Molnupiravir versus favipiravir in at-risk outpatients with COVID-19: A randomized controlled trial in Thailand.

OBJECTIVES: Evaluate and compare the efficacy and safety of molnupiravir and favipiravir in outpatients with mild to moderate COVID-19 and at risk of severe COVID-19. METHODS: In an open-label, parallel-group, multicenter trial in Thailand, participants with moderate COVID-19 and at least one factor associated with severe COVID-19 were randomly assigned 1:1 to receive oral molnupiravir or oral favipiravir (standard of care). Phone calls for remote symptom assessment were made on Days 6, 15, and 29. Participants with worsening symptoms were instructed to return to the hospital. The primary endpoint was pulmonary involvement by Day 29, as evidenced by ≥2 of the following: dyspnea, oxygen saturation <92% or imaging. RESULTS: Nine hundred seventy-seven participants (487 molnupiravir, 490 favipiravir) were enrolled from 8 July 2022 to 19 January 2023. 98% had received ≥1 dose of COVID-19 vaccine and 83% ≥3 doses. By Day 29, pulmonary involvement occurred in 0% (0/483) in molnupiravir arm versus 1% (5/482) in favipiravir arm (-1.0%; Newcombe 95.2% CI: -2.4% to -0.0%; P = 0.021); all-cause death in 0% (0/483) and <1% (1/482); COVID-19 related hospitalization in <1% (1/483) and 1% (3/482); treatment-related adverse event in 1% (5/483) and 1% (4/486); and serious adverse event in 1% (4/483) and 1% (4/486). CONCLUSIONS: Favipiravir and molnupiravir had a similar efficacy and safety profile. Whether either of the two reduced the risk of complications during the omicron era in this population with a low risk of pulmonary involvement and a high vaccine coverage remains unclear. There were no differences in any of the safety endpoints. THAI CLINICAL TRIALS REGISTRY ID: TCTR20230111009.

Discovery, Development, and Patent Trends on Molnupiravir: A Prospective Oral Treatment for COVID-19.

The COVID-19 pandemic needs no introduction at present. Only a few treatments are available for this disease, including remdesivir and favipiravir. Accordingly, the pharmaceutical industry is striving to develop new treatments for COVID-19. Molnupiravir, an orally active RdRp inhibitor, is in a phase 3 clinical trial against COVID-19. The objective of this review article is to enlighten the researchers working on COVID-19 about the discovery, recent developments, and patents related to molnupiravir. Molnupiravir was originally developed for the treatment of influenza at Emory University, USA. However, this drug has also demonstrated activity against a variety of viruses, including SARS-CoV-2. Now it is being jointly developed by Emory University, Ridgeback Biotherapeutics, and Merck to treat COVID-19. The published clinical data indicate a good safety profile, tolerability, and oral bioavailability of molnupiravir in humans. The patient-compliant oral dosage form of molnupiravir may hit the market in the first or second quarter of 2022. The patent data of molnupiravir revealed its granted compound patent and process-related patent applications. We also anticipate patent filing related to oral dosage forms, inhalers, and a combination of molnupiravir with marketed drugs like remdesivir, favipiravir, and baricitinib. The current pandemic demands a patient compliant, safe, tolerable, and orally effective COVID-19 treatment. The authors believe that molnupiravir meets these requirements and is a breakthrough COVID-19 treatment.

Remdesivir and EIDD-1931 Interact with Human Equilibrative Nucleoside Transporters 1 and 2: Implications for Reaching SARS-CoV-2 Viral Sanctuary Sites.

Equilibrative nucleoside transporters (ENTs) are present at the blood-testis barrier (BTB), where they can facilitate antiviral drug disposition to eliminate a sanctuary site for viruses detectable in semen. The purpose of this study was to investigate ENT-drug interactions with three nucleoside analogs, remdesivir, molnupiravir, and molnupiravir's active metabolite, ß-d-N4-hydroxycytidine (EIDD-1931), and four non-nucleoside molecules repurposed as antivirals for coronavirus disease 2019 (COVID-19). The study used three-dimensional pharmacophores for ENT1 and ENT2 substrates and inhibitors and Bayesian machine learning models to identify potential interactions with these transporters. In vitro transport experiments demonstrated that remdesivir was the most potent inhibitor of ENT-mediated [3H]uridine uptake (ENT1 IC50: 39 µM; ENT2 IC50: 77 µM), followed by EIDD-1931 (ENT1 IC50: 259 µM; ENT2 IC50: 467 µM), whereas molnupiravir was a modest inhibitor (ENT1 IC50: 701 µM; ENT2 IC50: 851 µM). Other proposed antivirals failed to inhibit ENT-mediated [3H]uridine uptake below 1 mM. Remdesivir accumulation decreased in the presence of 6-S-[(4-nitrophenyl)methyl]-6-thioinosine (NBMPR) by 30% in ENT1 cells (P = 0.0248) and 27% in ENT2 cells (P = 0.0054). EIDD-1931 accumulation decreased in the presence of NBMPR by 77% in ENT1 cells (P = 0.0463) and by 64% in ENT2 cells (P = 0.0132), which supported computational predictions that both are ENT substrates that may be important for efficacy against COVID-19. NBMPR failed to decrease molnupiravir uptake, suggesting that ENT interaction is likely inhibitory. Our combined computational and in vitro data can be used to identify additional ENT-drug interactions to improve our understanding of drugs that can circumvent the BTB. SIGNIFICANCE STATEMENT: This study identified remdesivir and EIDD-1931 as substrates of equilibrative nucleoside transporters 1 and 2. This provides a potential mechanism for uptake of these drugs into cells and may be important for antiviral potential in the testes and other tissues expressing these transporters.

AI-guided discovery of the invariant host response to viral pandemics.

BACKGROUND: Coronavirus Disease 2019 (Covid-19) continues to challenge the limits of our knowledge and our healthcare system. Here we sought to define the host immune response, a.k.a, the "cytokine storm" that has been implicated in fatal COVID-19 using an AI-based approach. METHOD: Over 45,000 transcriptomic datasets of viral pandemics were analyzed to extract a 166-gene signature using ACE2 as a 'seed' gene; ACE2 was rationalized because it encodes the receptor that facilitates the entry of SARS-CoV-2 (the virus that causes COVID-19) into host cells. An AI-based approach was used to explore the utility of the signature in navigating the uncharted territory of Covid-19, setting therapeutic goals, and finding therapeutic solutions. FINDINGS: The 166-gene signature was surprisingly conserved across all viral pandemics, including COVID-19, and a subset of 20-genes classified disease severity, inspiring the nomenclatures ViP and severe-ViP signatures, respectively. The ViP signatures pinpointed a paradoxical phenomenon wherein lung epithelial and myeloid cells mount an IL15 cytokine storm, and epithelial and NK cell senescence and apoptosis determine severity/fatality. Precise therapeutic goals could be formulated; these goals were met in high-dose SARS-CoV-2-challenged hamsters using either neutralizing antibodies that abrogate SARS-CoV-2•ACE2 engagement or a directly acting antiviral agent, EIDD-2801. IL15/IL15RA were elevated in the lungs of patients with fatal disease, and plasma levels of the cytokine prognosticated disease severity. INTERPRETATION: The ViP signatures provide a quantitative and qualitative framework for titrating the immune response in viral pandemics and may serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs. FUNDING: This work was supported by the National Institutes for Health (NIH) [grants CA151673 and GM138385 (to DS) and AI141630 (to P.G), DK107585-05S1 (SD) and AI155696 (to P.G, D.S and S.D), U19-AI142742 (to S. C, CCHI: Cooperative Centers for Human Immunology)]; Research Grants Program Office (RGPO) from the University of California Office of the President (UCOP) (R00RG2628 & R00RG2642 to P.G, D.S and S.D); the UC San Diego Sanford Stem Cell Clinical Center (to P.G, D.S and S.D); LJI Institutional Funds (to S.C); the VA San Diego Healthcare System Institutional funds (to L.C.A). GDK was supported through The American Association of Immunologists Intersect Fellowship Program for Computational Scientists and Immunologists. ONE SENTENCE SUMMARY: The host immune response in COVID-19.

Drug repurposing screens identify chemical entities for the development of COVID-19 interventions.

The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets.

Intergenerational effects of manipulating DNA methylation in the early life of an iconic invader.

In response to novel environments, invasive populations often evolve rapidly. Standing genetic variation is an important predictor of evolutionary response but epigenetic variation may also play a role. Here, we use an iconic invader, the cane toad (Rhinella marina), to investigate how manipulating epigenetic status affects phenotypic traits. We collected wild toads from across Australia, bred them, and experimentally manipulated DNA methylation of the subsequent two generations (G1, G2) through exposure to the DNA methylation inhibitor zebularine and/or conspecific tadpole alarm cues. Direct exposure to alarm cues (an indicator of predation risk) increased the potency of G2 tadpole chemical cues, but this was accompanied by reductions in survival. Exposure to alarm cues during G1 also increased the potency of G2 tadpole cues, indicating intergenerational plasticity in this inducible defence. In addition, the negative effects of alarm cues on tadpole viability (i.e. the costs of producing the inducible defence) were minimized in the second generation. Exposure to zebularine during G1 induced similar intergenerational effects, suggesting a role for alteration in DNA methylation. Accordingly, we identified intergenerational shifts in DNA methylation at some loci in response to alarm cue exposure. Substantial demethylation occurred within the sodium channel epithelial 1 subunit gamma gene (SCNN1G) in alarm cue exposed individuals and their offspring. This gene is a key to the regulation of sodium in epithelial cells and may help to maintain the protective epidermal barrier. These data suggest that early life experiences of tadpoles induce intergenerational effects through epigenetic mechanisms, which enhance larval fitness. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'

Orally delivered MK-4482 inhibits SARS-CoV-2 replication in the Syrian hamster model.

The COVID-19 pandemic progresses unabated in many regions of the world. An effective antiviral against SARS-CoV-2 that could be administered orally for use following high-risk exposure would be of substantial benefit in controlling the COVID-19 pandemic. Herein, we show that MK-4482, an orally administered nucleoside analog, inhibits SARS-CoV-2 replication in the Syrian hamster model. The inhibitory effect of MK-4482 on SARS-CoV-2 replication is observed in animals when the drug is administered either beginning 12 h before or 12 h following infection in a high-risk exposure model. These data support the potential utility of MK-4482 to control SARS-CoV-2 infection in humans following high-risk exposure as well as for treatment of COVID-19 patients.

Zebularine elevates STING expression and enhances cGAMP cancer immunotherapy in mice.

DNA methylation abnormality is closely related to tumor occurrence and development. Chemical inhibitors targeting DNA methyltransferase (DNMTis) have been used in treating cancer. However, the impact of DNMTis on antitumor immunity has not been well elucidated. In this study, we show that zebularine (a demethylating agent) treatment of cancer cells led to increased levels of interferon response in a cyclic guanosine monophosphate-AMP (cGAMP) synthase (cGAS)- and stimulator of interferon genes (STING)-dependent manner. This treatment also specifically sensitized the cGAS-STING pathway in response to DNA stimulation. Incorporation of zebularine into genomic DNA caused demethylation and elevated expression of a group of genes, including STING. Without causing DNA damage, zebularine led to accumulation of DNA species in the cytoplasm of treated cells. In syngeneic tumor models, administration of zebularine alone reduced tumor burden and extended mice survival. This effect synergized with cGAMP and immune checkpoint blockade therapy. The efficacy of zebularine was abolished in nude mice and in cGAS-/- or STING-/- mice, indicating its dependency on host immunity. Analysis of tumor cells indicates upregulation of interferon-stimulated genes (ISGs) following zebularine administration. Zebularine promoted infiltration of CD8 T cells and natural killer (NK) cells into tumor and therefore suppressed tumor growth. This study unveils the role of zebularine in sensitizing the cGAS-STING pathway to promote anti-tumor immunity and provides the foundation for further therapeutic development.

SARS-CoV-2 infection is effectively treated and prevented by EIDD-2801.

All coronaviruses known to have recently emerged as human pathogens probably originated in bats1. Here we use a single experimental platform based on immunodeficient mice implanted with human lung tissue (hereafter, human lung-only mice (LoM)) to demonstrate the efficient in vivo replication of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as two endogenous SARS-like bat coronaviruses that show potential for emergence as human pathogens. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats contain endogenous coronaviruses that are capable of direct transmission to humans. Our detailed analysis of in vivo infection with SARS-CoV-2 in human lung tissue from LoM showed a predominant infection of human lung epithelial cells, including type-2 pneumocytes that are present in alveoli and ciliated airway cells. Acute infection with SARS-CoV-2 was highly cytopathic and induced a robust and sustained type-I interferon and inflammatory cytokine and chemokine response. Finally, we evaluated a therapeutic and pre-exposure prophylaxis strategy for SARS-CoV-2 infection. Our results show that therapeutic and prophylactic administration of EIDD-2801-an oral broad-spectrum antiviral agent that is currently in phase II/III clinical trials-markedly inhibited SARS-CoV-2 replication in vivo, and thus has considerable potential for the prevention and treatment of COVID-19.

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice.

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog ß-d-N4-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (ß-d-N4-hydroxycytidine-5'-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.

Neonatal Inhibition of DNA Methylation Disrupts Testosterone-Dependent Masculinization of Neurochemical Phenotype.

Many neural sex differences are differences in the number of neurons of a particular phenotype. For example, male rodents have more calbindin-expressing neurons in the medial preoptic area (mPOA) and bed nucleus of the stria terminalis (BNST), and females have more neurons expressing estrogen receptor alpha (ERα) and kisspeptin in the ventromedial nucleus of the hypothalamus (VMH) and the anteroventral periventricular nucleus (AVPV), respectively. These sex differences depend on neonatal exposure to testosterone, but the underlying molecular mechanisms are unknown. DNA methylation is important for cell phenotype differentiation throughout the developing organism. We hypothesized that testosterone causes sex differences in neurochemical phenotype via changes in DNA methylation, and tested this by inhibiting DNA methylation neonatally in male and female mice, and in females given a masculinizing dose of testosterone. Neonatal testosterone treatment masculinized calbindin, ERα and kisspeptin cell number of females at weaning. Inhibiting DNA methylation with zebularine increased calbindin cell number only in control females, thus eliminating sex differences in calbindin in the mPOA and BNST. Zebularine also reduced the sex difference in ERα cell number in the VMH, in this case by increasing ERα neuron number in males and testosterone-treated females. In contrast, the neonatal inhibition of DNA methylation had no effect on kisspeptin cell number. We conclude that testosterone normally increases the number of calbindin cells and reduces ERα cells in males through orchestrated changes in DNA methylation, contributing to, or causing, the sex differences in both cell types.

Clinical evaluation of the lightening effect of cytidine on hyperpigmented skin.

BACKGROUND: Melanocytes, which reside in the basal layer of the epidermis, produce the pigment melanin in cytoplasmic organelles known as melanosomes. Melanosomes are transferred to keratinocytes which provide the color in our skin. Recently, Diwakar et  al reported the crucial roles of protein glycosylation in both melanogenesis and melanosome transfer to keratinocytes, and each was inhibited by the nucleotide cytidine. OBJECTIVE: The main objective of this study was to determine the clinical effects of topical application of cytidine to the hyperpigmented regions of the face in a group of human volunteers. METHODS: A randomized, vehicle-controlled study was conducted for 12 weeks on healthy Korean female subjects. Cytidine was formulated into the lotion at concentrations of 2%, 3%, and 4% (w/w) and compared to the vehicle control formulation. The clinical outcomes were evaluated by performing visual assessment grading, measuring melanin index, skin brightness, and skin color parameters. In vitro skin penetration studies were conducted using Franz cell chambers for the 2% cytidine test formulation. RESULTS: The test group showed significant improvements in the visual assessment scores, melanin index, skin brightness, and skin color compared to the control group. Although significant dose-dependent improvements were seen in the clinical study, the in vitro Franz cell studies indicated that the clinical efficacy and potency of cytidine might be further enhanced by formulating a better topical delivery system, which will be the goal of our future studies. CONCLUSIONS: This randomized, double-blind, 12-week clinical study successfully demonstrated the efficacy of cytidine on skin depigmentation in a dose-dependent manner.

Continuous zebularine treatment enhances hepatic differentiation of mesenchymal stem cells under liver-specific factors induction in vitro.

AIMS: To investigate the effect of zebularine, a stable inhibitor of DNA methylation, on hepatic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) under liver-specific factors induction in vitro. MAIN METHODS: BM-MSCs were isolated from the mononuclear cell fraction of rabbit bone marrow samples. The identification of these cells was carried out by immunophenotype analysis. The three hepatic differentiation protocols of BM-MSCs were as follows: liver-specific factors (hepatocyte growth factor and epidermal growth factor) without zebularine, liver-specific factors combined with a 24 h zebularine pre-treatment, and liver-specific factors combined with continuous zebularine treatment. BM-MSCs cultured in basic medium without the differentiation stimuli were set as the control. Morphological features, liver-specific gene and protein expression, and functional analyses were assessed to evaluate hepatic differentiation of BM-MSCs. Global DNA methylation status was tested for investigating the underlying mechanism. KEY FINDINGS: Flow cytometry immunophenotyping proved the isolated cells with plastic adherence and a spindle shape were CD29, CD90 positive and CD34, CD45 negative. Albumin (ALB) and alpha-fetoprotein (AFP) messenger RNA and protein expression, glycogen storage and urea production were significantly higher in the continuous zebularine-treated group than the other groups while the differences between the zebularine-untreated group and 24 h zebularine pre-treated group were not significant. Meanwhile, significant decrease of global DNA methylation was observed in the continuous zebularine-treated group. SIGNIFICANCE: We conclude that continuous zebularine treatment can improve hepatic differentiation of BM-MSCs under liver-specific factors induction in vitro, and the decrease of global DNA methylation maybe involved in this process.

Co-delivery of nucleoside-modified mRNA and TLR agonists for cancer immunotherapy: Restoring the immunogenicity of immunosilent mRNA.

This study reports on the design of mRNA and adjuvant-loaded lipid nanoparticles for therapeutic cancer vaccination. The use of nucleoside-modified mRNA has previously been shown to improve the translational capacity and safety of mRNA-therapeutics, as it prevents the induction of type I interferons (IFNs). However, type I IFNs were identified as the key molecules that trigger the activation of antigen presenting cells, and as such drive T cell immunity. We demonstrate that nucleoside-modified mRNA can be co-delivered with the clinically approved TLR agonist monophosphoryl lipid A (MPLA). As such, we simultaneously allow high antigen expression in vivo while substituting the type I IFN response by a more controllable adjuvant. This strategy shows promise to induce effective antigen-specific T cell immunity and may be useful to enhance the safety of mRNA vaccines.

A novel cytidine analog, RX-3117, shows potent efficacy in xenograft models, even in tumors that are resistant to gemcitabine.

RX-3117 (fluorocyclopentenylcytosine) is a cytidine analog and this class of drugs, including gemcitabine, has been widely used for the treatment of various types of cancers. However, there is no oral formulation of gemcitabine and drug resistance to gemcitabine is common. In this study, the efficacy of orally-administered RX-3117 was examined in 9 different human tumor xenograft models (colon, non-small cell lung, small cell lung, pancreatic, renal and cervical), grown subcutaneously in athymic nude mice. In the Colo 205, H460, H69 and CaSki models, gemcitabine treatment resulted in 28%, 30%, 25% and 0% tumor growth inhibition (TGI), respectively, whereas oral treatment with RX-3117 induced 100%, 78%, 62% and 66% TGI, respectively. This indicates that RX-3117 may have the potential to be used for the treatment of tumors that do not respond to gemcitabine. RX-3117 was also evaluated in a single primary low-passage human pancreatic Tumorgraft™CTG-0298 (TGI 76%), which is relatively resistant to gemcitabine (TGI 38%) and has a favorable RX-3117-activating enzyme profile. These studies demonstrated the therapeutic potential and anticancer efficacy of RX-3117.