Effect of combination of hydrophilic and lipophilic permeation enhancers on the skin permeation of kahalalide F.

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Research Paper

Journal of Pharmacy And Pharmacology

Effect of combination of hydrophilic and lipophilic permeation enhancers on the skin permeation of kahalalide F Punit P. Shaha, Pinaki R. Desaia, Ram Patlollab, Larry Klevansc and Mandip Singha a College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL, cMedimetriks Pharmaceuticals, Inc., Fairfield, NJ, USA and bIntegrated Product Development, Dr Reddys Laboratories, Hyderabad, AP, India

Keywords dermatitis; hydrophilic and lipophilic chemical enhancers; kahalalide F; skin inflammation; skin permeation Correspondence Mandip Singh, College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, 1520 S Martin Luther King Jr Boulevard, Dyson Pharmacy Building (Room 021), Tallahassee, FL 32307, USA. E-mail: [email protected]; [email protected] Received 2013 June 7, 2013 Accepted 2013 November 30, 2013 doi: 10.1111/jphp.12206

Abstract Objectives The purpose of this study was to investigate the influence of combination of various lipophilic and hydrophilic chemical enhancers on skin delivery of kahalalide F (KF). Methods KF formulations comprising a combination of lipophilic and hydrophilic chemical enhancers with varied per cent were prepared and evaluated for skin permeation studies. In vitro skin permeation of KF formulations was performed using Franz diffusion cell. Stability studies of KF formulations were performed according to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guideline, and the therapeutic efficacy of KF formulation was evaluated using allergic contact dermatitis animal model. Key findings The efficacy of KF formulations to improve skin delivery of KF was sequenced in the order of: formulation #4 > formulation #2 > formulation #1 > formulation #3, where formulation #4 contains labrasol (40% w/v), ethyl oleate (5% w/v) and span 80 (5% w/v) along with transcutol (40% w/v) and ethanol (10% w/v). Further, all the formulations were stable for 1 month when stored at 30°C/65% relative humidity. Conclusions The results of present study suggest that therapeutically effective concentrations of KF can be delivered in the skin using combination of lipophilic and hydrophilic chemical enhancers.

Introduction Kahalalide F (KF) (C75H124N14O16) is a novel antitumour agent, originally discovered and isolated from an extract of the Hawaiian marine mollusk Elysia rufescens (Plakobranchidae).[1] KF is tridecapeptide with a ring-shape side and lateral side, containing a fatty acid group connected to a latter. KF has shown to exhibit significant in vitro and in vivo antitumour activity against a variety of human cancer cell lines (colon, prostate and breast cancer cell) with half maximal inhibitory concentration of less than 1 μm.[2–4] These findings demonstrate that KF is a potent cytotoxic agent with promising activity against variety of solid tumours. Further, exploratory clinical trials were conducted with a parenteral formulation of KF in cancer patients and patients with other proliferative diseases, and it was discovered that KF is effective in treating various tumours.[3,5] Also, KF was found to be potent in patients with severe psoriasis, unresponsive to methotrex760

ate, or a combination of methotrexate and cyclosporine. These results were achieved by infusing approximately 1 mg of drug over an hour once a week for 8 weeks.[6] However, systemic delivery is known to be associated with several side effects. In addition, systemic delivery of a drug might not deliver sufficient amount of drug at the diseased site. Therefore, a percutaneous delivery of KF is a promising strategy to treat anti-inflammatory skin disorders like psoriasis and allergic contact dermatitis (ACD) that facilitates targeted delivery. The permeation of KF through the skin is extremely difficult because of its lipophilicity, large MW (1477.87 Da – free base) and cyclic molecular structure.[7,8] Further, stratum corneum (SC), the outermost layer of the skin, is the major obstacle in permeation of KF across the skin.[9] To overcome this barrier and to enhance skin permeation of a drug, different methodologies have been investigated and

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 760–768

Punit P. Shan et al.

developed, including use of prodrugs, drug-saturated systems, and physical and chemical enhancers that facilitate diffusion of drugs through the SC.[10] However, the most popular and commercially viable approach is the use of chemical penetration enhancers that can increase drug absorption either into the systemic circulation or to the deeper, viable skin layers that results in improved therapeutic responses.[11] Chemical enhancers modulate some of the properties of the SC. However, based on the lipophilicity and lipophilicity of drug, chemical enhancers are generally selected. Thus, an effective chemical enhancer may increase the diffusion coefficient of the drug into the SC (i.e. disrupt the barrier) or could improve partitioning between the formulation and the SC (perhaps by altering the solvent nature of the skin membrane to improve partitioning into the tissue). However, SC of skin has lipophilic environment, while dermis has hydrophilic environment. Therefore, there is a need for a combination of lipophilic and hydrophilic chemical enhancers to improve skin permeation of active drugs to the deep skin layers. Ethanol is widely used as a chemical enhancer and act by altering the organization of intercellular lipids of SC and thus increasing skin permeability.[12] Propylene glycol (PG) is another potent hydrophilic chemical enhancer. Various studies involving the measurement of solvent and solute have suggested that PG causes a ‘drag’ effect[13,14] where the solvent was believed to modulate the barrier of the skin allowing easier passage for the migration of the solute. This migration of solute can persist throughout the skin to the receptor compartment.[15,16] Further, Bowen and Heard suggested that PG forms a solvated complex with drug.[17] Therefore, the solvent permeates through the skin and carries the drug, present as a dispersion of solvated complexes across the skin layers[17,18] and thus results in sudden increase in the permeated amount of drug in the receiver compartment during initial hour of skin permeation study. Transcutol, diethylene glycol monoethyl ether, is reported to enhance the skin permeation by increasing the solubility of the applied compound and formation of depot in the skin.[19] n-Methyl-2-pyrrolidone (NMP) exerts its direct influence on the aqueous regions between the polar lipid head groups of the bilayer. It penetrates into this region of tissue in such amounts that they alter the solubilizing ability of this site and thus promoting drug partition into skin, which subsequently results in increased flux of the penetrant.[20] Further, labrasol (glycolysed ethoxylated C8/C10 glycerides)[21] and polyethylene glycol 400 (PEG400)[22,23] are reported to increase the drug deposition within the skin layers. Isopropyl myristate (IPM) is known to increase fluidity of lipid bilayers of the SC.[24] Ethyl oleate, a lipophilic chemical enhancer, is reported to function by partitioning into the lipid region of the SC, disrupting the structure and

Kahalalide F for skin inflammation treatment

lipid fluidity of the SC.[25] Further, span 80 affects the intercellular lipids of the SC by incorporating its C12 alkyl chain between them,[26] thus disrupting their packed structure, and as a consequence, the SC permeability and the diffusivity of the drug increase.[27,28] Because various lipophilic and hydrophilic chemical enhancers mentioned earlier are reported to increase the drug transport through the skin via different mechanisms, a combination of these chemical enhancers will result in improved drug transport across the skin because of involvement of different mechanisms based on nature of chemical enhancers. Thus, the selection of an appropriate combination of hydrophilic and lipophilic chemical enhancers is a vital part of the percutaneous formulations. The aim of present research work was to identify a suitable combination of chemical enhancers for improving skin permeation of KF. The enhancing effects of various hydrophilic chemical enhancer such as ethanol, PEG400, PG, NMP, transcutol and Tween 80 in combination with lipophilic chemical enhancers such as ethyl oleate, span 80, labrasol and IPM on the permeation of the KF were evaluated with in vitro percutaneous experiment using dermatomed human skin. The prototype percutaneous formulations of KF were prepared for initial formulation development using a combination of hydrophilic and lipophilic chemical enhancers. The skin permeation study was performed using procedures adapted from the FDA and American Association of Pharmaceutical Scientists report of the workshop on principles and practices of in vitro percutaneous penetration studies: relevance to bioavailability and bioequivalence.[29] Further, the therapeutic efficacy of KF formulation was evaluated using ACD mouse model where dexamethasone was used as a positive control.

Materials and Methods Ethanol, PEG 400, NMP, PG, span 80, IPM, phosphate buffer saline (PBS) sachets (pH 7.4), HPLC-grade water, HPLC-grade acetonitrile, trifluoroacetic acid (TFA) and octanol were purchased from Sigma (St Louis, MO, USA). Ethyl oleate, Tween 80, bovine serum albumin (BSA) and dimethyl sulfoxide (DMSO) were purchased from Spectrum Chemical Manufacturing Co. (Gardena, GA, USA). Brij O20-SS-(MH), formerly known as Volpo 20, was generously gifted by Croda, Inc. (Parsipanny, NJ, USA). Labroasol® and Transcutol® were generously gifted by Gatteffosse (Paramus, NJ, USA). KF was generously provided by PharmaMar USA, Inc. (Cambridge, MA, USA).

High-performance liquid chromatography analysis An HPLC system (Waters Corp, Milford, MA, USA) along with a Vydac reverse phase C18 (300 Å pore size silica) ana-


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lytical column (5 μm, 4.6 × 150 mm) (GraceVydac, Columbia, MD, USA) were used for the analysis of KF. The HPLC system with a photodiode array UV detector interfaced with Empower software (Waters Corp) was used. The mobile phase used was 0.08% v/v TFA in acetonitrile : water (50 : 50) for 25 min, with a flow rate of 0.6 ml/min. The column temperature was kept at 80°C. KF content in the samples was determined at 215 nm. The selectivity and accuracy were performed along with the limit of detection (LOD) and limit of quantification (LOQ) using this method.

Determination of Log P The logarithm of the octanol-water partition coefficient (Log P) value was determined using modification in the method described by Takács-Novák and Avdeef.[30] The Log P value is known as a measure of lipophilicity. In brief, 15 ml of each, octanol and water, were mixed and equilibrated in a separating funnel for 24 h. After equilibration, 5 mg of KF was added to the octanol-water mixture. The mixture was shaken to partition the drug between two phases. These two phases were allowed to separate. The mixture was then kept for 24 h to ensure complete solubility of KF in both the phases. The octanol and water phases were further analysed by HPLC method. The octanol phase was diluted using mobile phase before HPLC analysis.

Preparation of percutaneous formulations KF formulations (F# 1–4) were prepared by blending the mixture of chemical enhancers with ethanolic solution of KF (Table 1). Further, KF was dissolved in DMSO with gentle shaking to make the KF containing DMSO solution.

Determination of drug content KF content in various formulations was evaluated by diluting 100 μl of KF formulation with 900 μl of ethanol in a Table 1 Composition of the prototype kahalalide F formulations from 1 through 4 Ingredients

F#1

F#2

F#3

F#4

DMSO

KF (g) Ethanol (g) PEG400 (g) Transcutol (g) NMP (g) Ethyl oleate (g) Span 80 (g) Propylene glycol (g) Labrasol (g) Isopropyl myristate (g) DMSO (g)

0.25 10.00 30.00 34.75 10.00 5.00 5.00 5.00 – – –

0.25 10.00 30.00 – 10.00 – – 29.75 20.00 – –

0.25 10.00 – – 10.00 – – 35.00 39.75 5.00 –

0.25 10.00 – 39.75 – 5.00 5.00 – 40.00 – –

0.25 – – – – – – – – – 99.75

DMSO, dimethyl sulfoxide; KF, kahalalide F; NMP, n-methyl-2pyrrolidone; PEG400, polyethylene glycol 400.

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volumetric flask, and KF was extracted by shaking it overnight in ethanol. Then, an aliquot of the extracted sample was filtered through 0.45 μm polytetrafluoroethylene filters, and drug content was determined by HPLC.

Selection of receptor fluid To maintain the sink condition, KF was dissolved by gentle shaking in various receptor fluids such as 10% v/v ethanol in PBS (pH 7.4), 0.1–1% w/v Volpo 20 in PBS (pH 7.4) and 5% w/v BSA in PBS (pH 7.4). The final concentration of KF in receptor fluid was 1 mg/ml.

In vitro skin permeation studies Human skin permeation studies were performed as described by Shah et al.[31] Dermatomed human skin was obtained from Allosource (Centennial, CO, USA) in normal saline containing 10% glycerol with a thickness of 0.5 ± 0.1 mm. Skin was then stored at −80°C for a week based on our earlier work where we have shown that the skin permeation of melatonin and nimesulide was similar to the fresh rat skin even after storing it at −80°C for 60 days.[32] The dermatomed human skin was thawed and washed with water for 30 min to remove excess of glycerol before use. In vitro skin permeation studies were performed using established procedures[31] by mounting dermatomed human skin in Franz diffusion cell setup (Permegear, Inc., Riegelsville, PA, USA). The surface area of the dermatomed human skin exposed to the formulation in the donor chamber was 0.64 cm2, and the receiver fluid volume was 5 ml. 100 μl of KF formulations were applied evenly on the surface of the human skin in the donor compartment. The skin permeation study was performed using six diffusion cells and represented as an average of six cells. The receiver compartment was filled with 0.5% w/v volpo 20 in PBS (pH 7.4) and stirred at 300 rpm. The temperature of receiver compartment was maintained at 32 ± 0.5°C using a circulating water bath to simulate the skin temperature at physiological level. To replicate the clinical conditions, a non-occlusive method was followed, and the surface of the skin was exposed to the surrounding air. At predetermined time intervals (1, 2, 4, 6, 8, 12, 22 and 24 h), 0.3 ml of samples were taken from the receiver compartment and replaced with fresh buffer solution. The samples collected from receiver compartment were centrifuged at 13 500 rpm for 15 min and analysed for KF content using HPLC method.

Mass balance studies Mass balance studies were performed to evaluate the recovery of applied dose of KF in receiver compartment, skin



layers and donor compartment. For mass balance of KF formulations, the amount of KF present in receiver compartment, skin and donor compartment were investigated separately. To evaluate the amount of KF present in donor compartment, the donor compartment was removed, and the excess formulation was removed from the surface of the skin using a cotton swab. The skin was then washed with cotton swab dipped in 50% v/v ethanol : water and blotted dry with dry cotton swab. The donor compartment and cotton swab (dry and wet) were collected in a bottle sonicated for 30 min with 50% v/v ethanol : water. The bottles were covered with its lid tightly to avoid evaporation of ethanol. To investigate the amount of KF retained in dermatomed human skin, the entire dosing area (0.64 cm2) was collected using a biopsy punch (George Tiemann & Co., New York, NY, USA). The collected skin was homogenized with 250 μl PBS (pH 7.4) using a tissue homogenizer for 5 min at 5000 rpm. The homogenate was boiled for 10 min and centrifuged at 13 500 rpm for 20 min. The supernatant was analysed for KF content by HPLC.

Stability studies The formulations were filled in amber-coloured HPLC glass vials and incubated as per the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guideline at 30°C/65% relative humidity (RH) for 2 weeks and analysed every alternative day (0, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12 and 14 days). Appearance and clarity were analysed by visual inspection. The pH metre (VWR, Radnor, PA, USA) was used to measure pH. The amount of KF remaining after storage in each vial was further analysed using HPLC method. The stability study at each time point was performed in triplicate and represented as an average of three separate vials of each formulation.

In vivo model for allergic contact dermatitis Animals C57BL/6 mice of 6-week age (Charles River Laboratories, Wilmington, MA, USA) were grouped and housed (n = 6 per cage) in cages with Tek-Fresh bedding. The animals were kept under controlled conditions of 12 : 12 h light : dark cycle, 22 ± 2°C and 50 ± 15% RH. The mice were fed (Harlan Teklad) and water ad libitum. The animals were housed at Florida A&M University in accordance with the standards of the Guide for the Care and Use of Laboratory Animals, and the Association for Assessment and Accreditation of Laboratory Animal Care. The animals were acclimatized to laboratory conditions for 1 week before experiments. The protocol of animal study was


approved by the Institutional Animal Care and Use Committee, Florida A&M University (ACUC Protocol #017.10). Allergic contact dermatitis The ACD model was developed as described by Shah et al.[31,33,34] Briefly, the C57BL/6 mice were sensitized on day 0 by applying 25 μl of 0.5% v/v 2,4-dinitro-1-fluorobenzene (DNFB) in acetone : olive oil (4 : 1) on the shaved abdomen. Mice were then challenged on day 5 by epicutaneous application of 25 μl of 0.2% v/v DNFB in acetone : olive oil (4 : 1) on the right ear to induce an ACD response. The left ears were treated with vehicle alone (acetone : olive oil 4 : 1) and served as an internal control. The ACD response was determined by the degree of ear swelling compared with that of the vehicle-treated contralateral ear before DNFB challenge. The increase in ear thickness was measured with a vernier caliper (Fraction+ Digital Fractional Caliper, General Tools & Instruments Co., LLC., New York, NY, USA) at 0, 24, 48 and 72 h. Right ears of the mice were treated with KF formulations, 2 h after antigen challenge and three times a day thereafter for 3 days. Dexamethasone solution (0.5 mm), prepared in 10% v/v ethanolic PEG400, was used as a positive control. The ear swelling was measured before the application of KF formulations. This was considered as 0 h ear thickness. Then, the KF formulations were applied, and the ear thickness was measured at 24, 48 and 72 h. The ACD response was determined by taking a difference between 0 h and other time points.[35]

Statistical analysis The KF content in the skin tissue was expressed as mg/g of the tissue. Differences between the KF retained in skin, flux for different formulations and ear thickness were examined using analysis of variance (ANOVA) and Tukey multiple comparison test. Means were compared between two groups by Student’s t-test and between three dose groups by one-way ANOVA. Mean differences with P < 0.05 (at the 95% confidence interval) were considered to be significant.

Results and Discussion Development of high-performance liquid chromatography method The HPLC method was modified as described by Nuijen et al.[7] Correlation coefficient was found to be 0.9991, signifying that a linear relation exist between absorbance and KF concentration. Beer’s law was found to be obeyed between 0.1 and 25 μg/ml. Estimation of KF was performed in presence of various commonly used excipients at the levels they are normally used. None of the excipients interfered, signi-


763



fying the selectivity of the HPLC method for KF estimation. Accuracy was performed using recovery studies. The recovery was found to be near 100%, signifying the accuracy of the HPLC method. The LOD and LOQ for the developed HPLC method were 0.05 and 0.1 μg/ml, respectively.

Determination of Log P The concentration of KF in octanol and water phase was 166.3 and 0.32 μg/ml, respectively (Table 2). The Log P value was calculated by logarithm of the ratio of the concentrations of KF in the aqueous and octanol phase. Thus, the Log P value for KF was 2.71, signifying that KF is more lipophilic and has poor water solubility that makes it difficult to permeate to the deep skin layers.

Selection of receptor fluid KF was soluble in ethanol because of its lipophilicity. However, precipitation was observed in 10% v/v ethanol containing PBS (pH 7.4). Further, KF was soluble in 0.1–1% w/v volpo 20 and 5% w/v BSA in PBS (pH 7.4). However, BSA showed interference at the retention peak of KF during HPLC analysis, while 1% w/v volpo 20 in PBS (pH 7.4)

Table 2

showed no interference around the retention peak of KF along with excellent solubility. The presence of excess amount of surfactant like volpo 20 into the receptor fluid could lead to the emulsification of skin that could result in oozing of skin components in the receptor fluid. Further, these components could interfere with the retention peak of KF. Therefore, to avoid interference of skin components, 0.1% w/v volpo 20 in PBS (pH 7.4) was selected as receptor fluid for in vitro skin permeation studies of KF formulations.

In vitro skin permeation studies Figure 1 shows the combined effect of lipophilic and hydrophilic chemical enhancers on skin permeation of KF. All formulations were comprised of ethanol in addition to combination of lipophilic and hydrophilic chemical enhancers (Table 1). Ethanol was used as solubilizer and penetration enhancer for KF.[12] The ethanol concentration in all the formulations was kept constant at 10% w/w considering prior reports that demonstrated that the permeation enhancing effect of ethanol may reach a plateau level at higher concentrations[10] and, in some cases, a reduction of

Concentration of kahalalide F in octanol and water phase Average ± standard deviation (μg/ml)

Concentration (μg/ml) Octanol phase Water phase

164.52 0.32

166.43 0.31

166.3 ± 1.71 0.32 ± 0.01

167.95 0.33

DMSO, dimethyl sulfoxide.

(a)

F#1

F#2

F#3

(b)

F#4

40

3

35

2.5 μg of KF/g of skin

Permeated amount of KF per unit area (μg/cm2)

45

2 1.5 1

30 25 20 15 10

0.5 5 0 0

5

10

15

20

25

0 F#1

F#2

F#3

F#4

Time (h) Figure 1 (a) Permeated amount of kahalalide F (KF) per unit area as a function of time for prototype formulations. The data are presented as μg/cm2. (b) Skin retention performed after 24 h of skin permeation study for prototype formulations of kahalalide F. The skin permeation represented as μg of kahalalide F/g of skin. Data represent mean ± standard deviation, n = 6.

764




Table 3 In vitro human skin flux of kahalalide F formulations in comparison with dimethyl sulfoxide (DMSO) control formulation Formulation

Flux (μg/cm2/h)

F#1 F#2 F#3 F#4 DMSO

0.0448 0.0452 0.0335 0.0512 0.3231

the enhancement activity because of its ability to dehydrate the skin.[36] Further, for preparation of KF formulations, PEG400, transcutol, NMP, PG were used as hydrophilic chemical enhancers, while ethyl oleate, IPM, span 80 and labrasol were used as lipophilic chemical enhancers. The per cent of lipophilic and hydrophilic chemical enhancers were varied to prepare four different formulations and further evaluated by in vitro skin permeation studies. The amount of KF available in receiver compartment for KF formulations ranged from 1.52 to 2.68 μg/cm2 (Figure 1a). Further, the amount of KF retained in skin for KF formulations ranged from 18 to 36 μg/g of skin (Figure 1b). Among all formulations, F# 4 demonstrated the highest amount of KF retention in the skin, leading to increase in skin permeation across the dermatomed human skin by passive diffusion and thus increase in flux (Table 3). This could be because of the presence of transcutol and labrasol in the formulation, as they are known to increase drug deposition within the skin layers.[19,21] F#4 comprised of 50% w/w lipophilic chemical enhancers such as labrasol, ethyl oleate along with span 80 and 40% w/w of hydrophilic chemical enhancer like transcutol in addition to 10% w/w ethanol. Similar to F#4, F#2 showed increase in amount of KF retained and permeated through human dermatomed skin. This could be because of the synergistic effect of PEG400 and PG as well as presence of labrasol. However, F#2 showed significantly less (P < 0.001) amount of KF retained in and permeated through dermatomed human skin than F#4. This could be because of increase in hydrophilicity of F#2. Further, F#3 and F#1 failed to show increase in KF retention and permeation through human skin. This could be because of increase in hydrophilicity and use of lesser amount of labrasol or absence of labrasol, suggesting a need for a lipophilic chemical enhancer like labrasol in combination with hydrophilic chemical enhancer, transcutol, to improve skin permeation, retention and flux of KF. Because F#4 was superior in enhancing the amount of KF retained in skin and permeated through skin, F#4 was compared with DMSO to estimate maximum amount of KF permeated through the human skin. The amount of KF

retained and permeated through the skin for DMSO was significantly higher (P < 0.001) than F#4 (Figure 2). This was expected because DMSO is known to have greater ability to enhance the skin permeation of active drugs than other known penetration enhancers. In addition, the per cent of DMSO used was much higher than other permeation enhancers used. However, use of 100% DMSO showed detrimental effect on the skin integrity. SC was separated from the intact skin after application of DMSO that was expected.

Mass balance studies Mass balance for all formulations was performed to determine the recovery of applied dose of KF in the skin, receiver and donor compartments. The recovery from donor compartment was nearly 93–96% of the applied dose, as shown in Table 4. For all formulations, the total recovery was not statistically different.

Stability studies During the stability studies, the physical appearance, pH and the amount KF remaining on storage was unaffected, signifying that all the formulation are stable for 1 month (Figure 3) and can be used for further clinical studies.

In vivo model for allergic contact dermatitis To investigate the anti-inflammatory effect of KF, an in vivo ACD model was used as a representative animal model. The effect of KF containing formulations to treat the inflammation was evaluated on the reduction of ear swelling is shown in Figure 4. Two different concentrations of KF were prepared for F#4 as 0.25% and 0.5% w/w to investigate the dose-dependent therapeutic activity. After application of DNFP, the ear thickness was increased from 109.42 to 132.16 μm with time for control (untreated) animals. Further, the swelling was reduced significantly for mice treated with KF formulations. After 72 h, the ear thickness was found to be 4.42 and 4.02 μm for formulations comprising 0.25 and 0.5% w/w KF in F#4, respectively. These results also suggest that the therapeutic activity of the KF is not dependent on the loading dose of KF, as there was no significant difference between the two of the tested doses (0.25 and 0.5% w/w). This could be because of necessity of lower amount of KF to show therapeutic activity. On the other hand, dexamethasone (positive control) also showed very effective reduction in the inflammation. This was further confirmed by cutaneous histological examination after 72 h of treatment, and the results are presented in Figure 5. As illustrated in Figure 5, KF formulation F#4 in both tested strength (0.25% and 0.5% w/w) was highly effective in the treatment of ACD by the reduction of ear


765


F#4

(b)

DMSO

25

300

20

250

μg of KF/g of skin

Permeated amount of KF per unit area (μg/cm2)

(a)


15 10 5 0

0

5

10 15 Time (h)

20

∗

200 150 100 50 0

25

F#4

DMSO

Figure 2 (a) Comparison of permeated amount of kahalalide F (KF) per unit area as a function of time for F#4 and dimethyl sulfoxide (DMSO). The data are presented as μg/cm2. (b) Comparison of skin retention performed after 24 h of skin permeation study for F#4 and DMSO. The skin permeation represented as μg of kahalalide F/g of skin. Data represent mean ± standard deviation, n = 6; significance DMSO against F#4, *P < 0.05.

Table 4

Mass balance of the applied dose for kahalalide F formulations performed after in vitro permeation study Percent of applied dose

Formulation

Receptor fluid ± SD

Skin retention ± SD

Donor compartment ± SD

Total ± SD

F#1 F#2 F#3 F#4

0.50 ± 0.06 0.54 ± 0.04 0.41 ± 0.03 0.63 ± 0.07

0.093 ± 0.001 0.122 ± 0.002 0.074 ± 0.001 0.152 ± 0.003

94.36 ± 0.43 95.64 ± 0.19 95.3 ± 0.51 94.36 ± 0.34

94.95 ± 0.491 96.31 ± 0.551 95.78 ± 0.541 95.14 ± 0.413

SD, standard deviation. The results are represented as per cent of the applied dose recovered.

F#4

102 101 100 99 98

24 h 48 h 72 h

* * *

* * *

* * *

tr

97 2

4 6 8 10 12 Number of days of storage

14

16

o

0

dr

ug

96

Figure 3 Per cent of kahalalide F (KF) remaining after storage for prototype formulations. Data represent mean ± standard deviation, n = 6.

swelling as compared with untreated control. Many studies have revealed that multiple cytokines, chemokines, eicosanoids[37] and neuropeptides (substance P)[38] are also involved in the regulation of the process in ACD. KF might be involved in the inhibition of cytokines, chemokines or eicosanoids. However, the exact mechanism of antiinflammatory action of KF is not yet elucidated. However, increase in the therapeutic response of KF might be because of the increase in the skin permeation. 766

160 140 120 100 80 60 40 20 0

ea Con m tr en ol Co t) nt ro lV eh ic le F# 4 (0 .2 5% w /w ) F# 4 (0 .5 % w /w D ) ex am et ha so ne

F#3

Ear Thickness (μm)

F#2

(n

% of KF remaining after storage

F#1

Figure 4 Effect of F#4 containing 0.25% and 0.5% w/w kahalalide F treatment on the response of allergic contact dermatitis model in C57BL/6 mice. Data represent mean ± standard deviation, n = 6; *P < 0.05 versus control.

Conclusions Among all formulations, F#4 comprising ethanol, transcutol, ethyl oleate, span 80 and labrasol was considered as the best formulation based on permeated amount of KF in the receiver compartment. Also, skin retention of KF for F#4 was superior to other formulations. This might be because of appropriate percent as well as combination of




Control (No drug treatment)

Control Vehicle

500 μm

F#4 (0.25% w/w)

500 μm

F#4 (0.5% w/w)

500 μm

Dexamethasone

500 μm

500 μm

Figure 5 Haematoxylin and eosin staining of inflammation induced by topical application of 2,4-dinitro-1-fluorobenzene, and after 72 h treatment of F#4 (0.25% and 0.5% w/w of kahalalide F), control and a positive control, dexamethasone. The images were captured using 10× magnifications.

lipophilic and hydrophilic permeation enhancers. However, F#4 showed very less permeation compared with DMSO. This may be because of high permeation enhancing effect of DMSO compared with other permeation enhancers. DMSO represented maximum amount of KF permeated through the skin. Further, the ACD response of F#4 was comparable with that of dexamethasone. The dosedependent therapeutic activity of KF was not statistically different. Considering the potency of KF against cancer cell lines in vitro and its potency in an exploratory clinical trial in patients with severe psoriasis, a prototype formulation described herein such as F#4 might deliver sufficient of drug to ameliorate cell proliferation. Additional preclinical work in an animal model is required to support this hypothesis.

References 1. Hamann M et al. Kahalalides: bioactive peptides from a marine mollusk Elysia rufescens and its algal diet Bryopsis sp. (1). J Org Chem 1996; 61: 6594–6600. 2. Shilabin A et al. Lysosome and HER3 (ErbB3) selective anticancer agent kahalalide F: semisynthetic modifications and antifungal lead-exploration

Declarations Conflict of interest The Author(s) declare(s) that they have no conflicts of interest to disclose. The authors alone are responsible for the content and writing of the paper.

Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.

Acknowledgement The authors acknowledge the financial assistance provided by PharmaMar USA, Inc.. (Cambridge, MA, USA).

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The effect of ion-pair formation combined with penetration enhancers on the skin permeation of loxoprofen.

Effect of Enhancers on in vitro and in vivo Skin Permeation and Deposition of S-Methyl-L-Methionine.

Combining Chemical Permeation Enhancers for Synergistic Effects.

The effects of chemical and physical penetration enhancers on the percutaneous permeation of lidocaine through equine skin.

The effect of mangiferin on skin: Penetration, permeation and inhibition of ECM enzymes.

Meloxicam transdermal delivery: effect of eutectic point on the rate and extent of skin permeation.

Pharmacokinetics of drug permeation through human skin.

Investigating the sonophoresis effect on the permeation of diclofenac sodium using 3D skin equivalent.

Vehicle influence on permeation through intact and compromised skin.

Transport parameters in the human red cell membrane: solute-membrane interactions of hydrophilic alcohols and their effect on permeation.

Effect of liposomal fluidity on skin permeation of sodium fluorescein entrapped in liposomes.

In vitro permeation of platinum and rhodium through Caucasian skin.

Progestin permeation through polymer membranes III: Polymerization solvent effect on progesterone permeation through hydrogel membranes.

In vitro permeation of platinum through African and Caucasian skin.

Nanoemulsions (NEs), liposomes (LPs) and solid lipid nanoparticles (SLNs) for retinyl palmitate: effect on skin permeation.

Permeation of chromium salts through human skin in vitro.

Safety concerns over the use of intestinal permeation enhancers: A mini-review.

Quantitative Analysis of the Enhanced Permeation and Retention (EPR) Effect.

Improved skin permeation of methotrexate via nanosized ultradeformable liposomes.

Formulation of cellulose film containing permeation enhancers for prolonged delivery of propranolol hydrocloride.

Co-encapsulation of lipophilic antioxidants into niosomal carriers: percutaneous permeation studies for cosmeceutical applications.

Enhanced Permeation of an Antiemetic Drug from Buccoadhesive Tablets by Using Bile Salts as Permeation Enhancers: Formulation Characterization, In Vitro, and Ex Vivo Studies.

gel microgel formulation in microneedle-pierced skin: vertical (depth averaged) and horizontal permeation profiles.

Saturated long-chain esters of isopulegol as novel permeation enhancers for transdermal drug delivery.