Agent Verapamil Research Articles

A Mechanistic, Enantioselective, Physiologically Based Pharmacokinetic Model of Verapamil and Norverapamil, Built and Evaluated for Drug-Drug Interaction Studies.

The calcium channel blocker and antiarrhythmic agent verapamil is recommended by the FDA for drug–drug interaction (DDI) studies as a moderate clinical CYP3A4 index inhibitor and as a clinical Pgp inhibitor. The purpose of the presented work was to develop a mechanistic whole-body physiologically based pharmacokinetic (PBPK) model to investigate and predict DDIs with verapamil. The model was established in PK-Sim®, using 45 clinical studies (dosing range 0.1–250 mg), including literature as well as unpublished Boehringer Ingelheim data. The verapamil R- and S-enantiomers and their main metabolites R- and S-norverapamil are represented in the model. The processes implemented to describe the pharmacokinetics of verapamil and norverapamil include enantioselective plasma protein binding, enantioselective metabolism by CYP3A4, non-stereospecific Pgp transport, and passive glomerular filtration. To describe the auto-inhibitory and DDI potential, mechanism-based inactivation of CYP3A4 and non-competitive inhibition of Pgp by the verapamil and norverapamil enantiomers were incorporated based on in vitro literature. The resulting DDI performance was demonstrated by prediction of DDIs with midazolam, digoxin, rifampicin, and cimetidine, with 21/22 predicted DDI AUC ratios or Ctrough ratios within 1.5-fold of the observed values. The thoroughly built and qualified model will be freely available in the Open Systems Pharmacology model repository to support model-informed drug discovery and development.

Electrophysiological Examination of the Effects of Classical Pharmacological Agent Verapamil in Plant Based Model System

Homologs of human two pore channels (TPC) were documented in plants thus the examination of the effects of medicines against TPC-related diseases could be performed in plant model systems. Examination of electrical signalling properties - generation of Action Potentials (AP) - in Characeaen macroalgae Nitellopsis obtusa is an established method offering insights into effects of ion channel modulators in vivo. Standardized current clamp and voltage clamp approaches have been used for this purpose for decades. Verapamil is a well-known L-type Ca2+ channel blocker, also it was demonstrated to inhibit Kv channels in a variety of tissues and cells. Investigation of changes in ion currents during AP generation after verapamil treatment was carried out by the application of the C-C, V-C and patch clamp techniques in Nitellopsis obtusa model system. High discretization frequency allowed high temporal resolution analysis of real-time processes such as changes in excitation threshold, AP amplitude and velocity of repolarization phase along with activation/inactivation dynamics of ion channels. These parameters reflected modulation of activity of ion channels involved in AP generation. The effect of verapamil was found to be concentration dependent and manifested as a depolarization of membrane resting potential and excitation threshold, also as an increase of AP duration as well as a prolongation of inactivation times of transient currents during excitation. Our investigation demonstrate direct and rapid effect of verapamil on electrical signalling in plants on single cell level. The research was funded by DAINA – Polish-Lithuanian joint project S-LL-18-1.

Extinction of Antimicrobial Resistant Pathogens Using Silver Embedded Silica Nanoparticles and an Efflux Pump Blocker.

Control measures against antimicrobial resistant bacterial pathogens are important challenges in our daily life. In this study, we discuss the sensitivity and resistance of four bacterial pathogens, Vibrio alginolyticus, Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, to silver-silica hybrid nanoparticles. Successively, by combining with an efflux pump blocking agent Verapamil, we find that these hybrid nanoparticles induce complete mortality to even the most resistive S. aureus. The above pathogens are selected from a pool of 100 bacterial strains resistant to silver nitrate. While S. aureus shows increased resistance to the nanoparticles, the cell wall integrity and genetic stability of V. alginolyticus and E. coli are compromised in the presence of the hybrid nanoparticles. These studies suggest that the antimicrobial properties of the nanoparticles against Gram-negative pathogens originate from increased oxidative stress, which is confirmed by the blocking of reactive oxygen species (ROS) using scavengers such as ascorbic acid and observing DNA damage. The antimicrobial property of the nanoparticle when combined with its nontoxic nature to mammalian cells makes it a promising agent for controlling drug-resistant Gram-negative pathogens.

Modulation of DEG/ENaCs by Amphiphiles Suggests Sensitivity to Membrane Alterations

The bile acid-sensitive ion channel is activated by amphiphilic substances such as bile acids or artificial detergents via membrane alterations; however, the mechanism of membrane sensitivity of the bile acid-sensitive ion channel is not known. It has also not been systematically investigated whether other members of the degenerin/epithelial Na+ channel (DEG/ENaC) gene family are affected by amphiphilic compounds. Here, we show that DEG/ENaCs ASIC1a, ASIC3, ENaC, and the purinergic receptor P2X2 are modulated by a large number of different, structurally unrelated amphiphilic substances, namely the detergents N-lauroylsarcosine, Triton X-100, and β-octylglucoside; the fenamate flufenamic acid; the antipsychotic drug chlorpromazine; the natural phenol resveratrol; the chili pepper compound capsaicin; the loop diuretic furosemide; and the antiarrythmic agent verapamil. We determined the modification of membrane properties using large-angle x-ray diffraction experiments on model lipid bilayers, revealing that the amphiphilic compounds are positioned in a characteristic fashion either in the lipid tail group region or in the lipid head group region, demonstrating that they perturbed the membrane structure. Collectively, our results show that DEG/ENaCs and structurally related P2X receptors are modulated by diverse amphiphilic molecules. Furthermore, they suggest alterations of membrane properties by amphiphilic compounds as a mechanism contributing to modulation.

MP67-09 HIGH-THROUGHPUT AND NON-INVASIVE FUNCTIONAL DRUG SCREENING PLATFORM FOR DROSOPHILA MELANOGASTER MODELS OF NEPHROLITHIASIS

INTRODUCTION AND OBJECTIVES: An experimental study in rats was performed to evaluate the potential protective effects of a calcium channel blocking agent (Verapamil) on the ischemia induced oxidative stress related changes during hyperoxaluric phase. METHODS: A total of 32 rats have been included into the study program. Hyperoxaluria was induced by continuous administration of ethyleneglycol (EG) (0.75%). In addition to hyperoxaluria induction applied in Group1 animals in Groups 2 did receive a calcium channel blocking agent (verapamil) and animals in Group 3 consituted the control group. Histologic alterations of the kidneys including crystal formation were evaluated on days 1,7 and 28, respectively. In addition to the scavenger enzymes (catalase (CAT), superoxide dismutase (SOD), total glutathione (GSH)),malonil dialdehid (MDA) levels, (nitric oxide)NO levelsand the total antioxidant capacity (TAOC) of the kidney were also assessed both in the serum as well as the kidney tissue specimens of the animals in all groups. RESULTS: While the evlautaion of the antioxidant enzyme levels did reveal a significant reduction in animals receiving EG only; application of verapamil was found to be protective with limited reduction with a statistical significance when compared with the control group animals (Sidak-Bonferroni method, alpha1⁄41.000%). Moreover, comparison of kidney tissue T-AOC as well as NO levels in a comparative manner between the groups agan showed a statistically significant difefrence between Group 1 and the other group animals where the antioxidant capacity of the kidney seemed to be protected by verapamil application (Sidak-Bonferroni method, alpha1⁄4 1.000%). Lastly, both serum as well as tissue MDA levels were also statistically significant high in animals receiving EG only when compared with Group 2 and control animals during day7 as well as day 28 evaluation. CONCLUSIONS: Our current results indicate that as a potent calcium channel blocking agent Verapamil may limit the extent of oxidative stress related changes in renal tubules following hyperoxaluria induction and preservation of antioxidant capacity of the kidney along with the limitation of crystallization by this agent may allow us to understand the pathophysiology of stone formation as well as the possible use of this agent to limit both stone formation and recurrence.

Preparation of a multifunctional verapamil-loaded nano-carrier based on a self-assembling PEGylated prodrug

In an effort to prove the inherent side effects of doxorubicin (DOX) and potentially revoke the effects of drug resistance exhibited by cancer cells, we have designed a multifunctional DOX-delivery nano-carrier system able to encapsulate the drug resistance reversal agent Verapamil HCl (VRP·HCl). Hydrophilic short-chain polyethylene glycol (i.e., mPEG) was covalently linked to hydrophobic DOX and a benzoic imine linkage was used to form a linear amphiphilic PEGylated prodrug, namely mPEG-b-DOX. In aqueous solution, the amphiphilic PEG-b-DOX is able to self-assemble to form stable nanoparticles with a DOX loading content of approximately 40wt% and a diameter of ∼143nm. The resulting nanoparticles can simultaneously serve as an anticancer drug conjugate and as a drug carrier system. Here, the hydrophilic VRP could be encapsulated into the nano-carriers via a conventional dialysis method. The loading efficiency in mPEG-b-DOX nano-carrier was determined to be 53.97% and the loading content was found to be 7.71wt%. The VRP-loaded nano-carriers grew slightly in size, to a diameter of ∼177nm. We found that the release of DOX and VRP was much faster at a lower pH value. The biological activity of the nano-carriers were evaluated in vitro and compared with the DOX-loaded system. In doing so we found that the VRP-loaded nano-carrier features a much higher antitumor activity. Furthermore, the combined-system exhibits a significantly enhanced cytotoxicity with an elevated apoptosis rate observed for MCF-7/ADR used as a cell line in this in vitro study. This combinatory system and promising candidate for applications involving DOX chemotherapy proved to be easy to prepare and could be characterized in terms of biocompatibility, biodegradability, loading capacity, pH responsiveness and reversal of drug resistance.

Abstract 206: Intraarterial (IA) Therapy of Cerebral Vasospasm: Comparison of Single Agent vs Multiple Agent Infusion

Background: Cerebral vasospasm remains a major cause of morbidity and mortality in patients with subarachnoid hemorrhage. Vasospasm treatment typically includes IA infusion of vasodilators; however the optimal agent/s have not been elucidated. National surveys suggest that IA treatment algorithms vary between centers and commonly involve use of a single agent. We hypothesize that IA infusion of a cocktail of multiple vasodilators is more efficacious than single agent treatment. Methods: A prospective case series of consecutive patients with cerebral vasospasm (Group 2, N=50 patients, 106 vessels) treated between 2010-13 with IA infusion of a specific cocktail of multiple agents (nitroglycerin, verapamil and nicardipine) at a tertiary center, were compared to a historic control group (Group 1, N=54 patients, 116 vessels) at the same center between 2008-10 treated with a single agent (nicardipine or verapamil). Patient demographics, age, and modified Rankin score (mRS) at discharge and 3 months were collected in the cerebrovascular database. Arterial luminal diameters were measured on cerebral angiograms both pre-infusion (PrID) and post-infusion (PoID). The Improvement Ratio (IR) = (PoID - PrID/PrID) x 100 was calculated and statistically compared between groups using the T test. Results: Group 2 demonstrated statistically significant improvement IR 45.8% (SD 36.6) than Group 1 IR 10.9% (SD 12.0), P<0.001. Multiple agent infusion resulted in an average of 34.9% greater vessel diameter improvement than single agent therapy. Comparison of IR between different single agents within group 1 (nicardipine and verapamil) demonstrated no difference in vessel diameter change. Age of the patient had no effect on efficacy in either group. Patient outcomes measured by mRS were similar at discharge and at 3 months follow-up. Discussion: Treatment of cerebral vasospasm with an IA cocktail of nitroglycerine, verapamil, and nicardipine provides significantly better angiographic improvement of vasospasm than single agent therapy. This effect appears independent of patient age. Outcomes measured by mRS at discharge and 3 months were not significantly different, however will be examined in an ongoing prospective multi-center trial.

Study protocol of Prednisone in episodic Cluster Headache (PredCH): a randomized, double-blind, placebo-controlled parallel group trial to evaluate the efficacy and safety of oral prednisone as an add-on therapy in the prophylactic treatment of episodic cluster headache with verapamil

BackgroundEpisodic cluster headache (ECH) is a primary headache disorder that severely impairs patient’s quality of life. First-line therapy in the initiation of a prophylactic treatment is verapamil. Due to its delayed onset of efficacy and the necessary slow titration of dosage for tolerability reasons prednisone is frequently added by clinicians to the initial prophylactic treatment of a cluster episode. This treatment strategy is thought to effectively reduce the number and intensity of cluster attacks in the beginning of a cluster episode (before verapamil is effective). This study will assess the efficacy and safety of oral prednisone as an add-on therapy to verapamil and compare it to a monotherapy with verapamil in the initial prophylactic treatment of a cluster episode.Methods and designPredCH is a prospective, randomized, double-blind, placebo-controlled trial with parallel study arms. Eligible patients with episodic cluster headache will be randomized to a treatment intervention with prednisone or a placebo arm. The multi-center trial will be conducted in eight German headache clinics that specialize in the treatment of ECH.DiscussionPredCH is designed to assess whether oral prednisone added to first-line agent verapamil helps reduce the number and intensity of cluster attacks in the beginning of a cluster episode as compared to monotherapy with verapamil.Trial registrationGerman Clinical Trials Register DRKS00004716

New recipes for old ingredients: high doses of methylprednisolone and verapamil in cluster headache

Corticosteroids (C) rapidly suppress Cluster Headache (CH) attacks during the time required for the preventative agent verapamil (V), to have effects [1]. However, both drugs are often unsatisfactory. We present the case of a woman, affected by Chronic Cluster Headache (CCH), successfully treated with high doses of C and V. Moreover, we treated similarly 20 Episodic Cluster Headache (ECH) patients with satisfactory results. A 62-years old housewife, in 2006 had an isolated cluster (ICHD-II) of 40 days duration, unresponsive to NSAIDs. In 2008, she had a second cluster, responsive to 6 mg sc sumatriptan; oxygen inhalation was ineffective. Patient was successfully treated with V 240 mg and prednisone (P) 50 mg/day per os for 7 days, tapered in a month.In January 2009, she had a new cluster, that became chronic with 2-8 attacks/24 hours, not responsive to P 50 mg/day, V 320 mg/day, lithium (750 mg/day), valproate (1000 mg/day). When we saw her, in August 2011, she had 5 attacks/24 h, despite taking V per os 320 mg/day. Patient was administered methylprednisolone (MP) 500 mg iv/day for 2 days, then 250 mg for 3 days, followed by P 25 mg per os for 2 days, tapered in 8 days. V was increased gradually to 600 mg/day. In the following month there were no attacks. In September, she presented 1-3 attacks/24h nocturnal and mild, lasting 15 min. V was increased to 680 mg/day with disappearance of attacks. In November, V was slowly reduced to 320 with no recurrence. To the best of our knowledge this is the first report about high doses of iv MP associated with high doses of V per os being effective in CCH. Data are being elaborated on a group of 20 ECH patients treated similarly with good results. If confirmed, our findings warrant the reevaluation of the doses and timing of these drugs in CH with appropriate clinical trials.

Overcoming human P-glycoprotein-dependent multidrug resistance with novel dihydro-β-agarofuran sesquiterpenes

Sixteen ( 1– 16) dihydro-β-agarofuran sesquiterpenes were isolated from the fruits of Maytenus jelskii and evaluated against mammalian cells with a multidrug resistance phenotype mediated by the overexpression of the human P-glycoprotein. Their stereostructures have been elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques, CD studies, chemical correlations and biogenetic means. Eight compounds from this series were discovered as potent chemosensitizers ( 1, 2, 4, 6, 8, 9, 11 and 14), showing similar effectiveness to or higher than the classical P-glycoprotein reversal agent verapamil, a first-generation chemosensitizer, when reversing resistance to daunomycin and vinblastine. Detailed structure–activity relationships revealed that aromatic substituents at the 6 and 9-position of the sesquiterpene scaffold were able to modulate the intensity of inhibition.

Hyperoxaluria-induced tubular ischemia: the effects of verapamil and vitamin E on apoptotic changes with an emphasis on renal papilla in rat model

An experimental study in rats was performed to evaluate the presence and the degree of both tubular apoptotic changes and crystallization at cortical, medullar and papillary regions of the kidney during hyperoxaluric phase and assess the possible protective effects of vitamin E and verapamil on these pathologic changes (particularly in papillary part of the affected kidneys). A total of 32 rats have been included into the study program. Hyperoxaluria was induced by continuous administration of ethylene glycol (0.75%). In addition to hyperoxaluria induction, animals in Groups 2 and 3 did receive a calcium channel-blocking agent (verapamil) and vitamin E, respectively. Histologic alterations of the kidneys including crystal formation together with apoptotic changes were evaluated on days 1, 14 and 28, respectively. Both apoptotic changes and the presence and degree of crystallization were assessed separately in renal cortical region, medulla and particularly papillary parts of the removed kidneys. Although verapamil did well limit the degree of crystal formation and apoptosis and brought it to the same levels observed in control group animals in all parts of the kidneys during intermediate phase, addition of vitamin E was failed to show the same protective effect during both intermediate and late phase evaluations. As demonstrated in our study, the limitation of both crystal deposition and apoptotic changes might be instituted by calcium channel-blocking agents. Clinical application of such agents in the prophylaxis of stone disease might limit the formation of urinary calculi, especially in recurrent stone formers.

A programmed cell death pathway in the malaria parasite Plasmodium falciparum has general features of mammalian apoptosis but is mediated by clan CA cysteine proteases

Several recent discoveries of the hallmark features of programmed cell death (PCD) in Plasmodium falciparum have presented the possibility of revealing novel targets for antimalarial therapy. Using a combination of cell-based assays, flow cytometry and fluorescence microscopy, we detected features including mitochondrial dysregulation, activation of cysteine proteases and in situ DNA fragmentation in parasites induced with chloroquine (CQ) and staurosporine (ST). The use of the pan-caspase inhibitor, z-Val-Ala-Asp-fmk (zVAD), and the mitochondria outer membrane permeabilization (MOMP) inhibitor, 4-hydroxy-tamoxifen, enabled the characterization of a novel CQ-induced pathway linking cysteine protease activation to downstream mitochondrial dysregulation, amplified protease activity and DNA fragmentation. The PCD features were observed only at high (μM) concentrations of CQ. The use of a new synthetic coumarin-labeled chloroquine (CM-CQ) showed that these features may be associated with concentration-dependent differences in drug localization. By further using cysteine protease inhibitors z-Asp-Glu-Val-Asp-fmk (zDEVD), z-Phe-Ala-fmk (zFA), z-Phe-Phe-fmk (zFF), z-Leu-Leu-Leu-fmk (zLLL), E64d and CA-074, we were able to implicate clan CA cysteine proteases in CQ-mediated PCD. Finally, CQ induction of two CQ-resistant parasite strains, 7G8 and K1, reveals the existence of PCD features in these parasites, the extent of which was less than 3D7. The use of the chemoreversal agent verapamil implicates the parasite digestive vacuole in mediating CQ-induced PCD.

The side population of T-cell acute lymphoblasticleukemia cells is sensitive to modulators of PI3K/AKT/ mTOR signaling

The side population (SP), which overexpresses ABC-family membrane transporters including ABCG2 (also referred to as Breast Cancer Resistance Protein or BRCP), is thought to be enriched in cancer stem cells (CSC). SP cells are characterized by the capacity of extruding the Hoechst 33342 dye. The difficulty in eradicating tumors might be due to the fact that conventional treatments target the bulk of the tumor cells leaving behind the CSCs. Since CSCs may be responsible for aggressive behaviour of certain tumors as well as their sustained growth, strategies that target these cells will have significant clinical implications. So far the SP of T-cell acute lymphoblastic leukemia (T-ALL) has not been identified. Here, we have investigated the possible existence of SP cells in T-ALL cell lines and patients. In addition, we have studied if SP cells were sensitive to modulators of the PI3K/Akt/mTOR pathway. SP cells were analyzed using a Cell Lab Quanta SC flow cytometer equipped with an UV lamp and a 488 solid state laser. The Hoechst 33342 dye was excited at 366 nm. SP cells were gated on the FL1/FL3 histogram, while ABCG2 staining was evaluated on the FL2 channel. Hoechst staining specificity was demonstrated by the use of ABC-family transporter blocking agents (verapamil, fumitremorgin C, Ko143). The analysis documented the existence of cells displaying SP features (2.7-7.5%). Flow cytometric analysis demonstrated that the T-ALL SP expresses high levels of ABCG2, whereas the expression of two other ABC-family member transporters, P-gp or MRP1, was not detected. SP cells disappeared in samples treated with BEZ-235 (a dual inhibitor of PI3K/mTOR) or rapamycin (an mTORC1 inhibitor). Our findings indicate that SP cells exist also in T-ALL and are sensitive to PI3K/Akt/mTOR inhibition. Taken together, our preclinical findings strongly suggest that modulators of PI3K/AKT/mTOR could be valuable compounds for treating T-ALL because of their capability of targeting a population enriched in CSCs.

Novel dihydro-β-agarofuran sesquiterpenes as potent modulators of human P-glycoprotein dependent multidrug resistance

P-Glycoprotein (Pgp) overexpression is one factor contributing to multidrug resistance (MDR) in cancer cells and represents one drawback in the treatment of cancer. In an attempt to find more specific and less toxic anticancer MDR-reversal agents, we report herein the isolation, structure elucidation and biological activity of nine new (, and ) and seven known (, and ) dihydro-beta-agarofuran sesquiterpenes from the leaves of Celastrus vulcanicola. Their stereostructures were elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques, CD studies and biogenetic means. All the compounds were assayed on human MDR1-transfected NIH-3T3 cells, in order to determine their ability to reverse the MDR phenotype due to Pgp overexpression. Six compounds from these series (, , , , and ) showed an effectiveness that was similar to (or higher than) the classical Pgp reversal agent verapamil for the reversal of resistance to daunomycin and vinblastine. The structure-activity relationships are discussed.

Photoaffinity Labeling of the Plasmodium falciparum Chloroquine Resistance Transporter with a Novel Perfluorophenylazido Chloroquine

Several models describing how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Further progress requires molecular analysis of interactions between purified reconstituted PfCRT protein and these drugs. We have thus designed and synthesized several perfluorophenyl azido (pfpa) CQ analogues for PfCRT photolabeling studies. One particularly useful probe (AzBCQ) places the pfpa group at the terminal aliphatic N of CQ via a flexible four-carbon ester linker and includes a convenient biotin tag. This probe photolabels PfCRT in situ with high specificity. Using reconstituted proteoliposomes harboring partially purified recombinant PfCRT, we analyze AzBCQ photolabeling versus competition with CQ and other drugs to probe the nature of the CQ binding site. We also inspect how pH, the chemoreversal agent verapamil (VPL), and various amino acid mutations in PfCRT that cause CQ resistance (CQR) affect the efficiency of AzBCQ photolabeling. Upon gel isolation of AzBCQ-labeled PfCRT followed by trypsin digestion and mass spectrometry analysis, we are able to define a single AzBCQ covalent attachment site lying within the digestive vacuolar-disposed loop between putative helices 9 and 10 of PfCRT. Taken together, the data provide important new insight into PfCRT function and, along with previous results, allow us to propose a model for a single CQ binding site in the PfCRT protein.

A verapamil-sensitive chloroquine-associated H+ leak from the digestive vacuole in chloroquine-resistant malaria parasites

Chloroquine resistance in the malaria parasite Plasmodium falciparum has made malaria increasingly difficult to control. Chloroquine-resistant parasites accumulate less chloroquine than their chloroquine-sensitive counterparts; however, the mechanism underlying this remains unclear. The primary site of accumulation and antimalarial action of chloroquine is the internal acidic digestive vacuole of the parasite, the acidity of which is maintained by inwardly-directed H+ pumps, working against the (outward) leak of H+. In this study we have investigated the leak of H+ from the digestive vacuole of the parasite by monitoring the alkalinisation of the vacuole following inhibition of the H+-pumping V-type ATPase by concanamycin A. The rates of alkalinisation observed in three chloroquine-resistant strains were two- to fourfold higher than those measured in three chloroquine-sensitive strains. On addition of chloroquine there was a dramatic increase in the rate of alkalinisation in the chloroquine-resistant strains, whereas chloroquine caused the rate of alkalinisation to decrease in the chloroquine-sensitive strains. The chloroquine-associated increase in the rate of alkalinisation seen in chloroquine-resistant parasites was inhibited by the chloroquine-resistance reversal agent verapamil. The data are consistent with the hypothesis that in chloroquine-resistant parasites chloroquine effluxes from the digestive vacuole, in association with H+, via a verapamil-sensitive pathway.

Synergistic effects of verapamil on pingyangmycin‐induced cytotoxicity and apoptosis in KB cells

Previous studies have shown that pingyangmycin (PYM; bleomycin A5) can induce two distinct modes of cell death (necrosis, apoptosis). At high concentrations, PYM can be considered as an apoptosis mimetic drug. In this study, we explored the possibility that the membrane-modifying agent verapamil might affect the transport function of PYM through the plasma membrane, resulting in inducing apoptosis of tumor cells at low concentration of PYM. Cytotoxicity, flow cytometry and DNA fragmentation assays were used to detect the interaction of verapamil and PYM in human oral carcinoma cell line KB cells. Our results indicated that verapamil can enhance the cytotoxicity of PYM against KB cells with the non-toxic doses (P<0.05). The cell viability at a concentration of 500 microg ml-1 of PYM was 35+/-2% compared with control and 10 microg ml-1 verapamil decreased the cell viability lower to 28+/-1%. In addition, because of the synergistic effect of verapamil, KB cells apoptosis was found to be induced when treated with a lower concentration of PYM (50 microg ml-1) for 24 h by flow cytometry and DNA fragmentation assays. Verapamil was found to enhance PYM-induced cytotoxicity and apoptosis in KB cells. The responsiveness of PYM might be explained by the effective accumulation of PYM by verapamil in KB cells mediated by the inhibition of PYM efflux function of the cells.

Effect of verapamil on urinary stone-forming risk factors

Prevention of recurrent stone formation will only be possible with careful metabolic evaluation and appropriate management. In this present prospective study, a total of 95 patients with calcium oxalate (CaOx) stone disease were evaluated with respect to the effects of a calcium channel blocking agent (verapamil) therapy on stone-forming risk factors. A total of 95 patients with CaOx urolithiasis were well evaluated for the possible specific effects of verapamil administration on stone-forming risk factors during long-term follow-up. All patients had calcium-containing stones with normal renal morphology and function without any urinary tract infection. The follow-up period ranged from 12 to 36.6 months, with a mean value of 24.4 months. The age of the patients (54 male and 41 female; M/F: 1.31) ranged from 20 to 46 years (mean 34.3 years). On metabolic evaluation all patients had some kind of risk factors and patients were independently randomized into two groups, namely group 1 (n = 49): patients receiving calcium entry blocker, verapamil hydrochloride (isoptin 240 mg KKH tablets, oral t.i.d.); group 2 (n = 46): patients receiving no specific therapy (control patients) that were matched for sex and age. Follow-up results (at least 1 year) with respect to the changes in urinary stone-forming risk factors were recorded in both groups. During long-term follow-up patients undergoing no specific therapy did not show a significant change with respect to the urinary levels of stone-forming risk factors when compared with the others receiving verapamil on a regular basis. In the light of our results as well as the literature data, we believe that the pathophysiological mechanisms underlying the effect of verapamil on stone formation (as a result of enhanced crystal deposition) and on the excretion of the urinary stone-forming risk factors have to be well evaluated in further experimental as well as clinical studies. Although the exact mechanism of action is not clear; we may claim that the limitation of internal calcium shift by these agents may also well effect the tubular process related to oxalate handling which ultimately limits its excretion in urine.

The effects of α1-acid glycoprotein on the reversal of chloroquine resistance inPlasmodium falciparum

An in-vitro model based on the semi-automated microdilution technique has been developed for selecting compounds that might be used clinically for the reversal of chloroquine resistance. This was used initially to test the susceptibility of Plasmodium falciparum clone W2 to chloroquine (CQ). The model was then employed to investigate the effects of each of four resistance-reversing agents (verapamil, desipramine, chlorpheniramine and promethazine, at 1 microM) on this parasite's susceptibility to CQ, with and without alpha(1)-acid glycoprotein (AGP), at a patho-physiological concentration (1.25 g/litre), in the culture medium. In the absence of AGP, each of the resistance-reversing agents reduced the median inhibitory concentrations of CQ by 82%-97%, from a baseline value of about 94 ng/ml. In the presence of AGP, however, most of the resistance-reversing agents had much less effect. There appears to be competitive interaction between CQ, the resistance-reversing agents and AGP. The binding kinetics between CQ, resistance-reversing agents, AGP and other plasma proteins will clearly need to elucidated if clinically effective resistance-reversing agents are to be selected in vitro.

Verapamil Induces Upregulation of P-glycoprotein Expression on Human Monocyte Derived Dendritic Cells

Overexpression of P-glycoprotein, a transmembrane drug efflux pump that mediates efflux of chemotherapeutic agents contributes to drug resistance in many leukaemia and other cancerous cells. Non-malignant cells including leukocytes also express P‐glycoprotein, but physiologic functions for P-glycoprotein are poorly defined. Recently, P-glycoprotein expression has been described in human mononuclear phagocytes and Langerhans cells. It has been shown to play a role in phagocytic cell transmigration through endothelial-lined vessels in an ablumenal-lumenal direction, a process that mimics their migration into lymphatic vessels. Using the monoclonal antibody 4E3, and the P-glycoprotein antagonist, verapamil, the expression of P-glycoprotein on human monocyte-derived dendritic cells was evaluated. Dendritic cells used in this study were CD1a+, CD11c+, CD14−, CD80+, CD83+, CD86+ and MHC-IIHigh. The expression of these markers increased significantly as the cells matured. P-glycoprotein expression was upregulated as the dendritic cells matured as well as in the presence of the “inflammatory stress” of the pathogenic bacteria Strept. pyogenes. Addition of verapamil or Strept. pyogenes to the culture medium during the final 24 hours significantly upregulated P-glycoprotein expression. Immortalized cell lines did not upregulate P‐glycoprotein in the presence of verapamil. Evaluation of other normal cells showed that P-glycoprotein upregulation in the presence of verapamil was also a characteristic of macrophages. This novel observation of the upregulation of P-glycoprotein in the presence of verapamil appears to be a characteristic of activated myeloid derived antigen presenting cells and suggest that P-glycoprotein is essential for these cells as when it is blocked, they respond by increasing expression of this protein. In summary, this work describes that human dendritic cells generated from plastic-adherent monocytes rapidly upregulate expression of P-glycoprotein as they mature, and in the presence of inflammatory stress and the pharmacological agent verapamil, which blocks P-glycoprotein activity, suggesting that P-glycoprotein may play a role in activation as well as in migration of dendritic cells.