Studies on tissue specific carcinogenicity and on metabolism of N-nitrosamines (SC05 to FWC EMA/2020/46/TDA/L1.02 – ROC38))

Study type

• Study topic:
  Other
• Study topic, other:
  Evaluate tissue specific carcinogenicity and on metabolism of N-nitrosamines
• Study type:
  Not applicable
• Scope of the study:
  Method development or testing
• If ‘other’, further details on the scope of the study:
  Research project to better define threshold for nitrosamine impurities
• Data collection methods:
  Combined primary data collection and secondary use of data

Non-interventional study

No information provided.

Study drug and medical condition

• Medicinal product name, other:
  Several model compounds with nitrosmine substructure

Population studied

• Short description of the study population:
  none
• Special population of interest:
  Other
• Special population of interest, other:
  no patients involved
• Estimated number of subjects:
  0

Study design details

• Study design:
  The project investigated the tissue-specific tumour formation by N-nitrosamines in existing in vivo cancer studies. ; Further, metabolic activation and detoxification was investigated using in silico and in vitro methods.
• Main study objective:
  The Mutamind 5 project has two main objectives, each subdivided to several subobjectives. These are briefly described in the following:; Objective 1: Review of quantitative organ-specific carcinogenicity for N-nitrosamines; Objective 1a: Creation of a database of tissue-specific TD50s for N-nitrosamines ; Objective 1b: Analyse any relationships between structure, functional groups and/or physicochemical properties and tissue-specific carcinogenicity ; Objective 1c: Analyse the contributions of organ-specific metabolism, tissue distribution, repair mechanisms and stability of the activated impurity to tissue-specific carcinogenicity ; Objective 1d: Conclude on the implications of findings from objectives 1a, 1b and 1c on requirements for tissue sampling and analysis for in vivo mutagenicity studies with N-nitrosamines ; ; Objective 2: Mechanistic study on the metabolism of N-nitrosamines; Objective 2a: Selection of NDSRIs for empirical evaluation; Objective 2b: Pre-experimental in silico based generation of metabolites from its structure; Objective 2c: Experimental phase to assess metabolism of NDSRIs and corresponding active substances; Objective 2d: In silico to in vitro comparison; ;
• Data analysis plan:
  Objective 1: Review of quantitative organ-specific carcinogenicity for N-nitrosamines; The project investigated the tissue-specific tumour formation by N-nitrosamines with diverse physicochemical properties. ; A systematic review of peer-reviewed publications from 2010 onwards was combined with data from the (updated) Carcinogenic Potency Database (CPDB). Relationships between tissue-specific carcinogenicity and chemical structure, functional groups, CPCA categories, physicochemical properties and ADME parameters were analysed. ; The analysis identified the most frequent target organs of Nitrosamines and examined the influence of study design and physicochemical properties on target-organ prevalence. It also evaluated the tissue-specific expression of phase I and II enzymes using data from the Human Protein Atlas and peer-reviewed literature. ; ; Objective 2: Mechanistic study on the metabolism of N-nitrosamines; The study evaluated how metabolic activation and detoxification influenced the genotoxicity of NDSRIs using a diverse set of NDSRI compounds covering different structural features and mutagenic potencies. ; In silico tools predicted sites of metabolism, metabolite structures, and competing activation and detoxification pathways. Experimental studies incubated the selected NDSRIs with in vitro assays like S9 liver fractions or primary hepatocytes from humans/rats to identify metabolites and degradation products, including those formed through CYP-mediated activation, denitrosation and phase II conjugation.; Targeted LC–MS/MS methods quantified parent NDSRIs and selected metabolites over time, to calculate their metabolic half-lives and intrinsic clearance. Predicted and experimentally detected metabolites were integrated into compound-specific metabolic maps. Comparison of the in silico predictions with the experimental results assessed the performance of the prediction tools for phase I and II metabolism
• Summary results:
  N-Nitrosamine drug substance-related impurities (NDSRIs) pose considerable health risk due to their mutagenic and carcinogenic properties. One important aspects of NAs carcinogenicity is its tissue specificity. The analysis of prevalent target organs revealed liver as the predominant target organ following oral NAs exposure. Also organs of the upper aerodigestive tract, most notably the esophagus was observed. The organ level pattern aligned with cytochrome P450 (CYP) expression data, showing relevant CYP450 enzyme expression in the liver and esophageal tissue. These findings supported esophagus as target organ alongside the liver and offer suggestion for the tissue selection in the future toxicological in vivo studies of NDSRIs.; NDSRIs require a metabolic activation, which is influenced by their structural properties and enzyme accessibility. The second part of the project evaluated whether an integrated computational and experimental workflow could improve the prediction of NDSRI metabolism, with particular emphasis on cytochrome P450-mediated α-hydroxylation adjacent to the N-nitroso group. Seven structurally diverse NDSRIs spanning N-nitrosamine carcinogenic potency categorization approach categories 1–5 and three negative controls were investigated. MetaSite predictions, molecular docking of six human CYP isoforms, and quantum mechanical approaches were combined with clearance and metabolite profiling in induced rat liver S9, human liver S9 and primary human hepatocytes. The approaches provided complementary information: MetaSite identified overall plausible sites and pathways of metabolism, docking evaluated whether α-carbons could adopt catalytically competent orientations, and quantum-chemical calculations characterised intrinsic reactivity and energetic feasibility. Experimental studies demonstrated marked compound- and system-dependent differences in clearance and metabolite formation and showed that α-hydroxylation was only one of several competing routes