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This study investigated the ability of natural nanotubular clay mineral (Halloysite) and cellulose ether based biocomposite matrix as a controlled release agent for Verapamil HCl (BCS Class-I). Drug-loaded halloysite was prepared and tablet formulations were designed by varying amount of hydroxy propyl methyl cellulose (HPMC K4M). Physical characterization was carried out using SEM, FTIR, and DSC. Tabletability profiles were evaluated using USP1062 guidelines. Drug release kinetics were studied, and physiologically based pharmacokinetic (PBPK) modeling was performed. Compressed tablets possess satisfactory yield pressure of 625 MPa with adequate hardness and disintegration within 30 min. The initial release of the drug was due to surface drug on tablets, while the prolonged release at later time points (around 80 % drug release at 12 h) were due to halloysite loading. The FTIR spectra exhibited electrostatic attraction between the positively charged drug and the negatively charged Si-O-Si functional group of halloysite, while the thermogram showed Verapamil HCl melting point at ~146 °C with enthalpy change of -126.82 J/g. PBPK modeling exhibited PK parameters of optimized matrix formulation (VER-HNT 3% ) comparable to in vivo data. The study effectively demonstrated the potential of prepared biocomposite matrix as a commercially viable oral release modifying agent for highly soluble drugs., Competing Interests: Declaration of competing interest There is no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The purpose of this investigation was to formulate and evaluate the interaction between cation exchange resins and verapamil hydrochloride. The uptake studies were conducted using the rotating bottle apparatus. The Langmuir-like equation was applied to the experimental data and the maximum drug loading was determined from the Langmuir-like parameters. The drug-resin complexes were evaluated using XRD, SEM, and particle size analysis. Release studies were performed using USP dissolution apparatus 2. The resin with the lowest percentage of cross-linking had the highest uptake capacity. The percent increase in particle size due to complexation was found to be associated with drug loading; the highest drug loading had the highest increase in particle size. The X-ray diffraction patterns of the resins and the drug-resin complexes showed that they were both amorphous. The maximum drug release was approximately 40% when conventional dissolution testing was used. Results showed that sink conditions could not be maintained using conventional dissolution methods. Maximum drug release increased dramatically by increasing the volume of samples withdrawn and fresh dissolution medium added. Excellent correlation was obtained between sample volume and drug release rate with an R-value of 0.988. Particle diffusion-controlled model and film diffusion-controlled model were both applied to the experimental data. The results indicated that the rate-limiting step is the diffusion of the exchanging cations through the liquid film. The modified release formulation was prepared successfully and correlated very well with the USP monograph for verapamil hydrochloride extended release capsules., (© 2023. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Overexpression of drug efflux transporters is commonly associated with multidrug-resistance in cancer therapy. Here for the first time, we investigated the ability of diindolylmethane (DIM), a dietary bioactive rich in cruciferous vegetables, in enhancing the efficacy of Centchroman (CC) by modulating the drug efflux transporters in human breast cancer cells. CC is a selective estrogen receptor modulator, having promising therapeutic efficacy against breast cancer. The combination of DIM and CC synergistically inhibited cell proliferation and induced apoptosis in breast cancer cells. This novel combination has also hindered the stemness of human breast cancer cells. Molecular docking analysis revealed that DIM had shown a strong binding affinity with the substrate-binding sites of ABCB1 (P-gp) and ABCC1 (MRP1) drug-efflux transporters. DIM has increased the intracellular accumulation of Hoechst and Calcein, the substrates of P-gp and MRP1, respectively, in breast cancer cells. Further, DIM stimulates P-gp ATPase activity, which indicates that DIM binds at the substrate-binding domain of P-gp, and thereby inhibits its efflux activity. Intriguingly, DIM enhanced the intracellular concentration of CC by inhibiting the P-gp and MRP1 expression as well as activity. The intracellular retaining of CC has increased its efficacy against breast cancer. Overall, DIM, a dietary bioactive, enhances the anticancer efficiency of CC through modulation of drug efflux ABC-transporters in breast cancer cells. Therefore, DIM-based nutraceuticals and functional foods can be developed as adjuvant therapy against human breast cancer., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest, (Copyright © 2021. Published by Elsevier Inc.)

A novel, fast and sensitive enantioselective HPLC assay with a new core-shell isopropyl carbamate cyclofructan 6 (superficially porous particle, SPP) chiral column (LarihcShell-P, LSP) was developed and validated for the enantiomeric separation and quantification of verapamil (VER) in rat plasma. The polar organic mobile phase composed of acetonitrile/methanol/trifluoroacetic acid/triethylamine (98:2:0.05: 0.025, v/v/v/v ) and a flow rate of 0.5 mL/min was applied. Fluorescence detection set at excitation/emission wavelengths 280/313 nm was used and the whole analysis process was within 3.5 min, which is 10-fold lower than the previous reported HPLC methods in the literature. Propranolol was selected as the internal standard. The S-(-)- and R-(+)-VER enantiomers with the IS were extracted from rat plasma by utilizing Waters Oasis HLB C18 solid phase extraction cartridges without interference from endogenous compounds. The developed assay was validated following the US-FDA guidelines over the concentration range of 1-450 ng/mL (r 2 ≥ 0.997) for each enantiomer (plasma) and the lower limit of quantification was 1 ng/mL for both isomers. The intra- and inter-day precisions were not more than 11.6% and the recoveries of S-(-)- and R-(+)-VER at all quality control levels ranged from 92.3% to 98.2%. The developed approach was successfully applied to the stereoselective pharmacokinetic study of VER enantiomers after oral administration of 10 mg/kg racemic VER to Wistar rats. It was found that S-(-)-VER established higher C max and area under the concentration-time curve (AUC) values than the R-(+)-enantiomer. The newly developed approach is the first chiral HPLC for the enantiomeric separation and quantification of verapamil utilizing a core-shell isopropyl carbamate cyclofructan 6 chiral column in rat plasma within 3.5 min after solid phase extraction (SPE).

Radioresistance significantly decreases the efficacy of radiotherapy, which can ultimately lead to tumor recurrence and metastasis. As a novel type of nano-radiosensitizer, silver nanoparticles (AgNPs) have shown promising radiosensitizing properties in the radiotherapy of glioma, but their ability to efficiently enter and accumulate in tumor cells needs to be improved. In the current study, AS1411 and verapamil (VRP) conjugated bovine serum albumin (BSA) coated AgNPs (AgNPs@BSA-AS-VRP) were synthesized and characterized. Dark-field imaging and inductively coupled plasma mass spectrometry were applied to investigate the accumulation of AgNPs@BSA-AS and AgNPs@BSA-AS-VRP mixed in different ratios in U251 glioma cells. To assess the influences of 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP on the P-glycoprotein (P-gp) efflux activity, rhodamine 123 accumulation assay was carried out. Colony formation assay and tumor-bearing nude mice model were employed to examine the radiosensitizing potential of 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP. Thioredoxin Reductase (TrxR) Assay Kit was used to detect the TrxR activity in cells treated with different functionally modified AgNPs. Characterization results revealed that AgNPs@BSA-AS-VRP were successfully constructed. When AgNPs@BSA-AS and AgNPs@BSA-AS-VRP were mixed in a ratio of 19:1, the amount of intracellular nanoparticles increased greatly through AS1411-mediated active targeting and inhibition of P-gp activity. In vitro and in vivo experiments clearly showed that the radiosensitization efficacy of 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP was much stronger than that of AgNPs@BSA and AgNPs@BSA-AS. It was also found that 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP significantly inhibited intracellular TrxR activity. These results indicate that 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP can effectively accumulate in tumor cells and have great potential as high-efficiency nano-radiosensitizers in the radiotherapy of glioma.

In this study, we developed a new method for simultaneous determination of verapamil hydrochloride (VerHCl) and its metabolite norverapamil hydrochloride (NorHCl) by using the capillary electrophoresis-electrochemiluminescence. Under optimized experimental conditions, the linear ranges of the VerHCl and NorHCl concentrations were 0.015-10.0 and 0.060-10.0 μg/mL, respectively. The linearity relations were determined using the respective regression equations y = 581.2x + 19.94 and y = 339.4x + 29.16. The respective limits of detection (S/N = 3) were 0.006 and 0.024 μg/mL. The proposed method was used to study the pharmacokinetics of both agents in rat plasma. The maximum concentration (Cmax), half-life time (T1/2) and time to peak (Tmax) were 683.21 ± 74.81 ng/mL, 0.52 ± 0.21 h and 2.49 ± 0.32 h for VerHCl and 698.42 ± 71.45 ng/mL, 1.14 ± 0.26 h and 2.83 ± 0.23 h for NorHCl, respectively, following oral administration of 10 mg/kg VerHCl., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: This study aimed to investigate the reversal effect of verapamil (VER) on the chemoresistance to cisplatin of esophageal squamous cell carcinoma (ESCC) cells., Patients and Methods: The reversal effect of VER on cisplatin resistance in ESCC cells was evaluated via CCK-8 assay, colony formation assessment, and flow cytometry. The key genes that mediate this effect were screened via high-throughput transcriptome se¬quencing. The mRNA and protein expression levels of potassium calcium-activated channel subfamily M alpha 1 (KCNMA1) in ESCC cells were examined via quantitative real-time PCR and Western blot analysis, respectively. The protein expressions of KCNMA1 in tissue samples from patients with either positive or negative responses to the therapeutic regimen of VER were determined via immunohistochemistry assay. Cell models with KCNMA1 knockdown and overexpression were es¬tablished to examine the role of KCNMA1 in mediating the reversal effect of VER on the chemoresistance to cisplatin of ESCC cells., Results: Results revealed that VER significantly decreased the 50% inhibitory concentration of cisplatin, inhibited colony formation, and induced apoptosis in ESCC cells. The curative effects of VER combined with chemotherapeutic drugs in KCNMA1-positive patients were better than those in KCNMA1-negative patients. KCNMA1 upregulation enhanced the reversal effect of VER on the chemoresistance to cisplatin of ESCC cells., Conclusions: KCNMA1 facilitated the reversal effect of VER on cisplatin resistance in ESCC cells.

Multidrug resistance (MDR) caused by overexpressed permeability-glycoprotein (P-gp) in cancer cells is the main barrier for the cure of cancers. P-gp can pump many chemotherapeutic drugs, which is a viable target to overcome P-gp-mediated MDR by efficient inhibitors of P-gp. However, limited understanding of the efflux mechanism by human P-gp hinders the development of efficient inhibitors. Herein, the transport of a P-gp inhibitor, verapamil, by human P-gp has been investigated using targeted molecular dynamics simulations and energetics analysis based on our previous research on the transport of a drug (doxorubicin). The energetics analysis identifies that the driving forces for the transport of verapamil are electrostatic repulsions contributed by the positively charged residues in the initial stage and then hydrophobic interactions contributed by the important residues in the later stage. This scenario is generally consistent with that in the transport of doxorubicin. However, the positively charged residues and the important residues for the transport of verapamil are incompletely consistent with the relative residues for the transport of doxorubicin. Moreover, the binding free energy contributions of the positively charged residues for the transport of verapamil are generally higher than them for the transport of doxorubicin, while the important residues constitute significantly different binding free energy compositions in the transports of the two substrates. Consequently, the pathway for the transport of verapamil is identified, which shares only two residues (F336 and M986) with the pathway of doxorubicin. This may imply the weak competitiveness of verapamil with doxorubicin in the substrate efflux. Taken together, this work provided new insights into the efflux mechanisms by human P-gp and would be beneficial in the design of potent P-gp inhibitors., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Experimental 3D structures of calcium channels with phenylalkylamines (PAAs) provide basis for further analysis of atomic mechanisms of these important cardiovascular drugs. In the crystal structure of the engineered calcium channel CavAb with Br-verapamil and in the cryo-EM structure of the Cav1.1 channel with verapamil, the ligands bind in the inner pore. However, there are significant differences between these structures. In the crystal structure the ligand ammonium group is much closer to the ion in the selectivity-filter region Site 3, which is most proximal to the inner pore, than in the cryo-EM structure. Here we used Monte Carlo energy minimizations to dock PAAs in calcium channels. Our computations suggest that in the crystal structure Site 3 is occupied by a water molecule rather than by a calcium ion. Analysis of the published electron density map does not rule out this possibility. In the cryo-EM structures the ammonium group of verapamil is shifted from the calcium ion in Site 3 either along the pore axis, towards the cytoplasm or away from the axis. Our unbiased docking reproduced these binding modes. However, in the cryo-EM structures detergent and lipid molecules interact with verapamil. When we removed these molecules, the nitrile group of verapamil bound to the calcium ion in Site 3. Models of Cav1.2 with different PAAs suggest similar binding modes and direct contacts of the ligands electronegative atoms with the calcium ion in Site 3. Such interactions explain paradoxes in structure-activity relationships of PAAs.

Femtosecond laser desorption postionization mass spectrometry using 7.9 eV single-photon ionization (7.9 eV fs-LDPI-MS) detected three of four drug compounds previously found to have very low ionization efficiencies by secondary ion mass spectrometry. Electronic structure calculations of the ionization energies and other properties of these four drug compounds predicted that all display ionization energies below the 7.9 eV photon energy, as required for single-photon ionization. The 7.9 eV fs-LDPI-MS of carbamazepine, imipramine, and verapamil all showed significant precursor (M + ) ion signal, but no representative signal was observed for ciprofloxacin. Furthermore, 7.9 eV fs-LDPI-MS displayed higher M + signals and mostly similar fragment ions compared with 70 eV electron impact mass spectrometry. Ionization and fragmentation patterns are discussed in terms of calculated wave functions for the highest occupied molecular orbitals. The implications for improving lateral resolution and sensitivity of MS imaging of drug compounds are also considered.

Mobility shift-affinity capillary electrophoresis was employed for enantioseparation and simultaneous binding constant determination. Human serum albumin was used as a chiral selector in the background electrolyte composed of 20 mM phosphate buffer, pH 7.4. The applied setup supports a high mobility shift since albumin and the drug-albumin complex hold negative net charges, while model compounds of amlodipine and verapamil are positively charged. In order to have an accurate effective mobility determination, the Haarhoff-van der Linde function was utilized. Subsequently, the association constant was determined by nonlinear regression analysis of the dependence of effective mobilities on the total protein concentration. Differences in the apparent binding status between the enantiomers lead to mobility shifts of different extends (α). This resulted in enantioresolutions of Rs = 1.05-3.63 for both drug models. R-(+)-Verapamil (K A 1844 M -1 ) proved to bind stronger to human serum albumin compared to S-(-)-verapamil (K A 6.6 M -1 ). The association constant of S-(-)-amlodipine (K A 25 073 M -1 ) was found to be slightly higher compared to its antipode (K A 22 620 M -1 ) when applying the racemic mixture. The low measurement uncertainty of this approach was demonstrated by the close agreement of the association constant of the enantiopure S-(-)-form (K A 25 101 M -1 )., (© 2020 The Authors. Journal of Separation Science published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Drug-drug interactions (DDI) have been examined for various drugs for oral use, but less for non-oral applications. This study provides DDI prediction methods for non-orally administered CYP3A4 substrates based on clinical DDI data of oral dosages. Gut availability (F g ) and fraction contribution of CYP3A4 to hepatic intrinsic clearance (fm CYP3A4 ) were predicted by AUC ratio (AUCR) in oral DDI study with/without grapefruit juice, and alteration in intrinsic clearances with/without ketoconazole, respectively. AUCRs of non-orally administered CYP3A4 substrates with/without inhibitors or inducers were predicted with the estimated F g , fm CYP3A4 and changes in liver CYP3A4 activities with inhibitors/inducers predicted using Simcyp library. DDIs of intravenously administered midazolam and alfentanil with CYP3A4 inhibitors/inducers could be predicted well by this method with predicted AUCRs within ±64% of observed values. Moreover, maximum DDIs with strong CYP3A4 inducers could be predicted by comparing hepatic clearance with hepatic blood flow, as hepatic blood flow indicates the possible maximum hepatic clearance after strong enzyme induction. Predicted AUCRs of midazolam, alfentanil and R- and S-verapamil were less than, but not far from observed ratios, suggesting good conservative prediction. These methods were applied to blonanserin transdermal patch, suggesting much smaller interaction with CYP3A4 inhibitors/inducers compared to oral dosage of blonanserin., Competing Interests: Declaration of competing interest The authors declared no conflict of interst. All authors are or were employees of Sumitomo Dainippon Pharma Co., Ltd., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Membrane permeability through passive diffusion is one of the important pathways for passage of drugs across the blood brain barrier (BBB). The present study describes the development of biomimetic unilamellar lipopolymeric nanovesicles of size 268 ± 37 nm, consisting of polar brain lipids in conjunction with polydiacetylene and validation of their application for an abbreviated yet accurate membrane permeability assay with high-throughput and rapid identification of BBB permeability of drugs. The nanovesicle suspension was tested with drugs of known permeability across the BBB to validate the detection of changes in hue, absorbance and fluorescence in response to permeation across the nanovesicles. A simple device was developed based on the nanovesicle sensors along with a mobile application which allowed for the determination of hue corresponding to qualitative identification of whether a drug is BBB permeable (BBB+) or not (BBB-). With respect to determination of a suitable endpoint in this assay, a hue cut off of 275°, reduction in %blueness by less than 59% and a fluorescence intensity of ≥0.22 a.u. at 560 nm accurately differentiated between drugs which are permeable and impermeable across the BBB within 5 minutes. Further quantification of BBB permeability can be done through the concentration at which the above end-points are achieved. For the quantification of the permeability, absorbance and fluorescence measurements were performed. The device thus developed allows the rapid determination of BBB permeability of various agents in drug discovery especially in smaller set-ups with minimal equipment through changes in color, absorbance and fluorescence.

Over the past few years, cardiac tissue engineering has undergone tremendous progress. Various in vitro methods have been developed to improve the accuracy in the result of drug-induced cardiac toxicity screening. Herein, we propose a novel SU-8 cantilever integrated with an electromechanical-stimulator to enhance the maturation of cultured cardiac cells. The simultaneous electromechanical stimulation significantly enhances the contraction force of the cardiomyocytes, thereby increasing cantilever displacement. Fluorescence microscopy analysis was performed to confirm the improved maturation of the cardiomyocytes. After the initial experiments, the contractile behaviors of the cultured cardiomyocytes were investigated by measuring the mechanical deformation of the SU-8 cantilever. Finally, the proposed electromechanical-stimulator-integrated SU-8 cantilever was used to evaluate the adverse effects of different cardiac vascular drugs, i.e., verapamil, lidocaine, and isoproterenol, on the cultured cardiomyocytes. The physiology of the cardiac-drug-treated cardiomyocytes was examined with and without electrical stimulation of the cardiomyocytes. The experimental results indicate that the proposed cantilever platform can be used as a predictive assay system for preliminary cardiac drug toxicity screening applications., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

High-resolution mass spectrometry (HRMS) is an important technology for studying biotransformations of drugs in biological systems. In order to process complex HRMS data, bioinformatics, including data-mining techniques for identifying drug metabolites from liquid chromatography/high-resolution mass spectrometry (LC/HRMS) or multistage mass spectrometry (MS n ) datasets as well as elucidating the detected metabolites' structure by spectral interpretation software, are important tools. Data-mining technologies have widely been used in drug metabolite identification, including mass defect filters, product ion filters, neutral-loss filters, control sample comparisons and extracted ion chromatographic analysis. However, the metabolites identified by current different technologies are not the same, indicating the importance of technique integration for efficient and complete identification of metabolic products. In this study, a universal, high-throughput workflow for identifying and verifying metabolites by applying the drug metabolite identification software UNIFI is reported, to study the biotransformation of verapamil in rats. A total of 71 verapamil metabolites were found in rat plasma, urine and faeces, including two metabolites that have not been reported in the literature. Phase I metabolites of verapamil were identified as N-demethylation, O-demethylation, N-dealkylation and oxidation and dehydrogenation metabolites; phase II metabolites were mainly glucuronidation and sulfate conjugates, indicating that UNIFI software could be effective and valuable in identifying drug metabolites., (© 2019 John Wiley & Sons, Ltd.)

Background: Multidrug resistance (MDR) is a pressing obstacle in clinical chemotherapy for breast cancer. Based on the fact that the drug efflux is an important factor in MDR, we designed a codelivery system to guide the drug efflux inhibitor verapamil (VRP) and the chemotherapeutic agent novantrone (NVT) synergistically into breast cancer cells to reverse MDR., Results: This co-delivery system consists of following components: the active targeting peptide RGD, an inorganic calcium phosphate (CaP) shell and an organic inner core. VRP and NVT were loaded into CaP shell and phosphatidylserine polyethylene glycol (PS-PEG) core of nanoparticles (NPs) separately to obtain NVT- and VRP-loaded NPs (NV@CaP-RGD). These codelivered NPs allowed VRP to prevent the efflux of NVT from breast cancer cells by competitively combining with drug efflux pumps. Additionally, NV@CaP-RGD was effectively internalized into breast cancer cells by precise delivery through the effects of the active targeting peptides RGD and EPR. The pH-triggered profile of CaP was also able to assist the NPs to successfully escape from lysosomes, leading to a greatly increased effective intracellular drug concentration., Conclusion: The concurrent administration of VRP and NVT by organic/inorganic NPs is a promising therapeutic approach to reverse MDR in breast cancer.

ZSM-5/KIT-6 and ZSM-5/SBA-15 nanoparticles were synthesized and further modified by a post-synthesis method with (CH 2 ) 3 SO 3 H and (CH 2 ) 3 NHCO(CH 2 ) 2 COOH groups to optimize their drug loading and release kinetic profiles. The verapamil cargo drug was loaded by incipient wetness impregnation both on the parent and modified nanoporous supports. Nanocarriers were then coated with a three-layer polymeric shell composed of chitosan-k-carrageenan-chitosan with grafted polysulfobetaine chains. The parent and drug loaded formulations were characterized by powder XRD, N 2 physisorption, thermal analysis, AFM, DLS, TEM, ATR-FT-IR and solid state NMR spectroscopies. Loading of verapamil on such nanoporous carriers and their subsequent polymer coating resulted in a prolonged in vitro release of the drug molecules. Quantum-chemical calculations were performed to investigate the strength of the interaction between the specific functional groups of the drug molecule and (CH 2 ) 3 SO 3 H and CH 2 ) 3 NHCO(CH 2 ) 2 COOH groups of the drug carrier. Furthermore, the ability of the developed nanocomposites to positively modulate the intracellular internalization and thereby augment the antitumor activity of the p-gp substrate drug doxorubicin was investigated in a comparative manner vs. free drug in a panel of MDR positive (HL-60/Dox, HT-29) and MDR negative (HL-60) human cancer cell lines using the Chou-Talalay method., (Copyright © 2019 Elsevier B.V. All rights reserved.)

P-glycoprotein (P-gp) is a multidrug transporter that is expressed on the luminal surface of epithelial cells in the kidney, intestine, bile-canalicular membrane in the liver, blood-brain barrier, and adrenal gland. This transporter uses energy of ATP hydrolysis to efflux from cells a variety of structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs. In this regard, understanding the interaction with P-gp of drug entities in development is important and highly recommended in current US Food and Drug Administration guidelines. Here we tested the P-gp interaction of some A 3 adenosine receptor agonists that are being developed for the treatment of chronic diseases, including rheumatoid arthritis, psoriasis, chronic pain, and hepatocellular carcinoma. Biochemical assays of the ATPase activity of P-gp and by photolabeling P-gp with its transport substrate [ 125 I]-iodoarylazidoprazosin led to the identification of rigidified (N)-methanocarba nucleosides (i.e., compound 3 as a stimulator and compound 8 as a partial inhibitor of P-gp ATPase activity). Compound 8 significantly inhibited boron-dipyrromethene (BODIPY)-verapamil transport mediated by human P-gp (IC 50 2.4 ± 0.6 µ M); however, the BODIPY-conjugated derivative of 8 (compound 24 ) was not transported by P-gp. In silico docking of compounds 3 and 8 was performed using the recently solved atomic structure of paclitaxel (Taxol)-bound human P-gp. Molecular modeling studies revealed that both compounds 3 and 8 bind in the same region of the drug-binding pocket as Taxol. Thus, this study indicates that nucleoside derivatives can exhibit varied modulatory effects on P-gp activity, depending on structural functionalization. SIGNIFICANCE STATEMENT: Certain A 3 adenosine receptor agonists are being developed for the treatment of chronic diseases. The goal of this study was to test the interaction of these agonists with the human multidrug resistance-linked transporter P-glycoprotein (P-gp). ATPase and photolabeling assays demonstrated that compounds with rigidified (N)-methanocarba nucleosides inhibit the activity of P-gp; however, a fluorescent derivative of one of the compounds was not transported by P-gp. Furthermore, molecular docking studies revealed that the binding site for these compounds overlaps with the site for paclitaxel in the drug-binding pocket. These results suggest that nucleoside derivatives, depending on structural functionalization, can modulate the function of P-gp., (U.S. Government work not protected by U.S. copyright.)

In drug discovery, it is important to identify phase I metabolic modifications as early as possible to screen for inactivation of drugs and/or activation of prodrugs. As the major class of reactions in phase I metabolism is oxidation reactions, oxidation of drugs with TiO 2 photocatalysis can be used as a simple non-biological method to initially eliminate (pro)drug candidates with an undesired phase I oxidation metabolism. Analysis of reaction products is commonly achieved with mass spectrometry coupled to chromatography. However, sample throughput can be substantially increased by eliminating pretreatment steps and exploiting the potential of ambient ionization mass spectrometry (MS). Furthermore, online monitoring of reactions in a time-resolved way would identify sequential modification steps. Here, we introduce a novel (time-resolved) TiO 2 -photocatalysis laser ablation electrospray ionization (LAESI) MS method for the analysis of drug candidates. This method was proven to be compatible with both TiO 2 -coated glass slides as well as solutions containing suspended TiO 2 nanoparticles, and the results were in excellent agreement with studies on biological oxidation of verapamil, buspirone, testosterone, andarine, and ostarine. Finally, a time-resolved LAESI MS setup was developed and initial results for verapamil showed excellent analytical stability for online photocatalyzed oxidation reactions within the set-up up to at least 1 h. Graphical Abstract.

Objective: Extended release (ER) tablets/capsules in massive ingestion overdoses are prone to form pharmacobezoars potentially increasing the risk of late-appearing toxic effects and prolonged symptoms. Oral activated charcoal is often sufficient to prevent drug absorption, but in a recent massive ingestion of highly toxic substances, prior orogastric lavage might be considered. The disintegration characteristics of ER preparations in overdose situations is valuable to understand if the time line and course of the intoxication might be prolonged, but information on these characteristics are unavailable. Slow disintegration and/or pharmacobezoar formation, and the large size makes ER preparation impossible to evacuate using a 30F orogastric lavage tube. This study evaluates the disintegration and pharmacobezoar formation of a simulated massive ER tablet ingestion in an in vitro model, using a selection of extended release tablets, with different disintegrating characteristics when present in therapeutic numbers. Furthermore, the sizes of the formed pharmacobezoars were compared with the dimensions of a 30F orogastric lavage tube., Method: A standardized model mimicking the physical effects on pharmaceutical preparations in simulated gastric fluid (SGF) was developed and tested on three mono-depot ER tablets (quetiapine/Seroquel ® XR 50 mg, paracetamol/Pinex ® Retard 500 mg, verapamil/Isoptin ® Retard 240 mg), one poly-depot ER tablet (carbamazepine/Tegretol ® Retard 200 mg), and one immediate-release tablet (paracetamol/Panodil ® 500mg). Thirty tablets were placed in polyamide mesh bags, either together in one bag or in separate bags, immersed in 1 L SGF, and incubated at 37 °C for 48 h. Released drugs were quantified at 0.5-48 h., Results: Visual inspection showed that Seroquel ® XR, Pinex ® Retard, and Isoptin ® Retard tablets formed firm pharmacobezoars stable for more than 4 h and intact fractions remained for up to 24 h. Drug releases were reduced by 53%, 40%, and 31%, respectively, for up to 8 h compared to separated tablets. Light microscopy showed that contact with SGF transformed the coating of Seroquel ® XR and Pinex ® Retard to a diffusion-controlled swelled gel-layer, and the Isoptin ® Retard tablets into a rigid and slow-releasing matrix. Tegretol ® Retard disintegrated into microspheres within 30 min, and Panodil ® disintegrated within minutes., Discussion: The developed pharmacobezoars of mono-depot ER tablets demonstrated prolonged drug release. Neither the formed pharmacobezoars, nor the single tablets of the tested mono-depot ER preparations, would pass through the lumen of a standard orogastric lavage tube, rendering this modality ineffective for tablet removal in gastrointestinal decontamination.

Chitosan-based hydrogel nanoparticles provide a higher brain concentration of methotrexate (MTX) following IV administration in comparison with the drug's simple solution. The present study investigates the mechanism of this phenomenon, focusing on the possible role of P-gp. A previously developed MTX containing chitosan nanogel was fabricated and characterised. Then 48 rats were divided into four groups: two receiving nanogels and two receiving solution of MTX, while 1 of each two had received a verapamil dose 30 min before MTX. Then, rats were sacrificed in four time points in triplicate, and MTX concentration in their plasma and brains were quantified and were compared statistically. Following IV injection, spherical nanogels with a mean diameter of <200 nm, zeta potential of 22.8 ± 6.55 mv, Loading efficiency of 72.03 ± 0.85, and loading capacity of 1.41 ± 0.02 produce a significantly higher brain concentration compared with the simple solution. Furthermore, those receiving verapamil presented a higher brain concentration. It seems that in the short term after drug administration (<1 h) nanogels facilitate MTX passage by providing a higher concentration of drug in contact with BBB, but in a more extended period nanogels pass the BBB and release their content beyond that., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background/aims: The phenylalkylamine class of L-type Ca 2+ channel antagonist verapamil prolongs the effective refractory period (ERP) of human atrium, which appears to contribute to the efficacy of verapamil in preventing reentrant-based atrial arrhythmias including atrial fibrillation. This study was designed to investigate the molecular and electrophysiological mechanism underlying the action of verapamil on human Kv1.5 (hKv1.5) channel that determines action potential duration and ERP in human atrium., Methods: Site-directed mutagenesis created 10 single-point mutations within pore region of hKv1.5 channel. Wholecell patch-clamp method investigated the effect of verapamil on wild-type and mutant hKv1.5 channels heterologously expressed in Chinese hamster ovary cells. Docking simulation was conducted using open-state homology model of hKv1.5 channel pore., Results: Verapamil preferentially blocked hKv1.5 channel in its open state with IC 50 of 2.4±0.6 μM (n = 6). The blocking effect of verapamil was significantly attenuated in T479A, T480A, I502A, V505A, I508A, L510A, V512A and V516A mutants, compared with wild-type hKv1.5 channel. Computer docking simulation predicted that verapamil is positioned within central cavity of channel pore and has contact with Thr479, Thr480, Val505, Ile508, Ala509, Val512, Pro513 and Val516., Conclusion: Verapamil acts as an open-channel blocker of hKv1.5 channel, presumably due to direct binding to specific amino acids within pore region of hKv1.5 channel, such as Thr479, Thr480, Val505, Ile508, Val512 and Val516. This blocking effect of verapamil on hKv1.5 channel appears to contribute at least partly to prolongation of atrial ERP and resultant antiarrhythmic action on atrial fibrillation in humans., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

In this study, the application of a mixture of organic solvents as a supported liquid membrane for improving the efficiency of the electromembrane extraction procedure was investigated. The extraction process was followed by high-performance liquid chromatography analysis of two model drugs (verapamil and riluzole). In this research, four organic solvents, including 1-heptanol, 1-octanol, 2-nitrophenyl octyl ether, and 2-ethyl hexanol, were selected as model solvents and different binary mixtures (v/v 2:1, 1:1 and 1:2) were used as the supported liquid membrane. The mixture of 2-ethyl hexanol and 1-otanol (v/v, 2:1) improved the extraction efficiency of model drugs by 1.5 to 12 times. It was found that extraction efficiency is greatly influenced by the level of electric current. In this study, for various mixtures of organic solvents, the electric current fluctuated between 50 and 2500 μA, and the highest extraction efficiencies were obtained with low and stable electric currents. Finally, the optimized extraction condition was validated and applied for the determination of model drugs in urine and wastewater samples., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We present a simple approach to calculate the kinetic properties of lipid membrane crossing processes from biased molecular dynamics simulations. We demonstrate that by using biased simulations, one can obtain highly accurate kinetic information with significantly reduced computational time with respect to unbiased simulations. We describe how to conveniently calculate the transition rates to enter, cross, and exit the membrane in terms of the mean first passage times. To obtain free energy barriers and relaxation times from biased simulations only, we constructed Markov models using the dynamic histogram analysis method (DHAM). The permeability coefficients that are calculated from the relaxation times are found to correlate highly with experimentally evaluated values. We show that more generally, certain calculated kinetic properties linked to the crossing of the membrane layer (e.g., barrier height and barrier crossing rates) are good indicators of ordering drugs by permeability. Extending the analysis to a 2D Markov model provides a physical description of the membrane crossing mechanism.

Purpose: Ovarian cancer is usually treated with transurethral resection or systemic chemotherapy in clinic. However, the development of drug resistance in ovarian cancer is frequently observed in ovarian cancer patients, leading to failure of tumor inhibition and recurrence. In this study, we aimed to efficiently reverse the drug resistance and enhance the anticancer effects by co-delivery of chemotherapeutic agents and multi-drugs resistant proteins inhibitor in ovarian cancer treatment., Methods: The cell viability was measured by using MTT or flow cytometry (Annexin V/PI staining) under different culture conditions. Western blot was used to detect the expression of P-gp. We employed confocol to visualize the drug distribution under different culture systems. Using flow cytometry, we examined the drug absorption. MPEG-PLA was used to load chemotherapeutic drugs. We also applied mice model to evaluate the killing ability and side effects of free or methoxy poly (ethylene glycol)-poly (l-lactic acid) (MPEG-PLA) loaded drugs., Results: We found that pre-treatment of verapamil, a multi-drugs resistant proteins inhibitor, could efficiently reverse the drug resistant in ovarian cancer. To further improve the pharmacokinetics profiles and avoid the systemic toxicity caused by agents, we encapsulated verapamil and doxorubicin (DOX) by polymeric nanoparticles MPEG-PLA. Co-delivery of verapamil and DOX by nano-carrier revealed reduced drug resistance and enhanced anticancer effects compared with the free drug delivery. More importantly, accumulated drugs, prolonged drug circulation and reduced systemic were observed in nanoparticles encapsulation group., Conclusion: Co-delivery of verapamil and chemotherapeutic drugs by MPEG-PLA efficiently reversed the drug resistance, resulting in enhanced anticancer effects along with reduced systemic toxicity, which provides potential clinical applications for drug resistant ovarian cancer treatment., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Mycobacterial efflux pumps play a major role in the emergence of antimycobacterial drug resistance. Of particular interest is the proteinaceous multi-drug efflux pump protein Rv1258c that encodes a tetracycline/aminoglycoside resistance (TAP-2)-like efflux pump which is active in susceptible and drug resistant Mycobacterium tuberculosis. Rv1258c is implicated in drug resistance to numerous antimycobacterials including first line drugs rifampicin and isoniazid as well as fluoroquinolone and aminoglycoside antibiotic classes. To date, compounds like verapamil and piperine have been shown to inhibit Rv1258c but no direct evidence for binding or mode of action exist. Therefore in the present study we generated an accurate 3D model of Rv1258c using MODELLER and validated its structure using molecular dynamic simulation studies with GROMACS software. The 3D-structures of Rv1258c and the homologous template 1pw4 were simulated within a POPE/POPG lipid bilayer and found to behave similar. Another important finding was the identification of one local energy minima state of the apo protein, which speaks to the flexibility of the protein and will be investigated further. Extraction of one of the open channel conformations of Rv1258c and blind docking of various structurally diverse putative inhibitors and substrates, allowed for the identification of a probable binding site. Spectinamide was found to bind to a different location on the outside surface of the protein suggesting its ability to avoid the efflux channel. We further identified 246 putative compounds that showed higher binding affinity values to Rv1258c compared to piperine and verapamil. Interaction analysis of the top 20 purchasable compounds identified crucial hydrogen bond interactions with Ser26, Ser45 and Glu243 as well as a pi-pi stacking interaction with Trp32 that accounted for the strong affinity of these compounds for Rv1258c. Future studies will entail purchasing a number of compounds for in vitro activity testing against Mycobacterium tuberculosis., Competing Interests: The authors have declared that no competing interests exist.

Controlled-release multiparticulate systems of hydrophilic drugs usually suffer from poor encapsulation and rapid-release rate. In the present study, ultra-high loaded controlled release polymeric beads containing verapamil hydrochloride (VP) as hydrophilic model drug were efficiently prepared using superamphiphobic substrates aiming to improve patient compliance by reducing dosing frequency. Superamphiphobic substrates were fabricated using clean aluminum sheets etched with ammonia solution and were treated with 1.5% (w/v) perfluorodecyltriethoxysilane (PFDTS) alcoholic solution. The effect of the main polymer type (lactide/glycolide (PLGA) 5004A, PLGA 5010, and polycaprolactone (PCL)), copolymer (Eudragit RS100) content together with the effect of drug load on encapsulation efficiency (EE%) and in vitro drug release was statistically studied and optimized via D-optimal statistical design. In vivo pharmacokinetic study was carried out to compare the optimized system relative to the market product (Isoptin ® ). Results revealed that superamphiphobic substrates were successfully prepared showing a rough micro-sized hierarchical structured surface upon observing with scanning electron microscope and were confirmed by high contact angles of 151.60 ± 2.42 and 142.80°±05.23° for water and olive oil, respectively. The fabricated VP-loaded beads showed extremely high encapsulation efficiency exceeding 92.31% w/w. All the prepared systems exhibited a controlled release behavior with Q12 h ranging between 5.46 and 95.90%w/w. The optimized VP-loaded system composed of 150 mg (1.5% w/v) PCL without Eudragit RS100 together with 160 mg VP showed 2.7-folds mean residence time compared to the market product allowing once daily administration instead of three times per day.

Introduction: Fluorine-18 labeled positron emission tomography (PET) tracers were developed to obtain more insight into the function of P-glycoprotein (P-gp) in relation to various conditions. They allow research in facilities without a cyclotron as they can be transported with a half-life of 110 min. As the metabolic stability of previously reported tracers [ 18 F]1 and [ 18 F]2 was poor, the purpose of this study was to improve this stability using deuterium substitution, creating verapamil analogs [ 18 F]1-d 4 , [ 18 F]2-d 4 , [ 18 F]3-d 3 and [ 18 F]3-d 7 ., Methods: The following deuterium containing tracers were synthesized and evaluated in mice and rats: [ 18 F]1-d 4 , [ 18 F]2-d 4 , [ 18 F]3-d 3 and [ 18 F]3-d 7 ., Results: The deuterated analogs [ 18 F]2-d 4 , [ 18 F]3-d 3 and [ 18 F]3-d 7 showed increased metabolic stability compared with their non-deuterated counterparts. The increased metabolic stability of the methyl containing analogs [ 18 F]3-d 3 and [ 18 F]3-d 7 might be caused by steric hindrance for enzymes., Conclusion: The striking similar in vivo behavior of [ 18 F]3-d 7 to that of (R)-[ 11 C]verapamil, and its improved metabolic stability compared with the other fluorine-18 labeled tracers synthesized, supports the potential clinical translation of [ 18 F]3-d 7 as a PET radiopharmaceutical for P-gp evaluation., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

A series of aminoisopropanoloxy derivatives of xanthone has been synthesized and their pharmacological properties regarding the cardiovascular system has been evaluated. Radioligand binding and functional studies in isolated organs revealed that title compounds present high affinity and antagonistic potency for α 1 -(compound 2 and 8), β-(compounds 1, 3, 4, 7), α 1 /β-(compounds 5 and 6) adrenoceptors. Furthermore, compound 7, the structural analogue of verapamil, possesses calcium entry blocking activity. The title compounds showed hypotensive and antiarrhythmic properties due to their adrenoceptor blocking effect. Moreover, they did not affect QRS and QT intervals, and they did not have proarrhythmic potential at tested doses. In addition they exerted anti-aggregation effect. The results of this study suggest that new compounds with multidirectional activity in cardiovascular system might be found in the group of xanthone derivatives., (Copyright © 2018. Published by Elsevier Ltd.)

Purpose: To investigate the blood-to-retina verapamil transport at the blood-retinal barrier (BRB)., Methods: EverFluor FL Verapamil (EFV) was adopted as the fluorescent probe of verapamil, and its transport across the BRB was investigated with common carotid artery infusion in rats. EFV transport at the inner and outer BRB was investigated with TR-iBRB2 cells and RPE-J cells, respectively., Results: The signal of EFV was detected in the retinal tissue during the weak signal of cell impermeable compound. In TR-iBRB2 cells, the localization of EFV differed from that of LysoTracker ® Red, a lysosomotropic agent, and was not altered by acute treatment with NH 4 Cl. In RPE-J cells, the punctate distribution of EFV was partially observed, and this was reduced by acute treatment with NH 4 Cl. EFV uptake by TR-iBRB2 cells was temperature-dependent and membrane potential- and pH-independent, and was significantly reduced by NH 4 Cl treatment during no significant effect obtained by different extracellular pH and V-ATPase inhibitor. The EFV uptake by TR-iBRB2 cells was inhibited by cationic drugs, and inhibited by verapamil in a concentration-dependent manner with an IC 50 of 98.0 μM., Conclusions: Our findings provide visual evidence to support the significance of carrier-mediated transport in the blood-to-retina verapamil transport at the BRB.

Because P-glycoprotein (P-gp) plays an absorptive role in the skin, its pharmacological inhibition represents a strategy to promote cutaneous localization of anticancer agents that serve as its substrates, improving local efficacy while reducing systemic exposure. Here, we evaluated the ability of a nanoemulsion (NE) coencapsulating a P-gp inhibitor (elacridar) with the antitumor drug paclitaxel to promote epidermal targeting. Loaded NE displayed a nanometric size (45.2 ± 4.0 nm) and negative zeta potential (-4.2 ± 0.8 mV). Elacridar improved NE ability to inhibit verapamil-induced ATPase activity of P-gp; unloaded NE-inhibited P-gp when used at a concentration of 1500 μM, while elacridar encapsulation decreased this concentration by 3-fold (p <0.05). Elacridar-loaded NE reduced paclitaxel penetration into the dermis of freshly excised mice skin and its percutaneous permeation by 1.5- and 1.7-fold (p <0.05), respectively at 6 h, whereas larger drug amounts (1.4-fold, p <0.05) were obtained in viable epidermis. Assessment of cutaneous distribution of a fluorescent paclitaxel derivative confirmed the smaller delivery into the dermis at elacridar presence. In conclusion, we have provided novel evidence that NE containing elacridar exhibited a clear potential for P-gp inhibition and enabled epidermal targeting of paclitaxel, which in turn, can potentially reduce adverse effects associated with systemic exposure to anticancer therapy., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

P-glycoprotein (Pgp) is an efflux pump important in multidrug resistance of cancer cells and in determining drug pharmacokinetics. Pgp is a prototype ATP-binding cassette transporter with two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. Conformational changes at the NBDs (the Pgp engines) lead to changes across Pgp transmembrane domains that result in substrate translocation. According to current alternating access models (substrate-binding pocket accessible only to one side of the membrane at a time), binding of ATP promotes NBD dimerization, resulting in external accessibility of the drug-binding site (outward-facing, closed NBD conformation), and ATP hydrolysis leads to dissociation of the NBDs with the subsequent return of the accessibility of the binding site to the cytoplasmic side (inward-facing, open NBD conformation). However, previous work has not investigated these events under near-physiological conditions in a lipid bilayer and in the presence of transport substrate. Here, we used luminescence resonance energy transfer (LRET) to measure the distances between the two Pgp NBDs. Pgp was labeled with LRET probes, reconstituted in lipid nanodiscs, and the distance between the NBDs was measured at 37 °C. In the presence of verapamil, a substrate that activates ATP hydrolysis, the NBDs of Pgp reconstituted in nanodiscs were never far apart during the hydrolysis cycle, and we never observed the NBD-NBD distances of tens of Å that have previously been reported. However, we found two main conformations that coexist in a dynamic equilibrium under all conditions studied. Our observations highlight the importance of performing studies of efflux pumps under near-physiological conditions, in a lipid bilayer, at 37 °C, and during substrate-stimulated hydrolysis., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

We present a new high-throughput platform for studying titanium dioxide (TiO 2 ) photocatalytic oxidation reactions by performing reactions on a TiO 2 -coated surface, followed by direct analysis of oxidation products from the surface by desorption electrospray ionization mass spectrometry (DESI-MS). For this purpose, we coated a round glass wafer with photocatalytically active anatase-phase TiO 2 using atomic layer deposition. Approximately 70 aqueous 1 μL samples can be injected onto the rim of the TiO 2 -coated glass wafer, before the entire wafer is exposed to UV irradiation. After evaporation of water, the oxidation products can be directly analyzed from the sample spots by DESI-MS, using a commercial rotating sample platform. The method was shown to provide fast photocatalytic oxidation reactions and analysis with throughput of about four samples per minute. The feasibility of the method was examined for mimicking phase I metabolism reactions of amodiaquine, buspirone and verapamil. Their main photocatalytic reaction products were mostly similar to the products observed earlier in TiO 2 photocatalysis and in in vitro phase I metabolism assays performed using human liver microsomes.

The efflux transporter P-glycoprotein (P-gp) significantly modulates drug transport across the intestinal mucosa, strongly reducing the systemic absorption of various active pharmaceutical ingredients. P-gp inhibitors could serve as helpful tools to enhance the oral bioavailability of those substances. As a membrane-associated protein P-gp is surrounded and influenced by phospholipids. Some synthetic phospholipids have been found to strongly reduce P-gp's activity. In this study two representative phospholipids, 1,2-dioctanoyl-sn-glycero-3-phosphocholine (8:0 PC) and 1,2-didecanoyl-sn-glycero-3-phosphocholine (10:0 PC), were compared with Tween® 80 and Cremophor® EL, both commonly used surfactants with P-gp inhibitory properties. Their influence on the cellular transport of the P-gp substrate rhodamine 123 (RH123) was examined using Caco-2 cell layers. In addition, fluorescence anisotropy measurements were performed in order to investigate their effect on membrane fluidity. Finally, we compared the phospholipids with Tween® 80 and the competitive P-gp inhibitor verapamil in an in vivo study, testing their effects on the oral bioavailability of the P-gp substrate drug ritonavir. Both phospholipids not only led to the strongest absorption of RH123, but a permeability enhancing effect was detected in addition to the P-gp inhibition. Their effects on membrane fluidity were not consistent with their P-gp inhibiting effects, and therefore suggested a more complex mode of action. Both phospholipids significantly increased the area under the ritonavir plasma level curve (AUC) within 150min by more than tenfold, but were inferior to Tween® 80, which showed superior solubilizing effects. Finally, these phospholipids represent a novel substance class showing a high permeabilization potential for P-gp substrates. Because of their physiological structure and intestinal degradability, good tolerability without systemic absorption is expected. Formulating P-gp substrates with an originally low oral bioavailability is a difficult task, requiring concerted interplay of all excipients. P-gp inhibiting phospholipids offer a new tool to help cope with these challenges., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of this study was to evaluate the potential of polyelectrolyte complex nanoparticles (PENPs) based on hyaluronic acid/chitosan hydrochloride (HA/HCS) for co-loading mitoxantrone (MTO) and verapamil (VRP) to overcome multidrug resistance in breast tumors. PENPs co-loaded with MTO and VRP (MTO-VRP-PENPs) were affected by the method of preparation, molecular weight of HA, mass ratios and initial concentrations of HA/HCS, pH, and drug quantities. Optimized MTO-VRP-PENPs were ~209 nm in size with a zeta potential of approximately -24 mV. Encapsulation efficiencies (%) of MTO and VRP were 98.33%±0.27% and 44.21%±8.62%, respectively. MTO and VRP were successfully encapsulated in PENPs in a molecular or amorphous state. MTO-VRP-PENPs showed significant cytotoxicity in MCF-7/ADR cells in contrast to MTO-loaded PENPs (MTO-PENPs). The reversal index of MTO-VRP-PENPs was 13.25 and 10.33 times greater than that of the free MTO and MTO-PENPs, respectively. In conclusion, MTO-VRP-PENPs may serve as a promising carrier to overcome tumor drug resistance., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Developing green methods for analyte extraction is one of the most important topics in the field of sample preparation. In this study, for the first time, agarose gel was used as membrane in electromembrane extraction (EME) without using any organic solvent, for the extraction of four model basic drugs (rivastigmine (RIV), verapamil (VER), amlodipine (AML), and morphine (MOR)) with a wide polarity window (log P from 0.43 to 3.7). Different variables playing vital roles in the proposed method were evaluated and optimized. As a driving force, a 25V electrical field was applied to make the analyte migrate from sample solution with pH 7.0, through the agarose gel 3% (w/v) with 5mm thickness, into an acceptor phase (AP) with pH 2.0. The best extraction efficiency was obtained with an extraction duration of 25min. With this new methodology, MOR with high polarity (log P=0.43) was efficiently extracted without using any carrier or ion pair reagents. Limits of detection (LODs) and quantification (LOQs) were in the ranges of 1.5-1.8ngmL -1 and 5.0-6.0ngmL -1 , respectively. Finally, the proposed method was successfully applied to determine concentrations of the model drugs in the wastewater sample., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Interactions with serum proteins such as alpha 1 -acid glycoprotein (AGP) can have a significant effect on the behavior and pharmacokinetics of drugs. Ultrafast affinity extraction and peak profiling were used with AGP microcolumns to examine these processes for several model drugs (i.e., chlorpromazine, disopyramide, imipramine, lidocaine, propranolol and verapamil). The association equilibrium constants measured for these drugs with soluble AGP by ultrafast affinity extraction were in the general range of 10 4 -10 6 M -1 at pH 7.4 and 37°C and gave good agreement with literature values. Some of these values were dependent on the relative drug and protein concentrations that were present when using a single-site binding model; these results suggested a more complex mixed-mode interaction was actually present, which was also then used to analyze the data. The apparent dissociation rate constants that were obtained by ultrafast affinity extraction when using a single-site model varied from 0.14 to 7.0s -1 and were dependent on the relative drug and protein concentrations. Lower apparent dissociation rate constants were obtained by this approach as the relative amount of drug versus protein was decreased, with the results approaching those measured by peak profiling at low drug concentrations. This information should be useful in better understanding how these and other drugs interact with AGP in the circulation. In addition, the chromatographic approaches that were optimized and used in this report to examine these systems can be adapted for the analysis of other solute-protein interactions of biomedical interest., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The solubility of weakly basic drugs in passage through gastrointestinal tract leads to their pH-dependent release from extended release formulations and to lower drug absorption and bioavailability. The aim of this study was to modulate the micro-environmental pH of hypromellose/montanglycol wax matrices and to observe its influence on the release of weakly basic drug verapamil hydrochloride (VH) with a pH-dependent solubility with respect to gel layer formation and its dynamics. For this study, malic and succinic acids differing in their solubility and pKa were selected as pH modifiers. The dissolution studies were performed by the method of changing pH. Within the same conditions, pH, thickness, and penetration force of the gel layer were measured as well. From the PCA sub-model, it is evident that a higher acid concentration ensured lower gel pH and conditions for higher drug solubility, thus creating larger gel layer with smaller rigidity, resulting in higher VH release during the dissolution test. Incorporation of stronger and more soluble malic acid (100 mg/tablet) created the most acidic and the thickest gel layer through which a total of 74% of VH was released. Despite having lower strength and solubility, matrices containing succinic acid (100 mg/tablet) released a comparable 71% of VH in a manner close to zero-order kinetics. The thinner and less rigid gel layers of the succinic acid matrices allowed an even slightly faster VH release at pH 6.8 than from matrices containing malic acid. Thus acid solubility is more parametrically significant than acid pKa for drug release at pH 6.8.

P-glycoprotein (P-gp) is a multidrug transporter that uses energy from ATP hydrolysis to export many structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs from cells. Several structural studies on purified P-gp have been reported, but only limited and sometimes conflicting information is available on ligand interactions with the isolated transporter in a dodecyl-maltoside detergent environment. In this report we compared the biochemical properties of P-gp in native membranes, detergent micelles, and when reconstituted in artificial membranes. We found that the modulators zosuquidar, tariquidar, and elacridar stimulated the ATPase activity of purified human or mouse P-gp in a detergent micelle environment. In contrast, these drugs inhibited ATPase activity in native membranes or in proteoliposomes, with IC 50 values in the 10-40 nm range. Similarly, a 30-150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS was observed in detergent micelles compared with native or artificial membranes. Together, these findings demonstrate that the high-affinity site is inaccessible because of either a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. We, therefore, recommend studying structural and functional aspects of ligand interactions with purified P-gp and other ATP-binding cassette transporters that transport amphipathic or hydrophobic substrates in a detergent-free native or artificial membrane environment., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

A novel, economic, and time-efficient stability-indicating, reverse-phase ultra-performance liquid chromatographic (RP-UPLC) method has been developed for the analysis of verapamil hydrochloride in the presence of both impurities and degradation products generated by forced degradation. When verapamil hydrochloride was subjected to acid hydrolysis, oxidative, base hydrolysis, photolytic, and thermal stress, degradation was observed only in oxidative and base hydrolysis. The drug was found to be stable to other stress conditions. Successful chromatographic separation of the drug from impurities formed during synthesis and from degradation products formed under stress conditions was achieved on a Shimpak XR ODS, 75mm×3.0mm, 1.7μ particle size column, UV detection at 278nm and a gradient elution of ammonium formate, orthophosphoric acid and acetonitrile as mobile phase. The method was validated for specificity, precision, linearity, accuracy, robustness and can be used in quality control during manufacture and for assessment of the stability samples of verapamil hydrochloride. To the best of our knowledge, a validated UPLC method which separates all the sixteen impurities disclosed in this investigation has not been published elsewhere. Total elution time was about 18min which allowed quantification of more than 100 samples per day. The analytical method discussed in British Pharmacopeia was pH sensitive and not compatible to LC-MS analysis but the method reported in this study is not involved any pH adjustment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Multidrug resistance (MDR) poses a major burden to cancer treatment. As one important factor contributing to MDR, overexpression of P-glycoprotein (P-gp) results in a reduced intracellular drug accumulation. Hence, the ability to effectively block the efflux protein and to accumulate the therapeutics in cancer cells is of great significance in clinical practice. In this work, we successfully developed a smart stimulus-responsive short peptide-assembled system, termed as PD/VER nanogels, which synergistically combined the acid-activatable antitumor prodrug doxorubicin (Dox) with the P-gp inhibitor verapamil (VER) for reversing MDR. Systematic studies demonstrated that such an inhibitor-encapsulated nanogel could effectively enhance the accumulation of Dox in resistant cancer cells, thereby revealing significantly higher antitumor activity compared to free Dox molecules. This work showed that the assembly of bioactive agents with a synergistic effect into nano-drugs could provide a useful strategy to overcome cancer drug resistance., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Resistance to the anticancer antibiotics, doxorubicin and daunorubicin, in the producer organism Streptomyces peucetius is conferred by an ABC transporter made of two proteins, DrrA and DrrB, which together form a dedicated exporter for these two antibiotics. Surprisingly, however, the DrrAB system exhibits broad substrate specificity overlapping with well-studied multidrug resistance transporters, including P-glycoprotein. Therefore, it provides an excellent model for studying the molecular basis of multispecificity in a prototype efflux system with the potential to unravel the origin and evolution of multidrug resistance. It has been suggested that multispecificity in multidrug exporters may be generally determined by the number and location of aromatic residues. Strategically placed negatively charged residues may also be critical for binding of cationic lipophilic drugs. We selected 13 aromatic and four negatively charged residues on the basis of their location in and/or near the predicted drug-binding pocket of DrrB for analysis. Indeed, mutations of most tested residues drastically inhibited doxorubicin efflux. Interestingly, several mutants lost resistance to doxorubicin and verapamil simultaneously but retained resistance to Hoechst 33342 and/or ethidium bromide, suggesting the presence of overlapping as well as independent drug-binding sites in a common drug-binding pocket of DrrB. This study provides the first comprehensive analysis of residues involved in drug binding in a bacterial multidrug resistance protein of the ABC superfamily, and it shows strong similarity in the molecular mechanism of polyspecific drug recognition between DrrAB and Pgp. Altogether, we conclude that aromatic residue-based multidrug specificity is conserved across domains and over long evolutionary periods. The significance of these findings is discussed.

The clinical usage of docetaxel (DTX) has been blocked in the clinic because of its poor solubility and tumour multi-drug resistance (MDR). The dominating mechanism of MDR is the over-expression of p-gp on tumour cells. Traditional nano-medicines, such as nanoparticles and micelles, have been used to physically entrap DTX to improve their solubility, while the drug loading content was very low and the tumour resistance was neglected. In this study, the synthesized reduction-sensitive mPEG-PLGA-SS-DTX conjugate was utilized to load the p-gp inhibitor veraparmil (VRP) to prepare DTX and VRP co-delivered mPEG-PLGA-SS-DTX/VRP (PP-SS-DTX/VRP) multi-functional micelles to reverse MDR and enhance the anti-tumour effect of DTX. The micelles had a high drug loading content and showed an obvious reduction-sensitive release property for both DTX and VRP. In addition, an in vitro anti-tumour assay revealed that the micelles markedly inhibited the efflux activity of p-gp and accelerated cell apoptosis, resulting in the improvement of anti-tumour activity and reversal of MDR. The PP-SS-DTX micelles markedly enhanced the in vivo circulation time and increased the drug accumulation in tumour tissues. Therefore, the PP-SS-DTX/VRP micelle is a desirable drug delivery system for multi-drug resistance therapy of DTX and is very promising for clinical usage., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Photocatalytic degradation of pharmaceuticals (hydrocortisone, estradiol, and verapamil) and personal care product additives (parabens-methyl, ethyl, and propyl derivatives) was investigated in the homogeneous phase (with ferric ions as the catalyst) and on TiO 2 . Ferric ions in concentrations corresponding to concentrations in natural water bodies were shown to be a significant accelerator of the degradation in homogeneous reaction mixtures. In heterogeneous photocatalytic reactions on TiO 2 , lower reaction rates, but mineralisation to higher extents, were observed.

Background/aims: The human-voltage gated Kv1.3 channel (hKv1.3) is expressed in T- and B lymphocytes. Verapamil is able to block hKv1.3 channels. We characterized the effect of verapamil on currents through hKv1.3 channels paying special attention to the on-rate (kon) of verapamil. By comparing on-rates obtained in wild-type (wt) and mutant channels a binding pocket for verapamil and impacts of different amino acid residues should be investigated., Methods: Using the whole-cell patch clamp technique the action of verapamil on currents through wild-type and six hKv1.3 mutant channels in the open state was investigated by measuring the time course of the open channel block in order to calculate kon of verapamil., Results: The on-rate of verapamil to block current through hKv1.3&#95;T419C mutant channels is similar to that obtained for hKv1.3&#95;wt channels whereas the on-rate of verapamil to block currents through hKv1.3&#95;L417C and hKv1.3&#95;L418C mutant channels was ∼ 3 times slower compared to in wt channels. The on-rate of verapamil to block currents through hKv1.3&#95;L346C and the double mutant hKv1.3&#95;L346C&#95;L418C channel was ∼ 2 times slower compared to that obtained in the wt channel. The hKv1.3&#95;I420C mutant channel reduced the on-rate of verapamil to block currents ∼ 6 fold., Conclusions: We conclude that position 420 in hKv1.3 channels maximally interferes with verapamil reaching its binding site to block the channel. Positions 417 and 418 in hKv1.3 channels partially hinder verapamil reaching its binding site to block the channel whereas position 419 may not interfere with verapamil at all. Mutant hKv1.3&#95;L346C and hKv1.3&#95;L346C&#95;L418C mutant channels might indirectly influence the ability of verapamil reaching its binding site to block current., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

The creation of composite systems has become an emerging field in drug delivery. Chitosan has demonstrated several pharmaceutical advantages, especially in intranasal delivery. In this manuscript, a comparative study was conducted between regular vesicles (transfersomes and penetration enhancer vesicles) and composite vesicles (chitosan containing transfersomes and penetration enhancer vesicles) loaded with a model antihypertensive drug; verapamil hydrochloride VRP. Composite vesicles displayed larger particle size than regular vesicles owing to the coating potential of chitosan on the vesicular bilayer as displayed by transmission electron microscopy, with an increased viscosity of composite vesicles and a shift in the zeta potential values from negative to positive. The entrapment efficiency of VRP in the vesicles ranged from 24 to 64%, with best physical stability displayed with transfersomal vesicles prepared using sodium deoxycholate. Chitosan slowed the in vitro release of VRP from the selected formulation but managed to achieve high penetrability across sheep nasal mucosa as displayed by confocal laser microscopy. The chitosan composite transfersomal formulation exhibited absolute bioavailability of 81.83% compared to the oral solution which displayed only 13.04%. Findings of this manuscript highly recommend chitosan as a promising functional additive in vesicular formulations to improve the intranasal delivery of drugs with low oral bioavailability., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Here we examine the mechanisms by which nanoparticles enable the oral absorption of water-insoluble, P-glycoprotein efflux pump (P-gp) substrates, without recourse to P-gp inhibitors. Both 200nm paclitaxel N-(2-phenoxyacetyl)-6-O-glycolchitosan (GCPh) nanoparticles (GCPh-PTX) and a simulated Taxol formulation, facilitate drug dissolution in biorelevant media, unlike paclitaxel nanocrystals. Verapamil (40mgkg -1 ) increased the oral absorption from low dose Taxol (6 or 10mgkg -1 ) by 100%, whereas the oral absorption from high dose Taxol (20mgkg -1 ) or low dose GCPh-PTX (6 or 10mgkg -1 ) was largely unchanged by verapamil. There was virtually no absorption from control paclitaxel nanocrystals (20mgkg -1 ). Imaging of ex-vivo rat ileum samples showed that fluorescently labelled GCPh nanoparticles are mucoadhesive and are taken up by ileum epithelial cells. GCPh nanoparticles were also found to open Caco-2 cell tight junctions. In conclusion, mucoadhesive, drug solubilising GCPh nanoparticles enable the oral absorption of paclitaxel via the saturation of the P-gp pump (by high local drug concentrations) and by particle uptake and tight junction opening mechanisms., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The P-glycoprotein (Pgp) transporter plays a central role in drug disposition by effluxing a chemically diverse range of drugs from cells through conformational changes and ATP hydrolysis. A number of drugs are known to activate ATP hydrolysis of Pgp, but coupling between ATP and drug binding is not well understood. The cardiovascular drug verapamil is one of the most widely studied Pgp substrates and therefore, represents an ideal drug to investigate the drug-induced ATPase activation of Pgp. As previously noted, verapamil-induced Pgp-mediated ATP hydrolysis kinetics was biphasic at saturating ATP concentrations. However, at subsaturating ATP concentrations, verapamil-induced ATPase activation kinetics became monophasic. To further understand this switch in kinetic behavior, the Pgp-coupled ATPase activity kinetics was checked with a panel of verapamil and ATP concentrations and fit with the substrate inhibition equation and the kinetic fitting software COPASI. The fits suggested that cooperativity between ATP and verapamil switched between low and high verapamil concentration. Fluorescence spectroscopy of Pgp revealed that cooperativity between verapamil and a non-hydrolyzable ATP analog leads to distinct global conformational changes of Pgp. NMR of Pgp reconstituted in liposomes showed that cooperativity between verapamil and the non-hydrolyzable ATP analog modulate each other's interactions. This information was used to produce a conformationally-gated model of drug-induced activation of Pgp-mediated ATP hydrolysis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ca 2+ antagonist drugs are widely used in therapy of cardiovascular disorders. Three chemical classes of drugs bind to three separate, but allosterically interacting, receptor sites on Ca V 1.2 channels, the most prominent voltage-gated Ca 2+ (Ca V ) channel type in myocytes in cardiac and vascular smooth muscle. The 1,4-dihydropyridines are used primarily for treatment of hypertension and angina pectoris and are thought to act as allosteric modulators of voltage-dependent Ca 2+ channel activation, whereas phenylalkylamines and benzothiazepines are used primarily for treatment of cardiac arrhythmias and are thought to physically block the pore. The structural basis for the different binding, action, and therapeutic uses of these drugs remains unknown. Here we present crystallographic and functional analyses of drug binding to the bacterial homotetrameric model Ca V channel Ca V Ab, which is inhibited by dihydropyridines and phenylalkylamines with nanomolar affinity in a state-dependent manner. The binding site for amlodipine and other dihydropyridines is located on the external, lipid-facing surface of the pore module, positioned at the interface of two subunits. Dihydropyridine binding allosterically induces an asymmetric conformation of the selectivity filter, in which partially dehydrated Ca 2+ interacts directly with one subunit and blocks the pore. In contrast, the phenylalkylamine Br-verapamil binds in the central cavity of the pore on the intracellular side of the selectivity filter, physically blocking the ion-conducting pathway. Structure-based mutations of key amino-acid residues confirm drug binding at both sites. Our results define the structural basis for binding of dihydropyridines and phenylalkylamines at their distinct receptor sites on Ca V channels and offer key insights into their fundamental mechanisms of action and differential therapeutic uses in cardiovascular diseases., Competing Interests: The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper.