METHODS FOR TREATING ANGIOEDEMA

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/701,294, filed July 20, 2018; and U.S. Provisional Application No. 62/713,855, filed August 2, 2018; each of which is incorporated by reference herein in its entirety.

BACKGROUND

Angioedema is the swelling of deep dermis, subcutaneous, or submucosal tissue due to vascular leakage. The swelling may occur in the face, tongue, larynx, abdomen, or arms and legs. Angioedema of the throat, tongue or lungs can block or restrict the airways, causing difficulty breathing, which may become life threatening.

Angioedema is classified as either hereditary or acquired. Acquired angioedema (AAE) can be immunologic, nonimmunologic, or idiopathic. It is usually caused by allergy and occurs with other allergic symptoms and urticaria. It can also occur as a side effect to certain medications, particularly angiotensin converting enzyme (ACE) inhibitors.

Hereditary angioedema (HAE) exists in three forms, all of which are caused by a genetic mutation inherited in an autosomal dominant form. Types I and II are caused by mutations in the SERPING1 gene, which result in either diminished levels of the Cl -inhibitor protein (type I HAE) or dysfunctional forms of the same protein (type II HAE). Type III HAE has been linked with mutations in the F 12 gene, which encodes the coagulation protein factor XII.

The underlying mechanism of angioedema typically involves histamine or bradykinin. Histamine related angioedema is usually due to an allergic reaction to agents such as insect bites, foods, or medications. It can be treated with antihistamines,

corticosteroids, and/or epinephrine. On the other hand, angioedema related to bradykinin may be caused by an inherited problem known as Cl esterase inhibitor deficiency, medications known as ACE inhibitors, or a lymphoproliferative disorder. For bradykinin related angioedema, a Cl esterase inhibitor, ecallantide (plasma kallikrein inhibitor), or icatibant (bradykinin B2 receptor antagonist) may be used. In an emergency, fresh frozen plasma may also be used. However, current studies have not demonstrated consistent benefit of bradykinin antagonists, kallikrein inhibitor, and Cl inhibitor to patients with ACE inhibitor-induced angioedema. The efficacy and mechanism of action of standard pharmacotherapy such as corticosteroids and antihistamines in treatment of ACE inhibitor-

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B5015329.1 WO 2020/018785 Atty. IpCT/US2019/042393>325 induced angioedema also warrants further study. Current therapies are not particularly effective for ACE inhibitor induced angioedema, likely because they only impact one protein where there are probably multiple proteins involved. Accordingly, despite the variety of current treatment options, there remains a great need to develop more effective treatments of angioedema, especially of ACE inhibitor-induced angioedema or angioedema due to acquired Cl inhibitor deficiency.

SUMMARY

The present disclosure features methods of treating angioedema in a subject. The methods include administering a composition containing an angiotensin therapeutic agent at an initial rate to the subject. In some embodiments, the angioedema is angiotensin converting enzyme (ACE) inhibitor induced angioedema. In other embodiments, the angioedema is due to acquired Cl inhibitor deficiency.

In some embodiments, the angiotensin therapeutic agent is angiotensinogen, angiotensin I, angiotensin II, angiotensin III, or angiotensin IV. In certain embodiments, the angiotensin therapeutic agent is angiotensinogen, l-L-aspartate-5-L-valine angiotensin I, l-L- asparagine-5-L-valine angiotensin I, l-L-asparagine-5-L-isoleucine angiotensin I, l-L- aspartate-5-L-isoleucine angiotensin I, l-L-aspartate-5-L-valine angiotensin II, l-L- asparagine-5-L-valine angiotensin II, l-L-asparagine-5-L-isoleucine angiotensin II, l-L- aspartate-5-L-isoleucine angiotensin II, 4-L-valine angiotensin III, 4-L-isoleucine

angiotensin III, 3-L-valine angiotensin IV, or 3-L-isoleucine angiotensin IV. In certain embodiments, the angiotensin therapeutic agent is l-L-aspartate-5-L-isoleucine angiotensin I, l-L-aspartate-5-L-isoleucine angiotensin II, 4-L-isoleucine angiotensin III, or 3-L- isoleucine angiotensin IV.

In some embodiments, the angiotensin therapeutic agent is a peptide or protein, and the N-terminus of the peptide or protein has the amino acid sequence set forth in any one of SEQ ID NO: 1-26 or 28. In some embodiments, the peptide or protein has at least about 95% sequence homology with the amino acid sequence set forth in SEQ ID NO:27.

In some embodiments, the methods further comprise determining whether the subject has been taking an ACE inhibitor prior to administration of the composition to the subject. If the subject has been taking an ACE inhibitor, the subject is treated with a composition comprising an angiotensin therapeutic agent, and the angiotensin therapeutic agent is angiotensin II, angiotensin III, or angiotensin IV. In certain such embodiments,

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B5015329.1 WO 2020/018785 Atty. IpCT/US2019/042393>325 these methods further comprise administering an agent that decreases the level or inhibits the activity of an ACE substrate, as discussed in greater detail below.

In some embodiments, the methods further comprise determining whether the subject has acquired Cl inhibitor deficiency prior to administration of the composition to the subject. If the subject has acquired Cl inhibitor deficiency, the subject is treated with a composition comprising an angiotensin therapeutic agent, and the angiotensin therapeutic agent is angiotensin II, angiotensin III, or angiotensin IV. In certain such embodiments, these methods further comprise administering an agent that decreases the level or inhibits the activity of an ACE substrate, as discussed in greater detail below. In some

embodiments, the angiotensin therapeutic agent is administered at a rate of at least about 0.5 ng/kg/min ( e.g ., at least about 1.25 ng/kg/min or at least about 2.5 ng/kg/min). In certain embodiments, the angiotensin therapeutic agent is administered at a rate of from about 0.5 ng/kg/min to about 20 ng/kg/min (e.g., from about 1.25 ng/kg/min to about 10 ng/kg/min, or from about 1.25 ng/kg/min to about 5 ng/kg/min).

In some embodiments, the angiotensin therapeutic agent (e.g, angiotensin I or angiotensin II) is administered at an initial rate of at least about 0.5 ng/kg/min or at least about 1.25 ng/kg/min (e.g, about 2.5 ng/kg/min, about 5 ng/kg/min or about 10 ng/kg/min). In some embodiments, the angiotensin therapeutic agent (e.g, angiotensinogen, angiotensin III, or angiotensin IV) is administered at an initial rate of at least about 2.5 ng/kg/min (e.g, about 5 ng/kg/min or about 10 ng/kg/min). Preferably, the initial rate is no more than 15 ng/kg/min, more preferably no more than 10 ng/kg/min, or even no more than 5 ng/kg/min.

In some embodiments, the method also includes increasing the rate at which the angiotensin therapeutic agent is administered. In some embodiments, the rate at which the angiotensin therapeutic agent (e.g, angiotensin I or angiotensin II) is administered is increased to a final rate of less than or equal to 15 ng/kg/min. In some embodiments, the rate at which the angiotensin therapeutic agent (e.g, angiotensinogen, angiotensin III, or angiotensin IV) is administered is increased to a final rate of less than or equal to 20 ng/kg/min. In certain embodiments, the rate at which the angiotensin therapeutic agent is administered is increased over the course of no more than six hours.

In some embodiments, the method also includes decreasing the rate at which the angiotensin therapeutic agent is administered. In some embodiments, the rate at which the angiotensin therapeutic agent (e.g, angiotensin I or angiotensin II) is administered is decreased to a final rate of less than or equal to 0.5 ng/kg/min. In some embodiments, the

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B5015329.1 WO 2020/018785 Atty. IpCT/US2019/042393>325 rate at which the angiotensin therapeutic agent ( e.g ., angiotensin I or angiotensin II) is administered is decreased to a final rate of less than or equal to 2 ng/kg/min (e.g., about 0.5 or 1.25 ng/kg/min). In some embodiments, the rate at which the angiotensin therapeutic agent (e.g, angiotensinogen, angiotensin III, or angiotensin IV) is administered is decreased to a final rate of less than or equal to 4 ng/kg/min (e.g, about 0.5 or 1.25 ng/kg/min). In certain embodiments, the angiotensin therapeutic agent (e.g, angiotensin I or angiotensin II) is administered at an initial rate of about 2.5 ng/kg/min, and the rate is further decreased to about 1.25 ng/kg/min. In certain embodiments, the rate at which the angiotensin therapeutic agent is administered is decreased over the course of no more than six hours.

In some embodiments, the composition is administered continuously for at least 1-2 hours. In some embodiments, the composition is administered continuously over a period of time selected from less than 6 hours; from 6 hours to 12 hours; or at least 12 hours. In some embodiments, the composition is administered continuously for up to 24 hours.

In some embodiments, administering comprises parenteral administration, e.g, injection, subcutaneous infusion, intravenous infusion, peripheral administration, or pump administration.

In some embodiments, the angiotensin therapeutic agent (e.g, angiotensin II) is administered as a subcutaneous infusion over a period of time (e.g, via a pump), or as a bolus dose by subcutaneous injection. In certain embodiments, the angiotensin therapeutic agent (e.g, angiotensin II) is administered subcutaneously every 2-6 hours, e.g, about 0.5 mg to about 1.5 mg per injection.

In preferred embodiments, the subject is human.

In some embodiments, especially for treating ACE inhibitor-induced angioedema or angioedema due to acquired Cl inhibitor deficiency, the methods provided herein further comprise conjointly administering to the subject an agent that decreases the level or inhibits the activity of an ACE substrate (e.g, bradykinin, angiotensin I, or angiotensin-(l-7)). The agent may be, for example, a peptide, a small molecule, an inhibitory nucleic acid, a nucleic acid therapeutic (e.g, an mRNA therapeutic), or an antibody.

In some embodiments, the agent increases the level or activity of angiotensin converting enzyme (ACE). In some embodiments, the agent is angiotensin converting enzyme (ACE). In some other embodiments, the agent is a nucleic acid molecule encoding ACE. In certain specific embodiments, the agent is a vector comprising a nucleic acid molecule encoding ACE. In some embodiments, the ACE comprises an amino acid

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B5015329.1 WO 2020/018785 Atty. IpCT/US2019/042393>325 sequence having at least 70% identity to the ACE amino acid sequence of any one of SEQ ID NOs: 29-31. In some embodiments, the ACE comprises an amino acid sequence having at least 90% identity to the ACE amino acid sequence of any one of SEQ ID NOs: 29-31. In some preferred embodiments, the ACE comprises an amino acid sequence identical to the ACE amino acid sequence of any one of SEQ ID NOs: 29-31. In some embodiments, the ACE comprises at least one catalytic domain. The agent that increases the level or activity of ACE may also be, for example, a peptide or a small molecule.

In some embodiments, the agent increases the level or activity of angiotensin converting enzyme-2 (ACE-2). In some embodiments, the agent is angiotensin converting enzyme-2 (ACE-2). In some other embodiments, the agent is a nucleic acid molecule encoding ACE-2. In certain specific embodiments, the agent is a vector comprising a nucleic acid molecule encoding ACE-2. In some embodiments, the ACE-2 comprises an amino acid sequence having at least 70% identity to the ACE-2 amino acid sequence of SEQ ID NO: 32. In some embodiments, the ACE-2 comprises an amino acid sequence having at least 90% identity to the ACE-2 amino acid sequence of SEQ ID NO: 32. In some preferred embodiments, the ACE-2 comprises an amino acid sequence identical to the ACE amino acid sequence of SEQ ID NO: 32. In some embodiments, the ACE-2 comprises at least one catalytic domain. The agent that increases the level or activity of ACE-2 may also be, for example, a peptide or a small molecule.

In some embodiments, the agent is an agent that inhibits the activity or expression of renin. The agent that inhibits the activity or expression of renin may be, for example, a peptide, a small molecule, or an inhibitory nucleic acid ( e.g ., an antisense oligonucleotide, CRISPR single-guide RNA (sgRNA), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwi-interacting RNA (piRNA)). Exemplary agents that inhibit the activity or expression of renin include aliskiren, pepstatin, CGP2928, remikiren, enalkiren, TAK-272, VTP-27999, ciprokiren, ditekiren, A-65317, A-74273, CP- 80794, CGP-38560, zankiren, and a derivative thereof. In certain embodiments, the agent is an antibody, or an antigen-binding fragment thereof, which specifically binds to renin and/or a substrate of renin.

The angiotensin therapeutic agent (e.g., angiotensin II) may be administered before, after, or concurrently with the agent that decreases the level or inhibits the activity of an ACE substrate.

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BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A - 1B show the proposed mechanism of angiotensin metabolism during ACE inhibition and with the addition of exogenous angiotensin II. FIG. 1 A shows that when ACE is inhibited by ACE inhibitors or during vasodilatory shock, bradykinin, angiotensin I, and angiotensin-(l-7) increase. Angiotensin-(l-7) has effects that are the opposite of angiotensin II. Both angiotensin-(l-7) and bradykinin are vasodilatory, and they may build up when ACE is not functional, compounding the issue of angiotensin II insufficiency. FIG. 1B shows that the addition of exogenous angiotensin II provides a direct benefit by ameliorating the angiotensin II insufficiency. In addition, it may also provide benefit via biofeedback, and reduce vasodilatory angiotensins and bradykinin.

PET ATT ED DESCRIPTION

Disclosed herein are methods of treating angioedema in a subject by administering a composition containing an angiotensin therapeutic agent at an initial rate to the subject. The angiotensin therapeutic agent may be, for example, angiotensinogen, angiotensin I, angiotensin II, angiotensin III, angiotensin IV, or a peptide or protein comprising the sequence set forth in any one of SEQ ID NO: 1-28. The angiotensin therapeutic agent may be formulated as a pharmaceutically acceptable salt. The methods disclosed herein may use any suitable form or analog of angiotensinogen, angiotensin I, angiotensin II, angiotensin III, or angiotensin IV that exhibits the desired therapeutic effect in the subject.

Administering the composition containing an angiotensin therapeutic agent may comprise parenteral administration, such as injection ( e.g ., subcutaneous injection), subcutaneous infusion, peripheral administration, pump administration, or intravenous infusion. The angiotensin therapeutic agent described herein may be conjointly administered with an agent that decreases the level or inhibits the activity of an ACE substrate (e.g., bradykinin, angiotensin I, or angiotensin-(l-7)) to treat a subject with angioedema (e.g, ACE inhibitor- induced angioedema).

I. DEFINITIONS

For convenience, certain terms employed in the specification, and appended claims are collected here.

The term“comprise” or variations such as“comprises” or“comprising” will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).

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The singular forms“a,”“an,” and“the” include the plurals unless the context clearly dictates otherwise.

The term“including” is used to mean“including but not limited to.”“Including” and“including but not limited to” are used interchangeably.

The term“subject” refers to either a human or a non-human animal. This term includes mammals such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice, rabbits and rats).

The term“angiotensin therapeutic agent”, without further specificity, in this disclosure refers to angiotensinogen, angiotensin I, angiotensin II, angiotensin III, and/or angiotensin IV and analogs and mixtures thereof.

“About” and“approximately” 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%, preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms“about” and“approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term“about” or“approximately” can be inferred when not expressly stated.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term“pharmaceutically acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of a therapeutically effective substance (e.g., angiotensin II) of this disclosure. These salts can be prepared in situ during the final isolation and purification of the respiration uncoupling agents, or by separately reacting a purified respiration uncoupling agent in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate

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B5015329.1 WO 2020/018785 Atty. IpCT/US2019/042393>325 salts and the like (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66: 1-19).

The phrase "pharmaceutically acceptable carrier" as used herein means a

pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be

"acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as

pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and

suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

“Treating” a disease in a subject or“treating” a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is decreased or prevented from worsening.

As used herein, a therapeutic that "prevents" a condition (e.g., angioedema) refers to a composition that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

As used herein, the term“conjoint administration” or“conjointly administered” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the subject, which may include synergistic effects of the two agents). For example, the different therapeutic agents can be administered either in the same formulation or in separate formulations, either

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B5015329.1 WO 2020/018785 Atty. IpCT/US2019/042393>325 concomitantly or sequentially. Thus, a subject who receives such treatment can benefit from a combined effect of different therapeutic agents.

Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature and techniques relating to chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry, described herein, are those well-known and commonly used in the art.

II COMPOSITIONS

Angiotensin Therapeutics

Angiotensinogen, angiotensin I, angiotensin II, angiotensin III, and angiotensin IV are hormones naturally produced by the body that regulate blood pressure via

vasoconstriction and sodium reabsorption. Angiotensinogen is a polypeptide produced in the liver that is converted into angiotensin I by renin. Subsequently, angiotensin I may be cleaved and converted to angiotensin II by angiotensin converting enzyme (ACE).

Angiotensin II is converted to angiotensin III through the removal of an N-terminal aspartate of angiotensin II by aminopeptidase A (APA). Angiotensin III is converted to angiotensin IV by the removal of an N-terminal arginine by aminopeptidase N.

Hemodynamic effects of angiotensin II administration have been evaluated in numerous clinical studies, demonstrating significant effects on systemic and renal blood flow

(Harrison-Bemard, L.M., The renal renin-angiotensin system. Adv Physiol Educ, (2009) 33(4):270-74). In addition to its systemic effects, angiotensin therapeutic agents display a pronounced effect on the efferent arterioles of the kidney, maintaining glomerular filtration when blood flow is decreased. Angiotensin II also regulates sodium reabsorption in the kidney by stimulating Na ⁺ /H ⁺ exchangers in the proximal tubule and inducing the release of aldosterone and vasopressin (Harrison-Bemard, L.M., The renal renin-angiotensin system. Adv Physiol Educ, (2009) 33(4):270-4.).

The angiotensin therapeutic agent that may be used in the compositions and methods of this disclosure may be Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 1) also called 5-isoleucine angiotensin II, 5-L-isoleucine angiotensin II, l-aspartate-5-isoleucine angiotensin II, and l-L-aspartate-5-L-isoleucine angiotensin II. SEQ ID NO: l is an octa- peptide naturally present in humans and other species, such as equines, hogs, etc.

Isoleucine may be substituted by valine to result in 5-L-valine angiotensin II, Asp-Arg-Val-

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Tyr-Val-His-Pro-Phe (SEQ ID NO:2). Other angiotensin II analogs such as [Asn '-Phe ⁴ ]- angiotensin II (SEQ ID NO:3), nonapeptide Asn-Arg-Val-Tyr-Tyr-Val-His-Pro-Phe (SEQ ID NO:4), [Asn ¹ -He ⁵ -Ile ⁸ ]-angiotensin II (SEQ ID NO:5), [Asn ¹ -He ⁵ -Ala ⁸ ]-angiotensin II (SEQ ID NO:6), and [Asn ¹ -diiodoTyr ⁴ -Ile ⁵ ]-angiotensin II (SEQ ID NO:7) may also be used. Angiotensin II may be synthesized, for example, by solid phase peptide synthesis to incorporate modifications, such as C-terminal amidation, N-terminal acetylation, or diiodo- tyrosine. The term“angiotensin II,” without further specificity, is intended to refer to any of these various forms, as well as combinations thereof.

In some embodiments, the angiotensin therapeutic agent may be selected from 5-L- valine angiotensin II, 5-L-valine angiotensin II amide, 5-L-isoleucine angiotensin II, and 5- L-isoleucine angiotensin II amide, or a pharmaceutically acceptable salt thereof, preferably manufactured under current good manufacturing conditions (cGMP). In some

embodiments, the composition may include different forms of angiotensin II in different percentages, e.g ., a mixture 5-L-valine angiotensin II and 5-L-isoleucine angiotensin II. In some embodiments, the composition includes a mixture of angiotensinogen, angiotensin I, angiotensin II, angiotensin, III, and/or angiotensin IV. For example, the composition may include a mixture of different forms of angiotensinogen, angiotensin I, angiotensin II, angiotensin III, and/or angiotensin IV at varying percentages. The composition comprising the angiotensin therapeutic agent may be suitable for parenteral administration, e.g. , for injection or intravenous infusion.

An angiotensin III therapeutic that may be used in the compositions and methods of this disclosure may be Arg-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO:8). SEQ ID NO:8 is a hepta-peptide naturally present in humans and other species, such as equines, hogs, etc. Isoleucine may be substituted by valine to result in Arg-Val-Tyr-Val-His-Pro-Phe (SEQ ID NO:9). Other angiotensin III analogs such as [Phe ³ ]-angiotensin III (SEQ ID NO: 10), [Ile ⁴ - Ala ⁷ ]-angiotensin III (SEQ ID NO: 11), and [diiodoTyr ³ -Ile ⁴ ]-angiotensin III (SEQ ID NO: 12) may also be used. Angiotensin III may be synthesized, for example, by solid phase peptide synthesis to incorporate modifications, such as C-terminal amidation, N-terminal acetylation, or diiodo-tyrosine. The term“angiotensin III,” without further specificity, is intended to refer to any of these various forms, as well as combinations thereof.

In some embodiments, the angiotensin therapeutic agent may be selected from 4-L- valine angiotensin III, 4-L-valine angiotensin III amide, 4-L-isoleucine angiotensin III, and

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4-L-isoleucine angiotensin III amide, or a pharmaceutically acceptable salt thereof, preferably manufactured under current good manufacturing conditions (cGMP).

Angiotensin IV is a metabolite of angiotensin III. An angiotensin IV therapeutic that may be used in the compositions and methods of this disclosure may be Val-Tyr-Ile- His-Pro-Phe (SEQ ID NO: 13). SEQ ID NO: 13 is a hexa-peptide naturally present in humans and other species, such as equines, hogs, etc. Isoleucine may be substituted by valine to result in Val-Tyr-Val-His-Pro-Phe (SEQ ID NO: 14). Other angiotensin IV analogs such as [Phe ² ] -angiotensin IV (SEQ ID NO: 15), [Ile ³ -Ala ⁶ ]-angiotensin IV (SEQ ID NO: 16), and [diiodoTyr ² -Ile ³ ]-angiotensin IV (SEQ ID NO: 17) may also be used.

Angiotensin IV may be synthesized, for example, by solid phase peptide synthesis to incorporate modifications, such as C-terminal amidation, N-terminal acetylation, or diiodo- tyrosine. The term“angiotensin IV,” without further specificity, is intended to refer to any of these various forms, as well as combinations thereof.

In some embodiments, a composition comprising angiotensin IV may be selected from 3-L-valine angiotensin IV, 3-L-valine angiotensin IV amide, 3-L-isoleucine angiotensin IV, and 3-L-isoleucine angiotensin IV amide, or a pharmaceutically acceptable salt thereof, preferably manufactured under current good manufacturing conditions

(cGMP).

The angiotensin therapeutic agent that may be used in the compositions and methods of this disclosure may be Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu (SEQ ID NO: 18) also called 5-L-isoleucine angiotensin I. SEQ ID NO: 18 is an deca-peptide naturally present in humans and other species, such as equines, hogs, etc. Isoleucine may be substituted by valine to result in 5-L-valine angiotensin I, Asp-Arg-Val-Tyr-Val-His- Pro-Phe-His-Leu (SEQ ID NO: 19). Other angiotensin I analogs such as [Asn'-Phe ⁴ ]- angiotensin I (SEQ ID NO:20), nona-peptide Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu (SEQ ID NO:2l), octa-peptide Val-Tyr-Ile-His-Pro-Phe-His-Leu (SEQ ID NO:22), [Asn ¹ ]- angiotensin I Asn-Arg-Val-Tyr-Tyr-Val-His-Pro-Phe-His-Leu (SEQ ID NO:23), [Asn'-He ⁵ - Ile ⁸ ]-angiotensin I (SEQ ID NO:24), [Asn ¹ -He ⁵ -Ala ⁸ ]-angiotensin I (SEQ ID NO:25), and [Asn ¹ -diiodoTyr ⁴ -Ile ⁵ ]-angiotensin I (SEQ ID NO:26) may also be used. Angiotensin I may be synthesized, for example, by solid phase peptide synthesis to incorporate modifications, such as C-terminal amidation, N-terminal acetylation, or diiodo-tyrosine.

The term“angiotensin I,” without further specificity, is intended to refer to any of these various forms, as well as combinations thereof.

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An angiotensin therapeutic may be used as any suitable salt, deprotected form, acetylated form, deacetylated form, and/or prodrug form of the above-mentioned peptides, including pegylated forms of the peptides or conjugates as disclosed in US Patent

Publication 2011/0081371 (hereby incorporated by reference in its entirety). The term "prodrug" refers to any precursor compound which is able to generate or to release the above-mentioned peptide under physiological conditions. Such prodrugs may be larger peptides which are selectively cleaved in order to form the peptide of the disclosure. For example, in some embodiments, the prodrug may be angiotensinogen, angiotensin I, or its analogs that may result in the production of angiotensin II by the action of certain endogenous or exogenous enzymes. In another example, the prodrug may be angiotensin II, angiotensin III, or homologs thereof that may result in angiotensin III and angiotensin IV, respectively. Further prodrugs include peptides with protected amino acids, e.g ., having protecting groups at one or more carboxylic acid and/or amino groups. Suitable protecting groups for amino groups are the benzyloxycarbonyl, t-butyloxycarbonyl (BOC), fluorenylmethyloxycarbonyl (FMOC), formyl, and acetyl or acyl group. Suitable protecting groups for the carboxylic acid group are esters such as benzyl esters or t-butyl esters. The present disclosure also contemplates the use of angiotensinogen, angiotensin I, angiotensin II, angiotensin III, and/or angiotensin IV and/or precursor peptides having amino acid substitutions, deletions, additions, the substitutions and additions including the standard D and L amino acids and modified amino acids, such as, for example, amidated and acetylated amino acids, wherein the therapeutic activity of the base peptide sequence is maintained at a pharmacologically useful level.

In some embodiments, the angiotensin therapeutic agent is a peptide or protein, wherein the N-terminus of the peptide or protein consists of the amino acid sequence set forth in any one of SEQ ID NO: 1-26. In preferred embodiments, the N-terminus of the peptide or protein consists of the amino acid sequence set forth in SEQ ID NO: 18. In more preferred embodiments, the N-terminus of the peptide or protein consists of the amino acid sequence set forth in SEQ ID NO:28 (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile). In certain preferred embodiments, the peptide or protein has at least 95% sequence homology with the sequence set forth in SEQ ID NO:27, which corresponds to human angiotensinogen.

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SEQ ID NO:27 (Angiotensinogen, Homo sapiens ; GenBank: AAA51679.1)

MRKRAPQSEMAPAGVSLRAT I LCLLAWAGLAAGDRVYIHPFHLVIHNES TCEQLAKANAGK PKDPT FI PAP I QAKTS PVDEKALQDQLVLVAAKLDTEDKLRAAMVGMLANFLGFRI YGMHS ELWGWHGATVLS PTAVFGTLASLYLGALDHTADRLQAI LGVPWKDKNCTSRLDAHKVLSA LQAVQGLLVAQGRADSQAQLLLS TWGVFTAPGLHLKQPFVQGLALYTPWLPRSLDFTEL DVAAEKI DRFMQAVTGWKTGCSLMGASVDS TLAFNTYVHFQGKMKGFSLLAEPQE FWVDNS TSVSVPMLSGMGT FQHWSDI QDNFSVTEVPFTESACLLL I QPHYASDLDKVEGLT FQQNSL NWMKKLS PRT IHLTMPQLVLQGSYDLQDLLAQAELPAI LHTELNLQKLSNDRIRVGEVLNS I FFELEADEREPTES TQQLNKPEVLEVTLNRPFLFAVYDQSATALHFLGRVANPLS TA

For example, the peptide or protein may have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology with the sequence set forth in SEQ ID NO:27. The N-terminus of the peptide or protein may consist of the amino acid sequence set forth in any one of SEQ ID NO: 1-26 or SEQ ID NO:28, and the peptide or protein may have at least 95%, 96%, 97%, 98%, or 99% sequence homology with the sequence set forth in SEQ ID NO:27. The peptide or protein may be longer or shorter than the sequence set forth in SEQ ID NO:27, such as about 10 amino acids to about 2000 amino acids in length, about 100 to about 2000, about 100 to about 1500, about 100 to about 1000, about 200 to about 2000, about 200 to about 1500, about 200 to about 1000, about 500 to about 2000, about 500 to about 1500, about 500 to about 1000, about 10 to about 1000 amino acids, about 10 to about 500 amino acids, about 10 to about 400 amino acids, about 10 to about 300 amino acids, about 10 to about 200 amino acids, about 10 to about 100 amino acids, about 10 to about 50 amino acids, about 20 to about 500 amino acids, about 20 to about 400 amino acids, about 20 to about 300 amino acids, about 20 to about 200 amino acids, about 20 to about 100 amino acids, about 20 to about 50 amino acids, about 25 to about 500 amino acids, about 25 to about 400 amino acids, about 25 to about 300 amino acids, about 25 to about 200 amino acids, about 25 to about 100 amino acids, or about 25 to about 50 amino acids in length. For example, the N-terminus of the peptide or protein may consist of the amino acid sequence set forth in any one of SEQ ID NO: 1-26 or SEQ ID NO:28; the peptide or protein may have at least 95%, 96%, 97%, 98%, or 99% sequence homology with the sequence set forth in SEQ ID NO:27; and the peptide or protein may be about 10 amino acids to about 2000 amino acids in length. The peptide or protein may comprise an antibody Fc fragment ( e.g ., at its C-terminus), for example, to increase the

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B5015329.1 WO 2020/018785 Atty. IpCT/US2019/042393>325 half-life of the therapeutic angiotensin agent in vivo. Such fusion proteins would be expected to have less than 10% sequence homology with SEQ ID NO:27. The peptide or protein may comprise one or more modifications, such as glycosylation and/or pegylation, e.g ., which may result in more favorable pharmacokinetics and/or pharmacodynamics in certain subjects relative to angiotensinogen, angiotensin I, angiotensin II, angiotensin III, and/or angiotensin IV.

In some embodiments, the peptide or protein has about 1% to about 1000% of the activity as angiotensin II, such as about 2% to about 500% or about 5% to about 200%. In preferred embodiments, the peptide or protein has about 10% to about 1000% of the activity as angiotensin II, such as about 20% to about 500% or about 30% to about 300%. Activity refers to the ability of the angiotensin therapeutic agent to treat angioedema of a subject. For example, an angiotensin therapeutic agent that requires an administration rate that is 3 times greater than for angiotensin II, by weight, to treat angioedema of a subject by the same amount has about 33% of the activity of angiotensin II. The molecular weight of angiotensinogen is approximately 60 times the molecular weight of angiotensin II, for example, and thus, the activity of angiotensinogen is approximately 2% that of angiotensin II. A fusion peptide consisting of angiotensin II and the Fc fragment of an antibody has a similar molecular weight as angiotensinogen, and such a fusion peptide would be expected to display an activity greater than about 2% for fusions that display favorable

pharmacokinetics.

The composition may be formulated with varying concentrations of the angiotensin therapeutic agent. In certain embodiments, the composition comprises angiotensin I or angiotensin II at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 25, 30, 35,

40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180,

190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,

370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 600, 650, 700,

750, 800, 850, 900, 950, 1000 pg/ml. In certain embodiments, the composition comprises angiotensin III at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 25, 30, 35,

40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180,

190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,

370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 600, 650, 700,

750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or

2000 pg/ml. In some embodiments, the composition comprises angiotensin IV or

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B5015329.1 WO 2020/018785 Atty. IpCT/US2019/042393>325 angiotensinogen at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500,

1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 pg/ml. In other embodiments, the composition comprises the angiotensin therapeutic agent ( e.g ., angiotensin II) at a concentration of about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,

2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0,

10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0,

18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, or 25.0 mg/ml. In certain preferred embodiments, the composition comprises angiotensin II at a concentration of about 2.5 mg/mL.

Excipients

The pharmaceutical compositions of the present disclosure may also contain diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.

One of skill in the art would appreciate that the choice of any one excipient may influence the choice of any other excipient. For example, the choice of a particular excipient may preclude the use of one or more additional excipients because the

combination of excipients would produce undesirable effects. Methods of formulating the compositions of the present disclosure are described in the art, e.g., Patent No: US

9,220,745 B2, which is incorporated by reference herein in its entirety.

Additional pharmaceutical agents

The composition including the angiotensin therapeutic agent may further include one or more additional pharmaceutical agents. For example, the angiotensin therapeutic agent may be administered with agents such as albumin, clonidine, and/or a beta blocker. The quantity of the additional pharmaceutical agent administered may vary depending on the cumulative therapeutic effect of the treatment including the angiotensin therapeutic agent and the additional pharmaceutical agent. For example, the quantity of albumin administered may be 1 gram of albumin per kilogram of body weight given intravenously on the first day, followed by 20 to 40 grams daily. In some embodiments, antihistamines, corticosteroids, and/or epinephrine may also be used in combination with the angiotensin

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B5015329.1 WO 2020/018785 Atty. IpCT/US2019/042393>325 therapeutic. A Cl esterase inhibitor, a kallikrein inhibitor ( e.g ., ecallantide), or an antagonist to bradykinin or its receptor (e.g., icatibant) may also be used in combination with the angiotensin therapeutic agent to treat a subject with angioedema.

Studies have shown that ACE inhibition with captopril and other ACE inhibitors cause increases in bradykinin, Angiotensin I and other angiotensins, such as angiotensin-(l- 7) (Luque et al. (1996) J Hypertens 14:799-805). Angiotensin-(l-7) has effects that are the opposite of angiotensin II, and angiotensin-(l-7) has been shown to cause vasodilation and to decrease blood pressure (Ferrario et al. (1991) Hypertension 18:111126-133) (FIG. 1A). Similarly, bradykinin, an ACE substrate, has vasodilatory properties (Cherry et al. (1982) Proc Natl Acad Sci USA 1982:2106-2110; Cockcroft et al. (1994) Br J Clin Pharmacol 38:317-321; Margolius et al. (1996) Diabetes 45:Sl4-Sl9). These data indicate that patients with ACE inhibitor associated angioedema may suffer both an excess of vasodilatory mediators that are normally metabolized by ACE and an angiotensin II insufficiency. The addition of exogenous angiotensin II in this subset of patients may provide a dual benefit by ameliorating the angiotensin II insufficiency thereby improving blood pressure and may also prove beneficial by biofeedback resulting in less vasodilatory angiotensins. The decrease in vasodilatory angiotensins, which are also ACE substrates, may improve ACE availability and increase bradykinin degradation (FIG. 1B). Therefore, exogenous angiotensin II may treat angioedema, especially ACE inhibitor associated angioedema, by decreasing the levels of vasodilatory angiotensins and bradykinin. Moreover, agents that decrease vasodilatory ACE substrates may be combined with exogenous angiotensin II to further increase the therapeutic effects.

In some embodiments, particularly for treating ACE inhibitor-induced angioedema or angioedema due to acquired Cl inhibitor deficiency, the angiotensin therapeutic agent described herein may be administered conjointly with an agent that decreases the level or inhibits the activity of an ACE substrate (e.g, Bradykinin, Angiotensin I, or Angiotensin- (1-7)) to treat a subject with angioedema (e.g, ACE inhibitor-induced angioedema). For example, the angiotensin therapeutic agent may be administered conjointly with an agent that increases the level or activity of angiotensin converting enzyme (ACE). ACE is an enzyme involved in catalyzing the conversion of angiotensin I into a physiologically active peptide angiotensin II. Angiotensin II is a potent vasopressor and aldosterone-stimulating peptide that controls blood pressure and fluid-electrolyte balance. This enzyme plays a key role in the renin-angiotensin system. ACE can also inactivate the vasodilator,

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B5015329.1 WO 2020/018785 Atty. IpCT/US2019/042393>325 proinflammatory peptide, bradykinin. The term“angiotensin converting enzyme (ACE)” is intended to include fragments, variants ( e.g ., allelic variants), and derivatives thereof. Representative cDNA and protein sequences of angiotensin converting enzyme (ACE) orthologs are well-known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). Representative protein sequences of human angiotensin converting enzyme (ACE) isoforms are presented below.

SEQ ID NO: 29 _ Human ACE Isoform 1 Amino Acid Sequence (NP 002280.1)

1 mgaasgrrgp glllplplll llppqpalal dpglqpgnfs adeagaqlfa qsynssaeqv

61 lfqsvaaswa hdtnitaena rrqeeaalls qefaeawgqk akelyepiwq

nftdpqlrri

121 igavrtlgsa nlplakrqqy nallsnmsri ystakvclpn ktatcwsldp

dltnilassr

181 syamllfawe gwhnaagipl kplyedftal sneaykqdgf tdtgaywrsw

ynsptfeddl

241 ehlyqqlepl ylnlhafvrr alhrrygdry inlrgpipah llgdmwaqsw

eniydmvvpf

301 pdkpnldvts tmlqqgwnat hmfrvaeeff tslelspmpp efwegsmlek

padgrevvch

361 asawdfynrk dfrikqctrv tmdqlstvhh emghiqyylq ykdlpvslrr

ganpgfheai

421 gdvlalsvst pehlhkigll drvtndtesd inyllkmale kiaflpfgyl

vdqwrwgvfs

481 grtppsrynf dwwylrtkyq gicppvtrne thfdagakfh vpnvtpyiry

fvs fvlqfqf

541 healckeagy egplhqcdiy rstkagaklr kvlqagssrp wqevlkdmvg

ldaldaqpll

601 kyfqpvtqwl qeqnqqngev lgwpeyqwhp plpdnypegi dlvtdeaeas

kfveeydrts

661 qvvwneyaea nwnyntnitt etskillqkn mqianhtlky gtqarkfdvn

qlqnttikri

721 ikkvqdlera alpaqeleey nkilldmett ysvatvchpn gsclqlepdl

tnvmatsrky

781 edllwawegw rdkagrailq fypkyvelin qaarlngyvd agdswrsmye

tpsleqdler

841 lfqelqplyl nlhayvrral hrhygaqhin legpipahll gnmwaqtwsn

iydlvvpfps

901 apsmdtteam lkqgwtprrm fkeaddffts lgllpvppef wnksmlekpt

dgrevvchas

961 awdfyngkdf rikqcttvnl edlvvahhem ghiqyfmqyk dlpvalrega

npgfheaigd

1021 vlalsvstpk hlhslnllss eggsdehdin flmkmaldki afipfsylvd

qwrwrvfdgs

1081 itkenynqew wslrlkyqgl cppvprtqgd fdpgakfhip ssvpyiryfv

s fiiqfqfhe

1141 alcqaaghtg plhkcdiyqs keagqrlata mklgfsrpwp eamqlitgqp

nmsasamlsy

1201 fkplldwlrt enelhgeklg wpqynwtpns arsegplpds grvsflgldl

daqqarvgqw

1261 lllflgiall vatlglsqrl fsirhrslhr hshgpqfgse velrhs

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SEQ ID NO: 30 _ Human ACE Isoform 2 Amino Acid Sequence (HP 690043.1)

1 mgqgwatagl psllflllcy ghpllvpsqe asqqvtvthg tssqattssq ttthqatahq

61 tsaqspnlvt deaeaskfve eydrtsqvvw neyaeanwny ntnittetsk

illqknmqia

121 nhtlkygtqa rkfdvnqlqn ttikriikkv qdleraalpa qeleeynkil

ldmettysva

181 tvchpngscl qlepdltnvm atsrkyedll wawegwrdka grailqfypk

yvelinqaar

241 lngyvdagds wrsmyetpsl eqdlerlfqe lqplylnlha yvrralhrhy

gaqhinlegp

301 ipahllgnmw aqtwsniydl vvpfpsapsm dtteamlkqg wtprrmfkea

ddfftslgll

361 pvppefwnks mlekptdgre vvchasawdf yngkdfrikq cttvnledlv

vahhemghiq

421 yfmqykdlpv alreganpgf heaigdvlal svstpkhlhs lnllsseggs

dehdinflmk

481 maldkiafip fsylvdqwrw rvfdgsitke nynqewwslr lkyqglcppv

prtqgdfdpg

541 akfhipssvp yiryfvsfii qfqfhealcq aaghtgplhk cdiyqskeag

qrlatamklg

601 fsrpwpeamq litgqpnmsa samlsyfkpl ldwlrtenel hgeklgwpqy

nwtpnsarse

661 gplpdsgrvs flgldldaqq arvgqwlllf lgiallvatl glsqrlfsir

hrslhrhshg

721 pqfgsevelr hs

SEQ ID NO: 31 _ Human ACE Isoform 3 Amino Acid Sequence (NP 0017528.1)

1 mgqgwatagl psllflllcy ghpllvpsqe asqqvtvthg tssqattssq ttthqatahq

61 tsaqspnlvt deaeaskfve eydrtsqvvw neyaeanwny ntnittetsk

illqknmqia

121 nhtlkygtqa rkfdvnqlqn ttikriikkv qdleraalpa qeleeynkil

ldmettysva

181 tvchpngscl qlepdltnvm atsrkyedll wawegwrdka grailqfypk

yvelinqaar

241 lngyvdagds wrsmyetpsl eqdlerlfqe lqplylnlha yvrralhrhy

gaqhinlegp

301 ipahllgnmw aqtwsniydl vvpfpsapsm dtteamlkqg wtprrmfkea

ddfftslgll

361 pvppefwnks mlekptdgre vvchasawdf yngkdfrikq cttvnledlv

vahhemghiq

421 yfmqykdlpv alreganpgf heaigdvlal svstpkhlhs lnllsseggs

dehdinflmk

481 maldkiafip fsylvdqwrw rvfdgsitke nynqewwslr lkyqglcppv

prtqgdfdpg

541 akfhipssvp yirtamklgf srpwpeamql itgqpnmsas amlsyfkpll

dwlrtenelh

601 geklgwpqyn wtpnsarseg plpdsgrvsf lgldldaqqa rvgqwlllfl

giallvatlg

661 lsqrlfsirh rslhrhshgp qfgsevelrh s

In certain embodiments, the agent that increases the level or the activity of ACE is an ACE polypeptide, or a nucleic acid molecule ( e.g an mRNA, a DNA fragment, a vector) encoding an ACE polypeptide. In some embodiments, the ACE polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,

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93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the ACE amino acid sequence of any one of SEQ ID NOs: 29-31. In some embodiments, the ACE polypeptide comprises an amino acid sequence identical to the ACE amino acid sequence of any one of SEQ ID NOs: 29-31. In some embodiments, the ACE polypeptide consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the ACE amino acid sequence of any one of SEQ ID NOs: 29-31. In some preferred embodiments, the ACE polypeptide consists of an amino acid sequence identical to the ACE amino acid sequence of any one of SEQ ID NOs: 29-31. In certain embodiments, the ACE polypeptide comprises at least one catalytic domain. In some embodiments, the ACE polypeptide may further comprises a heterologous sequence, such as a peptide tag ( e.g ., Flag-tag, GST-tag, or 6xHis tag) or a fluorescent protein (e.g., GFP) to facilitate the expression, detection, and/or purification of the ACE polypeptide. In some embodiments, the agent that increases the level or activity of ACE is a small molecule.

In some embodiments, the angiotensin therapeutic agent may be administered conjointly with an agent that increases the level or the activity of angiotensin converting enzyme 2 (ACE-2) to treat a subject with angioedema (e.g, ACE inhibitor-induced angioedema). Angiotensin converting enzyme 2 (ACE-2) belongs to the angiotensin converting enzyme family of dipeptidyl carboxydipeptidases and has considerable homology to human angiotensin converting enzyme (ACE). This secreted protein catalyzes the cleavage of angiotensin I into angiotensin-(l-9), and angiotensin II into the vasodilator angiotensin-(l-7). The organ- and cell-specific expression of ACE-2 indicates that it plays a role in the regulation of cardiovascular and renal function, as well as fertility. In addition, the encoded protein is a functional receptor for the spike glycoprotein of the human coronaviruses SARS and HCoV-NL63. The term“angiotensin converting enzyme 2 (ACE- 2)” is intended to include fragments, variants (e.g, allelic variants), and derivatives thereof. Representative cDNA and protein sequences of angiotensin converting enzyme 2 (ACE-2) orthologs are well-known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). Representative protein sequence of human angiotensin converting enzyme 2 (ACE-2) is presented below.

SEP ID NO: 32 Human ACE-2 Amino Acid Sequence (NP 068576.0

1 msssswllls lvavtaaqst ieeqaktfld kfnheaedlf yqsslaswny ntniteenvq

61 nmnnagdkws aflkeqstla qmyplqeiqn ltvklqlqal qqngssvlse

dkskrlntil

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B5015329.1 WO 2020/018785 Atty. IpCT/US2019/042393>325

121 ntmstiystg kvcnpdnpqe clllepglne imansldyne rlwaweswrs

evgkqlrply

181 eeyvvlknem aranhyedyg dywrgdyevn gvdgydysrg qliedvehtf

eeikplyehl

241 hayvraklmn aypsyispig clpahllgdm wgrfwtnlys ltvpfgqkpn

idvtdamvdq

301 awdaqrifke aekffvsvgl pnmtqgfwen smltdpgnvq kavchptawd

lgkgdfrilm

361 ctkvtmddfl tahhemghiq ydmayaaqpf llrnganegf heavgeimsl

saatpkhlks

421 igllspdfqe dneteinf11 kqaltivgtl pftymlekwr wmvfkgeipk

dqwmkkwwem

481 kreivgvvep vphdetycdp aslfhvsndy s firyytrtl yqfqfqealc

qaakhegplh

541 kcdisnstea gqklfnmlrl gksepwtlal envvgaknmn vrpllnyfep

lftwlkdqnk

601 nsfvgwstdw spyadqsikv rislksalgd kayewndnem ylfrssvaya

mrqyflkvkn

661 qmilfgeedv rvanlkpris fnffvtapkn vsdiiprtev ekairmsrsr

indafrlndn

721 sleflgiqpt lgppnqppvs iwlivfgvvm gvivvgivil iftgirdrkk

knkarsgenp

781 yasidiskge nnpgfqntdd vqts f In certain embodiments, the agent that increases the level or activity of ACE-2 is an

ACE-2 polypeptide, or a nucleic acid molecule ( e.g ., an mRNA, a DNA fragment, a vector) encoding an ACE-2 polypeptide. In some embodiments, the ACE-2 polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the ACE-2 amino acid sequence of SEQ ID NO: 32. In some embodiments, the ACE polypeptide comprises an amino acid sequence identical to the ACE-2 amino acid sequence of SEQ ID NO: 32. In some embodiments, the ACE-2 polypeptide consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the ACE-2 amino acid sequence of SEQ ID NO: 32. In some preferred embodiments, the ACE-2 polypeptide consists of an amino acid sequence identical to the ACE-2 amino acid sequence of SEQ ID NO: 32. In certain embodiments, the ACE polypeptide comprises at least one catalytic domain. In some embodiments, the ACE-2 polypeptide may further comprises a heterologous sequence, such as a peptide tag (e.g., Flag-tag, GST-tag, or 6xHis tag) or a fluorescent protein (e.g, GFP) to facilitate the expression, detection, and/or purification of the ACE-2 polypeptide. In some embodiments, the agent that increases the level or activity of ACE-2 is a small molecule.

In some embodiments, an agent that inhibits the activity or expression of Renin may be administered conjointly with the angiotensin therapeutic agent to treat a subject with angioedema (e.g, ACE inhibitor-induced angioedema). Renin, an aspartyl protease, cleaves

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B5015329.1 WO 2020/018785 Atty. IpCT/US2019/042393>325 angiotensinogen to form angiotensin I in the plasma. Renin catalyzes the first step in the activation pathway of angiotensinogen, a cascade of reactions that can result in aldosterone release, vasoconstriction, elevation of blood pressure and increased sodium retention by the kidney. The agent that inhibits the expression or activity of renin may be, for example, a peptide, a small molecule, an inhibitory nucleic acid ( e.g ., an antisense oligonucleotide, CRISPR single-guide RNA (sgRNA), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwi-interacting RNA (piRNA)), or an antibody that specifically binds to renin and/or a substrate of renin. Exemplary agents that inhibit the activity or expression of renin include aliskiren, pepstatin, CGP2928, remikiren, enalkiren, TAK-272, VTP-27999, ciprokiren, ditekiren, A-65317, A-74273, CP-80794, CGP-38560, zankiren, or a derivative thereof.

The list of additional pharmaceutical agents described above is merely illustrative and may include any other pharmaceutical agents that may be useful for the treatment of angioedema and related conditions.

III. DOSING

Dosing of the angiotensin therapeutic agent according to the invention can be initiated and adjusted according to the individual subject’s physical condition, presentation, and response to therapy. Factors such as the subject’s blood pressure, self-reported response, or results of physical examination may affect either the initial dose or rate of angiotensin administration, and changes to or titration of (increasing or decreasing) the rate of administration. For example, if a subject is administered angiotensin at an initial rate of 2.5 ng/kg/min, changes to his or her mean arterial pressure (MAP) can indicate whether or not the dose or rate should be adjusted up or down. Administration of angiotensin is likely to increase MAP; when MAP exceeds a desired level, the dose or rate of administration can be titrated down to a level where MAP is in a desired range. Alternatively or in addition, if MAP increases, it can be treated with a short acting antihypertensive like clonidine or a beta blocker in order to maintain or increase angiotensin dose or rate for therapeutic effect. The subject’s tolerance for the therapy, safety indicators, and therapeutic effect will determine dosing, rate of administration, and duration of therapy.

In certain embodiments, the angiotensin therapeutic agent is angiotensin I, and angiotensin I is administered at a rate of at least 0.5, 0.75, 1, 1.25, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 ng/kg/min. In some embodiments, angiotensin I is administered at a rate of from about 0.5 ng/kg/min to about 20 ng/kg/min, e.g., from about

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0.5 ng/kg/min to about 15 ng/kg/min, from about 1.25 ng/kg/min to about 10 ng/kg/min, or from about 1.25 ng/kg/min to about 5 ng/kg/min. For example, in some embodiments, angiotensin I is administered at a rate of about 0.5 ng/kg/min, about 1.25 ng/kg/min, about 2.5 ng/kg/min, about 5 ng/kg/min, about 7.5 ng/kg/min, or about 10 ng/kg/min. In certain embodiments, the angiotensin therapeutic agent is angiotensin II, and angiotensin II is administered at a rate of at least 0.5, 0.75, 1, 1.25, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 ng/kg/min. In some embodiments, angiotensin II is administered at a rate of from about 0.5 ng/kg/min to about 20 ng/kg/min, e.g., from about 0.5 ng/kg/min to about 15 ng/kg/min, from about 1.25 ng/kg/min to about 10 ng/kg/min, or from about 1.25 ng/kg/min to about 5 ng/kg/min. For example, in some embodiments, the angiotensin II is administered at a rate of about 0.5 ng/kg/min, about 1.25 ng/kg/min, about 2.5 ng/kg/min, about 5 ng/kg/min, about 7.5 ng/kg/min, or about 10 ng/kg/min. In some embodiments, the angiotensin therapeutic agent is angiotensin III, and angiotensin III is administered at a rate of at least 0.5, 0.75, 1, 1.25, 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, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/kg/min. In some embodiments, angiotensin III is administered at a rate of from about 0.5 ng/kg/min to about 20 ng/kg/min, e.g, from about 0.5 ng/kg/min to about 15 ng/kg/min, from about 1.25 ng/kg/min to about 10 ng/kg/min, or from about 1.25 ng/kg/min to about 5 ng/kg/min. For example, angiotensin III may be administered at a rate of about 0.5 ng/kg/min, about 1.25 ng/kg/min, about 2.5 ng/kg/min, about 5 ng/kg/min, about 10 ng/kg/min, or about 20 ng/kg/min. In some embodiments, the angiotensin therapeutic agent is angiotensin IV, and angiotensin IV is administered at a rate of at least 0.5, 0.75, 1, 1.25, 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, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 ng/kg/min. In some embodiments, angiotensin IV is administered at a rate of from about 0.5 ng/kg/min to about 40 ng/kg/min, e.g, from about 0.5 ng/kg/min to about 20 ng/kg/min, from about 0.5 ng/kg/min to about 15 ng/kg/min, from about 1.25 ng/kg/min to about 10 ng/kg/min, or from about 1.25 ng/kg/min to about 5 ng/kg/min. For example, angiotensin IV may be administered at a rate of about 0.5 ng/kg/min, about 1.25 ng/kg/min, about 2.5 ng/kg/min, about 5 ng/kg/min, about 10 ng/kg/min, about 20 ng/kg/min, or about 40 ng/kg/min. In some embodiments, the angiotensin therapeutic agent is angiotensinogen, and angiotensinogen is administered at a rate of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ng/kg/min. In some embodiments, angiotensinogen is administered at a rate of from about 0.5 ng/kg/min to about 100 ng/kg/min, e.g, from about 0.5 ng/kg/min to

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B5015329.1 WO 2020/018785 Atty. IpCT/US2019/042393>325 about 50 ng/kg/min, from about 0.5 ng/kg/min to about 20 ng/kg/min, from about 0.5 ng/kg/min to about 15 ng/kg/min, from about 1.25 ng/kg/min to about 10 ng/kg/min, or from about 1.25 ng/kg/min to about 5 ng/kg/min. For example, angiotensinogen may be administered at a rate of about 0.5 ng/kg/min, about 1.25 ng/kg/min, about 2.5 ng/kg/min, about 5 ng/kg/min, about 10 ng/kg/min, about 20 ng/kg/min, about 50 ng/kg/min, or about 100 ng/kg/min.

In certain embodiments, the angiotensin therapeutic agent is angiotensin II, and is administered by a bolus dose, such as by subcutaneous injection, in single or divided injections. The angiotensin II may be injected subcutaneously every 2 to 6 hours (e.g., every 2 hours, every 3 hours, every 4 hours, every 5 hours, or every 6 hours). The amount of angiotensin II per dose may be from about 0.5 mg to about 1.5 mg (e.g., about 0.5 mg, about 0.75 mg, about 1 mg, about 1.25 mg, or about 1.5 mg). For example, angiotensin II may be injected at a dose of about 0.5 mg or less every 2 hours, about 0.75 mg or less every 3 hours, about 1 mg or less every 4 hours, about 1.25 mg or less every 5 hours, or about 1.5 mg or less every 6 hours.

Different subjects may require higher or lower rates or doses of administration of an angiotensin therapeutic agent to achieve a treatment goal. The rate of administration may be optimized for different subjects by administering an angiotensin therapeutic agent and the increasing or decreasing the rate of administration. In some cases, the subject may be administered an initial bolus of an angiotensin therapeutic agent followed by the administration of the angiotensin therapeutic agent at a lower rate or dose. Alternatively, the subject may be administered an angiotensin therapeutic agent at a low rate or dose followed by gradual, elevated rates/doses. Thus, in some embodiments, the method further comprises increasing the rate or dose at which an angiotensin therapeutic agent is administered, and in other embodiments, the method further comprises decreasing the rate or dose at which the angiotensin therapeutic agent is administered.

Angiotensin I or angiotensin II may be administered at an initial rate of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,

2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,

4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,

6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5,

8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 ng/kg/min, and the rate may be increased to a final rate of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,

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6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1,

8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12,

13, 14, 15, 16, 17, 18, 19, or 20 ng/kg/min. For example, angiotensin I or angiotensin II may be administered at an initial rate of about 1 ng/kg/min to about 5 ng/kg/min, and the rate may be increased by about 2.5 ng/kg/min to about 10 ng/kg/min ( e.g ., from about 2.5 ng/kg/min to about 5 ng/kg/min, from about 2.5 ng/kg/min to about 7.5 ng/kg/min, from about 2.5 ng/kg/min to about 10 ng/kg/min, from about 5 ng/kg/min to about 10 ng/kg/min, or from about 5 ng/kg/min to about 15 ng/kg/min).

Similarly, a peptide or protein comprising any one of SEQ ID NO: 1-7, SEQ ID NO: 18-26, or SEQ ID NO:28 at its N-terminus may be administered at an initial rate of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,

4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,

6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,

8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 ng/kg/min, and the rate may be increased to a final rate of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,

5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10,

11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/kg/min. For example, the peptide or protein may be administered at an initial rate of about 1 ng/kg/min to about 5 ng/kg/min, and the rate may be increased by about 2.5 ng/kg/min to about 10 ng/kg/min (e.g., from about 2.5 ng/kg/min to about 5 ng/kg/min, from about 2.5 ng/kg/min to about 7.5 ng/kg/min, from about 2.5 ng/kg/min to about 10 ng/kg/min, from about 5 ng/kg/min to about 10 ng/kg/min, or from about 5 ng/kg/min to about 15 ng/kg/min).

Angiotensin I or angiotensin II may be administered at an initial rate of about 1.0,

1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,

3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,

5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,

7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,

9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/kg/min, and the rate may be decreased to a final rate of about 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,

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3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,

5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,

7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,

9.5, 9.6, 9.7, 9.8, 9.9, or 10 ng/kg/min. For example, angiotensin I or angiotensin II may be administered at an initial rate of about 2.5 ng/kg/min to about 10 ng/kg/min, and the rate may be decreased by about 2.5 ng/kg/min to about 10 ng/kg/min ( e.g ., from about 10 ng/kg/min to about 5 ng/kg/min, from about 7.5 ng/kg/min to about 5 ng/kg/min, from about 10 ng/kg/min to about 2.5 ng/kg/min, from about 7.5 ng/kg/min to about 2.5 ng/kg/min, or from about 5 ng/kg/min to about 2.5 ng/kg/min). In certain embodiments, angiotensin I or angiotensin II may be administered at an initial rate of about 2.5 ng/kg/min, and the rate may be decreased to about 1.25 ng/kg/min.

Similarly, a peptide or protein comprising any one of SEQ ID NO: 1-7, SEQ ID NO: 18-26, or SEQ ID NO:28 at its N-terminus may be administered at an initial rate of about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,

5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,

7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1,

9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/kg/min, and the rate may be decreased to a final rate of about 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,

5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,

7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1,

9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 ng/kg/min. For example, the peptide or protein may be administered at an initial rate of about 2.5 ng/kg/min to about 10 ng/kg/min, and the rate may be decreased by about 2.5 ng/kg/min to about 10 ng/kg/min (e.g., from about 10 ng/kg/min to about 5 ng/kg/min, from about 7.5 ng/kg/min to about 5 ng/kg/min, from about 10 ng/kg/min to about 2.5 ng/kg/min, from about 7.5 ng/kg/min to about 2.5 ng/kg/min, or from about 5 ng/kg/min to about 2.5 ng/kg/min). In certain embodiments, the peptide or protein may be administered at an initial rate of about 2.5 ng/kg/min, and the rate may be decreased to about 1.25 ng/kg/min.

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Angiotensin III or angiotensin IV may be administered at an initial rate of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,

2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,

4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,

6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5,

8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17,

18, 19, or 20 ng/kg/min, and the rate may be increased to a final rate of about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,

2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,

4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7,

6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,

8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 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.

For example, angiotensin III or angiotensin IV may be administered at an initial rate of about 5 ng/kg/min to about 10 ng/kg/min, and the rate may be increased by about 5 ng/kg/min to about 10 ng/kg/min ( e.g ., from about 5 ng/kg/min to about 10 ng/kg/min, from about 5 ng/kg/min to about 15 ng/kg/min, from about 10 ng/kg/min to about 15 ng/kg/min, or from about 10 ng/kg/min to about 20 ng/kg/min).

Similarly, a peptide or protein comprising any one of SEQ ID NO:8-l7 at its N- terminus may be administered at an initial rate of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,

0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,

3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,

5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,

7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2,

9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/kg/min, and the rate may be increased to a final rate of about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,

1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2,

3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,

5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,

7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5,

9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 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. For example, the peptide or protein may be administered at an initial rate of about 5 ng/kg/min to about 10 ng/kg/min, and the

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B5015329.1 WO 2020/018785 Atty. IpCT/US2019/042393>325 rate may be increased by about 5 ng/kg/min to about 10 ng/kg/min ( e.g ., from about 5 ng/kg/min to about 10 ng/kg/min, from about 5 ng/kg/min to about 15 ng/kg/min, from about 10 ng/kg/min to about 15 ng/kg/min, or from about 10 ng/kg/min to about 20 ng/kg/min).

Angiotensin III or angiotensin IV may be administered at an initial rate of about 0.2,

0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,

2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4,

4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,

6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,

8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18,

19, 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, and the rate may be decreased to a final rate of about 0.10, 0.15, 0.20, 0.25,

0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,

2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5,

4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,

6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,

8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or

20 ng/kg/min. For example, angiotensin III or angiotensin IV may be administered at an initial rate of about 10 ng/kg/min to about 20 ng/kg/min, and the rate may be decreased by about 5 ng/kg/min to about 15 ng/kg/min (e.g., from about 20 ng/kg/min to about 10 ng/kg/min, from about 15 ng/kg/min to about 10 ng/kg/min, from about 20 ng/kg/min to about 5 ng/kg/min, from about 15 ng/kg/min to about 5 ng/kg/min, or from about 10 ng/kg/min to about 5 ng/kg/min).

Similarly, a peptide or protein comprising any one of SEQ ID NO:8-l7 at its N- terminus may be administered at an initial rate of about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,

1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0,

3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,

5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,

7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,

9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 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, and the rate may be decreased to a final rate of about 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55,

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0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2,

3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,

5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,

7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5,

9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/kg/min. For example, the peptide or protein may be administered at an initial rate of about 10 ng/kg/min to about 20 ng/kg/min, and the rate may be decreased by about 5 ng/kg/min to about 15 ng/kg/min ( e.g ., from about 20 ng/kg/min to about 10 ng/kg/min, from about 15 ng/kg/min to about 10 ng/kg/min, from about 20 ng/kg/min to about 5 ng/kg/min, from about 15 ng/kg/min to about 5 ng/kg/min, or from about 10 ng/kg/min to about 5 ng/kg/min).

Those of skill in the art will recognize that in the context of the present disclosure, a composition comprising an angiotensin therapeutic agent can be administered in any suitable way, but is typically administered by continuous infusion. Accordingly, increasing or decreasing a rate of administration can be accomplished by changing the rate of flow of an intravenous drip, changing the concentration of the agent in an intravenous drip, etc. However, the manner in which the rate of administration is changed will depend on the mode of administration of the therapeutic. Other modes of administration (via

subcutaneous injection pump, suppository, etc.) can be modulated in analogous fashions, and decreasing the rate of administration can be accomplished by doing the opposite of an action that would increase the rate of administration of the therapeutic.

An angiotensin therapeutic agent may be titrated while monitoring the therapeutic effect in a subject. The therapeutic effect of the angiotensin therapeutic agent may be determined based on, for example, patient’s self-reported response and physical exam. In some embodiments, the therapeutic effect is determined after a period of time (e.g., 1-2 hours) after administering the composition. If there is a therapeutic effect, the rate of administering the angiotensin therapeutic agent is maintained or increased. Administration of the angiotensin therapeutic agent may be decreased or stopped upon improvement or full resolution of angioedema symptoms. An angiotensin therapeutic agent may also be titrated while monitoring the safety of a subject. One aspect of the safety monitoring may involve measuring the mean arterial pressure (MAP) of the subject after a period of time (e.g, within 5-10 minutes, and again after e.g., 1-2 hours) after administering the angiotensin therapeutic agent. If the measured mean arterial pressure is below 110 mm Hg, e.g, below

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100 mm Hg, 90 mm Hg, or 80 mm Hg, the health care provider may elect to increase the rate of administering the angiotensin therapeutic agent. If the measured mean arterial pressure is at or above 90 mm Hg, the health care provider may elect to reduce the rate at which the angiotensin therapeutic agent is administered to the subject. In certain embodiments, if the measure mean arterial pressure exceeds a desired threshold for the subject, e.g., above 100 mm Hg, the subject may be co-administered with a short-acting antihypertensive drugs, such as clonidine or a beta blocker. The titration may occur over the course of minutes to hours. Thus, the rate at which an angiotensin therapeutic agent is administered may be increased or decreased over the course of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,

11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,

35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,

59, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 135, 150, 165, 180, 195, 210,

225, or 240 minutes, or over the course of about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,

9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24 hours or longer.

An angiotensin therapeutic agent may be administered as long as necessary to achieve or maintain the desired therapeutic effect. In some embodiments, the composition is administered continuously for at least 1-2 hours. In some embodiments, the composition is administered continuously over a period of time selected from less than 6 hours; from 6 hours to 12 hours; or at least 12 hours. In certain embodiments, the composition is administered continuously for up to 24 hours.

IV. SUBJECTS

The subject may be a mammal. The subject may be a rodent, lagomorph, feline, canine, porcine, ovine, bovine, equine, or primate. In preferred embodiments, the subject is a human. The subject may be a female or male. The subject may be an infant, child, or adult.

V. ROUTES OF ADMINISTRATION

The compositions of the disclosure can be administered in a variety of ways. In some embodiments, the compositions of the disclosure are suitable for parenteral administration. These compositions may be administered, for example, intraperitoneally, intravenously, intrarenally, intramuscularly, or intrathecally. In some embodiments, the compositions of the disclosure are injected or infused intravenously. In other embodiments,

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B5015329.1 WO 2020/018785 Atty. IpCT/US2019/042393>325 the compositions of the disclosure are injected or infused subcutaneously. In certain embodiments, the parenteral administration may comprise peripheral administration ( e.g ., intravenous administration via a peripheral venous line (PVL)) or pump administration (e.g., subcutaneous administration via a pump). One of skill in the art would appreciate that a method of administering a therapeutically effective substance formulation or composition of the disclosure would depend on factors such as the age, weight, and physical condition of the subject being treated, and the disease or condition being treated. The skilled worker would, thus, be able to select a method of administration optimal for a subject on a case-by- case basis.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present specification, including its specific definitions, will control.

While specific aspects of the subject matter have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

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