Broad-Spectrum, Patient-Adaptable Inhaled Niclosamide-Lysozyme Particles are Efficacious Against Coronaviruses in Lethal Murine Infection Models
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Abstract
Niclosamide (NIC) has demonstrated promising in vitro antiviral efficacy against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Though NIC is already FDA-approved, the oral formulation produces systemic drug levels that are too low to inhibit SARS-CoV-2. As an alternative, direct delivery of NIC to the respiratory tract as an aerosol could target the primary site of for SARS-CoV-2 acquisition and spread. We have developed a niclosamide powder suitable for delivery via dry powder inhaler, nebulizer, and nasal spray through the incorporation of human lysozyme (hLYS) as a carrier molecule. This novel formulation exhibits potent in vitro and in vivo activity against MERS-CoV and SARS-CoV-2 and may protect against methicillin-resistance staphylococcus aureus pneumonia and inflammatory lung damage occurring secondary to CoV infections. The suitability of the formulation for all stages of the disease and low-cost development approach will ensure wide-spread utilization
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SciScore for 10.1101/2020.09.24.310490: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources In vitro efficacy: All antiviral efficacy experiments were performed using Vero-E6 cells obtained from American Type Culture Collection (Manassas, Virginia, USA) Vero-E6suggested: NoneSoftware and Algorithms Sentences Resources Secondary structure analysis was performed in OriginPro (OrginLab Corporation) using the second derivative of the amide I band region (1580-1720 nm) and the peak analyzer function. OriginProsuggested: NoneImage … SciScore for 10.1101/2020.09.24.310490: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources In vitro efficacy: All antiviral efficacy experiments were performed using Vero-E6 cells obtained from American Type Culture Collection (Manassas, Virginia, USA) Vero-E6suggested: NoneSoftware and Algorithms Sentences Resources Secondary structure analysis was performed in OriginPro (OrginLab Corporation) using the second derivative of the amide I band region (1580-1720 nm) and the peak analyzer function. OriginProsuggested: NoneImage analysis of the plume geometry and spray pattern were permed in Fiji. Fijisuggested: (Fiji, RRID:SCR_002285)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:We acknowledge limitations within our study, namely the lack of pharmacokinetic data which may explain the mechanism of viral load reduction in brain, kidney, and lung tissue, as well as the utilization of only one dosing level of NIC-hLYS in the SARS-CoV-2 model. In our study, we utilized a “worst-case scenario” of efficacy evaluation by administering lethal inoculums of CoVs and initiating treatment 24 hours after the infection was established(78). The effect of the timing of treatment initiation, i.e., prophylactic versus 24 or 48 hours p.i., was not examined. Incorporation of prophylactic dosing may result in the increased efficacy of NIC-hLYS, as the antiviral mechanism of action of NIC may be related to the prevention of viral entry into host cells.(9) Future studies will examine these variables, based upon the notable activity of the novel NIC-hLYS formulation in these initial proof-of-concept efficacy studies. In conclusion, a novel formulation of NIC-hLYS optimized for delivery to the upper and lower respiratory tracts as a powder or stable suspension was developed. In vitro, the incorporation of hLYS into the formulation was noted to improve potency against MERS-CoV and SARS-CoV-2, as well as MRSA, which is an important causative agent for secondary bacterial pneumonias associated with COVID-19. The NIC-hLYS particles exhibited suppression of the inflammatory cytokines TNF-α and IL-6, which have been implicated in the development of more severe COVID-19, while eleva...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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SciScore for 10.1101/2020.09.24.310490: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Ethics approval Protocols for the study of SARS-CoV-2 drug efficacy (202003-CNU-023) and MERS-CoV drug efficacy (CNU-01192) in mice was approved by the Internal Animal Use Committee at Chungnam National University (CNU). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Goat anti-Mouse IgG Cross-Adsorbed Secondary Antibody HRP (Invitrogen, MA, USA) was diluted (1:5000) in blocking buffer, and 100 µL was added to each well and incubated for 1hr at room temperature. anti-Mou…SciScore for 10.1101/2020.09.24.310490: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Ethics approval Protocols for the study of SARS-CoV-2 drug efficacy (202003-CNU-023) and MERS-CoV drug efficacy (CNU-01192) in mice was approved by the Internal Animal Use Committee at Chungnam National University (CNU). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Goat anti-Mouse IgG Cross-Adsorbed Secondary Antibody HRP (Invitrogen, MA, USA) was diluted (1:5000) in blocking buffer, and 100 µL was added to each well and incubated for 1hr at room temperature. anti-Mouse IgGsuggested: (LifeSpan Cat# LS-C69682-5000, RRID:AB_1653096)Experimental Models: Cell Lines Sentences Resources Cycle threshold (Ct) values were converted to plaque forming units (pfu) using a standard curve generated from data using stock viruses with known pfu titers by plaque assay. 7.2. EC50 determination Vero-E6 cells grown in tissue culture flasks were detached by treatment with trypsin-EDTA and were seeded in 96-well tissue culture plates. Vero-E6suggested: NoneThe half maximal effective concentration (EC50) of the formulations was assessed by dosing infected Vero E6 cells plated in 96-wells with NIC-hLYS suspensions with NIC content ranging from 0.25 µg/mL to 0.004 µg/mL once daily over the course of 72 hours. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Secondary structure analysis was performed in OriginPro (OrginLab Corporation) using the second derivative of the amide I band region (1580-1720 nm) and the peak analyzer function. OriginProsuggested: NoneImage analysis of the plume geometry and spray pattern were permed in Fiji. Fijisuggested: (Fiji, RRID:SCR_002285)(GraphPad Software, San Diego, CA, USA). 8.7. GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
We acknowledge limitations within our study, namely the lack of pharmacokinetic data which may explain the mechanism of viral load reduction in brain, kidney, and lung tissue, as well as the utilization of only one dosing level of NIC-hLYS in the SARS-CoV-2 model. In our study, we utilized a “worst-case scenario” of efficacy evaluation by administering lethal inoculums of CoVs and initiating treatment 24 hours after the infection was established(83). The effect of the timing of treatment initiation, i.e., prophylactic versus 24 or 48 hours p.i., was not examined. Incorporation of prophylactic dosing may result in the increased efficacy of NIC-hLYS, as the antiviral mechanism of action of NIC may be related to the prevention of viral entry into host cells.(17) Future studies will examine these variables, based upon the notable activity of the novel NIC-hLYS formulation in these initial proof-of-concept efficacy studies. In conclusion, a novel formulation of NIC-hLYS optimized for delivery to the upper and lower respiratory tracts as a powder or stable suspension was developed. In vitro, the incorporation of hLYS into the formulation was noted to improve potency against MERS-CoV and SARS-CoV-2, as well as MRSA, which is an important causative agent for secondary bacterial pneumonias associated with COVID-19. The NIC-hLYS particles exhibited suppression of the inflammatory cytokines TNF-a and IL-6, which have been implicated in the development of more severe COVID-19, while elev...
Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04480333 Recruiting Safety, Tolerability and Pharmacokinetics of Inhaled Nanopar... NCT04292730 Completed Study to Evaluate the Safety and Antiviral Activity of Remde... Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
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