WO2021189038A1 - Ang 1-7 as a therapeutic for coronavirus - Google Patents

Abstract

The present disclosure relates to therapy for humans or animals with specific viral infections, e.g., caused by a coronavirus such as SARS-CoV-2 or SARS-CoV.

Description

ANG 1-7 AS A THERAPEUTIC FOR CORONA VIRUS

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application Serial Number 62/992,768, filed on March 20, 2020, which is incorporated herein by reference in its entirety.

FIELD

[0002] The present disclosure relates to therapy for humans or animals with specific viral infections, e.g., caused by a coronavirus such as SARS-CoV-2 or SARS-CoV.

BACKGROUND

[0003] The death rate from the current coronavirus pandemic in the United States has now surpassed 530,000, and we do not yet have any treatments that substantially improve survival. While most coronaviruses cause little more than a common cold, the COVTD-19 virus is so deadly because it stimulates the release of an inflammatory cascade that results in severe lung and heart injury, particularly for older individuals and those with underlying conditions including high blood pressure, diabetes mellitus, heart disease, and obesity.

[0004] In severe cases, SARS-CoV-2 infection has been shown to cause acute respiratory distress syndrome (ARDS) and myocardial inflammation and dysfunction - the 2 leading causes of death in COVTD-19.¹ The pathophysiologic findings in humans with COVTD-19 include excessive release of inflammatory cytokines; damage to the endothelial lining of blood vessels; excessive leakage of fluid into the lungs and other tissues; pro-thrombotic state with deep venous thrombosis, stroke, and thrombosis and emboli in the pulmonary circulation resulting in severe ventilation/perfusion mismatching and hypoxemia.^{2 7}

[0005] Ang (1-7) is formed by the action of the carboxypeptidase angiotensin converting enzyme 2 (ACE2) on Ang II, a hormone involved most importantly in blood pressure regulation. The balance between Ang II and Ang (1-7) is critical in normal homeostasis. Increased Ang II can be beneficial in situations such as dehydration, hemorrhage, and infection that result in inadequate intravascular volume and reduced blood flow. Ang II results in vasoconstriction and release of aldosterone from the adrenal gland, which causes the kidneys to reabsorb sodium and water. Both of these mechanisms help a mammalian host to maintain an adequate blood pressure (BP). Excessive levels of Ang II, however, can result in reduced tissue perfusion, hyperinflammatory state, apoptosis of endothelial cells, leaky blood vessels, and a prothrombotic state.^{8 16} Unrestrained Ang II causes acute lung injury and myocardial inflammation in preclinical models.^{17 18}

[0006] Ang (1-7) binds to a receptor called Mas and provides a counter-regulatory effect to Ang II. Ang (1-7) is anti-inflammatory, anti-thrombotic, and cytoprotective, and causes mild vasodilatory effects. The ACE2 -> Ang (1-7) -> Mas pathway is expressed in tissues with high levels of metabolism, such as the heart, lungs, brain, kidneys, and intestines, that will not tolerate diminished blood flow induced by excessive vasoconstriction.^{19 21} Ang (1-7) reduces or prevents acute lung injury in preclinical models.

[0007] Thus, the ratio of Ang II and Ang (1-7) is essential in maintaining proper health. Studies have shown that bronchoalveolar fluid from patients with ARDS has elevated levels of Ang II and decreased levels of Ang (1-7).²² Individuals at high risk for severe COVTD-19, including the elderly, males, and those with heart disease, hypertension, and diabetes, have reduced ACE2 levels or increased ratios of Ang II to Ang (1-7) at baseline.^{23 28} Elevated ACE2 levels have been shown to be beneficial in murine models of lung injury caused by acid administration or sepsis.

[0008] P38 MAPK is central to many cellular functions. Both Ang II and SARS-CoV-2 cause p38 MAPK activation, while Ang (1-7) has an inhibitory effect on this kinase.^{29 30} Excessive activation of P38 MAPK has pleiomorphic effects including i) increased activity of AD AMI 7, which causes activation and release of TNFa and cleavage of the ACE2 ectodomain, ii) internalization of ACE2 from the cell membrane, iii) increased release of inflammatory cytokines, iv) vasoconstriction, v) excessive fluid leak, and vi) pro-thrombotic state.³¹

[0009] Similar to the SARS-CoV coronavirus, SARS-CoV-2 enters cells via the ACE2 receptor. ACE2 levels and activity are reduced by the SARS-CoV after binding through shedding of the ectodomain of ACE2, thereby eliminating the protein’s protective functions. Given the homology of the ACE2 binding domain of the viral spike protein for SARS-CoV and SARS- CoV-2, the latter would also be predicted to reduce ACE2 levels and activity. [0010] Despite the high rate of hospitalization, ICU admissions, excess mortality, and worldwide recession caused by SARS-CoV-2, current therapies for hospitalized patients with severe SARS-CoV-2 have limited efficacy and there are no proven treatments for SARS-CoV or other coronaviruses. The present disclosure addresses this need and provides other advantages as well.

SUMMARY

[0011] The present disclosure provides methods and compositions for the treatment of subjects having an infection by a coronavirus that binds to and/or enters cells through the ACE2 receptor. The present methods and compositions can be used to improve one or more symptoms of such an infection, to prevent the development of severe symptoms, to promote recovery from respiratory failure in infected subjects, and to provide other beneficial effects.

[0012] In one aspect, the present disclosure provides a pharmaceutical composition comprising an angiotensin-(l-7) base, a sugar alcohol, and a pharmaceutically acceptable carrier, wherein the pH of the composition is about 5.5.

[0013] In some embodiments, the composition comprises a concentration of the angiotensin- (1-7) base effective for the treatment of a subject with an ACE2 receptor-binding coronavirus infection. In some embodiments, the ACE2 receptor-binding coronavirus infection is a SARS- CoV or SARS-CoV-2 infection. In some embodiments, the concentration of the angiotensin-(l- 7) base is about 2.5 mg/mL. In some embodiments, the angiotensin-(l-7) base is angiotensin-(l- 7) acetate. In some embodiments, the sugar alcohol is mannitol. In some embodiments, the concentration of mannitol in the solution is about 25 mg/mL. In some embodiments, the composition comprises 2.5 mg/mL angiotensin-(l-7) acetate and 25 mg/mL mannitol. In some embodiments, the pharmaceutically acceptable carrier is sterile water. In some embodiments, the solution is formulated for intravenous administration. In some embodiments, the solution is formulated for subcutaneous administration.

[0014] In another aspect, the present disclosure provides a method of treating a subject with an ACE2 receptor-binding coronavirus infection, the method comprising administering to the subject a pharmaceutical composition comprising an angiotensin-(l-7) base. [0015] In some embodiments of the method, the angiotensin-(l-7) base is angiotensin-(l-7) acetate. In some embodiments, the ACE2 receptor-binding coronavirus infection is a SARS-CoV or SARS-CoV-2 infection. In some embodiments, the subject is a human. In some embodiments, the pharmaceutical composition is administered by continuous intravenous infusion. In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the angiotensin-(l-7) base is administered to the subject at a concentration of from about 2.5 ng/kg/min. to about 40 ng/kg/min. In some embodiments, the angiotensin-(l-7) base is administered to the subject at a concentration of from about 5 ng/kg/min. to about 20 ng/kg/min. In some embodiments, the angiotensin-(l-7) base is administered to the subject at a concentration selected from the group consisting of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 ng/kg/min. In some embodiments, the pharmaceutical composition further comprises a sugar alcohol. In some embodiments, the sugar alcohol is mannitol. In some embodiments, the pH of the pharmaceutical composition is about 5.5. In some embodiments, the pharmaceutical composition is administered to the subject for about 1, 2, 3, 4, 5, 6, or 7 days.

[0016] In some embodiments, the subject has one or more symptoms of COVID-19. In some embodiments, the one or more symptoms of COVID-19 comprise a symptom selected from the group consisting of dyspnea at rest or with activity, cough, fatigue, muscle weakness, body aches, joint pain, loss of smell or taste, brain fog, headaches, and insomnia. In some embodiments, the subject has been hospitalized. In some embodiments, the subject has suffered from respiratory failure. In some embodiments, the respiratory failure involves acute respiratory distress syndrome (ARDS). In some embodiments, the subject requires high-flow oxygen, non- invasive ventilation, mechanical ventilation, or extracorporeal membrane oxygenation (ECMO).

[0017] In some embodiments, the angiotensin-(l-7) base reduces the time to recovery from the respiratory failure as compared to a subject with COVID-19 and respiratory failure who has not been administered the pharmaceutical composition. In some embodiments, the subject is asymptomatic for COVID-19. In some embodiments, the subject has a reduced ACE2 level and/or increased Ang II to Ang (1-7) ratio relative to a control value typical of a young, healthy adult. In some embodiments, the subject is elderly, is male, has heart disease, has hypertension, is obese, and/or has diabetes. [0018] In some embodiments, the angiotensin-(l-7) base leads to a reduction in the one or more symptoms of COVID-19 in the subject. In some embodiments, the angiotensin-(l-7) base leads to a reduction in p38 MAPK activity in one or more tissues of the subject. In some embodiments, the reduction in p38 MAPK activity leads to reduced replication of the coronavirus. In some embodiments, the angiotensin-(l-7) base leads to reduced inflammation, reduced thrombosis, and/or increased vasodilation in the subject. In some embodiments, the angiotensin-(l-7) base leads to a change in the level of one or more cytokines in the subject. In some embodiments, the one or more cytokines comprise a cytokine selected from the group consisting of tumor necrosis factor (TNF)-a, interleukin (IL)- l b, IL-6, IL-8, IL-10, and interferon (IFN)-y.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 shows the molecular structure of the heptapeptide Ang (1-7).

[0020] FIG. 2. Overview of the study phases and their duration.

[0021] FIG. 3. The assumed distribution of the primary endpoint in both arms of the clinical trial described in Examples 1 and 2, with day 29 and 30 representing the outcome score being 29 and +00, respectively, i.e., alive but not recovered by day 29 and death before day 29.

DETAILED DESCRIPTION

1. Overview

[0022] The present disclosure pertains to the administration of the heptapeptide Ang 1-7 and/or modified forms or formulations of Ang 1-7 that enhance the peptide’s stability, affinity to its receptor, bioavailability, pharmacokinetics, and/or tissue delivery, as a treatment to reduce or prevent the morbidity and mortality of SARS-CoV-2, SARS-CoV, and other viruses that enter cells via the ACE2 receptor. Ang 1-7 may be administered therapeutically in those exhibiting symptoms, or prophylactically for those who have had viral exposure, but have not yet developed symptoms. In some embodiments, Ang 1-7 is administered to critically ill patients, e.g., COVID- 19 patients, including patients that are hospitalized, that have respiratory failure, that have acute respiratory distress syndrome (ARDS), that have reduced levels of ACE2 receptor or ACE2 receptor activity, that have reduced levels of Ang 1-7, that have elevated levels of Ang II, that have elevated ratios of Ang II to Ang 1-7, or that have elevated levels of p38 MAPK. In some embodiments, the patients are elderly. In some embodiments, the patients have a disorder or condition associated with severe COVID-19 symptoms and/or lower ACE2 receptor levels, including diabetes, obesity, hypertension, heart disease, and others.

[0023] The therapeutic may be administered intravenously, subcutaneously, intramuscularly, orally, intranasally, or by inhalation. In particular embodiments, the Ang 1-7 is administered by continuous IV infusion, e.g., for up to 7 days. In some embodiments, the Ang 1-7 is administered subcutaneously.

[0024] The present disclosure also provides pharmaceutical compositions comprising Ang 1-7, e.g., comprising Ang 1-7 base such as Ang 1-7 acetate, and a pharmaceutically acceptable carrier. In particular embodiments, the pharmaceutical compositions also comprise a sugar alcohol, e.g., mannitol. In particular embodiments, the pharmaceutically acceptable carrier is sterile water. In some embodiments, the pharmaceutical composition has a pH of about 5.5.

[0025] The present methods and compositions can be used to improve any of a number of symptoms of an ACE2 receptor-binding coronavirus infection, e.g., SARS-CoV-2 infection.

2. Definitions

[0026] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0027] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the agent” includes reference to one or more agents known to those skilled in the art, and so forth.

[0028] The terms “about” and “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Any reference to “about X” specifically indicates at least the values X, 0.8X, 0.8 IX, 0.82X, 0.83X, 0.84X, 0.85X, 0.86X, 0.87X, 0.88X, 0.89X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, 1.11X, 1.12X, 1.13X, 1.14X, 1.15X, 1.16X, 1.17X, 1.18X, 1.19X, and 1.2X. Thus, “about X” is intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.”

[0029] The term “administer,” “administering,” or “administration” refers to the methods that may be used to enable delivery of agents or compositions such as Ang 1-7 to a desired site of biological action. These methods include, but are not limited to, parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intra-arterial, intravascular, intracardiac, intrathecal, intranasal, intradermal, intravitreal, and the like), transmucosal injection, oral administration, administration as a suppository, and topical administration. In particular embodiments of the present disclosure, the compositions are formulated for intravenous infusion or for subcutaneous administration.

[0030] The term “treating” or “treatment” refers to any one of the following: ameliorating one or more symptoms of a disease or condition; preventing the manifestation of such symptoms before they occur; slowing down or completely preventing the progression of the disease or condition (as may be evident by longer periods between reoccurrence episodes, slowing down or prevention of the deterioration of symptoms, etc.); enhancing the onset of a remission period; slowing down the irreversible damage caused in the progressive-chronic stage of the disease or condition (both in the primary and secondary stages); delaying the onset of said progressive stage; or any combination thereof. Those in need of treatment include those already with the disorder, as well as those prone to have the disorder, or those in whom the disorder is to be prevented. A therapeutic benefit can refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject can still be afflicted with the underlying disorder. A prophylactic effect can include delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease can undergo treatment, even though a diagnosis of this disease cannot have been made. In the context of the present disclosure, the present methods and compositions can be used, e.g., to improve, prevent, slow the development of, or eliminate any symptom or effect of an ACE2 receptor-binding coronavirus infection, e.g., SARS-CoV-2 infection, including, but not limited to, dyspnea at rest or with activity, cough, fatigue, muscle weakness, body aches, joint pain, loss of smell or taste, brain fog, headaches, insomnia, excessive release of inflammatory cytokines, damage to the endothelial lining of blood vessels, excessive leakage of fluid into the lungs and other tissues, pro-thrombotic state with deep venous thrombosis, stroke, and thrombosis and emboli in the pulmonary circulation resulting in severe ventilation/perfusion mismatching and hypoxemia.

[0031] The term “effective amount” or “effective dose” or “therapeutically effective amount” or “therapeutically effective dose” refers to an amount of a compound (e.g., Ang 1-7) that is sufficient to bring about a beneficial or desired clinical or physiological effect. For example, in the present disclosure a therapeutically effective amount or dose of a compound could be any amount or dose that improves, prevents, slows the development of, or eliminates any symptom of an ACE2 receptor-binding coronavirus infection, e.g., SARS-CoV-2 infection, including, but not limited to, dyspnea at rest or with activity, cough, fatigue, muscle weakness, body aches, joint pain, loss of smell or taste, brain fog, headaches, insomnia, excessive release of inflammatory cytokines, damage to the endothelial lining of blood vessels, excessive leakage of fluid into the lungs and other tissues, pro-thrombotic state with deep venous thrombosis, stroke, and thrombosis and emboli in the pulmonary circulation resulting in severe ventilation/perfusion mismatching and hypoxemia, in a subject. An effective amount or dose may be based on factors individual to each subject, including, but not limited to, the subject’s age, size, level of physical fitness, diet, genetic background, presence of any disease or condition, route of administration, the type or extent of any supplemental therapies used, etc.

[0032] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate such as a mammal. Mammals include, but are not limited to, humans and non-human animals such as murines, rats, simians, farm animals or livestock for human consumption such as pigs, cattle, and ovines, as well as sport animals and pets. In one aspect, the mammal is a human, e.g., a human with COVTD-19. [0033] The term “viral infection” generally refers to any stage of an infection by a virus in a host (e.g., a cell, an animal, a human, etc.), including, but not limited to, attachment of the virus to a cell, penetration of the virus to the cell (e.g., fusion of the virus to the cell membrane, uncoating of the virus), uncoating of capsid of the virus, replication (e.g., transcription or reverse transcription of the viral genome, translation of the viral genome or a derivative thereof, viral particle assembly), and lysis (e.g., release of new viral particles from the cell). The term viral infection can also refer to any period of viral infection, including, but not limited to, incubation phase, latent phase, dormant phase, acute phase, and development and maintenance of immunity towards a virus.

[0034] “Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds. A protein can refer to a full-length polypeptide as translated from a coding open reading frame, or as processed to its mature form, while a polypeptide or peptide can refer to a degradation fragment or a processing fragment of a protein that nonetheless uniquely or identifiably maps to a particular protein. A polypeptide can be a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Polypeptides can be modified, for example, by the addition of carbohydrate, phosphorylation, etc. Proteins can comprise one or more polypeptides.

[0035] As used herein, the terms “fragment,” or equivalent terms can refer to a portion of a protein that has less than the full length of the protein and optionally maintains the function of the protein. Further, when the portion of the protein is blasted against the protein, the portion of the protein sequence can align, for example, at least with 80% identity to a part of the protein sequence.

[0036] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. In some cases, conservatively modified variants of a protein can have an increased stability, assembly, or activity as described herein.

[0037] The following eight groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (L), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins, W. H. Lreeman and Co., N. Y. (1984)).

[0038] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0039] In the present application, amino acid residues are numbered according to their relative positions from the left most residue, which is numbered 1, in an unmodified wild-type polypeptide sequence.

[0040] As used in herein, the terms “identical” or percent “identity,” in the context of describing two or more or amino acid sequences, refer to two or more sequences or specified subsequences that are the same. Two sequences that are “substantially identical” have at least 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection where a specific region is not designated. With regard to amino acid sequences, in some cases, the identity exists over a region that is at least about 50 amino acids in length, or more preferably over a region that is 75-100 amino acids in length.

[0041] The terms “increased,” or “increase” are used herein to generally mean an increase by a statically significant amount. In some embodiments, the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10%-100% as compared to a reference level, standard, or control. Other examples of “increase” include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.

[0042] The terms “decreased” or “decrease” are used herein generally to mean a decrease by a statistically significant amount. In some embodiments, “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10%-100% as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.

[0043] The term “ACE2” refers to angiotensin converting enzyme 2, a gene that encodes a protein belonging to the angiotensin-converting enzyme family of dipeptidyl carboxydipeptidases. The ACE2 protein catalyzes the cleavage of angiotensin II (Ang II) to form angiotensin 1-7 (Ang 1-7), and angiotensin I (Ang I) to form angiotensin 1-9 (Ang 1-9). ACE2 also serves as the receptor for the spike glycoprotein of, e.g., SARS-CoV and SARS-CoV-2. The human ACE2 gene has the NCBI Gene ID 59272, and the encoded protein has the UniProt ID Q9BYF1.

[0044] The term “Ang (1-7)” (also referred to herein as angiotensin 1-7, angiotensin-(l-7), or Ang 1-7) refers to a heptapeptide formed by the ACE2-catalyzed cleavage of Ang II, and includes salts, analogs, and derivatives thereof. As a non-limiting example, Ang (1-7) acetate is the acetate salt of a water-soluble endogenous human heptapeptide. The amino acid sequence of Ang (1-7) is: H - Asp - Arg - Val - Tyr - lie - His - Pro - OH, and it has the molecular formula: C41H62N12O11. Its molecular mass (average) is 899.0, and its molecular structure is shown in FIG. 1. The balance between Ang II and Ang (1-7) is critical in normal homeostasis. Ang (1-7) binds to a receptor called Mas and provides a counter-regulatory effect to Ang II.

3. Compositions

Ang (1-7)

[0045] The present disclosure provides compositions and methods for the treatment and prevention of infections by coronaviruses that bind to and enter cells via the ACE2 receptor. Such coronaviruses include SARS-CoV and SARS-CoV-2. Without being bound by the following theory, it is believed that numerous features of the diseases caused by such infections, e.g., COVTD-19, are the result of dysfunction in the ACE2 and p38 MAPK pathways, and that this dysfunction can be corrected, entirely or partially, through the administration of Ang (1-7), e.g., using the herein described methods.

[0046] Any form of Ang (1-7) can be used in the present methods. In particular embodiments, Ang (1-7) is used in an aqueous solution, formulated for, e.g., IV administration or subcutaneous administration. In particular embodiments, a base salt of Ang (1-7) is used in the formulation, e.g., Ang (1-7) acetate, sulfate, carbonate, bicarbonate, phosphate, diphosphate, maleate, citrate, mesylate, nitrate, tartrate, or gluconate. In particular embodiments, the basic salt is Ang (1-7) acetate (the acetate salt of the water-soluble endogenous human heptapeptide). The amino acid sequence of Ang (1-7) is: H - Asp - Arg - Val - Tyr - lie - His - Pro - OH. The molecular structure of Ang (1-7) is shown in FIG. 1.

[0047] The present disclosure also provides analogs and derivatives of Ang (1-7) and salts thereof. For example, Ang (1-7) can comprise entirely naturally occurring amino acids, e.g., as in the endogenous peptide, or it can comprise one or more modified, non-proteinogenic, non standard, or non-natural amino acids, so long that the peptide retains some or all of the properties of endogenous Ang (1-7), e.g., binding to the Mas receptor, inhibiting p38 MAPK, etc. Such modifications can be used, e.g., to enhance the stability, receptor affinity, bioavailability, pharmacokinetics, and/or tissue delivery relative to the endogenous peptide. In some embodiments, the Ang (1-7) peptide comprises one or more amino acids such as D-amino acids, homo amino acids, beta homo amino acids, N-methyl amino acids, alpha-methyl amino acids, beta amino acids, gamma amino acids, backbone modifications such as peptoids, hydroxyproline, beta-alanine, citrulline, ornithine, norleucine, 3-nitrotyrosine, nitroarginine, pyroglutamic acid, selenocysteine, pyrrolysine, naphtylalanine, Abu, DAB, methionin sulfoxide, methionine sulfone, dehydroamino acids, aminobutyric acid, proline and pyruvic acid derivatives, 3 -substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, amino acids with modifications such as phosphorylation, methylation, biotinylation, glycosylation, and others.

Preparation

[0048] The Ang (1-7) peptide can be obtained from a variety of sources, including from natural sources, through chemical synthesis, and from commercial vendors. Peptides such as Ang (1-7) and Ang (1-7) analogs and derivatives may also be synthesized by solid-phase peptide synthesis methods using procedures similar to those described by Merrifield et al., J Am. Chem. Soc., 85:2149-2156 (1963); Barany and Merrifield, Solid-Phase Peptide Synthesis, in The Peptides: Analysis, Synthesis, Biology Gross and Meienhofer (eds.), Academic Press, N.Y., vol. 2, pp. 3-284 (1980); and Stewart et ah, Solid Phase Peptide Synthesis 2nd ed., Pierce Chem. Co., Rockford, Ill. (1984). During synthesis, N-a-protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to a solid support, i.e., polystyrene beads. The peptides are synthesized by linking an amino group of an N- a-deprotected amino acid to an a-carboxy group of an N-a-protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide. The attachment of a free amino group to the activated carboxyl leads to peptide bond formation. The most commonly used N-a-protecting groups include Boc, which is acid labile, and Fmoc, which is base labile.

[0049] In particular embodiments, the Ang (1-7) peptide (or analog, derivative or salt thereof) is obtained from a commercial source, such as Bachem Americas, Inc., Pepscan, Thermo Scientific, GenScript, Peptide, Biomatik, or others.

Pharmaceutical compositions

[0050] Ang (1-7) can be formulated in any of a number of ways. In some embodiments, Ang (1-7) is formulated as a pharmaceutical composition, i.e., comprising a pharmaceutically acceptable carrier. In certain aspects, pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions for the Ang (1-7) peptide described herein (see, e.g., REMINGTON’S PHARMACEUTICAL SCIENCES, 18TH ED., Mack Publishing Co., Easton, PA (1990)).

[0051] As used herein, “pharmaceutically acceptable carrier” comprises any of standard pharmaceutically accepted carriers known to those of ordinary skill in the art in formulating pharmaceutical compositions. Thus, the Ang (1-7) peptide by itself, such as being present as a pharmaceutically acceptable salt or as a conjugate, may be prepared as formulations in pharmaceutically acceptable diluents; for example, saline, phosphate buffer saline (PBS), aqueous ethanol, or solutions of glucose, mannitol, dextran, propylene glycol, oils (e.g., vegetable oils, animal oils, synthetic oils, etc.), microcrystalline cellulose, carboxymethyl cellulose, hydroxylpropyl methyl cellulose, magnesium stearate, calcium phosphate, gelatin, polysorbate 80 or the like, or as solid formulations in appropriate excipients.

[0052] In particular embodiments, the pharmaceutical composition comprises sterile water. The pharmaceutical composition can be any of a number of pH values. For example, in some embodiments the pH of the composition is about 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6. In particular embodiments, the pH is about 5.5. [0053] In particular embodiments, the pharmaceutical compositions comprise an Ang (1-7) base, e.g., Ang (1-7) acetate. In some embodiments, the Ang (1-7) base is present at about, e.g.,

1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more mg/ml. In some embodiments, the Ang (1-7) base is present at about 1-5, 1-10, 10-20, 20-30, 30-40, or 40-50, mg/ml. Such compositions can be used to administer, e.g., by continuous IV, the doses described elsewhere herein.

[0054] In particular embodiments, the pharmaceutical composition comprises a sugar alcohol. Examples of sugar alcohols include, but are not limited to, ethylene glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, and polyglycitol. In particular embodiments, the sugar alcohol is mannitol. In some embodiments, the mannitol is present at about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg/ml. In particular embodiments, the mannitol is present at about 25 mg/ml.

[0055] The pharmaceutical compositions may further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene, butylated hydroxyanisole, etc.), bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents, preservatives, flavoring agents, sweetening agents, and coloring compounds as appropriate. Other potential components include carriers, excipients, binders, filling agents, suspending agents, flavoring agents, sweetening agents, disintegrating agents, dispersing agents, surfactants, lubricants, colorants, diluents, solubilizers, moistening agents, plasticizers, stabilizers, penetration enhancers, wetting agents, anti-foaming agents, antioxidants, preservatives, or one or more combination thereof.

[0056] The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

[0057] In one aspect, the Ang (1-7) peptide is formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection. In one aspect, formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0058] In some embodiments, formulations suitable for subcutaneous injection also contain additives such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the growth of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In some cases it is desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin.

[0059] For intravenous injections or drips or infusions, the Ang (1-7) peptide described herein is formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For other parenteral injections, appropriate formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are known.

[0060] Parenteral injections can involve bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The pharmaceutical composition described herein can be in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. In one aspect, the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0061] For administration by inhalation, the peptide is formulated for use as an aerosol, a mist or a powder. Pharmaceutical compositions described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, such as, by way of example only, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the peptides described herein and a suitable powder base such as lactose or starch.

[0062] Representative intranasal formulations including the Ang (1-7) peptide are prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Preferably these formulations are prepared with suitable nontoxic pharmaceutically acceptable ingredients. The choice of suitable carriers is dependent upon the exact nature of the nasal dosage form desired, e.g., solutions, suspensions, ointments, or gels. Nasal dosage forms generally contain large amounts of water in addition to the active ingredient. Minor amounts of other ingredients such as pH adjusters, emulsifiers or dispersing agents, preservatives, surfactants, gelling agents, or buffering and other stabilizing and solubilizing agents are optionally present. Preferably, the nasal dosage form should be isotonic with nasal secretions.

[0063] Pharmaceutical compositions as described herein are manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes. Conventional formulation techniques include, e.g., one or a combination of methods: (1) dry mixing, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion. Other methods include, e.g., spray drying, pan coating, melt granulation, granulation, fluidized bed spray drying or coating (e.g., wurster coating), tangential coating, top spraying, tableting, extruding and the like.

[0064] As used herein, the term “unit dosage form” refers to physically discrete units suitable as unitary dosages for humans and other mammals, each unit containing a predetermined quantity of a therapeutic agent calculated to produce the desired onset, tolerability, and/or therapeutic effects, in association with a suitable pharmaceutical excipient (e.g., an ampoule). In addition, more concentrated dosage forms may be prepared, from which the more dilute unit dosage forms may then be produced. The more concentrated dosage forms thus will contain substantially more than, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times, the amount of the therapeutic compound.

[0065] Methods for preparing such dosage forms are known to those skilled in the art (see, e.g., REMINGTON’S PHARMACEUTICAL SCIENCES, supra). The dosage forms typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, adjuvants, diluents, tissue permeation enhancers, solubilizers, and the like. Appropriate excipients can be tailored to the particular dosage form and route of administration by methods well known in the art (see, e.g., REMINGTON’S PHARMACEUTICAL SCIENCES, supra).

[0066] Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols, e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc. The dosage forms can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preserving agents such as methyl-, ethyl-, and propyl-hydroxy-benzoates (i.e., the parabens); pH adjusting agents such as inorganic and organic acids and bases; sweetening agents; and flavoring agents. The dosage forms may also comprise biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.

[0067] In some embodiments, additional compounds or medications can be co-administered to the subject. Such compounds or medications can be co-administered for the purpose of alleviating signs or symptoms of the disease being treated, e.g., anti-inflammatory agents, etc. 4. Methods of Treatment

Subjects

[0068] Subjects can be any subject, e.g., human or other mammal, that has been infected with an ACE2 receptor-binding coronavirus, such as SARS-CoV or SARS-CoV-2. The infection can be detected in any of a number of ways, including by PCR test or antigen test. In some embodiments, the infection is detected by virtue of the presence of one or more symptoms of the infection, e.g., symptoms of COVID-19, together with knowledge of prior exposure to an infected individual.

[0069] In some embodiments, the subject is human. In some embodiments, the subject is an adult. In some embodiments, the subject is elderly, e.g., is at least 65, 70, 75, 80, or 85 years old. In some embodiments, the subject is a child. In some embodiments, the subject is an adolescent. In some embodiments, the subject is female. In some embodiments, the subject is male.

[0070] In some embodiments, the subject is asymptomatic for the infection, e.g., SARS-CoV-2 infection. In some embodiments, the subject has one or more symptoms of an ACE2 receptor binding coronavirus infection, e.g., of COVID-19. For example, in some embodiments, the subject has one or more symptoms such as fever or chills, dyspnea at rest or with activity, cough, fatigue, muscle weakness, sore throat, congestion, runny nose, nausea, vomiting, diarrhea, pain or pressure in chest, body aches, joint pain, loss of smell or taste, brain fog, confusion, inability to wake or stay awake, pale, gray, or blue-colored skin, lips, or nail beds, headaches, or insomnia.

[0071] In some embodiments, the subject has severe symptoms of the coronavirus infection, e.g., of COVID-19. For example, in some embodiments, the subject has been hospitalized, or is in an emergency department with planned hospitalization. In some embodiments, the subject has suffered from respiratory failure. In some embodiments, the subject requires a high flow oxygen (e.g., > 10 L/min) device. In some embodiments, the subject requires noninvasive ventilation. In some embodiments, the subject requires mechanical ventilation. In some embodiments, the subject requires Extracorporeal Membrane Oxygenation (ECMO). In some embodiments, the subject has acute respiratory distress syndrome (ARDS). [0072] In some embodiments, the subject has a decrease in ACE2 levels or activity, a decrease in the ratio of Ang (1-7) to Ang II, and/or an increase in p38 MAPK levels or activity. In some embodiments, the subject has a disease or condition associated with a decrease in ACE2 levels or activity, a decrease in the ratio of Ang (1-7) to Ang II, and/or an increase in p38 MAPK levels or activity, such as heart disease, hypertension, diabetes, or obesity.

5. Dosing and Administration

[0073] The herein described pharmaceutical compositions can be administered locally in the subject or systemically. In some embodiments, the compounds can be administered, for example, intraperitoneally, intramuscularly, intra-arterially, orally, intravenously, intracranially, intrathecally, intraspinally, intralesionally, intranasally, subcutaneously, intracerebroventricularly, topically, transdermally, sublingually, buccally, and/or by inhalation. In particular embodiments, the compounds are administered intravenously, e.g., by continuous IV infusion. In some embodiments, the compounds are administered subcutaneously.

[0074] The herein-described pharmaceutical compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective. The quantity to be administered depends on a variety of factors including, e.g., the age, body weight, physical activity, and diet of the individual, any conditions or diseases to be treated, and the stage or severity of any potential conditions or diseases. In certain embodiments, the size of the dose may also be determined by the existence, nature, and extent of any adverse side effects that accompany the administration of a therapeutic agent(s) in a particular individual.

[0075] It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, hereditary characteristics, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the presence and severity of any particular condition, and any other potential therapies that are being administered.

[0076] In some embodiments, the Ang (1-7) peptide is formulated and administered such that the subject receives, e.g., by continuous IV infusion, from about 2.5 ng/kg/min to about 40 ng/kg/min Ang (1-7) (e.g., Ang (1-7) acetate). In some embodiments, the Ang (1-7) peptide is administered such that the subject receives, e.g., by continuous IV infusion, from about 5 ng/kg/min to about 20 ng/kg/min Ang (1-7) (e.g., Ang (1-7) acetate). In some embodiments, the subject is administered an initial dose of about 5 ng/kg/min Ang (1-7) (e.g., Ang (1-7) acetate), and the dose is increased in 5 ng/kg/min increments as tolerated by the patient’s blood pressure up to, e.g., about 20 ng/kg/min Ang (1-7) (e.g., Ang (1-7) acetate). In some embodiments, the subject receives about 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 ng/kg/min Ang (1-7) (e.g., Ang (1-7) acetate).

[0077] In some embodiments, the Ang (1-7) peptide is administered by continuous IV infusion, for 1, 2, 3, 4, 5, 6, or 7 days. In some embodiments, the Ang (1-7) peptide is administered by continuous IV infusion, for up to 7 days or until hospital discharge, whichever occurs first.

Assessing efficacy

[0078] The efficacy of the administration of the Ang (1-7) peptide can be assessed in any of a number of ways. For example, in some embodiments, the Ang (1-7) peptide reduces, slows the development of, eliminates, prevents, or in any way improves one or more symptom of the infection, e.g., COVTD-19. For example, the symptom can be fever or chills, dyspnea at rest or with activity, cough, fatigue, muscle weakness, sore throat, congestion, runny nose, nausea, vomiting, diarrhea, pain or pressure in chest, body aches, joint pain, loss of smell or taste, brain fog, confusion, inability to wake or stay awake, pale, gray, or blue-colored skin, lips, or nail beds, headaches, insomnia, excessive release of inflammatory cytokines, damage to the endothelial lining of blood vessels, excessive leakage of fluid into the lungs and other tissues, pro-thrombotic state with deep venous thrombosis, stroke, and thrombosis and emboli in the pulmonary circulation resulting in severe ventilation/perfusion mismatching and hypoxemia.

[0079] In some embodiments, e.g., where the subject has suffered respiratory failure, the Ang (1-7) peptide can decrease the time it takes for the subject to recover from the respiratory failure, e.g., decrease relative to a subject with an equivalent infection and with respiratory failure but who has not been administered Ang (1-7). In some embodiments, the Ang (1-7) peptide leads to a decrease of, e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more relative to the time it takes for the subject to recover from the respiratory failure relative to a subject with an equivalent infection and with respiratory failure but who has not been administered Ang (1-7).

[0080] In some embodiments, the Ang (1-7) peptide leads to a reduction in the level of inflammation and/or thrombosis in the subject, and/or an increase in vasodilation in the subject.

[0081] In some embodiments, the Ang (1-7) peptide leads to a decrease in the level or activity of p38 MAPK in one or more tissues of the subject. In some embodiments, the Ang (1-7) administration leads to a decrease in the level or activity of p38 MAPK of, e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more relative to the level or activity in the absence of Ang (1-7) administration.

[0082] In some embodiments, the Ang (1-7) peptide leads to a reduction in the replication of the coronavirus. Without being bound to the following theory, it is believed that the reduction in viral replication is a result of the Ang (l-7)-mediated reduction in p38 MAPK activity. In some embodiments, the Ang (1-7) administration leads to a reduction in viral replication of, e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more relative to the amount of replication in the absence of Ang (1-7) administration.

[0083] In some embodiments, the Ang (1-7) peptide leads to a higher ratio of Ang (1-7) to Ang II in the subject. In some embodiments, the ratio of Ang (1-7) : Ang II increases by, e.g., about 10%, 20%, 30%, 40%, 50%, or more as a result of the Ang (1-7) administration.

[0084] In some embodiments, the Ang (1-7) peptide leads to a change in the level of one or more cytokines in the subject, e.g., tumor necrosis factor (TNF) alpha, interleukin (IL) 1 beta, IL-6, IL-8, IL-10, or interferon (IFN) gamma. In some embodiments, the Ang (1-7) peptide leads to a reduction in the level of one or more pro-inflammatory cytokines. In some embodiments, the Ang (1-7) peptide leads to an increase or decrease of, e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more in the level of one or more cytokines relative to the level in the absence of Ang (1-7) administration.

6. Kits

[0085] Other embodiments of the compositions described herein are kits comprising the herein- described compositions. The kit typically contains containers, which may be formed from a variety of materials such as glass or plastic, and can include for example, bottles, vials, syringes, and test tubes. A label typically accompanies the kit, and includes any writing or recorded material, which may be electronic or computer readable form providing instructions or other information for use of the kit contents.

[0086] In some embodiments, the kit comprises one or more reagents for the administration of Ang (1-7), e.g., IV or subcutaneous administration. In some embodiments, the kit comprises one or more containers comprising 2.5 mg/ml Ang (1-7).

[0087] In some embodiments, the kits can further comprise instructional materials containing directions (i.e., protocols) for the practice of the present methods (e.g., instructions for administering Ang (1-7) intravenously or subcutaneously). While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

7. Examples

Example 1. Chemistry Manufacturing and Control (CMC! information for Clinical Trial Introduction

[0088] Angiotensin-(l-7) [Ang 1-7] is a naturally occurring human endogenous heptapeptide. Each vial of drug product contains 1.2 mL of Ang (1-7) acetate study drug. Each 1 mL of drug product contains 2.5 mg of angiotensin-(l-7) base and 25 mg of mannitol in sterile water, with the pH adjusted to 5.5. The drug product is a clear, colorless liquid for injection. Storage temperature is 2° C to 8° C. The study drug vials are maintained at 2° C to 8° C at all times during transport and storage until prepared for patient administration by the hospital research pharmacist.

[0089] The drug was prepared as follows:

[0090] Bachem Americas, Inc. has manufactured Ang (1-7) acetate active pharmaceutical ingredient (API) using solid phase synthesis under current Good Manufacturing Practices (cGMP). Integrity Bio, a cGMP formulation and fill/finish company, manufactured the Drug Product and conducted release testing.

Drug Substance

[0091] The Drug Substance, Ang (1-7) acetate, was manufactured under cGMP by Bachem Americas using solid phase peptide synthesis. Ang (1-7) acetate is stored in vials containing 1 gram of Drug Substance. The Drug Substance is stored and shipped at -20° C until ready for formulation.

Drug Product

[0092] The final formulation of Drug Product was manufactured by Integrity Bio, Inc under cGMP. Each vial of Drug Product contains 1.2 mL of Ang (1-7) acetate study drug. Each 1 mL of drug product contains 2.5 mg of angiotensin-(l-7) base and 25 mg of mannitol in sterile water for injection, with the pH adjusted to 5.5. The drug product is a clear, colorless liquid for injection. Storage temperature is 2° C to 8° C.

[0093] All operations performed by Integrity Bio were performed per internal Standard Operating Procedures in accordance with the principles and guidelines of the USFDA cGMP. The final drug product underwent quality testing and met the release criteria. The Certificate of Analysis showed that the drug product met the following specifications:

Table 1.

Description and Composition

[0094] Each vial contains 3 mg of Ang (1-7) as an acetate salt and 30 mg of mannitol in 1.2 mL of sterile water, with pH adjusted to 5.5. The final Ang (1-7) base and mannitol concentrations are 2.5 mg/mL and 25 mg/mL, respectively.

Dosage Preparation and Storage Scheme

[0095] The study drug is shipped to the clinical sites from the central pharmacy or from other designated drug repositories in accordance with regulatory requirements. All other supplies are provided by the site. Study drug is shipped at 2° C to 8° C in temperature-monitored containers. Upon arrival at the clinical sites, study drug is stored at 2° C to 8° C and protected from light.

[0096] Study drug is prepared as follows: 1 mL of study drug is removed from the vial and injected into a 250- mL bag of 0.9% sodium chloride solution for intravenous infusion. The final concentration is 10,000 ng/mL. The 250-mL bag containing study drug may be stored at room temperature or under refrigeration.

[0097] 0.9% sodium chloride solution (normal saline) is used as placebo. Thus, the only preparation of placebo is the placing of the appropriate label.

[0098] Drug and placebo preparation is performed by the participating site’s designated unblinded research pharmacist. The patient, medical team, and all other research staff remain blinded. Detailed information on the preparation, labeling, storage, and administration of Ang (1- 7) acetate and placebo is provided in a protocol-specific Pharmacy Manual. Depending upon body weight, approximately 7-14 vials are used per patient.

Stability Testing

[0099] Stability testing is performed concurrently with the clinical trial. Stability testing is conducted at 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 36 months, and 48 months. Container closure integrity testing is conducted annually.

Placebo

[0100] 0.9% sodium chloride solution (normal saline) is used as placebo.

Labeling

[0101] Drug and placebo preparation is performed by the participating site’s designated unblinded research pharmacist on the same day of administration to the patient.

Example 2 A Multi-Center. Randomized. Double-Blind. Placebo Controlled Study of the Safety and Efficacy of Amnotensin-P -7) for the Treatment of COVID-19 in Critically Ill Patients

[0102] This is a randomized, double-blind, placebo-controlled study to evaluate the safety and efficacy of angiotensin-(l-7) [Ang (1-7)] in 250 critically ill patients diagnosed with COVID-19. The primary objective is to evaluate the safety and efficacy of intravenous (IV) infusion of Ang (1-7) compared to placebo with respect to time to recovery from respiratory failure in critically ill patients with COVTD 19.

Endpoints

Primary Endpoint

[0103] Time to recovery from respiratory failure, defined as a score of 4, 5, 6, 7, or 8 on the COVTD 19 disease severity 8-point ordinal scale used in the National Institutes of Health (NIH) Adaptive COVID-19 Treatment Trial (ACTT) [ClinicalTrials.gov Identifier: NCT04280705]:

1. Death

2. Hospitalized, on invasive mechanical ventilation or ECMO

3. Hospitalized, on non-invasive ventilation or high-flow oxygen devices 4. Hospitalized, requiring supplemental oxygen

5. Hospitalized, not requiring supplemental oxygen - requiring ongoing medical care (COVTD- 19-related or otherwise)

6. Hospitalized, not requiring supplemental oxygen - no longer requires ongoing medical care

7. Not hospitalized, limitation on activities and/or requiring home oxygen

8. Not hospitalized, no limitation on activities The score is assigned as follows:

-If the patient recovers from respiratory failure by Day 29, the score is the first Day on which the patient attains and then maintains a score of 4, 5, 6, 7, or 8 on the ordinal scale

-If the patient dies by Day 29, the score is ¥

-If the patient neither recovers from respiratory failure nor dies by Day 29, the score is 29 Secondary Endpoints:

-Mortality on Day 29 -Mortality on Day 57

-Proportion of patients alive and free of respiratory failure, defined as score of 4, 5, 6, 7, or 8 on the 8-point ordinal scale at Day 29

Exploratory Endpoints

-Score on the 8-point ordinal scale on Days 8, 15, 22, 29, and 57

-Proportion of patients on high-flow oxygen devices or noninvasive ventilation at enrollment who progress to require mechanical ventilation or ECMO, or dying, through Day 29

-Days alive and not on mechanical ventilator or ECMO through Day 29

-Days alive and not on mechanical ventilator or ECMO through Day 57 -Persistent symptoms of COVID-19 at Day 57 including dyspnea at rest or with activity, cough, fatigue, muscle weakness, body aches, joint pain, loss of smell or taste, brain fog, headaches, or insomnia

-Duration of mechanical ventilation -Duration of ECMO -Duration of noninvasive ventilation -Duration of requirement for high-flow oxygen -Duration of intensive care unit (ICU) stay -Duration of hospital stay

-Change in cardiac troponin, n-terminal pro-BNP, and D-dimer from baseline to Day 5 or day of discharge if before Day 5

-Nasopharyngeal swab for SARS-CoV-2 RNA on Day 5 or day of discharge if before Day 5

-For prespecified sites, change in angiotensin- 1-7 and angiotensin II levels from baseline to Day 5 or day of discharge if before Day 5

-For prespecified sites, change in cytokine levels including tumor necrosis factor (TNF)-alpha, interleukin (IF)- 1 beta, IF-6, IF-8, IF- 10, and interferon (IFN) gamma from baseline to Day 5 or day of discharge if before Day 5

Safety Endpoints

[0104] Proportion of patients with treatment-emergent serious adverse events (SAEs), Grade 3 or Grade 4 adverse events (AEs), or AEs leading to study drug discontinuation.

Study population

[0105] Adults (>18 years old) with COVID-19 who are hospitalized and meet the criteria for critical illness at the time of enrollment. Patients receiving anti-viral medications and other standard-of-care treatments for COVID-19 are allowed to enroll in the study, and treating physicians are allowed to continue or initiate treatment with antiviral drugs. [0106] In order to be eligible to participate in this study, an individual must meet all of the following criteria:

-Written informed consent must be provided by the patient or the patient’s legally authorized representative prior to performing study procedures

-Age >18 years

-Respiratory failure thought to be the result of COVID-19 infection

-Laboratory-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection as determined by polymerase chain reaction (PCR) test <10 days before randomization

-Currently hospitalized or in an emergency department with planned hospitalization

-Meet one of the following criteria: 1) require high-flow oxygen (> 10 L/min) device; 2) require noninvasive ventilation; 3) require mechanical ventilation for < 72 hours at the time of enrollment

[0107] An individual who meets any of the following criteria will be excluded from participation in this study:

-Simultaneous participation in any other clinical study incompatible with this one -Requirement for ECMO at Screening

-Hypotension at Screening, defined as supine blood pressure (BP) <85 mm Hg systolic or <50 mm Hg diastolic despite vasopressor support

-AST or ALT >5x the upper limit of normal

-Estimated GFR (eGFR) <30 mL/min

-Any known immune deficiency

-Pregnancy or breastfeeding

-Anticipated transfer to another facility that is not a study site ->7 days between hospital admission and start of Screening ->24 hours between start of Screening and Randomization

-On hospice care or presumption that death is imminent with a lack of commitment to full supportive therapy

Description of Study Intervention

[0108] Enrolled patients are randomized 1:1 to receive Ang (1-7) or placebo according to the dosing schedule below.

[0109] Treatments:

[0110] Ang (1-7) by continuous IV infusion for up to 7 days or until hospital discharge, whichever occurs first. Ang (1-7) is prepared by injecting 1 mL (2.5 mg) into a 250 mL bag of 0.9% sodium chloride (normal saline). Dosing starts at 5 ng/kg/min and then escalates in 5 ng/kg/min increments no more frequently than every 30 minutes as tolerated by the patient’s BP up to the maximum dose of 20 ng/kg/min; or Placebo — 0.9% sodium chloride (normal saline) by continuous IV infusion for up to 7 days or until hospital discharge, whichever occurs first. Supplied as 250 mL IV bags. Infusion rate matches that for Ang (1-7), as described above.

[0111] Study duration: Approximately 6 months (including 12 weeks recruitment per site and 2 months of treatment + follow-up). See, e.g., FIG. 2.

[0112] Participant duration: Approximately 2 months (57 days)

[0113] Screening laboratory tests include: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, creatinine, calculation of estimated glomerular filtration rate (eGFR), and C-reactive protein, and pregnancy test for females of childbearing potential.

[0114] Medication review includes: medications for treatment of COVTD-19 and vasopressors.

[0115] Safety laboratory tests include: complete blood count (CBC) with differential, hemoglobin, platelets, creatinine, glucose, total bilirubin, ALT, AST, and prothrombin time (PT).

[0116] All follow-up visits are performed via telephone call to assess clinical status (NIH ACTT ordinal scale), readmission to a hospital, and mortality. Day 57 assessment also includes questions regarding persistent symptoms associated with COVTD-19 including dyspnea at rest or with activity, cough, fatigue, muscle weakness, body aches, joint pain, loss of smell or taste, brain fog, headaches, or insomnia.

[0117] Angiotensin (1-7) levels, angiotensin II levels, and cytokine panel are collected at a prespecified subset of clinical trial sites. Cytokine panel includes tumor necrosis factor (TNF)- alpha, interleukin (IL)-lbeta, IL-6, IL-8, IL-10, interferon (IFN) gamma.

Overall design

[0118] This is a multi-center, randomized, double-blind, placebo-controlled study to evaluate the safety and efficacy of Ang (1-7) in 250 critically ill patients diagnosed with COVTD-19.

[0119] The primary endpoint is the time to recovery from respiratory failure, defined as a score of 4, 5, 6, 7, or 8 score on the 8 point ordinal COVID-19 disease severity scale used in the NIH ACTT. Secondary endpoints are: mortality on Day 29; mortality on Day 57; and proportion of patients alive and free of respiratory failure, defined as a score of 4, 5, 6, 7, or 8 on the 8-point ordinal scale on Day 29.

[0120] Exploratory endpoints include score on the 8-point ordinal scale on Days 8, 15, 22, 29, and 57; proportion of patients on high-flow oxygen devices or noninvasive ventilation at enrollment who progress to require mechanical ventilation or ECMO, or dying, through Day 29; days alive and not on mechanical ventilator or ECMO through Day 29; days alive and not on mechanical ventilator or ECMO through Day 57; persistent symptoms of COVID-19 at Day 57 including dyspnea at rest or with activity, cough, fatigue, body aches, joint pain, loss of smell or taste, brain fog, headaches, or insomnia; duration of requirements for mechanical ventilation, ECMO, noninvasive ventilation, and high-flow oxygen device; ICU stay; duration of hospital stay; change in levels of cardiac troponin, n-terminal-proBNP, and D-dimer from baseline to Day 5; results of nasopharyngeal swab for SARS-CoV-2 RNA on Day 5; at prespecified sites, change in levels of angiotensin II and angiotensin-(l-7) from baseline to Day 5; and at prespecified sites; change in levels of cytokine levels from baseline to Day 5.

[0121] There are several safety advantages associated with IV dosing. First, the Ang (1-7) levels can be maintained in a physiologic range that avoids wide swings in circulating drug levels. Second, if a patient were to experience a drug-related SAE, the infusion could be discontinued and Ang (1-7) levels would quickly drop towards pre- infusion levels. Third, it allows continuous exposure throughout the treatment period while minimizing the total drug exposure, reducing area under the curve (AUC) for blood levels. Lastly, continuous IV infusion requires nearly 10-fold less drug in a 24-hour period than daily subcutaneous injection.

[0122] Because of the increased Ang II levels in seriously ill patients with COVTD-19, they may require higher doses of Ang (1-7) to achieve the desired therapeutic effect. Dosing starts at 5 ng/kg/min and is escalated in 5 ng/kg/min increments no more frequently than every 30 minutes as tolerated by the patient’s BP up to a maximum allowable dose of 20 ng/kg/min. Dosing continues for 7 days or until hospital discharge, whichever occurs first.

Dosing and administration

[0123] Enrolled patients are randomized 1:1 to receive Ang (1-7) or placebo according to the dosing schedule. Note: The study drug infusion should not be started if the patient’s BP is <85 mm Hg systolic or <50 mm Hg diastolic.

End of Treatment

[0124] When a patient has reached seven 7 days of therapy or has recovered sufficiently that hospital discharge is planned, the infusion of Ang (1-7) is tapered off. Suggested regimen: decrease the infusion rate by 5 mg/kg/min no more frequently than every 60 minutes. The patient is then observed for at least 4 hours after the infusion has been discontinued to ensure stability of cardiovascular and pulmonary function.

Acquisition

[0125] Ang (1-7) is supplied in sterile vials containing 3.0 mg of drug in 1.2 mL of sterile water (i.e., 2.5 mg/mL). Based upon the patient’s body weight, each course of treatment requires from seven to fourteen vials. Study drug is shipped to the clinical sites from a central pharmacy and is stored under refrigeration at 2-8 degrees C.

[0126] Drug preparation is performed by the participating site’s designated unblinded research pharmacist on the same day of administration to the patient. The patient and all other research staff remain blinded. Formulation

[0127] Each vial contains 1.2 mL of Ang (1-7) study drug. Each 1 mL of Ang (1-7) study drug contains 2.5 mg of angiotensin-(l-7) base and 25 mg of mannitol in sterile water, with the pH adjusted to 5.5. Ang (1-7) acetate in sterile water is a clear colorless liquid for injection.

[0128] The placebo control is 0.9% normal saline and is a clear, colorless solution.

Product storage

[0129] Ang (1-7) in vials is stored at 2°C - 8°C and protected from light. Vials are thawed prior to preparation for injection. Any unused portion of study drug is discarded.

Randomization and blinding

[0130] This double blind study randomizes patients to receive either Ang (1-7) (125 patients) or matching placebo (125 patients). Randomization is stratified by site and requirement for mechanical ventilation. Randomization lists are generated for each stratum using block randomization with random block sizes of 2, 4, and 6.

[0131] At Screening (Day -1 or Day 1), the following assessments or procedures are performed:

-Informed consent

-Collection of demographics and medical history: medical history includes day of onset of COVID-19 symptoms, history of chronic medical conditions related to eligibility, medication allergies, and a review of medications and therapies taken for this current illness in the 7 days prior to Day 1

-Symptom-directed physical examination (including measurement vital signs and measurement of SpCh). Height (can be self-reported) and weight is obtained. PaC is not a study-mandated measurement; it can be used in the assessment of inclusion criterion number 6 if it was obtained as a clinically indicated test.

-Review of SARS-CoV-2 results: if the original sample was collected more than 7 days prior to planned randomization, the PCR can be repeated as part of Screening

-Blood sample collection for hematology, chemistry, liver tests, and C-reactive protein (CRP) -Pregnancy test for females of childbearing potential: women are counseled to either practice abstinence or use at least 1 primary form of contraception from the time of Screening through the end of the study

[0132] Note: Screening laboratory tests include ALT, AST, total bilirubin, creatinine, calculation of eGFR, CRP, and pregnancy tests. Laboratory tests performed in the 48 hours prior to enrollment are accepted for determination of eligibility.

Baseline

At baseline (Day 1), the following assessments or procedures are performed:

-Randomization

-Clinical data collection

-APACHE II score calculation

-Vital sign measurement, including Sp02

-Medication review

-AE evaluation

-Blood sample collection for hematology, chemistry, and liver tests -Blood sample for cardiac troponin, n-terminal pro-BNP, and D-dimer At prespecified sites:

-Blood sample for plasma Ang 1 -7 and Ang II levels -Blood sample for cytokine panel

[0133] Note: baseline assessments are performed before randomization. Laboratory tests performed as part of routine clinical care in the 24 hours prior to first dose are accepted for the baseline safety laboratory tests. Baseline may be the same as the screening laboratory tests. Safety laboratory tests at baseline include complete blood count (CBC) with differential, hemoglobin, platelets, creatinine, glucose, total bilirubin, ALT, AST, and prothrombin time (PT). The cytokine panel at baseline includes TNF-alpha, IL-lbeta, IL-6, IL-8, IL-10, IFN gamma. Power Justification

[0134] The primary endpoint of time to recovery from respiratory failure is based on an 8- point ordinal scale for post-treatment clinical status. The outcome score is assigned as follows:

-If the patient recovers from respiratory failure by Day 29, the score is the first day on which the patient attains and then maintains a score of 4, 5, 6, 7, or 8 on the ordinal scale. In this case, the outcome score may take a value of 1, 2, 3, ... , 28

-If the patient neither recovers from respiratory failure nor dies by Day 29, the outcome score is 29

-If the patient dies by Day 29, the outcome score is +¥.

[0135] This outcome score (primary endpoint) summarizes the time to recovery from respiratory failure as well as time to death observed within the 28-day window. A Wilcoxon- Mann- Whitney rank test is conducted to test whether the probability that a patient in the treatment arm has a better outcome than a patient in the control arm is greater the probability that a patient in the control arm has a better outcome than a patient in the treatment arm.

[0136] In a recent REMAP-CAP IL-6 receptor antagonist study with a similar patient population, we observed 36%, 52%, and 60% of the patients in the treatment arm experiencing recovery from respiratory failure by days 7, 14, and 21, respectively.⁴¹ The linearly extrapolated proportion of patients recovered from respiratory failure by day 28 was 67%. The 29-day and 57- day mortality in this population was 25% and 28%, respectively. In this study, we assume the same distribution for time to recovery and a 29-day mortality rate of 22% anticipating continuing improvement in COVTD patient outcomes. Under these assumptions, we expect to observe 84 recoveries and 28 deaths during 28-day follow-up (i.e., to study Day 29) among 125 patients randomized to the standard care arm. We assume a proportional hazards alternative with a hazard ratio of 1.55 for time to recovery, i.e., 50%, 68%, 76%, and 82% of the patients in the treatment arm will recover by days 7, 14, 21, and 28, respectively. We also assume that the 28-day mortality rate in the treatment arm is reduced to 12%. In total, we expect to observe 103 recoveries and 15 deaths from 125 patients in the treatment arm. The assumed distribution of the primary endpoint in both arms are summarized in FIG. 3, where day 29 and 30 representing the outcome score being 29 and +¥, respectively, i.e., alive but not recovered by day 29 and death before day 29.

[0137] Under these assumptions, the probability that a patient in the treatment arm has a better outcome than a patient in the control arm is 56.4%, and the probability that a patient in the control arm has a better outcome than a patient in the treatment arm is 35.9%. The Mann- Whitney rank test based on 125 patients per arm will have 81% power for detecting such a difference at the 2-sided significance level of 0.05.

[0138] For comparing secondary endpoints (29-day mortality, 57-day mortality, and proportion of patients alive and free of respiratory failure at day 29 following the first dose), we use Mantel-Haenszel test stratified by site and mechanical ventilation status at the baseline. Based on the REMAP-CAP IL-6 receptor antagonist study, we assume that the 29-day mortality, 57-day morality, and proportion of patients alive and free of respiratory failure at day 29 in the control arm are 22%, 25% and 67%, respectively.⁴¹ As a conservative estimate, the simple chi- squared test based on the proposed sample size (125 patients per arm) provides approximately 80% power for detecting a difference between 22% and 9.2% for 29-day mortality, a difference between 25% and 11.5% for 57-day mortality, and a difference between 67% and 82.2% for proportion of patients alive and free of respiratory failure at day 29, at the significance level of 0.05.

Populations for Analyses

[0139] The enrolled population includes 125 COVTD-19 patients in the treatment arm and 125 COVID 19 patients in the control arm.

[0140] The intent-to-treat (ITT) population includes all 125 COVID- 19 patients in the treatment arm and 125 COVID- 19 patients in the control arm. The proposed comparisons of endpoints are made based on the ITT principle, i.e., the patients assigned to the treatment arm are compared with patients assigned to the control arm regardless of the actual treatment received.

[0141] The safety population includes all 125 COVID- 19 patients in the treatment arm and 125 COVID 19 patients in the control arm. Statistical Analyses

General Approach

[0142] The 2-sided statistical significance level of all the tests is set at 0.05. There is no formal adjustment of multiple testing but the treatment effect on the primary and all second endpoints is reported and evaluated together as a totality of evidence for the potential treatment benefit under investigation.

[0143] Patients are followed to Day 29 unless they die earlier and complete information is available before discharge, and thus, we don’t expect missing data for primary and secondary endpoints. If there are missing data due to loss of follow-up, transferring hospital or missing record, the reasons for missing data or loss of follow-up is documented to further determine if missing data are random. In the presence of missing data for important endpoints, the multiple imputation is used to handle missing data and the main comparison is made based on imputed outcomes for all randomized patients. In implementing the multiple imputations, all available baseline and follow-up information is included to assist imputing missing values and 50 imputed complete data sets are created for making statistical inference, which appropriately accounts for the variability from imputations.

Analysis of the Primary Endpoint

[0144] The distribution of the primary endpoint is compared between 2 treatment arms using the Wilcoxon-Mann- Whitney rank test. The comparison is based on all randomized patients pr the intention-to-treat principle. The test is stratified by site and sex. The treatment effect is quantified by the difference between: the probability that a patient in the treatment arm has a better outcome than a patient in the control arm; and the probability that a patient in the control arm has a better outcome than a patient in the treatment arm.

[0145] This probability is the area under the receiver operating characteristics curve (ROC) between patients receiving the treatment and patients receiving the standard care and can be estimated by the Mann- Whitney U-statistics following an asymptotic normal distribution with a variance, that can be estimated analytically.⁴² The 95% confidence interval based on large sample approximation is constructed accordingly. Additional sensitivity analysis is performed to construct the 95% confidence interval based on the nonparametric bootstrap method. We further characterize the distribution of this composite score in 2 arms, separately.

[0146] As a sensitivity analysis, we repeat the comparison after nonparametrically adjusting the important baseline covariates selected a priori for bias correction and precision improvement.⁴³

Analysis of the Secondary Endpoints

[0147] For secondary endpoints, we use the Mantel-Haenszel (MH) test stratified by site and mechanical ventilation status at the baseline to compare the 29-day mortality, 57-day mortality, and proportion of patients alive and free of respiratory failure at day 29 following the first dose. The between group difference is summarized by the MH odds ratio (OR) and its 95% confidence interval.

[0148] As sensitivity analysis, we repeat the comparison of the secondary endpoints after nonparametrically adjusting the important baseline co variates selected a priori for bias correction and precision improvement.

Baseline Descriptive Statistics

[0149] Baseline patient characteristics are summarized by treatment arm to examine the balance in important risk factors between the 2 arms.

[0150] For the futility analysis, the conditional power assuming the observed treatment effect at interim analysis as true treatment effect is estimated. If the conditional power is less than 33%, the DSMB recommends to stop the study for futility; if the conditional power is between 33% and 66%, the DSMB recommends to increase the sample size to ensure 66% power. If the conditional power is above 66% and the comparison result doesn’t cross the efficacy boundary specified below, the DSMB recommends to continue the study with original sample size. If the sample size adjustment is made, the final comparison to evaluate the treatment effect is based on combining two Wald z-statistics based on observations before and after the sample size adjustment with equal weights.

[0151] For efficacy analysis, the type one error allocated to the interim analysis is 0.0054 and consequently, the type one error at the final analysis is 0.0446 based on O’Brien-Fleming stopping boundary. Under the alternatives assumed in the power calculation, there is a 26.2% chance of early stopping for efficacy and the overall power is 78%. If the study doesn’t stop at the interim analysis, the comparison of the primary endpoint at the final analysis is adjusted for the fact that the interim comparison is not statistically significant at the 0.0054 level.

Sub-Group Analyses

[0152] Analysis is also repeated in subgroups based on site, age, gender, baseline SpC level, and other factors selected by investigators prior to unblinding data.

Tabulation of Individual participant Data

[0153] Continuous measures are summarized by mean and standard deviation or median and inter quartile range. Categorical or ordinal measures are summarized by counts and proportions.

Exploratory Analyses

[0154] For the endpoint of the outcome score on the 8-point ordinal scale, the scores at Days 8, 15, 22, 29, and 57 are compared separately between treatment and control arms using the Wilcoxon-Mann-Whitney rank test stratified by site and mechanical ventilation status. We present the distribution of the 8-point scales in two arms, separately. The contrast is summarized by the difference between: The probability that a patient in the treatment arm has a better outcome than a patient in the control arm; and the probability that a patient in the control arm has a better outcome than a patient in the treatment arm.

[0155] We further characterize the distribution of this ordinal score by the proportion of each category in two arms, separately.

[0156] We use the Mantel-Haenszel test stratified by site and mechanical ventilation status at the baseline to compare the proportion of patients on high-flow oxygen devices or noninvasive ventilation at enrollment who progress to require mechanical ventilation or ECMO, or dying, through Day 29. If the sample size is too small and sparse frequency tables are observed, the exact p-value based on hypergeometric distribution is calculated.

[0157] A two-sample t-test is used to compare days alive and not requiring mechanical ventilation or ECMO through day 29, and day 57. As a supplementary comparison, Mantel- Haenszel (MH) test stratified by site and mechanical ventilation status is also used treating death as the worst response to avoid shortened days alive and ventilator or ECMO free due to death. The same tests are used to compare the duration of mechanical ventilation, duration of ECMO, duration of noninvasive ventilation, duration of requirement for high-flow oxygen, duration of ICU stay, and duration of hospital stay. All randomized patients are compared and patients without experiencing the clinical condition of interest is assigned a zero duration in the corresponding comparison.

[0158] For prespecified sites, we also compare the cardiac troponin, n-terminal pro-BNP, D- dimer, angiotensin 1-7, angiotensin II levels and cytokine panels including tumor necrosis factor (TNF)-alpha, interleukin (IL)-lbeta, IL-6, IL-8, IL-10, interferon (IFN) gamma at baseline and Day 5 or day of discharge if before Day 5 using mixed effects repeated measurement (MMRM) regression analysis adjusting for site and mechanical ventilation status. Unstructured variance- covariance matrix is used to account for within-person correlations. The association between changes in these endpoints and the time to recovery from respiratory failure is examined. Lastly, the Fisher’s exact test is used to compare the proportion of patients who have negative nasopharyngeal swab test results for SARS-CoV-2 RNA at Day 5 or day of discharge if before Day 5.

[0159] The treatment effect by actual treatments received is examined as a supplementary analysis to the main ITT analysis. Note that all endpoints are well defined for all patients. For example, for patients not on mechanical ventilation, the duration of mechanical ventilation is zero, and the comparison includes all randomized patients.

8. References

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9. Exemplary Embodiments

[0160] Exemplary embodiments provided in accordance with the presently disclosed subject matter include, but are not limited to, the claims and the following embodiments:

1. A pharmaceutical composition comprising an angiotensin-(l-7) base, a sugar alcohol, and a pharmaceutically acceptable carrier, wherein the pH of the composition is about 5.5. 2. The pharmaceutical composition of embodiment 1, wherein the composition comprises a concentration of the angiotensin-(l-7) base effective for the treatment of a subject with an ACE2 receptor-binding coronavirus infection.

3. The pharmaceutical composition of embodiment 2, wherein the ACE2 receptor-binding coronavirus infection is a SARS-CoV or SARS-CoV-2 infection.

4. The pharmaceutical composition of any one of embodiments 1 to 3, wherein the concentration of the angiotensin-(l-7) base is about 2.5 mg/mL.

5 . The pharmaceutical composition of any one of embodiments 1 to 4, wherein the angiotensin-(l-7) base is angiotensin-(l-7) acetate.

6. The pharmaceutical composition of any one of embodiments 1 to 5, wherein the sugar alcohol is mannitol.

7. The pharmaceutical composition of embodiment 6, wherein the concentration of mannitol in the solution is about 25 mg/mL.

8. The pharmaceutical composition of embodiment 7, wherein the composition comprises 2.5 mg/mL angiotensin-(l-7) acetate and 25 mg/mL mannitol.

9. The pharmaceutical composition of any one of embodiments 1 to 8, wherein the pharmaceutically acceptable carrier is sterile water.

10. The pharmaceutical composition of any one of embodiments 1 to 9, wherein the solution is formulated for intravenous administration.

11. The pharmaceutical composition of any one of embodiments 1 to 9, wherein the solution is formulated for subcutaneous administration.

12. A method of treating a subject with an ACE2 receptor-binding coronavirus infection, the method comprising administering to the subject a pharmaceutical composition comprising an angiotensin-(l-7) base.

13. The method of embodiment 12, wherein the angiotensin-(l-7) base is angiotensin-(l-7) acetate. 14. The method of embodiment 12 or 13, wherein the ACE2 receptor-binding coronavirus infection is a SARS-CoV or SARS-CoV-2 infection.

15. The method of any one of embodiments 12 to 14, wherein the subject is a human.

16. The method of any one of embodiments 12 to 15, wherein the pharmaceutical composition is administered by continuous intravenous infusion.

17. The method of any one of embodiments 12 to 15, wherein the pharmaceutical composition is administered subcutaneously.

18 . The method of any one of embodiments 12 to 17, wherein the angiotensin-(l-7) base is administered to the subject at a concentration of from about 2.5 ng/kg/min. to about 40 ng/kg/min.

19. The method of embodiment 18, wherein the angiotensin-(l-7) base is administered to the subject at a concentration of from about 5 ng/kg/min. to about 20 ng/kg/min.

20. The method of embodiment 19, wherein the angiotensin-(l-7) base is administered to the subject at a concentration selected from the group consisting of about 5, 6, 7, 8, 9, 10, 11, 12,

13, 14, 15, 16, 17, 18, 19, and 20 ng/kg/min.

21. The method of any one of embodiments 12 to 20, wherein the pharmaceutical composition further comprises a sugar alcohol.

22. The method of embodiment 21, wherein the sugar alcohol is mannitol.

23. The method of any one of embodiments 12 to 22, wherein the pH of the pharmaceutical composition is about 5.5.

24. The method of any one of embodiments 12 to 20, wherein the pharmaceutical composition is administered to the subject for about 1, 2, 3, 4, 5, 6, or 7 days.

25. The method of any one of embodiments 12 to 24, wherein the subject has one or more symptoms of COVTD-19.

26. The method of embodiment 25, wherein the one or more symptoms of COVTD-19 comprise a symptom selected from the group consisting of dyspnea at rest or with activity, cough, fatigue, muscle weakness, body aches, joint pain, loss of smell or taste, brain fog, headaches, and insomnia.

27. The method of embodiment 25 or 26, wherein the subject has been hospitalized.

28. The method of embodiment 27, wherein the subject has suffered from respiratory failure.

29. The method of embodiment 28, wherein the respiratory failure involves acute respiratory distress syndrome (ARDS).

30. The method of embodiment 28 or 29, wherein the subject requires high-flow oxygen, non-invasive ventilation, mechanical ventilation, or extracorporeal membrane oxygenation (ECMO).

31 . The method of any one of embodiments 28 to 30, wherein the angiotensin-(l-7) base reduces the time to recovery from the respiratory failure as compared to a subject with COVTD- 19 and respiratory failure who has not been administered the pharmaceutical composition.

32. The method of any one of embodiments 12 to 24, wherein the subject is asymptomatic for COVTD-19.

33. The method of any one of embodiments 12 to 32, wherein the subject has a reduced ACE2 level and/or increased Ang II to Ang (1-7) ratio relative to a control value typical of a young, healthy adult.

34. The method of embodiment 33, wherein the subject is elderly, is male, has heart disease, has hypertension, is obese, and/or has diabetes.

35. The method of any one of embodiments 25 to 34, wherein the angiotensin-(l-7) base leads to a reduction in the one or more symptoms of COVTD-19 in the subject.

36. The method of any one of embodiments 12 to 35, wherein the angiotensin-(l-7) base leads to a reduction in p38 MAPK activity in one or more tissues of the subject.

37. The method of embodiment 36, wherein the reduction in p38 MAPK activity leads to reduced replication of the coronavirus. 38. The method of any one of embodiments 12 to 37, wherein the angiotensin-(l-7) base leads to reduced inflammation, reduced thrombosis, and/or increased vasodilation in the subject.

39. The method of any one of embodiments 12 to 38, wherein the angiotensin-(l-7) base leads to a change in the level of one or more cytokines in the subject.

40. The method of embodiment 39, wherein the one or more cytokines comprise a cytokine selected from the group consisting of tumor necrosis factor (TNF)-a, interleukin (IL)- l b, IL-6, IL-8, IL-10, and interferon (IFN)-y.

[0161] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

Claims

WHAT IS CLAIMED IS: 1. A pharmaceutical composition comprising an angiotensin-(l-7) base, a sugar alcohol, and a pharmaceutically acceptable carrier, wherein the pH of the composition is about 5.5.

2. The pharmaceutical composition of claim 1, wherein the composition comprises a concentration of the angiotensin-(l-7) base effective for the treatment of a subject with an ACE2 receptor-binding coronavirus infection.

3. The pharmaceutical composition of claim 2, wherein the ACE2 receptor- binding coronavirus infection is a SARS-CoV or SARS-CoV-2 infection.

4. The pharmaceutical composition of claim 1 , wherein the concentration of the angiotensin-(l-7) base is about 2.5 mg/mL.

5 . The pharmaceutical composition of claim 1, wherein the angiotensin-(l-7) base is angiotensin-( 1-7) acetate.

6. The pharmaceutical composition of claim 1, wherein the sugar alcohol is mannitol.

7. The pharmaceutical composition of claim 6, wherein the concentration of mannitol in the solution is about 25 mg/mL.

8. The pharmaceutical composition of claim 7, wherein the composition comprises 2.5 mg/mL angiotensin-(l-7) acetate and 25 mg/mL mannitol.

9. The pharmaceutical composition of claim 1 , wherein the pharmaceutically acceptable carrier is sterile water.

10. The pharmaceutical composition of claim 1, wherein the solution is formulated for intravenous administration.

11. The pharmaceutical composition of claim 1, wherein the solution is formulated for subcutaneous administration.

12. A method of treating a subject with an ACE2 receptor-binding coronavirus infection, the method comprising administering to the subject a pharmaceutical composition comprising an angiotensin-(l-7) base.

13. The method of claim 12, wherein the angiotensin-(l-7) base is angiotensin-(l -7) acetate.

14. The method of claim 12, wherein the ACE2 receptor-binding coronavirus infection is a SARS-CoV or SARS-CoV-2 infection.

15. The method of claim 12, wherein the subject is a human.

16. The method of claim 12, wherein the pharmaceutical composition is administered by continuous intravenous infusion.

17 . The method of claim 12, wherein the pharmaceutical composition is administered subcutaneously.

18 . The method of claim 12, wherein the angiotensin-(l-7) base is administered to the subject at a concentration of from about 2.5 ng/kg/min. to about 40 ng/kg/min.

19. The method of claim 18, wherein the angiotensin-(l-7) base is administered to the subject at a concentration of from about 5 ng/kg/min. to about 20 ng/kg/min.

20. The method of claim 19, wherein the angiotensin-(l-7) base is administered to the subject at a concentration selected from the group consisting of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 ng/kg/min.

21. The method of any one of claim 12, wherein the pharmaceutical composition further comprises a sugar alcohol.

22. The method of claim 21, wherein the sugar alcohol is mannitol.

23. The method of claim 12, wherein the pH of the pharmaceutical composition is about 5.5.

24. The method of claim 12, wherein the pharmaceutical composition is administered to the subject for about 1, 2, 3, 4, 5, 6, or 7 days.

25. The method of claim 12, wherein the subject has one or more symptoms of COVTD-19.

26. The method of claim 25, wherein the one or more symptoms of COVTD- 19 comprise a symptom selected from the group consisting of dyspnea at rest or with activity, cough, fatigue, muscle weakness, body aches, joint pain, loss of smell or taste, brain fog, headaches, and insomnia.

27. The method of claim 25, wherein the subject has been hospitalized.

28. The method of claim 27, wherein the subject has suffered from respiratory failure.

29. The method of claim 28, wherein the respiratory failure involves acute respiratory distress syndrome (ARDS).

30. The method of claim 28, wherein the subject requires high-flow oxygen, non-invasive ventilation, mechanical ventilation, or extracorporeal membrane oxygenation (ECMO).

31 . The method of claim 28, wherein the angiotensin-(l-7) base reduces the time to recovery from the respiratory failure as compared to a subject with COVID-19 and respiratory failure who has not been administered the pharmaceutical composition.

32. The method of claim 12, wherein the subject is asymptomatic for COVTD- 19.

33. The method of any claim 12, wherein the subject has a reduced ACE2 level and/or increased Ang II to Ang (1-7) ratio relative to a control value typical of a young, healthy adult.

34. The method of claim 33, wherein the subject is elderly, is male, has heart disease, has hypertension, is obese, and/or has diabetes.

35. The method of claim 25, wherein the angiotensin-(l-7) base leads to a reduction in the one or more symptoms of COVTD-19 in the subject.

36. The method of claim 12, wherein the angiotensin-(l-7) base leads to a reduction in p38 MAPK activity in one or more tissues of the subject.

37. The method of claim 36, wherein the reduction in p38 MAPK activity leads to reduced replication of the coronavirus.

38. The method of claim 12, wherein the angiotensin-(l-7) base leads to reduced inflammation, reduced thrombosis, and/or increased vasodilation in the subject.

39. The method of claim 12, wherein the angiotensin-(l-7) base leads to a change in the level of one or more cytokines in the subject.

40. The method of claim 39, wherein the one or more cytokines comprise a cytokine selected from the group consisting of tumor necrosis factor (TNF)-a, interleukin (IL)- 1 b, IL-6, IL-8, IL- 10, and interferon (IFN)-y.