Potential Cholestatic Activity of Various Therapeutic Agents Assessed by Bile Canalicular Membrane Vesicles Isolated from Rats and Humans

doi:10.2133/dmpk.18.16

Drug Metabolism and Pharmacokinetics

Volume 18, Issue 1, 2003, Pages 16-22

https://doi.org/10.2133/dmpk.18.16 Get rights and content

Summary:

The active transport of solutes mediated by the bile salt export pump (BSEP/ABCB11) and multidrug resistance associated protein-2 (MRP2/ABCC2) are thought to involve bile acid-dependent and -independent bile formation, respectively. To evaluate the potential of therapeutic agents as inhibitors of such transporters on bile canalicular membranes, we examined the inhibition of the primary active transport of typical substrates by 15 drugs, clinically known to cause cholestasis in canalicular membrane vesicles. The inhibition by most of the compounds in rat canalicular membrane vesicles (CMVs) was minimal or observed at much higher concentrations than obtained in clinical situations. However, cloxacillin, cyclosporin A and midecamycin inhibited BSEP, and cyclosporin A and midecamycin inhibited MRP2 with an inhibition constant close to the clinical concentration. By comparing the inhibition potential between rat and human CMVs, the inhibition of BSEP- and MRP2-mediated transport by midecamycin and cyclosporin A was relatively similar whereas the inhibitory effect on BSEP-mediated transport by cloxacillin and glibenclamide was more marked in humans than in rats. These results suggest that the majority of cholestasis-inducing drugs have a minimal inhibitory effect on rat BSEP and MRP2 although species differences in inhibitory potential should be considered, especially in the case of BSEP.

References (37)

M. Trauner et al.
Molecular pathogenesis of cholestasis
New Engl. J. Med.
(1998)
S.S. Strautnieks et al.
A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis
Nat. Genet.
(1998)
C.C. Paulusma et al.
A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome
Hepatology
(1997)
M. Bohme et al.
Differential inhibition by cyclosporins of primary-active ATP-dependent transporters in the hepatocyte canalicular membrane
FEBS lett.
(1994)
B. Stieger et al.
Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver
Gastroenterology
(2000)
M. Saxena et al.
ATP-dependent efflux of 2,4-dinitrophenyl-S-glutathione
J. Biol. Chem.
(1995)
K. Niinuma et al.
Primary active transport of organic anions on bile canalicular membrane in humans
Am. J. Physiol.
(1999)
M. Horikawa et al.
The potential for an interaction between MRP2 (ABCC2) and various therapeutic agents: Probenecid as a candidate inhibitor of the biliary excretion of irinotecan metabolites
Drug Metab. Pharmacokin.
(2002)
Y. Yamaoka et al.
A pharmacokinetic analysis program (MULTI) for microcomputer
J. Pharmacobio.-Dyn.
(1981)
K. Ito et al.
Quantitative prediction of in vivo drug clearance and drug interactions from in vitro data on metabolism, together with binding and transport
Annu Rev Pharmacol Toxicol.
(1998)

K. Komatsu et al.

Prediction of in vivo drug-drug interactions between tolbutamide and various sulfonamides in humans based on in vitro experiments

Drug Metab Dispos.

(2000)

K. Ito et al.

Prediction of pharmacokinetic alterations caused by drug-drug interactions: Metabolic interaction in the liver

Pharmacol. Rev.

(1998)

J.F. Cadranel et al.

Chronic administration of cyclosporin A induces a decrease in hepatic excretory function in man

Dig. Dis. Sci.

(1992)

R.P.G. Watson et al.

A proposed mechanism for chlorpromazine jaundice-defective hepatic sulphoxidation combined with rapid hydroxylation

J. Hepatol.

(1988)

F.A. Crocenzi et al.

Role of bile salts in colchicine-induced hepatotoxicity. Implication for hepatocellular integrity and function

Toxicology

(1997)

B.J. Bruns

Colchicine toxicity

Australasian annals of Medicine.

(1969)

C. Funk et al.

Cholestatic potential of troglitazone as a possible factor contributing to troglitazone-induced hepatotoxicity: In vivo and in vitro interaction at the canalicular bile salt export pump (Bsep) in the rat

Mol. Pharmacol.

(2000)

J.G. Hardman et al.

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Cholestatic drug-induced liver injury (DILI) is a type of hepatotoxicity. Its underlying mechanisms are dysfunction of bile salt export pump (BSEP) and multidrug resistance-associated protein 2/3/4 (MRP2/3/4), which play major roles in bile acid (BA) excretion into the bile canaliculi and blood, resulting in accumulation of BAs in hepatocytes. The sandwich-cultured hepatocyte (SCH) model can simultaneously analyze hepatic uptake and biliary excretion. Therefore, we investigated whether sandwich-cultured human induced pluripotent stem cell (iPS cell)-derived hepatocytes (SCHiHs) are suitable for evaluating cholestatic DILI. Fluorescent N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2′-aminoethanesulfonate (tauro-nor-THCA-24-DBD, a BSEP substrate) was accumulated in bile canaliculi, which supports the presence of a functional bile canaliculi lumen. MRP2 was highly expressed in the Western blot analysis, whereas the mRNA expression of BSEP was hardly detectable. MRP3/4 mRNA levels were maintained. Of the 22 compounds known to cause DILI with BAs, 7 showed significant cytotoxicity. Most high-risk drugs were detected using the developed SCHiH system. However, a shortcoming was the considerably low expression level of BSEP, which prevented the detection of some relevant drugs whose risks should be detected in primary human hepatocytes.
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Idiosyncratic drug reactions (IDRs) are a significant problem, and a better understanding would greatly facilitate their prevention and treatment. Unfortunately, mechanistic studies of such idiosyncratic reactions are very difficult. Evidence suggests that most IDRs are caused by chemically reactive species, either because the drug is intrinsically reactive or because it forms a reactive metabolite. There is also evidence that most IDRs are immune mediated. There are several hypotheses about how drugs or their reactive metabolites can induce an immune response that leads to an IDR. The dominant hypotheses are the hapten and danger hypotheses; however, the immune response is very complex, and these simple hypotheses are insufficient to provide a complete picture. In some cases, there are specific HLA associations that are important risk factors, but there are clearly other unknown factors that determine who will have an IDR to a specific drug. It appears that the dominant response that prevents most patients from having a serious IDR is immune tolerance, and an animal model of idiosyncratic drug-induced liver injury (IDILI) was developed by the use of checkpoint inhibition to impair immune tolerance. This model may greatly facilitate mechanistic studies and provide better methods to prevent and treat these adverse reactions.
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Regular ArticlePotential Cholestatic Activity of Various Therapeutic Agents Assessed by Bile Canalicular Membrane Vesicles Isolated from Rats and Humans

Summary:

Molecular pathogenesis of cholestasis

New Engl. J. Med.

A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis

Nat. Genet.

A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome

Hepatology

Differential inhibition by cyclosporins of primary-active ATP-dependent transporters in the hepatocyte canalicular membrane

FEBS lett.

Drug- and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver

Gastroenterology

ATP-dependent efflux of 2,4-dinitrophenyl-S-glutathione

J. Biol. Chem.

Primary active transport of organic anions on bile canalicular membrane in humans

Am. J. Physiol.

The potential for an interaction between MRP2 (ABCC2) and various therapeutic agents: Probenecid as a candidate inhibitor of the biliary excretion of irinotecan metabolites

Drug Metab. Pharmacokin.

A pharmacokinetic analysis program (MULTI) for microcomputer

J. Pharmacobio.-Dyn.

Quantitative prediction of in vivo drug clearance and drug interactions from in vitro data on metabolism, together with binding and transport

Annu Rev Pharmacol Toxicol.