Clinical antiviral efficacy of favipiravir in early COVID-19 (PLATCOV): an open-label, randomised, controlled, adaptive platform trial.

In early symptomatic COVID-19 treatment, high dose oral favipiravir did not accelerate viral clearance. BACKGROUND: Favipiravir, an anti-influenza drug, has in vitro antiviral activity against SARS-CoV-2. Clinical trial evidence to date is inconclusive. Favipiravir has been recommended for the treatment of COVID-19 in some countries. METHODS: In a multicentre open-label, randomised, controlled, adaptive platform trial, low-risk adult patients with early symptomatic COVID-19 were randomised to one of ten treatment arms including high dose oral favipiravir (3.6g on day 0 followed by 1.6g daily to complete 7 days treatment) or no study drug. The primary outcome was the rate of viral clearance (derived under a linear mixed-effects model from the daily log10 viral densities in standardised duplicate oropharyngeal swab eluates taken daily over 8 days [18 swabs per patient]), assessed in a modified intention-to-treat population (mITT). The safety population included all patients who received at least one dose of the allocated intervention. This ongoing adaptive platform trial was registered at ClinicalTrials.gov (NCT05041907) on 13/09/2021. RESULTS: In the final analysis, the mITT population contained data from 114 patients randomised to favipiravir and 126 patients randomised concurrently to no study drug. Under the linear mixed-effects model fitted to all oropharyngeal viral density estimates in the first 8 days from randomisation (4,318 swabs), there was no difference in the rate of viral clearance between patients given favipiravir and patients receiving no study drug; a -1% (95% credible interval: -14 to 14%) difference. High dose favipiravir was well-tolerated. INTERPRETATION: Favipiravir does not accelerate viral clearance in early symptomatic COVID-19. The viral clearance rate estimated from quantitative measurements of oropharyngeal eluate viral densities assesses the antiviral efficacy of drugs in vivo with comparatively few studied patients.

Clinical Antiviral Efficacy of Remdesivir in Coronavirus Disease 2019: An Open-Label, Randomized Controlled Adaptive Platform Trial (PLATCOV).

BACKGROUND: Uncertainty over the therapeutic benefit of parenteral remdesivir in coronavirus disease 2019 (COVID-19) has resulted in varying treatment guidelines. METHODS: In a multicenter open-label, controlled, adaptive, pharmacometric platform trial, low-risk adult patients with early symptomatic COVID-19 were randomized to 1 of 8 treatment arms including intravenous remdesivir (200 mg followed by 100 mg daily for 5 days) or no study drug. The primary outcome was the rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clearance (estimated under a linear model fit to the daily log10 viral densities, days 0-7) in standardized duplicate oropharyngeal swab eluates, in a modified intention-to-treat population. This ongoing adaptive trial is registered at ClinicalTrials.gov (NCT05041907). RESULTS: The 2 study arms enrolled 131 patients (remdesivir n = 67, no study drug n = 64) and estimated viral clearance rates from a median of 18 swab samples per patient (a total of 2356 quantitative polymerase chain reactions). Under the linear model, compared with the contemporaneous control arm (no study drug), remdesivir accelerated mean estimated viral clearance by 42% (95% credible interval, 18%-73%). CONCLUSIONS: Parenteral remdesivir accelerates viral clearance in early symptomatic COVID-19. Pharmacometric assessment of therapeutics using the method described can determine in vivo clinical antiviral efficacy rapidly and efficiently.

Pharmacometrics of high-dose ivermectin in early COVID-19 from an open label, randomized, controlled adaptive platform trial (PLATCOV).

Background: There is no generally accepted methodology for in vivo assessment of antiviral activity in SARS-CoV-2 infections. Ivermectin has been recommended widely as a treatment of COVID-19, but whether it has clinically significant antiviral activity in vivo is uncertain. Methods: In a multicentre open label, randomized, controlled adaptive platform trial, adult patients with early symptomatic COVID-19 were randomized to one of six treatment arms including high-dose oral ivermectin (600 µg/kg daily for 7 days), the monoclonal antibodies casirivimab and imdevimab (600 mg/600 mg), and no study drug. The primary outcome was the comparison of viral clearance rates in the modified intention-to-treat population. This was derived from daily log10 viral densities in standardized duplicate oropharyngeal swab eluates. This ongoing trial is registered at https://clinicaltrials.gov/ (NCT05041907). Results: Randomization to the ivermectin arm was stopped after enrolling 205 patients into all arms, as the prespecified futility threshold was reached. Following ivermectin, the mean estimated rate of SARS-CoV-2 viral clearance was 9.1% slower (95% confidence interval [CI] -27.2% to +11.8%; n=45) than in the no drug arm (n=41), whereas in a preliminary analysis of the casirivimab/imdevimab arm it was 52.3% faster (95% CI +7.0% to +115.1%; n=10 (Delta variant) vs. n=41). Conclusions: High-dose ivermectin did not have measurable antiviral activity in early symptomatic COVID-19. Pharmacometric evaluation of viral clearance rate from frequent serial oropharyngeal qPCR viral density estimates is a highly efficient and well-tolerated method of assessing SARS-CoV-2 antiviral therapeutics in vitro. Funding: 'Finding treatments for COVID-19: A phase 2 multi-centre adaptive platform trial to assess antiviral pharmacodynamics in early symptomatic COVID-19 (PLAT-COV)' is supported by the Wellcome Trust Grant ref: 223195/Z/21/Z through the COVID-19 Therapeutics Accelerator. Clinical trial number: NCT05041907.

Protection from UVB Toxicity in Human Keratinocytes by Thailand Native Herbs Extracts.

Thai traditional medicine employs a wide range of indigenous herbs in the forms of tincture or tea for the cure of skin and systemic inflammatory diseases. The protection by Thai plants extracts against UVB DNA damage and cytotoxicity was investigated in human keratinocytes. Petroleum ether, dichloromethane and ethanol extracts were prepared from 15 Thai herb species, and the total phenolic and flavonoid contents, the antioxidant and UV-absorbing properties were assessed by standard procedures. Cytoprotective effects were evaluated on the basis of cell survival, caspase-3 activity and pyrimidine dimers determination. High total phenolic and flavonoid contents were found in the ethanol and dichloromethane fractions. Dichloromethane extract of turmeric was shown to possess the highest antioxidant activity. The maximum UV absorptions were found in the ethanol extract of turmeric and in the dichloromethane extract of ginger. These extracts stimulated the synthesis of Thioredoxin 1, an antioxidant protein, and could protect human HaCaT keratinocytes from UV-induced DNA damage and cytotoxicity. The present data support the utilization of turmeric and ginger extracts in anti-UV cosmetic pharmaceuticals.

Similarities and differences in the biogenesis, processing and lysosomal targeting between zebrafish and human pro-Cathepsin D: functional implications.

The lysosomal protease Cathepsin D (CD) plays a role in neurodegenerative diseases, cancer, and embryo-fetus abnormalities. It is therefore of interest to know how this protein is synthesized in animal species used for modeling human diseases. Zebrafish (Danio rerio) is emerging as a valuable 'in vivo' vertebrate model for several human diseases. We have characterized the biogenetic pathways of zebrafish and human CD transgenically expressed in both human SH-SY5Y cells and zebrafish PAC2 cells. Differently from human CD, zebrafish CD was synthesized as a mono-glycosylated precursor (pro-CD) that was eventually processed into a single-chain mature polypeptide. In PAC2 cells, ammonium chloride and chloroquine impaired the N-glycosylation, and greatly stimulated the secretion, of pro-CD; still, a portion of un-glycosylated pro-CD reached the lysosomes and was processed to mature CD. The treatment with tunicamycin, which abrogates N-glycosylation, resulted in a similar effect. Zebrafish pro-CD was correctly processed when expressed in human cells, and its glycosylation, transport and maturation were not impaired by ammonium chloride. On the contrary, the transport and processing of human pro-CD expressed in zebrafish cells were profoundly altered: while the intermediate single-chain was not detectable, a small amount of double-chain mature CD still formed. This fact indicates that the enzyme machinery for single- to double-chain processing of mammal CD is present in zebrafish. Our data highlight the respective impact of the information imparted by the primary sequence and of the cellular transport and processing machineries in the biogenesis of lysosomal CD.

Biocompatibility, endocytosis, and intracellular trafficking of mesoporous silica and polystyrene nanoparticles in ovarian cancer cells: effects of size and surface charge groups.

BACKGROUND AND METHODS: Nanoparticles engineered to carry both a chemotherapeutic drug and a sensitive imaging probe are valid tools for early detection of cancer cells and to monitor the cytotoxic effects of anticancer treatment simultaneously. Here we report on the effect of size (10-30 nm versus 50 nm), type of material (mesoporous silica versus polystyrene), and surface charge functionalization (none, amine groups, or carboxyl groups) on biocompatibility, uptake, compartmentalization, and intracellular retention of fluorescently labeled nanoparticles in cultured human ovarian cancer cells. We also investigated the involvement of caveolae in the mechanism of uptake of nanoparticles. RESULTS: We found that mesoporous silica nanoparticles entered via caveolae-mediated endocytosis and reached the lysosomes; however, while the 50 nm nanoparticles permanently resided within these organelles, the 10 nm nanoparticles soon relocated in the cytoplasm. Naked 10 nm mesoporous silica nanoparticles showed the highest and 50 nm carboxyl-modified mesoporous silica nanoparticles the lowest uptake rates, respectively. Polystyrene nanoparticle uptake also occurred via a caveolae-independent pathway, and was negatively affected by serum. The 30 nm carboxyl-modified polystyrene nanoparticles did not localize in lysosomes and were not toxic, while the 50 nm amine-modified polystyrene nanoparticles accumulated within lysosomes and eventually caused cell death. Ovarian cancer cells expressing caveolin-1 were more likely to endocytose these nanoparticles. CONCLUSION: These data highlight the importance of considering both the physicochemical characteristics (ie, material, size and surface charge on chemical groups) of nanoparticles and the biochemical composition of the cell membrane when choosing the most suitable nanotheranostics for targeting cancer cells.

Labeling and exocytosis of secretory compartments in RBL mastocytes by polystyrene and mesoporous silica nanoparticles.

BACKGROUND: For a safe 'in vivo' biomedical utilization of nanoparticles, it is essential to assess not only biocompatibility, but also the potential to trigger unwanted side effects at both cellular and tissue levels. Mastocytes (cells having secretory granules containing cytokines, vasoactive amine, and proteases) play a pivotal role in the immune and inflammatory responses against exogenous toxins. Mastocytes are also recruited in the tumor stroma and are involved in tumor vascularization and growth. AIM AND METHODS: In this work, mastocyte-like rat basophilic leukemia (RBL) cells were used to investigate whether carboxyl-modified 30 nm polystyrene (PS) nanoparticles (NPs) and naked mesoporous silica (MPS) 10 nm NPs are able to label the secretory inflammatory granules, and possibly induce exocytosis of these granules. Uptake, cellular retention and localization of fluorescent NPs were analyzed by cytofluorometry and microscope imaging. RESULTS: OUR FINDINGS WERE THAT: (1) secretory granules of mastocytes are accessible by NPs via endocytosis; (2) PS and MPS silica NPs label two distinct subpopulations of inflammatory granules in RBL mastocytes; and (3) PS NPs induce calcium-dependent exocytosis of inflammatory granules. CONCLUSION: These findings highlight the value of NPs for live imaging of inflammatory processes, and also have important implications for the clinical use of PS-based NPs, due to their potential to trigger the unwanted activation of mastocytes.

cDNA-AFLP analysis of differential gene expression in human hepatoma cells (HepG2) upon dengue virus infection.

In infectious diseases, the disease pathogenesis is the outcome of the interaction between the genome of the host and the genome of the pathogen. Despite the wide distribution of dengue infections in the world, and the large number of annual infections, few studies have investigated how the dengue genome alters the global transcriptional profile of the host cell. To investigate alterations in the liver cell transcriptome in response to dengue virus infection, liver cells (HepG2) were infected with dengue serotype 2 at MOI 5 and at 3 days post-infection RNA extracted and analyzed by cDNA-AFLP in parallel with mock-infected cells. From 73 primer combinations over 5,000 transcription-derived fragments (TDFs) were observed, of which approximately 10% were regulated differentially in response to infection. Sixty-five TDFs were subsequently cloned and sequenced and 27 unique gene transcripts identified. Semi-quantitative reverse transcription (RT)-PCR was used to validate the expression of 12 of these genes and 10 transcripts (CK2, KIAA509, HSP70, AK3L, NIPA, PHIP, RiboS4, JEM-1, MALT1, and HSI12044) were confirmed to be differentially regulated, with four transcripts (HSP70, NIPA, RiboS4, and JEM-1) showing a greater than twofold regulation. These results suggest that the expression of a large number of genes is altered in response to dengue virus infection of liver cells, and that cDNA-AFLP is a useful tool for obtaining information on both characterized and as yet uncharacterized transcripts whose expression is altered during the infection process.

A simplified PCR methodology for semiquantitatively analyzing dengue viruses.

The standard methodology for titrating dengue viruses, the plaque assay, is slow, time consuming and relatively expensive. Other methods require machinery that may not be routinely accessible to all researchers, particularly those in developing nations. We therefore sought to develop a rapid, simplified semiquantitative polymerase chain reaction (PCR) methodology based on the use of a template mimic. In particular, it was desired that the mimic should be applicable for use a DNA template to avoid the requirement for producing an in vitro RNA transcript. A 511 base pair fragment of the capsid-PrM junction of dengue serotype 4 was cloned into pGEM-T Easy vector and subjected to splicing overlap extension-PCR to generate a 160 base pair deletion. The deleted plasmid mimic competed competitively against the parent plasmid as well as the first strand cDNA of all four dengue viruses. The primers used are specific for the dengue virus, and no product was seen with first strand cDNA from a closely related flavivirus, Japanese encephalitis virus. Under the conditions used, accurate quantitation of the dengue viruses in the range of 10(3) to 10(6) pfu can be achieved in a single day, as opposed to the 7 days required for conventional plaque assay.