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.

Epigenetic TET-Catalyzed Oxidative Products of 5-Methylcytosine Impede Z-DNA Formation of CG Decamers.

Methylation of cytosine has been known to play a significant role in epigenetic regulation. 5-Methylcytosine was among the first base modification that was discovered for the capability to facilitate B/Z-DNA transition as observed in CG repeated tracks. A study on gene repression by Z-DNA prone sequence as in ADAM-12 has ignited our research interest for the Z-DNA role in epigenetics. Ten eleven translocation family proteins are responsible to catalyze 5-methylcytosine to produce oxidative products including 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine, which each may have unique function rather than the sole purpose of 5-methylcytosine clearance. Although the Z-DNA-promoting effect of 5-methylcytosine was well established, the effect of its oxidative products on Z-DNA remain unknown. In this study, the Z-DNA-promoting effect of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine on the CG decamer model were investigated along with known Z-DNA stabilizers, 5-methylcytosine and 8-oxoguanine. Experimental results from circular dichroism (CD) and NMR indicates that all oxidative products of 5-methylcytosine hinder B/Z-DNA transition as high salt concentration suitable to stabilize and convert unmodified CG decamer to Z-DNA conformation is insufficient to facilitate the B/Z-DNA transition of CG decamer containing 5-hydroxymethylcytosine, 5-formylcytosine, or 5-carboxycytosine. Molecular dynamic simulation and free energy calculation by MM-PBSA are in agreement with the experimental finding that 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine destabilize Z-DNA conformation of CG decamer, in contrast to its precursor. Investigation of Z-DNA switch-on/switch-off regulated by 5-methylcytosine and its oxidative products is a further step to elucidate the potential of epigenetic regulated via Z-DNA.

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.

Pharmaceutical Equivalence of Distributed Generic Antiretroviral (ARV) in Asian Settings: The Cross-Sectional Surveillance Study - PEDA Study.

OBJECTIVES: Ensuring that medicines meet quality standards is mandatory for ensuring safety and efficacy. There have been occasional reports of substandard generic medicines, especially in resource-limiting settings where policies to control quality may be less rigorous. As HIV treatment in Thailand depends mostly on affordable generic antiretrovirals (ARV), we performed quality assurance testing of several generic ARV available from different sources in Thailand and a source from Vietnam. METHODS: We sampled Tenofovir 300mg, Efavirenz 600mg and Lopinavir/ritonavir 200/50mg from 10 primary hospitals randomly selected from those participating in the National AIDS Program, 2 non-government organization ARV clinics, and 3 private drug stores. Quality of ARV was analyzed by blinded investigators at the Faculty of Pharmaceutical Science, Chulalongkorn University. The analysis included an identification test for drug molecules, a chemical composition assay to quantitate the active ingredients, a uniformity of mass test and a dissolution test to assess in-vitro drug release. Comparisons were made against the standards described in the WHO international pharmacopeia. RESULTS: A total of 42 batches of ARV from 15 sources were sampled from January-March 2015. Among those generics, 23, 17, 1, and 1 were Thai-made, Indian-made, Vietnamese-made and Chinese-made, respectively. All sampled products, regardless of manufacturers or sources, met the International Pharmacopeia standards for composition assay, mass uniformity and dissolution. Although local regulations restrict ARV supply to hospitals and clinics, samples of ARV could be bought from private drug stores even without formal prescription. CONCLUSION: Sampled generic ARVs distributed within Thailand and 1 Vietnamese pharmacy showed consistent quality. However some products were illegally supplied without prescription, highlighting the importance of dispensing ARV for treatment or prevention in facilities where continuity along the HIV treatment and care cascade is available.

Single C8-Arylguanine modifications render oligonucleotides in the Z-DNA conformation under physiological conditions.

Z-DNA is the only DNA conformation that has a left-handed helical twist. Although Z-DNA has been implicated in both carcinogenesis and mutagenesis, its specific biological role remains uncertain. We have demonstrated that the formation of C8-arylguanine DNA adducts, derived from arylhydrazines, shifts the B/Z-DNA equilibrium toward the Z-DNA conformation in d(CG)5 sequences. However, our previous work examined the effect of two adducts in the duplex, and it was unclear whether the two base modifications were working together to cause the equilibrium shift toward the Z-DNA conformation. Here we report the synthesis and characterization of a hairpin oligonucleotide sequence (d(CG)5T4(CG)5) containing only one C8-arylguanine modified base. The unmodified hairpin and the previously studied unmodified double-stranded oligonucleotide were conformationally similar, and each required ∼3 M NaCl to yield a B-/Z-DNA ratio of 1:1. The introduction of a single C8-arylguanine modification significantly reduced the NaCl concentration needed to produce a 1:1 B-/Z-DNA ratio in the hairpin. Further, the addition of MgCl2 and spermine to the C8-arylguanine-modified hairpin shifts the B/Z-DNA equilibrium such that the Z form predominated under physiological conditions. NMR and molecular modeling indicated the conformational effects produced by the C8-arylguanine modification occurred locally at the site of modification while CD data demonstrated that the C8-arylguanine-modified base destabilized the B form. Additionally, our data show that adopting the Z-DNA conformation is preferred over denaturation to the single-stranded form. Finally, the conformational effects of the C8-arylguanine modifications were not additive and the introduction of any such modifications drive Z-DNA formation under physiological conditions, which may provide a novel carcinogenesis mechanism where DNA adducts confer their carcinogenicity through a Z-DNA-mediated mechanism.

The synthesis of C8-aryl purines, nucleosides and phosphoramidites.

C8-Aryl purines, their nucleosides, and phosphoramidites has been synthetic targets for more than 60 years. Interest in these compounds stems from their utility as fluorescent markers, they have therapeutic uses, are biomarkers, biomolecular probes, supramolecular building blocks, and for conformational studies. Until recently, the selective arylation of the C8-position of purines has been a challenging task. Several approaches have been explored including building them up from a pyrimidine or selective C8-modification of an unsubstituted purine. Neither of these approaches has proven to have broad scope. The discovery that C8-aryl purine nucleosides can be made via the Suzuki cross-coupling reaction has allowed a diverse array of analogues to be prepared and, in turn, the corresponding phosphoramidites. The latter is particularly significant as C8-aryl purine adducts are a major mutation observed from aromatic carcinogens and ready access to C8-aryl phosphoramidites will facilitate the synthesis and study of C8-aryl purine biomarkers and modified oligonucleotides.

The conformational effect of para-substituted C8-arylguanine adducts on the B/Z-DNA equilibrium.

The B form of DNA exists in equilibrium with the Z form and is mainly affected by sequence, electrostatic interactions, and steric effects. C8-purine substitution shifts the equilibrium toward the Z form though how this interaction overcomes the unfavorable electrostatic interactions and decrease in stacking in the Z form has not been determined. Here, a series of C8-arylguanine derivatives, bearing a para-substituent were prepared and the B/Z equilibrium determined. B/Z ratios were measured by CD and conformational effects of the aryl substitution determined by NMR spectroscopy and molecular modeling. The para-substituent was found to have a significant effect on the B/Z DNA equilibrium caused by altering base-pair stacking of the B form and modifying the hydration/ion shell of the B form. A unique melting temperature versus salt concentration was observed and provides evidence relevant to the mechanism of B/Z conformational interconversion.

A general synthesis of C8-arylpurine phosphoramidites.

A general scheme for the synthesis of C8-arylpurine phosphoramidites has been developed. C8-Arylation of C8-bromo-2'-deoxyguanosine is the key step and has been achieved through the use of a Suzuki coupling. Since the coupling reaction is conducted under aqueous conditions, it is unnecessary to protect and then deprotect the hydroxyl groups, thus saving several steps and improving overall yields. Once the C8-arylgroup is introduced, the glycosidic bond becomes very sensitive to acid catalyzed cleavage. Protection of the amino groups as the corresponding N,N-dimethylformamidine derivative improves stability of the derivatives. Synthetic C8-arylpurines were successfully used to prepare synthetic oligonucleotides.