Serial viral load analysis by DDPCR to evaluate FNC efficacy and safety in the treatment of mild cases of COVID-19
\r\nRenato Martins da Silva&#x;Renato Martins da Silva1†Paula Gebe Abreu Cabral*&#x;Paula Gebe Abreu Cabral1*†Svio Bastos de SouzaSávio Bastos de Souza1Raul Ferraz ArrudaRaul Ferraz Arruda1Sheila Passos de Figueiredo CabralSheila Passos de Figueiredo Cabral1Arícia Leone Evangelista Monteiro de AssisArícia Leone Evangelista Monteiro de Assis1Yolanda Porto Muniz MartinsYolanda Porto Muniz Martins1Carlos Augusto de Araújo TavaresCarlos Augusto de Araújo Tavares1Antnio Brazil Viana JuniorAntônio Brazil Viana Junior2Junbiao ChangJunbiao Chang3Pingsheng LeiPingsheng Lei4
1High Complexity Center, Galzu Institute, Campos dos Goytacazes, Rio de Janeiro, Brazil
2Universidade Federal do Ceará (UFC)/Ebserh University Hospital Complex, Fortaleza, Brazil
3NMPA Key Laboratory for Research and Evaluation of Innovative Drug, Henan Normal University, Xinxiang, China
4Institute of Material Medical, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
Introduction: The SARS-CoV-2 outbreak has threatened the human population globally as the numbers of reinfection cases even after large-scale vaccination. Trials have been carried out to find drugs effective in fighting the disease, as COVID-19 is being considered a treatable disease only after we have antivirals. A clinical candidate originally developed for HIV treatment, AZVUDINE (FNC), is a promising drug in the treatment of COVID-19.
Methods: To predict the clinical outcome of COVID-19, we examined the course of viral load, every 48 h, by RT-PCR, and disease severity using an antiviral drug, FNC, with 281 participants. A randomized clinical trial was performed to evaluate the efficacy of FNC added to standard treatment, compared with placebo group added to standard treatment, for patients with mild COVID-19. RT-qPCR and ddPCR were applied to estimate the viral load in samples from patients. Also, the clinical improvement was evaluated as well as the liver and kidney function.
Results and discussion: Notably, the FNC treatment in the mild COVID-19 patients may shorten the time of the nucleic acid negative conversion (NANC) versus placebo group. In addition, the FNC was effective in reducing the viral load of these participants. The present clinical trial results showed that the FNC accelerate the elimination of the virus in and could reduce treatment time of mild patients and save a lot of medical resources, making it a strong candidate for the outpatient and home treatment of COVID-19.
A new coronavirus disease-2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) occurred as a pandemic resulting in serious disease burden in almost all countries (1, 2). After 3 years since the first cases of coronavirus disease 2019 (COVID-19) were reported the people have been striving to turn COVID-19 into a preventable and treatable disease through various measures, including vaccines (3, 4), small-molecule inhibitors (5, 6), bioactive natural products (7–9), and traditional medicine (10, 11). The triumph of vaccines has led to a significant decrease in symptomatic illness, severe and critical disease, and death. Nonetheless, the efficacy of vaccines has been affected by virus evolution and the emergence of new variants, and global access is sub-optimal (2).
The development of small molecules to treat COVID-19 has been achieved by several strategies, including computer-aided lead compound design and screening, natural product discovery, drug repurposing, and combination therapy. Antiviral drugs offer opportunities at various stages of SARS- CoV-2 infection, including pre- or post-exposure prophylaxis, early treatment, and late treatment. However, current small molecule antivirals have major limitations, such as remdesivir, in which the efficacy has varied among reports (12–15), the Paxlovid which is expensive, US $530 for each 5-day course (16), and has multiple drug-drug interactions, and the molnupiravir, costing US $700 per 5-day course (16), and has mutagenic potential (17).
Recently published studies illustrated the efficacy and safety of early use of a small-molecule antiviral in reducing hospitalization or death among the high-risk population with mild to moderate COVID-19 (12, 18–20). This promising antiviral, AZVUDINE (FNC), is the first double-target nucleoside drug that has demonstrated significant and broad-spectrum in vitro antiviral effects against targets such as HIV (21), HCV (22), EV71 (23), HBV (24), and recently a randomized, open-Label, controlled clinical trial of FNC tablets was performed in the treatment of mild and common COVID-19, showing that FNC treatment in the mild and common COVID-19 may shorten the mean times of the first nucleic acid negative conversion (NANC) versus standard antiviral treatment (25).
To detect and diagnose the virus, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is applied in many countries, and this method can estimate the viral load in samples from patients with this viral infection (13). Recently, Tsukagoshi et al. (18) examined viral loads of SARS-CoV-2 in fatal (15 cases), symptomatic/survived (133 cases), and asymptomatic cases (138 cases) using RT-qPCR. Notably, the viral load in the fatal cases was significantly higher than in symptomatic or asymptomatic cases (p < 0.05). The authors conclude that intervene early to prevent a severe stage of the disease in such cases.
Therefore, clinical trials of FNC treating COVID-19 with larger sample size were required. Thus, the objective of this randomized clinical trial was to evaluate the efficacy of FNC, evaluating the clinical improvement, the liver and kidney function, the NANC time, and the viral load in mild COVID-19 participants.
2. Methodology
2.1. Study design
This randomized, double-blind, placebo-controlled clinical trial (IGZ-2) was carried out in five co-participating research centers, distributed in four municipalities in the state of Rio de Janeiro, Brazil, as a strategic decision due to the need to concentrate molecular biology analyzes to maintain their standardization and quality, remembering that each RT-PCR equipment has its own sensitivity and different kits of reagents for RT-PCR have different performances. Two research centers were located in the city of Campos dos Goytacazes, namely Hospital São José and Santa Casa de Miseriocordia de Campos. In the municipality of São fidelis, the third research center was at Hospital Armando Vidal. In the municipality of Itaocara, the fourth research center was located at the Hospital de Itaocara. And the fifth research center was located in the municipality of Cambuci at Hospital Moacyr Gomes de Azevedo. However, the research centers served as an outpatient clinic for the evaluation of the participants, since this study treated mild patients contaminated with SARS-COV-2, all of whom were followed in their homes. This study took place in January 2022 when the Centers for Combating COVID-19 (CCC) provided the first care for infected patients. Interestingly, in this study, 88% of participants were vaccinated and 12% had natural immunity. Participants from four co-participating centers in the north and northwest regions of Rio de Janeiro were randomized. This trial was approved by the institutional review board of the National Health Surveillance Agency CE 0937457/21-4. The study was approved by the National Council for Research Ethics, CAAE 52176421.8.0000.5244. The study was also published in clinical trials (NCT05033145). All enrolled participants provided written informed consent. The methods are described in detail in the Supplementary material.
Patients in the FNC group were treated with oral AZVUDINE tablets 5 mg (five tablets once a night) and standard treatment. For the 5 mg dose of AZVUDINE, the mean half-life is 13.8 h, with the intact drug and metabolites being excreted in the urine within 24 h. Patients in the control group were treated with placebo added standard treatment.
Patients: Patients meeting the following criteria were enrolled in the study: (1) age 18 and over, regardless of gender; (2) respiratory or blood samples that were tested positive for SARS-CoV-2 nucleic acid by RT-PCR, or respiratory or blood samples that were tested highly homologous with the known SARS-CoV-2 by viral gene sequencing; (3) the confirmation of COVID-19 according to the diagnostic criteria of “the latest Clinical guide-lines for novel coronavirus” issued by the World Health Organization (WHO) on 28 January 2020. All enrolled patients signed informed consent forms.
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Serial viral load analysis by DDPCR to evaluate FNC efficacy and safety in the treatment of mild cases of COVID-19
\r\nRenato Martins da Silva&#x;Renato Martins da Silva1†Paula Gebe Abreu Cabral*&#x;Paula Gebe Abreu Cabral1*†Svio Bastos de SouzaSávio Bastos de Souza1Raul Ferraz ArrudaRaul Ferraz Arruda1Sheila Passos de Figueiredo CabralSheila Passos de Figueiredo Cabral1Arícia Leone Evangelista Monteiro de AssisArícia Leone Evangelista Monteiro de Assis1Yolanda Porto Muniz MartinsYolanda Porto Muniz Martins1Carlos Augusto de Araújo TavaresCarlos Augusto de Araújo Tavares1Antnio Brazil Viana JuniorAntônio Brazil Viana Junior2Junbiao ChangJunbiao Chang3Pingsheng LeiPingsheng Lei4
1High Complexity Center, Galzu Institute, Campos dos Goytacazes, Rio de Janeiro, Brazil
2Universidade Federal do Ceará (UFC)/Ebserh University Hospital Complex, Fortaleza, Brazil
3NMPA Key Laboratory for Research and Evaluation of Innovative Drug, Henan Normal University, Xinxiang, China
4Institute of Material Medical, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China
Introduction: The SARS-CoV-2 outbreak has threatened the human population globally as the numbers of reinfection cases even after large-scale vaccination. Trials have been carried out to find drugs effective in fighting the disease, as COVID-19 is being considered a treatable disease only after we have antivirals. A clinical candidate originally developed for HIV treatment, AZVUDINE (FNC), is a promising drug in the treatment of COVID-19.
Methods: To predict the clinical outcome of COVID-19, we examined the course of viral load, every 48 h, by RT-PCR, and disease severity using an antiviral drug, FNC, with 281 participants. A randomized clinical trial was performed to evaluate the efficacy of FNC added to standard treatment, compared with placebo group added to standard treatment, for patients with mild COVID-19. RT-qPCR and ddPCR were applied to estimate the viral load in samples from patients. Also, the clinical improvement was evaluated as well as the liver and kidney function.
Results and discussion: Notably, the FNC treatment in the mild COVID-19 patients may shorten the time of the nucleic acid negative conversion (NANC) versus placebo group. In addition, the FNC was effective in reducing the viral load of these participants. The present clinical trial results showed that the FNC accelerate the elimination of the virus in and could reduce treatment time of mild patients and save a lot of medical resources, making it a strong candidate for the outpatient and home treatment of COVID-19.
Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT05033145, identifier NCT05033145.
1. Introduction
A new coronavirus disease-2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) occurred as a pandemic resulting in serious disease burden in almost all countries (1, 2). After 3 years since the first cases of coronavirus disease 2019 (COVID-19) were reported the people have been striving to turn COVID-19 into a preventable and treatable disease through various measures, including vaccines (3, 4), small-molecule inhibitors (5, 6), bioactive natural products (7–9), and traditional medicine (10, 11). The triumph of vaccines has led to a significant decrease in symptomatic illness, severe and critical disease, and death. Nonetheless, the efficacy of vaccines has been affected by virus evolution and the emergence of new variants, and global access is sub-optimal (2).
The development of small molecules to treat COVID-19 has been achieved by several strategies, including computer-aided lead compound design and screening, natural product discovery, drug repurposing, and combination therapy. Antiviral drugs offer opportunities at various stages of SARS- CoV-2 infection, including pre- or post-exposure prophylaxis, early treatment, and late treatment. However, current small molecule antivirals have major limitations, such as remdesivir, in which the efficacy has varied among reports (12–15), the Paxlovid which is expensive, US $530 for each 5-day course (16), and has multiple drug-drug interactions, and the molnupiravir, costing US $700 per 5-day course (16), and has mutagenic potential (17).
Recently published studies illustrated the efficacy and safety of early use of a small-molecule antiviral in reducing hospitalization or death among the high-risk population with mild to moderate COVID-19 (12, 18–20). This promising antiviral, AZVUDINE (FNC), is the first double-target nucleoside drug that has demonstrated significant and broad-spectrum in vitro antiviral effects against targets such as HIV (21), HCV (22), EV71 (23), HBV (24), and recently a randomized, open-Label, controlled clinical trial of FNC tablets was performed in the treatment of mild and common COVID-19, showing that FNC treatment in the mild and common COVID-19 may shorten the mean times of the first nucleic acid negative conversion (NANC) versus standard antiviral treatment (25).
To detect and diagnose the virus, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is applied in many countries, and this method can estimate the viral load in samples from patients with this viral infection (13). Recently, Tsukagoshi et al. (18) examined viral loads of SARS-CoV-2 in fatal (15 cases), symptomatic/survived (133 cases), and asymptomatic cases (138 cases) using RT-qPCR. Notably, the viral load in the fatal cases was significantly higher than in symptomatic or asymptomatic cases (p < 0.05). The authors conclude that intervene early to prevent a severe stage of the disease in such cases.
Therefore, clinical trials of FNC treating COVID-19 with larger sample size were required. Thus, the objective of this randomized clinical trial was to evaluate the efficacy of FNC, evaluating the clinical improvement, the liver and kidney function, the NANC time, and the viral load in mild COVID-19 participants.
2. Methodology
2.1. Study design
This randomized, double-blind, placebo-controlled clinical trial (IGZ-2) was carried out in five co-participating research centers, distributed in four municipalities in the state of Rio de Janeiro, Brazil, as a strategic decision due to the need to concentrate molecular biology analyzes to maintain their standardization and quality, remembering that each RT-PCR equipment has its own sensitivity and different kits of reagents for RT-PCR have different performances. Two research centers were located in the city of Campos dos Goytacazes, namely Hospital São José and Santa Casa de Miseriocordia de Campos. In the municipality of São fidelis, the third research center was at Hospital Armando Vidal. In the municipality of Itaocara, the fourth research center was located at the Hospital de Itaocara. And the fifth research center was located in the municipality of Cambuci at Hospital Moacyr Gomes de Azevedo. However, the research centers served as an outpatient clinic for the evaluation of the participants, since this study treated mild patients contaminated with SARS-COV-2, all of whom were followed in their homes. This study took place in January 2022 when the Centers for Combating COVID-19 (CCC) provided the first care for infected patients. Interestingly, in this study, 88% of participants were vaccinated and 12% had natural immunity. Participants from four co-participating centers in the north and northwest regions of Rio de Janeiro were randomized. This trial was approved by the institutional review board of the National Health Surveillance Agency CE 0937457/21-4. The study was approved by the National Council for Research Ethics, CAAE 52176421.8.0000.5244. The study was also published in clinical trials (NCT05033145). All enrolled participants provided written informed consent. The methods are described in detail in the Supplementary material.
Patients in the FNC group were treated with oral AZVUDINE tablets 5 mg (five tablets once a night) and standard treatment. For the 5 mg dose of AZVUDINE, the mean half-life is 13.8 h, with the intact drug and metabolites being excreted in the urine within 24 h. Patients in the control group were treated with placebo added standard treatment.
Patients: Patients meeting the following criteria were enrolled in the study: (1) age 18 and over, regardless of gender; (2) respiratory or blood samples that were tested positive for SARS-CoV-2 nucleic acid by RT-PCR, or respiratory or blood samples that were tested highly homologous with the known SARS-CoV-2 by viral gene sequencing; (3) the confirmation of COVID-19 according to the diagnostic criteria of “the latest Clinical guide-lines for novel coronavirus” issued by the World Health Organization (WHO) on 28 January 2020. All enrolled patients signed informed consent forms.