Design and Development of Topical Microemulsion for Poorly Water-Soluble Antifungal Agents*

P. Puranajoti

R. T. Patil

P. D. Sheth

G. Bommareddy

P. Dondeti

K. Egbaria

*This study was sponsored by Research and Development,
Morton Grove Pharmaceuticals, Inc., Morton Grove, IL

 

KEY WORDS: topical microemulsion, water-soluable antifungal agents

ABSTRACT

Topical microemulsions for poorly soluble antifungal agents (miconazole, ketoconazole, and itraconazole) were designed and developed using either mineral oil or olive oil as an oil phase. Various combinations of surfactant and cosurfactant were used, including Labrafil^® M 1944 CS and Plurol^® Oleique (1:1); Labrafil^® M 1944 CS and Plurol^® Oleique (1:2); or Labrafil^® M 1944 CS, Capmul^® MCM C-8, Pluro^® Oleique, and dehydrated ethyl alcohol (3:3:1:1), to design microemulsions by constructing pseudoternary phase diagrams. Water-in-oil microemulsions were obtained using oil and surfactant concentrations that ranged from 8.3% to 33.3% v/v and 16.7% to 75.8% v/v, respectively. The surfactant:cosurfactant combination of Labrafil^® M 1944 CS and Plurol^® Oleique (1:1) and the oil phase of olive oil were chosen for preparing ketoconazole microemulsion containing 10% v/v alcohol. The ketoconazole gel was prepared using Carbopol^® 974P and 90% v/w alcohol. The release profiles of ketoconazole from both formulations were investigated using Franz diffusion apparatus. The release rates of ketoconazole from microemulsion and gel formulation were 766.8 and 677.6 µg/hour, respectively (n = 6, P . .05). No significant difference was seen between the release rates of ketoconazole from both formulations despite their differences in alcohol content. Microemulsions of poorly water-soluble antifungal agents were successfully developed with in vitro release rates comparable to that of the gel formulation.

INTRODUCTION

Most pharmaceutical drug substances are lipophilic compounds, which are practically insoluble in water. Researchers have developed drug delivery systems, such as tablets, capsules, ointments, creams, gels, suspensions, solutions, and emulsions, in many dosage forms to deliver these lipophilic substances to patients. A microemulsion,1-5 one of the pharmaceutical interests for new drug delivery, is normally composed of oil, water, surfactant, and cosurfactant. Hoar and Schulman6 were the first to introduce the word microemulsion, which they defined as a transparent solution obtained by titrating a normal coarse emulsion with medium-chain alcohols. The short to medium-chain alcohols are generally considered as cosurfactants in the microemulsion system.

The presence of surfactant and cosurfactant in the system makes the interfacial tension very low. Therefore, the microemulsion is thermodynamically stable and forms spontaneously, with an average droplet diameter of 1 to 100 µm.7-9 Advantages of microemulsion over coarse emulsion include its ease of preparation due to spontaneous formation, thermodynamic stability, transparent and elegant appearance, increased drug loading, enhanced penetration through the biological membranes, increased bioavailability,4,10 and less inter- and intra-individual variability in drug pharmacokinetics.11 These advantages make microemulsions attractive drug delivery systems.

Recently, microemulsions were reviewed for several applications, such as topical use, oral use, parenteral use, and cosmetics.1-5 The objective of the present study was to design and develop topical microemulsions for poorly water-soluble compounds using antifungal agents such as miconazole, ketoconazole, and itraconazole as model drugs. Three sets of surfactant and cosurfactant phase were used: Labrafil^® M 1944 CS and Plurol^® Oleique (1:1); Labrafil^® M 1944 CS and Plurol^® Oleique (1:2); Labrafil^® M 1944 CS, Capmul^® MCM C-8, Plurol^® Oleique, and dehydrated ethyl alcohol (3:3:1:1). These were evaluated individually. Either olive oil or mineral oil was used as the oil phase. A pseudoternary phase diagram was constructed for each microemulsion system. Additionally, a gel formulation of ketoconazole (about 1% w/w) containing 90% v/w of alcohol was also developed. The release profiles of ketoconazole from the microemulsion (containing about 10% v/v alcohol) and the gel formulation were compared using Franz diffusion apparatus, and the release rates were calculated.

EXPERIMENTAL SECTION

Materials and Reagents

Olive oil and mineral oil were obtained from Croda Inc. (Itasca, IL) while Labrafil^® M 1944 CS was purchased from Gatefosse (Westwood, NJ). Plurol^® Oleique, Capmul^® MCM C-8, and Carbopol^® 974P were generously given by Gatefosse (Westwood, NJ), Abitec Corporation (Columbus, OH), and B F Goodrich Specialty Chemicals (Cleveland, OH), respectively. Hydrochloric acid and dehydrated alcohol were of reagent grade, but methanol, acetonitrile, and all other reagents were of HPLC grade.

Pseudoternary Phase Diagram

The oil phase was mixed with the surfactant:cosurfactant phase, and the mixture was titrated with water until it turned turbid. The volume of water used was then recorded. Water titration was continued until the mixture turned clear and, again, the water volume was recorded. The pseudoternary phase diagram was constructed by plotting the amounts of water phase, oil phase, and surfactant:cosurfactant phase used in the experiment.

Preparation of Microemulsion

A water-in-oil microemulsion of ketoconazole was prepared by mixing about 1.1 g of ketoconazole with the proportion of olive oil, water, and Labrafil^® M 1944 CS and Plurol^® Oleique (1:1) in the microemulsion region to obtain 100 mL in volume as shown in Figure 1. Then, 11.1 mL dehydrated alcohol was added to help make ketoconazole soluble in the system.

Preparation of Gel

A gel formulation of ketoconazole was prepared by dissolving about 1 g of ketoconazole in 90 mL of dehydrated alcohol. Then 30% w/w Carbopol^® 974P solution in water was used to make up 100 g of gel formulation.

HPLC Analysis of Ketoconazole

HPLC apparatus (Hewlett Packard Series 1050) was set at the wavelength of 254 nm. The analysis was performed using a 3.9 x 300 mm stainless steel column packed with 10-micron particles (µBondapack C18, Alltech, Eke, Belgium). A composition of 60% v/v Acetonitrile, 40% v/v deionized water containing 0.2% v/v diethylamine was used as a mobile phase to elute ketoconazole. A 25-µL volume each of standard and sample solutions was injected, and ketoconazole was eluted isocratically using a flow rate of 1.0 mL/min at room temperature.

Standard Preparations

USP standard stock solution was prepared by weighing approximately 10 mg of ketoconazole into a 10-mL volumetric flask containing 8 mL of methanol. The volume was then adjusted to 10 mL with methanol, and this solution was used as the standard stock solution. Standard solutions with ketoconazole concentrations of 10, 50, 100, 150 and 200 µg/mL were prepared accordingly by diluting the standard stock solution with methanol. Each standard solution was filtered through a 0.45 µm membrane filter before injection onto the HPLC column.

Sample Preparations

About 0.1 mL of the microemulsion was transferred into a 10-mL volumetric flask and adjusted to volume with methanol. For the gel formulation, 0.1 g of gel was weighed into a 10-mL volumetric flask and adjusted to volume with methanol. Each sample solution was filtered through a 0.45 µm membrane filter before injection onto the HPLC column.

Drug Release Studies

The release profiles of ketoconazole from both microemulsion and gel formulations were generated from the percentage of ketoconazole released into the receptor chamber of the Franz Diffusion Apparatus (Crown Glass Company, Inc.) at each sampling time point. A test formulation (either microemulsion or gel formulation) at an equivalent amount of ketoconazole, approximately 10 mg, was placed on the Durapore Membrane (Millipore) in each donor chamber of the Franz Diffusion cell (n56). A 20% v/v of methanol in water containing 0.04% v/v of hydrochloric acid was used as a receptor medium for all tests. The temperature of the receptor medium was maintained at 37 6 0.2ºC throughout the experiment. Samples were taken at 1, 2, 3.5, 4, 5, 6, 7, 8, 22, and 24 hours and injected onto the HPLC column to determine the content of ketoconazole in the receptor medium at each time point.

RESULTS AND DISCUSSION

Pseudoternary Phase Diagram

In general, a pseudoternary phase diagram was constructed to determine the composition of an aqueous phase, an oil phase, and a surfactant:cosurfactant phase that will yield a microemulsion (transparent solution). Microemulsion preparation requires adjusting the HLB (hydrophilic lipophilic balance) value of the formulation by including a cosurfactant, which makes the polar solvent less hydrophilic. In this study, the common non-ionic cosurfactant in all the microemulsion systems was Plurol^® Oleique. It is a polyglyceryl-6-dioleate, which is a short chain alcohol with an HLB value of 10. Dehydrated alcohol was also incorporated into the microemulsion system as another cosurfactant to increase the curvature of the oil layer4. Labrafil^® M 1944 CS and Capmul^® MCM C-8 function as surfactants due to their self-emulsifying characteristics. For simplicity, the microemulsion is assumed to be a three-component system (oil, water, and the mixture of surfactant and cosurfactant). Any combination of the three components can be plotted as a percent on a pseudoternary phase diagram.4 Pseudoternary phase diagrams were constructed and the corresponding microemulsion regions were identified as shown in Figures 1 through 6. The results indicate that the area of the microemulsion region increased in the system containing dehydrated alcohol. Due to the low water-solubility of ketoconazole and the rigidity of oily surface, some amount of alcohol was added to dissolve the drug and increase the curvature of the oil layer4. The alcohol incorporated into the microemulsion system not only reduces the interfacial tension between the oil phase and the aqueous phase but also makes the lipophilic drug soluble in the system. However, alcohol evaporates easily; therefore, formulations containing alcohol may destabilize if their packages are not tightly closed.

According to the pseudoternary phase diagrams shown in Figures 1 through 6, the water-in-oil microemulsion systems of miconazole, ketoconazole, and itraconazole were obtained at oil concentrations ranging from 8.3 to 33.3% v/v and surfactant concentrations ranging from 16.7 to 75.8% v/v.

Microemulsion and Gel Formulation

A microemulsion of ketoconazole containing approximately 60% v/v surfactant and cosurfactant, 20% v/v olive oil, and 10% v/v dehydrated alcohol was prepared. In this case, alcohol was added to help solubilize ketoconazole in the system. Hydrochloric acid may be added, if necessary, to incorporate a sufficient amount of the drug (up to 2% w/v) into a microemulsion formulation. A gel formulation of ketoconazole containing about 3% w/w Carbopol^® 974P and 90% v/w dehydrated alcohol was also formulated. The release profiles of ketoconazole from both microemulsion and gel formulations were generated using Franz diffusion apparatus, and the corresponding release rates were calculated and evaluated for their in vitro release characteristics.

HPLC Analysis of Ketoconazole

To determine the amount of ketoconazole in the microemulsion and gel formulations, a series of standard solutions were prepared, filtered, and injected onto the HPLC column. Peak ketoconazole elution from the C-18 column occurred at about 5.1 minutes (Figure 7). The peak area obtained from a series of standard solutions and the corresponding concentrations were plotted and used as a standard curve. Figure 8 shows the standard curve of ketoconazole with a correlation coefficient (R²) of 0.999.

Release Studies

Each of the microemulsion and gel formulations, with the equivalent amount of ketoconazole, was placed separately on the membrane sandwiched between donor chamber and receptor chamber of a Franz diffusion cell (n56). The amount of ketoconazole released at each sampling time point was determined by HPLC, and the results are reported in Table 1. The release profiles of ketoconazole from each formulation were constructed by plotting the percentage of ketoconazole released against time in hours as shown in Figures 9 through 11.

As shown in the figures, the release rates of ketoconazole from both formulations reached steady state at approximately 22 hours, and the percentage of ketoconazole retained in both formulations was about the same. As shown in Figure 11, the release rate of ketoconazole from microemulsion at the beginning was slightly slower than the release rate from the gel formulation. This might be because some amount of the drug has partitioned into the oil phase. Therefore, it takes some time for the drug to partition out of the oil phase into the receptor medium. Since the gel formulation has no partitioning of the drug, the release rate is slightly faster. To compare the release rates of the drug from both formulations, the rate of drug released was calculated using the slope of each release profile (R² . 0.98) from zero to the 4-hour point. The results indicate ketoconazole release rates from microemulsion and gel formulation of 766.8 and 677.6 µg/hour, respectively.

No significant difference between the release rates of ketoconazole from both formulations was found (P . 0.05). The gel formulation does not contain a surfactant; hence, there is no partition of drug. Although partition of the drug to the oil phase did not occur in the gel formulation as in a microemulsion system, ketoconazole release from the cross-link of the carbopol polymer requires some lag time. This might be one reason that the ketoconazole release profiles from the microemulsion and the gel formulation were not much different (Figure 11). Additionally, the percentages of alcohol used in the gel formulation and the microemulsion were significantly different (90% v/w in gel versus 10% v/v in microemulsion). Alcohol can help break down the bonds between the cross-links of the polymer and thus facilitate the release of drug from the polymer. This may also contribute to the similar release rates. This also implies a microemulsion delivery system for a highly lipophilic drug such as ketoconazole would require a very low amount of alcohol, but a gel formulation made for the same concentration would require a very high amount of alcohol.

One major advantage of a microemulsion over traditional emulsion is the ease of preparation, especially with regard to large batch manufacturing. In general, several factors have to be considered for a coarse emulsion, such as intensity and duration of mixing, emulsification time (including rate and temperature), order of adding and mixing each ingredient, heating and cooling rates, and so on. Because microemulsion forms spontaneously with only gentle agitation, some of these factors can be avoided. Another advantage is the physical stability of the formulation. In a traditional emulsion system, the larger droplet size favors a decrease in the surface area, which in turn favors a decrease in the free energy of the system.4 However, a microemulsion system has lower interfacial tension between water and oil due to the presence of surfactant and cosurfactant; therefore, the surface area of the dispersed droplets is very large.4,12 The lower interfacial tension compensates the dispersion entropy; therefore, the microemulsion system becomes thermodynamically stable due to the low free energy of the system.

As mentioned earlier, a microemulsion normally contains droplet size diameters ranging from 1 to 100 µm; they appear transparent or clear because the light-scattering capability of the small droplets is weak.4 A microemulsion has a more elegant appearance, which may increase patient compliance. In addition, the presence of surfactant and cosurfactant in the system enables achievement of high drug loading, especially for lipophilic compounds. Regarding bioavailabiity,4,10,13-24 a lipophilic drug in a microemulsion system has better penetration of the physiological membrane due to several factors. These include the enhancing effect of surfactants, the proper balance between hydrophilicity and lipophilicity of the formulation, smaller particle size, the in vivo partition coefficient of the drug between the two immiscible phases, the presence of drug in an emulsified form, site of absorption, metabolism of the oil in the microemulsion, effect of lipid vehicle on gastric emptying, and drug solubility in the microemulsion excipients.

CONCLUSIONS

Microemulsions are potential drug delivery systems for several applications especially oral, nasal, topical, and transdermal. They represent an easy to manufacture, thermodynamically stable system with improved bioavailability, less alcohol content, and a transparent and elegant appearance. However, the toxicity of the surfactant and cosurfactant has to be investigated thoroughly for oral and nasal applications, especially for drugs requiring chronic use.9

REFERENCES

 1. Attwood D: Microemulsions, in Kreuter H (ed): Colloidal Drug Delivery Systems. New York, Marcel Decker, 1994, p 31.

 2. Ogino K, Abe M: Microemulsion formation with some typical surfactants, in Matijevic E (ed): Surface and Colloid Science. New York, Plenum Press, 1993, p 85.

 3. Paul BK, Moulik SP: Microemulsions: An overview. J Disp Sci 18(4):301, 1997.

 4. Tenjarla SN: Microemulsions: An overview and pharmaceutical applications. Critical Reviews ^TM in Therapeutic Drug Carrier Systems 16:461-521, 1999.

 5. Jayakrishnan A, Kalaiarasi K, Shah DO: Microemulsions: Evolving technologies for cosmetic application. J Soc Cosmetic Chem 34:335, 1983.

 6. Hoar TP, Schulman JH: Transparent water-in-oil dispersions: The oleopathic hydromicelle. Nature 102,152, 1943.

 7. Prince: Microemulsions, in Theory and Practice. New York, Academic Press, 1977.

 8. Prince: Microemulsions. J Soc Cosmetic Chem 21:193, 1970.

 9. Baviere, et al: The influence of alcohols on microemulsion composition. J Colloid Interf Sci 81:266, 1981.

10. Lieberman HA, Rieger MM, Banker GS: Pharmaceutical Dosage Forms: Disperse Systems, second ed, Vol 1. New York, Marcel Decker, 1996, 211-281, 315-370.

11. Kovarik JM, Muller EA, Van Bree JB, Tetzloff W, and Kathz K: Reduced inter- and intra-individual variability in cyclosporine pharmacokinetics from a microemulsion formulation. J Pharm Sci 83:444, 1994.

12. De Gennes PG, Taupin C: Microemulsions and the flexibility of oil/water interfaces. J Phys Chem 86:2294, 1982.

13. Osborne DW, Ward AJ, O'Neil K: Microemulsions as delivery of vehicles: 1. Characterization of a model system. Drug Dev Ind Pharm 14:1203, 1988.

14. Friedman D, Benita S: A mathematical model for drug release from o/w emulsions: Application to controlled release morphine emulsions. Drug Dev Ind Pharm 13:2067, 1987.

15. Gallarate M, Gasco MR, Trotta M: Influence of octanoic acid on membrane permeability of timolol from solutions and from microemulsions. Acta Pharm Technol 34:102, 1988.

16. Kriwet K, Muller-Goymann CC: Diclofenac release from phospholipid drug systems and permeation through excised human stratum corneum. Int J Pharm 125:231, 1995.

17. Ktistis G: Effect of polysorbate 80 and sorbitol concentration on in vitro release of indomethacin from microemulsions. J Disp Sci 18(1):49, 1997.

18. Osborne DW, Ward AJ, O'Neil K: Microemulsions as topical delivery vehicles: In vitro transdermal studies of a model hydrophilic drug. J Pharm Pharmacol 43:451, 1991.

19. Fevrier F, Bobin MF, Lafforgue C, Martii MC: Advances in microemulsions and transepidermal penetration of tyrosine. STP Pharm Sci 1:60, 1991.

20. Tenjarla SN, Puranajoti P, Kasina R, Mandal T: Preparation, characterization, and evaluation of miconazole-cyclodextrin complexes for improved oral and topical delivery. J Pharm Sci 87:425-429, 1998.

21. Tarr B, Yalkowsky SH: Enhanced intestinal absorption of cyclosporine in rats through the reduction of emulsion droplet size. Pharm Res 6:40, 1989.

22. Ritschel WA, Ritschel GB, Sabouni A, Wolochuk D, Schroeder T: Study on the peroral absorption of the endekapeptide cyclosporin A. Meth Find E Clin Pharm 11:281, 1989.

23. Constantinides PP, Scalart JP, Lancaster C, Marcello J, Marks G, Ellens H, Smith PL: Formulation and intestinal absorption enhancement evaluation of water-in-oil microemulsions incorporating medium-chain glycerides. Pharm Res 11:385, 1994.

24. Constantinides PP, Lancaster CM, Marcello J, et al: Enhanced intestinal absorption of an RGD peptide from water-in-oil microemulsions of different composition and particle size. J Contr Rel 34:109, 1995.

 

 

 

 

Table 1. Comparison of Ketoconazole Release from Microemulsion and Gel Formulation

Time (hr)       Ketoconazole Released, %     Microemulsion Gel    (Mean ± SD, n = 6)      (Mean ± SD, n = 6)

                         1 8.2 ± 2.70 11.16 ± 2.59

                         2            15.15 ± 3.13          19.16 ± 2.89

                      3.5-4   25.02 ± 3.01 (3.5 hr) 31.57 ± 3.10 (4 hr)

                         5            33.66 ± 3.01          34.50 ± 2.95

                         7            44.04 ± 4.24          40.55 ± 2.09

                         8            47.01 ± 5.51          42.64 ± 1.98

                        22            73.59 ± 2.28          64.77 ± 2.87

                        24            73.74 ± 1.91       65.29 ± 4.81