Nateglinide is an amino-acid derivative that lowers blood glucose levels by stimulating insulin secretion from the pancreas. This action is dependent upon functioning beta-cells in the pancreatic islets.1 Nateglinide interacts with the ATPsensitive potassium (K+ATP) channel on pancreatic beta-cells. The subsequent depolarization of the beta cell opens the calcium channel, producing calcium influx and insulin secretion.2 The extent of insulin release is glucose dependent and diminishes at low glucose levels. Nateglinide is highly tissue selective with low affinity for heart and skeletal muscle.3  The drug is widely used for the management of type-2 diabetes. It has short biological half life (1.5 ± 0.7 h) and bioavailability is 73%. The conventional therapies to treat existent diabetes consist of complex drug regimen that leads to increased pill burden, frequent and high daily dosing, and dose-associated side effects and ultimately results in poor patients’ compliance with worsening of such patients’ condition. 4,5 Lipospheres can enhance patient’s compliance by reducing drug dosing frequency, lowering adverse effects, and limiting fluctuation in plasma drug levels.6 Size of lipospheres varies from 1 to 1000 ?m, and various methods like solvent evaporation, spray drying, ionotropic gelation, coacervation, and melt emulsification congealing method have been employed for lipospheres preparation.7 Lipids have exhibited efficient biodegradability, biocompatibility, and ease of utilization in production process due to their lower melting points. 8,9 So, the idea of sustained delivery of an antidiabetic agent was generated because the mode of drug administration has been contributing a lot in improving therapeutic efficacy and decreasing adverse effects of a drug. So, to overcome noncompliance, discovery of novel controlled release methods of drug delivery like lipospheres for delivery of drug has been proven to be more beneficial. So aim of present work to formulate and evaluates Nateglinide loaded lipospheres for effective absorption and increase bioavailability.

MATERIAL AND METHODS

Preformulation Studies

Characterization of Nateglinide

Physical evaluation: It refers to the evaluation by sensory characters-taste, appearance, odor, feel of the drug, etc.

 Solubility of the drug was determined by taking some quantity of drug (about 1-2 mg) in the test tube separately and added the 5 ml of the solvent (Water, ethanol, methanol, 0.1 N HCl, 0.1 N NaOH, chloroform and phosphate buffer pH 6.8) Shake vigorously and kept for some time. Note the solubility of the drug in various solvents (at room temperature). 10

It is one of the parameters to judge the purity of drugs. In case of pure chemicals, melting points are very sharp and constant. Since the drugs contain the mixed chemicals, they are described with certain range of melting point.

Identification test using FT-IR Spectroscopy: Infra- red spectrum is an important record which gives sufficient information about the structure of a compound. This technique provides a spectrum containing a large number of absorption band from which a wealth of information can be derived about the structure of an organic compound. The region from 0.8 µ to 2.5 µ is called Near Infra-red and that from 15 µ to 200 µ is called Far infra-red region. Identification of Nateglinide was done by FTIR Spectroscopy with respect to marker compound. Nateglinide was obtained as white powder.

The moisture in a solid can be expressed on a wet weight or dry wet basis. On a wet weight basis, the water content of a material is calculated as a percentage of the weight of the weight solid. The term loss on drying is an expression of moisture content on a wet weight basis.

Moisture content determination:

The titrimetric determination of water is based upon the quantitative reaction of water with an anhydrous solution of sulphur dioxide and iodine in the presence of a buffer that reacts with hydrogen ions.

Determination of ? max of Nateglinide

 The ?max of Nateglinide was determined by running the spectrum of drug solution in double beam ultraviolet spectrophotometer.

 Drug encapsulated Lipospheres were developed by melt dispersion technique. 11 The formulation of different batches is depicted in Table 04. Briefly, the lipid core was melted on a water bath maintained at 70-72°C. Finely powdered drug was dispersed into the molten lipidic phase. The aqueous phase was prepared by heating a blend of water and surfactant to 70-72°C with a stabilizer. The molten lipidic phase was slowly transferred to the hot aqueous phase (o/w emulsion) and the emulsification was assisted by stirring the content on a sonicator continuously. The milky dispersion was then rapidly cooled to 20°C by immersing the formulation in an ice bath without stopping the agitation to yield a uniform dispersion of lipospheres. The obtained lipospheres were then washed with water and isolated by filtration.

Table 04: Preparation of Liposphere of Nateglinide

Percentage Yield of Lipospheres:

Yield of Lipospheres percent w/w is calculated according to the following formula:

% Yield = Weight of lipospheres /Wt. of Drug + Wt. of Excipients

Drug loading and Entrapment efficiency 12

The amount of Nateglinide present in lipospheres is determined by taking the known amount of lipospheres in which 10mg of drug should be present theoretically. Then the lipospheres crush and the powdered microspheres are taken and dissolve in 10 ml of methanol and stirred for 15 minutes with an interval of 5 minutes and allow keeping for 24 hours. Then the solution was filtered through whatmann filter paper. Then the absorbance after appropriate dilution was measured spectrophotometrically at 244nm by UV-Visible spectrophotometer.

Drug entrapment efficiency (%) = (Experimental drug content/Initial drug content in the formulation) × 100

Measurement of mean particle size

 The mean size of the lipospheres was determined by Photo Correlation Spectroscopy (PCS) on a submicron particle size analyzer (Malvern Instruments) at a scattering angle of 90°. A sample (0.5mg) of the lipospheres suspended in 5 ml of distilled water was used for the measurement. 13

Determination of zeta potential:

The zeta potential of the drug-loaded lipospheres was measured on a zeta sizer (Malvern zetasizer instruments) by determining the electrophoretic mobility in a micro electrophoresis flow cell. All the samples were measured in water.

Surface morphology (Scanning electron microscopy):

Morphology and surface topography of the lipospheres were examined by scanning electron microscopy. The lipospheres from the optimized batch were mounted on the SEM sample stab using a double-sided sticking tape and coated with gold (~200 nm) under reduced pressure (0.133 Pa) for 5 min using an Ion sputtering device. The gold coated lipospheres were observed under the scanning electron microscope and photomicrographs of suitable magnifications were obtained.

Flow property determination of the Lipospheres: Bulk density:

Both loose bulk density (LBD) and tapped bulk density (TBD) were determined. Accurately weighed amount of granules taken in a 50 ml capacity measuring cylinder was tapped for 100 times on a plane hard wooden surface and estimated the LBD and TBD, calculated by using following formulas. 14

RESULTS AND DISCUSSION

Characterization of Nateglinide encapsulated lipospheres

Percentage Yield of lipospheres: Yield of lipospheres percent w/w was calculated according to the following formula:       

% Yield = Weight of lipospheres /Wt. of Drug + Wt. of Excipients× 100

An optical microscope (MSW) with a camera attachment (Olympus) was used to observe the shape of the prepared liposphere formulation F4.

Figure 04: Microscopic observation of prepared optimized liposphere formulation F4

Particle size:

The mean size of the lipospheres was determined by photo correlation spectroscopy (PCS) on a submicron particle size analyzer (Horiba Instruments) at a scattering angle of 90°. A sample (0.5mg) of the lipospheres suspended in 5 ml of distilled water was used for the measurement. The results of measurement of mean particle size of optimized formulation F4 lipospheres were found 196.65 nm.

The zeta potential of the drug-loaded lipospheres was measured on a zeta sizer (Malvern Instruments) by determining the electrophoretic mobility in a micro electrophoresis flow cell. All the samples were measured in water Results of zeta potential of optimized formulation F4 lipospheres was found -36.45 mV respectively.

Scanning electron microscopy: Morphology and surface topography of the lipospheres were examined by scanning electron microscopy. The lipospheres from the optimized batch were mounted on the SEM sample stab using a double-sided sticking tape and coated with gold (~200 nm) under reduced pressure (0.133 Pa) for 5 min using an Ion sputtering device. The gold coated lipospheres were observed under the scanning electron microscope and photomicrographs of suitable magnifications were obtained.