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Summary: Five fatty acids (oleic, linoleic, myristic, lauric and capric) were incorporated in 10% (w/w) into ointment formulation and their influence on model drug tolnaftate release in vitro and enhancing effect on tolnaftate penetration into epidermis and dermis of human skin ex vivo were investigated. The prepared ointments were tested for homogeneity, pH and rheological properties. In vitro release studies and ex vivo skin penetration experiments were carried out using Hanson and Bronaugh-type flow-through diffusion cells, respectively. Tolnaftate cumulative amount liberated from semisolids was assayed using UV-Vis spectrophotometer. After in vitro skin penetration studies, appropriately extracted human skin layers were analysed for tolnaftate content using a validated HPLC method. Statistical analysis revealed that release rate of tolnaftate from control ointment and ointments with fatty acids was not significantly different and only 7.34 - 8.98% of drug was liberated into an acceptor medium after 6 hours. Tolnaftate amount penetrating into 1 cm2 of epidermis from ointments having oleic, linoleic, myristic and lauric acids was significantly greater (P < 0.05) than from the control ointment. Enhancing ratios of these fatty acids for tolnaftate penetration into epidermis ranged from 1.48 to 1.75. In conclusion, fatty acids did not increase the liberation of tolnaftate from ointment formulation, but exerted their enhancing effect on tolnaftate penetration into human epidermis in vitro. Results from in vitro release experiments do not forecast the situation on the skin in vitro, if chemical penetration enhancers are incorporated into the formulation.
Intact stratum corneum (SC), the outermost layer of the skin, functions as the main barrier for the penetration to and permeation through the skin for drug molecules. In the SC, corneocytes are surrounded by continuous matrix of neutral lipids. Independently of the molecule passive diffusion by the polar transcellular or lipoidal intercellular route, it has to diffuse through the lipid layers in order to cross the barrier. Therefore, alteration of SC lipid organization by disordering the hydrophobic lipid tails influences the rate and extent of SC resistance to the diffusion process. This reversible alteration might be achieved by adding variuos lipophilic chemical penetration enhancers (CPEs), which are pharmaceutically acceptable, to the drug formulation.
The SC lipids, which are arranged into lamellar bilayers (1), are mainly composed of ceramides (40-50%), free fatty acids (10-15%) and cholesterol (25%) in an approximately 1 : 0.9 : 0.4 mol ratio (2-7). Lipophilic CPEs interact with lipid hydrocarbon chains, disrupt the order of lipid packing and increase fluidization in the bilayers. In this way CPEs are capable to locally enhance the penetration of active compounds into SC and through it (8).
Fatty acids belong to the lipophilic CPEs and their penetration enhancing properties are related to the hydrocarbon chain length and the presence of double bonds. Obviously, the vehicle in which the CPE is dissolved also influences the magnitude of enhancement (8). Oleic acid is considered to be the most potential CPE among fatty acids due to its cis-monounsaturation and â€žkink" formation (2). Oleic acid creates a highly permeable, fluid-like phase within the SC lipids (9).
Tolnaftate was chosen as a model drug for the investigation of the enhancing effect of five fatty acids (oleic, linoleic, myristic, lauric and capric) on its penetration from 1% (w/w) ointment formulation into epidermis and dermis of human skin after in vitro skin penetration experiments. Tolnaftate is a topical antifungal drug of thiocarbamate class, acting against dermatophytes, which mainly invade the superficial layers of the skin. Tolnaftate is a lipophilic compound (XLogP = 5.5) of low molecular weight (307.4 Da), having weak basic properties and melting point of 109 °C-112 °C (Eur. Pharm.6.0; 01/2008:1158).
MATERIALS AND METHODS
Chemicals and reagents
Tolnaftate (O-naphthalen-2-yl methyl(3-methylphenyl)thiocarbamate; Eur. Pharm. 6.0) with purity of 99.7% was a gift from pharmaceutical company Sanitas AB (Kaunas, Lithuania). Polyethylene glycol 400 (PEG 400) was purchased from Carl Roth GmbH (Karlsruhe, Germany). Polyethylene glycol 1500 (PEG 1500) and capric acid (decanoic acid, C10:0) were obtained from Merck Schuchardt OHG (Hohenbrunn, Germany); white vaseline, anhydrous lanolin, oleic acid (cis-9-octadecenoic acid, C18:1) and methanol (Chromasolv®) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Ethanol (96.3%) was obtained from Stumbras AB (Kaunas, Lithuania). Linoleic acid (cis, cis-9,12-octadecadienoic acid, C18:2), myristic acid (tetradecanoic acid, C14:0) and lauric acid (dodecanoic acid, C12:0) were purchased from Alfa Aesar GmbH (Karlsruhe, Germany). Sodium azide (NaN3) was obtained from POCh (Gliwice, Poland). All other reagents were of analytical grade.
Human skin preparation
Caucasian women (of age 25-40) abdominal skin was obtained after cosmetic surgery in the Department of Reconstructive and Plastic Surgery, Hospital of Kaunas University of Medicine. The studies with human skin were approved by Kaunas Region Bioethical Committee. The appropriately treated skin was wrapped in aluminum foil and stored at -20 °C for not longer than 6 months before use.
The prepared ointments were evaluated microscopically, using Motic® B3-series (Meyer Instruments, Texas, USA) with integrated Moticam 1000 digital microscope camera. pH values and rheological properties were assessed using portable pH-meter HD2105.1 (Delta OHM, Italy) and rotary viscometer ST-2010 (JP Selecta S.A., Spain), respectively.
In vitro release studies were carried out using an assembly constructed in our laboratory. Hanson vertical diffusion cells, having semisolid formulation deposited in the donor chamber and covered by Cuprophane® cellulose-dialysis membrane (Medicell International Ltd, London, United Kingdom), were inserted into the glass beaker and overlayered with an appropriate acceptor medium. The glass beaker was put into the thermostated water bath (Grant GD120, Grant Instruments Ltd, Cambridge, Great Britain) and efficient mixing of the acceptor medium was achieved using Heidolph RZR 2021 mechanical stirrer with propeller-type impeller (Heidolph UK, Essex, United Kingdom).
The equipment for in vitro skin penetration experiments consisted of thermostated water bath (Grant GD120, Grant Instruments Ltd, Cambridge, Great Britain), peristaltic pump (Masterflex® L/S® pump drive (model type 7524-45) with multichannel pump head (model type 07535-08), Cole-Parmer Instrument Co., Illinois, USA) and Bronaugh-type flow-through diffusion cells, made from Teflon. Extraction procedure of human skin layers was performed in the Bandelin Sonorex Digitec Ultrasonic Bath (DT 156, Bandelin electronic GmbH & Co. KG, Berlin, Germany), having ultrasonic peak output of 640 W.
UV spectrophotometrical analysis was done using UV/Vis Unicam Helios-Î± spectrophotometer (Unicam, Analitical Technology INC, Cambridge, Great Britain).
High peformance liquid chromatography (HPLC) analysis was carried out using Shimadzu Liquid Chromatograph (Shimadzu Corporation, Kyoto, Japan) equipped with two Shimadzu LC-10AD VP pumps, degasser DGU-14A, auto injector SIL-10AD VP, system controller SCL-10A VP, UV-vis detector SPD-10A and column oven CTO-10AC VP.
Preparation of ointments
1% (w/w) tolnaftate ointments were prepared by dissolving tolnaftate in PEG 400 and incorporating the solution into the absorption base (fusion technique). The absorption base was composed of white vaseline, anhydrous lanolin and PEG 1500. These components were melted together (70 °C) on a steam bath and the hot (70 °C) solution of tolnaftate in PEG 400 was slowly added. Stirring was maintained until congealed.
Ointments having 10% (w/w) of different fatty acids were prepared in the same manner as the control ointment except that an appropriate amount of fatty acid was melted together with the components of the absorption base (myristic, lauric and capric acids) or poured into the molten phase (oleic and linoleic acids). The precise composition of the ointments prepared is presented in Table 1.
Physico-chemical properties of prepared ointments
Homogeneity, pH and rheological properties of prepared ointments were evaluated. For homogeneity assessment, the ointments were smeared on the glass slide and examined under the microscope. For pH evaluation, each ointment (2.5 g) was heated (50 °C) in 50 ml of distilled water and stirred on the magnetic stirrer for 50 min. Cooled solutions were filtered through ashless filter paper (Albet® 145, pore diameter 7-11 µm) and their pH was evaluated using pH-meter. Dynamic viscosity Î· (Pa·s) of each ointment was assessed at ambient temperature (21 °C) using rotary viscometer coupled with cylindrical spindle (R7), rotating in the selected speed interval of 1.5-10.0 rpm (each measurement lasted for 50 sec, n=3).
In vitro release studies
In vitro release tests were carried out using Hanson vertical diffusion cells with effective diffusion area of 1.77 cm2. Accurately weight amount of ointment (approximately 0.8 g) was spread evenly in the donor compartment and covered with the Cuprophan® cellulose-dialysis membrane, which was pre-hydrated in distilled water at 37 °C for 24 hours. 100 ml of acceptor medium, composed of 50/50 PEG 400/96.3% ethanol, provided sink conditions (solubility of tolnaftate at saturation was determined to be 18.75 ± 1.06 mg/ml, n=3). The acceptor medium was mixed at 100 rpm, using mechanical stirrer with propeller-type impeller. The whole assembly was kept in the water bath (37 °C). Aliquots of 3 ml were collected after 1, 2, 4 and 6 hours. After withdrawal of an aliquot, an equal volume (3 ml) of an acceptor medium (37 °C) was immediately added to maintain a constant volume. The samples were analyzed using UV spectrophotometer.
In vitro skin penetration experiments
Bronaugh-type flow-through diffusion cells, mounted with full-thickness human skin (the diffusional area being 0.64 cm2) and having the receptor volume of 0.13 ml, were placed on the heating block (37 °C). 12 hours equilibration period was followed, circulating physiological solution having 0.005% NaN3 underneath the skin. After the equilibration period, about 200 mg of the donor phase (infinite dose) was applied on the SC side of the skin surface for 24 hours. The cells were covered with aluminum foil. The acceptor fluid, which was pumped at a rate of 0.6 ml/min by the peristaltic pump, was composed of 4 ml of 0.9% NaCl + 0.005% NaN3 and it was entirely replaced after 4, 8 and 24 hours. After 24 hours the donor phase was removed and the skin surface was rinsed 2 times with 0.5 ml of 96.3% ethanol and then 2 times with 1 ml of physiological solution. The outer residuals of skin samples were trimmed off, leaving the central circles with area of 0.64 cm2.
Separation of skin layers and their extraction
Epidermis was separated from the rest of the skin (dermis) using dry heat separation method. The skin sample was placed (on the epidermis side) on the hot surface (60 °C) for 1-2 seconds and epidermis was peeled off. Then epidermis and dermis were separately extracted with 1 ml of pure methanol, following bath sonication for 30 min. The supernatant was filtered through nylon membrane filter (0.45 µm, Carl Roth GmbH, Karlsruhe, Germany) and injected into HPLC.
UV/Vis spectrophotometrical method
Samples obtained after in vitro release studies were analyzed using UV/Vis spectrophotometer. The absorbance peaks of tolnaftate were measured at Î» = 257 nm and the calibration curve (in the concentration range of 1.5-7.5 µg/ml) was prepared in the acceptor medium, composed of 50/50 PEG 400/96.3% ethanol (R2 = 0.9984, y = 59.77x + 0.4247). UV spectrophotometrical method was also used to quantify tolnaftate content remaining in the donor phase and skin washings, after in vitro skin penetration experiments. The donor phase, remaining on the skin surface, was carefully removed, appropriately extracted (using 96.3% ethanol and sonication at 60 °C for 30 min), filtered through the nylon membrane filter (0.45 µm) and analyzed according to the calibration curve (in the concentration range of 3.0-15.0 µg/ml) prepared in 96.3% ethanol (R2 = 0.9948, y = 58.47x + 0.09089)
A validated HPLC method with UV detection (10) was used to determine tolnaftate amount, penetrating into epidermis and dermis from 1% tolnaftate ointments after 24 hours of topical application. The samples of receptor fluid were also analysed using HPLC.
Separation of tolnaftate from endogenous compounds, deriving from skin matrix, was accomplished on a LiChrospher®100 RP-18 Endcapped column, 125 x 4 mm, i.d., packed with 5 µm size particles (Merck KGaA, Darmstadt, Germany). A LiChrospher 100 RP-18e (5 µm) (LiChroCART 4-4) was used as a guard column.
Isocratic elution, using 70% methanol and 30% bi-distilled water as the mobile phase, resulted in tolnaftate retention time of 13.2 min and the overall running time of 15.0 min. The flow rate of the mobile phase was set to 0.8 ml/min and the injection volume was 10 µl. The column was thermostated at 40 °C and tolnaftate detection was set at Î» = 257 nm.
For the statistical analysis, one-way analysis of variance (ANOVA) together with Tukey's HSD test were applied using SPSS software version 12.0. The level of significance was determined as P < 0.05.
Quality control tests of prepared ointments
The prepared ointments were all uniform in appearance, having light yellow colour and an odour of oil (in case of oleic or linoleic acid) or a specific odour of saturated fatty acid added. Observation of prepared ointments under the microscope revealed, that no crystals were present and the pattern of solidifying (mainly caused by PEG 1500) was seen.
pH values of 1% (w/w) tolnaftate control ointment and ointments with fatty acids are presented in Table 2. Ointments with 10% of fatty acids had lower pH values comparing to the control, but the pH was apropriate for application of these ointments on the skin surface (pH of SC and upper viable epidermis is considered to be 4.0-4.5 and 5.0-7.0, respectively (11)).
The rheograms, showing the relationship between dynamic viscosity and selected speed interval of cylindrical spindle rotation, are presented in Figure 1. The investigated ointments exhibited non-Newtonian pseudoplastic (shear-thinning) flow.
In vitro release profiles of tolnaftate from prepared ointments
The percentage of the cumulative amount of tolnaftate released from each ointment after 1, 2, 4 and 6 hours is presented in Figure 2. The cumulative amount (µg) of tolnaftate released per unit membrane area (1 cm2) was plotted against square root of time (Higuchi-plot; Figure 3). Higuchi-plots yielded straight lines, the slopes of which were taken as release rates. Table 3 summarizes the parameters of tolnaftate release from prepared ointments in vitro, comprising the cumulative amounts (in %) of drug released after 6 h, release rates and coefficients of determination (R2). The linearity (expressed as R2 in Table 3) of Higuchi-plots indicates, that diffusion of tolnaftate from ointments is a rate limiting step for drug liberation. Moreover, it shows, that Cuprophan® membranes used were not limiting drug diffusion from the donor to receptor compartment. The lag time for drug release, corresponding to x intercepts of the Higuchi-plots, were not exceeding 15 min for all ointments tested.
Analysis of variance (ANOVA) showed that release rate of tolnaftate from ointments was not significantly different (P â‰¥ 0.05).
Enhancing effect of fatty acids on tolnaftate penetration into human skin layers
Figure 4 and 5 present the amounts (µg/cm2) of tolnaftate penetrating into epidermis and dermis, respectively, from the control ointment (n=3) and from ointments having 10% (w/w) of different fatty acids (n=3) after in vitro skin penetration experiments. The enhancing effect of five different fatty acids on tolnaftate penetration into 1cm2 of epidermis (E) or dermis (D) was calculated as the enhancing ratio (ER) using the following formula:
The obtained ER values are presented in Table 4.
Statistical analysis revealed that tolnaftate amount penetrating into cm2 of epidermis from the ointments having 10% of oleic, linoleic, myristic and lauric acids was significantly greater (P < 0.05) than from the control ointment. Capric acid did not significantly enhance tolnaftate penetration into epidermis comparing to the control. According to Tukey HSD test, three homogenous subsets of groups whose means do not differ from one another, were found: ointments with (i) oleic, linoleic and lauric acid, (ii) linoleic, myristic and lauric acid, (iii) capric acid and control.
As tolnaftate is a hydrophobic substance, its high amounts in hydrophilic dermis are unexpectable. Tolnaftate penetration into dermis was not significantly enhanced from either ointment having 10% of fatty acid comparing to the control (P â‰¥ 0.05). These findings prove that fatty acids exert their enhancing effect only in the superficial layers of skin.
Mass balance of tolnaftate after in vitro skin penetration studies
In order to check the experimental conditions used for in vitro skin penetration experiments, mass balance of tolnaftate was calculated. After in vitro skin penetration experiments the donor phase, remaining on the skin surface, was carefully removed, appropriately extracted with 96.3% of ethanol and analyzed by UV spectrophotometer. The tolnaftate amounts remaining in the donor phase and found in the epidermis and dermis extracts were sumed up (no drug was detected in the acceptor fluid) and compared to the nominal amount of tolnaftate added on each skin sample. The relative error (RE) was calculated according to the formula:
The REs were calculated for each skin sample and was in the range from -4.19% to 8.45%. This is an indication of apropriately chosen experimental conditions.
For the formulation of ointment, white vaseline was chosen as the main vehicle for model drug tolnaftate. Other ingredients were added to improve formulation's consistency, spreadability, appearance and tolnaftate solubility. PEG 400 was chosen as an appropriate solvent for tolnaftate (AHFS Drug Information, 1999). 1% tolnaftate solution and creams, available over-the-counter, are also usually formulated in PEG 400 or PEG 400 - propylene glycol vehicle, respectively. PEG 1500 was added as a plasticizing and thickening agent. Anhydrous lanolin acted as emulsifier. Fatty acids were chosen as potent CPEs for a hydrophobic tolnaftate and they also exhibited emulsifying properties. It is worth mentioning that polyethylene glycols are also considered to be good CPEs (12), thus synergistic enhancing action of PEGs and fatty acids could be observed. Microscopic pictures of tolnaftate control ointment showed clear boundaries of PEG 400 droplets, as anhydrous lanolin alone was not able to efficiently emulsify solution of tolnaftate in PEG 400. Addition of 10% (w/w) fatty acids resulted in better emulsification: no clear boundaries of PEG 400 droplets, but solidification pattern of PEG 1500 could be observed in the microscopic pictures. Model drug tolnaftate was completely soluble in the prepared ointments.
The pH values of prepared ointments were compatible with the skin's surface pH. Dynamic viscosity measurement of prepared ointments revealed that semisolid preprations exhibited non-Newtonian shear-thinning flow. Addition of solid fatty acids (capric, lauric, myristic) obviously resulted in higher dynamic viscosity range comparing to the ointments with oleic or linoleic acid. Control ointment was formulated with higher amount of white vaseline (instead of 10% fatty acid), and only formulation with 10% myristic acid had higher dynamic viscosity range comparing to the control.
Drug release from a semisolid dosage form allows for drug availability on the skin surface and is evaluated using in vitro release tests. These tests characterize the performance of the vehicle in drug solubility, diffusion and release processes. Three important considerations have to be made before carrying out this type of experiments: (i) the chosen acceptor medium should provide "sink conditions" (the amount of substance in acceptor medium after experiment should not exceed 30% of it's saturation concentration), (ii) the diffusion membrane should not limit drug diffusion from donor to receptor phase and act as an inert physical support, (iii) the dose of semisolid applied should be infinite. The chosen acceptor medium (50/50 PEG 400/96.3% ethanol) provided sink conditions and the cellulose membrane with nominal molecular weight cut-off of 10 kDa was not limiting drug diffusion. Moreover, Cuprophan® membranes are recommended by USP 30 for transdermal delivery system release studies. About 0.8 g (0.45 g / cm2) of product were applied to the donor chamber and this corresponds to the infinite dose conditions (13).
The liberation of drug from semisolid formulation mainly depends on (i) the solubility of drug in the formulation and (ii) the viscosity of semisolid. Tolnaftate was soluble in the ointments prepared by fusion technique: microscopic observation of ointments prepared showed no crystals present. Increase in the viscosity usually results in the lower release of drug from semisolid. According to the results obtained, addition of liquid fatty acids (oleic and linoleic acid) to the ointments, resulted in higher release of tolnaftate, and this might be due to decrease of viscosity of the formulation and possibly due to solubilizing effect of these fatty acids on hydrophobic tolnaftate.
ANOVA as well as Kruskal-Wallis test showed that there is no significant difference among the ointments tested on the release of tolnaftate (P â‰¥ 0.05). It means that CPEs do not have a significant effect on the liberation of tolnaftate from ointment formulations and their enhancing effect is only observed after making in vitro skin penetration experiments. Similar observations were made by Wagner et al. (14) after investigation of CPEs effect on the liberation and penetration of flufenamic acid. The insertion and interaction of fatty acid molecules with the lipid domains in SC, the increase of fluidization and disruption of lipid structure in the superficial layers of skin, results in significantly higher penetration of tolnaftate molecules to epidermis from ointment formulations having 10% of oleic, linoleic, lauric and myristic acid comparing to the control.
Fatty acids increase the penetration of active drugs into the skin through the non-polar route, which is very important for the penetration of hydrophobic drugs, such as tolnaftate. The formulation of ointment itself, being the lipophilic base, has occlusive properties on the skin. Thus skin hydration might be increased also resulting in higher penetration of active molecules.
The dependence of fatty acid hydrocarbon chain length and presence of double bonds on its skin penetration enhancing properties is observed with our model drug tolnaftate. Among fatty acids tested, capric acid has the shortest chain length of C10 and this fatty acid did not significantly enhance the penetration of tolnaftate to epidermis comparing to the control. As stated by Kravchenko et al. (15), fatty acids having hydrocarbon chain length of less than C11, produce insufficient disruption of lipid alkyl radicals. Oleic and linoleic acids are unsaturated and have the longest chain length of C18. These two fatty acids were the strongest CPEs among others tested, but oleic acid was the most potential CPE due to its cis-unsaturation and 'kink' formation (2), which is responsible for the fluidization of SC lipids (9, 16). Lauric acid (C12) together with oleic and linoleic acids belonged to the same homogenous subset (Tukey HSD test) and had the very strong effect on enhancing tolnaftate penetration into epidermis. Similar results were obtained by Nair et al. (17) after testing oleic, linoleic and lauric acids enhancing effect on Arginine Vasopressin. Lauric acid may have a spatial form that is conformationally similar to the framework of cholesterol (15). Thus Nair et al. (17) hypothesized that in the presence of lauric acid disruption of ceramide-cholesterol or cholesterol-cholesterol interaction might occur. Myristic acid, which has the chain length of C14, also significantly enhanced tolnaftate penetration into epidermis.
The ointments tested release tolnaftate in almost the same manner (about 8% of tolnaftate was released after 6 hours from all formulations). The effect of the semisolid vehicle on the absorption kinetics of tolnaftate into the skin might be associated only with the hydration process of skin surface (8). However, fatty acids, presented in the ointments, penetrated into the skin and interacted with the intercellular lipids of the SC, disturbing their structure and enhancing the diffusion process of hydrophobic tolnaftate into human epidermis. Thus in vitro release experiments are not predicting the situation on the skin surface (14).
The formulated topical ointments, having 10% (w/w) of fatty acids, were intended to increase the bioavailability of tolnaftate in human skin layers in vitro. Addition of fatty acids to the ointments, having 1% (w/w) of tolnaftate as a model drug, does not significantly improve the liberation of active drug to the chosen acceptor medium comparing to the control, but significantly enhances drug penetration into human epidermis. This observation indicates that fatty acids exert their effect only when they are applied on the skin, and consequently no presumptions could be made about the enhancing effects of CPE's on drug penetration into the skin after in vitro release experiments.
Table 1. Composition of 1% (w/w) tolnaftate ointment, having 10% of fatty acid
Table 2. pH values of prepared ointments
1% (w/w) tolnaftate ointment
Ointment with 10% oleic acid
Ointment with 10% linoleic acid
Ointment with 10% myristic acid
Ointment with 10% lauric acid
Ointment with 10% capric acid
Table 3. Cumulative amount (%) of tolnaftate released after 6 h, mean release rates and determination coefficients (R2) of lines, yielded by Higuchi-plots
Cumulative amount of drug released after 6 h (% ± SD, n=3)
Release rate (µg/cm2âˆšh ± SD, n=3)
161,09 ± 10,62
With oleic acid
8,19 ± 0,23
189,5 ± 8,71
With linoleic acid
8,98 ± 1,18
192,98 ± 10,92
With lauric acid
8,49 ± 0,27
185,53 ± 18,17
With capric acid
7,34 ± 0,51
168,05 ± 14,69
With myristic acid
7,63 ± 0,07
166,26 ± 10,54
Table 4. ERs of fatty acids for tolnaftate penetration into epidermis and dermis
Figure 1. Relationship between ointments dynamic viscosity (Pa · s) and rotation speed of the spindle (rpm); the bars represent ±SD, n=3.
Figure 2. Percentage release of tolnaftate from ointment formulations; the bars represent ±SD.
Figure 3. Higuchi - plots of tolnaftate release from ointments; the bars represent ±SD.
Figure 4. Tolnaftate amount (µg/cm2) penetrating into epidermis from control ointment (n=3) and ointments having 10% of fatty acids (n=3). The bars represent ± SD.
Figure 5. Tolnaftate amount (µg/cm2) penetrating into dermis from control ointment (n=3) and ointments having 10% of fatty acids (n=3). The bars represent ± SD.