Dihydropyridine-type Calcium Channel Antagonists Research Articles

In vitro phototoxicity of dihydropyridine derivatives: A photochemical and photobiological study

Some photosensitizing drugs can cause phototoxic skin responses even after systemic administration; therefore, avoidance of undesired side-effects is a key consideration in drug discovery and development. As a prediction tool for phototoxic risk, we previously proposed the monitoring of reactive oxygen species (ROS) generated from compounds irradiated with UVA/B, which can be effective for understanding photochemical/photobiological properties. In this investigation, we evaluated the photosensitizing properties of a novel dihydropyridine derivative, with bradykinin B 2 receptor antagonist activity (compound A) using our ROS assay and several analytical/biochemical techniques. Exposure of compound A, and several dihydropyridine-type calcium channel antagonists to simulated sunlight resulted in the significant production of singlet oxygen, superoxide, or both, which indicates their photosensitive/phototoxic potential. This is consistent with the observation that compound A under UVA/B light exposure caused significant photodegradation and even peroxidation of fatty acid, which could lead to phototoxic dermatitis. Interestingly, the addition of radical scavengers, especially GSH, MPG and BHA, could attenuate the lipid peroxidation, suggesting the involvement of ROS generation in the phototoxic pathways of compound A. In the 3T3 neutral red uptake phototoxicity test, compound A also showed a phototoxic effect on 3T3 mouse fibroblast cells. These findings also support the usefulness of the ROS assay for the risk assessment studies on the drug-induced phototoxicity even at the early stages of pharmaceutical development.

Inhibition of human equilibrative nucleoside transporters by dihydropyridine-type calcium channel antagonists

Dihydropyridine-type calcium channel antagonists, in addition to having a vasodilatory effect, are known to inhibit cellular uptake of nucleosides such as adenosine. However, the nucleoside transporter subtypes involved and the mechanism by which this occurs are not known. Therefore, we have studied the inhibitory effects of dihydropyridines on both human equilibrative nucleoside transporters, hENT-1 and hENT-2, which are the major transporters mediating nucleoside transport in most tissues. Among the dihydropyridines tested, nimodipine proved to be the most potent inhibitor of hENT-1, with an IC 50 value of 60 ± 31 μM, whereas nifedipine, nicardipine, nitrendipine, and felodipine exhibited 100-fold less effective inhibitory activity. Nifedipine, nitrendipine, and nimodipine inhibited hENT-2 with IC 50 values in the micromolar range; however, nicardipine and felodipine had no significant effect on hENT-2. Removal of the 4-aryl ring or changing the nitro group at the 4-aryl ring proved not to be detrimental to the inhibitory effects of dihydropyridines on hENT-1, but resulted in a drastic decrease in their inhibitory effects on hENT-2. Kinetic studies revealed that nimodipine and nifedipine reduced V max of [ 3H]uridine transport without affecting K m. The inhibitory effects of nimodipine and nifedipine could be washed out. In addition, nimodipine and nifedipine inhibited the rate of NBTGR-induced dissociation of [ 3H]NBMPR from hENT-1 cell membrane. We conclude that dihydropyridines are non-competitive inhibitors of hENT-1 and hENT-2, probably working through reversible interactions with the allosteric sites. The inhibitory potencies of dihydropyridines may be associated with the structure of the 4-aryl ring, as well as the ester groups at the C-3 and C-5 positions.

Labedipinedilol-C: A Third-Generation Dihydropyridine-Type Calcium Channel Antagonist Displaying K+ Channel Opening, NO-Dependent and Adrenergic Antagonist Activities

Intravenous and oral labedipinedilol-C showed a dose-dependent long-lasting hypotension and a decrease of heart rate in normotensive and conscious spontaneously hypertensive rats (SHR). In isolated Wistar rat and guinea pig tissues, labedipinedilol-C competitively antagonized (-)isoproterenol-induced cardiac stimulation, tracheal relaxation, and phenylephrine-, CaCl2-, and high-K-induced aorta contractions in a concentration-dependent manner. The estimated pA2 and pKCa values were 8.22+/-0.04 and 7.11+/-0.52, respectively. [H]CGP-12177 binding to ventricle and lung tissues as well as [H]prazosin and [H]nitrendipine binding to brain membranes were inhibited by labedipinedilol-C with Ki values of 2.86, 9.03, 0.39, and 0.05 muM, respectively. The vasorelaxant effects of labedipinedilol-C on phenylephrine (10 microM)-induced contractions were attenuated by removing endothelium, by pretreatment with soluble guanylyl cyclase (sGC) inhibitors ODQ (10 microM) and methylene blue (10 microM), a NOS inhibitor L-NAME (100 microM), a K channel blocker TEA (10 mM), a KATP channel blocker glibenclamide (1 microM), and Ca-dependent K channel blockers apamin (1 microM) and charybdotoxin (0.1 microM). In human umbilical vein endothelial cells (HUVECs), labedipinedilol-C increased NO release, which was significantly inhibited by L-NAME. The Western blot analysis on HUVECs indicated that labedipinedilol-C increased the expression of eNOS. These results indicate that hypotension effects of labedipinedilol-C result from alpha-adrenoceptor and Ca entry-blocking activities and release of NO or NO-related substance from vascular endothelium. The endothelium-independent relaxation of vascular smooth muscle is probably linked to K channel opening and alpha-adrenoceptor-blocking activities.

Nifedipine protects against overproduction of superoxide anion by monocytes from patients with systemic sclerosis

We have reported previously that dihydropyridine-type calcium-channel antagonists (DTCCA) such as nifedipine decrease plasma markers of oxidative stress damage in systemic sclerosis (SSc). To clarify the cellular basis of these beneficial effects, we investigated the effects in vivo and in vitro of nifedipine on superoxide anion (O2•-) production by peripheral blood monocytes. We compared 10 healthy controls with 12 patients with SSc, first after interruption of treatment with DTCCA and second after 2 weeks of treatment with nifedipine (60 mg/day). O2•- production by monocytes stimulated with phorbol myristate acetate (PMA) was quantified by the cytochrome c reduction method. We also investigated the effects in vitro of DTCCA on O2•- production and protein phosphorylation in healthy monocytes and on protein kinase C (PKC) activity using recombinant PKC. After DTCCA had been washed out, monocytes from patients with SSc produced more O2•- than those from controls. Nifedipine treatment considerably decreased O2•- production by PMA-stimulated monocytes. Treatment of healthy monocytes with nifedipine in vitro inhibited PMA-induced O2•- production and protein phosphorylation in a dose-dependent manner. Finally, nifedipine strongly inhibited the activity of recombinant PKC in vitro. Thus, the oxidative stress damage observed in SSc is consistent with O2•- overproduction by primed monocytes. This was decreased by nifedipine treatment both in vivo and in vitro. This beneficial property of nifedipine seems to be mediated by its cellular action and by the inhibition of PKC activity. This supports the hypothesis that this drug could be useful for the treatment of diseases associated with oxidative stress.

Acute and sustained effects of dihydropyridine-type calcium channel antagonists on oxidative stress in systemic sclerosis

Purpose To evaluate the potential antioxidant properties of dihydropyridine calcium channel antagonists in systemic sclerosis. Methods Forty-two patients with systemic sclerosis were included (mean [± SD] age, 54 ± 12 years; mean disease duration, 8 ± 7 years). Plasma markers of oxidative stress (carbonyl residues, advanced oxidation protein products, malondialdehyde, nitrosothiols, and total thiol groups) were determined 72 hours after the discontinuation of usual dihydropyridine treatment (with either nifedipine or nicardipine), shortly after reinitiation of treatment, and 9 to 12 months later (long-term treatment) in 19 of the patients. Baseline values were compared with those in 23 healthy volunteers. Results Mean levels of plasma markers of oxidative stress were much higher in patients with systemic sclerosis than in controls (carbonyls, 0.4 ± 0.1 nmol/mg protein vs. 0.3 ± 0.1 nmol/mg protein, P = 0.0001; advanced oxidation protein products, 111 ± 13 μmol/L vs. 47 ± 7 μmol/L, p = 0.003; malondialdehyde, 11.3 ± 3.3 μmol/L vs. 5.5 ± 1.3 μmol/L, P <0.0001; nitrosothiols, 1.6 ± 0.2 μmol/L vs. 0.6 ± 0.2 μmol/L, P <0.0001). In contrast, thiol levels were lower in systemic sclerosis patients (264 ± 80 μmol/L vs. 435 ± 50 μmol/L, P <0.0001). Short-term treatment led to a significant decrease in oxidative stress markers (carbonyls, 0.3 ± 0.1 nmol/mg protein, P <0.0001), advanced oxidation protein products (60 ± 3 μmol/L, P <0.0001), malondialdehyde (8.8 ± 5.6 μmol/L, p = 0.0002), and nitrosothiols (1.4 ± 0.2 μmol/L, p = 0.0001), but an increase in thiol levels (340 ± 84 μmol/L, P <0.0001). These decreases persisted with long-term treatment. Conclusion Dihydropyridines significantly decrease oxidative stress in systemic sclerosis patients, in both the short and long term.

Modulation by dihydropyridine-type calcium channel antagonists of cytokine-inducible gene expression in vascular smooth muscle cells.

1. The 1,4-dihydropyridine nifedipine is frequently used in the therapy of hypertension and heart failure. In addition, nifedipine has been shown to exert distinct anti-arteriosclerotic effects both in experimental animal models and in patients. In the present study we have investigated the hypothesis that the latter effect of this class of drugs is mediated by an interference with the expression of pro-arteriosclerotic gene products in the vessel wall. Moreover, to elucidate as to whether nifedipine acts via L-type calcium channel blockade, its effects were compared to those of another dihydropyridine, Bay w 9798, which has no calcium-antagonistic properties in concentrations up to 10 microM as verified by superfusion bioassay. 2. Both, nifedipine and Bay w 9798, in concentrations ranging from 0.01 to 1 microM, augmented the interleukin-1beta/tumour necrosis factor-alpha (IL-1beta/TNF-alpha)-induced expression of the inducible isoform of nitric oxide synthase (iNOS) in rat aortic cultured smooth muscle cells (raSMC) 2 - 3 fold, as judged by RT - PCR and Western blot analyses. 3. In contrast, cytokine-induced mRNA expression of monocyte chemoattractant protein 1 (MCP-1) in these cells was down-regulated by more than 60% in the presence of both dihydropyridines, as judged by RT - PCR and Northern blot analyses. 4. Nuclear run-on assays and incubation with the transcription-terminating drug actinomycin D revealed that both drugs acted at the level of mRNA synthesis rather than stability. 5. These findings suggest that 1,4-dihydropyridines such as nifedipine affect the expression of both potentially pro-arteriosclerotic (MCP-1) and anti-arteriosclerotic (iNOS) gene products in the vessel wall at the level of transcription, and that these effects are unrelated to their calcium channel-blocking properties.

Amlodipine Treatment of Cocaine Dependence

Preclinical studies indicate that dihydropyridine-type calcium channel antagonists modulate dopamine neurotransmitter function and can reduce cocaine-reinforced behaviors. Amlodipine, a long-acting dihydropyridine-type calcium channel antagonist related to isradipine and nifedipine, was administered in open label fashion for 12 weeks to 26 cocaine-dependent patients. In subjects expressing cocaine craving, craving significantly declined during the course of the 12 weeks. Five individuals reported flushing, headache, fatigue, nocturia, nausea, and lightheadedness. No conclusions regarding efficacy can be made due to the small number of subjects and the openlabel design.

Nifedipine increases microvascular permeability via a direct local effect on postcapillary venules.

Calcium-channel antagonist drugs are prescribed widely for angina and hypertension. A limiting side effect is edema, which can make heart failure worse. We show that nifedipine, a dihydropyridine-type calcium-channel antagonist, can increase vascular permeability in rat skeletal muscle and skin when injected locally. In nifedipine-injected cremaster muscle, the copper content, used to quantify Monastral blue dye accumulation, was 15.0 +/- 2.4 microgram/g compared with 5.3 +/- 0.7 microgram/g in control preparations (P < 0. 05). The injection of nifedipine in rat skin in vivo increased local plasma leakage in injected sites from 5.5 +/- 1.1 microliter in control sites to 9.9 +/- 2.5, 17.0 +/- 2.4, 24.3 +/- 5.9, and 23.3 +/- 5.4 microliter in sites injected with 10(-10), 10(-9), 10(-8), or 10(-7.2) mol/site, respectively (P < 0.05 in each case compared with control). Vascular labeling techniques using light microscopy, electron microscopy, and microanalysis show that the microvascular site of leakage is not from capillaries but from postcapillary venules of 12-36 micrometer in diameter, the same site that controls the edema response in inflammation. Nifedipine can act within the microcirculation to increase the permeability of the postcapillary venule.

Distinct calcium channel isoforms mediate parathyroid hormone and chlorothiazide-stimulated calcium entry in transporting epithelial cells.

Some cells express multiple calcium channel isoforms that are likely to have distinct functions. The present study used molecular cloning and antisense techniques to identify calcium channel isoforms mediating calcium entry in mouse distal convoluted tubule (DCT) cells. The DCT is the major site of hormone- and diuretic-regulated calcium transport in the kidney. Cellular calcium absorption involves entry through apical membrane calcium channels that are sensitive to dihydropyridine-type calcium channel antagonists. Partial cDNA clones corresponding to one isoform of the calcium channel alpha1 pore-forming subunit, alpha1C, and one isoform of the calcium channel beta accessory subunit, beta3, were isolated by RT-PCR. Full-length transcripts were detected by Northern blot analysis in immortalized DCT cells. Antisense oligonucleotides complementary to the alpha1C sequence inhibited the rise of intracellular calcium ([Ca2+]i) induced by the thiazide diuretic, chlorothiazide (CTZ), but not that induced by parathyroid hormone (PTH). However, antisense oligonucleotides complementary to the beta3 sequence inhibited both CTZ- and PTH-induced rises of [Ca2+]i. beta3 antisense oligonucleotides also inhibited the membrane hyperpolarization induced by CTZ but not that triggered by PTH. Thus, members of the voltage-gated calcium channel family are expressed in DCT cells, where they are responsible for hormone- and drug-induced calcium uptake. The results suggest that DCT cells contain multiple calcium channels with distinct roles in the regulation of cellular calcium.

Intranasal delivery of RS-93522, a dihydropyridine-type calcium-channel antagonist.

Intranasal delivery of RS-93522, a dihydropyridine-type calcium-channel antagonist.

Endothelin-1 enhances responses to sympathetic nerve stimulation in endothelium-denuded and endothelium-intact rabbit ear arteries

In the rabbit isolated and endothelium-denuded ear artery, endothelin-1 (1-10 nM) elicited concentration-dependent vasoconstrictor responses. Lower concentrations of endothelin-1 (0.1, 0.3 and 1 nM) with little or no direct vasoconstrictor action significantly enhanced responses to sympathetic nerve stimulation in both endothelium-denuded and endothelium-intact arteries. The vasoconstrictor action of endothelin-1 and its enhancing effect on stimulation-induced responses were significantly decreased by the presence of the dihydropyridine-type calcium channel antagonist nicardipine (10 nM). The enhancing effect of low concentrations of endothelin-1 on responses to sympathetic nerve stimulation may play a role in the regulation of vascular tone.

Chromatographic separation of the diastereomers of a dihydropyridine-type calcium channel antagonist as the bis-3,5-dinitrobenzoates

The diastereomeric components of the calcium channel antagonist RS-93522-004 are separated as the bis-3,5-dinitrobenzoate ester by reversed-phase high-performance liquid chromatography. The diastereomer ratio determination is shown to be precise, accurate and sensitive, and is not affected by the reaction yield of the derivatization with 3,5-dinitrobenzoyl chloride. The four individual stereoisomers of RS-93522-004 were independently shown to have equal reactivity toward 3,5-dinitrobenzoyl chloride.