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Background: Reliable biomarkers of COVID-19 severity and outcomes are critically needed for clinical and research applications. We evaluated associations between anti-Spike IgG and SARS-COV-2 nucleocapsid antigen (N Ag) in plasma with clinical outcomes in outpatients with COVID-19. Method(s): We used data from 229 non-hospitalized, US-based adults with COVID-19 who enrolled between January and July 2021 into the placebo arm of the ACTIV-2/A5401 platform trial within 10 days of symptom onset. Pretreatment (day 0) plasma was analyzed by the quantitative Simoa SARS-CoV-2 IgG antibody (anti-Spike) assay (lower limit of quantification [LLoQ] 0.77ug/ mL), and the quantitative Simoa SARS-CoV-2 N Protein Advantage (Quanterix) measuring N Ag (LLoQ 3pg/mL). In addition to analyses for < LLoQ vs >=LLoQ anti-Spike and N Ag, we categorized participants into five N Ag groups (< 3 pg/ml;3-< 100 pg/ml;100-< 1,000 pg/ml;1,000-< 2,500 pg/ml;>=2,500 pg/ ml). Associations between SARS-CoV-2 anti-Spike and N Ag levels and clinical outcomes (all-cause hospitalization/death through day 28 and time to symptom improvement or resolution for two consecutive days from day 0 status) were estimated using log-binomial and Cox regression models, respectively. Result(s): At day 0, 40% had anti-Spike levels >=LLoQ and 64% of participants had plasma N Ag levels >=LLoQ. Participants with anti-Spike levels < LLoQ compared to those who had quantifiable anti-Spike at day 0, had an increased risk of hospitalization/death (16% vs 2%, RR [95% confidence interval (CI)]: 7.3 [1.8, 30.1]), and a significantly longer time to symptom improvement (median [Q1, Q3] 14 days [8, >27] vs 9 days [4, 16], hazard ratio [HR]: 0.6 [95%: CI: 0.4, 0.8], p< 0.001). Participants with higher N Ag levels at day 0 had an increased risk of hospitalization or death, ranging from 1% for < 3 pg/ml to 70% for >=2500 pg/ml (Figure). Compared to individuals who had N Ag levels < LLoQ at day 0, those in the highest category of N Ag levels (>=2500 pg/mL) experienced a significantly longer time to symptom improvement (median [Q1, Q3]: 25 days [13, >27] vs 10 days [5, 20];HR: 0.4 [95% CI: 0.2, 0.7];p=0.04). Conclusion(s): At study entry, the absence of Spike antibodies and higher levels of plasma SARS-CoV-2 N Ag predicted hospitalizations and death in untreated outpatients with COVID-19. These parameters may serve as informative biomarkers for risk stratification in the evaluation of outpatients with COVID-19. (Figure Presented).
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Background: SARS-CoV-2 variants resistant to monoclonal antibodies, and drug-drug interactions and potential mutagenicity of direct acting antivirals, heightens the need for additional therapeutics to prevent progression to severe COVID-19. Exogenous interferon beta is a promising therapeutic option against SARS-CoV-2 given its broad-spectrum antiviral activity and data suggesting impaired endogenous IFN production in individuals with severe disease. Method(s): The safety and efficacy of orally inhaled nebulized interferon-beta1a (SNG001) was evaluated in a Phase II randomized controlled trial on the ACTIV-2/ A5401 platform (NCT04518410). Adult outpatients with confirmed SARS-CoV-2 infection within 10 days of symptom onset were randomized to SNG001 once daily for 14 days or blinded pooled placebo. Primary outcomes included treatment-emergent Grade >=3 adverse event (TEAE) through day 28;time to symptom improvement of 13 targeted COVID-19 symptoms collected by daily study diary through day 28;and SARS-CoV-2 RNA < lower limit of quantification (LLoQ) from nasopharyngeal (NP) swabs at days 3, 7, and 14. All-cause hospitalization or death through day 28 was a key secondary outcome. Result(s): Of 221 participants enrolled at 25 US sites between February and August 2021, 220 (110 SNG001, 110 placebo) initiated study intervention, with a median age of 40 years, 55% female, and 20% SARS-CoV-2 vaccinated. There was no significant difference between SNG001 and placebo in Grade >=3 TEAEs (4% vs 8%, Fisher's exact test p=0.25). Median time to symptom improvement was 13 days for SNG001 and 9 days for placebo (Gehan-Wilcoxon test p=0.17). There was no difference in the proportion of participants with SARS-CoV-2 RNA < LLoQ at day 3, 7 or 14 (SNG001 vs placebo, Day 3: 28% vs. 39%;Day 7: 65% vs. 66%;Day 10: 91% vs. 91%;joint Wald test p=0.41). There were fewer hospitalizations with SNG001 (n=1;1%) compared with placebo (n=7;6%), but this difference was not statistically significant (Fisher's exact test p=0.07;Figure). All hospitalizations were due to COVID-19 and occurred among unvaccinated participants without protocol-defined high-risk factors. Conclusion(s): Inhaled nebulized SNG001 was safe and well tolerated but did not reduce SARS-CoV-2 RNA levels in the nasopharynx nor decrease time to improvement of COVID-19 symptoms in outpatients with mild-to-moderate COVID-19. The non-statistically significant decrease in hospitalizations among SNG001 participants warrants further investigation in a phase 3 clinical trial. Cumulative incidence of hospitalization or death comparing SNG001 vs. placebo.
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Background: Within the ACTIV-2/A5401 platform (NCT04518410), the safety and efficacy of tixagevimab/cilgavimab (T/C), an anti-SARS-CoV-2 monoclonal antibody combination, was studied in outpatients with COVID-19. Intravenous (IV) and intramuscular (IM) administration of T/C were assessed. Method(s): Non-hospitalized adults >=18 years enrolled within 10 days of positive SARS-CoV-2 test and symptom onset. Participants at higher risk of disease progression were eligible for IV T/C 300mg (150mg each component) or placebo;all were eligible for IM T/C 600mg (300mg each) administered to the lateral thigh or placebo. Co-primary outcomes were: time to symptom improvement through day 28;nasopharyngeal (NP) SARS-CoV-2 RNA below lower limit of quantification (LLoQ) on days 3, 7 or 14;and treatment emergent Grade >=3 adverse events. Result(s): Between February and May 2021, 223 participants (106 T/C, 117 placebo) initiated study intervention and were included in the IM analysis and 114 participants (58 T/C, 56 placebo) in the IV analysis;the IV study was stopped early for administrative reasons. Both studies enrolled 45% Latinx;the IM and IV populations included 12% and 19% Black participants, 49% and 59% female sex at birth, and median age was 39 and 44 years, respectively, all of which were balanced between active vs placebo for each. Median (IQR) days from symptom onset at enrollment was 6 (4, 7). There were no differences in time to symptom improvement comparing IM T/C to placebo (median 8 (IQR 7, 12) vs 10 (8, 13) days;p=0.35) or IV T/C to placebo (11 (9, 15) vs 10 (7, 15) days;p=0.71). A significantly greater proportion (80%) in the IM T/C arm had NP SARS-CoV-2 RNA below LLoQ at day 7 compared to placebo (65%), but not days 3 or 14, overall p=0.003 across visits. Secondary and post-hoc analyses revealed antiviral effects within the smaller IV study. There was no difference in Grade >=3 AEs with either administration route. Fewer participants were hospitalized who received T/C vs placebo (4 vs 7 in IM group;0 vs 4 in IV group), neither group reaching statistical significance. Conclusion(s): Tixagevimab/cilgavimab administered IM or IV was well-tolerated and demonstrated antiviral activity and a trend towards fewer hospitalizations, but did not change time to symptom improvement in mild-to-moderate COVID-19 compared to placebo. Monoclonal antibodies administered intramuscularly to the thigh may present a valuable alternative for early SARSCoV-2 infection. Virologic Outcomes of Tixagevimab/Cilgavimab treatment 600mg IM (panels A and B) or 300mg IV (panels C and D) versus placebo.
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Molnupiravir, a broad-spectrum antiviral is an isopropyl ester prodrug of beta-D-N4-hydroxycytidine. Molnupiravir targets RNA-dependent RNA-polymerase enzyme of the viruses. A new stability-indicating HPLC-method was developed to determine related substances and assay of molnupiravir. Separation was achieved by using Shim-pack GWS C18 column. The method was validated according to current ICH requirements. The calibration plot gave a linear relationship for all known analytes over the concentration range from LOQ to 200%. LOD and LOQ for all known analytes were found in 0.05-0.08 microg mL-1 and 0.12-0.20 microg mL-1, respectively, the mean recovery was found to be 97.79-102.44 %. Study showed that the method, results of robustness, solution stability studies are precise and within the acceptable limits. Molnupiravir was found to degrade in acid, alkali, and oxidative conditions, and was stable in thermal, moisture, and photolytic degradation condition. The method is simple, accurate, precise, and reproducible for routine purity analysis of drug-samples.Copyright © 2022 Indian Drug Manufacturers' Association. All rights reserved.
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Effective drug delivery to the brain is critical for the treatment of glioblastoma (GBM), an aggressive and invasive primary brain tumor that has a dismal prognosis. Radiation therapy, the mainstay of brain tumor treatment, works by inducing DNA damage. Therefore, inhibiting DNA damage response (DDR) pathways can sensitize tumor cells to radiation and enhance cytotoxicity. AZD1390 is an inhibitor of ataxia-telangiectasia mutated kinase, a critical regulator of DDR. Our in vivo studies in the mouse indicate that delivery of AZD1390 to the central nervous system (CNS) is restricted due to active efflux by P-glycoprotein (P-gp). The free fraction of AZD1390 in brain and spinal cord were found to be low, thereby reducing the partitioning of free drug to these organs. Coadministration of an efflux inhibitor significantly increased CNS exposure of AZD1390. No differences were observed in distribution of AZD1390 within different anatomic regions of CNS, and the functional activity of P-gp and breast cancer resistance protein also remained the same across brain regions. In an intracranial GBM patient-derived xenograft model, AZD1390 accumulation was higher in the tumor core and rim compared with surrounding brain. Despite this heterogenous delivery within tumor-bearing brain, AZD1390 concentrations in normal brain, tumor rim, and tumor core were above in vitro effective radiosensitizing concentrations. These results indicate that despite being a substrate of efflux in the mouse brain, sufficient AZD1390 exposure is anticipated even in regions of normal brain. SIGNIFICANCE STATEMENT Given the invasive nature of glioblastoma (GBM), tumor cells are often protected by an intact blood-brain barrier, requiring the development of brain-penetrant molecules for effective treatment. We show that efflux mediated by P-glycoprotein (P-gp) limits central nervous system (CNS) distribution of AZD1390 and that there are no distributional differences within anatomical regions of CNS. Despite efflux by P-gp, concentrations effective for potent radiosensitization are achieved in GBM tumor-bearing mouse brains, indicating that AZD1390 is an attractive molecule for clinical development of brain tumors.Copyright © 2022 American Society for Pharmacology and Experimental Therapy. All rights reserved.
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Kabasura Kudineer is a polyherbal decoction of the Siddha medical system (an Indian system of medicine), traditionally used to cure fever, colds, coughs, and respiratory ailments. The government of India had recommended Kabasura Kudineer as one of many preventive/treatment measures for COVID-19. Kabasura Kudineer Choornam is an admixed coarse powder of 15 herbs and its decoction is Kabasura Kudineer. The chemical constituents in the 15 herbs used for the preparation of the Choornam are known but the constituents present in the Kabasura Kudineer (decoction) are unidentified. Piperine, vasicine and eugenol are known for their potent activity against respiratory tract infections;hence, they were selected as marker compounds. The present work was planned to simultaneously quantify piperine, vasicine and eugenol in Kabasura Kudineer by the HPTLC method. The optimised mobile phase was toluene: ethyl acetate: methanol: ammonia (5:9:3:0.5, v/v/v/v), and the scanning was carried out at 287 nm. The Rf values of piperine, vasicine and eugenol were found to be 0.70, 0.32 and 0.82, respectively. The linearity range of piperine and vasicine was 500-3000 ng spot-1 and it was 10-60 ng spot-1 for eugenol. The quantities of piperine, vasicine and eugenol in Kabasura Kudineer (100 mL) were 0.03, 0.056 and 0.035 % w/v, respectively. This developed method can be used to simultaneously quantify piperine, vasicine and eugenol in any polyherbal formulation.Copyright © 2023, Informatics Publishing Limited and Society for Biocontrol Advancement. All rights reserved.
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Herein, we establish a novel isothermal digital amplification system termed digital nicking and extension chain reaction system-based amplification (dNESBA) by utilizing the isothermal NESBA technique and the newly developed miniaturized fluorescence monitoring system (mFMS). dNESBA enables parallel isothermal NESBA reactions in more than 10,000 localized droplet microreactors and read the fluorescence signals rapidly in 150 s by mFMS. This system could identify the genomic RNA (gRNA) extracted from target respiratory syncytial virus A (RSV A) as low as 10 copies with remarkable specificity. The practical applicability of dNESBA was also successfully verified by reliably detecting the gRNA in the artificial sputum samples with excellent reproducibility and accuracy. Due to the intrinsic advantages of isothermal amplifying technique including the elimination of the requirement of thermocycling device and the enhanced portability of the miniaturized read-out equipment, the dNESBA technique equipped with mFMS could serve as a promising platform system to achieve point-of-care (POC) digital molecular diagnostics, enabling absolute and ultra-sensitive quantification of various infectious pathogens even in an early stage.Copyright © 2023
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Favipiravir is a potential repurpose moiety to treat COVID-19 by depletion of virus load in infectious patients. To analyze and separate Favipiravir with remarkable efficiency, X-Bridge C8 column (150 x 4.6 mm, 5 µ) and a solvent phase of 0.1% TEA and acetonitrile (40:60 v/v) with 1-mL/min flow rate were used. The eluted favipiravir and possible degradants were detected at 225 nm. Further, the process was validated by using ICH (Q2R1) guidelines to ensure the method's suitability in the pharmaceutical sector. The RT of Favipiravir was observed at 3.7 min with good linearity of 2 to 30 µg/mL. %RSD of both system and method precision was assessed in the series of 0.32 to 0.98. The mean percentage recovery of Favipiravir was in the range of 99.0–100.4%. The limit of detection (LoD) and limit of quantification (LoQ) were assessed to be 0.024 and 0.084 μg/mL for favipiravir. The outcomes confirmed that the projected approach was economical, insightful, simple and precise with better sensitivity. Investigation of Favipiravir in the incidence of a variety of stressed or forced degradation environments ensures stability indicating quality of the developed approach. © 2023, Dr. Yashwant Research Labs Pvt. Ltd.. All rights reserved.
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A fast procedure obtained by the combination of fabric phase extraction (FPSE) with high performance liquid chromatography (HPLC) has been developed and validated for the quantification of favipiravir (FVP) in human plasma and breast milk. A sol-gel polycaprolactone-block-polydimethylsiloxane-block-polycaprolactone (sol-gel PCAP-PDMS-PCAP) coated on 100% cellose cotton fabric was selected as the most efficient membrane for FPSE in human plasma and breast milk samples. HPLC-UV analysis were performed using a RP C18 column under isocratic conditions. Under these optimezed settings, the overall chromatographic analysis time was limited to only 5 min without encountering any observable matrix interferences. Following the method validation procedure, the herein assay shows a linear calibration curve over the range of 0.2–50 µg/mL and 0.5–25 µg/mL for plasma and breast milk, respectively. The method sensitivities in terms of limit of detection (LOD) and limit of quantification (LOQ), validated in both the matrices, have been found to be 0.06 and 0.2 µg/mL for plasma and 0.15 and 0.5 µg/mL for milk, respectively. Intraday and interday precision and trueness, accordingly to the International Guidelines, were validated and were below 3.61% for both the matrices. The herein method was further tested on real samples in order to highlight the applicability and the advantage for therapeutic drug monitoring (TDM) applications. To the best of our knowledge, this is the first validated FPSE-HPLC-UV method in human plasma and breast milk for TDM purposes applied on real samples. The validated method provides fast, simple, cost reduced, and sensitive assay for the direct quantification of favipiravir in real biological matrices, also appliyng a well-known rugged and cheap instrument configuration. © 2022 Elsevier B.V.
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This study explored the degradation behavior of three types of disposable face masks in simulated seawater via the accelerated aging experiments. Microplastics (MPs) and dissolved organic carbon (DOC) were monitored in UV- and thermal-treated mask suspensions and their concentrations increased slowly in the early stage at 50 ℃ and 58 ℃. Owing to the high energy supply, the release rates of MPs and DOC at 76 ℃ were much faster than the above two temperatures. The time-temperature superposition principle (TTSP) was used to superpose the MPs/DOC release kinetics from other tested temperatures to the reference temperature and its applicability was verified by the similar activation energy. Then, a release kinetics model was established and fitted well with the superposed MP data (R2 ≥ 0.96). Since less than 0.1 % of carbon was leached, the superposed DOC data was roughly modelled by the exponential function (R2 ≥ 0.90). According to the TTSP and the established kinetics models, about 15 years were estimated to decompose half of a certain marine mask waste, together with leaching 0.21 ± 0.02 mg∙g-mask−1 of DOC. If mask consumption remains the same before 2025, they would contribute 40000–230000 tonnes of MPs and 13–97 tonnes of DOC to the ocean by 2040. © 2022
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Background. Predictors of SARS-CoV-2 RNA levels and changes over time during early COVID-19 are not well characterized. Methods. ACTIV-2 is a phase II/III randomized, placebo-controlled, platform trial to evaluate investigational agents for treatment of COVID-19 in non-hospitalized adults. Participants enrolled within 10 days of symptom onset. Nasopharyngeal samples were collected for SARS-CoV-2 RNA testing on Days 0, 3, 7, 14 and 28;RNA was quantified with qPCR assay. SARS-CoV-2 seropositivity was defined as detectable IgG to any of nucleocapsid, receptor binding domain, S1 and S2 antigens by Bio-Plex multiplex assay. Censored linear regression and repeated measures Poisson models evaluated predictors of RNA including age, sex, race, ethnicity, risk of severe COVID-19, diabetes, BMI, obesity (BMI > 35 kg/m2) and serostatus. Results. The study enrolled 537 participants from Aug 2020 to July 2021 at US sites. Median age was 48 years;49% were female sex, >99% cis-gender, 83% white, 29% Hispanic/Latino, and 21% had BMI > 35 kg/m2. At Day 0, median symptom duration was 6 days, 50% were seropositive (2 were vaccinated) and 17% had RNA below the lower limit of quantification (LLoQ). Higher Day 0 RNA was associated with shorter symptom duration (Spearman correlation = -0.40, p< 0.001), as well as older age, white race, lower BMI and seronegativity, even when adjusting for symptom duration (all p< 0.03). Among the 203 on placebo with Day 0 RNA >= LLoQ, female sex had larger decreases in RNA at Day 3 vs male sex (difference in mean change: -0.8 log10 copies/mL (95% CI: -1.2, -0.4), p< 0.001) when adjusted for symptom duration and Day 0 RNA;this difference was also observed when evaluating the proportion with RNA < LLoQ at Day 3 (Risk Ratio (95% CI): 2.38 (1.11, 5.09)). Seropositivity at Day 0 was associated with higher probability of RNA < LLoQ at Days 3 and 7 (p< 0.001) in adjusted models. Seropositivity at Day 0 did not differ by sex. Conclusion. In this well characterized clinical trial cohort, shorter symptom duration, older age, white race, lower BMI and seronegativity were associated with higher RNA in early infection. Female sex and seropositivity were associated with earlier viral clearance. Further research is needed to determine if viral decay differences mediated by these host factors influence clinical outcomes.
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Background: Azathioprine is a purine analog metabolized to 6- mercaptopurine (6-MP) utilizing glutathione. Its high oral bioavailability and longer duration of action make it viable as a treatment for ulcerative colitis or as an anti-rejection medication for renal transplant patients. Specific experience in overdose with this agent is limited although toxicity mimics 6-MP including hepatotoxicity, delayed leukopenia, and acute interstitial nephritis. Case report: A 46 year old female (64 kg) with a history of ulcerative colitis, migraines, and anxiety presented with a selfreported intentional ingestion of 1000mg azathioprine and presented to care approximately 8 h post-ingestion. Her compliance with azathioprine preceding the ingestion was unclear. She reported taking her other medications as prescribed (tadalafil, sulfasalazine, fioricet, alprazolam) the day prior to presentation. Other than one episode of emesis without pill fragments, myalgias, headache she had no other symptoms. Her presenting vital signs were HR 84, RR 22, BP 90/63, T 36.2 degreeC. Initial labs included a normal chemistry profile, undetectable serum acetaminophen and salicylates, an ethanol level of 50 mg/dL and venous lactate of 1.6mmol/L. She received a total of 3 L of crystalloid IV fluids with improvement in blood pressure to 125/66 and was transferred for higher level of care. Due to the delay in presentation and well appearance, activated charcoal and hemodialysis were considered but deferred. While inpatient she had laboratory evaluation including CBC and differential every 8 h. In the ED she developed a fever, 38.1 degreeC. PCR testing for COVID-19 was negative. Whole blood thiopurine metabolites (Prometheus Biosciences, Test 3200) were sent approximately 33 h from time of ingestion. 6-thioguanine levels were 108 pmol/8x10degree8 RBC, below the therapeutic reference range (230-400 pmol/8x10degree8 RBC). 6-methylmercaptopurine metabolites were below the lower limit of quantification (761pmol/8x10degree8 RBC). Genetic testing for thiopurine S-methyltransferase was declined by the patient. She was hospitalized for 4 days and did not develop any substantial vital sign abnormalities or creatinine elevation. Her absolute neutrophil count dropped to 500/mm3 approximately 76 h post-ingestion, but started to improve 84 h post-ingestion and granulocyte-macrophage colony-stimulating factor was deferred. Her peak AST was 113 IU/L, approximately 46 h post-ingestion and returned to normal (16 IU/L) upon follow-up 7 days postingestion. White blood cell count 7 days post-ingestion was 4.3 K/mm3. Discussion(s): Azathioprine overdose is rarely reported in the literature. Case reports describe delayed leukopenia and hepatotoxicity from repeat supratherapeutic ingestions, however, based upon limited experience serious toxicity from single acute ingestions appears rare. A report of a single acute ingestion of 7500mg of azathioprine resulted in moderate leukopenia (4.1 K/ mm3) 3 days post-ingestion. Peak immunosuppressive effects can take up to 2 weeks from initiation or change in dose. Symptoms in this case are consistent with effects from azathioprine including vomiting, transient hypotension, and myalgias. Conclusion(s): Intentional ingestions of azathioprine are infrequently reported and can result in serious delayed myelosuppression. We report a case of a single acute ingestion of >15 mg/kg resulting in delayed myelosuppression managed conservatively.
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Bauxite residue (BR), simultaneously an environmental challenge as well as known to be a secondary resource for resources various valuable metals like Ti, V, Ga, and rare earth metal (REM). Lack of understanding and technology detects BR to be stockpiled which is counterproductive considering the environment, land scarcity, and management of BR inventories. As BR remains unexploited, significant amounts of REMs in BR remain unlocked, which are critical metals from green energy, environmental sustainability, and supply chain bottleneck perspective. Our current investigation analyses the potential of BR as secondary resources and quantity and worth of REM being remains unlocked. The quantitative content of global bauxite, alumina, and BR production during the last 5 decades have been analyzed. Also, plausible BR generation in the next 3 decades has been estimated. Considering the content of REM in BR amount of REM either stockpiled or to be stockpiled along with BR has been analyzed. Our study indicated about 9.14 million tons of REM remain locked in the stockpiled BR, 31.24 million tons of REM remain locked in the bauxite reserve. The worth of worldwide REM oxide remains unexploited in bauxite reserves and locked in stockpiled BR could be approximately $5000 billion, potentially can meet current and project demand of REM abundantly.
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Introduction: Remdesivir (GS-5734) is a nucleoside analog prodrug with antiviral activity against several single-stranded RNA viruses, including the novel severe respiratory distress syndrome virus 2 (SARS-CoV-2). It is currently the only FDA-approved antiviral agent for the treatment of individuals with COVID-19 caused by SARS-CoV-2. However, remdesivir pharmacokinetics/pharmacodynamics (PK/PD) and toxicity data in humans are extremely limited. It is imperative that precise analytical methods for the quantification of remdesivir and its active metabolite, GS-441524, are developed for use in further studies. We report, herein, the first validated anti-viral paper spray-mass spectrometry (PS-MS/MS) assay for the quantification of remdesivir and GS-441524 in human plasma. We seek to highlight the utility of PS-MS/MS technology and automation advancements for its potential future use in clinical research and the clinical laboratory setting. Methods: Calibration curves for remdesivir and GS-441524 were created utilizing seven plasma-based calibrants of varying concentrations and two isotopic internal standards of set concentrations. Four plasma-based quality controls were prepared in a similar fashion to the calibrants and utilized for validation. No sample preparation was needed. Briefly, plasma samples were spotted on a paper substrate contained within pre-manufactured plastic cassette plates, and the spots were dried for 1 h. The samples were then analyzed directly for 1.2 min utilizing PS-MS/MS. All experiments were performed on a Thermo Scientific Altis triple quadrupole mass spectrometer utilizing automated technology. Results: The calibration ranges were 20 - 5000 and 100 - 25000 ng/mL for remdesivir and GS-441524, respectively. The calibration curves for the two antiviral agents showed excellent linearity (average R2 = 0.99-1.00). The inter- and intra-day precision (%CV) across validation runs at four QC levels for both analytes was less than 11.2% and accuracy (%bias) was within ± 15%. Plasma calibrant stability was assessed and degradation for the 4 °C and room temperature samples were seen beginning at Day 7. The plasma calibrants were stable at -20 °C. No interference, matrix effects, or carryover was discovered during the validation process. Conclusions: PS-MS/MS represents a useful methodology for rapidly quantifying remdesivir and GS-441524, which may be useful for clinical PK/PD, therapeutic drug monitoring (TDM), and toxicity assessment, particularly during the current COVID-19 pandemic and future viral outbreaks.
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Background: The discovery and development of SARS-CoV-2 therapies remains a priority. SAB-185 is a Transchromosomic, bovine-derived, fully human polyclonal immunoglobulin product for SARS-CoV-2 being studied in ACTIV-2, randomized controlled platform trial evaluating the safety and efficacy of investigational agents for non-hospitalized adults with mild-moderate COVID-19 Methods: This Phase II trial was a superiority comparison of SAB-185 vs. placebo. Participants with confirmed SAR-CoV-2 infection received intravenous infusion of SAB-185 (3,840 Units/kg) or placebo. Primary outcome measures were proportion of participants with SARS-CoV-2 RNA < lower limit of quantification (LLoQ) in nasopharyngeal (NP) swab, time to improvement in targeted symptoms for 2 consecutive days after Day 0, and safety through Day 28. Secondary outcomes included quantitative NP RNA levels and all-cause hospitalizations and deaths. Antiviral or clinical efficacy and safety criteria for graduation to Phase III were pre-specified. Results: From April to August 2021, randomized participants from 42 sites in the US received SAB-185 (N=107) or placebo (N=106). Median age was 38 years (quartiles: 30,48), 54% female, >98% cis-gender, 7% Black/African-American, 50% Hispanic, and 11% were classified as high-risk for COVID-19 progression, with median 4 days (3,6) from symptom onset. Day 0 NP SARS-CoV-2 RNA levels were similar between SAB-185 and placebo: 4.80 vs 4.80 log10 copies/ml. No differences were observed in the proportion with NP SARS-CoV-2 RNA< lower limit of quantification (LLoQ) in nasopharyngeal (NP) swab, time to improvement in targeted symptoms for 2 consecutive days after Day 0, and safety through Day 28. Secondary outcomes included quantitative NP RNA levels and all-cause hospitalizations and deaths. Antiviral or clinical efficacy and safety criteria for graduation to phase 3 were pre-specified. Conclusion: SAB-185 was safe in this Phase II study. While no significant differences to placebo were seen in symptom duration and proportion of participants with NP SARS-CoV-2 RNA< lower limit of quantification (LLoQ) in nasopharyngeal (NP) swab, time to improvement in targeted symptoms for 2 consecutive days after Day 0, and safety through Day 28. Secondary outcomes included quantitative NP RNA levels and all-cause hospitalizations and deaths. Antiviral or clinical efficacy and safety criteria for graduation to phase 3 were pre-specified.
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Background: SARS-CoV-2 viremia is associated with adverse outcomes in COVID-19. The immunologic mediators of this relationship remain under-explored. In this study, we aimed to evaluate the correlation between immune exhaustion markers, SARS-CoV-2 viremia clearance and clinical outcomes. Methods: We included 126 participants with confirmed SARS-CoV-2 infection who were hospitalized at an urban hospital in Boston, Massachusetts, during the first surge of the COVID-19 pandemic in early 2020. Plasma samples from days 0, 3, and 7 of hospitalization were available for analyses. The plasma SARS-CoV-2 viral load was determined by reverse transcription quantitative PCR (RT-qPCR). Proteomics data were generated using the Olink platform and neutralization level was assessed using a pseudovirus neutralization assay. Viremia persistence was defined as >40 copies/ml (detection limit) if the baseline detectable viremia was <1000 copies/ml, or >100 copies/ml (quantification limit) if the baseline viremia was ≥1000 copies/ml at day 7 of admission. Partial least-squares discriminant analysis (PLS-DA) was used to select exhaustion markers that could distinguish viremia persistence and clearance. An exhaustion score was generated based on features selected by PLS-DA and was divided into four quartiles. Differentially expressed proteins between 1st and 4th quartiles were determined by linear model adjusting for baseline characteristics. R (4.1.0) was used for statistics. Results: Viremia persistence was associated with a higher level of baseline viremia, a higher rate of severe diseases and mortality within 28 days of follow-up. Viremia persistence was associated with elevation of certain exhaustion protein markers including TIM3, PDL1, LGALS9, LAG3 and IL2RA. With PLS-DA, we selected TIM3, PDL1, and LGALS9 into the exhaustion score modeling. A higher exhaustion score was associated with higher baseline viremia, persistent viremia, severe disease, and death (Figure). When compared to the lowest exhaustion score (1st quartile), the highest exhaustion score (4th quartile) was associated with elevation in proteins belonging to IL-18 signaling pathway, lung fibrosis, and immune evasion in COVID-19. The immune exhaustion level was not associated with the neutralization level. Conclusion: In participants with COVID-19, soluble exhaustion markers are associated with delayed viremia clearance, immune evasion independent of humoral immunity development, and adverse outcomes.
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Background: Camostat, a serine protease inhibitor, prevents activation of the SARS-CoV-2 spike protein and blocks SARS-CoV-2 infection in vitro. We studied the safety and antiviral and clinical efficacy of orally administered camostat in non-hospitalized adults with mild-moderate COVID-19. Methods: ACTIV-2/A5401 is a platform trial to evaluate therapies for non-hospitalized adults with mild-moderate COVID-19. In a Phase II portion of the study, participants were enrolled within 10 days of COVID-19 related symptom onset and randomized to camostat 200 mg orally every 6 hours for 7 days or the pooled placebo group. Objectives were to evaluate the safety and efficacy of camostat to reduce the duration of COVID-19 symptoms and increase the proportion of participants with SARS-CoV-2 RNA below the lower limit of quantification (LLoQ) from nasopharyngeal (NP) swabs on days 3, 7, and 14. Participants completed a study diary from day 0 to day 28 scoring COVID-19 symptoms as absent, mild, moderate, or severe. Results: Of the 224 participants enrolled from 54 US sites, 215 participants (108 camostat, 107 placebo) initiated study intervention and formed the modified intent-to-treat population. Fifty-four percent were female, >99% cis-gender, 85% White, 9% Black, and 51% Latinx. Median age was 37 years;47% reported ≤5 days of symptoms at study entry and 26% met the protocol definition of higher risk of progression to severe COVID-19. Most frequent symptoms on day 0 were cough (86%), fatigue (85%), nasal obstruction/congestion (71%) and body/muscle aches (71%). There was no significant difference between camostat and placebo arms in grade 3 or higher adverse events (7.4% vs. 6.5%, respectively). Median (Q1, Q3) time to symptom improvement was 9 days for both camostat (5, 20) and placebo (6, 19). There were no significant differences in the proportion of participants with NP SARS-CoV-2 RNA<="" div=""> Conclusion: Camostat was well-tolerated. Despite compelling in vitro data, camostat did not show evidence of antiviral or clinical efficacy in ACTIV-2/A5401. This highlights the critical importance of randomized controlled trials in the evaluation of therapies for COVID-19.
ABSTRACT
Background: Molnupiravir, a prodrug of the broadly active, direct-acting antiviral, ribonucleoside analogue EIDD-1931, is a promising COVID-19 drug. Given the primary route of SARS-CoV-2 transmission through respiratory droplets we evaluated EIDD-1931 PK in saliva, nasal secretions and tears of patients with mild-to-moderate COVID-19 through the phase Ib/IIa AGILE platform (NCT04746183). Methods: Patients with PCR-confirmed SARS-CoV-2 infection, within 5 days of symptom onset with mild-to-moderate disease were randomised to oral molnupiravir 300, 600 or 800 mg twice daily. Plasma and non-plasma (saliva, nasal and tear swabs) samples were collected pre-dose, 0.5, 1, 2, and 4 hours post-dose on study days 1 and 5 and molnupiravir and EIDD-1931 measured by LC/MS (lower limit of quantification, 2.5 ng/mL). PK parameters were determined (Phoenix 64, WinNonlin, v. 8.3) and non-plasma:plasma (NP:P) ratios (based on AUC0-4) calculated. Relationships between paired non-plasma and plasma samples were evaluated by linear regression. Results: Twelve participants (n=4 per dose;75% female) completed the study contributing 111, 112 and 97 saliva, nasal and tear samples, respectively. Molnupiravir was detected in 11% of saliva samples [median (range) 4.86 ng/mL (2.63-31.44)] and not evaluated in swabs. Quantifiable EIDD-1931, following molnupiravir 300, 600 and 800 mg twice daily were i) saliva: 17.7 (2.8-133), 16.6 (2.9-469), 25.8 (4.0-230) ng/mL, ii) nasal swabs: 182 (18-1700), 136 (18-917), 295 (24-1879) ng/mL and iii) tears: 297 (24-1650), 176 (16-1260), 307 (32-2760) ng/mL. PK parameters are shown (Table 1). Median (range, CV%) pooled NP:P ratio for saliva was 0.03 (0.01-0.11, 60%;n=16). Nasal and tear ratios were 6-fold higher with values of 0.21 (0.05-0.73, 70%;n=17) and 0.22 (0.09-1.05, 92%;n=12), respectively. Non-plasma and plasma concentrations were significantly correlated (r2: 0.360-0.677;p<0.0001). Of measured saliva, nasal and tear samples, 6, 50 and 61%, respectively were within or above the EIDD-1931 EC90 against SARS-CoV-2 in primary human airway epithelia cultures (approximately 0.5-1 μ M ≈ 130-260 ng/mL). Conclusion: This is the first report of EIDD-1931 PK at sites of initial SARS-CoV-2 exposure in patients with COVID-19. Investigations of PK/PD relationships are warranted;however, these data suggest therapeutic concentrations are potentially achieved in nasal and tear compartments, but not saliva and have important implications for prophylactic coverage.
ABSTRACT
Background: Casirivimab+imdevimab (hereinafter referred to as drug) remains vital in reducing hospitalization/death by 70% when administered early in the course of the infection. Our aim was to illustrate the mechanism of drug action in vivo and determine the magnitude of antiviral efficacy of various dose regimens given to outpatients with COVID-19, evaluating the presence of SARS-CoV-2 sero-antibody and ≥1 high-risk factor for developing severe COVID-19 illness as predictors of viral kinetics. Methods: Analysis data came from 2 clinical studies in SARS-CoV-2 infected outpatients with no or ≥1 risk factor for severe COVID-19 (NCT04425629 and NCT04666441), who received single dose of placebo or drug IV (300mg to 8g) or SC (600mg to 1.2g), had assessed viral load in nasopharyngeal swab and drug concentrations in serum (N=4500). The median number of viral load assessments per patient was 5 (range 1-8) within up to 14 days of follow-up time. Drug concentrations were predicted using the individual pharmacokinetic parameters yielded by a population model. The median patient age was 42 years, with similar proportion of males and females. The median viral load at baseline was 6.79 log10 copies/mL, and the median time of symptom onset was 3 days before study baseline. A standard target cell-limited model was used to estimate the time of infection and reconstruct viral kinetic profiles. Various relationships between exposure and resulting antiviral response were evaluated, where the drug could block de novo infection, increase the elimination rate of infected cells, or reduce viral production from infected cells. Results: The results support that the main mechanism of drug action is blocking de novo infection with an estimated decrease in the infectivity rate of 96.6%, for all dose regimens evaluated herein. High-risk factor for severe COVID-19 and baseline sero-antibody-positive/other status were associated with a 4.71% decrease and a 4.96% increase in the elimination rate of infected cells, respectively. The estimated median and 95th percentile of time to viral clearance (ie, viral count reaches below assay quantification limit) were 1.4 and 3.4 days shorter in drug vs placebo (median 10.6 vs 12.0 days, and 95th percentile 15.2 vs 18.6 days). Conclusion: All IV and SC casirivimab+imdevimab dose regimens evaluated herein showed similar near-maximal antiviral activity by blocking de novo infection;hence, shortening the time to virus clearance.