ANGIOTENSIN-1-7 FOR THE TREATMENT OF COVID-19

Cross Reference

This application claims priority to U.S. Provisional Patent Application Serial No.

63/108272. filed October 30, 2020, incorporated by reference herein in its entirety

Federal Funding Statement

This invention was made with government support under Grant No. PR202594 awarded by the Department of Defense. The government has certain rights in the invention.

Sequence Listing Statement:

A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on October 27, 2021 having the file name “20-1696-WO-SeqList ST25.txt” and is 1 kb in size.

Background

COVID-19 leads to induced respiratory distress which progresses from shortness of breath to requiring hospitalization and supplemental oxygen by nasal cannula, high flow oxygen delivered by a mask with <15 liters/minute of oxygen, BiPAP with a mask that delivers high pressure 100% oxygen at 5 mmHg peak inspiratory pressure, and ultimately to intubation and assisted ventilation with a respirator. With the number of COVID-19 cases in the United States since January of 2020 exceeding all other countries, COVID- 19 presents an acute and urgent threat to global health security . Therapeutic interventions capable of reducing pulmonary distress would reduce the fatality rate, improve long term outcomes, and free up scarce ICU resources.

Summary

In one aspect, the disclosure provides methods for treating a severe acute respiratory syndrome (SARS) coronavirus infection, comprising administering to a subject with a SARS coronavirus infection a composition comprising an amount effective to treat the infection of a polypeptide consisting of at least 4 consecutive amino acids of angiotensin 1-7 (A( 1-7)) [Asp-Arg-Val-Tyr-Ile-His-Pro (SEQ ID NO: 1)] or a pharmaceutically acceptable salt thereof, wherein the administering comprises continuous infusion of the composition for at least 4, 5, 6. or 7 consecutive days. In one embodiment, the SARS coronavirus infection comprises SARS-CoV or SARS COVID-19. In another embodiment, the composition comprises one or more polypeptides consisting of the ammo acid sequence:

Asp-Arg-Val-Tyr-Ile-His-Pro (SEQ ID NO: 1) A(l-7);

Asp-Arg-Val-Tyr-Ile (SEQ ID NO:2) A(1 -5);

Arg-Val-Tyr-Ile-His-Pro (SEQ ID NO:3) A(2-7); and/or

Val-Tyr-Ile-His-Pro (SEQ ID NO:4) A(3-7).

In another embodiment, the administering comprises continuous intravenous infusion. In a further embodiment, the administering comprises continuous infusion of the composition for at least 8, 9, 10, 11 , 12, 13, 14, or more consecutive days. In various embodiments, the polypeptide is administered at a concentration of between about 0.05 ug/mg (w/w) and about 500 ug/mg (w/w); the polypeptide is administered at a concentration of between about 1 ug/mg (w/w) and about 100 ug/mg (w/w); and/or the polypeptide is continuously infused at an infusion rate of at least 6.25 ng/kg/min (9 ug/kg/day); at least 25 ng/kg/min (36 ug/kg/day); at least 42 ng/kg/min (60.48 ug/kg/day), or at least 62.5 ng/kg/min (90 ug/kg/day).

In one embodiment, the subject is a human subject. In other embodiments, the method improves lung function, bone marrow function, kidney function, coagulation parameters, brain function, and/or heart function; and/or the method reduces local organ system production of angiotensin II,

In various further embodiments, the subject is a human subject that is hospitalized; the subject has one or both of shortness of breath and respiratory distress requiring oxygen; and/or the subject has severe acute respiratory’ distress.

In a further embodiment, the composition further comprises one or more other active agents selected including but not limited to anti-viral agents, corticosteroids, anti-coagulants, and agents that affect autophagy (including but not limited to hydroxychloroquine).

In one embodiment, the polypeptide consists of SEQ ID NO:1 and the administering comprises continuous infusion of the composition at 90 ug/kg/day for at least 4 consecutive days.

Detailed Description As used herein, the singular fonns "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. All embodiments of any aspect of the invention can be used in combination, unless the context clearly dictates otherwise.

As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Giu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Vai; V).

In one aspect, the disclosure provides methods for treating a severe acute respiratory syndrome (SARS) coronavirus infection, comprising administering to a subject with a SARS coronavirus infection a composition comprising an amount effective to treat the infection of a polypeptide consisting of at least 4 consecutive amino acids of angiotensin 1-7 (A(l-7)) [Asp-Arg-Val-Tyr-Iie-His-Pro (SEQ ID NO: 1)] or a pharmaceutically’ acceptable salt thereof, wherein the administering comprises continuous infusion of the composition for at least 7 consecutive days.

In one embodiment, the SARS coronavirus infection comprises SARS-CoV or SARS CO VID-19. In another embodiment, the SARS coronavirus infection comprises SARS COVID-19.

CO VID-19, like SARS-CoV, binds to the cell surface protein receptor, ACE2, a member of the ACE2/A(l-7)/Mas axis. SARS pathology may emerge, in part, due to the reduced ability of ACE2 to cleave Angiotensin-II (Ang-II), a pro-inflammatory, fibrotic peptide. Ang-II is a potent vasoconstrictor that can increase lung injury and lung edema, increases blood pressure, and excessive concentrations are thought to be harmful to a number of organ systems, A(l- 7) is the endogenous ligand of the Mas receptor and a member of the ACE2/A(l-7)/Mas axis. A(l-7) counteracts the effects of A-II in some situations. The methods of the invention can thus be used to treat a SARS coronavirus infection (i.e.: any SARS coronavirus that binds to cell surface protein receptor ACE2). While not being bound by a specific mechanism, the methods may provide enhanced local concentrations of A(l-7) resulting in more normal function of affected organ systems, and reduce local organ system production of Ang-II to further improve organ system function. A( 1-7) is rapidly metabolized in the blood into fragments that are biologically active.

Thus, in various embodiments, the composition comprises one or more polypeptides consisting of the amino acid sequence:

Asp-Arg-Val-Tyr-Ile-His-Pro (SEQ ID NO: 1) A(l-7);

Asp-Arg-Val-Tyr-Ile (SEQ ID NO:2) A( 1 -5);

Arg-Val-Tyr-Ile-His-Pro (SEQ ID NO:3) A(2-7); and/or

Val-Tyr-Ile-His-Pro (SEQ ID NO:4) A(3-7).

The composition may be continuously infused by any suitable route that permits continuous infusion. In one embodiment, the administering comprises continuous intravenous infusion.

In one embodiment, the administering comprises continuous infusion of the composition for at least 8, 9, 10, 11, 12, 13, 14, or more consecutive days. In another embodiment the administering comprises continuous intravenous infusion of the polypeptide for at least 14 consecutive days.

In one embodiment, the polypeptide is administered at a concentration of between about 0.05 ug/mg (w/w) and about 500 ug/mg (w/w). In another embodiment, the polypeptide is administered at a concentration of between about 1 ug/mg (w/w) and about 100 ug/mg (w/w). In various further embodiments, the polypeptide is continuously infused at an infusion rate of at least 6.25 ng/kg/min (9 ug/kg/day); at least 25 ng/kg/min (36 ug/kg/day); at least 42 ng/kg/min (60.48 ug/kg/day), or at least 62.5 ng/kg/min (90 ug/kg/day).

As used herein, "treat" or "treating" means accomplishing one or more of the following: (a) reducing the severity or progression of the infection; (b) limiting or preventing development or progression of symptoms characteristic of the infection; (c) inhibiting worsening of symptoms characteristic of the infection; (d) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s), (e) reducing local organ system production of angiotensin II compared to control (i.e.: subject prior to infection, or baseline determined from population of non-infected subjects), (f) reducing mortality, and/or (g) reducing length of hospital stay required.

Symptoms and features characteristic of SARS coronavirus infection that the methods may be used to treat or limit include, but are not limited to shortness of breath, reduced oxygen saturation, (SPO2), respiratory distress requiring oxygen, severe acute respiratory' distress, acute respiratory' distress syndrome (ARDS), need for hospitalization, need for intubation or other mechanical ventilation, reduction in need for subject stay in the intensive care unit, reduction in positive end expiratory pressure (PEEP), respiratory failure, widespread inflammation and damage to multiple organ systems including the heart, kidney, liver, lungs, brain, and bone marrow; multiple organ failure, and death.

In one embodiment, the subject is a human subject that is hospitalized. In further embodiments, the subject has one or both of shortness of breath and respiratory distress requiring oxygen; and/or the subject has severe acute respiratory distress.

The subject may be any suitable subject that may be infected with a SARS coronavirus. In one embodiment the subject is a human subject. In various embodiments, the methods result in one or more of (compared to a subject with a SARS coronavirus infection who receives no treatment, and/or compared to previous symptoms of the subject prior to treatment): o Improved lung function: As used herein, ‘"improved lung function” means one or more of increased oxygenation of the blood, reduced pulmonary infiltrates, reduced dyspnea, reduced cough, reduced pain, reduced inflammation, reduced shortness of breath, reduced death rate due (at least in part) to lung complications, reduced time on ventilator, reduced time in ICU due (at least in part) to lung complications, reduced time in hospital, and/or reduced return visits to health care providers both in patient and hospitalized; o Improved bone marrow function: As used herein, “improved bone marrow function” means one or more of reduction of anemia, reduction of thrombocytopenia, reduction of lymphopenia, increases white blood cell (WBC) formation, increased lymphocyte formation, increased platelet formation, and/or increased red blood cell (RBC) production; o Improved kidney function: As used herein, “improved kidney function” means one or more of improved glomerular filtration, reduced glomerular leakages of protein, reduced pain, reduced need for dialysis, reduced proteinuria, reduced hematuria, reduced dysuria, reduced acute kidney injury, reduced renal failure, improved maintenance of glomerular filtration rate, and/or reduced inflammation; o Improved coagulation parameters. SARS coronavirus patients often have hypercoagulation so they form clots in limbs which require amputation, in the brain (stroke), and lungs as well as kidneys. As used herein, “Improved coagulation parameters means one or more of reduced embolism, reduced embolic disease (time of onset, extent, duration), normalized coagulation, and/or reduced coagulopathy; o Improved brain function: As used herein, ‘Improved brain function” means one or more of improved cognition, reduced fuzzy brain syndrome, reduced headaches, reduced lassitude and irritability, reduced somnolence, improved attention span, reduced loss of smell and/or taste, reduced stroke, reduced inflammation, and/or reduced delirium ; and/or o Improved heart function in the subject: As used herein, “Improved brain function” means one or more of reduced incidence and/or severity of heart attack, reduced incidence and/or duration of arrhythmias, reduced congestive heart failure, improved cardiac output, reduced incidence and/or duration of vasculitis, reduced alterations in blood pressure, reduced hypertension; reduced myocarditis, reduced heart failure, improved maintenance of left ventricular function, and/or reduced inflammation.

In another embodiment, the method reduces local organ system production of angiotensin II. As noted above, Ang-II is a potent vasoconstrictor that can increase lung injury’ and lung edema, increases blood pressure, and excessive concentrations are thought to be harmful to a number of organ systems. Thus, this embodiment provides a significant treatment benefit to the subject.

In all embodiments herein, reference to the polypeptide includes salts of the polypeptide. Suitable acids which are capable of forming salts with the polypeptides include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and the like. Suitable bases capable of forming salts with the peptides include, but are not limited to, inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally' substituted ethanol-amines (e.g., ethanolamine, diethanolamine and the like).

The polypeptide can further be derivatized to provide enhanced half-life, for example, by linking to polyethylene glycol. The peptides or salts thereof may comprise L-amino acids. D-amino acids (which are resistant to L-amino acid-specific proteases in vivo) to convey special properties as deemed appropriate.

The compositions may comprise other components as deemed appropriate by attending medical personnel. In various embodiments, the compositions may comprise one or more (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and/or (g) a buffer. In some embodiments, the buffer in the composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. In one embodiment, the composition may comprise a lyoprotectant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the composition may comprise a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidme, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the composition may comprise a bulking agent, like glycine . In yet other embodiments, the composition may comprise a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate- 60, polysorbate-65, polysorbate-80 polysorbate-85, poloxamer- 188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. In one embodiment, the composition may comprise a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood. Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the composition may comprise a stabilizer, e.g., a molecule which, when combined with the polypeptide substantially prevents or reduces chemical and/or physical instability of the polypeptide in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride, paraben, and combinations of methyl paraben and propyl paraben.

Tire polypeptides may be tire sole active agent in the compositions, or the composition may further comprise one or more other active agents suitable for the desired treatment, including but not limited to anti-viral agents, corticosteroids, anti-coagulants, and agents that affect autophagy (including but not limited to hydroxychloroquine).

Example 1. A Phase 2 Randomized Double-Blinded Placebo Control (i.e. Standard of

Care) Trial to Evaluate the Safety, Tolerability, and Preliminary Efficacy of Intravenous

USB002 to Treat Patients with Severe Respiratory Distress due to COVID-19 Infection LIST OF ABBREVIATIONS

A(l-7) Angiotensin 1-7

A -II Angiotensin-II

AA Gradient Alveolar-to-arterial gradient

ACE Angiotensin-converting

Enzyme

ACE2 Angiotensin-converting Enzyme 2

ADR Ad verse drug reaction

AE Adverse event

ALI Acute Lung Injury'

APACHE III Acute Physiology and Chronic Health Evaluation

III

ARDS Acute Respiratory Distress Syndrome

ARF Acute respiratory failure

ATiR Angiotensin II Receptor Type 1

AT2 Angiotensin II Receptor Type 2

AUC Area under the curve

BAL Bronchoalveolar lavage

BiPap Bilevel positive airway pressure

BMI Body Mass Index

BP Blood pressure

C Celsius

CFU-Meg Colony-forming unit - megakaryocyte

Cmax Maximum concentration

CPAP Continuous positive airway pressure

CNS Central nervous system

CO Cardiac output

COPD Chronic obstructive pulmonary disease

CRF Case report form

CT A Clinical trial agreement CTCAE Common Tenninology Criteria for Adverse Events

CVP Central venous pressure

CVS Cardiovascular system

CXRs Chest X-Rays dL Deciliter

DLT Dose-limiting toxicity

DSMB Data Safety Monitoring Board

ECG Electrocardiogram

ECMO Extracorporeal mem brane eCRF Electronic case report form

EPO Erythropoietin

FDA Food and Drug Administration

GCP Good clinical practices

HR Heart rate

ICF Informed Consent Form

International Conference on Harmonization of Technical

Requirements for Registration of Pharmaceuticals for Human Use

ICU Intensive care unit

IP Investigational Product

IRE Institutional Review Board

IV Intravenous

IVRS Interactive Voice Response System kg Kilogram

LIS Lung Injury Score

MAP Mean arterial pressure ug Microgram

MedDRA Medical Dictionary’ of Drag Regulatory'

Activities

MEG-1 Megakaryo blasts mg Milligrams mL Milliliter

MM Medical monitor mm Millimeters mM Millimolar mm Hg Millimeters mercury mOsm Milliosmole

MPO Myeloperoxidase mRNA Messenger ribonucleic acid

MID Maximum tolerated dose

NCI National Cancer Institute

NDA New' Drug .Application

NF National Formulary'

NO Nitric oxide

NOAEL No-observed adverse effect level

OA Oleic acid

PaCO ₂ Partial arterial pressure of carbon dioxide

PAI-1 Plasminogen activator inhibitor type 1

PAO ₂ Partial pressure of alveolar oxygen

PBW Predicted body weight

PCWP Pulmonary capillary wedge pressure

PEEP Positive end expiratory pressure

PH Pulmonary hypertension pH Hydrogen ion concentration

PK Ph armacoki n etics

PR Pulse rate

Pt Patient

PVR Pulmonary vascular resistance

QTc QT interval corrected for heart rate

QTcB Manual correction of QT values

QTcF Automatic correction of QT values using Fridericia's

Formula

RR Respiratory rate RRT Renal replacemen t therapy

RV pressure Right ventricular pressure SAE Serious adverse event

SaO ₂ Arterial oxygen saturation

SAP Statistical Analysis Plan

SBT Spontaneous breathing trial

SC Subcutaneous SD rat Sprague-Dawley rat

SD Standard deviation

SHR Spontaneously hypertensive

SOB Shortness of breath soc Standard of care SpO ₂ Oxygen saturation

TEAE Treatment-emergent adverse event

TF Tissue factor

TG+ Renin transgenic

TGF-1 Transforming Growth Factor 1 V _t Tidal volume

USP United States Pharmacopeia

VDI Vasopressor dependency index

VFD Ventilator-free days

VILI Ventilator-induced lung injury WBC White blood cell

WHO World Health Organization

Investigational Product: USB002

USB002 is pharmaceutically formulated Angiotensin 1-7 [A(1-7)], a non- hypertensive derivative of Angiotensin-II and is suspended in a vehicle as a sterile solution in individual vials containing 3.2 ml of USB002 (30 mg/ml) which is stable at room temperature tor 90 days.

Clinical Experience with USB002

The safety of USB002 has been demonstrated in seven clinical trials whose indications include breast cancer and chronic kidney' disease. USB002 has also been studied in healthy volunteers. To date, 98 subjects have been treated with USB002 (referred to in studies as TXA127). Doses administered ranged from 2.5 ug/kg/day to 300 ug/kg/'day administered as a subcutaneous (SC) dose tor up to 28 days. USB002 has been well tolerated with no drug-related serious adverse events (SAEs) noted to date. A Phase 1/2 randomized, double-blinded, placebo-controlled study of USB002 given IV alone and with erythropoietin (EPO) was conducted in subjects with anemia associated with end-stage renal disease. The trial had two arms: EPO plus TXA127 at one of three doses (10, 25, or 50 ug/kg), and EPO plus placebo three times weekly, administered IV following each hemodialy sis session. No dose limiting toxicity (DLT) was observed in any dosing group. There were no differences between TXA127 and EPO in adverse events (AE) by body system. As of 01 May 2020, there were seven clinical trials listed in ClinicalTrials.gov evaluating A(l-7) for various indications.

Rationale for Clinical Trial

Due to the current pandemic, COVID-19 ieads to induced respiratory distress which progresses from SOB requiring hospitalization and supplemental oxygen by nasal cannula, high flow oxygen delivered by a mask with <15 liters/mmute of oxygen, BiPAP with a mask that delivers high pressure 100% oxygen at 5 mmHg peak inspiratory pressure, and ultimately to intubation and assisted ventilation with a respirator. With the number of COVID-19 cases in the United States since January of 2020 exceeding all other countries and the number of COVID-19 related deaths exceeding those of the Vietnam War w hich occurred over 19-years (as of May 13, 2020) and increasing, COVID- 19 pre sents an acute and urgent threat to global health security. Therapeutic interventions capable of reducing pulmonary distress would reduce the fatality rate, improve long term outcomes, and free up scarce ICU resources.

Respiratory distress in untreated COVID-19 infected patients presents as a progressive continuum from SOB to cardiopulmonary collapse and death. It is diagnosed in symptomatic patients with bilateral lung infiltrates in patients with confirmed positive tests for COVID- 19. Without supportive therapy, pulmonary distress rapidly progresses to hospitalization where supplemental oxygen can be given by nasal cannula, high-flow mask, BiPAP mask, and eventually’ intubation and assisted ventilation. Overall, the physiological damage to cells and structures of the lung can begin within hours of exposure and progress within days (Fahy 2012). The pathologic hallmarks of COVID-19- induced pulmonary distress include neutrophilic alveolitis, hyaline membranes secondary' to protein transudation and precipitation in the airspace, microthrombi secondary to the generation of procoagulant mediators as well as endothelial and epithelial injury. For these reasons, there is considerable interest in the use of A(1 -7) to treat patients who have developed respiratory distress from COVID- 19 infection (Soto 2020, Piero 2020, NCT 04332666).

Route of Administration

USB002 or placebo will be administered IV at a continuous rate of assigned drug delivery over 96 hours in Part 1 of this protocol and over 14 days in Part 2 of the protocol. Subjects will continue to receive USB002 or placebo for the duration of the protocol- specified period of time or until there is:

1 . Sa O ₂ ≥ 94% on room air.

2. A perceived safety issue prevents further dose administration, or

3. Sponsor’s request to terminate the subject’s participation in the study.

Dose Regimen

IV Administration:

IJSB002 or placebo will be diluted in up to 250 ml of saline and infused continuously by a dedicated line for 96 hours in Part 1 of this protocol and for 14 days in part 2 of this protocol. New infusion set and medication will be changed based on institutional standards

Part 1 : Dose Escalation

In the Part 1 dose-escalation trial, there will be six subjects at each dose (three

USB002 and placebo; up to 24 subjects) of which 12 will receive USB002 and 12 will receive placebo. Dose escalation will follow the traditional “3+3” design for four sequential doses. If none of the three active subjects experience a DLT that is different from the placebo subjects, another three subjects will be administered USB002 at the next higher dose level and another 3 subjects will receive placebo, lire dose escalation will be performed in a randomized, double-blinded manner. However, if 1 of the three active subjects experiences a DLT different from the placebo treated subjects, three more subjects will be treated at the same USB002 dose level and another three subjects will receive placebo. The dose escalation will continue until the maximum dose of 900 ug/kg/day is reached. The focus for the safety evaluation of each cohort will be the relative rate and type of AEs reported for the SOC plus USB002 group as compared to the SOC plus placebo group. This approach has been adopted knowing that many severe AEs will be reported due to the disease and the addition of remdesivir to the SOC

The DLT is expected to be hypotension (blood pressure [BP] < 90/60 mmHg; warranting the administration of fluid boluses or IV pressor agents). BP will be measured every ⁷ 8 hours during administration.

Infusion of USB002 or placebo will continue over 96 hrs for each subject. Dose escalation of USB002 will start at 90 ug/kg, then up to 300 ug/kg, then up to 600 ug/kg, and the maximum dose will be 900 ug/kg which is the maximum dose in the protocol for USB002 treatment of ARDS due to sepsis. Dose escalation will occur by increasing the infusion rate of 6.25 ng/kg/min (9 ug/kg/day), to 25 ng/kg/min (36 ug/kg/day), 42 ng/kg/min (60.48 ug/kg/day) and 62.5 ng/kg/min (90 ug/kg/day). The infusion rate for the SOC plus placebo will be the rate at which infusion would have occurred if the subject had randomized to SOC plus USB002.

After 96 hours the investigator will review the WBC data to determine the change from baseline. If the WBC has dropped 15% and/or lymphocytes increase by 15% after 96 hours, the treatment will be continued at the discretion ofthe investigator to Day 14.

Part 2: Preliminary Efficacy

The Part 2 preliminary efficacy determination will be randomized, double-blinded, and placebo controlled. The dose of USB002 will be the dose chosen in Part 1 ofthe protocol. Subjects will be randomized to SOC plus USB002 or SOC plus placebo (n==68 subjects/cohort, total n=136 subjects). The infusion rate of the placebo plus SOC will be the same infusion rate as the USB002 plus SOC.

Treatment Period

For Part 1 of the protocol, all subjects will receive SOC plus USB002 or SOC plus placebo for up to 96 hours: any who demonstrate a possible response to therapy will be continued to Day 14. For Part 2 of the protocol subjects will receive SOC plus USB002 or SOC plus placebo for 14 days. Infusion will be discontinued when SaOi > 94% on room air.

Vital signs will be closely monitored. Vital signs (BP, heart rate, respiration rate) will be taken at least every 4 hours for the first 12 hrs after dosing, and every' 8 hours for the remainder of the duration of dosing with Investigational Product, or more frequently according to the institution’s standard of care.

STUDY OBJECTIVES AND PURPOSE

Primary Objective

Parts 1 and 2

To determine the safety and tolerability of USB002 in treatment of severe respiratory distress from COVID-19 infection and ascertain die existence and strength of an early efficacy signal . Part 1:

● To determine the maximum tolerated dose (MTD) of USB002 (up to 900 ug/kg/day) for 96-hours to be administered in Part 2.

Part 2

● To determine the preliminary’ efficacy of USB002 in treatment of severe respiratory' distress from COVID- 19 infection.

Study Endpoints

Part 1

Tire safety of SOC plus USB002 will focus on the rate of occurrence and severity of AEs resulting from changes in clinical monitoring criteria as compared to SOC plus placebo group:

● Cardiovascular parameters as indicated by' BP, HR, ECG, requirements for inotropes and vasopressors;

● Assessment of changes in hematological parameters, kidney’ function, and liver function (serum albumin, total and direct bilimbin, and prothrombin time).

Clinical Laboratory Evaluation (Baseline, 48-hours, 96-hours):

● Clinical chemistry'

● Hematology

● Coagulation

● LLver function

Indications of efficacy' will be assessed: ● Duration of time of unassisted breathing

● Duration of time without mechanical ventilator support

Part 2

Overall safety will be the occurrence and severity of AEs based on the clinical monitoring criteria, as in Part 1.

The primary focus of efficacy will be:

● Subjects with respiratory rate ≤ 16 breaths per minute on room air and at rest for 15 minutes on Day 28

Secondary outcomes for efficacy will be:

● All -cause mortality;

● Subjects alive and free of invasive mechanical ventilation;

● Subjects alive and discharged from the hospital;

● Subjects alive and free of respiratory failure. Respiratory failure defined based on resource utilization requiring at least 1 of the following:

● Endotrachealintubation and mechanical ventilation

● Oxygen delivered by high-flow nasal cannula (heated, humidified, oxygen delivered via reinforced, nasal cannula, at flow rates > 20 L/min with fraction of delivered oxygen < 0.5)

● Noninvasive positive pressure ventilation

● Extracorporeal membrane oxygenation (ECMO)

● Change in the WHO nine-point ordinal scale of clinical status;

● Recovery landmarks: time to meeting weaning criteria, 30-minute spontaneous breathing trial and time to discontinuation of ventilatory assistance, if applicable.

The WHO nine-point ordinal scale of clinical improvement (OSCI score) is defined as the following:

1 . Uninfected: No clinical or virological evidence of infection

2. Ambulatory: No limitation of activities

3. Ambulatory: Limitation of activities

4. Hospitalized; mild disease: Hospitalized, no oxygen therapy

5. Hospitalized; mild disease: Oxygen by mask or nasal prongs 6. Hospitalized; severe disease: Non-invasive ventilation or high-flow oxygen

7. Hospitalized; severe disease: Intubation and mechanical ventilation

8. Hospitalized; severe disease: Ventilation and additional organ support - pressors, RRT, ECMO

9. Dead: Death

Additional Outcomes

● Changes in Sp O ₂ (measured by pulseoximetry)

● Changes in positive end expiratory pressure (PEEP)

● Duration assisted ventilation

● Duration of time of unassisted breathing

● Duration of time without mechanical ventilator support

STUDY DESIGN

Part 1

Dose-escalation will be randomized, double-blinded, placebo-controlled study performed in up to 24 subjects, up to six subjects (in addition to SOC, three will receive USB002 and three will receive placebo) in each of 4 doses. Subjects will receive a continuous IV infusion of USB002 for 96-hours followed by an additional 24-hours to assess safety. For subjects who show signs of a possible response as indicated by a decrease in WBC from baseline of 15% and/or increase in lymphocytes by 15%, the investigator may continue therapy until Day 14. Infusion will be discontinued if Sa O ₂ ≥ 94% on room air.

The decision to dose-escalate, de-escalate, or increase the number of subjects in a Group to further examine safety variables will be made by a Data Safety Monitoring Board (DSMB). Minutes will be recorded at the decision meetings and circulated for final approval before being placed on file. The dose escalation will use the traditional 3-1-3 design to assess dose-response as described in section 1 .4.3 Dose Regimen.

Early and regular assessment of the progress and safety will be conducted by the DSMB. If there is any concern, a delay to future enrollment may be instituted at the discretion of the DSMB, who will have access to study' data via the electronic case report forms (eCRFs).

If there is toxicity at the lowest level (6.25 ng/kg/min; 90 ug/day), the option for de- escalation will be discussed and determined by the DSMB.

If a subject withdraws or is withdrawn for reasons oilier than DLT prior to completing the 48-hr treatment period, the subject will not be replaced.

Toxicity will be graded according to the NCI-CTCAE Version 5.0 criteria. Limiting toxicities will be considered as any SAE or Grade 3 to 5 toxicity judged to be probably or definitely related to USB002 or any of the predetermined study specific criteria, as determined by the DSMB.

Definition of a cardiovascular DLT: Systolic pressure, diastolic pressure, heart rate, and inotropes and doses thereof will be recorded just prior to administration of study drug, to establish an appropriate baseline in the context of the individual patient's situation. A reduction of: 15% in BP or HR in conjunction with other symptoms and/or signs will be flagged as a potential DLT to be reviewed by the Medical Monitor (MM) and/or DSMB.

Part 2

Part 2, preliminary efficacy assessment will be performed in a randomized, double- blinded, placebo-controlled study which will begin enrolling after Part 1 is complete through Day 28. In Part 2, subjects will continuously receive for 14 days the dose of USB002 selected in Part 1 . Clinical status of subjects will be evaluated once weekly using the nine-level ordinal scale (WHO Master Protocol R&D Blueprint, version 2, February 18, 2020; NCT04292899). Subject participation in Part 2 will terminate on Day 60. Follow- up for the assessment of any AEs and mortality to Day 60 regardless of treatment or hospitalization status. Infusion will be discontinued before 14 days if Sa O ₂ > 94% on room air.

Toxicity will be graded according to the NCI-CTCAE Version 5.0 criteria. Limiting toxicities will be considered as any SAE or Grade 3 to 5 toxicity judged to be probably or definitely related to USB002 or any of the predetermined study specific criteria, as determined by the DMSB.

Description of Treatment(s); Drug Packaging and Labeling

USB002 is formulated as a sterile, preservative-free, non-pyrogenic solution for injection. Formulated solution for injection consists of the following (Table 1):

Table 1. USB002 Formulated Solution

The parenteral formulation is produced as a pH buffered solution, adjusted for the proper osmolality with the addition of 4% mannitol to provide a final osmolality of 295-415 mOsm for the dosing concentrations of A(l-7). The product is packaged in a 30 mg/ml, single- use, stoppered vial with a 3.2-mL fill. Tire individual components used to manufacture the drug product are commonly used in other parenteral drug products.

Sterilization of USB002 is achieved via 0.22-micron filtration of the final bulk solution into the appropriate number of vials filled under aseptic conditions.

Placebo will be the same formulation as USB002 without angiotensin 1-7.

Investigational Product Administration

The investigational product (IP) will be held secure in the site pharmacy, where it will be prepared for infusion and provided to the study staff for subject infusion/injection. The IP will be provided to the study staff indicating the date, subject ID#, and dose.

Dosage concentrations are standardized so that a 10-pL volume of solution will be dispensed per kilogram of subject body mass, based on the subject’s baseline weight.

Preparation for Administration

USB002 is a single-use parenteral product, supplied in sterile, pre-mixed, ready-to- use 3.2-mL vials. A new, unused vial should be used for each dose. Used vials should be retained for accountability monitoring by the Sponsor.

Invert the vial gently several times before use, to ensure uniformity of the solution prior to withdrawing the amount required based on the subject s weight. Maintain aseptic technique while handling USB002, and inspect for particulate matter and discoloration prior to administration whenever the solution and container permit. Do not use the study drug if contamination is suspected .

Withdraw the required volume from the vial (via appropriate gauge needle and syringe) and make up the volume to 5 mL with sterile saline. Cap the needle for delivery to the study staff. Labels to be placed on the syringe will be provided for completion to identify the subject #, dose #, and time of preparation. Tire drug must be used (infusion or SC administration completed) within 36 hours of preparation.

For IV administration, USB002 should not be administered at the same time with other medications in the line and in some instances may' require a dedicated line however, USB002 can be administered with the following:

● Water for injection, USP

● Sodium Chloride (0.9%) Injection, USP

● Dextrose (5%) Injection USP

● Dextrose (5%) in Sodium Chloride (0.9%) Injection, USP

● Dextrose in Ringers Lactate Injection, USP

● Lactated Ringers Injection, USP

As far as possible, for a period of 90 minutes after initiation ofUSB002 infusion the patient should not be scheduled for any procedures, administered any medications that may reduce B P or transported out of the ward or ICU for any reason.

Study Duration and Tria! Periods

Subjects will be enrolled to the trial once they are diagnosed with COVID-19 and pulmonary distress requiring supplemental oxygen, consent has been obtained from the subjects or their legal representative and eligibility' has been confirmed. Baseline laboratory' assessments will be obtained, if not already completed prior to subject consent. Baseline data will include time of admission to hospital, ICU, and time of supplemental oxygen therapy, nasal high-flow oxygen therapy, non-invasive mechanical ventilation or mechanical ventilator via intubation initiation. Registration will be performed by the Study Coordinator, the drug will be requested from the pharmacy, and dosing will begin within 6 hours (study Dav 1).

PATIENT SELECTION CRITERIA

Inclusion Criteria

1 . Prior to any study-related procedures Informed Consent must be obtained from tire patient or from the legal representative;

2. Women of childbearing po tential who use highly effective me thods of birth control defined as those, alone or in combination, that result in a low failure rate when used consistently and correctly;

3. Patients with a confirmed severe diagnosis of COVID-19 by FDA-approved testing within 1 week of enrollment. Severe disease is defined as patients with an oxygen saturation (SaO2) < 94% on room air or requiring supplemental oxygen or requiring invasive mechanical ventilation, or requiring ECMO;

4. Patients who are admitted to a hospital and under the care or consultation of a licensed clinician (skilled in the diagnosis and management of patients with potentially life-threatening illness and the ability to recognize and manage medication-related AE): Patients for whom use of an IV agent is clinically appropriate;

5. Age 18 or greater (no upper limit); males and females;

6. Subjects who receive SOC appropriate to treat respiratory' distress associated with COVID- 19 infection including remdesivir, dosed as per the FDA approved Emergency Use Authorization Instructions for Use, anticoagulants, dexamethasone or other medications found to benefit subjects with COVID- 19 induced pulmonary distress as per the recommendation of FDA.

Exclusion Criteria

1. Terminally ill patients due to underlying cardiac, cancer or severe debilitating neurological disease including coma;

2. Pregnant and lactating women;

3. Patients who are imminently being discharged from the hospital due to medical improvement;

4. Interstitial lung disease, pulmonary embolism, pneumothorax, cerebrovascular accident, cardiac valve pathology;

5. QTcF > 450 ms;

6. History of hypotension;

7. Alanine aminotransferase (ALT) or aspartate aminotransferase (AST) > 3 times upper limit of normal;

8. Participating in any other clinical trial of an experimental treatment for COVID-19. ASSESSMENT OF EFFICACY

The primary measure of efficacy is:

● Subjects with respiratory rate ≤ 16 breaths per minute on room air and at rest for 15 minutes on Day 28.

Secondary outcomes for efficacy will be:

● All -cause mortality;

● Subjects alive and free of invasive mechanical ventilation; ● Subjects alive and discharged from the hospital;

● Subjects alive and free of respiratory failure. Respiratory failure defined based on resource utilization requiring at least 1 of the following:

● Endotracheal intubation and mechanical ventilation;

● Oxygen delivered by high-flow nasal cannula (heated, humidified, oxygen delivered via reinforced nasal cannula at flow rates > 20 L/min with fraction of delivered oxygen < 0.5);

● Noninvasive positive pressure ventilation;

● Extracorporeal membrane oxygenation (ECMO);

● Change in the WHO nine-point ordinal scale of clinical improvement;

● Recovery landmarks: time to meeting weaning criteria, 30-minute spontaneous breathing trial and time to discontinuation of ventilatory assistance, if applicable.

Adverse Events (AE)

An AE is the development of an undesirable medical condition or the deterioration of a pre-existing medical condition following or during exposure to a pharmaceutical product, whether considered causally related to the product. An undesirable medical condition can be symptoms (e.g., nausea, chest pain), signs (e.g., tachycardia, enlarged liver), or the abnormal results of an investigation (e.g., laboratory findings, ECG). In clinical studies, an AE can include an undesirable medical condition occurring at any time, including run- in or washout periods, even if no study treatment has been administered. Treatment- emergent sepsis-related symptoms and events (i.e., not present at baseline or worsened in severity following start of treatment) should be reported as AEs.

Serious Adverse Events (SAE) A SAE is an AE occurring during any study phase (i.e., run-in, treatment, washout, follow'- up), and at any dose of the IP, comparator, or placebo, that meets one or more of the following criteria:

● Results in death;

● Is immediately life-threatening;

● Requires in-patient hospitalization or prolongation of existing hospitalization;

● Results m persistent or significant disability or incapacity;

● Is a congenital abnormality or birth defect;

● Is an important medical event that may jeopardize the patient or may require medical intervention to prevent one of the outcomes listed above.

STATISTICAL METHODS

Detailed statistical analysis information will be provided separately in the SAP which will be signed off prior to database lock; Part 1 and Part 2 will have separate SAPs as the research question to be addressed is different for the two Parts. The focus for Part 1 will be reviewing the safety and possible efficacy signals for the escalating dose for the SOC plus USB002 subjects as compared to the SOC plus placebo group, while for Part 2 the interest is in the comparison of the efficacy outcomes of the randomized groups (i.e. SOC plus USB002 vs. SOC plus placebo). Any deviations to the planned analyses specified within the SAP will be justified in writing and presented within the final clinical study report.

In Part 1, the data summaries, which will be supported with by-subject listings, will focus on the dose group for SOC phis USB002 as compared to the SOC plus placebo as well as time in trial. Any subjects who are allowed to re-initiate therapy will be highlighted and presented separately.

Part 2 efficacy assessments will focus on comparing the endpoints for the two treatment groups for a signal of efficacy including: subjects with respiratory rate ≤ 16 breaths per minute on room air and at rest for 15 minutes on Day 28, all-cause mortality, time without mechanical ventilation, subjects discharge alive, subjects free of respiratory failure, responders according the change in clinical status using a 9-point ordinal scale (WHO Master Protocol R&D Blueprint, version 2, February 18, 2020, NCT04292899). Data relating to efficacy response will be listed and summarized by dose cohort and time point, as appropriate. On each measurement day, the worst score from the previous day of measure will be recorded. Efficacy Analysis

The primary outcome, proportion of subjects with respiratory rate < 16 breaths per minute on room air and at rest for 15 minutes at Day 28, will be compared between the two treatment groups using Fisher’s exact test. Subjects who die or on ventilator at the time of the Day 28 assessment will be considered as a failure, and subjects who are alive and discharged from hospital by Day 28 will be considered as a success for this outcome, A logistic regression analysis will also be performed to adjust for the baseline covariates: age baseline severity, and comorbidity.

The secondary outcomes that are binary, e.g. subjects alive and free of invasive mechanical ventilation, the proportion will be compared be compared using Fisher’s exact test. As in the primary outcome analysis, subjects who die on or before the Day 28 assessment will be considered as failure for these outcomes. For outcomes related to time to event, they will be analyzed using a Cox proportional hazards model incorporating the same baseline covariates as in the primary outcome analysis; summary statistics will be presented with survival curves. The proportion of subjects who remained free of respiratory failure will be analyzed with logistic regression as will the secondary outcome change in the WHO scale by one category.

All other outcomes will be presented descriptively.

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1 1 . NCT 04292899 Study to Evaluate the Safety and Antiviral Activity of Remdesivir(GS- 5734) in Participants with Severe Coronaviraus Disease (COVID- 19)

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15. Papinska AM, Rodgers KE. Long-Term Administration of Angiotensin (1-7) to db/db Mice Reduces Oxidative Stress Damage in the Kidneys and Prevents Renal Dysfunction. Oxid Med Cell Longev. Hindawi; 2018;2018: 1841046-10.

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18. Rodgers KE, Oliver J, & diZerega GS. Phase I/II dose escalation study of Angiotensin 1-7 [A( 1-7)] administered before and after chemotherapy in patients with newly- diagnosed breast cancer. Can Chemother Pharmacol May 2006 57(5); 559-568. 19. Shenoy V, Ferreira AJ, Qi Y, et al. The angiotensin-converting enzyme 2/angiogenesis-(l-7)/Mas axis confers cardiopulmonary protection against lung fibrosis and pulmonary' hypertension. American Journal of Respiratory’ and Critical Care Medicine. American Thoracic Society; 2010 Oct 15;182(8): 1065— 7:2.

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Example 2

Part 1 of the study detailed in Example 1 was carried out on patients having severe respiratory' distress from COVID-19 infection and requiring mechanical ventilation or noninvasive ventilation support to survive. To date, the number of patients is 6. with 3 on USB002 and 3 on placebo.

The first dose was 90 ug/kg infused intravenously over 24-hours for 4-days (96 hrs). Thus, each day the study subject received 90 ug/kg in a constant infusion. The placebo was the vehicle (essentially saline) the USB002 was dissolved in. Dose escalation is ongoing, and results from the first 6 subjects at the first dose is provided. All subjects were also provided standard of care therapy (antiviral medication, oxygen ventilation, IV crystalloid support, anti-inflammatory medication).

Blinded data to date is provided in Table 1.

MV mechanical ventilation (respirator and intubation), NIV noninvasive ventilation

(usually high oxygen flow mask), The data in Table 1 is believed to show that subjects receiving USB002 treatment had significantly improved survival, improved clinical status as measure by OSCI score, improvement in spontaneous ventilation, and/or reduction in C -reactive protein levels in their blood relative to placebo treated subjects, over the course the study, and it is believed that such therapeutic benefits will be maintained or increase with longer term treatment. Reduction in serum CRP (measure of overall body inflammation) is a major indicator of improved organ function. CO VID-19 effects most organs, not just lungs.