Combined Metabolic Activators Accelerates Recovery in Mild‐to‐Moderate COVID‐19

Abstract COVID‐19 is associated with mitochondrial dysfunction and metabolic abnormalities, including the deficiencies in nicotinamide adenine dinucleotide (NAD+) and glutathione metabolism. Here it is investigated if administration of a mixture of combined metabolic activators (CMAs) consisting of glutathione and NAD+ precursors can restore metabolic function and thus aid the recovery of COVID‐19 patients. CMAs include l‐serine, N‐acetyl‐l‐cysteine, nicotinamide riboside, and l‐carnitine tartrate, salt form of l‐carnitine. Placebo‐controlled, open‐label phase 2 study and double‐blinded phase 3 clinical trials are conducted to investigate the time of symptom‐free recovery on ambulatory patients using CMAs. The results of both studies show that the time to complete recovery is significantly shorter in the CMA group (6.6 vs 9.3 d) in phase 2 and (5.7 vs 9.2 d) in phase 3 trials compared to placebo group. A comprehensive analysis of the plasma metabolome and proteome reveals major metabolic changes. Plasma levels of proteins and metabolites associated with inflammation and antioxidant metabolism are significantly improved in patients treated with CMAs as compared to placebo. The results show that treating patients infected with COVID‐19 with CMAs lead to a more rapid symptom‐free recovery, suggesting a role for such a therapeutic regime in the treatment of infections leading to respiratory problems.


Supplementary Appendix
This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Combined metabolic activators accelerate recovery in mild-to-moderate COVID-19

Methodology
Phase II: Open label, randomized controlled design Phase III: Double blinded, randomized controlled design Treatment arm: At home-treatment with standard therapy + CMAs consisting of serine, Lcarnitine tartrate, N-acetylcysteine, and nicotinamide riboside as compared to standard therapy + placebo. Standard therapy includes only hydroxychloroquine in phase II and either hydroxychloroquine or favipiravir in phase III.

Primary objective
Studies are planned as Phase II and Phase III clinical drug research to be conducted in patients diagnosed with COVID-19. Patients will be ambulatory and after the diagnosis/confirmation of diagnosis, will be sent home with their treatment.
The primary objective is to assess the clinical efficacy of the combination of metabolic activators administration and standard therapy in COVID-19 patients.
For the primary purpose, the proportion of patients who were recovered during the course of disease until 14 days after the initial diagnosis of COVID-19 disease.

Secondary objectives
The secondary aim is to evaluate the safety and tolerability of combined metabolic activators administration and standard therapy combination.
The following secondary safety objectives have been identified both in phase II and phase III studies: • Number / characteristics of adverse event (AE), Serious Adverse Event (SAE) and treatment discontinuation due to study drug from the beginning of the study to the end of the follow-up period • Number / features of all changes in hematology parameters evaluated as AE from the beginning of the treatment to the end of the follow-up period.
• Number / characteristics of all changes in selected biochemical parameters evaluated as AE from the beginning of the treatment to the end of the follow-up period.
• The changes in vital signs (systolic and diastolic blood pressures, pulse, respiratory rate, body temperature, pulse oximetry values), baseline values, and the status of treatment and follow-up visits

Number of subjects
A total of 100 COVID-19 disease patients in phase II will be enrolled and randomized on a 3:1 basis to the combined metabolic activators administration + standard therapy or placebo + standard therapy in Turkey. Standard therapy includes only hydroxychloroquine treatment.
A total of 300 COVID-19 disease patients in phase III will be enrolled and randomized on a 3:1 basis to the combined metabolic activators administration + standard therapy or placebo + standard therapy in Turkey. Standard therapy includes either hydroxychloroquine or favipiravir treatment.

Main inclusion criteria
To be included in the study, patients should meet all the following criteria: • Patients of both genders (females and males) over 18 years of age • Written informed consent obtained from the subjects prior to any procedures related to the study.

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Understand all procedures to be applied within the scope of the study protocol • Ambulatory patients with symptoms diagnosed with COVID-19 with real time PCR test result positivity in the last 24 hours

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Patients with stable clinical course and who could be treated on an ambulatory basis

Main exclusion criteria
Subjects cannot be included in the study if any of the following criteria is met: • Patients who has partial oxygen saturation below 90% or with severe clinical status requiring admission to critical care unit • Inability or unwillingness to give written informed consent • Physician makes a decision that trial involvement is not in patients' best interest, or any condition that does not allow the protocol to be followed safely.

Dosage and duration of therapy
Phase II study was planned as a parallel group, randomized and open label study. The study subjects in phase II will be randomized on a 3:1 basis to the combined metabolic activators administration+ standard therapy or placebo + standard therapy in Turkey. Standard therapy includes only hydroxychloroquine treatment. Standard therapy + placebo group will include 25 volunteers; the standard therapy + combined metabolic activators administration group will consist of 75 volunteers.
Phase III study was planned as a parallel group, randomized and double-blinded study. The study subjects in phase III will be randomized on a 3:1 basis to the combined metabolic activators administration+ standard therapy or placebo + standard therapy in Turkey. Standard therapy includes one of the hydroxychloroquine or favipiravir treatment. Standard therapy + placebo group will include 75 volunteers; the standard therapy + combined metabolic activators administration group will consist of 225 volunteers.
In both studies, after the volunteers sign the informed consent forms, the treatment will be administered as the first by the responsible investigator. All other treatments will be administered by the patient at home. Hydroxychloroquine treatment will be administered at an initial dose of 800 mg/day (2x400 mg oral) followed by 400 mg/day (2x200 mg oral) for a total of 5 days. The dosage of favipiravir will be 1600 mg orally twice daily for 1 day followed by 600 mg orally twice daily for 4 days.
The standard therapy + combined metabolic activators administration group will be applied the same dosage and duration of the standard therapy. Additionally, CMAs will be given for two weeks in doses indicated below (was given orally, twice/day; one dose in the morning, one dose after dinner): The total treatment period for the standard therapy will be 5 days and administration of CMAs will be for 14 days. On the 14th day, patients will perform a last visit which will be the follow-up visit.

Investigational Products
Investigational products will be produced as the test product and will be packed as individual dosages in identical HDPE plastic bottles of 60 mL with a screw cap. Test product will be in the form of powder and will be dissolved in 200 ml preferably cold water before use. Placebo treatment will include lactose only in identical HDPE plastic bottles of 60 mL with a screw cap.

Study design
The study comprises two visits: Visit 1 (Screening; Day 0): The subjects will get the informed consent form and oral information on the study protocol. Blood samples and will be taken to check out the eligibility to the study.
After clinical and physical examination blood samples will be obtained for routine analysis outlined in the study protocol. Chest tomography (CT) and virus load assessment by PCR will be performed. An ECG will also be performed. Plasma samples will be taken for clinical chemistry analysis.
Eligible study subjects will be invited to the clinic for randomization to the active treatment and standard therapy groups. They will be asked to take the first dose and be observed for development of side effects. Patients who can tolerate the study agents will start to take combined metabolic activators (i.e., 2 dosage daily just after breakfast and dinner) for two weeks.
After initial dosing at the hospital patients will be instructed to stay at home during their treatment and will be asked to attend to the investigational site if symptoms are worsened or diminished during the 14-day period.
Visit 2 (Week 2, Day 14): Clinical and physical examination, adverse events recording, virus load assessment by PCR will be performed. Chest tomography will be performed. Plasma samples will be taken for clinical chemistry analysis.
After the visit 2, subjects will stop taking their study drugs.

Study duration
The active treatment duration will be 2 weeks for each subject and the total study duration is estimated as 6 months.

Efficacy evaluation
The primary efficacy evaluation will be on the percentage (proportion) of patients recovered during the treatment period for both arms in COVID-19 patients.
The primary objective is to assess the clinical efficacy of the combined metabolic activators administration and standard therapy in COVID-19 patients based on the proportion of recovery.

Safety evaluation
Safety will be assessed by monitoring of adverse events, physical examination, vital signs measurements and clinical laboratory tests. 3. Laboratory safety parameters will include complete blood count, alanine aminotransferase, aspartate aminotransferase, creatinine, C-reactive protein, triglycerides, cholesterol, glucose, LDH, ferritin, D-dimer. 4. Eligible study subjects at Visit 1 will be offered to be enrolled in the study and to take the first dose at the hospital. 5. Two doses taken just after breakfast and dinner. Patients will be treated on an ambulatory basis. If patients' symptoms worsen, patient will be instructed to attend the hospital for an examination.

Visit
6. The study participants will be observed for the development of any allergic reactions or intolerance after taking the first dose at the hospital. 7. Adverse events (AE) and serious adverse events (SEA) will be monitored continuously and all AEs that occur at any time during the study will be reported in Case Report Forms.    Figure S1. The effect of combined metabolic activators on the recovery of the patients is presented on the patient groups received standard therapy with A) hydroxychloroquine and B) favipiravir separately.

Table S2
Baseline demographics of the study population

SUPPLEMENTARY DATASETS
Dataset S1 The characteristics of each patient involved in the Phase-2 and Phase-3 studies.
Dataset S2 Summary of laboratory and physical variables before and after treatment in the CMA and placebo groups in A) phase-2, B) phase-3 and C) all patients.
Dataset S3 Untargeted metabolomics data for each patient before and after treatment.
Dataset S4 Plasma level of metabolites that are significantly different on Days 14 vs Day 0 in the CMA and placebo groups. Only metabolites detected in >50% of samples were analyzed.
Dataset S5 Plasma level of metabolites that are significantly different between the CMA and placebo groups on Days 0 and Day 14. Only metabolites detected in >50% of samples were analyzed.
Dataset S6 Association between the plasma level all metabolites with the plasma levels of metabolic activators including serine, carnitine, nicotinamide riboside, and cysteine.

Dataset S7
The Olink multiplex inflammation panel used to detect the dynamic range of 72 proteins in plasma samples of the 93 patients participated in the study.
Dataset S8 Plasma level of inflammation related proteins that are significantly different on Days 14 vs Day 0 in the CMA and placebo groups. Only proteins detected in >50% of samples were analyzed.
Dataset S9 Plasma level of inflammation related proteins that are significantly different between the CMA and placebo groups on Days 0 and Day 14. Only metabolites detected in >50% of samples were analyzed.
Dataset S10 Association between the plasma level all inflammation related proteins with the plasma levels of metabolic activators including serine, carnitine, nicotinamide riboside, and cysteine.
Dataset S11 Association between the plasma level of clinical variables including ALT, AST, LDH, Triglycerides (TGs) and hemoglobin with plasma level of all metabolites.
Dataset S12 Association between the plasma level of clinical variables including ALT, AST, LDH, Triglycerides (TGs) and hemoglobin with plasma level of all inflammation related proteins.
Dataset S13 Association between the plasma level of key inflammation related proteins including MCP-4, IL-18R1, HGF, CXCL10 and CSF-1 with plasma level of all metabolites.