WO2022235234A2

WO2022235234A2 - Preparation of new formulations usable in covid-19 treatment and determination of clinical effects thereof

Info

Publication number: WO2022235234A2
Application number: PCT/TR2022/050181
Authority: WO
Inventors: Mustafa Güzel; Muhammet Davut Arpa; Neslihan ÜSTÜNDAĞ OKUR; Hasan Önal; Emre Şefik ÇAĞLAR
Original assignee: Istanbul Medipol Universitesi
Current assignee: Istanbul Medipol Universitesi
Priority date: 2021-05-04
Filing date: 2022-03-01
Publication date: 2022-11-10
Anticipated expiration: 2023-11-04
Also published as: WO2022235234A3

Abstract

The present invention involves synthesizing Angiotensin-1-7 which is a medicine active ingredient on both laboratory and industrial scale in order to be used effectively in Covid-19 treatment, and its new formulations.

Description

PREPARATION OF NEW FORMULATIONS USABLE IN COVID-19 TREATMENT AND DETERMINATION OF CLINICAL EFFECTS THEREOF

Field of the Invention

The invention relates to a medicine that is effective in Covid-19 treatment and comprises Angiotensin- 1-7 as the active ingredient. The present invention involves synthesizing Angiotensin- 1-7 which is a medicine active ingredient on both laboratory and industrial scale in order to be used effectively in Covid-19 treatment, and its new formulations.

Background of the Invention (Prior Art) The proprietary medicines against Covid-19, a viral disease that was declared as a pandemic by the World Health Organization on March 11, 2020, are very limited. Therefore, it is essential to propose and implement new treatment methods and different solutions against this relentless disease that affects the whole world. Existing studies conducted with Angiotensin 1-7 (AT 1-7) were mainly carried out by considering the antihypertensive property of this peptide. In the state of the art, the medicine formulation comprising AT 1-7 and its effect on Covid-19 disease are unknown.

In chronic diseases, the Renin-Angiotensin system (RAS) is active. The RAS system has two branches. While vasoconstriction, proliferation and inflammation occur over ATI receptor via the Renin / Angiotensin I / Angiotensin II pathway (inflammation-producing pathway), angiotensin I and II are degraded to Angiotensin (1-7) [Ang (1-7)] via the ACE2/ Angiotensin (1-7) pathway (anti-inflammatory). Ang (1-7) has the opposite effect of Angiotensin II and binds to Mas receptors, thereby showing vasodilation, antiproliferative and anti-inflammatory effects.

RAS plays a role in the pathogenesis of various cardiovascular diseases such as hypertension, atherosclerosis, myocardial infarction, cardiomyopathy, heart failure and metabolic syndrome. Excessive RAS activation is thought to directly cause damage to the vascular endothelium in diabetes. RAS activation and subsequent upregulation of angiotensin II (Ang II) cause increased renal fluid and sodium retention as well as increased vascular tone, thereby making patients vulnerable to endothelial damage and damage in kidneys, heart, and nervous system. Healthy individuals have very low ACE2 activity. In patients with type ID, ACE2 activity is increased with microvascular or macrovascular complications. This indicates that ACE2 may play a role as a compensatory mechanism in the regulation of vascular and renal function in a patient with T1D. Serum ACE2 activity is almost 30% higher in men than in women. Accordingly, they were significantly older than those in the lowest quartile, had a longer duration of diabetes, higher SBP, and lower eGFR. Therefore, ACE2 activity has been positively associated with such clinical features associated with aging, cardiovascular risk, and proinflammatory status.

The ACE2/Ang (l-7)/Mas receptor pathway creates a protective balance in chronic diseases in which the RAS system is overactive.

It gives rise to think that increasing plasma Ang (1-7) may be beneficial in many disease states, particularly those associated with elevated Ang II / Ang (1-7).

Angiotensin (1-7) Peptide

Angiotensin (1-7) shows vasodilation, myocardial protection, anti arrhythmia, antihypertensive effects on the cardiovascular system, and prevents pathological cardiac remodeling. In addition, it reduces insulin resistance. It acts through Ang (1-7) Mas receptors. In Mas 1 -knockout animal studies, myocardial contractile dysfunction, cardiac fibrosis, hypertension, endothelial dysfunction, renal fibrosis, glomerular dysfunction, insulin resistance and dyslipidemia were observed.

Ang (1-7) also has a wide spectrum of effects in different organs and tissues that go beyond the above-mentioned cardiovascular and renal effects. These effects are mediated by Mas and can compensate for many of the detrimental effects of Ang II. Through these mechanisms, Ang (1-7) can ameliorate pathological conditions, including fibrosis and inflammation in organs such as the lung, liver, and kidney. In addition, this heptapeptide has positive effects on metabolism, increasing glucose uptake and lipolysis, while reducing insulin resistance and dyslipidemia. Ang (1-7) has effects on learning and memory as well as may improve cerebroprotection against ischemic stroke. The reproductive system may also be affected by Ang (1-7) treatment, with increased ovulation, spermatogenesis, and sexual steroid synthesis. Finally, Ang (1-7) is considered a potential anti-cancer treatment since it can inhibit cell proliferation and angiogenesis. Therefore, ACE2 / Ang (1-7) / Mas pathway appears to be involved in many physiological and pathophysiological processes in many systems and organs, especially by opposing the detrimental effects of inappropriate overactivation of the ACE / Ang II / AT1R axis.

Angiotensin (1-7) is mainly metabolized by ACE in the lungs.

Recently, Ang (1-7) has been shown to play a protective role in blood-brain barrier damage. In cerebral ischemia-reperfusion injury and hypoxia-induced blood-brain barrier injury, Ang (1-7) promotes the expression of zonula occludens-1 and claudin-5 which are tight junction- related proteins in brain endothelial cells. Ang (1-7) does not function as a brain protector when given only in the brain. It regulates perfusion pressure in different parts of the brain. Chronic Ang (1-7) infusion attenuated the development of hypertension and ameliorated baroreflex control of heart rate and glucose metabolism in fructose-fed rats.

Ang (1-7) and ACE2 activities have been shown to have a protective effect on the lung. In animal studies, experimental models that modulate Ang (1-7) infusion in ARDS have been shown to reduce the pro-inflammatory response and ameliorate lung injury scores and lung function. A randomized controlled study conducted on recombinant humans increased NO synthesis. In humans with ARDS, ACE2 has been shown to reduce Ang II levels and proinflammatory mediators and increase plasmatic surfactant protein D without hemodynamic changes.

Angiotensin II/Ang (1-7) Balance in SARS-CoV-2 Infection

Following SARS-CoV-2 binding, ACE2 function is reduced and Ang II levels are elevated in the blood of patients with COVID-19. These effects, together with the “cytokine storm”, cause a worse prognosis. Current researches show that the poor prognosis in COVID-19 patients is associated with factors such as gender (male), age (>60 years), underlying diseases (hypertension, diabetes, and cardiovascular disease), secondary ARDS, and other relevant factors. Possible pathophysiologies that may occur with disruption of Angiotensin II / Ang (1-7) balance are as follows: Cardiacfibrosis and hypertrophy, increase in vascular resistance, endothelium, increase in thrombosis tendency, increase in inflammation and oxidative stress, increase in tissue Fibrosis, atrial arrhythmia, impaired baroreceptor sensitivity, weakening of blood-brain barrier integrity, ovulation, decrease in spermatogenesis, decrease in sexual steroid synthesis, insulin resistance, dyslipidemia, facilitating the formation and spread of cancer.

Ang (1-7) increases the baroreflex control of heart rate by acting on the vagal. The virus itself can also increase proBNP levels in COVID-19 patients. SARS-CoV-2 binds with ACE2, causing the uncontrolled release of angiotensin 2 (ANG II) and decreased synthesis of Ang (1-7). Secondly, Ang II plays an opposing role and facilitates the secretion of proBNP while exerting an anti-inflammatory effect to protect tissue. In addition, a high incidence of thrombosis and thromboembolism has been reported in patients suffering from COVID-19. ACE2 expression has been shown to occur in endothelial cells of arterial and venous vessels. There is clear evidence that endothelial cells are prone to acquire SARS-CoV-2 infection, and then endothelium develops. Patients with acute respiratory failure suffering from COVID-19 exhibit a very severe hypercoagulation state from a consumptive coagulopathy, massive endothelial stimulation causes a large von Willebrand Factor elevation equivalent to damage.

While pulmonary involvement due to COVID-19 infection is rare and mild in children who do not have a chronic disease in the past, a severe inflammatory picture called MIS-C may occur 4 weeks after having COVID infection. The main reason for this clinical difference between children and adults is that the distribution of ACE-2 receptors is concentrated in arteries, venous endothelial cells and arterial smooth muscle cells instead of the lung.

It may be due to the pathophysiology of different clinical pictures seen in the postinfectious period in adult patients and the inability to restore the angiotensin II/ Ang (1-7) balance of the chronic diseases they have.

The heart, brain and kidney are the main sources of angiotensin (1-7) production. In the human coronary circulation, NEP appears to have a more prominent role in ang (1-7) production than ACE2. Pharmacokinetic experiments had determined that ang (1-7) in humans has a short half-life of approximately 0.5 hours. Following subcutaneous injection, the peptide rises rapidly in the blood, reaching its peak plasma concentration in approximately 1 hour. The plasma half-life of ang (1-7) in rats is only 9 seconds. Ang (1-7) level is 5-20 times higher in people over the age of 50 (3), suffering from chronic diseases such as diabetes/COPD/Asthma, taking ACE inhibitor/ARB type blood pressure medicine for at least 3 years, smoking, and not having COVID-19 infection compared to healthy people. Safety Profile of Angiotensin (1-7)

Phase I and Pharmacokinetic Study of Angiotensin (1-7) which is an Endogenous Antiangiogenic Hormone It is administered 400 mcg/kg/day to 6 patients suffering from metastatic pancreatic cancer and 700 mcg/kg/day Angiotensin (1-7) to 6 patients with metastatic lung cancer and 6 patients with prostate cancer for 5 days. In the patient group administered 700 mcg/kg/day, one patient had a stroke and one patient had reversible cranial neuropathy. As a result, 400 mcg/kg/dose was recommended for the phase II study. The values of angiotensin administration at different doses on the 1st and 5th days in cancer patients are given in Tables 1 and 2, respectively.

Table 1. 1st day of angiotensin administration at different doses in cancer patients

Table 2. 5th day of angiotensin administration at different doses in cancer patients

Phase I/II Study of Angiotensin 1-7 Before and After Chemo treatment in Patients Suffering from Newly Diagnosed Breast Cancer Five different doses (2.5, 10, 50, 75, and 100 mcg/kg) of subcutaneous angiotensin 1-7 treatment were administered to 55 newly diagnosed breast cancer patients for 7 days before chemo treatment and 10 days after chemo treatment. Toxicity did not occur in any of the preferred doses, therefore no toxicity-related chemo treatment modification occurred. 100 mcg/kg/dose angiotensin (1-7) administration significantly reduced the incidence of thrombocytopenia, anemia, and lymphopenia secondary to chemo treatment.

Pharmacokinetic Study of Angiotensin 1-7

The relation of dose with AUC and Cmax demonstrates first-order plasma elimination kinetics. Comparison of pharmacokinetics with hematological pharmacodynamics demonstrates that doses of D50 mcg/kg/day would be appropriate for optimal effect. The table of pharmacokinetic values of the study is given in Table 3.

Table 3. Pharmacokinetic study of Angiotensin 1-7

Phase II Angiotensin (1-7) Treatment in the Treatment of Patients with Metastatic Sarcoma In this study, it was reported that 20 mg/day (400 mcg/kg/dose) Angiotensin (1-7) treatment administered subcutaneously to 20 metastatic sarcoma patients in order to benefit from its anti-angiogenetic effect was well tolerated and did not show toxicity. In chronic diseases, serum Angiotensin (1-7) levels can reach 40-60 pg/ml. It was thought that these levels should be reached in order to stop the cytokine storm. Therefore, in our study, it was planned to administer 400 mcg/kg angiotensin (1-7) peptide treatment subcutaneously for 7 days. There are 5 studies registered in clinical studies on the treatment of SARS-CoV-2 and Angiotensin (1-7) (Table 4).

Table 4. Studies with Clinical study Records on SARS-CoV2 and Angiotensin (1- 7) Treatment

Brief Description and Aims of the Invention

The present invention relates to a medicine comprising Angiotensin- 1-7 as the active ingredient, which meets the above-mentioned requirements, eliminates the disadvantages and provides some additional advantages, as well as its preparation and its activity against Covid-19.

The primary aim of the invention is to propose new solutions against the Covid-19 pandemic which also seriously affects our country and to contribute to the country’s economy with new medicine formulations that can be developed.

The aim of the invention is to prepare a new medicine effective against the Covid-19 pandemic or new supplementary medicines that will not make a medicine-medicine interaction with the active ingredient Angiotensin and that will stimulate the immune system.

Another aim of the invention is to use this medicine in the treatment of not only Covid-19 but also other diseases that threaten the immune system along with viral infections similar to this disease.

With the invention, it is aimed to reduce the hospitalization rate and to prevent the severe course of the disease in the treatment of Covid-19 by using AT 1-7. With the invention, it is also aimed to significantly reduce the mortality and morbidity rate of COVID-19 with the medicine comprising Angiotensin-1-7.

In the invention, it is envisaged that the structure of Angiotensin 1-7 acetate (AT 1-7) is administered subcutaneously to the patient. Therefore, the prepared formulation must be an isotonic formulation. The solubility of angiotensin 1-7 acetate in isotonic solution is quite low. Therefore, the solubility of AT 1-7 will be increased in the prepared formulation and at the same time, it will be converted into isotonic injection preparation form. It is lyophilized by adding 2-Hydroxypropyl-Beta-Cyclodextrin (HPBS) material to increase the solubility.

The invention is a lyophilized powder type formulation that can be used in the treatment of Covid-19 and is re-dissolved with water for injection, thereby taking the form of a parenteral solution. Lyophilized preparations also have advantages in terms of their high stability and ease of production in the pharmaceutical industry.

Also, since AT-1-7 which is an endogenous ligand, does not contain any harmful active compound in its toxicological profile, it is certain that it will provide a serious advantage over existing medicines.

Description of the Figures

Figure 1: Formation of angiotensin (1-7) in RAAS

Figure 2: Cytokine Storm

Figure 3: Impairment of angiotensin II/ Angiotensin (1-7) balance with the effect of SARS- CoV-2 virus

Detailed Description of the Invention

The medicine of the invention which can be used in the treatment of cerebral ischemia, stroke, ARDS, hypertension, heart failure, myocardial infarction, cardiac arrhythmia, type 1 and type 2 diabetes, complications of diabetes, male and female infertility, cirrhosis, cancer, cancer metastasis or COVID-19 infection, comprises

• 1-35 mg Angiotensin- 1-7 (AT 1-7) as the active ingredient, 24-480 mg 2-Hydroxypropyl- Beta-Cyclodextrin (HPBS) as the excipient to increase the solubility therein and active ingredient stabilization, 1- 30 pL Sodium hydroxide (NaOH) solution to adjust the pH, and

• 0.4-0.6% Sodium chloride (NaCl) solution or 2-4% Dextrose solution or 2-4% Mannitol solution to adjust the osmotic pressure.

The renin-angiotensin system (RAS) is a hormonal system that regulates blood pressure and fluid balance. While the Renin/Angiotensin II pathway (the pathway that produces inflammation) constricts the vessels and produces an inflammatory response, the angiotensin II is degraded to Angiotensin (1-7) via the ACE 2/Angiotensin (1-7) pathway (anti inflammatory). These two pathways are like the two pans of a scale that must be balanced.

In chronic diseases such as diabetes and cardiovascular diseases, as the disease progresses and the vascular structure deteriorates, the Renin-angiotensin system begins to work harder to maintain blood pressure. As a result of the overactivity of the Angiotensin II pathway, which raises blood pressure and produces inflammation, a part of Angiotensin II is balanced by the degradation of Angiotensin (1-7) via the ACE2 enzyme (Figure 1). Ang (1-7) level is 5-20 times higher in people over the age of 50, suffering from chronic diseases such as diabetes/COPD/Asthma, taking ACE inhibitor/ ARB type blood pressure medicine for at least 3 years, smoking, and not having COVID-19 infection compared to healthy people. Table 5 shows how the Angiotensin IE Angiotensin 1-7 balance is established in different clinical situations.

Table 5. Balance of Angiotensin II/Ang-1-7 in Different Conditions

The virus uses the ACE2 receptor, which is an important part of this system, as the gateway to the body. The ACE2 receptor is concentrated in the lungs and veins in adults, and in veins in children. Angiotensin 1-7 level decreases significantly when ACE2 enzyme becomes inoperative, and angiotensin II level increases. The balance between the two branches of the renin-angiotensin system is impaired. With the increase of angiotensin II, an excessive inflammatory response is produced, which causes a situation called cytokine storm (Figure 2)·

The invention is a lyophilized parenteral preparation using the active ingredient Angiotensin 1-7 acetate.

Ideal medicine formulations for the invention:

Ideal formulation 1 for the invention: ATI-7: 1-35 mg

HPBS: 24-480 mg NaOH solution: 1- 30 gL

0.1-1% NaCl solution: 1 mL

Ideal formulation 2 for the invention:

ATI-7: 1-35 mg HPBS: 24-480 mg NaOH solution: 1- 30 gL

1-8% Dextrose solution: 1 mL

Ideal formulation 3 for the invention:

AT 1-7 1-35 mg HPBS 24-480 mg NaOH solution 1- 30 gL

1-8% Mannitol solution 1 mL

2-Hydroxypropyl-Beta-Cyclodextrin (HPBS) as the excipient to increase the solubility therein and active ingredient stabilization, NaOH solution at low concentration to adjust the pH and 0.4-0.6% NaCl solution or 2-4% dextrose solution or 2-4% mannitol solution as a solvent to adjust the osmotic pressure are used. In a preparation method of the inventive formulations, AT 1-7 and HPBS are first weighed in the ideal ratio and dissolved with 0.4-0.6% NaCl solution. The mixing speed is between 50- 1000 rpm. The sterilization operation is conducted by filtering through a membrane filter. pH is adjusted to a range of 4-8. NaOH solution is added to adjust the pH. Then, the prepared solution is lyophilized. The powder lyophilisate is re-solutionized by reconstituting with water for injection before use.

In another preparation method of the invention, AT 1-7 and HPBS are weighed in the ideal ratio and dissolved with water for injection. The sterilization operation is conducted by filtering through a membrane filter. NaOH solution is added to adjust the pH. Then, the prepared solution is lyophilized. The powder lyophilisate is re-solutionized by reconstituting with 0.4-0.9% NaCl solution or 2-4% dextrose solution or 2-4% mannitol solution before use.

In another preparation method of the invention, 0.4-0.9% NaCl solution, 2-4% dextrose solution and 2-4% mannitol solution can be used together as a mixture to solubilize the AT 1-7 and HPBS mixture and adjust the osmotic pressure or reconstitute lyophilized powder.

The invention is an inventive formulation and provides innovation on the indication for Covid-19. Thanks to the invention, it is thought to prepare new proprietary medicines that can show anti -viral effects against the Covid-19 virus. The new formulation and plurality of medicines that can be good for the treatment of Covid-19 show a synergistic effect without interacting with each other.

Covid-19 infection binds to the ACE2 receptor, reduces the level of Angiotensin (1-7) and impairs the balance of Angiotensin II / Angiotensin (1-7) in the body, thereby causing a cytokine storm. The invention will provide the Angiotensin II/ Angiotensin (1-7) balance by increasing the level of Angiotensin (1-7), which is decreased due to Covid-19. Our invention will provide the necessary time for the immune system to defend itself, to restore this disturbed balance in the body and to calm the cytokine storm. It is unique in terms of its mechanism of action, it has no similarity with any medicine.

GLOSSARY OF ABBREVIATIONS

ACE2 Angiotensin-converting enzyme 2

ALT Alanineamino transferase

Ang (1-7) Angiotensin 1-7

ARDS Acute Respiratory Distress Syndrome

AST Aspartate Aminotransferase

CAO Serious Adverse Event

WHO World Health Organization

ECMO Extracorporeal Membrane Oxygenation cGFR Estimated Glomerular Filtration Rate

ECG Electrocardiogram

RAS Renin Angiotensin System

ST Standard Treatment

Study Protocol

8. Female patients who are not pregnant and are not likely to be pregnant for 4 weeks and are not breastfeeding.

Exclusion Criteria

1. Patients diagnosed with severe or critical COVID-19,

2. Patients with respiratory rate > 30 /min

3. Patients with heart rate > 125 /min

4. Patients with respiratory failure defined as a clinical need for high-flow oxygen treatment, non-invasive positive pressure ventilation or endotracheal intubation and mechanical ventilation

5. Patients with shock, defined systolic pressure <90 mm Hg or diastolic blood pressure <60 mm Hg, or in need of vasopressor

6. Patients with multiple organ dysfunction/deficiency

7. Patients with a WHO Clinical Progression Scale score >6

8. Patients hospitalized for reasons other than the severity of COVID-19 or comorbidities (e.g. Social reasons, local policies, isolation/quarantine)

9. Patients of another clinical research study

10. Patients receiving immune plasma outside of standard COVID-19 treatment

11. Patients who need mechanical ventilators or ECMO

12. Patients with severe liver disease, AST or ALT level 5 times or more

13. Patients with severe renal impairment (GFR <30 mL/min)

14. Patients with immunodeficiency, Tuberculosis

15. Patients with hematological cancer

16. Patients who are pregnant or Postpartum

17. Patients with a history of poor compliance or whose ability to comply with research procedures are questionable or unable to give informed consent at the discretion of the researcher, or patients with a past psychiatric illness or hospitalized by court order.

Primary Endpoint: · The primary endpoint of this study is that patient worsening with a WHO clinical progression score >6 within 1-28 days.

Secondary Endpoints: Recovery of the patient with a WHO Clinical Progress Score < 2 within 28 days,

Secondary points

• WHO Progress Scale Score change from baseline within 1 to 28 days,

• Daily shortness of breath, cough, and sputum scale in 1 to 28 days (BCSS) (including disaggregated scores),

• Percentage of patients who need mechanical ventilator with a worsening clinical condition,

• Percentage of patients sent to intensive care,

• Recovery time,

• Time of discharge from the hospital,

• National Early Warning Score change from baseline within 1 to 28 days (NEWS 2),

• All causes of mortality within 28 days,

• QoL (EQ-5D-5L respiratory survey),

Study Evaluation Chart

(1) An informed consent form is taken from the patient before the study.

(2) Baseline values should be taken prior to medicine study.

(3) Bioindicator: Hemogram, D-dimer, Ferritin, CRP, LDH, troponin, pro BNP, von Willebrand antigen); Biochemistry: Glucose, creatinine, ALT, AST, HbAlc (Checked only in scanned); coagulation: aPTT, PT; inflammation marker: IL-6 (4) In women of childbearing potential

(5) WHO clinical follow-up score, BCSS, EQ5DL, adverse event, rehospitalization status will be questioned by phone on days 14, 21 and 28 in early discharged patients. The NEWS2 score is for hospitalized patients only.

(6) Only while in the hospital.

Research Product (IMP) Angiotensin (1-7) peptide produced under GMP (Good Manufacturing Practices) conditions will be administered subcutaneously. Angiotensin (1-7) was prepared as a sterile solution at concentrations of 0.25, 1.0, 5.0 and 10.0 mg/ml, packaged in a 1 ml glass vial with a pH of 6.0 and an osmolality adjusted with mannitol. Angiotensin (1-7) is stable at room temperature for up to 2 weeks and can be stored at refrigerated temperatures (2-8°C).

Aim and Hypothesis

Primary Aim:

The overall aim of the study is to research whether Angiotensin 1-7 is superior to placebo in improving the clinical course and outcome of hospitalized adult patients suffering from SARS-CoV-2 infection confirmed with mild/moderate symptoms of COVID-19.

Secondary Aim:

To evaluate the efficacy and safety of angiotensin 1-7 for COVID-19.

The hypothesis of this study is that Ang (1-7) supportive treatment will also be important in the prevention and/or treatment of complications that may occur in SARS-CoV-2 infection and in the postinfectious period.

Study Design

This study is a randomized, double-blind, placebo-controlled, phase II clinical study.

Inclusion and Exclusion Criteria for Volunteers

Inclusion Criteria

• Adult, patients aged 18-59 years,

• SARS-CoV-2 patients confirmed by PCR/RT-PCR.

• Patients with the presence of symptoms consistent with COVID-19 (e.g. Shortness of breath, cough, sputum, muscle aches, headache, fatigue, sore throat, loss or change in sense of smell and/or taste, nasal flow and anorexia).

• Patients with a WHO 10-point Clinical Progress Score of 4 to 5,

• Patients with a WHO 10-point Clinical Progress Score of 3 and in need of hospitalization due to COVID-19 severity or comorbidity,

• Patients with a mobility score of <6, that is, not bedridden,

• Patients who accepted the patient informed consent form,

• Female patients who are not pregnant and are not likely to be pregnant for 4 weeks and are not breastfeeding.

Exclusion Criteria

• Patients diagnosed with severe or critical COVID-19,

• Patients with respiratory rate > 30 /min

• Patients with heart rate > 125 /min

• Patients with respiratory failure defined as a clinical need for high-flow oxygen treatment, non-invasive positive pressure ventilation or endotracheal intubation and mechanical ventilation

• Patients with shock, defined systolic pressure <90 mm Hg or diastolic blood pressure <60 mm Hg, or in need of vasopressor

• Patients with multiple organ dysfunction/deficiency

Patients with a WHO Clinical Progression Scale score >6 Patients hospitalized for reasons other than the severity of COVID-19 or comorbidities (e.g. Social reasons, local policies, isolation/quarantine)

• Patients of another clinical research study

• Patients receiving immune plasma outside of standard COVID-19 treatment

• Patients who need mechanical ventilators or ECMO

• Patients with severe liver disease, AST or ALT level 5 times or more

• Patients with severe renal impairment (GFR <30 mL/min)

• Patients with immunodeficiency, Tuberculosis

• Patients with hematological cancer

• Patients who are pregnant or Postpartum

• Patients with a history of poor compliance or whose ability to comply with research procedures are questionable or unable to give informed consent at the discretion of the researcher, or patients with a past psychiatric illness or hospitalized by court order.

Exclusion Criteria

Patients may withdraw from the study voluntarily or may be excluded with the discretion of the researcher. In the following situations, the researcher may decide to exclude the patient from the study.

• The occurrence of any serious adverse event (CAO), laboratory abnormality, or other medical condition that affects patient health, or the occurrence of other medical conditions in which it is not in the patient’s best interest to continue to participate in the study. • The patient meets exclusion criteria developed during the study or previously unrecognized, which would prevent the patient from staying in the study anymore.

• The volunteer’ s non-compliance with the study requirements.

• Pregnancy, breastfeeding.

• Admission of the patient to the intensive care unit (patients admitted to the intensive care unit will enter the final visit and will be monitored for clinical progression or improvement only according to the WHO clinical progression scale until the 28th day).

• If the patient cannot tolerate the side effects.

• Grade 4 serious adverse event.

• The researcher decides that it is in the patient’ s best interest to withdraw from the study.

• Patient withdrawal of consent.

• There are protocol violations, including non-compliance.

• The occurrence of administrative reasons.

Patient Withdrawal from the Study/Stopping the Study

Patients who discontinued the study early for any reason will undergo a final visit. All evaluations listed in the study evaluation chart for the final visit (Day 28) will be performed at this time.

Patients will be informed that they have the right to withdraw from the study at any time and that they do not have to state their reasons. Any withdrawal will be fully documented. The researcher will record the main reason for the early discontinuation of the treatment on the case report form within 48 hours of stopping the treatment, and if there is more than one condition, the primary reason will be stated.

If the patient is discontinued due to an adverse event, they should be monitored weekly for four weeks or until the event resolves or stabilizes (whichever comes first).

In case of early withdrawal, the patient will undergo all scheduled examinations.

Study Operations and Flow Chart Study Operations

In this randomized, double-blind, placebo-controlled, phase II study, 200 patients who met the inclusion criteria will be included the study. The visits to be made in the study and the procedures to be done in each visit are explained below.

Scanning Visit and Start (Day 0)

• An informed consent form is taken from the patient with a sign before the study.

• Medical history, demographic information, physical examination procedures will be performed.

• Nasal swab will be taken from the patients and RT-PCR test will be performed.

• Blood samples will be taken.

• If you are a woman, a urinary pregnancy test will be done.

• Inclusion/exclusion criteria will be questioned.

• Concomitant medicines will be asked. If the patient is found suitable after the scan;

• Vital signs will be taken.

• EQ-5D-5L respiratory survey and BCSS survey will be administered to patients.

• Medicine assignments will be made by randomization.

• Blood samples will be taken.

• Lung X-Ray/Lung CT will be taken.

• Medicine will be administered from the assigned arm (Research product or placebo) after medicine assignment.

• Acceptance criteria will be checked.

• Concomitant medicines will be asked.

• Clinical status BCSS and NEWS2 score will be determined.

• Side effects will be monitored.

• Bleeding/Thrombosis will be checked.

Visits on Day 3 and 5; Day 9 and 11; Day 16 and 18; Day 23 and 25

• Blood samples will be taken on these days (Only while in hospital).

• Concomitant medicines, clinical status questioning (BCSS and NEWS2 score), side effects follow-up and Bleeding/Thrombosis control will be performed. Visits on Day 7, 14, 21

• Nasal swab will be taken from the patients and RT-PCR test will be performed (Only while in the hospital).

• Blood samples will be taken (Only while in hospital).

• ECG will be taken (Only while in hospital).

• Concomitant medicines, clinical status questioning (BCSS and NEWS2 score), side effects follow-up and Bleeding/Thrombosis control will be performed.

Visit on day 28 (Final Visit)

• The operations on days 7, 14 and 21 will also be performed at the visit on day 28.

• In addition to these, a physical examination will be performed and vital signs will be determined.

Volunteer patients included in the study will be treated for up to 7 days. Discharge from the hospital will be made according to the following criteria. The doctor may decide that hospital care is no longer needed depending on the patient’s condition improves. The patients who were discharged from the hospital before the 28th day will have their final visit on day 28, and the necessary procedures will be applied.

Study Evaluation Chart

(1) An informed consent form is taken from the patient before the study.

(2) Baseline values should be taken prior to medicine study.

(3) Bioindicator: Hemogram, D-dimer, Ferritin, CRP, LDH, troponin, pro BNP, von Willebrand antigen); Biochemistry: Glucose, creatinine, ALT, AST, HbAlc (Checked only in scanned); coagulation: aPTT, PT; inflammation marker: IL-6

(4) In women of childbearing potential

(6) Only while in the hospital.

Endpoints Primary Endpoint The patient worsened with a WHO clinical progression score >6 within 1-28 days.

Secondary Endpoints

Recovery of the patient with a WHO Clinical Progress Score < 2 within 28 days,

• WHO Progress Scale Score change from baseline within 1 to 28 days,

• Percentage of patients sent to intensive care,

• Recovery time,

• Time of discharge from the hospital,

• All causes of mortality within 28 days,

• Qol (EQ-5D-5L respiratory survey),

8. Security -Related Definitions and Procedures

Definitions

Adverse Event (AO)

An adverse event is defined as any adverse medical event in a patient administered a medicinal product or in a clinical study patient and is not necessarily causally related to treatment. Thus, an adverse event may be any adverse or undesirable sign (e.g. an Abnormal laboratory finding), symptom or disease temporally associated with the use of a medicinal product, whether related to the medicinal product or not.

Causality of Adverse Events

The researcher should evaluate the relationship of AOs to the study product (IMP) using the following definitions: 1. Related: An adverse event is considered IMP -related when there is a reasonable possibility that the event may have been caused by the IMP.

2. Not related: An adverse event is considered unrelated to IMP when other more likely causes are documented (but not limited to underlying diseases, risk factors, concomitant medicines) and/or when there is no reasonable probability that the event will occur.

Serious Adverse Events (CAO)

A serious adverse event (CAO) is an adverse medical event resulting in at least one of the following:

• Resulting in death,

• Posing a life-threatening situation (defined as a patient at risk of sudden death at the time of the event),

• Imposing hospitalization or prolonging the period of hospitalization,

• Resulting in permanent and serious disability/limitation;

• Occurrence of congenital anomaly, neoplasm or birth defect,

• Medical events that do not result in death, are not life-threatening and do not require hospitalization, but are medically serious or require medical and surgical intervention to prevent the consequences listed above,

• Worsening of the patient’s condition because of COVID-19 due to admission to the intensive care unit or requiring mechanical ventilation will be reported as CAO.

Reporting Adverse Events

Researchers need to evaluate the AOs encountered, their severity and intensity, the cause- effect relationship with the medicines that patients are using, and their clinical outcomes.

Researchers will report adverse reactions that occur and are thought to be related to the medicines that patients are using, to the Turkish Pharmacovigilance Center (TLJFAM) within fifteen days, either directly or through the pharmacovigilance contact point in the health institutions where they work. Legal Regulations and Ethical Issues

The study will be carried out in accordance with the International Conference on Harmonization Good Clinical Practices (ICH ICU) Guidelines and the Declaration of Helsinki. At the same time, all current local regulations will be complied with.

For ethical approval of the study, an application will be made to the Istanbul Medipol University Clinical Research Ethics Committee. The study will be started after the approval of the Ethics Committee.

The study protocol and other relevant documents and any revisions to these documents will be submitted to the Ethics Committee for approval before implementation.

Informed Voluntary Consent Form

The researcher or a person designated by the researcher will explain to each patient or patient’s legal guardian the benefits and risks of participating in the study and written informed consent will be obtained prior to enrollment of the patient. Confidentiality

All necessary precautions will be taken by the responsible researcher and his staff in order to protect the confidentiality of all patient data collected and processed during the conduct of this study, in accordance with local laws and regulations on the protection of personal data.

Storing Records

The researcher should keep the documents related to the research in accordance with the relevant legislation; should take measures to prevent accidental or premature destruction of such documents.

Study Method

The planned study will be carried out under Prof. Dr. Hasan Onal’s coordination. Medipol Hospitals are the sponsor of the study. The study will be coordinated by Omega Research Organization Training and Consultancy Company (SAK), the legal representative of the sponsor.

Responsibilities of the Coordinator

Responsibilities of the Coordinator Researcher; evaluating the scientific content of the study, preparing the study protocol, study form and other relevant study documents, preparing the final report of the study, ensuring that the study is carried out in accordance with the protocol, presenting the results to the scientific circles after the study is completed, and communicating with the official authorities regarding the study.

Also, keeping the research records appropriately is under the responsibility of the researcher. All documents must be kept in a safe place in accordance with confidentiality rules.

Responsibilities of the Supporter

Designing the documents related to the study and providing the necessary support to the Contracted Research Organization (SAK) authorized for the application of the relevant Ethics Committee, delivering the documents related to the operation of the study to the study centers, ensuring coordination between the study centers are under the responsibility of the supporter.

Responsibilities of the Contracted Research Organization (SAK)

SAK will be responsible for the preparation of the study protocol, Ethics Committee application file, the final report of the study and publications to be made based on the results of the study for the Coordinator Researcher, transferring the collected data to the Statistics Department for data entry, conducting data management operations, querying the erroneous records and sending them to the centers, performing the necessary statistical analyzes and reporting them.

SURVEY TABLES TO BE USED IN THE STUDY

WHO Clinical Progress Scale

NEWS 2 score system

EQ-5D-5L

EQ-5D-5L In this section, individuals give values between 0 and 100 about their current health status and mark it on a thermometer-like scale.

Nicotinamide (NAD) and its derivatives

The effects of nicotinamide are listed below.

1. Reduces the DNA damage response.

2. Improves mitochondrial activity.

3. Slows down aging.

4. Plays a vital role in controlling the immune system overactivation and cytokine storm of many viruses, including COVID-19.

As the organism ages, with a decrease in sirtuin activity, the level of NAD decreases, telomeres shorten, and cells gradually deteriorate, stop dividing and die. This aging-related decline in NAD is believed to be an enormous risk for diseases and/or disabilities such as hearing and vision loss, cognitive dysfunction, auto-immunity, dysregulation of the immune response. Stabilizing telomeres has a beneficial effect on immune cell functions.

NAD depletion mediated by uncontrolled PARP activity in many viral families, including Togaviridae and Coronaviridae, indirectly leads to decreased sirtuin 1 (SIRT1) activity. This PARP-1 overactivation process can be reversed by exogenous NAD administration. Moreover, NAD supplementation with sequential inhibition of PARP-1 prevents NF-KB activation. NAD modulates NF-kB transcriptional activity, thereby having a vital role in controlling the ongoing inflammatory state, overactivation of the immune system and even cytokine storm.

Elderly people and those with pre-existing conditions are greatly affected by COVID-19 and are more susceptible to poor clinical prognosis. The pathophysiological changes associated with aging, decreased immune function, and comorbidity make them suffer from a severe disease of COVID-19 and more serious complications. Aging-related reductions in NAD and subsequent decline in telomere length adversely affect the immune response and COVID-19 outcome. The use of NAD as an immunomodulator for COVID-19 in elderly patients may be important. NAD has a direct inhibitory effect on PARP-1 and can prevent excessive activation of proinflammatory cytokines. Increasing the level of NAD will also cause telomeres to be stabilized and this has a positive effect on the function of immune cells. Therefore, NAD involvement in DNA repair and immune response can be targeted in therapeutic trials for aging.

It is thought that nicotinamide will potentiate the effect of angiotensin (1-7) due to its effect on oxidative stress.

Nicotinamide and its precursor derivatives can be found in formulations 1, 2 and 3, which are ideal formulations for the invention in the range of 50 to 250 mg. The formulation comprising Nicotinamide and its precursor derivatives in the range of 50 to 250 mg may comprise nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR) and/or nicotinamide mononucleotide (NMN), which increase the NAD level in the body.

Claims

1. A medicine characterized by comprising:

• 1-35 mg Angiotensin- 1-7 (AT 1-7) as the active ingredient, 24-480 mg 2- Hydroxypropyl-Beta-Cyclodextrin (HPBS) as the excipient, 1- 30 pL Sodium hydroxide (NaOH) solution, and

2. The medicine according to claim 1, characterized in that it is used in the treatment of cerebral ischemia, stroke, ARDS, hypertension, heart failure, myocardial infarction, cardiac arrhythmia, type 1 and type 2 diabetes, complications of diabetes, male and female infertility, cirrhosis, cancer, cancer metastasis or COVID-19 infection.

3. The medicine according to claim 1, characterized by comprising Nicotinamide (NAD) and/or its derivatives in a range of 50 to 250 mg.

4. The medicine according to claim 3, characterized by comprising nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR) and/or nicotinamide mononucleotide (NMN) to increase the level of NAD in the body.

5. A method for preparing the medicine according to claim 1, characterized by comprising the following operation steps:

• Dissolving 1-35 mg Angiotensin- 1-7 (ATI-7) and 24-480 mg 2-Hydroxypropyl- Beta-Cyclodextrin (HPBS) with 0.4-0.6% NaCl chloride solution,

• Mixing,

Sterilizing by filtering through a membrane filter, Adjusting the pH to a range of 4-8 by adding NaOH,

• Lyophilizing the obtained solution,

• Reconstituting with water for injection and re-solutionizing before use in the case of a powder lyophilisate.

6. The method for preparing the medicine according to claim 1, characterized by comprising the following operation steps:

• Dissolving 1-35 mg Angiotensin- 1-7 (AT 1-7) and 24-480 mg 2-Hydroxypropyl- Beta-Cyclodextrin (HPBS) with water for injection,

• Sterilizing by filtering through a membrane filter,

• Adjusting the pH by adding NaOH,

• Lyophilizing the obtained solution,

• Re-solutionizing by reconstituting with 0.4-0.9% NaCl solution or 2-4% dextrose solution or 2-4% mannitol solution before use in the case of a powder lyophilisate.

7. The method for preparing the medicine according to claim 1, characterized by comprising the operation steps of dissolving 1-35 mg Angiotensin- 1-7 (ATI-7) and 24-480 mg 2-Hydroxypropyl-Beta-Cyclodextrin (HPBS) mixture and adjusting the osmotic pressure or adding 0.4-0.9% NaCl solution, 2-4% dextrose solution and 2-4% mannitol solution together as a mixture to reconstitute lyophilized powder.