Reversal of multidrug resistance by the inhibition of ATP-binding cassette pumps employing “Generally Recognized As Safe” (GRAS) nanopharmaceuticals: A review

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Abstract

Pumps of the ATP-binding cassette superfamily (ABCs) regulate the access of drugs to the intracellular space. In this context, the overexpression of ABCs is a well-known mechanism of multidrug resistance (MDR) in cancer and infectious diseases (e.g., viral hepatitis and the human immunodeficiency virus) and is associated with therapeutic failure. Since their discovery, ABCs have emerged as attractive therapeutic targets and the search of compounds that inhibit their genetic expression and/or their functional activity has gained growing interest. Different generations of pharmacological ABC inhibitors have been explored over the last four decades to address resistance in cancer, though clinical results have been somehow disappointing. “Generally Recognized As Safe” (GRAS) is a U.S. Food and Drug Administration designation for substances that are accepted as safe for addition in food. Far from being “inert”, some amphiphilic excipients used in the production of pharmaceutical products have been shown to inhibit the activity of ABCs in MDR tumors, emerging as a clinically translatable approach to overcome resistance. The present article initially overviews the classification, structure and function of the different ABCs, with emphasis on those pumps related to drug resistance. Then, the different attempts to capitalize on the activity of GRAS nanopharmaceuticals as ABC inhibitors are discussed.

Introduction

Pumps of the ATP-binding cassette superfamily, ABCs [1], are the largest group of transmembrane proteins. According to their sequence and homology, they are classified into seven subfamilies named from A to G [2]. ABCs are involved in the active efflux of a broad variety of endogenous molecules (e.g., lipids, proteins and products of the metabolism) and xenobiotics (e.g. drugs) out of the cell against a concentration gradient [3]. The energy source is the hydrolysis of ATP.

There are 49 human ABCs [4], [5], [6], [7], [8] distributed ubiquitously or tissue-specifically in liver, kidney, lung, pancreas, stomach [7], intestine [9], central nervous system [10], [11], and different anatomical barriers [9], [10], [12], [13], [14], [15], [16]. Since ABCs regulate the access of drugs to the intracellular space [17], their overexpression is a mechanism of multidrug resistance (MDR) in cancer [2], [18], [19], [20], hepatitis B [21], [22] and the human immunodeficiency virus (HIV) [23], [24], [25]. ABCs also preclude the intestinal absorption of drugs administered by the oral route, reducing their bioavailability [26], [27], [28], [29]. In addition, they might alter the renal excretion of drugs and their metabolites [30], [31], [32].

P-glycoprotein (P-gp, ABCB1, MDR1) was the first ever identified ABC [33], [34], followed by the multidrug resistance-associated protein-1 (MRP-1, ABCC1) [35], [36] and the breast cancer resistance protein (BCRP, ABCG2) [37], [38].

It is estimated that at least 50% of the compounds listed in the Sigma catalogue are substrates of P-gp or MRP (ABCC) [39]. Drug/ABC interactions are quite specific and they need to be individually assessed. In addition, drugs might be substrates of more than one pump. For example, antiretrovirals of the family of nucleoside reverse transcriptase inhibitors (NRTIs) are known substrates of BCRP [40]. However, the intracellular accumulation and brain distribution of abacavir, an NRTI, is also reduced by P-gp [41]. This phenomenon makes the screening very laborious and predictions very difficult.

Since their discovery, ABCs have emerged as attractive therapeutic targets [42]. In this context, the search of inhibitors of the genetic expression and/or the functional activity of ABCs as pharmacotherapy adjuvants in cancer [17] and HIV [43] has gained interest in the scientific community. In addition, patient pharmacogenetic polymorphism is being studied to correlate underexpression or overexpression of specific ABCs to altered pharmacological outcomes [32]; polymorphism is established when there are changes in the nucleotide sequence of a specific gene in the general population with a frequency greater than 1%.

Different generations of pharmacological ABC inhibitors have been explored over the last four decades to overcome MDR in cancer [44], [45], [46], [47]. Several of them, such as verapamil, cyclosporine A and quinidine [48] belong to a first generation of ABC blockers. The main common characteristic is that they are drugs already approved by the regulatory agencies for other uses and they were clinically evaluated as ABC inhibitors in a new intended use. Conversely, second (e.g., valspodar [49]) and third generation inhibitors (e.g., elacridar [50], tariquidar [51], laniquidar [52], zosuquidar [53]) were specifically designed for this application and do not display other pharmacodynamic effects. In general, clinical results were disappointing due to insufficient therapeutic benefit, unacceptable systemic toxicity, lack of validated, reproducible and sensitive methods to measure ABC concentrations in different cells, tissues and organs, and the co-existence of other pumps that distort the analysis [18], [54], [55].

“Generally Recognized As Safe” (GRAS) is a U.S. Food and Drug Administration (US-FDA) designation for substances that are accepted as safe for addition in food (under the conditions of use) by qualified experts [56]. GRAS compounds are listed in the Code of Federal Regulations Title 21 (21 CFR) parts 182 and 184. Pharmaceutical excipients are conceived as inert constituents incorporated into a pharmaceutical product to improve the performance of a drug (e.g., physicochemical stability, dissolution rate, bioavailability), to make the manufacturing process more efficient and to prolong the product shelf life [57], [58]. For example, a broad spectrum of lipidic and polymeric drug carriers [59], [60] have been used to overcome the poor aqueous solubility of approximately 50% of the drugs on the market [61], [62], [63]. The application of amphiphilic polymeric excipients that self-assemble into nanoscopic micelles has emerged as one of the most appealing and popular technology platforms [64], [65]. However, far from being “inert”, some of these amphiphiles have been shown to inhibit the activity of ABCs in MDR tumors [66]. Over the last two decades, Kabanov and colleagues have investigated the relationship between the structure of different poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) block copolymers (poloxamer, Pluronic®) and the inhibition of the functional activity of ABCs [67], [68]. Their investigations revealed that the inhibitory activity relies not only on the direct interaction of the copolymer with the transporter and the plasma cell membrane but also from the hindrance of several cellular pathways [67], [68]. This comprehensive work led to the development of an innovative formulation of the antitumoral drug doxorubicin (Dox), namely SP-1049C (Supratek Pharma Inc., Canada), that incorporates a combination of poloxamers as inhibitors of P-gp [69], [70]. This product showed increased effectiveness in preclinical and clinical phases and it was granted Orphan Drug status for the treatment of gastric cancer by the US-FDA in 2008 [71]. These investigations constitute a solid basis for the implementation of new therapeutic approaches to overcome ABC-mediated resistance in other types of cancer and diseases.

First, the present article succinctly describes the classification, structure and function of the different ABCs. Those pumps directly associated with resistance to drugs are emphasized. Then, the attempts to capitalize on the activity of GRAS nanopharmaceuticals as ABC inhibitors are overviewed. Finally, the clinical impact and the perspectives of the field are discussed.

Section snippets

Pumps of the ATP-binding cassette superfamily: classification, structure and function

ABCs are integral membrane proteins that play a fundamental role in the active translocation of amino acids, proteins, lipids, lipopolysaccharides, inorganic ions, peptides, sugars, metal ions, metabolism products and drugs across the plasma cellular membrane [1], [2], [3]. Forty nine genes that encode for the corresponding ABC have been identified in humans and classified into seven subfamilies from ABCA to ABCG according to genomic organization, order of domains and sequence conservation [72]

Pharmacological inhibition of ABCs

Chemotherapy remains the most effective approach to treat disseminated and hematological tumors. However, drug resistance accounts for about 90% of the therapeutic failures [103]. In fact, 50% of the cancers express detectable levels of P-gp [2]. Evidence on the relationship between MRP-1 and BCRP expression and MDR is more limited. There is solid evidence showing that these ABCs mediate resistance in non-small cell lung cancer and leukaemia, respectively, resulting in poor clinical response

Inhibition of ABCs by “Generally Recognized As Safe” (GRAS) nanopharmaceuticals

The first report on the use of a macromolecular or polymeric pharmaceutical excipient as ABC blocker most likely appeared in the literature in 1972, when Rhiem and Biedlerm observed that polysorbate 80 (Tween® 80) enhanced the response of MDR Chinese hamster cells to daunorubicin and actinomycin D [116]. It is remarkable that regardless of the fact that the overexpression of P-gp in this cell type was discovered four years later (1976), there was already indirect evidence that its inhibition

Clinical status and future perspectives

ABC-mediated drug resistance has become a great hurdle towards the attainment of therapeutic success in many different types of cancer. More recently, these mechanisms were also unveiled in infectious diseases that claim millions of lives every year. After the discovery of P-gp, several transmembrane proteins involved in MDR were proposed as valuable cellular targets to improve the treatment of cancer [261], [262], [263], [264], [265], [266], [267], [268]. In this context, the co-administration

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    This review is part of the Advanced Drug Delivery Reviews theme issue on “Nanotechnology and drug resistance”.

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