Delivery of Amphotericin B for Effective Treatment of Candida Albicans Induced Dermal Mycosis in Rats via Emulgel System: Formulation and Evaluation.

BACKGROUND: Amphotericin B (AmB) is among the gold standard antifungal agents used for the treatment of the wide range of fungal infections. However, the drug has various side- effects. Transdermal approach for the delivery of drug is one of the accepted and convenient modes of drug delivery. AIM: The current work was designed to formulate and to evaluate the AmB emulgel system.
MATERIALS AND METHODS: In the preparation of AmB emulgel, Carbopol 930 was used as a gel in this study. The formulation was evaluated for viscosity, spreadability, drug content, drug release and in vitro and in vivo antifungal testing.
RESULTS: AmB emulgel was found to penetrate skin effectively and without any irritation. Further, in vivo studies revealed effective therapeutic potential against Candida albicans induced dermal mycosis.
CONCLUSIONS: The current work, for the first time, revealed effective delivery of AmB across the skin.

What was known?

Amphotericin B (AmB) is one of the highly recognized antifungal agents used for treatment of wide range of fungal diseases. This work for the first time has established the role of AmB is treatment of dermal mycosis.

Introduction

Transdermal route of drug delivery is one of the most preferred routes as it offers a lot of advantages such as the ease of drug application, self-administration, reduced drug metabolism etc., still, the route faces a lot of challenges. Stratum corneum, one of the uppermost layers of the skin is one of the rate limiting steps for transport of drug across the skin.[1]

Amphotericin B (AmB) is one of the widely used antifungal agents used in the treatment of infections caused by Candida, Aspergillus and Fusarium. The drug is also given by intravenous route, but it has a lot of side-effects such as chills, nausea, hemolysis and nephrotoxicity.[23] Current advanced lipid formulations find an important use in patients with impaired renal function and those who do not respond to AmB.[45] Conventional formulations of AmB with brand name fungizone cream, fungizone lotion, gels, ointments were being used, but these formulations resulted in severe blistering, itching, redness, peeling, dryness or irritation of the skin, systemic absorption and failed to achieve mycological eradication.[67]

Gellified emulsion is a stable and better vehicle for delivery of hydrophobic or water insoluble drugs. Emulgels are emulsions, either of the oil-in-water or water in - oil type, which are gelled by mixing with a gelling agent. They have a high patient acceptability since they possess the previously mentioned advantages of both emulsions and gels. Therefore, they have been recently used as vehicles to deliver various drugs to the skin.[8910] The objective of the present study was to formulate and to evaluate the emulgel formulation of AmB.

Materials and Methods

Chemicals

Carbopol 930, Tween 20, Span 20, methyl and propyl parabens, light liquid paraffin, propylene glycol, triethanolamine and ethyl alcohol were purchased from Central Drug House, India. Candida albicans strain (Microbial Type Culture Collection) was obtained from Institute of Microbial Technology, Chandigarh.

Animals

Healthy albino Wistar rats (100-150 g) and rabbits of either sex were obtained from Animal House, Shri Ram Institute of Technology-Pharmacy, Jabalpur, M.P., India. The rats of either sex were housed in groups of five in each cage (standard plastic cages) and rabbits individually in standard rabbit cage with 12/12 h light and dark cycle in institutional animal house prior to pharmacological studies. The animals were fed with standard diet and provided water ad libitum. After 1 week of acclimatization, the animals were used for further experiments. All animals were fasted overnight before test; tap water was supplied ad libitum. The usage of animals in the experiments was approved from the Institutional Animal Ethics Committee. Animals were maintained under standard conditions in an animal house approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India.

Formulation

The composition of AmB emulgel formulations is shown in Table 1. The gels in all the formulations were prepared by dispersing Carbopol 934 in purified water with constant stirring at a moderate speed and pH was adjusted to 6-6.5 using triethanolamine.[11]

Table 1

Quantitative composition of AmB emulgel formulations (% wt/wt)

The oil phase of the emulsion was prepared by dissolving Span 20 in light liquid paraffin while the aqueous phase was prepared by dissolving Tween 20 in purified water. Methyl and propyl paraben were dissolved in propylene glycol whereas AmB was dissolved in ethanol and both solutions were mixed with the aqueous phase. Both the oily and aqueous phases were separately heated to 70°C to 80°C; then the oily phase was added to the aqueous phase with continuous stirring until cooled to room temperature. Obtained emulsion was mixed with the gel in 1:1 ratio with gentle stirring to obtain the emulgel.

Characterization

pH measurements

The apparent pH of the formulations was measured by a pH meter (Accument AB15, Fisher Scientific, USA) in triplicate at 25°C.

Centrifugation

Thermodynamic stability of the samples was studied by ultracentrifugation using a 70Ti rotor (Optima L-70, Beckman Instruments, USA) at 40,000 rpm (or 117,734 × g) for 30 min at 4°C.

Viscosity measurements

The viscosity of different gel formulations was determined using a Brook Field Viscometer (Brookfield LVT Viscometer) with spindle #1. Readings were observed after 1 min, when the pointer was stabilized.

Spreadability

One of the criteria for an emulgel to meet the ideal quantities is that it should possess good spreadability. It is a term expressed to denote the extent of the area to which gel readily spread on application to skin or affected part. The therapeutic efficacy of a formulation also depends upon its spreading value. Spreadability is expressed in terms of time in seconds taken by two slides to slip off from emulgel that is placed in between the slides under the direction of certain load. Lesser the time taken for separation of two slides, better the spreadability. It is calculated by using the formula:

S = M. L/T

where,

M = wt. tied to upper slide

L = length of glass slides

T = time taken to separate the slides

Drug content determination

Drug concentration in emulgel was measured by ultraviolet (UV) spectrophotometer. AmB content in emulgel was measured by dissolving known quantity of emulgel in solvent (methanol) by sonication. Absorbance was measured after suitable dilution at UV/visible spectrophotometry spectrophotometer (UV-1700, Shimadzu Corporation, Japan).

In vitro skin permeation, stripping and retentivity studies

In vitro skin permeation studies for AmB emulgel formulation was performed using hairless abdominal skin of rats (male Wistar rats, 150-200 g). Skins with a surface area 3.14 cm2 eq were mounted on Franz diffusion cells. Receptor compartment comprised of receptor fluid (ethanol: Phosphate-buffered saline; pH 6.4, 30:70) whereby stratum corneum was in contact with donor compartment. AmB ethanolic solution and AmB emulgel were placed on donor compartment so that skin surface was covered evenly. Temperature was maintained at 37 ± 1°C. Sampling was done at various intervals up to 24 h and assayed for AmB content spectrophtometrically. Franz diffusion cells were then dismantled; skin surface was washed 10 times with a cotton swab and stripped using a fixed diameter and length of scotch magic tape. Same 10 tape strips were mixed in 2-propanol and 0.1 M hydrochloric acid (90:10) vortexed and centrifuged to allow AmB to dissolve in order to analyze drug concentration.

Microbiological assay

Ditch plate technique was used for the present study. It is a technique used for evaluation of fungistatic activity of a compound. It is mainly applied for semisolid formulations. Previously prepared sabouraud's agar dried plates were used. was placed in a ditch cut in the plate. Freshly prepared culture loops were streaked across the agar at a right angle from the ditch to the edge of the plate. After incubation for 18-24 h at 25°C, the fungal growth was observed and the percentage inhibition was measured as follows:[11]

% inhibition = (L2/L1) ×100

where,

L1 = total length of the streaked culture

L2 = length of inhibition.

Skin-irritation studies

Skin irritation studies on AmB emulgels were evaluated by Drazie patch test on albino rabbits (1.5-2 kg).[1213] All the experiments were approved by Institutional Animal Ethics Committee. Animals were divided into three groups with three animals (rabbits 1.5-2 kg) in each as:

· Group I: Control (no drug treatment);

· Group II: AmB ethanolic solution;

· Group III: AmB emulgel (Formulation code: Carbopol Emulgel 3 = CEG 3)

Back of rabbits was clipped and hair was removed 48 h prior to application of formulation. AmB ethanolic solution and AmB emulgel was applied on hair free skin of rabbits uniformly. The skins were observed for any gross visible change after 24 h, 48 h and 72 h of administration. The mean erythemal scores were recorded (ranging from 0 to 4) depending on the degree of erythema as follows: No erythema = 0, slight erythema (barely perceptible-light pink) = 1, moderate erythema (dark pink) = 2, moderate-to-severe erythema (light red) = 3 and severe erythema (extreme redness) = 4.

In vivo performance studies

In order to determine in vivo efficacy of the formulation, C. albicans induced mycosis model was used for the studies. All the experiments were conducted with prior permission of Institutional Animal Ethics Committee. Briefly, rat's hair (Wistar albino rats, 100-150 g) were removed using the depilatory cream and an area of 3 cm2 × 3 cm2 was marked. On next day, skin of animal was slightly abraded using sandpaper. Previously prepared inoculum of C. albicans was applied using a glass rod. Animals were divided into three groups containing five animals each. First group served as control, which received no treatment. Second group received ethanolic solution of AmB. Emulgel formulation was applied to animals of third group. Animals received the treatment for 6 consecutive days post-infection except control group. The animals were observed for any gross morphological changes. After 6 days, in order to determine efficacy of treatment, animal skin was wiped with cotton swab (ethanol 70%). Skin was excised from treated site, homogenized in 5 ml saline in tissue homogenizer. The so formed homogenate was streaked on solid yeast extract-peptone-dextrose medium and incubated at 25°C for 4 days. Numbers of colony forming units (CFUs) on agar plate were counted and the logarithm of the number of CFUs per infected site was calculated. An animal with more than one fungal colony was considered as positive.[1415]

Statistical analysis

Data were expressed as Mean ± standard error of the mean from six rats and three rabbits in each group respectively. Results were analyzed statistically using one-way analysis of variance test followed by post-hoc Dunnet test for a level of significance at *P < 0.05; **P < 0.01; ***P < 0.001.

Results

Physical examination

The prepared AmB emulgel formulations were white viscous creamy preparations with a smooth and homogeneous appearance. They were easily spreadable with acceptable bioadhesion and fair mechanical properties. The pH values of all the prepared formulations ranged from 6.3 to 6.5, which is considered acceptable to avoid the risk of irritation upon application to the skin.

Centrifugation

None of the formulation showed breakage justifying its stability during the centrifugation.

Spreadability

The values of spreadability indicate that the emulgel is easily spreadable by small amount of shear. Spreadability of CEG1 and CEG2 was 11.21 ± 0.87 g.cm/s and 13.42 ± 0.93 g.cm/s while CEG3 and CEG4 was 17.31 ± 0.74 g.cm/s and 18.68 ± 0.57 g.cm/s, indicating spreadability of emulgel containing AmB was good as compared to the marketed gel. This may be due to low level of liquid paraffin concentration in CEG1and CEG3.

Rheological studies

The measurement of viscosity of the prepared emulgel was done with Brookfield viscometer (Brookfield DV-E viscometer). The highest viscosity was found in emulgel CEG3, it may be due to high level of the Carbopol and emulsifier concentration. The lowest viscosity was found in formulation CEG2 due to the low level of emulsifying agent concentration [Figure 1].

Figure 1

Viscosities (in poise) of amphotericin B emulgel formulations at low and high rates of shear

The drug content in emulgel was found in range of 65.5 ± 1.82% to 83.87 ± 1.20%. Higher drug content in CEG3 i.e., 83.87 ± 1.20%, may be due to the low concentration of liquid paraffin and emulsifying agent and lower drug content was found in CEG4 i.e., 65.5 ± 1.82%, which may be due to the high concentration of liquid paraffin and emulsifying agent.

A very less amount of AmB was found in receptor compartment fluid from emulgel CEG1, among all the formulations, CEG3 emulgel showed best drug release in receptor compartment [Figure 2]. In tape stripping experiments content of AmB was significantly greater (P < 0.05) in skin treated with ethanolic solution of AmB (159.24 ± 5.71 μg/cm2) as compared with AmB emulgel (CEG 3 emulgel, 12.59 ± 4.68 μg/cm2). This study revealed optimum penetration of AmB in the skin as compared to AmB ethanol solution, which could be beneficial in treatment of superficial mycosis. Skin retentivity of AmB from emulgel (CEG 3) was more as compared to AmB ethanol solution (156.23 ± 5.49 μg/cm2 vs. 12.57 ± 3.87 μg/cm2). This retentivity could be attributed due to occlusive potential of emulgel system. All these studies proved that the system caused accumulation of AmB in skin, restricted flux of drug and produced a reservoir in the skin. Such an approach could be beneficial in topical mycoses.

Figure 2

In vitro drug release of amphotericin B from emulgel. All values are reported as mean ± standard error of the mean

The use of control plates showed that the plain emulgel bases were microbiologically inert toward the C. albicans strain. The antifungal activity of AmB in its different emulgel formulations was found to be good. The emulgel formulations were found to have the same rank order in their antifungal activities as in the in vitro release studies. Thus, the highest activity was observed with CEG3 and CEG2, where the percentage inhibition was found to be 54.5 ± 1.20% (P < 0.05) and 51.6 ± 1.34% (P < 0.05) respectively while the lowest activity was found with CEG4, where the percentage inhibition was 32.14 ± 1.10%, ethanol solution of AmB showed 46.4 ± 1.174% inhibition.

Primary skin irritation studies

Shaved skin rabbit model is one of the useful approach to access skin irritation of topical/transdermal pharmaceutical dosage form. In the current work, ethanolic solution of AmB produced a slight degree of irritation on rabbit skin, whereas, relatively, no such adverse effect was observed on animals treated with AmB emulgel. Such skin tolerability is advantageous leading to increased patient acceptability [Table 2 and Figure 3].

Table 2

Mean erythema scores observed for AmB emulgel

Figure 3

Skin irritation studies of amphotericin B emulgels as evaluated by Drazie patch test on albino rabbits

In vivo studies

C. albicans induced rat mycosis model was used to determine in vivo efficacy of the emulgel formulation. Table 3 demonstrates the significant efficacy (P < 0.05) of emulgel formulation in treating Candida infections when compared with AmB-ethanol solution treated group. Isolates of Candida were removed from the skin and tested for their viability. In emulgel treated group only one out of five animals exhibited positive culture test. Rapid recovery of infection was observed with AmB loaded emulgel.

Table 3

Colony forming unit of Candidia albicans in skin of rats after treatment with AmB emulgel

Discussion

AmB is one of the macrocyclic antibiotics, which has been used since 40 years. It is the drug of choice for “invasive fungal infections,” it is also used for a large number of non-fungal indications.[16] AmB is insoluble in water and high dose of it shows hemolytic activity. All these adverse effects restrict the use of AmB. To reduce all these adverse effects, AmB is used in a lipid formulation, still these have low margin of safety.[17] Based on all these aspects, aim of current work was to develop emulgel formulation of AmB for topical delivery.

Emulgel are versatile transdermal drug delivery approach, which has merits of both emulsion and gels, making it suitable for delivery of water soluble and insoluble drugs. The current emulgel system comprised of Carbopol 930 as gelling agent. Carbopol gels demonstrate pseudoplastic behavior. When influenced with high shear rate, their viscosity decreases and emulgels becomes fluidy. Such a behavior is desirable for adequate removal of formulation from container and better spreadability is observed.[18] Thus, Carbopols could be considered as one of the adequate gelling agent for formulation of emulgels.

The amount of liquid paraffin in semisolid formulation directly affects release of drug. Thus in current study, such fact could be a possible reason for good release profile for emulgel CEG3 than CEG4. Increased concentration of liquid paraffin (7.5%) in CEG4 along with Carbopol (1%) may be possible reason for the poor release of drug, which is also evident from previous work.[19] In this work, formulation CEG1 showed poor drug release as compared to CEG3. This could be attributed due to difference in emulsifier concentration. Thus, it could be said that, emulsifier and not liquid paraffin has more pronounced effect on drug release.

In vitro drug permeation studies reflected CEG3 emulgel had best permeation profile, which could be due to effective concentration of Carbopol and paraffin. Thus CEG3 emulgel was used to access skin irritancy and therapeutic potential of the formulation. Drazie test done for skin irritation studies is one of the important evaluation parameter used to study behavior of formulation on intact skin. Emulgel system, virtually, was found to be non-irritant to the rabbit skin. Rat topical mycoses model was used to evaluate antifungal efficacy of the formulation. AmB Emulgel (CEG3) demonstrated excellent therapeutic potential when compared to AmB ethanol solution treated group, which is evident from less CFUs generation in emulgel treated group.

Emulsion when incorporated in gel behave as reservoir of drugs, the drug being released from inner to outer pseudophase and ultimately reaching the skin. According to another view, these droplets break on stratum corneum surface. Along with this, emulsion droplet filled with drug could also hasten drug permeation. Movement of emulsion droplets is also one of the reasons for permeation of drug from skin.[20] In addition, a hydrophobic drug is preferentially encapsulated in the oil droplet and the highly drug loaded droplets favor partitioning into the epidermis, resulting in the enhanced drug permeation. This phenomenon confirms that the oil droplet nature of an o/w microemulsion is a crucial factor for flux of drugs, especially hydrophobic substances. When a hydrophobic drug is encapsulated in oil droplet, drug partitioning is favored in epidermis, leading to increased drug accumulation in skin. In the present work, increased accumulation of AmB across the skin could be due to such fact. In vitro drug release followed by in vitro antifungal studies dictates the efficacy of AmB emulgel against fungal infection, which is further confirmed by in vivo studies. Thus, it could be concluded that AmB[2122] in emulgel system represent an excellent means of delivery of antifungal agent. However, systematic clinical studies are further required to extrapolate its effects on humans.

What is new?

Amphotericin B (AmB) is one of the highly recognized antifungal agents used for treatment of wide range of fungal diseases. This work has, for the first time established the role of AmB in the treatment of dermal mycosis.