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Objectives: Isradipine is a dihydropyridine calcium channel inhibitor that has demonstrated concentration-dependent neuroprotective effects in animal models of Parkinson's disease (PD) but failed to show efficacy in a phase 3 clinical trial. The objectives of this study were to model the plasma pharmacokinetics of isradipine in study participants from the phase 3 trial; and, to investigate associations between drug exposure and longitudinal clinical outcome measures of PD progression., Methods: Plasma samples from nearly all study participants randomized to immediate-release isradipine 5-mg twice daily (166 of 170) were collected for population pharmacokinetic modeling. Estimates of isradipine exposure included apparent oral clearance and area under the concentration-time curve. Isradipine exposure parameters were tested for correlations with 36-month changes in disease severity clinical assessment scores, and time-to-event analyses for initiation of antiparkinson therapy., Results: Isradipine exposures did not correlate with the primary clinical outcome, changes in the antiparkinson therapy-adjusted Unified Parkinson's Disease Rating Scale parts I-III score over 36 months (Spearman rank correlation coefficient, r s : 0.09, P = 0.23). Cumulative levodopa equivalent dose at month 36 was weakly correlated with isradipine plasma clearance (r s : 0.18, P = 0.035). This correlation was sex dependent and significant in males, but not females. Those with higher isradipine exposure had decreased risk of needing antiparkinson treatment over 36 months compared with placebo (hazard ratio: 0.87, 95% CI: 0.78-0.98, P = 0.02)., Interpretation: In this clinical trial, higher isradipine plasma exposure did not affect clinical assessment measures of PD severity but modestly decreased cumulative levodopa equivalent dose and the time needed for antiparkinson treatment initiation., Trial Registration: ClinicalTrials.gov NCT02168842., (© 2021 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Isradipine is a dihydropyridine calcium channel blocker (CCB) commonly used as vasodilator with antihypertensive properties. A remote-controlled release formulation for isradipine would substantially improve the clinical outcomes of the patients requiring chronic long-term treatment. In this work, sustained release (SR) tablets of isradipine, composed of hydroxypropylmethyl cellulose (HPMC), have been produced by wet granulation and their in vitro and in vivo characterization was compared to a conventional tablet dosage form of immediate release (IR) as preliminary assessment. Tablets composed of 15.0% (wt/wt) HPMC exhibited a SR profile over a period of 24 hours. The release of isradipine followed a Fickian diffusion pattern obeying to the first order kinetics and the extent of absorption was even higher in comparison to the developed conventional tablets, which showed immediate drug release. In vivo studies were carried out in rabbits, showing that the extent of isradipine absorption from the developed tablets was higher in comparison to IR tablets due to the modified release profile obtained for the former ( p  < 0.05). Our results suggest that SR tablets of isradipine are an efficient solid dosage form to overcome the limitations encountered in conventional IR tablets.

The present study demonstrated the systematic adaptation of quality by design-integrated approach for the development of novel nanostructured lipid carrier (NLC) of an anti-hypertensive drug isradipine (ISD) to address the inherent challenges such as low solubility and low oral bioavailability. Plackett-Burman design was used for preliminary screening of significant process and formulation variables (p <0.05), which were further processed using Box-Behnken design for the attainment of optimization goal that is, mean particle size (85.7 ± 7.3 nm), drug entrapment efficiency (87.4 ± 3.29%), and in vitro drug release characteristics (92.89 ± 5.47%). The optimized ISD-NLC formulation also demonstrated well-dispersed uniform-shaped particles (polydispersity index 0.207 ± 0.029), high gastrointestinal fluid stability (zeta potential -10.17 ± 0.59 mV), and higher in vitro gut permeation (21.69 ± 2.38 μg/cm 2 of ISD-NLC as compared to 11.23 ± 1.74 μg/cm 2 in ISD suspension). Furthermore, lipolysis studies were performed for the purpose of in vivo fate, and significantly higher drug content of ISD from ISD-NLC in aqueous phase was found (72.34 ± 4.62%) as compared to drug suspension (3.01 ± 0.91%). Relative bioavailability of ISD-NLC and ISD suspension was increased by 4.2-fold and 1.78-fold in the absence and presence of cycloheximide which is a lymphatic uptake inhibitor revealing lymphatic uptake of ISD-NLC in bioavailability improvement. Hence, systematic adaptation of quality by design integrated approach improved gut permeation and potential solubilizaton fate (dynamic lipolysis) of ISD-NLC, which further improved the lymphatic uptake and biodistribution of drug thereby promisingits in vivo prospect and clinical efficacy., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Context Isradipine is an effective calcium channel blocker used in the management of hypertension. It undergoes extensive first pass metabolism and has low oral bioavailability. Hence we attempted to develop isradipine-loaded invasomes. Objective The purpose of this work was to prepare and characterize invasomes carrier for isradipine, and to evaluate the optimized formulation obtained for pharmacodynamic study. Materials and methods Isradipine-loaded invasomes were prepared by conventional thin layer evaporation technique using Phospholipon® 90G, β-citronellene (terpene) and ethanol. Prepared formulations were characterized in terms of size, size distribution, morphology, entrapment efficiency, and antihypertensive activity. Results and discussion It was observed that prepared isradipine-loaded invasomes delivers ameliorated flux, reasonable entrapment efficiency, and more effectiveness for transdermal delivery. The optimized formulation presented the particle size of 194 ± 18 nm, entrapment efficiency (88.46%), and attained mean transdermal flux of 22.80 ± 2.10 μg/cm 2 /h through rat skin. Confocal laser scanning microscopy revealed an enhanced permeation of Rhodamine-Red-loaded isradipine invasomes to the deeper layers of the rat skin. During antihypertensive study, the treatment group showed a substantial and constant decrease in blood pressure, for up to 24 h. The isradipine invasomes formulation was found to be effective, with a 20% reduction in blood pressure by virtue of better permeation through Wistar rat skin. Conclusion It was concluded that the developed isradipine invasomes accentuate the transdermal flux and the results obtained encouraged the use of the isradipine-loaded invasomes as the formulation for the potential management of hypertension.

In the interest of developing in vivo positron emission tomography (PET) probes for neuroimaging of calcium channels, we have prepared a carbon-11 isotopologue of a dihydropyridine Ca2+-channel antagonist, isradipine. Desmethyl isradipine (4-(benzo[c][1,2,5]oxadiazol-4-yl)-5-(isopropoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridine -3-carboxylic acid) was reacted with [11C]CH3I in the presence of tetrabutylammonium hydroxide in DMF in an HPLC injector loop to produce the radiotracer in a good yield (6 ± 3% uncorrected radiochemical yield) and high specific activity (143 ± 90 GBq·µmol-1 at end-of-synthesis). PET imaging of normal rats revealed rapid brain uptake at baseline (0.37 ± 0.08% ID/cc (percent of injected dose per cubic centimeter) at peak, 15-60 s), which was followed by fast washout. After pretreatment with isradipine (2 mg·kg-1, i.p.), whole brain radioactivity uptake was diminished by 25%-40%. This preliminary study confirms that [11C]isradipine can be synthesized routinely for research studies and is brain penetrating. Further work on Ca2+-channel radiotracer development is planned.

Isradipine (ISR) is a potent calcium channel blocker with low oral bioavailability due to low aqueous solubility, extensive first-pass metabolism and P-glycoprotein (P-gp)-mediated efflux transport. In the present investigation, an attempt was made to develop isradipine-loaded self-nano emulsifying powders (SNEP) for improved oral delivery. The liquid self-nano emulsifying formulations (L-SNEF/SNEF) of isradipine were developed using vehicles with highest drug solubility, i.e. Labrafil® M 2125 CS as oil phase, Capmul® MCM L8 and Cremophor® EL as surfactant/co-surfactant mixture. The developed formulations revealed desirable characteristics of self-emulsifying system such as nano-size globules ranging from 32.7 to 40.2 nm, rapid emulsification (around 60 s), thermodynamic stability and robustness to dilution. The optimized stable self-nano emulsifying formulation (SNEF2) was transformed into SNEP using Neusilin US2 (SNEP(N)) as adsorbent inert carrier, which exhibited similar characteristics of liquid SNEF. The solid state characterization of SNEP(N) by Fourier transform infrared spectroscopy, differential scanning calorimetry, powder X-ray diffraction and scanning electron microscopic studies shown transformation of crystalline drug into amorphous form or molecular state without any chemical interaction. The in vitro dissolution of SNEP(N) compared to pure drug was indicated by 18-fold increased drug release within 5 min. In vivo pharmacokinetic studies in Wistar rats showed significant improvement of oral bioavailability of isradipine from SNEP(N) with 3- and 2.5-fold increments in peak drug concentration (C(max)), area under curve (AUC(0-∞)) compared to pure isradipine. In conclusion, these results signify the improved oral delivery of isradipine from developed SNEP.

Isradipine could be used for the treatment of high blood pressure or Parkinsonism, yet the study on pharmacokinetics (PK) of isradipine is lacking in the Chinese population. The current study aims to assess the dose proportionality, pharmacokinetics and gender effect of isradipine following oral single and multiple doses in Chinese subjects. A randomized, open-label, parallel-group trial was conducted in 30 healthy Chinese volunteers. Subjects randomly received a single dose of 2.5, 5 or 10 mg, and multiple doses (2.5 mg) of isradipine. Blood samples were collected pre-dose (0 h) and 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 9, 12, 24, 36 and 48 h post-dose. Isradipine was rapidly absorbed with the time to maximum concentration <1.27 h for all dosage groups. The maximum concentrations were 2.46, 5.34, 10.93 and 3.32 ng/mL and area under the concentration-time curve from time zero to the last time point (AUClast ) were 7.05, 12.58, 24.68 and 5.31 ng/ml · h for the 2.5, 5 and 10 mg single-dose and 2.5 mg multiple-dose groups, respectively. The half-life ranged from 5.76 to 7.94 h. The maximum concentration and AUC were found to increase linearly and dose-dependently for isradipine. No statistical gender differences were found. These findings indicated that the pharmacokinetic parameters of isradipine in Chinese population were dose-proportional and predictable over a range of 2.5-10 mg isradipine oral doses., (Copyright © 2013 John Wiley & Sons, Ltd.)

Poorly water-soluble drug with a short half-life such as isradipine (IDP) offer challenges in the controlled release formulation because of low dissolution rate and poor bioavailability. Self-emulsifying solid dispersions (SESD) of IDP consisted of surfactant and fatty acid in poloxamer 407 (POX 407) as a carrier and were manufactured by the melting method. Then, controlled release HPMC matrix tablet containing SESD were prepared via direct compression. The dissolution behaviors and in vivo bioavailability of controlled release matrix tablet in healthy human volunteers were investigated. The physical properties of solid dispersion were also examined using differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and scanning electron microscopy (SEM). It was shown that structure of IDP was amorphous in the solid dispersion. The dissolution rate of IDP from SESD was markedly enhanced because of increased solubility and wetting effect. Controlled release HPMC matrix tablets containing SESD released drug in a controlled manner and were stable during storage over 3 months at 40 °C/75% RH. Furthermore, the tablet containing 5mg IDP SESD showed significantly increased oral bioavailability and extended plasma concentration compared with the marketed 5 mg Dynacirc(®) capsule. A combined method of solid dispersion and controlled release technology could provide versatile dosage formulations containing IDP with poor water solubility and short half-life., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Proliposomes loaded with isradipine were prepared successfully to enhance the oral bioavailability of isradipine. In this study, proliposomes were prepared by film deposition by the carrier method with varying ratios of hydrogenated soy phosphatidyl choline (HSPC) and cholesterol using spray-dried mannitol (Pearlitol SD 200) as the carrier. The formulation containing an equimolar ratio of HSPC and cholesterol showed smaller vesicle size, high surface charge, and entrapment efficiency. The formation of liposomes and surface morphology of optimized proliposome formulation was studied by optical and scanning electron microscopy, respectively. Fourier transform infrared, differential scanning calorimetry, and powder X-ray diffractometry studies were performed to assess the solid-state characteristics of the formulation. Ex vivo permeation enhancement assessed from flux, permeability coefficient, and enhancement ratio were significantly higher for proliposomes, compared to control. The pharmacokinetic parameters were evaluated in male albino Wistar rats, and a significant improvement in bioavailability (2.4-fold) was observed from the optimized proliposome formulation, compared to control (oral suspension). The stability study revealed that the formulations are stable when stored at 4°C.

Alzheimer's disease is the most devastating neurodegenerative disorder in the elderly, yet treatment options are severely limited. The drug development effort to modify Alzheimer's disease pathology by intervention at beta amyloid production sites has been largely ineffective or inconclusive. The greatest challenge has been to identify and define downstream mechanisms reliably predictive of clinical symptoms. Beta amyloid accumulation leads to dysregulation of intracellular calcium by plasma membrane L-type calcium channels located on neuronal somatodendrites and axons in the hippocampus and cortex. Paradoxically, L-type calcium channel subtype Ca(v)1.2 also promotes synaptic plasticity and spatial memory. Increased intracellular calcium modulates amyloid precursor protein processing and affects multiple downstream pathways including increased hyperphosphorylated tau and suppression of autophagy. Isradipine is a Federal Drug Administration-approved dihydropyridine calcium channel blocker that binds selectively to Ca(v)1.2 in the hippocampus. Our studies have shown that isradipine in vitro attenuates beta amyloid oligomer toxicity by suppressing calcium influx into cytoplasm and by suppressing Ca(v)1.2 expression. We have previously shown that administration of isradipine to triple transgenic animal model for Alzheimer's disease was well-tolerated. Our results further suggest that isradipine became bioavailable, lowered tau burden, and improved autophagy function in the brain. A better understanding of brain pharmacokinetics of calcium channel blockers will be critical for designing new experiments with appropriate drug doses in any future clinical trials for Alzheimer's disease. This review highlights the importance of Ca(v)1.2 channel overexpression, the accumulation of hyperphosphorylated tau and suppression of autophagy in Alzheimer's disease and modulation of this pathway by isradipine., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Isradipine (ISDP) is an effective calcium channel blocker used in the treatment of hypertension. It undergoes extensive first pass metabolism and bioavailability through the oral route is only about 15 to 24%. Hence we attempted to develop a matrix type controlled transdermal drug delivery system for ISDP. Formulations A1, A2, A3 were composed of Eudragit RL100 and hydroxypropyl methyl cellulose (HPMC) in 1:3, 1:1, 3:1 ratios; A4, A5, A6 were composed of Eudragit RS100 and HPMC in 1:3, 1:1, 3:1 ratios. All six formulations carried 5 mg of ISDP/patch area, 5% v/w of D-limonene, 15 % v/w of propylene glycol in methanol:dichloromethane (1:1). The physicochemical compatibility of the drug and the polymers was studied by infrared spectroscopy and differential scanning calorimetry. The results suggested no physicochemical incompatibility between the drug and the polymers. The prepared transdermal drug delivery system were evaluated for physicochemical characteristics, mainly in vitro release and ex vivo permeation. The ex vivo permeation studies were carried out across excised rat skin using Franz diffusion cell. All the formulations exhibited satisfactory physicochemical characteristics. Cumulative amount of the drug released in 36 h from the six formulations were 1695.32, 1527.89, 1455.54, 1485.65, 1282.81 and 916.88 microg/cm(2) respectively. Corresponding values for the cumulative amounts of drug permeated across the rat skin for the above matrix films were 1456.29, 1284.70, 1182.99, 1212.72, 1046.05, and 782.60 microg/cm(2) respectively. By fitting the data into zero order, first order and Higuchi models, it was concluded that drug release from matrix films followed Higuchi model and the mechanism of drug release was diffusion mediated. Based on the physical evaluation, in vitro drug release and ex vivo permeation characteristics, it was concluded that for potential therapeutic use, monolithic drug matrix films A1, may be suitable for the development of a transdermal drug delivery system of ISDP.

Despite more than 30 years of clinical use, only few studies have been published reporting on the release mechanism underlying the drug delivery from push-pull osmotic pumps (PPOP). The aim of this study is to understand which factors have an effect on the drug delivery for modelling the drug release and to develop a mathematical model predictive of the drug release kinetics. The influence of the drug property was tested on two model drugs, isradipine (ISR) and chlorpheniramine (CPA) which are respectively practically insoluble and freely soluble. Results show that, regardless of the drug properties which do not significantly affect the drug delivery, the release kinetics is mainly controlled by four factors, (i) the PEG proportion in the membrane, (ii) the tablet surface area, (iii) the osmotic agent proportion and (iv) the drug layer polymer grade. The influence of each key formulation factors on the release mechanism was investigated defining their applicability range. A mathematical approach was developed to predict the drug delivery kinetics varying the PPOP controlling factors and helps to more efficiently design PPOP.

The mechanism of drug release from push-pull osmotic systems (PPOS) has been investigated by Magnetic Resonance Imaging (MRI) using a new benchtop apparatus. The signal intensity profiles of both PPOS layers were monitored non-invasively over time to characterize the hydration and swelling kinetics. The drug release performance was well-correlated to the hydration kinetics. The results show that (i) hydration and swelling critically depend on the tablet core composition, (ii) high osmotic pressure developed by the push layer may lead to bypassing the drug layer and incomplete drug release and (iii) the hydration of both the drug and the push layers needs to be properly balanced to efficiently deliver the drug. MRI is therefore a powerful tool to get insights on the drug delivery mechanism of push-pull osmotic systems, which enable a more efficient optimization of such formulations.

A highly sensitive and rapid method for the analysis of isradipine in human plasma using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed. The procedure involves a simple liquid-liquid extraction of isradipine and amlodipine (IS, internal standard) with methyl-t-butyl ether after alkaline treatment and separation by RP-HPLC. Detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 372.1-->m/z 312.2 and m/z 408.8-->m/z 237.9, for quantification of isradipine and IS, respectively. The standard calibration curves showed good linearity within the range of 10 to 5000 pg/mL (r(2)>or=0.9998). The lower limit of quantitation (LLOQ) was 10 pg/mL. The retention times of isradipine (0.81 min) and IS (0.65 min) suggested the potential for high throughput of the proposed method. In addition, no significant metabolic compounds were found to interfere with the analysis. This method offered good precision and accuracy and was successfully applied for the pharmacokinetic and bioequivalence studies of 5 mg of sustained-release isradipine in 24 healthy Korean volunteers.

Voltage-dependent calcium channels are important for calcium influx and the ensuing intracellular calcium signal in various excitable membranes. The beta subunits of these channels modify calcium currents through pore-forming alpha1 subunits of the high-voltage- activated calcium channels. In the present study, beta3 subunit-null mice were used to investigate the importance of the beta3 subunit of the voltage-dependent calcium channel, which couples with the CaV2.2 (alpha1B) subunit to form the major component of neuronal N-type calcium channels in the brain. Western blot analysis revealed a significant decrease in N-type calcium channels in beta3 subunit-null mice, while protein levels of other high-voltage-activated calcium channel alpha1 subunits were unchanged. Immunoprecipitation analysis with an anti-CaV2.2 antibody showed that reshuffling of the assembly of N-type channels had occurred in the beta3 subunit-null mice. Ablation of this subunit resulted in modified nociception, decreased anxiety, and increased aggression. The beta3 subunit-null mice also showed impaired learning ability. These results suggest the importance of voltage-dependent calcium channels and the key role of the beta3 subunit in memory formation, nociceptive sensory transduction, and various neurological signal transduction pathways.

The authors sought to determine whether sustained-release (SR) isradipine provided comparable systemic availability to that of immediate-release (IR) isradipine in non-treatment-seeking, cocaine-dependent individuals. This information could be used to design a rational dosage regimen for additional isradipine clinical trials in the treatment of stimulant dependence and related neurovascular disorders. Eight male volunteers who met Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria for cocaine dependence participated in a randomized, double-blind, crossover study. Subjects received a 15-mg dose of an IR isradipine formulation and a 30-mg dose of an SR isradipine formulation, separated by a 2-day interval. Vital signs and blood sampling for isradipine serum levels were performed before and at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 8, 10, 12, and 24 h after each isradipine dose administration. Neither the 15-mg dose of IR isradipine nor the 30-mg dose of SR isradipine produced significant adverse effects on cardiovascular parameters, but the IR formulation was more likely to produce marked short-term decreases in pressor response. Significant intersubject variability in serum concentrations and pharmacokinetic parameters occurred for both formulations. The relative bioavailability of the SR formulation was 55.5% of that of the IR formulation. Both formulations cumulatively may deliver about the same amount of drug, but IR isradipine achieves a higher peak concentration than SR isradipine. The more favorable cardiovascular profile of SR isradipine would, however, make it more appropriate as an investigational medication for the treatment of stimulant dependence and related neurovascular disorders.

This study examines the influence of chronic treatment with inorganic lead (Pb(2+)) on the presence of dihydropyridine receptors in two types of excitable cells, primary cultures of skeletal muscle and neural retina from embryonic chick. As assessed through binding of the dihydropyridine antagonist [3H] PN 200-110, treatment of skeletal muscle cultures with Pb2+ caused measurable reductions in the numbers of dihydropyridine receptors. The reductions were dose-dependent, requiring concentrations greater than 1microM, slow in onset, requiring incubation times greater than 12h, and tissue specific, being pronounced in skeletal muscle but absent from neural retina. The reduction in dihydropyridine receptors is attributable to enhanced rates of receptor degradation or reductions in biosynthesis, without activation or inhibition of protein kinase C, and are discussed with respect to the role of dihydropyridine receptors in excitation-contraction coupling.

Traumatic brain injury (TBI) often causes a persistent and debilitating impairment of cognitive function. Although the neurochemical basis for TBI-induced cognitive dysfunction is not well characterized, some studies suggest prominent involvement of the CNS cholinergic system. Previous studies from our laboratories have shown that alpha 7* nicotinic cholinergic receptors (nAChrs) are especially vulnerable to the pathophysiological effects of TBI. Hippocampal and cortical alpha-[(125)I]-bungarotoxin (BTX) expression of alpha 7* nAChrs is significantly decreased in many brain regions following TBI and this reduction persists for at least 3 weeks following injury. In the present study we evaluated whether chronic nicotine infusion could attenuate TBI-induced deficits in alpha 7* nAChr expression. Male Sprague-Dawley rats were sham-operated, or subjected to mild or moderate unilateral cortical contusion injury. Immediately following brain injury, osmotic mini-pumps that delivered chronic saline or nicotine (0.125 or 0.25 mg/kg/h) were implanted. The animals were euthanatized and the brains prepared for nAChr quantitative autoradiography, 7 days following surgery. Brain injury caused significant decreases in BTX binding in several regions of the hippocampus. TBI-induced deficits in alpha 7* nAChr density were reversed in four of the six hippocampal brain regions evaluated following chronic nicotine administration. If TBI-induced deficits in alpha 7* nAChr expression play a role in post-injury cognitive impairment, pharmacological treatments which restore nAChr binding to control levels may be therapeutically useful.

Neuronal L-type calcium channels have been implicated in pain perception and neuronal synaptic plasticity. To investigate this we have examined the effect of disrupting the gene encoding the CaV1.3 (alpha 1D) alpha subunit of L-type Ca2+ channels on neurological function, acute nociceptive behavior, and hippocampal synaptic function in mice. CaV1.3 alpha 1 subunit knockout (CaV1.3 alpha 1(-/-)) mice had relatively normal neurological function with the exception of reduced auditory evoked behavioral responses and lower body weight. Baseline thermal and mechanical thresholds were unaltered in these animals. CaV1.3 alpha 1(-/-) mice were also examined for differences in N-methyl-D-aspartate (NMDA) receptor-dependent (100 Hz tetanization for 1 s) and NMDA receptor-independent (200 Hz in 100 microM DL-2-amino-5-phosphopentanoic acid) long-term potentiation within the CA1 region of the hippocampus. Both NMDA receptor-dependent and NMDA receptor-independent forms of long-term potentiation were expressed normally. Radioligand binding studies revealed that the density of (+)[3H]isradipine binding sites in brain homogenates was reduced by 20-25% in CaV1.3 alpha 1(-/-) mice, without any detectable change in CaV1.2 (alpha 1C) protein levels as detected using Western blot analysis. Taken together these data indicate that following loss of CaV1.3 alpha 1 subunit expression there is sufficient residual activity of other Ca2+ channel subtypes to support NMDA receptor-independent long-term potentiation and some forms of sensory behavior/function.

It was established that a regular administration of isradipine is accompanied by an increase in the mean values of Cmax (by 21.7%) and AUC (by 21.8%), which leads to extension of the pharmacodynamics effects without cumulation (the values of Clt, T1/2, and Tmax remain unchanged). The maximum hemodynamic effect of isradipine upon a single administration in a single dose of 5 mg coincides with the time or reaching the maximum drug concentration in the blood (Tmax). There is a reliable correlation between the hypotensive effect, the peripheral vascular resistance, and the concentration of isradipine.

Previous studies have shown that the kappa-opioid effects are sensitive to pertussis toxin (PTX) and affected by Ca(2+) fluxes. However, the possible involvement of Ca(2+) channels in PTX-induced inhibition of kappa-opioid effects has not been reported. The effect of intracerebroventricular (i.c.v.) treatment of pertussis toxin (1 microg/rat, PTX) or saline on the kappa-opioid agonist, U-50,488H (U5H) induced tail-flick analgesia and hypothermia in rats was determined. The effect of nimodipine (NIM), a dihydropyridine (DHP)-sensitive Ca(2+) channel blocker (CCB), on PTX-induced modulation of U5H effects was examined. The DHP ligand, [3H]PN200-110 binding was also determined in both PTX and saline treated rats to study the possible involvement of L-type Ca(2+) channels in PTX modulation of kappa-opioid agonist effects. The analgesia and change in colonic temperature were determined using tail-flick analgesiometer and telethermometer, respectively. U5H (40 mg/kg, i.p.) produced significant analgesic and hypothermic responses. PTX treatment significantly (P<0.01) antagonized the analgesic and hypothermic effects of U5H. Acute pretreatment of NIM (1 mg/kg, i.p.) 15 min prior significantly (P<0.01) reversed the PTX-induced antagonism of U5H effects. In the binding study, PTX treatment (72 h before) resulted in a significant (P<0.005) upregulation (+45% vs. saline control) of DHP binding (B(max)) with no change in affinity (K(d)). The results showed significant upregulation of DHP binding in accordance with PTX-induced antagonism of U5H effects and this blockade was reversed by NIM. Thus, present results suggest that U5H-induced analgesia and hypothermia may be mediated through PTX-sensitive transducer G-proteins (G(i/o)) coupled to L-type Ca(2+) channels.

Age-related changes in the relative contribution of voltage-dependent calcium channel (VDCC) subtypes to depolarization-induced Ca(2+) influx and in the density of VDCC subtypes in cortical synapses were investigated using synaptosomes and their membrane preparations from brain cortices of Wistar rats. The relative contribution of VDCC subtypes to Ca(2+) influx was determined by measuring the inhibition of depolarization-induced Ca(2+) influx with four VDCC subtype-specific peptide blockers. In adult rat synaptosomes, L-, N-, P- and Q-type channels accounted for 24, 32, 27 and 12% of the total Ca(2+) influx, respectively. Brain aging significantly reduced the relative contributions of N- and P-type channels and increased the contribution of the channels resistant to the four blockers used. The densities of VDCC subtypes, determined by binding experiments using radiolabeled PN200 -110, omega-conotoxin GVIA and omega-conotoxin MVIIC, were found to be significantly decreased in aged synaptic plasma membranes. On the contrary, the dissociation constants of the blockers were not changed except for PN200-110-sensitive L-type channels. These results suggest that aging alters the relative contributions of each VDCC subtype to depolarization-induced Ca(2+) influx and decreases the number of VDCCs in rat brain cortical synapses. These changes in VDCCs may lead to age-related hypofunction of synaptic neurotransmission in brain cortices.

Purpose: To characterize the in vivo specific binding and pharmacokinetics of a 1,4-dihydropyridine (DHP) calcium channel antagonist, PN 200-110, in the senescent brain, using senescence-accelerated prone mice (SAMP8) and senescence-resistant mice (SAMR1)., Methods: Blood, brain, and heart samples were taken periodically from SAMR1 and SAMP8 following intravenous injection of (+)-[3H]PN 200-110, and the concentration of (+)-[3H]PN 200-110 in the plasma and tissues was determined. In addition, the in vivo specific binding of (+)-[3H]PN 200-110 in the brains of SAMRI and SAMP8 was measured periodically after intravenous injection of the radioligand., Results: There was very little significant difference between SAMR1 and SAMP8 in terms of the half-life (t(1/2)), total body clearance (CL(tot)), steady-state volume of distribution (Vd(ss)). and AUC for the plasma concentration of (+)-[3H]PN 200-110 after intravenous injection of the radioligand. The brain concentration (AUCbrain) for (+)-[3H]PN 200-110 and the brain/plasma AUC ratio (AUCbrain/AUCplasma) were significantly lower in SAMP8 than in SAMR1, and the heart concentration (AUCheart) and the heart/plasma AUC ratio (AUCheart/AUCplasma) were similar in both strains. Also, the brain/plasma unbound AUC ratio (AUCbrain/AUCplasma-free) for (+)-[3H]PN 200-110 was significantly lower in SAMP8 than in SAMRI. The in vivo specific binding (AUCspecific binding, maximal number of binding sites: Bmax) of (+)-[3H]PN 200-110 was significantly lower in brain particulate fractions of SAMP8 than SAMR1., Conclusions: The concentration and in vivo specific binding of (+)-[3H]PN 200-110 was significantly reduced in the senescent brain. The simultaneous analysis of the concentrations of centrally acting drugs and the in vivo specific binding in the brain in relation to their pharmacokinetics may be valuable in evaluating their CNS effects.

Isradipine is a calcium channel-blocking agent of the dihydropyridine type, used in the treatment of hypertension. A terminal half-life of 8-9 hr has been reported, in several pharmacokinetic studies after oral administration of isradipine. In a yet unpublished study a much shorter half-life was observed, and the present trial was therefore conducted in order to estimate the half-life after intravenous administration of isradipine. The bioavailability was estimated as well. In a randomised cross-over design ten healthy young volunteers were given either isradipine orally or an intravenous infusion. The two study periods were separated by at least 3 days. Blood samples for measurement of isradipine concentration were collected for 10-12 hr after administration and half-life and bioavailability were estimated. Mean terminal half-life after intravenous administration was calculated to be 2.8 hr, and the bioavailability to be 0.28. None of the 10 subjects suffered from side effects. In the present intravenous study the half-life of isradipine seems to be of much shorter than demonstrated in previous oral studies.

Previous studies from our laboratory demonstrated the up-regulation of cardiac dihydropyridine (DHP) receptors in rabbits chronically treated with nifedipine (NIFE). The goal of the present study was to further examine the functionality of this increased number of receptors by analysing different steps of excitation contraction coupling mechanism in adult rats chronically treated with NIFE (a single 10-mg oral dose/kg/day for 28 days). Ca2+ channel density was assessed by specific binding at the DHP receptors with [methyl-(3)H]PN 200-110 in rat ventricular membranes. Chronic NIFE treatment produced up-regulation of Ca2+ channels, being the maximal binding capacities 222+/-19 fmol/mg protein (n=14) and 310+/-21 fmol/mg protein (n=11) in untreated and treated animals, respectively (P<0.05). The functional consequences of this up-regulation of Ca2+ channels were determined in isolated ventricular myocytes by measuring L-type Ca2+ currents (I(Ca)) with the whole-cell configuration of patch-clamp technique and by intracellular Ca2+ (Ca2+(i)) transients estimated by the Indo-1/AM fluorescence ratio (410/482) simultaneously monitored with cell shortening. Peak I(Ca) density recorded at 0 mV was 32% greater in myocytes isolated from the treated group than in those obtained from the untreated group (-10.43+/-0.73 pA/pF (n=13) vs-7.10+/-0.59 pA/pF (n=12) P<0.05). Ca2+(i) transient amplitude and cell shortening, explored at 1 and 2 mM extracellular calcium ([Ca]0) were significantly higher in ventricular myocytes obtained fom NIFE-treated rats than in myocytes isolated from untreated animals. At 2 mM [Ca]0, the values of Ca2+(i) transient and shortening were 460+/-61 nM and 11+/-1 % of resting length (L(0)) in myocytes from treated rats (n=9) and 212+/-22 nM and 5.3+/-0.5% of L(0) in myocytes from control rats (n=6, P<0.05). The results demonstrate an up-regulation of functionally-active cardiac Ca2+ channels after NIFE treatment, and offer a possible explanation for a "withdrawal effect" at myocardial level after the suppression of the treatment with this drug., (Copyright 1999 Academic Press.)

We have previously identified the binding region of a new Ca2+ antagonist semotiadil in the skeletal muscle Ca2+ channel. To the same semotiadil derivatives, the cardiac counterpart showed distinct and different binding characteristics: semotiadil and its photoaffinity analog D51-4700 inhibited [3H]PN200-110 binding to cardiac membrane preparations with IC50 values of 13-20 microM, which are 10 times higher than those in skeletal muscle. Hill slopes of the binding inhibition were 0.94-1.0 for the cardiac channels compared to 0.63-0.67 for the skeletal muscle channels. A possible explanation for the difference is that the semotiadil binding site is differently conferred in cardiac and skeletal muscle Ca2+ channels. To reveal this within the primary structure, photoaffinity labeling of cardiac membranes was employed. [3H]D51-4700 was photo-incorporated in several polypeptides but only the alpha1 subunit of the Ca2+ channel was photolabeled in a specific manner. Antibody mapping of the [3H]D51-4700-labeled alpha1 subunit with several anti-peptide antibodies revealed that the labeled site was located solely in a peptide fragment between Cys1461 and Lys1529. This region encompasses the labeled site of skeletal muscle, but contains several non-identical amino acid residues, which may participate in expressing different binding characteristics between the two muscle type Ca2+ channels.

Raynaud's phenomenon (RP) is a peripheral circulatory disorder characterized by sudden episodes of digital artery spasm, often precipitated by cold temperature or emotional stress. Although the cause of RP is not fully known, it appears to involve inappropriate adrenergic response to cold stimuli. Treatment of RP is conservative in most patients, but in patients with severe disease includes the use of agents that promote digital vasodilation. The calcium-channel antagonists, particularly the dihydropyridine derivative nifedipine, are the most thoroughly studied drug class for the treatment of RP. Approximately two thirds of patients respond favorably, with significant reductions in the frequency and severity of vasospastic attacks. Nifedipine use is often limited by the appearance of adverse vasodilatory effects such as headache or peripheral edema. The newer second-generation dihydropyridines such as amlodipine, isradipine, nicardipine, and felodipine also appear to be effective in patients with RP and may be associated with fewer adverse effects.

1. The aim of this study was to investigate the possible anticonvulsant effect of a dihydropyridine calcium antagonist, isradipine, which easily crosses the blood-brain barrier displaying high affinity and specificity for the brain L-type voltage-sensitive calcium channel, on maximal electroshock seizures in mice. 2. Isradipine at i.p. doses of 2.5 mg/kg and 5.0 mg/kg was found to cause a statistically significant increase in the convulsion threshold of maximal electroshock seizures in a dose-dependent manner (P = 0.047 and P = 0.022, respectively). 3. It was concluded that the mode of action of the anticonvulsant effect of isradipine is related to blockade of the intraneuronal calcium currents, which play an important role in epileptic activity.

This work represents a study of the binding and distribution of three different calcium channel blockers in the Sprague-Dawley rat liver, using an in situ perfusion technique. For this purpose, [3H] desmethoxyverapamil, [3H] PN200-110 (isradipine) and [3H] azidopine were used as binding probes interacting with calcium channels. The perfusion steps of the liver involved both portal vein and thoracic inferior vena cava cannulations as inlet and outlet respectively. The subhepatic inferior vena cava was ligated to prevent leakage of the perfusate. Buffer, containing the tracer drug, was administered via the portal vein at a rate of 1 mL/min and perfusate collected at the same rate within specified time intervals during 50 min. The concentration of the tracer solutes in the perfusate's outlet increased with time, and steady state was observed for all tracers at > or = 40 min. The effect of adding cold isradipine to tracer desmethoxyverapamil, or cold verapamil to tracer PN200-110 were also assessed. First order rate constants for hepatocellular influx, efflux and calcium channel binding of the tracer substances were obtained using a simplified model from Goresky et al. These constants were mathematically manipulated and changed into permeability constants, second order binding constants, and residency times. Tracer solute influx across hepatocellular membranes is solubility-diffusion controlled, is inversely related to the molecular weights and is different in value from the efflux constants. Cold isradipine reduced the binding constant of desmethoxyverapamil by 36%, while cold verapamil reduced the binding constant of PN200-110 by 23%. Azidopine cellular distribution was low, however, binding to its receptor was analogous to desmethoxyverapamil and PN200-110. Moreover, PN200-110 had the highest residency time with no effect of cold verapamil on its receptor binding, while desmethoxyverapamil had the lowest residency time which significantly increased in the presence of cold isradipine.

In this review, several deficiencies of published bioequivalence studies for controlled-release calcium antagonists have become apparent. As a consequence, some of the published conclusions based on such studies must be viewed with care. A proper statistical analysis of bioequivalence is not frequently reported. A proper statistical analysis of the pharmacokinetic variables involves the calculation of 90% confidence intervals (CI) for the test: reference ratio of the means of the pharmacokinetic variables of the test and reference product. The CI must fall completely within the predetermined bioequivalence range (usually 0.8 to 1.25) for the products to be declared bioequivalent. Serious methodological errors, such as a conclusion of bioequivalence based on a lack of statistically significant difference between products, and conversely, a conclusion of bioequivalence because of a statistically significant difference, or because of a mere failure to show bioequivalence, are still made. With calcium antagonists in particular, an assessment of the rate of absorption and of the maximum concentration is important, as those characteristics may have implications for the safety profile with this class of drugs. As a minimum, in single doses studies the maximum concentration (Cmax), and the time to the maximum concentration (tmax), and in multiple-dose studies the Cmax, and the peak-trough fluctuation (%PTF) must be considered. Some bioequivalence studies of calcium antagonists are deficient in this respect. To show bioequivalence for controlled-release formulations, multiple-dose studies are required but some published bioequivalence studies contain only single-dose assessments. Similarly, bioequivalence studies under fed conditions are rarely published, although food may have a significant effect on the absorption rate of these drugs. Some calcium antagonists, such as verpamil, show stereo-selective pharmacokinetics, so that enantiomers may have to be investigated. Unfortunately, few of the published studies of controlled-release calcium antagonists satisfy all requirements. One would expect that data submitted to regulatory authorities for approval of generic formulations are more complete; published data are in many cases not satisfactory.

Withdrawal hyperexcitability was seen in isolated mouse hippocampal slices, prepared after chronic treatment with ethanol, by inhalation for 2 weeks. The pattern of hyperexcitability differed from those seen previously when a different method of ethanol administration and a different strain of mice were used. Thresholds for field potentials were decreased, but the transient increase in paired pulse potentiation, reported earlier, was not evident. Chronic administration of the calcium channel antagonist, isradipine (PN-200-110) during ethanol treatment significantly decreased the withdrawal syndrome, both in vivo and in vitro. Brain concentrations of isradipine during the test period were found to be sufficient to produce acute effects on the withdrawal hyperexcitability. No changes were seen in the field potentials when slices were prepared after treatment with isradipine alone. A small, but significant, increase in excitability was seen in vivo after the treatment with isradipine alone. Previous studies showed that isradipine did not protect against the hyperexcitability due to gamma-aminobutyric acid (GABA)A antagonism, so the results suggest that neuronal calcium channels may be involved in ethanol withdrawal hyperexcitability, but decreases in GABAA inhibition may not be important.

Long-lasting myocardial ischaemia reduces the density of sarcolemmal L-type calcium channels (LCC). Ischaemic preconditioning protects the myocardium against development of infarction. The aim of this study was to investigate if ischaemia-induced loss in LCC is affected by ischaemic preconditioning. Specific (+) - [3H]isradipine binding to LCC was compared in membranes and homogenates from control and ischaemic regions of non-preconditioned and ischaemically preconditioned hearts [two 10 min left anterior descending coronary artery (LAD) occlusions, each followed by 30 min reperfusion]. Biopsies were sampled after 60 min mid LAD occlusion from ischaemic and control (supplied by circumflex artery) regions. Sixty min ischaemia reduced binding density of specific (+) - [3H]isradipine in membranes by 23 +/- 11% (n = 7, P < 0.05) in the non-preconditioned group and by 20 +/- 8% (n = 6, P < 0.05) in the preconditioned group. Binding density in homogenates was reduced by 36 +/- 5% (n = 5. P < 0.05) in the non-preconditioned group and by 21 +/- 5% (n = 5. P < 0.05) in the preconditioned group. The reductions in the two groups and reductions in membranes and homogenates were not statistically different. The dissociation constant of binding was similar in the groups. In conclusion, 60 min of ischaemia reduced the binding density of (+)-[3H]isradipine in membranes and homogenates by 20-36%. The reduction in density of binding sites was not caused by redistribution of sarcolemmal LCC to an intracellular compartment. Ischaemic preconditioning did not affect the decline in binding density as hypothesized.

Ca2+ channels of the L-type were characterised in intact human peripheral blood lymphocytes using a radioligand binding technique and the dihydropyridine-type Ca2+ channel antagonist [3H](+)-PN 200-110 (isopropyl-4-(2,1,3-benzoxadiazol-4-yl)1,4-dihydro-5-methoxycarbon yl-2, 6-dimethyl-3-pyridine carboxylate) as a ligand. [3H](+)-PN 200-110 binding to human peripheral blood lymphocytes was time-, temperature-, concentration-dependent and of high affinity. The dissociation constant (Kd) value was 0.4 +/- 0.02 nM and the maximum binding capacity (Bmax) was 33.5 +/- 1.6 fmol/10(6) cells. Pharmacological analysis of [3H](+)-PN 200-110 binding to human peripheral blood lymphocytes was consistent with the labelling of a Ca2+ channel of the L-type. In fact, dihydropyridine derivatives were the most potent competitors of [3H](+)-PN 200-110 binding, whereas phenylalkylamine and benzothiazepine compounds or non-selective Ca2+ channel modulators were weak or ineffective displacers. These findings are the first observation that human peripheral blood lymphocytes express Ca2+ channels of the L-type. The possibility that Ca2+ channel antagonists may interfere with immune system function is discussed.

We investigated if and how stable guanosine 5'-triphosphate-analogues affect (+)-[3H]isradipine binding in rat hearts. Gpp(NH)p and GTP-gamma-S inhibit specific (+)-[3H]isradipine binding in membranes and cell-homogenates by reducing the binding density without changing the Kd of the k-1. Inhibition by Gpp(NH)p was less in crude tissue homogenates than in membranes apparently due to a soluble factor. Pretreatment of cardiomyocytes with cholera toxin or the presence of the protein kinase A inhibitor, PKI6-22, did not influence the effect of 10(-3) M Gpp(NH)p on binding. The inhibitory effect of 10(-3) M Gpp(NH)p was not significantly altered in membranes from in vivo pertussis toxin treated rats. The addition of 10(-3) M Ca2+ or Mg2+ abolished the inhibitions. Gpp(NH)p in the concentration that inhibits binding, reduced the free concentration of Ca2+. The Ca(2+)-lowering effect of 10(-3) M Gpp(NH)p produced 70%, 60% and 100% of the inhibition in membranes, sonicated and unsonicated cell homogenates. Thus, Gpp(NH)p inhibited specific (+)-[3H] isradipine binding mainly by lowering the free concentration of Ca2+ by chelation and not by activation of cholera toxin or pertussis toxin-sensitive G proteins or protein kinase A.

We have examined the pharmacokinetic interaction between isradipine and lovastatin in six male and six female, healthy, normotensive, human subjects after a single dose and after treatment for 5 days. The isradipine plasma concentrations were determined by a radioimmunoassay and the lovastatin serum concentrations by gas chromatography-mass spectrometry (GC/MS) and by the inhibition of the 3-hydroxy-3-methylglutaric-coenzyme reductase activity. We found that the apparent serum concentrations of lovastatin were 4- to 6-fold higher in the reductase-inhibition assay than the GC/MS assay, suggesting that the bulk of the reductase inhibition is due to active metabolites. The peak and the time-to-peak concentrations were unaffected by the treatments, either after the first dose or after continued administration. In male subjects, after repeated doses of isradipine, the lovastatin area under the time-concentration curves (AUCs) decreased by 40% as determined by the GC/MS assay (P < .001) and 20% as determined by the reductase-inhibition assay (P < .0022). In the female subjects, isradipine treatment decreased the lovastatin AUCs as determined by the GC/MS assay, but this was not statistically significant due to a high variance. Furthermore, in the female subjects, isradipine had no effect on the lovastatin AUCs as determined by the reductase-inhibition assay. Because the lovastatin peak and the time-to-peak concentrations were unaffected by isradipine treatment, the decreased lovastatin AUCs were probably not due to altered intestinal absorption. More likely, because lovastatin has a high hepatic clearance, the decreased AUCs seen after isradipine treatment could be due to increases in the clearance of lovastatin secondary to increased hepatic blood flow.

Since the earlier review in Drugs substantial additional data have accumulated regarding the antihypertensive efficacy of isradipine in various clinical situations, as well as data on its clinical effects in atherosclerosis. Recent therapeutic trials confirm that the efficacy of isradipine in the treatment of patients with mainly mild to moderate hypertension, when administered orally as a conventional or modified release preparation, is similar to that of titrated dosages of amlodipine, felodipine, nifedipine, diltiazem, captopril, methyldopa, metoprolol, prazosin and hydrochlorothiazide. A further decrease in blood pressure can be expected when isradipine is combined with another antihypertensive drug in patients who have not responded adequately to monotherapy. Initial studies have shown that intravenous isradipine is effective in controlling hypertension following coronary artery bypass graft surgery and that it appears useful in the treatment of intraoperative hypertension and hypertensive crisis, and in hypertensive disorders in pregnancy, when administered orally or intravenously. A large study, the Multicentre Isradipine Diuretic Atherosclerosis Study (MIDAS), was designed to compare the efficacy of isradipine and hydrochlorothiazide in reducing the rate of progression of carotid artery wall thickness, measured by B-mode ultrasound, as a surrogate for early atherosclerosis. Results indicated that wall thickness increased significantly less with isradipine than hydrochlorothiazide after 6 months of therapy. Thereafter the rate of progression remained parallel for the remainder of the 3-year trial. The confirmation of its antihypertensive efficacy, along with its favourable haemodynamic profile and reversal of left ventricular hypertrophy, minimal effect on glucose and lipid metabolism, preservation of quality of life and good tolerability, makes isradipine a suitable drug for the treatment of most patients with mild to moderate hypertension.

We describe here the effects of PCA50941 (a novel 1,4-dihydropyridine derivative) comparatively with Bay K 8644 on various parameters in bovine adrenal chromaffin cells. The binding of [3H](+)-isradipine to bovine adrenal medulla plasma membranes was inhibited similarly by PCA50941 and Bay K 8644 at various [Ca2+]o suggesting a common binding site for both compounds on the dihydropyridine receptor. In voltage-clamped chromaffin cells PCA50941 (1 microM) and Bay K 8644 (1 microM) shifted the I-V relationship of whole-cell Ca2+ currents by about 5-10 mV towards more hyperpolarizing potentials. At -20 mV, PCA50941 enhanced ICa by 195 +/- 16% and Bay K 8644 by 288 +/- 51%. Stimulation of fura 2-loaded chromaffin cell suspensions with 17.7 K+/0.5 Ca2+ increased 3-fold the basal [Ca2+]i. PCA50941 increased further the K(+)-evoked peak to 655 nM, and Bay K 8644 to 1129 nM. In the presence of 5 mM Ca2+, PCA50941 or Bay K 8644 increased the [Ca2+] peaks to 427 and 350 nM, respectively. PCA50941 potentiated the release of catecholamines from perfused bovine adrenal glands evoked by 30 s pulses of 17.7 mM K+ in a manner dependent on the [Ca2+]o. Thus at 1, 2.5, 5 and 10 mM Ca2+, secretion was 2.3-, 3.8-, 5- and 4-fold greater than in control glands. Bay K 8644 enhanced the K(+)-induced response 3- and 9-fold at [Ca2+]o of 0.25 or 0.5 mM, respectively; at higher [Ca2+]o the potentiation was similar to that of PCA50941.(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied binding of isradipine to A7r5 vascular smooth muscle cells as a function of membrane potential and cell proliferation. Consistent with a voltage-modulated receptor model, two classes of binding sites were detected in confluent cultures: high-affinity sites under depolarizing (50 mM K+) conditions (Kd = 45 +/- 3 pM), and lower affinity sites under resting (5 mM K+) conditions (Kd = 181 +/- 20 pM). However, proliferating cells also displayed the high-affinity state at rest (Kd = 29 +/- 9 pM) in addition to a low-affinity site (Kd = 869 +/- 383 pM). Analysis of dissociation rates also revealed two receptor classes during proliferation. Proliferating cells showed a single class of high-affinity sites (Kd = 39 +/- 6 pM) when depolarized, similar to confluent cells. Receptor density in confluent monolayers increased from 15 +/- 3 fmol/10(6) cells at 5 days to 72 +/- 6 fmol/10(6) cells after 10 days. These results suggest (i) that some L-type Ca2+ channels are spontaneously active in proliferating vascular smooth muscle cells, but require depolarization to activate in a confluent monolayer, and (ii) that the density of dihydropyridine receptors increases after a monolayer becomes confluent.

Isradipine, a 1,4 dihydropyridine calcium channel antagonist, is a potent coronary artery dilator that increases coronary blood flow with little effect on cardiac contractility. Isradipine is an approved antihypertensive agent, but its antianginal effects have not been well documented. In this placebo-controlled, double-blind, parallel-group design study we evaluated the duration of effects and safety of isradipine 10 mg bid in male patients with chronic stable angina pectoris. Seventy-two patients experiencing moderately severe angina between 3 and 7.5 minutes during a standard Bruce exercise test received placebo in a single-blind manner for 8-14 days. Sixty-one of these patients had reproducible treadmill exercise test results on three consecutive occasions and underwent further exercise tests at 3, 8, and 12 hours after a placebo period. Patients were then randomized (double blind) to either placebo or isradipine 10 mg bid for 2 weeks. Symptom-limited exercise tests were repeated predose and at 3, 8, and 12 hours after the 0800 hour dose dosing. Exercise duration increased significantly from baseline (last qualifying test during the single-blind placebo therapy, i.e., 0800 hours predose at visit 4) in the isradipine group compared to the placebo group prior to the administration of the 0800 hour dose (i.e., 12 hours after the 2000 hour dose) by 51 vs. 18 seconds, p = 0.04; and after the administration of the 0800 hour dose at 3 hours by 78 vs. 29 seconds, p = 0.005; and at 8 hours by 54 vs. 18 seconds, p = 0.04. Similarly, statistical significance was achieved when exercise data were analyzed using visit 4 (single-blind placebo therapy) corresponding time points as baseline. At 12 hours after the 0800 hour dose, exercise tolerance did not increase significantly after isradipine compared to placebo. Time to 1-mm ST-segment depression increased significantly after isradipine at 3 hours post 0800 hour dose compared to placebo (87 vs. 7 seconds, p < 0.01) but not at the 0, 8, or 12-hour postdose time points, regardless of which baseline was used. Isradipine therapy did not affect the rate-pressure double product. A significant correlation between the mean increase in total exercise time and mean plasma isradipine concentration was also present (p = 0.0295). During double-blind treatment, drug-related adverse events were experienced by four patients in the isradipine group and two patients in the placebo group. None of the patients experienced ischemic complications during the study.(ABSTRACT TRUNCATED AT 400 WORDS)

We characterized the occupancy of dihydropyridine (DHP) calcium antagonist receptors by amlodipine in spontaneously hypertensive rats (SHR) in relation to its pharmacokinetics. Oral administration of amlodipine (10 mg/kg) in SHR produced a significant (20-70%) decrease in the number of specific (+)[3H]PN 200-110 binding sites in cardiac tissues 0.5-18 h later, and the effect was greatest 3 h later. In these rats, there was little change in cerebral cortical (+)[3H]PN 200-110 binding. Occupancy of cardiac calcium antagonist receptors after oral administration of amlodipine correlated well with its plasma concentration. In vitro blockade of cardiac (+)[3H]PN 200-110 binding sites induced by amlodipine also persisted after the tissues were washed by centrifugation and suspension, whereas that induced by nifedipine was reversible under these conditions. Thus, our results suggest that the gradual onset and long-lasting pharmacologic effect of amlodipine are due to its slow binding kinetics (association and dissociation) of cardiovascular receptor sites in addition to its slow pharmacokinetics.

The in vivo determination of the density of dihydropyridine (DHP) binding sites will allow the assessment of pathophysiological changes associated with heart disease. The calcium channel antagonist S 11568: (+/-)(amino-7 dioxa-2,5 heptyl)-2(dichloro -2,3 phenyl) -4 methyl-6dihydro -1,4 pyridine has an in vitro profile of high potency and of high selectivity for the L-type Ca2+ channel. S 11568 was labelled by a reaction between 11C-diazomethane and the precursor 6-(7-amino-2,5-dioxa heptyl)-4-(2,3-dichloro phenyl)-5-(ethoxycarbonyl)-2 methyl-1,4 dihydro nicotinic acid. (+)-PN 200 110, a DHP with in vitro high affinity for the L-type Ca2+ channel, was also radiolabeled. Positron emission tomographic (PET) studies of both 11C-DHP myocardial uptake were performed in Beagle dogs. 11C-(+)-PN 200 110 had a rapid wash-out from myocardium. In contrary, after a bolus injection, 11C-S 11568 myocardial concentration increased to reach a maximum in 1-2 minutes and then remained in a plateau with a slight downslope while the blood concentration fell rapidly. Myocardial uptake was 2 to 4 fold higher than lung uptake, leading to a good contrast on PET images. Pre-treatment with unlabeled S 11568 (2 mumol/kg or 6 mumol/kg over 15 minutes) reduced myocardial uptake by 60% and 80%, respectively. Specific binding was estimated during a displacement experiment: bolus of unlabeled S 11568: 1 mumol/kg followed by a continuous infusion of 3 mumol/kg over 2 hours. It was found to represent 80% of the total binding. To assess influence of S 11568 on coronary blood flow and therefore on the myocardial tracer delivery, coronary blood flow was measured using 15O-H2O and PET at baseline and following bolus injections of 0.4, 0.8, 2 mumol/kg of S 11568. Only the higher dose increased coronary blood flow. This is the in vivo demonstration of the binding characteristics to myocardial tissue of a DHP ligand. Such properties make S 11568 suitable for PET experiments. The studies of DHP binding sites will provided new insights concerning physiological situations as well as heart disease.

Since the magnitude of the response to a drug may depend upon the drug input rate, the concentration-effect relationship of the new dihydropyridine (+/-)-isradipine was investigated using different administration modalities. Ten normotensive healthy volunteers were given, double-blind and in a crossover fashion, isradipine as a 1 mg iv infusion, 5 mg oral solution, 5 mg standard tablet, 10 mg slow release formulation, and a placebo. Blood pressure, heart rate, and plasma isradipine concentrations were recorded for 24 h. The maximal fall in diastolic blood pressure was similar after the infusion (-11.40 mmHg), the oral solution (-15.20 mmHg), and the standard tablet (-12.50 mmHg). In healthy volunteers the slow release form had no significant effect on blood pressure. The concentration-effect plots showed an increasing slope in the order infusion, solution, and table, and anticlockwise hysteresis. This was partly due to marked heart rate counter-regulation, the corresponding mean maximal heart rate increases being 24, 19, and 17 beats.min-1. The pronounced counter-regulation of the heart rate implies that a slow isradipine input rate would be more effective in decreasing blood pressure.

The effects of S-312-d (S-(+)-methyl-4,7-dihydro-3-isobutyl-6-methyl-4-(3- nitrophenyl)thieno[2,3-b]pyridine-5-carboxylate, CAS 120056-57-7) on the 1,4-dihydropyridine binding sites using membrane fractions prepared from monkey cerebral cortex, hippocampus, and cerebellum and on isolated monkey arteries were investigated. Specific binding of [3H]-(+)-isradipine was saturable and reversible, and of high affinity. 1,4-Dihydropyridine calcium channel antagonists competed for the binding in the order of S-312-d = nilvadipine = nicardipine > nifedipine. The effect of S-312-d on the binding was due mainly to alterations in the dissociation constant (Kd), without alterations in the binding density (Bmax). In the helical strips of cerebral, coronary, renal mesenteric, and femoral arteries contracted with K+ or prostaglandin (PG) F2 alpha, the addition of S-312-d caused a concentration-dependent relaxation. Relaxant activities of S-312-d in the cerebral arteries contracted with U-46619 (a thromboxane A2 mimetic) or endothelin-1 did not differ significantly from those in the arteries contracted with K+ or PGF2 alpha. Potencies of relaxations induced by S-312-d and 1,4-dihydropyridine calcium channel antagonists in the K(+)-depolarized mesenteric arteries correlated well with those of the binding experiment. Ca(2+)-induced contractions in the mesenteric arteries previously exposed to Ca(2+)-free medium and depolarized by excess K+ were attenuated by S-312-d, whereas PGF2 alpha-induced contractions of the arteries exposed to Ca(2+)-free medium were unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

The pharmacokinetic variables and antihypertensive effect of the calcium antagonist isradipine, were investigated in 30 hypertensive patients. Isradipine was given orally in parallel group design in plain and slow release formulations in doses of 2.5 mg twice daily and 5.0 mg once daily, respectively. Isradipine concentration in serum was measured by a sensitive RIA method after the first dose and after 6 weeks of treatment. The pharmacokinetics and concentration/effect relationship after the first dose and after 6 weeks of treatment were compared. No differences in pharmacokinetics were observed between single and multiple dosing. Data were in accordance with results from studies in healthy volunteers. Rate, but not extent of bioavailability differs between the two isradipine formulations. Antihypertensive efficacy of the two formulations was similar (16/11 and 19/15 mmHg), and a significant time dependent increase in Emax from 10/3 mmHg to 23/14 mmHg after 6 weeks of treatment was observed.

The newly developed sustained-release form of isradipine (Vascal uno/Lomir SRO) releases the active substance continuously from a gel matrix over a period of 24 hours. In comparison with the standard formulation, the absolute bioavailability of the sustained-release preparation is somewhat increased at about 22.5%. Maximum plasma levels are reached five to seven hours after oral administration of a capsule, and then level off. Both maximum plasma levels and bioavailability increase linearly with dose. At therapeutic dosage isradipine does not accumulate. Twenty-four hours after ingestion of the capsule, plasma levels are still half the maximum. As a result of slow absorption and minimal fluctuations in the concentration of the active agent, side effects of the sustained-release version of isradipine are even rarer than with the standard preparation.

The relationship between the plasma concentration of mepirodipine (1,4-dihydropyridine Ca2+ antagonist) and its occupancy of cardiac and cerebral Ca2+ antagonist receptors in rats has been characterized by a radioreceptor assay technique using (+)-[3H]PN 200-110. Oral administration of mepirodipine in rats produced a dose-dependent and sustained decrease in the number of specific (+)-[3H]PN 200-110 binding sites in both tissues, and the effect was more pronounced in the cardiac tissue than in the cerebral cortex. The occupancy of cardiac and cerebral Ca2+ antagonist receptors by mepirodipine correlated well with its plasma concentration, whereas a 20-fold higher plasma concentration of this drug was necessary to occupy Ca2+ antagonist receptors in the cerebral cortex. Thus, these data suggest that mepirodipine occupies Ca2+ antagonist receptors in cardiovascular tissue selectively over those in brain tissue.

1. The present experiments were undertaken in order to characterize further the apparently irreversible inhibition of the contraction of depolarized rat aorta caused by lacidipine, a 1,4-dihydropyridine calcium antagonist. 2. We studied the effect of lacidipine on contraction evoked by 100 mM KCl solution in rat aorta, treated by N omega-nitro-L-arginine (0.1 mM), an inhibitor of nitric oxide (NO) synthesis. We compared the effect of prolonged depolarization on lacidipine and (+)-isradipine inhibition and the reversal of this inhibition after washout in the absence of dihydropyridines. Assuming that the onset of lacidipine-evoked inhibition was a pseudo-first order association kinetics, we estimated the dissociation rate constant (k-1 = 0.031 min-1), the association rate constant (k1 = 2.70 x 10(8) M-1 min-1) and the dissociation constant (KD = k-1/k1 = 115 pM) which was close to the IC50 value in steady-state conditions (160 pM). 3. The inhibitory effects of lacidipine and (+)-isradipine on rat aorta contraction were reversibly enhanced after preincubation with the drug in a 40 mM KCl-solution. Washout with drug-free 40 mM K(+)-depolarizing solution reversed inhibition in the (+)-isradipine-treated preparations, but not in the lacidipine-treated ones. 4. Radioligand binding studies were performed with [3H]-lacidipine and [3H]-isradipine in microsomes from rat aorta and rat ileum. Both ligands bound to a homogeneous population of binding sites (for[3H]-lacidipine: KD = 23 +/- 2.6 pM, Bmax = 380 +/- 21 fmol mg-1 protein in membranes from aorta; KD =23 +/- 3.1 pM, Bmax = 790 +/- 60 fmol mg-1 protein in membranes from ileum; for [3H]-isradipine:KD = 140 +/- 46 pM, Bmax = 350 +/- 64 fmol mg-1 protein in membrane from aorta; KD = 68 +/- 14 pM,Bmax = 760 +/- 75 fmol mg-1 protein in membranes from ileum). After isotopic dilution, [3H]-lacidipine and [3H]-isradipine dissociated according to a monoexponential kinetics. In membranes from ileum, the calculated dissociation rate constants (kappa&#95; 1) were 0.0257 min-1 and 0.0595 min-1, for [3H]-lacidipine and[3H]-isradipine, respectively.5. The non specific binding of [3H]-lacidipine and [3H]-isradipine, was measured in intact rat aorta preparations incubated under the conditions of the functional experiments, in the presence of nifedipine(1 microM). After incubation with [3H]-lacidipine 77.6 +/- 1.9 pM for 2 h the concentration of drug in the tissue was 15.15 +/- 1.18 fmol mg-1 w.wt. and still amounted to 7.24 +/- 0.61 fmol mg-1 w.wt. after 3.5 h washout in drug-free solution. After incubation with [3H]-isradipine 47.2 +/- 0.4 pM for 2 h it was 2.26 +/-0.07 fmol mg-1 w.wt. and was undetectable after 3.5 h washout in a drug-free solution.6. It is concluded that lacidipine interacts reversibly with dihydropyridine binding sites and that the apparent irreversible inhibition of contraction in depolarized preparations could be related to a nonspecific binding in a tissue compartment different from the plasma membrane.