Introduction
Acne is a very common inflammatory skin disease, which is also known as pimples. They are usually found on the face, chest, upper back and neck. Propionibacterium and Staphylococcus aureus are acne-causing bacteria [1]. The occurrence of acne is closely related to many factors, including excessive sebum production, hormone levels, bacterial infection, and inflammatory reactions, among which excessive sebum production is a more important cause [2]. The main activity of mature sebaceous glands is the production and secretion of sebum, which contains a complex mixture of lipids. Sebum composition is different between species, and this difference is probably due to the diverse functions of sebum [3]. Other functions of sebaceous glands, including the secretion of pro-inflammatory sebaceous lipids, various locally produced cytokines, peptides and neuropeptides around the sebaceous glands, are related to the occurrence of acne [2].
Acne rosacea is an inflammatory and chronic skin disease that often affects the cheeks, nose, chin and forehead. Manifestations of this disease include persistent facial erythema, papules, pimples, telangiectasia and frequent flushing [4]. The prevalence of rosacea in light-skinned populations varies between 2 and 22% [5]. Rosacea has episodes of exacerbation and remission and is seen in people aged between 30-50 years. Women are more affected by this disease than men [4].
Topical drugs approved by the US Food and Drug Administration (FDA) for the treatment of acne rosacea include topical formulations of sodium acetamide sulfate, metronidazole, and azelaic acid [6]. Azelaic acid is a straight chain dicarboxylic acid with two pKa values of 4.53 and 5.33 and limited solubility in water. Azelaic acid has antimicrobial activity against Propionibacterium acnes and Staphylococcus epidermidis. This antimicrobial activity may be due to inhibition of the synthesis of microbial cell proteins. With this mechanism, azelaic acid reduces the bacterial population of propionibacterium on the skin and sebaceous follicles. Also, azelaic acid has anti-tyrosinase and anti-mitochondrial activities, and by reducing free radicals, it reduces the activity of melanocytes and their growth in melasma, and stops the local hyperpigmentation of the macula in the skin [7].
Azelaic acid gel formulation has advantages over its cream formulation which include the loading of a lower dose of azelaic acid and longer duration on the skin, which increases the drug release and biological supply. In addition, the gel formulation can penetrate into the skin without damaging the skin or reducing the epidermal water content. Therefore, it is a suitable treatment option for acne rosacea [11]. This study aims to prepare an azelaic acid gel formulation and evaluate its physicochemical properties and perform preliminary stability testing on the formulation.

Methods
Azelaic acid, sodium hydroxide, potassium dihydrogen phosphate, polysorbate, benzoic acid, propylene glycol, ethylenediaminetetraacetic acid and ethanol 96% were purchased from Merck Company, Germany. Moreover, carbomer P940 was purchased from Corel Pharma Chem, India; lecithin from DUKSAN company, South Korea; triglyceride from Nutricia company, the Netherlands; and methanol from Dr. Majeli Company, Iran. Azelaic acid gel formulation was prepared in several steps using the method presented in a previous study [14].
To draw the standard curve of azelaic acid, concentrations of 10, 20, 40, 60, 80 and 100 μg/ml of azelaic acid were prepared in methanol, and according to the λmax obtained from the photodiode array (PDA) detector, the area under the curve (AUC) during was read at a wavelength of 212 nm [15]. Phosphate buffer with pH 4.8 and molarity 0.05 was prepared. To determine the amount of azelaic acid, 18C column and HPLC methanol-phosphate buffer (20:80) were used as the mobile phase. To stabilize the device pressure, the column was washed by the mobile phase for 60 minutes. Then, the area under the peak curve was measured at a wavelength of 212 nm. This test was repeated on F3, F4 and F5 formulations (each for 3 times). 
Five grams of each of F3, F4 and F5 formulations were weighed and made up to 50 ml with distilled water. The pH of the sample was measured by a pH meter (Metrohm) at a temperature of 25°C. A viscometer (DV-II, Brookfield) with spindle 5 was used to determine the viscosity of the gel formulation (at 2 shear speeds of 30 and 50 rpm). To investigate the long-term stability of azelaic acid in the gel formulation for 3 months from the day of manufacture, the amount of azelaic acid in the F5 gel formulation was measures in this period. The long-term stability study was conducted at a temperature of 25°C and a humidity of 60°C. The required temperature and humidity were provided by a stability climate chamber (Memmert company).
The amounts of azelaic acid at different times were compared using t-test in SPSS software, version 17. To study the release of azelaic acid from the gel, the standard curve of azelaic acid was drawn in a medium containing monobasic phosphate buffer solution with a pH of 4.8 and 96% ethanol with a ratio of 1:1. Then, the release profile of azelaic acid from the F5 gel formulation was investigated during 6 hours in this environment. 

Results
The F1 formulation had a sandpaper-like appearance. In the F2 formulation, the sandpaper-like state was eliminated, but the gel formulation was very smooth. In formulation F3, which was made by increasing the concentration of lecithin and using an overhead mixer, the appearance of the formulation was suitable. The F4 and F5 formulations also had a suitable appearance.
The results of determining the amount of azelaic acid in different formulations at the end of the first, second and third months showed that the amount of azelaic acid in the F3 formulation was 99.72% in the first month, 96.25% in the second month, and 100.5% in the third month. In the F4 formulation, the amounts of azelaic acid in the first, second and third months were 100.39, 97.85 and 98.76%, respectively. In the F5 formulation, these amounts were 100.25, 102.9, and 99.5%, respectively. The amount of azelaic acid in the F5 gel formulation was more than 99% of the determined amount during three months. The results of the study on the release of azelaic acid from the gel, showed that about 30% of the drug was released in the first hour. After 2 hours, the release rate reached about 50%, and the release did not increase significantly until 6 hours.

Conclusion
According to the obtained results, in the F5 gel formulation, where three stages of homogenizing were used in its manufacturing process and the formulation was well homogenized, the amount of azelaic acid did not change significantly during the storage period and a lower standard deviation was observed compared to other formulations. It can be concluded that the use of a homogenizer when making the azelaic acid gel formulation is effective in making the gel more homogenous, and the F5 formulation is more homogeneous than the other formulations. Thus, it is suggested as the optimal formulation.

Ethical Considerations

Compliance with ethical guidelines
This study was approved by Ethics Committee of Guilan University of Medical Sciences (Code: IR.GUMS.REC.1399.511).

Funding
This article is taken from PhD dissertation of Zahra Sabour, approved by Ethics Committee of Guilan University of Medical Sciences.  

Authors' contributions
Conceptualization and study design: Saeed Manoochehri; Data collection, analysis and interpretation: Saeed Manoochehri; Mohammad Moazen and Saeed Ghasemi;  initial draft preparation and statistical analysis: Saeed Ghasemi and Saeed Manoochehri; Experiments: Zahra Sabour.

Conflicts of interest
The authors declared no conflict of interest.

Acknowledgements
The authors would like to thank Gita Alkan Saberi, (an expert in the Pharmaceutics Laboratory) for her assistance.

References