The Potential of L-Type Calcium Channels as a Drug Target for Neuroprotective Therapy in Parkinson's Disease.

The motor symptoms of Parkinson's disease (PD) mainly arise from degeneration of dopamine neurons within the substantia nigra. As no disease-modifying PD therapies are available, and side effects limit long-term benefits of current symptomatic therapies, novel treatment approaches are needed. The ongoing phase III clinical study STEADY-PD is investigating the potential of the dihydropyridine isradipine, an L-type Ca2+ channel (LTCC) blocker, for neuroprotective PD therapy. Here we review the clinical and preclinical rationale for this trial and discuss potential reasons for the ambiguous outcomes of in vivo animal model studies that address PD-protective dihydropyridine effects. We summarize current views about the roles of Cav1.2 and Cav1.3 LTCC isoforms for substantia nigra neuron function, and their high vulnerability to degenerative stressors, and for PD pathophysiology. We discuss different dihydropyridine sensitivities of LTCC isoforms in view of their potential as drug targets for PD neuroprotection, and we conclude by considering how these aspects could guide further drug development.

Role of L-Type Voltage-Gated Calcium Channels in Epileptiform Activity of Neurons.

Epileptic discharges manifest in individual neurons as abnormal membrane potential fluctuations called paroxysmal depolarization shift (PDS). PDSs can combine into clusters that are accompanied by synchronous oscillations of the intracellular Ca2+ concentration ([Ca2+]i) in neurons. Here, we investigate the contribution of L-type voltage-gated calcium channels (VGCC) to epileptiform activity induced in cultured hippocampal neurons by GABA(A)R antagonist, bicuculline. Using KCl-induced depolarization, we determined the optimal effective doses of the blockers. Dihydropyridines (nifedipine and isradipine) at concentrations ≤ 10 µM demonstrate greater selectivity than the blockers from other groups (phenylalkylamines and benzothiazepines). However, high doses of dihydropyridines evoke an irreversible increase in [Ca2+]i in neurons and astrocytes. In turn, verapamil and diltiazem selectively block L-type VGCC in the range of 1-10 µM, whereas high doses of these drugs block other types of VGCC. We show that L-type VGCC blockade decreases the half-width and amplitude of bicuculline-induced [Ca2+]i oscillations. We also observe a decrease in the number of PDSs in a cluster and cluster duration. However, the pattern of individual PDSs and the frequency of the cluster occurrence change insignificantly. Thus, our results demonstrate that L-type VGCC contributes to maintaining the required [Ca2+]i level during oscillations, which appears to determine the number of PDSs in the cluster.

Physicochemical, pharmacokinetic, and pharmacodynamic characterization of isradipine tablets for controlled release.

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.

Harnessing ionic mechanisms to achieve disease modification in neurodegenerative disorders.

Progressive neuronal death is the key pathogenic event leading to clinical symptoms in neurodegenerative disorders (NDDs). Neuroprotective treatments are virtually unavailable, partly because of the marked internal heterogeneity of the mechanisms underlying pathology. Targeted neuroprotection would require deep mechanistic knowledge across the entire aetiological spectrum of each NDD and the development of tailored treatments. Although ideal, this strategy appears challenging, as it would require a degree of characterization of both the disease and the patient that is currently unavailable. The alternate strategy is to search for commonalities across molecularly distinct NDD forms and exploit these for the development of drugs with broad-spectrum efficacy. In this view, mounting evidence points to ionic mechanisms (IMs) as targets with potential therapeutic efficacy across distinct NDD subtypes. The scope of this review is to present clinical and preclinical evidence supporting the link between disruption of IMs and neuronal death in specific NDDs and to critically revise past and ongoing attempts of harnessing IMs for the development of neuroprotective treatments.

Lower Affinity of Isradipine for L-Type Ca2+ Channels during Substantia Nigra Dopamine Neuron-Like Activity: Implications for Neuroprotection in Parkinson's Disease.

Ca2+-influx through L-type Ca2+-channels (LTCCs) is associated with activity-related stressful oscillations of Ca2+ levels within dopaminergic (DA) neurons in the substantia nigra (SN), which may contribute to their selective degeneration in Parkinson's disease (PD). LTCC blockers were neuroprotective in mouse neurotoxin models of PD, and isradipine is currently undergoing testing in a phase III clinical trial in early PD. We report no evidence for neuroprotection by in vivo pretreatment with therapeutically relevant isradipine plasma levels, or Cav1.3 LTCC deficiency in 6-OHDA-treated male mice. To explain this finding, we investigated the pharmacological properties of human LTCCs during SN DA-like and arterial smooth muscle (aSM)-like activity patterns using whole-cell patch-clamp recordings in HEK293 cells (Cav1.2 α1-subunit, long and short Cav1.3 α1-subunit splice variants; ß3/α2δ1). During SN DA-like pacemaking, only Cav1.3 variants conducted Ca2+ current (ICa) at subthreshold potentials between action potentials. SN DA-like burst activity increased integrated ICa during (Cav1.2 plus Cav1.3) and after (Cav1.3) the burst. Isradipine inhibition was splice variant and isoform dependent, with a 5- to 11-fold lower sensitivity to Cav1.3 variants during SN DA-like pacemaking compared with Cav1.2 during aSM-like activity. Supratherapeutic isradipine concentrations reduced the pacemaker precision of adult mouse SN DA neurons but did not affect their somatic Ca2+ oscillations. Our data predict that Cav1.2 and Cav1.3 splice variants contribute differentially to Ca2+ load in SN DA neurons, with prominent Cav1.3-mediated ICa between action potentials and after bursts. The failure of therapeutically relevant isradipine levels to protect SN DA neurons can be explained by weaker state-dependent inhibition of SN DA LTCCs compared with aSM Cav1.2.SIGNIFICANCE STATEMENT The high vulnerability of dopamine (DA) neurons in the substantia nigra (SN) to neurodegenerative stressors causes Parkinson's disease (PD). Ca2+ influx through voltage-gated L-type Ca2+ channels (LTCCs), in particular Cav1.3, appears to contribute to this vulnerability, and the LTCC inhibitor isradipine is currently being tested as a neuroprotective agent for PD in a phase III clinical trial. However, in our study isradipine plasma concentrations approved for therapy were not neuroprotective in a PD mouse model. We provide an explanation for this observation by demonstrating that during SN DA-like neuronal activity LTCCs are less sensitive to isradipine than Cav1.2 LTCCs in resistance blood vessels (mediating dose-limiting vasodilating effects) and even at supratherapeutic concentrations isradipine fails to reduce somatic Ca2+ oscillations of SN DA neurons.

A concerted action of L- and T-type Ca(2+) channels regulates locus coeruleus pacemaking.

Dysfunction of noradrenergic locus coeruleus (LC) neurons is involved in psychiatric and neurodegenerative diseases and is an early hallmark of Parkinson's disease (PD). The analysis of ion channels underlying the autonomous electrical activity of LC neurons, which is ultimately coupled to cell survival signaling pathways, can lead to a better understanding of the vulnerability of these neurons. In LC neurons somatodendritic Ca(2+) oscillations, mediated by L-type Ca(2+) channels, accompany spontaneous spiking and are linked to mitochondrial oxidant stress. However, the expression and functional implication of low-threshold activated T-type Ca(2+) channels in LC neurons were not yet studied. To this end we performed RT-PCR expression analysis in LC neurons. In addition, we utilized slice patch clamp recordings of in vitro brainstem slices in combination with L-type and T-type Ca(2+) channel blockers. We found the expression of a distinct set of L-type and T-type Ca(2+) channel subtypes mediating a pronounced low-threshold activated Ca(2+) current component. Analyzing spike trains, we revealed that neither L-type Ca(2+) channel nor T-type Ca(2+) channel blockade alone leads to a change in firing properties. In contrast, a combined application of antagonists significantly decreased the afterhyperpolarization amplitude, resulting in an increased firing frequency. Hence, we report the functional expression of T-type Ca(2+) channels in LC neurons and demonstrate their role in increasing the robustness of LC pacemaking by working in concert with Cav1 channels.

Functional segregation of voltage-activated calcium channels in motoneurons of the dorsal motor nucleus of the vagus.

Calcium influx elevates mitochondrial oxidant stress (mOS) in dorsal motor nucleus of the vagus (DMV) neurons that are prone to Lewy body pathologies in presymptomatic Parkinson's disease (PD) patients. In experimental PD models, treatment with isradipine, the dihydropyridine with the highest affinity to Cav1.3 channels, prevents subthreshold calcium influx via Cav1.3 channels into midbrain dopamine neurons and protects them from mOS. In DMV neurons, isradipine is also effective in reducing mOS despite overwhelming evidence that subthreshold calcium influx is negligible compared with spike-triggered influx. To solve this conundrum we combined slice electrophysiology, two-photon laser scanning microscopy, mRNA profiling, and computational modeling. We find that the unusually depolarized subthreshold voltage trajectory of DMV neurons is positioned between the relatively hyperpolarized activation curve of Cav1.3 channels and that of other high-voltage activated (HVA) calcium channels, thus creating a functional segregation between Cav1.3 and HVA calcium channels. The HVA channels flux the bulk of calcium during spikes but can only influence pacemaking through their coupling to calcium-activated potassium currents. In contrast, Cav1.3 currents, which we show to be more than an order-of-magnitude smaller than the HVA calcium currents, are able to introduce sufficient inward current to speed up firing. However, Kv4 channels that are constitutively open in the subthreshold range guarantee slow pacemaking, despite the depolarizing action of Cav1.3 and other pacemaking currents. We propose that the efficacy of isradipine in preventing mOS in DMV neurons arises from its mixed effect on Cav1.3 channels and on HVA Cav1.2 channels.

Cav1.3 channels control D2-autoreceptor responses via NCS-1 in substantia nigra dopamine neurons.

Dopamine midbrain neurons within the substantia nigra are particularly prone to degeneration in Parkinson's disease. Their selective loss causes the major motor symptoms of Parkinson's disease, but the causes for the high vulnerability of SN DA neurons, compared to neighbouring, more resistant ventral tegmental area dopamine neurons, are still unclear. Consequently, there is still no cure available for Parkinson's disease. Current therapies compensate the progressive loss of dopamine by administering its precursor l-DOPA and/or dopamine D2-receptor agonists. D2-autoreceptors and Cav1.3-containing L-type Ca(2+) channels both contribute to Parkinson's disease pathology. L-type Ca(2+) channel blockers protect SN DA neurons from degeneration in Parkinson's disease and its mouse models, and they are in clinical trials for neuroprotective Parkinson's disease therapy. However, their physiological functions in SN DA neurons remain unclear. D2-autoreceptors tune firing rates and dopamine release of SN DA neurons in a negative feedback loop through activation of G-protein coupled potassium channels (GIRK2, or KCNJ6). Mature SN DA neurons display prominent, non-desensitizing somatodendritic D2-autoreceptor responses that show pronounced desensitization in PARK-gene Parkinson's disease mouse models. We analysed surviving human SN DA neurons from patients with Parkinson's disease and from controls, and detected elevated messenger RNA levels of D2-autoreceptors and GIRK2 in Parkinson's disease. By electrophysiological analysis of postnatal juvenile and adult mouse SN DA neurons in in vitro brain-slices, we observed that D2-autoreceptor desensitization is reduced with postnatal maturation. Furthermore, a transient high-dopamine state in vivo, caused by one injection of either l-DOPA or cocaine, induced adult-like, non-desensitizing D2-autoreceptor responses, selectively in juvenile SN DA neurons, but not ventral tegmental area dopamine neurons. With pharmacological and genetic tools, we identified that the expression of this sensitized D2-autoreceptor phenotype required Cav1.3 L-type Ca(2+) channel activity, internal Ca(2+), and the interaction of the neuronal calcium sensor NCS-1 with D2-autoreceptors. Thus, we identified a first physiological function of Cav1.3 L-type Ca(2+) channels in SN DA neurons for homeostatic modulation of their D2-autoreceptor responses. L-type Ca(2+) channel activity however, was not important for pacemaker activity of mouse SN DA neurons. Furthermore, we detected elevated substantia nigra dopamine messenger RNA levels of NCS-1 (but not Cav1.2 or Cav1.3) after cocaine in mice, as well as in remaining human SN DA neurons in Parkinson's disease. Thus, our findings provide a novel homeostatic functional link in SN DA neurons between Cav1.3- L-type-Ca(2+) channels and D2-autoreceptor activity, controlled by NCS-1, and indicate that this adaptive signalling network (Cav1.3/NCS-1/D2/GIRK2) is also active in human SN DA neurons, and contributes to Parkinson's disease pathology. As it is accessible to pharmacological modulation, it provides a novel promising target for tuning substantia nigra dopamine neuron activity, and their vulnerability to degeneration.

Nuclear membrane R-type calcium channels mediate cytosolic ET-1-induced increase of nuclear calcium in human vascular smooth muscle cells.

The objective of this work was to verify whether, as in the case of the plasma membrane of human vascular smooth muscle cells (hVSMCs), cytosolic ET-1-induced increase of nuclear calcium is mediated via the activation of calcium influx through the steady-state R-type calcium channel. Pharmacological tools to identify the R-type calcium channels, as well as real 3-D confocal microscopy imaging techniques coupled to calcium fluorescent probes, were used to study the effect of cytosolic ET-1 on nuclear calcium in isolated nuclei of human hepatocytes and plasma membrane perforated hVSMCs. Our results showed that pre-treatment with pertussis toxin (PTX) or cholera toxin (CTX) prevented cytosolic ET-1 (10(-9) mol/L) from inducing a sustained increase in nuclear calcium. Furthermore, the L-type calcium channel blocker nifedipine did not prevent cytosolic ET-1 from inducing an increase in nuclear calcium, as opposed to the dual L- and R-type calcium channel blocker isradipine (PN200-110) (in the presence of nifedipine). In conclusion, the preventative effect with PTX and CTX, and the absence of an effect with nifedipine, as well as the blockade by isradipine on cytosolic ET-1-induced increase in nuclear calcium, suggest that this nuclear calcium influx in hVSMCs is due to activation of the steady-state R-type calcium channel. The sarcolemmal and nuclear membrane R-type calcium channels in hVSMCs are involved in ET-1 modulation of vascular tone in physiology and pathology.

Development of isradipine loaded self-nano emulsifying powders for improved oral delivery: in vitro and in vivo evaluation.

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.

Practical Radiosynthesis and Preclinical Neuroimaging of [11C]isradipine, a Calcium Channel Antagonist.

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.

Pilot investigation of isradipine in the treatment of bipolar depression motivated by genome-wide association.

OBJECTIVES: Motivated by genetic association data implicating L-type calcium channels in bipolar disorder liability, we sought to estimate the tolerability, safety, and efficacy of isradipine in the adjunctive treatment of bipolar depression. METHODS: A total of 12 patients with bipolar I or II depression entered this pilot, proof-of-concept eight-week investigation and 10 returned for at least one post-baseline visit. They were initiated on isradipine at 2.5 mg and titrated up to 10 mg daily, with blinded assessments of depression using the Montgomery-Åsberg Depression Rating Scale (MADRS) as well as adverse effects. RESULTS: Among the 10 patients, three had bipolar II disorder; all but two reported current episode duration longer than six months. In all, four of 10 completed the study; no significant adverse events were observed, although one subject discontinued treatment per protocol because of possible hypomanic symptoms which had resolved prior to study visit. In a mixed-effects model, mean improvement in depression severity, assessed by MADRS, was 2.1 (standard error = 0.36) points/week (p < 0.001). Two of the 10 subjects remitted and four of the 10 subjects experienced 50% or greater symptomatic improvement with treatment. CONCLUSIONS: Isradipine merits further investigation for the treatment of bipolar depression. This preliminary trial illustrates the potential utility of genetic investigation in identifying psychiatric treatment targets.

Enhanced isradipine sensitivity in vascular smooth muscle cells due to hypoxia-induced Cav1.2 splicing and RbFox1/Fox2 downregulation.

Calcium influx via the L-type voltage-gated Cav1.2 calcium channel in smooth muscle cells regulates vascular contraction. Calcium channel blockers (CCBs) are widely used to treat hypertension by inhibiting Cav1.2 channels. Using the vascular smooth muscle cell line, A7r5 and primary culture of cerebral vascular smooth muscle cells, we found that the expression and function of Cav1.2 channels are downregulated during hypoxia. Furthermore, hypoxia induces structural changes in Cav1.2 channels via alternative splicing. The expression of exon 9* is upregulated, whereas exon 33 is downregulated. Such structural alterations of Cav1.2 channels are caused by the decreased expression of RNA-binding proteins RNA-binding protein fox-1 homolog 1 and 2 (RbFox1 and RbFox2). Overexpression of RbFox1 and RbFox2 prevents hypoxia-induced exon 9* inclusion and exon 33 exclusion. Importantly, such structural alterations of the Cav1.2 channel partly contribute to the enhanced sensitivity of Cav1.2 to isradipine (a CCB) under hypoxia. Overexpression of RbFox1 and RbFox2 successfully reduces isradipine sensitivity in hypoxic smooth muscle cells. Our results suggest a new strategy to manage ischemic diseases such as stroke and myocardial infarction.

L-type calcium channel regulation of depression, anxiety and anhedonia-related behavioral phenotypes following chronic stress exposure.

Exposure to chronic and unpredictable stressors can precipitate mood-related disorders in humans, particularly in individuals with pre-existing mental health challenges. L-type calcium channels (LTCCs) have been implicated in numerous neuropsychiatric disorders, as LTCC encoding genes have been identified as candidate risk factors for neuropsychiatric illnesses. In these sets of experiments, we sought to examine the ability of LTCC blockade to alter depression, anxiety, and anhedonic-related behavioral responses to chronic unpredictable stress (CUS) exposure in female and male rats. Rats first underwent either 21 days of CUS or no exposure to chronic stressors, serving as home cage controls (HCC). Then rats were examined for anhedonia-related behavior, anxiety and depression-like behavioral responses as measured by the sucrose preference test (SPT), elevated plus maze (EPM), and forced swim test (FST). CUS exposed females and males showed anhedonic and anxiogenic-like behavioral responses on the SPT and EPM, respectively, when compared to HCCs. In female and male rats, systemic administration of the LTCC blocker isradipine (0.4 mg/kg and 1.2 mg/kg, I.P.) attenuated the CUS-induced decrease in sucrose preference and reversed the CUS-induced decrease in open arm time. In the FST, systemic isradipine decreased immobility time across all groups, consistent with an antidepressant-like response. However, there were no significant differences in forced swim test immobility time between HCC and CUS exposed animals. Taken together, these data point to a role of LTCCs in the regulation of mood disorder-related behavioral phenotype responses to chronic stress exposure.

Inactivation induced by pathogenic Cav1.3 L-type Ca2+-channel variants enhances sensitivity for dihydropyridine Ca2+ channel blockers.

BACKGROUND AND PURPOSE: Pathogenic gain-of-function mutations in Cav1.3 L-type voltage-gated Ca2+-channels (CACNA1D) cause neurodevelopmental disorders with or without endocrine symptoms. We aimed to confirm a pathogenic gain-of function phenotype of CACNA1D de novo missense mutations A749T and L271H, and investigated the molecular mechanism causing their enhanced sensitivity for the Ca2+-channel blocker isradipine, a potential therapeutic for affected patients. EXPERIMENTAL APPROACH: Wildtype and mutant channels were expressed in tsA-201 cells and their gating analysed using whole-cell and single-channel patch-clamp recordings. The voltage-dependence of isradipine action was quantified using protocols inducing variable fractions of inactivated channels. The molecular basis for altered channel gating in the mutants was investigated using in silico modelling and molecular dynamics simulations. KEY RESULTS: Both mutations were confirmed pathogenic due to characteristic shifts of voltage-dependent activation and inactivation towards negative potentials (~20 mV). At negative holding potentials both mutations showed significantly higher isradipine sensitivity compared to wildtype. The affinity for wildtype and mutant channels increased with channel inactivation as predicted by the modulated receptor hypothesis (30- to 40-fold). The IC50 was indistinguishable for wildtype and mutants when >50% of channels were inactivated. CONCLUSIONS AND IMPLICATIONS: Mutations A749T and L271H induce pathogenic gating changes. Like wildtype, isradipine inhibition is strongly voltage-dependent. Our data explains their apparent higher drug sensitivity at a given negative voltage by the availability of more inactivated channels due to their more negative inactivation voltage range. Low nanomolar isradipine concentrations will only inhibit Cav1.3 channels in neurons during prolonged depolarized states without selectivity for mutant channels.

Phase II safety, tolerability, and dose selection study of isradipine as a potential disease-modifying intervention in early Parkinson's disease (STEADY-PD).

Isradipine, a dihydropyridine calcium channel antagonist, has been shown to be neuroprotective in animal models of Parkinson's disease (PD). To establish a dosage of isradipine controlled-release (CR) that is tolerable and demonstrates preliminary efficacy for use in a future pivotal efficacy trial a Phase 2, randomized, double-blind, parallel group trial (Safety, Tolerability and Efficacy Assessment of Dynacirc CR in Parkinson Disease [STEADY-PD]) was undertaken in subjects with early PD not requiring dopaminergic therapy (dopamine agonists or levodopa) randomized 1:1:1:1 to 5, 10, or 20 mg of isradipine CR or matching placebo daily. The primary outcome was tolerability defined as no more than a 30% difference in the proportion of patients completing the study on the originally assigned dosage between an active and placebo group. If more than one isradipine dosage was tolerable, then a 3-point difference in total Unified Parkinson's Disease Rating Scale (UPDRS) change between baseline and week 52 (or time to sufficient disability to require dopaminergic therapy) was taken as a criterion for selection of the most desirable dosage for future study. STEADY-PD enrolled 99 subjects. The tolerability of isradipine was dose dependent: placebo, 25 of 26 patients (96%); 5 mg, 19 of 23 patients (83%); 10 mg 19 of 26 patients (73%); and 20 mg 9 of 24 patients (37%). There was no difference in change in UPDRS among dosages. The most common adverse events were peripheral edema (30) and dizziness (24). Isradipine 10 mg daily was the maximal tolerable dosage in this study of early PD. A large placebo-controlled trial will be necessary and is planned to assess efficacy of isradipine 10 mg daily to slow progression of PD disability.

Pharmacokinetic properties of isradipine after single-dose and multiple-dose oral administration in Chinese volunteers: a randomized, open-label, parallel-group phase I study.

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.

L-type calcium channel antagonist isradipine age-dependently decreases plaque associated dystrophic neurites in 5XFAD mouse model.

Epidemiological studies suggest that L-type calcium channel (LTCC) antagonists may reduce the incidence of age-associated neurodegenerative diseases including Alzheimer's disease (AD). However, the neuroprotective mechanism of LTCC antagonists is unknown. Amyloid-ß (Aß) pathology disrupts intracellular calcium signaling, which regulates lysosomes and microglial responses. Neurons near Aß plaques develop dystrophic neurites, which are abnormal swellings that accumulate lysosomes. Further, microglia accumulate around Aß plaques and secrete inflammatory cytokines. We hypothesized that antagonism of LTCCs with isradipine would reduce Aß plaque-associated dystrophic neurites and inflammatory microglia in the 5XFAD mouse model by restoring normal intracellular calcium regulation. To test this hypothesis, we treated 6- and 9-month-old 5XFAD mice with isradipine and tested behavior, examined Aß plaques, microglia, and dystrophic neurites. We found that isradipine treatment age-dependently reduces dystrophic neurites and leads to trending decreases in Aß but does not modulate plaque associated microglia regardless of age. Our findings provide insight into how antagonizing LTCCs alters specific cell types in the Aß plaque environment, providing valuable information for potential treatment targets in future AD studies.

Usage of L-type calcium channel blockers to suppress drug reward and memory driving addiction: Past, present, and future.

Over the past three decades, L-type Ca2+ channel (LTCC) blockers have been considered a potential therapeutic drug to alleviate the symptoms of drug addiction. This idea has been supported, in part, by 1) expression of LTCCs in the brain dopaminergic circuits that are thought to play critical roles in the development and expression of addictive behaviors and 2) common usage of LTCC blockers in treating hypertension, which may enable off-label use of these drugs with good brain penetration as therapeutics for brain disorders. Addiction can be viewed as a maladaptive form of learning where powerful memories of drug-associated stimuli and actions drive compulsive drug intake. Largely under this framework, we will focus on the dopaminergic system that is thought be critically involved in drug-associated learning and memory and provide a brief overview of the past and recent studies testing the therapeutic potential of LTCC blockers for addictive disorders in animal models and humans and offer a future perspective on the use of LTCC blockers in drug addiction and, possibly, addiction to other non-drug rewards (e.g., gambling, eating, shopping). Interested readers can refer to other related articles in this issue and a comprehensive review available elsewhere (Little, 2021) to gain further insights into the roles of LTCCs in drug addiction and withdrawal symptoms associated with dependence. This article is part of the Special Issue on 'L-type calcium channel mechanisms in neuropsychiatric disorders'.

Formulation and Evaluation of Isradipine Nanosuspension and Exploring its Role as a Potential Anticancer Drug by Computational Approach.

BACKGROUND: T-type calcium channels are aberrantly expressed in different human cancers and regulate cell cycle progression, proliferation, migration, and survival. FAK-1 can promote tumor protein degradation (p53) through ubiquitination, leading to cancer cell growth and proliferation. Similar findings are obtained regarding protease inhibitors' effect on cytokine-induced neutrophil activation that suppresses Granulocyte-macrophage colony-stimulatingfactor (GM-CSF) TNF-α-induced O2 release and adherence in human neutrophils without affecting phosphorylation of Extracellular signal-regulated kinase (ERK) and p38. Nanosuspensions are carrier-free, submicron colloidal dispersions, which consist of pure drugs and stabilizers. Incorporating drug loaded in nanosuspensions offer a great advantages of passive drug targeting with improved solubility, stability, and bioavailability, as well as lower systemic toxicity. OBJECTIVE: The present investigation objective was to establish a molecular association of Protease and Focal Adhesion Kinase 1 as cancer targets for isradipine, a calcium channel blocker (CCB). Furthermore, the study also aimed to formulate its optimized nanosuspension and how the physical, morphological, and dissolution properties of isradipine impact nanosuspension stability. METHODS: Five different molecular targets, namely Cysteine Proteases (Cathepsin B), Serine Proteases (Matriptase), Aspartate Proteases, Matrix Metalloproteases (MMP), and FAK-1 were obtained from RCSB-PDB, which has some potential associations with inhibition in cancer pathogenesis. Molecular interactions of these targets with CCB isradipine were identified and established by molecular simulation docking studies. Isradipine-loaded nanosuspension was prepared by precipitation technique by employing a 23 factorial design. PVP K-30, poloxamer 188, and sodium lauryl sulfate (SLS) were used as polymer, co-polymer, and surfactant, respectively. The nanosuspension particles were assessed for particle size, zeta potential, viscosity, polydispersity index (PDI), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), In-vitro drug release kinetics, and short-term stability study. RESULTS: Considerable interactions were found with Cysteine, Serine, Aspartate, Threonine, and Matrix metalloproteases with binding energies of -3.91, -6.7, -3.48, -8.42, respectively. Furthermore, the interaction of isradipine with FAK-1 was compared with 7 native ligands and was found to show significant interaction with binding energies of - 8.62, -7.27, -7.69, -5.67, -5.41, -7.44, -8.21, respectively. The optimized nanosuspension was evaluated and exhibited a particle size of 754.9 nm, zeta potential of 32.5 mV, viscosity of 1.287 cp, and PDI of 1.000. The In-vitro dissolution of the optimized formulation (F8) was found to be higher (96.57%) as compared to other formulations. CONCLUSION: Isradipine could act as a potential inhibitor of different proteases and FAK-1 associated with tumor growth initiation, progression, and metastasis. Furthermore, isradipine-loaded nanosuspension with optimized release could be utilized to deliver the anticancer drug in a more targeted way as emerging cancer nanotechnology.