Treatment with FIASMA prevented cellular entry of these viruses (25,?30, 31, 32) indicating the significance of the acid sphingomyelinase/ceramide system for viral infection. is used for the treatment of diseases of the upper and lower respiratory tract. It has almost no side effects. As an infection system, we used replication-deficient VSV pseudoviral particles (pp-VSV) presenting SARS-CoV-2 spike protein on their surface, abbreviated pp-VSV-SARS-CoV-2 spike (22). Several previous studies showed that these particles accurately reflect key 6H05 (trifluoroacetate salt) aspects of the entry of coronavirus into host cells (14, 22). We demonstrate that entry of pp-VSV-SARS-CoV-2 spike into cultured epithelial cells or freshly isolated nasal epithelial cells results in activation of the acid sphingomyelinase and a release of ceramide. These events were blocked by pretreatment with low doses of ambroxol. In accordance, ambroxol prevented cellular entry of pp-VSV-SARS-CoV-2 spike. More importantly, we obtained nasal epithelial cells from volunteers prior and after inhalation with ambroxol and infected the cells with pp-VSV-SARS-CoV-2 spike. These experiments demonstrated that inhalation of ambroxol is sufficient to reduce acid sphingomyelinase activity in nasal epithelial cells and to prevent infection with pp-VSV-SARS-CoV-2 spike tests. tests. Results are presented in arbitrary units (a.u.). NP-40, Nonidet P40. Next, we investigated whether pp-VSV-SARS-CoV-2 spike induces an activation of the acid sphingomyelinase in Vero-E6 cells and whether this increase is prevented by pretreatment of 6H05 (trifluoroacetate salt) the cells with ambroxol. Infection of the cells with pp-VSV-SARS-CoV-2 spike resulted in a rapid activation of the acid sphingomyelinase that peaked 30?min after infection (Fig.?2tests. give the time course without ambroxol, and give the time course with 25?M ambroxol. 0.05, ???tests, as indicated 6H05 (trifluoroacetate salt) or compared with the corresponding value without inhibitor. represent 16/C18 ceramides, and represent C22/24 ceramides. NP-40, Nonidet P40; pp-VSV-SARS-CoV-2, vesicular stomatitis virus pseudoviral particles presenting SARS-CoV-2 spike protein on their surface. We have previously shown that incubation of epithelial cells with pp-VSV-SARS-CoV-2 spike triggers a release of ceramide that is essential for cellular infection with the virus (14). Here, we confirm these data 6H05 (trifluoroacetate salt) and show that infection of Vero-E6 cells with pp-VSV-SARS-CoV-2 spike results in a release of C16/C18 ceramide as well as C22/C24 ceramide (Fig.?2these receptors (10). Here, we demonstrate that infection of Vero-E6 cells with pp-VSV-SARS-CoV-2 spike results in the formation of ceramide-enriched membrane domains that cluster ACE2 (Fig.?3, and and and inhibition of the acid sphingomyelinase and the release of ceramide. In contrast, addition of sphingomyelin did not alter infection with pp-SARS-CoV-2 (Fig.?4tests. tests. are untreated and ambroxol-treated cells, and represent cells constituted with anti-ceramide antibodies or PPIA sphingomyelin. tests. are untransfected cells, and represent transfected cells. eGFP, enhanced GFP; pp-VSV-SARS-CoV-2, vesicular stomatitis virus pseudoviral particles presenting SARS-CoV-2 spike protein on their surface; SARS-CoV-2, severe acute respiratory syndrome coronavirus?2. Treatment of Caco-2 cells with ambroxol or genetic downregulation of the acid sphingomyelinase in Caco-2 cells also resulted in an inhibition of pp-VSV-SARS-CoV-2 uptake, which was restored by C16 ceramide (Fig.?4tests. tests. eGFP, enhanced GFP; pp-VSV-SARS-Cov-2, vesicular stomatitis virus pseudoviral particles presenting SARS-CoV-2 spike protein on their surface. To simulate the effects of ambroxol on infection as closely as possible to treatment of patients, we isolated nasal epithelial cells from volunteers. The volunteers then inhaled 2?ml ambroxol (7.5?mg/ml), and nasal epithelial cells were again isolated from the opposite nasal cavity 60?min after inhalation and inoculated with pp-VSV-SARS-CoV-2 spike for 60?min. Cells were washed, and infectious entry was determined after incubation for 24?h. In addition, we determined the activity of the acid sphingomyelinase in.