Universal ion chromatography method for anions in active pharmaceutical ingredients enabled by computer-assisted separation modeling.

Ion Chromatography (IC) is one of the most widely used methods for analyzing ionic species in pharmaceutical samples. A universal IC method that can separate a wide range of different analytes is highly desired as it can save a lot of time for method development and validation processes. Herein we report the development of a universal method for anions in active pharmaceutical ingredients (APIs) using computer-assisted chromatography modeling tools. We have screened three different IC columns (Dionex IonPac AS28-Fast 4 µm, AS19 4 µm and AS11-HC 4 µm) to determine the best suitable column for universal IC method development. A universal IC method was then developed using an AS11-HC 4 µm column to separate 31 most common anionic substances in 36 mins. This method was optimized using LC Simulator and a model which precisely predicts the retention behavior of 31 anions was established. This model demonstrated an excellent match between predicted and experimental analyte retention time (R2 =0.999). To validate this universal IC method, we have studied the stability of sulfite and sulfide analytes in ambient conditions. The method was then validated for a subset of 29 anions using water and organic solvent/water binary solvents as diluents for commercial APIs. This universal IC method provides an efficient and simple way to separate and analyze common anions in APIs. In addition, the method development process combined with LC simulator modeling can be effectively used as a starting point during method development for other ions beyond those investigated in this study.

Development of a Robust Manufacturing Route for Molnupiravir, an Antiviral for the Treatment of COVID-19.

Herein is described the development of a large-scale manufacturing process for molnupiravir, an orally dosed antiviral that was recently demonstrated to be efficacious for the treatment of patients with COVID-19. The yield, robustness, and efficiency of each of the five steps were improved, ultimately culminating in a 1.6-fold improvement in overall yield and a dramatic increase in the overall throughput compared to the baseline process.

Description of the cytotoxic effect of a novel drug Abietyl-Isothiocyanate on endometrial cancer cell lines.

The objective of the present study was to determine the in-vitro effect of Abietyl-Isothiocyanate (ABITC), a representative of a new class of anti-cancer drugs, on endometrial cancer (EC) cell lines. ABITC at concentrations ≥1 µM displayed dose-dependent and selective cytotoxicity to EC cell lines (ECC-1, AN3CA, RL95-2) in comparison to other cancer cell lines. After treatment with ABITC, ECC-1 unlike control cells displayed hallmark features of apoptosis including chromatin condensation and nuclear fragmentation. At concentrations below the IC50, ABITC exerted anti-proliferative effects by blocking cell-cycle progression through G0/G1 and S-phase. In addition, cells attempted to counteract drug treatment by pro-survival signaling such as deactivation of JNK/SAPK and p38 MAPK and activation of AKT and ErK1/2. ABITC also altered EGF-receptor phosphorylation. At a concentration of 5 µM ABITC generated an excess amount of reactive oxygen species (ROS) and displayed pro-apoptotic signaling such as activation of caspase-8, JNK-SAPK and deactivation of PARP-1. Co-treatment with an antioxidant blocked the drug effects by reducing ROS generation, cytotoxicity and pro-apoptotic signaling. In summary, novel isothiocyanate ABITC is an anti-proliferative and selectively cytotoxic drug to EC cells in-vitro. Key mechanisms during cell death are predominantly correlated to excess generation of ROS. We suggest the further development of ABITC as a potential therapeutic by studying the drug efficacy in EC in-vivo models.

Nucleolar binding of an anti-NMDA receptor antibody in hydra: a non-canonical role for an NMDA receptor protein?

Two emerging concepts in cell biology are the back-and-forth trafficking of receptor proteins and nuclear transcription factors between the nucleus and the cell membrane, and the alternative splicing of messenger RNA to produce similar proteins targeted to different cell sites. Recent evidence suggests that the nucleolus is a dynamic structure whose components may be involved in both types of trafficking. In the nervous system of higher animals, the N-methyl-D-aspartate (NMDA)-specific glutamate receptor has various roles in development and cell communication. It is involved in learning, memory, axonal guidance and nerve regeneration. We have reported earlier that the NR1 subunit of the NMDA receptor is present in the cell periphery and the nucleus of stem cells, neurons and epitheliomuscular cells of the early-evolved cnidarian, Hydra vulgaris (Scappaticci et al., 2004. Cell Tissue Res 316:263-270); it is involved in coordinating hydra's neuroeffector systems (Kass-Simon and Scappaticci, 2003. Hydrobiologia 530/531:67-71; Pierobon et al., 2004. Eur J Neurosci 20:2598-2604; Scappaticci and Kass-Simon, 2008. Comp Biochem Physiol A 150:415-422; Kay and Kass-Simon, 2009. Bio Bull 216:113-129). Here we report immunocytochemical experiments, using a mouse monoclonal antibody raised against the mammalian NR1 receptor subunit, and an in silico genomic and gene expression analysis identifying the homologues in hydra of mammalian NR1 and fibrillarin (FBL) genes, and their expressed proteins. The experiments reveal that the NR1 antibody specifically labels the nucleoli of large and small interstitial cells (stem cells), nematoblasts, neuroblasts, and epitheliomusclar cells; antibody labeling of the nucleolar marker, FBL, confirms the nucleolar localization of NR1 antibody labeling. Genomic analysis reveals that NR1 and FBL genes are conserved in hydra, and suggests that there are at least two NR1 splice variants, one of which contains both nuclear and nucleolar targeting signals. The finding that an NR1 receptor subunit (or a portion of it) appears in nucleoli of hydra cells is unique, and has not been reported for any other organism. Its presence in nucleoli of hydra may signal the existence of a yet-undescribed shuttle mechanism between the cell surface and the nucleous, or the alternative deployment of NR1 splice variants to different cell sites.

Cis-regulatory organization of the Pax6 gene in the ascidian Ciona intestinalis.

The Pax6 gene has attracted intense research interest due to its apparently important role in the development of eyes and the central nervous system (CNS) in many animal groups. Pax6 is also of interest for comparative genomics since it has not been duplicated in tetrapods, making for a direct orthology between the Ciona intestinalis gene CiPax6 and Pax6 in mammals. CiPax6 has been shown to be expressed in the anterior brain, caudal nerve cord, and in parts of the brain associated with the photoreceptive ocellus. This information was extended here using in-situ hybridization, and shows that CiPax6 transcripts mark the lateral regions of the nerve cord, remarkably similar to Pax6 expression in the mouse. As a means of dissecting the cis-regulation of CiPax6 we tested 8 kb of sequence using transient reporter transgene assays. Three separate regions were found that work together to drive the overall CiPax6 expression pattern. A 211 bp sequence 2 kb upstream of the first exon was found to be a major enhancer driving expression in the sensory vesicle (the anterior portion of the ascidian brain). Other upstream sequences were shown to work with the sensory vesicle enhancer to drive expression in the remainder of the CNS. An "eye enhancer" was localized to the first intron, which controls specific expression in the central portion of the sensory vesicle, including photoreceptor cells. The fourth intron was found to repress ectopic expression of the reporter gene in middle portions of the embryonic brain. Aspects of this overall regulatory organization are similar to the organization of the Pax6 homologs in mice and Drosophila, particularly the presence of intronic elements driving expression in the eye, brain and nerve cord.