MRP3

MRP3 (multidrug resistance-associated protein 3)

Aliases: ABC31, EST90757, MLP2, MOAT-D, cMOAT2
Gene name: ATP binding cassette subfamily C member 3 (ABCC3)

Summary

ABCC3/MRP3 is an ATP-dependent, unidirectional efflux transporter that plays relatively inconspicuous yet important roles in the disposition of bilirubin and xenobiotics, in hepatocyte relief (especially in cholestatic conditions), and in multidrug resistance. MRP3 was first identified in the liver at the sinusoidal membrane, where it was shown to transport xenobiotics from the liver into the blood, but it is also expressed in other tissues including the gastrointestinal tract (GIT). Typical substrates of MRP3 are bile salts, as well as glucuronide conjugates of bilirubin and drugs. It also facilitates the oral absorption of conjugated forms of some dietary estrogens and antioxidants. It is upregulated in cholestasis and/or conditions with impaired MRP2 function, thus compensating for reduced biliary elimination of bilirubin. In lack of reports on MRP3-mediated DDI, MRP3 is not specifically mentioned in the FDA or EMA regulatory guidances; however, investigation may be indicated for some molecules, e.g. where active oral absorption is suspected, circulating glucuronide conjugates are observed, or changes in liver chemistry are noted. 

Localization

Unlike other efflux transporters such as MRP2, BCRP, or MDR1, MRP3 is basolaterally expressed in polarized cells; hence, in the liver it effluxes its substrates into the blood instead of the bile, and it can facilitate rather than hinder absorption of its substrates from the GIT. It is highly expressed in the liver – both in hepatocytes and cholangiocytes –, in enterocytes [1, 2], as well as in the adrenal gland, kidney, pancreas, placenta, and gallbladder, with lower expression in the lung, spleen, stomach, brain, and tonsils [3]. In addition, MRP3 is overexpressed in some tumor types, and in the liver under cholestatic conditions [4, 5]. Caco-2 cells, a widely used cell line derived from human colon adenocarcinoma cells, also express the MRP3 transporter at the basolateral membrane [6].

Function, physiology, and clinically significant polymorphisms

MRP3, a 170-kDa protein encoded by the ABCC3 gene (chromosome 17q21.3), groups with the ‘long’ MRPs that, like MRP1 and MRP2, contain an extra N-terminal domain in addition to two membrane-spanning and two nucleotide-binding domains [7]. Substrates of MRP3 include endogenous compounds (estradiol-17β-glucuronide, androgen glucuronides [8], leukotriene C4, monovalent bile salts such as cholate and glycocholate), glucuronides of plant-derived compounds (e.g. of phytoestrogens, resveratrol, and curcumin) [9, 10], chemotherapeutic agents, as well as other drugs and drug conjugates including fexofenadine, (acetaminophen glucuronide, and morphine-3-glucuronide [11, 12]. As opposed to MRP1 and MRP2, MRP3 has a higher affinity for glucuronide conjugates than for glutathione conjugates, and does not require glutathione to transport substrates [13]. Efficient basolateral transport by hepatic MDR3 followed by rapid renal clearance through OATs and MRPs may explain low systemic exposure to certain drug conjugates, as it was observed in the case of cabotegravir glucuronide [14].
MRP3 is involved in the physiological regulation of bile salt enterohepatic circulation, and acts as an overflow pump for bile acids in all forms of cholestasis [12]. MRP3 expression is induced under cholestatic conditions, and/or where MRP2 function is impaired (e.g. Dubin-Johnson syndrome), functioning as an alternative hepatocellular protection pathway when normal canalicular bile salt transport is compromised [15, 16]. Cholestatic liver injury in mice can be further alleviated by administration the vitamin A metabolite 9-cis-retinoic acid that elevates MRP3 expression by inhibiting the sumoylation of retinoid X receptor α (RXRα) [17].
Known pharmacological inhibitors of MRP3 include tenofovir, indomethacin, furosemide, and probenecid, as well as non-nucleoside reverse transcriptase inhibitors (delavirdine, efavirenz, and nevirapine), and nucleoside reverse transcriptase inhibitors (emtricitabine and lamivudine). Fidaxomicin, suramin and dronedarone were predicted to inhibit MRP3 in an in silico screen of 1470 drugs, and model predictions were validated in a vesicular transport assay [18]. In in vitro studies, the highly potent pan-MRP inhibitor MK571 is often used [19, 20].
Over 100 genetic variants of MRP3 have been identified [21]. Some polymorphisms showed high inter-ethnic variability, but none of the 27 non-synonymous coding SNPs had an allele frequency greater than 5% in any ethnic population. A transfected cell-based in vitro assessment of the functional consequences of several non-synonymous polymorphisms (R1381S, S346F, and S607N) in MRP3 indicated that these may be risk factors in the development of hepatotoxicity [22], but this has not been explored in vivo.

Clinical significance

To date, no clinically relevant DDIs involving MRP3 have been reported, and there is limited information on its role in the clinical ADME of drugs. This is perhaps not surprising, given that MRP3 often transports conjugated metabolites of drugs (e.g. morphine-3-glucuronide and acetaminophen-glucuronide), which are not generally monitored clinically. However, Mrp3 (-/-) mice show decreased AUC of conjugates of resveratrol, phytoestrogens, 4-methylumbelliferon, gemfibrozil, methotrexate, and diclofenac [23, 24], which underlines the contribution of MRP3 to the oral absorption of these compounds.
MRP3 appears to play a compensatory role for MRP2 in bile acid transport, especially in cholestasis, as evidenced by its up-regulation in Dubin-Johnson patients, as well as TR- and EHBR (transport-deficient and Eisai hyperbilirubinemic) rats, all of whom are genetically deficient in MRP2. MRP3 also transports bilirubin glucuronides into the blood under conditions of impaired biliary bilirubin excretion. 
Although with a narrower range of transported chemotherapeutics compared to MRP1 or MRP2, high expression of MRP3 has also been associated with resistance to cancer treatment with methotrexate, vincristine, and epipodophyllotoxins (e.g. teniposide and etoposide) [25]. MRP3 was overexpressed in HER2-positive breast cancers [5], but its involvement in chemotherapy resistance is very limited compared to that of MRP1 or MRP2. Diphenhydramine and its methylated derivatives were shown to sensitize ovarian cancer cells to cisplatin by diminishing the cisplatin efflux capacity of multiple pumps including MRP2, MRP3, and MRP5 [26].
Several polymorphic variants of MRP3 have been implicated in drug disposition and response to therapy. The rs4793665 SNP of MRP3 was shown to influence morphine clearance in pediatric patients [27]. The 189A>T regulatory polymorphism was associated with poor treatment responses in childhood acute lymphoblastic leukemia [28]; carriers of this allele had higher plasma levels of methotrexate, and gene reporter assays indicated enhanced promoter activity, indicating increased efflux by MRP3. Small cell lung cancer patients carrying the ABCC3 -211T allele showed significantly worse progression-free survival [29].

Regulatory requirements

Currently, neither the FDA nor the EMA guidance specifically recommends the study of MRP3 interactions for NCEs. This is probably due to the lack of clear clinical reports of MRP3-mediated DDIs. However, this transporter may be relevant to NCEs where active oral absorption is suspected, where conjugated (particularly glucuronide) metabolites circulate systemically, or changes in circulating levels of bile salts or bilirubin are observed.

References

1.    Ortiz, D.F., et al., MRP3, a new ATP-binding cassette protein localized to the canalicular domain of the hepatocyte. Am J Physiol., 1999. 276(6 Pt 1): p. G1493-500.
2.    van de Wetering, K., et al., Intestinal breast cancer resistance protein (BCRP)/Bcrp1 and multidrug resistance protein 3 (MRP3)/Mrp3 are involved in the pharmacokinetics of resveratrol. Mol Pharmacol., 2009. 75(4): p. 876-85. Epub 2008 Dec 29.
3.    Scheffer, G.L., et al., Tissue distribution and induction of human multidrug resistant protein 3. Lab Invest., 2002. 82(2): p. 193-201.
4.    Rau, S., et al., Expression of the multidrug resistance proteins MRP2 and MRP3 in human cholangiocellular carcinomas. Eur J Clin Invest., 2008. 38(2): p. 134-42.
5.    Partanen, L., et al., Amplification and overexpression of the ABCC3 (MRP3) gene in primary breast cancer. Genes Chromosomes Cancer., 2012. 51(9): p. 832-40. doi: 10.1002/gcc.21967. Epub 2012 May 14.
6.    Hirohashi, T., et al., Function and expression of multidrug resistance-associated protein family in human colon adenocarcinoma cells (Caco-2). J Pharmacol Exp Ther., 2000. 292(1): p. 265-70.
7.    Slot, A.J., S.V. Molinski, and S.P. Cole, Mammalian multidrug-resistance proteins (MRPs). Essays Biochem, 2011. 50(1): p. 179-207.
8.    Li, C.Y., et al., Major glucuronide metabolites of testosterone are primarily transported by MRP2 and MRP3 in human liver, intestine and kidney. J Steroid Biochem Mol Biol, 2019. 191: p. 105350.
9.    van de Wetering, K., et al., Targeted metabolomics identifies glucuronides of dietary phytoestrogens as a major class of MRP3 substrates in vivo. Gastroenterology., 2009. 137(5): p. 1725-35. Epub 2009 Jul 3.
10.    Jia, Y.M., et al., Multidrug Resistance-Associated Protein 3 Is Responsible for the Efflux Transport of Curcumin Glucuronide from Hepatocytes to the Blood. Drug Metab Dispos, 2020. 48(10): p. 966-971.
11.    Zhou, S.F., et al., Substrates and inhibitors of human multidrug resistance associated proteins and the implications in drug development. Curr Med Chem., 2008. 15(20): p. 1981-2039.
12.    Patel, M., K.S. Taskar, and M.J. Zamek-Gliszczynski, Importance of Hepatic Transporters in Clinical Disposition of Drugs and Their Metabolites. J Clin Pharmacol, 2016. 56 Suppl 7: p. S23-39.
13.    Zelcer, N., et al., Characterization of drug transport by the human multidrug resistance protein 3 (ABCC3). J Biol Chem., 2001. 276(49): p. 46400-7.
14.    Patel, M., et al., Mechanistic Basis of Cabotegravir-Glucuronide Disposition in Humans. J Pharmacol Exp Ther, 2019. 370(2): p. 269-277.
15.    Kullak-Ublick, G.A., B. Stieger, and P.J. Meier, Enterohepatic bile salt transporters in normal physiology and liver disease. Gastroenterology., 2004. 126(1): p. 322-42.
16.    Zollner, G., et al., Adaptive changes in hepatobiliary transporter expression in primary biliary cirrhosis. J Hepatol., 2003. 38(6): p. 717-27.
17.    Yuan, Z., et al., 9-cis-retinoic acid elevates MRP3 expression by inhibiting sumoylation of RXRalpha to alleviate cholestatic liver injury. Biochem Biophys Res Commun, 2018. 503(1): p. 188-194.
18.    Ali, I., et al., Identification of novel MRP3 inhibitors based on computational models and validation using an in vitro membrane vesicle assay. Eur J Pharm Sci, 2017. 103: p. 52-59.
19.    Weiss, J., et al., Inhibition of MRP1/ABCC1, MRP2/ABCC2, and MRP3/ABCC3 by nucleoside, nucleotide, and non-nucleoside reverse transcriptase inhibitors. Drug Metab Dispos., 2007. 35(3): p. 340-4. Epub 2006 Dec 15.
20.    Bodo, A., et al., Differential modulation of the human liver conjugate transporters MRP2 and MRP3 by bile acids and organic anions. J Biol Chem., 2003. 278(26): p. 23529-37. Epub 2003 Apr 19.
21.    Bruhn, O. and I. Cascorbi, Polymorphisms of the drug transporters ABCB1, ABCG2, ABCC2 and ABCC3 and their impact on drug bioavailability and clinical relevance. Expert Opin Drug Metab Toxicol, 2014. 10(10): p. 1337-54.
22.    Kobayashi, K., et al., Functional analysis of nonsynonymous single nucleotide polymorphism type ATP-binding cassette transmembrane transporter subfamily C member 3. Pharmacogenet Genomics., 2008. 18(9): p. 823-33.
23.    Tang, S.C., et al., Genetically modified mouse models for oral drug absorption and disposition. Curr Opin Pharmacol, 2013. 13(6): p. 853-8.
24.    Scialis, R.J., et al., Identification and Characterization of Efflux Transporters That Modulate the Subtoxic Disposition of Diclofenac and Its Metabolites. Drug Metab Dispos, 2019. 47(10): p. 1080-1092.
25.    Kool, M., et al., MRP3, an organic anion transporter able to transport anti-cancer drugs. Proc Natl Acad Sci U S A., 1999. 96(12): p. 6914-9.
26.    Melnikova, M., et al., Diphenhydramine increases the therapeutic window for platinum drugs by simultaneously sensitizing tumor cells and protecting normal cells. Mol Oncol, 2020. 14(4): p. 686-703.
27.    Hahn, D., et al., Influence of MRP3 Genetics and Hepatic Expression Ontogeny for Morphine Disposition in Neonatal and Pediatric Patients. J Clin Pharmacol, 2020. 60(8): p. 992-998.
28.    Ansari, M., et al., Polymorphism in multidrug resistance-associated protein gene 3 is associated with outcomes in childhood acute lymphoblastic leukemia. Pharmacogenomics J., 2012. 12(5): p. 386-94. doi: 10.1038/tpj.2011.17. Epub 2011 May 24.
29.    Muller, P.J., et al., Polymorphisms in ABCG2, ABCC3 and CNT1 genes and their possible impact on chemotherapy outcome of lung cancer patients. Int J Cancer, 2009. 124(7): p. 1669-74.