Clbiliary
(ml/min/kg)
21 ± 7
19 ± 9
7.0 ± 2.0
4.1 ± 2.2
B
*
E217G Accumulation
(pmol/mg protein)
160
*
140
120
100
80
60
40
20
0
CTL
1
10
100
S-Sulfone (µM)
BEI (%)
18 ± 3
10 ± 4
in vitro Clbiliary
(ml/min/kg)
20 ± 3
10 ± 2
*
160
13 ± 12
*
*
C
E217G Accumulation
(pmol/mg protein)
10 ± 3
*
6.4 ± 0.8 3.4 ± 2.0
*
140
120
100
80
60
40
20
0
CTL
1
10
100
S-Sulfone (µM)
BEI (%)
18 ± 3
10 ± 4
in vitro Clbiliary
(ml/min/kg)
20 ± 3
10 ± 2
*
10 ± 3
13 ± 12
*
*
6.4 ± 0.8 3.4 ± 2.0
Figure 5.3. Effect of Sulindac and Metabolites on Hepatobiliary Disposition of
E217G in Rat SCH. Accumulation of [3H]E217G (1 µM, 100 nCi/mL, 10 min) in
cells+bile (solid bars) and cells (white bars) was measured in the presence of
increasing concentrations (1-100 µM) of sulindac (A), S-sulfone (B) or S-sulfide (C)
in rat SCH. BEI and in vitro Clbiliary of E217G were calculated as described in
Materials and Methods. Data are presented as the mean ± S.E.M (n=3-4 livers in
triplicate); *, statistically different from vehicle control (CTL) by one-way ANOVA with
Bonferroni’s post-hoc test.
167
*
NF Accumulation
(pmol/mg protein)
80
60
40
20
0
CTL
Sulindac S-sulfone S-sulfide
BEI (%)
64 ± 1
45 ± 1*
40 ± 1*
27 ± 1*
in vitro Clbiliary
(ml/min/kg)
5.9 ± 0.7
4.3 ± 0.1*
3.6 ± 0.1*
2.9 ± 0.2*
Figure 5.4. Effect of Sulindac and Metabolites on Hepatobiliary Disposition of NF in
Rat SCH. Accumulation of NF (5 µM, 10 min) in cells+bile (solid bars) and cells
(white bars) was measured in the presence of 100 µM sulindac, S-sulfone or Ssulfide in rat SCH. BEI and in vitro Clbiliary of NF were calculated as described in the
Materials and Methods. Data are presented as the mean ± S.E.M (n=3 livers in
triplicate); *, statistically different from vehicle control (CTL) by one-way ANOVA with
Bonferroni’s post-hoc test.
168
IC50 = 25.6 ± 10.7 µM
100
80
60
40
20
+
Na -dependent TC uptake
(% control)
A
0
0.1
1
10
100
1000
S-sulfone (µM)
120
IC50 = 12.7 ± 5.3 µM
100
80
60
40
20
+
Na -dependent TC uptake
(% control)
B
0
0.1
1
10
100
1000
S-sulfide (µM)
Figure 5.5. Inhibition of Na+-dependent TC Initial Uptake in Suspended Rat
Hepatocytes by S-sulfone (A) and S-sulfide (B). Na+-dependent [3H]TC initial uptake
(1 µM, 60 nCi/ml, 30 sec) was determined by subtracting [3H]TC uptake in Na+-free
buffer from [3H]TC uptake in Na+-containing buffer in the presence of increasing
concentrations of S-sulfone (0.5-100 µM) and S-sulfide (0.5-100 µM). Symbols and
error bars represent the mean ± S.E.M. of n=3 livers in duplicate. The dotted curve
represents the best fit of the sigmoidal inhibitory Emax model to the data using
WinNonlin; the curve shape factor (r) was estimated as 0.4±0.1 and 0.8±0.2 for Ssulfone and S-sulfide, respectively.
169
E217G uptake (% control)
A
120
IC50 = 12.6 ± 5.1 µM
100
80
60
40
20
0
0.1
1
10
100
1000
S-Sulfone (µM)
E217G uptake (% control)
B
100
IC50 = 6.5 ± 1.2 µM
80
60
40
20
0
0.1
1
10
100
1000
S-Sulfide (µM)
Figure 5.6. Inhibition of Na+-independent E217G Initial Uptake in Suspended Rat
Hepatocytes by S-sulfone (A) and S-sulfide (B). Na+-independent [3H]E217G initial
uptake (1 µM, 60 nCi/ml, 90 sec) was determined in Na+-free buffer in the presence
of increasing concentrations of S-sulfone (0.5-100 µM) and S-sulfide (0.5-100 µM).
Symbols and error bars represent the mean ± S.E.M. of n=3 livers in duplicate. The
dotted curve represents the best fit of the sigmoidal inhibitory Emax model to the data
using WinNonlin; the curve shape factor (r) was estimated as 0.8±0.2 and 1±0.2 for
S-sulfone and S-sulfide, respectively.
170
TC Accumulation
(pmol/mg protein)
350
300
250
200
150
100
50
0
CTL
10
100
10
100
10
100
Sulindac (µ
µM) S-sulfone (µ
µM) S-sulfide (µ
µM)
BEI (%)
59
57
44
54
52
60
49
in vitro Clbiliary
(ml/min/kg)
27
22
18
19
21
13
3.3
Figure 5.7. Effect of Sulindac and Metabolites on Hepatobiliary Disposition of TC in
Human SCH. Accumulation of [3H]TC (1 µM, 100 nCi/mL, 10 min) in cells+bile (solid
bars) and cells (white bars) was measured in the presence of 10 and 100 µM
sulindac, S-sulfone or S-sulfide in human SCH. BEI and in vitro Clbiliary of TC were
calculated as described in Materials and Methods. Bars and error bars denote mean
± one-half of the range from n=2 livers in duplicate.
171
IC50 = 42.2 µM
100
80
60
40
20
+
Na -dependent TC uptake
(% control)
A
0
0.1
1
10
100
1000
IC50 = 3.1 µM
100
80
60
40
20
+
B
Na -dependent TC uptake
(% control)
S-sulfone (µM)
0
0.01
0.1
1
10
100
1000
S-sulfide (µM)
Figure 5.8. Inhibition of Na+-dependent TC Initial Uptake in Suspended Human
Hepatocytes by S-sulfone (A) and S-sulfide (B). Na+-dependent [3H]TC initial uptake
(1 µM, 60 nCi/ml, 30 sec) was determined by subtracting [3H]TC uptake in Na+-free
buffer from those values in Na+-containing buffer in the presence of increasing
concentrations of S-sulfone (1-300 µM) and S-sulfide (0.1-100 µM). Open and
closed circles denote individual data from n=2 livers in duplicate. The dotted curve
represents the best fit of the sigmoidal inhibitory Emax model to the data using
WinNonlin; the curve shape factor (r) was estimated as 0.6 and 0.9 for S-sulfone and
S-sulfide, respectively.
172
CHAPTER 6
Summary and Future Directions
The objective of this dissertation research was to examine the role of hepatic
transport proteins in drug disposition and drug-induced liver injury. Although a
number of studies have focused on elucidating the transport mechanisms
responsible for hepatic uptake and biliary excretion of drugs, the effect of one or
more transport protein(s) on overall drug disposition and drug-induced liver injury
has not been fully characterized. To address these questions, a series of
experiments was designed using a multi-experimental approach.
Sandwich-cultured hepatocytes (SCH) have been used instead of primary
hepatocytes cultured in monolayer to examine the hepatobiliary disposition of
substrates because SCH exhibit polarized architecture, form extensive canalicular
networks over time in culture, and maintain function of metabolic enzymes and
transport proteins. The maintenance of tight junction integrity in the presence of Ca2+
enables substrate accumulation in the canalicular spaces. Biliary clearance data
estimated in rat and human SCH have been correlated with in vivo biliary clearance
for several therapeutic compounds (Liu et al., 1999a; Ghibellini et al., 2007b; Abe et
al., 2008). However, the use of SCH is limited when determining the contribution of a
specific transport protein to overall drug hepatobiliary disposition because specific
inhibitors or probe substrates for individual transport proteins have not been
identified. Alternatively, membrane vesicles overexpressing a single transport
protein can be used to determine substrate specificity or affinity for transport proteins.
Isolated liver perfusion is an established technique. This approach enables careful
and comprehensive assessment of hepatic disposition of drugs and metabolites by
examining the rate of appearance of substrate in bile and in the fluid perfusing the
174
liver vasculature. In mice, this technique presents a number of technical challenges
due to the small size of the liver. In recent years, our laboratory has modified
successfully the technique in rats for use in mice (Nezasa et al., 2006).
The underlying hypothesis of this doctoral dissertation research was that
multiple transport proteins are involved in drug disposition and drug-induced liver
injury. This hypothesis was examined by a series of experiments described below:
1. Elucidate the mechanism of differences in the hepatobiliary disposition of
acetaminophen and metabolites in male and female mice (Chapter 2). In situ
mouse liver perfusion using male and female wild-type and Bcrp-knockout
mice was performed to test the hypothesis that the biliary excretion of phase
II conjugates was influenced by higher Bcrp expression in male mouse livers
than female mouse livers. Acetaminophen and phase II metabolites (i.e,
acetaminophen sulfate and glucuronide) were chosen based on previous
findings demonstrating that Bcrp plays an important role in the biliary
excretion of these conjugates in mice. Comprehensive pharmacokinetic
models were developed to describe the hepatobiliary disposition of
acetaminophen and metabolites in mouse livers.
2. Assess the utility of an integrated in vitro approach [SCH and
modeling/simulation] to predict alterations in drug hepatobiliary disposition
(Chapter 3). These experiments tested the hypothesis that rat and human
SCH can be used to mimic the in vivo disposition of troglitazone (TGZ) and
175
generated metabolites in rats and humans. Additionally, pharmacokinetic
modeling and Monte Carlo simulation approaches coupled with data from
SCH were utilized to examine the consequences of altered biliary excretion,
which might be due to drug interactions or disease states, on hepatic
exposure in hypothetical “populations” of hepatocytes.
3. Examine the potential role of hepatic transport proteins in drug-induced
liver injury (Chapters 4 and 5). Trabectedin (a promising anticancer agent)
and sulindac (a non-steroidal anti-inflammatory drug) were chosen based on
previously published findings regarding the incidence of liver injury mediated
by these drugs in humans.
1) Dexamethasone (DEX) decreased liver injury mediated by trabectedin in
rats and humans, but this was not mimicked in rat hepatocytes cultured on a
fibronectin matrix. The hypothesis underlying this aim (Chapter 4) was that
Mrp2 and CYP3A1/2 played a role in the hepato-protective effect of DEX
against
trabectedin-mediated
hepatotoxicity.
Trabectedin-mediated
cytotoxicity was examined in SCH. The role of Mrp2 in biliary excretion of
trabectedin was evaluated in isolated perfused livers from wild-type and
Mrp2-deficient TR- rats.
2) The hypothesis underlying this aim (Chapter 5) was that the interaction of
sulindac as well as its metabolites with hepatic transport proteins altered the
hepatobiliary disposition of bile acids and other endogenous or exogenous
compounds, and these interactions played a role in sulindac-mediated liver
176
injury. Probe substrates including nitrofurantoin (NF), taurocholate (TC), and
estradiol-17β-glucuronide (E217G) were used to examine the interaction
between sulindac/metabolites and Bcrp-, Ntcp/Oatp/Bsep-, Oatp/Mrp2mediated transport, respectively, in rat or human SCH, and in suspended
hepatocytes.
Mechanism of differences in the hepatobiliary disposition of phase II
conjugates in male and female mice (Chapter 2).
Sex-related variations in pharmacokinetics have been considered as a
potential determinant for the clinical effectiveness of therapeutic agents by affecting
ADME (absorption, disposition, metabolism and elimination). Emerging evidence
suggests that sex-related variations in the expression and activity of drug transport
proteins may contribute to differences in therapeutic response or toxicity.
BCRP is highly expressed in the liver and mediates the excretion of a wide
variety of drugs and environmental toxicants into bile, as well as specific metabolites
(e.g., sulfate and glucuronide conjugates) formed in the liver. Interestingly, it has
been reported that BCRP protein expression in liver is sex-dependent in mice and
humans (Merino et al., 2005b). Furthermore, several anticancer drugs that are
known BCRP substrates (e.g., topotecan, methotrexate, and doxorubicin) exhibit
sex-dependent disposition in humans. Only a limited number of studies have
explored relationships between BCRP and sex-related treatment efficacy; the
specific role of BCRP in this therapeutically-important phenomenon has not been
elucidated.
177
In situ mouse liver perfusion was applied to four different groups of mice;
male and female wild-type and Bcrp-deficient mice (C57BL/6) were used to compare
the perfusate disposition and biliary excretion of acetaminophen sulfate (AS) and
acetaminophen glucuronide (AG) in the presence of differential BCRP expression
(i.e, higher in males than in females). Male and female Bcrp-deficient mice, obtained
through collaboration with Eli Lilly and Co., provided a unique opportunity to
ascertain whether mechanisms other than Bcrp expression modulate hepatic
disposition of acetaminophen metabolites between male and female mice. AG and
AS biliary excretion in female wild-type mice was 3- and 2.5-fold lower, respectively,
compared to male wild-type mice, reflecting male-predominant Bcrp expression. In
Bcrp-deficient mice, sex-dependent differences in AG biliary excretion were not
observed, indicating that Bcrp was likely responsible for the sex difference in wildtype mice. Unexpectedly, excretion of AG and AS into the perfusate was also sexdependent; basolateral excretion of AG was higher in male wild-type mouse livers
while AS basolateral excretion was higher in female wild-type mouse livers.
Pharmacokinetic modeling was used to describe the disposition of
acetaminophen, AG and AS in perfused mouse livers. The AG biliary excretion rate
constant (Kbile,AG) was ~7-fold higher in male wild-type mouse livers, but was similar
between male and female Bcrp-deficient mouse livers. Likewise, the rate constant
for AS biliary excretion (Kbile,AS) in male livers was ~2.5-fold higher than in female
wild-type mouse livers. Sex-dependent differences in metabolite formation rate
constants were observed for AG (~2-fold higher in males) and AS (~2-4-fold higher
in females). The basolateral excretion rate constant of AS (Kperf,AS), but not AG
178
(Kperf,AG), differed based on sex; the AS basolateral excretion rate constant was ~2fold higher in females, potentially due to higher Mrp4 expression in female livers.
Overall, these results demonstrated that sex-dependent differences in the
hepatic disposition of AG and AS were due, in part, to sex-related BCRP expression.
Other factors relating to formation of conjugates as well as conjugate transport from
the liver into blood likely played a role in disposition of AG and AS. Sex-related
differences in BCRP expression may cause clinically significant alterations in the
disposition of drugs that are BCRP substrates. These exciting results provide a new
framework for investigations directed towards elaborating the mechanisms
underlying sex-related differences in drug disposition and drug interactions resulting
in altered drug response and/or toxicity associated with therapeutic agents and
environmental chemicals.
An integrated in vitro approach [SCH and modeling/simulation] to predict
alterations in hepatobiliary drug disposition (Chapter 3).
Troglitazone (Rezulin; TGZ), an anti-diabetic and anti-inflammatory drug,
was withdrawn from the market in 2000 due to severe hepatotoxicity. Inhibition of
BSEP by TGZ and TGZ-sulfate (TS) has been hypothesized to be responsible for
the cholestatic effect; BSEP was inhibited more potently by TS than by TGZ,
demonstrating the importance of metabolite-mediated hepatotoxicity in addition to
that of the parent drug. Unfortunately, preclinical toxicological studies failed to
predict this hepatotoxicity (Smith, 2003).
The aim of this study was to evaluate the capability of an in vitro model, rat
and human SCH, to mimic the in vivo metabolism and hepatobiliary disposition of
179
TGZ in rats and humans. Consistent with the in vivo metabolism of TGZ, TGZ was
extensively metabolized to TS and to a lesser extent to TG and TGZ-quinone (TQ) in
rat and human SCH. Moreover, TS was the predominant metabolite in medium and
hepatocytes in both rat and human SCH.
A secondary objective of this specific aim was to examine the impact of
impaired biliary excretion on the hepatobiliary disposition of TS and TG using data
from SCH coupled with pharmacokinetic modeling and Monte Carlo simulations.
Parameters governing the hepatobiliary disposition of TGZ and its metabolites in
SCH were estimated by compartmental pharmacokinetic modeling. Subsequently,
Monte Carlo simulations were performed by utilizing parameter estimates and
variance associated with nonlinear least-squares regression and uncontrolled error
(e.g., experimental or analytical error; 15%) to examine the impact of altered biliary
excretion on accumulation of TS and TG in hepatocytes and medium. In a
hypothetical “population” of hepatocytes, cellular accumulation of substrate
[representative of liver] was highly sensitive to altered biliary transport, but changes
in biliary excretion exhibited only a modest effect on substrate in medium
[representative of blood]. Data from Monte Carlo simulations at each time point were
utilized to estimate the number of observations necessary to detect a statistically
significant difference in TS or TG accumulation in hepatocytes and medium when
the rate constant for biliary excretion of TS or TG was modulated [2-fold increase or
decrease from experimentally-determined parameter estimates]. The number of
observations required to detect a statistically significant difference for TS and TG
was less in hepatocytes than in medium at all time points, supporting the hypothesis
180
that cellular accumulation of these substrates is more sensitive to changes in altered
biliary excretion than accumulation in medium. In clinical studies, plasma
concentrations are typically used to examine the effect of altered hepatic transport
function (e.g., drug interactions in biliary excretion, polymorphisms of canalicular
transport proteins). However, Monte Carlo simulations suggest that plasma
concentrations may not always reflect changes in hepatic exposure when biliary
excretion pathways are modulated. Previous findings have
demonstrated that
BCRP plays an important role in TS biliary excretion in humans (Enokizono et al.,
2007). Simulations suggest that TS concentrations in the liver would be higher in
subjects with less functional BCRP (due to drug-drug interactions, disease state
alterations, and/or polymorphisms), resulting in more potent BSEP inhibition, thereby
increasing the susceptibility of some patients to TS-mediated cholestatic effects.
The potential role of hepatic transport proteins in drug-induced liver injury.
Drug-induced liver injury has been the most common reason for the
withdrawal of approved drugs from the market. Although drug-induced liver injury
has been attributed to many factors including covalent adduct formation,
immunotoxicity, and impaired bioenergetics, accumulating evidence suggests that
inhibition of hepatic transport proteins by a drug and/or derived metabolite may
mediate liver injury. Many studies have focused on interactions with BSEP because
inhibition of BSEP results in accumulation of detergent-like bile acids in the liver
which may lead to hepatic injury. However, when drugs or metabolites are
intrinsically hepatotoxic, impaired function of one or more transport proteins other
181
than BSEP which are responsible for hepatobiliary disposition of a drug or its
metabolites can also contribute to drug-induced liver injury.
1) Trabectedin (Chapter 4)
Liver injury mediated by trabectedin (ET-743), a promising anticancer agent,
has been the most common side effect during phase I/II clinical trials (Beumer et al.,
2005b). Interestingly, pretreatment with DEX in rats and humans markedly
decreased ET-743-mediated hepatotoxicity, which was attributed to DEX-mediated
CYP3A4 induction. However, the protection by DEX against trabectedin-mediated
hepatotoxicty could not be mimicked in primary rat hepatocytes cultured on a
fibronectin matrix. The objective of this specific aim was to examine whether SCH
could predict the hepatoprotective effect of DEX against trabectedin-mediated
hepatotoxicity, and to further examine the involvement of Mrp2 in ET-743 biliary
excretion using isolated perfused rat livers (IPL).
Pretreatment with DEX in wild-type rat SCH significantly decreased
trabectedin-mediated cytotoxicity compared to vehicle control. In addition to inducing
CYP3A1/2, DEX also induces Mrp2.
To investigate the involvement of Mrp2,
trabectedin-mediated cytotoxicity was compared between wild-type and Mrp2deficient TR- rats. Surprisingly, trabectedin-mediated cytotoxicity was less extensive
in TR- than wild-type rat SCH. One possible explanation for this unexpected finding
was
that
TR-
rat
hepatocytes
were
protected
from
trabectedin-mediated
hepatotoxicity because of a higher level of glutathione in these hepatocytes due to
impaired Mrp2-mediated excretion. To examine this hypothesis, trabectedinmediated cytotoxicity was examined in wild-type rat SCH pretreated with buthionine
182
sulfoximine (BSO), a glutathione depleting agent. Trabectedin-mediated cytotoxicity
was significantly decreased in BSO-pretreated compared to vehicle-pretreated SCH.
Consistent with previous findings (Turncliff et al., 2004), immunoblot analysis
demonstrated that Mrp2 and CYP3A1/2 protein expression in rat SCH was
increased by DEX pretreatment. Interestingly, BSO pretreatment markedly increased
expression of Mrp4 and to a lesser extent Mrp2 and Mrp3 compared to vehicle
control. To examine the involvement of Mrp2 in trabectedin hepatobiliary disposition,
IPL studies were performed in wild-type and TR- rats. Trabectedin biliary excretion in
IPLs from TR- rats was decreased by ~75% compared to wild-type IPLs, suggesting
that Mrp2 plays a significant role in trabectedin biliary excretion.
Collectively, the hepatoprotection that DEX provides in trabectedin-mediated
hepatotoxicity may be due to the combined effect of increased metabolism by
CYP3A1/2 and enhanced biliary excretion by Mrp2.
Increased expression of
basolateral transport proteins such as Mrp3 and/or Mrp4 due to BSO-pretreatment in
TR- rat SCH may be one mechanism underlying the unexpected attenuation of
trabectedin-mediated hepatotoxicity.
2) Sulindac, sulindac sulfone and sulindac sulfide (Chapter 5)
Sulindac is converted to two major metabolites; sulindac sulfide (S-sulfide)
has anti-inflammatory activity and sulindac sulfone (S-sulfone) has been recognized
recently as a chemopreventive agent in colon cancer. After oral administration of
sulindac, plasma concentrations of sulindac and its metabolites were comparable in
humans (Davies and Watson, 1997). Among other NSAIDS, sulindac was
associated most consistently with hepatotoxicity (Aithal and Day, 2007), but the
183
mechanism(s) of sulindac-mediated hepatotoxicity are not fully characterized. It has
been shown that S-sulfide concentrations in plasma correlated in a linear manner
with the incidence of hepatotoxicity, suggesting that S-sulfide may be involved in
sulindac-mediated hepatotoxicity (Duggan et al., 1978; Laffi et al., 1986). The
potential role of transport proteins in drug-induced liver injury has been hypothesized
recently and some studies have demonstrated the interaction between sulindac and
transport proteins (e.g., MRP2, MRP4, OAT1, OAT3, and OCT3) (Khamdang et al.,
2002; El-Sheikh et al., 2006). However, only limited information about the interaction
between sulindac metabolites and hepatic transport proteins is available (e.g., Ssulfide-mediated MRP4 inhibition) (Rius et al., 2008). Therefore, the aim of this study
was to determine the interaction of hepatic transport proteins with sulindac as well
as its metabolites using nitrofurantoin (NF), taurocholate (TC) and estradiol-17βglucuronide (E217G) as probe substrates for Bcrp-, Ntcp/Oatp/Bsep- and
Oatp/Mrp2-mediated transport.
In rat SCH, the in vitro Clbiliary of NF was decreased significantly by 100 µM
sulindac (33% decrease), S-sulfone (33% decrease) and S-sulfide (42% decrease)
without affecting NF accumulation in the hepatocyte and bile compartments,
suggesting that Bcrp-mediated transport was inhibited by sulindac and its
metabolites. Sulindac (10 µM; 53% decrease), S-sulfone (100 µM; 76% decrease)
and S-sulfide (100 µM; 84% decrease) significantly inhibited in vitro Clbiliary of TC; the
biliary excretion index (BEI) of TC was inhibited significantly by 100 µM S-sulfone
(43% decrease) and S-sulfide (55% decrease) but not by sulindac. The BEI of
E217G was not decreased in the presence of sulindac, S-sulfone and S-sulfide, but
184
in vitro Clbiliary of E217G was significantly decreased by S-sulfone and S-sulfide,
suggesting more potent inhibition in the hepatic uptake process than biliary excretion.
Suspended hepatocytes were selected as the model system to further characterize
the inhibitory kinetics of sulindac metabolites on TC and E217G uptake, because it is
easier to determine initial uptake rates using this system compared to SCH. In
suspended rat hepatocytes, S-sulfone and S-sulfide inhibited Na+-dependent TC
initial uptake (IC50 of 25.6±10.7 µM and 12.7±5.3 µM, respectively), and Na+independent E217G initial uptake (IC50 of 12.6±5.1 µM and 6.5±1.2 µM, respectively).
In addition, S-sulfide markedly decreased TC accumulation in the hepatocytes and
bile compartments as well as in vitro TC Clbiliary, but did not affect TC BEI in human
SCH. The IC50 of S-sulfide (3.1 µM) on Na+-dependent TC uptake determined in
suspended human hepatocytes was ~14-fold lower than for S-sulfone (42.2 µM).
Collectively, studies conducted to address this specific aim revealed that
hepatic transport proteins (Ntcp/NTCP, Oatp, Bsep or Bcrp) are inhibited by sulindac
and its metabolites. Considering the important role of Ntcp/NTCP, Oatp and Bsep in
hepatic uptake and excretion of bile acids in the liver, impaired hepatic transport of
bile acids by sulindac and its metabolites may disturb bile acid homeostasis,
resulting in sulindac-mediated hepatotoxicity. Although the contribution of BCRP to
bile acid transport is minor, inhibition of BCRP-mediated transport by sulindac and
its metabolites may result in increased hepatic accumulation of potentially toxic
metabolites such as S-sulfide or other co-administered drugs. Several studies with
sulindac alone or combined with other drugs (e.g., lovastatin, celecoxib, doxetaxel,
carboplatin, and vinorelbine) have been conducted recently to examine the
185
therapeutic effect of sulindac as an antiproliferative agent for colon cancer (Jones et
al., 2005; Hoang et al., 2006; Xiao and Yang, 2008). Based on the present study
demonstrating inhibition of multiple transport proteins by sulindac and its metabolites,
the possibility of drug-drug interactions should be examined carefully when sulindac
is coadministered with other drugs, and careful monitoring for hepatotoxicity would
be warranted.
Future directions
This dissertation research has prompted a number of proposed future studies
regarding the impact of sex-dependent transport protein expression on overall drug
pharmacokinetics, the utility of pharmacokinetic modeling and simulation coupled
with SCH to examine the impact of transport modulation on drug disposition, and
assessing multiple types of interactions between compounds and transport proteins.
These topics are detailed below.
Sex differences in drug transport
Discrepancies in drug metabolism between sexes are currently thought to
play a role in determining sex-differences in pharmacokinetic parameters. For
example, significant sex-differences in some key CYP450 enzymes have been
demonstrated.
A recent study analyzed 38 data sets containing the clearance
values of 10 xenobiotics which were CYP3A [but not P-glycoprotein (P-gp)]
substrates in healthy young male and female subjects; the overall mean of weightednormalized clearance values was significantly different between males and females
186
(Greenblatt and von Moltke, 2008). Although some animal and human data suggest
sex differences in expression of transport proteins, evidence for sex-dependent
pharmacokinetic differences in relation to transport proteins in humans are less
apparent. For example, despite higher P-gp expression in livers of men compared to
women, phenotyping with fexofenadine did not demonstrate sex differences in
clearance (Schuetz et al., 1995; Kim et al., 2001).
There are several potential reasons to explain the less apparent transportermediated pharmacokinetic differences in male and female subjects. First, the
overlapping substrate specificity among hepatic transport proteins may be a
confounding factor in assessing sex-dependent differences in drug transport. For
example, multiple transport mechanisms such as BSEP and MRP4 are involved in
the hepatobiliary disposition of fexofenadine which was used in P-gp phenotyping
(Matsushima et al., 2008). Although higher P-gp expression in male subjects results
in decreased biliary excretion of fexofenadine in the liver, BSEP-mediated biliary
excretion and/or MRP3-mediated basolateral excretion of fexofenadine could
completely offset the impact of differences in P-gp expression. Second, tissuespecific sex-dependent expression of transport proteins can be another confounding
factor. Sex-dependent hepatic Bcrp/BCRP expression in mice and humans was not
observed in rats (Merino et al., 2005b), but Bcrp expression in the kidney was higher
in male rats (Tanaka et al., 2005). Although this finding may suggest apparent
species differences in sex-dependent expression of transport proteins, it also
suggests that differential mechanisms may be involved in the tissue-specific
regulation of transport proteins. Therefore, sex-dependent expression of transport
187
proteins in one organ may not change the overall pharmacokinetics due to equal
expression of the same transport protein in other organs between males and
females.
Overall, it is still challenging to examine the possible influence of sex on
transporter-mediated drug disposition due to the apparent complexity of transport
mechanisms as mentioned above. It is plausible that transporter-dependent sex
differences in drug disposition between males and females may influence
therapeutic outcome and/or toxicological response if a drug is targeting one specific
organ for its therapeutic efficacy (e.g., statins).
Data regarding sex-dependent
differences in transport proteins in clearance organs (e.g., liver, kidney) can be
incorporated in physiologically-based pharmacokinetic modeling to predict the
impact of sex-dependent transport functions on the overall drug disposition.
Pharmacokinetic modeling and simulation coupled with data from SCH
The utility of SCH as an in vitro model to examine phase I and II metabolism
and hepatic transport mechanisms has been established (Chapter 3, (Liu et al.,
1999c; Hoffmaster et al., 2005; Turncliff et al., 2006; Ghibellini et al., 2007a).
Pharmacokinetic modeling has been employed to describe the hepatobiliary
disposition of model substrates, and subsequent simulation studies utilizing
parameter estimates from pharmacokinetic modeling have been performed to further
examine the impact of altered function of one or more hepatic transport proteins on
overall hepatobiliary disposition (Chandra et al., 2005; Hoffmaster et al., 2005;
Turncliff et al., 2006; Ghibellini et al., 2008). However, these simulations did not
188
incorporate
uncertainty
associated
with
parameter
estimates
or
experimental/analytical error, thereby resulting in a less reliable prediction of the true
phenomenon. A Monte Carlo simulation is a stochastic method using random
numbers and probability to analyze the behavior of a complex system that involves
uncertainty and to determine how random variation or error influences the sensitivity,
performance, or reliability of a model (Rubinstein, 2008). Recently, Monte Carlo
simulations have been applied to the design and execution of clinical trials, and to
evaluating methods to predict drug-drug interactions (Kato et al., 2003; Michelson et
al., 2006). Monte Carlo simulations using data from rat SCH were employed to
predict the impact of altered biliary excretion on the hepatobiliary disposition of TS
and TG in a hypothetical “population” of hepatocytes, and to examine the impact of a
first-order leakage process of TS and TG from the bile canaliculi of SCH into the
incubation medium on the accumulation of TS and TG in hepatocytes and medium.
Interestingly, pharmacokinetic modeling revealed the presence of a first-order
leakage process for TS and TG from bile canaliculi to the incubation medium in SCH
after 20 min of incubation. The mechanism(s) responsible for this leakage process in
SCH have not been characterized. In future studies, it would be beneficial to
examine the hepatobiliary disposition of drugs which undergo enterohepatic
recycling as the parent drug, or without deconjugation, [e.g., rosuvastatin, TS (Kawai
et al., 2000; Nezasa et al., 2002)] because it may reflect a fundamental physiological
process of bile excretion in vivo and subsequent reuptake into the liver after
intestinal absorption. On the other hand, when the time-course data with more than
189
a 20-min incubation time in SCH is used to predict in vivo drug biliary clearance, the
leakage process in SCH may result in underestimation of biliary clearance.
As shown in Chapters 4 and 5, the SCH system is a useful in vitro model to
examine the potential role of transport proteins in the hepatobiliary disposition of a
drug that is associated with liver injury due to its intrinsically toxic properties, or its
interaction with hepatic transport proteins. However, elucidating the role of hepatic
transport proteins in the hepatobiliary disposition of hepatotoxic drugs does not
answer the important question of how impaired hepatic transport mechanisms result
in hepatotoxicity. Although increased accumulation of detergent-like bile acids in the
liver due to drug/metabolite-mediated BSEP inhibition has been proposed as one
mechanism for liver injury associated with some hepatotoxic drugs, the role of other
transport proteins in drug-induced hepatotoxicity remains to be determined.
In order to answer these questions, SCH can be used to measure the activity
of alanine aminotransferase (ALT) and alkaline phosphatase (ALP) along with the
accumulation
of
drugs/metabolites
in
medium,
hepatocytes,
and
bile
at
predetermined time points. Pharmacokinetic modeling can be constructed to explain
the flux of drug/metabolites in SCH. Changes in the activity of ALT and ALP due to
drug exposure can be linked to pharmacokinetic parameters as toxicodynamic
responses when developing an integrated pharmacokinetic/toxicodynamic model.
This approach may provide a mechanistic understanding of the role of hepatic
transport proteins in drug-mediated hepatotoxicity.
Several clinical studies have demonstrated that genetic polymorphisms in
BCRP (ABCG2) can influence drug disposition. For example, the ABCG2 C421A
190
polymorphism, which is associated with decreased protein expression and function
in vitro (Imai et al., 2002), increased plasma concentrations of several BCRP
substrates such as rosuvastatin, diflomotecan, and sulfasalazine (Morisaki et al.,
2005; Zhang et al., 2006a; Yamasaki et al., 2008). However, the ABCG2 C421A
polymorphism had no effect on the plasma pharmacokinetics of NF, which was
tested as a potential clinical probe substrate to assess BCRP activity in humans
(Adkison et al., 2008). Hepatobiliary disposition of the BCRP substrates, whose
pharmacokinetics were examined in subjects, can be assessed in human SCH.
Pharmacokinetic modeling/simulation of the data from human SCH can be utilized to
test whether the impact of hypothetical changes in the biliary excretion pathway on
hepatobiliary disposition of BCRP substrates is consistent with previously reported in
vivo findings in humans. SCH with partial and/or complete BCRP knockdown using
RNAi, which has been recently optimized for Bcrp knockdown in rat SCH (Yue,
2008), can be used to confirm the reliability of predicting the consequences of
altered biliary excretion.
Interaction between compounds and hepatic transport proteins
The results of this dissertation research demonstrate that compounds interact
with transport proteins in multiple ways. For example, BSO increased protein
expression of Mrp4 in rat SCH (Chapter 4).
BSO inhibits γ-glutamylcysteine
synthetase, the rate-limiting step in GSH synthesis (Griffith and Meister, 1979), and
has been used as a glutathione (GSH)-depleting agent. Considering that GSH is
excreted with monoanionic bile acids by Mrp4 (Rius et al., 2003), increased Mrp4
191
expression by BSO may augment GSH depletion in hepatocytes. Interestingly,
Aleksunes et al. demonstrated that the transcription factor NFE2-related factor 2
(Nrf2) was involved in the upregulation of Mrp3 and Mrp4 as one mechanism of
neutralizing oxidative stress during acetaminophen hepatotoxicity (Aleksunes et al.,
2008). Therefore, BSO-mediated upregulation of Mrp4 may be mediated by an
indirect response associated with oxidative stress due to GSH depletion.
Pharmacokinetic modeling of the hepatobiliary disposition of acetaminophen
and metabolites in mouse livers (Chapter 2) required an intervening compartment
between the sites of conjugate formation and excretion into bile to describe the
prolonged biliary excretion of AG and AS during the washout phase. These data
suggest the presence of a discrete compartment with cytosolic localization and
functional Bcrp activity that explains the unidirectional flux of AG and AS prior to
biliary excretion. This hypothesis may be supported by a recent study demonstrating
subcellular localization of BCRP in folate-deprived MCF cells as a process of cellular
adaptation to folate deprivation. (Ifergan et al., 2005). Additionally, the presence of
functional P-gp in the Golgi and the mitochondrial membrane has been reported in
doxorubicin-resistant K562 leukemia cells (Munteanu et al., 2006).
Intracellular
sequestration of substrates mediated by transport proteins, which are functionally
expressed in cytosolic organelles, may affect overall hepatobiliary disposition of
substrates, and further studies are warranted.
192
193
Summary
This dissertation work furthered our understanding of the role of hepatic
transport proteins in drug disposition and drug-induced liver injury using several
model compounds. First, the hepatobiliary disposition of AS and AG was influenced
by sex-dependent Bcrp expression in mice. Clinically-significant sex-dependent
disposition of drugs/metabolites that are BCRP substrates is plausible due to malepredominant BCRP expression in humans. Second, the similarity of TGZ metabolism
and disposition in rat and human SCH with those in rats and humans in vivo further
established the utility of the SCH model as an in vitro tool to predict in vivo
disposition in rats and humans. In addition, pharmacokinetic modeling and
simulation coupled with data obtained from the SCH model predicted likely
alterations in drug/metabolite hepatobiliary disposition in humans.
Third, the
potential involvement of transport proteins other than BSEP in drug-induced liver
injury was proposed for trabectedin (Mrps) and sulindac/metabolites (Ntcp, Oatp or
Bcrp).
194
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199
APPENDIX 1
GF120918 (Elacridar) Inhibits Multidrug ResistanceAssociated Protein 3 (MRP3)- and MRP4-Mediated Transport.
This Chapter will be submitted to Drug Metabolism and Disposition as Short
Communication.
ABSTRACT
GF120918 (GW918) has been used extensively as a potent and selective
inhibitor of P-glycoprotein-(P-gp; MDR1) and breast cancer resistance protein(BCRP; ABCG2) mediated transport. Interestingly, pharmacokinetic modeling of
data generated in our laboratory supports a potential interaction between GW918
and hepatic basolateral efflux proteins. The inhibition, by GW918, of both biliary
and basolateral excretion routes may cause unexpected hepatic accumulation of
drugs. The aim of the present study was to examine the interaction between
GW918 and hepatic basolateral efflux transport proteins, specifically MRP3 and
MRP4. Effects of GW918 (P-gp/BCRP inhibitor) and MK571 (MRP inhibitor;
positive control) on MRP3- and MRP4-mediated transport were examined in
inside-out HEK293 membrane vesicles overexpressing MRP3 or MRP4 using two
model substrates (Estradiol-17-β-glucuronide, E217G and methotrexate, MTX). In
the presence of physiologic concentration of reduced glutathione (GSH, 3 mM),
MK571 (18 µM) significantly inhibited MRP3- and MRP4-mediated E217G and
MTX transport. Interestingly, GW918 (18 µM) significantly inhibited MRP3mediated E217G transport, and MRP4-mediated E217G (30%) and MTX (60%)
transport when GSH was coadministered. In conclusion, GW918, which previously
was designated as a specific canalicular transport inhibitor, also inhibits MRP3and MRP4-mediated hepatic basolateral transport.
201
INTRODUCTION
GW918 (GF120918 ; elacridar; N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine
carboxamide) has been used extensively as a potent and selective inhibitor for Pglycoprotein (P-gp; MDR1) and breast cancer resistance protein (BCRP; ABCG2)
in vitro as well as in vivo. For example, the systemic exposure of topotean was
increased 10-fold in mice in the presence of GW918 (Jonker et al., 2000); the
apparent oral bioavailability of topotecan combined with GW918 was improved
from 40% to 97.1% in patients (Kruijtzer et al., 2002). Recently, a phase I clinical
study was conducted to determine an optimal oral dosing schedule for
coadministration of topotecan and GW918 (Kuppens et al., 2007).
Although GW918 has been designated as a canalicular transport inhibitor,
pharmacokinetic modeling of in vitro/in vivo data generated in our laboratory
indicated that GW918 inhibits a hepatic basolateral efflux process. However, the
exact mechanism of an interaction between GW918 and hepatic basolateral
export proteins remains to be elucidated. Dual inhibition of biliary and basolateral
excretion by GW918 may cause unexpected hepatic accumulation of drugs,
thereby impacting drug efficacy and/or toxicity.
A previous study suggested GW918-mediated inhibition of
basolateral
transport of [D-Pen2, D-Pen5]-enkephalin (DPDPE), an opioid peptide, in isolated
perfused rat liver experiments (Hoffmaster et al., 2004b). In this study, GW918
decreased steady-state perfusate concentrations of DPDPE due to increased
perfusate clearance (Hoffmaster et al., 2004b).
202
These unexpected findings,
particularly in Mrp2-deficient TR- rat livers where DPDPE biliary clearance was
decreased, could be explained by the increased liver/perfusate partitioning and
inhibition of hepatic basolateral efflux of DPDPE in the presence of GW918.
Furthermore, Tian et al. also observed a significant decrease in the rate constant
representing basolateral efflux of spiramycin by GW918 in isolated perfused
mouse livers (Tian et al., 2007). An interaction between GW918 and MRP4 was
proposed by Lee et al. who examined the role of Bcrp in brain disposition of
mitoxantrone and dehydroepiandrosterone sulfate (DHEAS) in mice (Lee et al.,
2005b). The brain uptake of [3H]DHEAS and [3H]mitoxantrone in Bcrp-deficient
mice was comparable with that in wild-type and P-gp-deficient mice, indicating a
minor role of Bcrp and P-gp in the active efflux transport of these substrates at the
blood brain barrier (BBB). Interestingly, the inhibitory effect of GW918 on the
brain disposition of [3H]DHEAS and [3H]mitoxantrone was still observed in P-gpdeficient mice, suggesting a possible contribution of one or more GW918-sensitive
efflux transporters that are distinct from Bcrp and/or P-gp at the BBB. Based on
the abundant expression of MRP4 mRNA at the BBB, it was proposed that MRP4
plays a role in efflux of DHEAS and mitoxantrone at the BBB, and that GW918
may affect the brain uptake of these substrates by inhibiting MRP4.
This study was aimed to identify interactions between GW918 and hepatic
basolateral transport proteins MRP3 and MRP4.
Estradiol 17-β-glucuronide
(E217G) and methotrexate (MTX) were selected as model substrates for MRP3
and MRP4 since the kinetics of both compounds have been characterized
previously in MRP3- and MRP4-overexpressing membrane vesicles; E217G is
203
transported with moderate affinity and low capacity for MRP3 [Km = 25.6 µM; Vmax
= 75.6 pmol/mg protein/min] and for MRP4 [Km = 30.3 µM; Vmax = 102 pmol/mg
protein/min] (Zeng et al., 2000; Chen et al., 2001); MTX is transported with low
affinity and high capacity for MRP3 [Km = 776 µM; Vmax = 288 pmol/mg
protein/min] and for MRP4 [Km = 220 µM; Vmax = 240 pmol/mg protein/min] (Zeng
et al., 2000; Chen et al., 2002) .
204
MATERIALS AND METHODS
Chemicals. Estradiol-17-β-glucuronide (E217G), rifampicin, AMP, ATP, pyruvic
acid and gentamicin sulfate were purchased from Sigma Chemical Co (St. Louis,
MO, USA); creatine phosphate and creatine kinase were purchased from Roche
Diagnostics (Indianapolis, IN, USA), and MK571 was purchased from Cayman
Chemical Co (Ann Arbor, MI, USA). [3H]E217G (46.9 Ci/mmol; purity>97%) were
purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA).
[3H]Methotrexate, disodium salt (MTX; 24 mCi/mmol; purity>99%) was purchased
from Moravek Biochemicals (Brea, CA). All other chemicals and reagents were of
analytical grade and readily available from commercial sources.
Transient Transfection of HEK293 Cells with MRP3 or MRP4. The
pcDNA3.1(+)/MRP3 was kindly provided by Dr. Susan Cole, Queen’s University,
Ontario, Canada.
Recombinant pcDNA3.1/Hygro plasmids containing human
MRP4 cDNA were kindly provided by Dr. Dietrich Keppler, German Cancer
Research Center, Heidelberg, Germany. HEK293 cells were cultured in T-150
flasks with DMEM (high glucose with phenol red containing 10% FBS). After cellcounting with trypan blue in 1:10 dilutions, 4.6 × 106 cells in serum-free DMEM
were seeded in T-150 flasks. After 24-h of cell seeding, HEK293 cells were
transiently transfected using FuGENE 6 kit (Roche Molecular Biochemicals, Laval,
PQ, Canada) per the manufacturer’s instructions.
Briefly, 46.77 µl of Fugene
reagent was diluted with 1553 µl of serum free DMEM without antibiotics and
incubated for 5 min at room temperature per transfection. DNA was added to the
mixture of serum free medium and Fugene reagent and incubated for 45 min at
205
room temperature. After incubation, the total mixture was added directly to the T150 flask. Incubation was continued until cells were confluent (~60-72 hr).
Membrane Vesicle Preparation. MRP3- or MRP4-overexpressing membrane
vesicles were prepared as described previously (Leslie et al., 2001). Transfected
HEK293 cells were homogenized in buffer (250 mM sucrose/50 mM Tris, 0.25 mM
CaCl2, and EDTA-free protease inhibitor, pH 7.4). Cells were disrupted by N2
cavitation (200 PSI, 5 min). The exploded cell suspension was mixed with 0.5 M
EDTA and centrifuged at 1900 rpm for 10 min at 4˚C to remove nuclei. The
supernatant was layered onto 10-12 ml of 35% (w/w) sucrose/1 mM EDTA/50 mM
Tris, pH 7.4 in high speed centrifuge tubes. After centrifugation at 100,000g at
4˚C for 1 hr using SW28 rotor (Beckman), the interface was placed in a 25 mM
sucrose/50 mM Tris, pH 7.4 buffer solution and centrifuged at 100,000g at 4˚C for
30 min. The pellets were resuspended with 250 mM sucrose/50 mM Tris, pH 7.4
and centrifuged at 100,000g at 4˚C for 30 min. The membranes were washed
with buffer (250 mM sucrose, 50 mM Tris, pH 7.4) and resuspended by passing
them through a 27-gauge needle (10-20 times) to make the suspension
homogeneous and aid in vesicle formation. The membranes were aliquoted and
stored at – 80˚C until use.
Western Blot Analysis. Overexpression of MRP3 and MRP4 in membrane
vesicles was confirmed by Western blot analysis. After determination of protein
concentration using the BCA protein assay reagent kit (Pierce Biotechnology, Inc.,
Rockford, IL), samples were diluted to a final protein concentration of 2 µg/10 µl
with 4X NuPAGE sample buffer, dithiothreitol (DTT; 10% final sample volume) and
206
water. Samples were loaded onto a NuPAGE 4-12% 10-well SDS polyacrylamide
gel and separated by electrophoresis in a NOVEX Minicell II blot with NuPAGE
running buffer at 180 V until the marker reached the bottom of the gel (~2 hr).
Proteins in the SDS polyacrylamide gel were transferred onto a PVDF membrane
at 30V for 90 min with NuPAGE transfer buffer. Following transfer, the membrane
was incubated in 5% nonfat dry milk solution in Tris-buffered saline (TBS) with
0.1% (v:v) Tween-20 for 30-60 min at room temperature.
After blocking, the
membrane was incubated with anti-MRP3 antibody (M3II-9; Alexis Biochemicals)
and anti-MRP4 antibody (M4I-10; Alexis Biochemicals) for 2 hr at room
temperature or over-night at 4˚C and rinsed three times at 10 min intervals.
Subsequently, the membrane was incubated with 15 mL horseradish peroxidaselinked secondary antibodies, and rinsed three times. MRP3 and MRP4 in
membranes were treated with SuperSignal West Dura (Pierce Biotechnology) and
detected with a VersaDoc 1000 molecular imager (Bio-Rad Laboratories, Hercules,
CA).
Membrane Vesicle Transport Studies. Uptake of [3H]E217G and [3H]MTX into
membrane vesicles was performed by a rapid filtration method as described
previously (Leslie et al., 2001). Membrane vesicles (5 µg of protein per time point)
were pre-warmed for 1 min at 37˚C and added to Tris/sucrose (TS) buffer,
supplemented with an ATP-regeneration mixture (4 mM ATP or AMP, 10 mM
MgCl2, 10 mM creatine phosphate and 100 µg/ml creatine kinase) in a final
volume of 40 µl. The reaction mixture was incubated with [3H]E217G or [3H]MTX
at 37˚C in the presence of 10 mM DTT or 3 mM GSH solubilized in 10 mM DTT.
207
Initial uptake for E217G and MTX was determined at 1.5 min and 5 min,
respectively, based on previously published studies (Zeng et al., 2000; Chen et al.,
2001; Chen et al., 2002). At designated times, 800 µl of ice-cold TS buffer was
added to the sample mixture. Diluted samples were filtered through Type A/E
glass fiber filters (Pall Life Sciences, East Hills, NY) by a rapid filtration method
and washed twice with TS buffer. Radioactivity associated with the filters was
measured by liquid scintillation counting.
substituted with AMP.
In control experiments, ATP was
Net ATP-dependent transport was calculated by
subtracting uptake measured in the presence of AMP from uptake measured in
the presence of ATP. Measurements were corrected for the amount of ligand
bound to the filters (usually <2% of total radioactivity). To evaluate the inhibitory
effects of GW918 or MK571 (18 µM) on [3H]E217G and [3H]MTX uptake mediated
by MRP3 and MRP4, GW918 or MK571 was added into the reaction mixture, and
then the transport assay was performed as described previously. MK571 was
utilized as a positive control to ensure functional inhibition of MRP3- and MRP4mediated uptake. GW918 and MK571 were dissolved in dimethyl sulfoxide
(DMSO) and diluted with incubation medium. The final DMSO content was less
than 0.1 % in the reaction mixture. In all transport assays, the same concentration
of DMSO was used as a control.
Data Analysis. All data are expressed as mean ± SE unless otherwise indicated.
The effect of GW918 and MK571 on MRP3- and MRP4-mediated uptake was
assessed using a one-way analysis of variance followed by Bonferroni’s post hoc
test. Differences were considered to be statistically significant when p < 0.05
208
RESULTS
Western blot analysis confirmed overexpression of MRP3 and MRP4 in
membrane vesicles (Figure A.1).
To examine functional activity of MRP3- or
MRP4-overexpressing membrane vesicles, [3H]E217G was used as a model
substrate at initial concentrations of 5 µM.
At the 0.5-min time point, ATP-
dependent [3H]E217G uptake was markedly higher in MRP3- and MRP4membrane vesicles with 56.7 ± 15.3 pmol/mg protein and 29.8 ± 6.1 pmol/mg
protein, respectively, compared to 7.4 ± 4.5 pmol/mg protein in control (CTL)
membrane vesicles prepared from nontransfected HEK293 cells (Figure A.2).
ATP-dependent MRP3-mediated [3H]E217G uptake rate in the absence of
GSH was ~26% stimulated by GW918, compared to CTL (Figure A.3.A). In MRP4overexpressing membrane vesicles, uptake of [3H]E217G in the absence of GSH
was ~30% decreased in the presence of GW918, compared to in the absence of
GW918 (Figure A.3.B). MK571 (18 µM), a known inhibitor of MRPs, was used as a
positive control to ensure functional inhibition of MRP-mediated [3H]E217G uptake.
MK571 inhibited ~47% of MRP3-mediated [3H]E217G uptake while transport of
MRP4-mediated [3H]E217G uptake was completely inhibited in the presence of
MK571 in the absence of GSH (Figure A.3.A and A.3.B).
MTX was employed as a substrate with low affinity and high capacity for
MRP3 and MRP4 to examine the interaction between GW918 and basolateral
transport proteins. In MRP3-overexpressing membrane vesicles, ATP-dependent
MRP3-mediated [3H]MTX uptake in the absence of GSH was not affected by
GW918 (Figure A.3.C). Transport of MRP4-mediated [3H]MTX uptake in the
209
absence of GSH was ~15% decreased by GW918 (Figure A.3.D). The degree of
inhibition of [3H]MTX uptake by MK571 was comparable between values obtained
in MRP3-overexpressing membrane vesicles (75% inhibition) and those in MRP4overexpressing membrane vesicles (82% inhibition).
The effect of GSH on MRP3- and MRP4-mediated [3H]E217G and [3H]MTX
uptake was examined in the presence of GW918 or MK571 because previous
reports indicated that GSH is necessary for MRP4-mediated transport of some
substrates (Rius et al., 2003; Rius et al., 2008). DTT (10 mM) was added to the
reaction mixture as a vehicle control since GSH was solubilized in DTT. ATPdependent MRP3- and MRP4-mediated [3H]E217G and MRP3-mediated [3H]MTX
uptake was comparable in the absence or presence of GSH, but MRP4-mediated
[3H]MTX uptake was significantly decreased In the presence of GSH, compared to
DTT control (Figure A. 4.A-D).
ATP-dependent MRP3-mediated [3H]E217G uptake in the presence of
GSH was not affected by GW918 (Figure A.4.A). In MRP4-overexpressing
membrane vesicles, transport of [3H]E217G in the presence of GSH was ~30%
decreased by GW918, compared to without GW918 (Figure A.4.B). MRP3mediated [3H]E217G uptake and MRP4-mediated [3H]E217G uptake was inhibited
~50% and ~100% when MK571 and GSH were included in the incubation medium
(Figure A.4.A and A.4.B).
In MRP3-overexpressing membrane vesicles, ATP-dependent [3H]MTX
uptake in the presence of GSH was significantly decreased (15%) by GW918,
compared to without GW918 (Figure A.4.C). Transport of MRP4-mediated
210
[3H]MTX uptake in the presence of GSH was ~60% decreased by GW918 (Figure
A.4.D). The degree of inhibition of [3H]MTX uptake by MK571 in the presence of
GSH was comparable (~90% inhibition) between values obtained in MRP3overexpressing
membrane
vesicles
and
membrane vesicles (Figure A.4.C and A.4.C).
211
those
in
MRP4-overexpressing
DISCUSSION
The present study demonstrated that GW918 inhibited MRP3-mediated
[3H]MTX transport (~15% inhibition) and MRP4-mediated transport of [3H]E217G
(~30% inhibition) and [3H]MTX (~60% inhibition) when GSH was present. In
contrast, GW918 did not inhibit MRP3-mediated [3H]E217G transport. Compoundspecific GW918 inhibition of MRP3-mediated transport may be due to different
affinities of the substrate for MRP3; E217G represents a substrate with moderate
affinity and MTX a substrate with low affinity.
GW918-mediated inhibition of MRP4 transport may cause organ-specific
differences in drug disposition, depending on the localization of MRP4 throughout
the body. Considering the sinusoidal localization of MRP4 in the liver, GW918mediated inhibition of MRP4 transport coupled with inhibition of canalicular
transport proteins including P-gp and BCRP, may increase hepatic accumulation
of drug, thus causing unexpected hepatic toxicity or compromised therapeutic
efficacy. In contrast to the liver, MRP4 along with P-gp and BCRP are expressed
on the apical domain in the kidney and brain. Therefore, inhibition of MRP4
transport by GW918 may result in more potent inhibition of drug efflux in the
kidney and brain than expected.
While the bile salt export pump (BSEP) is the major canalicular bile salt
transport proteins in hepatocytes, MRP4, but not MRP3, plays an important role in
hepatic basolateral efflux of bile acids in the presence of GSH (Zeng et al., 2000;
Byrne et al., 2002; Rius et al., 2003). Interestingly, affinities of bile acids for MRP4
in the presence of GSH were comparable to those obtained for BSEP,
212
emphasizing the importance of MRP4 as an alternate route of bile acid excretion
from hepatocytes into blood (Byrne et al., 2002; Rius et al., 2003). Up-regulation
of MRP4 under cholestatic conditions has been observed in mice and rats, as well
as in BSEP-deficient patients suffering from progressive familial intrahepatic
cholestasis 2 (PFIC2) (Schuetz et al., 2001; Denk et al., 2004; Keitel et al., 2005).
Therefore, up-regulation of MRP4 under cholestatic conditions has been
recognized as a protective mechanism against hepatotoxicity by preventing
accumulation of bile acids in hepatocytes through MRP4-mediated basolateral
efflux of bile acids. Considering the hepatoprotective role of MRP4, GW918mediated inhibition of MRP4 transport may predispose the hepatocyte to toxicity
when canalicular excretion of bile acids also is impaired due to disease states or
drug interactions.
In conclusion, the present study demonstrated that GW918 significantly
inhibited MRP4-mediated transport in the presence of GSH in inside-out
membrane vesicles. GW918 also impaired MRP3-mediated transport, but the
extent of inhibition was less than for MRP4. Although GW918 has been used
extensively as a P-gp and BCRP inhibitor, the potential inhibition of other hepatic
basolateral transport proteins such as MRP4 and MRP3 may confound data
interpretation when this inhibitor is employed.
213
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of the beta -O-glucuronide conjugate of the tobacco-specific carcinogen 4(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by the multidrug
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Pharmacol Exp Ther 324:86-94.
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Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized
to the basolateral hepatocyte membrane. Hepatology 38:374-384.
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Ramachandran V, Komoroski BJ, Venkataramanan R, Cai H, Sinal CJ,
Gonzalez FJ and Schuetz JD (2001) Disrupted bile acid homeostasis reveals
an unexpected interaction among nuclear hormone receptors, transporters,
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Tian X, Li J, Zamek-Gliszczynski MJ, Bridges AS, Zhang P, Patel NJ, Raub TJ,
Pollack GM and Brouwer KL (2007) Roles of P-glycoprotein, Bcrp, and Mrp2
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215
A.
CTL
vesicle
MRP3
vesicle
B.
MRP3
CTL
vesicle
MRP4
vesicle
MRP4
Figure A.1. Representative western blot of MRP3 (A) and MRP4 (B) in MRP3and MRP4- overexpressing membrane vesicle preparations. Membrane vesicles
were prepared from HEK293 cells transiently transfected with MRP3 or MRP4
cDNA. Equal amounts of protein (7 µg/lane) were used for immunoblot (n=3).
216
ATP dependent E217G uptake
(pmol/mg protein/0.5 min)
80
60
40
20
0
CTL
MRP3
MRP4
Figure A.2. Functional activity of MRP3- or MRP4-overexpressing membrane
vesicles. Membrane vesicles were incubated with 5 µM of [3H]E217G for 0.5 min
at 37˚C. Net ATP-dependent transport was calculated by subtracting uptake
measured in the presence of AMP from uptake measured in the presence of ATP
(mean ± SD from a single experiment in triplicate).
217
80
60
40
20
0
CTL
GW918
MK571
*
180
D
160
140
120
100
80
60
40
20
0
CTL
GW918
MRP4-mediated E217G uptake rate
(pmol/mg protein/1.5 min)
MRP3-mediated E217G uptake rate
(pmol/mg protein/1.5 min)
B
100
35
30
25
20
15
10
5
0
CTL
MK571
100
80
60
40
20
0
CTL
MK571
GW918
*
MRP4-mediated MTX uptake rate
(pmol/mg protein/5 min)
C
MRP3-mediated MTX uptake rate
(pmol/mg protien/5 min)
A
GW918
MK571
Figure A.3. Inhibitory effect of GW918 and MK571 on MRP3- and MRP4mediated [3H]E217G and [3H]MTX uptake in the absence of GSH. Membrane
vesicles overexpressing MRP3 (A) or MRP4 (B) were incubated with 0.5 µM of
[3H]E217G for 1.5 min at 37˚C in the presence or absence of 18 µM GW918 or
MK571 (mean ± S.E; n=3 in triplicate). Membrane vesicles overexpressing MRP3
(C) or MRP4 (D) were incubated with 10 µM of [3H]MTX for 5 min at 37˚C in the
presence or absence of 18 µM GW918 or MK571 (mean ± SD from a single
experiment in triplicate). Net ATP-dependent uptake was calculated by subtracting
values in the presence of 4 mM AMP from those in the presence of 4 mM ATP.
218
60
40
20
0
CTL
CTL
MRP3-mediated MTX uptake rate
(pmol/mg protein/5 min)
GW918
15
10
5
0
CTL
D
200
150
100
50
0
GW918
CTL
GW918
DTT
250
DTT
20
MK571
*
CTL
*
25
GSH
300
CTL
MRP4-mediated E217G uptake rate
(pmol/mg protein/1.5 min)
80
DTT
C
B
*
100
MK571
MRP4-mediated MTX uptake rate
(pmol/mg protein/5 min)
MRP3-mediated E217G uptake rate
(pmol/mg protein/1.5 min)
A
*
250
200
150
100
50
0
CTL
DTT
GSH
MK571
GSH
CTL
GW918
MK571
GSH
Figure A.4. Inhibitory effect of GW918 and MK571 on MRP3- and MRP4mediated [3H]E217G and [3H]MTX uptake in the presence of GSH (3 mM).
Membrane vesicles overexpressing MRP3 (A) or MRP4 (B) were incubated with
0.5 µM of [3H]E217G for 1.5 min at 37˚C in the presence or absence of 18 µM
GW918 or MK571 (mean ± SD from a single experiment in triplicate). Membrane
vesicles overexpressing MRP3 (C) or MRP4 (D) were incubated with 10 µM of
[3H]MTX for 5 min at 37˚C in the presence or absence of 18 µM GW918 or MK571
(mean ± SD from a single experiment in triplicate). GSH (3 mM), which is
solubilized in 10 mM DTT (vehicle control), was added into the reaction mixture.
Net ATP-dependent uptake was calculated by subtracting values in the presence
of 4 mM AMP from those in the presence of 4 mM ATP.
219
APPENDIX 2
Data Summary
Figure 2.2. APAP concentrations (nM) in outflow perfusate from male wild-type (●)
and Bcrp-deficient (○), and female wild-type (▼) and Bcrp-deficient (∆) mouse liver
perfusions. Livers were perfused with Krebs-Henseleit buffer containing 500 nM
APAP for 40 min followed by an additional 30-min infusion with drug-free buffer (n =
3-4/group).
Wild-type mice
Time
(min)
0
10
20
30
35
40
40.3
42
46
50
60
70
Bcrp-deficient mice
Male
Female
Male
Female
0
334 ± 48
372 ± 19
343 ± 18
356 ± 23
332 ± 28
338 ± 88
229 ± 87
57 ± 41
0
0
0
0
297 ± 32
332 ± 116
379 ± 63
358 ± 41
357 ± 49
345 ± 34
206 ± 20
22 ± 26
0
0
0
0
324 ± 20
333 ± 29
349 ± 29
336 ± 39
355 ± 14
321 ± 49
153 ± 10
22 ± 25
0
0
0
0
354 ± 53
412 ± 8
406 ± 11
376 ± 39
384 ± 32
346 ± 91
188 ± 40
37 ± 33
0
0
0
221
Figure 2.3. Cumulative biliary excretion and basolateral excretion of AG (A and B,
respectively; nmol/g liver) in male and female wild-type and Bcrp-deficient perfused
mouse livers through 70 min as described in Figure 2.2 legend (n = 3-4/group; p <
0.05).
AG
(nmol/g liver)
Cumulative Biliary Excretion
Cumulative Basolateral Excretion
Wild-type mice
Male
Female
1.02 ± 0.40
19.06 ± 2.09
222
0.42 ± 0.19
12.47 ± 1.58
Bcrp-deficient mice
Male
Female
0.32 ± 0.02
18.42 ± 1.92
0.22 ± 0.08
12.35 ± 2.19
Figure 2.4. Cumulative biliary excretion and basolateral excretion of AS (A and B,
respectively; nmol/g liver) in male and female wild-type and Bcrp-deficient perfused
mouse livers through 70 min as described in Figure 2.2 legend (n = 3-4/group;
N.D.:not detected; p < 0.05).
AS
(nmol/g liver)
Cumulative Biliary Excretion
Cumulative Basolateral Excretion
Wild-type mice
Male
Female
0.14 ± 0.05
1.02 ± 0.31
223
0.07 ± 0.03
3.37 ± 0.73
Bcrp-deficient mice
Male
Female
0
1.09 ± 0.50
0
2.91 ± 0.96
Figure. 2.6. Representative outflow perfusate rate (pmol/min/g liver) of AG (●) and
AS (■), and biliary excretion rate (pmol/min/g liver) of AG (○) and AS (□) in male and
female wild-type (solid symbols) and Bcrp-deficient (open symbols) mouse livers
(N/A, not available; N/D, not detectable).
Male Wild-type Mouse Liver
Outflow Perfusate Rate
(pmol/min/g liver)
Observed
Time
(min)
0
5
15
25
37.5
44
48
55
65
Biliary Excretion Rate
(pmol/min/g liver)
Predicted
Observed
AG
AS
AG
AS
0
296.87
599.62
579.43
619.80
566.82
315.37
145.49
62.91
0
6.40
44.05
35.71
50.54
19.16
0.00
0.00
0.00
0
218.09
545.92
644.76
676.33
516.10
326.50
137.90
39.74
0
26.50
40.24
40.67
40.68
19.03
5.35
0.43
0.01
Time
(min)
0
5
15
25
35
45
55
65
AG
AS
AG
AS
0
0
2.02
8.44
16.21
28.44
25.28
14.40
0
0
2.71
3.52
3.02
5.54
1.95
1.51
0
0.40
3.99
9.23
14.91
20.26
22.39
22.83
0
0.38
2.04
3.13
3.73
3.68
2.20
1.21
Female Wild-type Mouse Liver
Outflow Perfusate Rate
(pmol/min/g liver)
Observed
Predicted
Time
(min)
0
5
15
25
37.5
44
48
55
65
AG
AS
AG
AS
0
220.34
464.22
460.86
501.22
259.86
116.06
30.36
21.78
0
68.75
111.48
119.32
125.00
28.52
0
0
0
0
222.76
443.40
476.05
480.90
307.03
148.33
37.94
5.30
0
102.62
123.24
123.33
123.33
33.37
4.44
0.09
0
224
Time
(min)
0
5
15
25
35
45
55
65
Predicted
Biliary Excretion Rate
(pmol/min/g liver)
Observed
Predicted
AG
AS
AG
AS
0
0
1.47
3.92
6.61
10.13
11.79
6.05
0
0
1.57
1.84
1.69
1.66
1.27
0
0
0.24
1.99
4.21
6.48
8.51
9.02
9.03
0
0.31
1.31
1.89
2.21
2.07
1.17
0.64
Male Bcrp-deficient Mouse Liver
Outflow Perfusate Rate
(pmol/min/g liver)
Observed
Time
(min)
0
5
15
25
37.5
44
48
55
65
Predicted
AG
AS
AG
AS
0
294.34
516.36
486.93
451.61
295.18
65.85
37.09
0
0
1.44
19.87
20.31
16.67
13.69
0
0
0
0
279.21
483.85
499.94
501.13
278.11
106.70
17.32
1.24
0
11.45
19.27
19.80
19.83
10.65
3.87
0.56
0.03
Biliary Excretion Rate
(pmol/min/g liver)
Observed
Time
(min)
0
5
15
25
35
45
55
65
Predicted
AG
AS
AG
AS
0
0.00
1.57
3.83
7.34
6.84
8.90
3.86
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
0
0.36
2.55
4.84
6.85
8.22
7.52
6.53
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Female Bcrp-deficient Mouse Liver
Biliary Excretion Rate
Outflow Perfusate Rate
(pmol/min/g liver)
(pmol/min/g liver)
Observed
Predicted
Observed
Predicted
Time
(min)
0
5
15
25
37.5
44
48
55
65
AG
AS
AG
AS
0
16.31
146.33
428.06
488.61
374.24
338.91
181.65
49.53
0
41.36
80.18
126.21
91.74
26.83
0.00
0.00
0.00
0
89.67
275.19
374.86
435.64
386.06
303.41
193.53
101.36
0
67.67
85.18
85.31
85.31
27.01
4.42
0.13
0.00
225
Time
(min)
0
5
15
25
35
45
55
65
AG
AS
AG
AS
0
0
1.13
2.98
4.31
7.00
8.67
3.97
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
0
0.10
1.06
2.62
4.40
6.10
6.86
6.90
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Figure 3.2. Accumulation of TGZ, TS, and TG in rat SCH. Rat SCH were treated for
10 min with 5 µM TGZ, TS, or TG and accumulation (pmol/mg protein) was
measured in cells + bile (solid bars) and in cells (open bars). Values represent the
mean of triplicate determinations from n=1 experiment (TGZ) or mean ± SEM of
triplicate determinations in n=3 experiments (TS and TG).
TGZ
TS
TG
Accumulation (pmol/mg protein)
Cells + Bile
Cells
1577
1560.2
2274.8 ± 547.9
1939.3 ± 461.5
907.8 ± 247.0
605.4 ± 171.2
226
Figure 3.3. Mass vs. time profiles of TGZ (●), TS (○), TG (▼) and TQ (∆) in the
medium initially containing 10 µM TGZ in day 4 rat SCH (Mean ± S.E.M.; n=3 livers
in triplicate). Dashed lines represent the best fit of the pharmacokinetic model
derived from the scheme depicted in Figure 3.1 to the data.
Accumulation in Medium (pmol/well)
Observed
Predicted
Time
(min)
TGZ
TS
TG
TQ
TGZ
TS
TG
TQ
10
20
30
60
90
120
14141±1394
12956±513
12165±552
9533±748
7035±461
5294±431
72±10
446±88
922±85
2782±302
4489±397
5670±404
29±2
178±14
361±24
579±117
881±172
1034±221
150±11
204±35
190±13
151±19
128±19
98±21
13773
12646
11612
8989
6958
5386
80
324
977
2849
4488
5794
45
139
278
622
887
1092
115
166
184
170
135
105
227
Figure 3.4. Mass vs. time profiles of TGZ (●), TS (○) and TG (▼) in the hepatocytes
in day 4 rat SCH; incubation medium initially contained 10 µM TGZ (Mean ± S.E.M.;
n=3 livers in triplicate). Dashed lines represent the best fit of the pharmacokinetic
model derived from the scheme depicted in Figure 3.1 to the data.
Accumulation in Hepatocytes (pmol/well)
Observed
Predicted
Time
(min)
TGZ
TS
TG
TGZ
TS
TG
10
20
30
60
90
120
226 ± 49
296 ± 34
235 ± 14
206 ± 58
110 ± 38
45 ± 17
819 ± 179
1072 ± 264
1187 ± 333
1378 ± 276
1296 ± 280
1182 ± 243
59 ± 5
76 ± 7
64 ± 15
46 ± 6
37 ± 11
24 ± 3
233
217
199
154
119
92
582
1013
1229
1360
1285
1197
53
61
58
47
38
31
228
Figure 3.5. Mass vs. time profiles of TS (○) and TG (▼) in the bile canaliculi of day 4
rat SCH; incubation medium initially contained 10 µM TGZ (Mean ± S.E.M.; n=3
livers in triplicate). Dashed lines represent the best fit of the pharmacokinetic model
derived from the scheme depicted in Figure 3.1 to the data.
Accumulation in Bile (pmol/well)
Observed
Predicted
Time
(min)
TS
TG
TS
TG
10
20
30
60
90
120
138 ± 70
234 ± 22
202 ± 40
174 ± 68
191 ± 51
112 ± 18
28 ± 2
50 ± 2
49 ± 10
36 ± 4
26 ± 13
20 ± 5
60
247
172
182
174
162
17
53
44
33
26
22
229
Figure 4.1. Effect of DEX on trabectedin-mediated cytotoxicity in SCRH isolated
from wild-type (WT) Wistar rats. Cytotoxicity was determined by measuring LDH
release into the culture medium after 24 hr trabectedin (1 - 1000 nM) exposure.
Each bar represents the mean ± S.E.M. (n=3) for samples from vehicle- (0.1 µM
DEX; solid bars) or additional DEX (1 µM ; white bars); *, p < 0.05.
Trabectedin Concentration
(nM)
Vehicle-treated WT SCRH
(0.1 µM DEX)
DEX-treated WT SCRH
(1 µM DEX)
1
10
50
100
1000
0.1 ± 0.1
1.3 ± 0.8
18.9 ± 4.6
21.5 ± 4.1
25.7 ± 3.2
0.1± 0.1
0.1 ± 0.3
5.1 ± 1.1
6.9 ± 0.8
11.6 ± 2.3
230
Figure 4.2. Trabectedin-mediated cytotoxicity in SCRH from WT and TR- rats.
Cytotoxicity was determined by measuring LDH release into the culture medium
after 24 hr trabectedin (1 - 100 nM) exposure. Each bar represents the mean ±
S.E.M. (n=3) for samples from wild-type (solid bars) or TR- (white bars) SCRH; *, p <
0.05.
-
Trabectedin Concentration
(nM)
WT SCRH
(0.1 µM DEX)
TR SCRH
(0.1 µM DEX)
1
10
50
100
0.4 ± 0.2
0.9 ± 0.4
23.0 ± 1.7
28.3 ± 0.7
0.2 ± 0.2
0.6 ± 0.6
7.5 ± 3.5
10.2 ± 0.2
231
Figure 4.3. Effect of BSO on trabectedin-mediated cytotoxicity. Cytotoxicity was
determined by measuring LDH release into the culture medium after 24 hr
trabectedin (50, 100, and 1000 nM) exposure. BSO (500 µM) was added to the
culture medium 24 h prior to trabectedin exposure. Each bar represents the mean ±
S.E.M. (n=3) for samples from vehicle- (solid bars) or BSO-pretreated (white bars)
SCRH; *, p < 0.05.
Trabectedin Concentration
(nM)
Vehicle-treated WT SCRH
BSO-treated WT SCRH
(500 µM BSO)
50
100
1000
18.9 ± 4.6
21.5 ± 4.1
25.7 ± 3.2
3.8 ± 1.9
7.8 ± 2.1
13.4 ± 2.0
232
Figure 4.5. Trabectedin perfusate concentrations (Figure 4.5.a) and cumulative
biliary excretion (Figure 4.5.b) in isolated perfused livers from WT (solid circle) and
TR- (white triangle) rats. Data are presented as mean ± S.D., n= 3/group.
Perfusate Concentrations (nM)
Time
WT Rat Livers
TR- Rat Livers
(min)
5
35.1 ± 1.2
38.2 ± 11.5
10
7.3 ± 6.4
13.9 ± 3.2
20
0.5 ± 0.8
2.2 ± 2.0
30
0
0.2 ± 0.3
40
0
0
50
0
0
60
0
0
70
0
0
80
0
0
90
0
0
Cumulative Biliary Excretion (% of dose)
Time
WT Rat Livers
TR- Rat Livers
(min)
5
0.08 ± 0.09
0
10
0.84 ± 0.54
0.08 ± 0.10
20
1.79 ± 0.84
0.37 ± 0.29
30
2.13 ± 0.96
0.49 ± 0.37
40
2.26 ± 1.00
0.53 ± 0.39
50
2.31 ± 1.01
0.54 ± 0.41
60
2.34 ± 1.02
0.56 ± 0.41
70
2.36 ± 1.01
0.56 ± 0.42
80
2.38 ± 1.00
0.56 ± 0.42
90
2.39 ± 0.99
0.57 ± 0.42
233
Figure 5.2. Effect of Sulindac and Metabolites on Hepatobiliary Disposition of TC in
Rat SCH. Accumulation of [3H]TC (1 µM, 100 nCi/mL, 10 min) in cells+bile (solid
bars) and cells (white bars) was measured in the presence of increasing
concentrations (1-100 µM) of sulindac (A), S-sulfone (B) or S-sulfide (C) in rat SCH.
BEI and in vitro Clbiliary of TC were calculated as described in Materials and Methods.
Data are presented as the mean ± S.E.M (n=3-4 livers in triplicate); *, statistically
different from vehicle control (CTL) by one-way ANOVA with Bonferroni’s post-hoc
test.
TC accumulation (pmol/mg protein) in rat SCH
Cells+bile
Cells
Concentration
Mean SEM Mean SEM
(µM)
0
50.6
4.0
10.6
1.7
1
41.6
3.5
9.0
2.3
Sulindac
10
31.0
6.5
12.6
4.8
100
35.5
4.1
11.0
3.2
0
67.2 13.6 11.2
1.4
1
46.2 16.9
7.6
1.4
S-Sulfone
10
33.5
7.1
8.2
2.2
100
22.9
8.8
9.6
2.7
0
75.2
9.1
11.1
1.5
1
50.4
8.3
10.8
2.1
S-Sulfide
10
48.2 13.1
9.4
2.3
100
17.6
7.7
8.1
1.8
234
Figure 5.3. Effect of sulindac and metabolites on hepatobiliary disposition of E217G
in rat SCH. Accumulation of [3H]E217G (1 µM, 100 nCi/mL, 10 min) in cells+bile
(solid bars) and cells (white bars) was measured in the presence of increasing
concentrations (1-100 µM) of sulindac (A), S-sulfone (B) or S-sulfide (C) in rat SCH.
BEI and in vitro Clbiliary of E217G were calculated as described in Materials and
Methods. Data are presented as the mean ± S.E.M (n=3-4 livers in triplicate); *,
statistically different from vehicle control (CTL) by one-way ANOVA with Bonferroni’s
post-hoc test.
E217G accumulation (pmol/mg protein) in rat SCH
Cells+bile
Cells
Concentration
Mean SEM Mean SEM
(µM)
0
143.2 24.0 116.4 14.9
1
146.9 15.4 123.2 3.9
Sulindac
10
110.2 9.9 101.4 12.4
100
42.2
0.5
37.1
2.2
0
141.1 10.0 116.0 6.1
1
120.0 6.7 107.9 7.8
S-Sulfone
10
80.9
3.5
78.8
5.5
100
30.9
3.2
26.7
2.9
0
141.1 10.0 116.0 6.1
1
113.2 8.2 104.2 5.8
S-Sulfide
10
71.9
8.6
59.3
4.9
100
8.7
1.9
7.7
1.8
235
Figure 5.4. Effect of Sulindac and Metabolites on Hepatobiliary Disposition of NF in
Rat SCH. Accumulation of NF (5 µM, 10 min) in cells+bile (solid bars) and cells
(white bars) was measured in the presence of 100 µM sulindac, S-sulfone or Ssulfide in rat SCH. BEI and in vitro Clbiliary of NF were calculated as described in the
Materials and Methods. Data are presented as the mean ± S.E.M (n=3 livers in
triplicate); *, statistically different from vehicle control (CTL) by one-way ANOVA with
Bonferroni’s post-hoc test.
NF accumulation (pmol/mg protein) in rat SCH
Cells+bile
Cells
Concentration
Mean SEM Mean SEM
(µM)
CTL
58.4
4.9
20.9
1.1
Sulindac
100
61.4
0.3
34.0
0.0
S-Sulfone
100
59.7
1.4
36.5
0.6
S-Sulfide
100
68.9
8.6
50.7
7.1
236
Figure 5.5. Inhibition of Na+-dependent TC initial uptake in suspended rat
hepatocytes by S-sulfone (A) and S-sulfide (B). Na+-dependent [3H]TC initial uptake
(1 µM, 60 nCi/ml, 30 sec) was determined by subtracting [3H]TC uptake in Na+-free
buffer from [3H]TC uptake in Na+-containing buffer in the presence of increasing
concentrations of S-sulfone (0.5-100 µM) and S-sulfide (0.5-100 µM). Symbols and
error bars represent the means ± S.E.M. of n=3 livers in duplicate. The dotted curve
represents the best fit of the sigmoidal inhibitory Emax model to the data using
WinNonlin; the curve shape factor (r) was estimated as 0.4±0.1 and 0.8±0.2 for Ssulfone and S-sulfide, respectively.
S-Sulfone
Observed
Predicted
Concentration
(µM)
Mean
SEM
0.5
1
5
10
30
50
75
100
72.4
71.4
68.7
53.0
51.3
39.1
47.6
36.3
21.2
10.6
17.1
8.8
19.3
0.7
0.0
7.1
82.8
78.5
65.8
59.3
48.4
43.3
39.4
36.7
237
S-Sulfide
Observed
Predicted
Mean
SEM
89.5
84.6
69.9
54.2
43.0
21.1
13.3
12.0
22.1
19.5
22.2
6.7
15.4
7.4
0.0
6.5
93.0
88.4
67.8
54.8
33.5
25.0
19.5
16.1
Figure 5.6. Inhibition of Na+-independent E217G unitial uptake in suspended rat
hepatocytes by S-sulfone (A) and S-sulfide (B). Na+-independent [3H]E217G initial
uptake (1 µM, 60 nCi/ml, 90 sec) was determined in Na+-free buffer in the presence
of increasing concentrations of S-sulfone (0.5-100 µM) and S-sulfide (0.5-100 µM).
Symbols and error bars represent the means ± S.E.M. of n=3 livers in duplicate. The
dotted curve represents the best fit of the sigmoidal inhibitory Emax model to the data
using WinNonlin; the curve shape factor (r) was estimated as 0.8±0.2 and 1±0.2 for
S-sulfone and S-sulfide, respectively.
S-Sulfone
Observed
Predicted
Concentration
(µM)
Mean
SEM
0.5
1
5
10
30
50
75
100
103.3
86.7
66.4
50.3
34.0
23.2
16.3
20.2
0.0
14.9
20.3
14.7
6.2
5.6
0.0
7.4
93.0
88.4
67.8
54.7
33.4
25.0
19.4
16.1
238
S-Sulfide
Observed
Predicted
Mean
SEM
93.1
84.5
56.7
56.8
16.0
12.4
5.5
8.1
0.0
5.6
13.3
6.7
5.5
2.6
0.0
2.2
93.0
86.9
56.5
39.3
17.6
11.3
7.8
5.9
Figure 5.7. Effect of sulindac and metabolites on hepatobiliary disposition of TC in
human SCH. Accumulation of [3H]TC (1 µM, 100 nCi/mL, 10 min) in cells+bile (solid
bars) and cells (white bars) was measured in the presence of 10 and 100 µM
sulindac, S-sulfone or S-sulfide in human SCH. BEI and in vitro Clbiliary of TC were
calculated as described in Materials and Methods. Bars and error bars denote
means ± one-half of the range from n=2 livers in duplicate.
TC accumulation (pmol/mg protein) in human SCH
Cells+bile
Cells
Concentration
Mean
Range/2
Mean Range/2
(µM)
CTL
Sulindac
S-Sulfone
S-Sulfide
10
100
10
100
10
100
255.1
61.2
108.0
49.5
209.5
226.8
190.6
215.9
119.9
33.5
22.3
33.4
21.0
24.7
13.8
19.7
90.9
127.8
88.7
103.7
48.4
15.4
20.2
17.3
20.9
8.3
13.4
7.1
239
Figure 5.8. Inhibition of Na+-dependent TC initial uptake in suspended human
hepatocytes by S-sulfone (A) and S-sulfide (B). Na+-dependent [3H]TC initial uptake
(1 µM, 60 nCi/ml, 30 sec) was determined by subtracting [3H]TC uptake in Na+-free
buffer from those values in Na+-containing buffer in the presence of increasing
concentrations of S-sulfone (1-300 µM) and S-sulfide (0.1-100 µM). Open and
closed circles denote individual data from n=2 livers in duplicate. The dotted curve
represents the best fit of the sigmoidal inhibitory Emax model to the data using
WinNonlin; the curve shape factor (r) was estimated as 0.6 and 0.9 for S-sulfone and
S-sulfide, respectively.
S-Sulfone
Concentration
(µM)
0.1
0.5
1
5
10
30
50
100
300
Observed
(n=1)
95.8
91.2
69.9
55.3
55.3
32.8
Observed
(n=2)
90.8
87.7
72.9
74.8
47.8
36.1
19.7
S-Sulfide
Predicted
90.7
76.6
71.9
55.2
47.1
37.3
23.2
240
Observed
(n=1)
55.6
37.2
16.6
0.0
0.0
0.0
Observed
(n=2)
Predicted
95.1
96.1
87.6
59.1
37.6
11.1
6.2
0.0
74.2
38.8
24.9
10.6
6.8
3.7
Figure A.2. Functional activity of MRP3- or MRP4-overexpressing membrane
vesicles. Membrane vesicles were incubated with 5 µM of [3H]E217G for 0.5 min
at 37˚C. Net ATP-dependent transport was calculated by subtracting uptake
measured in the presence of AMP from uptake measured in the presence of ATP
(mean ± SD from a single experiment in triplicate).
CTL
MRP3
MRP4
E217G transport (pmol/mg protein/0.5 min)
Mean
SD
7.4
4.5
56.7
15.3
29.8
6.1
241
Figure A.3. Inhibitory Effect of GW918 and MK571 on MRP3- and MRP4-
mediated [3H]E217G and [3H]MTX uptake in the absence of GSH. Membrane
vesicles overexpressing MRP3 (A) or MRP4 (B) were incubated with 0.5 µM of
[3H]E217G for 1.5 min at 37˚C in the presence or absence of 18 µM GW918 or
MK571 (mean ± S.E; n=3 in triplicate). Membrane vesicles overexpressing MRP3
(C) or MRP4 (D) were incubated with 10 µM of [3H]MTX for 5 min at 37˚C in the
presence or absence of 18 µM GW918 or MK571 (mean ± SD from a single
experiment in triplicate). Net ATP-dependent uptake was calculated by subtracting
values in the presence of 4 mM AMP from those in the presence of 4 mM ATP.
CTL
GW918
MK571
CTL
GW918
MK571
E217G transport (pmol/mg protein/1.5 min)
MRP3
MRP4
Mean
SEM
Mean
SEM
64.7
7.1
22.4
6.4
81.4
9.3
15.9
4.8
34.5
2.0
0.5
0.5
MTX transport (pmol/mg protein/5 min)
MRP3
MRP4
Mean
SD
Mean
SD
128.1
36.6
78.6
15.6
128.7
35.1
67.9
7.5
32.2
25.1
14.0
15.4
242
Figure A.4. Inhibitory Effect of GW918 and MK571 on MRP3- and MRP4-
mediated [3H]E217G and [3H]MTX uptake in the presence of GSH (3 mM).
Membrane vesicles overexpressing MRP3 (A) or MRP4 (B) were incubated with
0.5 µM of [3H]E217G for 1.5 min at 37˚C in the presence or absence of 18 µM
GW918 or MK571 (mean ± SD from a single experiment in triplicate). Membrane
vesicles overexpressing MRP3 (C) or MRP4 (D) were incubated with 10 µM of
[3H]MTX for 5 min at 37˚C in the presence or absence of 18 µM GW918 or MK571
(mean ± SD from a single experiment in triplicate). GSH (3 mM), which is
solubilized in 10 mM DTT (vehicle control), was added into the reaction mixture.
Net ATP-dependent uptake was calculated by subtracting values in the presence
of 4 mM AMP from those in the presence of 4 mM ATP.
E217G transport (pmol/mg protein/1.5 min)
MRP3
MRP4
Mean
SD
Mean
SD
CTL (DTT)
71.4
6.9
20.8
2.2
CTL
69.4
9.5
20.7
2.8
GW918
74.4
1.6
14.7
3.2
MK571
32.9
3.3
0.0
0.4
MTX transport (pmol/mg protein/5 min)
MRP3
MRP4
Mean
SD
Mean
SD
CTL (DTT)
234.3
7.6
221.2
10.4
CTL
237.4
15.7
184.4
4.2
GW918
199.3
7.2
79.1
8.9
MK571
18.4
2.2
26.5
2.0
243