This medicine is known as naproxen. Natural home remedies and over-the-counter OTC products can be used to temporarily relieve toothache pain until you can visit your dentist.
Certain over-the-counter pain medications can be used alongside antibiotics and other self care options to help alleviate the pain caused by a UTI. The generation of these syncytia results from activation of the SARS-CoV-2 spike protein at the cell plasma membrane level. On the basis of these observations, we performed two high-content microscopy-based screenings with more than 3, approved drugs to search for inhibitors of spike-driven syncytia. We converged on the identification of 83 drugs that inhibited spike-mediated cell fusion, several of which belonged to defined pharmacological classes.
We focused our attention on effective drugs that also protected against virus replication and associated cytopathicity. One of the most effective molecules was the antihelminthic drug niclosamide, which markedly blunted calcium oscillations and membrane conductance in spike-expressing cells by suppressing the activity of TMEM16F also known as anoctamin 6 , a calcium-activated ion channel and scramblase that is responsible for exposure of phosphatidylserine on the cell surface.
These findings suggest a potential mechanism for COVID disease pathogenesis and support the repurposing of niclosamide for therapy. One of the defining features of coronavirus biology is the coordinated process by which the virus binds and enters the host cell, which involves both docking to receptors at the cell surface ACE2 for SARS-CoV2 5 , and proteolytic activation of the spike protein by host encoded proteases at two distinct sites 6.
One activation step is spike cleavage at the S1—S2 boundary, which can occur either before or after receptor binding. A second proteolytic activation exposes the S2 portion, and primes S2 for fusion of virus and cellular membranes.
Detailed post-mortem analysis of these patients has previously been reported Most of these syncytial cells were bona fide pneumocytes, as they expressed two pneumocyte-specific markers napsin and surfactant B , and were positive for viral RNA by in situ hybridization Extended Data Fig.
Further characterization of these patients has previously been reported Nuclei in white; cell contours in red using wheat-germ agglutinin WGA ; spike protein in green. S, spike. The percentage of syncytia normalized on total cells is plotted as a z score. Four drugs that were further studied with viral infection are indicated. The percentage of nuclei within in syncytia over total nuclei is shown at the bottom. Numbers on some of the images indicate the screening well.
Images are representative of 25 per well; screening was performed in duplicate. In vitro, the expression of codon-optimized spike cDNA in Vero cells led to the conspicuous presence of syncytia Fig. The presence of spike protein on the plasma membrane also triggered the fusion of heterologous cells when these expressed the ACE2 receptor. Supplementary Video 1 shows the effect of co-culturing, for 12 h, spike-transfected human U2OS cells which have undetectable levels of ACE2 with enhanced green fluorescent protein eGFP -transfected Vero cells.
Progressive, heterologous cell fusion continued during the h observation period. At higher magnification, spike-expressing Vero cells and spike-positive syncytia projected a notable number of plasma membrane protrusions, showing filopodia extensions and contacting the plasma membrane of neighbouring cells Extended Data Fig.
We developed two assays for high-throughput screening HTS using high-content imaging. The first was based on heterologous fusion between Vero and spike-expressing U2OS cells cell fusion inhibition assay CFIA , whereas the second was based on direct expression of spike protein in Vero cells syncytia inhibition assay SIA. As drugs are common to the two libraries, this amounts to 3, different small molecules.
For the SIA screening, we expressed spike protein for 15 h, incubated the cells with the drugs for an additional 24 h and then imaged and quantified syncytia Fig. The distribution of the results for each of the two libraries is shown in Extended Data Fig. Representative images from the screening are shown in Fig. Dose—response curves for syncytia inhibition for three selected drugs niclosamide, clofazimine and salinomycin are shown in Extended Data Figs. Extended Data Fig. The choice for further studies took in consideration the following criteria: 1 common inhibitory effect in the two screenings; 2 relative efficacy within specific drug classes; and 3 pharmacological suitability to clinical application.
The ability of the drugs to protect cells from viral cytopathic effects was analysed after 5 days Extended Data Fig. Of the most effective drugs, we selected one antihistamine deptropine , one antidepressant sertraline and the antileprotic antibiotic clofazimine for further studies.
The five selected drugs were further assessed for cell protection against virus-induced cell death in a range of doses, followed by the analysis of cell survival after five days. Niclosamide, clofazimine and salinomycin were the most effective in this cell protection assay Extended Data Fig. Niclosamide and salinomycin displayed similar half-maximal inhibitory concentration IC 50 values of 0.
All three drugs also inhibited viral replication in respiratory Calu-3 cells Extended Data Fig. Of note, the cells still infected in the presence of niclosamide were no longer syncytial Extended Data Fig. After 1 h, cells were washed and cultured in drug-containing medium for 48 h. Virus production in the culture supernatants was quantified by plaque assay using Vero E6 cells. See Supplementary Video 4 for time-lapse movie. Representative frames from of a total of from a h time lapse movie of spike-expressing Vero cells.
Vero cells were co-transfected with GCaMP6s and either a control or a spike-expressing vector and treated with the indicated drugs. See Supplementary Video 5 for representative movies. We wanted to understand the mechanism s by which the selected drugs inhibited syncytia formation. We were intrigued by the observation that our assays had selected for specific drug classes.
In particular, there were 11 antipsychotics, 8 antidepressants and 5 first-generation histamine 1 H 1 receptor antagonists among the top selected drugs. Besides H 1 receptor antagonists and anticholinergics, phenothiazine antipsychotics and other tricyclics also act, to a different extent, on these receptors. We performed time-lapse imaging of Vero cells that express the calcium indicator GCaMP6s 20 , co-cultured with U2OS cells expressing spike and mCherry fluorescent protein.
The oscillation traces for more than 50 cells or syncytia in the case of fused cells are overlaid for each condition control or spike in Fig. Salinomycin, while still inhibiting syncytia formation, was ineffective in these experiments. Representative movies showing these results are in Supplementary Video 5. Of note, cell treatment with thapsigargin or cyclopiazonic acid also inhibited the expansion of syncytia while not affecting cell viability Fig.
Currents were measured using a voltage ramp in HEK cells. Statistics as in b. One hour after infection, cells were washed and incubated with fresh medium. After 48 h, cells were immunostained for spike and nucleocapsid N proteins. As a consequence, chloride blue secretion is increased, and phosphatidylserine PS pink is externalized. We investigated the levels of the 10 members of this family in several cell lines and in primary bronchial human airway epithelial cells Extended Data Fig.
We found that TMEM16F, which functions both as a non-specific ion channel and a lipid scramblase responsible for phosphatidylserine externalization onto the outer leaflet of the plasma membrane 23 , was expressed in all cells and that its levels further increased after spike expression Extended Data Fig. We performed whole-cell voltage-clamp recordings of HEK cells to measure the endogenous currents carried by this channel. We recorded small but clearly detectable outwardly rectifying currents in response to voltage ramps.
We thus wanted to explore the relationship between this effect and the suppression of syncytia. In line with these observations, treatment with either niclosamide or clofazimine for 1 h significantly reduced the levels of externalized phosphatidylserine in response to ionomycin Fig. We envisage at least three mechanisms by which spike can activate TMEM16 proteins.
Macrophages fusing into inflammatory giant cells 26 or to form bone-reabsorbing osteoclasts 27 , myoblasts committed to fuse into myotubes 28 , cytotrophoblasts becoming syncytiotrophoblasts 29 and, finally, sperm cells during egg fertilization 30 all expose phosphatidylserine at the cell surface. From a mechanistic point of view, these finding are consistent with a model Fig. Although the former event is required for plasma membrane fusion, chloride secretion might have relevance in COVID pathogenesis.
In addition to niclosamide, other drugs known to inhibit the TMEM16 family were also able to inhibit spike-induced syncytia in our assays, including nitazoxanide, hexachlorophene and dichlorophen. In addition, it seems likely that, similar to niclosamide and nitazoxanide 22 , other drugs that target TMEM16A in particular, trifluoperazine 34 , serotonin reuptake inhibitors 35 , 36 and ivermectin 37 , all of which inhibited syncytia formation might also inhibit TMEM16F.
Thus, our screening for inhibition of syncytia seems to have disclosed a common mechanism for spike-dependent cell—cell fusion. In particular, niclosamide is a synthetic salicylanilide developed in the s as a molluscicide against snails 42 and later approved in humans against tapeworm infection Niclosamide has previously been reported to be active against various enveloped and non-enveloped viruses, including SARS-CoV-2 Together, our findings provide a mechanism and a rationale for repurposing of niclosamide to treat patients with COVID No statistical methods were used to predetermine sample size.
The investigators were not blinded to allocation during experiments and outcome assessment, except where specified below. The experiments were not randomized. Of these, 25 were male and 16 were female; the average age was 77 for men and 84 for women. Extensive characterization of these patients has previously been reported Use of these post-mortem samples for investigation was approved by the competent Joint Ethical Committee of the Regione Friuli Venezia Giulia, Italy re.
Vero E6 cells were provided by A. Davidson and D. The stable U2OS cell clone expressing mCherry was obtained by transduction with lentiviral particles for constitutive expression of mCherry rLV. The medium was replaced after 4 h. Expression of the transgene was verified by fluorescence microscopy. Primary bronchial human airway epithelial cells were purchased from Epithelix and maintained in Mucilair cell culture medium Epithelix. Cells were seeded in cm Petri dishes 1. After extensive washes with PBS, cells were detached with 0.
Images were subsequently analysed, using the Harmony software package v. The thresholds for image segmentation were adjusted according to the signal-to-background ratio. Splitting coefficient was set to avoid splitting of overlapping nuclei fused cells. All the cells that had a nuclear area greater than four times the average area of a single nucleus and were simultaneously positive for at least two red and two green dots were considered as fused.
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What should I do if I missed a dose of Naproxen Aleve? If you think you or someone else may have overdosed on: Naproxen Aleve , call your doctor or the Poison Control center. If someone collapses or isn't breathing after taking Naproxen Aleve , call What to Expect You should start feeling better about one hour after you take naproxen.
Additional Dosage Information Your dosage will depend on the formulation you use, your medical condition, the severity of your pain, your age, and other factors. Doctors may suggest a lower dose for elderly people or those with liver or kidney problems.
Secondary Uses Naproxen is used for numerous medical conditions. West-ward Color : white Shape : capsule Imprint : West-ward See More. Medical Disclaimer Drugs A-Z provides drug information from Everyday Health and our partners, as well as ratings from our members, all in one place. The information within all other sections is proprietary to Everyday Health. Read more.
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