Preparation of Sustained Release Formulation of Verapamil Hydrochloride Using Ion Exchange Resins.

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 Maximum drug release increased dramatically by increasing the volume of samples withdrawn and fresh dissolution medium added.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.

Dietary bioactive diindolylmethane enhances the therapeutic efficacy of centchroman in breast cancer cells by regulating ABCB1/P-gp efflux transporter.

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.

Development and Validation of a Chiral Liquid Chromatographic Assay for Enantiomeric Separation and Quantification of Verapamil in Rat Plasma: Stereoselective Pharmacokinetic Application.

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 (r2 ≥ 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 Cmax 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).

Upregulation of KCNMA1 facilitates the reversal effect of verapamil on the chemoresistance to cisplatin of esophageal squamous cell carcinoma cells.

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.

Determination of Verapamil Hydrochloride and Norverapamil Hydrochloride in Rat Plasma by Capillary Electrophoresis With End-Column Electrochemiluminescence Detection and Their Pharmacokinetics Study.

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.

Increasing the accumulation of aptamer AS1411 and verapamil conjugated silver nanoparticles in tumor cells to enhance the radiosensitivity of glioma.

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.

Efflux mechanism and pathway of verapamil pumping by human P-glycoprotein.

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.

An Experimental and Theoretical Study of Laser Postionization of Femtosecond-Laser-Desorbed Drug Molecules.

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.

Investigation of the enantioselective interaction between selected drug enantiomers and human serum albumin by mobility shift-affinity capillary electrophoresis.

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 (KA 1844 M-1 ) proved to bind stronger to human serum albumin compared to S-(-)-verapamil (KA 6.6 M-1 ). The association constant of S-(-)-amlodipine (KA 25 073 M-1 ) was found to be slightly higher compared to its antipode (KA 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 (KA 25 101 M-1 ).

Phenylalkylamines in calcium channels: computational analysis of experimental structures.

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.

Prediction methods of drug-drug interactions of non-oral CYP3A4 substrates based on clinical interaction data after oral administrations - Validation with midazolam, alfentanil, and verapamil after intravenous administration and prediction for blonanserin transdermal patch.

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 (Fg) and fraction contribution of CYP3A4 to hepatic intrinsic clearance (fmCYP3A4) 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 Fg, fmCYP3A4 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.

Development of color changing polydiacetylene-based biomimetic nanovesicle platforms for quick detection of membrane permeability across the blood brain barrier.

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.

Rapid detection and structural characterization of verapamil metabolites in rats by UPLC-MSE and UNIFI platform.

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 (MSn ) 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.

Micro-patterned SU-8 cantilever integrated with metal electrode for enhanced electromechanical stimulation of cardiac cells.

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.

Incorporation of drug efflux inhibitor and chemotherapeutic agent into an inorganic/organic platform for the effective treatment of multidrug resistant breast cancer.

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.

Verapamil delivery systems on the basis of mesoporous ZSM-5/KIT-6 and ZSM-5/SBA-15 polymer nanocomposites as a potential tool to overcome MDR in cancer cells.

ZSM-5/KIT-6 and ZSM-5/SBA-15 nanoparticles were synthesized and further modified by a post-synthesis method with (CH2)3SO3H and (CH2)3NHCO(CH2)2COOH 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, N2 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 (CH2)3SO3H and CH2)3NHCO(CH2)2COOH 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.

Evidence for the Interaction of A3 Adenosine Receptor Agonists at the Drug-Binding Site(s) of Human P-glycoprotein (ABCB1).

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 A3 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 [125I]-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 (IC50 2.4 ± 0.6 µM); 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 A3 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.

Identification of Verapamil Binding Sites Within Human Kv1.5 Channel Using Mutagenesis and Docking Simulation.

BACKGROUND/AIMS: The phenylalkylamine class of L-type Ca2+ 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 IC50 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.

TiO2 Photocatalyzed Oxidation of Drugs Studied by Laser Ablation Electrospray Ionization Mass Spectrometry.

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 TiO2 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) TiO2-photocatalysis laser ablation electrospray ionization (LAESI) MS method for the analysis of drug candidates. This method was proven to be compatible with both TiO2-coated glass slides as well as solutions containing suspended TiO2 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.