Antioxidants as Adjuvant Therapy to Standard Therapy in Patients With COVID-19
Antioxidants as Adjuvant Therapy to Standard Therapy in Patients With COVID-19
Introduction: SARS-CoV2 infection produces severe pneumonia with pulmonary alveolar collapse.
 There is no specific treatment to date. In experimental models and humans with septic shock,
 there is a high production of nitric oxide (NO) and reactive nitrogen species (RNS) and can
 cause multiple organ failure. The administration of antioxidants such as n-acetylcysteine
 (NAC), vitamin C, melatonin, and vitamin E participate in increasing the intracellular
 content of GSH, ROS sequestration, protection of the lipids of cell membranes, cytosol
 proteins, nuclear DNA, mitochondrial and decrease LPO.

 Justification: as there is no specific antiviral therapy, the therapeutic options are
 limited, complications and mortality are high; It is intended to evaluate the effect of
 antioxidants on the storm outcome of the dysregulation of oxidative stress.

 Hypothesis: It is postulated that adjuvant therapy with antioxidants and Pentoxifylline
 reduces the use of ventilators in patients with or without septic shock secondary to severe
 SARS-COV2 pneumonia as decreases lipoperoxidation, and corrects dysregulation of oxidative
 stress by increasing the antioxidant capacity.

 Objectives: To evaluate whether it is possible to avoid intubation or decrease assisted
 mechanical ventilation days, improve oxidative stress dysregulation in patients with
 SARS-COV2 infection with severe pneumonia with or without septic shock.

 Methodology: Quasi-experimental, open analytical, prospective, and longitudinal study
 (before-after). In patients over 18 years of age who are admitted to the CITIBANAMEX Center
 with or without septic shock secondary to severe SARS-COV2 pneumonia. There will be two
 groups: 1) patients without septic shock and 2) patients with septic shock secondary to
 severe pneumonia due to SARS-COV2. A single antioxidant will be applied following the
 clinical decision tree (NAC, Vit C, Vit E, melatonin) more Pentoxifylline orally or by
 orogastric tube for a total of 5 days from the start of the protocol. APACHE II will
 calculate the risk, SOFA, MEXSOFA, measurements of IL-8, vitamin C, NO3 / NO2, LOP, total
 antioxidant capacity will be carried out at baseline and 48 hours. SOFA will be calculated
 for seven days, in addition to days of hospitalization, days of mechanical ventilation. It
 was evaluated 28 days after discharge by telephone.
I. Background

 The SARS-CoV-2 virus has a positive-sense RNA, with a genome of approximately 27-32 kb in
 length. COVID-2019 infection causes severe pneumonia that turns into a pulmonary alveolar
 collapse within a few hours and leads to the cessation of oxygen exchange. The incubation
 period for the virus is 2 to 10 days, and the clinical spectrum of the disease ranges from
 asymptomatic infection to severe respiratory failure. There is elevated lymphopenia, lactate,
 creatinine, and kinase dehydrogenase, and higher concentrations of interleukins such as
 IL-1β, IL-5, IL-7, IL-8, IL-9, IL-10, IL-15, IL-12p70, FGF, GCSF, GMCSF, IFNγ, IP10, MCP1,
 MIP1A, MIP1B, PDGF, TNF-α and VEGF.

 There is no treatment for the definitive cure of COVID-19, and there is no vaccine that
 allows prevention. Considering that the best management choice is to reestablish hemodynamic
 status, stop organ failure, improve anti-inflammatory conditions, and improve redox status,
 management strategies could not be randomized since individual conditions change, and
 patients may have comorbidities at first. The studies that support antioxidant therapy in
 septic management range from those carried out in vitro, in vivo in an animal model and
 humans, so the evidence makes it necessary that patients treated with specific antiviral
 drugs, or antibiotics, receive at the same time nutritional supplement and antioxidants.

 The data that support each of the antioxidants as therapy in septic shock are mentioned
 below.

 1. N-ACETYL CYSTEINE.

 The administration of N-acetylcysteine (NAC), a glutathione (GSH) precursor, as a
 strategy to limit oxidative lung injury has been proposed since it increases the
 intracellular content of GSH. Alterations in GSH metabolism, in alveoli and lung tissue,
 are a central feature in many lung diseases. NAC increases the synthesis of GSH,
 increases glutathione-S-transferase (GST) activity, and has a direct action on free
 radicals (ROS). The application of NAC reduces levels of IL-8, IL-6, ICAM. NAC in
 patients with septic shock is associated with a shorter time on mechanical ventilation
 and fewer days of stay in the ICU.

 The application of NAC reduces levels of IL-8, IL-6, ICAM. NAC in patients with septic
 shock is associated with a shorter time on mechanical ventilation and fewer days of stay
 in the ICU. NAC uptake and intracellular concentration can be increased through the use
 of liposomes (L-NAC). NAC supplementation in animals exposed to lipopolysaccharides
 (LPS) reduced lung edema, lipoperoxidation (OLP), ACE damage, chloramine concentration,
 and concentrations of the eicosanoids thromboxane and leukotrienes (LTB2 and LTB4) in
 the lung. In clinical trials, supplementation with a bolus of 150 mg/kg NAC followed by
 50 mg/kg/day of NAC for four days in patients with acute lung injury ALI or ARDS
 improved the oxygenation rate from day 1 to 4 and reduced mortality.

 2. MELATONIN

 Melatonin (MT) has been shown to possess ROS-sequestering properties, protects lipids in
 cell membranes, cytosol proteins, and nuclear and mitochondrial DNA.

 Furthermore, in another study, MT demonstrated anti-apoptotic, antioxidant, and
 pleiotropic anti-inflammatory effects in vitro and in vivo as direct elimination
 activity against ROS and stimulation of antioxidant enzymes, such as CAT, SOD, GPx, GR,
 and gamma-glutamylcysteine synthase, MT can accumulate within the mitochondria and thus
 reduce the local excess production of ROS, which is typical in dysfunctional
 mitochondria during sepsis. Based on these favorable preliminary data, randomized
 control trials are warranted to assess TM's efficacy and safety as an add-on treatment
 in COVID-19 sepsis. The previously mentioned studies have recommended its use in sepsis,
 which should be considered in COVID-19 since it is also accessible, and its cost is low,
 making it possible to weigh the risk/benefit in the event of a pandemic.

 3. VITAMIN C

 Ascorbic acid, or vitamin C, is a water-soluble antioxidant that functions as a cofactor
 for multiple enzymes. It is absorbed at the intestinal level through the
 sodium-dependent transporter of vitamin C, filtered freely in the glomerulus, and
 reabsorbed at the proximal tubule level through the same transporter. Ascorbic acid
 inhibits the production of superoxide (O2-) and peroxynitrite (OONO-) by inhibiting
 superoxide-producing NADPH oxidase (O2-) and inducible nitric oxide (iNOS) mRNA
 expression, which prevents the abundant production of nitric oxide (NO) that generates
 peroxynitrite (OONO-) in the presence of O2-.

 4. PENTOXIFILINA.

 Pentoxifylline is a xanthine drug indicated in some severe alcoholic hepatitis; it also acts
 on the plasma membrane of red blood cells and makes it more malleable, thus improving blood
 perfusion. Pentoxifylline exerts several antioxidant and anti-inflammatory activities, such
 as reducing the restoration of GSH levels, maintaining mitochondrial viability, inhibiting
 the production of TNF-α, preserving vascular endothelial functions, and also supplementation
 with antioxidants has been reported better oxygenation rates, higher GSH, and more robust
 immune response. Also, there was a reduction in hospital stay length, the time of mechanical
 ventilation, the length of the ICU stays, the multiple organ dysfunction rate, and the
 mortality rate in patients with ALI / ARDS.

 II. Research question

 Will the administration of adjuvant therapy with specific antioxidant and pentoxifylline in
 patients with or without septic shock secondary to severe pneumonia due to COVID-19, will it
 avoid the use of mechanical ventilation, reduce the time of use of a mechanical ventilator,
 days of hospital stay, decrease the lipoperoxidation and will it increase the antioxidant
 capacity in patients admitted to intensive care?

 III. Justification

 In this COVID-19 pandemic, severe pneumonia and septic shock are the leading cause of
 morbidity and mortality in intensive care units worldwide. In this sense, and based on the
 discoveries of recent years in the field of oxidative stress, including those recently found
 in our group, it is necessary to report results on new treatments capable of reducing the
 deleterious inflammatory response and the redox state. In patients with pneumonia and septic
 shock. The situation that currently occurs in patients who progress to severity due to
 infection with COVID-19.

 Septic shock has been presented in other viral diseases such as Middle East Respiratory
 Syndrome Coronavirus (MERS-CoV) detected for the first time in Saudi Arabia, in which it
 exhibited a wide range of presentations at the time of diagnosis, similar to SARS-COv2 from
 patients without symptoms, subtle signs of pneumonia or multiorgan failure, with the capacity
 to cause the death of which the possible therapeutic interventions with antioxidants have
 been proposed since then that have been proposed for the new virus through the conclusions
 based on systematic reviews.

 Many viral diseases such as SARS-CoV, although clinical data are limited, can develop
 moderate and severe septic shock and increase ROS and RNS production, which is associated
 with overexpression of iNOS, NADP oxidases, cyclooxygenase two, and xanthine oxidase, which
 activates transcription factors such as NF-B resulting in an exacerbated pro-inflammatory
 host response. Also, O2 and ONOO participate as an essential mediator of pro-inflammatory
 interleukin production. These will continue to stimulate the production and release of more
 ROS and RNS that can interfere with mitochondrial respiration since mitochondrial dysfunction
 is commonly induced in an environment of septic shock. Therefore, antioxidant treatment may
 be a way to avoid excessive inflammation associated with a history of high oxidation in
 COVID-19 patients.

 With this study, we intend to evaluate the effect of the use of antioxidants on outcomes in
 storm regulation due to dysregulation of oxidative stress, shortening of ventilator use, days
 of stay, and clinical repercussion through the measurement of organ dysfunction in six
 different systems, using the SOFA score before and after the intervention, in critically ill
 patients due to SARS-Cov2 infection.

 IV. Hypothesis

 It is hypothesized that adjuvant therapy with antioxidants and pentoxifylline reduces
 ventilator use in patients with or without septic shock secondary to severe COVID-19
 pneumonia and decreases lipoperoxidation and corrects dysregulation of oxidative stress
 through the increase of antioxidant capacity.

 V. Primary objective

 Provide combined antioxidant therapy as an adjunct to standard therapy for patients with or
 without septic shock secondary to severe SARS-COV2 pneumonia to evaluate whether it is
 possible to avoid intubation, reduce days of assisted mechanical ventilation, and improve
 stress dysregulation oxidant leading to multiple organ failure.

 VI. Secondary objective

 1. Evaluate the prevalence of comorbidity in patients with or without septic shock and
 severe SARS-CoV2 pneumonia in the ICU.

 2. To evaluate the effect of adjuvant antioxidant therapy in reducing days with the
 ventilator and days of hospital stay in patients

 3. Analyze the effect on organ failure in five devices and systems (neurological,
 respiratory, hemodynamic, hepatic, hematological) of each of the therapies implemented
 in the different systems evaluated with the SOFA score.

 4. Measure lipoperoxidation in basal and post-therapy samples

 5. Measure the antioxidant capacity in basal and post-therapy samples.

 6. Measure IL-6 in basal and post-therapy samples.

 7. Measure procalcitonin, CRP, troponin, pro-BNP, ferritin, and D-dimer.

 8. Determine the status of outcomes by comorbidity strata.

 9. Document the use of ARA, ACE, SGLT2 inhibitors in patients with COVID-19.

 10. Analyze the previous use of steroids and those who did not have it in a stratified way

 VII. Methodology

 Study design

 It is a quasi-experimental, open analytical, prospective, and longitudinal (before-after)
 study.

 Sample size

 The sample size calculation was based on studies that currently have mortality using Vitamin
 C because there is no history of antioxidants in the clinical context. The sample size was
 calculated using X2 to compare two independent proportions.

 Therefore, it will be necessary to include 11 patients in each group if desired to obtain 80%
 possibility (80% power) or 32 if the power is 99% to detect a mean difference of ≥3 in SOFA
 between the groups. On the other hand, treatment will be possible in these patients, it will
 be possible to measure the basal state of oxidative stress, and state three after the therapy
 allows the use of small samples, as the patient is his control.

 Statistic analysis

 Continuous variables will be expressed as mean ± standard deviation or median with minimum
 and maximum, depending on their distribution. Categorical variables will be expressed as
 frequencies and percentages. The normality of the variables will be evaluated using the
 Shapiro-Wilk or Shapiro-France test, depending on the sample size. Variables with normal
 distribution will be analyzed with parametric tests (Student's t-test for independent
 measurements or paired t-test for before-after measurements). While various non-parametric
 tests were used (Mann-Whitney test, Kruskal-Wallis or Wilcoxon signed rank test, depending on
 the particular case) to contrast variables without Gaussian distribution. Analysis of paired
 samples (before-after) will be performed with Friedman or Wilcoxon and paired t-test,
 depending on the distribution of the data. For multivariate analysis, a binary logistic
 regression analysis will be performed. Also an analysis of repeated samples and panel data
 testing different models (grouped model, model for longitudinal data, marginal approximation
 model and multilevel model).
Inclusion Criteria:

 -  Patients admitted to the UTC in the Temporary COVID-19 Citibanamex Center with
 suspected or diagnosed severe pneumonia due to SARS-COV2 with or without septic shock.

 -  Patients who accept and sign informed consent. If the patient is clinically unable to
 authorize, acceptance by a first-degree relative will be requested.

 -  Diagnosis of septic shock in the last 24 hours characterized by refractory hypotension
 and vasopressor requirement despite adequate fluid resuscitation (20 mL/kg of colloids
 or 40 mL/kg of crystalloids) to maintain a blood pressure ≥ 65 mmHg with lactate> two
 mmol / L.

 Exclusion Criteria:

 -  Patients with an advance directive format.

 -  Chronic use of steroids in the past six months or recent.

 -  Use of statins before admission.

 -  Patients who are under some antioxidant treatment.

 -  Any contraindication for the use of Vit C, Vit E, NAC, and melatonin.

 -  Pregnant women.