RESUMEN
Endocytosis, or internalization through endosomes, is a major cell entry mechanism used by respiratory viruses. Phosphoinositide 5-kinase (PIKfyve) is a critical enzyme for the synthesis of phosphatidylinositol (3, 5)biphosphate (PtdIns (3, 5)P2) and has been implicated in virus trafficking via the endocytic pathway. In fact, antiviral effects of PIKfyve inhibitors against SARS-CoV-2 and Ebola have been reported, but there is little evidence regarding other respiratory viruses. In this study, we demonstrated the antiviral effects of PIKfyve inhibitors on influenza virus and respiratory syncytial virus in vitro and in vivo. PIKfyve inhibitors Apilimod mesylate (AM) and YM201636 concentration-dependently inhibited several influenza strains in an MDCK cell-cytopathic assay. AM also reduced the viral load and cytokine release, while improving the cell integrity of human nasal air-liquid interface cultured epithelium infected with influenza PR8. In PR8-infected mice, AM (2 mg/mL), when intranasally treated, exhibited a significant reduction of viral load and inflammation and inhibited weight loss caused by influenza infection, with effects being similar to oral oseltamivir (10 mg/kg). In addition, AM demonstrated antiviral effects in RSV A2-infected human nasal epithelium in vitro and mouse in vivo, with an equivalent effect to that of ribavirin. AM also showed antiviral effects against human rhinovirus and seasonal coronavirus in vitro. Thus, PIKfyve is found to be involved in influenza and RSV infection, and PIKfyve inhibitor is a promising molecule for a pan-viral approach against respiratory viruses.
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Fiebre Hemorrágica Ebola , Gripe Humana , Humanos , Animales , Ratones , Oseltamivir , Antivirales/farmacología , Antivirales/uso terapéutico , Mucosa NasalRESUMEN
Background: Elderly people are known to be vulnerable to virus infection. However, this has not been appropriately tested in in vitro studies due to a lack of appropriate virus infection models. In this report, we investigated the impact of age on respiratory syncytial virus (RSV) in pseudostratified air-liquid-interface (ALI) culture bronchial epithelium, which more closely mimic human airway epithelium morphologically and physiologically, than submerged cancer cell line cultures. Methods: RSV A2 was inoculated apically to the bronchial epithelium obtained from 8 donors with different ages (28-72 years old), and time-profiles of viral load and inflammatory cytokines were analyzed. Results: RSV A2 replicated well in ALI-culture bronchial epithelium. The viral peak day and peak viral load were similar between donors at ≤60 years old (n = 4) and > 65 years old (n = 4; elderly group), but virus clearance was impaired in the elderly group. Furthermore, area under the curve (AUC) analysis, calculated from viral load peak to the end of sample collection (from Day 3 to 10 post inoculation), revealed statistically higher live viral load (PFU assay) and viral genome copies (PCR assay) in the elderly group, and a positive correlation between viral load and age was observed. In addition, the AUCs of RANTES, LDH, and dsDNA (cell damage marker) were statistically higher in the elderly group, and the elderly group showed a trend of higher AUC of CXCL8, CXCL10 and mucin production. The gene expression of p21CDKN1A (cellular senescence marker) at baseline was also higher in the elderly group, and there was a good positive correlation between basal p21 expression and viral load or RANTES (AUC). Conclusion: Age was found to be a key factor affecting viral kinetics and biomarkers post virus infection in an ALI-culture model. Currently, novel or innovative in vitro cell models are introduced for virus research, but when virus studies are conducted, similarly to working with other clinical samples, the age balance is important to obtain more accurate results.
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BACKGROUND: PC786 is a nebulized nonnucleoside respiratory syncytial virus (RSV) polymerase inhibitor designed to treat RSV, which replicates in the superficial layer of epithelial cells lining the airways. METHODS: Fifty-six healthy volunteers inoculated with RSV-A (Memphis 37b) were randomly dosed with either nebulized PC786 (5 mg) or placebo, twice daily for 5 days, from either 12 hours after confirmation of RSV infection or 6 days after virus inoculation. Viral load (VL), disease severity, pharmacokinetics, and safety were assessed until discharge. RSV infection was confirmed by reverse-transcription quantitative polymerase chain reaction with any positive value (intention-to-treat infected [ITT-I] population) or RSV RNA ≥1 log10 plaque-forming unit equivalents (PFUe)/mL (specific intention-to-treat infection [ITT-IS] population) in nasal wash samples. RESULTS: In the ITT-I population, the mean VL area under the curve (AUC) was lower in the PC786 group than the placebo group (274.1 vs 406.6 log10 PFUe/mL × hour; Pâ =â .0359). PC786 showed a trend toward reduction of symptom score and mucous weight. In ITT-IS (post hoc analysis), the latter was statistically significant as well as VL AUC (Pâ =â .0126). PC786 showed an early time to maximum plasma concentration, limited systemic exposure, and long half-life and consequently a 2-fold accumulation over the 5-day dosing period. PC786 was well tolerated. CONCLUSIONS: Nebulized PC786 demonstrated a significant antiviral effect against RSV, warranting further clinical study. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov: NCT03382431; EudraCT: 2017-002563-18.
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Antivirales , Infecciones por Virus Sincitial Respiratorio , Antivirales/efectos adversos , Benzamidas/efectos adversos , Benzazepinas/efectos adversos , Humanos , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Compuestos de Espiro/efectos adversos , Resultado del TratamientoRESUMEN
PC945 is a novel antifungal agent, optimised for inhaled treatment. In this study, the relationship between antifungal effects of PC945 and its exposure in the lungs was investigated in Aspergillus fumigatus intranasally infected, temporarily neutropenic mice. Mice were given prophylactic PC945 intranasally once daily (0.56 µg/mouse) on either Day -7 to 0 (8 doses) or Day -1 to 0 (2 doses). Lung tissue, plasma and bronchoalveolar lavage (BAL) fluid were collected 24 or 72 h post A. fumigatus inoculation for biomarker and pharmacokinetic analyses. BAL cell pellets and supernatants were prepared separately by centrifugation. 8 prophylactic doses of PC945 were found to demonstrate significantly stronger antifungal effects (lung fungal burden and galactomannan (GM) in BAL and plasma) than prophylaxis with 2 doses. PC945 concentrations were below the limit of detection in plasma but readily measured in lung extracts. The concentrations were much higher after extended prophylaxis (709 and 312 ng/g of lung) than short prophylaxis (301 and 195 ng/g of lung) at 24 and 72 h post last dose, respectively, suggesting PC945 accumulation in whole lung after repeat dosing although it was likely to be a mixture of dissolved and undissolved PC945, meaning that the data should be interpreted with caution. Interestingly, low concentrations of PC945 were detected in BAL supernatant (6.6 and 1.9 ng/ml) whereas high levels of PC945 were measured in BAL cell pellets (626 and 406 ng/ml) at 24 and 72 h post last dose, respectively, in extended prophylaxis. In addition, the PC945 concentrations in BAL cells showed a statistically significant correlation with measured anti-fungal activities. These observations will be pursued, and it is intended that BAL cell concentrations of PC945 be measured in future clinical studies rather than standard measurement in BAL itself. Thus, PC945's profile makes it an attractive potential prophylactic agent for the prevention of pulmonary fungal infections.
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Antifúngicos , Aspergillus fumigatus , Animales , Antifúngicos/farmacología , Líquido del Lavado Bronquioalveolar , Modelos Animales de Enfermedad , Pulmón , Mananos , RatonesRESUMEN
BACKGROUND: Sestrin 2, Endocan, and Sirtuin 1 are distinct molecules with some biologic actions associated with asthma pathophysiology. The aim of the present study was to determine the molecular level differences attributable to underlying asthma severity. METHODS: We initially recruited 85 asthmatics with a wide spectrum of severity. All of the patients were optimally treated according to current guidelines. Demographics, test results of lung function, and treatment regimes of all patients were recorded. Sestrin 2, Endocan, and Sirtuin 1 were measured in different biological samples (sputum with two processing methods and serum). RESULTS: A total of 60 patients (35 with severe asthma) were analyzed, since 25 patients failed to produce an adequate sample of sputum. Patients with severe asthma showed significantly higher values for Sestrin 2 [pg/mL], measured in both sputum supernatant and cell pellet, compared to those with mild to moderate asthma [9524 (5696, 12,373) vs. 7476 (4265, 9273) p = 0.029, and 23,748 (15,280, 32,742) vs. 10,084 (3349, 21,784), p = 0.008, respectively]. No other significant differences were observed. No significant associations were observed between biomarkers, inflammatory cells, and lung function. CONCLUSION: Sestrin 2 is increased in patients with severe asthma as part of a mechanism that may modify structural alterations through the imbalance between oxidative stress and antioxidant activity.
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Invasive pulmonary Aspergillosis is a leading cause of morbidity and mortality in immunosuppressed patients and treatment outcomes using oral antifungal triazoles remain suboptimal. Here we show that combining topical treatment using PC945, a novel inhaled triazole, with systemic treatment using known triazoles demonstrated synergistic antifungal effects against Aspergillus fumigatus (AF) in an in vitro human alveolus bilayer model and in the lungs of neutropenic immunocompromised mice. Combination treatment with apical PC945 and either basolateral posaconazole or voriconazole resulted in a synergistic interaction with potency improved over either compound as a monotherapy against both azole-susceptible and resistant AF invasion in vitro. Surprisingly there was little, or no synergistic interaction observed when apical and basolateral posaconazole or voriconazole were combined. In addition, repeated prophylactic treatment with PC945, but not posaconazole or voriconazole, showed superior effects to single prophylactic dose, suggesting tissue retention and/or accumulation of PC945. Furthermore, in mice infected with AF intranasally, 83% of animals treated with a combination of intranasal PC945 and oral posaconazole survived until day 7, while little protective effects were observed by either compound alone. Thus, the combination of a highly optimised topical triazole with oral triazoles potentially induces synergistic effects against AF infection.
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Antifúngicos/farmacología , Aspergillus fumigatus/crecimiento & desarrollo , Benzamidas/farmacología , Alveolos Pulmonares , Aspergilosis Pulmonar/tratamiento farmacológico , Triazoles/farmacología , Voriconazol/farmacología , Administración Tópica , Benzamidas/agonistas , Línea Celular , Sinergismo Farmacológico , Humanos , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/microbiología , Alveolos Pulmonares/patología , Aspergilosis Pulmonar/metabolismo , Aspergilosis Pulmonar/patología , Triazoles/agonistas , Voriconazol/agonistasRESUMEN
OBJECTIVES: The growing emergence of azole-resistant Aspergillus fumigatus strains worldwide is a major concern for current systemic antifungal treatment. Here we report antifungal activities of a novel inhaled triazole, PC1244, against a collection of multi-azole-resistant A. fumigatus strains. METHODS: MICs of PC1244 were determined for A. fumigatus carrying TR34/L98H (nâ=â81), TR46/Y121F/T289A (nâ=â24), M220 (nâ=â6), G54 (nâ=â11), TR53 (nâ=â1), TR463/Y121F/T289A (nâ=â2), G448S (nâ=â1), G432C (nâ=â1) and P216S (nâ=â1) resistance alleles originating from either India, the Netherlands or France. The effects of PC1244 were confirmed in an in vitro model of the human alveolus and in vivo in temporarily neutropenic, immunocompromised mice. RESULTS: PC1244 exhibited potent inhibition [geometric mean MIC (range), 1.0 mg/L (0.125 to >8 mg/L)] of growth of A. fumigatus strains carrying cyp51A gene mutations, showing much greater potency than voriconazole [15 mg/L (0.5 to >16 mg/L)], and an effect similar to those on other azole-susceptible Aspergillus spp. (Aspergillus flavus, Aspergillus terreus, Aspergillus tubingensis, Aspergillus nidulans, Aspergillus niger, Aspergillus nomius, Aspergillus tamarii) (0.18-1 mg/L). In TR34/L98H and TR46/Y121F/T289A A. fumigatus-infected in vitro human alveolus models, PC1244 achieved superior inhibition (IC50, 0.25 and 0.34 mg/L, respectively) compared with that of voriconazole (IC90, >3 mg/L and >10 mg/L, respectively). In vivo, once-daily intranasal administration of PC1244 (0.56-70 µg/mouse) to the A. fumigatus (AF91 with M220V)-infected mice reduced pulmonary fungal load and serum galactomannan more than intranasal posaconazole. CONCLUSIONS: PC1244 has the potential to become a novel topical treatment of azole-resistant pulmonary aspergillosis.
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Antifúngicos/farmacología , Aspergillus/efectos de los fármacos , Triazoles/farmacología , Animales , Aspergillus/clasificación , Aspergillus/aislamiento & purificación , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Francia , Galactosa/análogos & derivados , Humanos , India , Pulmón/microbiología , Mananos/sangre , Ratones , Pruebas de Sensibilidad Microbiana , Países Bajos , Aspergilosis Pulmonar/microbiología , Resultado del Tratamiento , Triazoles/administración & dosificaciónRESUMEN
BACKGROUND AND PURPOSE: Effective anti-respiratory syncytial virus (RSV) agents are still not available for clinical use. Current major targets are virus surface proteins, such as a fusion protein involved in viral entry, but agents effective after RSV infection is established are required. Here we have investigated the effects of late therapeutic intervention with a novel inhaled RSV polymerase inhibitor, PC786, on RSV infection in human airway epithelium. EXPERIMENTAL APPROACH: Air liquid interface-cultured bronchial or small airway epithelium was infected with RSVA2. PC786 was applied apically or basolaterally once daily following peak virus load on Day 3 post inoculation. Apical wash was collected daily for determination of viral burden by PCR and plaque assay (primary endpoints) and biomarker analyses. The effects were compared with those of ALS-8112, an anti-RSV nucleoside analogue, and GS-5806, a fusion-protein inhibitor, which were treated basolaterally. KEY RESULTS: Late intervention with GS-5806 did not show significant anti-viral effects, but PC786 produced potent, concentration-dependent inhibition of viral replication with viral load falling below detectable limits 3 days after treatment commenced in airway epithelium. These effects were superior to those of ALS-8112. PC786 showed inhibitory activities against RSV-induced increases of CCL5, IL-6, double-strand DNA and mucin. The effects of PC786 were also confirmed in small airway epithelium. CONCLUSION AND IMPLICATIONS: Late therapeutic intervention with the RSV polymerase inhibitor, PC786, reduced the viral burden quickly in human airway epithelium. Thus, PC786 demonstrates the potential to be an effective therapeutic agent to treat active RSV infection.