Development And Evaluation Of Hydrolyzed Graft Copolymer Based Hydrogel Of Ornidazole For Treatment Of Periodontal Disease

Various surgical approaches are used for the treatment of periodontal diseases like Scaling and root planning, Bone/Tissue grafts, Flap surgery etc5,6. The surgical therapies are unable to remove microorganisms completely as they remain in the debriments after surgery. This emerges a need to combine various systemic and/or local delivery systems with the surgical one5. Various systemic delivery systems of Tetracyclin Hcl, Metronidazole, Doxycycline etc. are available having various disadvantages of systemic side effects, toxicities, higher dose and dose frequency, low patient compliance, first pass metabolism etc5. “These disadvantages of systemic therapy directed us to go for the local therapies which avoid these many disadvantages7,8. In comparison to solid devices, semisolid (gel) formulations have an advantages, such as, relatively faster release of the incorporated drug (particularly with respect to powders, fibres or microparticles); ease of preparation; ease of administration; high biocompatibility and mucoadhesivity (which allow the adhesion to the mucosa in the dental pocket and rapid elimination through normal catabolic pathways), decreasing the risk of irritative or allergic host reactions at the application site9. Controlled/sustained release formulations such as bio/muco- adhesive systems, prepared using various natural polysaccharides have gained an immense attention due to their sustainability, biodegradability, economy and safety over comparable synthetic materials. This study uses modified natural polysaccharide starch.

MATERIALS AND METHODS

Materials

Drug Ornidazole was obtained as a gift sample from Veteran Pharma, Ahmedabad. All the other ingredients used were of analytical grade, and were used as procured.

Methods

Formulation Development

Determination of ?max:

A drug solution of 14µg/ml was prepared in phosphate buffer pH 6.6 and UV spectra were performed between 400-200 nm to determine absorption maxima (?max).

Calibration study of Ornidazole:

Accurately 100 mg of Ornidazole was weighed and dissolved in 100 ml of phosphate buffer pH 6.6 to prepare a stock solution A. From it, sequential dilutions were performed to prepare working standards; 2µg/ml to 20µg/ml concentrations. The absorbance was measured using UV spectrophotometer at 317 nm.

Preparation of drug loaded gel:

Calculated amount of hydrolyzed copolymer was dissolved in deionized water to prepare 6% w/v polymeric solution. After complete dissolution, the drug (10% w/w of polymer) was added to polymeric solution; mixed properly; and the whole mixture was kept overnight to equilibrate.

pH11:

Each formulation was evaluated for its pH by using a digital glass electrode pH meter. The pH meter was first calibrated using standard buffer capsules pH 4.0 and 9.0. Then the pH was measured by bridging the electrode near the surface of gel and allowing it to equilibrate for a minute.

Rheological study12:

The Brookfield rheometer was used to study viscosity of each formulation. The viscosities have been measured using spindle S 61 at different rpm at 27oC.

Compatibility study13:

To ensure the compatibility in formulation, FTIR spectra of drug ornidazole and formulation was carried out using KBr pallet method.

Thermal analysis10,11:

Differential scanning calorimetry of pure drug and formulation was performed up to the temperature of 600oC. The heating rate was uniform in all cases at 10oC/min.

Syringeability study14:

The time required to squeeze out 1 ml of gel from the syringe with 22 guaze needle was estimated. The more viscous the gel, higher will be the duration. The viscosity of gel must be such that it should comfortably squeezed out during syringe application.

Spreadability study15:

1 g of gel was placed on the centre of glass plate (20 cm × 20 cm). The other glass plate of the same size was then carefully placed upon it and a weight of 100g was applied for a minute to remove the entrapped air. The diameter of spreaded gel (d mm) was accurately measured and the spreading area was calculated by using following equation:

                                                Spreading area (mm2) = d2×?/4

Mucoadhesive strength9:

This study was performed using modified apparatus. It is comprised of a two arm balance, one side of which contained two glass plates and the other side contained a container. One of the two glass plates was attached permanently to the base of the stage, and the other was attached to the arm of the balance by a thick strong thread. The membrane used for mucoadhesive testing was fresh rabbit intestinal membrane. Fresh rabbit intestine was glued to the upper side of the lower plate and another was glued to the lower side of the upper plate by using cyanoacrylate adhesive. The weighed gel (0.1 g) was placed on the rabbit intestine glued to the upper side of the lower plate. Then, the upper plate was placed over the lower plate and 50 g preload force was applied for 5 min (preload time). After removal of the preload force, the water, kept in a burette at some height, was siphoned into the container at a rate of 10 ml per minute till the plates were detached from each other. The weight of water (g) required for the detachment of the glass plates was considered as the mucoadhesion force of the applied gel.

Drug content analysis:

Accurately 1g of gel was weighed and dissolved in 100 ml of phosphate buffer pH 6.6. Drug content analysis was carried out by preparing stock solution as per the below mentioned scheme by further dilutions, to achieve the concentrations in calibration range using UV spectrophotometer. The drug content was found by using following equation:

Concentration (µg/ml) = (Absorbance/ slope) × Dilution factor

In vitro diffusion study9:

Diffusion study was carried out using Dialysis sac method. Dialysis sac of average diameter 15.9 mm was taken. Briefly 2 ml of formulation was placed in the dialysis sac hermetically sealed at one end and open at another. The sac was then folded and hermetically sealed to close both the ends. This “dialysis bag” was then emerged with the help of a thread into a beaker containing 50 ml of phosphate buffer pH 6.6 as diffusion media maintained at 37±0.5oC on a magnetic stirrer. Aliquots of 5 ml was withdrawn periodically at suitable time intervals and every time equal volume was replaced with fresh phosphate buffer pH 6.6 maintained at 37±0.5oC. The amount of drug release was measured by taking absorbance using UV spectrophotometer at 317 nm.

Kinetic models application16:

To understand the drug release mechanism from the developed formulation following kinetic models were applied.

1. Higuchi model 
2. Zero order release model
3. First order release model 
4. Kosmeyer-Peppas model

RESULTS AND DISCUSSION:

Determination of ?max and calibration curve:

The UV spectrum run for a drug solution (14 µg/mL) shows a sharp peak at 317 nm in phosphate buffer pH 6.6. Hence this is taken as a working ?max throughout the study for drug estimation. Further a calibration curve plotted taking concentration on X axis and absorbance on a Y axis shows a linear relationship for drug solution with regression coefficient of 0.9991. The drug working standards between the range 2-20 µg/ml followed the Beer-Lamberts law.

The peaks at 3165.29 cm-1 and 3311.89 cm-1 are attributed to O-H stretching. The characteristic peaks, at 3114.18 cm-1 and 3091.03 cm-1 are due to C-H stretching. As this drug is from the class 5-nitroimidaole, the distinctive sharp peaks at 1536.35 cm-1 ,1362.75 cm-1 , 1272.10 cm-1 and 829.42 cm-1 are due to -NO2 stretching. The same peak at wave number 829.42 cm-1 also indicates C-N stretching. Further a sharp peak at 1151.54 cm-1 is assigned to C-O stretching.

IR spectra of Formulation:

All the characteristic peaks of drug ornidazole i.e., for O-H stretching (3313.82 cm-1), C-H stretching (3090.07 cm-1 ), -NO stretching (1536.35 cm-1,1361.79 cm-1, 1268.24 cm-1 and 828.45 cm-1), C-N stretching (828.45 cm-1) and C-O stretching (1150.58 cm-1) is present in IR spectrum of formulation along with the peaks of polymer such as 1675.23 cm-1 for C=O stretching and at 1427.37 cm-1 for COO- stretching. This concludes that there is no interaction between drug and polymer and thus are compatible with each other.

Differential scanning calorimetry of Ornidazole and Formulation has been performed up to the temperature of 350oC. The heating rate was uniform in all cases at 10oC/min.

DSC spectra of pure drug (Ornidazole)11,19

DSC Spectra of Formulation

With respect to literature review, the endothermic peak of pure drug Ornidazole has been obtained at 90.87oC. The endothermic peak of Ornidazole in formulation shows no significant change indicating compatibility.

Preparation of gel formulation:

Four sets of gel formulations F1 to F4 were prepared using graft copolymer G1-G4 and evaluated for various parameters.The physical appearance of gels has been shown in the figure

Rheological study17:

Pseudoplastic flow is typically exhibited by polymers in solution. The viscosity of a pseudoplastic material cannot be explained by a single value, because no part of the curve is linear. The most satisfactory representation for a pseudoplastic material however is probably a graphical plot of the entire consistency curve. In this study, the rheology of prepared modified polymers has been performed by measuring the shear stress at different shear rates. Rheological data of 2% solutions of each hydrolyzed copolymer has been obtained as below:

In vitro diffusion study: