Antitumor agents. 293. Nontoxic dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylenedioxybiphenyl-2,2'-dicarboxylate (DDB) analogues chemosensitize multidrug-resistant cancer cells to clinical anticancer drugs

Novel dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylenedioxybiphenyl-2,2'-dicarboxylate (DDB) analogues were designed and synthesized to improve their chemosensitizing action on KBvin (vincristine-resistant nasopharyngeal carcinoma) cells, a multidrug resistant cell line overexpressing P-glycoprotein (P-gp). Structure-activity relationship analysis showed that aromatic and bulky aliphatic side chains at the 2,2'-positions effectively and significantly sensitized P-gp overexpressing multidrug resistant (MDR) cells to anticancer drugs, such as paclitaxel (TAX), vincristine (VCR), and doxorubicin (DOX). DDB derivatives 16 and 23 showed 5-10 times more effective reversal ability than verapamil (VRP) for TAX and VCR. Analogue 6 also exhibited five times greater chemosensitizing effect against DOX than VRP. Importantly, no cytotoxicity was observed by the active DDB analogues against both non-MDR and MDR cells, suggesting that DDB analogues serve as novel lead compounds for the development of chemosensitizers to overcome the MDR phenotype. The mechanism of action studies demonstrated that effective inhibition of P-glycoprotein by DDB analogues dramatically elevated the cellular concentration of anticancer drugs.     

Synthesis and evaluation of dihydropyrroloquinolines that selectively antagonize P-glycoprotein

In a search for improved multiple drug resistance (MDR) modulators, we identified a novel series of substituted pyrroloquinolines that selectively inhibits the function of P-glycoprotein (Pgp) without modulating multidrug resistance-related protein 1 (MRP1). These compounds were evaluated for their toxicity toward drug-sensitive tumor cells (i.e. MCF-7, T24) and for their ability to antagonize Pgp-mediated drug-resistant cells (i.e. NCI/ADR) and MRP1-mediated resistant cells (i.e. MCF-7/VP). Cytotoxicity and drug accumulation assays demonstrated that the dihydropyrroloquinolines inhibit Pgp to varying degrees, without any significant inhibition of MRP1. The compound termed PGP-4008 was the most effective at inhibiting Pgp in vitro and was further evaluated in vivo. PGP-4008 inhibited tumor growth in a murine syngeneic Pgp-mediated MDR solid tumor model when given in combination with doxorubicin. PGP-4008 was rapidly absorbed after intraperitoneal administration, with its plasma concentrations exceeding the in vitro effective dose for more than 2 h. PGP-4008 did not alter the plasma distribution of concomitantly administered anticancer drugs and did not cause systemic toxicity as was observed for cyclosporin A. Because of their enhanced selectivity toward Pgp, these substituted dihydropyrroloquinolines may be effective MDR modulators in a clinical setting.     

The effect of survivin on multidrug resistance mediated by P-glycoprotein in MCF-7 and its adriamycin resistant cells

Although anticancer chemotherapeutic drugs have been designed to inhibit the growth of tumor cells, chemotherapy frequently fails due to the development of multidrug resistance (MDR). In this paper, the effect of survivin on multidrug resistance mediated by P-glycoprotein (Pgp) was investigated in breast cancer cells. Overexpression of survivin in MCF-7 cells transfected with survivin expression vector pEGFP/survivin results in decreasing sensitivity to anticancer drugs and activation of Pgp to export drug out of cells. Down regulation of survivin in MCF-7/adriamycin (ADR) transfected with RNAi directed against survivin vector psh1/survivin could increase the drug accumulation in cells by inhibiting Pgp. Downregulation of the expression of the Pgp with the specific inhibitor verapamil could markedly suppress the survivin mRNA expression, whereas the reverse impact was not observed. Survivin might modulate the turnover of Pgp or transport by Pgp in cells, which result in anti-apoptosis and drug resistance. Our results suggest that survivin might play a key role in MDR in the presence of Pgp, and this might represent a novel strategy for modulating MDR in cancer cells.     

SDZ PSC 833 and SDZ 280-446 are the most active of various resistance-modifying agents in restoring rhodamine-123 retention within multidrug resistant P388 cells.

Multidrug resistance (MDR) of tumor cells may result from overexpression of P-glycoprotein (Pgp) but may be down-modulated by resistance-modifying agents (RMAs). The cyclosporin SDZ PSC 833 and the cyclopeptolide SDZ 280-446 were found to be the strongest RMAs known to date for restoring the sensitivity of MDR cells to anticancer drugs, as well as for restoring their retention of daunomycin, a fluorescent anthracycline. Using rhodamine-123 (Rhod-123), another fluorescent probe of Pgp function which also differentiates sensitive and MDR cells, several RMAs were compared for their capacity to inhibit Pgp function. At variance with the data obtained with the daunomycin probe, a series of RMAs did not detectably restore Rhod-123 retention by the MDR cells. With the remaining RMAs, achieving the same levels of Rhod-123 retention required 3 times lower RMA concentrations when the RMA was added to the MDR cells for both the initial uptake and the efflux of Rhod-123 rather than for its uptake only. Nevertheless, the data emphasized the large superiority of SDZ PSC 833 and SDZ 280-446 over all other RMAs.     

3'-O, 4'-O-aromatic acyl substituted 7,8-pyranocoumarins: a new class of P-glycoprotein modulators

Objectives P‐glycoprotein (Pgp) overexpression in tumour cells leads to multidrug resistance (MDR) and causes failure in cancer chemotherapy. We have previously identified (±)‐praeruptorin A (PA) as a potential lead compound for Pgp modulators. In this study we investigated the MDR‐reversing activities of PA derivatives. Methods Series 7,8‐pyranocoumarins with various C‐3′ and C‐4′ side chains had been semi‐synthesized and their MDR‐reversing activity was investigated in Pgp‐overexpressing MDR tumour cell line HepG2/Dox and in a KB V1 xenograft animal model. Key findings All 7,8‐pyranocoumarins exhibited equal or higher activity in modulating Pgp. DCK ( 12 ), DMDCK ( 15 ), 16 , 21 , 23 and 24 at 4 µm achieved 91%～99% decrease in IC50 value (concentration inhibiting cell growth by 50%) of anticancer agents vinblastine, doxorubicin, puromycin and paclitaxel, and were more active than others. DMDCK also remarkably enhanced the growth inhibitory effect of paclitaxel on KB V1 xenografts (P < 0.05), showing a potency required for clinical usage. Mechanistic studies suggested that these 7,8‐pyranocoumarins might reverse Pgp‐MDR through directly binding to substrate binding site(s) or allosteric site(s) on Pgp therefore impairing Pgp‐mediated drug transport. Conclusions Results from the study suggested that 3′‐O, 4′‐O‐aromatic acyl substituted 7,8‐pyranocoumarins could serve as a new class of Pgp modulator. Acyls play an important role in maintaining and enhancing the Pgp‐modulating ability of pyranocoumarins. 3,4‐Dimethoxyl substituted aromatic acyls, bearing a methoxy that might interact with Pgp as hydrogen bond accepter, were shown to be the most potent for reversing MDR.     

Reversal of P-glycoprotein-mediated multidrug resistance by protopanaxatriol ginsenosides from Korean red ginseng

The overexpression of P-glycoprotein (Pgp) or the multidrug resistance-associated protein (MRP) confers multidrug resistance (MDR) to cancer cells. MDR cells can be sensitized to anticancer drugs when treated concomitantly with an MDR modulator. In this study, we investigated whether or not ginseng saponins could reverse MDR mediated by Pgp or MRP. The chemosensitization and drug accumulation effects of ginseng saponins such as the total saponin, protopanaxadiol ginsenosides (PDG), protopanaxatriol ginsenosides (PTG), ginsenosides-Rb 1, -Rb 2, -Rc, -Rg 1 and -Re were tested on the daunorubicin- and doxorubicin-resistant acute myelogenous leukemia sublines (AML-2/D100 and AML-2/DX100), which overexpress Pgp and MRP, respectively. PTG showed cytotoxicity in both sublines and was able to reverse resistance in the AML-2/D100 subline in a concentration-dependent manner. Conversely, other ginseng saponins at concentrations less than 300 microg/mL showed neither cytotoxicity nor chemosensitizing activity in both resistant sublines. Flow cytometry analysis showed that the effect of PTG (100 microg/mL) on drug accumulation of daunorubicin in the AML-2/D100 subline was 2-fold higher than that observed in the presence of verapamil (5 microg/mL) and 1.5 times less than cyclosporin A (3 microg/mL). The maximum non-cytotoxic concentrations of PTG did not appear to increase the Pgp levels, which is in contrast to verapamil and cyclosporin A. PTG at 200 microg/mL or more completely inhibited the azidopine photolabeling of Pgp. The results suggest that PTG has a chemosensitizing effect on Pgp-mediated MDR cells by increasing the intracellular accumulation of drugs through direct interaction with Pgp at the azidopine site. In addition, PTG may have a beneficial effect on cancer chemotherapy with respect to the possibility of long-term use without the concern of Pgp activation.     

Apoptosis-based drug screening and detection of selective toxicity to cancer cells

The goal of our study was to determine whether an apoptosis assay used after short-term drug exposure could predict selective toxicity to cancer cells. To this end we compared the effect of eight anticancer drugs and 10 toxic compounds without known antitumor activity in cultures of human breast cancer cells and normal diploid fibroblasts by Apoptosis ELISA and growth inhibition assays. There was an overlap in concentration values of drugs and toxins inhibiting proliferation in cancer cells. In contrast, Apoptosis ELISA clearly distinguished between the two groups of compounds. Anticancer drugs induced apoptosis in cancer cells at 0.0015-0.5 microM, while toxins were effective at much higher concentrations of 8.0-50.0 microM. Moreover, six out of the 10 toxins did not induce apoptosis in cancer cells. The normal:cancer cell (N:C) ratio for growth inhibiting concentrations was in a similar range for anticancer drugs and toxins. The N:C ratio for apoptosis inducing concentrations was 33-200 for anticancer drugs and 1.3-3.0 for toxins. Our data indicate that apoptosis assays could be used to detect selective toxicity of anticancer drugs by determining apoptosis induction in cancer cells or through a comparison of apoptosis-inducing concentrations in normal and cancer cells.     

Cyclosporins: structure-activity relationships for the inhibition of the human MDR1 P-glycoprotein ABC transporter

Cyclic undecapeptide cyclo-[MeBmt(1)-Abu(2)-MeGly(3)-MeLeu(4)-Val(5)-MeLeu(6)-Ala(7)-D-Ala(8)-MeLeu(9)-MeLeu(10)-MeVal(11)], the immunosuppressive and antifungal antibiotic cyclosporin A (CsA), was reported to interfere with the MDR1 P-glycoprotein (Pgp), a transmembranous adenosine 5'-triphosphate binding cassette (ABC) transporter with phospholipid flippase or "hydrophobic vacuum cleaner" properties that mediate multidrug resistance (MDR) of cancer cells. By use of photoaffinity-labeled cyclosporins and membranes from Pgp-expressing cells, it was recently shown that in vitro, Pgp molecules could bind a large cyclosporin domain involving residues 4-9 as well as the side chain of residue 1. Tumor cell MDR can also be reversed by a product more distantly related to cyclosporin with the structure [Thr(2), Leu(5), D-Hiv(8), Leu(10)]-CsA (SDZ 214-103). In a standardized assay that measures Pgp function in vivo (on intact live cells) by the Pgp-mediated efflux of the calcein-AM Pgp substrate and uses human lymphoblastoid MDR-CEM (VBL(100)) cells as highly resistant Pgp-expressing cells, SDZ 214-103 was found to be one of the most active Pgp inhibitors among naturally occurring cyclosporins, with an IC(50) of 1.6 microM in an assay where CsA gives an IC(50) of 3.4 microM. Using the in vivo assay, 60, mostly natural, cyclosporin analogues were analyzed to establish structure-activity relationships (SAR). Our SAR are compatible with the in vitro-defined Pgp binding domain model and further disclose that in vivo Pgp inhibition is favored by larger hydrophobic side chains on cyclosporin residues 1, 4, 6, and 8 and a smaller one on residue 7, although with no effect on the residue 5 side chain; moreover, larger hydrophobic side chains on other residues 2, 3, 10, and 11 (outside the in vitro-defined Pgp binding domain) also favor the eventual inhibition of Pgp function. The N-desmethylation of any of the seven N-methylated amides, as naturally occurring in numerous cyclosporins, regularly leads to a decreased Pgp inhibitory activity (Pgp-InhA), up to its abrogation if it occurs at residues 4 and 9. Nevertheless, despite unfavorable use of [Thr(2)] and [Leu(10)] residues, all [D-Hiv(8)] analogues whose lead is SDZ 214-103 show a large Pgp-InhA. The SAR for Pgp inhibition by cyclosporins are thus very complex. Because CsA and SDZ 214-103 show largely different conformations when free in solution, but remarkably similar ones when bound to the cytosolic cyclophilins, SAR for Pgp inhibition must similarly include requirements for occurrence of suitable conformers for insertion in the cell membrane, sufficient conformational plasticity for gaining access to Pgp binding sites, and an adequate conformer structure there to achieve such binding with a high enough affinity and possibly escape from sequestration on cyclophilins.     

Interaction of the P-glycoprotein multidrug transporter (MDR1) with high affinity peptide chemosensitizers in isolated membranes, reconstituted systems, and intact cells.

P-glycoprotein-mediated multidrug resistance can be reversed by the action of a group of compounds known as chemosensitizers. The interactions with P-glycoprotein of two novel hydrophobic peptide chemosensitizers (reversins 121 and 205) have been studied in model systems in vitro, and in a variety of MDR1-expressing intact tumor cells. The reversins bound to purified P-glycoprotein with high affinity (77-154 nM), as assessed by a quenching assay using fluorescently labeled purified protein. The peptides modulated P-glycoprotein ATPase activity in Sf9 insect cell membranes expressing human MDR1, plasma membrane vesicles from multidrug-resistant cells, and reconstituted proteoliposomes. Both peptides induced a large stimulation of ATPase activity; however, higher concentrations, especially of reversin 205, led to inhibition. This pattern was different from that of simple linear peptides, and resembled that of chemosensitizers such as verapamil. In both membrane vesicles and reconstituted proteoliposomes, 1-2 microM reversins were more effective than cyclosporin A at blocking colchicine transport. Reversin 121 and reversin 205 restored the uptake of [3H]daunorubicin and rhodamine 123 in MDR1-expressing cells to the level observed in the drug-sensitive parent cell lines, and also effectively inhibited the extrusion of calcein acetoxymethyl ester from intact cells. In cytotoxicity assays, reversin 121 and reversin 205 eliminated the resistance of MDR1-expressing tumor cells against MDR1-substrate anticancer drugs, and they had no toxic effects in MDR1-negative control cells. We suggest that peptides of the reversin type interact with the MDR1 protein with high affinity and specificity, and thus they may be good candidates for the development of MDR1-modulating agents to sensitize drug resistance in cancer.     

P-glycoprotein as a drug target in the treatment of multidrug resistant cancer

Multidrug resistance (MDR) is a major obstacle to successful cancer chemotherapy. One important mechanism of MDR involves the multidrug transporter, P-glycoprotein (Pgp), which confers upon cancer cells the ability to resist lethal doses of certain cytotoxic drugs by pumping the drugs out of the cells and thus reducing their cytotoxicity. Pgp belongs to the ATP-binding cassette (ABC) family of transporter molecules which require hydrolysis of ATP to run the transport mechanism. The substrates of Pgp may be endogenous (steroid hormones, cytokines) or exogenous (cytostatic drugs). A number of studies have demonstrated a negative correlation between Pgp expression levels and chemosensitivity or survival in a range of human malignancies. In principle, Pgp mediated drug resistance can be circumvented by treatment regimens that either exclude Pgp substrate drugs or include Pgp inhibitory agents. Experimental studies have demonstrated that certain structural modifications of anthracyclines confer the ability to escape Pgp transport. The therapeutic benefit of Pgp inhibitors as chemosensitizers is currently being explored in phase III clinical trials, and the first promising results have already been reported. Another therapeutic option for Pgp inhibitors has recently evolved as several Pgp inhibitors, many of which are generally low-toxic substances, by themselves constrain proliferation and cause cell death by apoptosis in certain MDR cancer cell lines. The dual effect of Pgp inhibitors, targeting MDR cancer cells selectively, may translate into improved efficacy of cancer chemotherapy and perhaps new and less toxic drug treatment strategies in human MDR cancer.     

Studies on pyrrolopyrimidines as selective inhibitors of multidrug-resistance-associated protein in multidrug resistance

Multidrug resistance mediated by P-glycoprotein (Pgp) or multidrug-resistance-associated protein (MRP) remains a major obstacle for successful treatment of cancer. Inhibition of Pgp and MRP transport is important for high efficacy of anticancer drugs. While several Pgp inhibitors have entered clinical trials, the development of specific MRP1 inhibitors is still in its infancy. In our screening program, we have identified a pyrrolopyrimidine (4) as a novel and selective MRP1 inhibitor. Subsequent SAR work on the 4-position of the template revealed the phenethylpiperazine side chain as a potent replacement of the benzylthio group of the lead molecule. Introduction of groups at the 2-position seems to have no detrimental effect on activity. Modifications to the nitrile group at the 7-position resulted in the identification of analogues with groups, such as amides, with superior pharmacokinetic profiles. In vivo efficacy has been demonstrated by xenograft studies on selected compounds.     

The medicinal chemistry of multidrug resistance (MDR) reversing drugs

Multidrug resistance (MDR) is a kind of resistance of cancer cells to multiple classes of chemotherapic drugs that can be structurally and mechanistically unrelated. Classical MDR regards altered membrane transport that results in lower cell concentrations of cytotoxic drug and is related to the over expression of a variety of proteins that act as ATP-dependent extrusion pumps. P-glycoprotein (Pgp) and multidrug resistance protein (MRP1) are the most important and widely studied members of the family that belongs to the ABC superfamily of transporters. It is apparent that, besides their role in cancer cell resistance, these proteins have multiple physiological functions as well, since they are expressed also in many important non-tumoural tissues and are largely present in prokaryotic organisms. A number of drugs have been identified which are able to reverse the effects of Pgp, MRPI and sister proteins, on multidrug resistance. The first MDR modulators discovered and studied in clinical trials were endowed with definite pharmacological actions so that the doses required to overcome MDR were associated with unacceptably high side effects. As a consequence, much attention has been focused on developing more potent and selective modulators with proper potency, selectivity and pharmacokinetics that can be used at lower doses. Several novel MDR reversing agents (also known as chemosensitisers) are currently undergoing clinical evaluation for the treatment of resistant tumours. This review is concerned with the medicinal chemistry of MDR reversers, with particular attention to the drugs that are presently in development.     

Multidrug resistance: retrospect and prospects in anti-cancer drug treatment

Conventional cancer chemotherapy is seriously limited by the multidrug resistance (MDR) commonly exhibited by tumour cells. One mechanism by which a living cell can achieve multiple resistances is via the active efflux of a broad range of anticancer drugs through the cellular membrane by MDR proteins. Such drugs are exported in both ATP-dependent and -independent manners, and can occur despite considerable concentration gradients. To the ATP-dependent group belongs the ATP-binding cassette (ABC) transporter family, which includes P-gp, MRP, BCRP, etc. Another protein related to MDR, though not belonging to the ABC transporter family, is lung resistance-related protein (LRP). All of these proteins are involved in diverse physiological processes, and are responsible for the uptake and efflux of a multitude of substances from cancer cells. Many inhibitors of MDR transporters have been identified over the years. Firstly, MDR drugs were not specifically developed for inhibiting MDR; in fact, they had other pharmacological properties, as well as a relatively low affinity for MDR transporters. They included compounds of diverse structure and function, such as verapamil and cyclosporine, and caused side effects. Secondly, the new drugs were more inhibitor-specific, in terms of MDR transport, and were designed to reduce such side effects (e.g., R-verapamil, dexniguldipine, etc.). Unfortunately, they displayed poor response in clinical studies. Recently, new compounds obtained from drug development programs conducted by the pharmaceutical industry are characterized by a high affinity to MDR transporters and are efficient at nanomolar concentrations. Some of these compounds (e.g., MS-209) are currently under clinical trials for specific forms of advanced cancers. We aim to provide an overview of the properties associated with those mammalian MDR transporters known to mediate significant transport of relevant drugs in cancer treatments. We also summarize recent advances concerning resistance to cancer drug therapies with respect to the function and overexpression of ABC and LRP multidrug transporters.     

Multidrug resistance-associated protein: A protein distinct from P-glycoprotein involved in cytotoxic drug expulsion

• 1.1. Multidrug resistance (MDR) is a phenomenon originally seen in cultured tumor cells that, following selection for resistance to a single anticancer agent, become resistant to a range of chemically diverse anticancer agents. These MDR cells show a decrease in intracellular drug accumulation due to active efflux by transporter proteins. The transporter best characterized is P-glycoprotein (Pgp). This protein has been identified in many cancers and has been the target for agents able to inhibit its action, thereby reversing resistance.
• 2.2. More recently, another transporter, multidrug resistance-associated protein (MRP) has been identified in a number of MDR human tumor cell lines that do not apparently express Pgp. The presence of MRP at the cell surface of these cells is associated with alterations in drug accumulation and distribution.
• 3.3. The gene-encoding MRP has been cloned and sequenced and shown by transfection studies to be able to confer resistance and changes in drug accumulation in sensitive tumor cells. The profile of anticancer drugs expelled in the presence of MRP is similar, but not identical, to that of Pgp.
• 4.4. MRP has been identified in a number of different types of cancers, but it is not yet clear to what extent it is involved with clinical resistance. Furthermore, resistance modulators useful against Pgp are less effective in reversing MRP-mediated resistance.
• 5.5. It is not fully understood how MRP brings about drug efflux, but it is clear that the underlying mechanisms are different from those responsible for Pgp-mediated drug efflux. In particular, glutathione (GSH) is required for the effective expulsion of the anticancer agents.
• 6.6. Unlike Pgp, MRP is able to transport metallic oxyanions and glutathione and other conjugates, including peptidyl leukotrienes. Agents that inhibit organic anion transport, such as probenecid, can block MRP activity.
• 7.7. Like Pgp, MRP is expressed not only in resistant tumor cells, but also in normal human tissues. These include the epithelial cells lining the airways and the gastrointestinal tract. In cells in normal tissues, MRP appears to be located within the cytoplasm, which may mean that it functions here in a manner slightly different to that in malignant cells. It is now also recognized in cells and tissues from other species, such as the rat and mouse.

     

Enhancement of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) toxicity by acetohydroxamic acid analogues of 3-nitropyrazole in vitro

A series of acetohydroxamic acid derivatives of 3-nitropyrazole were synthesized and evaluated for their ability to potentiate (chemosensitization) the activity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) against EMT-6 mouse mammary tumor cells in vitro. The compounds were designed to test the hypothesis that the chemosensitizing activity of the analogues would be proportional to the rate of isocyanate formation via a Lossen rearrangement, in part a function of the leaving group at the N terminus of each acetohydroxamate. Substitution of acetohydroxamic acid side chains at the N-1 position of the parent 3-nitropyrazole resulted in compounds which were preferentially toxic to cells treated under hypoxic conditions, and which were capable of enhancing the toxicity of CCNU in hypoxia. As was observed for cytotoxicity, the enhancement of CCNU toxicity by these sensitizing agents was significantly reduced under aerobic treatment conditions. A strong correlation was established between hypoxic toxicity and chemosensitizing potency. The activity of the analogue, however, was not proportional to their excepted rates of Lossen rearrangement. Nevertheless, several potent chemosensitizing compounds were identified; some of which were 10–50 x 's more potent on a molar basis than Misonidazole, the reference chemosensitizing compound.     

Reversal of multidrug resistance in mouse lymphoma cells by phenothiazines

Various compounds were tested with regard to their reversal of multidrug resistance (MDR) in mouse tumor cells transfected with the human MDR1 gene. Phenothiazines containing aromatic moieties were bound through stacking interaction involving the polarization of the aromatic aminoacid substituents at the target site of p-glycoprotein (Pgp) 170, as a consequence of their large dipoles (as in the binding of phenothiazine to calmodulin-like structures). Acting as a calcium channel blocker, verapamil may induce conformational changes in the calcium channel-like structures of the transmembrane regions of Pgp. Most probably the tyrosine moieties of Pgp are involved in the action of verapamil and phenothiazines. Tomato lectin specifically binds to the polylactosamine moiety of Pgp170 at the first loop of Pgp. Other targets in the membrane may exist in close proximity to Pgp170, such as conA-reactive glycoproteins with terminal mannosyl residues. WGA-reactive N-acetyl glucosamine residues can also be modified resulting in conformational changes in trans-membrane regions of the ABC transporter. Our results demonstrate that MDR can be reversed by interaction of various compounds with Pgp or by modification of the membrane structure around the Pgp.     

Three decades of P-gp inhibitors: skimming through several generations and scaffolds

Many tumor cells become resistant to commonly used cytotoxic drugs due to the overexpression of ATP-binding cassette (ABC) transporters, namely P-glycoprotein (P-gp). The discovery of the reversal of multidrug resistance (MDR) by verapamil occured in 1981, and in 1968 MDR Chinese hamster cell lines were isolated for the first time. Since then, P-gp inhibitors have been intensively studied as potential MDR reversers. Initially, drugs to reverse MDR were not specifically developed for inhibiting P-gp; in fact, they had other pharmacological properties, as well as a relatively low affinity for MDR transporters. An example of this first generation P-gp inhibitors is verapamil. The second generation included more specific with less side-effect inhibitors, such as dexverapamil or dexniguldipine. A third generation of P-gp inhibitors comprised compounds such as tariquidar, with high affinity to P-gp at nanomolar concentrations. These generations of inhibitors of P-gp have been examined in preclinical and clinical studies; however, these trials have largely failed to demonstrate an improvement in therapeutic efficacy. Therefore, new and innovative strategies, such as the fallback to natural products, the design of peptidomimetics and dual activity ligands emerged as a fourth generation of P-gp inhibitors. The chemistry of P-gp inhibitors, as well as their in vitro, in vivo and clinical trials are discussed, and the most recent advances concerning Pgp modulators are reviewed.     

New triazine derivatives as potent modulators of multidrug resistance.

A series of 70 triazine derivatives have been synthesized and tested for their capacity to modulate multidrug resistance (MDR) in DC-3F/AD and KB-A1 tumor cells in vitro, in comparison with verapamil (VRP), a calcium channel antagonist currently used in therapy as an antihypertensive drug, which also shows MDR modulating activity. Among the 12 selected compounds, 16 (S9788) showed high MDR reversing properties in vitro (300- and 6-fold VRP at 5 microM in DC-3F/AD and KB-A1 cells, respectively) and induced a strong accumulation of adriamycin. The relationship between the increase of ADR accumulation and the fold reversal induced by these compounds and their lack of effects on the sensitive DC-3F cells suggest that they act mainly by inhibiting the P-glycoprotein (Pgp) catalyzed efflux of cytotoxic agents, as already described for a majority of MDR modulators. In vivo, in association with the antitumor drug vincristine (0.25 mg/kg), 16 (100 mg/kg) increased the T/C by 39% in mice bearing the resistant tumor cell line P388/VCR. According to these interesting properties, 16 was selected for a clinical development because it was more bioavailable than 34, even though it was less active.     

Structure-activity studies of substituted quinoxalinones as multiple-drug-resistance antagonists

A significant problem in the clinical treatment of cancer relates to the development of tumor resistance to many chemotherapeutic agents. Acquired drug resistance is often mediated through overexpression of membrane transport proteins that effectively efflux anticancer agents. Two of the best-studied transporters, P-glycoprotein (Pgp) and MRP1, have pharmacological properties that only partially overlap. In our search for improved drug-resistance antagonists, we have identified a family of substituted quinoxalines that selectively antagonizes Pgp over MRP1. Consequently, a focused library of congeners was designed and synthesized starting with a parent bromomethylquinoxalinone. This parent quinoxalinone was then condensed with a series of phenols to yield a family of substituted phenoxymethylquinoxalinones. These compounds were evaluated for their toxicity toward drug-sensitive MCF-7 breast carcinoma cells and for their abilities to antagonize Pgp and MRP1 in drug-resistant cell lines (NCI/ADR and MCF-7/VP, respectively). The results of this structure-activity study indicate that compounds with carbonyl substitutions of the phenoxy group (ester, amide, or ketone moieties) demonstrate excellent antagonism of Pgp while having relatively low toxicity toward drug-sensitive cells. Importantly, none of these compounds antagonized MRP1. Because of their transporter selectivity, we predict that substituted quinoxalinones may be more effective MDR modulators in vivo than are nonselective transporter antagonists.