(T1530-09-56) Antioxidant Screening for the Inhibition of N-nitroso Propranolol Formation in Solution and Solid-State

Purpose: Nitrosamine drug substance-related impurities (NDSIRs) are a group of complex nitrosamines formed by N-nitrosation of a vulnerable amine in the active drug molecule. While the genotoxicity of such impurities is generally not well studied, this is a critical issue that needs prompt attention, given that many active pharmaceutical ingredients (APIs) contain an amine fragment that could potentially lead to NDSIR formation. Propranolol can react with sodium nitrite, and upon nitrosation forms N-nitroso propranolol (NNP). NNP was found to be a mutagenic and genotoxic nitrosamine in a variety of bacterial and mammalian systems, and is a potential human carcinogen [1]. According to the FDA Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs) guidance issued in August 2023, NNP is a Category 5 nitrosamine with an acceptable intake (AI) limit of 1500 ng/day [2]. Previous studies have proven the utility of antioxidants and pH modifiers in mitigating nitrosamine formation and even decrease the existing amount of nitrosamine in tablets [3–6]. The objective of the present study is to evaluate the inhibition efficiency of three antioxidants in solution state, and five in solid state using propranolol as model drug. Ultimately, this study aims to inform and identify the best antioxidant/inhibitor to use for the prevention of NNP formation in tablets.

Methods: A solution-state study was performed in 20mM ammonium formate buffer (pH 3.0) with propranolol, sodium nitrite and antioxidant/inhibitor molar concentration of 1:1:20 at room temperature and 50°C. The antioxidants include ascorbic acid, maltol and cysteine. Initially, the reactants were dissolved in reaction buffer, making 10, 20 and 80mM of propranolol, sodium nitrite and antioxidants, respectively. The appropriate amount of reactant solutions were added to a vial, making 2mM of propranolol and sodium nitrite and 40mM of antioxidants with a final reaction volume of 4mL. The reaction was carried out for 24 hours, sampling at 0, 1, 3, 6, 8 and 24hr time points and stopping reaction with 3mM ammonium hydroxide. Reaction pH was monitored throughout the experiment. A solid-state study was conducted with five antioxidants/inhibitors including sodium bicarbonate, ascorbic acid, sodium ascorbate, maltol and cysteine. Final powder contained 20% propranolol, 0, 0.5 or 1% antioxidant/inhibitor and 79-80% MCC spiked with 0, 100 or 500ppm sodium nitrite. In total, there were 33 mixtures evaluated. The powder blends were stored at 40°C/75%RH for 14 days, and NNP was extracted from the solids with acetonitrile and the mother liquor was filtered with 0.22µm PVDF syringe filter. Samples from solution and solid-state studies were analyzed by LC-MS/MS for NNP content.

Results: Antioxidants in solution and solid-state studies were effective in mitigating NNP formation. In solution study, cysteine was the best inhibitor at both RT and 50°C. The inhibition effectiveness over 24 hr was 96.2% in RT, and 98.8% in 50°C. Ascorbic acid effectiveness was nearly comparable with cysteine, affording 88.1% and 96.5% effectiveness at RT and 50°C, respectively. Results from solid-state study indicated that 1% antioxidant was more effective at inhibiting NNP formation than 0.5%. Among the antioxidants tested, sodium ascorbate, sodium bicarbonate, ascorbic acid and cysteine were equally effective. Maltol was the least effective in all cases. Across a range of studies, the average effectiveness of sodium bicarbonate, sodium ascorbate, ascorbic acid, cysteine, and maltol were 86.4, 97.4, 98.5, 98.1 and 80.4%, respectively. Based upon both solution and solid-state studies, ascorbic acid and cysteine were the most effective.

Conclusion: Overall, results from solution and solid-state studies showed that ascorbic acid and cysteine were most effective in mitigating the formation of NNP from propranolol.

References: 1. Li X, Le Y, Seo J-E, Guo X, Li Y, Chen S, et al. Revisiting the mutagenicity and genotoxicity of N-nitroso propranolol in bacterial and human in vitro assays. Regulatory Toxicology and Pharmacology. 2023;141:105410.
2. Food and Drug Administration. Guidance for Industry: Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs) [Internet]. FDA.
3. Nanda KK, Tignor S, Clancy J, Marota MJ, Allain LR, D’Addio SM. Inhibition of N-Nitrosamine Formation in Drug Products: A Model Study. J Pharm Sci. 2021;110:3773–5.
4. Shakleya D, Asmelash B, Alayoubi A, Abrigo N, Mohammad A, Wang J, et al. Bumetanide as a Model NDSRI Substrate: N-nitrosobumetanide Impurity Formation and its Inhibition in Bumetanide Tablets. Journal of Pharmaceutical Sciences [Internet]. 2023
5. Homšak M, Trampuž M, Naveršnik K, Kitanovski Z, Žnidarič M, Kiefer M, et al. Assessment of a Diverse Array of Nitrite Scavengers in Solution and Solid State: A Study of Inhibitory Effect on the Formation of Alkyl-Aryl and Dialkyl N-Nitrosamine Derivatives. Processes. 2022;10:2428.
6. Shakleya D, Alayoubi A, Brown D, Mokbel A, Abrigo N, Mohammad A, et al. Nitrosamine mitigation: NDMA impurity formation and its inhibition in metformin hydrochloride tablets. International Journal of Pharmaceutics. 2024;666:124832.

Acknowledgements: This work was funded by the University of Maryland Strategic Partnership: MPowering the State (MPower). Conflict of interest: There are no conflicts of interest of these authors to declare