Safety and Effectiveness of High-Dose Vitamin C in Patients with COVID-19; A Randomized Controlled open-label Clinical Trial

Background: To assess the effectiveness of vitamin C treatment against coronavirus disease 2019 (COVID-19) Methods: An open-label, randomized, and controlled trial was conducted on patients with severe COVID-19 infection. The case and control treatment groups each consisted of 30 patients. The control group received lopinavir/ritonavir and hydroxychloroquine and the case group received high-dose of vitamin C (six gr daily) added to the same regimen. Results: There were no statistically signicant differences between two groups with respect to age and gender, laboratory results, and underlying diseases. On the 3 rd day of hospitalization, the mean core body temperatures was signicantly lower and SpO2 was higher In the case group (p value = 0.001, and 0.014, respectively). The median length of hospitalization in case group which was signicantly longer than the control group (8.5 days vs. 6.5 days) (p value = 0.0280). There was no signicant difference in SpO2 levels at discharge time, the length of ICU stay, and mortality between the two groups. Conclusions: We did not nd signicantly better outcomes in the group who were treated with high-dose vitamin C in addition to the main treatment regimen at discharge. Trial registration: The project was registered by Iranian Registry of Clinical Trials. function 4 , cellular immune function 5 , anti-oxidative capacity, neutrophil function 6 , and treatment of cancer and pancreatitis 7, 8 . Intravenous (IV) administration increases the plasma ascorbate concentrations more than oral supplementation (30 mM vs 0.2 mM, respectively) 9, 10 . The evidence behind theoretical possible effect of vitamin C against COVID-19 is promising. In a clinical study of the role of ascorbic acid against Ebstein Barr Virus (EBV) infection showed the EBV IgG and IgM antibody levels reduced during IV vitamin C therapy 11 . Also in a case report of enterovirus/rhinovirus induced acute respiratory distress syndrome (ARDS) in 2017, infusion of high-dose IV vitamin C was associated with rapid resolution of lung injury 12 . The impact of vitamin C administration on alleviating lung injury has also been investigated and supported in other studies 13 . There are other studies expressing the positive effect of IV vitamin C on patients with severe sepsis 14–16 . A meta-analysis also reported the impact of vitamin C on decreasing the duration of ICU-admission and mechanical ventilation care in patients with ARDS 17–19 . Since the impact of IV vitamin C on treatment of viral-induced ARDS and important role of this vitamin on the immune and endothelial system, we to investigate the correlation of the high-dose IV vitamin C administration with improvement of 2019-nCoV-induced ARDS. There is lack of data and clinical trials which studied this correlation recently. was performed to evaluate the effect of time on body temperature. Chi-square and Fisher's exact tests were used to assess the statistical relationships between categorical variables. The level of signicance was set as P-value < 0.05 for all analyses.


Introduction
The coronavirus disease 2019 (COVID- 19) pandemic which started at late 2019 and spread the world outrageously is caused by infection with Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a member of the coronaviridae family. By September 2020, almost one million lives have been sacri ced by this disease, even more deaths are expected unless proper management does not take into place soon, in terms of prevention, transmission, and treatment. Ascorbic acid or Ascorbate (vitamin C) is an essential water soluble nutrient that functions as a key antioxidant and is involved in the synthesis of collagen and neurotransmitters, and affects wound healing, energy metabolism, nervous system function, and immune cell health [1][2][3] . The serum level of this vitamin has been correlated with its effect on the endothelial function 4 , cellular immune function 5 , anti-oxidative capacity, neutrophil function 6 , and treatment of cancer and pancreatitis 7,8 . Intravenous (IV) administration increases the plasma ascorbate concentrations more than oral supplementation (30 mM vs 0.2 mM, respectively) 9, 10 .
The evidence behind theoretical possible effect of vitamin C against COVID-19 is promising. In a clinical study of the role of ascorbic acid against Ebstein Barr Virus (EBV) infection showed the EBV IgG and IgM antibody levels reduced during IV vitamin C therapy 11 . Also in a case report of enterovirus/rhinovirus induced acute respiratory distress syndrome (ARDS) in 2017, infusion of high-dose IV vitamin C was associated with rapid resolution of lung injury 12 . The impact of vitamin C administration on alleviating lung injury has also been investigated and supported in other studies 13 . There are other studies expressing the positive effect of IV vitamin C on patients with severe sepsis [14][15][16] . A meta-analysis also reported the impact of vitamin C on decreasing the duration of ICU-admission and mechanical ventilation care in patients with ARDS [17][18][19] .
Since the impact of IV vitamin C observed on the treatment of viral-induced ARDS and the important role of this vitamin on the immune and endothelial system, we aimed to investigate the correlation of the high-dose IV vitamin C administration with improvement of 2019-nCoV-induced ARDS. There is lack of data and clinical trials which studied this correlation recently.

Participants
Between April and May 2020, 85 patients with compelling clinical symptoms for diagnosis of COVID-19 were admitted to Ziaeian Hospital, Tehran, Iran. Based on the eligibility criteria ( Fig. 1), 25 patients were excluded and 60 patients were included in the study. The inclusion criteria were age older than 18 years, positive COVID-19 polymerase chain reaction (PCR) test or COVID-19 suspicion based on clinical ndings (mainly fever, dyspnea, dry cough), imaging ndings of COVID-19 on spiral chest computer tomography (CT) or high resolution CT (HRCT) imagings validated by a trained radiologist, clinical manifestations of ARDS or myocarditis, and oxygen saturation lower than 93% from admission or after 48 hours from the rst COVID-19 treatment. The exclusion criteria were receiving anti-retroviral therapy or immune system booster medications in the last three months, no proven and con rmed COVID-19 disease based on the inclusion criteria, patients with Glucose-6-phosphate dehydrogenase (G6PD) de ciency, patients with end stage renal diseases (ESRD), and pregnancy.

Study arms and treatment plans
The patients were divided into two subgroups equally by block randomization; the case group included 30 patients receiving 1.5 grams vitamin C IV every six hours for ve days and the control group included 30 patients who did not receive vitamin C. All of the participants were also treated with oral Lopinavir/Ritonavir (Kaletra, Abbott Laboratories) 400/100 mg twice daily and daily dose of oral Hydroxychloroquine (400 mg) according to the Iranian COVID-19 treatment protocol at time of this study (it should be noted that based on the vast number of studies for COVID-19, hydroxychloroquine is not considered as mainstay in the protocol for COVID-19 in Iran). On the rst day of hospitalization, laboratory studies including complete blood count (CBC), Creactive protein (CRP), and erythrocyte sedimentation rate (ESR) were obtained. Patients were assessed by daily measurements of core body temperature, respiratory rate (RR), heart rate (HR), and peripheral capillary oxygen saturations (SpO2). The treatment subsided whenever any kind of drug side effects appeared. Some of the patients deteriorated during the admission and received corticosteroid (methylprednisolone 125 mg daily for three days) and IVIG (5 to 10 gr daily for three to ve days). Patients were discharged when they achieved a stable SpO2 > 92%, no evidence of respiratory distress was remaining, and were afebrile for at least three consecutive days.
Sample size calculation was performed for non-inferiority tests of difference between two group proportions. We assumed an effectiveness of 65% for the intervention group and effectiveness of 50% for the control group. We also assumed a margin of noninferiority of at least 10% between the two groups. The power of the study was determined as 90% (G * Power, Erdfelder, Faul, & Buchner, 1996).

Ethical considerations
In accordance to the declaration of Helsinki, written informed consent was obtained from all participants before initiation of the study. The patients were assured that declining to participate in the study or leaving the study at any point would not affect the quality of their treatment and that they would thereafter receive the standard care. The study protocol was approved by the institutional review board (IRB) of Tehran University of Medical Sciences (TUMS) with Ethical code (IR.TUMS.VCR.REC.1399.078).

Measurements and statistical analysis
In this open label and nonblinded study, distribution of age, gender, initial clinical symptoms, and vital signs of the rst day of admission were compared between the two groups. The vital signs including body temperature, RR, HR and SpO2 were also compared on the 3rd and last day of treatment between the two groups as an outcome measure. Differences in duration of hospitalization, number of patients whose condition deteriorated and needed ICU admission, length of ICU admission, and difference between mortality rates were measured.
Data was analyzed using SPSS software (IBM Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp.). Quantitative variables are reported by mean and standard deviation (SD) and qualitative variables are reported using frequency and percentage. Because of the normal distribution of our data via Shapiro-Wilk test, the independent t-test was used to assess the means differences and a mixed-design analysis of variance model (ANOVA) was performed to evaluate the effect of time on body temperature. Chi-square and Fisher's exact tests were used to assess the statistical relationships between categorical variables. The level of signi cance was set as P-value < 0.05 for all analyses.

Results
Demographic characteristics, underlying diseases, and clinical and laboratory ndings Demographic characteristics, underlying diseases, and clinical and laboratory ndings are presented in Table 1. Male to female ratio was 1:1. There were no statistically signi cant differences between two groups considering age and gender, laboratory results and underlying diseases (p value > 0.05). All clinical ndings except for fever (23.33% vs. 63.33% in case and control groups, respectively, p value = 0.002) and myalgia (13.33% vs. 60.0% in case and control groups, respectively, p value < 0.001)were not signi cantly different between the two groups.  There was no signi cant difference in body temperature at the time of discharge between the two groups (p value > 0.05)( Table 1). The mean body temperatures upon admission and on the 3rd day of admission were signi cantly higher in the control group (p value = 0.001). The mixed-design analysis of variance model (ANOVA) performed to evaluate the effect of time on body temperature at the time of admission, on the 3rd day of admission and discharge, showed a signi cant effect of time on body temperature (Wilks' Lambda = 0.589, F (2,57) = 19.879, p value < 0.001) (Fig. 2). Post hoc comparison indicated a signi cant difference between body temperatures at time of admission, discharge, and on the 3rd day of hospitalization (p value < 0.001).
SpO2 at admission and discharge were not signi cantly different between the two groups (p value > 0.05) ( Table 1). SpO2 on the 3rd day of admission was higher in the case group compared to the control group (median, 90.5% vs. 88.0% respectively, p value = 0.014) ( Table 1). A non-parametric Friedman test of difference among repeated measures of SpO2 was conducted and there was a signi cant difference in mean ranks in both groups with the oxygen saturation increasing signi cantly in both groups (p value < 0.001). The case group had a median length of admission in the hospital of 8.5 (range: 7.0-12.0) days which was signi cantly longer than the control group with a median length of admission of 6.5 (range: 4.0-12.0) days. There was no signi cant difference in the length of ICU stay between the two groups (p value > 0.05, Table 1). There was a non-signi cant higher rate of intubation in the case group (p value > 0.05) ( Table 1). Death rate was equal in both groups (three deaths in each group, p value > 0.05). During treatment with high-dose vitamin C, none of the patients experienced adverse events such as headache, nausea, bloating, or abdominal discomfort.

Discussion
Until the time of this study, no de nite treatment option has been suggested and cleared for COVID-19. While this pandemic is still responsible for death of almost a million people and infection of many more, search for better treatment options should never be delayed. 20,21 . Vitamin C is an essential water soluble nutrient that has different important roles in our body, especially in immune cell functions 1,2,4 . Studies report that vitamin C can be effective in treatment of bacterial and viral infection. These studies showed vitamin C weakly inhibits the multiplication of viruses such as in uenza type A, Herpes simplex virus type 1 (HSV-1) and poliovirus type 1 [22][23][24] . A clinical study showed the effect of IV vitamin C therapy on reduction of IgG and IgM antibody levels in EBV infection 11 .
There is also a report of a case of enterovirus/rhinovirus induced ARDS where the infusion of high-dose intravenous vitamin C was associated with rapid resolution of lung injury 12 .
Some studies showed that serum vitamin C levels may plummet in some patients especially in the critically ill during the course of infection 25,26 ; and vitamin C de ciency may contribute to organ injury and immune paralysis which leads us to assume high-doses of vitamin C might improve clinical outcomes of critically ill patients 25 . There is also some evidence that shows vitamin C may reduce patients' susceptibility to lower respiratory tract infections such as pneumonia and it may have a protective role in lung infections but, further studies need to evaluate the e cacy of treatment with vitamin C in severe viral respiratory tract infections [25][26][27][28][29] .
A number of meta-analysis demonstrated that the use of intravenous vitamin C as a therapy for sepsis and ARDS has bene ts such as a lower rate of vasopressor requirements, shorter duration of both mechanical ventilation and admission in the ICUs; along with a shorter hospital admission in critically ill patients 18,[30][31][32] . Lin et al., found that administration of more than 50 mg/kg daily vitamin C had a signi cant effect in reduction of mortality rate in patients with severe sepsis. They concluded that a better survival rate correlated with administration of high doses of vitamin C 33 . Fowler et al., reported in their randomized, double blind, placebo-controlled, multicenter trial that high doses of vitamin C did not signi cantly improve organ dysfunction scores in patients with severe sepsis or ARDS but in three secondary outcomes, use of vitamin C was associated with a signi cantly lower risk of mortality on the 28th day after diagnosis of the infection (29.8% vs. 46.3%), a higher number of ventilator-free days (13.1 vs. 10.6 days) and a higher number of ICU-free days (10.7 vs. 7.7 days) 34 .
All these ndings emphasize possible bene cial effects of vitamin C as a treatment for COVID-19. Here, we conducted a randomized clinical trial with 60 patients in two groups. Thirty patients were treated with 1.5 grams of IV vitamin C, every 6 hours for 5 days in addition to the main treatment regimen (case group), whereas the other 30 patients were treated only with the standard regimen. Demographic characteristics, underlying diseases, and clinical and laboratory ndings were not signi cantly different between the two groups. Fever and myalgia were signi cantly more frequent in the control group but, other clinical ndings were not notably different. SpO2 was improved in all patients. There is a similar report of SpO2 improvement in China associated with treatment with high doses of intravenous vitamin C (doses range from 2 to 10 grams per day in 8-10-hour IV infusions) in 50 moderate to severe COVID-19 patients. They also reported that all patients were cured and discharged 35 . The absence of a control group weakened the conclusions based on this report.
In the present study, there was no signi cant difference in oxygen SpO2 levels between the two groups at discharge but the median of SpO2 levels were signi cantly higher in the case group on the 3rd day of admission. The mean body temperature signi cantly decreased during the admission in both groups and there was no signi cant difference between two groups regarding the core body temperature at discharge but, on the 3rd day of treatment, the mean of patients' body temperature was signi cantly lower in the case group. Length of stay in the hospital had a median of 8.5 days and it was unexpectedly higher in the case group (8.5 vs. 6.5, p value = 0.028). Other outcomes including number of deaths, number of intubations and duration of ICU admission were not signi cantly different between two groups. We did not nd any side effects in the patients. Other studies also reported good tolerance of high-dose vitamin C in their trials 36 .
There are not enough data and clinical trials that have evaluated the correlation between high-dose vitamin C treatment in COVID-19 patients with ARDS and improvement of their status but, there are several ongoing studies that aim to investigate the impact of high-dose vitamin C on COVID-19 patients (details of ongoing studies are presented in Table 2). Investigators in these studies will assess primary outcomes such as 50% reduction in symptoms score in 28 days, incidence of adverse effects (including severe adverse reactions), time to clinical improvement (TTIC), TTIC of NEWS2 (National Early Warning Score 2), number of hospital admission days, the rate of decline in lung infection rate, in-hospital mortality rates and number of ventilator-free days. Based on estimated dates, none of these studies will be completed before August 1st 2020. The ndings of these studies will be valuable and we hope to see promising results in their studies.
Our study has its own limitations which can be covered in the future studies. The open label design of the study and relatively small patient population are the main limitations. Further randomized double-blind clinical trials with more patient population can be bene cial.

Conclusion
In this study, we found that there were improvements in peripheral oxygen saturation and body temperature in both groups during the time of admission but we did not nd signi cantly better outcomes in the group who were treated with high-dose vitamin C in addition to the main treatment regimen at discharge. Randomization and treatment assignment.