Medicinal Chemistry

Review

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Medicinal Chemistry

Modulators of the human ABCC2: hope from natural sources?

Human ABCC2 is an ATP-binding cassette transporter involved in the export of endobiotics and xenobiotics. It is involved in cisplatin resistance in cancer cells, particularly in ovarian cancer. The few known ABCC2 modulators are poorly efficient, so it is necessary to explore new ways to select and optimize efficient compounds ABCC2. Natural products offer an original scaffold for such a strategy and brings hope for this aim. This review covers basic knowledge about ABCC2, from distribution and topology aspects to physiological and pathological functions. It summarizes the effect of natural products as ABCC2 modulators. Certain plant metabolites act on different ABCC2 regulation levels and therefore are promising candidates to block the multidrug resistance mediated by ABCC2 in cancer cells.

Human ABCC2 The controlled transfer of molecules by membrane integrated transport proteins became necessary early in archaic cell-like structures for the uptake of nutrients, the excretion of metabolic products, protection against xenobiotics and detoxification of endobiotics [1] . It is widely accepted that drug permeation across membrane barriers is regulated by the basic physical characteristics of the drugs as well as their interactions with membrane transporters. In cancer chemotherapy, the ultimate membrane barrier is the plasma membrane of the cancer cell. ABC transporters are active components of this barrier, and on the basis of their overlapping substrate recognition patterns, some of them act as a shield for drug-resistant cancer cells [2] . The ABC transporter gene family is one group of evolutionary old and highly conserved genes [1] . The designation ABC was based on the highly conserved ATP-binding cassette domain, the most characteristic feature of the superfamily [3] . These proteins use the energy of the bound ATP to transport various molecules across all cell membranes [4,5] . Members of the ABC C subfamily, multidrug resistance protein family

10.4155/fmc.15.131 © 2015 Future Science Ltd

(MRP), MRP1 to MRP7, are extensively studied due to their implication in drug resistance. MRP1 (ABCC1) was discovered in 1992 [6,7] and a spontaneous mutation in hyperbilirubinemic rats with a deficiency in biliary excretion led to the cloning of the liver homolog of ABCC1, termed ABCC2 (or MRP2, Multidrug Resistance Protein 2, also referred to as cMOAT) [8] . Distribution

Elisabeta Baiceanu*,1,2, Gianina Crisan2, Felicia Loghin3 & Pierre Falson**,1 1 Drug Resistance Modulation & Membrane Proteins Laboratory, Molecular & Structural Basis of Infectious Systems, Mixed Research Unit between the National Centre for Scientific Research & Lyon I University n 5086, Institute of Biology & Chemistry of Proteins, 7 passage du Vercors 69367, Lyon, Cedex, France 2 Pharmaceutical Botany Department, Faculty of Pharmacy, University of Medicine & Pharmacy ‘Iuliu Haţieganu’ Cluj-Napoca, 23 Marinescu Street, Cluj-Napoca, Romania 3 Toxicology Department, Faculty of Pharmacy, University of Medicine & Pharmacy ‘Iuliu Haţieganu’ Cluj-Napoca, 5–9 Louis Pasteur Street, Cluj-Napoca, Romania *Author for correspondence: [email protected] **Author for correspondence: [email protected]

In contrast to most ABCC subfamily members, that are typically expressed in basolateral membranes, but similarly to ABCB1 (or P-gp, P-glycoprotein), ABCC2 is always localized in the apical membranes of polarized cells [7,9–11] . It is poorly understood why ABCC2 traffics exclusively to the apical membrane, and literature offers conflicting reports [12,13] . ABCC2 was identified in hepatocytes and at major physiological barriers, such as kidney proximal tubular epithelia enterocytes of the small and large intestine and syncitiotrophoblast of the placenta [14] , and lung [15] Also, ABCC2 mRNA was detected in peripheral nerves, brain blood barrier [16] , gallbladder, placental trophoblasts and CD4 + lymphocytes (Figure 1) [10] .

Future Med. Chem. (2015) 7(15), 2041–2063

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ISSN 1756-8919

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Review Baiceanu, Crisan, Loghin & Falson

Key terms ABC transporters: ATP-binding cassette transporters are ubiquitous membrane-bound proteins, present in all prokaryotes, as well as plants, fungi, yeast and animals. These pumps can move substrates in (influx) or out (efflux) of cells. ABC proteins transport a number of endogenous substrates, including inorganic anions, metal ions, peptides, amino acids, sugars and a large number of hydrophobic compounds and metabolites across the plasma membrane, and also across intracellular membranes. The human genome contains 49 ABC genes, arranged in eight subfamilies and named via divergent evolution. Multidrug resistance: Phenomenon characterized by the ability of drug resistant tumors to exhibit simultaneous resistance to a number of structurally and functionally unrelated chemotherapeutic agents. Out of the various mechanisms described, much research focused on the involvement of ABC transporters in the acquisition of the resistant phenotype. ABCC2: Second member of the subfamily C of the ABC transporter superfamily. It plays an important role in detoxification and chemoprotection by transporting a wide range of compounds, especially conjugates with glutathione, glucuronate and sulfate. Mutations of the ABCC2 gene are associated with Dubin–Johnson syndrome, a condition in which the lack of hepatobiliary transport of nonbile salt organic anions results in conjugated hyperbilirubinemia.

The abundance of ABCC2 has been recently quantified using quantitative proteomics approaches. These studies show that ABCC2 represents 10% of the small intestine membrane proteins, more abundant than ABCB1 (8%), ABCC3 (7%) and ABCG2 (or Breast Cancer Resistance Protein, BCRP [4%]). In the colon, ABCC2 is highly represented with 25%, although less abundant than ABCC3 (36%) [17] . In human liver, ABCC2 is also the most abundant ABC protein (9%)

Brain

Liver

Lungs

Gall bladder Kidney

Reproductive system Placenta

Intestine MRP2 tissue expression Figure 1. ABCC2 distribution in the human body.

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Future Med. Chem. (2015) 7(15)

along with BSEP (13%) [18] . Overall, this suggests the major role that ABCC2 plays on drug disposition and direct or indirect toxicity (Figure 2) . So far, the abundancy of ABCC2 in kidney has not been measured by quantitative proteomics, but there is consistent proof that ABCC2 is highly expressed at renal level too. Of interest is also the fact that ABCC2 is not sufficiently represented in the brain–blood barrier to be quantified using quantitative proteomics [19] . In addition to normal human tissues, ABCC2 protein is also present in some human malignant tumors, as shown by immunostaining of specimens from hepatocellular, renal clear-cell, colorectal, ovarian, leukemia, mesothelioma, lung, breast, bladder and gastric cancer [9] . In contrast, ABCC2 protein expression is negligible or absent in primary testicular tumors, pancreatic adenocarcinomas and gliomas [10,20] . Topology

ABC transporters share a common topology and motifs in the nucleotide-binding domains (NBD). NBDs contain three highly conserved motifs: the Walker A/P-loop, a signature motif/C-loop and the Walker B motif. In mammals, functional ABC transporters contain two transmembrane domains (TMDs), each consisting of six transmembrane (TM) α-helices. TMDs likely provide substrate polyspecificity and translocation. NBDs are located in the cytoplasm and couple the energy to transport the substrates from cytosolic side of the membrane to the other [5,21] . ABCC2, encoded by a gene comprising 32 exons in humans, is thought to contain 17 TM segments organized in three TMD, TMD0, TMD1 and TMD2. The N-terminus has an extracellular location. TMD0 is made of approximately 200 residues defining five putative TM spans and linked to the TMD1 domain via a cytoplasmic loop L0. TMD1 and TMD2 exhibit six TM spans each and are connected via an intracellular segment L1. ABCC2 contains two cytosolic domains 1 and 2, which are located on the L1 segment and the intracellular C-terminus of the TMD2 domain, respectively (Figure 3) [12] . For a better understanding of the physiologic and pathologic functions of ABCC2, there is a pressing need to elucidate its structure. The crystallization of membrane proteins remains a major challenge despite the growing interest and the technological advancements. In this context, ABCB1 is the best known ABC pump, with three solved crystal structures, including structures with binded ligands [22–25] . Transport mechanism

Structural and biochemical data from a number of ABC transporters fundament the ‘ATP-switch’ model.

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36 25

Membrane proteins (%)

40 30 10

20

8

5

3

10

2

13

7

4

9

Colon 3

1

Small intestine Liver

0 MRP2

P-gp

BCRP

MRP3

BSEP

ABC transporter Figure 2. Abundancy of ATP-binding cassette transporters measured by quantitative proteomics. Data taken from [17,18] .

Transport is considered a multistep process involving conformational changes between the NBDs and TMDs. The driver for transport is an on–off switch between two main conformations of the NBDs: a ‘closed dimer’ formed by binding two ATP molecules at the dimer interface, and dissociation of the ‘closed dimer’ to an ‘open dimer’ facilitated by ATP hydrolysis and the release of Pi/ ADP. The ‘switch’ from the ‘open’ to ‘closed’ conformation of the NBD dimer induces changes in the conformation of the TMDs. The hydrolysis of the ATP reverses the switch, from the ‘closed’ dimer to ‘open’ dimer and resets the transporter ready for the next transport cycle [26,27] . How MRPs transport their substrates is not known in detail, due to the lack of structural information, but more information is known from the 3D-structure of the mouse P-glycoprotein for which several structures have been released [22–25] , improved by the release of ABCB1 Cyanidioschyzon merolae and ABCB1 Caenorhabditis elegans structures [24,28–29] . Data suggest the existence of a large cavity in which drugs bind [23,24] . Three to four drug-binding sites are proposed, among which the H and R sites could be precisely located, thanks to the cocrystallization of two inhibitors together with the elucidation of their inhibition mechanism in our laboratory [23,30] . It has been proposed that ABCC2 contains two distinct binding sites: one site for substrate transport and a second site for allosteric regulation of the affinity of the transport site for the substrate [31] . A mutagenesis study revealed four basic residues within the TMD important for transport activity [32] . Models of ABCC2 were built to predict the binding of inhibitors and a series of quercetin glucuronide conjugates [31–33] . Suggested by preliminary success, homology modeling could be a


more amenable approach to derive 3D interactions for ABCC2 than the other ABC family members [31,32] . Pedersen et al. suspect that depending on the physicochemical properties of a drug, it can interact with ABCC2 at several different sites: transported inhibitors compete with the substrate on the cytosolic binding site, while stimulators bind to a second cytosolic binding site similar to the transport site. Yet, lipophilic cationic inhibitors cannot bind to these two cytosolic binding sites because of the charge repulsion, so their transport is mediated by a third binding site accessible from the lipid bilayer [34] . Clinical relevance

ABC proteins have an important influence on the absorption, distribution and/or elimination of drugs and other xenobiotics from different tissues in the body and are believed to be involved in protecting tissues from xenobiotic accumulation and resulting toxicity [35,36] . A well-defined role in the transport of clinically relevant drugs was especially described for the ABCB1, the MRPs 1–5 and ABCG2 [37] . ABCC2 has several physiological and pharmacological activities that makes it an important efflux pumps in living organisms [11,38] . Physiological substrates

ABCC2 has an important role in the export of organic anions, unconjugated bile acids and xenobiotics into the bile, and also contributes to protection against drugs. Mutations of the ABCC2 gene are associated with Dubin–Johnson syndrome, a condition in which the lack of hepatobiliary transport of nonbile salt organic anions results in conjugated hyperbilirubinemia [37] . The overall clinical phenotype of the disease

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NH2

NH2

TMD0

TMD0

TMD1

TMD2

TMD1

TMD2

COOH NBD1

NBD2

NBD1

COOH

NBD2

Figure 3. Topology of ABCC2 protein. Adapted with permission from [13].

is relatively mild, affected individuals suffering from a recessively inherited conjugated hyperbilirubinemia, which can result in observable jaundice. Different approaches helped identify the substrates transported by ABCC2: analysis of compounds transported into bile of normal rats, but not of ABCC2(-) mutant rats; differential uptake of compounds into inside-out bile canalicular membrane vesicles isolated from normal and ABCC2(-) mutant rats; transfection or transduction of human ABCC2 cDNA into cell lines followed by analysis of drug resistance; accumulation of compounds into cells or isolated inside-out membrane vesicles, and transepithelial transport of compounds [37] . The apical localization of ABCC2 in polarized cells involved in the transport of conjugated endogenous and xenobiotic substances suggests the importance of this efflux pump in the terminal Phase of detoxification [12] . ABCC2, rather than ABCB1 or ABCG2, is the optimal elimination pump for conjugates of various toxins and carcinogens with glutathione (GSH), glucuronate or sulfate from hepatocytes into bile, from kidney proximal tubules into urine, or from intestinal epithelial cells into the intestinal lumen [39] . A combination of an efflux pump substrate with an inhibitor/modulator could be used to increase the intestinal absorption and penetration into specific tissues, but it can also determine adverse effects because of the impaired elimination of the drug. The co-administration of two drug substrates for the same transporter may have unexpected and/or unwanted results. Transporter-mediated drug interactions are not easy to predict in vivo, and they are often understood a posteriori. For example, in the case of ABCC2 – diclofenac inhibits transport of anionic substrates, but it stimulates the transport of amphiphilic substrates [40–42] . This is one more reason why it is so important to understand the transport mediated by ABCC2. The

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importance of the ABC transporters is considered as part of the evaluation of the pharmacokinetic profile of a drug, US FDA and also EMA, recommend to test for transport, inhibition or induction of ABC transporters, a tool to predict drug–drug interaction. Some rug transporters have a broad substrate specificity and they are very likely to be involved in drug–drug interactions [43] . The substrate specificities and the tissue- and cell-specific localization of multidrug ABC transporters (including ABCC2), together with data from in silico, in vitro and in vivo experiments, show that these drug transporters play a crucial role in the pharmaco- and toxico-kinetics of xenobiotics. Due to its high expression in the liver, kidney and intestine, it is increasingly recognized that ABCC2 has a major impact in absorption, distribution, metabolism and excretion of drugs and toxins. Anticancer drugs & other therapeutics as ABCC2 substrates

With 7.6 million deaths worldwide (13% of all deaths) in 2008, cancer is one of the most deadly diseases worldwide. Chemotherapy is one of the pillars of cancer treatment, but efficiency is limited by drug resistant phenotypes. Drug resistance arises through several mechanisms, it is either inherent (i.e., at the first treatment), or acquired (i.e., after subsequent treatments) [44] . After a long-term drug use, resistance appears not only to the respective drug, but also to a series of structurally-unrelated drugs. This phenomenon is known as multidrug resistance (MDR) [44,45] . About 50% of all cancers are resistant to therapy and the main hurdle to treating resistant cancer cells is the developing of multidrug resistance [44–46] . The commonly accepted cellular processes that contribute to resistance against chemotherapy are dimin-



ished drug accumulation, reduced drug activation and increased drug metabolization, DNA repair or elevated DNA damage tolerance, modification/alteration of the lethal target, enhanced expression of anti-apoptotic genes and inactivation of the p53 pathway [47,48] . An important MDR mechanism is the increased efflux of chemotherapy drugs from cancer cells by proteins of the superfamily of ABC transporters [49] . ABC transporters generate MDR in tumor cells by actively effluxing antineoplastic drugs, which in turn reduces the intracellular concentration of the drug below the effective therapeutic threshold [50] . It became evident over the last two decades that ABCB1 is the main but not the only human ABC transporter that confers resistance to clinically important chemotherapy agents. MRP-related proteins confer in vitro resistance to natural-derived anticancer drugs and their metabolites, folate antimetabolites, nucleoside and nucleotide analogs, peptide-based agents and alkylating agents [13] . In this subfamily, ABCC2, as well as the other members, recognizes and transports many hydrophobic and hydrophilic antineoplastic agents. ABCC2 confers resistance in cultured cancer cells to methotrexate, paclitaxel, docetaxel, epipodophyllotoxins, vincristine, vinblastine, doxorubicin, as well as glutathione-derived alkylating agents [36,51] . Studies on knock-out mice prove that ABCC2 is involved in the pharmacokinetics of docetaxel [52] , methotrexate and its metabolite 7-hydroxymethotrexate [53] . In addition to these common functions, ABCC2 has a specific role in cisplatin resistance of ovarian, hepatocarcinoma and esophageal cancers [54–57] . Moreover, its downregulation in these cells increases their sensitivity to cisplatin [12,58] . A lot of evidence acquired in vitro indicates that ABCC2 has a role in the detoxification of metalloid salts such as arsenic, antimony or platinum compounds, chemical carcinogens (as shown in ABCC2

Review

knockout mice for [14C]PhIP (2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine) and [14C]IQ (2-amino3-methylimidazo[4,5-f]quinolone [59]) or herbicides which have been shown to upregulate the expression of the transporter (mRNA and protein) [11,53] . Some of the compounds that are most relevant from a pharmacological and toxicological perspective are listed in Table 1. A comprehensive list of all the ABCC2 substrates is given in the recent review published by Van der Schoor [60] . Phytochemicals as ABCC2 modulators Modulation of ABCC2: where are we?

Even for the newest anticancer therapeutics such as tyrosine kinase inhibitors or antibodies, an MDR phenotype development was observed [61] . Several approaches have been developed to restore the drug sensitivity of cells expressing MDR ABC pumps. Drug resistance resulting from ABC multidrug transporters are prevented by a group of compounds known as MDR modulators, inhibitors or chemosensitizers [62] . Reversal agents for ABCC2-induced chemoresistance have not been as readily identified as for ABCB1. Most of ABCB1 antagonists have little or no effect on the drug efflux mediated by MRPs [63] . Some inhibitors of ABCC2 have been established, most of which do not have high selectivity to ABCC2. For instance, MK-571 has an IC50 (50% inhibitory concentration) of 10 μM in membrane vesicle models (in cell models it is used at around 50 μM to inhibit ABCC2 function) [64,65] , but it also inhibits ABCC1 with higher affinity (5 μM in MDCKII-MRP1 cells). Probenecid and furosemide are inhibitors, whereas under certain conditions, sulfinpyrazone, penicillin G and indomethacin considerably stimulate ABCC2 transport activity. The non-nucleoside reverse transcriptase inhibitors (delavirdine, efavirenz and nevirapine), nucleoside reverse transcriptase inhibitors (abacavir,

Table 1. Relevant ABCC2 substrates. Physiological compounds

Glutathione, leukotriene C4, conjugated bile salts, ilirubin glucuronides, steroides (e.g., β-estradiol 3-β- d - glucuronide, ethinylestradiol-3-Oglucuronide and estrone 3-sulfate)

Exogenous compounds

– Anticancer drugs: doxorubicin, etoposide, methotrexate, cisplatin, vincristine, vinblastine – HIV drugs: indinavir, ritonavir, saquinavir, adevovir, cidofovir – Antibiotics: ampicillin, cetodizime, ceftriaxone, grepafloxacine, irinotecan – Other drugs: pravastatine, temocaprilate, drug conjugates (e.g., glucuronide conjugates of grepafloxacin, diclofenac and acetaminophen) – Toxicants: S-glutathionyl-1,2,4-dinitrobenzene, S-glutathionyl ethacrynic acid, ochratoxin A, heavy metal complexes – Fluorescent dyes: fluo-3, carboxydichlorofluorescein, sulfobromaphtalein, calcein

Adapted with permission from [11]; Data taken from [11,51–53,59].



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Review Baiceanu, Crisan, Loghin & Falson emtricitabine and lamivudine) and tenofovir may act as ABCC2 inhibitors in vitro [66] . Although probenecid inhibits both ABCC1 and ABCC2, it has also been shown stimulating the ABCC2 substrates transport. Probenecid has dual effects on ABCC2 transport: when testing the uptake of valproic acid in bovine brain microvascular endothelial cells, it was observed that a low range inhibitor concentration increases uptake, while high range concentrations decrease it [39,67] . This suggests that probenecid binds to different sites of ABCC2 promoting either inhibition or activation of the transport of the drug depending on the concentration of the modulator. Of the note, the same effect was observed on the human ABCB1 on Hoechst transport modulated by QZ59SSS [30] . Other ABCC2 inhibitors tested in cell-based models are included in Table 2. Contrary to ABCB1, quite few ABCC2 inhibitors are known, and all display a limited inhibition efficiency as shown by the poor inhibition constant estimated. Modulators from natural sources

Although many innovative approaches are currently available, studies to design and find a modulator that is selective, not toxic and highly potent appear to be a valid way to resolve the problem of MDR. Screening for modulators from natural sources answers to this aim [90,91] . Identifying compounds that are ABCC2 modulators and understanding their structure–activity relationships are important considerations in the selection and optimization of new drug candidates [92] . For this review, we summarized the results for natural compounds that serve as ABCC2 modulators and that could be used in strategies to reverse drug resistance. All natural compounds/extracts known to modulate the function of ABCC2 are presented in Table 3. Table 2. ABCC2 inhibitors. Inhibitor

Ki (μM) or IC50 (μM)

Substrate

Cyclosporine A

8 (Ki)

Vinblastine

Ref. [63]

Cyclosporine A

20 (IC50 )

Calcein AM

[68]

Delavirdine

ND

CMFDA

[69]

Efavirenz

ND

CMFDA

[69]

Emtricitabine

ND

CMFDA

[69]

Etoposide

750 (Ki)

Vinblastine

[63]

Gemifloxacin

16 (IC50 )

Erythromycin

[70]

MK-571

26 (Ki)

Vinblastine

[63]

Reserpine

295 (Ki)

Vinblastine

[63]

CMFDA: 5-chloromethylfluorescein diacetate; Experimental system: MDCKII-MRP2 cells; IC50 : Concentration to reach 50% transport inhibition; Ki: Inhibition constant;

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It is known that natural compounds found in vegetables, fruits, plant-derived beverages and herbal dietary supplements modulate the activity of ABC transporters (ABCB1, ABCG2, ABCC1) [134] . Sometimes referred to as ‘Fourth Generation Inhibitors’ [90] , natural products offer an original and one of the largest scaffold variety for the development of new candidates. Unlocking the opportunity to screen in these libraries brings hope for the aim of identifying (experimentally or in silico) potent ABCC2 modulators. This is an effort to identify potentially interesting lead structures that, after chemical optimization, would have a higher affinity. The search of better ABCC2 inhibitors remains a promising opportunity to modulate more efficiently the resistance of drugs transported by this protein [90] . The secondary metabolites of plants, which include alkaloids, phenolics and terpenoids, interfere with the activity of efflux pumps; they either interact directly with the protein, disturbing the tertiary structure, or compete with therapeutic drugs [134] . There is extensive evidence that natural substances such as flavonoids, chromones, chalchones [135] , stilbenes [136] , have a very good inhibition profile in the case of ABCB1, ABCG2 and ABCC1 [137] . Recent preclinical developments of a chromone derivative are indeed promising in the case of ABCG2 MDR [138] . One approach to investigate the effect of natural products on ABC proteins is to interrogate in targeted chemolibraries of selected compounds. Such libraries can be used for in silico and in vitro high-throughput screening. Examples of such libraries are given in Table 4. The screening for new drug candidates directed at a molecular target proved to be challenging in practice. Now, it is recognized that diversity within biologically relevant ‘chemical space’ is more important than library size. Natural product collections exhibit a wide range of pharmacophores and a high degree of stereochemistry, and these properties contribute to the possibility to provide positive hits – even against difficult screening targets, such as ABCC2 [139] . Virtual screening uses computer-based methods to discover new ligands on the basis of biological structures. After the hype it received in the 1970s and 1980s, virtual screening struggled to deliver to its initial promise [140] . In silico methods have regained interest because they are easy to set up, require little financial investment and are relatively fast. Most often, these methods are used as preliminary filters of large compound collections, so that only the most promising molecules are tested experimentally [141] . With the improvement of the quality of the benchmarking datasets, virtual screening methods can become more reliable. The elucidation of ABCC2 structure could allow



Dietary fats

Ochratoxin A

Toxins

Miroestrol and deoxymiroestrol de Pueraria candollei

Turpentine

Steroidal saponin of Trillium tschonoskii

Steroids and phytosterols

Varia

Hesperidin and orange juice

3′,4′,7-trihydroxyisoflavone Polyphenols and derivatives (daidzein metabolite)

ABCC2 mRNA decrease

Transcriptional Natural substance/extract regulation

HO HO

H3C O

HO HO

O

HO

HO

HO

OH HO

O

Structure

O

N H

O

O

H O

OH

OH O

Table 3. Natural extracts/compounds modulating ABCC2.

Cl

OH

H

O

O

O

O

OH

OH O

H

OH

O

CH3

OH

OH

OH

O CH3

↓ the expression of ABCC2 mRNA

Effect on ABCC2

HFD feeding only in female, but not male, C57BL/6J mice; wild-type mice and mice lacking liver X receptors; male Sprague–Dawley rats; (highfat diet for 4 weeks)

IL-6-deficient mice liver (livers were harvested 14 and 24 h after injection 100 μl of turpentine subcutaneously)

Caco2 cells

C57BL/6 mice (subcutaneously administration in corn oil at a dose of 0.5 mg/kg/day once a day for 7 days)

HepG2 and R-HepG2 cells (24 h treatment, 15 μM)

↓ expression of mRNA ABCC2; fatty acids, omega-3 ↓ the expression of ABCC2 mRNA; soybean oil affects the expression of ABCC2 mRNA

↓ the expression of ABCC2 mRNA

ABCC2 affects the export of ochratoxin A; ochratoxin A might ↓ the expression of ABCC2 mRNA;

↓ the expression of BSEP and ABCC2 mRNA

Chemosensitization in the cells; ↓ the expression of ABCB1, ABCC1, ABCC2, MRP3, MRP5, MVP and GST- π genes

Male Sprague–Dawley rats (orange ↓ ABCC2 protein level and juice or a 0.079% hesperidin mRNA level in the small suspension – oral administration for intestine and liver 2 days)

HeLa cells (48h treatment, 25 μM)

Experimental model

[99–101]

[98]

[97]

[96]

[95]

[94]

[93]

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Genistein

Forskolin

Diosgenin

Pilocarpine

Rutaecarpine from Evodia rutacarpa

Praeruptorin A and praeruptorin C from Peucedanum praeruptorum

Terpenes

Steroids

Alkaloids

Coumarins

Polyphenols β-naphtoflavone and derivatives

ABCC2 mRNA increase


HO

Structure

Future Med. Chem. (2015) 7(15) O

O

O

N H

O

O

O

CH3

N

N

O

O

N

O

O

O

O O

N

CH3

OH

HO

O

O

CH3

H

O

O

HO

HO

H3C

O

CH3

HO

O

O

Table 3. Natural extracts/compounds modulating ABCC2 (cont.).

OH

CH3

HepG2 cells

Mice – oral administration (at the doses of 10, 20 and 30 mg/kg for consecutive 7 days)

Rats (10 mg/kg every 30 min until the onset of a convulsive status epilepticus)

Rats (fed with diosgenin 1% wt/wt)

BeWo cell line

HepG2, Huh 7, Caco2 cell lines

HepG2, HepaRG cells and fresh primary human hepatocytes male C57BL/6 mice (200 mg/kg CO i.p.)

Experimental model

Praeruptorin A and praeruptorin C significantly induces the ABCC2 mRNA and protein expression, and enhanced the transport activity ABCC2

↑ the expression of ABCC2 mRNA

↑ the expression of ABCC2 protein

↑ the expression of ABCC2 mRNA; ethinyl-estradiol cotreated with diosgenin ↑ the expression of ABCC2 mRNA

↑ expression of the ABCC2 mRNA (10 μM)

↑ the expression of ABCC2 mRNA and protein level + ↑ transport activity (at 10 μM)


Effect on ABCC2

[108]

[107]

[106]

[105]

[104]

[58,103]

[102]

Ref.




Butylated hydroxyanisole

Soybean oil

Apple polyphenol extract

Ginkgo biloba 761 extract

Kojic acid

Erucin

Varia

Chinese herbal drugs (Schisandra chinensis Baill and Glycyrrhiza uralensis Fisch), and selected constituents

Resveratrol

Stilbens

ABCC2 mRNA increase (cont.).


Structure

X

OH

OMe

HO

S C N

HO

OH

CH3

S

OH

CH3

CH3

O

O


CH3

OH

Chinese herbal drugs ↑ the expression of ABCC2 mRNA


Soybean oil affects ABCC2 mRNA expression



↑ the expression of ABCC2 mRNA (5–20 μM; decline to control level between 20–40 μM)

ABCC2 and ABCG2 identified as the two apical transporters involved in the efflux of resveratrol conjugates

Induction of ABCC2 luciferase activity after genistein treatment (50 μM); diminished by resveratrol cotreatment (50 μM)

Effect on ABCC2

HepG2 cells (treatment with extract Bioactive terpenoids and at 100 μg/ml, ginkgolide A and flavanoids Ginkgo biloba 761 ginkgolide A at 50 μM) extract ↑ the expression of ABCC2 mRNA

Hepatocyte cultures (treatment with extract – concentration not given)

Preneoplastic cells derived from colon adenoma (LT97) (128 and 255 μg/ml)

Rats (20% of the total parenteral nutrition for 4 days)

Caco2 cells (350 mg/kg CO i.p.)

Male F344 rats (2% kojic acid in drinking water for 7 weeks)

Caco2 cells

Rats

HepG2 cells (genistein-induced ABCC2 expression)

Experimental model

[115]

[114]

[113]

[101]

[102]

[112]

[111]

[110]

[109]

Ref.



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Genistein

Myricetin

Morin

Robinetin

Luteolin

Chrysin, quercetin, Polyphenols and derivatives genistein, biochanin A, resveratrol

Direct or indirect effect on ABCC2

Structure

HO

HO

HO

HO

HO

HO

OH

OH

OH

OH

OH

O

O

O

O

O

O

HO

O

O

O

O

O

O

OH

OH

OH

OH

OH




OH

OH

OH

OH

OH

OH

OH

OH

Mice (intraperitoneal injections)

MDCKII-MRP2 cells (monolayer)

Inverted Sf9 membrane vesicles expressing ABCC2; estradiol-17βglucuronide as substrate; tested concentration: 80 μM

MDCKII-MRP2 cells (monolayer)

Rats (intravenous injections with irinotecan)


ABCC2 Sf9 inside-out vesicles

Caco2 cells (bicameral system) (polyphenols at 50 μM; ochratoxin A at 0.75 and 7.5 nM)

Experimental model

Luteolin inhibits the reaction (cisplatin results in increased expression of ABCC2 protein)

Inhibits the transport of calcein (IC50 15 mM)

Inhibits ABCC2-mediated transport of the substrate

25 mM enhances the sensitivity of the cells towards vincristine (IC50 7.6 to 5.8 mM)

Genistein suppresses the ABCC2-mediated biliary excretion of irinotecan hydrochloride and its metabolites


50% inhibition of calcein uptake at 50 μM quercetin; better inhibition for the metabolites

Impairment of the transport of ochratoxin A (ABCC2 substrate) through inhibition of the pump; chrysin, genistein and resveratrol inhibit the ABCC2 export of ochratoxin A

Effect on ABCC2

[120]

[118]

[34]

[118,119]

[117]

[34]

[116]

[97]

Ref.



Silymarin

2”-O-β l - galactopyranosylorientin

Green tea extracts and components (epigallocatechin gallate, epigallocatechin, theanine, caffeine)

Curcumin

Polyphenols Baicalin and derivatives (cont.)

Direct or indirect effect on ABCC2 (cont.)


HO

HO

Structure

O

HO

HO

O

HO

OH

O

CH3

OH

OH

O

O

O

O

O

O

O

OH

O

OH OH

OH


CH3

O

OH

OH

OCH3

OH

OH

OH

OH

OH

OH

OH

CH2OH

OH

OH

OH

O

O

OH

O

O

HO

OH O

OH

O


MDCKII-MRP2 membrane vesicles and monolayers

MDCKII-MRP2 cells (monolayers) and LS-180 cells

Caco2 cell lines (25, 50 and 100 μmol/l)



Rats

Experimental model

Slight inhibition but at much higher doses than in the vesicle model (IC50 = 5 μM for membrane vesicles; not significant inhibition in intact cells)

Inhibition of ABCC2 function at 1 mg/ml GTE, none of the components had separate effects

2”-O-β l - galactopyranosylorientin rather than 2”-O-β l - galactopyranosylvitexin is an ABCC2 substrate.



ABCC2 affects the export of baicalin

Effect on ABCC2

[68]

[123]

[122]

[34]

[34]

[121]

Ref.


Review

2051

2052

Reserpine

Chelerythrine

Sanguinarine chloride

Polydatin

Stilbenes

Aconitum alkaloids

Alkaloids


H3C

O

Structure

Future Med. Chem. (2015) 7(15) O

O

+ N

OH

O

O

+ N

N

H3C

O

O

H

O CH3

H

H

O

H

OH

O

HO

H3C

O

N H H

N

O

O

HO

HO

O

O

H3C

HO

H3C

H3C

OH

O

CH3

Cl O

O

O

OH

O O

CH3

O

CH3

H



O

O

CH3

OH

O

O

O CH3

CH3

Effect on ABCC2

Caco2 (uptake across the apical membrane) (25, 50 and 100 μmol/l)




MDCKII-MRP2 cells

ABCC2 protein affects the export of polydatin.




ABCC2 is involved in the efflux of aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine

Caco2 (bidirectional ABCC2 ABCC2 might transport transport assay), MDCKII-MDR1 and aconitine, mesaconitine, MDCKII-BCRP hypaconitine, hydrolates such as benzoylaconine, benzoylmesaconine and benzoylhypaconine

Experimental model

[126]

[34]

[34]

[34]

[125]

[124]

Ref.




Glycyrrhizin

Tetrahydroquinolines derivatives

Terpenoids

Terpenes

Varia

Phalloidin

Mycotoxins



HO

O

O

HO

H N

HO

O

S

N

O

N H

O

NH

OH

H

O CH3

CH3

CH3

CH3

H

H3C

CH3

CH3

CH3

OH

O

OH

CH3

O

CH3

NH

O

OH

OH

HN

O

HO

O

H3C

H3C

H

O

N H

N H

H H3C CH 3 O

OH

O

H

O CH3

OH

H H3C CH 3

NH

OH

HO

O

O

OH

H3C

Structure


Inverted Sf9 membrane vesicles expressing ABCC2; estradiol-17βglucuronide and CDCF substrates; tested concentration: 80 μM

Rats (choline-deficient l -amino acid-defined diet) Inverted Sf9 membrane vesicles expressing ABCC2; estradiol-17βglucuronide substrate; tested concentration: 80 μM

Inverted Sf9 membrane vesicles expressing ABCC2; estradiol-17βglucuronide substrate;

Rats (0.5 mg/kg injections)

Experimental model

Four derivatives that may act as ABCC2 inhibitors (with IC50 < 30 μM)

Suppresion of the ABCC2 protein level (immunoblot and immunohistochemical analyses of liver ABCC2) Glycyrrhizinic acid inhibits ABCC2-mediated transport

↓ of the substrate accumulation observed in the presence of glycyrrhetic acid (IC50 20 mM) and abietic acid (IC50 51 mM)

↓ the biliary excretion of taurocholate, leukotriene C4, pravastatin, vinblastine and glutathione suggesting an effect on ABCC2-mediated transport.

Effect on ABCC2

[130]

[34]

[129]

[128]

[127]

Ref.



Review

2053

2054


Extracts of Kaempferia parviflora and components

Rhei Rhizoma extract

Timosaponin BII

Varia (cont.)


Structure

Gal 2 Glu

O



O

OH

O

Glu

Rat everted intestine

A549 cells expressing ABCC1 and ABCC2, but not Pgp; cellular accumulation of calcein

Organic anion-transporting polypeptide (OATP) 1B1- and 1B3-transfected 293 cells (human embryonic kidney); ABCG2 and ABCC2 membrane vesicles

Experimental model

Suppressed ABCB1 mediated efflux transport of rhodamine 123 (at 300 μg/ml); suppressed in vitro and in vivo ABCC2 mediated intestinal efflux of 2,4-dinitrophenyl-Sglutathione (1000 μg/ml)

Extracts and flavone derivatives from the rhizome of K. parviflora suppress MRP function, and therefore may be useful as modulators of multidrug resistance in cancer cells

Influx (OATP) and efflux transporters (ABCG2/ABCC2), but not metabolic enzymes, contribute significantly to rat timosaponin-2 hepatobiliary disposition

Effect on ABCC2

[133]

[132]

[131]

Ref.




this approach to find new, promising modulators by interrogating in specialized chemolibraries. Studies indicate that ABCC2 function regulation occurs at least at three distinct levels, comprising endocytic retrieval from the apical membrane, transcriptional and translational regulation as well as direct interaction with the protein [142] . The experimental models used for the evaluation of ABCC2 modulation reflect this, focusing on these levels, this is why the models vary – from vesicle to cell and animal models, from gene screening to proteomics and functional transport mediated by ABCC2 protein. Yet, the complexity of the methods used and their variety does not allow to draw a simple conclusion on which modulation strategy works best, nor which compound or class of compounds hold most promise. Caution is required for the interpretation of these results and the limitations of the different assays must be considered. For example, when data refer only to mRNA levels, discrepancies between mRNA and protein levels should be noted. In the case of ABCG2, its expression in kidney is low at mRNA level but higher at protein level. What is more, spliced mRNA variants may not code the entire, functional protein [143] . Similarly, if a substrate of an efflux transporter has poor apparent permeability, membrane vesicle assays will still detect an effect. On the other hand, membrane vesicles are not suitable for transport measurements of compounds with high permeability or high nonspecific binding [144] . Our findings show that a large number of plant secondary metabolites (such as alkaloids, terpenoids, steroids and phenolic acids, flavonoids, catechins, chalcones, stilbenes, mycotoxins, fatty acids and complex herbal extracts) act on the regulation of the expression and ABCC2 protein interaction. Successful experiments using modulators of natural origin are regarded as proof of concept that plant secondary products are interesting candidates for chemosensitization [145] (Figure 4) .

Review

Table 4. Natural product chemolibraries. Name

Compounds (n)

Natural-product chemolibraries for virtual screening

Ref.

SuperNatural II

400,000

[71]

Universal Natural Product Database

200,000

[72]

Chinese Natural Product Database

50,000

[73]

Drug Discovery Portal

40,000

[74]

iSmart

20,000

[75]

NuBBE Database

700

[76]

Drug Bank

8000

[77]

Natural-product chemolibraries for experimental screening Natural Compound Library

800

Screen-Well Natural Product library, Enzo

500

[79]

Drug Compound Libraries, Albany Molecular Research

300,000

[80]

MEGx: Natural Product Screening Products, AnalytiCon discovery

5000

[81]

Natural product-like library

1500

[82]

Greenpharma Natural Compound Library

150,000

[83]

The Natural Products Library Initiative (NRPLI) at The Scripps Research Institute (TSRI)

10,000

[84]

ChemBridge

1 million

[85]

ChemDiv Libraries

1.5 million

[86]

Enamine Collections

1.9 million

[87]

Life Chemicals Library

1 million

[88]

The Specs Library

1.5 million

[89]

[78]

Most of the studies focus on post-transcriptional regulation of the protein, more specifically on transport assays. The data on mRNA expression sometimes emerged serendipitously while investigating other subjects. For example, while researching the effect of

No modulators

15

10 5 Post-translational 0 Polyphenols

mRNA Steroids and phytosterols

mRNA

Terpenes Alkaloids

Varia

Figure 4. Classes of phytochemicals tested as ABCC2 modulators (according to data presented in Table 3).



2055


Polyphenols

Direct or indirect effects on MRP2

Steroids and phytosterols Terpenes

mRNA

18%

15%

Alkaloids Varia

mRNA

28% 8% 43% 18%

8%

54%

0%

29%

55%

15%

9% 0% Figure 5. ABCC2 levels of regulation and most important classes of phytochemicals tested (expressed in % of compounds affecting each level) (according to data presented in Table 3).

essential fatty acids and their influence on the fatty liver disease, it was discovered that omega-3 acids decrease the expression of ABCC2 mRNA [99] . The evaluation of the effect of different phytochemicals on ABCC2 transport is more targeted – mostly polyphenols, terpenes and alkaloids were tested and identified as modulators or inhibitors (Figure 5) . In a comparative study of ABCC1 and ABCC2mediated calcein transport in MDCKII transfected cells, van Zanden et al. analyzed large series of flavonoids to define the structural requirements needed for potent inhibition. The angle between the B- and C-ring of flavonoids was suggested to be one of the three structural characteristics for ABCC1 inhibition, along with the total number of methoxylate and hydroxyl groups. In this study the authors conclude that ABCC2 recognition demonstrates higher selectivity than ABCC1, exhibiting a strong reliance on the presence of a flavonol B-ring pyrogallol group. High specificity of ABCC2 interactions is also observed in a group of biphenyl-substituted heterocyclic compounds. The ABCC2 transport and inhibition profile of this series of compounds correlate well with the torsion angle between the two phenyl rings. As the torsion angle increases, the compounds transit from nonsubstrate and/or noninhibitor to substrate and/or weak inhibitor, and eventually to substrate and/or potent inhibitor [32,116,118,146] .

2056


ABCC2 transports various constituents in food, such as dietary flavonoids quercetin 4′-β-glucoside [147] and sulphate conjugates of epicatechin [148] . Several flavonoid β-d-glycosides including phloridzin, quercetin 4′-β-glycoside and genistein-7-glycoside are substrates for ABCC2 [149] . Similar results were obtained for the glycosides of the flavonoids quercetin, genistein and diosmetin [39] . Curcumin inhibits both the ABCC1 and 2 mediated transport in cells [68] . A further functional interaction was described between flavopiridol and bilirubin. The interactions between cross- stimulatory or inhibitory compounds appear very complex. ABC transporters can transport multiple different substrates at the same time, and the cotransport results in a modification of the transport efficacy [11] . Some polyphenols act on ABCC2 mRNA regulation and they also have inhibitory properties on the function of the protein. However, the concentrations needed to inhibit ABCC2 transport are high and these compounds cannot be considered efficient. In addition, many of the studies that report ABCC2 inhibitors were conducted on inverted membrane vesicles. While this is a good model for the screening of the modulatory effect (inhibition, stimulation), transport itself is not evaluated in this assay, and further investigations are needed to confirm the results – such as cell-based models, in vivo experiments, etc. Also, QSAR analysis is only efficient when addressing simple, well-defined active sites. This is not



the case of ABCC2 (and other multidrug transporters), which displays several different binding sites. Finding a specific ABCC2 inhibitor opens the possibility to assess the actual role of ABCC2 in drug resistance. For example, upregulation of ABCC2 is present in many cisplatin resistant cell lines and tumor tissues [54–55,150–151] . An inhibitor with high specificity for ABCC2 could help elucidate what is the link between ABCC2 overexpression and cisplatin resistance. It was recently shown that a new technology, mass cytometry, can answer specifically to this question as it is possible to evaluate the accumulation of platinum compounds in cells [152] . Moreover, given the high expression of ABCC2 in liver, intestine and kidney, and its importance in drug trafficking across membranes, these inhibitors would allow to build reliable in vitro experiments to predict the pharmacokinetics of different drugs in the organism. Renewed interest gives the spotlight of drug discovery to natural products. Compounds of natural origin continue to enter clinical trials, particularly as

Review

anticancer and antimicrobial agents. 34% of the drugs approved by FDA between 1981 and 2001 are medicines based on natural products (such as statins, antitubulin anticancer drugs, immunosuppressants) [139] . Natural product and/or natural product structures continue to play a highly significant role in the drug discovery and development process [153] . With the improvement of high-throughput screening methods molecularly targeting ABCC2 and by using new library collections of natural compounds, new ABCC2 modulators can lead the way to innovating applications in MDR treatment. Conclusion & future perspective Given the prevalence of ABC transporters, surprisingly few systems have been characterized, either biochemically, structurally or mechanistically. Broad generalizations about the functioning of these systems are extrapolated from just a few observations, and therefore a pressing need exists to extend these characterizations.

Executive summary Human ABCC2 • ABCC2 (multidrug resistance protein 2) is a major ATP-binding cassette (ABC) protein, with a large distribution in the human organism. • It is most abundant in the liver, kidney and intestine, where it transports glutathione-, glucuronate- or sulfate-conjugated endobiotics and xenobiotics from the cytoplasm across the membrane to the extracellular compartment. Its 3D structure and exact transport mechanism are still not fully elucidated.

Clinical relevance • ABCC2 has an important role in the export of organic anions, unconjugated bile acids and xenobiotics into the bile, and also contributes to protection against drugs. Localized on the apical side of polarized cells, ABCC2 is an important efflux pump in the terminal phase of detoxification. • ABCC2 recognizes and transports many hydrophobic and hydrophilic antineoplastic agents and confers resistance in cultured cancer cells to methotrexate, paclitaxel, docetaxel, epipodophyllotoxins, vincristine, vinblastine, doxorubicin, cisplatin, etc.

Phytochemicals as ABCC2 modulators • Even for the newest anticancer therapeutics, a multidrug resistant (MDR) phenotype development was observed. Several approaches have been developed to restore the drug sensitivity of cells expressing MDR ABC pumps, such as by using MDR modulators, inhibitors or chemosensitizers. • So far there are few known ABCC2 inhibitors, with poor efficiency and selectivity. • The secondary metabolites of plants interfere with the activity of efflux pumps, either through direct interaction with the protein, or distortion of the tertiary structure, or competition with therapeutic drugs. • Our review shows that a large number of plant secondary metabolites (such as alkaloids, terpenoids, steroids and phenolic acids, flavonoids, catechins, chalcones, stilbenes, mycotoxins, fatty acids and complex extracts) act on the regulation of the expression and ABCC2 protein interaction, making these compounds interesting candidates for chemosensitization. • Natural products continue to play a highly significant role in the drug discovery and development process. With the improvement of high-throughput screening methods molecularly targeting ABCC2 and by using new library collections of natural compounds, new ABCC2 modulators can lead the way to innovating applications in MDR treatment.

Conclusion • The use of a combination of cytotoxic agent and natural modulator may provide a new strategy to overcome MDR in cancer patients and to improve chemotherapy efficiency. Recent technological advances could revitalize the exploitation of the value of natural products as starting points for drug discovery, and bring hope to the efforts of finding new and efficient ABCC2 modulators and tackling the problem of MDR where ABCC2 has a significant impact.



2057

Review Baiceanu, Crisan, Loghin & Falson The high-resolution structural characterization of eukaryotic ABC transporters, particularly in humans, is a fertile area for research. The interest resides in the elucidation of the intrinsic mechanisms, and for the therapeutic potential that comes from the characterization of substrate binding, nucleotide and regulatory sites. The goal is to understand in quantitative detail the molecular and kinetic mechanisms of ABC transporter function and ABC proteins in general. ABCC2 is one of the major drug efflux pumps present in the organism. It plays a crucial role in the elimination of various drugs and contributes to their pharmacokinetic features. It participates mainly to biliary secretion of endogenous compounds and its overexpression in tumors may confer an MDR phenotype. ABCC2 appears to play a direct role in platinum compounds resistance. There are several strategies to modulate the response to chemotherapy, but a selective inhibition or modulation of the ABC protein function and/or expression with a wide range of nontoxic modulators is, in principle, direct and straightforward. Learning more about ABCC2 could bring answers to some research problems related to ABC transporters. Aside from the general topologic structure of ABCC2, not much is known about the detailed structure of ABCC2. Similarly, the exact transport mechanisms remains enigmatic. So far, we lack true ABCC2 modulators and inhibitors and it remains elusive on what strategy could bring best results. Even though ABCC2

is vastly distributed in tissues and cells, and some of its roles are documented, solving the ABCC2 puzzle can arm us with better tools to fight MDR, especially in those cancer cases where ABCC2 seems to play a major part. This review sums up the efforts made in the research of ABCC2 modulators from natural sources. We summarize the evidence that certain plant secondary metabolites act on different ABCC2 regulation levels. A combination of a cytotoxic agent with a natural modulator (not necessarily an inhibitor of ABCC2) may provide a new strategy to overcome MDR in cancer patients and to improve the efficiency of chemotherapy. Recent technological advances could revitalize the exploitation of the value of natural products as starting points for drug discovery, and bring hope to the efforts of finding new and efficient ABCC2 modulators.

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Financial & competing interests disclosure This work was financed by ANR-13-BSV5-0001-01, received by P Falson and E Baiceanu. E Baiceanu was also supported by the research contract no. 1491/29/28.01.2014 financed by the University of Medicine and Pharmacy ‘Iuliu Hatieganu,’ ClujNapoca, Romania. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.



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