Background Development of an effective antiviral drug for Coronavirus Disease 2019 (COVID-19) is a global health priority. Although several candidate drugs have been identified through in vitro and in vivo models, consistent and compelling evidence from clinical studies is limited. The lack of evidence from clinical trials may stem in part from the imperfect design of the trials. We investigated how clinical trials for antivirals need to be designed, especially focusing on the sample size in randomized controlled trials. Methods and findings A modeling study was conducted to help understand the reasons behind inconsistent clinical trial findings and to design better clinical trials. We first analyzed longitudinal viral load data for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) without antiviral treatment by use of a within-host virus dynamics model. The fitted viral load was categorized into 3 different groups by a clustering approach. Comparison of the estimated parameters showed that the 3 distinct groups were characterized by different virus decay rates (p-value < 0.001). The mean decay rates were 1.17 d−1 (95% CI: 1.06 to 1.27 d−1), 0.777 d−1 (0.716 to 0.838 d−1), and 0.450 d−1 (0.378 to 0.522 d−1) for the 3 groups, respectively. Such heterogeneity in virus dynamics could be a confounding variable if it is associated with treatment allocation in compassionate use programs (i.e., observational studies). Subsequently, we mimicked randomized controlled trials of antivirals by simulation. An antiviral effect causing a 95% to 99% reduction in viral replication was added to the model. To be realistic, we assumed that randomization and treatment are initiated with some time lag after symptom onset. Using the duration of virus shedding as an outcome, the sample size to detect a statistically significant mean difference between the treatment and placebo groups (1:1 allocation) was 13,603 and 11,670 (when the antiviral effect was 95% and 99%, respectively) per group if all patients are enrolled regardless of timing of randomization. The sample size was reduced to 584 and 458 (when the antiviral effect was 95% and 99%, respectively) if only patients who are treated within 1 day of symptom onset are enrolled. We confirmed the sample size was similarly reduced when using cumulative viral load in log scale as an outcome. We used a conventional virus dynamics model, which may not fully reflect the detailed mechanisms of viral dynamics of SARS-CoV-2. The model needs to be calibrated in terms of both parameter settings and model structure, which would yield more reliable sample size calculation. Conclusions In this study, we found that estimated association in observational studies can be biased due to large heterogeneity in viral dynamics among infected individuals, and statistically significant effect in randomized controlled trials may be difficult to be detected due to small sample size. The sample size can be dramatically reduced by recruiting patients immediately after developing symptoms. We believe this is the first study investigated the study design of clinical trials for antiviral treatment using the viral dynamics model.
Aims The prognostic value of genetic variants for predicting lethal arrhythmic events (LAEs) in Brugada syndrome (BrS) remains controversial. We investigated whether the functional curation of SCN5A variations improves prognostic predictability. Methods and results Using a heterologous expression system and whole-cell patch clamping, we functionally characterized 22 variants of unknown significance (VUSs) among 55 SCN5A mutations previously curated using in silico prediction algorithms in the Japanese BrS registry (n = 415). According to the loss-of-function (LOF) properties, SCN5A mutation carriers (n = 60) were divided into two groups: LOF-SCN5A mutations and non-LOF SCN5A variations. Functionally proven LOF-SCN5A mutation carriers (n = 45) showed significantly severer electrocardiographic conduction abnormalities and worse prognosis associated with earlier manifestations of LAEs (7.9%/year) than in silico algorithm-predicted SCN5A carriers (5.1%/year) or all BrS probands (2.5%/year). Notably, non-LOF SCN5A variation carriers (n = 15) exhibited no LAEs during the follow-up period. Multivariate analysis demonstrated that only LOF-SCN5A mutations and a history of aborted cardiac arrest were significant predictors of LAEs. Gene-based association studies using whole-exome sequencing data on another independent SCN5A mutation-negative BrS cohort (n = 288) showed no significant enrichment of rare variants in 16 985 genes including 22 non-SCN5A BrS-associated genes as compared with controls (n = 372). Furthermore, rare variations of non-SCN5A BrS-associated genes did not affect LAE-free survival curves. Conclusion In vitro functional validation is key to classifying the pathogenicity of SCN5A VUSs and for risk stratification of genetic predictors of LAEs. Functionally proven LOF-SCN5A mutations are genetic burdens of sudden death in BrS, but evidence for other BrS-associated genes is elusive.
Objective. To clarify the significance of immunometabolism in systemic lupus erythematosus (SLE), and to determine the effect of calcium/calmodulin-dependent protein kinase 4 (CaMK4) on T cell metabolism.Methods. Metabolomic profiling was performed using capillary electrophoresis mass spectrometry in naive T cells from MRL/lpr mice treated with anti-CD3/CD28 antibodies in the absence or presence of a CaMK4 inhibitor (KN-93). The expression of GLUT1 and CaMK4 in CD4+ T cells from healthy controls (n = 16), patients with inactive SLE (n = 13), and patients with active SLE (n = 14) was examined by flow cytometry and quantitative polymerase chain reaction. In vitro experiments were performed to determine the effect of KN-93 on the expression of GLUT1 during Th17 cell differentiation in T cells from patients with SLE.Results. CaMK4 inhibition significantly decreased the levels of glycolytic intermediates such as glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, pyruvate, and lactate (P < 0.05), whereas it did not affect the levels of the pentose phosphate pathway intermediates such as 6-phosphod-gluconate, ribulose-5-phosphate, ribose-5-phosphate, and phosphoribosyl pyrophosphate. The expression levels of GLUT1 and CaMK4 in effector memory CD4+ T cells were significantly higher in patients with active SLE compared to healthy controls (P < 0.01 and P < 0.05, respectively) and patients with inactive SLE (P < 0.05 and P < 0.01, respectively). A functional analysis revealed that CaMK4 inhibition decreased the expression of GLUT1 during Th17 cell differentiation (P < 0.01), followed by a reduction of interleukin-17 (IL-17) production (P < 0.05).Conclusion. The results of the study indicate that the activity of CaMK4 could be responsible for glycolysis, which contributes to the production of IL-17, and CaMK4 may contribute to aberrant expression of GLUT1 in T cells from patients with active SLE.
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