Evaluating the Impact of Fatty Alcohols on Permeation of Clotrimazole from Topical Creams

December 3, 2020
Supriya Bhide

Supriya Bhide is a graduate student at the Department of Pharmaceutics and Drug Delivery, University of Mississippi, MS.

Apoorva Panda

Apoorva Panda is a graduate student at the Department of Pharmaceutics and Drug Delivery, University of Mississippi, MS.

S. Rangappa

S. Rangappa is a research scientist at the Department of Pharmaceutics and Drug Delivery, University of Mississippi, MS.

Abhishek Shettar

Abhishek Shettar is a research scientist at the Department of Pharmaceutics and Drug Delivery, University of Mississippi, MS.

M.A. Repka

M.A. Repka is professor and chair at the Department of Pharmaceutics and Drug Delivery, University of Mississippi, MS.

Rosa Maria Badani Prado

Rosa Maria Badani Prado is a graduate student at the Dave C. Swalm School of Chemical Engineering, Mississippi State University, MS.

S. Kundu

S. Kundu is associate professor at the Dave C. Swalm School of Chemical Engineering, Mississippi State University, MS.

Norman Richardson

Norman Richardson is technical sales manager at BASF Pharma Solutions, Florham Park, NJ.

S. Narasimha Murthy

S. Narasimha Murthy is professor of Pharmaceutics and Drug Delivery at the University of Mississippi, MS, founder-director of the Institute for Drug Delivery and Biomedical Research, Bangalore, Karnataka, India, and chief scientific officer, Topical Products Testing LLC, University, MS.

Pharmaceutical Technology, Pharmaceutical Technology-12-02-2020, Volume 44, Issue 12
Page Number: 24–31

Study results indicate that fatty alcohols may plausibly enhance the skin permeation of clotrimazole.

Pharmaceutical creams are one of the most common types of formulations used for the topical delivery of drugs. Creams are either oil in water (o/w) or water in oil (w/o) type emulsions with a semisolid consistency (1). They are generally formulated using ingredients like emollients, viscosity builders, emulsifying agents, and permeation enhancers. Fatty alcohols have been reported to play different roles in a cream formulation. For example, fatty alcohols are used as viscosity building agents, stabilizing agents, pH modifiers, and counter ions. They also could be used to enhance the solubility of drugs in a nonpolar medium. Fatty alcohols are known to play the role of permeation enhancers for various drugs (2,3).

In the present study, topical o/w cream formulations, incorporated with a poorly permeable model molecule clotrimazole, were prepared using different fatty alcohols. Clotrimazole is an imidazole derivative and is a broad-spectrum anti-fungal agent (4). All the formulations were identical in composition except that different fatty alcohols were used in different products. The objective of the study was to investigate the influence of fatty alcohols on the permeation of clotrimazole from these formulations. The permeation studies revealed an interesting correlation between the physicochemical characteristics of the fatty alcohols and the rate of delivery of clotrimazole.

Materials and methods

Materials. Clotrimazole (≥ 98%), an antifungal agent was gifted from Yarrow Chem Products (Mumbai, India). Kolliwax CSA 50 (cetostearyl alcohol 50), Kolliwax CSA 70 (cetostearyl alcohol 70), Kolliwax MA (myristyl alcohol), Kolliwax CA (cetyl alcohol), Kolliwax SA (stearyl alcohol), Kollisolv PG (propylene glycol), Kollicream OD (octyldodecanol) and Kolliphor CS 20 (macrogol cetostearyl ether 20) were gifted by BASF (Florham Park, NJ). Euxyl 320 and Nile Red were purchased from Sigma Aldrich (St. Louis, MO). High-performance liquid chromatography (HPLC)-grade solvents, like methanol and ethanol, were purchased from Fisher Chemicals (Pittsburgh, PA). Cryopreserved Human cadaver dermatomed skin was obtained from Science Care (Phoenix, AZ).

Preparation of clotrimazole containing topical o/w cream formulation. Five different fatty alcohols were considered for this study, such as cetostearyl alcohol 50 (CSA 50), cetostearyl alcohol 70 (CSA 70), cetyl alcohol (CA), myristyl alcohol (MA), and stearyl alcohol (SA), respectively. The formula for the cream preparation was used as mentioned in Table I. The aqueous phase and the oil phase were heated and maintained at 60 °C. The oil phase was added to the aqueous phase and they were homogenized for 30 minutes at 3000 RPM in Silverson Homogenizer L5M-A (Silverson Machines, East Longmeadow, MA). Propylene glycol along with the clotrimazole was added near to the end of the homogenization process. All formulations were subjected to a cooling program 60 °C to 25 °C in 20 minutes using the VWR chiller system (VWR International, Radnor, PA).

Measurement of melting point using DSC. The melting point of all five fatty alcohols were determined using differential scanning calorimetry (DSC) using DSC 25 (TA Instruments, New Castle, DE). The melting point of the API was also determined. Approximately 7 mg of powdered sample was placed in Tzero Hermetic Pan and was sealed with Tzero lid (TA Instruments). The pan was placed in the sample chamber along with the reference pan and analysis was done by using the ramp method with the rate of 10 °C/min.

Characterization of quality attributes. To assess the impact of different quality attributes on the permeation of drugs from topical formulations at infinite dose conditions, different tests such as pH, globule size distribution, viscosity at a low shear rate, and phase distribution of drugs, were performed and evaluated.

pH measurement. The pH of the creams was measured using a Mettler Toledo (Columbus, OH) pH meter. The probes used in the study were Mettler Toledo InLab Micro electrodes. The pH meter was calibrated using standard buffers with a known pH of 4.0, 7.0, and 10.0, respectively. The pH of each cream formulation was measured thrice. In between every individual measurement, the accuracy of pH measurement was checked using buffer standard of pH 4.0 and using buffer standard of pH 10.0.

Phase separation and drug quantitation. One gram of cream sample in Eppendorf tubes was subjected to centrifugation at 50,000 RPM in the Beckman Coulter centrifugation instrument (Beckman Coulter, Brea, CA). All formulations were centrifuged for 8 h. The separated oil phase and aqueous phase were weighed separately. The 2 mg of the oil phase was diluted to 50 mL with methanol and the aqueous phase was diluted by adding 2 mL of methanol. The samples were subjected to HPLC analysis. The distribution of the drug in both phases was evaluated.

Globule size measurement. The globule size of the cream formulation was measured by using Axio Cam HR3 (Carl Zeiss Microscopy, Germany) bright-field microscopy at a magnification of 125x. A thin smear of the cream was prepared with a coverslip on a glass slide and observed immediately under the microscope. For each cream, the mean diameter of randomly distributed 100 globules was measured using the Zen Lite software (Carl Zeiss Microscopy). The globule size measurement was done by calculating d10, d50, and d90 values for each cream.

Rheological study. The rheology of all cream samples was studied using TA Instruments HR2 Rheometer at 32 °C with a 20 mm parallel plate with 600-grit sandpaper and solvent trap. Viscosity of each cream was measured at shear rate 0.01 (1/s).

In-vitro skin permeation study. The in-vitro skin permeation study was performed using Franz diffusion cells with an effective diffusion area of 1.77 cm2. Cryopreserved human cadaver dermatomed skin devoid of any disease was obtained from Science Care and was stored in water-impermeable plastic bags at below –80 °C until the time of use. Before use, the skin was thawed at room temperature for 10 min and was cut in sections large enough to fit the 1.77 cm2 active permeation area. A hairless thigh region of 250 µm was sandwiched between the donor and receiver medium with the stratum corneum (SC) side facing the donor compartment. The skin barrier integrity was checked before starting the experiment by measuring the resistance at a frequency of 1 kHz and a low voltage of 100 mV. The skin having a resistance value greater than 10 KΩ/cm2 was only used for permeation studies. The receiver compartment (7 mL) was filled with 2% Tween 80 in phosphate-buffered saline (PBS) solution and was maintained at 37 °C. As clotrimazole is insoluble in water, Tween 80 was used as a solubilizer in the release medium within the receiver compartment to maintain the sink conditions (5). For the skin permeation study, an infinite dose of the formulation was placed on the skin using a syringe. During the in-vitro permeation testing (IVPT) studies, the samples were withdrawn from the receiver compartment at the time points of 2 h, 4 h, 8 h, 10 h, 12 h, and replaced by equal quantities of fresh medium. The amount of clotrimazole in the aliquots was quantified and determined using HPLC.

Determination of saturation solubility of clotrimazole in different fatty alcohols. Different percentages (0, 10, 25, 50, and 75% w/w) of clotrimazole were added to 5 different fatty alcohols (Kolliwax CSA 50, CSA 70, MA, CA, and SA) contained in glass vials. The drug-excipient mixture in glass vials was heated on a hot plate at a temperature slightly above the melting point of the excipient to prepare a molten state mixture. After 30 minutes of mixing drug-excipient, the molten mixture was transferred into the DSC pans. The drug-excipient mixture in the molten state was weighed inside the aluminum DSC pan and the pan was sealed with the aluminum hermetic lid tightly. DSC pans were allowed to equilibrate for 1 h. After 1 h, DSC analysis was performed over the temperature range of 20 °C to 200 °C at 20 °C/min increments. DSC was performed using the TA instruments discovery series DSC 25. TRIOS software was used to analyze the enthalpy of melting.

Analytical method. An isocratic HPLC method was used for the quantification of clotrimazole (6). The analysis of clotrimazole containing samples was performed using a Waters HPLC system (Waters 600 Controller, Milford, MA) equipped with a 600-pump unit, a 717 plus autosampler with an injection valve with a sample loop of 50 µL, and a 2487 dual absorbance UV detector. The mobile phase comprised of a 10 mM solution of dibasic potassium phosphate and acetonitrile. (3:1 v/v) with a flow rate of 1 mL/min and an injection volume of 20 µL.

Results and discussion

Melting point determination using DSC. The melting point was determined by using the DSC technique. Melting points for different fatty alcohols were found to be as shown in Figure 1. The physicochemical characteristics of different fatty alcohols shown in Table II were pooled up from BASF technical data and literature reports (7–10). The melting point was determined by DSC. It is evident from the DSC graph that myristyl, cetyl, and stearyl alcohols all had a single endothermic peak of enthalpy of fusion. As expected, cetostearyl alcohols 50 and 70 both showed a broader peak or a bimodal peak because they are mixtures of cetyl and stearyl alcohols in different ratios. Therefore, an average value of melting point and log POct/water based on their molar ratios was considered for comparison and correlations.

Characterization of quality attributes. As mentioned in Table III, the amount of drug in the aqueous phase is below detectable levels in the cream. Moreover, the pKa of clotrimazole is known to be 4.7 as per the literature (11), so if any dissolved clotrimazole were present it would be almost 96–99% unionized in the aqueous phase at pH 6–7.86. Therefore, as the pH differences did not lead to any significant difference in the extent of the percent ionized/unionized ratio, it is not likely the pH differences would affect the permeation of drugs from the topical cream products prepared with different fatty alcohols.

The drug distribution in different phases is one of the major factors that could influence transdermal absorption. The creams were separated into aqueous and lipid phases by the process of ultracentrifugation. The amount of clotrimazole was negligible and below detectable levels in the aqueous phase of the product. The entire drug was essentially found in the lipid phase. The amount of drug in the oil phase was between 8.6–10.3 mg, which essentially is a narrow range. The drug concentration in the lipid phase could translate into differences in thermodynamic activity. It is the thermodynamic potential that drives drug diffusion into and across skin and not only the drug concentration in the applied product. In some cases, lipid phases were saturated (degree of saturation of 1) as we could see some drug crystals in creams prepared with cetostearyl alcohol. Therefore, our observations suggest that although the amount of drug in the lipid phase differed marginally, the degree of saturation that translates into the thermodynamic force remained identical across all the products.

The globule size distribution for the cream formulations was determined by microscopy. The photomicrographs of all the cream products are shown in Figure 2. The globule size distribution could have a significant impact on the permeation since the contact specific area increases with decrease in globule size. However, as shown in Figure 2 and Table III, the d10, d50, and d90 values did not differ significantly for all cream formulations under the study. The creams containing SA did not have any globules as it did not form a rigid microstructure to stabilize the dispersed phase. As all products were found to be shear thinning. The viscosity at low shear values represents the inherent viscosity of the formulations. As mentioned in Table III, the values ranged between 1200–3100. A significant difference in the viscosity was observed and could potentially influence the permeation of drugs. Decreasing rank order of fatty acid creams concerning their viscosity was CA>CSA50>CSA70>MA>SA. This did not correlate with the increasing transdermal flux of clotrimazole from different topical creams (MA>CA>CSA50>CSA70>SA) as one would expect if there was a predominant influence of viscosity on the permeation.

In-vitro skin permeation study. Finite dose conditions are generally recommended for the evaluation of topical products as they are near the therapeutic dose of the product. However, at finite dose conditions, several factors would come into play in determining the performance of topical products. In infinite dose studies, the dose used in the study is generally several-fold higher than the clinically relevant dose. The use of infinite dose eliminates the influence of the factors associated with evaporative metamorphosis. Hence, the permeation studies in the present project were performed with infinite dose in the donor under the occluded condition to prevent loss of contents due to evaporation. The permeation studies were performed on two cadaver donors (n=3 on each donor) to accommodate all the products on the same donors. The cumulative permeation- time profile of six replicates with standard error of the mean (SEM) is represented in Figure 3. The control trials were performed using clotrimazole solution in 1% Tween-80. The permeation from the control was below detectable quantities. The transdermal flux of clotrimazole from different topical creams was in the following rank order: MA>CA>CSA50>CSA70>SA.

As discussed earlier, most of the critical quality attributes did not differ significantly across the creams. Therefore, one can conclude that the differences in the transdermal drug flux are not attributable to significant differences in the quality attributes of the products. The other reason to which differences in the performance of the products can be attributed is the interaction of excipients with the skin, either altering its permeability by the way of disorganizing the organized lipid structures or by the way of increasing the solubility of the API in the lipid domains of the SC.

Fatty alcohols are reported to enhance the permeation of drugs by the way of penetrating the lipid barriers and/or by disorganizing the lipid bilayers (12). Given the fact that the clotrimazole predominantly exists in the lipid phase of the cream and is highly lipophilic, (log P = 6.1) (4), one can anticipate the drug to partition into the SC lipids and propagate through the SC layers. The penetration of SC lipid domains can only have a favorable effect on the drug permeation if it does not affect the pathways of absorption of clotrimazole. If the drug was hydrophilic, disruption of lipid domains would have facilitated the drug’s permeation. Therefore, it was hypothesized that the differences in permeation profile from different fatty alcohol containing topical creams are attributable to the relative ability of fatty alcohols to enhancing the partitioning and solubility of the drug in the stratum corneum.

There are several reports regarding the penetrability of fatty alcohols into the SC. It is known from earlier reports that the physicochemical characteristics of substrates such as melting point, log P, and molecular weight would play a major role in determining the extent of penetration of any substrate into the skin (13,14). In this study, the fatty alcohols that we used did not differ significantly in their molecular weight. The only property that significantly differed was log P values as shown in Table II. Therefore, the steady-state flux of the drug was correlated with the log P of the fatty alcohol as shown in Figure 4. As shown in Figure 4, there was a significantly good correlation between the two variables with a correlation coefficient of R2 = 0.8745. However, the negative slope indicated an inverse relationship between the partition coefficient of enhancer and the permeation flux of the drug.

Melting point is another physical property that correlates well with the skin permeability of molecules. As mentioned earlier in Table II, the melting points of the fatty alcohols used in this project were found to be in the range between 38–54 °C. Therefore, just to understand the trend in the relationship between the melting point and the steady-state flux of clotrimazole, the graph of steady-state flux values of clotrimazole from different topical creams vs melting point of fatty alcohols was plotted, as shown in Figure 5. The correlation coefficient was found to be 0.9371. This relationship supports the interpretation that the lower the melting point, the better is the ability to penetrate the SC.

From all the above results, it is evident that the fatty alcohols have a significant impact on the drug permeation from o/w creams. Based on the literature reports and our observation, it is likely that the fatty alcohols penetrate the skin lipids and act as a solvent for the API to form a reservoir and permeate across the skin eventually. This means that the API should be soluble in the fatty alcohols in significant quantities.

To ascertain this assumption, the solubility of the drug in the fatty alcohol was investigated using DSC. All the fatty alcohols were able to dissolve clotrimazole in significant amounts (about 50% and over). When API was mixed with the fatty alcohol at a 1:1 ratio, none of the mixtures showed the endothermic peak for the drug except SA (Figure 6A). Whereas, in the case of 3:1 mixture of drug:fatty alcohol mixture, the endothermic peak of the API was observed in case of all the fatty alcohols (Figure 6B). The enthalpy of fusion of the drug differed in different fatty alcohols. As the enthalpy of fusion is a function of the mole fraction of the drug undissolved in the mixture, one can at least approximate the solubility of the drug in the fatty alcohol relatively. Based on the enthalpy of fusion of the undissolved drug, one can conclude that the drug was soluble to different extents in different fatty alcohols. For example, the solubility of API highest in MA and lowest in SA. This is correlating well with the transdermal flux data, in which the rate and extent of permeation of relatively higher compared to other creams, from the cream containing MA. Whereas, the transdermal flux was least from topical cream with SA and so was the API solubility in SA as well. As shown in Table IV, it is noteworthy that the rank order of transdermal flux of clotrimazole from different fatty alcohol creams matched well with the extent of the solubility of the drug in the fatty alcohol supporting our hypothesis that the fatty alcohol acted as a solvent/carrier for the API in the SC. The difference in transdermal flux across different products could be either due to difference in the extent of the solubility of the drug in the fatty alcohol or due to different extents of penetration of fatty alcohol or both.


The difference in quality attributes across the topical products prepared using different fatty alcohols could not be solely attributed to the difference in the performance of the topical products of clotrimazole. A good correlation was found between the steady-state flux of clotrimazole and the physicochemical characteristics of the chosen fatty alcohols. Observations on the solubility of clotrimazole in the different fatty alcohol excipients and the different log P of the fatty alcohol excipients suggests that the penetration of fatty alcohol into the SC and solvent behaviour for the API, were found to be the predominant mechanism of permeation of clotrimazole from the topical creams with the specified compositions.


The authors acknowledge BASF Pharma Solutions, Florham Park, NJ for the funding support and supply of lipid excipients for the project.


Kolliwax, Kollisolv, Kollicream, and Kolliphor are all registered trademarks of BASF.


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About the Authors

Supriya Bhide and Apoorva Panda are both graduate students, S. Rangappa and Abhishek Shettar are both research scientists, and M.A. Repka is professor and chair, all at the Department of Pharmaceutics and Drug Delivery, University of Mississippi, MS; Rosa Maria Badani Prado is a graduate student, and S. Kundu is associate professor, both at the Dave C. Swalm School of Chemical Engineering, Mississippi State University, MS; Norman Richardson is technical sales manager at BASF Pharma Solutions, Florham Park, NJ; and S. Narasimha Murthy* is professor of Pharmaceutics and Drug Delivery at the University of Mississippi, MS, founder-director of the Institute for Drug Delivery and Biomedical Research, Bangalore, Karnataka, India, and chief scientific officer, Topical Products Testing LLC, University, MS, murthygroup@gmail.com, testingtopicals@gmail.com.

*To whom all correspondence should be addressed.

Article Details

Pharmaceutical Technology
Vol. 44, No. 12
December 2020
Pages: 24–31


When referring to this article, please cite it as S. Bhide, et al., “Evaluating the Impact of Fatty Alcohols on Permeation of Clotrimazole from Topical Creams,” Pharmaceutical Technology 44 (12) 2020.

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