Development and Optimisation of Ofloxacin and Ornidazole Dental Films for Periodontitis

INTRODUCTION

Periodontitis can be defined as “an apical extension of gingival inflammation to involve the tissues supporting the tooth, including periodontal ligament and bone”. Periodontitis is a localized inflammatory response caused by bacterial infection of a periodontal pocket associated with subgingival plaque. This periodontal pocket provides ideal conditions for the proliferation of microorganisms.¹ Local drug delivery has been viewed as a promising route to overcome various complications of orally administered antibiotics such as poor bioavailability at the site of periodontal infection, high systemic dosage, toxicity and adverse effects. High bioavailability, longer duration of antibiotic activity at the site of infection and low systemic toxicity are few benefits of local drug delivery system.² Periodontal disease is an infection that involves the inflammatory process and the immune response. The presence of periodontal pathogens such as Porphyromonas gingivalis, Actionobacillus actinmycetemcomitans are responsible for periodontal destruction. It refers to acute and chronic disorder of the soft tissues surrounding the teeth which eventually leads to loss of supporting bone. Periodontitis is an inflammatory response in which the structural support to the tooth is destroyed. The disease results in resorption of the alveolar bone, detachment of the periodontal ligament supporting the tooth and formation of periodontal pocket .Pocket provides an ideal environment for the proliferation of a variety of pathogenic bacteria.³ The microorganisms colonizing the subgingival area represent the principal etiological factor in the development of the inflammation and tissue destruction. Periodontal pathogens have been shown to produce a large number of biological molecules that may act directly on host tissue and destroy its integrity.Dental films are most widely used form of medicated intra-pocket dental drug delivery device. Films are matrix type delivery systems wherein drugs are distributed throughout the polymer films and drug release through the films occur by diffusion and matrix dissolution or erosion. In the form of films, we can directly place the drug into the pocket so as to attain therapeutic drug levels in the gingival crevicular fluid and thus reduce the adverse effects of an antibacterial agent on other non-oral body parts. Advantages of dental films over conventional dosages form are ease of insertion, minimum pain on insertion, dimension and shape of the film can be controlled to corresponding to the dimension of the pocket, less storage space and minimal or no side effects.⁴ Systemically applied antimicrobials have been used for the treatment of severe forms of periodontitis. But side effects including hypersensitivity, gastrointestinal intolerance and the development of bacterial resistance have been described. These drawbacks would be markedly reduced if antimicrobial agents applied locally could be used. The local tissue concentration of a drug can be enhanced by incorporating the active agent into controlled release delivery systems to be placed directly in the periodontal pocket.⁵

The aim of current study is to formulate and evaluate ideal dental films that will remove the bacterial deposits from the tooth surface and to shift the pathogenic microbiota to one compatible with periodontal health. Dental films are made by solvent casting and direct milling methods. Suitable polymers are used that dissolves in periodontal cavity and achieve rapid disintegration. Plasticizer are used to improve the flexibility of the films. Evaluation and characterization will be carried out determine the effectiveness of the films.⁵

MATERIALS AND METHODS

MATERIALS USED:

Ofloxacin and ornidazole was purchased from yarrow chem, Ethyl cellulose was purchased from Rolex chemicals Industries, HPMC was purchased from Central drug house (P) Ltd, glycerin was purchased from Aldrich chemicals pvt. Ltd, Methanol was purchased from Vasa scientific co, PEG 400 was purchased from yarrow chem.

PREFORMULATION STUDY

Infrared (IR) absorption spectroscopy:

To investigate any possible interaction between the drug and utilized polymers, IR spectrum of pure drug and its physical mixture was carried by using FTIR the range selected was from 400cm-1 to 4000 cm-1

OPTIMIZATION

The runs or formulation, which are designed based on Response surface method, are evaluated for the response. The responses values are subjected to multiple regression analysis to find out the relationship between the variables used and the response values obtained. For optimization concentrations of Ethyl cellulose, HPMC were selected as variables and swelling index, folding endurance, 6h Drug release were selected as responses.

Preparation of cast film containing ofloxacin and ornidazole:

The method used for the preparation of dental implants was solvent casting technique. Dental inserts were prepared by dissolving Ethyl cellulose and HPMC alone and in combination with methanol as solvent. Plasticizer (PEG-400) provide good strength to films and therefore added to the preparation with respect to the concentration of polymer.

Consequently, ethyl cellulose and HPMC were dissolved in the suitable solvent methanol, containing PEG-400. Into this, Ofloxacin and Ornidazole of required concentration was added and stirred the polymeric solution homogeneously by using a magnetic stirrer. After complete mixing, the solution was poured into a clean Petri dish containing mercury and placed on a horizontal plane. The solvent was allowed to evaporate slowly by inverting a glass funnel with a cotton plug closed in the stem of the funnel on Petri dish at 24° for 24 h. After complete evaporation of a solvent, cast films were obtained. Dental films were then cut into pieces of 0.5×0.5 cm and wrapped in an aluminium foil and stored in desiccators at the relative humidity at room temperature in a dark place until further use.⁶

EVALUATION OF FILMS:

PREFORMULATION STUDIES OF THE SELECTED DRUG

Infrared (IR) absorption spectroscopy:

To investigate any possible interaction between the drug and utilized polymers, IR spectrum of pure drug and its physical mixture was carried by using FTIR the range selected was from 400cm-1 to 4000 cm-1.

Differential Scanning Calorimetry (DSC):

The DSC thermo gram was recorded using differential scanning calorimeter (Shimadzu DSC-60 Calorimeter, Tokyo, Japan). Thermo gram of ofloxacin and ornidazole, mixture of ofloxacin and ornidazole with different polymer was obtained at a scanning rate of 10°C/min conducted over a temperature range of 35-250° C in the environment of liquid nitrogen.

1.Physical appearance:

All the oral films were visually inspected for color, clarity, flexibility and smoothness

2.Surface pH study:

Films were left to swell for 1h on the surface of agar plate, prepared by dissolving 2% (m/V) agar in warmed phosphate buffer of pH 6.8 under stirring and then set aside till gelling at room temperature. The surface pH was measured by means of a pH paper placed on the surface of the swollen films. The mean of three reading was recorded.⁷

3.Weight uniformity of the films:

Film (size of 1 cm2 ) was taken from different areas of film. The weight variation of each film was calculated.

4.Thickness uniformity of the films:

The thickness of each film was measured using screw guage (thickness tester) at different positions of the film and the average was calculated.⁸

5.Folding Endurance:

Folding endurance was determined by repeatedly folding a small strip of film at the same place till it broke. The number of times, the film could be folded at the same place without breaking gave the value of folding endurance.⁹

6.Percentage Moisture loss

The films were weighed individually and kept in desiccators containing calcium chloride at room temperature for 24 h. Individual films were weighed repeatedly until they showed a constant weight. The percentage of moisture loss was calculated as the difference between initial and final weight with respect to initial weight.¹⁰

%MoistureLoss= (IntialWeight-FinalWeight) / Finalweight X 100

7.Percentage of Moisture Uptake:

A weighed film kept in desiccators at room temperature for 24h was taken out and exposed to 84% relative humidity (calcium chloride) in desiccators until a constant weight for the film was obtained. The percentage of moisture uptake was calculated as the difference between final and initial weight with respect to initial weight.¹¹

%Moistureloss=(Finalweight-Initialweight) / Initialweigh X100 t

8.Swelling index determination:

The films were weighed and immersed in phosphate buffer pH 6.8 solution (5 ml). At predetermined time intervals, films were removed from the medium, blotted to remove the excess liquid and weighed immediately. The swellingindex was calculated using the following equation.

Swelling Index= (W2-W1) / W1

Where, W1 and W2 are the weights of film before and after immersion in the medium, respectively.¹²

9.Drug Content uniformity in the Film:

The oral films were tested for the content uniformity. Oral film of 1cm2 size was cut andplaced in a 10 mL volumetric flask; 10ml of methanol was added and kept aside till the implant dissolve completely. From this solution, 1 ml was pipetted out and diluted to 10 ml with phosphate buffer pH 6.8. The absorbance of the solution was measured at 288.8 nm. The contents were kept for 24 hours to complete dissolve the film.¹³

10.In Vitro drug release:

Film of known weight and dimensions were taken separately into small test tubes containing 10 ml of pH 6.8 phosphate buffer. The test tubes were sealed with the aluminium foil and kept at room temperature. The sample was withdrawn and replaced with fresh 1ml of pH 6.8 for every 1 hour up to 6 hours. The concentration of drug in the buffer was measured at 278 nm by using a UV-spectrophotometer.¹⁴

RELEASE KINETICS FOR OPTIMIZED FORMULA

For the determination of the drug release kinetics from the Polymeric nanoparticles, the in vitro release data were analyzed by zero order, first order, Higuchi Equations, Peppas model

Zero order kinetics: Drug dissolution from Pharmaceutical dosage forms that do not disaggregate and release the drug slowly, assuming that the area does not change and no equilibrium conditions are obtained can be represented by the following equation.

Qt = Q 0 + K0t, Where Qt= amount of drug dissolved in time t, Q0 = initial amount of drug in the solution and K0 = zero order release constant.

First order kinetics: In order to study the first order release rate kinetics the release rate data were fitted to the following equation.Log Qt = log Q0+ K10/2.303 ,Where Qt is the amount of drug released in time t, Q0 is the amount of drug in the solution and K1 is the first order release constant.

Higuchi model: Higuchi developed several theoretical models to study the release of waterSoluble and low soluble drugs incorporated in semisolids and or solid matrices. Mathematical expressions were obtained for drug particles dispersed in a uniform matrix behaving as the Diffusion media, the equation is:

Qt = Kh. T1/2, Where Qt = Amount of drug released in t, Kh= Higuchi dissolution constant.

Krosmeyer and Pappas release model: To study this model the release rate data are fitted to the following equation:

Mt/Mα = Ktn ,Where Mt/M∞ is the faction of drug release constant, t is the release time and n is the Diffusion exponent for the drug release that is dependent on the shape of the matrix dosage form.

Characterization

DSC:

Differential Scanning Calorimetry (DSC) Analysis. (F9) sample was taken in aluminium pan, sealed with aluminium cap and kept under nitrogen purging (atmosphere). The samples were scanned from 30-300°C with the scanning rate of 10°C rise/ min using differential scanning calorimeter. Both the sample and reference are maintained at nearly the same temperature throughout the experiment. Generally, the temperature program for a DSC analysis is designed such that the sample holder temperature increases linearly as a function of time. The reference sample should have a well-defined heat capacity over the range of temperatures to be scanned.

SEM:

Formulation samples were mounted on double sided tape on aluminum stubs and micrographs were taken using a Leo VP-435 SEM. Images were taken of either surface of the film. Films were cut transversally to image the cross section of the film by SEM. An automatic threshold was used to convert grey scale to binary images.

RESULTS AND DISCUSSION

Fourier Transform Infrared spectroscopy

The IR spectra of ofloxacin and ornidazole with combination of polymers are shown in table-(9 & 10). FTIR spectra of Ofloxacin and Ornidazole pure drug, polymer with drug and excipients with drug was recorded. Functional group for the pure drug and functional group of polymer was found to be dentical according to the pharmacopoeia. Physical mixture of drug with other polymers were observed with identical functional group with minimal variation ranges. The IR spectrum of the formulation showed that there is no significant evidence for interaction between drug and the polymer. Peaks of both drug as well as formulation were observed are same. So this clearly suggest that the drug has not undergone any interaction with the polymer in the formulation, as there is no any shift in the positions of the characteristic absorption bands of drug in the formulation. So from the study we can conclude that the materials selected was feasible for preparation of films.

Formulation study

The schematic representation for the formulation process is given in the Table 1. In the present work total of nine formulations were designed by response surface design then prepared and evaluated for IR studies, DSC, entrapment efficiency, drug content, and In-vitro drug release studies. Based on evaluation, JMP gave best formulation F9 as optimized formulation.

Optimization

The formulations which are designed based on response surface design are evaluated for the response.The response values are subjected to multiple regression analysis to find out the relationship between the input factors used and the response values obtained. The response values subjected for this analysis are

Swelling Index (R1 )

Percentage of drug release at 6hr (R2 )

Folding Endurance.(R3 )

PHYSICOCHEMICAL PROPERTIES:

The films prepared by general procedure were evaluated for the following properties:

1.Physicalappearance:

All the oral films were visually inspected for colour, flexibility and smoothness. The results were observed that colour, flexibility and smoothness was similar with respect to pharmacopoeia.

2.Weight uniformity of the films:

Drug loaded films (1cm X 1 cm) were tested for uniformity of weight, and the results are given in table (11). The results were observed in the range 10.13 to 11.94 for all the trials formulations. The optimized formulation results of F9 was found to be within standard.

3.Thickness uniformity of the films:

The thickness of each film was measured at 10 different points, and the average thickness with S.D was calculatedand the results are given in table (11). The data of films thickness indicate that there was no much difference in the thickness among the formulations. The results were observed in the range 0.40 to 0.43 mm for all the trials formulations. The optimized formulation results of F9 showed desired thickness which is optimum for the preparation of films.

4.Folding Endurance:

Folding endurance of dental films was >100 times indicated that the formulation have good film properties. Folding endurance results were shown in table (11) and fig no (8). The results were observed in the range 129 to 156 for all the trials formulations which was satisfactory. The optimized formulation results of F9 was found to be 142 which is >100. The optimized formulation results of F9 was found to be within standard.

5.Drug Content uniformity in the Film:

The results of content uniformity indicated that the drug was uniformly dispersed. The percentage drug content in various trial formulations ranged from 92.76 to 98.54 given in table (11). Drug content more than 91% in formulations, ensuring that the methods of formulation and the ingredients selected are not affecting the stability of the drug. High drug content also gives the assurance that a good therapeutic outcome can be expected. The optimized formulation results of F9 was found to be within the range of pharmacopoeial standards.

6.Percentage Moisture loss:

Moisture loss studies were conducted in all formulations and reported in table (11). For all the formulations, the percentage moisture loss varied Between 5.97 to 12.93. It indicates that films can maintain its intrigity at dry conditions. The optimized formulation F9 showed good percentage moisture loss and proved that it can maintain its intrigity at dry conditions.

7.Surface pH study:

Surface pH study was conducted in all formulations and reported in table (11). Surface pH of all the trial formulations was found to be in between 6.2 to 6.9, and hence, no periodontal pocket irritation is expected. Surface pH study on optimized formulation F9 revealed that it has good surface pH and the formulation will not cause any irritation on periodontal pocket.

8.Percentage Moisture Uptake:

Percentage Moisture uptake studies were conducted in all formulations and reported in table (11). For all the formulations, the percentage moisture uptake varied Between 7.53 to 12.95. Formulation (F2) showed lowest moisture uptake whereas formulation (F4) showed highest moisture uptake 12.95. Study of percentage moisture uptake on Optimized formulation F9 showed satisfactory results according to the standard.

9.Swelling index determination:

Swelling index determination studies were conducted in all formulations and reported in table (11) and fig no (7). All the prepared trial formulations were evaluated for swelling index, the swelling index was found to be 14.3 to 15.6. Swelling index study on optimised formulation F9 showed 15.02.

10.In vitro Drug Diffusion Studies:

Formulations F4 and F5 containing higher concentrations of ethyl cellulose exhibited greatest (85.20% and 91.80% respectively) percentage of drug diffusion values, which are significantly different compared to the lowest values observed with the formulations containing lower concentration ethyl cellulose (F1 and F6 formulations).In the present study it was observed that as the concentrations of ethyl cellulose increased in the formulations, the drug diffusion rate increased substantially.

CONCLUSION

The main aim of the research work was to formulation and evaluation of the Ofloxacin and Ornidazole antibiotic by solvent evaporation method. When antibiotics are given by conventional route it contains complications related to orally administered antibiotics such as poor bioavailability at the site of periodontal infection, high systemic dosage, toxicity and adverse effects. Therefore to overcome from various complications antibiotics are administered by local route in form of dental film. Therefore, it is finally concluded that antibiotics can be administered locally through dental films.