Introduction
Dry eye is one of the age-related diseases, which is considered one of the most common eye diseases with more than 7 million affected people (9% of the population) in Iran. One of the most important known treatments for eye diseases is the use of eye drops, which in most cases can improve the patient’s condition. Among the effective drops, we can indicate Artelac Rebalance and Vitadrop eye drops, which contain vitamin B12 and hyaluronic acid. Vitamin B12 is an essential factor in maintaining eye health and has antioxidant properties that protect the surface of the eye from damage caused by harmful factors such as reactive oxygen species (ROS). Recently, injectable and topical forms of vitamin B12 are used for eye pain and dry eye treatment [4].
One of the most important factors determining clinical effectiveness and increasing patient acceptance is the drug’s efficacy duration. The short shelf life of eye drops can increase the need to use drops and reduce patient satisfaction [8, 9, 10, 11]. The high prevalence of eye diseases and the significant need for specific medicines and considering the economic conditions of the country make it necessary to prepare new eye treatment products with optimal effectiveness inside the country, which, while meeting the medical needs of the society, can help the economic development of the country. Due to the positive effects of vitamin B12 and hyaluronic acid, preparation and formulation of an eye drop with the presence of these components is necessary for the treatment of dry eye disease in Iranian people, which can provide a platform for competition even with international companies.

Methods
Vitamin B12 was obtained from Raha Pharmaceutical Company (Iran). Hyaluronic acid, boric acid and benzalkonium chloride were obtained from Sigma Aldrich Company (USA). The rest materials were obtained from Merck Company (Germany). To prepare eye drops containing 40 µg/ml vitamin B12 and 0.2% hyaluronic acid, the materials shown in Table 1 were mixed.


UV spectroscopy was used to check the changes in the concentration of vitamin B12 in the prepared formulation. The efficacy of the protective agent (benzalkonium chloride) was investigated according to the USP 43 pharmacopoeia and using the services of the Food and Drug Laboratory (Tavan Institute, Iran) after 6 months of stability testing. To check the stability of the prepared formulation, the eye drops were kept at a temperature of 40 and a humidity of 75% for 6 months. During this period, properties such as color, uniformity, the presence of particles in the formulation, the amount of B12 based on UV absorption, and the amount of osmolarity and viscosity were examined every month, and at the end of the study, the effectiveness of the protective agent was tested.

Results
According to the results vitamin B12 had the maximum absorption at the wavelength of 361 nm. The standard curve for B12 is plotted. In the stability test, according to the obtained equation, the amount of vitamin B12 was evaluated at different times. The formulations prepared at different times were evaluated and the results of the accelerated stability test according to World Health Organization (WHO) guidelines after 6 months are presented in Table 2.


According to the results in Table 3, the selected protective agent (benzalkonium chloride) in the prepared concentration can be a suitable option to protect the ophthalmic formulation and the results are in accordance with the USP pharmacopoeia.



Conclusion
The results obtained from the formulation prepared in this study after the accelerated stability test according to the WHO guidelines showed that the eye drops containing vitamin B12 have good stability after 6 months and the amount of the effective substance has not changed. On the other hand, in this study, the minimum concentration of the protective agent benzalkonium chloride was used, which was able to maintain its effectiveness after 6 months of stability study. In this study, a 0.2% hyaluronic acid was used in the formulation, which has been shown in studies to effectively remove eye inflammation and to be effective in dry eye disease, with no any complications.
According to the obtained results, it seems that the formulation has the necessary efficiency as an artificial eye drop and can effectively deliver vitamin B12 to the eyes. This formulation with the minimum concentration of benzalkonium chloride can be a suitable option for the treatment of dry eyes with osmolarity, viscosity and pH similar to commercial and acceptable formulations in the pharmaceutical industry.

Ethical Considerations
Compliance with ethical guidelines
This study was approved by the Ethics Committee of Iran University of Medical Sciences, Tehran, Iran. (Code: IR.IUMS.REC.1399.1374).

Funding
The present study was financially supported by the Vice-President of Research and Technology, Iran University of Medical Sciences, Tehran, Iran.

Authors' contributions
Conceptualization and study design: Ali Rastegari, Zohreh Mohammadi, and Homa Faghihi; Data acquisition, analysis and curation: Ali Rastegari, Zohreh Mohammadi, and Sanaz Rezaei; Writing: Ali Rastegari, and Zohreh Mohammadi.

Conflicts of interest
The authors declared no conflict of interest.

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