Colloids and Surfaces B: Biointerfaces 116 (2014) 351–358
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Amphotericin B topical microemulsion: Formulation, characterization and evaluation Dhruv Butani, Chetan Yewale, Ambikanandan Misra ∗ Pharmacy Department, Faculty of Technology & Engineering, The Maharaja Sayajirao University of Baroda, Kalabhavan, Vadodara 390001, Gujarat State, India
a r t i c l e
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Article history: Received 10 October 2013 Received in revised form 6 January 2014 Accepted 9 January 2014 Available online 19 January 2014 Keywords: Amphotericin B Antifungal Microemulsion Topical Permeation
a b s t r a c t The present studies were designed to develop a microemulsion (ME) formulation of Amphotericin B (Amp B) for the treatment of invasive fungal infections. The oil phase was selected on the basis of drug solubility whereas the surfactant and co-surfactant were screened and selected on the basis of their oil solubilizing capacity as well as their efficiency to form ME. Pseudo-ternary phase diagrams were constructed and on the basis of ME existence ranges various formulations of Amp B were developed. The influence of surfactant and co-surfactant mass ratio (Smix) on the ME formation and permeation of ME through excised rat skin was studied. The optimized formulation (ME 7) consisting of 0.1% (w/w) Amp B, 5% (w/w) Isopropyl Myristate and 35% (w/w) Smix (3:1, Tween 80 and Propylene glycol), has shown a globule size of 84.20 ± 2.13 nm, a polydispersity index of 0.164 ± 0.031, pH 7.36 ± 0.02 and conductance of 229.3 ± 1.95 S. ME 7 exhibited 2-fold higher drug permeation as compared to plain drug solution. Besides this, the formulation was also evaluated for drug content, stability, skin retention, skin sensitivity and anti-fungal activity. In vitro anti-fungal activity in Trichophyton rubrum fungal species have shown that ME7 has higher zone of inhibition and the formulation was found stable at 2–8 ◦ C and at room temperature (25 ± 2 ◦ C) for the period of three months. The results indicate that, the investigated ME may be used as a promising alternative for Amp B therapy. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Fungus is normally found on the skin, in the mouth, throat, stomach, colon, vagina, and rectum. It causes health problems when there is an over growth in one of these areas of the body. When this organism proliferates, it can produce symptomatic infections of the mouth, intestine, vagina or skin. Amphotericin B (Amp B) is a broad spectrum polyene macrolide antifungal antibiotic mainly used for the treatment of invasive fungal infections [1,2]. Amp B remains “gold standard” drug of choice for the treatment of disseminated mycosis in immunodepressed patients e.g. AIDS, organ transplants, cancer chemotherapy [3–5] and against the antimony-resistant visceral leshmeniasis [6]. The fungicidal and leishmanicidal activities of Amp B are closely related to its unusual chemical structure characterized by the hydrophilic polyhydroxyl and hydrophobic
∗ Corresponding author at: Pharmacy Department, Faculty of Technology & Engineering, The Maharaja Sayajirao University of Baroda, Post Box No.: 51, Kalabhavan, Vadodara 390001, Gujarat State, India Tel.: +91 265 2419231; fax: +91 265 2418927. E-mail addresses: [email protected], [email protected], [email protected] (A. Misra). http://dx.doi.org/10.1016/j.colsurfb.2014.01.014 0927-7765/© 2014 Elsevier B.V. All rights reserved.
´˚ of the molecule polyene faces on the long axis (approximately 25 A) [7]. It interacts selectively with 24-substituted sterols, such as ergosterol and episterol found in fungal and leishmania cells [8], respectively. Amp B acts on ergosterol, a steroid present in the membrane of the fungal cells [9] by increasing its permeability that promotes an ion efflux into the parasite, thus leading to its death [10,11]. Amp B has poor bioavailability by oral route and its usefulness is compromised by a high incidence of adverse reactions including fever, chills, nausea, vomiting, headache and renal dysfunction with associated anemia, hypokalemia and hypomagnesaemia when administered via parenteral route [12]. These efforts have led to the development of commercial preparations of phospholipid vesicles for therapeutic use such as AmBisome, Amphotec and Abelcet [13,14]. However, the utility of these new products is greatly limited by their high costs and side effects. So, there is need to develop low cost formulations. There has been increased interest during recent years in the use of topical vehicles that may modify drug penetration into the skin. Optimal vehicles have to exert a high capacity for incorporating both lipophilic and hydrophilic drugs as well as high skin permeability. Many of the dermal vehicles contain chemical enhancers
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and strong solvents to achieve these goals [15]. Irritation is the major disadvantage of chronic application. Therefore, it is desirable to develop a topical vehicle system that does not require chemical enhancers or alcohols to facilitate drug penetration into and through the skin. In this study, the cutaneous drug delivery potential of an alcohol-free MEs composed of non-irritating and pharmaceutically acceptable ingredients was evaluated. In topical drug delivery, diffusion takes place mainly through the stratum corneum (lipoidal barrier) and the drug follows different paths to permeate through the stratum corneum. Owing to poor aqueous solubility, Amp B cannot permeate through the skin [16]. Thus, for Amp B optimum solubility in both aqueous and lipid phase is vital in order to maximize its flux. Therefore, with an aim to enhance the solubility and eventually the dermal bioavailability of Amp B, ME formulations were prepared. It increases the dermal penetration and permeation of the drug. Owing to the facile and low cost of preparation, ME was opted over other colloidal counter parts such as liposomes, niosomes and nanoparticles [17]. Colloidal systems and innovative drug-delivery systems such as MEs have been investigated as drug delivery and targeting systems, since they can modify bioavailability, stability and side effects of various drugs [18]. Several mechanisms have been proposed to explain the advantages of ME for the topical delivery of drugs [19–22]. Several factors affecting topical drug delivery include the affinity of a drug to the internal phase in ME, ingredients of ME reducing the barrier of the stratum corneum, increased concentration gradient towards skin and the dispersed phase acting as a reservoir, which makes it possible to maintain a constant concentration in continuous phase [23–25]. High cost, various side effects and lower drug loading capacity are the drawbacks of currently available formulations and to avoid these drawbacks the development of new pharmaceutical formulations of Amp B for topical administration is desirable. Considering the above drawbacks the present study was aimed to develop a stable topical ME formulation of Amp B for the treatment of invasive fungal infections.
Table 1 Saturation solubility of Amp B in different oils, surfactants and co-surfactants at 25 ◦ C (mean ± SD; n = 3). Sr. no.
Components
Solubility (mg/ml)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Capmul MCM C8 Labrafac Lipofile Oleic acid Isopropyl myristate Lemon oil Tween 80 Tween 20 Labrasol Transcutol PEG 400 Isopropyl alcohol Propylene glycol
0.138 ± 0.052 0.216 ± 0.046 0.027 ± 0.009 0.819 ± 0.095 0.043 ± 0.0022 4.261 ± 0.155 2.379 ± 0.123 2.152 ± 0.140 0.825 ± 0.078 0.547 ± 0.032 0.127 ± 0.036 2.943 ± 0.263
at 405.5 nm by appropriate dilution of filtrate with methanol. Solubility results of Amp B in various oils, surfactants and co-surfactants are given in Table 1. 2.3. Construction of pseudo-ternary phase diagrams In order to find out the concentration ranges of components in ME existing range, pseudo-ternary phase diagrams were constructed using water titration method at room temperature (25 ◦ C). Three different phase diagrams were prepared with the 1:1, 2:1 and 3:1 weight ratios of tween 80 to PG respectively. For each phase diagram at a specific weight ratio, the ratios of oil to the mixture of surfactant and co-surfactant were varied as 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1. The mixtures of oil, surfactant and co-surfactant at certain weight ratios were diluted with drop wise addition of water under moderate magnetic stirring until the mixture became clear. The concentration of components was recorded in order to complete the pseudo-ternary phase diagram. Based on these diagrams, appropriate concentration of materials were selected and used in the preparation of Amp B ME.
2. Materials and methods 2.4. Preparation of ME 2.1. Materials Amp B was obtained as a gift sample from Lyka Labs. Pvt. Ltd, Mumbai, India. Capmul MCM C8 was obtained as a gift sample from Abitec Corporation Limited, Columbus, Ohio. Tween 80 and Isopropyl myristate (IPM) were purchased from Loba Chem., Mumbai, India. Labrafac Lipofile, Labrasol and Transcutol were obtained as a gift sample from Colorcon Asia Pvt. Ltd., Goa, India. Oleic acid (OA), Lemon oil, Tween 20, Polyethylene Glycol 400 (PEG 400), Isopropyl alcohol (IPA) and Propylene Glycol (PG) were purchased from Himedia Pvt. Ltd., Mumbai, India. Fungal strain Trichophyton rubrum, ATCC Code 28188TM (KWIK-STIK 0444 P) was purchased from ATCC Microbiologics, Minnesota, USA. 2.2. Screening of oils, surfactants and co-surfactants for ME The solubility of Amp B in various oils, surfactants and cosurfactants were determined to find out the appropriate oils, surfactants and co-surfactants with good solubilizing capacity for Amp B in ME. Oils employed were OA, IPM, Labrafac Lipofile and Capmul MCM C8. Surfactants and co-surfactants employed were tween 80, tween 20, labrasol, transcutol, IPA, PG and PEG 400. An excess amount of Amp B was added into 10 ml of each oil, surfactants and co-surfactants [3] and the resultant mixture was shaken reciprocally at 25 ◦ C for 72 h followed by centrifugation for 10 min at 9000 rpm. The supernatant was filtered through a membrane filter (0.45 m) and Amp B was determined spectro-photometrically
All MEs were prepared by using double distilled water in order to avoid surface-active impurities. According to the ME areas in the phase diagrams, different Amp B ME formulations were prepared by varying the ratios between surfactant/co-surfactant and at Km = 3:1 given in Table 2. Different Amp B o/w MEs were prepared by dissolving 0.1% (w/w) Amp B in 5% (w/w) IPM and different quantities of 3:1 Smix (Tween 80 and PG) with the aid of vortex mixer. The mixture was made up to 100% (w/w) with drop wise addition of double distilled water with continuous stirring. In order to verify the effect of Smix on MEs, all other components and process variables were kept constant. ME was optimized with respect to Smix ratios and its concentration effect on ex-vivo permeation characteristics. Amp B loaded MEs were protected from the light by storing in amber colored bottles wrapped with aluminium foil. 2.5. Characterization of ME According to the ME regions in the phase diagrams, seven ME formulations were prepared and evaluated for following parameters: 2.5.1. Droplet size and polydispersity index (PDI) The average droplet size and PDI of MEs were evaluated by photon correlation spectroscopy (Malvern, UK). All the samples were analyzed in triplicates by dilution in 1:5 ratios with water and filtered through 0.22 m filter. The samples were analysed by using
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Table 2 Compositions and characterization (mean ± S.D.; n = 3) of selected Amp B ME formulations. Formulation code
Composition [IPM: Smix (3:1) (Tween 80:propylene glycol):water:Amp B]
Droplet diameter (nm)
Polydispersity Index (PDI)
Viscosity (mP S)
pH
Conductance (S)
% Transparency
ME1 ME2 ME3 ME4 ME5 ME6 ME7
5.0: 65.0: 29.9: 0.1 5.0: 60.0:34.9: 0.1 5.0: 55.0:39.9: 0.1 5.0:50.0:44.9:0.1 5.0:45.0:49.9:0.1 5.0:40.0:54.9:0.1 5.0:35.0:59.9:0.1
25.50 ± 1.22 34.29 ± 0.86 49.17 ± 1.39 53.79 ± 1.06 61.55 ± 2.67 69.89 ± 2.32 84.20 ± 2.13
0.281 ± 0.006 0.328 ± 0.014 0.286 ± 0.012 0.299 ± 0.029 0.207 ± 0.010 0.358 ± 0.043 0.164 ± 0.031
723 ± 0.6 701 ± 0.7 618 ± 1.8 587 ± 0.9 498 ± 1.3 394 ± 2.2 340 ± 0.7
7.62 ± 0.02 7.55 ± 0.04 7.49 ± 0.01 7.52 ± 0.02 7.41 ± 0.03 7.45 ± 0.01 7.36 ± 0.02
97.5 ± 2.23 115.6 ± 2.85 135.4 ± 1.91 169.2 ± 1.67 181.6 ± 2.19 208.1 ± 3.95 229.3 ± 1.95
99.1 ± 0.19 98.4 ± 0.22 97.4 ± 0.19 98.0 ± 0.06 98.5 ± 0.13 96.8 ± 0.20 98.3 ± 0.28
1 ml cuvettes in a thermostatic chamber at 25 ◦ C and the measurements were performed using a He–Ne laser.
washed and examined for integrity and then stored in a refrigerator at 4 ◦ C overnight for later use [20,28].
2.5.2. Measurement of viscosity The ME formulations were evaluated for their rheological behavior at 25 ± 2 ◦ C using Brookfield viscometer model DV-III equipped with spindle number 40 (Engineering Laboratories, Inc., Middleboro, MA, USA).
2.6.2. In vitro permeation study The extent and rate of skin permeation of Amp B from prepared MEs and plain drug solution was determined by using diffusion cell with effective diffusion area of 1.54 cm2 . ME containing 0.1% (w/w) of Amp B was applied on to the skin surface and mounted between the two half of the cells of which stratum corneum faces the donor compartment and was sealed with parafilm to provide occlusive conditions in order to prevent evaporation of water from the formulations [27]. The receptor chamber was filled with 20 ml of mixture of phosphate buffer solution pH 7.4 and methanol (80:20%, v/v) [1], temperature was maintained at 32 ± 0.5 ◦ C [28,29] and was stirred at 400 rpm throughout the experiment. 0.5 ml of the ME was applied on the epidermal surface of the skin and 0.5 ml [30] of the receptor medium was extracted at 1 h, 2 h, 3 h, 4 h, 5 h, 6 h and 24 h for Amp B determination using UV spectroscopy at max 408.5 nm. Extracted volume was replaced immediately with an equal volume of mixture of phosphate buffer (pH 7.4) and methanol (80: 20%, v/v). All samples were filtered through a filter paper and analyzed by UV spectroscopy (UV-1700, Shimadzu, Japan) at max 408.5 nm.
2.5.3. Centrifugation The centrifugation of formulations at 13,000 rpm for 30 min and at 4000 rpm for 4 h was carried out to assess the physical stability of ME. 2.5.4. Measurement of electrical conductivity Electrical conductivity of the formulations was measured using a conductivity meter (DDS-11C, Shanghai Instrument, China) and based on the electrical conductivity, the phase systems of the MEs were determined. The electrode was dipped in the ME sample until equilibrium was reached. Before conductivity measurement, the conductivity cell was calibrated using standard KCl solution. 2.5.5. Measurement of pH The pH of the systems was measured by direct immersion of pH meter electrode (Lab India, Mumbai, India) in the formulations at room temperature [5] and all the measurements were carried out in triplicates. 2.5.6. Macroscopic appearance The color, isotropy and homogeneity of the MEs and the presence of precipitates or phase separation were scored after visual and cross polarizers examination at room temperature [26]. 2.5.7. % Transparency The % transparencies of MEs were measured by UV spectrophotometer (UV-1700, Shimadzu, Japan) at a max of 700.00 nm. 2.5.8. Dye solubility test A water soluble dye (methyl orange) was added to the ME system and the phase system was evaluated.
2.6.3. Data analysis The cumulative amount of Amp B permeated through excised rat skins was calculated by Eq. (1): [3,31,32] Qs =
2.6.1. Preparation of skin In order to evaluate the effect of Amp B ME on skin permeation as compared to plain drug solution, formulations were subjected to in vitro permeation study. The animal study protocol to conduct skin permeation was approved by the Institutional Animal Ethics Committee. Abdominal skins were obtained from female Albino Wistar rats weighing 200 ± 30 g [27]. A 2.5 cm × 2.5 cm patch of skin from the abdominal region was excised after hair removal with a depilatory, and then the subcutaneous fat and connective tissue were removed. To achieve higher reproducibility, the excised skins were
n−1 s
n−i
Vi × Ci
(1)
where Cn is the drug concentration of the receiver solution at each sampling time, Ci is the drug concentration of the sample, and V0 and Vi are the volumes of the receiver solution and the sample, respectively. S is the effective diffusion area (S = 1.54 cm2 ). The permeation rate of Amp B at a steady-state (Jss , g cm−2 h−1 ) through the rat skin was calculated from the slope of linear portion of the plots of Qt versus time [33,34]. The experiments were carried out in triplicate for each sample, and the results were presented as an average ± SD. The permeability coefficient (Kp , cm/h) was calculated according to following Eq. (2): [3,27,33] Kp =
2.6. Ex-vivo diffusion study
V0 × Cn +
Jss C0
(2)
where Kp is the permeability coefficient, Jss is the flux calculated at steady-state and C0 represents the drug concentration which remains constant in the vehicle. 2.7. Skin retention study Protocol for skin retention study was approved by the Institutional Animal Ethics Committee. Skin was obtained from the abdomen of female Albino Wister rat weighing 200 ± 30 g. The full-thickness skin was excised after hair was removed with a depilatory. Subcutaneous fat and other extraneous tissues were
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trimmed; the skin was washed with physiological saline followed by phosphate buffered saline (pH 7.4) and then visually inspected for integrity to ensure the absence of holes or other imperfections. The excised rat skin were stored at −20 ◦ C and used within 1 week of harvest. The skin was placed with stratum corneum facing upward (inside of the tube) and dermal side downward (to face the medium). The position-fixed skin was made water tight by a rubber band. Assembly was adjusted as previously mentioned under the ex-vivo diffusion study. After 24 h, the effective diffusion area of the skin was separated, washed several times with distilled water, to remove formulation excess, and then cut into small pieces. The segments obtained were vortexed with methanol and then left for soaking for 24 hrs to ensure effective extraction of the retained drug from the skin. The resulting mixture was then filtered using 0.45 m syringe filter and 1 ml from the filtrate was diluted with receptor fluid then filtered and Amp B was quantified using UV spectrophotometry at max 408.5 nm [5]. 2.8. Skin sensitivity studies This study was performed on rabbits to evaluate the irritant potential of the developed formulation after topical application [35,36]. The rabbits (weighing 2.0–2.5 kg) were purchased from Department of Biochemistry, The Maharaja Sayajirao University of Baroda (Vadodara, India). The hairs on the back of rabbit were removed 24 h prior to the administration of formulations [32] whereas the control group was treated with normal saline. ME1, ME3, ME5, ME7 and the saline solution containing 0.1% Amp B was applied to the treatment group twice a day for 5 days consecutively (n = 3) [6]. The animals were observed for any signs of itching or change in skin such as erythema, papule, flakiness and dryness for a period of 5 days [37]. After withdrawal, observation for single or multiple administrations was continued for 3 days [38]. The irritation scores of the test area were obtained by judging the extent of erythema and edema according to the criteria [39]. Erythema and edema were graded as follows: 0 for no visible reaction, 1 for just present reaction, 2 for slight reaction, 3 for moderate reaction, and 4 for severe reaction. Eventually, the total scores for irritation test in each condition were calculated using the following Eq. (3): [32]. Average irritation scores =
erythema reaction scores + dropsy reaction no. of animals
(3)
2.9. In vitro antifungal activity The various ME formulations ME1, ME3, ME5, ME7 and plain drug solution were assayed for antifungal activity against the fungal strain Trichophyton rubrum. This fungus was grown on Sabouraud’s agar plate at 25 ± 2 ◦ C. The plates were first sterilized in hot air oven at 160 ◦ C for 60 min. The fungal culture suspensions were made according to the ATCC protocol of microbiologics [40] and were allowed to stand for 20 min before transferring on to the solid agar medium as the strain was available in lyophilized form. To the sterile petri-dishes in which solidified agar growth medium was taken, the fungal culture suspension was spread with the help of stick. The inoculums were spread uniformly over the solid agar surface by spreader glass rod and incubated at 25 ± 2 ◦ C for 7 days to grow fungus. Then wells were made in the middle of the plates with the help of sterile cork borer and the wells were filled with the formulations and the plates were incubated at 25 ± 2 ◦ C. Clear rings appeared around the wells in 48 h. The rings were called the zone of inhibition. Larger the zone of inhibition, more effective is the formulation. The antifungal activity was evaluated by measuring
zones of inhibition of fungal growth surrounding the formulations. The zones of inhibition were measured with scale in mm and the complete antifungal analysis was carried out under strict aseptic conditions and the mean inhibition zone from three plates was calculated [5]. 2.10. Stability of ME The physical stability of ME containing Amp B was investigated via clarity, particle size analysis and phase separation, which was observed at 2–8 ◦ C and at room temperature upto 3 months. The centrifugation was carried out at 13,000 rpm for 30 min to assess the physical stability of ME [41]. Clarity, phase separation and concentration of Amp B were investigated monthly to judge the optimal storage temperature. Chemical stability was evaluated on drug loaded formulations, stored at 2–8 ◦ C and at room temperature by determining Amp B content by UV spectrophotometry at max 405.5 nm. 2.11. Statistical analysis All skin permeation experiments were repeated three times and data were expressed as the mean value ± S.D. (n = 3). Statistical data were analyzed by one way analysis of variance (ANOVA). A multiple comparison test was used to compare different formulations, and a P value of ME5 > ME3 > ME1 > plain drug solution. ME7 showed highest zone of inhibition due to its high fluidity. Plain drug solution showed lowest zone of inhibition due to its less penetration effect than ME. The water content was more in ME7, so the drug released from it was more as compared to ME1, ME3 and ME5, in which the drug molecules were more rigid in viscous structure. This indicated that the ME7 was more effective as compared to another formulation. 3.8. Stability Study Stability studies were carried out to detect any changes in pH, droplet size, and drug content of formulation for the period of 3 months at 2–8 ◦ C and room temperature. All formulations were physically stable, retaining homogeneity with no phase separation after 3 months. The major changes of droplet size and degradation of Amp B were not observed during 3 months. The centrifuge tests showed that all MEs had good physical stability. No marked changes were recorded in the stored MEs except for slight decrease in viscosity. This decrease may be due to loss of some water during storage. After 3 months, there was a very little change in pH, droplet size, drug content and viscosity at 2–8 ◦ C than room temperature. So, the ME formulations were more stable at 2–8 ◦ C than room temperature. 4. Conclusion The Amp B ME was formulated for topical application and the results of present study clearly demonstrated the role of ME in effective topical delivery of Amp B. ME7 has shown higher skin deposition, lower skin irritation and better anti-fungal activity. The developed system may provide better remission from the disease due to localized delivery with minimal side effects and the data from in vitro study has been encouraging but further evaluation is needed to elucidate the clinical efficacy of this topical dosage form. Conflicts of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Amphotericin B topical microemulsion: Formulation, characterization and evaluation Dhruv Butani, Chetan Yewale, Ambikanandan Misra ∗ Pharmacy Department, Faculty of Technology & Engineering, The Maharaja Sayajirao University of Baroda, Kalabhavan, Vadodara 390001, Gujarat State, India
a r t i c l e
i n f o
Article history: Received 10 October 2013 Received in revised form 6 January 2014 Accepted 9 January 2014 Available online 19 January 2014 Keywords: Amphotericin B Antifungal Microemulsion Topical Permeation
a b s t r a c t The present studies were designed to develop a microemulsion (ME) formulation of Amphotericin B (Amp B) for the treatment of invasive fungal infections. The oil phase was selected on the basis of drug solubility whereas the surfactant and co-surfactant were screened and selected on the basis of their oil solubilizing capacity as well as their efficiency to form ME. Pseudo-ternary phase diagrams were constructed and on the basis of ME existence ranges various formulations of Amp B were developed. The influence of surfactant and co-surfactant mass ratio (Smix) on the ME formation and permeation of ME through excised rat skin was studied. The optimized formulation (ME 7) consisting of 0.1% (w/w) Amp B, 5% (w/w) Isopropyl Myristate and 35% (w/w) Smix (3:1, Tween 80 and Propylene glycol), has shown a globule size of 84.20 ± 2.13 nm, a polydispersity index of 0.164 ± 0.031, pH 7.36 ± 0.02 and conductance of 229.3 ± 1.95 S. ME 7 exhibited 2-fold higher drug permeation as compared to plain drug solution. Besides this, the formulation was also evaluated for drug content, stability, skin retention, skin sensitivity and anti-fungal activity. In vitro anti-fungal activity in Trichophyton rubrum fungal species have shown that ME7 has higher zone of inhibition and the formulation was found stable at 2–8 ◦ C and at room temperature (25 ± 2 ◦ C) for the period of three months. The results indicate that, the investigated ME may be used as a promising alternative for Amp B therapy. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Fungus is normally found on the skin, in the mouth, throat, stomach, colon, vagina, and rectum. It causes health problems when there is an over growth in one of these areas of the body. When this organism proliferates, it can produce symptomatic infections of the mouth, intestine, vagina or skin. Amphotericin B (Amp B) is a broad spectrum polyene macrolide antifungal antibiotic mainly used for the treatment of invasive fungal infections [1,2]. Amp B remains “gold standard” drug of choice for the treatment of disseminated mycosis in immunodepressed patients e.g. AIDS, organ transplants, cancer chemotherapy [3–5] and against the antimony-resistant visceral leshmeniasis [6]. The fungicidal and leishmanicidal activities of Amp B are closely related to its unusual chemical structure characterized by the hydrophilic polyhydroxyl and hydrophobic
∗ Corresponding author at: Pharmacy Department, Faculty of Technology & Engineering, The Maharaja Sayajirao University of Baroda, Post Box No.: 51, Kalabhavan, Vadodara 390001, Gujarat State, India Tel.: +91 265 2419231; fax: +91 265 2418927. E-mail addresses: [email protected], [email protected], [email protected] (A. Misra). http://dx.doi.org/10.1016/j.colsurfb.2014.01.014 0927-7765/© 2014 Elsevier B.V. All rights reserved.
´˚ of the molecule polyene faces on the long axis (approximately 25 A) [7]. It interacts selectively with 24-substituted sterols, such as ergosterol and episterol found in fungal and leishmania cells [8], respectively. Amp B acts on ergosterol, a steroid present in the membrane of the fungal cells [9] by increasing its permeability that promotes an ion efflux into the parasite, thus leading to its death [10,11]. Amp B has poor bioavailability by oral route and its usefulness is compromised by a high incidence of adverse reactions including fever, chills, nausea, vomiting, headache and renal dysfunction with associated anemia, hypokalemia and hypomagnesaemia when administered via parenteral route [12]. These efforts have led to the development of commercial preparations of phospholipid vesicles for therapeutic use such as AmBisome, Amphotec and Abelcet [13,14]. However, the utility of these new products is greatly limited by their high costs and side effects. So, there is need to develop low cost formulations. There has been increased interest during recent years in the use of topical vehicles that may modify drug penetration into the skin. Optimal vehicles have to exert a high capacity for incorporating both lipophilic and hydrophilic drugs as well as high skin permeability. Many of the dermal vehicles contain chemical enhancers
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and strong solvents to achieve these goals [15]. Irritation is the major disadvantage of chronic application. Therefore, it is desirable to develop a topical vehicle system that does not require chemical enhancers or alcohols to facilitate drug penetration into and through the skin. In this study, the cutaneous drug delivery potential of an alcohol-free MEs composed of non-irritating and pharmaceutically acceptable ingredients was evaluated. In topical drug delivery, diffusion takes place mainly through the stratum corneum (lipoidal barrier) and the drug follows different paths to permeate through the stratum corneum. Owing to poor aqueous solubility, Amp B cannot permeate through the skin [16]. Thus, for Amp B optimum solubility in both aqueous and lipid phase is vital in order to maximize its flux. Therefore, with an aim to enhance the solubility and eventually the dermal bioavailability of Amp B, ME formulations were prepared. It increases the dermal penetration and permeation of the drug. Owing to the facile and low cost of preparation, ME was opted over other colloidal counter parts such as liposomes, niosomes and nanoparticles [17]. Colloidal systems and innovative drug-delivery systems such as MEs have been investigated as drug delivery and targeting systems, since they can modify bioavailability, stability and side effects of various drugs [18]. Several mechanisms have been proposed to explain the advantages of ME for the topical delivery of drugs [19–22]. Several factors affecting topical drug delivery include the affinity of a drug to the internal phase in ME, ingredients of ME reducing the barrier of the stratum corneum, increased concentration gradient towards skin and the dispersed phase acting as a reservoir, which makes it possible to maintain a constant concentration in continuous phase [23–25]. High cost, various side effects and lower drug loading capacity are the drawbacks of currently available formulations and to avoid these drawbacks the development of new pharmaceutical formulations of Amp B for topical administration is desirable. Considering the above drawbacks the present study was aimed to develop a stable topical ME formulation of Amp B for the treatment of invasive fungal infections.
Table 1 Saturation solubility of Amp B in different oils, surfactants and co-surfactants at 25 ◦ C (mean ± SD; n = 3). Sr. no.
Components
Solubility (mg/ml)
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Capmul MCM C8 Labrafac Lipofile Oleic acid Isopropyl myristate Lemon oil Tween 80 Tween 20 Labrasol Transcutol PEG 400 Isopropyl alcohol Propylene glycol
0.138 ± 0.052 0.216 ± 0.046 0.027 ± 0.009 0.819 ± 0.095 0.043 ± 0.0022 4.261 ± 0.155 2.379 ± 0.123 2.152 ± 0.140 0.825 ± 0.078 0.547 ± 0.032 0.127 ± 0.036 2.943 ± 0.263
at 405.5 nm by appropriate dilution of filtrate with methanol. Solubility results of Amp B in various oils, surfactants and co-surfactants are given in Table 1. 2.3. Construction of pseudo-ternary phase diagrams In order to find out the concentration ranges of components in ME existing range, pseudo-ternary phase diagrams were constructed using water titration method at room temperature (25 ◦ C). Three different phase diagrams were prepared with the 1:1, 2:1 and 3:1 weight ratios of tween 80 to PG respectively. For each phase diagram at a specific weight ratio, the ratios of oil to the mixture of surfactant and co-surfactant were varied as 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1. The mixtures of oil, surfactant and co-surfactant at certain weight ratios were diluted with drop wise addition of water under moderate magnetic stirring until the mixture became clear. The concentration of components was recorded in order to complete the pseudo-ternary phase diagram. Based on these diagrams, appropriate concentration of materials were selected and used in the preparation of Amp B ME.
2. Materials and methods 2.4. Preparation of ME 2.1. Materials Amp B was obtained as a gift sample from Lyka Labs. Pvt. Ltd, Mumbai, India. Capmul MCM C8 was obtained as a gift sample from Abitec Corporation Limited, Columbus, Ohio. Tween 80 and Isopropyl myristate (IPM) were purchased from Loba Chem., Mumbai, India. Labrafac Lipofile, Labrasol and Transcutol were obtained as a gift sample from Colorcon Asia Pvt. Ltd., Goa, India. Oleic acid (OA), Lemon oil, Tween 20, Polyethylene Glycol 400 (PEG 400), Isopropyl alcohol (IPA) and Propylene Glycol (PG) were purchased from Himedia Pvt. Ltd., Mumbai, India. Fungal strain Trichophyton rubrum, ATCC Code 28188TM (KWIK-STIK 0444 P) was purchased from ATCC Microbiologics, Minnesota, USA. 2.2. Screening of oils, surfactants and co-surfactants for ME The solubility of Amp B in various oils, surfactants and cosurfactants were determined to find out the appropriate oils, surfactants and co-surfactants with good solubilizing capacity for Amp B in ME. Oils employed were OA, IPM, Labrafac Lipofile and Capmul MCM C8. Surfactants and co-surfactants employed were tween 80, tween 20, labrasol, transcutol, IPA, PG and PEG 400. An excess amount of Amp B was added into 10 ml of each oil, surfactants and co-surfactants [3] and the resultant mixture was shaken reciprocally at 25 ◦ C for 72 h followed by centrifugation for 10 min at 9000 rpm. The supernatant was filtered through a membrane filter (0.45 m) and Amp B was determined spectro-photometrically
All MEs were prepared by using double distilled water in order to avoid surface-active impurities. According to the ME areas in the phase diagrams, different Amp B ME formulations were prepared by varying the ratios between surfactant/co-surfactant and at Km = 3:1 given in Table 2. Different Amp B o/w MEs were prepared by dissolving 0.1% (w/w) Amp B in 5% (w/w) IPM and different quantities of 3:1 Smix (Tween 80 and PG) with the aid of vortex mixer. The mixture was made up to 100% (w/w) with drop wise addition of double distilled water with continuous stirring. In order to verify the effect of Smix on MEs, all other components and process variables were kept constant. ME was optimized with respect to Smix ratios and its concentration effect on ex-vivo permeation characteristics. Amp B loaded MEs were protected from the light by storing in amber colored bottles wrapped with aluminium foil. 2.5. Characterization of ME According to the ME regions in the phase diagrams, seven ME formulations were prepared and evaluated for following parameters: 2.5.1. Droplet size and polydispersity index (PDI) The average droplet size and PDI of MEs were evaluated by photon correlation spectroscopy (Malvern, UK). All the samples were analyzed in triplicates by dilution in 1:5 ratios with water and filtered through 0.22 m filter. The samples were analysed by using
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Table 2 Compositions and characterization (mean ± S.D.; n = 3) of selected Amp B ME formulations. Formulation code
Composition [IPM: Smix (3:1) (Tween 80:propylene glycol):water:Amp B]
Droplet diameter (nm)
Polydispersity Index (PDI)
Viscosity (mP S)
pH
Conductance (S)
% Transparency
ME1 ME2 ME3 ME4 ME5 ME6 ME7
5.0: 65.0: 29.9: 0.1 5.0: 60.0:34.9: 0.1 5.0: 55.0:39.9: 0.1 5.0:50.0:44.9:0.1 5.0:45.0:49.9:0.1 5.0:40.0:54.9:0.1 5.0:35.0:59.9:0.1
25.50 ± 1.22 34.29 ± 0.86 49.17 ± 1.39 53.79 ± 1.06 61.55 ± 2.67 69.89 ± 2.32 84.20 ± 2.13
0.281 ± 0.006 0.328 ± 0.014 0.286 ± 0.012 0.299 ± 0.029 0.207 ± 0.010 0.358 ± 0.043 0.164 ± 0.031
723 ± 0.6 701 ± 0.7 618 ± 1.8 587 ± 0.9 498 ± 1.3 394 ± 2.2 340 ± 0.7
7.62 ± 0.02 7.55 ± 0.04 7.49 ± 0.01 7.52 ± 0.02 7.41 ± 0.03 7.45 ± 0.01 7.36 ± 0.02
97.5 ± 2.23 115.6 ± 2.85 135.4 ± 1.91 169.2 ± 1.67 181.6 ± 2.19 208.1 ± 3.95 229.3 ± 1.95
99.1 ± 0.19 98.4 ± 0.22 97.4 ± 0.19 98.0 ± 0.06 98.5 ± 0.13 96.8 ± 0.20 98.3 ± 0.28
1 ml cuvettes in a thermostatic chamber at 25 ◦ C and the measurements were performed using a He–Ne laser.
washed and examined for integrity and then stored in a refrigerator at 4 ◦ C overnight for later use [20,28].
2.5.2. Measurement of viscosity The ME formulations were evaluated for their rheological behavior at 25 ± 2 ◦ C using Brookfield viscometer model DV-III equipped with spindle number 40 (Engineering Laboratories, Inc., Middleboro, MA, USA).
2.6.2. In vitro permeation study The extent and rate of skin permeation of Amp B from prepared MEs and plain drug solution was determined by using diffusion cell with effective diffusion area of 1.54 cm2 . ME containing 0.1% (w/w) of Amp B was applied on to the skin surface and mounted between the two half of the cells of which stratum corneum faces the donor compartment and was sealed with parafilm to provide occlusive conditions in order to prevent evaporation of water from the formulations [27]. The receptor chamber was filled with 20 ml of mixture of phosphate buffer solution pH 7.4 and methanol (80:20%, v/v) [1], temperature was maintained at 32 ± 0.5 ◦ C [28,29] and was stirred at 400 rpm throughout the experiment. 0.5 ml of the ME was applied on the epidermal surface of the skin and 0.5 ml [30] of the receptor medium was extracted at 1 h, 2 h, 3 h, 4 h, 5 h, 6 h and 24 h for Amp B determination using UV spectroscopy at max 408.5 nm. Extracted volume was replaced immediately with an equal volume of mixture of phosphate buffer (pH 7.4) and methanol (80: 20%, v/v). All samples were filtered through a filter paper and analyzed by UV spectroscopy (UV-1700, Shimadzu, Japan) at max 408.5 nm.
2.5.3. Centrifugation The centrifugation of formulations at 13,000 rpm for 30 min and at 4000 rpm for 4 h was carried out to assess the physical stability of ME. 2.5.4. Measurement of electrical conductivity Electrical conductivity of the formulations was measured using a conductivity meter (DDS-11C, Shanghai Instrument, China) and based on the electrical conductivity, the phase systems of the MEs were determined. The electrode was dipped in the ME sample until equilibrium was reached. Before conductivity measurement, the conductivity cell was calibrated using standard KCl solution. 2.5.5. Measurement of pH The pH of the systems was measured by direct immersion of pH meter electrode (Lab India, Mumbai, India) in the formulations at room temperature [5] and all the measurements were carried out in triplicates. 2.5.6. Macroscopic appearance The color, isotropy and homogeneity of the MEs and the presence of precipitates or phase separation were scored after visual and cross polarizers examination at room temperature [26]. 2.5.7. % Transparency The % transparencies of MEs were measured by UV spectrophotometer (UV-1700, Shimadzu, Japan) at a max of 700.00 nm. 2.5.8. Dye solubility test A water soluble dye (methyl orange) was added to the ME system and the phase system was evaluated.
2.6.3. Data analysis The cumulative amount of Amp B permeated through excised rat skins was calculated by Eq. (1): [3,31,32] Qs =
2.6.1. Preparation of skin In order to evaluate the effect of Amp B ME on skin permeation as compared to plain drug solution, formulations were subjected to in vitro permeation study. The animal study protocol to conduct skin permeation was approved by the Institutional Animal Ethics Committee. Abdominal skins were obtained from female Albino Wistar rats weighing 200 ± 30 g [27]. A 2.5 cm × 2.5 cm patch of skin from the abdominal region was excised after hair removal with a depilatory, and then the subcutaneous fat and connective tissue were removed. To achieve higher reproducibility, the excised skins were
n−1 s
n−i
Vi × Ci
(1)
where Cn is the drug concentration of the receiver solution at each sampling time, Ci is the drug concentration of the sample, and V0 and Vi are the volumes of the receiver solution and the sample, respectively. S is the effective diffusion area (S = 1.54 cm2 ). The permeation rate of Amp B at a steady-state (Jss , g cm−2 h−1 ) through the rat skin was calculated from the slope of linear portion of the plots of Qt versus time [33,34]. The experiments were carried out in triplicate for each sample, and the results were presented as an average ± SD. The permeability coefficient (Kp , cm/h) was calculated according to following Eq. (2): [3,27,33] Kp =
2.6. Ex-vivo diffusion study
V0 × Cn +
Jss C0
(2)
where Kp is the permeability coefficient, Jss is the flux calculated at steady-state and C0 represents the drug concentration which remains constant in the vehicle. 2.7. Skin retention study Protocol for skin retention study was approved by the Institutional Animal Ethics Committee. Skin was obtained from the abdomen of female Albino Wister rat weighing 200 ± 30 g. The full-thickness skin was excised after hair was removed with a depilatory. Subcutaneous fat and other extraneous tissues were
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trimmed; the skin was washed with physiological saline followed by phosphate buffered saline (pH 7.4) and then visually inspected for integrity to ensure the absence of holes or other imperfections. The excised rat skin were stored at −20 ◦ C and used within 1 week of harvest. The skin was placed with stratum corneum facing upward (inside of the tube) and dermal side downward (to face the medium). The position-fixed skin was made water tight by a rubber band. Assembly was adjusted as previously mentioned under the ex-vivo diffusion study. After 24 h, the effective diffusion area of the skin was separated, washed several times with distilled water, to remove formulation excess, and then cut into small pieces. The segments obtained were vortexed with methanol and then left for soaking for 24 hrs to ensure effective extraction of the retained drug from the skin. The resulting mixture was then filtered using 0.45 m syringe filter and 1 ml from the filtrate was diluted with receptor fluid then filtered and Amp B was quantified using UV spectrophotometry at max 408.5 nm [5]. 2.8. Skin sensitivity studies This study was performed on rabbits to evaluate the irritant potential of the developed formulation after topical application [35,36]. The rabbits (weighing 2.0–2.5 kg) were purchased from Department of Biochemistry, The Maharaja Sayajirao University of Baroda (Vadodara, India). The hairs on the back of rabbit were removed 24 h prior to the administration of formulations [32] whereas the control group was treated with normal saline. ME1, ME3, ME5, ME7 and the saline solution containing 0.1% Amp B was applied to the treatment group twice a day for 5 days consecutively (n = 3) [6]. The animals were observed for any signs of itching or change in skin such as erythema, papule, flakiness and dryness for a period of 5 days [37]. After withdrawal, observation for single or multiple administrations was continued for 3 days [38]. The irritation scores of the test area were obtained by judging the extent of erythema and edema according to the criteria [39]. Erythema and edema were graded as follows: 0 for no visible reaction, 1 for just present reaction, 2 for slight reaction, 3 for moderate reaction, and 4 for severe reaction. Eventually, the total scores for irritation test in each condition were calculated using the following Eq. (3): [32]. Average irritation scores =
erythema reaction scores + dropsy reaction no. of animals
(3)
2.9. In vitro antifungal activity The various ME formulations ME1, ME3, ME5, ME7 and plain drug solution were assayed for antifungal activity against the fungal strain Trichophyton rubrum. This fungus was grown on Sabouraud’s agar plate at 25 ± 2 ◦ C. The plates were first sterilized in hot air oven at 160 ◦ C for 60 min. The fungal culture suspensions were made according to the ATCC protocol of microbiologics [40] and were allowed to stand for 20 min before transferring on to the solid agar medium as the strain was available in lyophilized form. To the sterile petri-dishes in which solidified agar growth medium was taken, the fungal culture suspension was spread with the help of stick. The inoculums were spread uniformly over the solid agar surface by spreader glass rod and incubated at 25 ± 2 ◦ C for 7 days to grow fungus. Then wells were made in the middle of the plates with the help of sterile cork borer and the wells were filled with the formulations and the plates were incubated at 25 ± 2 ◦ C. Clear rings appeared around the wells in 48 h. The rings were called the zone of inhibition. Larger the zone of inhibition, more effective is the formulation. The antifungal activity was evaluated by measuring
zones of inhibition of fungal growth surrounding the formulations. The zones of inhibition were measured with scale in mm and the complete antifungal analysis was carried out under strict aseptic conditions and the mean inhibition zone from three plates was calculated [5]. 2.10. Stability of ME The physical stability of ME containing Amp B was investigated via clarity, particle size analysis and phase separation, which was observed at 2–8 ◦ C and at room temperature upto 3 months. The centrifugation was carried out at 13,000 rpm for 30 min to assess the physical stability of ME [41]. Clarity, phase separation and concentration of Amp B were investigated monthly to judge the optimal storage temperature. Chemical stability was evaluated on drug loaded formulations, stored at 2–8 ◦ C and at room temperature by determining Amp B content by UV spectrophotometry at max 405.5 nm. 2.11. Statistical analysis All skin permeation experiments were repeated three times and data were expressed as the mean value ± S.D. (n = 3). Statistical data were analyzed by one way analysis of variance (ANOVA). A multiple comparison test was used to compare different formulations, and a P value of ME5 > ME3 > ME1 > plain drug solution. ME7 showed highest zone of inhibition due to its high fluidity. Plain drug solution showed lowest zone of inhibition due to its less penetration effect than ME. The water content was more in ME7, so the drug released from it was more as compared to ME1, ME3 and ME5, in which the drug molecules were more rigid in viscous structure. This indicated that the ME7 was more effective as compared to another formulation. 3.8. Stability Study Stability studies were carried out to detect any changes in pH, droplet size, and drug content of formulation for the period of 3 months at 2–8 ◦ C and room temperature. All formulations were physically stable, retaining homogeneity with no phase separation after 3 months. The major changes of droplet size and degradation of Amp B were not observed during 3 months. The centrifuge tests showed that all MEs had good physical stability. No marked changes were recorded in the stored MEs except for slight decrease in viscosity. This decrease may be due to loss of some water during storage. After 3 months, there was a very little change in pH, droplet size, drug content and viscosity at 2–8 ◦ C than room temperature. So, the ME formulations were more stable at 2–8 ◦ C than room temperature. 4. Conclusion The Amp B ME was formulated for topical application and the results of present study clearly demonstrated the role of ME in effective topical delivery of Amp B. ME7 has shown higher skin deposition, lower skin irritation and better anti-fungal activity. The developed system may provide better remission from the disease due to localized delivery with minimal side effects and the data from in vitro study has been encouraging but further evaluation is needed to elucidate the clinical efficacy of this topical dosage form. Conflicts of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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