Mariangela Cantore

Perspectives of P-Glycoprotein Modulating Agents in Oncology and Neurodegenerative Diseases: Pharmaceutical, Biological, and Diagnostic Potentials

Human ATP binding cassette (ABC) transporters belong to a family of 49 genes classified into seven subfamilies: ABCA, ABC-B, ABC-C, ABC-D, ABC-E, ABC-F, ABC-G. Some of these transporters are involved in multidrug resistance (MDR), in particular ABC-B1, better known as Pglycoprotein (P-gp), ABC-G2, better known as breast cancer resistance protein (BCRP), and ABC-C1-6, also known as multidrug resistance associated proteins (MRP1-6). 3 These transporters are overexpressed in several tumor cell lines and are responsible for drug efflux out of the cells. They use the energyofATPhydrolysis to extrude compoundsbya complex translocation process. Three models for P-gp translocation have been suggested: (1) pore, (2) flippase, and (3) hydrophobic vacuum cleaner models. In the pore model, drugs binding P-gp to the cytosol are transported out through a protein channel. In the flippase model, P-gp flips drugs that are transported from the inner to the outer compartment of the plasmamembrane against a concentration gradient. In the hydrophobic vacuum cleaner model, molecules recognized by P-gp in the lipid bilayer enter the protein from the membranous site and exit through the central cavity. P-gp contains 12 transmembrane helices organized in two membrane spanning domains (MSDs), each containing six transmembrane helices, and two nucleotide binding domains (NBDs) responsible for ATP binding. BCRP is a “half transporter” because it is formed by only one MSD and one NBD although it dimerizes to be fully active. MRPs differ from P-gp because they display three MSDs, and the additional domain contains five transmembrane domains. This review will focus on the physiological and pathological role of P-gp and will highlight the involvement of this protein both in MDR of tumors and in the physiological function of several barriers. P-gp overexpression is a significant factor in chemotherapy failure due to the ability of this pump to limit the cell accumulation of antineoplastic drugs. Moreover, P-gp is expressed in barriers such as the blood-brain barrier (BBB), bloodcerebro spinal fluid (B-CSF) barrier, and blood-testis barrier (BTB). It modulates the absorption and excretion of xenobiotics across these barriers. P-gp is localized at the apical membranes of liver, kidney, placenta, and the villus tip of enterocytes in the gut. In the gut, P-gp displays a strategic activity modulating access of drugs to the CYP3A4 enzyme, thereby regulating drug metabolism and absorption. 1.1. P-gp Involvement in Cancer and CNS Diseases.MDR is a complex phenomenon that is caused by tumor microenvironment changes or cancer cell-specific factors. Cancer cell-specific factors can occur at different levels: (i) increased drug efflux or decreased drug influx; (ii) drug inactivation; (iii) drug target modification; (iv) apoptosis evasion. The first of these mechanisms can be mediated by plasma membrane transporters such as P-gp. Asmentioned above, high levels of P-gp are reported in the luminal membrane of the endothelial cells constituting the BBB, B-CSF, and BTB. This strategic localization gives P-gp a crucial physiologically role in keeping drugs in the blood. P-gp exerts a protective function in the BBB; indeed, recent studies have reported a potential correlation between P-gp activity and/or expression in CNS disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and epilepsy. AD, a neurodegenerative disorder characterized by a progressive loss of cognitive function, evolves as several forms of dementia that display insoluble β-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs). Aβ secretion is *To whom correspondence should be addressed. Phone: þ39-0805442727. Fax þ39-080-5442231. E-mail: colabufo@farmchim.uniba.it. Abbreviations: Aβ, β-amyloid; AB, apical-basolateral; ABC, ATP binding cassette; AD, Alzheimer’s disease; AML, acute myelogenous leukemia; ATP, adenosine 50-triphosphate; BA, basolateral-apical; Bmax, maximal bound; BBB, blood-brain barrier; BCRP, breast cancer resistance protein; B-CSF, blood-cerebro spinal fluid; BTB, blood-testis barrier; Caco-2, human colonic carcinoma; calcein-AM, calcein acethoxymethyl ester; CD56þ, peripheral blood mononuclear cells; CHOP, cyclophosphamide, hydroxydaunorubicin (adriamycin), oncovin (vincristine), prednisone/prednisolone; CNS, central nervous system; C-PIB, C-Pittsburgh compound B; CYP3A4, cytochrome P-4503A4; DV, distribution volume; FDA, Food and Drug Administration; F-FDDNP, F(2-(1-{6-[(2-[F]fluoroethyl(methyl)amino]-2-naphthyl}ethylidene)malononitrile; FMZ, flumazenil; GI, gastrointestinal; HCT-8, human colorectale adenocarcinoma; ISF, interstitial fluid; Kd, dissociation constant; LBs, Lewy bodies; LLC-PK1, porcine kidney cell line; LRP1, low density lipoprotein receptor-related protein; MDCK, Madin-Darby canine kidney; MDE, multidrug efflux; MDR, multidrug resistance;MPPþ, 1-methyl-4-phenylpyridinium;MRP,multidrug resistance associated proteins;MSA, multisystem atrophy;MSD, membrane spanning domain; MTD, maximum tolerated dose; MTL, medial temporal lobe; NBD, nucleotide binding domain; NFTs, neurofibrillary tangles; NSCLC, non-small-cell lung cancer; PAHG, hippocampus, parahippocampal, ambient gyrus; Papp, apparent permeability; PD, Parkinson’s disease; PET, positron emission tomography; P-gp, Pglycoprotein; PSP, progressive supranuclear palsy; SCLC, small-cell lung cancer; SNP, single nucleotide polymorphism; SPECT, single photon emission computed tomography; T8, T-suppressor cells; TLE, temporal lobe epilepsy.

Small and Innovative Molecules as New Strategy to Revert MDR

Multidrug resistance (MDR) is a complex phenomenon principally due to the overexpression of some transmembrane proteins belonging to the ATP binding cassette (ABC) transporter family. Among these transporters, P-glycoprotein (P-gp) is mostly involved in MDR and its overexpression is the major cause of cancer therapy failure. The classical approach used to overcome MDR is the co-administration of a P-gp inhibitor and the classic antineoplastic drugs, although the results were often unsatisfactory. Different classes of P-gp ligands have been developed and, among them, Tariquidar has been extensively studied both in vitro and in vivo. Although Tariquidar has been considered for several years as the lead compound for the development of P-gp inhibitors, recent studies demonstrated it to be a substrate and inhibitor, in a dose-dependent manner. Moreover, Tariquidar structure–activity relationship studies were difficult to carry out because of the complexity of the structure that does not allow establishing the role of each moiety for P-gp activity. For this purpose, SMALL molecules bearing different scaffolds such as tetralin, biphenyl, arylthiazole, furoxane, furazan have been developed. Many of these ligands have been tested both in in vitro assays and in in vivo PET studies. These preliminary evaluations lead to obtain a library of P-gp interacting agents useful to conjugate chemotherapeutic agents displaying reduced pharmacological activity and appropriate small molecules. These molecules could get over the limits due to the antineoplastic-P-gp inhibitor co-administration since pharmacokinetic and pharmacodynamic profiles are related to a dual innovative drug.

4-Biphenyl and 2-naphthyl substituted 6,7-dimethoxytetrahydroisoquinoline derivatives as potent P-gp modulators

Starting from lead compound 1 (EC(50)=1.64 microM), its non-basic nucleus has been conformationally restricted by 4-biphenyl and 2-naphthyl moieties. In each series we investigated if the presence of H-bond donor or acceptor substituents, the basicity and the lipophilicity (clogP) were correlated with the P-gp inhibiting activity of tested compounds. In the biphenyl series, derivative 4d displayed the best results (EC(50)=0.05 microM). The corresponding amide 3d was found less active (EC(50)=3.5 microM) ascertaining the importance of basicity in this series whilst the presence of hydroxy or methoxy substituents seems to be negligible. In the naphthyl series, both the basicity and the presence of H-bond donor or acceptor groups seem to be negligible. Moreover, the lipophilicity did not influence the P-gp inhibition activity of each series. Specific biological assays have been carried out to establish the P-gp interacting mechanism of tested compounds discriminating between substrates and inhibitors. Moreover, compound 4d displayed a potent P-gp inhibition activity with good selectivity towards BCRP pump.

Synthesis and Preclinical Evaluation of Novel PET Probes for P-Glycoprotein Function and Expression

UNLABELLED P-glycoprotein (P-gp) is an ATP-dependent efflux pump protecting the body against xenobiotics. A P-gp substrate (7) and an inhibitor (6) were labeled with (11)C, resulting in potential tracers of P-gp function and expression. METHODS 6 and 7 were labeled using (11)CH(3)I. (11)C-verapamil was prepared as published previously, using (11)C-methyl triflate. MicroPET scans (with arterial sampling) and biodistribution studies were performed in rats pretreated with saline, cyclosporin A (CsA, 50 mg/kg), or cold 6 (15 mg/kg). RESULTS The radiochemical yields of (11)C-6 and (11)C-7 were approximately 30% with a total synthesis time of 45 min. Cerebral distribution volumes (DV) of (11)C-6 (2.35 +/- 0.11) and (11)C-7 (1.86 +/- 0.15) in saline-treated rats were higher than of (11)C-verapamil (0.64 +/- 0.12). DVs of (11)C-7 and (11)C-verapamil were significantly increased by CsA (to 5.26 +/- 0.14 and 5.85 +/- 0.32, respectively). The DV of (11)C-6 was reduced by cold 6 (to 1.65 +/- 0.03). Its uptake was also reduced (up to 67%) in several peripheral organs that express P-gp. CONCLUSIONS (11)C-7 is a novel tracer of P-gp function with higher baseline uptake than (11)C-verapamil. Upregulation of P-gp function in response to treatment (which is hard to detect with (11)C-verapamil) may be detectable using (11)C-7 and PET. Because (11)C-6 shows specific binding in target organs, this compound is the first PET tracer allowing measurement of P-gp expression.

Substrates, Inhibitors and Activators of P-glycoprotein: Candidates for Radiolabeling and Imaging Perspectives

In recent years, several PET tracers for monitoring the activity and expression of P-gp at the BBB have been tested. P-gp substrates such as [(11)C]verapamil and [(11)C]loperamide can be employed to visualize P-gp activity, but they display a moderate baseline uptake in the brain and formation of radiolabeled metabolites which hamper the interpretation of PET data. P-gp inhibitors such as [(11)C]elacridar, [(11)C]laniquidar and [(11)C]tariquidar have been tested to investigate P-gp expression and the results need further investigation. Recently, we developed MC18, MC266 and MC80, that have been characterized as an inhibitor, substrate and inducer of P-gp both by in vitro assays and in the everted gut sac method. These compounds have been radiolabelled with (11)C and been evaluated in vivo. In the present review, we compare the outcome of biological in vitro assays and the corresponding in vivo PET data for the P-gp inhibitors [(11)C]MC18 and [(11)C]elacridar, the P-gp substrates [(11)C]MC266 and [(11)C]verapamil, the P-gp inducer [(11)C]MC80 and the P-gp modulator cyclosporin A. Since a satisfactory overlap was found comparing in vivo results and the corresponding in vitro findings, the proposed biological in vitro assays could be predictive for the in vivo PET data of novel radiotracers. PET tracers could be employed for various purposes: radiolabeled P-gp inhibitors to monitor decreased expression of P-gp at the BBB in neurodegenerative disorders such as Alzheimers and Parkinsons disease; and radiolabeled P-gp substrates with a high baseline uptake to monitor increased expression of P-gp in epileptic foci.

Multi-drug-resistance-reverting agents: 2-aryloxazole and 2-arylthiazole derivatives as potent BCRP or MRP1 inhibitors.

The 2‐aryloxazole and 2‐arylthiazole scaffolds were used for generating compounds that we characterized for their inhibitory activity toward ATP binding cassette transporters involved in multi‐drug resistance, such as BCRP and MRP1, by using tumor cell lines overexpressing each transporter. These SAR studies are a significant step toward improving the inhibitory potency against P‐glycoprotein, BCRP, and MRP1.

ABC transporters in CSCs membranes as a novel target for treating tumor relapse

CSCs are responsible for the high rate of recurrence and chemoresistance of different types of cancer. The current antineoplastic agents able to inhibit bulk replicating cancer cells and radiation treatment are not efficacious toward CSCs since this subpopulation has several intrinsic mechanisms of resistance. Among these mechanisms, the expression of ATP-Binding Cassette (ABC) transporters family and the activation of different signaling pathways (such as Wnt/β-catenin signaling, Hedgehog, Notch, Akt/PKB) are reported. Therefore, considering ABC transporters expression on CSCs membranes, compounds able to modulate MDR could induce cytotoxicity in these cells disclosing an exciting and alternative strategy for targeting CSCs in tumor therapy. The next challenge in the cure of cancer relapse may be a multimodal strategy, an approach where specific CSCs targeting drugs exert simultaneously the ability to circumvent tumor drug resistance (ABC transporters modulation) and cytotoxic activity toward CSCs and the corresponding differentiated tumor cells. The efficacy of suggested multimodal strategy could be probed by using several scaffolds active toward MDR pumps on CSCs isolated by tumor specimens.

SAR Studies on Tetrahydroisoquinoline Derivatives: The Role of Flexibility and Bioisosterism To Raise Potency and Selectivity toward P-glycoprotein

The development of P-glycoprotein (P-gp) ligands remains of considerable interest, mostly for investigating the proteins structure and transport mechanism. In recent years, many different generations of ligands have been tested for their ability to modulate P-gp activity. The aim of the present work is to perform SAR studies on tetrahydroisoquinoline derivatives in order to design potent and selective P-gp ligands. For this purpose, the effect of bioisosteric replacement and the role of flexibility have been investigated, and four series of tetrahydroisoquinoline ligands have been developed: (a) 2-aryloxazole bioisosteres, (b) elongated analogues, (c) 2H-chromene, and (d) 2-biphenyl derivatives. The results showed that both 2-biphenyl derivative 20b and elongated derivative 6g behaved as strong P-gp substrates. In conclusion, important aspects for developing potent and selective P-gp ligands have been highlighted, providing a solid starting point for further optimization.

Naphthalenyl derivatives for hitting P-gp/MRP1/BCRP transporters.

Substituted naphthalenyl derivatives bearing oxazole, or thiazole or furyl heteronuclei have been carried out as bioisosters of aryl-oxazoles and -thiazoles derivatives previously reported in order to investigate the role of the hindrance on the activity towards P-gp/BCRP/and MRP1 transporters. In addition, the role of naphthalenyl group to modulate P-gp intrinsic activity of these compounds was ascertained. The results demonstrated that all naphthalenyl derivatives displayed comparable P-gp activity with respect to lead compounds previously characterized in our SAR studies but were less active towards BCRP and MRP1 pumps. In terms of intrinsic activity, the replacement of aryl with naphthalenyl moiety led to P-gp inhibitors, unambiguous or ambiguous substrates on the base of the heteronucleus and the substituent on the naphthalenyl fragment. Indeed, oxazole derivatives were: inhibitors (R=H, F, OH), unambiguous substrates (R=OCH(3)), or ambiguous substrate (R=Br); thiazole derivatives were: unambiguous substrates (R=OCH(3), Br), or ambiguous substrates (R=H, F). Finally furyl derivatives were ambiguous substrates.

A benzopyrane derivative as a P-glycoprotein stimulator: a potential agent to decrease β-amyloid accumulation in Alzheimer's disease.

Neurodegenerative disorders such as Alzheimer’s disease (AD) are characterized by slow and progressive loss of one or more functions of the central nervous system (CNS). Since many factors, often little-known, are involved in the pathogenesis of these diseases, and their diagnosis is related to the clinical symptoms, AD is usually diagnosed in an advanced stage precluding the possibility of an effective disease-modifying agent rather than a therapy that treats only the symptoms. Recently, P-glycoprotein (P-gp) has been suggested as a new biomarker for the diagnosis of AD in the early stages, using imaging techniques such as positron emission tomography (PET) and Single-photon emission computed tomography (SPECT). 2] Pgp belongs to the ATP binding cassette (ABC) transporter family, which are localized at the cell membranes of liver, kidney and placenta cells, and on the villus tip of enterocytes in the gut, and they are important constituents of the blood– brain barrier (BBB), blood–cerebro spinal fluid (B-CSF), and the blood–testis barrier (BTB). 4] These pumps use ATP hydrolysis energy to efflux toxins, xenobiotics and antineoplastic agents out of the cells. Breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs) also belong to this family of transporters. These efflux proteins, differently localized with respect to P-gp, are critical components of several biological barriers and are able to efflux different substrates. 6] MRPs bear an additional and specific five transmembrane domain compared with P-gp, and as such, they can efflux organic ions with high-molecular weights. In contrast, BCRP is a P-gp monomer, and so is considered a “half transporter”; it effluxes the same substrates as P-gp. P-gp is a protein involved in the active transport of molecules across biological membranes, and it has been shown that alterations of its expression and/or activity are related to the onset of neurodegenerative disorders. 2] Indeed, it has been reported that up-regulation of this efflux pump is responsible for a decrease in b-amyloid intracellular accumulation, which is an important hallmark in AD. Therefore, targeting bamyloid clearance by P-gp stimulation could be an useful strategy to prevent AD progression. For this purpose, P-gp ligands are useful for the diagnosis of neurodegenerative diseases by PET or SPECT techniques. In particular, P-gp substrates are needed to detect the activity whereas P-gp inhibitors to measure the expression of the efflux protein. Furthermore, P-gp stimulators could be employed for the treatment of disorders where decreased P-gp expression and/or activity have been proven to play a role in the progression or etiology of the disease. The most recently reported P-gp inhibitors are tariquidar, elacridar, and our ligand MC18. However, these molecules have limitations: 1) elacridar shows similar activity towards P-