Inhibitor Verapamil Research Articles

Systematic Development and Verification of a Physiologically Based Pharmacokinetic Model of Rivaroxaban.

Rivaroxaban is indicated for stroke prevention in nonvalvular atrial fibrillation (AF). Its elimination is mediated by both hepatic metabolism and renal excretion. Consequently, its clearance is susceptible to both intrinsic (pathophysiological) and extrinsic (concomitant drugs) variabilities that in turn implicate bleeding risks. Upon systematic model verification, physiologically based pharmacokinetic (PBPK) models are qualified for the quantitative rationalization of complex drug-drug-disease interactions (DDDIs). Hence, this study aimed to develop and verify a PBPK model of rivaroxaban systematically. Key parameters required to define rivaroxaban's disposition were either obtained from in vivo data or generated via in vitro metabolism and transport kinetic assays. Our developed PBPK model successfully predicted rivaroxaban's clinical pharmacokinetic parameters within predefined success metrics. Consideration of basolateral organic anion transporter 3 (OAT3)-mediated proximal tubular uptake in tandem with apical P-glycoprotein (P-gp)-mediated efflux facilitated mechanistic characterization of the renal elimination of rivaroxaban in both healthy and renal impaired patients. Retrospective drug-drug interaction (DDI) simulations, incorporating in vitro metabolic inhibitory parameters, accurately recapitulated clinically observed attenuation of rivaroxaban's hepatic clearance due to enzyme-mediated DDIs with CYP3A4/2J2 inhibitors (verapamil and ketoconazole). Notably, transporter-mediated DDI simulations between rivaroxaban and the P-gp inhibitor ketoconazole yielded minimal increases in rivaroxaban's systemic exposure when P-gp-mediated efflux was solely inhibited, but were successfully characterized when concomitant basolateral uptake inhibition was incorporated in the simulation. In conclusion, our developed PBPK model of rivaroxaban is systematically verified for prospective interrogation and management of untested yet clinically relevant DDDIs pertinent to AF management using rivaroxaban. SIGNIFICANCE STATEMENT: Rivaroxaban is susceptible to DDDIs comprising renal impairment and P-gp and CYP3A4/2J2 inhibition. Here, systematic construction and verification of a PBPK model of rivaroxaban, with the inclusion of a mechanistic kidney component, provided insight into the previously arcane role of OAT3-mediated basolateral uptake in influencing both clinically observed renal elimination of rivaroxaban and differential extents of transporter-mediated DDIs. The verified model holds potential for investigating clinically relevant DDDIs involving rivaroxaban and designing dosing adjustments to optimize its pharmacotherapy in atrial fibrillation.

In silico and in vitro evaluation of tetrahydropyridine compounds as efflux inhibitors in Mycobacterium abscessus

Herein, we evaluated tetrahydropyridine (THP) compounds (NUNM) as antimicrobials and inhibitors of the efflux mechanism in M. abscessus. subsp. abscessus. The modulation factor (MF) of efflux inhibitors was calculated from the minimum inhibitory concentrations (MICs) of amikacin (AMI), ciprofloxacin (CIP) and clarithromycin (CLA) in the absence and presence of subinhibitory concentrations of the NUNM compounds and canonical inhibitors carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and verapamil (VP). The kinetics of the intracellular accumulation of the fluorimetric substrate ethidium bromide (EtBr) was evaluated and calculated by the relative final fluorescence (RFF). In addition, molecular modeling simulations for the MmpL5 and Tap efflux transporters with ligands (CLA, NUNM, CCCP, VP and EtBr) were performed to better understand the efflux mechanism. We highlight the NUNM01 compound because it reduced the MICs of AMI, CIP and CLA by 4-, 4- and 16-fold, respectively, had the highest effect on EtBr accumulation (RFF = 3.1) and showed a significant in silico affinity for the evaluated proteins in docking simulations. Based on the analyses performed in vitro and in silico, we propose that NUNM01 is a potential pharmacophore candidate for the development of a therapeutic adjuvant for M. abscessus infections.

Abstract 1254: Predictive models for tumor cell targeting with plinabulin, derived from in vitro screening and Affymetrix mRNA expression data

Abstract Tubulin binding drugs are approved for the treatment of many cancer types, without the use of molecular markers to select patients likely to respond. Plinabulin binds β-tubulin in a differentiated pocket and is being tested in a Phase 3 clinical study for the treatment of NSCLC. Additional indications are being considered for plinabulin and an algorithm for selecting especially responsive cancers and patient subgroups would be of significant value. With this in mind, Affymetrix HG-U133 Plus 2.0 array mRNA expression data for 43 human breast, lung, prostate, ovarian or CNS cancer cell lines were utilized to develop mathematical models to predict in vitro plinabulin potency against the same cell lines. Cells were treated for only 24 hours with plinabulin and then cultured for another 48 hours without plinabulin. Viable cell number was then measured with a Cell Titer-Blue Assay, and the plinabulin concentration causing a 70% reduction in viable cells (IC70) versus vehicle treated controls was derived. Cell lines were clearly separable into plinabulin Active (21 cell lines with IC70<1.0 μM) and Inactive (IC70>9.5 μM) groups. Log2 transformed Affymetrix gene probeset signal values, preprocessed with the GeneChip robust multi-array average analysis algorithm, were selected and ranked as predictors of plinabulin activity with a bootstrap forest partitioning technique, utilizing JMP 14.1 statistical software. 56 HIT probesets were identified that also had significantly different expression in responding versus non-responding cell lines (p<0.01, uncorrected t-test). For probesets with gene annotation, only the probeset for each gene with the highest Jetset score was utilized (Li et al., 2011). Top HIT predictor genes include CTNNB1 (β-catenin; oncogene), CALD1 (caldesmon; inhibits myosin ATPase activity), ERI1 (RNA processing), LGR5 (adult stem cell biomarker), SECISBP2L (SLAN; prolongs mitosis), and TRAK1 (mitochondrial/endosome trafficking). Models were constructed from HIT gene probesets in JMP to identify plinabulin responding cell lines, utilizing either one-layer TanH multimode fit neural networks or binary logistic regression. Surprisingly, models incorporating approximately 4-10 probeset values were derived that perfectly predicted plinabulin activity. Importantly, the cell lines tested in the above analyses were not those known to express high levels of multi-drug resistant (MDR) transporters. The importance of MDR status was therefore evaluated separately. Plinabulin activity, unlike that of taxanes, was not significantly affected by the MDR transporter inhibitor verapamil (10 μM), in ovarian cancer cell lines with a known MDR phenotype. To conclude, our work provides novel algorithms that may be of value in selecting cancer patients with tumor cells that are particularly susceptible to the direct cytotoxic effects of plinabulin. Citation Format: James R. Tonra, Hagen Klett, Chenghao Shen, Gerhard Kelter, Ramon W. Mohanlal, G. Kenneth Lloyd, Lan Huang. Predictive models for tumor cell targeting with plinabulin, derived from in vitro screening and Affymetrix mRNA expression data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1254.

Endogenous, cholesterol-activated ATP-dependent transport in membrane vesicles from Spodoptera frugiperda cells

Transport proteins of the ATP-binding cassette (ABC) family are found in all kingdoms of life. In humans, several ABC efflux transporters play a role in drug disposition and excretion. Therefore, in vitro methods have been developed to characterize the substrate and inhibitor properties of drugs with respect to these transporters. In the vesicular transport assay, transport is studied using inverted membrane vesicles produced from transporter overexpressing cell lines of both mammalian and insect origin. Insect cell expression systems benefit from a higher expression compared to background, but are not as well characterized as their mammalian counterparts regarding endogenous transport. Therefore, the contribution of this transport in the assay might be underappreciated. In this study, endogenous transport in membrane vesicles from Spodoptera frugiperda -derived Sf9 cells was characterized using four typical substrates of human ABC transporters: 5(6)-carboxy-2,′7′-dichlorofluorescein (CDCF), estradiol-17β-glucuronide, estrone sulfate and N-methyl-quinidine. Significant ATP-dependent transport was observed for three of the substrates with cholesterol-loading of the vesicles, which is sometimes used to improve the activity of human transporters expressed in Sf9 cells. The highest effect of cholesterol was on CDCF transport, and this transport in the cholesterol-loaded Sf9 vesicles was time and concentration dependent with a Km of 8.06 ± 1.11 μM. The observed CDCF transport was inhibited by known inhibitors of human ABCC transporters, but not by ABCB1 and ABCG2 inhibitors verapamil and Ko143, respectively. Two candidate genes for ABCC-type transporters in the S. frugiperda genome (SfABCC2 and SfABCC3) were identified based on sequence analysis as a hypothesis to explain the observed endogenous ABCC-type transport in Sf9 vesicles. Although further studies are needed to verify the role of SfABCC2 and SfABCC3 in Sf9 vesicles, the findings of this study highlight the need to carefully characterize background transport in Sf9 derived membrane vesicles to avoid false positive substrate findings for human ABC transporters studied with this overexpression system.

Regulation mechanism of ρ-Hydroxybenzoic acid on grapevine root secretion characteristics

ρ-Hydroxybenzoic acid (4-HBA) is a major autotoxin that can regulate grapevine secretion characteristics and cause replant disease. In our study, we aimed to investigate the involvement of various signal transduction pathways (calcium, Ca2+ and reactive oxygen species) in 4-HBA-induced grapevine root secretions. The roots of grapevine seedlings were treated with specific calcium channel inhibitor (Verapamil, Vp), and inhibitors of superoxide dismutase (SOD) (diethyldithiocarbamate, DETC) and NADPH oxidase (diphenylene iodonium, DPI). The distribution of Ca2+ in the root tip tissues was observed, and the phenylalanine ammonia-lyase (PAL) activity and phenolic acid content (ρ-Hydroxybenzoic acid and salicylic acid) in the secretions were measured. The results showed that 1000 μM of 4-HBA had a significant allelopathic effect on seedling growth, leading to Ca2+ flow from the cell wall to the cytoplasm, and that Vp hindered this process. The treatments with Vp and 4-HBA or DETC and 4-HBA decreased the PAL activity of roots and 4-HBA content in root exudates compared to the 4-HBA treatment. The salicylic acid (SA) content in root exudates decreased after co-treating with Vp and 4-HBA, DPI and 4-HBA. This result indicates that Ca2+ and SOD play major roles in 4-HBA induced root secretions.

Scrutinizing the drug resistance mechanism of multi- and extensively-drug resistant Mycobacterium tuberculosis: mutations versus efflux pumps

BackgroundIn order to shorten the course of treatment and its effectiveness, it is essential to gain an in-depth insight into the drug resistance mechanisms of Mycobacterium tuberculosis (M. tuberculosis).MethodsIn this study, we evaluated the contribution of 26 drug efflux pumps plus target gene mutations to the drug resistance levels in multi-drug resistant (MDR)/pre-extensively drug-resistant (pre-XDR)/extensively drug-resistant (XDR) and mono-drug resistant clinical isolates of M. tuberculosis. The panels of 25 M. tuberculosis clinical strains were characterized for drug resistance-associated mutations with whole-genome sequencing and antibiotic profiles in the presence and absence of efflux inhibitor verapamil (VP).ResultsDifferent MICs were observed for the same target gene mutations. Out of the 16 MDR/pre-XDR/XDR isolates, 6 (37.5%) and 3 (18.8%) isolates demonstrated a significant decrease in rifampicin (RIF) MIC and isoniazid (INH) MIC due to the VP exposure (64 μg/mL), respectively. Susceptibility to RIF was fully restored in two isolates after VP exposure. Moreover, the efflux pump genes of Rv2938, Rv2936, Rv1145, Rv1146, Rv933, Rv1250, Rv876, Rv2333, Rv2459, Rv849, and Rv1819 were overexpressed in the presence of anti-TB drugs, showing the contribution of these efflux pumps to the overall resistance phenotype.ConclusionsOur results clearly showed that efflux systems, besides spontaneous mutations, play a role in the development of INH/RIF resistance. In addition, although VP was effective in reducing the expression of some efflux pumps, it was not very successful at the phenotypic level.

In Vitro and In Situ Characterization of the Intestinal Absorption of Capilliposide B and Capilliposide C from Lysimachia capillipes Hemsl.

The goal of this investigation was to determine the processes and mechanism of intestinal absorption for capilliposide B (CAPB) and capilliposide C (CAPC) from the Chinese herb, Lysimachia capillipes Hemsl. An analysis of basic parameters, such as drug concentrations, time, and behavior in different intestinal segments was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS). The susceptibility of CAPB and CAPC to various inhibitors such as P-glycoprotein (P-gp) inhibitor (verapamil); multidrug resistance-associated protein 2 (MRP2) inhibitor (indomethacin); cytochrome P450 protein 3A4 (CYP3A4) inhibitor (ketoconazole); and the co-inhibitor of P-gp, MRP2 and CYP3A4 (cyclosporine A) were assessed using both caco-2 cell monolayer and single-pass intestinal perfusion (SPIP) models. As a result, CAPB and CAPC are both poorly absorbed in the intestines and exhibited segment-dependent permeability. The intestinal permeability of CAPB and CAPC were significantly increased by the co-treatment of verapamil, indomethacin. In addition, the intestinal permeability of CAPB was also enhanced by ketoconazole and cyclosporine A. It can be concluded that the intestinal absorption mechanisms of CAPB and CAPC involve processes such as facilitated passive diffusion, efflux transporters, and enzyme-mediated metabolism. Both CAPB and CAPC are suggested to be substrates of P-gp and MRP2. However, CAPB may interact with the CYP3A4 system.

Drug Transport across Porcine Intestine Using an Ussing Chamber System: Regional Differences and the Effect of P-Glycoprotein and CYP3A4 Activity on Drug Absorption.

Drug absorption across viable porcine intestines was investigated using an Ussing chamber system. The apparent permeability coefficients, Papp,pig, were compared to the permeability coefficients determined in humans in vivo, Peff,human. Eleven drugs from the different Biopharmaceutical Classification System (BCS) categories absorbed by passive diffusion with published Peff,human values were used to test the system. The initial experiments measured Papp,pig for each drug after application in a Krebs–Bicarbonate Ringer (KBR) buffer and in biorelevant media FaSSIF V2 and FeSSIF V2, mimicking fasted and fed states. Strong sigmoidal correlations were observed between Peff,human and Papp,pig. Differences in the segmental Papp,pig of antipyrine, cimetidine and metoprolol confirmed the discrimination between drug uptake in the duodenum, jejunum and ileum (and colon); the results were in good agreement with human data in vivo. The presence of the P-gp inhibitor verapamil significantly increased Papp,pig across the ileum of the P-gp substrates cimetidine and ranitidine (p < 0.05). Clotrimazole, a potent CYP3A4 inhibitor, significantly increased Papp,pig of the CYP3A4 substrates midazolam, verapamil and tamoxifen and significantly decreased the formation of their main metabolites. In conclusion, the results showed that this is a robust technique to predict passive drug permeability under fasted and fed states, to identify regional differences in drug permeability and to demonstrate the activity of P-gp and CYP3A4.

MDR1 inhibition increases sensitivity to doxorubicin and etoposide in adrenocortical cancer.

Chemotherapy for adrenocortical carcinoma (ACC) has limited efficacy and is accompanied by severe toxicity. This lack of effectiveness has been associated with high tumoral levels of the multidrug resistance (MDR) pump P-glycoprotein (P-gp), encoded by the MDR1 gene. In this study, effects of P-gp inhibition on the sensitivity of ACC cells to cytotoxic drugs were evaluated. MDR1 mRNA and P-gp expression were determined in human adrenal tissues and cell lines. H295R, HAC15 and SW13 cells were treated with mitotane, doxorubicin, etoposide, cisplatin and streptozotocin, with or without the P-gp inhibitors verapamil and tariquidar. Cell growth and surviving fraction of colonies were assessed. MDR1 mRNA and P-gp protein expression were lower in ACCs than in adrenocortical adenomas (P < 0.0001; P < 0.01, respectively). MDR1 and P-gp expression were positively correlated in ACC (P < 0.0001, ρ = 0.723). Mitotane, doxorubicin, cisplatin and etoposide dose dependently inhibited cell growth in H295R, HAC15 and SW13. Tariquidar, and in H295R also verapamil, increased the response of HAC15 and H295R to doxorubicin (6.3- and 7.5-fold EC50 decrease in H295R, respectively; all P < 0.0001). Sensitivity to etoposide was increased in H295R and HAC15 by verapamil and tariquidar (all P < 0.0001). Findings were confirmed when assessing colony formation. We show that cytotoxic drugs, except streptozotocin, used for ACC treatment, inhibit ACC cell growth and colony formation at clinically achievable concentrations. P-gp inhibition increases sensitivity to doxorubicin and etoposide, suggesting that MDR1 is involved in sensitivity to these drugs and could be a potential target for cytotoxic treatment improvement in ACC.

Acid diterpenes from Copaiba oleoresin (Copaifera langsdorffii): Chemical and plasma stability and intestinal permeability using Caco-2 cells

Ethnopharmacological relevanceCopaiba oleoresin has been used in folk medicine in the treatment of bronchitis, syphilis, skin diseases and ulcers due to its anti-inflammatory and antiseptic activities, but there is no information about major compounds oral absorption to support the traditional use. Aim of studyConsidering the potential of copalic (CA) and kaurenoic acid (KA) – major biological activity (in vitro) diterpenes found in the oleoresin, this study aimed to evaluate the intestinal permeability of CA and KA using Caco-2 cells model as predictive test for oral drug absorption. Materials and methodsChemical stability at pH 1.2 and 7.4 and plasma stability were evaluated to mimic physiological conditions of the gastrointestinal tract. The intestinal permeability of CA and KA was evaluated in Caco-2 cells in the presence and absence of the P-glycoprotein inhibitor verapamil. ResultsCA and KA were rapidly degraded at pH 1.2 (0.2 M Clark-Lubs buffer). At pH 7.4 (0.1 M phosphate buffer), CA was stable for up to 24 h and KA for up to 6 h. In human plasma, CA and KA can be considered stable for 24 h and 12 h at 37 °C, respectively. Caco-2 cells were considered viable when incubated with CA or KA in the range of 3.9–250 μM for 24 h. CA and KA exhibited moderate apparent permeability (Papp) of 4.67 (±0.08) × 10−6 cm/s and 4.66 (±0.04) × 10−6 cm/s, respectively. Simultaneous incubation with verapamil showed that P-glycoprotein does not play a relevant role on CA and KA oral absorption, with Papp of 4.48 (±0.26) × 10−6 cm/s and 5.37 (±0.72) × 10−6 cm/s observed for CA and KA, respectively. ConclusionThe oral absorption of both CA and KA is driven by mainly passive permeability, is not limited by p-glycoprotein, but enteric-coated dosage forms should be used to avoid chemical instability in the gastric pH.

Assessing Potential Drug-Drug Interactions Between Dabigatran Etexilate and a P-Glycoprotein Inhibitor in Renal Impairment Populations Using Physiologically Based Pharmacokinetic Modeling.

Plasma concentrations of dabigatran, an active principle of prodrug dabigatran etexilate (DABE), are increased by renal impairment (RI) or coadministration of a P‐glycoprotein inhibitor. Because the combined effects of drug–drug interactions and RI have not been evaluated by means of clinical studies, the decision of DABE dosing for RI patients receiving P‐glycoprotein inhibitors is empirical at its best. We conducted virtual drug–drug interactions studies between DABE and the P‐glycoprotein inhibitor verapamil in RI populations using physiologically based pharmacokinetic modeling. The developed physiologically based pharmacokinetic model for DABE and dabigatran was used to predict trough dabigatran concentrations in the presence and absence of verapamil in virtual RI populations. The population‐based physiologically based pharmacokinetic model provided the most appropriate dosing regimen of DABE for likely clinical scenarios, such as drug–drug interactions in this RI population based on available knowledge of the systems changes and in the absence of actual clinical studies.

Telemetered common marmosets is useful for the assessment of electrocardiogram parameters changes induced by multiple cardiac ion channel inhibitors.

The objective of this study is to assess the response of telemetered common marmosets to multiple cardiac ion channel inhibitors and to clarify the usefulness of this animal model in evaluating the effects of drug candidates on electrocardiogram (ECG). Six multiple cardiac ion channel inhibitors (sotalol, astemizole, flecainide, quinidine, verapamil and terfenadine) were orally administered to telemetered common marmosets and changes in QTc, PR interval and QRS duration were evaluated. Drugs plasma levels were determined to compare the sensitivity in common marmosets to that in humans. QTc prolongation was observed in the marmosets dosed with sotalol, astemizole, flecainide, quinidine, verapamil and terfenadine. PR prolongation was noted after flecainide and verapamil administration, and QRS widening occurred following treatment with flecainide and quinidine. Drugs plasma levels associated with ECG changes in marmosets were similar to those in humans, except for verapamil-induced QTc prolongation. Verapamil-induced change is suggested due to body temperature decrease. These results indicate that telemetered common marmoset is a useful animal for evaluation of the ECG effects of multiple cardiac ion channel inhibitors and the influence of body temperature change should be considered in the assessment.

Regulation of ABCB1 activity by microRNA-200c and microRNA-203a in breast cancer cells: the quest for microRNAs' involvement in cancer drug resistance.

Aim: ABCB1 is a major player in cancer drug resistance. The purpose of this study was to functionally assess the regulation of ABCB1 activity in a doxorubicin-resistant breast cancer cell line by miR-200c and miR-203.Methods: Human breast carcinoma cell lines MCF-7 (Doxorubicin-sensitive and not expressing ABCB1) and KCR (Doxorubicin-resistant and expressing ABCB1) were used to evaluate the expression levels of miR-200c and miR-203 by Real-time quantitative PCR (RT-qPCR). The effects of transient ectopic expression of miRNA-200c and miR-203 on the expression of ABCB1 in KCR and MCF-7 cells was verified by RT-qPCR and Western Blot. The extrusion activity of the ABCB1 pump was analyzed by fluorescence microscopy and flow cytometry through fluorescence substrate retention assays (DiOC2) in the presence and absence of the ABCB1 inhibitor verapamil.Results: RT-qPCR results indicated a 100,000-fold increase in ABCB1 mRNA expression levels in KCR cells compared to MCF-7 cells, and is inversely correlated with the expression of miR-203 and miR-200c. The insertion of miR-200c and miR-203 led to a higher retention of DiOC2 within KCR cells, and slightly reduced the protein levels of ABCB1 in KCR cells, although the high initial expression of ABCB1 masked the reduction in protein levels. The increased intracellular accumulation of the fluorescent due DiOC2 in the presence of the ABCB1 inhibitor verapamil correlated with the inhibition caused by miR-203 and miR-200c in transfected cells.Conclusion: The present study demonstrates that miR-200c and miR-203 exert a negative modulating effect on the activity of ABCB1 associated with doxorubicin resistance.

Reversal of multidrug resistance in breast cancer cells by a combination of ursolic acid with doxorubicin

Multidrug resistance (MDR) has seriously affected or hindered the effect of chemotherapy. Ursolic acid (UA) as a natural compound exhibits a number of potential biological effects including antitumor. Searching for the reversal agents from the natural products has been an effective strategy recently applied in overcoming the MDR. So in this study, the reversal effect of UA on the MDR and involved mechanisms were investigated via a multidrug-resistant MCF-7/ADR cells model and ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analytical methods. The synergistic effects were yielded by the combination of UA and Dox based on the investigation of the intracellular accumulation, the P-glycoprotein (P-gp) mediated transport, the energy metabolism including glycolysis, tricarboxylic acid (TCA) cycle, and glutamine metabolism as well as related amino acid metabolism. Obtained results showed that the UA could increase amount of doxorubicin (Dox) entering the cell to accumulate in nuclei, decrease the efflux ratio of digoxin comparable to the effects of the known inhibitor verapamil by acting as a P-gp substrate, decrease the content of intracellular alanine, lactate, pyruvate, glucose, α-ketoglutarate, glutamate, glutamine, aspartate, serine, and glycine. Taken together, inhibition of P-gp function and disruption of the metabolism of energy and related amino acids could be the key mechanisms by which UA could reverse the MDR. The findings also indicated that UA could be a potential alternative adjuvant antitumour herbal medicine to resensitize cells with MDR to chemotherapeutic agents.

NOS1 upregulates ABCG2 expression contributing to DDP chemoresistance in ovarian cancer cells.

Nitric oxide synthase 1 (NOS1) has been reported to promote various cancer processes including chemoresistance. However, the role of NOS1 in chemoresistance has remained unclear. ATP-binding cassette, subfamily G, member 2 (ABCG2) has been identified as a molecular cause of multidrug resistance in a number of cancer types, including ovarian cancer. The present study observed that in ovarian cancer cells, the expression of ABCG2 was significantly upregulated in response to cis-diamminedichloroplatinum (cisplatin/DDP) treatment, in addition the expression of NOS1 exhibited an increasing trend. Additionally, the levels of NOS1 and ABCG2 in chemoresistant ovarian cancer profiles in Gene Expression Omnibus datasets (GSE26712 and GSE51373) were higher than in chemosensitive profiles. Furthermore, overexpression of NOS1 could upregulate ABCG2 expression, and expression of ABCG2 was inhibited by NOS1 selective inhibitor (N-PLA). In assays of cell survival, NOS1 appeared to increase the potential for DDP resistance, and this effect was reversed by addition of ABCG2 inhibitor (verapamil). The present study indicated that NOS1-induced chemoresistance was partly mediated by the upregulation of ABCG2 expression. This result suggests a link between the expression of NOS1 and the ABCG2-associated chemoresistance in ovarian cancer.

Benzoxazole-based Zn(II) and Cu(II) Complexes Overcome Multidrug-resistance in Cancer.

Multidrug resistance (MDR) represents a significant impediment to successful cancer treatment. In this study, novel metal [Zn(II), Cu(II), Mg(II), Ni(II), Pd(II), and Ag(I)] complexes of 2-trifluoroacetonylbenzoxazole previously synthesized and characterized by our group were tested for their MDR-reversing activity in comparison with the free ligands in L5178Y mouse T-lymphoma (MDR) cells transfected with human ATP-binding cassette sub-family B member 1 (ABCB1; P-glycoprotein) gene. Cytotoxic and antiproliferative effects of the complexes were assessed by the thiazolyl blue tetrazolium bromide (MTT) method. Modulation of ABCB1 activity was measured by rhodamine 123 accumulation assay using flow cytometry. The apoptosis-inducing activity of some complexes was also tested on the multidrug resistant L5178Y mouse T-lymphoma cells, using the annexin-V/propidium iodide assay. When compared to the free ligand, a remarkable enhancement in MDR reversal and cytotoxic activity was found for the Zn(II) and Cu(II) complexes. The activity of the complexes proved to be up to 29- and 5-fold higher than that of the ligands and the ABCB1 inhibitor verapamil as positive control, respectively. The complexes possessed a remarkable potential to induce apoptosis of MDR cells. Our results suggest that the Zn(II) and Cu(II) complexes display significant MDR-reversing activity in a dose-dependent manner and possess strong cytotoxic activity and a remarkable potential to induce apoptosis in MDR L5178Y mouse T-lymphoma cells.

In Vitro Assessment of Potential for CYP-Inhibition-Based Drug-Drug Interaction Between Vonoprazan and Clopidogrel.

It was recently proposed that CYP-mediated drug-drug interactions (DDIs) of vonoprazan with clopidogrel and prasugrel can attenuate the antiplatelet actions of the latter two drugs. Clopidogrel is metabolized to the pharmacologically active metabolite H4 and its isomers by multiple CYPs, including CYP2C19 and CYP3A4. Therefore, to investigate the possibility of CYP-based DDIs, in vitro metabolic inhibition studies using CYP probe substrates or radiolabeled clopidogrel and human liver microsomes (HLMs) were conducted in this work. Reversible inhibition studies focusing on the effects of vonoprazan on CYP marker activities and the formation of the [14C]clopidogrel metabolite H4 were conducted with and without pre-incubation using HLMs. Time-dependent inhibition (TDI) kinetics were also measured. It was found that vonoprazan is not a significant direct inhibitor of any CYP isoforms (IC50 ≥ 16μM), but shows the potential for TDI of CYP2B6, CYP2C19, and CYP3A4/5. This TDI was weaker than the inhibition induced by the corresponding reference inhibitors ticlopidine, esomeprazole, and verapamil, based on the measured potencies (kinact/KI ratio and the R2 value). In a more direct in vitro experiment, vonoprazan levels of up to 10µM (a 100-fold higher concentration than the plasma Cmax of 75.9nM after taking 20mg once daily for 7days) did not suppress the formation of the active metabolite H4 or other oxidative metabolites of [14C]clopidogrel in a reversible or time-dependent manner. Additionally, an assessment of clinical trials and post-marketing data suggested no evidence of a DDI between vonoprazan and clopidogrel. The body of evidence shows that the pharmacodynamic DDI reported between vonoprazan and clopidogrel is unlikely to be caused by the inhibition of CYP2B6, CYP2C19, or CYP3A4/5 by vonoprazan.

Neonicotinoid insecticides are potential substrates of the multixenobiotic resistance (MXR) mechanism in the non-target invertebrate, Dreissena sp.

Mussels are among the most frequently used invertebrate animals in aquatic toxicology to detect toxic exposure in the environment. The presence and activity of a cellular defence system, the multixenobiotic resistance (MXR) mechanism, was also established in these organisms.In isolated gill tissues of dreissenid mussels (D. bugensis) the MXR activity was assayed after treatment by commercially available insecticides (formulated products) which contain neonicotinoids as their active ingredients: Actara (thiamethoxam), Apacs (clothianidin), Calypso (thiacloprid) and Kohinor (imidacloprid), respectively. While applying the accumulation assay method, 0.5 μM rhodamine B was used as model substrate and 20 μM verapamil as model inhibitor of the MXR mechanism.In acute (in vitro) experiments when isolated gills were co-incubated in graded concentrations of insecticides and rhodamine B simultaneously, Calypso and Kohinor treatment resulted increasing rhodamine accumulation. Chemical analysis of gills in vitro incubated in insecticides demonstrated higher tissue concentrations of thiamethoxam, clothianidin and thiacloprid in the presence of verapamil suggesting that the active ingredients of Actara, Apacs and Calypso are potential substrates of the MXR mediated cellular efflux. In contrast, verapamil did significantly alter the accumulated imidacloprid concentrations in gills, suggesting that the active component of Kohinor is not transported by the MXR mechanism.Chronic (in vivo) exposures of the intact animals in lower, 1, 10 mg/L concentration of neonicotinoid products, resulted in a decreased level of both rhodamine accumulation and verapamil inhibition by the 12th–14th days of treatment. These results suggest an enhancement of MXR activity (chemostimulation), building up gradually in the animals exposed to Actara, Apacs and Kohinor, respectively.Neonicotinoid-type insecticides are generally considered as selective neurotoxins for insects, targeting the nicotinic type acetylcholine receptors (nAChRs) in their central nervous system. Our present results provide the first evidences that neonicotinoid insecticides are also able to alter the transmembrane transport mechanisms related to the MXR system.

P-gp Inhibition by the Anti-psychotic Drug Pimozide Increases Apoptosis, as well as Expression of pRb and pH2AX in Highly Drug-resistant KBV20C Cells.

The present study was designed to identify drugs that could sensitize P-glycoprotein (P-gp)-overexpressing resistant KBV20C cancer cells to treatment with Halaven (HAL) or vincristine (VIC). Using relatively low doses or IC50 concentrations of drugs to sensitize anti-mitotic drug-resistant KBV20C cells, pimozide (PIM) sensitized HAL-resistant KBV20C cancer cells, with higher P-gp inhibitory activity than another anti-psychotic drug, fluphenazine (FLU). The first-generation P-gp inhibitor verapamil required a dose that was similar to that of PIM for P-gp inhibition, suggesting that PIM has a similar specificity for binding P-gp to prevent efflux of anti-mitotic drugs. Furthermore, co-treatment with PIM and another anti-mitotic drug, VIC, also increased sensitization of KBV20C cells, suggesting that PIM can be combined with other anti-mitotic drugs to sensitize resistant cancer cells. PIM caused a reduction in cell viability and an increase in the number of cells arresting at the G2 phase of the cell cycle. PIM induced both early and late apoptosis in KBV20C cells in response to HAL treatment. Furthermore, the DNA damage marker pH2AX, the cell-cycle protein pRb, and the pro-apoptotic protein C-PARP levels increased after HAL-PIM co-treatment, indicative of a mechanism involving G2 phase arrest and an increase in the number of cells undergoing apoptosis. PIM may be a promising therapeutic agent for the treatment of cancers that are resistant to anti-mitotic drugs.

Assessment of HBEC-5i endothelial cell line cultivated in astrocyte conditioned medium as a human blood-brain barrier model for ABC drug transport studies

Endothelial cells are main components of the Blood-Brain Barrier (BBB) and form a tight monolayer that regulates the passage of molecules, with the ATP-Binding Cassette (ABC) transporters efflux pumps. We have developed a human in vitro model of HBEC-5i endothelial cells cultivated alone or with human astrocytes conditioned medium on insert. HBEC-5i cells showed a tight monolayer within 14 days, expressing ZO-1 and claudin 5, a low apparent permeability to small molecules, with a TEER stability during five days. The P-gp, BCRP, MRPs transporters were well expressed and functional. Accumulation and efflux ratio measurement with different ABC transporters substrates (Rhodamine 123, BCECF AM, Hoechst 33342) and inhibitors (verapamil, Ko143, probenecid and cyclosporin A) were conducted. At barrier level, the functionality of ABC transporters was three-fold enhanced in astrocyte conditioned medium. We validated our model by the transport of pharmacological substrates: caffeine, rivaroxaban, and methotrexate. The rivaroxaban and methotrexate were released with an efflux ratio >3 and were decreased by more than half with inhibitors. HBEC-5i model could be used as relevant tool in preclinical studies for assessing the permeability of therapeutic molecules to cross human BBB.