First-in-man study of ACT-709478, a novel selective triple T-type calcium channel blocker.

OBJECTIVE: Increased activity of T-type Ca2+ channels is linked to idiopathic generalized epilepsies, thus blocking these channels may be a new treatment option. ACT-709478 is an orally available triple T-type Ca2+ channel blocker. The aim of this first-in-man study was to investigate the pharmacokinetics, pharmacodynamics, tolerability, and safety of single doses of ACT-709478 in healthy subjects. METHODS: This double-blind, placebo-controlled, randomized study included 65 healthy male subjects. Ascending single oral doses of 1-400 mg ACT-709478 or placebo were administered to sequential groups of eight subjects (6 on active, 2 on placebo). Effect of food was tested in a crossover part at 60 mg. Blood and saliva sampling for pharmacokinetic evaluations and safety assessments was performed regularly. Effects on the central nervous system were assessed with a battery of pharmacodynamic tests. RESULTS: The maximum plasma concentration (Cmax ) was reached within 3 to 4 hours (≤60 mg) and within 20 to 28 hours (>60 mg), and across all dose levels the terminal half-life (95% confidence interval) ranged from 36 (29-45) to 43 (22-86) hours. Multiple peaks were observed and Cmax and area under the plasma concentration-time curve (AUC)0-∞ increased in a less than dose-proportional manner. A 1.6-fold increase in Cmax and no change in AUC0-∞ was observed in fed compared to fasted conditions. A significant correlation (P < 0.0001) between plasma and saliva concentrations was established using linear regression. All adverse events were transient and of mild or moderate intensity. No treatment-related effects on vital signs, clinical laboratory, telemetry, or electrocardiography were detected. The results of pharmacodynamic tests did not show relevant mean changes compared to baseline or placebo. SIGNIFICANCE: ACT-709478 exhibits good tolerability and safety after single-dose administration and its pharmacokinetic and pharmacodynamic properties warrant further investigations.

Selective tools for the solid-phase extraction of Ochratoxin A from various complex samples: immunosorbents, oligosorbents, and molecularly imprinted polymers.

The evolution of instrumentation in terms of separation and detection has allowed a real improvement of the sensitivity and the analysis time. However, the analysis of ultra-traces of toxins such as ochratoxin A (OTA) from complex samples (foodstuffs, biological fluids…) still requires a step of purification and of preconcentration before chromatographic determination. In this context, extraction sorbents leading to a molecular recognition mechanism appear as powerful tools for the selective extraction of OTA and of its structural analogs in order to obtain more reliable and sensitive quantitative analyses of these compounds in complex media. Indeed, immunosorbents and oligosorbents that are based on the use of immobilized antibodies and of aptamers, respectively, and that are specific to OTA allow its selective clean-up from complex samples with high enrichment factors. Similar molecular recognition mechanisms can also be obtained by developing molecularly imprinted polymers, the synthesis of which leads to the formation of cavities that are specific to OTA, thus mimicking the recognition site of the biomolecules. Therefore, the principle, the advantages, the limits of these different types of extraction tools, and their complementary behaviors will be presented. The introduction of these selective tools in miniaturized devices will also be discussed.

Use of DMPC and DSPC lipids for verapamil and naproxen permeability studies by PAMPA.

Verapamil and naproxen Parallel Artificial Membrane Permeability Assay (PAMPA) permeability was studied using lipids not yet reported for this model in order to facilitate the quantification of drug permeability. These lipids are 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and an equimolar mixture of DMPC/DSPC, both in the absence and in the presence of 33.3 mol% of cholesterol. PAMPA drug permeability using the lipids mentioned above was compared with lecithin-PC. The results show that verapamil permeability depends on the kind of lipid used, in the order DMPC > DMPC/DSPC > DSPC. The permeability of the drugs was between 1.3 and 3.5-times larger than those obtained in lecithin-PC for all the concentrations of the drug used. Naproxen shows similar permeability than verapamil; however, the permeability increased with respect to lecithin-PC only when DMPC and DMPC/DSPC were used. This behavior could be explained by a difference between the drug net charge at pH 7.4. On the other hand, in the presence of cholesterol, verapamil permeability increases in all lipid systems; however, the relative verapamil permeability respect to lecithin-PC did not show any significant increase. This result is likely due to the promoting effect of cholesterol, which is not able to compensate for the large increase in verapamil permeability observed in lecithin-PC. With respect to naproxen, its permeability value and relative permeability respect lecithin-PC not always increased in the presence of cholesterol. This result is probably attributed to the negative charge of naproxen rather than its molecular weight. The lipid systems studied have an advantage in drug permeability quantification, which is mainly related to the charge of the molecule and not to its molecular weight or to cholesterol used as an absorption promoter.

Simultaneous determination of valsartan, amlodipine besylate and hydrochlorothiazide using capillary zone electrophoresis (CZE).

A capillary zone electrophoresis method was developed for the simultaneous determination of valsartan (VAL), amlodipine besylate (AML) and hydrochlorothiazide (HCZ) in their combined tablets. Separation was achieved on a fused silica capillary by applying a potential of 15 kV (positive polarity) and a running background electrolyte containing 40 mM phosphate buffer at pH 7.5 with UV detection at 230 nm. The samples were injected hydrodynamically for 3s at 0.5 psi and the temperature of the capillary cartridge was kept at 25 degrees C. Pyrazinoic acid was used as an internal standard. The method was validated according to ICH guidelines regarding specificity, linearity, limits of detection and quantitation, accuracy and precision, (Supplementary materials, Table S2). The method showed satisfactory linearity in the ranges of 10-200, 2-20 and 2-20 µg mL(-1) with LODs of 1.82, 0.39, 0.65 µg mL(-1) and LOQs of 5.51, 1.17, 1.96 µg mL(-1) for VAL, AML and HCZ, respectively. The proposed method was successfully applied for the analysis of the studied drugs in their laboratory prepared mixtures and co-formulated tablets. The results were compared with reported methods and no significant differences were found. The proposed method can be used for quality control of the cited drugs in ordinary laboratories.

First-order derivative UV spectrophotometric method for simultaneous measurement of delapril and manidipine in tablets.

A first-order derivative spectrophotometric (1D-UV) method was developed and validated for simultaneous determination of delapril (DEL) and manidipine (MAN) in tablets. The 1D-UV spectra were obtained using change lambda = 4.0 nm and wavelength set at 228 nm for DEL and 246 nm for MAN. The method was validated in accordance with the ICH requirements, involving the specificity, linearity, precision, accuracy, robustness and limits of detection and quantitation. The method showed high specificity in the presence of two drugs and formulation excipients and was linear over the concentration range of 18-54 microg mL(-1) (r2 = 0.9994) for DEL and 6-18 microg mL(-1) (r2 = 0.9981) for MAN with adequate results for the precision (< or = 1.47%) and accuracy (98.98% for DEL and 100.50% for MAN). Moreover, the method proved to be robust by a Plackett-Burman experimental design evaluation. The proposed 'D-UV method was successfully applied for simultaneous analysis of DEL and MAN in tablets and can be used as alternative green method to separation techniques. The results were compared with the validated liquid chromatography, capillary electrophoresis and liquid chromatography-tandem mass spectrometry methods, showing non-significant difference.

Gut modulatory effects of Daphne oleoides are mediated through cholinergic and Ca++ antagonist mechanisms.

CONTEXT: The present study describes the spasmogenic and spasmolytic activities of Daphne oleoides Schreb. (Thymelaeaceae), exploring the possible underlying pharmacological mechanisms. AIM: Pharmacological investigation of Daphne oleoides to provide evidence for its therapeutic application in gastrointestinal motility disorders. MATERIALS AND METHODS: Methanol crude extract of Daphne oleoides (Do.Cr) was studied in gastrointestinal isolated tissues. RESULTS: In spontaneously contracting rabbit jejunum preparations, Do.Cr at 0.3-3.0 mg/mL caused moderate stimulation, followed by relaxant effect at the next higher concentrations (5.0-10 mg/mL). In presence of atropine, spasmogenic effect was blocked and the relaxation was emerged, suggesting that the spasmogenic effect of Daphne oleoides is mediated through activation of muscarinic receptors. When tested against the high K+ (80 mM)-induced contractions, Do.Cr (0.3-5.0 mg/mL), like verapamil, inhibited the induced contractions, suggesting Ca++ channel blockade (CCB) effect. The CCB effect was further confirmed when pre-treatment of the tissue with Do.Cr shifted the Ca++ concentration-response curves to the right, similar to that caused by verapamil. DISCUSSION AND CONCLUSION: These results indicate that Daphne oleoides exhibits gut excitatory and inhibitory effects, occurred via cholinergic and Ca++ antagonistic pathways, respectively.

An Investigative Review for Pharmaceutical Analysis of 1,4-Dihydropyridine-3,5-Dicarboxylic Acid Derivative: Cilnidipine.

Hypertension is commonly a quiet condition, and it expands the risk of heart diseases and stroke. Calcium delivers a substantial role in cardiovascular functions and hence is essential for cardiac automaticity and functioning. Calcium channel antagonists are the choice of drugs for the management of cardiovascular diseases; they precisely stop the introduction of calcium through L-type calcium channels are existing channels in the heart. Cilnidipine belongs to the class 4th generation calcium channel blockers as a foremost therapeutic agent used in the treatment of hypertension and heart diseases. This review article focuses on an inclusive account of crucial analytical methodologies used for the pharmaceutical analysis of cilnidipine in pure forms, biological samples and pharmaceuticals. According to literature reports several analytical techniques such as hyphenated techniques, high-performance thin-layer chromatography, high-performance liquid-chromatography, capillary electrophoresis, voltammetry, UV/Vis-spectrophotometry, and Fourier-transform infrared spectroscopy approaches have been used for determination of cilnidipine alone or in the combined dosage form. We have also discussed the pharmacopeial assay methods, physicochemical properties, and also depict the stacked column chart for year wise publication count for cilnidipine. From literature, concluded that the high-performance liquid-chromatography and UV/Vis-spectrophotometry methods are the most prevailing methods for the analysis of cilnidipine. The data presented in this review may provide a very significant base for further studies on cilnidipine in the area of drug analysis.

Thermal analysis applied to verapamil hydrochloride characterization in pharmaceutical formulations.

Thermogravimetry (TG) and differential scanning calorimetry (DSC) are useful techniques that have been successfully applied in the pharmaceutical industry to reveal important information regarding the physicochemical properties of drug and excipient molecules such as polymorphism, stability, purity, formulation compatibility among others. Verapamil hydrochloride shows thermal stability up to 180 degrees C and melts at 146 degrees C, followed by total degradation. The drug is compatible with all the excipients evaluated. The drug showed degradation when subjected to oxidizing conditions, suggesting that the degradation product is 3,4-dimethoxybenzoic acid derived from alkyl side chain oxidation. Verapamil hydrochloride does not present the phenomenon of polymorphism under the conditions evaluated. Assessing the drug degradation kinetics, the drug had a shelf life (t90) of 56.7 years and a pharmaceutical formulation showed t90 of 6.8 years showing their high stability.

Structural evidence for direct interaction between the molecular components of the transverse tubule/sarcoplasmic reticulum junction in skeletal muscle.

The architecture of the junctional sarcoplasmic reticulum (SR) and transverse tubule (T tubule) membranes and the morphology of the two major proteins isolated from these membranes, the ryanodine receptor (or foot protein) and the dihydropyridine receptor, have been examined in detail. Evidence for a direct interaction between the foot protein and a protein component of the junctional T tubule membrane is presented. Comparisons between freeze-fracture images of the junctional SR and rotary-shadowed images of isolated triads and of the isolated foot protein, show that the foot protein has two domains. One is the large hydrophilic foot which spans the junctional gap and is composed of four subunits. The other is a hydrophobic domain which presumably forms the SR Ca2+-release channel and which also has a fourfold symmetry. Freeze-fracture images of the junctional T tubule membranes demonstrate the presence of diamond-shaped clusters of particles that correspond exactly in position to the subunits of the feet protein. These results suggest the presence of a large junctional complex spanning the two junctional membranes and intervening gap. This junctional complex is an ideal candidate for a mechanical coupling hypothesis of excitation-contraction coupling at the triadic junction.

Determination of amlodipine enantiomers in pharmaceuticals using capillary electrophoresis separation and diode array detection.

The present work describes a capillary zone electrophoresis (CZE) separation technique coupled with on-capillary diode array detector (DAD) for highly reliable enantioselective determination of amlodipine (AML) in commercial tablets. For the separation of AML enantiomers, (2-hydroxypropyl)-beta-cyclodextrin (HP-beta-CD) was an appropriate chiral selector providing complete enantioresolution. Optimized separation conditions consisted of 50 mmol/l glycine-acetate buffer, pH 3.2, 50 mg/ml HP-beta-CD. Hydroxyethylcellulose (HEC, 0.2% w/v) served as an electroosmotic flow (EOF) suppressor in this buffer. DAD detection was used for the characterization of the composition of separated zones according to differences in corresponding UV-VIS spectra (scanned in interval 200-800 nm). It was demonstrated, comparing reference and real spectra of the analytes, that the proposed separation method was selective enough to produce pure (non-mixed, i.e. spectrally homogeneous) analyte zones without any interfering compound. Successful validation and application of the proposed CZE-DAD method suggest its routine use in enantioselective control of AML in pharmaceuticals.

Nifedipine preparations on the Belgian market: a comparative study.

Nifedipine preparations formulated as immediate-release capsules and prolonged-release tablets were evaluated with different tests, including in vitro dissolution, assay and content uniformity, and determination of related compounds. The analytical methods were previously validated according to international guidelines. All examined products complied with the postulated requirements but the dissolution test for prolonged-release tablets showed that these products cannot be interchanged.

Ultrasonication-assisted synthesis of sphere-like strontium cerate nanoparticles (SrCeO3 NPs) for the selective electrochemical detection of calcium channel antagonists nifedipine.

In this work, strontium cerate nanoparticles (SrCeO3 NPs, SC NPs) were developed through facile synthetic techniques (Ultrasound-Assisted (UA) and Stirring-Assisted (SA) synthesis) and utilized as an electrocatalyst for the selective and sensitive electrochemical detection of calcium channel blocker nifedipine (NDF). The as-prepared UASC NPs and SASC NPs were characterized using XRD, Raman, TEM, EDS, mapping, XPS and BET analysis which exposed the formation of SC NPs in the form of spherical in shape and well crystalline in nature. BET studies reveal that UASC NPs have maximum surface area than that of SASC NPs. Further, the use of the as-developed UASC NPs and SASC NPs as an electrocatalyst for the detection of NDF. Interestingly, the UASC NPs modified screen printed carbon electrode (UASC NPs/SPCE) exhibited an excellent electrocatalytic activity in terms of lower reduction potential and enhanced reduction peak current when compared to SASC NPs and unmodified SPCE. Moreover, as-prepared UASC NPs/SPCE displayed wide linear response range (LR, 0.02-174 µM), lower detection limit (LOD, 5 nM) and good sensitivity (1.31 µA µM-1 cm-2) than that of SASC NPs (LR = 0.02-157 µM, LOD = 6.4 nM, sensitivity - 1.27 µA µM-1cm-2). Furthermore, UASC NPs/SPCE showed an excellent selectivity even in the existence of potentially co-interfering compounds such as similar functional group containing drugs, pollutants, biological substances and some common cations/anions. The developed sensor was successfully employed for the determination of NDF in real lake water, commercial NDF tablet and urine samples with acceptable recovery.

Analytical techniques for the determination of verapamil in biological samples and dosage forms: an overview.

Verapamil (VER) is a calcium channel blocker that is widely used to treat various cardiovascular diseases and is also effective in migraine prophylaxis. As the therapeutic range of VER is very narrow and toxicity can occur in patients after oral administration, therapeutic drug monitoring is recommended to optimize pharmacotherapy. The choice of an appropriate bioanalytical method for therapeutic drug monitoring of VER in the biological samples is a very important step in achieving fast and reliable results. This review focuses on the various analytical methods reported between 1976 and 2019 for the determination of VER in different biological samples and pharmaceutical dosage forms along with their methodological limitations. This review provides an overview for pharmaceutical industry researchers, clinicians and clinical chemists.

Analysis of dihydropyridine calcium channel blockers using negative ion photoionization mass spectrometry.

The fragmentation of dihydropyridine calcium-channel antagonists are compared by electrospray ionization (ESI) and atmospheric pressure photonization (APPI). The results demonstrate that in ESI the preferred ionization process is in positive mode, with the mass spectra of [M+H]+ showing base peak ions probably formed by loss of alcohols from carboxyl groups. Conversely, in APPI, a high intense peak is observed in negative mode due to deprotonated molecule [M-H]- after two serial 1, 2-hydride shifts leading to a rearranged deprotonated molecule [M-H]-. These ions undergo another 1,2-hydride shifts to produce a nitro-phenyl product ion of m/z 122. The APPI is also used to develop a method for the quantitation of dihydropyridines (e.g., nifedipine) in human plasma.

Sub-second adsorption for the pico-molar monitoring of diltiazem in pharmaceutical preparations by fast Fourier transformation with the use of continuous cyclic voltammetry at an Au microelectrode in a flowing system.

Diltiazem was determined at the sub-nanomolar level for the first time by a new technique, involving fast Fourier continuous cyclic voltammetry in a flow-injection system. The best performance was achieved with the basic parameters being set at pH value of 2.0, scan rate value of 35 V/s, accumulation potential of 300 mV and accumulation time of 0.8 s. This paper additionally introduces a special computer based numerical method for the calculation of the analyte signal and the noise reduction. Concerning the electrode response calculations were carried out according to the partial and total charge exchanges on the electrode surface after subtraction of background current from that of noise. Furthermore, to obtain a sensitive determination, the currents integration range included all potential scan ranges, even oxidation and reduction of the Au surface electrode, during the measurements. In general, the potential waveform includes the potential steps for cleaning, accumulation and the step of the potential ramp of the analyte. This potential waveform was applied to an Au disk microelectrode in a continuous way. Finally, the method was found to be linear for the concentration range of 1-41450 pg/ml (r = 0.9986), while showing a limit of detection and quantitation of 0.29 and 1 pg/ml, respectively.

Determination of azelnidipine by LC-ESI-MS and its application to a pharmacokinetic study in healthy Chinese volunteers.

A simple, rapid and sensitive high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) assay for determination of azelnidipine in human plasma using perospirone as the internal standard (IS) was established. After adjustment to a basic pH with sodium hydroxide solution, plasma samples were extracted with diethyl ether and separated on a C18 column with a mobile phase of methanol-5 mM ammonium acetate solution (90:10, v/v). The lower limit of quantification (LLOQ) was 0.20 ng/ml. After administration of a single dose of azelnidipine 8mg and 16 mg, respectively; the area under the plasma concentration versus time curve from time 0 h to 96 h (AUC(0-96) were (186 +/- 47) ng ml(-1) h, (429 +/- 145) ng ml(-1) h, respectively; clearance rate (CL/F) were (45.94 +/- 11.61), (42.11 +/- 14.23) L/h, respectively; peak plasma concentration Cmax were (8.66 +/- 1.15), (19.17 +/- 4.13) ng/ml, respectively; apparent volume of distribution (Vd) were (1749 +/- 964), (2480 +/- 2212) L, respectively; time to Cmax (Tmax) were (2.8 +/- 1.2), (3.0 +/- 0.9) h, respectively; elimination half-life (t(1/2beta)) were (22.8 +/- 2.4), (23.5 +/- 4.2) h, respectively; and MRT were (25.7 +/- 1.3), (26.2 +/- 2.2) h, respectively; The essential pharmacokinetic parameters after oral multiple doses (8 mg, q.d.) were as follows: (Cmax) ss, (15.04 +/- 2.27) ng/ml; (Tmax) ss, (2.38 +/- 0.92) h; (Cmin) ss, (3.83 +/- 0.94) ng/ml; C(av), (7.05 +/- 1.54) ng/ml; DF, (1.62 +/- 0.26); AUCss, (169.19 +/- 36.87) ng ml(-1) h.

[Obtaining antibodies to 1,4-dihydropyridine calcium channel blockers].

Immunization of rabbits with amlodipine conjugated with horseradish peroxidase resulted in raising polyclonal antibodies that allowed group determination of 1,4-dihydropyridine calcium channel blockers in aqueous solutions by ELISA with a sensitivity of 0.1 to 1.0 ng/ml for amlodipine, felodipine, nifedipine, and isradipine.

Transcriptomic and Proteomic Analyses Reveal the Diversity of Venom Components from the Vaejovid Scorpion Serradigitus gertschi.

To understand the diversity of scorpion venom, RNA from venomous glands from a sawfinger scorpion, Serradigitus gertschi, of the family Vaejovidae, was extracted and used for transcriptomic analysis. A total of 84,835 transcripts were assembled after Illumina sequencing. From those, 119 transcripts were annotated and found to putatively code for peptides or proteins that share sequence similarities with the previously reported venom components of other species. In accordance with sequence similarity, the transcripts were classified as potentially coding for 37 ion channel toxins; 17 host defense peptides; 28 enzymes, including phospholipases, hyaluronidases, metalloproteases, and serine proteases; nine protease inhibitor-like peptides; 10 peptides of the cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 protein superfamily; seven La1-like peptides; and 11 sequences classified as "other venom components". A mass fingerprint performed by mass spectrometry identified 204 components with molecular masses varying from 444.26 Da to 12,432.80 Da, plus several higher molecular weight proteins whose precise masses were not determined. The LC-MS/MS analysis of a tryptic digestion of the soluble venom resulted in the de novo determination of 16,840 peptide sequences, 24 of which matched sequences predicted from the translated transcriptome. The database presented here increases our general knowledge of the biodiversity of venom components from neglected non-buthid scorpions.

A stability-indicating high performance liquid chromatographic (HPLC) assay for the simultaneous determination of atorvastatin and amlodipine in commercial tablets.

A simple, rapid, precise and accurate isocratic reversed-phase stability-indicating HPLC method was developed and validated for the simultaneous determination of atorvastatin (AT) and amlodipine (AM) in commercial tablets. The method has shown adequate separation for AM, AT from their associated main impurities and their degradation products. Separation was achieved on a Perfectsil Target ODS-3, 5 microm, 250 mm x 4.6 mm i.d. column using a mobile phase consisting of acetonitrile-0.025 M NaH(2)PO(4) buffer (pH 4.5) (55:45, v/v) at a flow rate of 1 ml/min and UV detection at 237 nm. The drugs were subjected to oxidation, hydrolysis, photolysis and heat to apply stress conditions. The linearity of the proposed method was investigated in the range of 2-30 microg/ml (r=0.9994) for AT and 1-20 microg/ml (r=0.9993) for AM. The limits of detection were 0.65 microg/ml and 0.35 microg/ml for AT and AM, respectively. The limits of quantitation were 2 microg/ml and 1 microg/ml for AT and AM, respectively. Degradation products produced as a result of stress studies did not interfere with the detection of AT and AM and the assay can thus be considered stability-indicating.

Analyses by GC-MS and GC-MS-MS of the Hantzsch synthesis products using hydroxy- and methoxy-aromatic aldehydes.

EI mass spectra of products of the dihydropyridine Hantzsch synthesis using hydroxy and methoxy aldehydes as starting materials are reported. The reaction products (C-4 hydroxy- and methoxyphenyl-1,4-dihydropyridines and chromeno[3,4,c]-pyridines) were derivatized with N-methyl-N-(trimethylsilyl)-trifluoracetamide to be analyzed by gas chromatographic techniques. Fragmentation pathways for 1,4-dihydropyridines and chromeno-pyridines are proposed. The study provides (mainly through MS-MS technique) useful data for the confirmation of the structure of the compounds and also is a valuable tool for further analytical purposes to follow both photostability and reactivity studies with free radicals for these types of compounds.