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ADME PK

BSEP Inhibition

Understand the potential drug-bile salt interaction liabilities of your compound by using our BSEP inhibition assay.

BSEP inhibition is in our portfolio of in vitro transporter services. Cyprotex deliver consistent, high quality BSEP inhibition data.

The effect of BSEP inhibition and its measurement in vitro

  • BSEP (bile salt export pump; ABCB11) is an ATP binding cassette (ABC) drug efflux transporter located in the canalicular membrane of hepatocytes1.
  • BSEP is the major transporter for the secretion of bile acids from hepatocytes into bile in humans1.
  • Bile secretory failure results in cholestasis. Bile secretory failure may be a result of;
    • Mutations in the gene encoding for BSEP which have been associated with cholestatic liver diseases of varying severity including progressive familial intrahepatic cholestasis type 2 (PFIC-2) and benign recurrent intrahepatic cholestasis type 2 (BRIC-2)2.
    • Direct inhibition of BSEP by xenobiotics or drugs leading to acquired cholestasis and drug-induced liver injury (DILI)3.
  • Because of the link between BSEP inhibition and cholestatic DILI, the European Medicines Agency Guideline on the Investigation of Drug Interactions (2012) recommends in vitro screening of BSEP inhibition. If inhibition is observed then adequate biochemical monitoring including serum bile salts should be assessed during drug development4.
  • Cyprotex offer a BSEP inhibition assay which investigates inhibition of uptake of the BSEP probe substrate taurocholic acid into inside-out vesicles containing the human BSEP transporter.
Active evaluation of compounds for their potential to interfere with BSEP function is recommended at the earliest stages of drug development.

5Morgan RE et al., (2010) Toxicol Sci 118(2); 485-500

Protocol

BSEP inhibition assay protocol

Substrate 1 μM taurocholic acid
Test Article Concentrations 5 point IC50
Incubation Time 30 min at 37°C
Test System ATP-dependent uptake into inside-out human BSEP vesicles (other species available on request)
Analysis Method LC-MS/MS
Controls 5mM AMP (to correct for non-active uptake)
Ketoconazole (positive control)
Data Delivery IC50

Data

Data from Cyprotex's BSEP Inhibition assay

Figure 1
Graph illustrating the inhibition of taurocholate uptake into inside out vesicles expressing human BSEP transporter by the BSEP inhibitor ketoconazole (n=2 replicates per concentration).
Figure 2
Inhibition of human BSEP uptake (substrate = taurocholate) into inside out vesicles for a number of known BSEP inhibitors. Data shown are the mean ± standard deviation of 3 replicates.

Q&A

Question and answers on BSEP inhibition

Why is it important to investigate BSEP inhibition?

Bile salt export pump (BSEP; ABCB11) is an ATP-binding cassette drug efflux transporter. It is located in the canalicular (apical) membrane of hepatocytes1. The major function of the BSEP transporter is the secretion of bile acids from hepatocytes into bile in humans1.

Genetic mutations in the gene encoding BSEP have been implicated in several cholestatic liver disorders such as the progressive familial intrahepatic cholestasis type 2 (PFIC-2) and the less serious benign recurrent intrahepatic cholestasis type 2 (BRIC-2)2.

The relationship between drug-induced cholestasis and inference with the BSEP transporter is well established with several published reports describing drug-induced BSEP as either the cause or a likely contributing factor for liver injury in late stage clinical trials5. The EMA guidance for drug interaction (2012)4 recommend investigating BSEP inhibition using in vitro methods, and suggest that adequate biochemical monitoring (including serum bile salts) should be evaluated during drug development4.

Please provide an overview of Cyprotex’s BSEP Inhibition assay.

The BSEP inhibition assay measures the inhibition of uptake of taurocholic acid into inverted vesicles containing the BSEP transporter. Cyprotex uses Corning® Gentest™ inside-out vesicles prepared by insect cells (Sf9) infected with a recombinant baculovirus encoding the cDNA for the human BSEP transporter. Briefly the BSEP substrate taurocholate and test compound are pre-incubated with the vesicles prior to initiation of the reaction by ATP. At the end of the incubation period the reaction is terminated by the addition of cold washing buffer followed by filtration and analysis of the samples by LC-MS/MS. AMP and vehicle incubations are included as negative controls. Ketoconazole is included as a positive control inhibitor.

How is the data analysed from the vesicle assay?

ATP-dependent uptake activity is calculated using the following equation;

The % remaining activity is calculated using the following equation:

The IC50 is calculated from the % activity remaining using the curve fitting software XLfit.

How did you decide on the incubation conditions for BSEP inhibition?

The BSEP vesicles were purchased as part of a kit from BD Gentest for which a protocol was provided. Despite this we performed our own internal validation to confirm the conditions were appropriate in terms of time linearity, protein concentration linearity and substrate concentration (at or below the Km).

References

1 Wang L et al., (2002) The role of bile salt export pump mutations in progressive familial intrahepatic cholestasis type II. J Clin Invest 110(7); 965-972
2 Dawson PA, Lan T, and Rao A. (2009) Bile acid transporters. Journal of Lipid Research 50(12); 2340-2357
3 Stieger B (2010) Role of the bile salt export pump, BSEP, in acquired forms of cholestasis. Drug Metab Rev 42(3); 437-445
4 The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (Adopted 2012)
5 Morgan RE et al., (2010) Interference with bile salt export pump function is a susceptibility factor for human liver injury in drug development. Toxicol Sci 118(2); 485-500

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