Preparation of Chitosan Okra Nanoparticles: Optimization and Evaluation as Mucoadhesive Drug Delivery System.

OBJECTIVE: To prepare chitosan-okra gum nanoparticles and their evaluation as mucoadhesive drug delivery system for intranasal delivery of esculin. METHOD: Esculin loaded chitosan-okra gum based nanoparticles were prepared using ionic gelation method. The preparation method was optimized using Box-Behnken experimental design employing okra gum concentration, chitosan concentration, pH and stirring speed as independent variables and particle size, encapsulation efficiency and zeta potential of the formulation were selected as dependent variables. The optimized formulation was characterized using FTIR, SEM and TEM. The nanoparticles were evaluated for their bioadhesive strength and in vitro drug release studies. The optimized intranasal formulation was administered to rats and the pharmacokinetic profile and biodistribution studies were carried out to calculate the targeting efficiency of the formulation in the brain. RESULTS: The nanoparticles were found to depict particle size in the range of 294.0 to 613.4 nm. The concentration of gums was found to significantly influence the particle size and encapsulation efficiency. The nanoparticles depicted bioadhesive strength of 32±2%. The in vitro drug release studies showed 96.4±4.2% release of esculin from nanoparticles in 4h. The drug targeting of NPs to brain was found to be nearly double that of esculin given as simple solution. CONCLUSION: The nanoparticles prepared using chitosan-okra gum were found to possess good mucoadhesive strength with and high brain targeting efficiency.

HPLC Determination of Esculin and Esculetin in Rat Plasma for Pharmacokinetic Studies.

An optimized, sensitive and validated reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV detection is described for simultaneous determination of esculin and its aglycone, esculetin, in rat plasma. After addition of internal standard (chrysin), plasma samples were pretreated by solid-phase extraction and introduced into the HPLC system. Analytes were separated on a RP C18 column with a mobile phase of 0.075% acetic acid in water (solvent A) and 90% acetonitrile in solvent A (solvent B) using gradient elution at a flow rate of 1.0 mL/min. The wavelength for UV detection was set at 338 nm. Calibration curves for esculin and esculetin were constructed over a range of 10-1,000 ng/mL. The developed method was found to be specific, precise and accurate. The method was successfully applied to study the pharmacokinetics of esculin and esculetin in rats. After oral administration of 120 mg/kg, the mean Cmax values were 340.3 and 316.5 ng/mL and the AUClast values were 377.3 and 1276.5 h ng/mL for esculin and esculetin, respectively. The bioavailability of esculin was calculated to be 0.62%.

Simultaneous determination of esculin and its metabolite esculetin in rat plasma by LC-ESI-MS/MS and its application in pharmacokinetic study.

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method operated in the negative electrospray ionization (ESI) switching mode has been developed and validated for the simultaneous determination of esculin and its metabolite esculetin in rat plasma. After addition of internal standards scopoletin, the plasma sample was pretreated by solid-phase extraction (SPE), and separated on a reversed phase C(18) column with a mobile phase of 0.01% formic acid in water (solvent A) and methanol (solvent B) using isocratic elution (A:B=20:80, v/v). The detection of target compounds was done in multiple reaction monitoring (MRM) mode. The MRM detection was operated in the negative ESI mode using the transitions of m/z 339.1 ([M-H](-))→176.7 for esculetin, m/z 176.9 ([M-H](-))→133.0 and m/z 191.0 ([M-H](-))→175.9 for scopoletin. The standard curves, which ranged from 25 to 3200 ng/mL for esculin with the lowest limit of quantification (LLOQ) of 0.25 ng/mL and from 1.25 to 160 ng/mL for esculetin with the LLOQ of 1.25 ng/mL, were fitted to a 1/x weighted quadratic regression model. The method also afforded satisfactory results in terms of the sensitivity, specificity, precision (intra- and inter-day, RSD<8.73%), accuracy, recovery as well as the stability of the analyte under various conditions. The method was successfully applied to study the pharmacokinetics of esculin and its metabolite esculetin in rat plasma after oral administration of esculin at a dose of 100mg/kg.

Quantification of aesculin in rabbit plasma and ocular tissues by high performance liquid chromatography using fluorescent detection: application to a pharmacokinetic study.

A simple and sensitive high performance liquid chromatography method with fluorescence detection (HPLC-FD) was described for the determination of aesculin (AL) at low concentrations in rabbit plasma and ocular tissues. After deproteinization by methanol using pazufloxacin mesilate (PM) as an internal standard (I.S.), supernatants were evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was reconstituted in mobile phase and a volume of 20µL was injected into the HPLC for analysis. Analytes were separated on an Ultimate XB-C18 column (250mm × 4.6mm i.d., 5µm particle size) and protected by a ODS guard column (10mm × 4.0mm i.d., 5µm particle size), using acetonitrile-0.1% triethylamine in water (adjusted to pH 3.0 using phosphoric acid) (12:88, v/v) as mobile phase with a flow rate of 1.0mL/min. The wavelengths of fluorescence detector (FD) were set at 344nm for excitation and 466nm for emission. The lower limit of quantitation (LOQ) for AL was 0.80ng/mL for plasma and vitreous body, 1.59ng/mL for aqueous humor, and 6.55ng/g for iris and 1.66ng/g for retina. The method was used in the study of AL concentrations in plasma and ocular tissues after topical administration of AL eye drops.

Determination of aesculin in rat plasma by high performance liquid chromatography method and its application to pharmacokinetics studies.

A sensitive and reproducible high performance liquid chromatography method with UV detection was described for the determination of aesculin in rat plasma. After deproteinization by methanol using metronidazole as internal standard (I.S.), solutes were evaporated to dryness at 40 degrees C under a gentle stream of nitrogen. The residue was reconstituted in 100 microl of mobile phase and a volume of 20 microl was injected into the HPLC for analysis. Solutes were separated on a Diamonsil C18 column (250 mm x 4.6 mm i.d., 5 microm particle size, Dikma) protected by a ODS guard column (10 mm x 4.0 mm i.d., 5 microm particle size), using acetonitrile-0.1% triethylamine solution (adjusted to pH 3.0 using phosphoric acid) (10:90, v/v) as mobile phase (flow-rate 1.0 ml/min), and wavelength of the UV detector was set at 338 nm. No interference from any endogenous substances was observed during the elution of aesculin and internal standard (I.S., metronidazole). The retention times for I.S and aesculin were 10.4 and 12.4 min, respectively. The limit of quantification was evaluated to be 57.4 ng/ml and the limit of detection was 24.0 ng/ml. The method was used in the study of pharmacokinetics of aesculin after intraperitoneal injection (i.p.) administration in rats.

Common mechanisms of inhibition for the Na+/glucose (hSGLT1) and Na+/Cl-/GABA (hGAT1) cotransporters.

1. Electrophysiological methods were used to investigate the interaction of inhibitors with the human Na(+)/glucose (hSGLT1) and Na(+)/Cl(-)/GABA (hGAT1) cotransporters. Inhibitor constants were estimated from both inhibition of substrate-dependent current and inhibitor-induced changes in cotransporter conformation. 2. The competitive, non-transported inhibitors are substrate derivatives with inhibition constants from 200 nM (phlorizin) to 17 mM (esculin) for hSGLT1, and 300 nM (SKF89976A) to 10 mM (baclofen) for hGAT1. At least for hSGLT1, values determined using either method were proportional over 5-orders of magnitude. 3. Correlation of inhibition to structure of the inhibitors resulted in a pharmacophore for glycoside binding to hSGLT1: the aglycone is coplanar with the pyranose ring, and binds to a hydrophobic/aromatic surface of at least 7x12A. Important hydrogen bond interactions occur at five positions bordering this surface. 4. In both hSGLT1 and hGAT1 the data suggests that there is a large, hydrophobic inhibitor binding site approximately 8A from the substrate binding site. This suggests an architectural similarity between hSGLT1 and hGAT1. There is also structural similarity between non-competitive and competitive inhibitors, e.g., phloretin is the aglycone of phlorizin (hSGLT1) and nortriptyline resembles SKF89976A without nipecotic acid (hGAT1). 5. Our studies establish that measurement of the effect of inhibitors on presteady state currents is a valid non-radioactive method for the determination of inhibitor binding constants. Furthermore, analysis of the presteady state currents provide novel insights into partial reactions of the transport cycle and mode of action of the inhibitors.