Fluconazole Topical Microemulsion: Preparation and Evaluation

 

Rohit R Shah*, Chandrakant S Magdum, Kiran A  Wadkar and Nilofar S  Naikwade

Appasaheb Birnale College of Pharmacy, Sangli

*Corresponding Author E-mail:  rohitrshah@yahoo.co.in

 

ABSTRACT

The aim of the present study was to prepare and evaluate different formulations of fluconazole in microemulsion base using isopropyl myristate as oil phase, Labrasol as surfactant and plurol oleique as co-surfactant. Isopropyl myristate was selected as oil phase due to its good solublising capacity. Microemulsion existence region was determined using the pseudo-ternary phase diagrams for preparing different formulations.

 

Five different formulations were formulated with various values of oil (5 – 25%), water (10 – 50%), and the mixture of surfactant and co-surfactant (at the ratio of 4) (45 – 65%). In-vitro permeation of fluconazole from the microemulsions was evaluated using Keshary Chien diffusion cells mounted with 0.45µ cellulose acetate membrane. The amount of drug permeated across was analyzed by HPLC and the droplet size and zeta potential of the microemulsions was characterized using a Zetasizer Nano-ZS. The globule size ranged between 122 - 418nm. The permeability of optimised microemulsion formulation was increased approximately five folds than that of the marketed formulation. The results indicated that the microemulsion system studied would be a promising tool for enhancing the percutaneous delivery of fluconazole.

 

KEYWORDS : Fluconazole, Topical Microemulsion, Skin permeation

 


INTRODUCTION:

Fungal infection of skin is now-a-days one of the common dermatological problem. The physicians have a wide choice for treatment from solid dosage forms to semisolid and to liquid formulations. Amongst the topical formulations clear transparent gels have been widely accepted in both cosmetics and pharmaceuticals.1-2

 

Fluconazole is a synthetic triazole antifungal drug used for the treatment of superficial and systemic fungal infections. It is available as tablets for oral administration, as a powder for oral suspension and as a sterile solution for intravenous use. It is widely used in vaginal candidiasis, oropharyngeal and esophageal candidiasis and cryptococcal meningitis. It is also effective for the treatment of Candida urinary tract infections, peritonitis, and systemic Candida infections including candidemia, disseminated candidiasis, and pneumonia.


The concept of microemulsions was first introduced by Hoar and Schulman in the 1940s. They are defined as a system of water, oil and amphiphile which is an optically isotropic and thermodynamically stable liquid solution3-5.

 

As compared to conventional formulations microemulsions are wide better as they have enhanced drug solubility, good thermodynamic stability, ease of manufacturing and enhancement effect on transdermal delivery5-6. This system is suitable for delivery of both water insoluble drugs and water soluble drugs. Water insoluble drugs may be delivered through oil-in-water (o/w) microemulsions 7-9, while water soluble drug may be delivered through water-in-oil (w/o) microemulsions.

 

Recently researchers have focused on microemulsions for transdermal delivery of various drugs of anti-inflammatory 10-16, anaesthetics 17-18, antifungals19-20, steroids 21-22, etc.

 

The present study is an attempt to develop new microemulsion formulation of fluconazole for topical application. Here we have evaluated the in-vitro penetration of fluconazole from microemulsion, also compared the same with the commercially available marketed gel formulation using Keshary-Chien diffusion cell.

 

MATERIALS AND METHODS:

Fluconazole was kindly gifted by Dr. Reddy’s Laboratory, Hyderabad; isopropyl myristate was received as gift sample from Rita Corporation USA. labrasol and plurol oleique were gifted by Gattefosse, France and all other chemicals used were of AR grade and used without further purification.

 

 

Table No. 1. Formulation of Fluconazole microemulsion

Ingredients

(in % w/w)

FILP-A

FILP-B

FILP-C

FILP-D

FILP-E

Isopropyl myristate

25

20

15

10

5

Labrasol/

Plurol oleique

65

60

55

50

45

Water

10

20

30

40

50

 

Screening of oils, surfactants and co-surfactants for microemulsion:

Solvents for the study were selected based on the good solublising capacity for fluconazole. In present study the solubility of fluconazole was investigated in different oils like isopropyl myristate, isopropyl palmitate, labrafil M 1944CS and surfactants and co-surfactants like tween 80, plurol oleique, cremophor RH40, labrasol etc.

 

Excess of fluconazole was added to 5 ml each, of oils, surfactants, and co-surfactants in screw capped tubes and shaken on orbital flask shaker at 100 RPM for 48 hours at ambient temperature. The suspension was centrifuged at 5000 RPM and clear supernatant liquid was decanted and filtered through 0.45µ nylon membrane filter (Whatmann). The solubility of fluconazole was estimated by HPLC method.

 

Construction of pseudo-ternary phase diagrams:

The microemulsion existence region was determined by constructing pseudo-ternary phase diagrams. Titration method was employed for its determination. These diagrams will be best suited for making different possible compositions of oil surfactant/co-surfactant and water.

 

Different mixtures of surfactant to co-surfactants were prepared and the weight ratios were fixed to 1:2, 1:1, 2:1 and 4:1. These mixtures (S/CoS) were mixed with oil phase to give weight ratio of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 and 1:9, water was added drop by drop and stirred using magnetic stirrer until homogeneous dispersion or solution was obtained. After each addition the system was examined for appearance and flow property. The end point of the titration was the point in which the solution becomes cloudy or turbid. The quantity of aqueous phase required to make the mixture turbid was noted.

The percentages of the different incorporated pseudo phases were then calculated and the same procedure was followed with other S/CoS ratio.

 

Preparation of Fluconazole Microemulsion:

Fluconazole containing microemulsions were formulated by mixing oil, surfactant, and co-surfactant with varying component ratio as described in Table 1 (FILP – A, B, C, D and E). 0.5 % w/w of fluconazole was dissolved in this mixture and then an appropriate amount of water was added to the mixture drop by drop with constant stirring on magnetic stirrer. Fluconazole containing microemulsion was obtained spontaneously on stirring the mixtures at ambient temperature. All microemulsions were stored at ambient temperature.

 

 

Table No. 2. Solubility of fluconazole in different oils, surfactant and co-surfactant

Phase type

Excipient

Solubility (mg/ml)

Oil

Isopropyl myristate

0.60 ± 0.044

 

Soya bean oil

0.44 ± 0.15

 

Cotton seed oil 2

0.41 ± 0.041

 

Olive oil 2

0.35 ± 0.032

surfactant

Labrasol

71 ± 1.0

 

Tween80

14.08 ± 0.532

 

Cremophor RH – 40

4.98 ± 0.197

co-surfactant

Labrafac Lipofile

3.21± 0.235

 

Plurol Oleique

6.89 ± 0.350

 

 

Measurement of droplet size and zeta potential:

The average droplet size and zeta potential of the microemulsions were measured using a Zetasizer Nano-ZS (Malvern Instruments, UK). The measurement was done at 25°C.

 

In-vitro permeation study 23-27:

The in-vitro permeation rate of fluconazole from various microemulsion formulations was determined to evaluate the effect of the formulation variables.

 

The permeation studies were performed using Keshary-

Chien diffusion cells fitted with 0.45µ cellulose acetate membrane (Sartorius) at 37 ± 0.1°C using a thermostatic water pump. (Cyberbath, CB 2000, Cyberlab Inc. USA.) The effective diffusion area was 2.54 cm2 (18mm orifice diameter), and the receptor compartment was filled with 13.5 ml of phosphate buffer pH 7.4. The receptor fluid was constantly stirred by externally driven teflon coated star head magnetic bars.

 

Accurately weighed 1gm of fluconazole was placed in the donor compartment. Samples (0.5ml) were withdrawn from the receptor fluid at predetermined time interval for upto 6 hrs after the application. An equal volume of the fresh phosphate buffer was immediately replenished after each sampling. All the collected samples were stored at -20°C until analysed by HPLC. The permeation study was done in triplicate.

 

 

Figure No 1: Pseudo ternary phase diagrams for microemulsion composed of oil (Isopropyl Myristate), surfactant (S, Labrasol), co-surfactant (Co-S, Plurol Oleique) and water.

   

Km = 0.5                                      Km = 1.0

    Km = 2.0                                          Km = 4.0

 

Table No. 3: Droplet size and zeta potential of formulations

Sr. No.

Formulation

Droplet Size (nm)

Zeta Potential (mv)

1.

FILP – A

418

-0.232

2.

FILP – B

274

-0.114

3.

FILP – C

187

-0.535

4.

FILP – D

122

-0.596

5.

FILP – E

214

-0.185

 

 

Estimation of Fluconazole by HPLC:

The amount of fluconazole in the receptor compartment was determined by HPLC method. The HPLC system consisted of a pump (model Jasco PU-2080 plus, intelligent HPLC pump) with 20 ml loop sample injector (#7725i, Rheodyne, USA) per injection was used. Detector consisted of a UV-Vis (Jasco UV-2075 intelligent UV-Vis detector model.) The equipment was operated through software Borwin version 1.5, LC-Net II/ADC system. The column used was Inertsil ODS, C18 column having dimensions 4.6mmf´250mm i.d. 5µm particle size (GL. Sciences INC, JAPAN.)

 

The samples were chromatographed using an isocratic mobile phase consisting of 45:55v/v mixture of 25mM TRIS hydroxyl-methyl amino methane in phosphate buffer pH 7.0 and acetonitrile. The pH of mobile phase was adjusted to 7.0. The flow rate was 1.5ml/min and the detection wavelength was 276 nm. All operations were carried out at ambient temperature.

 

Optical Birefringence 12, 28:

Birefringence is a light scattering phenomenon. The formulations were examined under polarized light microscopy (Polarizing microscope, Japan) in order to determine optical isotropy of the samples.

 

Determination of pH:

The pH values of the samples were measured by a pH meter (model HI 8417, Hanna Instruments Inc., Woonsocket, USA), at 20 ± 1 C.

 

Determination of Viscosity 12, 29-33:

The viscosities of microemulsions were measured with a Brookfield rotational viscometer (LV2, Brookfield Inc., USA) equipped with spindle no. 4. The measurement was done at ambient temperature. Viscosities were determined in triplicate.

 

Figure No 2: In-vitro cumulative percent fluconazole permeated from microemulsion formulations

 

Table No. 4: pH and viscosities of formulations

Sr. No.

Formulation

pH

Viscosity

1.

FILP – A

3.96

93.3

2.

FILP – B

3.77

85

3.

FILP – C

3.48

85

4.

FILP – D

3.37

85

5.

FILP – E

3.23

85

 

RESULTS AND DISCUSSION:

Screening of oils, surfactants and co-surfactants for microemulsion:

To develop microemulsion formulations for topical delivery of poorly water-soluble fluconazole, proper selection of oil is needed. The optimisation of the components to be used in formulating microemulsion was decided based on the solubility of fluconazole in the various oils, surfactants and co-surfactants. The solubility data is shown in Table 2.

 

The solubility of fluconazole amongst various oils investigated was found to be highest in isopropyl myristate (0.60 ± 0.044mg/ml), followed by cotton seed oil, olive oil and soya been oil. Amongst surfactant, tween 80 showed maximum solubility (14.08 ± 0.532mg/ml) followed by labrasol and cremophor RH-40. Plurol oleique showed highest solubility among the co-surfactants (6.89 ± 0.350mg/ml), followed by labrafac lipophile.

 

Based on the solubility studies of fluconazole in oil, surfactant and co-surfactant and the preformulation studies we found IPM, labrasol, plurol oleique could be the most appropriate combination for development of microemulsion.

 

Construction of pseudo-ternary phase diagrams and microemulsion formulation:

The microemulsion existence region was determined by constructing phase diagrams. Fig.1 describes the pseudo ternary phase diagrams with various weight ratios of labrasol to plurol oleique. The translucent region presented in phase diagram reveals the microemulsion existence region. No distinct conversion from water-in-oil (w/o) to oil-in-water (o/w) microemulsion was observed. The rest of region on the phase diagram represents the turbid and conventional emulsions based on visual inspection.

 

The phase study clearly revealed that microemulsion existence region increased with increase in the weight ratio of surfactant (0.5 - 4). The maximum proportion of oil was incorporated in weight ratio 4:1 of labrasol to plurol oleique.

 

Five different proportions of oil (25-5%), surfactant - co-surfactant (65-45%), and water (10-50%) were selected for formulation.

 

Measurement of droplet size and zeta potential:

The droplet size and zeta potential for the formulations are represented in Table 3. The result shows that the droplet diameter decreases with increasing ratio of oil: surfactant/co-surfactant. These results are in accordance with the report that the addition of surfactant to microemulsion system causes the interfacial film to condense and to be stable, while the co surfactant causes the film to expand 34.

The pH and viscosities of formulations are listed in table 4.

 

In-vitro permeation study:

Fig. 2 illustrates the permeation rates of fluconazole through the different microemulsion formulations. FILP – E showed the highest permeation rate (99.02 ± 1.15) among the formulations tested. The surfactant mixture content in the formulation affected the permeation rate significantly. As the composition of surfactant mixture was decreased from 65% to 45% at S/CoS = 4, the permeation rate of fluconazole increased approximately by 2-folds. The increase in fluconazole permeation with decrease in surfactant mixture may be signified as, the drug being poorly soluble in water and yet solublised may have been solublised in the surfactant mixture35.

 

The increase in percutaneous absorption of drug might also be affected by the globule size of the microemulsion. As the droplet size is very small the number of vesicles that interact on fixed area of stratum corneum also increases thereby increasing the efficiency of percutaneous uptake. This might be the reason why microemulsions of other batches whose particle sizes were larger than that of batch FILP – E showed relatively lower permeation rates. From the permeation studies, it clearly reveals that the permeation rate increases (70% to 99 %), as the concentration of both oil as well as S/CoS decreases.

 

Conclusion:

The fluconazole topical microemulsions were formulated and the different compositions of components were obtained by constructing pseudoternary phase diagrams. Their concentrations were optimised after the evaluation of their effect on permeation of drug. The optimum formulation of the microemulsion consisted of IPM 5%, labrasol/plurol oleique 45% (4:1) and water.

 

Acknowledgements:

Authors are grateful to Dr. Reddy’s Laboratory, Hyderabad, Rita Corporation USA and Gattefosse, France for providing the necessary gift samples. We are also thankful to the Principal and Management of Appasaheb Birnale College of Pharmacy, Sangli for providing the necessary facilities to carry out this work.

 

References:

1.      Nürnberg E. Welche galenischen Grundlagen werden heute für die Hautbehandlung eingesetzt? Hautarzt. 1978; 29: 61-67.

2.      Provost C. Transparent oil-water gels: a review. Int J Cosmet Sci. 1986; 8: 233-247.

3.      Danielson I., Lindmann B., The definition of microemulsion. Colloid Surf., 1981; 3: 391.

4.      Tenjarla S. Microemulsions: An Overview and Pharmaceutical Applications. Crit. Rev. Ther. Drug Carrier Syst. 1999; 16(5): 461-521.

5.      Lawrence J. and Rees G., Microemulsion-based media as novel drug delivery systems. Adv. Drug Deliv. Rev., 2000; 45: 89–121.

6.      Gasco M. R., Microemulsions in the Pharmaceutical Field. In: Perspectives and Applications, Industrial Applications of Microemulsions. Marcel Dekker Inc., New York, 1997; pp. 97–122.

7.      Jeppson R. and Ljunberg S., Anticonvulsant activity in mice of diazepam in an emulsion formulation for intravenous administration. Acta. Pharmacol. Toxicol. 1975; 36: 312-312.

8.      Mizushima Y., Hamano T and Yokoyama K., Use of a lipid emulsion as a novel carrier for corticosteroids. J. Pharm. Pharmacol., 1982, 34: 49-50.

9.      Kronevi T. and Ljunberg S., Sequel following intra-arterially injected diazepam formulations. Acta Pharm. Suecica. 1983; 20: 389-389.

10.    Mei Z., Chen H., Weng T., Yang Y., Yang X. Solid lipid nanoparticle and microemulsion for topical delivery of triptolide Eur. J. Pharm. Biopharm. 2003; 56: 189 –196.

11.    Kweon J. H., Chi S. C., and Park E. S., Transdermal Delivery of Diclofenac Using Microemulsions. Arch. Pharm. Res. 2004; 27(3): 351 – 356.

12.    Djordjevic L., Primorac M., Stupar M., Krajisnik D., Characterization of caprylocaproyl macrogolglycerides based microemulsion drug delivery vehicles for an amphiphilic drug, Int. J. Pharm., 2004; 271: 11–19.

13.    Chen H. B., Chang X. L., Weng T., et. al. A study of microemulsion systems for transdermal delivery of triptolide. J. Control. Rel. 2004; 98: 427–436.

14.    Yuan Y., Li S. M.., Moc F. K., Zhonga D. F., Investigation of microemulsion system for transdermal delivery of meloxicam Int. J. Pharm. 2006; 321: 117–123

15.    Goud K. Desai H. Enhanced Skin Permeation of Rofecoxib Using Topical Microemulsion Gel, Drug Development Research 2004; 63: 33–40.

16.    Park E. S., Cui Y., Yun B. J., Ko I. J., and Chi S. C., Transdermal Delivery of Piroxicam Using Microemulsions Arch. Pharm. Res. 2005; 28(2): 243-248.

17.    Zabka M., Benkova, M.: Microemulsions containing local anaesthetics. Part 6: Influence of microemulsion vehicle on in vivo effect of pentacaine. Pharmazie 1995; 50: 703- 704.

18.    Sintov AC.,  Brandys SR Facilitated skin penetration of lidocaine: Combination of a short-term iontophoresis and microemulsion formulation Int. J. Pharm. 2006; 316: 58–67

19.    El Laithy HM, El-Shaboury KM, The development of Cutina lipogels and gel microemulsion for topical administration of fluconazole AAPS PharmSciTech. 2002; 3(4): E35.

20.    Trotta M., Gallarate M., Carlotti ME., Morel S., Preparation of griseofulvin nanoparticles from water-dilutable microemulsions Int. J. Pharm. 2003; 254: 235–242

21.    Peltola S., Saarinen-Savolainen P., Kiesvaara J., Suhonen T.M., Urtti A., Microemulsions for topical delivery of estradiol International Journal of Pharmaceutics 2003; 254: 99–107

22.    Biruss B., Kahlig H., Valenta C. Evaluation of an eucalyptus oil containing topical drug delivery system for selected steroid hormones Int. J. Pharm. 2007; 328:142–151.

23.    Nasseri AA., Aboofazeli R., Zia H., Needham TE., Lecithin – Stabilized Microemulsion – Based Organogels for Topical Application of Ketorolac Tromethamine. II. In vitro Release Study. Iranian J. Pharmaceutical Research 2003; 117-123.

24.    Podlogar F., Rogaˇc M., Gaˇsperlin M., The effect of internal structure of selected water – Tween 40® – Imwitor 308® – IPM microemulsions on ketoprofen release, Int. J. Pharm., 2005; 302: 68–77.

25.    Thakker KD., and Chern WH., Development and Validation of In Vitro Release Tests for Semisolid Dosage Forms - Case Study. Dissolution Technologies, 2003; 5: 10 – 15.

26.    Shaikh IM., Jadhav KR., Gide PS., Kadam VJ., Pisal SS., Topical delivery of aceclofenac from lecithin organogels: preformulation study. Curr. Drug Deliv., 2006; 3(4): 417-27.

27.    Tomsic M., Podlogar F., Gasperlin M., Rogac M., Jamnik A. Water–Tween 40®/Imwitor 308®–isopropyl myristate microemulsions as delivery systems for ketoprofen: Small-angle X-ray scattering study. Int. J.  Pharm., 2006; 327: 170–177.

28.    Prince LM., In ‘Microemulsions: Theory and Practice,’ (Ed by L.M. Prince), London, Academic press Inc, 1977; pp 11.

29.    Špiclin P., Homar M., Zupanˇciˇc-Valant A., Gašperlin M., Sodium ascorbyl phosphate in topical microemulsions. Int. J. Pharm., 2003; 256: 65–73.

30.    Sintov A. C., Shapiro L., New microemulsion vehicle facilitates percutaneous penetration in vitro and cutaneous drug bioavailability in vivo, J. Control. Rel., 2004; 95: 173 – 183.

31.    Podlogar F., Gašperlin M., Tomšic M., Jamnik A., Rogac M., Structural characterisation of water – Tween 40® /Imwitor 308® isopropyl myristate microemulsions using different experimental methods. Int. J. Pharm., 2004; 276: 115–128.

32.    Chen H., Mou D., Du D., Chang X., Zhu D., Liu J., et.al. Hydrogel-thickened microemulsion for topical administration of drug molecule at an extremely low concentration. Int. J. Pharm., 2007; 341: 78–84.

33.    Mehta SK., Kaur G., Bhasin KK., Analysis of Tween based microemulsion in the presence of TB drug rifampicin”, Colloids and Surfaces B: Biointerfaces. 2007; 60: 95–104

34.    Kale NJ., Allen LV., Studies of microemulsion using Brij 96 as surfactant and glycerine, ethylene glycol and Propylene glycol as co surfactant. Int. J. Pharm., 1989; 57(2): 87 – 93

35.    Shah VP., Skin penetration enhancer: scientific prospective. In: Hsieh, DS (Ed.), Drug permeation enhancement: Theory and applications, Marcel Dekker Inc., New York, 1994; pp 19 – 24.

 

 

 

Received on 05.01.2009  Modified on 10. 02. 2009

Accepted on 02.03.2009  © RJPT All right reserved

Research J. Pharm. and Tech.2 (2): April.-June.2009; Page 353-357