INTRODUCTION:

Periodontitis, i.e., “peri”=around, “odont”=tooth, “itis”=inflammation, refers to a number of inflammatory diseases affecting the periodontium, the supporting tissues around the teeth. Oral cavity provides a diverse environment for the multiplication of wide range of microorganisms. Periodontitis has a multifactorial etiology, with primary etiological agents being pathogenic bacteria in the sub-gingival area.

Periodontal diseases consist of several pathological conditions such as gingivitis and periodontitis. Periodontitis is an inflammatory response to the overgrowth of anaerobic organisms in the subgingiva and if unchecked, results in the destruction of the bone and soft tissues supporting the tooth, and is characterized by periodontal pocket formation⁴. Periodontal disease treatment using a localized drug delivery system aims at delivering a therapeutic agent at a sufficient level inside the periodontal pocket and at the same time minimizes the side effects associated with systemic drug administration. Hence, drug delivery systems containing antibacterial agent are used for delivery to the periodontal pocket^1,2. Periodontal films are matrix delivery system from which drug is released either through diffusion or matrix dissolution and is applied to a periodontal pocket for the purpose of treating periodontal diseases. The pharmaceutical composition is provided in the form of a gel, sheet, film or bar-like formulation which releases a controlled and effective amount of an active ingredient at the periodontal pocket.³

MATERIALS AND METHODS:

The materials for the formulation were procured from different laboratories.

Preformulation Studies:

Identification of Drug⁴

The monograph of Tinidazole signified that the substance under examination was intimately mixed with potassium bromide. FTIR spectrum of the sample was taken using potassium bromide pellet method. The spectrum of test specimen was recorded over the range from 4000cm^-1 to 500cm^-1 and compared with the corresponding USP reference standard.

Organoleptic Evaluation⁵

Organoleptic properties of drug like color, appearance and odor was observed and recorded.

Determination of Melting Point and solubility of tinidazole:

The melting point of drug was determined by capillary tube method. The drug was filled to capillary tube which has one end sealed. The filled capillary tube was placed inside the melting point apparatus and the temperature at which drug melted was noted.⁶

Solubility of tinidazole was checked in various solvents like water, phosphate buffer saline pH 7.4, 6.8, methanol, acetone. 100mg of drug was accurately weighed and transferred into a stoppered tube containing 0.1ml of solvent. If completely dissolved, the drug is said to be very soluble. If insoluble, added 0.9ml of solvent to it and is said to be freely soluble on complete dissolution. Otherwise, added 2ml of solvent to the same. The drug, if completely dissolved in the solvent, then it is said to be soluble. If insoluble, further 7ml of solvent was added and observed to be sparingly soluble on complete dissolution. On further addition of 10ml of solvent it is said to be slightly soluble, if completely dissolved. If it is not completely dissolved in the above solution, accurately weighed 1mg of drug and added 10 ml of solvent. If the solvent dissolves the drug, it is said to be very slightly soluble.

Analytical Method Used in the Determination of tinidazole⁷

UV spectrophotometry method was developed for the analysis of drug using double beam Systronics-2202 spectrophotometer.

Determination of λmax of tinidazole:

10mg/ml solution of Tinidazole was taken in specific buffer solution (pH 6.6) using serial dilution technique and scanned in range 200-400nm using UV spectrometer to find out the wavelength of maximum absorbance.

Compatibility studies⁸

Excipients are any substance other than active or prodrug included in the manufacturing process or contained in the finished product.

FTIR Study:

The IR spectra were recorded using FTIR spectrophotometer. The samples were prepared by mixing the drug and the excipients in 1:1 ratio and the mixtures were stored in closed containers for 1 month. FTIR spectrum of the samples was taken using potassium bromide pellet method. The physical mixtures of tinidazole and excipients were scanned in the wavelength region between 4000 and 500 cm^-1 and compared to check compatibility of drug with excipient.

DSC Studies:

DSC study was carried out using DSC-60 instrument (Shimadzu) to check the compatibility of ingredients. The samples were prepared by mixing the drug and the excipients in 1:1 ratio. Accurately weighed samples were sealed in aluminum pans and analyzed in an inert atmosphere of nitrogen at flow rate of 25ml/min. A temperature range of 0°C to 300°C was used, and the heating rate was 10°C/min. DSC thermograms of pure drugs and physical mixtures of drugs and excipients were studied for their interactions.

Formulation of periodontal film⁹

Periodontal films were prepared by solvent casting technique. Glass moulds were used for casting of the films. Formulations were designed as shown in the Table‐6, in which Ethyl cellulose was taken as the main non-biodegradable polymer in combination with different co‐polymers of different concentrations for each cast films. Films were prepared by dissolving Ethyl cellulose and corresponding copolymer (PVP K‐30/ HPMC K4M/ Eudragit RL-100) in acetone, using dibutyl phthalate and PEG‐400 as plasticizers. Tinidazole was added to the polymeric solution and mixed homogenously using magnetic stirrer in a closed beaker. After complete mixing for about 30 minutes, it kept for 15 minutes in closed condition to remove the entrapped air bubbles. 10ml of the solution was poured into the clean leveled glass moulds of 9cm diameter. The solvent was allowed to evaporate slowly by inverting a glass funnel with a cotton plug closed into the stem of the funnel at room temperature for 24hours. After complete evaporation of solvent, cast films were obtained, which were then cut into pieces, wrapped in an aluminum foil and stored in a desiccator at room temperature in a dark place for further evaluation studies.

Evaluation of Peridontal film:

Characterization of the films^9,10

Formulated films were subjected to the preliminary evaluation tests. Films with any imperfections, entrapped air, or differing in thickness, weight (or) content uniformity were excluded from further studies. Physicochemical properties such as thickness, weight uniformity, percentage moisture loss, folding endurance, surface pH, swelling index and drug content uniformity of the prepared films were determined.

Physical appearance:

All the films were visually inspected for colour, clarity, flexibility and smoothness.

Thickness uniformity and uniformity of weight¹¹

The thickness of the patch was measured using calibrated vernier caliper at different spots of patch. Patch was inserted between the jaws after setting the pointer to zero. The readings from the main scale and vernier scale were measured and the mean thickness was calculated. Film pieces (size of 7x2mm) were taken from different areas of film. The weight variation of each film was calculated.

Estimation of percentage moisture lossand folding endurance studies^12,13

6 films of different concentrations of size (7x2mm) were weighed accurately and then they were kept in desiccators for 3 consecutive days and then reweighed. % moisture loss was calculated by formula:

Moisture loss = (Initial wt – Final wt/ Initial wt) ×100.

The folding endurance of the films was determined by repeatedly folding one film at the same place till it broke or folded up to 350 times, which is considered satisfactory to reveal good film properties. The film was folded number of times at the same place without breaking gave the value of the folding endurance. This test was done on all the films for six times.

Drug content uniformity¹⁴

The drug-loaded films of known weight (7 x 2mm) were dissolved in 5ml of pH 6.6 phosphate buffer in a beaker. The dispersion was kept in the dark place overnight. The dispersion was filtered. 0.1ml of the filtered solution was diluted to 10ml with pH 6.6 phosphate buffer in a 10ml volumetric flask. Drug concentrations were determined by taking three readings using a UV visible spectrophotometer at 320nm.

In vitro drug release studies¹⁴

The pH of gingival fluid lies between 6.5–6.8, so phosphate buffer pH 6.6 was used as simulated gingival fluid. Also, since the film should be immobile in the periodontal pocket, a static dissolution model was adopted for the dissolution studies. Sets of six films of known weight and dimension were placed separately in small sealed 1ml test tubes containing 1.0ml of phosphate buffer (pH 6.6) and kept at room temperature for 24 h. The buffer was then drained off and replaced with a fresh 1.0ml of buffer. The concentration of drug was determined by UV/VIS spectrophotometer (Shimadzu) at 276nm after suitable dilution with 0.1N HCl. The procedure was continued for 10 consecutive days for all formulations.

Scanning Electron Microscopy (SEM)¹⁴

A Scanning electron microscope (model JFC-1100 E, Jeol, Japan) was used to study the surface characteristics of the optimized filmF8 before and after dissolution. Samples (7x2mm) were mounted on the SEM sample stab using a double sided sticking tape. The samples were coated with gold (200 A⁰) under reduced pressure (0.001 torr) for 2 min using an ion sputtering device (model JFC-1100 E, Jeol, Japan). The gold coated samples were observed under the SEM at room temperature and photomicrographs of suitable magnifications were obtained

In-vitro Antibacterial studies¹⁵

80ml of nutrient agar media was prepared and sterilized at 15 lb pressure for 20 min in an autoclave. Under aseptic condition 20ml of nutrient agar media was transferred into 4 sterile petri plates. After solidification 0.1ml of microbial suspension of both E. coli and S. aureus of known concentration was spread on the media. Wells were prepared by using a sterile borer of diameter 6 mm and the samples were added in each well separately. The optimized film F8 and the standard drug solution sample were tested. The plates were then incubated at 37⁰C for 48 hrs. Then the zone of inhibition was measured and compared.

Kinetic Data Analysis: Drug release models¹⁵

To analyze the drug release rate kinetics and mechanism of drug release from the dental films, the in vitro drug release studies data was fitted into Zero order, First order, Hixson-Crowell Cube Root Law model, Higuchi model, and Korsmeyer peppas models. From these, best-fit models were selected. The quantitative interpretation of the values obtained in the dissolution assay is facilitated by the usage of a generic equation that mathematically translates the dissolution curve in the function of some other parameters related with the pharmaceutical dosage forms. The kind of drug, its polymorphic form, crystallinity, particle size, solubility and amount in the pharmaceutical dosage form can influence the release kinetics.

Stability studies¹⁶

The stability of the entire drug loaded polymer films were studied at different temperatures using the reported procedure. The films of size (7 x 2mm) were weighed in three sets (18 films in each set). The films were wrapped individually in aluminium foil and also in butter paper and placed in Petri dishes. These containers were stored at different temperatures like 27 ± 2⁰C, 5–8 ±2⁰C, 40±20 ⁰C for a period of 30 days. All the polymeric films were observed for any physical changes, such as color, appearance, flexibility, or texture, and the drug content was estimated at an interval of 10 days.

RESULTS AND DISCUSSIONS:

Preformulation Study:

Identification of Drug:

The sample spectrum was compared with the reference spectrum. There were no significant changes in the functional groups. The frequency of observed functional groups C=O, C=C and O-H are within the standard limits. The finger print region has not changed significantly. So the drug was identified as Tinidazole (Fig.-1).

Organoleptic Evaluation:

It is Pale yellow powder and it is Odourless or almost odourless and it appears to be fine crystalline powder.

Determination of Melting Point and solubility of drug:

The standard melting point of Tinidazole is in the range of 125-128⁰C. The observed value was 127⁰C which is within the range as per official monograph. So the drug was identified as Tinidazole.

The solubility was determined by dissolving the drug in different solvents like water, Phosphate buffer saline 6.6, chloroform and acetone. It was insoluble in water and soluble in acetone, phosphate buffer saline 6.6 and chloroform.

Analytical Method for The Determination of Tinidazole

Determination of λ max of Tinidazole in phosphate buffer saline pH 6.6

The 10µg/ml sample was prepared and scanned between 200 to 400nm. The drug showed maximum absorption at 318nm. So, the λ max of tinidazole was found to be 318nm.

FTIR Studies:

FTIR studies were carried out for drug [Tinidazole] and for the drug-excipients physical mixtures. There were no significant changes in the frequency of the functional groups of Tinidazole. So, the drug was compatible with Ethyl cellulose+ HPMC K4M+ PVP K30+EudragitRL 100

DSC studies:

The DSC studies were carried out for drug [Tinidazole] and drug-excipients physical mixtures. The recorded DSC thermograms showed the profile of Tinidazole with melting point at 127⁰C. Drug with excipients, showed melting point at 128⁰C. The melting point remains almost the same, indicated that the drug and excipients are compatible with each other.

Evaluation of Tinidazole Dental Films:

Physical Appearance:

All the polymer combinations used for preparation of periodontal films showed good film forming properties. The films were translucent, with good strength and visually smooth surfaced. The drug and polymer distribution was uniform all throughout the film.

Thickness of the Films and uniformity of weight

The thickness of drug-loaded films were measured with the help of vernier caliper and the average thickness with Standard deviation was calculated. The thickness of various films is given in table-1. The data of films thickness indicated that the values were almost uniform. Film F6 showed maximum thickness due to copolymer Eudragit-RL100.

Nine drug loaded films (7x2mm) were weighed, mean and standard deviation were calculated for each film and the results of weight uniformity are given in table-1. Lesser S.D. values indicated that the films were uniform in weight.

Table.1: Measurement of thickness

Formulation code	*Thickness(mm) (Mean±SD)**	*Weight Uniformity(mg) Mean±SD)**
F1	0.074±0.005	1.27±0.040
F2	0.084±0.008	0.95±0.320
F3	0.078±0.004	1.43±0.201
F4	0.104±0.004	1.84±0.104
F5	0.118±0.006	2.16±0.170
F6	0.126±0.002	0.98±0.129
F7	0.084±0.006	2.24±0.122
F8	0.116±0.002	2.14±0.180
F9	0.122±0.002	1.35±0.040

Percentage moisture loss and folding endurance:

Moisture loss studies were conducted on all the tinidazole formulations and reported in table -2. It was observed that formulation F2 showed maximum amount of moisture loss due to the more water vapour permeability of PVP K30. Formulation F5 showed minimum percentage moisture loss because of hydrophobic Eudragit RL 100.

The folding endurance of the films was > 250 times. It means all the formulations have good folding endurance.

Drug content uniformity:

The content uniformity test is commonly employed for unit dosage forms. In order to make sure about the uniform dispersion of drug in films, the drug content was carried out. The drug content was analyzed at 318 nm using suitable blank. All the formulations showed more than 80% of the drug loading indicating much of the drug is not lost. The results were expressed in AM ± SD and reported in Table-1. The results indicated that the drug was uniformly dispersed.

Table 2: Measurement of % Moisture loss

Formulation code	*% Moisture loss (Mean±SD)**	*Folding endurance (Mean±SD)**
F1	13.5±0.043	215.2±0.22
F2	14.5.±0.068	225.6±0.15
F3	14.01±0.055	238.0±0.24
F4	10.8±0.068	266.5±0.24
F5	7.3±0.025	283.0±0.32
F6	9.7±0.040	286.2±0.26
F7	11.6±0.043	240.2±0.42
F8	9.8±0.035	235.6±0.12
F9	8.7±0.045	254.0±0.28

In vitro drug release.

The releases of Tinidazole from the dental films were varied according to type and concentration of polymer. The release of the drugs from film formulations ranked in the order F5> F6>F2>F4>F3>F1>F9>F8>F7, where the amounts of the drug released after 7 days were 94.56%, 90.34%, 89.36%, 88.21%, 88.06%, 87.06%, 85.87%, 83.61%, 78.45% respectively. The cumulative % drug release profile of all the formulation batches has been shown in Table-3 and graph is plotted between cumulative % drug releases versus time.

Kinetic Study of Periodontal Film

To determine the release mechanism that gives the best description to the pattern of drug release, the in vitro release data were fitted to zero-order, first-order, Hixson Crowell equation and Higuchi matrix model. The release data were also kinetically analyzed using the Korsmeyer–Peppas model. The accuracy and prediction ability of the models were compared by calculation of R²as given in Table No.4. The model giving R² close to unity was taken as the best fit model. The value of ‘n’ indicates the drug release mechanism. The ‘n’ value is used to characterize different release mechanism concluding that value n=0.5 indicates fickian diffusion and values of n between 0.5 and 1.0 or n=1.0 indicate non- fickian mechanism. The release kinetics data indicates that the release of drug from periodontal film best fits to zero order release kinetics.

Table. 3. Percentage Cumulative drug release of formulations

Time (hours)	Cumulative % drug release
Time (hours)	F1	F2	F3	F4	F5	F6	F7	F8	F9
0	0	0	0	0	0	0	0	0	0
24	35.19	31.27	36.17	37.04	29.42	38.12	25.32	27.35	24.31
48	45.81	54.31	43.87	46.24	36.36	41.25	34.26	38.12	41.61
72	50.69	68.43	54.42	54.70	46.75	50.21	37.52	41.25	50.79
96	68.03	72.19	62.73	63.31	61.62	62.08	43.92	50.21	61.58
120	75.93	79.11	69.37	72.31	76.22	75.43	56.18	62.08	68.16
144	82.43	81.69	75.86	80.33	88.96	83.61	68.44	75.43	76.54
168	87.06	89.36	88.06	88.21	94.56	90.34	78.45	83.61	85.87

Table No. 4: Kinetic profile of periodontal film

Formulation Code	Zero order R²	First order R²	Higuchi model R²	Hixon-Crowell model R²	Kosmeyer- Peppas
Formulation Code	Zero order R²	First order R²	Higuchi model R²	Hixon-Crowell model R²	R²	N
F1	0.966	0.916	0.989	0.982	0.521	0.870
F2	0.853	0.977	0.981	0.954	0.525	0.886
F3	0.951	0.946	0.991	0.964	0.506	0.859
F4	0.961	0.970	0.995	0.977	0.504	0.865
F5	0.975	0.916	0.999	0.967	0.587	0.880
F6	0.971	0.954	0.976	0.974	0.517	0.867
F7	0.959	0.934	0.968	0.956	0.567	0.827
F8	0.961	0.939	0.961	0.964	0.562	0.846
F9	0.951	0.979	0.990	0.990	0.585	0.869

Scanning Electron Microscopy (SEM):

Scanning electron micrographs indicate that prior to drug release, the top surface of the films is smooth which exhibits pores as can be seen in Figure 2. These pores indicated that the film F8 had sufficient drug loading capacity. After the release of the drug, the surface became rougher and irregular as can be seen in Figure 2. The reason for this is the coagulation of polymers after the release of the drug from the pore.

Fig.2: SEM images of dental film F5 after drug release.

In vitro antibacterial study

In vitro antibacterial activity was performed as mentioned in methodology on E. coli organism.. The study indicates that the formulated polymeric film F5 containing Tinidazole retained their antibacterial activity and also it shows the same effect as the standard tinidazole drug.

Fig 3: Antibacterial zone with E. coli (With control and F5)

Stability Study

The selected formulation F5 was subjected to stability study. Initial and third month studies were carried. The results showed that there were no significant changes for physical appearance, % drug content, surface pH, folding endurance and in vitro drug release. The drug release profile of formulation. So, the drug product was found to be stable. The stability study will be continued further up to 6 months.

SUMMARY:

In this work an attempt was made to develop and evaluate Tinidazole dental films with ethyl cellulose as the main non-biodegradable polymer and various co- polymers in different ratio for the treatment of periodontitis and to select the best formulation among them. The main objective of the work is to formulate the dental film which can release the drug above MIC level through its treatment period. Preformulation study of the drug was carried by IR absorption method, solubility testing, melting point determination and organoleptic evaluation. The drug was identified by comparing the sample spectrum with that of the reference spectrum. The frequency of observed functional groups C=O, C=C and O-H are within the standard limits. FTIR and DSC studies were conducted to determine the compatibility between the drug and the excipients. The FTIR spectrum of Tinidazole exhibited peak signals at 3130, 2956, 1522, 1479, 1428, 1365, 1264, 1126 and 1026 cm^-1 due to =CH stretching, C-H stretching, and C=N stretching of imidazole ring, CH2 bending, C-C stretching, N=O symmetric stretching, C-O, S=O and C-N stretching. There were no significant changes in the frequency of functional groups of drug in the presence of other excipients. The DSC thermogram indicted that melting point of drug was 128⁰C which remained almost the same in the presence of polymers. The results of FTIR and DSC studies revealed that the drug and polymers were compatible without any significant changes in the nature of drug. From various evaluation parameters like, % moisture loss, percentage drug content and in vitro release studies, the formulation F5 was concluded as the best formulation among them. Measurement of thickness and uniformity of weight studies indicated that there was no much difference in thickness and weight within formulations. Percentage moisture loss studies showed a minimum % moisture loss for the formulation F5 which may be due to the hydrophobic character of eudragit RL100. Folding endurance studies ensures that all the formulations had good film properties. The pH of all the formulations was in the range of 6.1 to 6.9, which lies in the normal pH range of the periodontal mucosa and would not produce any mucosal irritation. As the concentration of polymer increased, the swelling index also increased. The formulation F5 showed comparatively high Percentage drug content than the others. This indicates the homogenous distribution of drug throughout the film which may be due to high entrapment of drug in the ethyl cellulose eudragit RL100 (10%w/w of ethyl cellulose) matrix. In vitro release studies showed a cumulative release of 78.37% after 7 days for the formulation containing an optimum concentration of eudragit RL100 (ie 10%w/w of ethyl cellulose).

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Received on 11.05.2020 Modified on 26.06.2020

Research J. Pharm. and Tech. 2021; 14(5):2750-2756.

DOI: 10.52711/0974-360X.2021.00485