Simultaneous Quantification of Nirmatrelvir/Ritonavir in Human Serum by Lc-Ms/Ms (preprint)

In December 2021, the U.S. Food and Drug Administration (FDA) granted emergency authorization for Paxlovid® as an antiviral treatment for COVID-19. Paxlovid® is composed of two tablets, nirmatrelvir and ritonavir. Dose adjustment is necessary in cases of renal insufficiency. The aim of present study is to establish a LC-MS/MS method for simultaneous determination of nirmatrelvir/ritonavir in human serum for therapeutic drug monitoring. Internal standard saquinavir was added in 25 μl human serum samples, and then the samples were precipitated with methanol. The analytes were separated by gradient elution on a C18 column, using a mobile phase of 0.1% formic acid-water and methanol, at a flow rate of 0.4 ml/min. The injection volume was 2 μl, and the analysis time was 5 min. The determination of the analytes was performed by electrospray ionization in positive mode by full mass monitoring. The detected ions of nirmatrelvir, ritonavir and saquinavir were m/z 500.24792, 721.32004 and 671.39155, respectively. The linear concentration range for nirmatrelvir was 78.13~20000 ng/ml, for ritonavir was 15.63~4000 ng/ml (r2>0.9900). The accuracy ranged from 87.45%~104.63%, and the intra-day and inter-day precision RSD was < 15%. The recovery of Nirmatrelvir ranged from 98.72%~109.83%, and that of ritonavir was 95.41%~112.36%. The matrix effect of Nirmatrelvir was 88.31%~97.73%, and that of ritonavir was 85.17%~103.05%. This method was used to measure the trough concentrations of nirmatrelvir/ritonavir in 17 patients. The trough concentration of nirmatrelvir was 1331.7~8352.5 ng/ml, and that of ritonavir was 53.4~1325.5 ng/ml, with large individual differences. The method is simple, sensitive, specific, and reproducible, and can be used for monitoring the blood concentration and pharmacokinetic study of nirmatrelvir/ritonavir in COVID-19 patients.

A Review of Potential Therapeutic Strategies for COVID-19.

Coronavirus disease 2019 is a rather heterogeneous disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The ongoing pandemic is a global threat with increasing death tolls worldwide. SARS-CoV-2 belongs to lineage B ß-CoV, a subgroup of Sarbecovirus. These enveloped, large, positive-sense single-stranded RNA viruses are easily spread among individuals, mainly via the respiratory system and droplets. Although the disease has been gradually controlled in many countries, once social restrictions are relaxed the virus may rebound, leading to a more severe and uncontrollable situation again, as occurred in Shanghai, China, in 2022. The current global health threat calls for the urgent development of effective therapeutic options for the treatment and prevention of SARS-CoV-2 infection. This systematic overview of possible SARS-CoV-2 therapeutic strategies from 2019 to 2022 indicates three potential targets: virus entry, virus replication, and the immune system. The information provided in this review will aid the development of more potent and specific antiviral compounds.

Small-Molecule RAF265 as an Antiviral Therapy Acts against PEDV Infection.

Porcine epidemic diarrhea virus (PEDV), a member of the family Coronaviridae, causes acute diarrhea, vomiting, dehydration, and high mortality in newborn piglets, and has caused significant economic losses in the pig industry. There are currently no specific drugs available to treat PEDV. Viruses depend exclusively on the cellular machinery to ensure an efficient replication cycle. In the present study, we found that small-molecule RAF265, an anticancer drug that has been shown to be a potent inhibitor of RAF, reduced viral loads of PEDV by 4 orders of magnitude in Vero cells, and protected piglets from virus challenge. RAF265 reduced PEDV production by mediating cytoskeleton arrangement and targeting the host cell's translation machinery. Treatment with RAF265 inhibited viral entry of PEDV S-glycoprotein pseudotyped viral vector particle (PEDV-pp), at half maximal effective concentrations (EC50) of 79.1 nM. RAF265 also presented potent inhibitory activity against viral infection by SARS-CoV-2-pp and SARS-CoV-pp. The present work may provide a starting point for further progress toward the development of antiviral strategies effective against coronavirus PEDV.

Differential susceptibility to SARS-CoV-2 in the normal nasal mucosa and in chronic sinusitis.

Human nasal mucosa is susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and serves as a reservoir for viral replication before spreading to other organs (e.g. the lung and brain) and transmission to other individuals. Chronic rhinosinusitis (CRS) is a common respiratory tract disease and there is evidence suggesting that susceptibility to SARS-CoV-2 infection differs between the two known subtypes, eosinophilic CRS and non-ECRS (NECRS). However, the mechanism of SARS-CoV-2 infection in the human nasal mucosa and its association with CRS has not been experimentally validated. In this study, we investigated whether the human nasal mucosa is susceptible to SARS-CoV-2 infection and how different endotypes of CRS impact on viral infection and progression. Primary human nasal mucosa tissue culture revealed highly efficient SARS-CoV-2 viral infection and production, with particularly high susceptibility in the NECRS group. The gene expression differences suggested that human nasal mucosa is highly susceptible to SARS-CoV-2 infection, presumably due to an increase in ACE2-expressing cells and a deficiency in antiviral immune response, especially for NECRS. Importantly, patients with NECRS may be at a particularly high risk of viral infection and transmission, and therefore, close monitoring should be considered.

Analysis of the characteristics of outpatient and emergency diseases in the department of otolaryngology during the "COVID-19" pandemic.

The pandemic of "Corona Virus Disease 2019" (COVID-19) has changed the lives of people. There have been changes in common outpatient and emergency cases in otolaryngology, so an analysis of data pertaining to this was completed. This study is to evaluate the impact of viral infection disease in otolaryngological common disease. This study uses the data of common diseases in the outpatient and emergency department during the "COVID-19" pandemic (from February to April 2020) and the same period in the past 3 years from the Department of Otolaryngology. During the "COVID-19" period compared with the same period last year, the ranking of cases by diseases has changed. Diseases such as chronic pharyngitis, allergic rhinitis, sudden deafness, and tinnitus increased, meanwhile acute pharyngitis and acute laryngopharyngitis decreased (p < 0.05). The viral infection has impacted the mental behaviors of people, therefore mental-related disease cases of the department of Otolaryngology have increased indirectly. This study provides real data to illustrate mental-related diseases. It also provides experience and shows the importance of keeping and maintaining mental health.

A Peptide-Based Magnetic Chemiluminescence Enzyme Immunoassay for Serological Diagnosis of Coronavirus Disease 2019.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel ß-coronavirus, causes severe pneumonia and has spread throughout the globe rapidly. The disease associated with SARS-CoV-2 infection is named coronavirus disease 2019 (COVID-19). To date, real-time reverse-transcription polymerase chain reaction (RT-PCR) is the only test able to confirm this infection. However, the accuracy of RT-PCR depends on several factors; variations in these factors might significantly lower the sensitivity of detection. METHODS: In this study, we developed a peptide-based luminescent immunoassay that detected immunoglobulin (Ig)G and IgM. The assay cutoff value was determined by evaluating the sera from healthy and infected patients for pathogens other than SARS-CoV-2. RESULTS: To evaluate assay performance, we detected IgG and IgM in the sera from confirmed patients. The positive rate of IgG and IgM was 71.4% and 57.2%, respectively. CONCLUSIONS: Therefore, combining our immunoassay with real-time RT-PCR might enhance the diagnostic accuracy of COVID-19.