(T1330-11-62) Laboratory Study in Gastric Nitrosamine Formation

Purpose: Nitrosamine reduction is a top priority for the entire industry, with recent FDA guidance setting ambitious timelines for nitrosamine drug substance-related impurities (NDSRI) compliance. Endogenous formation of nitrosamines from secondary amines has long been understood. However, there is no current in vitro test to approximate this phenomenon. This study specifically speaks to measuring the formation of NDSRIs from secondary and dimethyl tertiary amines, which the FDA has identified as most problematic. These functional groups can readily react with nitrite present in the gastric tract to form NDSRI impurities.

Methods: In vivo conditions were simulated utilizing in vitro USP Dissolution Apparatus 2 with a well characterized drug product known to form an NDSRI. Dissolution conditions were selected to mimic conditions at various states in the feeding cycle including biorelevant pH, salts, enzymes, and volume. The appropriate nitrite quantity was established based upon IARC Vol. 94, Ingested Nitrate and Nitrite, and Cyanobacterial Peptide Toxins and the extrapolated maximum nitrite quantity during a meal. This was coupled with LC/MS analysis to achieve the desired sensitivity and selectivity.

Results: Each set of biorelevant conditions performed showed NDSRI concentrations substantially higher than the regulatory limit of 2.3µg/g. Most experiments exceeded this threshold 50-fold. This was true whether a simple buffer was used or simulated gut fluids.

Conclusion: Nitrite concentrations and pH alone are sufficient to drive the reaction. Given the similarity in gastric conditions to the widely accepted NAP test, which is designed to show the possibility of nitrosamine formation, these results are not surprising. Future work should be performed based on product specific pharmacokinetics. Interspecies NOx conversion in the more complex in vivo environment may reduce the effect of nitrite concentration differences.

References: Raisfeld-Danse IH, Chen J. Drug interactions. III. Formation of nitrosamines from therapeutic drugs. Formation, mutagenic properties and safety assessment of propranolol hydrochloride with respect to the intragastric formation of N-nitrosopropranolol under conditions found in patients. J Pharmacol Exp Ther. 1983 Jun;225(3):713-9. PMID: 6345752.
IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. IARC monographs on the evaluation of carcinogenic risks to humans. Ingested nitrate and nitrite, and cyanobacterial peptide toxins.


Image 1: N-Nitrosopropranolol Mass Spectra


Image 2: NDSRI formation in reference to API mass, pH 3.0 Fasted State Simulated Gastric Fluid/pH 6.8 Fasted State Simulated Intestinal Fluid with Enzymes


Image 3: NDSRI formation in reference to API mass, pH 1.6 Fasted State Simulated Gastric Fluid/pH 6.8 Fasted State Simulated Intestinal Fluid