1. INTRODUCTION:

Fluconazole an antifungal agent applied topically for its action against the fungal infection in the form of semisolid formulation. It is commonly marketed under the trade name Diflucan^Ò or Trican^Ò. The necessity of physical characterization of drug and its suitability for particular type of formulation is must for its development.

Partition coefficient (P) is the ratio of concentrations of unionized compound in the two phases of a mixture of immiscible solvents at equilibrium. It is expressed as

K = C_o / C_w …….. (1)

Where, K is partition coefficient, C_o is concentration of solute in organic phase and C_w is concentration of solute in aqueous phase. Partition coefficient is a measure of differential solubility of the compound between two solvents¹. For ionizable substances measurements are made only on their non-ionized form (free acid or free base) produced by the use of an appropriate buffer with a pH of at least 1 pH unit below (free acid) or above (free base) the pKa. Usually solvent pair chosen is aqueous (water) and hydrophobic (n-octanol).

Partition coefficients are useful in estimating distribution of drugs within living organisms. Information on structural formula, dissociation constant, water solubility, hydrolysis, surface tension of the substance, etc, can be obtained from partition coefficient.

It is useful in the study of the equilibrium thermodynamics and is very useful to understand solute-solvent interactions in both aqueous and organic solvents. Hydrophobic drugs with high partition coefficients are preferentially distributed to hydrophobic compartments (lipid bilayers of cells) while hydrophilic drugs with low partition coefficients are preferentially found in hydrophilic compartments (blood serum). Most studies are concentrated on the water because water is the major and most important solvent in biological systems. The partition coefficient of a solute between octanol and water is widely used to predict drug pharmacokinetic properties². Various techniques used to measure the partition coefficients include the classical shake flask method and stir flask methods, dual-phase potentiometric titrations, reversed phase planer and liquid chromatographic procedures, cyclic voltammeter, centrifugal partition chromatography, counter-current distribution, rotating diffusion cells, etc. The most classical and reliable method of determination of partition coefficient is the shake flask method, which consists of dissolving some of the solute in question in equal volumes of aqueous and organic solvents and then measuring the concentration of the solute in each solvent by spectroscopic method³.

Adsorption is a phenomenon by which the molecules of gas, vapor and liquid spontaneously concentrate at a contacting surface without undergoing chemical reaction, thereby it forms the surface layer. Adsorption is one of the most important mechanisms of interaction between drugs and excipients. The forces involved in this type of interaction can be either physical or chemical in nature or combination of both. When the drug is in contact with adsorptive it can be adsorbed on it causing reduction in their systemic absorption and toxicity of drug⁴. Activated charcoal is commercially available adsorptive and activated by heating. The process of activating results in particles with small pores having a large surface area (typically 950-2000 m²/g), and are coated with carbon moieties that have varying ability to form complex with drug molecules. The in-vitro adsorption of several drugs onto various adsorptives has been studied with a variety of techniques⁵.

Fig. 1: Freundlich adsorption isotherm of Fluconazole on activated charcoal

Kim D. applied statistical adsorption isotherms for the multilayer adsorption of gas molecules on non-porous solid adsorbents.⁷ Robert et al. studied the adsorption of tramadol hydrochloride on activated charcoal in-vitro and in-vivo and found that 13 to 14 fold rightward antinociceptive dose-response curve⁵. Sanjuan et al studied the in-vitro adsorption of methotrexate and calcium leucovorin onto cholestyramine and concluded that it is possible to use cholestyramine by the oral route in order to prevent reabsorption of methotrexate which is excreted via bile, thus improving the elimination of drug in the faeces⁸. Oremusova et al. studied the adsorption, partition and release study of terbinafine hydrochloride and claimed that adsorption follows the both the adsorption isotherms.⁹ Teiko et al. studied the adsorption of drugs on the various adsorbents and found that medicinal carbon showed an excellent adsorption of all the tested drugs.¹⁰ Otero et al. studied the effect of temperature on adsorption process of salicylic acid on polymeric adsorbents and activated charcoal¹¹. Suhair et al studied the adsorption of ketotifen on some of the pharmaceutical excipients and suggested that the predominant mechanism of ketotifen adsorption is physical and exothermic in nature¹². Andrzej et al. studied the application of adsorption method to the chromatographic analysis of free drug concentration¹³.

The process of adsorption is affected by various factors, viz. time, pH, and specific surface area, cross linking time, surface free energy and nature of adsorbents used⁴. The equations to describe the adsorptions theoretically well are the Langmuir and Freundlich adsorption isotherms⁶. Fluconazole is a highly selective inhibitor of fungal cytochrome P-450 by sterol C-14 alpha-demethylation. Fluconazole a bis-triazole antifungal agent used in the treatment of superficial and systemic infection. Mammalian cell demethylation is much less sensitive to Fluconazole inhibition. It exhibits in vitro activity against Cryptococcus neoformans and Candida spp. In common with other azole antifungal agents, most fungi show a higher apparent sensitivity to Fluconazole in vivo than in vitro. There have been reports of cases of superinfection with Candida species other than C. albicans, which are often inherently not susceptible to oral formulation. In such cases requires alternative antifungal therapy. The drug has slight solubility in water (8mg/ml) and phosphate buffer (10 mg/ml), soluble in organic solvents at room temperature. It has melting point of 138-142 °C and is widely available as tablets and IV infusion. It is well absorbed following oral administration having bioavailability of 90% or more compared to intravenous administration¹⁴.

In the present work, the partition coefficient of Fluconazole in different organic solvents and phosphate buffer pH 7.4 has been determined by shake flask method where as adsorption studies on activated charcoal and talc were performed at different temperatures to fit the adsorption isotherm models to investigate the suitability of the drug for the topical effects.

2. EXPERIMENTAL:

2.1. Materials:

Fluconazole was a generous gift from Cipla Pharmaceutical Ltd. Mumbai. Dichloromethane, dichloroethane, hexanol and octanol were purchased from Spectrochem Pvt. Ltd. Mumbai. The Activated charcoal and Talc were purchased from Loba Chemie, Mumbai and all other ingredients were of analytical grade.

2.2 Calibration curve of Fluconazole:

A calibration curves as mean of five sets were obtained by UV-visible spectrophotometer (Jasco, Japan). An accurately weighed 10 mg of Fluconazole was dissolved in 100 ml of each solvent system as listed in Table 1. These solutions are further diluted to 5, 10, 15, 20, 25, 30, 35 and 40 µg/ml and absorbance was measured at l_max 260 nm.

Fig. 2: Langmuir adsorption isotherm of Fluconazole on activated charcoal

Table 1: Calibration curve - linear regression analysis values

Solvent system	r	Slope
Phosphate buffer pH 7.4 at 25°C	0.999	43.56
Phosphate buffer pH 7.4 at 35°C	0.999	42.55
Phosphate buffer pH 7.4 at 45°C	0.997	45.41
Dichloromethane	0.994	45.69
Dichloroethane	0.997	40.68
Hexanol	0.997	36.95
Octanol	0.998	31.96

2.3. Partition coefficient study¹⁵:

The partition coefficient of Fluconazole was studied using various solvent systems listed in Table 2 by shake flask method. Fluconazole was added continuously to non aqueous phase till saturated solution is obtained. The solution was filtered through Whatman filter paper. The filtrate was used as stock solution to prepare different strength solutions of which aliquots of 10, 20, 30 and 40 ml were transferred separately to different flasks already containing 50 ml of phosphate buffer and the final volumes were made to 100 ml using respective pure non aqueous solvent. These flasks were shaken on orbital shaker at room temperature (25°C) for 1 hour to attain chemical equilibrium. The solution mixtures were separated using separating funnel. At the end, aqueous and non-aqueous portions were analyzed for Fluconazole concentration using UV-visible spectrophotometer at l_max 260 nm. The reliability of the determined values of partition coefficients were tested by comparison of the means of triplicate determinations with overall mean.

2.4. Adsorption Study¹⁵:

The adsorption of Fluconazole was studied using hydrophobic adsorptives, activated charcoal and talc.

2.4.1 Activation of adsorptive:

Adsorptives were passed through sieve No.180 and evenly spreaded in a Petri-dish followed by heating in hot air oven at 100 °C for 24 hours to make the surface active for adsorption. The dried adsorptives were allowed to cool to room temperature and used for the study immediately.

2.4.2 Adsorption on talc and charcoal:

In each of stoppered glass bottle accurately weighed 1 g of adsorptive were transferred. An aqueous stock solution of Fluconazole (1 mg/ml) was prepared by dissolving small quantity of drug in methanol and the final volume was adjusted with phosphate buffer pH 7.4. A specified quantity of stock solution was transferred to each bottle to have a concentration of 1, 1.5, 2, 2.5, 3, 3.5, and 4 mg/ 100ml, respectively. The bottles were immediately placed in incubator maintained at specified temperatures (i.e. 25, 35 and 45°C) and intermittently agitated during the period of 1 hour to attain adsorption equilibrium.

2.4.3 UV spectrometric analysis:

After completion of 1 hour the content of bottles were filtered through the membrane filter (100 Å) and filtrates were analyzed by UV spectrophotometer at l_max = 260 nm.

Table 2: Results of partition coefficient of Fluconazole

Solvent Systems	Partition coefficient*
Dichloromethane/ phosphate buffer pH 7.4	1.08±0.01
Dichloroethane/ phosphate buffer pH 7.4	1.05±0.01
Hexanol/ phosphate buffer pH 7.4	1.03±0.02
Octanol/ phosphate buffer pH 7.4	1.01±0.01

(*Results are the mean of triplicate observations ± SD)

3. RESULTS AND DISCUSSION:

Adsorption, partition and releasing balances of drug are important properties which have to be known in the development of dosage form. The study of partition balances is useful in understanding the behavior of drugs in the organism. Investigations of temperature dependences of drug adsorption on hydrophilic or hydrophobic surfaces are often used to obtain information about the adsorption and adhesive mechanisms which are directly related to its physicochemical properties that also help in predicting their compatibility and suitability.

In this work, effect of length of carbon chain on partition process of Fluconazole in o/w system was studied. The effect of temperature on adsorption of Fluconazole on activated charcoal and talc at different temperatures was studied in order to find out which isotherm describe more appropriate experimental adsorption process. Before formulating the any formulation we have to study their physicochemical characteristics for their compatibility and suitability.

Calibration curve of Fluconazole:

Fluconazole absorbance spectrum has two maxima at 260 and 267 nm. Determination of calibration curve and all measurements for adsorption and partition were carried out at λ_max 260 nm. From the plot of absorbance at λ_max = 260 nm Vs concentration in various organic solvents and at temperatures of 25, 35, 45°C in phosphate buffer (pH 7.4) it shows linear regression and obeys Beer’s law. The linear regression line values in different solvents are given in Table 1.

Fig. 3: Freundlich adsorption isotherm of Fluconazole on talc

Partition coefficient:

The partition coefficient study is useful in understanding of the behavior of drug in the organism. Parameters of the calibration curve were used for determination of the experimental partition coefficients in the o/w partition solvent systems. All the obtained values of partition coefficient were given in Table 2.

The experimental observations showed that as the length of carbon chain of lipophilic solvents increases (octanol > hexanol > dichloroethane > dichloromethane) the partition coefficient values goes on decreasing. The observed partition coefficient values are very close to 1 suggesting the easy permeation through biological membrane of the microorganism. The partition coefficient of drug between octanol and water is a physicochemical properties with wide spread

applications in the assessment of its penetration and distribution.

Adsorption:

The effect of temperature on Fluconazole adsorption from non-aqueous buffered solutions on Activated charcoal and Talc I. P. at temperatures 25°C, 35°C and 45°C were studied. The objective of this study was to investigate which adsorption isotherm is more appropriate for the experimental adsorption process. From the observed values amount of solute adsorbed per gram of activated charcoal and talc at all temperatures were calculated. The calculated data indicate that Fluconazole adsorption was lower at high temperature than that at low temperature. It was also found that higher amounts of Fluconazole were adsorbed on activated charcoal than that of talc at all temperatures.

The experimental adsorption isotherms were expressed by the Freundlich (equation 1) and Langmuir model (equation 2) of adsorption⁶.

log w/m = log K + 1/n log C ………. (1)

Where, w/m is observed values of the Fluconazole in mg adsorbed per gram of adsorbent, C is concentration of Fluconazole in mg per 100 ml remaining in solution after equilibrium had been established, K is amount of Fluconazole adsorbed per one gram adsorbent at an equilibrium concentration dependent on character of adsorbent and adsorbate, and n is Freundlich parameter dependent on temperature and related to Fluconazole affinity to the adsorbent.

……. (2)

Where, ‘a’ is related to the surface area of the adsorbates as amount of maximum monolayer adsorption and ‘b’ is constant called adsorption coefficient related with enthalpy of adsorption. The experimental data was fitted to Freundlich isotherm model as shown in Fig. 1 and 3. The calculated data was also fitted to Langmuir isotherm model, Fig. 3 and 4.

According to Freundlich isotherm the plot at different temperatures for adsorption of Fluconazole on activated charcoal and talc is linear and provided n and K values as shown in Table 3 and 4. The value n increased with temperature of solutions and plot of n vs. temperature was not found linear. Plot of K vs. temperature was found to be nearly linear and decreased with increasing temperature. All Langmuir plots for different temperatures gave a and b values as listed in Table 3 and 4. The values a and b increased with increasing temperature of solution. This clearly suggests that adsorption of Fluconazole on activated charcoal and talc conveniently describes both models of adsorption.

Fig. 4: Langmuir adsorption isotherm of Fluconazole on talc

CONCLUSION:

Experimentally determined partition coefficients increased with the length of carbon chain in the partitioning system. Based on results obtained it could be concluded that n-octanol /water system is the most suitable than other organic solvent systems. This is because the partition coefficient obtained in n-octanol/water system is nearly equal to 1. The Freundlich and Langmuir adsorption isotherms showed the adsorption of Fluconazole on talc and activated charcoal at different temperatures conveniently describing both models. Partition and adsorption data suggest that Fluconazole can be formulated as topical dosage form for local effects for the increasing therapeutic action.

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Received on 26.03.2009 Modified on 10.04.2009

Asian J. Research Chem. 2(2): April.-June, 2009 page 213-219

Temperature (°C)	Freundlich adsorption isotherm			Langmuir adsorption isotherm
Temperature (°C)	n	K	r	b	a	r
25	8.32	0.0074	0.997	91849	61.56	0.998
35	10.32	0.0065	0.997	97405	76.43	0.998
45	13.65	0.0042	0.996	103200	87.46	0.995

Temperature (°C)	Freundlich adsorption isotherm			Langmuir adsorption isotherm
	n	K	r	b		a		r
25	9.71	0.0045	0.996		113891		137.66		0.993
35	11.65	0.0033	0.997		120879		143.31		0.992
45	12.87	0.0028	0.996		143446		162.68		0.994