Revisiting the Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceuticals.

N-Nitrosamines are a class of indirect acting mutagens, as their metabolic degradation leads to the formation of the DNA-alkylating diazonium ion. Following up on the in-silico identification of thousands of nitrosamines that can potentially be derived from small molecule drugs and their known impurities described in a previous publication, we have now re-analyzed this dataset to apply EMA's Carcinogenic Potency Categorization Approach (CPCA) introduced with the 16th revision of their Q&A document for Marketing Authorization Holders. We find that the majority of potential nitrosamines from secondary amine precursors belongs to potency categories 4 and 5, corresponding to an acceptable daily intake of 1500 ng, whereas nitrosamines from tertiary amine precursors distribute more evenly among all categories, resulting in a substantial number of structures that are assigned the more challenging acceptable intakes of 18 ng/day and 100 ng/day for potency categories 1 and 2, respectively. However, the nitrosative dealkylation pathway for tertiary amine is generally far slower than the direct nitrosation on secondary amines, with a direct nitrosation mechanism suspected only for structures featuring electron-rich (hetero)aromatic substituents. This allows for greater focus towards those structures that require further review, and we demonstrate that their number is not substantial. In addition, we reflect on the nitrosamine risk posed by secondary amine API impurities and demonstrate that based on the ICH Q3A/B identification threshold unknown impurities may exist that could be transformed to relevant amounts of NA. We also demonstrate that the analytical sensitivity required for the quantification of high potency nitrosamines can be problematic especially for high dose APIs. In summary, the regulatory framework rolled out with the latest Q&A document represents a substantial improvement compared with the previous situation, but further refinement through interaction between manufacturers, regulators, not-for-profit and academic institutions will be required to ensure patient access to vital medicines without compromising safety.

Nitrosamine acceptable intakes should consider variation in molecular weight: The implication of stoichiometric DNA damage.

N-nitrosamines (NAs) are a class of compounds of which many, especially of the small dialkyl type, are indirect acting DNA alkylating mutagens. Their presence in pharmaceuticals is subject to very strict acceptable daily intake (AI) limits, which are traditionally expressed on a mass basis. Here we demonstrate that AIs that are not experimentally derived for a specific compound, but via statistical extrapolation or read across to a suitable analog, should be expressed on a molar scale or corrected for the target substance's molecular weight. This would account for the mechanistic aspect that each nitroso group can, at maximum, account for a single DNA mutation and the number of molecules per mass unit is proportional to the molecular weight (MW). In this regard we have re-calculated the EMA 18 ng/day regulatory default AI for unknown nitrosamines on a molar scale and propose a revised default AI of 163 pmol/day. In addition, we provide MW-corrected AIs for those nitrosamine drug substance related impurities (NDSRIs) for which EMA has pre-assigned AIs by read-across. Regulatory acceptance of this fundamental scientific tenet would allow one to derive nitrosamine limits for NDSRIs that both meet the health-protection goals and are technically feasible.

A Nitrite Excipient Database: A Useful Tool to Support N-Nitrosamine Risk Assessments for Drug Products.

N-Nitrosamine risk assessment and control have become an integral part of pharmaceutical drug product development and quality evaluation. Initial reports of nitrosamine contamination were linked with the drug substance and its manufacturing process. Subsequently, the drug product and aspects of the formulation process have shown to be relevant. Regarding specific formulation contributions to nitrosamine content in a product, one risk lies in possible interactions between nitrosating agents, derived from nitrite in excipients, and vulnerable amines, either present as moieties of the active molecule or as impurities / degradants. However, the limited validated information on nitrite levels in excipients available until now, has been an obstacle for scientists to assess the risk of nitrosamine formation in pharmaceutical products. This has driven the creation of a database to store and share such validated information. The database, maintained by Lhasa Limited, constitutes a central platform to hold the data donated by the pharmaceutical company members on the nitrite concentrations in common excipients measured with validated analytical procedures. The goal of this data sharing initiative is to provide a common framework to contextualize and estimate the risk posed by presence of nitrites to contribute to the formation of nitrosamines in drug products. The major findings from the database analyses are: (1) average nitrite content and batch to batch variance differ among excipients, (2) for solid dosage forms, the nitrite contribution is dominated by the highest formula % excipients, e.g., the fillers (diluents), which are typically used in larger proportion, and are characterized by low nitrite levels and low variability, leading to an average value of 1 µg/g nitrite in a typical formulation, (3) substantial differences in average nitrite content in batches from different excipient vendors potentially reflecting differences in source materials or processing methods for excipient manufacturing. That final point suggests that future selection of raw materials or processing by excipient manufacturers may help reduce nitrite levels in finished drug product formulations, and thus the overall risk of nitrosamine formation in cases where the product contains vulnerable amines.

The Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceuticals.

This article reports the outcome of an in silico analysis of more than 12,000 small molecule drugs and drug impurities, identifying the nitrosatable structures, assessing their potential to form nitrosamines under relevant conditions and the challenges to determine compound-specific AIs based on data available or read-across approaches for these nitrosamines and their acceptance by health authorities. Our data indicate that the presence of nitrosamines in pharmaceuticals is likely more prevalent than originally expected. In total, 40.4 % of the analyzed APIs and 29.6 % of the API impurities are potential nitrosamine precursors. Most structures identified through our workflow could form complex API-related nitrosamines, so-called nitrosamine drug substance related impurities (NDSRIs), although we also found structures that could release the well-known small and potent nitrosamines NDMA, NDEA, and others. Due to common structural motifs including secondary or tertiary amine moieties, whole essential drug classes such as beta blockers and ACE inhibitors are at risk. To avoid the risk of drug shortages or even the complete loss of therapeutic options, it will be essential that the well-established ICH M7 principles remain applicable for nitrosamines and that that the industry and regulatory authorities keep an open communication not only about the science but also to make sure there is a good balance between risk and benefit to patients.

Letter to the editor of Heliyon re: determination of dimethylamine and nitrite in pharmaceuticals by ion chromatography to assess the likelihood of nitrosamine formation Heliyon. 2021; 77: e06179.

Our letter to the editor of Heliyon outlines queries on the methodology and sample preparation used in article e06179, published in 2021. The nitrite measurements reported are higher than those observed in our experience. In the interest of reporting nitrite levels that are fully accurate, we would like to discuss the findings with the article authors.

Avoiding N-nitrosodimethylamine formation in metformin pharmaceuticals by limiting dimethylamine and nitrite.

Since late 2019, concerns regarding trace levels of the probable human carcinogen N-dimethylnitrosamine (NDMA) in Metformin-containing pharmaceuticals have been an issue if they exceeded the maximum allowable intake of 96 ng/day for a medicine with long-term intake. Here, we report results from an extensive analysis of NDMA content along the active pharmaceutical ingredient (API) manufacturing process as well as two different drug product manufacturing processes. Our findings confirm that Metformin API is not a significant source of NDMA found in Metformin pharmaceuticals and that NDMA is created at those steps of the drug product manufacturing that introduce heat and nitrite. We demonstrate that reduction of nitrite from excipients is an effective means to reduce NDMA in the drug product. Limiting residual dimethylamine in the API has proven to be another important factor for NDMA control as dimethylamine leads to formation of NDMA in the drug products. Furthermore, analysis of historical batches of drug products has shown that NDMA may increase during storage, but the levels reached were not shelf-life limiting for the products under study.

NDMA analytics in metformin products: Comparison of methods and pitfalls.

BACKGROUND: For nearly three years, the concerns regarding trace levels of N-nitrosamines in pharmaceuticals and the associated cancer risk have significantly expanded and are a major issue facing the global pharmaceutical industry. N-nitrosodimethylamine (NDMA) found in formulations of the popular anti-diabetic drug metformin is a prominent example. This has resulted in product recalls raising the profile within the media. Issues of method robustness, sample preparation and several unexpected sources of nitrosamine contamination have been highlighted as false positive risks. It has become apparent that the identification of the root causes of artefactual formation of nitrosamines must be identified to mitigate risk associated with the analysis. METHODS: A comparison study between four laboratories, across three companies was designed, employing orthogonal mass spectrometric methods for the quantification of NDMA in two metformin immediate release (IR) formulations and one extended release (XR) formulation. These were 2x LC-MS/MS, GC-MS/MS and GC-HRMS. RESULTS: Good agreement of results was obtained for the IR formulations. However, we measured higher concentrations of NDMA in the XR formulation using GC-MS/MS compared to LC-MS/MS. We could show that this was due to artefactual (in situ) formation of NDMA when samples were extracted with dichloromethane. Removal of dimethylamine (DMA) and nitrite from the extracted sample or the addition of a nitrosation scavenger are shown to be effective remedies. NDMA in situ formation was not observed in 10% MeOH or acetonitrile. CONCLUSION: Metformin pharmaceuticals contain traces of the API impurity DMA as well as inorganic nitrite from excipients. This can lead to artefactual formation of NDMA and hence false positive results if DCM is used for sample extraction. Similar artefacts are likely also in other pharmaceuticals if these contain the secondary amine precursor of the respective nitrosamine analyte.

Novel means of viral antigen identification: improved detection of avian influenza viruses by proximity ligation.

Recent outbreaks of avian influenza in different parts of the world have caused major economic losses for the poultry industry, affected wildlife seriously and present a significant threat even to human public health, due to the risk for zoonotic transmission. The ability to recognize avian influenza viruses (AIVs) early is of paramount importance to ensure that appropriate measures can be taken quickly to contain the outbreak. In this study, the performance of a proximity ligation assay (PLA) for the detection of AIV antigens in biological specimens was evaluated. It is shown that PLA: (i) as a novel principle of highly sensitive antigen detection is extending the arsenal of tools for the diagnosis of AIV; (ii) is very specific, nearly as sensitive as a commonly used reference real-time PCR assay, and four orders of magnitude more sensitive than a sandwich ELISA, utilizing the same antibody; (iii) avoids the necessity of nucleic acids extraction, which greatly facilitates high-throughput implementations; (iv) allows the use of inactivated samples, which safely can be transported from the field to diagnostic laboratories for further analysis. In summary, the results demonstrate that PLA is suited for rapid, accurate and early detection of AIV.

Multiplexed proximity ligation assays to profile putative plasma biomarkers relevant to pancreatic and ovarian cancer.

BACKGROUND: Sensitive methods are needed for biomarker discovery and validation. We tested one promising technology, multiplex proximity ligation assay (PLA), in a pilot study profiling plasma biomarkers in pancreatic and ovarian cancer. METHODS: We used 4 panels of 6- and 7-plex PLAs to detect biomarkers, with each assay consuming 1 microL plasma and using either matched monoclonal antibody pairs or single batches of polyclonal antibody. Protein analytes were converted to unique DNA amplicons by proximity ligation and subsequently detected by quantitative PCR. We profiled 18 pancreatic cancer cases and 19 controls and 19 ovarian cancer cases and 20 controls for the following proteins: a disintegrin and metalloprotease 8, CA-125, CA 19-9, carboxypeptidase A1, carcinoembryonic antigen, connective tissue growth factor, epidermal growth factor receptor, epithelial cell adhesion molecule, Her2, galectin-1, insulin-like growth factor 2, interleukin-1alpha, interleukin-7, mesothelin, macrophage migration inhibitory factor, osteopontin, secretory leukocyte peptidase inhibitor, tumor necrosis factor alpha, vascular endothelial growth factor, and chitinase 3-like 1. Probes for CA-125 were present in 3 of the multiplex panels. We measured plasma concentrations of the CA-125-mesothelin complex by use of a triple-specific PLA with 2 ligation events among 3 probes. RESULTS: The assays displayed consistent measurements of CA-125 independent of which other markers were simultaneously detected and showed good correlation with Luminex data. In comparison to literature reports, we achieved expected results for other putative markers. CONCLUSION: Multiplex PLA using either matched monoclonal antibodies or single batches of polyclonal antibody should prove useful for identifying and validating sets of putative disease biomarkers and finding multimarker panels.

In vitro analysis of DNA-protein interactions by proximity ligation.

Protein-binding DNA sequence elements encode a variety of regulated functions of genomes. Information about such elements is currently in a state of rapid growth, but improved methods are required to characterize the sequence specificity of DNA-binding proteins. We have established an in vitro method for specific and sensitive solution-phase analysis of interactions between proteins and nucleic acids in nuclear extracts, based on the proximity ligation assay. The reagent consumption is very low, and the excellent sensitivity of the assay enables analysis of as few as 1-10 cells. We show that our results are highly reproducible, quantitative, and in good agreement with both EMSA and predictions obtained by using a motif finding software. This assay can be a valuable tool to characterize in-depth the sequence specificity of DNA-binding proteins and to evaluate effects of polymorphisms in known transcription factor binding sites.

Patient-based cross-platform comparison of oligonucleotide microarray expression profiles.

The comparison of gene expression measurements obtained with different technical approaches is of substantial interest in order to clarify whether inter-platform differences may conceal biologically significant information. To address this concern, we analyzed gene expression in a set of head and neck squamous cell carcinoma patients, using both spotted oligonucleotide microarrays made from a large collection of 70-mer probes and commercial arrays produced by in situ synthesis of sets of multiple 25-mer oligonucleotides per gene. Expression measurements were compared for 4425 genes represented on both platforms, which revealed strong correlations between the corresponding data sets. Of note, a global tendency towards smaller absolute ratios was observed when using the 70-mer probes. Real-time quantitative reverse transcription PCR measurements were conducted to verify expression ratios for a subset of genes and achieved good agreement regarding both array platforms. In conclusion, similar profiles of relative gene expression were obtained using arrays of either single 70-mer or multiple short 25-mer oligonucleotide probes per gene. Although qualitative assessments of the expression of individual genes have to be made with caution, our results indicate that the comparison of gene expression profiles generated on these platforms will help to discover disease-related gene signatures in general.

Effective transcriptome amplification for expression profiling on sense-oriented oligonucleotide microarrays.

Gene expression analysis using microarrays of synthetic long oligonucleotides is limited in that it requires substantial amounts of RNA. To obtain these quantities from minute amounts of starting material, protocols were developed that linearly amplify mRNA by cDNA synthesis and in vitro transcription. Since orientation of the product is antisense (aRNA), it is inapplicable for dye-labelling by reverse transcription and hybridization to sense-oriented oligonucleotide arrays. Here, we introduce a novel protocol in which aRNA labelling is achieved by a combination of two reverse and one forward transcription reactions followed by dye-incorporation using Klenow fragment, generating fluorescent antisense cDNA. We demonstrate high fidelity in arrays using up to 10(5)-fold amplification, starting from 2 ng total RNA. The generated data are highly reproducible and maintain relative gene expression levels between samples. These results demonstrate that our protocol describes an efficient and reliable technique to expand the applicability of oligonucleotide arrays to studies where RNA is the limited source material.

Optimization of high-density cDNA-microarray protocols by 'design of experiments'.

Expression analysis using microarray technology implies a complex experimental procedure with a large number of parameters affecting the final result. We have demonstrated that optimization of such a complex protocol can be far better handled using design of experiments (DOE) than by working on a single parameter at a time. Based on the results of a screening design, we developed a spotting buffer composed of formamide, betaine and nitrocellulose. This buffer provides a 2-fold increase in signal-to-background ratio compared to 3x SSC. Comparison to seven other buffers tested on 10 different substrates revealed it had the highest sensitivity. DNA dissolved in this buffer can be spotted on epoxysilane-coated microscope slides at a density of up to 70 000 spots per slide. A second DOE approach characterized the RNA labeling process with regard to the concentration of fluorescent dyes, dNTPs and reverse transcriptase. Adjust ments of the concentrations of dNTPs, as well as reverse transcriptase, towards the optimum, produced an improvement in the performance of the labeling procedure by a factor of 3 (Cy3) and 10 (Cy5). These results demonstrate that the process of establishing a stable expression profiling protocol and its further optimization can be significantly shortened and improved by DOE.

Profile of gene expression induced by the tumour promotor TPA in murine epithelial cells.

Malignant transformation of mouse skin by chemical carcinogens and tumour promoters, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), is a multistage process that leads to squamous cell carcinoma (SCC) formation. In an effort to identify tumour-associated genes, we studied the influence of short-term TPA-treatment on the gene expression profile of murine skin. A comprehensive microarray with some 5,000 murine gene specific cDNA fragments was established and hybridised with pooled RNA derived from control and TPA-treated dorsal skin samples. Of these genes, 54 were up- and 35 were down-regulated upon TPA application. Additionally, we performed suppression subtractive hybridisation (SSH) with respective RNA pools to generate and analyse a cDNA library enriched for TPA-inducible genes. Expression data of selected genes were confirmed by quantitative real-time PCR and Northern blot analysis. Comparison of microarray and SSH data revealed that 26% of up-regulated genes identified by expression profiling matched with those present in the SSH library. Besides numerous known genes, we identified a large set of unknown cDNAs that represent previously unrecognised TPA-regulated genes in murine skin with potential function in tumour promotion. Additionally, some TPA-induced genes, such as Sprr1A, Saa3, JunB, Il4ralpha, Gp38, RalGDS and Slpi exhibit high basal level in advanced stages of skin carcinogenesis, suggesting that at least a subgroup of the identified TPA-regulated genes may contribute to tumour progression and metastasis.