1. INTRODUCTION:

Oral drug administration has been one of the most convenient and widely accepted routes of delivery for most therapeutic agents. Traditionally, oral dosage forms refer to tablets, capsules, and liquid preparations taken orally, swallowed, and transiting the gastrointestinal tract (GIT) for post buccal absorption. However, some undesirable physiological properties of the gastrointestinal (GI) tract limit the feasibility of administration of some molecules by this route (i.e., proteins, polypeptides, etc.). The relatively poor absorption, presence of abundant digestive enzymes in the GI lumen and epithelium, post absorption efflux (i.e., by P-glycoprotein, etc.), and first-pass metabolism by the hepatic enzymes and subsequent elimination, limit the ability of many drugs to reach therapeutic levels by this route.^{1, 9}

The major disorders of the oral cavity are toothache, periodontal diseases, dental caries, bacterial and fungal infections and aphthous stomatitis. Candidiasis, commonly called yeast infection or thrush, occurs commonly in the mucous membranes of the mouth. Oral thrush refers to temporary candidiasis in the mouth of babies, whilst if occurring in the mouth or throat of adults, is a fungal infection (mycosis) of any of the Candida species, of which Candida albicans is the most common.²C. albicans is a resident commensal fungus of the normal oral flora.³

Oral thrush occurs in ~9.5 million people worldwide based on ~90% of HIV/AIDS patients⁵ not taking but needing anti-retroviral therapy, estimated at by the WHO in 2009.⁶ In Europe, North America, Mexico, Thailand, and South Africa, oral candidiasis is observed in 50–67 % of children, and is considered the most common oral manifestation of HIV infection. Oropharyngeal candidiasis occurs in up to 55 % of people with HIV infection and in over 90 % of those with advanced disease.²Oral candidiasis (OC) is also a frequent side effect of inhaled corticosteroids (iCSTs).⁷

Chronic antimycotic therapy in high doses is undesirable for treatment of oral infections due to potential side effects. Therefore, to minimize these adverse effects and the ominous risk of drug resistance, topical therapy should be considered the first-line candidate for the treatment of oral and pharyngeal candidiasis. ³

As the conven-tional formulations like mouth paints, rinses, troches, oral gels, etc., have been found to be incapable of maintaining the salivary concentration of drugs for a prolonged period of time , it was decided to develop a muco-adhesive buccal formulation which could be stuck on to the inner surface of the cheek and which would maintain the salivary concentration of the drug above the 'minimum inhibitory concentration' (MIC) against Candida albicans (2 mcg/ml) for a prolonged period of time.⁴

Clotrimazole (CTZ) is the first line broad-spectrum antifungal agent that has been extensively used for the prophylaxis and treatment of oral and vaginal candidiasis. It is known to be very effective locally and only a small percentage of the drug applied to the oral mucosa can be detected in the serum or urine. Presently, for the topical treatment for oral candidiasis, clotrimazole (CTZ) is available only in the form of troche (a common brand is Mycelex® troche, Miles Pharma, USA) which is required to be taken three to five times a day for 14days.³ In case of conventional formulation of clotrimazole some people may experience gastrointestinal (digestive) effects such as diarrhea, abdominal pain, nausea and vomiting.⁷

Therefore, there is a need for the development of CTZ buccal bio-adhesive controlled release formulation. Recent years have seen an increasing interest in the development of novel buccal bio-adhesive dosage forms. These are useful for both for systemic delivery of drugs, as well as for local targeting of drugs to a particular region in the body. A wide range of polymers of synthetic, semi synthetic and natural origin like polycarbophil, sodiumcarboxymethylcellulose (SCMC), hydroxypropylmethyl cellulose, chitosan and xanthan gum have been described for the formulation of bio adhesive systems but none of these polymer possess all the characteristics of an ideal polymer (nontoxic, nonirritant, strong non covalent adhesion, sustained release, stable and cheap) for a bio-adhesive drug delivery system. Therefore, the present study was aimed to design and develop buccal bio-adhesive novel buccoadhesive application of CTZ which would maintain the salivary concentration of the drug above the MIC against C. albicans for a prolonged period of time.³

Bio-adhesive formulations for buccal administration are used to release a drug either for local or systemic activity. Local activity can be investigated either for the treatment of mycosic infection, aphthous stomatitis and peridontal diseases, or for obtaining local anaesthesia. Systemic activity is researched through buccal administration, because of the possibility for the drug to escape the hepatic first-pass effect. This route can therefore be used for the administration of drugs such as propranolol, testosterone and peptides.⁸

2. MATERIAL AND METHOD:

2.1 Materials

CLT was a gift sample from Amoli organic ltd, Mumbai, Eudragit RLPO was obtained as a gift sample from Evonik Degussa India Pvt. Ltd., Carbomer 934(Loba chemie Pvt.Ltd.) and PVP K30(Research lab fine chem. Industries) were obtained from commercial sources. Other solvents and materials used in study were of reagent grade.

2.2 Preformulation

Spectral analysis of clotrimazole:

a. UV spectroscopy

The UV spectrum of clotrimazole indicates the value for λ_maxis 227nm (Shimadzu UV1800).

b. Infrared spectroscopy:

2.3 Formulation of NBF

The NBF were prepared using compositions as given in Table no. 1. The various components in each formula were mixed by rolling the bag containing the polymer and drug mixture. The mixture (100mg) was then compressed using a 13 mm diameter die on an KBR press (Techno search instruments) using a compression force of 200kg/cm² and a compression time of 5min. The NBF were 0.600-0.796mm thick, depending upon the polymer combination used.

Table No. 1: Formulation table

Formulation	F1	F2	F3	F4	F5	F6
Clotrimazole	10	10	10	10	10	10
Caarbomer 934	20	40	60	80	30	20
PVP K30	60	40	20	0	30	20
Eudragit RLPO	10	10	10	10	30	50
Total wt.	100	100	100	100	100	100

2.4 Evaluation

Thickness measurements¹⁰

The thickness of each NBF was measured at five different locations (centre and four corners) using an electronic digital micrometer (Mitutoyo Co., Japan). Data are represented as a mean±S.D. of five replicate determinations.

Diameter measurements¹⁰

The diameter of ten NBF was measured by using an electronic digital micrometer (Mitutoyo Co., Japan). Data are represented as a mean±S.D. of ten replicate determinations.

Weight Variation test²

Ten NBF were taken and weighed individually (Elder). Average weight was calculated and standard deviation was computed.

Drug Content Uniformity²

Randomly five NBF from each batch were weighed accurately and powdered; the equivalent weight of 10 mg of Clotrimazole was taken and made the volume up to 10.0mL with ethanol in 10.0 mL volumetric flask and kept aside with constant shaking for 24 h to extract the total drug present in the NBF. Then the solution was filtered and the volume was made with ethanol and analyzed for drug content at λmax of 227 nm(Shimadzu UV1800). Data are represented as a mean±S.D. of five replicate determinations.

Swelling study¹¹

The swelling study was carried out by using 1% agar gel plate method. After determination of the original weight, the samples were allowed to swell on the surface of agar plate kept in an incubator maintained at 37⁰C. Increase in the weight of the NBF (n = 5) was determined at preset time intervals (1–8 h). The percent swelling, %S, was calculated using the following equation:

%S = (Xt – Xo/Xo) × 100

Where Xt is the weight of the swollen NBF after time t, and Xo is the original NBF weight at zero time.

Surface pH evaluation⁴

The surface pH of the NBF of each formulation was determined in order to investigate the possibility of in vivo side effects. As an acidic or alkaline pH may cause irritation to buccal mucosa, it was our attempt to keep the surface pH as close to neutral as possible. The NBF were first allowed to swell by keeping them in contact with 1.0mL of distilled water (pH 6.5±0.05) for two hours in glass tubes. The surface pH was then noted by bringing glass micro electrode near the surface of NBF and allowing it to equilibrate for 1 min. Data are represented as a mean±S.D. of five replicate determinations.

Measurement of bio-adhesive strength⁴

Bio-adhesive strength of the NBF was measured on a modified physical balance using the method described by Gupta et al. The method used goat buccal mucosa as the model mucosal membrane and buffer pH 6.8 as the moistening fluid. The weight in g, required to detach the NBF from the mucosal surface gave the measure of bio-adhesive strength.

In vitro drug release studies ^{2, 14}

The release rate of clotrimazole from NBF was determined using USP Dissolution Testing Apparatus II (Paddle type). The dissolution test was done using 500 ml of 6.8 pH buffer+1% Tween 80 solution at 37 ± 0.5⁰C and at 50 rpm. A sample (10 ml) of the solution was withdrawn from the dissolution apparatus after every one hour the samples were replaced with fresh dissolution medium. The samples were filtered through Membrane filter no. 0.45µm. Absorbance of these solutions was measured at 227 nm using UV spectrophotometer Shimadzu 1800. Cumulative percentage drug release was calculated. Data are represented as a mean±S.D. of six replicate determinations.

Drug-excipients interaction studies^{12, 13}

Assessment of possible incompatibilities between an active drug substance and different excipients plays an important part of the formulation stage during the development of solid dosage form. Infra Red Spectrum (IR) can be used to assess possible drug excipients interaction. IR Study was performed for drug to determine the purity and for optimize formulation to determine any drug and excipients interaction.

3. RESULTS:

3.1 Thickness and diameter measurement:

The minimum thickness for NBF was found to be 0.600±0.012 and maximum will be 0.796±0.013mm, while the diameter of NBF varies from 12.99±0.011 to 13.01±0.008mm. The results are also represented in table no. 2.

3.2 Weight variation:

The weight of the formulated NBF of clotrimazole (F1 to F6) was found to be uniform with low standard deviation values from 98.8±0.919mg to maximum 99.1±0.994mg. The results are also represented in table no. 2.

3.3 Content uniformity:

The maximum % drug content for NBF was found to be 101.79±1.79% and minimum %drug content was found to be 95.83±0.421% .The results are also presented in fig. no. 5 and in table no. 2.

Fig no. 5: % Drug content

3.4 Surface pH:

The surface pH of all the formulation was found near to be neutral pH range and hence these formulations should not cause any irritation in the buccal cavity. The results are also represented in table no. 2.

Table no. 2

Sr. No.	Formulations	Thickness (mm)*	Diameter (mm)	Weight (mg)	% drug content	pH of formulations
1	F1	0.796±0.013	12.99±0.012	98.8±0.919	95.833±0.421	6.46±0.055
2	F2	0.700±0.01	13.00±0.013	98.9±0.876	97.381±0.679	6.32±0.045
3	F3	0.696±0.011	13.00±0.012	99.1±0.738	101.786±1.786	6.24±0.055
4	F4	0.606±0.009	13.01±0.008	98.9±0.876	100.595±1.882	5.82±0.048
5	F5	0.600±0.01	12.99±0.009	99.0±0.943	98.095±0.498	6.06±0.055
6	F6	0.600±0.012	12.99±0.011	99.1±0.994	101.905±0.978	6.24±0.055

3.5 Swelling index

Appropriate swelling behavior of NBF is essential for uniform and prolonged release of the drug and effective mucoadhesion. The %swelling index of all formulation was found to be in the range of 170% to 352% for 8h.

Fig No. 6: % swelling index

3.6 In-vitro drug release study:

These studies were primarily aimed at finding out whether the release patterns of drug from the formulation are satisfactory. Drug release tests were carried out for all six formulations (F1-F6). From drug release profile, it was observed that formulation F3 containing Carbomer934 as mucoadhesive agent, PVP K 30 as a hydrophilic polymer and Eudragit RLPO as sustained release polymer show maximum drug release of 94% in 5 hrs.

Fig No. 7: Comparative release data of F1-F6 formulation

3.7 In vitro bio-adhesive strength

Results of invitro bio adhesion of NBF were obtained. All the formulation was chosen to evaluate the bio-adhesive properties. In vitro bio adhesive strength and In vitro bio adhesive force for the F3 were found to be 6.788±0.070and 12.537±0.129 respectively.

Fig. N0. 8: Mucoadhesive strength (gm) measurement

3.8 IR studies

To study any interaction between drug and polymers used in the preparation of NBF, IR spectroscopy was carried out for the test preparations. The IR spectra of the NBF showed the same absorption bands as the physical mixtures, illustrating absence of interaction between clotrimazole and polymer. It presumably suggests that the drug molecule is present in an unchanged state in the formulation.IR spectra of Clotrimazole and mixture of clotrimazole, carbomer 934, PVP K30, Eudragit RLPO is shown in fig No. 1 to 4.

4. DISCUSSION:

The present study was conducted with a view to formulate and evaluate the novel bucco-adhesive formulation for buccal drug delivery system. Clotrimazole was used as a model drug on the basis of their physiochemical properties such as half-life, optimum oil/water partition coefficient, molecular weight and dose make it suitable candidate for buccal drug delivery system.

4.1 Preformulation study:

The procured sample of Clotrimazole was tested for its identification such as melting point, solubility, U.V.determination; results of all these tests were in compliance with specifications of I.P. In support of these identification tests, the quality of drug was also confirmed with IR spectral analysis, which showed presence of all prominent peaks and found identical with reference spectrum given in I.P.

4.2 Standard curves of Clotrimazole:

Standard curves of Clotrimazole were prepared separately in phosphate buffer pH 6.8 and in methanol. Clotrimazole followed Beer Lambert’s law in the concentration range of 0.5-6.5μg/ml. The linear correlation present between concentration and absorbance. The calculation of in-vitro drug release and drug content are based on respective standard curve.

4.3 Physical characterizations of Novel bucco-adhesive formulation:

The Novel bucco-adhesive formulation prepared by direct compression method were evaluated for diameter, thickness, weight variation, drug content, pH and % swelling index for all batches (F1 to F6).

4.4 In vitro bio-adhesive study

In vitro bio adhesive studies indicate the F3 and F4 formulation shows prominent mucoadhesive property.

4.5 In-vitro drug release study:

4.6 Drug and excipients interaction study:

The prominent peaks of drug were observed in the IR of formulation also therefore it can conclude that no significant interaction observed between the drug and excipients.

5. REFERENCES:

1. Ghosh Tapash K. and Pfister William R., Drug delivery to the oral cavity, Deker series(CRC press),2005,68-69.

2. Dhake A. S., Shinkara Dattatraya M, Shayleb Somashekar, Patil Sanjay B, Setty Chitral M, Development and evaluation of mucoadhesive tablets of clotrimazole and its βcyclodextrin complex for the treatment of candidiasis International journal of pharmacy and pharmaceutical sciences, 3(3), 2011, 159-164.

3. Singh S., Jain S., Muthu M. S., Tiwari S. and Tilak R., Preparation and Evaluation of Buccal Bioadhesive Films Containing Clotrimazole, AAPS, 9(2), 2008, 660-667.

4. Khanna Rajesh, Agarwal S.P., Ahuja Alka, Preparation and evaluation of bioerodible buccal tablets containing clotrimazole, International Journal of Pharmaceutics, 138, 1996, 67-73.

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9. Alagusundaram M., Chetty C.M., Umasankarai K., Anitha P., Gnanprakash K. and Dhachinamoorthi D., Buccal drug delivery system- an overview, Research J. Pharm and Tech., 2(4), 2009.

10. Perumal V.A., Formulation of monolayered films with drug and polymers of opposing solubilities, International Journal of Pharmaceutics, 358, 2008 184–191.

11. Ismail Fatma A., Mucoadhesive buccal patches of miconazole nitrate: in vitro/in vivo performance and effect of ageing, International Journal of Pharmaceutics, 264, 2003, 1–14.

12. Shojaei Amir H., Buccal Mucosa As A Route For Systemic Drug Delivery: A Review, J Pharm Pharmaceut Sci (www.ualberta.ca/~csps) 1 (1), 1998, 15-30.

13. Patel R, Shardul N; Patel J; Baria A. Arch Pharm Sci and Res, 1 (2), 2009, 212-217.

14. Nakhat PD, Kondawar AA, Babala IB, Rathi LG and Yeole PG. Studies on buccoadhesive tablets of terbutaline sulphate. Indian J Pharm Sci, 69(4), 2007, 505‐10.

Received on 22.06.2012 Modified on 18.07.2012

Research J. Pharm. and Tech. 5(7): July 2012; Page 921-926