Astodrimer sodium antiviral nasal spray for reducing respiratory infections is safe and well tolerated in a randomized controlled trial.

Astodrimer sodium is a dendrimer molecule with antiviral and virucidal activity against SARS-CoV-2 and other respiratory viruses in vitro, and has previously been shown to be safe and well tolerated, and not systemically absorbed, when applied to the vaginal mucosa. To investigate its potential utility as a topical antiviral, astodrimer sodium has been reformulated for application to the nasal mucosa to help reduce viral load before or after exposure to respiratory infection. The current investigation assessed the safety, tolerability and absorption of astodrimer sodium 1% antiviral nasal spray. This was a single-centre, double-blinded, randomized, placebo-controlled, exploratory clinical investigation. Forty healthy volunteers aged 18 to 65 years with no clinically significant nasal cavity examination findings were randomized 3:1 to astodrimer sodium nasal spray (N = 30) or placebo (N = 10) at an Australian clinical trials facility. An initial cohort of participants (N = 12 astodrimer, N = 4 placebo) received a single application (one spray per nostril) to assess any acute effects, followed by a washout period, before self-administering the spray four times daily for 14 days to represent an intensive application schedule. Extent of absorption of astodrimer sodium via the nasal mucosa was also assessed in this cohort. A second cohort of participants (N = 18 astodrimer, N = 6 placebo) self-administered the spray four times daily for 14 days. The primary endpoint was safety, measured by frequency and severity of treatment emergent adverse events (TEAEs), including clinically significant nasal cavity examination findings, in the safety population (all participants randomized who administered any spray). Participants were randomized between 6 January 2021 and 29 March 2021. TEAEs occurred in 8/10 (80%) participants in the placebo arm and 19/30 (63.3%) participants in the astodrimer sodium arm; all were of mild intensity. TEAEs considered potentially related to study product occurred in 5/10 (50%) participants receiving placebo and 10/30 (33.3%) of participants receiving astodrimer sodium. No participants experienced serious AEs, or TEAEs leading to withdrawal from the study. No systemic absorption of astodrimer sodium via the nasal mucosa was detected. Astodrimer sodium nasal spray was well tolerated and is a promising innovation warranting further investigation for nasal administration to potentially reduce infection and spread of community acquired respiratory virus infections.Trial Registration: ACTRN12620001371987, first registered 22-12-2020 (Australia New Zealand Clinical Trials Registry, https://anzctr.org.au/ ).

Protective Effects of Astodrimer Sodium 1% Nasal Spray Formulation against SARS-CoV-2 Nasal Challenge in K18-hACE2 Mice.

Strategies to combat COVID-19 require multiple ways to protect vulnerable people from infection. SARS-CoV-2 is an airborne pathogen and the nasal cavity is a primary target of infection. The K18-hACE2 mouse model was used to investigate the anti-SARS-CoV-2 efficacy of astodrimer sodium formulated in a mucoadhesive nasal spray. Animals received astodrimer sodium 1% nasal spray or PBS intranasally, or intranasally and intratracheally, for 7 days, and they were infected intranasally with SARS-CoV-2 after the first product administration on Day 0. Another group was infected intranasally with SARS-CoV-2 that had been pre-incubated with astodrimer sodium 1% nasal spray or PBS for 60 min before the neutralisation of test product activity. Astodrimer sodium 1% significantly reduced the viral genome copies (>99.9%) and the infectious virus (~95%) in the lung and trachea vs. PBS. The pre-incubation of SARS-CoV-2 with astodrimer sodium 1% resulted in a significant reduction in the viral genome copies (>99.9%) and the infectious virus (>99%) in the lung and trachea, and the infectious virus was not detected in the brain or liver. Astodrimer sodium 1% resulted in a significant reduction of viral genome copies in nasal secretions vs. PBS on Day 7 post-infection. A reduction in the viral shedding from the nasal cavity may result in lower virus transmission rates. Viraemia was low or undetectable in animals treated with astodrimer sodium 1% or infected with treated virus, correlating with the lack of detectable viral replication in the liver. Similarly, low virus replication in the nasal cavity after treatment with astodrimer sodium 1% potentially protected the brain from infection. Astodrimer sodium 1% significantly reduced the pro-inflammatory cytokines IL-6, IL-1α, IL-1ß, TNFα and TGFß and the chemokine MCP-1 in the serum, lung and trachea vs. PBS. Astodrimer sodium 1% nasal spray blocked or reduced SARS-CoV-2 replication and its sequelae in K18-hACE2 mice. These data indicate a potential role for the product in preventing SARS-CoV-2 infection or for reducing the severity of COVID-19.

Virucidal and antiviral activity of astodrimer sodium against SARS-CoV-2 in vitro.

An effective response to the ongoing coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will involve a range of complementary preventive modalities. The current studies were conducted to evaluate the in vitro SARS-CoV-2 antiviral and virucidal (irreversible) activity of astodrimer sodium, a dendrimer with broad spectrum antimicrobial activity, including against enveloped viruses in in vitro and in vivo models, that is marketed for antiviral and antibacterial applications. We report that astodrimer sodium inhibits replication of SARS-CoV-2 in Vero E6 and Calu-3 cells, with 50% effective concentrations (EC50) for i) reducing virus-induced cytopathic effect of 0.002-0.012 mg/mL in Vero E6 cells, and ii) infectious virus release by plaque assay of 0.019-0.032 mg/mL in Vero E6 cells and 0.030-0.037 mg/mL in Calu-3 cells. The selectivity index (SI) in these assays was as high as 2197. Astodrimer sodium was also virucidal, irreversibly reducing SARS-CoV-2 infectivity by >99.9% (>3 log10) within 1 min of exposure, and up to >99.999% (>5 log10) shown at astodrimer sodium concentrations of 10-30 mg/mL in Vero E6 and Calu-3 cell lines. Astodrimer sodium also inhibited infection in a primary human airway epithelial cell line. The data were similar for all investigations and were consistent with the potent antiviral and virucidal activity of astodrimer sodium being due to irreversible inhibition of virus-host cell interactions, as previously demonstrated for other viruses. Further studies will confirm if astodrimer sodium binds to SARS-CoV-2 spike protein and physically blocks initial attachment of the virus to the host cell. Given the in vitro effectiveness and significantly high SI, astodrimer sodium warrants further investigation for potential as a topically administered agent for SARS-CoV-2 therapeutic applications.

Human Immunodeficiency Virus Type 1 Vpu Inhibitor, BIT225, in Combination with 3-Drug Antiretroviral Therapy: Inflammation and Immune Cell Modulation.

BIT225 is a first-in-class inhibitor of human immunodeficiency virus (HIV) type 1 Vpu. A phase II trial enrolled 36 HIV-1-infected, treatment-naive participants in Thailand to receive standard-of-care antiretroviral therapy (ART), tenofovir disoproxil fumarate/emtricitabine/efavirenz (Atripla), with 100 or 200 mg of BIT225 or placebo (daily) for 12 weeks. Combined treatment with BIT225 and ART was found to be generally safe and well tolerated, with antiviral efficacy comparable to that of ART alone. The secondary end point-soluble CD163, a marker of monocyte/macrophage inflammation-was noted to be significantly decreased in the BIT225 arm. Plasma-derived activated CD4+ and CD8+ T cells, natural killer cells, and interleukin 21 were increased in those treated with BIT225. These findings are consistent with inhibition of the known effects of HIV Vpu and may reflect clinically important modulation of inflammatory and immune function. Further clinical study is planned to both confirm and extend these important findings in treatment-naive, and treatment-experienced individuals. Clinical Trials Registration. Australian New Zealand Clinical Trials Registry (Universal Trial Number U1111-1191-2194).

The antiviral compound BIT225 inhibits HIV-1 replication in myeloid dendritic cells.

BACKGROUND: Previous studies with BIT225 (N-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide) have demonstrated a unique antiviral activity that blocks the release of HIV-1 from monocyte-derived macrophages (MDM). Antagonising the ion channel formed by HIV-1 Vpu, BIT225 preferentially targets de novo intracellular virus produced in 'virus-containing compartments' of MDM. In primary infections, dendritic cells (DC) are one of the first cells infected by HIV-1 and can transfer virus to more permissive CD4(+) T cells, making these cells an important target for novel antiviral therapies. To extend previous findings with BIT225, we aimed to further characterise the antiviral activity of BIT225 on HIV-1 replication in monocyte-derived DC (MDDC). RESULTS: The anti-HIV-1 activity of BIT225 was evaluated in vitro within MDDC alone and in co-cultures with activated CD4(+) T cells to examine the effect of the drug on HIV-1 transfer. Antiviral activity was determined by measuring HIV-1 reverse transcriptase activity in the culture supernatant of BIT225 treated and DMSO control cultures. A single dose of BIT225 resulted in a mean (SE) peak inhibition of HIV-1 release from MDDC by 74.5 % (±0.6) following 14 days of culture and a 6-fold reduction of HIV-1 transfer to activated uninfected CD4(+) T cells in co-culture. CONCLUSIONS: HIV-1 release from MDDC was inhibited by BIT225. This data broadens the drug's antiviral activity profile within cells of the myeloid lineage. These findings suggest a potential role for BIT225 in reducing HIV-1 production and preventing viral dissemination in early and chronic infection and may assist in limiting virus spread with any ongoing viral replication during antiretroviral therapy.

A Phase 1b/2a study of the safety, pharmacokinetics and antiviral activity of BIT225 in patients with HIV-1 infection.

OBJECTIVES: BIT225 (N-carbamimidoyl-5-(1-methyl-1H-pyrazol-4-yl)-2-naphthamide), a novel acyl-guanidine, is a novel antiviral drug that blocks Vpu ion channel activity and has anti-HIV-1 activity in vitro. The antiviral effect of BIT225 is most pronounced in cells of the myeloid lineage. With infected circulating monocytes and tissue-resident macrophages representing a key cellular reservoir of HIV-1, BIT225 has a potential role in the eradication of the virus from the host. PATIENTS AND METHODS: BIT225-004 is a Phase 1b/2a, placebo-controlled, randomized study of the safety, pharmacokinetics and antiviral activity of BIT225 in 21 HIV-1-infected, ART-naive subjects. Twenty-one subjects were enrolled and received BIT225 (400 mg twice daily) or placebo treatment for 10 days (randomized 2:1). The anti-HIV-1 effect of BIT225 in the monocyte reservoir was measured in CD14+ monocytes isolated from the peripheral blood on days 1 (pre-dose), 5, 10 and 20; isolated monocytes were co-cultured ex vivo with MT4 T cells. De novo HIV-1 replication was measured by p24 activity of released virus into the culture supernatant to day 25 of co-culture. In addition, monocyte samples were collected for analysis by RT-PCR total HIV-1 DNA single-copy assay. RESULTS: Measurement of HIV-1 directly within the patient's monocyte population indicated that BIT225 treatment significantly reduced the viral burden in myeloid lineage cells, which was more evident in those individuals with the highest viral loads. In addition, BIT225-treated subjects demonstrated a significantly reduced level of monocyte activation (sCD163) compared with the placebo controls. CONCLUSIONS: This study's unique design demonstrates that BIT225 can significantly reduce the dissemination of HIV-1 from infected monocytes. This has important ramifications for diminishing the seeding/re-seeding of the viral reservoir.

Ligand-protein docking studies of potential HIV-1 drug compounds using the algorithm FlexX.

Four compounds are docked to a pentameric bundle representing the transmembrane part of the Vpu protein from HIV-1. Employing the docking algorithm FlexX, their free energy of binding is estimated leading to the conclusion that potential drug candidates need to form H-bonds either with neighbouring or with n + 2 helices at the site of the serines within the bundle.

A novel Hepatitis C virus p7 ion channel inhibitor, BIT225, inhibits bovine viral diarrhea virus in vitro and shows synergism with recombinant interferon-alpha-2b and nucleoside analogues.

The novel small molecule, BIT225 (N-[5-(1-methyl-1H-pyrazol-4-yl)-napthalene-2-carbonyl]-guanidine: CAS No. 917909-71-8), was initially identified using a screening strategy designed to detect inhibitors of Hepatitis C virus (HCV) p7 ion channel activity. Here we report that BIT225 has potent stand-alone antiviral activity against the HCV model pestivirus bovine viral diarrhea virus (BVDV) with an IC(50) of 314nM. Combinations of BIT225 with recombinant interferon alpha-2b (rIFNalpha-2b) show synergistic antiviral action against BVDV and the synergy is further enhanced by addition of ribavirin. Synergy was also observed between BIT225 and two nucleoside analogues known to inhibit the HCV RNA-dependent RNA polymerase. BIT225 has successfully completed a phase Ia dose escalating, single dose safety trial in healthy volunteers and a phase Ib/IIa trial to evaluate the safety and pharmacokinetics of repeated dosing for selected doses of BIT225 in HCV-infected persons. A modest, but statistically significant drop in patient viral load was detected over the 7 days of dosing (ref. www.biotron.com.au). Given the critical role of the p7 protein in the HCV life cycle and pathogenicity, our data indicate that molecules like BIT225, representing a new class of antiviral compounds, may be developable for therapeutic use against HCV infection, either as monotherapy, or in combination with other HCV drugs.

Antiviral efficacy of the novel compound BIT225 against HIV-1 release from human macrophages.

Building on previous findings that amiloride analogues inhibit HIV-1 replication in monocyte-derived macrophages (MDM), Biotron Limited has generated a library of over 300 small-molecule compounds with significant improvements in anti-HIV-1 activity. Our lead compound, BIT225, blocks Vpu ion channel activity and also shows anti-HIV-1 activity, with a 50% effective concentration of 2.25+/-0.23 microM (mean+/-the standard error) and minimal in vitro toxicity (50% toxic concentration, 284 microM) in infected MDM, resulting in a selectivity index of 126. In this study, we define the antiretroviral efficacy of BIT225 activity in macrophages, which are important drug targets because cells of the monocyte lineage are key reservoirs of HIV-1, disseminating virus to the peripheral tissues as they differentiate into macrophages. In assays with acutely and chronically HIV-1Ba-L-infected MDM, BIT225 resulted in significant reductions in viral integration and virus release as measured by real-time PCR and a reverse transcriptase (RT) activity assay at various stages of monocyte-to-macrophage differentiation. Further, the TZM-bl assay showed that the de novo virus produced at low levels in the presence of BIT225 was less infectious than virus produced in the absence of the compound. No antiviral activity was observed in MDM chronically infected with HIV-2, which lacks Vpu, confirming our initial targeting of and screening against this viral protein. The activity of BIT225 is post-virus integration, with no direct effects on the HIV-1 enzymes RT and protease. The findings of this study suggest that BIT225 is a late-phase inhibitor of the viral life cycle, targeting Vpu, and is a drug capable of significantly inhibiting HIV-1 release from both acute and chronically infected macrophages.

Recall from informed consent counselling for cataract surgery.

The authors investigated the effect of giving written material on information recall from informed consent counselling for cataract surgery. Fifty English-speaking patients who underwent non-urgent cataract extraction at Flinders Medical Centre, South Australia, were prospectively enrolled. Systematic counselling for cataract surgery was provided, with a written copy of the content given to a randomly selected group of patients (n = 24). All subjects completed a questionnaire after counselling and again at two weeks after surgery to test their satisfaction with, and recall of, information provided. Patients were found to be satisfied with the amount of information they received and most were able to recall details about the cataract surgery procedure. However, many could not recall success rates or complication rates and only a minority could list any complication. The provision of written information did not significantly alter recall (p>0.05). Recall was significantly better immediately after counselling than two weeks after surgery (p<0.05). Younger patients also had significantly better recall (p<0.05). Patients were happy with the information they had been given but did not remember enough from the informed consent process to satisfy legal requirements.

The synergistic effects of betulin with acyclovir against herpes simplex viruses.

Betulin, a pentacyclic triterpenoid, was isolated from the bark of Betula papyrifera. The antiviral efficacies of betulin on herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) were evaluated using viral plaque reduction assays on Vero cells. The results indicate that betulin is active against both HSV-1 and HSV-2 infections with the 50% effective concentrations (EC(50)) of 0.40 and 4.15 microg/ml, respectively. The cytotoxicity of betulin was examined on Vero cells using a neutral red uptake assay. The 50% cytotoxic concentration (CC(50)) of betulin was 73.1 microg/ml. A synergistic antiviral effect between betulin and acyclovir (ACV) was determined by drug combination studies. Strong and moderate synergistic antiviral effects were observed for betulin and ACV against HSV-1 when the concentrations of ACV and betulin were higher than 0.068 and 0.4 microg/ml, respectively. At the concentrations lower than these, additive effect was found. Synergistic antiviral effects were also found against HSV-2 at higher concentrations than for HSV-1, i.e. 0.45 microg/ml of ACV combined with 8.4 microg/ml of betulin.

Quantitation of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in the liver of HBV-infected patients by LightCycler real-time PCR.

Antivirals for hepatitis B virus (HBV) reduce viral load and improve liver histology, however, their effect on covalently closed circular DNA (cccDNA), the HBV transcriptional template, has not been extensively examined. This study evaluated a newly designed LightCycler based quantitative cccDNA PCR assay. A linear range of 2.5 x 10(1) to 1 x 10(9) copies/assay using primers specific for HBV cccDNA and 2.5 x 10(1) to 2.5 x 10(9) copies/assay using primers specific for total HBV DNA (tDNA) was established. beta-Globin was used to estimate the number of cells in each PCR reaction. Enzymatic digestion with an ATP-dependent DNase improved the analytic specificity to a greater than 1:10000 ratio of cccDNA:RC DNA (relaxed circular DNA). One-tenth of the extracted DNA from 1mg of liver biopsy, was analyzed from six patients, three HBV-infected and three uninfected individuals, under blinded conditions; three were found positive and three negative for cccDNA and tDNA. Approximately 6 x 10(3) copies of cccDNA/mg of tissue were detected in a pre-transplant biopsy from an HBV-infected patient treated with lamivudine. Sequential post-transplant liver biopsies were negative for both HBV cccDNA and tDNA. An HBV-infected patient with cirrhosis who was antiviral therapy naïve had 3.7 x 10(4) copies of cccDNA/mg of liver tissue. Another treatment-naïve patient with a history of high HBV viral load had 1 x 10(5) copies of cccDNA/mg of tissue (4 x 10 (6) copies of tDNA/mg of tissue). Further studies are warranted but the high level of sensitivity, specificity, rapidity and accuracy provided by this novel assay with the LightCycler system indicate that it could be useful for monitoring antiviral therapy.