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.

The Cardiomyocyte RNA-Binding Proteome: Links to Intermediary Metabolism and Heart Disease.

RNA functions through the dynamic formation of complexes with RNA-binding proteins (RBPs) in all clades of life. We determined the RBP repertoire of beating cardiomyocytic HL-1 cells by jointly employing two in vivo proteomic methods, mRNA interactome capture and RBDmap. Together, these yielded 1,148 RBPs, 391 of which are shared with all other available mammalian RBP repertoires, while 393 are thus far unique to cardiomyocytes. RBDmap further identified 568 regions of RNA contact within 368 RBPs. The cardiomyocyte mRNA interactome composition reflects their unique biology. Proteins with roles in cardiovascular physiology or disease, mitochondrial function, and intermediary metabolism are all highly represented. Notably, we identified 73 metabolic enzymes as RBPs. RNA-enzyme contacts frequently involve Rossmann fold domains with examples in evidence of both, mutual exclusivity of, or compatibility between RNA binding and enzymatic function. Our findings raise the prospect of previously hidden RNA-mediated regulatory interactions among cardiomyocyte gene expression, physiology, and metabolism.

Identification of Maturation-Specific Proteins by Single-Cell Proteomics of Human Oocytes.

Oocytes undergo a range of complex processes via oogenesis, maturation, fertilization, and early embryonic development, eventually giving rise to a fully functioning organism. To understand proteome composition and diversity during maturation of human oocytes, here we have addressed crucial aspects of oocyte collection and proteome analysis, resulting in the first proteome and secretome maps of human oocytes. Starting from 100 oocytes collected via a novel serum-free hanging drop culture system, we identified 2,154 proteins, whose function indicate that oocytes are largely resting cells with a proteome that is tailored for homeostasis, cellular attachment, and interaction with its environment via secretory factors. In addition, we have identified 158 oocyte-enriched proteins (such as ECAT1, PIWIL3, NLRP7)(1) not observed in high-coverage proteomics studies of other human cell lines or tissues. Exploiting SP3, a novel technology for proteomic sample preparation using magnetic beads, we scaled down proteome analysis to single cells. Despite the low protein content of only ∼100 ng per cell, we consistently identified ∼450 proteins from individual oocytes. When comparing individual oocytes at the germinal vesicle (GV) and metaphase II (MII) stage, we found that the Tudor and KH domain-containing protein (TDRKH) is preferentially expressed in immature oocytes, while Wee2, PCNA, and DNMT1 were enriched in mature cells, collectively indicating that maintenance of genome integrity is crucial during oocyte maturation. This study demonstrates that an innovative proteomics workflow facilitates analysis of single human oocytes to investigate human oocyte biology and preimplantation development. The approach presented here paves the way for quantitative proteomics in other quantity-limited tissues and cell types. Data associated with this study are available via ProteomeXchange with identifier PXD004142.

Proteomic Analysis Reveals Branch-specific Regulation of the Unfolded Protein Response by Nonsense-mediated mRNA Decay.

Nonsense-mediated mRNA decay (NMD) has originally been described as a surveillance mechanism to inhibit the expression of mRNAs with truncated open reading frames (ORFs) and to contribute to the fidelity of gene expression. It is now recognized that NMD also controls the expression of physiological genes with "intact" mRNA. Stress can decrease NMD efficiency and thus increase the mRNA levels of physiological NMD targets. As stress can also inhibit translation, the net outcome for shaping the proteome is difficult to predict. We have thus analyzed de novo protein synthesis in response to NMD inhibition or the induction of mild endoplasmic reticulum (ER) stress by treatment of cells with the reducing agent dithiotreitol (DTT). For this purpose, we combined pulsed azidohomoalanine (AHA) and stable isotope labeling by amino acids in cell culture (SILAC). Labeled proteins were purified by click chemistry-based covalent coupling to agarose beads, trypsinized, fractionated, and analyzed by mass spectrometry (MS). We find that mild ER stress up-regulates the de novo synthesis of components of all three branches of the unfolded protein response (PERK, IRE1 and ATF6) without increasing eIF2α phosphorylation or impairing of protein translation. In contrast, inhibition of NMD induces de novo protein synthesis of downstream targets of the PERK and IRE1 pathways, whereas we could not detect regulation of ATF6-responsive genes. These data thus support a model that implicates a positive feedback loop of ER stress inhibiting NMD efficiency which further promotes the ER stress response in a branch-specific manner.

Crystal Structure of the CTP1L Endolysin Reveals How Its Activity Is Regulated by a Secondary Translation Product.

Bacteriophages produce endolysins, which lyse the bacterial host cell to release newly produced virions. The timing of lysis is regulated and is thought to involve the activation of a molecular switch. We present a crystal structure of the activated endolysin CTP1L that targets Clostridium tyrobutyricum, consisting of a complex between the full-length protein and an N-terminally truncated C-terminal cell wall binding domain (CBD). The truncated CBD is produced through an internal translation start site within the endolysin gene. Mutants affecting the internal translation site change the oligomeric state of the endolysin and reduce lytic activity. The activity can be modulated by reconstitution of the full-length endolysin-CBD complex with free CBD. The same oligomerization mechanism applies to the CD27L endolysin that targets Clostridium difficile and the CS74L endolysin that targets Clostridium sporogenes. When the CTP1L endolysin gene is introduced into the commensal bacterium Lactococcus lactis, the truncated CBD is also produced, showing that the alternative start codon can be used in other bacterial species. The identification of a translational switch affecting oligomerization presented here has implications for the design of effective endolysins for the treatment of bacterial infections.

5-azacytidine inhibits nonsense-mediated decay in a MYC-dependent fashion.

Nonsense-mediated RNA decay (NMD) is an RNA-based quality control mechanism that eliminates transcripts bearing premature translation termination codons (PTC). Approximately, one-third of all inherited disorders and some forms of cancer are caused by nonsense or frame shift mutations that introduce PTCs, and NMD can modulate the clinical phenotype of these diseases. 5-azacytidine is an analogue of the naturally occurring pyrimidine nucleoside cytidine, which is approved for the treatment of myelodysplastic syndrome and myeloid leukemia. Here, we reveal that 5-azacytidine inhibits NMD in a dose-dependent fashion specifically upregulating the expression of both PTC-containing mutant and cellular NMD targets. Moreover, this activity of 5-azacytidine depends on the induction of MYC expression, thus providing a link between the effect of this drug and one of the key cellular pathways that are known to affect NMD activity. Furthermore, the effective concentration of 5-azacytidine in cells corresponds to drug levels used in patients, qualifying 5-azacytidine as a candidate drug that could potentially be repurposed for the treatment of Mendelian and acquired genetic diseases that are caused by PTC mutations.

Arginine deficiency leads to impaired cofilin dephosphorylation in activated human T lymphocytes.

The amino acid arginine is fundamentally involved in the regulation of the immune response during infection, inflammatory diseases and tumor growth. Arginine deficiency (e.g. due to the myeloid cell enzyme arginase) inhibits proliferation and effector functions of activated T lymphocytes. Here, we studied intracellular mechanisms mediating this suppression of human T lymphocytes. Our proteomic analysis revealed an impaired dephosphorylation of the actin-binding protein cofilin upon T-cell activation in the absence of arginine. We show that this correlates with alteration of actin polymerization and impaired accumulation of CD2 and CD3 in the evolving immunological synapse in T cell-antigen presenting cells conjugates. In contrast, T-cell cytokine synthesis is differentially regulated in human T lymphocytes in the absence of arginine. While the production of certain cytokines (e.g. IFN-γ) is severely reduced, T lymphocytes produce other cytokines (e.g. IL-2) independent of extracellular arginine. MEK and PI3K activity are reciprocally regulated in association with impaired cofilin dephosphorylation. Finally, we show that impaired cofilin dephosphorylation is also detectable in human T cells activated in a granulocyte-dominated purulent micromilieu due to arginase-mediated arginine depletion. Our novel results identify cofilin as a potential regulator of human T-cell activation under conditions of inflammatory arginine deficiency.

Madin-Darby canine kidney cells are increased in aerobic glycolysis when cultured on flat and stiff collagen-coated surfaces rather than in physiological 3-D cultures.

We investigate the influence of the dimensionality and the biochemistry of the culture system on the cellular functionality by analyzing the protein expression levels in Madin-Darby canine kidney (MDCK) cells grown in 3-D and 2-D substrates. We cultured MDCK cells on a hard and flat 2-D uncoated plastic surface, on a 2-D collagen-coated plastic surface and in 3-D collagen gel and employed 2-D gel electrophoresis, MALDI-TOF-MS, and LC-MS/MS analysis to identify the differentially regulated proteins. We found significant differences in the expression of antioxidant proteins, actin-binding proteins, glycolytic enzymes, and heat-shock proteins/chaperons among the three types of cultures. While MDCK cells cultured in 3-D collagen up-regulate antioxidant proteins and proteins involved in the dynamic remodeling of the actin cytoskeleton, 2-D collagen-coated plastic surfaces induce the up-regulation of glycolytic enzymes. Our data shows that the culture conditions have profound effects on the physiology of the cell. Culture in 3-D collagen induces a differentiated polarized phenotype. In contrast, collagen-coated 2-D substrates favor a tumor-like phenotype with increased glycolysis. Thus, the suitability of 2-D cultures to study the physiological behavior of cells, especially in drug discovery, bioprocessing, and toxicology, should be carefully reconsidered.

Molecular variability of FLT3/ITD mutants and their impact on the differentiation program of 32D cells: implications for the biological properties of AML blasts.

FLT3 is the most frequently mutated gene in acute myeloid leukemia (AML), with internal tandem duplications (ITDs) accounting for up to 30% of its mutations. To analyze the impact of individual ITDs on the expression profile of immature myeloid cells, we have established 32D cell lines expressing nine different FLT3/ITDs isolated from AML patients and subjected them to whole genome expression profiling and 2DE/LC/MS proteomics. Our data indicate that in comparison to the controls, FLT3/ITD-positive 32D cells exhibit less mature expression profiles resembling early hematopoietic progenitors. Moreover, our results suggest that there exist biological differences among individual ITD variants.

NagA-dependent uptake of N-acetyl-glucosamine and N-acetyl-chitin oligosaccharides across the outer membrane of Caulobacter crescentus.

Among the 67 predicted TonB-dependent outer membrane transporters of Caulobacter crescentus, NagA was found to be essential for growth on N-acetyl-beta-D-glucosamine (GlcNAc) and larger chitin oligosaccharides. NagA (93 kDa) has a predicted typical domain structure of an outer membrane transport protein: a signal sequence, the TonB box EQVVIT, a hatch domain of 147 residues, and a beta-barrel composed of 22 antiparallel beta-strands linked by large surface loops and very short periplasmic turns. Mutations in tonB1 and exbBD, known to be required for maltose transport via MalA in C. crescentus, and in two additional predicted tonB genes (open reading frames cc2327 and cc3508) did not affect NagA-mediated GlcNAc uptake. nagA is located in a gene cluster that encodes a predicted PTS sugar transport system and two enzymes that convert GlcNAc-6-P to fructose-6-P. Since a nagA insertion mutant did not grow on and transport GlcNAc, diffusion of GlcNAc through unspecific porins in the outer membrane is excluded. Uptake of GlcNAc into tonB and exbBD mutants and reduction but not abolishment of GlcNAc transport by agents which dissipate the electrochemical potential of the cytoplasmic membrane (0.1 mM carbonyl cyanide 3-chlorophenylhydrazone and 1 mM 2,4-dinitrophenol) suggest diffusion of GlcNAc through a permanently open pore of NagA. Growth on (GlcNAc)(3) and (GlcNAc)(5) requires ExbB and ExbD, indicating energy-coupled transport by NagA. We propose that NagA forms a small pore through which GlcNAc specifically diffuses into the periplasm and functions as an energy-coupled transporter for the larger chitin oligosaccharides.

Comparative 2-DE protein analysis in a 3-D geometry gel.

Two-dimensional gel electrophoresis (2-DE) separation has not been considered suitable for large-scale comparative protein expression studies due to its limited throughput. We present a high-throughput analysis method based on three-dimensional (3-D) geometry gel electrophoresis. Following conventional isoelectric focusing (IEF), up to 36 immobilized pH gradient (IPG) strips are arrayed on the top surface of a 3-D gel body, and the samples transferred electrokinetically to the gel. A specific thermal management ensures that sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) occurs under identical electrophoretic and thermal conditions, avoiding gel-to-gel variations and thereby providing immediate comparability of the separation patterns. Proteins are Cy3-labeled for online detection of laser-induced fluorescence (LIF). Images are acquired by a digital camera and recorded as a 3-D image stack during electrophoresis. Image processing software decomposes the 3-D image stack into vertical sections representing conventional 2-DE slab gels, making results immediately accessible without further gel processing. The large number of simultaneously analyzed samples (n = 36) allows treating the sample index as a quasi-continuous experimental parameter (e.g., concentration, time, dose). The method offers a wide range of applications in molecular discovery, clinical diagnosis, pharmacology, and toxicology, like protein monitoring during disease development and screening of drug candidates for their effect on protein expression.

Comparative proteome analysis of Staphylococcus aureus biofilm and planktonic cells and correlation with transcriptome profiling.

Pathogenic staphylococci can form biofilms in which they show a higher resistance to antibiotics and the immune defense system than their planktonic counterparts, which suggests that the cells in a biofilm have an altered metabolic activity. Here, 2-D PAGE was used to identify secreted, cell wall-associated and cytoplasmic proteins expressed in Staphylococcus aureus after 8 and 48 h of growth. The proteins were separated at pH ranges of 4-7 or 6-11. The protein patterns revealed significant differences in 427 protein spots; from these, 258 non-redundant proteins were identified using ESI-MS/MS. Biofilm cells expressed higher levels of proteins associated with cell attachment and peptidoglycan synthesis, and in particular fibrinogen-binding proteins. Enzymes involved in pyruvate and formate metabolism were upregulated. Furthermore, biofilm cells expressed more staphylococcal accessory regulator A protein (SarA), which corroborates the positive effect of SarA on the expression of the intercellular adhesion operon ica and biofilm growth. In contrast, proteins, such as proteases and particularly immunodominant antigen A (IsaA) and staphylococcal secretory antigen (SsaA), were found in lower amounts. The RNA expression profiling largely supports the proteomic data. The results were mapped onto KEGG pathways.