Egg-Derived Anti-SARS-CoV-2 Immunoglobulin Y (IgY) With Broad Variant Activity as Intranasal Prophylaxis Against COVID-19.

COVID-19 emergency use authorizations and approvals for vaccines were achieved in record time. However, there remains a need to develop additional safe, effective, easy-to-produce, and inexpensive prevention to reduce the risk of acquiring SARS-CoV-2 infection. This need is due to difficulties in vaccine manufacturing and distribution, vaccine hesitancy, and, critically, the increased prevalence of SARS-CoV-2 variants with greater contagiousness or reduced sensitivity to immunity. Antibodies from eggs of hens (immunoglobulin Y; IgY) that were administered the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein were developed for use as nasal drops to capture the virus on the nasal mucosa. Although initially raised against the 2019 novel coronavirus index strain (2019-nCoV), these anti-SARS-CoV-2 RBD IgY surprisingly had indistinguishable enzyme-linked immunosorbent assay binding against variants of concern that have emerged, including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529). This is different from sera of immunized or convalescent patients. Culture neutralization titers against available Alpha, Beta, and Delta were also indistinguishable from the index SARS-CoV-2 strain. Efforts to develop these IgY for clinical use demonstrated that the intranasal anti-SARS-CoV-2 RBD IgY preparation showed no binding (cross-reactivity) to a variety of human tissues and had an excellent safety profile in rats following 28-day intranasal delivery of the formulated IgY. A double-blind, randomized, placebo-controlled phase 1 study evaluating single-ascending and multiple doses of anti-SARS-CoV-2 RBD IgY administered intranasally for 14 days in 48 healthy adults also demonstrated an excellent safety and tolerability profile, and no evidence of systemic absorption. As these antiviral IgY have broad selectivity against many variants of concern, are fast to produce, and are a low-cost product, their use as prophylaxis to reduce SARS-CoV-2 viral transmission warrants further evaluation. Clinical Trial Registration: https://www.clinicaltrials.gov/ct2/show/NCT04567810, identifier NCT04567810.

Inflammation in Viral Vector-Mediated Ocular Gene Therapy: A Review and Report From a Workshop Hosted by the Foundation Fighting Blindness, 9/2020.

Translational Relevance: Subclinical or clinical inflammation often arises during ocular gene therapy with viral vectors. Understanding the biological bases and impacts on efficacy are important for clinical management and the improvement of future therapies.

RRx-001, a downregulator of the CD47- SIRPα checkpoint pathway, does not cause anemia or thrombocytopenia.

Introduction: The CD47 and SIRPα checkpoint pathway has garnered much interest within the anti-cancer research community, with multiple experimental checkpoint inhibitors targeting CD47 and SIRPα in development. The use of such checkpoint inhibitors may however be limited by hematologic toxicity.Areas covered: We report on RRx-001, the first known small molecule downregulator of CD47 and SIRPα, which has shown a lack of hematologic toxicity in clinical trials.Expert opinion: RRx-001 is the first reported small molecule downregulator of CD47 and SIRPα and lacks any notable hematologic or systemic toxicity as demonstrated in clinical trials to date. Small molecule RRx-001 could be used in combination with or in place of CD47 targeting antibodies for anti-cancer treatment.

Dapansutrile, an oral selective NLRP3 inflammasome inhibitor, for treatment of gout flares: an open-label, dose-adaptive, proof-of-concept, phase 2a trial.

BACKGROUND: Gout flares are driven by interleukin (IL)-1ß. Dapansutrile inhibits the NLRP3 inflammasome and subsequent activation of IL-1ß. In this study we aimed to investigate the safety and efficacy of orally administered dapansutrile in patients with a gout flare. METHODS: In this open-label, proof-of-concept, phase 2a trial, adult patients (aged 18-80 years) with a monoarticular monosodium urate crystal-proven gout flare were enrolled at an outpatient clinic in the Netherlands and sequentially assigned using a dose-adaptive design to receive 100 mg/day, 300 mg/day, 1000 mg/day, or 2000 mg/day oral dapansutrile for 8 days. The coprimary outcomes were change in patient-reported target joint pain from baseline to day 3 and from baseline to day 7, assessed in the per-protocol population (all patients who received at least 80% of the study drug and had no major protocol deviations). Safety was assessed in the intention-to-treat population. This trial is registered with the EU Clinical Trials Register, EudraCT 2016-000943-14, and is completed. FINDINGS: Between May 18, 2017, and Jan 21, 2019, 144 patients were assessed for eligibility, of whom 34 were enrolled and 29 were included in the per-protocol population (three patients were excluded due to receiving <80% of study drug and two had major protocol deviations): eight patients received 100 mg/day, seven received 300 mg/day, six received 1000 mg/day, and eight received 2000 mg/day. Between baseline and day 3, there was a mean reduction in patient-reported target joint pain of 52·4% (SD 32·94; p=0∙016) for the 100 mg/day group, 68·4% (34·29; p=0∙016) for the 300 mg/day group, 55·8% (44·90; p=0∙063) for the 1000 mg/day group, and 57·6% (38·72; p=0∙016) for the 2000 mg/day group. At day 7, there was a mean reduction of 82·1% (22·68; p=0∙031) for the 100 mg/day group, 84·2% (16·33; p=0∙016) for the 300 mg/day group, 68·9% (34·89; p=0∙031) for the 1000 mg/day group, and 83·9% (15·44; p=0∙008) for the 2000 mg/day group, compared to baseline. 25 (73·5%) of 34 patients reported a total of 45 treatment-emergent adverse events, most of which were metabolism and nutrition disorders (17 [37·8%]) and gastrointestinal disorders (ten [22·2%]). Two serious adverse events occurred during the study, admission to hospital because of worsening of gout flare at day 3, and admission to hospital because of coronary stenosis 18 days after the patient received their last dose; these were considered moderate in severity and unrelated to the study drug. INTERPRETATION: Dapansutrile is a specific NLRP3 inflammasome inhibitor with a satisfactory safety profile and efficacy in the reduction of target joint pain in this study. Future studies are needed to confirm the clinical potential of dapansutrile.

RRx-001, a first-in-class small molecule inhibitor of MYC and a downregulator of CD47, is an "erythrophagoimmunotherapeutic".

The main mechanism of action of RRx-001, a pharmaceutically unprecedented sui generis Phase 3 small molecule that is derived from the aerospace industry, is clarified. RRx-001 has demonstrated anticancer activity through antiangiogenic, immune, epigenetic, antioxidant, apoptotic and nitric oxide (NO) pathways, resulting in its pleiomorphic description as an antiangiogenic/vascular normalizer.

REPLATINUM Phase III randomized study: RRx-001 + platinum doublet versus platinum doublet in third-line small cell lung cancer.

RRx-001 is a cysteine-directed anticancer alkylating agent with activity in a Phase II study in platinum refractory small cell lung cancer. Here, we describe the design of REPLATINUM, an open-label, Phase III trial. 120 patients with previously platinum-treated small cell lung cancer in third line will be randomized 1:1 to receive RRx-001 followed by four cycles of a platinum doublet, and then alternating cycles of RRx-001 and single agent platinum until progression versus four cycles of a platinum doublet. At radiologic progression on the platinum doublet, patients may cross over to the RRx-001 arm. Primary objective: to demonstrate superior progression-free survival in the RRx-001 population. Secondary objectives: to demonstrate superiority for overall survival and objective response rate. Clinical Trial registration: NCT03699956.

RRx-001 protects normal tissues but not tumors via Nrf2 induction and Bcl-2 inhibition.

BACKGROUND: RRx-001, a minimally toxic next-generation checkpoint inhibitor that targets myeloid suppressor cells in the tumor microenvironment, has also been shown to protect normal tissues from the cytotoxic effects of chemotherapy and radiation. The following experiments were carried out to determine whether the cytoprotective functions of RRx-001 in normal cells were operative in tumor cells. DESIGN: The effects of RRx-001 on normal cells, and ovarian cancer A2780 and UWB1 cells were evaluated with a colony-forming assay. Western blot densitometry was used to measure Nrf2 nuclear translocation in Caco2 cells after exposure to RRx-001. Following incubation with RRx-001, levels of the antioxidant, NQO1, were determined in Caco2 cells by measuring absorbance over 300 min at 440 nm. RRx-001-mediated cytotoxicity in HCT-116 colorectal cancer cells was evaluated with an MTT assay. In addition, the effect of RRx-001 incubation on the protein expression of Nrf2, PARP, cleaved PARP, procaspases 3, 8, and 9, Bcl-2, and Bax in HCT-116 colorectal cells was determined by western blot analysis. RESULTS: RRx-001 is demonstrated to induce Nrf2 in normal tissues, mediating protection, and to downregulate the Nrf2-controlled antiapoptotic target gene, B-cell lymphoma 2 (Bcl-2) in tumors, mediating cytotoxicity. CONCLUSION: Through Nrf2 induction in normal cells and inhibition of Bcl-2 in tumor cells, RRx-001 selectively protects normal cells against lethality in normal cells, but induces apoptosis in tumor cells.

OLT1177, a ß-sulfonyl nitrile compound, safe in humans, inhibits the NLRP3 inflammasome and reverses the metabolic cost of inflammation.

Activation of the NLRP3 inflammasome induces maturation of IL-1ß and IL-18, both validated targets for treating acute and chronic inflammatory diseases. Here, we demonstrate that OLT1177, an orally active ß-sulfonyl nitrile molecule, inhibits activation of the NLRP3 inflammasome. In vitro, nanomolar concentrations of OLT1177 reduced IL-1ß and IL-18 release following canonical and noncanonical NLRP3 inflammasome activation. The molecule showed no effect on the NLRC4 and AIM2 inflammasomes, suggesting specificity for NLRP3. In LPS-stimulated human blood-derived macrophages, OLT1177 decreased IL-1ß levels by 60% and IL-18 by 70% at concentrations 100-fold lower in vitro than plasma concentrations safely reached in humans. OLT1177 also reduced IL-1ß release and caspase-1 activity in freshly obtained human blood neutrophils. In monocytes isolated from patients with cryopyrin-associated periodic syndrome (CAPS), OLT1177 inhibited LPS-induced IL-1ß release by 84% and 36%. Immunoprecipitation and FRET analysis demonstrated that OLT1177 prevented NLRP3-ASC, as well as NLRP3-caspase-1 interaction, thus inhibiting NLRP3 inflammasome oligomerization. In a cell-free assay, OLT1177 reduced ATPase activity of recombinant NLRP3, suggesting direct targeting of NLRP3. Mechanistically, OLT1177 did not affect potassium efflux, gene expression, or synthesis of the IL-1ß precursor. Steady-state levels of phosphorylated NF-κB and IkB kinase were significantly lowered in spleen cells from OLT1177-treated mice. We observed reduced IL-1ß content in tissue homogenates, limited oxidative stress, and increased muscle oxidative metabolism in OLT1177-treated mice challenged with LPS. Healthy humans receiving 1,000 mg of OLT1177 daily for 8 d exhibited neither adverse effects nor biochemical or hematological changes.

Antiviral activity of CHO-SS cell-derived human omega interferon and other human interferons against HCV RNA replicons and related viruses.

The fully glycosylated human omega interferon produced from CHO-SS cells (glycosylated IFN-omega) has been shown to be well-tolerated in man and to induce a sustained virologic response in patients infected with hepatitis C virus (HCV). We examined the antiviral activity of glycosylated IFN-omega and various human IFNs (IFN-alpha, -beta, -gamma and non-glycosylated bacterial (Escherichia coli) recombinant IFN-omega (non-glycosylated IFN-omega)) against HCV RNA replicons and several viruses related to HCV. Since none of the IFNs displayed cytotoxicity we compared their activities based on the effective concentration of the IFN that inhibited virus growth by 50% (EC50). Glycosylated IFN-omega was found to be the most potent antiviral agent of all the IFNs tested against bovine viral diarrhea virus (BVDV), yellow fever virus and West Nile virus. With HCV RNA replicons, non-glycosylated IFN-omega was comparable in activity to IFN-alpha while glycosylated IFN-omega was markedly more potent, indicating that glycosylation has an important effect on its activity. Drug combination analysis of glycosylated IFN-omega+ribavirin (RBV) in BVDV showed a synergy of antiviral effects similar to IFN-alpha+RBV, as well as a unique antagonism of RBV cytotoxic effects by glycosylated IFN-omega. Transcription factor (TF) profiling indicated that IFN-alpha or glycosylated IFN-omega treatment upregulated the same 17 TFs. IFN-alpha and glycosylated IFN-omega also upregulated 9 and 40 additional unique TFs, respectively. The differences in the expression of these TFs were modest, but statistically significantly different for eight of the TFs that were upregulated exclusively by glycosylated IFN-omega. The activation of these additional TFs by glycosylated IFN-omega might contribute to its high potency.

Safety pharmacology, toxicology and pharmacokinetic assessment of recombinant human omega-interferon produced from CHO-SS cells.

Human omega-interferon (IFN-omega) has been shown to be well-tolerated in man and to induce reductions of hepatitis C virus RNA levels in a series of human clinical trials. Here we provide an overview of our preclinical safety evaluation of the fully-glycosylated human IFN-omega produced from CHO-SS cells that is currently being evaluated clinically. IFN-omega was not associated with any biologically-relevant adverse effects in a series of 10 safety pharmacology experiments, in the Ames mutagenicity test, in the micronucleus test, or in intraarterial, intravenous, paravenous or subcutaneous local tolerance studies. Acute, subacute, subchronic and reproductive toxicity studies performed in cynomolgus monkeys and rats showed a toxicity profile similar to that of human alpha interferon (IFN-alpha). Except for the acute (single-dose) toxicology study, all of the other toxicity studies showed evidence for the formation of anti-IFN-omega antibodies over time in the animals. These antibodies were found to neutralize IFN-omega antiviral activity in vitro in a dose-dependent manner. The average pharmacokinetic parameters following a single subcutaneous dose of IFN-omega in rabbits, rats and monkeys were determined and found to be similar to that of human IFN-alpha. These findings demonstrate that IFN-omega has a safety profile consistent with that required for its use in man. IFN-omega might be beneficial for the treatment of patients infected with hepatitis C virus who fail to respond to IFN-alpha or as a first-line treatment option.