New Formulation and Characterization of Topical Films of Tioconazole and Evaluation of Their Antifungal Therapy

Purpose: New formulation of Scleroglucan (Sclg) films loaded with tioconazole, a medication typically applied for dermal treatments prepared. The feasibility of that treatment relies on the penetration of medications through the target layers of the skin in effective concentrations. Methods: Dynamic and mechanical characterization and swelling studies of the novel delivery system were analysed. An aqueous solution of Sclg (Cp=1% w/v) and glycerol (2% w/v) was prepared and kept at room temperature under magnetic stirring for 72 hrs. Tioconazole previously solubilised in Labrasol, was added to the polymer/glycerol solution. 4 ml of solution was poured in a plastic plate. The films were dried at 40°C for 1 hr and then allowed to dry at room temperature (about 25°C) for a week. Translucent films were obtained. The fungal strain used to test the film are CO23 sensitive to drugs, CO23 RFLC resistant to fluconazole, CO23 RFK resistant to micafungin, ATCC standard strain. Results: The water uptake of the films significantly increased up to 24 hrs. The optical microscope films images show that the presence of the drug did not significantly influence the appearance of the samples. The in vitro studies demonstrated the perceptible fungal activity of the new formulation against Candida albicans infections. Original Research Article Ansari; JPRI, 33(46A): 400-408, 2021; Article no.JPRI.75418 401 Conclusion: The patches showed antimicrobial activity against all tested strains. An evident inhibition zone diameter, about 40 mm, for the strains sensitive to azoles (CO23 RFK and CO23) in comparison to strain resistant to fluconazole (CO23 RFLC) was observed. After 48 hours the inhibition zone diameters were reduced of about 6-7 mm in comparison to those observed after 24 hours of incubation.


INTRODUCTION
Fungal contaminations on the skin are most regularly facing infections around the worldwide. Such huge numbers of individuals have experienced fungal infections in all countries. The development of fungal contaminations can be quick and caused serious effect due to the damage of immune functions [1,2]. Topical treatment is commonly used as an interesting alternativefor the treatment of the cutaneous infections because of its advantages, for example, focusing of medications to the site of infection and reducing the risk of systemic side effects [3,4]. Nowadays, antifungal medications are mostly used as traditional cream and gel preparations in topical treatment [5]. The feasibility of that treatment relies on the penetration of medications through the target layers of the skin at the effective concentrations. The physicochemical characteristics of drug molecules and the definite formulation are effective factors in topical drug delivery. Therefore, a number of formulation strategies have been examine for delivering antifungal compounds through targeted sites of the skin.
Dermatophytosis is the causative agent responsible for tinea and onychomycosis diseases. Cutaneous Candida infection is superficial fungal infection typically involving the skin [6,7] Even, Candida may penetrate and enters theblood and even more to deep tissue, a condition that prompts to life-threating systemic candidiasis [8], This is most common when the immune system is weakened [9]. Topical treatment of fungal infections has a number of advantages, including targeting of the infection site, decrease of the risk of systemic side effects, improvement of the efficacy of treatment and, high patient compliance.
Nowadays, the formulation may play a most significant role for penetration of drugs into skin [10,11].Topical delivery of drugs for the treatment of fungal infections of skin encompasses new carrier systems for approved and investigational compounds. Delivery of antifungal compounds into skin can be enhanced with the carriers including colloidal systems, vesicular carriers, and nanoparticles.
Furthermore, researchers have established and proposed various formulation techniques to improve the solubility and bioavailability of poorly water-soluble compounds [12]. One of the most common approach involves the use of a solubilizing agent, such as Labrasol (saturated polyglycolyzed C 8 -C 10 glyceride) [13] mixed with other pharmaceutical excipients, such as hydrophobic oils, lipids, and co solvents, to form micro emulsions or emulsion systems [14][15][16].The drugs are held tightly in the more hydrophobic cores of the emulsion system, thus avoiding drug precipitation.

Topical Delivery of Antifungal via Skin
Human skin is a well-organized membrane and, it has three main layers, epidermis, dermis and hypodermis. Stratum corneum, the outermost layer of epidermis, is shaped by dead and keratinized cells, and it is an outstanding barrier to penetration of drugs through the skin [17] drugs should penetrate into skin layers to confirm effective drug concentrations following topical administration. In topical administration, the entering of drugs to systemic circulation is prevented or minimized. Thus, the systemic adverse effects of drugs are avoided [18]. Moreover, topical preparations have improved patient compliance due to their noninvasiveness and they can be self-administered [19,20].

Materials
Sclg was provided by Cargill (Minneapolis, USA).Glycerol (analytical grade) and Labrasol was bought by Gattefosse. Tioconazole, was purchased by Sigma. For the sample preparations distilled water was used.

Preparation of sclg films
Sclg was cleaned by dialysis. An aqueous solution of Sclg (cp=1% w/v) and glycerol (2 % w/v) was prepared and kept at room temperature under magnetic stirring for 72 hrs. Tioconazole previously solubilised in Labrasol, was added to the polymer/glycerol solution. 4 ml of solution was poured in a plastic plate. The films were dried at 40°C for 1 hr and then allowed to dry at room temperature (about 25°C) for a week. Translucent films were obtained.
Films were prepared with three different amounts of tioconazole, 5g, 10g and 20g (Table 1). In For the swelling studies and for the fungal activity tests 1x1 cm 2 samples were used.

Dynamo-mechanical analysis
The mechanical properties were investigated with a software-controlled dynamometer, TA-XT2i (Stable Micro Systems, UK), equipped with a 5-kg load cell. The accuracy of the force measurement was 0.0025 %, and the distance resolution was 0.0025 mm.
The film was placed in a holder with a cylindrical hole (d= 8.50 mm) and the diameter of the spherical probe was 6.35mm. The pre-test and post-test speeds were fixed at 2.00 mm/s, the test speed at 1.00 mm/s and the trigger force at 0.098 N.
The following parameters were calculated: Where F max is the maximum applied force at film break, ACS is the cross-sectional area of the film above the hole of the film holder, ACS =2rδ, where r is the radius of the hole and δ is the thickness of the film.

2) Elongation
Where r is the radius of the film exposed in the cylindrical hole of the film holder and D represents the displacement of the probe from the point of contact to point of puncture. The maximum load and the maximum displacement of films were measured, and then converted to puncture strength, and elongation at break.

Susceptibility testing method
The antimicrobial activity of the patches was assessed against the test organisms C. albicans ATCC 24433, and the strain CO23 of C. albicans, which was isolated from a subject with vulvo-vaginal candidiasis, originally susceptible to micafungin (FK463, MIC 0.025 μg/ml) and fluconazole (FLC MIC 0.25 μg/ml). The strains CORFK and CO23 RFK were made resistant to FK463 or to FLC by ten growth passages in step wise-increasing concentrations in solidified agar, according to the National Committee of Clinical Laboratory Standards CLSI.;(2007).

Film thickness measurements
The thickness of the films was assessed by means of a posiTector 6000 digital thickness meter purchased by DeFelsko (USA). The thickness of the films was measured at five different positions and the obtained values always lay within 10% of the mean.

Water Uptake Studies
The Sclg films showed an extraordinary swelling. The water uptake behaviour was followed as a function of time. The experiments were carried out in distilled water at room temperature. At fixed time intervals the excess of water was removed from the films with soft filter paper for 5 sec, and then the corresponding weights were determined. All experiments were carried out in triplicate and the obtained values always lay within 10% of the mean.

Folding Endurance
The film folding endurances were manually measured. A strip of film was repeatedly folded at the same place till it broke. The number of times the film could be folded at the same place without breaking gave the value of folding endurance. The folding endurance of prepared films was measured in triplicate and the obtained values always lay within 10% of the mean.

Water Uptake Studies
The water uptake results are reported as a function of time in Fig 2. The water uptake of the films significantly increased up to 24 hrs and after 30 hrs the films completely dissolved in the swelling medium.
In Fig 3 the films images recorded by means of an optical microscope show that the presence of the drug did not significantly influenced the appearance of the samples.

Physical and dynamo-mechanical characterization of Sclg films
Different formulations of Sclg films were prepared, as listed in Table 1.  Table I In principle, an adequate film mechanical strength is required in order to withstand mechanical stress (e.g., filling and packaging processes, pushing through blister package, handling by patient).
Puncture strength is a measure of film toughness and is directly proportional to the resistance to break or fracture. Sclg films were capable to elongate the double of their initial size. In Table II the physical parameters evaluated for the Sclg films are reported.
As an example, the mechanical properties of FA and FE formulations are shown in Fig 4.

C. albicans activity tested on Sclg films loaded with tioconazole
Petri plates were set up by pouring 20 ml of Sabouraud medium that was allowed to solidify, and then dried for 30 min in a biological safety cabinet with vertical laminar flow. 10 l of a standardized inoculum suspension of 2.5x10 3 CFU/ml was poured and uniformly spread over the plates. Patches with different amounts of tioconazole (5, 10, 20 µg) were placed in the center of Petri plates. The Petri dishes were then incubated at 30 °C for 24 and 48 hrs. The antifungal activity was measured as the diameter of the inhibitory zone by a caliper and expressed in mm (disk diameter included) (Fig 5).
The sensitivity to the different patches was classified by the diameter of the inhibition zone. An inhibitory zone with diameter less than 10 mm corresponds to lack of activity.
The inhibitory zones of samples containing 5, 10 and 20 µg of tioconazole were measured. As reported in Table III, the patches showed an antimicrobial activity against all tested strains. An evident inhibition zone diameter, about 40 mm, for the strains sensitive to azoles (CO23 RFK and CO23) in comparison to strain resistant to fluconazole (CO23 RFLC) was observed. After 48 hours the inhibition zone diameters were reduced of about 6-7 mm in comparison to those observed after 24 hours of incubation. Patches without drug showed no activity; in some cases no significant differences of inhibition zone between the patches with different concentrations of tioconazole (5 and 10 µg) were observed.

CONCLUSIONS
A new formulation, a Scleroglucan (Sclg) films loaded with tioconazole. The films were prepared with Sclg and glycerol used as a plasticizer, and Labrasol a surfactant used for increasing the solubility and bioavailability of the low-soluble drug. Films were prepared with three different amounts of tioconazole, 5g, 10g and 20g . The in vitro studies demonstrated the perceptible fungal activity of the new formulation against Candida Albicans infections. The inhibitory zones of samples containing 5, 10 and 20 µg of tioconazole were measured. The patches showed an antimicrobial activity against all tested strains. An evident inhibition zone diameter, about 40 mm, for the strains sensitive to azoles (CO23 RFK and CO23) in comparison to strain resistant to fluconazole (CO23 RFLC) was observed. After 48 hours the inhibition zone diameters were reduced of about 6-7 mm in comparison to those observed after 24 hours of incubation. Patches without drug showed no activity; in some cases no significant differences of inhibition zone between the patches with different concentrations of tioconazole (5 and 10 µg) were observed.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

COMPETING INTERESTS
Author has declared that no competing interests exist.