Human Journals Research Article September 2016 Vol.:7, Issue:2 © All rights are reserved by Vaishali Potnis et al. Formulation and Evaluation of Dental Films for the Treatment of Periodontal Diseases Keywords: Periodontal diseases, Sterile films, Antibacterial activity, Sustained release ABSTRACT Vaishali Potnis*, Suchita Dhamne and Ajit Mane A novel film formulation was developed for concomitant delivery of NSAID, Ibuprofen (IBU 1.25% w/v) and * Department of Pharmaceutics, JSPM’s Jayawantrao antimicrobial, Metronidazole (MTZ 0.75% w/v) for local treatment of periodontal diseases. The plasticized, aqueous S awant college of Pharmacy and Research, Hadapsar, dispersions of Ethyl cellulose (EC), sodium carboxy methyl Pune- 411 028, India. cellulose (Sod CMC), HPMC K4M and Eudragit S100 were cast into thin films. Both the placebo and drug loaded, sterile films were characterized for various structural, physicochemical Su bmission: 25 August 2016 and mechanical properties. The formulation composed of Sod. Ac cepted: 1 September 2016 CMC (1.20%w/v) with the plasticizer propylene glycol (30%w/w) (DFSMBPG3) possessed superior film characteristics Published: 25 September 2016 such as smooth even surface, thickness (0.263mm), neutral pH (6.54-7.12), consistent average weight (78mg), negligible moisture loss <1.5% w/w, greater tensile strength (2.12 kg), folding endurance (> 290) and prolonged release of both the APIs viz: IBU (92.12%) and MTZ (95.82%) respectively over about 8 hours. The sterile films were devoid of any acute irritancy potential as revealed by intact and dense vasculature around CAM of incubated egg of hen. The selected film www.ijppr.humanjournals.com composition also demonstrated good antimicrobial efficacy (diameter of the zone of inhibition >20mm) which was also comparable with the bulk of MTZ used as a reference, against pathogenic bacterium S. mutans responsible for periodontal diseases. The stability data (30 days)of this film composition indicated only minimal changes in physicochemical, mechanical properties, % contents and release (in-vitro) profiles of both the APIs. Hence, the novel periodontal film composition is a promising alternative to conventional local therapy due to the multipronged control of major symptoms of periodontal diseases including gingivitis. www.ijppr.humanjournals.com INTRODUCTION: Periodontal diseases represent various periodontal tissue infections including gingivitis and periodontitis. These diseases have a direct association with dysbiosis of the bio-flora residing on the periodontal pockets. Unlike, gingivitis, a milder and relatively harmless inflammatory response, periodontal disease causes greater damage to the connective tissue and bone support. Other contributory factors of periodontitis include genetics, prolonged consumption of products containing tobacco and alcohol, malnourishment, immunodeficiency, osteoporosis, diabetes mellitus, emotional stress and antibiotic drug candidates belonging to tetracycline group. The typical therapy based on oral administration of conventional dosage forms of NSAIDs and antibiotics suffers from poor local concentrations within the affected periodontal pockets as a consequence of flushing action (replacement of gingival crevicular fluid at about 40times/hour within 5mm pocket). Moreover, it suffers from hyperacidity, gastric irritation and hypersensitivity manifestations. More serious disease condition, known as refractory periodontitis requires aggressive treatment with these medications delivered using novel approaches for prolonged residence at the target site. A few reports have cited the effective use of antibiotics including MTZ for control of infections caused by various aerobic and anaerobic pathogens associated with periodontitis. Manojkumar, Prabhushankar, and Satheshbabu have quoted effective use of MTZ against anaerobes Porphyromonas gingivalis and Provetella intermedia while MG Ahmed and co-workers have used Ciprofloxacin in (local treatment) for control of S. mutans associated with gingivitis.There is evidence citing effective alteration of progression of certain forms of periodontitis and reduction of gingival inflammation and alveolar bone resorption due to concomitant administration of antibiotic and NSAIDs. Luana Perioli and co-worker have developed buccal film formulation of a drug of choice for anti-inflammatory and analgesic activity in the oral cavity. Hence, the work was undertaken with the objective of prolonging concomitant delivery of antimicrobial and NSAID using hydrophobic and hydrophilic polymeric periodontal film compositions for the effective control of major symptoms of periodontitis. Citation: Vaishali Potnis et al. Ijppr.Human, 2016; Vol. 7 (2): 41-56. 42 www.ijppr.humanjournals.com MATERIALS AND METHODS: 1. Materials: The drugs IBU and MTZ were obtained as gift samples from Micro Lab Ltd., Bangalore, and Research Lab Fine Industries, Mumbai respectively. The polymer Eudragit S100 (Eu S100) was a generous donation from Evonik India Pvt. Ltd., Mumbai. All other materials used were of analytical reagent grade. An authentic culture of S. mutans was procured from MTCC Chandigarh; India. 2. Methods: 2.1Pre-formulation Studies: A stock solution (100µg/ ml) was prepared by dissolving accurately weighed the quantity of IBU (20mg) and MTZ (10mg) in an appropriate volume of methanol: distilled water (1:9). The λ max and linearity range values of appropriately diluted (2ml up to 10ml) solution were noted. 2.2 Compatibility studies: The compatibility of a mixture of APIs with or without polymeric addition was ascertained by exposing to the environmental conditions over the 15 days. The detection of changes in any of the physical or physicochemical characteristics of blends if any was carried out by visual and FT-IR spectrophotometry (BrukerALPHA-T 1.2.4). 2.3 Preparation of periodontal films: To determine the optimum concentrations of polymer, plasticizer and solvent placebo films were evaluated for structural, physicochemical and mechanical properties. The films, which exhibited all the characteristics, were selected for the inclusion of APIs and taken up for further studies. The polymeric films were prepared by solvent casting technique. Each of the polymers (Ethyl cellulose (EC), Sod. CMC and HPMC K4M were dissolved in distilled water (DW) where Eu S100 was dissolved in alcohol. The polymeric dispersions were stirred continuously and the required quantity of Propylene glycol (PG) added gradually with careful stirring. (Table 1 reports the typical composition of films selected for loading of IBU (1.50%w/w) and MTZ (0.75% w/w) Citation: Vaishali Potnis et al. Ijppr.Human, 2016; Vol. 7 (2): 41-56. 43 www.ijppr.humanjournals.com in a controlled manner were poured into a clean Petri plates placed on a horizontal plane which was previously lubricated with 2ml Glycerin. The solvent was allowed to evaporate slowly by placing inverted glass funnel with a cotton plug in the stem of the funnel on the Petri plates at room temperature for 24 hrs. The dried films were wrapped into a composite packing of butter paper and aluminum foil. Table 1: Formulation of selected periodontal medicated Film Sr. Formulation IBU MTZ EC EuS 100 HPMC Sod. CMC No. Code K4M 1. DFECBPG3 1.5% 0.75% 2.80% -- -- -- 2. DFEUBPG3 1.5% 0.75% -- 2.80% -- -- 3. DFHMBPG3 1.5% 0.75% -- -- 1.40% -- 4. DFSMBPG3 1.5% 0.75% -- -- -- 1.20% *PG 30% w/w of dry weight of film forming polymer Casting solvent up to 20ml-Alcohol(99.98%v/v) for EuS100 -DW for EC, HPMC K4M, Sod. CMC 2.4 Evaluation of medicated periodontal films: The periodontal films were assessed visually for appearance, presence of any surface imperfections and entrapment of air. The average weights of film samples of each formulation were noted in triplicate. a) Appearance: The appearance of each film sample was checked visually with or without the aid of magnifying glass (2X) against the dark and white background and surface homogeneity, transparency was noted. b) Texture: The texture characteristics of drug loaded strips ( 7×2 mm) of each film type were noted using scanning electron microscopy (JEOL,Model JFC-1100 E, Japan, room temperature, Citation: Vaishali Potnis et al. Ijppr.Human, 2016; Vol. 7 (2): 41-56. 44 www.ijppr.humanjournals.com magnification 200-20 mm ) before and after 8 hours of dissolution (in-vitro, medium simulated 0 gingival fluid, SGF, maintained at temp 36±2o C).Film samples were coated with gold (200 A ) under reduced pressure (5 torr) for 2 min using an ion sputtering device before recording the micrographs. c) Thickness uniformity: Thickness (mm) at different positions (at the margins and center) of strips of each film sample (square sample dimensions 1cm) was noted using digital vernier calipers d) Surface pH: The pH of aqueous dispersions (50 ml) of dissolved strips of each film formulation was noted in triplicate using the previously calibrated pH meter e) Tensile strength The tensile strength (gm) of blocks (10* 70 mm) cut from each film sample was noted at gradually increasing pull until the film sample tears off. f) Folding endurance: The repeated folding of each type of square sized medicated periodontal film samples (n=3) was carried out at the same point till it broke and the number was noted that which film were broken. g) Weight uniformity: The strips (square shape 1cm2 dimensions) cut off from different areas of Casted medicated periodontal films were weighed individually and the uniformity of weight of each film type was calculated. h) Moisture contents (%w/w): The moisture contents of 3 strips (square shape,1cm2) of each film sample were calculated Individually on the basis of difference in initial (W1) and final weight (W2) obtained after exposure to anhydrous calcium chloride for 72 hrs. Citation: Vaishali Potnis et al. Ijppr.Human, 2016; Vol. 7 (2): 41-56. 45 www.ijppr.humanjournals.com i) Sterilization and Sterility: The individually packed strips of each film type were irradiated by UV radiation (240nm- 280nm) for 120 minutes and tested using Method B of sterility testing with slight modification wherein the strips of dimensions (1 cm2 square sized) were dissolved in about 50 ml of sterile water for Injection I.P and 20 ml of dispersion of each of the film type was added aseptically, over the surface of each of the three sterile media viz: McConkey Agar (incubation at 30-35oC for 3 days), Chloramphenicol Yeast Glucose Agar (incubation at30-35oC for 3 days) and Plate Count Agar (incubation at 20-25oC for 5days). The colony count of each of the film was noted after specified conditions of incubation. j) Contents of MTZ and IBU: The method described by Naga Priya KR et al. was followed, where 2 strips of each type of medicated films (1cm2) were individually dissolved (mechanical stirring) using 5 ml of phosphate buffer (pH 6.8) contained in plain glass vials. The resulting solutions were clarified by passing through Whatman filter membrane ( #41) and 1 ml of filtrate was withdrawn, diluted suitably and assayed spectrophotometrically for contents of both APIs, using simultaneous estimation method (based on the method reported by Md. El-Ghobashyet al) , developed and validated in the laboratory . k) Release (in-vitro) of MTZ and IBU: Simulated Gingival Fluid (SGF, pH 6.6- 6.8, maintained at 37 ± 0.5 °C) contained in the dry test tube was used as dissolution fluid. Each of six strips of known weight and dimensions of selected medicated periodontal films was placed individually in the test tubes for 8 h. At the interval of 1 hr. the supernatant of dissolution medium from each tube was aspired using a disposable plastic syringe (2ml) and replaced with 1 ml volume of fresh SGF (maintained at conditions described above). The cumulative % release of IBU and MTZ was determined using the experimental method of estimation. Citation: Vaishali Potnis et al. Ijppr.Human, 2016; Vol. 7 (2): 41-56. 46 www.ijppr.humanjournals.com l) Antibacterial (in-vitro) efficacy: Antibacterial efficacy of medicated periodontal films described by Manojkumar et al. was used where the film samples of selected formulations (0.5 × 0.5 cm, square shaped) were placed aseptically onto the sterile Petri plates containing sheep blood agar previously seeded with S. mutans. Subsequently, the films were incubated at 37OC for 24 hours and their antibacterial efficacy was noted and expressed as (diameter of the zone of inhibition) and compared with the zone created by a solution of combined APIs (equivalent to concentrations used in casting solution) and that created by the placebo film formulation of the same composition. m) Mucosal irritancy potential (in vivo): Mucosal irritation study was carried out by HET-CAM (Hen’s Egg Chorioallantoic membrane) test. In this test fertilized three hen’s eggs for each formulation weighing between 50 and 60 g were selected and candled in order to discard the defective ones. These eggs were incubated in humidified incubator at a temperature 40ºC for 3 days. On day 3, egg albumin (3ml) was taken off from the sharpened end of the egg using sterile techniques. For the development of CAM away from the shell, the eggs were kept in the equatorial position. On the fifth day of incubation, the eggs were candled and non-feasible embryos were removed. On the tenth day, formulations (0.5ml) were installed through the window (2×2 cm) on the equator. A 0.9% Sodium Chloride (NaCl) solution was used as a control as it is reported to be practically non-irritant and the scores were recorded. n) Environmental stability: Three sets (5 strips in each set) of medicated periodontal films (1cm2) were weighed. The films were wrapped individually in butter paper followed by aluminum foil and placed in Petri dishes. The plates were stored at ambient humidity conditions at room temperature (25 ± 2ºC) and in a hot oven at 45 ± 2ºC for 30 days. The samples were analyzed for physical changes such as appearance, color, and texture. Drug content was estimated at regular intervals as described earlier. Citation: Vaishali Potnis et al. Ijppr.Human, 2016; Vol. 7 (2): 41-56. 47 www.ijppr.humanjournals.com RESULTS AND DISCUSSION: The organoleptic, physicochemical and solubility characteristics confirmed the identity and purity of the drug candidates IBU and MTZ. Moreover, the spectral characteristics (wavelength of maxima and linearity of concentration and absorbance range- 400nm- 200nm) further supported the identity and purity of APIs and formulation excipients. The IR spectra of physical mixtures of IBU and MTZ and excipients did not indicate gross changes in structure since the position and height of peaks associated with major functional groups in their structures (Figure 1a, 1b, 1c, 1d, and 1e). Figure 1a: IR spectrum of IBU and MTZ Citation: Vaishali Potnis et al. Ijppr.Human, 2016; Vol. 7 (2): 41-56. 48 www.ijppr.humanjournals.com Figure 1b: IR spectrum of IBU, MTZ, and EC Figure 1c: IR spectrum of IBU, MTZ and Eu S 100 Citation: Vaishali Potnis et al. Ijppr.Human, 2016; Vol. 7 (2): 41-56. 49 www.ijppr.humanjournals.com Figure 1d: IR spectrum mixture of IBU, MTZ, and HPMC K4M Figure 1e: IR spectrum of IBU, MTZ, and Sod.CMC Drug loaded periodontal films casted using aqueous dispersions of either Ethyl cellulose or Sodium CMC were opaque and with rough texture while those prepared using either Citation: Vaishali Potnis et al. Ijppr.Human, 2016; Vol. 7 (2): 41-56. 50