Dear Esteem Community Members,
May anyone assist or share publication and or protocol outline for an In Vitro Metabolism Study with Human hepatocytes or microsome to study metabolites generate by NDSRI ?
Any assistance ob this matter is appreciate!
Thanking you in anticipation.
Regards
This study, published by Cheung et al., might give you a good reference as a starting point.
Evaluation of the nitrosamine impurities of ACE inhibitors using computational, in vitro, and in vivo methods demonstrate no genotoxic potential
https://onlinelibrary.wiley.com/doi/10.1002/em.22618
Abstract:
Evaluation and mitigation of the potential carcinogenic risks associated with nitrosamines in marketed pharmaceutical products are areas of interest for pharmaceutical companies and health authorities alike. Significant progress has been made to establish acceptable intake (AI) levels for N -nitrosamine drug substance-related impurities (NDSRIs) using SAR, however some compounds require experimental data to support derivation of a recommended AI. Many angiotensin-converting enzyme inhibitors, identified by the suffix “pril,” have secondary amines that can potentially react to form nitrosamines. Here we consider a structural assessment and metabolism data, coupled with comprehensive in vitro and in vivo (mouse) genotoxicity testing to evaluate this particular class of nitrosamines. N -nitroso ramipril and N -nitroso quinapril, both of which are predicted to have inhibited nitrosamine bioactivation due to steric hinderance and branching at the α-position were non-genotoxic in the in vivo liver comet assay and non-mutagenic in the in vivo Big Blue® mutation and duplex sequencing assays. Predicted metabolism along with in vitro metabolism data and quantum chemical calculations related to DNA interactions offer a molecular basis for the negative results observed in both in vitro and in vivo testing. These nitrosamines are concluded to be non-mutagenic and non-carcinogenic; therefore, they should be controlled according to ICH Q3B guidance. Furthermore, these results for N -nitroso ramipril and N -nitroso quinapril should be considered when evaluating the appropriate AI and control strategy for other structurally similar “pril” NDSRIs.
The USFDA has specifically cited this article on their website ( CDER Nitrosamine Impurity Acceptable Intake Limits | FDA ) for the human in vitro assays:
Li X, et al. Revisiting the mutagenicity and genotoxicity of N-nitroso propranolol in bacterial and human in vitro assays. Regul Toxicol Pharmacol. 2023 Jun; 141:105410
doi:10.1016/j.yrtph.2023.105410
The following article is also available in the public domain,
Mutagenicity and genotoxicity evaluation of 15 nitrosamine drug substance-related impurities in human TK6 cells, 10.1016/j.yrtph.2024.105730
Both these articles have been published by USFDA and are free to download; they should serve as a basis for performing in vitro studies using human tissues.
Thank you for sharing valuable information!
It seems further research on in vitro metabolism of NDSRIs (using different systems) is planned at FDA NCTR (there is an open FDA postdoctoral fellowship on this online): “in vitro metabolism studies on nitrosamines using liver S9 fractions, microsomes and mammalian cell culture systems”; metabolite characterization with “HPLC-UV, LC-MSMS and 1H-NMR”.
Research Project: This fellowship will focus on investigating the metabolism of nitrosamine drug substance-related impurities (NDSRIs) and its potential impact on mutagenicity and carcinogenicity to address critical FDA regulatory knowledge gaps regarding drug safety. You will collaborate with an interdisciplinary team of scientists to elucidate metabolic activation pathways of structurally complex NDSRIs, with a particular emphasis on DNA adduct formation, using in vitro metabolic systems and mammalian cell models. These studies will provide mechanistic insights into the structure-activity relationships underlying the mutagenic and carcinogenic potency of NDSRIs. The research activities will also support improvements in (quantitative) structure-activity relationship ([Q]SAR) modeling and enhance the FDA’s ability to conduct regulatory safety assessments of NDSRIs in affected drug products.
Educational Activities Will Include
Gaining experience with in vitro metabolism studies using liver S9 fractions, microsomes, and mammalian cell culture systems.
Learning to characterize major intermediates and metabolites using high-performance liquid chromatography-ultraviolet spectroscopy (HPLC-UV), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and/or proton nuclear magnetic resonance spectroscopy (1H-NMR).
Developing skills in the identification and quantification of DNA adducts formed by NDSRIs in S9/microsomal incubations with DNA and in cell culture systems, using 1H-NMR and LC-MS/MS.
Presenting research findings at scientific conferences and publishing results in peer-reviewed journals.