ATP-dependent export pumps and their inhibition by cyclosporins

doi:10.1016/0065-2571(94)90023-X

Advances in Enzyme Regulation

Volume 34, 1994, Pages 371-380

https://doi.org/10.1016/0065-2571(94)90023-X Get rights and content

Abstract

Cyclosporins are potent tools to inhibit several primary-active, ATP-dependent export carriers. This has been demonstrated in membrane vesicle transport assays for CsA and for its non-immunosuppressive analog PSC 833. Inhibition in the low micromolar and in the nanomolar concentration range is shown for the three distinct ATP-dependent export carriers in the liver canalicular membrane mediating the secretion into bile of leukotrienes (LTC₄, other cysteinyl leukotrienes, and related conjugates), bile salts (taurocholate), and amphiphilic, mostly cationic substances (daunorubicin and other P-glycoprotein substrates).

Competitive inhibition by cyclosporins is most potent for ATP-dependent taurocholate transport with K_i values of 0.2 and 0.6 μm for CsA and PSC 833, respectively. This inhibition is in agreement with in vivo studies in the rat demonstrating a block at the canalicular membrane in the hepatobiliary elimination of labeled taurocholate. The data suggest that cholestasis, as a side effect during CsA therapy, is largely due to inhibition of the ATP-dependent bile salt export carrier in the canalicular membrane.

Inhibition by cyclosporins is less effective with respect to ATP-dependent leukotriene transport, both during biosynthetic release from mastocytoma cells and during hepatobiliary excretion. The K_i values for the former were 4.5 and 30 μm, and the K_m/K_i ratios only 0.015 and 0.002 for CsA and PSC 833, respectively.

Distinct transporters are inhibited by the cyclosporins with different potency and structurally modified cyclosporins may serve to induce preferential inhibition of a selected transporter. This is illustrated by the inhibition of the multidrug export carrier with daunorubicin as substrate using PSC 833 as inhibitor with a K_i value of 0.3 μm in an in vitro membrane transport system.

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Overall, these relatively low numbers of overlapping DEGs suggest different mechanisms for the three cholestatic drugs. CsA is a known inhibitor of the BSEP and several other ATP-dependent export pumps, which is suggested to be the mechanism underlying CsA-induced cholestasis (Böhme et al., 1994; Kis et al., 2012). The inhibition of the BSEP could not be assessed in HepG2 cells since expression of the BSEP is low in these cells and the expression values of the ABCB11 gene did not pass the PMA criteria (Weerachayaphorn et al., 2009; Yu et al., 2002).
Drug-induced liver injury remains the most common cause of acute liver failure and a frequently indicated reason for withdrawal of drugs. For the purpose of evaluating the relevance of liver cell models for assessing hepatotoxic risks in intact humans, we here aimed to benchmark 'omics-derived mechanistic data from three in vitro models for parenchymal liver function, intended for the investigation of drug-induced cholestasis, against 'omics data from cholestatic patients. Transcriptomic changes in HepG2 cells, primary mouse hepatocytes and primary human hepatocytes exposed to known cholestatic compounds were analyzed using microarrays. Some of the differentially expressed genes in HepG2 cells were also differentially expressed into the same direction in human cholestasis. The overlap between drug-induced transcriptomic responses in primary mouse hepatocytes and primary human hepatocytes appeared limited and no genes overlapping with in vivo cholestasis were found. Thereupon, a pathway for drug-induced cholestasis was used to map the drug-induced transcriptomic modifications involved in bile salt homeostasis. Indications of an adaptive response to prevent and reduce intracellular bile salt accumulation were observed in vivo as well as in the in vitro liver models. Furthermore, drug-specific changes were found, which may be indicative for their cholestatic properties. Furthermore, connectivity mapping was applied in order to investigate the predictive value of the in vitro models for in vivo cholestasis. This analysis resulted in a positive connection score for most compounds, which may indicate that for identifying cholestatic compounds the focus should be on gene expression signatures rather than on differentially expressed genes.
Integrating multiple omics to unravel mechanisms of Cyclosporin A induced hepatotoxicity in vitro
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Citation Excerpt :
This has been confirmed in animal experiments (Galán et al., 1992). Additionally, CsA inhibits several ATP-dependent export carriers, including the Bile Salt Export Pump (BSEP, ABCB11) (Böhme et al., 1994; Kis et al., 2009). The BSEP is responsible for the secretion of bile into the bile canaliculus and inhibition of these transport proteins by CsA will slow down or block the flow of bile, leading to either an intra-hepatocellular build-up of bile-acids or to an increase of serum bile acid levels.
In order to improve attrition rates of candidate-drugs there is a need for a better understanding of the mechanisms underlying drug-induced hepatotoxicity. We aim to further unravel the toxicological response of hepatocytes to a prototypical cholestatic compound by integrating transcriptomic and metabonomic profiling of HepG2 cells exposed to Cyclosporin A. Cyclosporin A exposure induced intracellular cholesterol accumulation and diminished intracellular bile acid levels. Performing pathway analyses of significant mRNAs and metabolites separately and integrated, resulted in more relevant pathways for the latter. Integrated analyses showed pathways involved in cell cycle and cellular metabolism to be significantly changed. Moreover, pathways involved in protein processing of the endoplasmic reticulum, bile acid biosynthesis and cholesterol metabolism were significantly affected. Our findings indicate that an integrated approach combining metabonomics and transcriptomics data derived from representative in vitro models, with bioinformatics can improve our understanding of the mechanisms of action underlying drug-induced hepatotoxicity. Furthermore, we showed that integrating multiple omics and thereby analyzing genes, microRNAs and metabolites of the opposed model for drug-induced cholestasis can give valuable information about mechanisms of drug-induced cholestasis in vitro and therefore could be used in toxicity screening of new drug candidates at an early stage of drug discovery.
A randomised phase III trial of the pharmacokinetic biomodulation of irinotecan using oral ciclosporin in advanced colorectal cancer: Results of the Panitumumab, Irinotecan & Ciclosporin in COLOrectal cancer therapy trial (PICCOLO)
2013, European Journal of Cancer
Citation Excerpt :
The canalicular transport of irinotecan, SN-38 and SN-38G is principally mediated by the transporter proteins ABCC2 and ABCB1. Ciclosporin binds to and inhibits ABCC2,8 binds ABCB19 and causes cholestasis.10 In pre-clinical studies, pre-treatment with intravenous ciclosporin increased the area under the curve (AUC) of irinotecan and reduced its non-renal clearance, consistent with inhibition of biliary excretion.11
The main toxicity of irinotecan in advanced colorectal cancer (CRC) is delayed diarrhoea. Intestinal SN-38, released by deconjugation of the parent glucuronide excreted into the bile or produced in situ by intestinal carboxylesterase, is toxic to the intestinal epithelium. The canalicular transport of irinotecan and SN-38G is mediated by ABCC2 (MRP2) and ABCB1 (MDR1) which are both inhibited by ciclosporin. We tested whether irinotecan and ciclosporin was non-inferior for anti-cancer efficacy and superior for toxicity compared with single-agent irinotecan.
Six hundred and seventy-two patients with advanced, measurable CRC following prior fluoropyrimidine-containing chemotherapy were randomised to either irinotecan 3-weekly 350 mg/m² (or 300 mg/m² if age >70 or performance status (PS) = 2) or 3-weekly irinotecan at 140 mg/m² (120 mg/m² if age >70 or PS = 2) with ciclosporin 3 mg/kg t.d.s. for three days by mouth starting on the morning before irinotecan. The primary end-point was the proportion of patients alive and progression-free at 12 weeks. The key secondary end-point was the incidence of grade ⩾3 diarrhoea within 12 weeks of randomisation.
The proportion of patients progression-free at 12 weeks with irinotecan was 53.4% compared to 47.2% with irinotecan plus ciclosporin (difference = −6.3%, 95% confidence interval (CI) [−13.8%, 1.3%]). Since the lower limit of the 95% CI crossed the pre-specified non-inferiority margin of −10.6%, non-inferiority of irinotecan plus ciclosporin compared to irinotecan alone was not statistically demonstrated. 15.0% patients developed severe diarrhoea on irinotecan compared to 13.8% on irinotecan plus ciclosporin, a non-significant difference.
The pharmacokinetic biomodulation of irinotecan using oral ciclosporin does not improve the therapeutic index of irinotecan in advanced CRC.
The trial was funded by Cancer Research UK and supported by Amgen Pharma.
Dose-dependent inhibition of transporter-mediated hepatic uptake and biliary excretion of methotrexate by cyclosporine A in an isolated perfused rat liver model
2010, Journal of Pharmaceutical Sciences
Citation Excerpt :
Nevertheless, these data suggest that the hepatic uptake and biliary excretion of the drug are important processes, which may affect the concentration, and consequently, the effects/toxicity of the drug in the liver and intestine. In the present study, we investigated the effects of two different doses of CyA, a drug known to inhibit the hepatic sinusoidal uptake transporters OATP/ Oatp22 and the biliary efflux transporter MRP2/ Mrp2,20,21 on the hepatobiliary disposition of MTX in a recirculating IPRL model. Both doses of CyA significantly reduced the hepatic (Tab. 1)
Methotrexate (MTX) and cyclosporine (CyA) are coadministered in a number of diseases. In this study, the effects of CyA on the hepatobiliary disposition of MTX were investigated in an isolated perfused rat liver model. A bolus 5-mg dose of MTX was added to a recirculating perfusate in the absence or presence of a relatively low (0.5 mg) or high (2.5 mg) dose of CyA or vehicle pretreatment, and perfusate, bile, and terminal liver samples were collected for analysis by high-performance liquid chromatography (HPLC). In control and vehicle groups, MTX showed a low hepatic extraction ratio (~ 0.1) and was almost completely eliminated by excretion into the bile. The low-dose CyA significantly reduced (60%) the hepatic extraction ratio and clearance of MTX, without affecting the bile/liver concentration ratio, suggesting inhibition of sinusoidal uptake of MTX only. In contrast, the high-dose CyA significantly reduced both hepatic uptake and Mrp2-mediated biliary excretion of MTX. Isolated rat hepatocyte uptake studies showed significant inhibition of [³H]MTX uptake in the presence of CyA. It is concluded that CyA significantly alters the hepatobiliary disposition of MTX by inhibiting its sinusoidal uptake and/or biliary transport, potentially reducing enterohepatic recirculation of the drug in vivo.
Cyclosporin A and atherosclerosis - Cellular pathways in atherogenesis
2010, Pharmacology and Therapeutics
Cyclosporin A (CsA) is an immunosuppressant drug widely used in organ transplant recipients and people with autoimmune disorders. Long term treatment with CsA is associated with many side effects including hyperlipidemia and an increased risk of atherosclerosis. While its immunosuppressive effects are closely linked to its effects on T cell activation via the inhibition of the nuclear factor of activated T cells (NFAT) pathway, the precise mechanisms underlying its cardiovascular effects appear to involve multiple pathways additional to those relevant for immunosuppression. These include inhibition of calcineurin activity and intracellular cyclophilin peptidylprolyl isomerase and chaperone activities, inhibition of pro-inflammatory extracellular cyclophilin A, and NFAT-independent transcriptional effects. CsA demonstrates complex effects on lipoprotein metabolism and bile acid production, and affects endothelial cells, smooth muscle cells and macrophages, all of which are critical to the atherosclerotic process. Interpretation of the available data is hampered as many experimental models are used to study the effects of CsA in vivo and in vitro, leading to diverse and often contradictory findings. In this review we will describe the cellular mechanisms related to CsA-induced hyperlipidemia and atherosclerosis, with a focus on identifying pro-atherogenic pathways that are distinct from those relevant to its immunosuppressant effects. The potential of CsA analogues to avoid such sequelae will be discussed.
DNA damage caused by benzo(a)pyrene in MCF-7 cells is increased by verapamil, probenecid and PSC833
2007, Toxicology Letters
The aim of this study was to clarify whether pharmaceutical drugs capable of inhibiting ABC-transporters affect the toxicity of benzo(a)pyrene (BP). MCF-7 breast adenocarcinoma cells were cultured for 24 and 48 h with benzo(a)pyrene (1 μM) and the transporter inhibitors verapamil (0.125–100 μM), PSC833 (0.05–5 μM) or probenecid (0.05–2 mM). DNA binding of benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) was analyzed by synchronous fluorescence spectrophotometry and p53 protein by immunoblotting. BP metabolism was studied using thin layer chromatography (TLC). MTT assay and ATP quantitation were used for the analysis of cell viability. At 24 h there was no statistically significant increase in the DNA–adduct formation by any of the used inhibitors. However, at 48 h all of the inhibitors, in concentrations known to effectively block ABC transporters, increased the BPDE–DNA adduct formation 1.5 to 2-fold compared to adduct formation with BP only. PSC833 and verapamil also increased p53 protein expression at 48 h (p < 0.05). Probenecid decreased glucuronidation of ³H-BP metabolites. Other inhibitors did not decrease statistically significantly the overall formation of water-soluble metabolites. BP alone slightly decreased viability of cells at 48 h according to ATP quantitation as compared to vehicle treated controls (86.4 ± 16.4%). Even though the used inhibitors showed some cytotoxicity, the combination of BP and inhibitors did not decrease cell viability in synergistic manner. According to these results certain pharmaceutical drugs may increase DNA damage caused by benzo(a)pyrene in MCF-7 cells at least partly through the inhibition of transporters. Taking into account the complex metabolism of BP and lack of specificity of the inhibitors used, it is likely that increased DNA damage seen in this study was the result of multiple interactions between the inhibitors, BP metabolism and the efflux of the compounds.

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Advances in Enzyme Regulation

Abstract

References (29)

Biochemistry of multidrug resistance mediated by the multidrug transporter

Annu. Rev. Biochem.

Multidrug resistance

Advan. Cancer Res.

The biochemistry of P-glycoprotein-mediated multidrug resistance

Annu. Rev. Biochem.

Expression of the multidrug-resistant gene in hepatocarcinogenesis and regenerating rat liver

Science

Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues

The function of Gp170, the multidrug resistance gene product, in rat liver canalicular membrane vesicles

J. Biol. Chem.

mdr2 Encodes P-glycoprotein expressed in the bile canalicular membrane as determined by isoform-specific antibodies

J. Biol. Chem.

Cloning and regulation of the rat mdr2 gene

Nucl. Acids Res.

ATP-dependent primary active transport of cysteinyl leukotrienes across liver canalicular membrane

J. Biol. Chem.

ATP-dependent transport of taurocholate across the hepatocyte canalicular membrane mediated by a 110-kDa glycoprotein binding ATP and bile salt

J. Biol. Chem.

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

FEBS Lett.

ATP-dependent leukotriene export from mastocytoma cells

FEBS Lett.

Inhibition by cyclosporin A of adenosine triphosphate-dependent transport from the hepatocyte into bile

Gastroenterology

Cyclosporins as drug resistance modifiers

Biochem. Pharmacol.

Cited by (56)

Validation of gene expression profiles from cholestatic hepatotoxicants in vitro against human in vivo cholestasis

Integrating multiple omics to unravel mechanisms of Cyclosporin A induced hepatotoxicity in vitro

A randomised phase III trial of the pharmacokinetic biomodulation of irinotecan using oral ciclosporin in advanced colorectal cancer: Results of the Panitumumab, Irinotecan & Ciclosporin in COLOrectal cancer therapy trial (PICCOLO)

Dose-dependent inhibition of transporter-mediated hepatic uptake and biliary excretion of methotrexate by cyclosporine A in an isolated perfused rat liver model

Cyclosporin A and atherosclerosis - Cellular pathways in atherogenesis

DNA damage caused by benzo(a)pyrene in MCF-7 cells is increased by verapamil, probenecid and PSC833