P-glycoprotein Modulators Research Articles

Simotinib as a modulator of P-glycoprotein: substrate, inhibitor, or inducer?

As a new antitumor drug, simotinib hydrochloride is prescribed for prolonged periods, often to patients with comorbidities. Therefore, the risk for developing drug resistance and drug-drug interactions between simotinib and other agents has to be taken into consideration. As P-glycoprotein (P-gp) is an efflux transporter, which plays a significant role in drug resistance and influences the pharmacological properties and toxicities of the drugs it interacts with, the interactions between simotinib and P-gp were investigated. Cytotoxicity was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Intracellular drug concentrations were detected by high-performance liquid chromatography, fluorescence-activated cell sorting and using a fluorescence reader. P-gp ATPase activity was measured using the Pgp-Glo assay, and intracellular pH was assessed using the fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl. The expression and transcription of P-gp were detected by western blotting and the luciferase assay. Simotinib has no cross-resistance to P-gp substrates, and its efflux rate was independent of either the P-gp expression or the coadministered P-gp substrate. Simotinib reversed chemotherapeutic agent resistance in a short time by increasing the intracellular concentration of the chemotherapeutic agent and blocked rhodamine 123 efflux. Further studies demonstrated that simotinib inhibited P-gp activity by modulating its ATPase activity and the intracellular pH. Although simotinib induced P-gp expression after extended treatment, the induced expression of P-gp had little impact on drug resistance. Simotinib is not a substrate of P-gp. As a modulator, it functions mainly as an inhibitor of P-gp by modulating the intracellular pH and ATPase activity, although it also induces P-gp expression after extended treatment.

Investigative Approaches for Oral Delivery of Anticancer Drugs: A Patent Review.

Typically, chemotherapy has been dominated by intravenous administration. Though, inclination towards oral ingestion of chemotherapeutics is increasing since it offers ample of fascinating opportunities including better quality of life, treatment advantages and low healthcare cost. However, low or moderate bioavailability along with significant inter-patient variability and narrow therapeutic window challenges their oral administration. Thus, optimization of oral route with maximized efficacy and miniminal adverse events is a challenging area. To surmount the challenges, a number of strategies like P-glycoprotein (P-gp) modulation, colloidal carrier etc. have been under investigation and scientists are exploring the utility of solid dispersions, prodrugs, biconjugates, complexes, microparticulate, and nanoparticulate systems (liposome, SLN, dendrimers, SEDDS, nanoparticles). Among these, nanoparticulate systems have shown promising results due to their targeting potential and ability to alter absorption pathways. Functional excipients with P-gp modulating activity are also being explored for more effective oral delivery of chemotherapeutics. This article explores the encouraging reports, recent patents and inventions on the feasibility and applicability of oral administration of chemotherapeutics.

Nuclear Transcription Factor Kappa B Downregulation Reduces Chemoresistance in Bone Marrow-derived Cells Through P-glycoprotein Modulation

Nuclear transcription factor kappa B (NF-κB) is associated with many types of refractory cancer. However, despite multiple strategies to treat cancer and novel target drugs, multidrug resistance still causes relapses. The best-characterized mechanism responsible for multidrug resistance involves the expression of the MDR-1 gene product, P-glycoprotein (P-gp). Because the direct inhibition of this protein is very toxic, other methods of multidrug resistance (MDR) regulation have been proposed. The MDR-1 promoter sequence contains a κB site, which is recognized by NF-κB. The aim of this work was to characterize whether NF-κB modulation changes the response of bone marrow-derived cells (BMDCs) to chemotherapy. We exposed BMDCs to etoposide and doxorubicin, two of the most used antineoplastic drugs. BMDCs presented high tolerance to these drugs, which correlated with high intrinsic P-gp activity and strong protein expression of NF-κB. To determine the mechanism behind the poor sensitivity of BMDCs to chemotherapy, we blocked the activity of the heterodimer protein NF-κB using the pharmacological inhibitor Bay 11-7085 and through the transfection of an adenovirus negative mutant of I kappa B alpha. The multidrug resistance phenotype of BMDCs was reversed by inhibiting the NF-κB pathway, and this change was accompanied by a decrease in P-gp activity. NF-κB is a possible target for improving the antineoplastic response.

Screening a Small Library of Xanthones for Antitumor Activity and Identification of a Hit Compound which Induces Apoptosis.

Our previous work has described a library of thioxanthones designed to have dual activity as P-glycoprotein modulators and antitumor agents. Some of these compounds had shown a significant cell growth inhibitory activity towards leukemia cell lines, without affecting the growth of non-tumor human fibroblasts. However, their effect in cell lines derived from solid tumors has not been previously studied. The present work aimed at: (i) screening this small series of compounds from an in-house library, for their in vitro cell growth inhibitory activity in human tumor cell lines derived from solid tumors; and (ii) initiate a study of the effect of the most potent compound on apoptosis. The tumor cell growth inhibitory effect of 27 compounds was first analysed in different human tumor cell lines, allowing the identification of a hit compound, TXA1. Its hydrochloride salt TXA1·HCl was then synthesized, to improve solubility and bioavailability. Both TXA1 and TXA1·HCl inhibited the growth of MCF-7, NCI-H460, A375-C5, HeLa, 786-O, Caki-2 and AGS cell lines. The effect of TXA1·HCl in MCF-7 cells was found to be irreversible and was associated, at least in part, with an increase in cellular apoptosis.

P-Glycoprotein Modulation Facilitates the Selective Inhibition of Oxidative Phosphorylation in Alloreactive T Cells to Prevent Graft-Versus-Host Disease after Hematopoietic Stem Cell Transplant

T lymphocytes play a central role in many human immunologic disorders including both autoimmune and alloimmune diseases. In hematopoietic stem cell transplant (HSCT), acute graft-versus-host-disease (GVHD) is caused by an attack on the recipient's tissues from donor allogeneic T cells. Ex vivo application of phototherapy to selectively deplete GVHD-causing cells prior to transplant may prevent GVHD. However, none of the photosensitive agents in use today have demonstrated selectivity without significant toxicity occurring in resting cells. We have designed the first-in-class, novel photosensitizer 2-Se-Cl with the ability to accumulate in pathogenic T cells in proportion to degree of oxidative phosphorylation (OXPHOS). Unique to 2-Se-Cl is the ability to potently stimulate P-glycoprotein (P-pg). Enhanced P-gp activity promotes the efficient removal of photosensitizer not sequestered in mitochondria, and protects resting lymphocytes essential for antipathogen and antitumor responses. To confirm that 2-Se-Cl selectively depletes antigen-specific immune responses, human lymphocytes were cultured with staphylococcal enterotoxin B (SEB) for 72 hours. Cells were then pulsed with 2-Se-Cl and exposed to 5 J/cm2 of light, which resulted in the impedance of OXPHOS and induction of apoptosis in stimulated T cells. After photodepletion (PD), resting cells remained intact with the ability to respond to stimulation by Toxic Shock Syndrome Toxin-1 (TSST-1) but not SEB. To evaluate the selective depletion of alloimmune responses, a well-established complete mHC antigen-mismatched murine model of HSCT was employed. To prepare the PD - treated primed splenocytes, donor C57BL/6 splenocytes were cocultured for 5 days with irradiated Balb/c splenocytes, and then photodepleted. In vitro analysis of PD - treated primed splenocytes demonstrated selective depletion of antihost immune responses by CFSE dilution, with complete retention of third-party responses (C3H/HeJ mice). On the day of HSCT, recipient Balb/c mice were irradiated with 900 cGy, and received 5x106 T cell-depleted bone marrow cells accompanied by 5x106 PD - treated (treatment group) or untreated (control group) primed splenocytes, and then monitored for signs of GVHD according to an established mouse GVHD grading system. Five animals underwent HSCT in each group in 3 independent experiments. All mice died of acute GVHD after the addition of 5x106 untreated primed C57BL/6 cells at a median of 25 days after HSCT. In contrast, > 90% of mice that received the same number of PD - treated cells survived > 60 days without evidence of GVHD (see figure). Additionally, third-party C3H/Hej mice that received the same number of PD-treated cells died of GVHD, confirming the selective depletion of the alloimmune response for BalB/c by 2-Se-Cl. The high selectivity of this novel photosensitizer may have broad applications, including targeting the bioenergetics in alloimmune, autoimmune, and malignant T cells to provide alternative treatment options and improve clinical outcomes for patients with diseases such as GVHD, systemic lupus erythematosus, or peripheral T cell lymphoma. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Functionalized nanocarrier combined seizure-specific vector with P-glycoprotein modulation property for antiepileptic drug delivery

Despite optimal therapeutic regimen with currently available antiepileptic drugs (AEDs), approximately a third of epilepsy patients remain drug refractory. Region-specific overexpression of multidrug efflux transporters at the blood–brain barrier, such as P-glycoprotein (P-gp), might contribute to multidrug resistance (MDR) by reducing target concentrations of AEDs. Therefore, development of nanomedicine that can modulate P-gp function as well as facilitate targeted AEDs delivery represents a promising strategy for epilepsy intervention. To achieve this, we sought to exploit the possibility of combination of active targeting function of tryptophan by transporter-mediated endocytosis and overcoming MDR by Pluronic block copolymers. Herein, a tryptophan derivate (TD) functionalized Pluronic P123/F127 mixed micelles encapsulating LTG (TD-PF/LTG) was developed to promote AEDs delivery to epileptogenic focus. TD-PF/LTG was about 20 nm in diameter with a spherical shape and high encapsulation efficiency. A rat epilepsy model with pilocarpine was established to evaluate the brain penetration efficiency of the LTG-incorporated polymeric micellar formulation, compared with free LTG formulations. Studies showed that TD-PF/LTG was more efficient than PF/LTG as well as free LTG in delivering the drug to the brain, especially the hippocampus. The enhanced targeted delivery could be ascribed to the increased tryptophan uptake at epileptogenic focus as well as P-gp modulation property of the nanomaterial. Taken together, TD-conjugated Pluronic micelles showed promising potential as a nanoplatform for the delivery of AEDs in refractory epilepsy.

Modulation of P-glycoprotein at the Human Blood-Brain Barrier by Quinidine or Rifampin Treatment: A Positron Emission Tomography Imaging Study.

Permeability-glycoprotein (P-glycoprotein, P-gp), an efflux transporter at the human blood-brain barrier (BBB), is a significant obstacle to central nervous system (CNS) delivery of P-gp substrate drugs. Using positron emission tomography imaging, we investigated P-gp modulation at the human BBB by an approved P-gp inhibitor, quinidine, or the P-gp inducer, rifampin. Cerebral blood flow (CBF) and BBB P-gp activity were respectively measured by administration of (15)O-water followed by (11)C-verapamil. In a crossover design, healthy volunteers received quinidine and 11-29 days of rifampin treatment during different study periods. CBF and P-gp activity was measured in the absence (control; prior to quinidine treatment) and presence of P-gp modulation. At clinically relevant quinidine plasma concentrations, P-gp inhibition resulted in a 60% increase in (11)C-radioactivity distribution across the human BBB as measured by the brain extraction ratio (ER) of (11)C-radioactivity. Furthermore, the magnitude of BBB P-gp inhibition by quinidine was successfully predicted by a combination of in vitro and macaque data, but not by rat data. Although our findings demonstrated that quinidine did not completely inhibit P-gp at the human BBB, it has the potential to produce clinically significant CNS drug interactions with P-gp substrate drugs that exhibit a narrow therapeutic window and are significantly excluded from the brain by P-gp. Rifampin treatment induced systemic CYP3A metabolism of (11)C-verapamil; however, it reduced the ER by 6%. Therefore, we conclude that rifampin, at its usual clinical dose, cannot be used to induce P-gp at the human BBB to a clinically meaningful extent and is unlikely to cause inadvertent BBB-inductive drug interactions.

A New Class of Safe, Potent, and Specific P-gp Modulator: Flavonoid Dimer FD18 Reverses P-gp-Mediated Multidrug Resistance in Human Breast Xenograft in Vivo

Flavonoid dimer FD18 is a new class of dimeric P-gp modulator that can reverse cancer drug resistance. FD18 is a potent (EC50 = 148 nM for paclitaxel), safe (selective index = 574), and selective P-glycoprotein (P-gp) modulator. FD18 can modulate multidrug resistance toward paclitaxel, vinblastine, vincristine, doxorubicin, daunorubicin, and mitoxantrone in human breast cancer LCC6MDR in vitro. FD18 (1 μM) can revert chemosensitivity of LCC6MDR back to parental LCC6 level. FD18 was 11- to 46-fold more potent than verapamil. FD18 (1 μM) can increase accumulation of doxorubicin by 2.7-fold, daunorubicin (2.1-fold), and rhodamine 123 (5.2-fold) in LCC6MDR. FD18 inhibited P-gp-mediated doxorubicin efflux and has no effect on influx. FD18 at 1 μM did not affect the protein expression level of P-gp. Pharmacokinetics studies indicated that intraperitoneal administration of 45 mg/kg FD18 was enough to maintain a plasma level above EC50 (148 nM) for more than 600 min. Toxicity studies with FD18 (90 mg/kg, i.p. for 12 times in 22 days) with paclitaxel (12 mg/kg, i.v. for 12 times in 22 days) revealed no obvious toxicity or death in mice. In vivo efficacy studies indicated that FD18 (45 mg/kg, i.p. for 12 times in 22 days) together with paclitaxel (12 mg/kg, i.v. for 12 times in 22 days) resulted in a 46% reduction in LCC6MDR xenograft volume (n = 11; 648 ± 84 mm(3)) compared to paclitaxel control (n = 8; 1201 ± 118 mm(3)). There were no animal deaths or significant drop in body weight and vital organ wet weight. FD18 can increase paclitaxel accumulation in LCC6MDR xenograft by 1.8- to 2.2-fold. The present study suggests that FD18 represents a new class of safe and potent P-gp modulator in vivo.

Assessment of drug interaction potential of Eschscholzia californica (California poppy) through modulation of CYPs, P-gp and PXR

Eschscholzia californica, a North American plant, is traditionally used for depression, neurasthenia, and various psychiatric conditions. With the rapid rise in the use of herbal supplements in combination with conventional drugs, the risk for potential herb-drug interactions is also increasing. Most of the clinically relevant pharmacokinetic drug interactions occur due to modulation of Cytochrome P450 enzymes (CYPs), P-glycoprotein (P-gp), and Pregnane X receptor (PXR). This study aimed to determine the effects of an EtOH extract, BuOH and CHCl3 fractions and alkaloids of California poppy on the activity of CYPs, P-gp and PXR. The extract and fractions showed strong inhibition of CYP3A4, 2D6, and 2C19 and a weak inhibition of 2C9 and 1A2. Among the alkaloids, escholtzine inhibited 3A4, 2D6 and 2C19 (IC50 3.0, 2.0 and 0.4µM) and protopine inhibited 2D6 and 2C19 (IC50 0.04 and 1.3µM). No inhibition of P-gp was observed with the extract, fractions or its constituents. A significant activation (> 2 fold) of PXR was observed with the extract and fractions (30 µg/mL) while the alkaloids were not as potent (> 1.5 fold at 60µM). Further studies are in progress to find out if the PXR activation would lead to an increased expression of drug metabolizing enzymes and transporters (CYPs and P-gp). Acknowledgement: Supported by the National Institute of General Medical Sciences, NIH Grant Number P20GM104932.

Arylamino Esters As P-Glycoprotein Modulators: SAR Studies to Establish Requirements for Potency and Selectivity.

A set of basic aryl-group-containing compounds was synthesized with the aim of developing potent and selective P-glycoprotein (P-gp) modulators that are able to reverse multidrug resistance (MDR). The natures of the spacer (dicyclohexylamine or dialkylamine) and the aryl moieties were modified to investigate selectivity and the mechanism of P-gp interaction. The inhibitory activities of the compounds toward P-gp, multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP), the most relevant ATP binding cassette (ABC) transporters for MDR, were evaluated. The mechanism of P-gp interaction for each compound was investigated with three biological assays: apparent permeability (Papp ) determination (B→A/A→B) in Caco-2 cell monolayers, ATP cell depletion, and inhibition of Calcein-AM transport in MDCK-MDR1 cells. These assays allowed us to estimate the selectivity of the compounds for the three efflux pumps and to identify the structural requirements that define the P-gp-interaction profile. All dicyclohexylamine derivatives were found to be P-gp substrates, whereas one dialkylamine derivative was shown to be a P-gp inhibitor. The good MRP1 activity of one cis/cis isomer highlighted this as a lead candidate for the development of MRP1 ligands.

Modulation of lipid rafts (LR), p-glycoprotein (P-gp) and proinflammatory cytokines (PIC) with HMG-Co A reductase inhibitor simvastatin (SV) in chronic myelogenous leukemia (CML) patients with imatinib mesylate (IM) resistance.

e18032 Background: Primarily monocyte-derived cytokines and soluble interleukin-2-receptor (sIL2R) form of a group of PIC which are significantly elevated in patients with CML-cp or CML-bc. C-react...

Effects of Muscone on the Expression of P-gp, MMP-9 on Blood-Brain Barrier Model In Vitro.

Muscone is the main chemical ingredient in Musk which is main crude drug in Tongqiaohuoxue decoction (TQHXD), and TQHXD has a protective effect on damaged neurons, so we hypothesize that muscone can alter blood-brain barrier (BBB) permeability via the modulation of P-glycoprotein (P-gp) and matrix metalloproteinase-9 (MMP-9) expression. In this study, astrocytes (AC) and human umbilical vein endothelial cells (ECV304) were co-cultured to simulate the BBB model in vitro. Leak testing, transmembrane resistance experiments, and BBB-specific enzyme testing were used to test whether the model was successful. Different concentrations of muscone permeating the BBB were detected by gas chromatography (GC). The change of the transendothelial electrical resistance (TEER) on the BBB in vitro after treating with muscone was detected by Millicell-ERS. The protein expression of P-gp, MMP-9 in normal, and oxygen/glucose deprivation (OGD) BBB model was determined by western blotting to inquire that the mechanism of muscone penetrates the BBB model in vitro. The results show that muscone was detected in the lower medium of the BBB model by GC; the values of TEER were no significant difference before and after muscone (8 μM) was added to the BBB model; the expression of P-gp significantly decreased after the BBB model treatment with muscone (4, 8, and 16 μM) for 24 h; the expression of P-gp and MMP-9 in different concentrations of muscone groups had different degrees of reduction compared with the BBB in the state of OGD. In conclusion, muscone could permeate the BBB model, and it was associated with the inhibition of P-gp and MMP-9 expression. An understanding of the mechanisms of muscone across the BBB is crucial to the development of therapeutic modalities for cerebral vascular diseases.

Pharmacokinetic Interaction between Paclitaxel and Polyoxypregnanes

Polyoxypregnanes (POPs) as novel P-glycoprotein (P-gp) modulators were derived from a Chinese medicinal herb, Marsdeniae Tenacissimae Caulis., which has long been used in combination anti-cancer therapy. Our previous study demonstrated that three most abundant POPs in the herb can reverse P-gp-mediated paclitaxel resistance. The objective of present study was to investigate potential pharmacokinetic (PK) interaction between paclitaxel and POPs in Sprague-Dawley (SD) rats. POPs were orally pretreated by 8 h before intravenous paclitaxel administration to jugular vein- and bile duct-cannulated male SD rats. Paclitaxel and its metabolites were analyzed with LC-MS in plasma, feces, urine, bile and different tissues. POPs significantly increased the area under plasma concentration-time curve of paclitaxel by 1.5 folds, without changing Cmax and elimination half-life of paclitaxel. POPs did not dramatically affect paclitaxel metabolism and tissue distribution profile. The excretions of the intact paclitaxel in the urine and bile were significantly decreased in the presence of POPs. These results indicated that enhanced systemic exposure of paclitaxel might be due to the inhibitory effects of POPs on the P-gp-mediated excretion of paclitaxel. Our study revealed minor PK interaction between paclitaxel and POPs, and provided basis for a potential therapeutic benefit of this combination therapy for cancer patients.

Xanthone analogues as potent modulators of intestinal P-glycoprotein

Intestinal P-glycoprotein (P-gp) is a limiting step for oral absorption of drugs. Therefore, P-gp inhibitors have been studied as enhancers of oral absorption of drugs that are P-gp substrates. We investigated the in vitro and in vivo P-gp inhibitory activity of synthesized xanthone analogues. With 3-(3-chloro-2-hydroxypropoxy)-1-hydroxy-9H-thioxanthen-9-one, compound 13, accumulation of daunomycin (DNM) increased 707% and efflux of DNM decreased 66% compared to DNM alone. Relative bioavailability (RB) of paclitaxel (PTX, 25 mg/kg) increased 2.5-fold after oral administration with 13 (5 mg/kg). In a xenograft animal model, oral administration of PTX (40 mg/kg) with 13 (10 mg/kg) significantly inhibited tumour growth and was more effective than intravenously administered PTX (10 mg/kg) alone. Therefore, the synthesized xanthone analogue 13 might have therapeutic benefits for oral absorption of P-gp substrate anticancer drugs.

Novel non-substrate modulators of the transmembrane efflux pump P-glycoprotein (ABCB1)

Novel non-substrate modulators of the transmembrane efflux pump P-gp have been profiled as nontoxic and mdr reversing agents.

芳香族エナミドメタセシス反応の開発：生物活性多置換インドール化合物の設計·合成·機能評価

We have established aromatic enamide-ene metathesis to give highly substituted indole derivatives. This is a new method for the preparation of chiral 2-trans-cyclopropyl indoles, which successfully led to our H4 antagonist candidates. Based on this findings, we also designed and synthesized conformationally restricted analogues and regioisomers of the nonsteroidal anti-inflammatory drug indomethacin. Evaluation of the inhibitory effects of these compounds on COX, P-glycoprotein, and multidrug-resistance indicated that NSAIDS modulation of multidrug resistant P-glycoprotein and multidrug-resistant protein-1 is not associated with COX-1 and COX-2 inhibitory activities.

Deconstruction of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline moiety to separate P-glycoprotein (P-gp) activity from σ2 receptor affinity in mixed P-gp/σ2 receptor agents.

6,7-Dimethoxytetrahydroisoquinoline is widely used as basic moiety in σ2 receptor ligands, in order to provide σ2versus σ1 selectivity. This same moiety is also widely exploited in modulators of P-glycoprotein (P-gp) efflux pump, so that mixed σ2/P-gp agents are often obtained. Deconstruction of 6,7-dimethoxytetrahydroisoquinoline moiety present in the potent mixed σ2/P-gp agent 6,7-dimethoxy-2-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]butyl]-1,2,3,4-tetrahydroisoquinoline (1) could lead to the separation of σ2 affinity from P-gp activity. Therefore, phenethylamino-, benzylamino- and indanamine series were obtained. The NH group was also methylated in the N-phenethylamino series, and ethylated in the benzylamino series, to better match 6,7-dimethoxytetrahydroisoquinoline. The σ2 affinity drastically decreased with the increase of conformational freedom, whereas alkylation of the NH-group was beneficial for σ2 receptor interaction. By contrast, deconstruction of 6,7-dimethoxytetrahydroisoquinoline slightly reduced P-gp activity, with dimethoxy-substituted derivatives displaying potent P-gp interaction. Therefore, 'ring-opened' 6,7-dimethoxytetrahydroisoquinoline derivatives represent a promising strategy to obtain P-gp selective agents devoid of σ2 receptor affinity.

Diterpenic compounds with selective antiproliferative activity in multidrug resistant cancer cells

The failure of chemotherapeutics in cancer is mainly imputed to multidrug resistance (MDR). Among several proposed approaches to overcome MDR, the modulation of P-glycoprotein (Pgp) is the most highlighted [1]. Another valuable strategy is to take advantage of the collateral sensitivity effect, where some compounds may selectively exert a more pronounced cytotoxicity on cells with MDR phenotype than in the parental ones [1]. Macrocyclic diterpenes isolated from Euphorbia species, have been recognized as potent Pgp modulators and as having a collateral sensitivity effect on MDR cell variants [2]. Our main goal is to explore these two approaches, since tackling more than one MDR mechanism simultaneously might be a solution to control MDR in its complexity. The fractionation of the methanol extract of the aerial parts of E. piscatoria led to the isolation of a macrocyclic diterpene that allowed to build a small library of derivatives [3]. In this work, eleven compounds were evaluated for their potential collateral sensitivity effect on nine cell lines, gastric, pancreatic and colon cancer cell models (drug sensitive and drug resistant sublines), using the SRB assay. Most of the derivatives exhibited significant collateral sensitivity effect towards the MDR gastric cell line (with Pgp overexpression) and the MDR colon cells. The most significant results were obtained with a benzoyl derivative that demonstrated ability to target different cellular contexts with concomitant high antiproliferative activity. These compounds were previously assessed as Pgp modulators, at non-cytotoxic doses, on MDR1-mouse lymphoma cells [3]. A regression analysis between the antiproliferative activity presented herein and the previously assessed Pgp modulatory effect showed a strong relation between the compounds that presented both high Pgp modulation and cytotoxicity. Acknowledgements: FCT, Portugal (PTDC/QUI-QUI/099815/2008; PTDC/QEQ-MED/0905/2012; SFRH/BD/72915/2010).

From mixed sigma-2 receptor/P-glycoprotein targeting agents to selective P-glycoprotein modulators: small structural changes address the mechanism of interaction at the efflux pump.

Generations of modulators of the efflux pump P-glycoprotein (P-gp) have been produced as tools to counteract the Multidrug Resistance (MDR) phenomenon in tumor therapy, but clinical trials were not successful so far. With the aim of contributing to the development of novel P-gp modulators, we started from recently studied high-affinity sigma-2 (σ2) receptor ligands that showed also potent interaction with P-gp. For σ2 receptors high-affinity binding, a basic N-atom is a strict requirement. Therefore, we reduced the basic character of the N-atom present in these ligands, and we obtained potent P-gp modulators with poor or null σ2 receptor affinity. We also evaluated whether modulation of P-gp by these novel compounds involved consumption of ATP (as P-gp substrates do), as a source of energy to support the efflux. Surprisingly, even small structural changes resulted in opposite behavior, with amide 13 depleting ATP, in contrast to its isomer 18. Two compounds, 15 and 25, emerged for their potent activity at P-gp, and deserve further investigations as tools for P-gp modulation.

Improving the MDR reversal activity of 6,17-epoxylathyrane diterpenes

Aiming to optimize macrocyclic lathyrane-type diterpenes as effective Pgp modulators, the phytochemical study of the methanolic extract of Euphorbia boetica aerial parts was carried out. Two new macrocyclic 6,17-epoxylathyrane-type diterpenes, named epoxyboetiranes A (1) and B (2), along with three known analogues (3–5) were isolated. Epoxyboetirane A (1), a triacetate isolated in large amounts, was hydrolyzed to give epoxylathyrol (6). In order to study the effect of the substitution pattern of the macrocyclic scaffold on MDR reversal, 6 was acylated with aroyl, phenylacetyl, cinnamoyl and alkanoyl chlorides/anhydrides, yielding eight new esters, epoxyboetiranes C–J (7–14). The ability of compounds 1–14 as P-glycoprotein (Pgp, ABCB1) modulators was evaluated through combination of transport and chemosensitivity assays, using L5178Y mouse T lymphoma cell line transfected with the human MDR1 gene. In the transport assay, excepting 1, 3 and 6, the compounds, at non-cytotoxic concentrations, displayed strong MDR reversing activity in a dose-dependent mode, exhibiting all the new acyl derivatives (7–14) a many fold increase in the activity when compared with 1. Apart from 11 and 12, all compounds exhibited remarkable synergistic effects in combination with doxorubicin. An ATPase assay, using membrane vesicles from mammalian cells overexpressing Pgp, was also performed with two representatives of the modulators (4 and 5). The results suggest that both compounds compete with substrates for the Pgp drug-binding sites.