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Although vaccination campaigns are currently being rolled out to prevent coronavirus disease (COVID-19), antivirals will remain an important adjunct to vaccination. Antivirals against coronaviruses do not exist, hence global drug repurposing efforts have been carried out to identify agents that may provide clinical benefit to patients with COVID-19. Itraconazole, an antifungal agent, has been reported to have activity against animal coronaviruses. Using cell-based phenotypic assays, the in vitro antiviral activity of itraconazole and 17-OH itraconazole was assessed against clinical isolates from a German and Belgian patient infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Itraconazole demonstrated antiviral activity in human Caco-2 cells (EC50 = 2.3 µM; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). Similarly, its primary metabolite, 17-OH itraconazole, showed inhibition of SARS-CoV-2 activity (EC50 = 3.6 µM). Remdesivir inhibited viral replication with an EC50 = 0.4 µM. Itraconazole and 17-OH itraconazole resulted in a viral yield reduction in vitro of approximately 2-log10 and approximately 1-log10, as measured in both Caco-2 cells and VeroE6-eGFP cells, respectively. The viral yield reduction brought about by remdesivir or GS-441524 (parent nucleoside of the antiviral prodrug remdesivir; positive control) was more pronounced, with an approximately 3-log10 drop and >4-log10 drop in Caco-2 cells and VeroE6-eGFP cells, respectively. Itraconazole and 17-OH itraconazole exert in vitro low micromolar activity against SARS-CoV-2. Despite the in vitro antiviral activity, itraconazole did not result in a beneficial effect in hospitalized COVID-19 patients in a clinical study (EudraCT Number: 2020-001243-15).
The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN2, which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication.
Six dentin adhesives were tested in vitro regarding their cytotoxicity on human fibroblasts. The adhesives Hybrid Bond, One-up Bond F Plus, AdheSE, Clearfil SE Bond, Optibond Solo Plus and Syntac were eluted with culture medium as single or sequentially applied adhesive part for 24 h. 75 Petri dishes were produced per group. They were evaluated triangulated, comprising the quantitative evaluation (105 ones) to determine “viable”, “dead” and “debris” cells with the use of a cell-counter and the reactivity index was also identified based on the qualitative assessment (420 ones). One-up Bond F Plus, AdheSE and Clearfil SE Bond showed a statistical difference of viable cells to the cell control. For One-up Bond F Plus, statistically, differences compared to hybrid bond and Syntac were also found. All the adhesives except One-up Bond F Plus showed significant differences between single and sequentially applied adhesive part regarding the quantitative evaluation. The test material showed a moderate grade of cytotoxicity. As a result, a statistically significant difference of the cytotoxicity between the self-etch and etch-and-rinse adhesives cannot be demonstrated regarding the qualitative evaluation and the reactivity index, but the differences between sequentially applied and single applied components can be proved.
Forty-two chemicals were tested for their ability to induce cytogenetic change in Chinese hamster ovary cells using assays for chromosome aberrations (ABS) and sister chromatid exchanges (SCE). These chemicals were included in the National Toxicology Program's evaluation of the ability of four in vitro short-term genetic toxicity assays to distinguish between rodent carcinogens and noncarcinogens. The conclusions of this comparison are presented in Zeiger et al. [Zeiger E, Haseman JK, Shelby MD, Margolin BH, Tennant RW (1990): [Environ Molec Mutagen 16(Suppl 18): 1-14]. The in vitro cytogenetic testing was conducted at four laboratories, each using a standard protocol to evaluate coded chemicals with and without exogenous metabolic activation. Most chemicals were tested in a single laboratory; however, two chemicals, tribromomethane and p-chloroaniline, were tested at two laboratories as part of an interlaboratory comparison. Four chemicals (CI. basic red 9 HCI, 2-mercaptobenzothiazole, oxytetracycline HCI, and rotenone) were tested for SCE in one laboratory and in a different laboratory for ABS. Tetrakis(hydroxymethyl)phosphonium sulfate was tested at one laboratory and the chloride form was tested at a different laboratory. Twenty-five of the 42 chemicals tested induced SCE. Sixteen of these also induced ABS; all chemicals that induced ABS also induced SCE. There was approximately 79"10 reproducibility of results in repeat tests, thus, we conclude that this protocol is effective and reproducible in detecting ABS and SCE.