Comparative study of nisoldipine-loaded nanostructured lipid carriers and solid lipid nanoparticles for oral delivery: preparation, characterization, permeation and pharmacokinetic evaluation.

Nisoldipine (ND) has low oral bioavailability (5%) due to first-pass metabolism. Previously, solid lipid nanoparticles (SLNs) of ND were reported. In this study, nanostructured lipid carriers (NLCs) of ND are developed to enhance the oral bioavailability. ND-NLCs were prepared using hot homogenization-ultrasonication method, using oleic acid and trimyristate as liquid lipid and solid lipid, respectively. Prepared NLCs are evaluated for an optimal system using measuring size, zeta potential, entrapment efficiency, in-vitro release and in-situ absorption studies. Further, in vivo pharmacokinetic (PK) studies of NLC were conducted in rats comparison with SLN and suspension as controls. Size, ZP and EE of optimized NLCs were found to be 110.4 ± 2.95 nm, -29.4 ± 2.05 mV and 97.07 ± 2.27%, respectively. Drug loaded into NLCs was converted to amorphous form revealed by differential scanning calorimeter (DSC) and X-ray diffractometry (XRD) technique and nearly spherical in shape by scanning electron microscopy (SEM) studies. Drug release and absorption of ND were prolonged from ND-NLCs and ND-SLNs. From the PK results, NLCs showed 2.46 and 1.09-folds improvement in oral bioavailability of ND compared with suspension and SLNs formulations, respectively. Taken together, the NLCs and SLNs are used as carriers for the enhancement of oral bioavailability of the ND.

Investigating the in vitro stereoselective metabolism of m-nisoldipine enantiomers: characterization of metabolites and cytochrome P450 isoforms involved.

m-Nisoldipine, as a novel 1,4-dihydropyridine calcium ion antagonist, was presented as a couple of enantiomers [(-), (+)-m-nisoldipine]. In this report, the in vitro metabolism of m-nisoldipine enantiomers was investigated in rat liver microsomes (RLM) by the combination of two liquid chromatography mass spectrometric techniques for the first time. The metabolites were separated and assayed by ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry and further identified by comparison of their mass and chromatographic behaviors with reference substances. A total of 18 metabolites of (-)-m-nisoldipine and 16 metabolites of (+)-m-nisoldipine were detected, respectively, which demonstrated that (+)-m-nisoldipine is more metabolically stable than (-)-m-nisoldipine. In addition, the identified metabolic pathways of m-nisoldipine enantiomers were involved in dehydrogenation, oxidation and ester hydrolysis. Afterwards, based on high-performance liquid chromatography coupled to triple quadrupole linear ion trap mass spectrometry, various selective cytochrome P450 (CYP) enzyme inhibitors were employed to evaluate CYP isoforms. The results indicated that the inhibitors of CYP1A1/2, CYP2B1/2, 2D and 2C11 had no obvious inhibitory effects, yet the inhibitor of CYP 3A had a significant inhibitory effect on metabolism of m-nisoldipine enantiomers. This showed that CYP 3A might primarily metabolize m-nisoldipine in RLM.

Study of in vitro metabolism of m-nisoldipine in human liver microsomes and recombinant cytochrome P450 enzymes by liquid chromatography-mass spectrometry.

This is a report about the investigation of the metabolic fate of m-nisoldipine in human liver microsomes and the recombinant cytochrome P450 enzymes by using LC-MS/MS. A sensitive and reliable LC-MS/MS method was developed to obtain a rapid and complete characterization of new metabolites and the metabolism pathways. The analytes were separated on a reversed phase C18 column with acetonitrile and 0.1% aqueous formic acid as the mobile phase. Tandem mass spectrometry with positive electrospray ionization was used to enable the structural characterization of the metabolites. A total of 10 metabolites were characterized with proposed structures in the incubation of human liver microsomes by comparing their retention times and spectral patterns with those of the parent drug. Dehydrogenation of the dihydropyridine core and reactions of side chains such as hydroxylation and hydrolysis of ester bonds were the major metabolic pathways. The specific cytochrome P450 (CYP) enzymes responsible for m-nisoldipine metabolites were identified using chemical inhibition and cDNA expressed CYP enzymes. The results indicated that CYP2C19 and CYP3A4 might play major roles in the metabolism of m-nisoldipine in human liver microsomes.

Simultaneous determination of m-nisoldipine and its three metabolites in rat plasma by liquid chromatography-mass spectrometry.

A simple, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of m-nisoldipine and its three metabolites in rat plasma has been developed using nitrendipine as an internal standard (IS). Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse phase C(18) column and analyzed by MS in the multiple reaction monitoring (MRM) mode. To avoid contamination by residual sample in the injection syringe, a special injection protocol was developed. We found that m-nisoldipine, metabolite M1 and IS could be ionized under positive or negative electrospray ionization conditions, whereas metabolite M and M2 could only be ionized in the positive mode. The mass spectrometry fragmentation pathways for these analytes are analyzed and discussed herein. The total analysis time required less than 5 min per sample. We employed this method successfully to study the metabolism of m-nisoldipine when it was orally administered to rats at a dose of 9 mg/kg. Three metabolites of m-nisoldipine and an unknown compound of molecular weight 386 were found for the first time in rat plasma. The concentration of the potentially active metabolite was approximately equal to its parent compound concentration.

T-type Ca2+ channel blockers prevent cardiac cell hypertrophy through an inhibition of calcineurin-NFAT3 activation as well as L-type Ca2+ channel blockers.

T-type Ca2+ channels (TCCs) are involved in cardiac cell growth and proliferation in cultured cardiomyocytes. Underlying molecular mechanisms are not well understood. In this study, we investigated the role of TCCs in signal transduction in cardiac hypertrophy compared with L-type Ca2+ channels (LCCs). Cardiomyocytes dissociated from neonatal mouse ventricles were cultured until stabilization. Cell hypertrophy was induced by reapplication of 1% fatal bovine serum (FBS) following a period (24 h) of FBS depletion. Cell surface area increased from 862+/-73 microm2 to 2153+/-131 microm2 by FBS stimulation in control (250+/-1.8%). T-type Ca2+ current (I(CaT)) was inhibited dose-dependently by kurtoxin (KT) and efonidipine (ED) with IC50 0.07 microM and 3.2 microM, respectively in whole-cell voltage clamp. On the other hand, 1 microM KT which inhibits I(CaT) over 90% did not effect on L-type Ca2+ current (I(CaL)). 10 microM ED had the ability of I(CaL) blockade as well as that of I(CaT) blockade. 3 microM nisoldipine (ND) suppressed I(CaL) by over 80%. The increase in cell surface area following reapplication of FBS as observed in control (250+/-1.8%) was significantly reduced in the presence of 1 microM KT (216+/-1.2%) and virtually abolished in the presence of 10 microM ED (97+/-0.8%) and 3 microM ND (80+/-1.1%). Hypertrophy was associated with an increase in BNP mRNA of 316+/-3.6% in control and this increase was reduced as well in the presence of 1 microM KT (254+/-1.8%) and almost abolished in the presence of 10 microM ED (116+/-1.1%) and 3 muM ND (93+/-0.8%). Immunolabeling showed that translocation of nuclear factor of activated T cells (NFAT3) into the nucleus in response to FBS stimulation was markedly inhibited by either KT or ED as well as ND. Calcineurin phosphatase activity was upregulated 2.2-fold by FBS, but KT, ED and ND decreased this upregulation (1.7-fold, 0.8-fold, and 0.7-fold with KT, ED and ND respectively). These results suggest that blockade of Ca2+ entry into cardiomyocytes via TCCs may block pathophysiological signaling pathways leading to hypertrophy as well as via LCCs. The mechanism may be the inhibition of calcineurin-mediated NFAT3 activation resulting in prevention of its translocation into the nucleus.

Photochemical degradation of solid-state nisoldipine monitored by HPLC.

The photochemical degradation of solid-state nisoldipine, 1,4-dihydropyridine calcium antagonist, was investigated under daylight and UV light conditions. Degradation products were identified by using the retention times of corresponding standards and quantified by high-performance liquid chromatographic method. The daylight illumination induced appearance of nitrosophenylpyridine, while formation of second degradation product, nitrophenylpyridine, was observed only upon UV light illumination. The photodegradation kinetics of solid-state nisoldipine under daylight and UV light illumination belongs to class of zero-order reactions. The rate constants of disappearance of nisoldipine upon illumination were determined for raw material as well as pharmaceuticals (tablets, film-tablets and capsules).

Scavenging of the one-electron reduction product from nisoldipine with relevant thiols: electrochemical and EPR spectroscopic evidences.

PURPOSE: To determine the formation of the one-electron reduction product from nisoldipine and its reactivity with relevant thiols in mixed medium. METHODS: Cyclic voltammetry (CV) and electron paramagnetic resonance (EPR) techniques were used to determine the one-electron reduction product corresponding to the nitro radical anion. CV was employed to assess both the rate constants corresponding to the decay of the radicals and its interaction with relevant thiols. RESULTS: The nisoldipine radical anion follows second order kinetics, with an association rate constant of 283+/-16 l mol(-1) sec(-1). Nitro radical anion from nisoldipine significantly reacted with thiol compounds. This reactivity was significantly higher than the natural decay of the radical in mixed medium. EPR spectra recorded in situ using DMF/ 0.1 N NaOH (pH 13) confirmed the formation of the nitro radical anion, giving a well-resolved spectra in 35 lines using 0.1 G modulation. CONCLUSIONS: Electrochemical and EPR data indicated that all the tested thiols scavenged the nitro radical anion from nisoldipine. The following tentative order of reactivity towards the thiols can be proposed: cysteamine approximately glutathione > N-acetylcysteine > captopril > penicillamine.

Characterization in rat aorta of the binding sites responsible for blockade of noradrenaline-evoked calcium entry by nisoldipine.

1. The effectiveness of the calcium antagonist, 1,4-dihydropyridine nisoldipine, as an inhibitor of contraction and 45Ca entry evoked by noradrenaline in rat aorta has been investigated and correlated with binding characteristics in intact artery. 2. Contractions evoked by noradrenaline were concentration-dependently depressed by nisoldipine (0.3-300 nM). About 60% of the response was resistant to inhibition, while KCl-induced contractions could be completely blocked. Noradrenaline-induced contractions were also less sensitive to nisoldipine inhibition than were KCl-induced contractions. 3. Preincubation of the aorta with nisoldipine in high KCl depolarizing solution increased the inhibition of the contraction evoked by a short application of noradrenaline or KCl to a similar extent. 4. The inhibition by nisoldipine of 45Ca influx evoked either by KCl depolarizing solution or by noradrenaline correlated well with the inhibition of the contractile responses. However, while KCl-stimulated 45Ca influx was totally abolished by nisoldipine (300 nM), 38% of the noradrenaline-stimulated 45Ca influx was resistant to inhibition by nisoldipine (300 nM). 5. The study of [3H]-(+)-PN 200-10 ([3H]-(+)-isradipine) binding in intact aorta showed the presence of a homogeneous population of specific binding sites. KD values were dependent on the KCl concentration in the bath while Bmax was unaffected. Binding of [3H]-(+)-isradipine was also increased in tissue exposed to noradrenaline; in the presence of 10(-5) M noradrenaline, binding parameters of [3H]-(+)-isradipine were close to the values obtained in aorta bathed in 20 mM KCl solution. 6. Displacement of [3H]-(+)-isradipine specific binding by nisoldipine was determined in segments of mesenteric artery and of aorta. The potency of nisoldipine was dependent on the incubation conditions applied to the vessel, as follows: KCl (100 mM) depolarizing solution greater than noradrenaline (10(-5) M) = KCl (25 mM) solution greater than physiological solution. The Ki value measured in aorta exposed to noradrenaline (10(-5) M) was close to the IC50 value of nisoldipine on the noradrenaline-evoked contraction. 7. The membrane potential value of rat aorta was estimated by the distribution of [3H]-tetraphenylphosphonium bromide ([3H]-TPP+), [3H]-TPP+ uptake concentration-dependently decreased when the KCl concentration in the bath was increased from 5.9 to 130 mM. Noradrenaline also concentration-dependently decreased [3H]-TPP+ uptake; the maximum effect (1-10 microns noradrenaline) was comparable in amplitude to the effect of 25 mM KCl solution. 8. It is concluded that in rat aorta, noradrenaline activates voltage-operated calcium channels that contain the specific, voltage-sensitive binding sites for calcium antagonistic dihydropyridines. The existence of a fraction of noradrenaline-stimulated '"Ca entry that is resistant to nisoldipine blockade suggests that another Ca2 + entry pathway is also opened by the agonist.