Characterisation and Evaluation of prepared Fluconazole Nanogel for Treating Seborrheic Dermatitis

Divya¹, Inderbir Singh², Upendra Nagaich¹*

¹Department of Pharmaceutics, Amity Institute of Pharmacy, Amity University, Noida,

Uttar Pradesh 201313, India.

²Department of Pharmaceutics, Chitkara University, Jansla, Rajpura, Punjab 140401.

*Corresponding Author E-mail: upendra_nagaich@hotmail.com

ABSTRACT:

Objective: This study aims to demonstrate antifungal activity and in-vitroanalysis of fluconazole nanogel with different polymer concentration for seborrheic dermatitis. Methods: The class of antifungal drug- Fluconazole was used to formulate the nanogel with different concentrations of polymers. The prepared batches of nanogel were analyzed for its antifungal activity, ex-vivo skin permeation study, and skin thickness study. The animals are used in four groups for the in vivo antifungal activity of nanogel as control, disease, standard, and test. The prepared fluconazole (FLZ) nanogel was compared with marketed fluconazole gel. Results: The prepared formulation having hydroxymethyl cellulose (HPMC=1.5mg) as the polymer showed up with best properties among eight different batches. It was effective with a 38 mm inhibition zone in culture media of yeast Malassezia strain. The skin permeation study ensured 95% cumulative drug release in 18 hr. The ex-vivo studies also give the best result for F5 batch (HPMC=1.5mg) with a minimum skin thickness of 1.4 micrometer. Conclusion: In-vivo and ex-vivo studies on the different batches of fluconazole, when compared with marketed gel, showed evidence that nanogel of FLZ (1mg with HPMC=1.5mg) can treat seborrheic dermatitis.

KEYWORDS: Seborrheic Dermatitis, Fluconazole nanogel, antifungal activity, skin permeation.

INTRODUCTION:

Seborrheic dermatitis (SD) is a peripheral fungal disease of the skin, mainly affects the areas rich in sebaceous glands. The etiology of the disease is unknown as it showed up with different symptoms depending on individual. Some relations were examined and resulted to be the associated with the species of the genus Malassezia (formerly, Pityrosporum). These yeasts are members of the human skin flora mainly in sebaceous area such as abdomen, upper arms, face, neck,back, chest^1,2, and maybe related to skin disorders such as pityriasis Versicolor and rarely with systemic infection. Although no interpretation has been made in respect of severity and the figure of fungal organisms of the disease, and the specific mechanism of action used by this organism for causing seborrheic dermatitis.

Antifungal agents are the mainstay of anti-seborrheic therapy. This decreases the number of Malassezia organisms present on the skin which are known to be effective in the treatment of SD. The ergosterol production which is present in the fungal cell wall and essential for cell growth, get inhibited by the P-450 (CYP 450) fungal cytochrome system. This blockage results in high accumulation of sterol precursors. This is a fungistatic process that stops the growth of fungus and inhibits it for further reproduction. Besides, the preponderance of disease in sebum-rich, the fungal metabolites produce an inflammatory mediator by reacting with triglycerides released from sebaceous glands. Another hypothesis believe that keratinocyte production of proinflammatory cytokines in the lipid layer of the fungus leads to inflammation and skin eruption⁴. There is no genetic heritance has been identified with SD. The different class of drug that is azoles inhibit the 5-LPG(lipoxygenase) production followed by leukotriene B4 synthesis in the fungus.

Table 1: Formulation table for preparing FLZ-NG²⁶

S. No.	Ingredients (mg)
S. No.		F1	F2	F3	F4	F5	F6	F7	F8
1.	FLZ	1	1	1	1	1	1	1	1
2.	Carbopol 940	0.5	1	-	-	-	-	-	-
3.	Methyl cellulose	-	-	2	4	-	-	-	-
4.	HPMC	-	-	-	-	1.5	2	-	-
5.	Pectin	-	-	-	-	-	-	3	4
6.	Glycerin	10	10	10	10	10	10	10	10
7.	Propylene glycol	20	20	20	20	20	20	20	20
8.	Methylparaben	0.03	0.03	0.03	0.03	0.03	0.03	0.03	0.03
9.	Propylparaben	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01
10.	Purified water to	100	100	100	100	100	100	100	100

Now-a days, nanogels are extensively used as carriers for all formulations due to its prime advantages over the conventional drug release. The pre-requisite for a successful Nano delivery system includes thefairly good drug-loading capacity, compared to conventional carriers. The mechanism of action is unique as it immediately converts into nanoparticle (self-aggregation ofamphiphilic polymers by intra- or intermolecular associations between the hydrophobic element). This added to the low surface tension of the particle due to nano size. It can be further explained as the property of these nanoparticle to spontaneously align as the hydrophobic element in the core surrounded by the hydrophilic moiety outside. This association of polar region outside is called the critical micelle concentration or the critical aggregation concentration.

In this research article, the effectiveness of antifungal drugs against allergic seborrheic dermatitis were studied when applied topically along with nanogel. The result shows that nanogel increases the penetration of the drug for the percutaneous delivery of drugs to the skin layers for the treatment of various skin inflammatory disorders.

MATERIALS AND METHODS:

Materials ²⁶

Fluconazole (FLZ) was received as a gift sample from Unichem Laboratory, Ghaziabad. Eudragit RS was purchased from Sigma-Aldrich Pvt Ltd. Ethyl-cellulose and tween 80 were purchased from CDH Pvt. Ltd., New Delhi. All the solvents used were of analytical grade.

Methods:

Preparation of FLZ nanogel²⁶

The different batches of the drug fluconazole were prepared with various concentration of polymer as mentioned in Table 1. First step is to prepare a hydrogel base to form a fluconazole nanogel (FLZ -NG). The polymers used in formulations are Carbopol 940, HPMC, and methylcellulose ^[4]. The two different concentrations of each polymer were used to prepare hydrogel. These are dispersed in luke warm water (antisolvent) with constant stirring. With the help of syringe hydrogel containing nanogel were added to anti-solvent polymer mixture.²⁶Methyl and propylparaben as preservatives were added slowly with continuous stirring until gel formation.⁵

Antifungal study:

The antifungal activity of FLZ against the yeast Malassezia was compared with marketed FLZ gel as control^[6]. Using the Kirby-Bauer test also known as the disk-diffusion method, antifungal activity was seen in agar plates as inhibition zone which depicts the relation with the dissolution model.

Ex-vivo skin permeation study:

The skin was obtained from the freshly sacrificed rat. The skin was cleaned with isopropyl alcohol and distilled water. this skin is stored at -18^∘C until further use to prevent decomposition. For evaluation the skin was placed between the cells of the Franz-Diffusion cell where the stratum corneum side was the donor compartment and the dermal side was the receiver compartment. One gram of each gel was placed in a donor compartment ⁷. The receptor compartment was filled with 10ml PBS (pH6.8), thermoregulated at 37^∘C, and magnetically stirred at 400 rpm. Two milliliters were withdrawn at an interval of 1, 2, 3, 5, 7, 12, 18, and 24 h. Each sample was filtered and then determined for drug content by UV spectrophotometer at 260 nm. The concentrations of all the formulations in withdrawn samples were calculated and then the percentage drug release was determined⁸.

Skin thickness study:

The skin scaling is one of the symptoms of SD. Thus, a formulation needs to reach the deepest layer of skin to treat the same. This test was performed to analyze the drug present in the skin after permeation study. The used skin was extracted from the FD cell ³and swabbed with phosphate buffer pH (6.5), and then by methanol. This step was repeated twice before cutting the skin in small pieces. these are then kept in methanol for extracting drugs present in the skin after 48 hours via UV at 265 nm ⁹.

In-vivo antifungal study:

Experimental Animals:

Three healthy male rats in each group, weighing 200-250g were selected for the study. Rats were divided into four groups (n=3) as follows: Group-I (control): There was no application on the surface of the rat skin. Group-II (disease group): the only disease is induced. Group-III (standard control): Marketed FLZ gel is applied after disease induction. Group-IV (test group): FLZ -NG was applied after induction of SD^10,11.

Experimental Protocol:

An area of 1 cm² was shaved for each rat. The disease was induced using DNCB model (1-cholro-2,4-dinitrobenzene)²⁷. Formulation F5 was applied on the exposed area of the rat labeled test whereas drug solution was applied to standard and control were untreated. The (3:1) solution of 200ml 0f 0.5% DNCB in acetone was applied to the exposed area of the animal of groups II, III, and IV. On the interval of 3 days starting from 14-day rats were treated with 200 ml of 1% DNCB on an area of 1 cm(sq)²⁷. after half an hour of application of 1-cholro-2,4-dinitrobenzene, the marketed gel was applied to group III whereas the formulated FLZ-NG was applied on group IV animals daily. This procedure was observed for erythema and edema daily for up to seven days and compared. Some topical gels produce skin irritation when applied. Thus, a skin irritation study was performed^12,13.

Skin irritation test:

This test was performed on a healthy white rat with an average weight of 1.5 to 2.5Kg. About 7-8 cm² areas on the upper surface of the rat in each group were shaved and cleaned with spirit. The test sites were visually observed for erythema and edema daily for up to seven days and compared¹⁴.

Stability studies:

The F5 batch were kept under two different temperatures, i.e., 4-8°C (Refrigerator; RF), 25±2 °C (Room temperature; RT), for 60 days¹⁵.

The samples from the different environmental conditions were withdrawn in specific interval andevaluated for drug deposition for gel such as entrapment efficiency and particle size distribution studies¹⁶.

RESULTS AND DISCUSSION:

Antifungal activity:

Table 2 shows the diameter of inhibition zone with different batches from F1 to F8. The highest value is 38mm for F5 batch (HPMC 1.5mg) with the lowest as 14mm in F2 (Carbopol 1mg)¹⁷. These results accord with the in-vitro release study.

Ex-vivo skin permeation study:

Permeation study was performed on eight batches of FLZ -NG using Franz-Diffusion cell with animal skin. The samples were withdrawn at different intervals and evaluated for 24 hrs.¹⁸. The drug release for formulation F5 were found to be best among all the batches since it showed the sustained release of more than 80% of drug in 24 hours¹⁹.

Figure 1: Cumulative percent ex-vivo skin permeation drug release of batches of FLZ -NG. Around 50% of drugs were released in the starting first 10 h of total release followed by drug releases of 90% in next 24 hours, with maximum release of in batch F5. The values were expressed as, n = 6, mean±SD.

Skin thickness test:

The skin thickness test was performed as mentioned above, the result shows a significant decrease in group IV as compared to standard drugs¹². The results were described graphically in Figure 2.

Figure 2: The skin thickness in micrometer in four groups of the animal model showing the 0.6 µm in control (group I), 2.3 µm in disease (group II), whereas 1.8 µm in standard (group III) and 1.4 µm in the test (group IV). The values were expressed as, n = 6, mean ± SD.

Histology:

The DNCB is applied on animal skin for seven days on the proposed protocol of groups of animals. The physical observations were done on the different groups in regular intervals. The FLZ-NG showed significant improvement on seventh day as compared to Group II (diseased group) and Group III (marketed FLZ gel)^20,21.

Stability studies:

The best formulation F5 batch was examined under two different condition and results are found to be satisfactory after 6 months of storage.

CONCLUSION:

Eight different batches of FLZ-NG were prepared and examined under various in-vivo and ex-vivo studies. The antifungal activity, skin permeation, and skin thickness test proves that the NG preparation of F5 formulation has better results²² with the HPMC polymer (0.5mg). due to the small size of nanoparticles present in the gel has high skin permeability with good antifungal activity as it reaches the target rapidly due to the small particle size property. Hence, when compared with the marketed FLZ- gel (1mg) on animal skin shows better results as it reaches the deeper layer faster and can be used as a carrier for treating SD^23-25.

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Received on 19.01.2022 Modified on 03.07.2022

Research J. Pharm. and Tech 2023; 16(6):2880-2884.

DOI: 10.52711/0974-360X.2023.00474

Formulations	F1	F2	F3	F4	F5	F6	F7	F8
Inhibition Zone	20 mm	14 mm	32 mm	29 mm	38 mm	35 mm	34 mm	37 mm