MRP2

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MRP2 (multidrug resistance-associated protein 2)

Aliases: ABC30, CMOAT, DJS, cMRP
Gene name: ATP binding cassette subfamily C member 2 (ABCC2)

Summary

ABCC2/MRP2 is a unidirectional efflux transporter that primarily transports organic anions, including drug conjugates and conjugated bilirubin. Although expressed in numerous tissues in humans, particularly in the liver, kidney, gastrointestinal tract (GIT) and placenta, MRP2 has a more restricted tissue distribution than MDR1 or BCRP. MRP2 is most highly expressed in the liver, where it facilitates the elimination of bilirubin glucuronides and positively charged drugs and conjugates into the bile. It plays a similar role in renal elimination, while in other organs such as the placenta and the GIT it restricts the distribution of its substrates. Therefore, the primary role of MRP2 is to limit cellular exposure to its substrates. Dubin-Johnson syndrome patients possess an inherited mutation of the ABCC2 gene, giving rise to non-functional MRP2, and suffer from chronic hyperbilirubinemia. Although there are clinical DDIs ascribed to MRP2, and several drug substrates and inhibitors are known, MRP2 receives little attention in the current FDA guidance, and it is not mentioned in the EMA guidance. Nevertheless, investigations for NCEs should be considered on a case-by-case basis, particularly if hyperbilirubinemia is observed. 

Localization

MRP2 localizes to the apical membrane domain of polarized cells such as hepatocytes, renal proximal tubule cells, and intestinal epithelia, where it mediates unidirectional transport of substrates to the luminal side of the organ, therefore acting as an ATP-dependent efflux pump. MRP2 is also present in the gallbladder, bronchi, and placenta. Of the nine MRPs, only MRP2 localizes exclusively to the apical membrane of polarized cells. 
In contrast with other efflux transporters like MDR1 and BCRP, MRP2 is either absent or below detectable limits in the human blood-testis and blood-brain barriers. It is, however, present in brain capillaries of hippocampus specimens from patients with temporal lobe epilepsy, and in malignant tumor cells from renal clear-cell, hepatocellular, ovarian, colorectal, lung, breast, and gastric carcinomas (reviewed in [1]).

Function, physiology, and clinically significant polymorphisms

The predicted membrane topology of MRP2 transporter includes 12 transmembrane domains with at least two substrate-binding sites. The localization of MRP2 supports a function in the terminal excretion and detoxification of endogenous and xenobiotic organic anions, particularly conjugates of glutathione (GSH), glucuronate or sulfate, as well as a contribution to resistance towards anticancer drugs targeting solid malignant tumors. MRP2 has a broad substrate specificity: it transports organic anions with the highest affinity for glucuronate, GSH conjugates of lipophilic substances (e.g. LTC4), sulfated bile salts [2], compounds without anionic conjugate residues (e.g. methotrexate and bromosulfophthalein), reduced and oxidized GSH, nucleotide analogues, and anticancer drugs. MRP2 transports conjugated endogenous and xenobiotic substances, including hormones, toxins and carcinogens, into the bile (from hepatocytes), urine (from renal proximal tubular cells), and the intestinal lumen (from enterocytes) [3]. Intriguingly, while some estrogen conjugates are transported highly actively by BCRP as well as MRPs, androgen glucuronides are excreted exclusively by MRPs, with MRP3 displaying higher affinity compared to MRP2 [4]. The hepatic MRP2 pump contributes to the driving forces of bile flow and is the major transporter responsible for the biliary excretion of bilirubin glucuronides. 
Known pharmacological inhibitors of MRP2 include probenecid, cyclosporine, vindesine, PSC833 and MK571. With relevance to potential food-drug interactions, flavonoids like quercetin may not only be substrates but also inhibitors of MRP2 [5, 6]; e.g., the flavone baicalein enhanced the oral bioavailability of silybin by inhibiting MRP2- and BCRP-mediated intestinal efflux [7].
MRP2 appears to have at least two substrate-binding sites, which demonstrate some inter-dependency. For example, in vitro glutathione (GSH) transport by MRP2 is stimulated by sulfinpyrazone at low concentrations, but at much higher concentrations, the latter occupies both transporter binding sites, preventing the co-transport of glutathione, yet allowing sulfinpyrazone to be transported alone [8].
While the majority of the >200 identified naturally occurring ABCC2 SNPs do not result in clinically relevant changes in function, selected variants have been shown to alter the PK of clinical drugs. The hereditary disorder Dubin-Johnson syndrome is characterized by complete loss of functionally active MRP2, resulting in chronic hyperbilirubinemia. In instances where sequence variants in the ABCC2 gene correlate with loss of MRP2 function, this is often compensated for by the upregulation of other membrane transporters with similar substrate specificities, notably MRP3 and MRP4, in the basolateral membrane of hepatocytes [9].
MRP2 expression is regulated at the transcriptional and post-transcriptional level by many endogenous and xenobiotic substances, and is altered in different disease states. Transcriptional levels may respond to changes in the intracellular concentrations of bile acids and other lipophilic compounds that are ligands for nuclear hormone receptors such as PXR, CAR, FXR and HNF4[10]. Induction of MRP2 mRNA by PXR/CAR ligand drugs, however, does not necessarily translate into elevated protein abundance: rifampin and carbamazepine both significantly induced ABCC2 transcription in the duodenum without affecting protein levels [11]. MRP2 is upregulated in acute liver injury and may mitigate the effects of ER stress [12], whereas MRP2 protein abundance declines in chronic liver diseases [13]. Interestingly, Mrp2 in mice was found to be regulated by the circadian clock, and its expression correlated inversely with the diurnal variation in plasma unconjugated bilirubin concentration [14].

Clinical significance

MRP2 transports numerous clinically important compounds across multiple drug classes. Examples of clinical drug substrates include antibiotics such as ampicillin, azithromycin, and ceftriaxone; anticancer agents such as cisplatin, doxorubicin, irinotecan, and methotrexate (MTX); antihypertensives such as olmesartan and temocaprilate; HIV drugs such as adefovir, lopinavir, and saquinavir [15]; and phytotherapeutic flavonolignan silybin [16]. MRP2 confers resistance to diverse chemoterapeutics such as cisplatin, MTX, mitoxantrone, and etoposide/teniposide. However, DDIs due to MRP2 modulation are difficult to describe for multiple reasons. To name a few, specific and potent clinical inhibitors and substrates are lacking; many MRP2 substrates are conjugated drug metabolites; and compensation by other MRPs (e.g. MRP3 and 4) may mask the effect of MRP2 on drug clearance. Thus, studies that discuss the involvement of MRP2 in DDIs typically refer to multiple transporters, including OATs, P-gp, BCRP, other MRPs, and OATPs. E.g., increased risk of kidney injury upon coadministration of the semisynthetic vinca alkaloid vindesine with high-dose MTX could be assigned to simultaneous inhibition of BCRP and MRP2 by vindesine and the resultant accumulation of MTX in proximal tubule cells [17]. As another example for the involvement of multiple transporters, leflunomide/teriflunomide increased the kidney/plasma ratio of acyclovir by inhibiting MRP2, but also increased plasma concentration by concomitant inhibition of OAT1 and OAT3 [18].
MRP2-mediated biliary excretion of estradiol metabolites such as the strongly cholestatic estradiol-17β-glucuronide (E217βG) is relevant to the development of cholestasis associated with oral contraceptive use or pregnancy, as E217βG can only exert its inhibitory effect on BSEP from the luminal side [19].
Roughly half a dozen polymorphisms in the ABCC2 gene have been shown to be of clinical significance. The SNPs -24C/T, 1249G/A, 2366C/T, 3563T/A, 3972C/T, 4544G/A, and 4348G/A are associated with altered PK of irinotecan, pravastatin, methotrexate (MTX), mycophenolic acid, telmisartan, talinolol, tacrolimus, lopinavir, doxorubicin, and deferasirox. In particular, pediatric cancer patients carrying the MRP2 -24T allele show a gender-specific reduced methotrexate clearance, with -24T allele females having approximately 2-fold higher AUC versus non-variant patients, which results in more severe toxicities [20, 21]. Renal allograft recipients carrying the -24T or the 3972T allele showed a 17% increase in the AUC of mycophenolic acid [22]. The -24T allele also predisposes to diclofenac-induced hepatotoxicity [23], increases the likelihood of resistance to antiepileptic drugs [24], and accelerates the clearance of the iron chelator deferasirox [25]. The 1249G>A polymorphism, on the other hand, appeared to decrease the risk of therapy resistance in Asian epileptic patients [26]. 
In addition to conjugated hyperbilirubinemia, Dubin-Johnson syndrome patients also have elevated urinary coproporphyrin ratios, which incidentally may provide quantitative information of in vivo MRP2 activity [27].

Regulatory requirements

Although there are several identified substrates and inhibitors of human MRP2, and a number of clinical citations on DDI and the impact of genetic polymorphisms, there is no specific recommendation for this transporter in the current FDA or EMA guidelines. Nonetheless, in vitro evaluation of the interactions of NCEs and particularly their conjugated metabolites should be considered on a case-by-case basis, especially where conjugated hyperbilirubinemia is observed.

References

1.    Nies, A.T. and D. Keppler, The apical conjugate efflux pump ABCC2 (MRP2). Pflugers Arch, 2007. 453(5): p. 643-59.
2.    Jemnitz, K., et al., ABCC2/Abcc2: a multispecific transporter with dominant excretory functions. Drug Metab Rev, 2010. 42(3): p. 402-36.
3.    Heredi-Szabo, K., et al., Potentiation of MRP2/Mrp2-mediated estradiol-17beta-glucuronide transport by drugs--a concise review. Chem Biodivers, 2009. 6(11): p. 1970-4.
4.    Jarvinen, E., H. Kidron, and M. Finel, Human efflux transport of testosterone, epitestosterone and other androgen glucuronides. J Steroid Biochem Mol Biol, 2020. 197: p. 105518.
5.    Fang, Y., et al., Structure affinity relationship and docking studies of flavonoids as substrates of multidrug-resistant associated protein 2 (MRP2) in MDCK/MRP2 cells. Food Chem, 2019. 291: p. 101-109.
6.    Oh, J.H., J.H. Lee, and Y.J. Lee, Evaluation of the Mrp2-mediated flavonoid-drug interaction potential of quercetin in rats and in vitro models. Asian J Pharm Sci, 2019. 14(6): p. 621-630.
7.    Xu, P., et al., Baicalein Enhances the Oral Bioavailability and Hepatoprotective Effects of Silybin Through the Inhibition of Efflux Transporters BCRP and MRP2. Front Pharmacol, 2018. 9: p. 1115.
8.    Evers, R., et al., Vinblastine and sulfinpyrazone export by the multidrug resistance protein MRP2 is associated with glutathione export. Br J Cancer, 2000. 83(3): p. 375-83.
9.    Donner, M.G. and D. Keppler, Up-regulation of basolateral multidrug resistance protein 3 (Mrp3) in cholestatic rat liver. Hepatology, 2001. 34(2): p. 351-9.
10.    Arana, M.R., et al., Physiological and pathophysiological factors affecting the expression and activity of the drug transporter MRP2 in intestine. Impact on its function as membrane barrier. Pharmacol Res, 2016. 109: p. 32-44.
11.    Brueck, S., et al., Transcriptional and Post-Transcriptional Regulation of Duodenal P-Glycoprotein and MRP2 in Healthy Human Subjects after Chronic Treatment with Rifampin and Carbamazepine. Mol Pharm, 2019. 16(9): p. 3823-3830.
12.    Huang, W.G., et al., Endoplasmic Reticulum Stress Increases Multidrug-resistance Protein 2 Expression and Mitigates Acute Liver Injury. Curr Mol Med, 2020. 20(7): p. 548-557.
13.    Drozdzik, M., et al., Protein Abundance of Hepatic Drug Transporters in Patients With Different Forms of Liver Damage. Clin Pharmacol Ther, 2020. 107(5): p. 1138-1148.
14.    Wang, S., et al., Circadian Clock Gene Bmal1 Regulates Bilirubin Detoxification: A Potential Mechanism of Feedback Control of Hyperbilirubinemia. Theranostics, 2019. 9(18): p. 5122-5133.
15.    Chen, Z., et al., Mammalian drug efflux transporters of the ATP binding cassette (ABC) family in multidrug resistance: A review of the past decade. Cancer Lett, 2016. 370(1): p. 153-64.
16.    Xie, Y., et al., Metabolism, Transport and Drug-Drug Interactions of Silymarin. Molecules, 2019. 24(20).
17.    Huang, C., et al., Coadministration of vindesine with high-dose methotrexate therapy increases acute kidney injury via BCRP, MRP2, and OAT1/OAT3. Cancer Chemother Pharmacol, 2020. 85(2): p. 433-441.
18.    Liao, X.Y., et al., Leflunomide increased the renal exposure of acyclovir by inhibiting OAT1/3 and MRP2. Acta Pharmacol Sin, 2020. 41(1): p. 129-137.
19.    Jetter, A. and G.A. Kullak-Ublick, Drugs and hepatic transporters: A review. Pharmacol Res, 2020. 154: p. 104234.
20.    Rau, T., et al., High-dose methotrexate in pediatric acute lymphoblastic leukemia: impact of ABCC2 polymorphisms on plasma concentrations. Clin Pharmacol Ther, 2006. 80(5): p. 468-76.
21.    Liu, Y., et al., Association of ABCC2 -24C>T polymorphism with high-dose methotrexate plasma concentrations and toxicities in childhood acute lymphoblastic leukemia. PLoS One, 2014. 9(1): p. e82681.
22.    Naesens, M., et al., Multidrug resistance protein 2 genetic polymorphisms influence mycophenolic acid exposure in renal allograft recipients. Transplantation, 2006. 82(8): p. 1074-84.
23.    Daly, A.K., et al., Genetic susceptibility to diclofenac-induced hepatotoxicity: contribution of UGT2B7, CYP2C8, and ABCC2 genotypes. Gastroenterology, 2007. 132(1): p. 272-81.
24.    Xue, T. and Z.N. Lu, Association between the polymorphisms in the ATP-binding cassette genes ABCB1 and ABCC2 and the risk of drug-resistant epilepsy in a Chinese Han population. Genet Mol Res, 2016. 15(4).
25.    Cao, K., et al., ABCC2 c.-24 C>T single-nucleotide polymorphism was associated with the pharmacokinetic variability of deferasirox in Chinese subjects. Eur J Clin Pharmacol, 2020. 76(1): p. 51-59.
26.    Wang, Y., et al., The recessive model of MRP2 G1249A polymorphism decrease the risk of drug-resistant in Asian Epilepsy: a systematic review and meta-analysis. Epilepsy Res, 2015. 112: p. 56-63.
27.    Benz-de Bretagne, I., et al., Urinary elimination of coproporphyrins is dependent on ABCC2 polymorphisms and represents a potential biomarker of MRP2 activity in humans. J Biomed Biotechnol, 2011. 2011: p. 498757.