INTRODUCTION:

Diabetes is a chronic disorder; various drugs having a different mechanism of action are available in the market. In this sulfonylurea play an important role. Gliclazide is an ant diabetic drug and comes under the category of second-generation sulfonyl urea [1]. The main mechanism of the Gliclazide is to bind with ATP sensitive potassium channel receptors, and reducing the potassium conductance and cause depolarization of the membrane, which increases the intracellular calcium ion concentration and induces the insulin secretion. The core aim of the study is to improve the aqueous solubility of poorly soluble water-soluble by the nanosuspension method [2-5]. Nan suspensions were found to be a promising methodology used to enhance the dissolution behavior of poorly water-soluble drugs [6-10].

Because of this nanosuspension technology, the absorption from absorption windows the drugs can be increased due to a reduction in particle size and which improve the bioavailability [11-17]. For the water-insoluble drug to increase the solubility and dissolution behavior, it can be converted into nanosuspension using different concentrations of polymers, solvents, surfactants, and stabilizers [18-23]. Nanosuspension of Gliclazide may be suitable for dose titration therapy for age-appropriate patients and better patient convenience for diabetic treatment [24-26].

MATERIALS AND METHODS:

Materials:

Drug Gliclazide was obtained as a gift sample from sun pharmaceuticals, New Delhi. HPMC was obtained from Lobachemic Laboratory Pvt Ltd, Mumbai. All other ingredients used were of Analytical grade.

Methods:

Description:

The pure drug sample was studied for their organoleptic properties like colour, odour, and nature.

pH Value:

0.1% solution was prepared in distilled water using the minimum amount of methanol and the pH of the solution was measured using pH meter [27].

Solubility studies:

The solubility of the drug sample was studied in various solvents.

Procedure:

10mg of the drug was suspended separately in 10ml of different solvents in test tubes. It is shaken vigorously and observed the solubility.

The solubility of the drug is determined using water and phosphate buffer (pH 7.4) and organic solvents like acetone, ethanol, chloroform, dichloromethane.

Determination of absorption maxima (λ) of Gliclazide

The identification of drugs was done by UV spectrophotometric by a simple quantitative method using Shimadzu Spectrophotometer UV1800 (Shimadzu Corp., Japan). 10mg of Gliclazide was dissolved in phosphate buffer pH 7.4 and diluted to 100ml with the buffer solution. 1ml of the drug solution was diluted to 10ml with the same solvent and detected between 200-400nm. The maximum absorbance shown in the graph was considered as a max of the drug [28].

Preparation of Calibration curve of Gliclazide in (pH 7.4) phosphate buffer by using the UV method.:

A stock solution was prepared by dissolving 100mg of Gliclazide in 100ml From this 10ml was withdraw and diluted with 100ml of phosphate buffer
(pH7.4). From this, 10ml was withdrawn and diluted with 100ml of phosphate buffer (pH 7.4) and the standard graph was plotted by using different concentrations (7µg/ml- 32µg/ml). The absorbance was measured spectrophotometrically at 226nm [29].

Fourier Transform Infrared Spectroscopy:

The FTIR spectrum of pure drug, Physical mixture of drugs and excipients and the Optimized formulations were taken in the range 4000-400cm^-1using (Jasco FTIR 410) KBr pellet method and the characteristic peaks of the drug are observed [30].

Formulation of Gliclazide Nanosuspension:

The Gliclazide nanosuspension was prepared by the nanoprecipitation method. In this 30mg of the drug is dissolved in the required quantity of ethanol and it is kept in magnetic stirrer. And the prepared solution is mixed with the antisolvent system (HPMC/PVP/PEG600) in the presence of surfactants. Rapid mixing of drug solution into the antisolvent system leads to sudden supersaturation forms with fine drug solids [23, 27].

Fig. 1: Trails of Gliclazide nanosuspension

Characterization of Optimized Nanosuspension

Total Drug Content:

The Drug content was determined by UV-Visible spectrophotometer at 226nm. About 1ml of nanosuspension preparation was taken and diluted with phosphate buffer pH 7.4[31].

Redispersibility:

Nanosuspension stored in vials was determined by tilting the vial bottle up and down with hand till the sediment was uniformly dispersed in the aqueous and the number of times tilted was noted.

Viscosity Measurement:

The viscosity of lipid-based formulations of several compositions can be measured at different temperatures using Brookfield viscometer.

Measurement of particle size and size distribution:

The polydispersibility index is determined by Photon Correlation Spectroscopy (PCS). Particle size and Polydispersibility index (PI) control the saturated solubility; dissolution velocity and biological performance. The 100µl of optimized nanosuspension was diluted to 5ml with double distilled water and this diluted dispersion was measured using Malvern zetasizer.

Dissolution velocity and saturation solubility:

Adding the excess amount of sample is added to distilled water (2ml) and the samples were shaking in screw-capped vials for 24hrs. The sample was centrifuged at 1000rpm for 10 min and filter. The filtrate was diluted with distilled water and drug content was estimated in UV spectroscopy.

Zeta potential:

Zeta potential of the formulation was measured using Malvern zetasizerZS (Nano series ZS 90 UK). The prepared formulations were diluted with water and placed in the electrophoretic cell. Each sample was measured 3 times at 25% C and average values were measuring the response.

The physical stable nanosuspension solely stabilized by electro stable repulsion, a zeta potential of ±30Mv is required as a minimum.

Scanning electron microscopy (SEM):

The morphological features of Gliclazide nanosuspension are observed by scanning electron microscope at different magnification.

In-vitro drug release study:

Nanosuspension was taken in a modified diffusion cell apparatus. The drug release from nanosuspension was determined using a dialysis bag containing 10ml of the nanosuspension in water- Jacketed beaker containing 300ml of phosphate buffer pH 7.4 at 37±1ºC for 24 hrs. Samples were withdrawn up to 24 hrs periodically and replaced with phosphate buffer pH 7.4 medium. The sample was filtrated and the drug content was determined at 226nm using UV spectroscopy [32].

Comparison of selected formulation with marketed formulation

The optimized nanosuspension formulation (F5) is compared with the marketed formulation which had the same dose and releases pattern.

Study of Release Kinetics:

The dissolution profile data obtained from optimized formulation (SR layer) to various kinetic studies such as Zero Order, First Order, Higuchi, and Korsmeyer-Peppas models. These models are used to characterize drug dissolution /release profiles [32,33].

Zero-order: Cumulative amount of drug released Vs time

First-order: Log cumulative percentage of drug remaining Vs time.

Higuchi’s: Cumulative percentage of drug released Vs square root of time.

Korsmeyer’s: Log cumulative percentage of drug released Vs log time.

Stability study:

Stability study of optimized Gliclazide nanosuspension was carried out by placing formulation in glass vials at different temperature conditions for 3 months at room temperature (25ºC), refrigerator (4ºC). After 3 months samples were visually noted and change in particle size distribution was observed using zeta sizer.

RESULTS:

Description:

Table 2: Description of Drug

Drug	Description of Drug
Gliclazide	Off-white, crystalline powder, and odourless

pH Value:

It is weakly acidic, the pH value was found to be 3.5

Solubility studies:

Table 3: Solubility Profile of Drug

S. No.	Medium	Gliclazide
1	Drug +Water	Insoluble
2	Drug + Acetone	Sparingly soluble
3	Drug + Ethanol	Slightly soluble
4	Drug + Dichloromethane	Soluble
5	Drug + Phosphate buffer pH 7.4	Soluble

It is soluble in phosphate buffer pH 7.4, sparingly soluble in Acetone and insoluble in water.

Determination of λ max:

The pure drug absorption spectrum was scanned between 200-400nm in pH 7.4 PBS. The peak was shown in Figure 2:

Fig. 2: Absorption Spectrum of Gliclazide

Preparation of Calibration curve for Gliclazide:

Table 4: Calibration Curve of Gliclazide measured using UV Spectrophotometry at 226 nm

S. No.	Concentration (µg/ml)	Absorbance at 226 nm
1	0	0
2	7	0.114
3	12	0.219
4	17	0.310
5	22	0.409
6	27	0.570
7	32	0.603

Fig. 3: Calibration Curve of Gliclazide measured using UV Spectrophotometry at 226 nm

The calibration curve was in the concentration range of 7-32µg/ml. The calibration curve value was 0.9991 indicating excellent linearity of the data.

Drug -Polymer Interaction Studies:

Fig. 4: FTIR Spectrum of Gliclazide

Fig.5: FTIR Spectrum of Gliclazide +Poly Vinyl Pyrrolidine

Fig. 6: FTIR Spectrum of Gliclazide +Poly Ethylene Glycol

Fig. 7: FTIR Spectrum of Gliclazide + Hydroxyl Propyl Methyl Cellulose

Fig. 8: FTIR Spectrum of Gliclazide Nanosuspension

From the FTIR spectra figures, 4 to 8, characteristic of the functional group of pure drug Gliclazide, Physical mixtures and optimized formulation of Gliclazide were identified. FTIR spectrum of optimized Gliclazide formulation showed due to the presence of C=C Str (aro), indicated N-H (Str) is present, represents S=O (Str), due to C=O (Str) amide, indicate C-N (Str) is present and showed that the characteristic bands of Gliclazide were not altered by the addition of excipients and after formulated into Nanosuspension dosage form with no change in their position and indicating no interaction between the drug and excipients.

Characterization of Optimized Nanosuspension:

Total Drug Content:

The total drug content of all the formulation ranges from 83% to 97%, the optimized formulation F5 showed 97% and the results are shown in Table 5.

Table 5: Total Drug Content of Gliclazide nanosuspension

Batch code	% Drug content
F1	85
F2	83
F3	90
F4	92
F5	97
F6	87
F7	87
F8	84
F9	88

Redispersibility:

Table 6: Redispersibility of different formulation

Formulation code	Redispersibility
F1	Fast
F2	Fast
F3	Medium
F4	Fast
F5	Very fast
F6	Fast
F7	Fast
F8	Fast
F9	Medium

Viscosity measurement:

Table 7: Viscosity measurement of different formulation

Formulation code	The viscosity of the dispersion medium
F1	0.858mPa-s
F2	0.865mPa-s
F3	0.876mPa-s
F4	0.848mPa-s
F5	0.898mPa-s
F6	0.859mPa-s
F7	0.862mPa-s
F8	0.870mPa-s
F9	0.874mPa-s

Particle size:

Table 8: Particle size of Gliclazide nanosuspension

Batch code	Particle size nm
F1	0.420
F2	0.461
F3	0.401
F4	0.448
F5	0.201
F6	0.490
F7	0.451
F8	0.361
F9	0.374

Polydispersibility Index gives the degree of Particle Size Distribution. The high value of the Polydispersibility Index showed broad particle distribution and this maintains the stability of the nanosuspension.

Polydispersibility index:

Table 9: Polydispersibility index of Gliclazide nanosuspension

Batch code	Polydispersibility index
F1	0.421
F2	0.481
F3	0.492
F4	0.488
F5	0.231
F6	0.501
F7	0.482
F8	0.384
F9	0.392

Fig. 9: Size Distribution by Intensity of Gliclazide nanosuspension

Dissolution velocity and saturation solubility study.:

The saturation solubility of the different formulation was shown in Table: 10. Saturation solubility of an optimized F5 formulation shown 0.951mg/ml. The improvement in saturation solubility is improved due to a reduction in particle size and an increase in surface area. This showed that this increase in saturation solubility may improve bioavailability.

Table 10: Saturation solubility of different formulation

Batch code	Saturation solubility in water mg/ml
F1	0.421
F2	0.538
F3	0.698
F4	0.773
F5	0.951
F6	0.566
F7	0.689
F8	0.495
F9	0.537

Zeta Potential:

The maximum zeta potential is ±30mV is required whereas, in the case of a combined electrostatic and stearic stabilization, a minimum of zeta potential is ±20 mV is desired able. The valve of F5 showed the desired value, and which produced the stable system and the results are shown in Table:11 and Figure:10.

Table 11: Zeta potential of Gliclazide nanosuspension

Batch code	Zeta potential
F1	-12.08
F2	-13.84
F3	-19.51
F4	-11.43
F5	-20.5
F6	-19.6
F7	-18.1
F8	-18.4
F9	-12.56

Fig. 10: Zeta Potential of optimized Gliclazide Nanosuspension formulation (F5)

Scanning Electron Microscopy (SEM):

SEM analysis was used to study the surface morphology of the particles. The SEM picture of F5 revealed a smooth texture. The surface structure of the nanosuspension in the SEM of F5 appeared good in shape and particle size. The results are shown in Figure:11.

Fig. 11: SEM of Gliclazide Nanosuspension

In-vitro Drug Release of Gliclazide Nanosuspension:

The % Cumulative Drug Release of F1 to F9 92%, 94%, 96%, 91%, 99%, 93%, 92%, 94%, 95% respectively, All formulation was developed with various concentration of polymers, from this, the optimal concentration of polymer in F5 showed maximum drug release and the results are shown in Figure:12.

Fig. 12: In Vitro Drug Release of Trial batches of Gliclazide Nanosuspension

Comparison of selected formulations with marketed formulations:

The in vitro release studies of the optimized formulation F5 is compared to the marketed tablets that have the same dose and release pattern. The results were depicted in Figure:13.

Fig. 13: In-vitro Dissolution profile of Gliclazide nanosuspension formulation (F5) and Marketed Gliclazide SR formulation

Study of Release Kinetics:

Gliclazide Nanosuspension release rate from the optimized formulation was fitted with various kinetics equations from the graphical representation it can be understood that this is the best fit into zero-order kinetics which has shown a regression coefficient () of 0.959. Peppas equation was used to analyze the release pattern of the drug from the polymeric System. The value of ‘’n’’ was found to be 0.4 indicating the drug release follows Fickian diffusion.

Stability study of Optimized Gliclazide Nanosuspension:

There was no change in the physical appearance of the F5up to 3 months (at 4ºC and temperature). A thin layer of sediment was observed and it is disappeared with slight handshaking. The average particle size diameters were 0.201nm and 0.361 when stored at room temperature and refrigerator (4ºC) respectively. Before performing the stability study for F5 was observed as 0.200nm, from this it cleared that the prepared nanosuspension was stable during the stability study period.

DISCUSSION:

The FTIR study confirmed that there is no chemical interaction between drug and excipients. Gliclazide is a poorly water-soluble drug, the nanoprecipitation method was employed to produce stable nanosuspension and which can increase the solubility and dissolution rate. In this method particle size of Gliclazide can be obtained in the nano ranges, by changing the surfactants and polymer concentration and agitation at (6000 rpm) and the time was 12 hours was constant. The optimal concentration of polymer and surfactant showed an effect on particle size reduction. The drug content was increased for F5 due to the optimal concentration of the polymer and also increases the rapid dissolution. For all the formulations, the Particle size, Polydispersibility index, and Zeta potential were done. This F5 showed the values were desirable and it indicates that the fabricated F5 is stable. The surface morphology was observed by Scanning electron Microscope photography, which showed that the prepared nanoparticles were spherical with a smooth surface. All the Formulated nanosuspensions were subjected to dissolution studies for drug release. Among all the formulations F5 was selected which showed the highest % of drug release (Maximum 99.46% at 24 hours) than other batches. The cumulative Percentage release of nanosuspension significantly increased with increase optimal concentration of the polymer. The in vitro release study of the optimized formulation (F5) were compared to marketed SR Gliclazide tablets. The marketed formulation had a similar dose and release pattern with that of the optimized formulation. The encouraging results revealed that the optimized formulations release pattern matches with the marketed formulation. The release kinetics confirmed that the optimized formulation followed zero-order kinetics. The stability study was conducted as per the ICH guidelines and showed that the prepared nanosuspension is stable during the stability study period.

CONCLUSION:

The study concluded that Gliclazide nanosuspension can be developed with various concentration polymers and surfactants and the results revealed that the F5 showed the controlled drug release mainly due to the formation of nanosized particles, thus objectives of the fabricated nanosuspension Gliclazide by nanoprecipitation method has been achieved with success. The proposed modified release liquid dosage form complies with the patient requirement and it is much more suitable for the dose titration therapy in diabetes treatment. In the future, an awareness to be developed scientist to deliver an anti-diabetic drug inpatient conventional dosage form and extend the same to industrial application.

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Received on 26.03.2020 Modified on 24.04.2020

Research J. Pharm. and Tech. 2021; 14(2):779-786.

DOI: 10.5958/0974-360X.2021.00136.0


Fig. 14: Zero Order Release Kinetics	Fig. 15: First order Release Kinetics

Fig. 16: Higuchi Model	Fig. 17: Korsmeyer Peppas model

Formulation code	Drug (mg)	Polymers HPMC(E50)/PVP/PEG6000(mg)	Surfactant SLS (mg)	Ethanol (ml)	Water (ml)
F1	30	10	10	50	100
F2	30	20	10	40	100
F3	30	30	10	30	100
F4	30	10	10	50	100
F5	30	20	10	40	100
F6	30	30	10	30	100
F7	30	10	10	50	100
F8	30	20	10	40	100
F9	30	30	10	30	100