Current Threat of Nitrosamines in Pharmaceuticals and Scientific Strategies for Risk Mitigation.

The current global situation of nitrosamine contamination has expanded from angiotensin-II receptor blockers (ARBs) to wide range of medicines as the risk of contamination via the drug substances, formulation, manufacturing process, and packaging is possible for many drug products. The understanding of chemistry, toxicology, and root causes of nitrosamines are mandatory to effectively evaluate and mitigate the risks associated with the contaminated mutagen. Lessons learnt and scientific findings from previously identified root causes are good examples on how to perform effective risk assessments and establish control strategies. Addressing the risk of nitrosamine contamination in pharmaceuticals requires significant knowledge and considerable resources to collect the necessary information for risk evaluation. Examples of the resources required include a reliable laboratory facility, reference material, highly specific and sensitive instrumentation able handle trace levels of contamination, data management, and the most limited resource - time. Therefore, the supporting tools to assist with risk assessment e.g., shared databases for drug and excipients in concern, screening models for the determination of nitrosamine formation potential, and an in silico model to help with toxicity estimation, have proven to be beneficial to tackle the risk and concern of nitrosamine contamination in pharmaceuticals.

Basis to Aid Crisis: Favipiravir Oral Solution for Hospital Compounding During COVID-19 Drug Shortage.

The COVID-19 pandemic outbreak has been overwhelming the healthcare system worldwide. A rapidly growing number of younger pediatric patients in Thailand necessitated the formulation of favipiravir, the most locally accessible antiviral agent against COVID-19, into a child-friendly dosage form as a safer alternative to a dispersion of crushed tablets in simple syrup. While striving to quickly develop a liquid formulation that is feasible for any local hospital production units, an oral solution was chosen due to its simplicity. Despite the large dose and poor aqueous solubility of favipiravir, a combination of pH control and use of poloxamer as a solubilizing agent has enabled us to streamline the manufacturing process of a 200 mg/15 mL oral solution for hospital compounding. To ensure its efficacy and safety, a specification for quality control was also established in accordance with the ICH quality guidelines and USP. The finished product stability was subsequently demonstrated under the conditions of 5°C ± 3°C, 25°C ± 2°C/75% RH ± 5% RH, 30°C ± 2°C/75% RH ± 5% RH, and 40°C ± 2°C/75% RH ± 5% RH. The results indicated that our formulation can be stored at 30°C ± 2°C/75% RH for 30 days, which will very well serve the need to allow drug distribution and patient use during the crisis, while the shelf-life can be extended to 60 days when stored at 5°C ± 3°C. Thus, accessibility to an essential medical treatment has been successfully enhanced for pediatric patients in Thailand and neighboring countries during the COVID-19 outbreak.

Nitrosamine Contamination in Pharmaceuticals: Threat, Impact, and Control.

Nitrosamine-contaminated medicinal products have raised safety concerns towards the use of various drugs, not only valsartan and all tetrazole-containing angiotensin II receptor blockers, but also ranitidine, metformin, and other medicines, many of which have been recalled and prone to shortage. At any stages, from drug substance synthesis throughout each product's lifetime, these impurities may evolve if an amine reacts with a nitrosating agent coexisting under appropriate conditions. Consequently, drug regulatory authorities worldwide have established stringent guidelines on nitrosamine contamination for all drug products in the market. This review encompasses various critical elements contributing to successful control measures against current and upcoming nitrosamine issues, ranging from accumulated knowledge of their toxicity concerns and potential root causes, precise risk evaluation, as well as suitable analytical techniques with sufficient sensitivity for impurity determination. With all these tools equipped, the impact of nitrosamine contamination in pharmaceuticals should be mitigated. An evaluation aid to tackle challenges in risk identification, as well as suitable industry-friendly analytical techniques to determine nitrosamines and other mutagenic impurities, are among unmet needs that will significantly simplify the risk assessment process.

5-Methylcytosine containing CG decamer as Z-DNA embedded sequence for a potential Z-DNA binding protein probe.

Attempting to elucidate biological significance of the left-handed Z-DNA is a research challenge due to Z-DNA potential role in many diseases. Discovery of Z-DNA binding proteins has ignited the interest in search for Z-DNA functions. Biosensor with Z-DNA forming probe can be useful to study the interaction between Z-DNA conformation and Z-DNA binding proteins. In this study, 5-methylcytosine (mC) containing CG decamers were characterized for their suitability to form Z-DNA and to be used in Z-DNA forming probe. The 5'-thiol oligonucleotide embedded with 5'-mCGmCGmCGmCGm CG-3' was designed and developed as a potential Z-DNA forming probe for Z-DNA binding protein screening.

Single-Molecule Manipulation of the Duplex Formation and Dissociation at the G-Quadruplex/i-Motif Site in the DNA Nanostructure.

We demonstrate the single-molecule operation and observation of the formation and resolution of double-stranded DNA (dsDNA) containing a G-quadruplex (GQ) forming and counterpart i-motif forming sequence in the DNA nanostructure. Sequential manipulation of DNA strands in the DNA frame was performed to prepare a topologically controlled GQ/i-motif dsDNA. Using strand displacement and the addition and removal of K(+), the topologically controlled GQ/i-motif dsDNA in the DNA frame was obtained in high yield. The dsDNA was resolved into the single-stranded DNA, GQ, and i-motif by the addition of K(+) and operation in acidic conditions. The dissociation of the dsDNA under the GQ and i-motif formation condition was monitored by high-speed atomic force microscopy. The results indicate that the dsDNA containing the GQ- and i-motif sequence is effectively dissolved when the duplex is helically loosened in the DNA nanoscaffold.

Real-time investigation of SV40 large T-antigen helicase activity using surface plasmon resonance.

The simian virus 40 (SV40) genome is a model system frequently employed for investigating eukaryotic replication. Large T-antigen (T-ag) is a viral protein responsible for unwinding the SV40 genome and recruiting necessary host factors prior to replication. In addition to duplex unwinding T-ag possesses G-quadruplex DNA helicase activity, the physiological consequence of which is unclear. However, formation of G-quadruplex DNA structures may be involved in genome maintenance and function, and helicase activity to resolve these structures may be necessary for efficient replication. We report the first real-time investigation of SV40 T-ag helicase activity using surface plasmon resonance (SPR). In the presence of ATP, T-ag was observed to bind to immobilized single-stranded DNA, forked duplex DNA, and the human telomeric foldover quadruplex DNA sequence. Inhibition of T-ag duplex helicase activity was observable in real-time and the intramolecular quadruplex was unwound.

Simian virus 40 large T-antigen G-quadruplex DNA helicase inhibition by G-quadruplex DNA-interactive agents.

On the basis of growing evidence for G-quadruplex DNA structures in genomic DNA and the presumed need to resolve these structures for DNA replication, the G-quadruplex DNA unwinding ability of a prototypical replicative helicase, SV40 large T-antigen (T-ag), was investigated. Here, we demonstrate that this G-quadruplex helicase activity is robust and comparable to the duplex helicase activity of T-ag. Analysis of the SV40 genome demonstrates the presence of sequences that may form intramolecular G-quadruplexes, which are the presumed natural substrates for the G-quadruplex helicase activity of T-ag. A number of G-quadruplex-interactive agents as well as new perylene diimide (PDI) derivatives have been investigated as inhibitors of both the G-quadruplex and the duplex DNA helicase activities of T-ag. A unique subset of these G-quadruplex-interactive agents inhibits the G-quadruplex DNA unwinding activity of T-ag, relative to those reported to inhibit G-quadruplex DNA unwinding by RecQ-family helicases. We also find that certain PDIs are both potent and selective inhibitors of the G-quadruplex DNA helicase activity of T-ag. Surface plasmon resonance and fluorescence spectroscopic G-quadruplex DNA binding studies of these T-ag G-quadruplex helicase inhibitors have been carried out, demonstrating the importance of attributes in addition to binding affinity for G-quadruplex DNA that may be important for inhibition. The identification of potent and selective inhibitors of the G-quadruplex helicase activity of T-ag provides tools for probing the specific role of this activity in SV40 replication.

Identification of the adduct between a 4-Aza-3-ene-1,6-diyne and DNA using electrospray ionization mass spectrometry.

The interactions between a novel enediyne [1-methyl-2-(phenylethynyl)-3-(3-phenylprop-2-ynyl)-3H-benzimidazolium] (1) and various cytosine-containing oligonucleotides were studied using electrospray ionization mass spectrometry (ESI-MS) in a flow injection analysis mode useful for small volumes. This enediyne ligand, developed as a potential alternative to the highly cytotoxic natural enediynes, some of which have been successfully used as anti-tumor agents, has previously been shown to interact with DNA through frank strand scission as well as via the formation of adducts that lead to 2'-deoxycytidine-specific cleavage. Through ESI-MS, the structures of these adducts were examined and a sequence dependence of the 2'-deoxycytidine-specific cleavage was noted. Collisionally activated dissociation of the observed adducts confirmed the strength of the interactions between the enediyne and DNA and supports a direct linkage between the enediyne and the cytosine nucleobase, likely the result of a nucleophilic attack of the phenylethynyl group by the cytosine amine.

2-Alkynyl-N-propargyl pyridinium salts: pyridinium-based heterocyclic skipped aza-enediynes that cleave DNA by deoxyribosyl hydrogen-atom abstraction and guanine oxidation.

Diradical-generating cyclizations such as the enediyne Bergman cyclization and the enyne allene Myers-Saito cyclization have been exploited by nature in the mechanism of DNA cleavage by a series of potent antitumor antibiotics. Alternative diradical-generating cyclizations have been proposed in the design of selective antitumor agents; however, little information is available concerning the utility of these alternative cyclizations in radical-based DNA cleavage chemistry. One such alternative diradical-generating cyclization, the aza-Myers-Saito cyclization of aza-enyne allenes that are derived from base-promoted isomerization of skipped aza-enediynes, has been recently reported. Here, we report the synthesis and DNA cleavage chemistry of a series of pyridinium skipped aza-enediynes (2-alkynyl-N-propargyl pyridinium salts). Efficient DNA cleavage requires the presence of the skipped aza-enediyne functionality, and optimal DNA cleavage occurs at basic pH. Within this series of compounds, the analogue bearing a p-methoxyphenyl group on the pyridinium 2-alkyne substituents was found to be the most effective DNA cleavage agent, displaying significant supercoiled DNA-nicking activity at concentrations as low as 1 microM. Detailed studies of this analogue show that DNA cleavage occurs through 4'-hydrogen-atom abstraction from the DNA backbone and oxidation of guanine bases. This is the first report of enediyne-like radical-based DNA cleavage by an agent designed to undergo an alternative diradical-generating cyclization.

Duplex and quadruplex DNA binding and photocleavage by trioxatriangulenium ion.

The stable trioxatriangulenium ion (TOTA) has previously been shown to bind to and photooxidize duplex DNA, leading to cleavage at G residues, particularly 5'-GG-3' repeats. Telomeric DNA consists of G-rich sequences that may exist in either duplex or G-quadruplex forms. We have employed electrospray ionization mass spectrometry (ESI-MS) to investigate the interactions between TOTA and duplex DNA or G-quadruplex DNA. A variety of duplex decamer oligodeoxynucleotides form complexes with TOTA that can be detected by ESI-MS, and the stoichiometry and fragmentation patterns observed are commensurate with an intercalative binding mode. TOTA also forms complexes with four-stranded and hairpin-dimer G-quadruplex oligodeoxynucleotides that can be detected by ESI-MS. Both the stoichiometry and the fragmentation patterns observed by ESI-MS are different than those observed for G-tetrad end-stacking binding ligands. We have carried out (1)H NMR titrations of a four-stranded G-quadruplex in the presence of TOTA. Addition of up to 1 equiv of TOTA is accompanied by pronounced upfield shifts of the G-tetrad imino proton resonances in the NMR, which is similar to the effect observed for G-tetrad end-stacking ligands. At higher ratios of added TOTA, there is evidence for additional binding modes. Duplex DNA containing either human telomeric repeats (T(2)AG(3))(4) or the Tetrahymena telomeric repeats (T(2)G(4))(4) are readily photooxidized by TOTA, the major sites of oxidation being the central guanine residues in each telomeric repeat. These telomeric repeats were incorporated into duplex/quadruplex chimeras in which the repeats adopt a G-quadruplex structure. Analysis by denaturing polyacrylamide gel electrophoresis reveals significantly less TOTA photocleavage of these quadruplex telomeric repeats when compared to the duplex repeats.