INTRDUCTION:

Buccal mucosa is a potential site for the delivery of drugs to the systemic circulation. A drug administered through the buccal mucosa enters directly in the systemic circulation, thereby minimizing the first-pass hepatic metabolism and adverse gastro-intestinal effect.¹ Buccal region of the oral cavity is an attractive target for administration of the drug of choice.² Buccal drug absorption can be promptly terminated in case of toxicity by removing the dosage form from the buccal cavity. It is also possible to administer drugs to patients who cannot be dosed orally to prevent accidental swallowing. Therefore adhesive mucosal dosage forms were suggested for oral delivery.³Buccal mucosa makes a more appropriate choice of site if prolonged drug delivery is desired because buccal site is less permeable than the sublingual site.

Buccal compacts or buccal bioadhesive drug devices designed to remain in contact with buccal mucosa and release the drug over a long period of time in a controlled fashion. In addition, there is excellent acceptability and the drug can be applied, localized and may be removed easily at any time during the treatment period.⁴ Hence buccoadhesive drug delivery systems have been developed basically to increase the retention of drug in the oral cavity.

The route provides intimate contact between a dosage forms and absorbing tissue thereby resulting in high drug concentration in a local area and hence continuous release of drug from the medication towards medium from where it is constantly removed. Such dosage forms are very much useful for the treatment of buccal diseases among which oral candidiasis in one.

Oral candidiasis is the most common, treatable oral mucosal infections seen in persons with human immunodeficiency virus (HIV) infection or acquired immune deficiency syndrome (AIDS).⁵ Oral Candida infections are observed in more than 90% of HIV-positive patients at some time during their disease, particularly in advanced immunosuppression. The most common causative pathogen is Candida albicans.⁶

Ketoconazole (KTZ) is a broad-spectrum imidazole derivative of antifungal agent developed for treatment of human mycotic infections and plays an essential role in the antifungal chemotherapy.⁷ It is weak base with poor water solubility.⁸

In recent years, hydrophilic matrices have attracted considerable attention as sustained drug release devices. Various types of polymers can be used in the hydrophilic matrix and the hydration of these polymers results in the formation of an outer gel layer that controls drugs release. HPMC, the nonionic cellulose ether, is commonly used in the formulation of hydrophilic matrix systems. On the other hand, acrylic acid derivatives Carbopols have also attracted interest in their use in controlled drug delivery.¹

Figure - 1: In-Vitro Bioadhesive strength measurement Test assembly.

Lipophilic drugs, although being well absorbed through oral epithelia, exhibit too low fluxes due to a low chemical potential gradient, which is the driving force for transport.⁹ In this regard, cyclodextrins (CD) are capable of forming inclusion complexes with many drugs by taking up a whole drug molecule, or a part of it, into the cavity of the cyclodextrin molecule. Drug cyclodextrins complexes can improve the clinical usage of drugs by increasing their aqueous solubility, dissolution rate, and pharmaceutical availability.¹

Solubilization of poorly water-soluble drugs by complexation with cyclodextrins and then delivery via the buccal or sublingual mucosa may be advantageous for increasing drug absorption. However, the buccal route of administration using drug- cyclodextrin complexes has not been evaluated extensively.¹

MATERIALS AND METHODS:

Materials:

Ketoconazole was generously gifted by Torrent Pharmaceuticals Ltd., Ahmadabad, India. β- Cyclodextrin was gifted by SA Pharmachem Pvt. Ltd., Mumbai. Hydroxypropyl methylcellulose K4M was obtained as a Gift Sample from Colorcon Asia Limited, Goa. Carbopol 934P was provided by Central Drug House India, Ethyl cellulose (10cps), Lactose DC was purchased from SD Fine Chem. Mumbai, India. All the reagents used for the study were of Analytical grade.

Figure- 2: In-Vitro Bioadhesion time measurement test assembly.

Methods:

Phase Solubility Analysis of Ketoconazole with β- Cyclodextrin:

Solubility measurement was carried out according to method described by Higuchi and Connors in phosphate buffer pH 6. For phase solubility studies of KTZ, an excess amount of drug (30 mg) was added to 10 ml portions of phosphate buffer pH 6, each containing variable amount of b-CD such as 0, 1.1, 2.2, 3.3 and 4.4 x 10^-3 moles/liter in vials. All the above solutions with variable amount of b-CD were shaken for 7 days at room temp. After equilibrium had been reached (7 days), the solutions were filtered and their absorbance was noted at 225 nm.⁷ The solubility of the KTZ in every b-CD solution was calculated and phase solubility diagram was drawn between the solubility of Ketoconazole and different concentrations of b- cyclodextrin.

The stability constant of KTZ b-CD complex was calculated using Higuchi and Connor’s equation.¹⁰

Slope

K_(1:1) =

S₀ (1 – slope)

Were, S₀ = Intrinsic solubility of KTZ in aqueous complexation media (phosphate buffer) “slope” was calculated from phase solubility diagram.

Preparation of inclusion complexes of Ketoconazole with β-Cyclodextrin in 1:1 molar ratio by Kneading Method ^{7, 11}:

In this method, a solution of distilled water and ethanol (1:1) was used as moistening agent. Ketoconazole with b-CD in 1:1 molar ratios were taken. First cyclodextrin is added to the mortar, small quantity of moistening agent is added while triturating to get slurry like consistency. Then slowly drug is incorporated into the slurry and trituration is further continued for one hour consistency of pastes was maintained using the appropriate quantities of moistening agent. This slurry was finally dried in a hot air oven at 40ºC for 24 hours. Finally dried complexes were then passed through sieve no. 100 and stored in decicator over fused calcium chloride until further use.

Figure- 3: Phase solubility diagram of Ketoconazole complex with β-Cyclodextrin.

R- Value = 0.99881; B- Value (slope) = 0.522

Stability constant K _(1:1) = = 6461.8355 M^-1

Formulation of Ketoconazole buccoadhesive tablets:

In this work, direct compression method has been employed to prepare buccal tablet with HPMC K4M and Carbopol 934P as polymers. For one tablet accurately weighted 32 mg “Ketoconazole inclusion complex powder” which is equivalent to 10 mg of Ketoconazole was used in the formulation. The powder blends of various proportions were compressed into tablets of diameter 8mm on Clit pilot press 10 Station machine. Using stainless steel flat surface dies and punches by maintaining individual tablet weight constant at 150 mg. The compression force was maintained in such a way that the hardness of resulting tablets ranged between 4 – 5 Kg/cm². Ethyl cellulose solution (10%w/v in ethanol) was cast on the prepared tablets from three sides as an impermeable backing layer which was aimed to provide unidirectional drug release.¹²

Table-1: Preliminary Trial Formulations

Ingredients	T₁	T₂	T₃	T₄	T₅	T₆	T₇
KTZ inclusion complex powder	32	32	32	32	32	32	32
HPMC K4M	10	20	-	-	30	20	10
Carbopol 934P	-	-	10	20	10	20	30
Lactose DC	106	96	106	96	76	76	76
Magnesium stearate	1	1	1	1	1	1	1
Talc	1	1	1	1	1	1	1

Evaluation of Ketoconazole Buccoadhesive tablets:

Physical evaluation:

Twenty tablets from each batch were evaluated for uniformity in tablet weight and thickness. Three tablets from each batch were evaluated for Hardness test using Monsanto hardness tester. Ten tablets from each batch were evaluated for Friability test using Electrolab, USP EF-2 Friability Tester.

Uniformity of drug content:

Five tablets were powdered in a glass mortar and the powder equivalent to 50 mg of drug was placed in a stoppered 100 ml conical flask. The drug was extracted with 40 ml methanol with vigorous shaking on a mechanical gyratory shaker (100 rpm) for 1 hour. Then heated on water bath with occasional shaking for 30 minutes and filtered into 50 ml volumetric flask through cotton wool and filtrate was made upto the mark by passing more methanol through filter, further appropriate dilution were made and absorbance was measured at 225nm against blank (methanol).

Evaluation of Bioadhesion ¹³:

In-Vitro Bioadhesive strength measurement Test:

(Methods Based on Measurement of Adhesion Strength)

A modified balance method was used for determining the mucoadhesive strength. An apparatus designed for determination of mucoadhesive bond strength was used. Schematic representation of bioadhesion test assembly is shown in figure-1

Bioadhesive strength expressed in Newton, required for detachment of the tablet from the mucosa was determined using the fresh sheep buccal mucosa as mucosal substrate. The working of the fabricated bioadhesion test apparatus was based on the principle of double beam physical balance.

Measurement of adhesion force ³:

The two sides of the balance were balanced with a 5 g weight on the right hand side. Fresh sheep buccal mucosa was obtained from a local slaughterhouse and used within 2 hours of slaughter. The mucosal membrane was separated by removing the underlying fat and loose tissues, washed with distilled water and then with phosphate buffer pH 6.8 at 37^oC.

A piece of buccal mucosa was tied with the mucosal side upwards using thread over the protrusion in the Teflon block. The block was then lowered into the glass beaker which was then filled with phosphate buffer pH 6.8 kept at 37 ± 1 ^oC to keep mucosal membrane moist. This was then kept below the left-hand setup of the balance. The tablet to be tested for Mucoadhesion was then stuck with a little moisture, on to the cylinder [E] hanging on the left-hand side. The balance beam was raised. The 5 g weight on the right pan was removed. This lowered the Teflon cylinder along with the tablet over the mucosa, with a force of 5 g.

Figure-4: Dissolution profiles of Ketoconazole and Ketoconazole - β-cyclodextrin Complex.

The balance was kept in this position for 3 minutes and then the weights were increased gradually on the right pan, till the tablet separated from the mucosal surface. The excess weights on the pan i.e. total weight minus 5 g, is the force required to separate the tablet from the mucosa. This study was performed for each tablet formulation in triplicate.

In-Vitro Bioadhesion time measurement test ¹³:

The fresh sheep buccal mucosa was fixed on the side of the beaker with glue. Before addition of the buffer, the tablet was attached to sheep buccal mucosa by applying light force with finger tip for 20 seconds. The beaker was then filled with 800ml of phosphate buffer pH 6.8 and was kept at 37⁰C. A stirring rate of 150 rpm was used to simulate buccal and saliva movement. The attachment of tablet was monitored until 12 hours. The time for tablet to detach from the sheep buccal mucosa was recorded as the Mucoadhesion time. The bioadhesion time measurement test assembly is shown in figure -2

The tablet hydration study ¹⁴:

The tablet hydration studies were carried out in Petri dishes with pH 6.8 phosphate buffer. Periodically, the tablets were withdrawn from the beaker and weighed on electronic balance after removal of surface water by light blotting with a lab tissue. The sampling times of hydration studies were 0.5, 1, 2, 4, 6, 8, 12 and 24 hours The rate of hydration was calculated according to the model describing the absorption of liquid into polymeric matrices via diffusion.

Tablet surface P^H evaluation¹⁵:

The surface pH of the tablets was determined in order to investigate the possibility of any side effects, in-vivo. As an acidic or alkaline pH may cause irritation to the buccal mucosa, it was our attempt to keep the surface pH as close to neutral as possible.

The tablets were first allowed to swell by keeping them in contact with 1 ml of distilled water (pH 6.5±0.05) for 2 hours in glass tubes. The surface pH was then noted by bringing glass micro electrode near the surface of tablet and allowing it to equilibrate for 1 min. Thereafter surface pH measurements were recorded and shown in Table- 5 and 6.

Table – 2: Factorial design formulations

Ingredients	F₁	F₂	F₃	F₄	F₅	F₆	F₇	F₈	F₉	C1	C2
KTZ inclusion complex powder	32	32	32	32	32	32	32	32	32	32	32
HPMC K4M	10	10	10	20	20	20	30	30	30	15	25
Carbopol 934P	5	10	15	5	10	15	5	10	15	7.5	12.5
Lactose DC	98.5	93.5	88.5	88.5	83.5	78.5	78.5	73.5	68.5	91	76
Magnesium stearate (2%)	3	3	3	3	3	3	3	3	3	3	3
Talc (1%)	1.5	1.5	1.5	1.5	1.5	1.5	1.5	1.5	1.5	1.5	1.5

* (All values are given in mg/tablet)

Table – 3: Factorial design Batches of Ketoconazole BDDS.

Variable	Formulations
Variable	F₁	F₂	F₃	F₄	F₅	F₆	F₇	F₈	F₉	C₁	C₂
X₁	-1	-1	-1	0	0	0	+1	+1	+1	-0.5	+0.5
X₂	-1	0	+1	-1	0	+1	-1	0	+1	-0.5	+0.5

Figure- 5: Bioadhesive strength measurement of Trial Formulations

In-vitro Dissolution studies^{3, 16}:

In vitro dissolution studies of buccal tablets of Ketoconazole were carried out in USP TDT 06P tablet dissolution test apparatus-II (Electrolab), employing a paddle stirrer at 50 rpm using 900ml of pH 6.8 Phosphate buffer + 0.5% w/v SLS solution at 37 ±0.5^oC as dissolution medium. One tablet was used in each test. The tablets were supposed to release drug from one side only; therefore an impermeable backing membrane side of tablet was fixed to a 2×2 cm glass slide with a solution of cyanoacrylate adhesive. Then it was placed in dissolution apparatus. At predetermined time intervals 5ml of the samples were withdrawn by means of a syringe fitted with a pre filter. The volume withdrawn at each interval was replaced with same quantity of fresh dissolution medium maintained at 37±0.5°C. The samples were analyzed for drug release by measuring the absorbance at 225 nrn using UV-Visible spectrophotometer after suitable dilutions.

In-vitro drug permeation through Sheep buccal mucosal membrane:

The Keshary- Chein cell with receptor side volume of 25 ml and diffusion area of 4.90 cm² was used. Fresh Sheep buccal mucosa was mounted between donor and receptor compartment of diffusion cell.

Optimization using Factorial Design Method.^17-19:

Optimization has been done by using 3² full factorial designs, where amount of HPMC K4M (X₁) and amount of Carbopol 934P (X₂) were taken as independent variables and t_50%, t_70% and swelling index taken as dependent variables. Step-wise backward linear regression analysis was used to develop polynomial equations for the dependent variables t_50%, t_70% and swelling index values by using PCP Disso V3 software. The validity of the developed polynomial regression equations was verified by preparing two check point formulations (C₁ and C₂), as shown in Tables- 3 and 4.

Stability studies:

Stability studies were performed at a temperature of 40⁰C and 75 % RH, over a period of three months (90 days) on the promising buccal tablets of ketoconazole formulation F₁. Sufficient number of tablets (15) were packed in amber colored screw capped bottles and kept in stability chamber maintained at 40⁰±1⁰C and 75 % RH. Samples were taken at 15 days intervals for drug content estimation upto three months.

RESULTS AND DISCUSSION:

Complexation of Ketoconazole with β-Cyclodextrins:

A. Phase solubility analysis of Ketoconazole with β- cyclodextrins-

The phase solubility diagram obtained for KTZ- β-CD is shown in Figure-5. This plot indicates linear rise in the solubility of the drug as a function of β- cyclodextrin concentration. Hence, the solubility plot of Ketoconazole in the presence of β- cyclodextrin can be classified as A_L type

The linear host – guest complexation plot with slope less than 1 also suggested formation of 1:1 (Ketoconazole: β-cyclodextrin) complex with β-cyclodextrin. The apparent solubility constant, K _(1:1), obtained from the slope of linear portion of phase solubility plot was 6461.8355 M^-1. These values suggest good stability of Ketoconazole - β-cyclodextrin complexes at 1:1 molar ratios.

Table- 4: Coded values and actual values for the independent Variables.

Coded values	Actual Values (mg)
Coded values	X₁ [ HPMC K4M ]	X₂ [ Carbopol 934P ]
-1	10	5
0	20	10
+1	30	15
-0.5	15	7.5
+0.5	25	12.5

Evaluation of Ketoconazole buccoadhesive tablets:

Table -5: Evaluation of Trial formulations

Formula-tion code	Mean Hardness Kg/cm²	Thickness (mm)	Friability % w/w	Average weight (mg)	Mean drug content %±SD	SI ± SD (after 6 hrs)	Mucoadhesion (time of detachment hrs)	Tablet Surface P^H
T₁	4.26	2.9	0.52	147.86	98.65 ± 0.76	20.08	1	7.12
T₂	4.62	2.9	0.64	148.37	95.36 ± 1.42	45.64	3	7.24
T₃	4.65	3.1	0.40	152.47	97.03 ± 0.55	21.17	>12	6.88
T₄	4.35	2.8	0.26	145.80	96.71 ± 3.07	51.17	> 12	6.17
T5	4.14	2.9	0.27	144.64	97.85 ± 2.23	97.83	> 12	6.32
T₆	4.68	2.8	0.39	152.18	96.92 ± 1.77	112.24	> 12	6.35
T₇	4.56	2.9	0.53	146.54	97.69 ± 2.05	114.61	10	6.04

* Each value represents the mean ± SD of 3 determinations

Table – 6: Evaluation of factorial design formulations.

Formula-tion code	Mean Hardness Kg/cm²	Thickness (mm)	Friability % w/w	Average weight (mg)	Mean drug content %±SD	SI ± SD (after 6 hrs)	Mucoadhesion (time of detachment hrs)	Tablet Suface P^H
F₁	4.52	3.1	0.51	149.58	99.52 ± 1.60	22.74	>12	7.02
F₂	4.68	2.8	0.64	145.37	98.12 ± 1.08	33.36	>12	6.71
F₃	4.32	3.2	0.68	152.62	94.90 ± 0.81	48.35	>12	6.12
F₄	4.46	3	0.52	148.38	95.77 ± 0.08	56.52	> 12	6.11
F₅	4.66	3.1	0.61	149.89	101.14 ± 1.35	63.21	> 12	6.58
F₆	4.51	3.2	0.67	151.12	98.44 ± 0.68	75.56	> 12	6.21
F₇	4.71	3.3	0.52	154.98	96.09 ± 2.13	82.30	>12	6.85
F₈	4.64	2.8	0.51	144.56	97.18 ± 0.87	89.48	>12	6.65
F₉	4.31	2.9	0.62	147.94	95.87 ± 0.15	93.86	> 12	6.16
C₁	4.21	3.2	0.63	155.56	96.33 ± 1.88	45.12	>12	6.81
C₂	4.69	3.1	0.67	154.04	96.68 ± 2.56	81.78	> 12	6.34

* Each value represents the mean ± SD of 3 determinations

Figure- 6: Bioadhesive strength measurement of Factorial design Formulations

Figure -7: Cumulative Percent Drug Released Vs Time Plots (Zero Order) of formulations T₁, T₂, T₃, T₄, T _5,T₆ and T₇

Preparation of Ketoconazole – β-Cyclodextrin inclusion complex:

From the phase solubility analysis data, the molar ratios of complexes were determined for Ketoconazole and β-Cyclodextrin. The inclusion complex of ketoconazole was prepared in 1:1M ratios with β-Cyclodextrin. These complexes were prepared using kneading method. The prepared complexes were free flowing and off white in colour.

Table - 7: Dissolution and Swelling Index Parameter for the Trial Formulations

Formulation code	t_50% (hours)	T_70% (hours)	SI (after 6 hours)	Cumulative percent drug release in 10 hours
T₁	0.4	1	20.08	98.63
T₂	0.5	2.7	45.64	99.45
T₃	2.1	4.1	21.17	87.16
T₄	3.2	4.9	51.17	96.61
T₅	6.3	8.9	97.83	74.93
T₆	7.2	9.9	112.24	71.88
T₇	6	8.4	114.61	77.37

Invitro dissolution studies of Ketoconazole and complex:

The KTZ complexes with β-CD presented better dissolution performance over pure drug in an In-vitro test. According to these results, inclusion complexes prepared using with β- Cyclodextrin, at 1:1M ratio showed about 100 % drug release in 80 min shown in figure-4.

Physicochemical Properties: It could be observed that all the prepared tablets fulfill the IP requirements for Physicochemical Properties. The hardness of prepared buccal tablets was found to be in the range of 4.14 to 4.71 kg/cm². Thickness was in the range of 2.8 to 3.3mm. The friability of all tablets was less than 1% i.e. in the range of 0.26 to 0.68 %. The percentage deviation from mean weights of all the batches of tablets was found to be within the prescribed limits as per IP. The low values in standard deviation indicates uniform drug content in all the batches prepared as observed from data table given in table 5 and 6.

Table – 8: Dissolution and Swelling Index Parameter for 3² full factorial Design Batches

Formulation code	Variable level in coded form		t_50%(hours)	t_70%(hours)	SI (after 6 hours)	Cumulative percent drug release in 10 hours
Formulation code	X₁	X₂	t_50%(hours)	t_70%(hours)	SI (after 6 hours)	Cumulative percent drug release in 10 hours
F₁	-1	-1	3.9	5.4	22.74	99.41
F₂	-1	0	4.9	6.9	33.36	91.87
F₃	-1	+1	5.8	8.2	48.35	83.95
F₄	0	-1	6.1	8.5	56.52	74.43
F₅	0	0	6	8.5	63.21	73.28
F₆	0	+1	6.3	8.9	75.56	70.02
F₇	1	-1	6.4	9	82.30	70.50
F₈	1	0	6.6	9.2	89.48	69.01
F₉	1	+1	7.6	10.4* (extrapol.)	93.86	65.18
C₁	-0.5	-0.5	5.4	7.9	45.12	75.07
C₂	+0.5	+0.5	6.9	9.9* (extrapol.)	81.78	64.88

C₁, C₂ check point batches, t_50%, t_70%,swelling index analyzed by matrix model fitting using PCP disso V3 Software

Figure -8: Cumulative Percent Drug Released Vs Time for F1,F2,F3,F4,F5,F6,F7,F8,F9,C1 and C2 of factorial formulations..

Table -9: Kinetic Data of Trial Formulations

FORMULATION	CORRELATION COEFFICIENT [R]
FORMULATION	Zero Order	First Order	Higuchi’s Equation	Peppas Equation
T₁	0.9112	-0.9486	0.9347	0.9575
T₂	0.9571	-0.9540	0.9862	0.9955
T₃	0.9956	-0.9793	0.9928	0.9938
T₄	0.9714	-0.9878	0.9881	0.9901
T₅	0.9902	-0.9770	0.9865	0.9645
T₆	0.9892	-0.9645	0.9788	0.9637
T₇	0.9823	-0.9933	0.9971	0.9903

Mucoadhesion time of tablets increases with increase in polymer content. Mucoadhesion test was performed using sheep buccal tissue. The time for tablet to detach from buccal tissue was recorded as mucoadhesion time. Formulations containing polymer HPMC alone like T₁, T₂ exhibited less mucoadhesion time (1 to 3 hrs) but the formulations containing carbopol alone and along with HPMC like T₃,T₄, T₅,T₆,T₇ and all the factorial formulations i.e. F₁ to F₉ exhibited mucoadhesion time more than 12 hours as shown in table 5 and 6.

Bioadhesive Strength Measurement:

Bioadhesive Strength Measurement of tablets indicated that the bioadhesive strength was proportional to carbopol content. The mean bioadhesive strength values after 3 min of contact time was 0.2934 N for formulation F₁.The values of bioadhesive strength Figure-5 and 6 were decreased in the following order:

T₇>F₉>T₆>T₄>F₆>C₂>F₃>T₅>C₁>F₈>F₅>T₃>F₂>F₇>F₄>F₁>T₂>T₁. Therefore, increasing carbopol concentration increases the bioadhesion. This increase in the bioadhesion could be due to the formation of secondary mucoadhesive bonds with mucin because of rapid swelling and interpenetration of the polymer chains in the interfacial region, while other polymers undergo only superficial bioadhesion. The peak detachment force was considered to be dependent on the formation of hydrogen bonds between the functional groups of the bioadhesive and the mucus. HPMC alone had poor adhesive properties, but when used in combination with Carbopol, its overall adhesion was increased. Very strong bioadhesion could damage the epithelial lining of buccal mucosa.

In-vitro water uptake studies

In-vitro water uptake studies are of great significance as variation in water content causes a significant variation in mechanical properties of formulations. The capacity of the formulation to take up water is an important intrinsic parameter of the polymeric system in consideration to the release of the drug on the mucosal surface. Water absorbing capacity of system (SI after 6 hours.) decreased in the following order T₇> T₆> T₅> F₉> F₈>F₇>C₂>F₆>F₅>F₄>T₄>F₃> T₂ >C₁> F₂ >F₁>T₃> T₁;

Figure -9: Cumulative Percent Drug permeated across sheep buccal mucosa Vs Time Plots (Zero Order) of formulations F₁

Table - 10: Kinetic Data of Factorial Formulations

FORMULATION	CORRELATION COEFFICIENT [R]
FORMULATION	Zero Order	First Order	Higuchi’s Equation	Peppas Equation
F₁	0.9959	-0.9006	0.9791	0.9812
F₂	0.9829	-0.9561	0.9844	0.9813
F₃	0.9960	-0.9655	0.9865	0.9962
F₄	0.9843	-0.9935	0.9857	0.9893
F₅	0.9830	-0.9955	0.9904	0.9965
F₆	0.9865	-0.9910	0.9895	0.9863
F₇	0.9903	-0.9849	0.9716	0.9741
F₈	0.9915	-0.9877	0.9801	0.9887
F₉	0.9916	-0.9758	0.9841	0.9748
C₁	0.9836	-0.9793	0.9846	0.9777
C₂	0.9948	-0.9886	0.9781	0.9830
F₁in Permeation study	0.9847	-0.9885	0.9921	0.9868

Table-11 Predicted Values And Observed Values

Formulations	Predicted values (hours)			Observed values (hours)
Formulations	t_50%	t_70%	Swelling index (after 6 hours)	t_50%	t_70%	Swelling index (after 6 hours)
C₁	5.12	7.27	44.70	5.4	7.9	45.12
C₂	6.78	9.39	80.93	6.9	9.9	81.78

The Surface pH:

The Surface pH of all formulations was found to be within ±1 units of neutral pH hence these formulations should not cause any irritation in buccal cavity.

In vitro drug release study

From the figure 7, it is evident that as the proportion of polymers in the formulation increases, cumulative percent drug released was found to be reduced. Among the seven trial batches, formulation T₁ to T₄ have released 87 to 100% drug in 10 hours, whereas T₅ to T₇ formulations have released 70 to 75% drug in 10 hours. A higher diffusive flux develops as a consequence of the higher Solubilization rate operated by β-CD, which increases the amount of mobile species. Both these effects result in an enhanced release rate of drug.It indicates that controlled release of drug can be obtained with increased in amount of polymers (HPMC K4M and carbopol 934P).

In seven trail formulations, T₅ formulation has shown promising dissolution parameters (t_50%=6.3 hours, t_70%=8.9 hours) and good mucoadhesion time (> 12 hours). Based on the composition of T₅ formulation, we have fixed the constraints for the level of independent variables (X₁ and X₂) i.e. 10 to 30 mg for HPMC K4M (X₁) and 5 to 15 mg for carbopol 934P (X₂) in designing formulation of 3² full factorial design.

Table - 12: Drug content Data of Stability Formulation (F₁)

Trial No.	1^st Day (%)	15^th Day (%)	30^th day (%)	45^thDay (%)	90^th Day (%)
I	99.32	98.87	98.51	97.25	95.38
II	101.22	97.80	97.94	97.01	97.2
III	98.02	98.99	98.12	98.5	96.65
Mean (X)	99.52	98.55333	98.19	97.58667	96.41
SD	1.60	0.655159	0.291376	0.800021	0.933435

In this 3² full factorial design, two factors (proportion of two polymers) are evaluated, each at three levels and experiments are performed on all nine possible combinations. Dissolution parameters i.e. t_50%, t_70% and swelling index values were selected as dependent variables. Formulation code of the nine batches of factorial formulations along with dissolution parameter values (t_50%, t_70%)swellingindex and cumulative percent drug released in 10 hours were shown in table 7 and 8.

From the data in the above table, it is evident that formulation F₁ has shown highly satisfactory values for dissolution parameters (t_50%=3.9 hours; t_70% = 5.4 hours and swelling index =22.74 hours) and has released approximately 99.41% drug in 10 hours.

Hence, formulation F₁ may be considered as the optimized buccal tablet containing KTZ inclusion complex with β-CD for improved bioavailability.

In-vitro Drug permeation study of formulation F₁:

Based on the results of factorial design of all formulations, the F₁ formulation was selected for In-vitro drug permeation studies. The oral mucosa of sheep resembles that of humans more closely in terms of structure and composition and therefore sheep buccal mucosa was selected for drug permeation studies. The results of drug permeation from buccal tablets through the sheep buccal mucosa reveal that Ketoconazole was released from the formulation and permeated through the sheep buccal membrane and could possibly permeate through the human buccal membrane. The drug permeation was slow and steady (Figure- 9) and 68.47 ± 2.11% of ketoconazole could permeate through the buccal membrane in 10 hours with average flux of 139.28 µg/cm^-2/min.

The results, reported show that KTZ permeation through mucosa was quite good and increased in the presence of β-Cyclodextrin. This effect, in principle, can be attributed to both an increase of driving force for permeation due to the increase of KTZ apparent solubility in the presence of β- CD as well as to enhancing effect of β- CD.

Drug Release Kinetics:

In-vitro drug release data of all the buccal tablet formulations was subjected to goodness of fit test by linear regression analysis according to zero order equations, Higuchi’s and Korsmeyer-Peppas models to ascertain the mechanism of drug release. The results of linear regression analysis including regression coefficients are summarized in tables 9 and 10.

From the data, it can be seen that except formulation T₁, T₂ and T₃ all the trial formulations containing combination of polymers HPMC and Carbopol have displayed zero order release kinetics (‘r’ values in the range of 0.996 to 0.911 ). From Higuchi’s and Peppas data, it is evident that the drug is released by non-Fickian diffusion mechanism except formulation containing HPMC and Carbopol alone. From the kinetic data of factorial formulations (table 10), it is evident that all the formulations have shown drug release by zero order kinetics.

The values of ‘r’ for Higuchi’s equation of factorial formulations range from 0.971 to 0.992. This data reveals that drug release follows non-Fickian diffusion mechanism. This is because as the proportion of polymers in the matrix increased there was an increase in the amount of water uptake and proportionally greater swelling leading to a thicker gel layer. Zero-order release from swellable hydrophilic matrices occurs as a result of constant diffusional pathlengths. When the thickness of the gelled layer and thus the diffusional pathlengths remain constant, zero-order release can be expected, as seen for formulations.

This analysis highlights that the introduction of β-CD in surface eroding controlled release tablets supplies an additive mean to tailor the release by modulating the dissolution rate of the drug in the swollen layer as well as the erosion rate of the matrix.

Development of Polynomial Equations

From the data of dissolution parameters shown in table-33 for factorial formulations F_l to F₉, polynomial equations for three dependent variables (t_50%, t_70% and swelling index) have been derived using “PCP Disso 2000 V3 software’. Polynomial equation for table-32 full factorial designs is:

Y= b₀+b₁ X₁+b₂X₂+b₁₂X₁X₂+b₁₁X₁²+b₂₂X₂² …….....1

Where, Y is dependent variable, b₀ arithmetic mean response of nine batches, and b₁ estimated coefficient for factor X₁. The main effects (X₁ and X₂) represent the average result of changing one factor at a time from its low to high value. The interaction term (X_1,X₂) shows how the response changes when two factors are simultaneously changed. The polynomial terms (X₁² and X₂²) are included to investigate non-linearity.

The equation derived for t_50% is:

Y₁ = 5.9556 + 1. 000 X₁ + 0.6667 X₂……………….2

The equation derived for t_70%is:

Y₂= 8.3333 +1.35X₁ + 0.7667X₂………………….3

The equation derived for swelling index is:

Y₃ = 62.82 + 26.8650X₁ + 9.36 X₂……….………...4

Validity of the above equations was verified by designing two check point formulations (C₁ and C₂) and studying the drug release profiles. The dissolution parameters predicted from the equations derived and those observed from experimental results are summarized in the table 11

The closeness of predicted and observed values for t_50%, t_70% and swelling index values indicates validity of derived equations for the dependent variables. The computer generated response surfaces and contour plots for the dependent variables are shown in figures- 23 to 25. The response surface and contour plot for reveal that it varies in a somewhat linear fashion with the amount of two polymer(s). However, the steeper ascent in the response surface with CP than with HPMC is clearly discernible, indicating that the effect of CP is comparatively more pronounced than that of HPMC

Stability study:

Stability study Stability study was performed on the promising formulation F₁ by storing the samples at 45±1°C for 3 months (90 days). The samples were tested for any changes in physical appearance and drug content at weekly intervals. Drug content studies were performed at the 15 days interval storage. These results indicate that there were no significant changes in drug content of the formulation F₁ shown in table-12.

CONCLUSION:

The β- Cyclodextrin has been suggested to act as penetration enhancers. They enhance the permeation of the drug by carrying the drug through the aqueous barrier towards the surface of the membrane, where the drug passes from the complex into the membrane. Addition of β- CD to the matrix increased the flux by increasing the solubility of ketoconazole, thus improving the diffusible form of the drug species at the tablet membrane interface. Though the complex did not penetrate the membrane, the drug in the complex was in rapid dynamic equilibrium with the “free” drug, thus continuously supplying the drug molecules to the membrane surface in a diffusible form. The role of dissolution enhancement in increasing the rate of delivery is more relevant when the tablet is employed as transmucosal system since, differently from solution conditions; a very limited contribution to delivery derives from matrix erosion.

It has been shown that the incorporation of β-cyclodextrin in buccoadhesive tablets of ketoconazole with hydrophilic matrix intended for the delivery of poorly soluble drugs can be a suitable strategy to optimize the release features of the system while maintaining good bioadhesive properties. Cyclodextrin are responsible for an increase in the erosion rate of the tablet and an improved dissolution of the drug inside the polymeric matrix. This latter effect is the crucial factor, which determines the increase of release rate from the tablets in solution as well as an increase in the amount of Ketoconazole permeated through sheep buccal mucosa. This systems turns to be of great potential as buccal delivery system; in view of the possibility of tailoring release features while maintaining good bioadhesive properties.

ACKNOWLEDGEMENT:

The Authors are thankful to Torrent Pharmaceuticals Ltd., Ahmadabad for providing gift sample of Ketoconazole and SA Pharmachem Pvt. Ltd., Mumbai for providing gift sample of β- Cyclodextrin.

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Received on 27.02.2009 Modified on 12.04.2009

Research J. Pharm. and Tech.2(2): April.-June.2009,;Page 396-404