Development and Validation of a Novel Stability Indicating Reverse Phase High Performance Liquid Chromatographic Method for Related Substances and Assay Analysis of Molnupiravir Drug Substance and Drug Product

Molnupiravir, a broad-spectrum antiviral is an isopropyl ester prodrug of beta-D-N4-hydroxycytidine. Molnupiravir targets RNA-dependent RNA-polymerase enzyme of the viruses. A new stability-indicating HPLC-method was developed to determine related substances and assay of molnupiravir. Separation was achieved by using Shim-pack GWS C18 column. The method was validated according to current ICH requirements. The calibration plot gave a linear relationship for all known analytes over the concentration range from LOQ to 200%. LOD and LOQ for all known analytes were found in 0.05-0.08 microg mL-1 and 0.12-0.20 microg mL-1, respectively, the mean recovery was found to be 97.79-102.44 %. Study showed that the method, results of robustness, solution stability studies are precise and within the acceptable limits. Molnupiravir was found to degrade in acid, alkali, and oxidative conditions, and was stable in thermal, moisture, and photolytic degradation condition. The method is simple, accurate, precise, and reproducible for routine purity analysis of drug-samples.Copyright © 2022 Indian Drug Manufacturers' Association. All rights reserved.

Key Design Features of Lipid Nanoparticles and Electrostatic Charge-Based Lipid Nanoparticle Targeting.

Lipid nanoparticles (LNP) have gained much attention after the approval of mRNA COVID-19 vaccines. The considerable number of currently ongoing clinical studies are testament to this fact. These efforts towards the development of LNPs warrant an insight into the fundamental developmental aspects of such systems. In this review, we discuss the key design aspects that confer efficacy to a LNP delivery system, i.e., potency, biodegradability, and immunogenicity. We also cover the underlying considerations regarding the route of administration and targeting of LNPs to hepatic and non-hepatic targets. Furthermore, since LNP efficacy is also a function of drug/nucleic acid release within endosomes, we take a holistic view of charged-based targeting approaches of LNPs not only in the context of endosomal escape but also in relation to other comparable target cell internalization strategies. Electrostatic charge-based interactions have been used in the past as a potential strategy to enhance the drug release from pH-sensitive liposomes. In this review, we cover such strategies around endosomal escape and cell internalization in low pH tumor micro-environments.

SARS-CoV-2 infection activates CREB/CBP in cellular cyclic AMP-dependent pathways.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global coronavirus disease 2019 (COVID-19) pandemic that has affected the lives of billions of individuals. However, the host-virus interactions still need further investigation to reveal the underling mechanism of SARS-CoV-2 pathogenesis. Here, transcriptomics analysis of SARS-CoV-2 infection highlighted possible correlation between host-associated signaling pathway and virus. In detail, cAMP-protein kinase (PKA) pathway has an essential role in SARS-CoV-2 infection, followed by the interaction between cyclic AMP response element binding protein (CREB) and CREB-binding protein (CBP) could be induced and leading to the enhancement of CREB/CBP transcriptional activity. The replication of Delta and Omicron BA.5 were inhibited by about 49.4% and 44.7% after knockdown of CREB and CBP with small interfering RNAs, respectively. Furthermore, a small organic molecule naphthol AS-E (nAS-E), which targets on the interaction between CREB and CBP, potently inhibited SARS-CoV-2 wild-type (WT) infection with comparable the half-maximal effective concentration (EC50 ) 1.04 µM to Remdesivir 0.57 µM. Compared with WT virus, EC50 in Calu-3 cells against Delta, Omicron BA.2, and Omicron BA.5 were, on average, 1.5-fold, 1.1-fold, and 1.5-fold higher, respectively, nAS-E had a satisfied antiviral effect against Omicron variants. Taken together, our study demonstrated the importance of CREB/CBP induced by cAMP-PKA pathway during SARS-CoV-2 infection, and further provided a novel CREB/CBP interaction therapeutic drug targets for COVID-19.

The role of lipid components in lipid nanoparticles for vaccines and gene therapy.

Lipid nanoparticles (LNPs) play an important role in mRNA vaccines against COVID-19. In addition, many preclinical and clinical studies, including the siRNA-LNP product, Onpattro®, highlight that LNPs unlock the potential of nucleic acid-based therapies and vaccines. To understand what is key to the success of LNPs, we need to understand the role of the building blocks that constitute them. In this Review, we discuss what each lipid component adds to the LNP delivery platform in terms of size, structure, stability, apparent pKa, nucleic acid encapsulation efficiency, cellular uptake, and endosomal escape. To explore this, we present findings from the liposome field as well as from landmark and recent articles in the LNP literature. We also discuss challenges and strategies related to in vitro/in vivo studies of LNPs based on fluorescence readouts, immunogenicity/reactogenicity, and LNP delivery beyond the liver. How these fundamental challenges are pursued, including what lipid components are added and combined, will likely determine the scope of LNP-based gene therapies and vaccines for treating various diseases.

Molecular interactions in remdesivir-cyclodextrin systems.

Remdesivir (REM) is the first antiviral drug (Veklury™) approved by the Food and Drug Administration for the therapy of COVID-19. Due to its poor water solubility, the preparation of Veklury™ requires a suitable solubilizing excipient at pH 2 conditions. For this purpose, the final formulation contains the randomly substituted sulfobutylether-ß-cyclodextrin (SBEßCD) as a complexing agent. Herein, extensive NMR spectroscopic study with various cyclodextrin (CD) derivatives were conducted to understand the interactions in SBEßCD - REM systems at the molecular level. The pKa value of REM has been determined experimentally for the first time, as the protonation state of the aminopyrrolo-triazine moiety can play a key role in CD-REM inclusion complex formation as SBEßCD has permanent negative charges. The UV-pH titration experiments yielded a pKa of 3.56, thus the majority of REM bears a positive charge at pH 2.0. NMR experiments were performed on ß- and γCD derivatives to determine complex stabilities, stoichiometries and structures. The stability constants were determined by nonlinear curve fitting based on 1H NMR titrations at pH 2.0, while Job's method was used to determine the stoichiometries. ßCD complexes were one order of magnitude more stable than their γCD counterparts. Sulfobutylation resulted in a significant increase in stability and the single isomer derivatives showed unexpectedly high stability values (logK = 4.35 for REM - per-6-SBEßCD). In the case of ßCDs, the ethylbutyl-moiety plays a key role in complexation immersing into the ßCD cavity, while the phenoxy-moiety overtakes and drives the inclusion of REM in the case of γCDs. This is the first comprehensive study of REM-CD complexation, allowing the design of new CD derivatives with tailored stabilities, thereby aiding the formulation or production and even the analytical characterization of REM.

The Importance of Apparent pKa in the Development of Nanoparticles Encapsulating siRNA and mRNA.

Polymer and lipid nanoparticles have been extensively used as carriers to address the biological barriers encountered in siRNA and mRNA delivery. We summarize the crucial role of nanoparticle charge and ionizability in complexing RNAs, binding to biological components, escaping from the endosome, and releasing RNAs into the cytoplasm. We highlight the significant impact of the apparent pKa of nanoparticles on their efficacy and toxicity, and the importance of optimizing pKa in the development of lead formulations for RNAs. We also discuss the feasibility of fine-tuning the pKa in nanoparticles and the applications of this approach in the optimization of delivery systems for RNAs.

The Potentially Therapeutic Role of EPAC in Curbing the Process of Idiopathic Pulmonary Fibrosis via Differential Cellular Pathways.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrosis disease caused by genetic susceptibility (causative) and other indirect risk factors such as smoking, micro-aspiration and air pollution. Repeated damage of lung epithelial cells can cause fibroblast activation and excessive collagen will lead the scar formation and severe fibrosis. It has been decades since drugs for the treatment of IPF were developed, but clinical choices were limited. Exchange Protein directly Activated by cAMP (EPAC), as a newly emerging cAMP (adenosine 3',5'-cyclic monophosphate) downstream molecule, plays a vital role in the cellular pathways of IPF such as inhibiting fibroblast proliferation, stress fiber formation and epithelium cell adhesion, so it may be a novel target for drug development and treatment for curbing IPF. Here, we hypothesize that EPAC may participate in the signaling pathways related to IPF in different cell types (fibroblasts; airway smooth muscle cells; vascular endothelial cells; lung epithelial cells; macrophages; mesenchymal stem cells; T cells), thereby playing a potentially therapeutic role in resisting the process of fibrosis. We summarize the current correlation between EPAC and IPF in these different cell types, and further insights into EPAC will help to optimize the pharmacological treatment for IPF.

Insights into forsythia honeysuckle (Lianhuaqingwen) capsules: A Chinese herbal medicine repurposed for COVID-19 pandemic.

Background: In December 2019, a novel coronavirus, SARS-CoV-2 caused a series of acute atypical respiratory diseases worldwide. However, there is still a lack of drugs with clear curative effects, and the clinical trial research of vaccines has not been completely finished. Purpose: LH capsules are approved TCM patent medicine that are widely used for the treatment of respiratory tract infectious diseases caused by colds and flu. On April 12, 2020, LH capsules and granules were officially repurposed by the China Food and Drug Administration (CFDA) for patients with mild COVID-19 based on their safety and efficacy demonstrated through multicentre, randomized, controlled clinical trials. We hope to conduct a comprehensive review of it through modern pharmacy methods, and try to explain its possible mechanism. Methods: Using the full names of LH capsules Lianhuaqingwen, Lianhua Qingwen andSARS-COV-2, COVID-19 as the keywords of the search terms, systemically search for existing related papers in various databases such as Web of Science and PubMed. And completed the collection of clinical data in ClinicalTrials.gov and Chinese Clinical Trial Registry. Last but not least, we have sorted out the anti-inflammatory and antiviral mechanisms of LH capsules through literature and Selleck. Results: This review systematically sorted out the active ingredients in LH capsules. Furthermore, the related pharmacological and clinical trials of LH capsule on SARS-CoV-2, IAV and IBV were discussed in detail. Moreover, the present review provides the first summary of the potential molecular mechanism of specific substances in LH capsules involved in resistance to SARS-COV-2 infection and the inhibition of cytokine storm syndrome (CSS) caused by IL-6. Conclusion: This review summarizes the available reports and evidence that support the use of LH capsules as potential drug candidates for the prevention and treatment of COVID-19. However, TCM exerts its effects through multiple targets and multiple pathways, and LH capsules are not an exception. Therefore, the relevant mechanisms need to be further improved and experimentally verified.

Targeting the sAC-Dependent cAMP Pool to Prevent SARS-Cov-2 Infection.

An outbreak of the novel coronavirus (CoV) SARS-CoV-2, the causative agent of COVID-19 respiratory disease, infected millions of people since the end of 2019, led to high-level morbidity and mortality and caused worldwide social and economic disruption. There are currently no antiviral drugs available with proven efficacy or vaccines for its prevention. An understanding of the underlying cellular mechanisms involved in virus replication is essential for repurposing the existing drugs and/or the discovery of new ones. Endocytosis is the important mechanism of entry of CoVs into host cells. Endosomal maturation followed by the fusion with lysosomes are crucial events in endocytosis. Late endosomes and lysosomes are characterized by their acidic pH, which is generated by a proton transporter V-ATPase and required for virus entry via endocytic pathway. The cytoplasmic cAMP pool produced by soluble adenylyl cyclase (sAC) promotes V-ATPase recruitment to endosomes/lysosomes and thus their acidification. In this review, we discuss targeting the sAC-specific cAMP pool as a potential strategy to impair the endocytic entry of the SARS-CoV-2 into the host cell. Furthermore, we consider the potential impact of sAC inhibition on CoV-induced disease via modulation of autophagy and apoptosis.