New phenotypic cytotoxicity assay for ROS-inducing compounds using rat renal epithelial cells.

An important mechanism of chemical toxicity is the induction of oxidative stress through the production of excess reactive oxygen species (ROS). In this study, we show that the level of drug-induced ROS production between NRK52E and HepG2 cells is significantly different for several marketed drugs and a number of Takeda's internal proprietary compounds. Nifedipine, a calcium channel blocker and the initial focus of the study, was demonstrated to promote in vitro ROS production and a decrease in cell viability in NRK52E cells but not HepG2 cells. ROS production after nifedipine treatment was inhibited by a NOX inhibitor (GKT136901) but not the mitochondrial NADH dehydrogenase inhibitor, rotenone, suggesting that nifedipine decreases NRK52E cell viability primarily through a NOX-mediated pathway. To understand the breadth of NOX-mediated ROS production, 12 commercially available compounds that are structurally and/or pharmacologically related to nifedipine as well as 172 internal Takeda candidate drugs, were also evaluated against these two cell types. Over 15 % of compounds not cytotoxic to HepG2 cells (below 50 µM) were cytotoxic to NRK52E cells. Our results suggest that a combination of cell viability data from both NRK52E and HepG2 cells was superior for the prediction of in vivo toxicity findings when compared to use of only one cell line. Further, the NRK52E cell viability assay is a good predictor of NOX-mediated ROS production and can be used as a follow up assay following a negative HepG2 response to aid in the selection of suitable compounds for in vivo toxicity studies.

Nifedipine toxicity is exacerbated by acetyl l-carnitine but alleviated by low-dose ketamine in zebrafish in vivo.

Calcium channel blocker (CCB) poisoning is a common and sometimes life-threatening emergency. Our previous studies have shown that acetyl l-carnitine (ALCAR) prevents cardiotoxicity and developmental toxicity induced by verapamil, a CCB used to treat patients with hypertension. Here, we tested whether toxicities of nifedipine, a dihydropyridine CCB used to treat hypertension, can also be mitigated by co-treatment with ALCAR. In the zebrafish embryos at three different developmental stages, nifedipine induced developmental toxicity with pericardial sac edema in a dose-dependent manner, which were surprisingly exacerbated with ALCAR co-treatment. Even with low-dose nifedipine (5 µm), when the pericardial sac looked normal, ALCAR co-treatment showed pericardial sac edema. We hypothesized that toxicity by nifedipine, a vasodilator, may be prevented by ketamine, a known vasoconstrictor. Nifedipine toxicity in the embryos was effectively prevented by co-treatment with low (subanesthetic) doses (25-100 µm added to the water) of ketamine, although a high dose of ketamine (2 mm added to the water) partially prevented the toxicity.As expected of a CCB, nifedipine either in the presence or absence of ketamine-reduced metabolic reactive oxygen species (ROS), a downstream product of calcium signaling, in the rapidly developing digestive system. However, nifedipine induced ROS in the trunk region that showed significantly stunted growth indicating that the tissues under stress potentially produced pathologic ROS. To the best of our knowledge, these studies for the first time show that nifedipine and the dietary supplement ALCAR together induce adverse effects while providing evidence on the therapeutic efficacy of subanesthetic doses of ketamine against nifedipine toxicity in vivo.

Lipid emulsion alleviates the vasodilation and mean blood pressure decrease induced by a toxic dose of verapamil in isolated rat aortae and an in vivo rat model.

This study aimed to examine the effects of lipid emulsion on the vasodilation and cardiovascular depression induced by toxic doses of calcium channel blockers. The effects of lipid emulsion on the vasodilation induced by bepridil, verapamil, nifedipine, and diltiazem were investigated in isolated endothelium-denuded rat aortae. The effect of lipid emulsion on the comparable hemodynamic depression induced by the continuous infusion of a toxic dose of either verapamil or diltiazem was examined in an in vivo rat model. The results showed the following decreasing order for the magnitude of lipid emulsion-mediated inhibition of vasodilation: bepridil, verapamil, nifedipine, and diltiazem. Lipid emulsion (0.5-2%) reversed the vasodilation induced by a toxic dose of verapamil, whereas only a higher concentration (2%) reversed the vasodilation induced by a toxic dose of diltiazem. Pretreatment with lipid emulsion alleviated the systolic and mean blood pressure decreases induced by a toxic dose of verapamil, whereas it had no effect on the decrease induced by diltiazem. Taken together, these results suggest that lipid emulsion alleviates the severe vasodilation and systolic blood pressure decrease induced by a toxic dose of verapamil, and this alleviation appears to be associated with the relatively high lipid solubility of verapamil.

OptoDyCE as an automated system for high-throughput all-optical dynamic cardiac electrophysiology.

The improvement of preclinical cardiotoxicity testing, discovery of new ion-channel-targeted drugs, and phenotyping and use of stem cell-derived cardiomyocytes and other biologics all necessitate high-throughput (HT), cellular-level electrophysiological interrogation tools. Optical techniques for actuation and sensing provide instant parallelism, enabling contactless dynamic HT testing of cells and small-tissue constructs, not affordable by other means. Here we show, computationally and experimentally, the limits of all-optical electrophysiology when applied to drug testing, then implement and validate OptoDyCE, a fully automated system for all-optical cardiac electrophysiology. We validate optical actuation by virally introducing optogenetic drivers in rat and human cardiomyocytes or through the modular use of dedicated light-sensitive somatic 'spark' cells. We show that this automated all-optical approach provides HT means of cellular interrogation, that is, allows for dynamic testing of >600 multicellular samples or compounds per hour, and yields high-content information about the action of a drug over time, space and doses.

A novel vesicular transdermal delivery of nifedipine - preparation, characterization and in vitro/in-vivo evaluation.

Nifedipine is a calcium channel blocker extensively used in the treatment of anginal and hypertension. On oral administration it undergoes extensive first pass metabolism, which outweighs its absorbance through gastrointestinal tract (GIT) and bioavailability of the drug in systemic circulation. As an alternative to oral route transdermal route of drug delivery was developed. In the present investigation, proniosomes are prepared by varying the ratio of span-40, lecithin, aqueous phase and polymer. Formulation containing span-40, lecithin, isopropyl alcohol, 0.1% glycerol (5:5:4) and HPMC (2%) showed smaller vesicle size, high entrapment efficiency. The niosomal formation after hydration and their surface morphology of optimized formulation was studied by Motic and transmission electron microscopy. FTIR and differential scanning calorimetry studies were performed to unravel and understand the solid state properties of the drug and chemical interaction with formulation excipients. The ex-vivo Franz-diffusion studies were carried out in pH 6.8 using rat skin and the results showed better permeability of niosomes with good steady state flux and enhancement ratio suggesting the potential of proniosomal carriers for improved transdermal delivery of nifedipine. Skin irritation studies for 7 days, showed that the drug when formulated as proniosomes to be non-irritant with no erythemia development compared to pure drug. From the bio-distribution studies, the vesicles prepared with hydroxy propyl methyl cellulose with span-40 was found to be ideal batch as the concentration of drug at target site was higher.

Glycidipine, a promising hypotensive and cardioprotective agent.

Toxicological pharmacological study of the molecular complex of nifedipine and glycyrrhizic acid 1:10 (glycidipine) obtained using mechanochemical technique was carried out. High hypotensive and cardioprotective effects of the agent were demonstrated. Chronic administration (45 days) produced no toxic effects in vital organs and systems of Wistar rats and ISIAH rats.

Therapy for nifedipine-induced gingival overgrowth by Saireito in rats.

OBJECTIVE: A calcium antagonist, nifedipine, causes gingival overgrowth as a side effect. It has been confirmed that the Japanese traditional medicine, Saireito, inhibits the nifedipine-induced proliferation of gingival fibroblasts in vitro. We performed an in vivo experiment to determine whether Saireito has a therapeutic use in the treatment of nifedipine-induced gingival overgrowth. METHODS: The rats had significant gingival overgrowth induced by the administration of nifedipine. The space between the submandibular incisors and the width of buccal gingiva of maxillary left first molar were macroscopically measured. The buccal gingiva was microscopically examined. RESULTS: Eight weeks after Saireito was administrated together with nifedipine, Saireito decreased both the incisor space and the gingiva width which had been enlarged by nifedipine and furthermore, the area of connective tissue of nifedipine + Saireito group was significantly smaller than that of nifedipine alone. CONCLUSION: In conclusion, Saireito may be clinically effective in therapy for calcium antagonist-induced gingival overgrowth.

Verapamil toxicity dysregulates the phosphatidylinositol 3-kinase pathway.

OBJECTIVES: Recent animal research and clinical case reports suggest benefit from high-dose insulin therapy (HDIT) for the treatment of calcium channel blocker (CCB) toxicity. One molecular signaling pathway, the phosphatidylinositol 3-kinase (PI3K) pathway, that contributes to CCB toxicity and the efficacy of HDIT, was examined for a role in this phenomenon. METHODS: A differentiated 3T3-L1 adipocyte model system was utilized to characterize metabolic and molecular signaling events dysregulated in response to acute CCB toxicity. Glucose uptake assays were performed in the presence of representatives of three classes of CCB drugs, and the ability of HDIT to reverse observed inhibition was assessed. Western blot analyses were utilized to probe which insulin-dependent signaling pathway was inhibited by CCB toxicity. RESULTS: Representative compounds from the dihydropyridine and phenylalkylamine classes of CCBs were more effective at inhibiting glucose uptake in differentiated 3T3-L1 adipocytes than a representative from the benzothiazepine class. Phosphorylation at serine 473 of the Akt protein (P-Akt), a protein representing a common pathway for insulin receptors (IR), insulinlike growth factor receptors (IGFR), and hybrid receptors formed by IR and IGFR subunits, was abolished in the presence of toxic doses of the phenylalkylamine CCB verapamil. Phosphorylation at serine 473 of Akt was rescued in the presence high concentrations of insulin. CONCLUSIONS: These data suggest that dysregulation of the insulin-dependent PI3K pathway is partially responsible for insulin resistance and the hyperglycemic state observed in response to acute CCB toxicity.

Pemphigus and pemphigoid-like effects of nifedipine on in vitro cultured normal human skin explants.

BACKGROUND: A variety of drugs have been implicated in the onset and exacerbation of pemphigus and bullous pemphigoid. The demonstration of biochemical acantholysis in skin explants to various drugs in the absence of autoantibodies, in which the tested drugs evoke a biochemical reaction that leads to desmosomal function loss, may be a valuable adjunct to patient management by confirming the suspicion of drug-related pemphigus or bullous pemphigoid. OBJECTIVE: To determine whether a skin explant model might serve as a possible in vitro correlate of drug-induced pemphigus and pemphigoid-like effects related to the calcium channel blocker nifedipine. METHODS: Normal human breast skin obtained from nonpemphigus and nonpemphigoid patients undergoing mastectomy was cultured with nifedipine at final concentrations of 2, 4, and 8 mM. The drug effect on skin explants evidenced by morphologic changes was evaluated by microscopy by three observers. RESULTS: Five out of seven explants cultured with nifedipine at concentrations ranging from 2 to 8 mM exhibited obvious morphologic changes of two types: intraepithelial (or pemphigus-type) splittings and subepithelial (or pemphigoid-type) splittings. Two explants showed no acantholysis and no subepithelial splittings. Control cultures without polyethylene glycol 200 (PEG) showed no changes. Skin control samples cultured in medium supplemented with 10% PEG displayed vacuolar degeneration throughout the entire epidermis, but no sign of cell-cell dyshesion or dermo-epidermal detachment. CONCLUSIONS: A type of skin susceptibility to nifedipine may be genetically determined, with some nifedipine-treated patients developing an acantholytic reaction and others a subepidermal bullous eruption.

A calcium agonist, Bay k 8644, suppresses the embryotoxic effects induced by dihydropyridines calcium channel blockers in cultured rat embryos.

Day 9 rat embryos were exposed to 1,4-dihydropyridine calcium channel blockers; nifedipine (NIF), nicardipine (NIC) or nitrendipine (NIT), for 48 hr in the whole embryo culture system. There were dose-dependent growth retardation and abnormalities, predominantly in cardiovascular system. The three compounds exhibited very similar pattern of dysmorphogenic effects, but the potency of these compounds were quantitatively different. The incidences of embryos with the abnormalities were 100%, 100% and 85% following either exposure of NIF, NIC or NIT at concentration of 300, 8 and 15 microM, respectively. This study was to investigate whether these blocker-induced embryotoxicity was due to calcium channel blocking properties themselves in the embryos. Day 9 rat embryos were co-exposed to 1,4-dihydropyridine calcium channel agonist, Bay k 8644 (BAY) and each calcium channel blocker under the same culture condition. The retarded embryonic growth induced by 200 or 300 microM of NIF, 8 microM of NIC and 15 microM of NIT nearly of completely ameliorated when embryos were co-exposed with BAY at one-third or half concentration of each calcium channel blocker. Supplementation of BAY reduced the incidence of abnormalities by NIF-, NIC- and NIT-alone. These results suggested that one of mechanisms for embryotoxicity induced by calcium channel blocker was directly related to channel blocking property of the chemicals.

Effects of nilvadipine (a dihydropyridine-type calcium entry blocker) on cerebral blood flow in acute experimental brain ischemia in rats.

The purpose of the present study was to examine the effect of nilvadipine (a dihydropyridine-type calcium entry blocker) on the cerebral circulation, experimental models of cerebral ischemia were used to measure abrupt changes in the cortical cerebral blood flow (CBF) at the 'penumbra' (the boundary region between the anterior cerebral artery (ACA) and the middle cerebral artery (MCA)). Left middle cerebral artery occlusion (MCAO) was performed in Sprague--Dawley rats, and then 8 or 16 micrograms kg-1 of nilvadipine was administered to two groups of the rats. The former group was referred to as the 8-MCAO(+) group (n = 8), and the latter group, the 16-MCAO(+) group (n = 8). To a control group (n = 8), only the solvent of nilvadipine was administered. After the administration in each group, CBF and the mean arterial blood pressure (MAP) were continuously measured for 60 min. The MAP in both the 8-MCAO(+) and 16-MCAO(+) groups was significantly lower than in the control group for 25 min after nilvadipine administration (Wilcoxon 2 sample test, p < 0.05). Also, the CBF in the 16-MCAO(+) group was significantly lower (about 20%) for 55 minutes than that in the control group. On the other hand, the CBF in the 8-MCAO(+) group three minutes after nilvadipine administration was not significantly lower than in the control group (Wilcoxon 2 sample test). Our findings confirmed that nilvadipine (8 micrograms kg-1) maintained CBF while lowering the blood pressure in the experimental model of acute cerebral ischemia.

Interaction between nifedipine and digoxin in rats.

Possible nifedipine-digoxin interaction was investigated in rats by comparing lethal doses of intravenously infused digoxin in control and experimental rats. In the experimental rats, nifedipine was administered intraperitoneally, 30 minutes prior to infusing digoxin at a constant rate of 40mcg per minute. Results indicate that nifedipine administered within the dosage range 0.5-2.0mg per kg rat body weight, lowered the lethal dose of intravenously infused digoxin by 26-38% compared with control rats, thus indicating a synergistic effect between the two drugs. There was very little dose dependence of this effect. It is concluded that concomitant administration of nifedipine and digoxin in humans may lead to drug interactions.

Modulation of cytostatic drugs by nifedipine in two heterotransplanted human testicular-cancer cell lines differing in their sensitivity to standard agents.

Drug resistance is an important clinical problem in testicular cancer patients with relapsed or refractory disease after first-line chemotherapy. Here we report that the relative reduction in tumour volume in nude mice heterotransplanted with either H 12.1 or H 23.1 human testicular cancer cell lines was significantly increased by addition of the calcium antagonist nifedipine to the maximum tolerated dose (MTD) of cisplatin (DDP). The mean reduction in relative tumour volume at day 30 (rVR) reached statistical significance for both cell lines following combination therapy of DDP with nifedipine compared to DDP alone (55 +/- 7% versus 12 +/- 4% for H 23.1 and 60 +/- 9% vs. DDP with nifedipine has also been confirmed in H 12.1 cells in vitro. However, in vivo, this combination was associated with a concordant increase in therapeutic toxicity. In contrast, no improvement in in vivo anti-tumour activity was obtained by combining similar dose-schedules of nifedipine with the MTD of epirubicin, or with MTDs of vinblastine or etoposide. These results are in agreement with our immunohistochemical finding that H 12.1 and H 23.1 do not over-express the Pgp 170 glycoprotein which mediates the multiple drug resistance (MDR) phenotype and involves both anthracyclines and vinblastine, but not DDP. We conclude that another Pgp-MDR modulator, nifedipine, is able to increase the anti-tumour activity of DDP in vivo and in vitro via a specific but as yet unknown mechanism, which is most likely not MDR-related. However, the increased anti-tumour activity is, in vivo, associated with a considerable increase in overall toxicity. Further studies are necessary to decrease therapeutic toxicity, before clinically relevant models for modifiers of DDP-resistance could possibly be applied to patients.

Safety and efficacy of amlodipine added to hydrochlorothiazide therapy in essential hypertension.

We compared amlodipine, a dihydropyridine calcium antagonist, to placebo as add-on therapy to hydrochlorothiazide in 91 hypertensive patients inadequately controlled on hydrochlorothiazide (50 mg/d for four weeks). This was a double-blind, randomized, multicenter, parallel group-trial; 45 patients received placebo and 46 received amlodipine in doses of 2.5 to 10 mg qd (mean 9 mg/d). Supine blood pressure systolic/diastolic, mean +/- SE mm Hg) 24-hour postdose was significantly reduced by 14.2 +/- 2.3/11.7 +/- 1, compared to placebo, 4.5 +/- 2.7/5 +/- 1.2. Standing blood pressure was similarly reduced: amlodipine by 14 +/- 2.7/12.5 +/- 1.2; placebo by 3 +/- 2.1/5.8 +/- 1.2. This reduction in blood pressure was attained without any significant changes in pulse rate, EKG, and serum lipids (triglycerides were reduced in the amlodipine group by 42.9 mg/dL, P = .023). Only two patients had side effects requiring discontinuation from the study (both in the amlodipine group). Side effects occurred in 27 amlodipine-treated patients (11 with peripheral edema) and 18 patients in the placebo (three with peripheral edema) group. Investigator's assessment of therapeutic effect and tolerability, and the percent of responders v nonresponders was also in favor of amlodipine. Thus amlodipine administered once daily is an effective and safe agent for second-step therapy in mild to moderate essential hypertension.

[Effect of nifedipine (corinfar) on cerebrovascular circulation in patients with exertion-induced stenocardia]. / Vliianie nifedipina (korinfara) na mozgovoe krovoobrashchenie u bol'nykh so stenokardiei napriazheniia.

Headache as a side effect of corinfar treatment courses for angina of effort was more commonly seen in patients with pretreatment dystonic rheoencephalographic changes. A single corinfar dose produced a drop of venous tone. Corinfar-associated headache was less common in patients after long-term treatment with high-dose nitroglycerin.

Diltiazem, nifedipine, and their combination in patients with stable angina pectoris: effects on angina, exercise tolerance, and the ambulatory electrocardiographic ST segment.

The efficacy and safety of oral nifedipine and diltiazem were compared in 20 patients with stable angina pectoris with use of a placebo run-in, randomized, double-blind titration to maximal effect crossover protocol. The effects of treatment withdrawal were also analyzed. All patients received placebo for 2 weeks and were then randomly assigned to receive either diltiazem or nifedipine. A 2 week drug titration phase in which patients received either diltiazem (180 to 360 mg/day) or nifedipine (30 to 120 mg/day) in three divided doses was followed by a 1 week maintenance phase. Patients then received placebo for 1 to 2 weeks, followed by crossover to the other treatment regimen and a second placebo washout period of 1 week. Patients (n = 13) who remained symptomatic on both diltiazem and nifedipine during the monotherapy periods entered a 3 week combination treatment phase, followed by a final 1 week placebo washout period. Frequency of angina, nitroglycerin consumption, exercise tolerance (Naughton protocol), and frequency of daily episodes of ST segment deviations on the electrocardiogram (1 mm of ST segment depression persisting for at least 1 min with and without chest pain) on an ambulatory electrocardiographic monitor were assessed during the baseline placebo, active monotherapy, placebo withdrawal, and combination treatment phases. Plasma drug levels were also measured. Compared with initial placebo values, the frequency of angina and the amount of nitroglycerin treatment were reduced by both diltiazem (p less than .001) and nifedipine (p less than .02). Diltiazem was more effective than nifedipine in reducing angina (p less than .02). Exercise duration increased with both drugs (p less than .0001). Diltiazem was significantly better than nifedipine in reducing the episodes of ST segment depression on the ambulatory monitor (p less than .01). Diltiazem reduced the resting heart rate (p less than .01); both drugs reduced the resting blood pressure and rate-pressure product. Overall, combination therapy was more effective in patients who did not maximally respond to diltiazem or nifedipine alone with respect to anginal and exercise variables and in reducing blood pressure at rest and during exercise. Plasma drug levels could not predict an individual patient's treatment response. Diltiazem may increase nifedipine drug levels when the drugs are combined. Fewer side effects were observed with diltiazem than nifedipine; the most side effects were seen with combination treatment. There were no apparent withdrawal effects observed with either treatment regimen.(ABSTRACT TRUNCATED AT 400 WORDS)

Hyperphalangeal bones induced in rat pups by maternal treatment with nifedipine.

Hyperphalangeal bones were found in postnatal rat pups of mothers treated with nifedipine during pregnancy. The anomaly occurred only at the region between the middle and distal phalanges of the 3rd and 4th fingers and toes. The critical periods of the anomaly were days 13 and 14 of pregnancy for the fingers, and days 14 and 15 of pregnancy for the toes. The incidences were dose-related, being more than 90% in both fingers and toes at a single dose of 150 mg/kg, and even more marked at the 4th digits than at the 3rd digits. Neither right/left difference nor sex difference was manifested in the incidence of the anomaly.

Enalapril in experimental hypertension and acute heart failure: comparison with calcium channel blockers.

The hemodynamic effects of enalaprilat (MK-422) and lisinopril (MK-521) were compared with the calcium channel blocker felodipine in dogs with ischemic left ventricular (LV) failure. The combination of nitrendipine plus enalapril was also examined in ischemic failure and in rats with spontaneous hypertension. In anesthetized dogs coronary embolization with 50 micron plastic microspheres reduced cardiac output and LV dP/dt max by approximately 40%, and LV end-diastolic pressure increased to greater than 13 mm Hg. Enalaprilat and lisinopril reduced mean arterial pressure by a maximum of 20 mm Hg and total peripheral resistance by approximately 30%. Left ventricular dP/dt:LVP, which was substantially decreased by embolization, was slightly increased by both angiotensin converting enzyme (ACE) inhibitors. The calcium entry blockers felodipine and nitrendipine qualitatively produced many of the same hemodynamic effects as the ACE inhibitors, but, in addition, they markedly reduced coronary resistance, increased myocardial blood flow, and did not alter cardiac contractility (LV dP/dt max). In spontaneously hypertensive rats single doses of nitrendipine (1.25 to 5.0 mg/kg per os) and enalapril (0.3 and 3.0 mg/kg per os) reduced mean arterial pressure, but differences were observed in the onset (enalapril 2 h versus nitrendipine 0.5 h), the duration of action, and magnitude of effect. In terms of blood pressure lowering, nitrendipine, 5.0 mg/kg per os, was clearly additive to 3.0 mg/kg per os of enalapril, but other combinations (enalapril, 3 mg/kg per os plus 0.625 mg/kg of nitrendipine or enalapril, 0.3 mg/kg per os plus 0.625 mg/kg nitrendipine) were not.(ABSTRACT TRUNCATED AT 250 WORDS)

Nifedipine therapy in angina pectoris: evaluation of safety and side effects.

Nifedipine, a calcium-channel blocker, differs in structure and mode of action not only from the nitrates and beta blockers but also from the other well-known calcium-channel blockers. These differences have important implications for the treatment of angina pectoris. Our clinical experience over a period of 6 years with all types of angina patients--mostly those with chronic stable angina but also those with Prinzmetal's and unstable angina-corroborates the efficacy reports published in the world literature. Although the safety profile of nifedipine has been generally regarded as favorable, a few reports of clinically significant adverse effects in specific patient groups have appeared in the literature. In order to provide a more comprehensive assessment of safety, the records of over 3000 patients treated with nifedipine in open and controlled multiple-dose studies were tabulated and analyzed. Of special concern were patients with clinically significant adverse experiences, patients with a concomitant diagnosis of congestive heart failure (CHF), patients who were being treated concurrently with beta blockers, and patients who had been taking nifedipine for more than 6 months. Results of this analysis confirm that nifedipine can be safely administered to a broad spectrum of angina patients, including those with a concomitant diagnosis of CHF and those receiving concurrent therapy with beta-blocking agents.