Study on Factors Influencing Drug Release from Fluconazole-Microsponges Prepared by Suspension Polymerization

John I D’souza*and Harinath N More

Bharati Vidyapeeth College of Pharmacy, Near Chitranagari, Kolhapur 416013, Maharashtra India

* Corresponding Author E-mail: johnsir4u@gmail.com

ABSTRACT

Controlled release of drugs onto the epidermis with assurance that the drug remains primarily localized and does not enter the systemic circulation in significant amounts is an area of research that has only recently been addressed with success. Microsponge delivery system comprised of a polymeric bead having network of pores with an active ingredient held within, was developed to provide controlled release of the active whose final target is skin itself. Macroporous microspheres of styrene (monomer) and divinylbenzene (cross-linker) were prepared by suspension polymerization using sodium polyacrylate and sodium sulphate as dispersion stabilizers and benzoyl peroxide as catalyst. Effect of dispersion stabilizers concentration and speed of rotation on particle size distribution of blank microsponges revealed optimized concentrations of dispersion stabilizers and speed of rotation; sodium polyacrylate: sodium sulphate (0.1:1.5) and 450 respectively to get the particles of size smaller than 30µ m, suitable for topical delivery without grittiness. Average production yield of blank microsponges was 82.63±1.34%. Fluconazole was then loaded in these microsponges by entrapment method. Drug release from microsponges was slowed down with increase in cross-linking density while very little influenced by the particle size. The entire active ingredient was released from the network of pores of the beads into the solvent during wetting test for entrapped microsponges. Drug released from all microspongic gel formulations was best fitted in zero-order kinetic model (correlation coefficient = 0.972).

KEY WORDS Fluconazole, microsponges, cross-linking density, wetting test

INTRODUCTION:

Superficial skin infections caused by fungus are among the most common diseases. Dermatophytes, which usually cause such infections can be classified in three genera; Microsporum, Trichophyton and Epidermophyton. These fungi are inherently not pathogenic, but when the host’s cellular defense or skin function is altered, colonization, infection and disease can occur. 1 Topical treatment is recommended in cases of dermatophytoses by non-inflammatory tinea Fluconazole, a hydrophilic bis-triazole, is an antimycotic with a broad spectrum that has been used in the treatment of dermatophytoses by oral administration.5 It is reliably absorbed after oral administration with systemic bioavailability over 90%. After oral dosing, fluconazole accumulates in eccrine sweat and diffuses rapidly and extensively in the SC. Fluconazole concentration in the skin is higher than in the serum and its elimination from SC is corporis, tinea cruris, tinea faciei, tinea manuum and considerably slower than from serum or plasma.

The tinea pedis.2 Clinical efficacy of topical antifungal therapy depends on the drug ability to penetrate into the stratum corneum (SC) and the duration of treatment.3 The efficacy of topical imidazoles, which are more fungistatic than fungicidal, is related to the highest concentration is achieved in SC and it is still high 1 week after completion of the treatment. This concentration is much higher than MIC for most dermatophytes. The prolonged skin retention of fluconazole has been attributed to its high affinity to SC due to an interaction between duration of treatment and, as a consequence, to the maintenance of the minimal inhibitory concentrations (MIC) for a prolonged time.4

The skin concentrations, higher than fluconazole MIC for most dermatophytes, might be obtained after topical administration of drug from dosage forms applied at clinically relevant doses using a skin model that can be related to human skin.8

The present study aimed to evaluate the controlled topical delivery of fluconazole after application of a fluconazole gels containing entrapped drug in microsponges. The potential of microsponges to control the delivery of candidate drug (fluconazole) was investigated to study factors influencing drug release. In vitro skin penetration studies were carried out in Franz diffusion cells using cellophane membrane.

MATERIAL AND METHODS:

Fluconazole was gift sample from Wallace Pharmaceuticals Ltd., Goa. Styrene and divinyl benzene was kindly gifted from Thermax Ltd., Pune. Carbopol was gifted from Lubrizol (Mumbai, India). Sodium CMC, sodium polyacrylate, sodium sulphate, Benzoyl peroxide were purchased from SD Fine chemicals (Mumbai, India). All other chemicals used were analytical reagent grade.

Preparation of blank microsponges9

Blank microsponges were prepared by suspension polymerization and drug was loaded by entrapment method. Polymerization of styrene-divinylbenzene was conducted in the presence of 2.4g of sodium CMC. The speeds of agitation were 400, 450 and 500 rpm. Two dispersion stabilizers sodium polyacrylate (0.05, 0.1, and 0.15%) and sodium sulphate (0.5, 1, and 1.5%) were incorporated. All formulations were prepared in 600 ml water using 100g of monomer and 1 g of benzoyl peroxide (BPO) as catalyst.

Table 1: Formulation to investigate effect of cross-linking density on drug release

Formu- lations	Weight of Styrene (g)	Weight of DVB (g)	Drug	Cross- linking density (%)
I	41	9	55	10.008
II	35.2	19.8	55	20.016
III	30.7	36	55	30.009
IV	14	36	55	40.032
V	5	45	55	50.04

To produce beads water was added to 1000ml reaction

Preparation of Fluconazole microsponges by entrapment process

Blank microsponges prepared by above procedure, with different cross-linking density and particle size were entrapped by following procedure: 1.5 g of fluconazole was dissolved in 3 g ethyl alcohol. The first half of the drug solution was added to the 1.5 g blank in an amber bottle. Bottle was arranged on a roller mill and mixed for 1 h. The mixture was dried in an oven at 65 °C for 2.5 h. This process was repeated for a second entrapment step for the remaining drug solution and the drying process was held at 50 °C for 24 h.10

Particle size determination 11

Particle size analysis of loaded and unloaded microsponges was performed by optical microscopy. The particle size range and values (d50) were studied expressed for all formulations as mean size range. Cumulative percentage drug release from microsponges of different particle size was plotted against time to study effect of particle size on drug release. Particles larger than 30 µm can impart gritty feeling and hence particles of sizes smaller than 30µ m were used to entrap fluconazole and were used to prepare Carbopol gels, containing 2% of drug.

Cross-linking density

Resiliency of microsponges can be modified by altering amount of cross-linking monomer (DVB). Softer or firmer microsponges, according to the need can be prepared. Effect of cross-linking density, with 5 different levels of DVB, on drug release from microsponges was studied. Theoretical cross-linking density was calculated from following equation:

Weight of DVB x purity of DVB

Cross – linking density = –––––––––––––––––––––––––––––

Total weight of monomer

Purity of DVB was 55.6%.

Determination of loading efficiency and production yield

The production yield of the microparticles can be determined by calculating accurately the initial weight of the raw materials and the last weight of the microsponge 12 obtained. flask and then placed in an 84°C water bath. While stirring the dispersion stabilizers were added to the vessel through the center neck and flask was flushed with nitrogen. When the contents were well dispersed, with continuous stirring, monomer, catalyst and medicament previously mixed were added through center neck. The reaction was allowed to continue for 24h at specified temperature and agitation. Following polymerization beads were filtered, washed with several portions of water and allowed to dry at 70°C.

Effect of cross-linking density, concentration of dispersion stabilizers and speed of rotation (rpm) on particle size and drug release was investigated. Table 1 represents formulations investigated to study effect of cross-linking density on drug release.

Practical mass of microsponges

Production yield (PY) = ––––––––––––––––––––––––––x 100

Theoretical mass

The loading efficiency (%) of the microsponges can be calculated according to the following equation:

Actual drug content in microsponges

Loading efficiency = ––––––––––––––––––––––––––x 100

Theoretical mass

Wetting test

1 g of the loaded microsponges was placed into a 250 ml glass stoppered flask. 100 ml of ethanol was added to the flask by a pipette. A stopper was then placed in the flask which was clamped and shaken on mechanical shaker for 5±1 minutes. The liquid was immediately filtered through Whatman paper. Aliquot sample was removed by pipette and placed into a tarred glass container which was placed on a steam bath until the solvent was evaporated. The container was then placed in an oven maintained at a temperature of 105°C for 30 minutes. The container was then cooled and weighed to calculate percent weight of drug released. 13

In vitro drug release studies

Accurately weighed loaded microsponges were placed within 40 ml 0.9% sodium chloride of pH 7.4, assuring perfect sink conditions, in 50 ml glass bottles. The latter were horizontally shaken at 37 °C at pre- determined time intervals. Aliquot samples were withdrawn (replaced with fresh medium) and analyzed UV-spectrophotometrically at λ = 264 nm. 14, 15

Table 2: Formulation of fluconazole gels

Ingredients	Formulations % w/w
Fluconazole (free or entrapped; equivalent to)	2
Propylene glycol	40
Methanol	8
Menthol	0.04
Methyl paraben	0.18
Propyl paraben	0.02
Sodium metabisulphite	0.10
Disodium edetate	0.10
Carbopol 934P NF	1.00
Triethanolamine	Qs
Lavender	Qs
Purified water qs to make	100

Preparation of gels

Gel formulations containing loaded microsponges equivalent to 2% of fluconazole were prepared, as shown in table 2. A clear dispersion of Carbopol was prepared in water using moderate agitation. Intermittent sprinkling of Carbopol prevents lump formation resulting clear homogenous dispersion. Drug was dissolved in propylene glycol and methanol. Various ingredients viz. parabens, sodium metabisulphite and disodium edetate were dissolved in water and added to the drug solvent system. Triethanolamine was used to neutralize and volume was made with water. Gels prepared were degassed by ultrasonication.16

Drug diffusion from microspongic gels

The in vitro measurements of drug permeation through cellophane membrane was performed. 3 g of gels containing free and entrapped fluconazole were placed in the donor compartment, while the receptor compartment contained 12 mL 0.9% sodium chloride of pH 7.4. Sodium lauryl sulphate (1%) was added to the receptor compartment to ensure sink condition. Aliquots of 0.5 mL samples were withdrawn at 0, 1, 3, 6, 12, and 24 hours from the receptor compartment, and fluconazole was assayed spectrophotometrically at 264 nm.

RESULT AND DISCUSSION:

Preparation of blank microsponges

Blank macroporous- microspheres as shown in figure 1, were successfully prepared by optimizing reaction conditions; by suspension polymerization and drug was loaded by entrapment method. Product obtained was free flowing powder with fairly narrow particle size distribution. Particle of the size range 30µ m and smaller, 30-100µ m and bigger than 100µ m were separated and loaded with the drug by entrapment method.

Figure 2 shows effect of particle size on drug release. Particle size distribution of microsponges was affected by rpm and concentration of dispersion stabilizers. Effect of speed of rotation on particle size distribution is shown in figure 3.

Figure 1: Photomicrograph of microsponges prepared by suspension polymerization after drug loading by entrapment

The values for d50 were observed to be 45.25, 64.32 and 72.50 for microsponges with 400, 450 and 500 speed of rotation (rpm) respectively. Particle size is also well affected by concentrations of both dispersion stabilizers. Table 3 shows average mean diameters of microsponges at different levels of dispersion stabilizers.

Figure 2: Effect of particle size on release rate of fluconazole from microsponges

Cross-linking density

Cross-linking in excess of 10% is used in most systems, which allows the structure to retain its shape after the release of active from the pore network. As shown in figure 4, increased cross-linking has slowed down the rate of release.

Determination of production yield and loading efficiency

The production yield of the microparticles was determined at different cross-linking densities, while keeping rpm; 450 and concentration of dispersion stabilizers constant (sodium polyacrylate: sodium sulphate; 0.15:1.5); as these conditions are favorite to obtain particles of size smaller than 30µ m, suitable for topical delivery of drugs without grittiness. Average production yield was 82.63±1.34.

The loading efficiency (%) of the microsponges, prepared by entrapment method was calculated. Loading efficiency was found affected by cross-linking density and were observed; 77.43±2.55, 64.93±1.36 and 57.83±3.25 for microsponges of 20, 30 and 40 cross-linking density respectively.

Figure 3: Effect of speed of rotation on particle size distribution

Wetting test

For the microsponges with the 20 % w/w drug added during entrapment per total weight of the sample, ≥ 95% of drug was found released after wetting test. It was shown that substantially the entire active ingredient was released from the network of pores of the beads into the solvent.

Drug diffusion from microspongic gels

In vitro drug permeation against cellophane membrane from the microspongic gels containing 2% equivalent of drug is as shown in figure 5. The drug release was more in first one hour, may be due to surface adhered drug. The amount of fluconazole released from all gel formulations was best fitted in zero-order kinetic model (correlation coefficient = 0.972).

CONCLUSION:

The polymeric microsponges with various cross- linking densities were prepared by hot suspension polymerization method for delivery of topical drugs e.g. fluconazole, which can avoid the systemic uptake of drug. Concentration of dispersion stabilizers and speed of rotation has profound effect on particle size of microsponges. The entrapment efficiency and the drug release profile depend on cross-linking densities of microsponges.

Table 3: Average mean diameter of microsponges at different concentrations of dispersion stabilizers

Sodium polyacrylate%	Sodium sulphate%	Average mean diameter of microsponges
0.05	0.5	127
0.05	1	88
0.05	1.5	87
0.1	0.5	93
0.1	1	82
0.1	1.5	78
0.15	0.5	65
0.15	1	37
0.15	1.5	37

Figure 4: Effect of cross-linking density on drug release from microsponges

Figure 5: Drug diffusion from microspongic gels of fluconazole

ACKNOWLEDGEMENT:

Authors are thankful to Wallace Pharmaceuticals Ltd. Goa, Thermax Ltd. Pune and Lubrizol Mumbai for generous gift sample of fluconazole, styrene & divinyl benzene and Carbopol respectively.

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