Improved methods and compositions for the treatment of pemphigus or pemphigoid disease

Abstract

The present invention provides, among other things, improved methods and compositions for treating pemphigus and pemphigoid comprising administering to a subject suffering from pemphigus or pemphigoid an angiotensin (1-7) peptide, wherein the administration results in a reduction in the intensity, severity, duration, or frequency of at least one symptom of pemphigus or pemphigoid.

Description

[0001] IMPROVED METHODS AND COMPOSITIONS FOR THE TREATMENT

[0002] OF PEMPHIGUS OR PEMPHIGOID DISEASE

[0003] BACKGROUND

[0004] Pemphigus and pemphigoid disease are autoimmune blistering skin diseases or disorders characterized by the formation of blisters in the skin or membranes of mouth, nose or eyes, for example, due to autoantibodies that attack normal body structures. Pemphigus is characterized by shallow ulcers in the epidermis causing fragile blisters that break quickly, while pemphigoid causes deeper, tense blisters in the layer between epidermis and dermis that are not easily disrupted. They are also associated with hives. Current therapy includes, for example, corticosteroid treatment, mycophenolate mofetil and rituximab. Pemphigus and pemphigoid disease may cause fatalities. Prolonged systemic immunosuppression remains challenging and there is a need for improved therapies. SUMMARY OF THE INVENTION

[0005] The present invention provides, among other things, improved methods and compositions for treating an autoimmune blistering skin disease, preferably pemphigus or pemphigoid disease, comprising administering an angiotensin (1-7) receptor agonist, in particular an angiotensin (1-7) peptide, to a subject suffering from the disease.

[0006] In preferred embodiments, the angiotensin (1-7) peptide has an amino acid sequence of Asp1-Arg2-Val3-Tyr4-He5- His6-Pro7(SEQ ID NO: 1), Asp1- Arg2-Val3- Ser4-Ile5-His6-Cys7(SEQ ID NO: 2), or Ala1- Arg2-Val3-Ser4-Ile5-His6-Cys7(SEQ ID NO: 3).

[0007] In some embodiments, the angiotensin (1-7) receptor agonist is a non- peptidic angiotensin (1-7) receptor agonist. In a preferred embodiment, the non- peptidic angiotensin (1-7) receptor agonist is a compound with the following structure: or a pharmaceutically acceptable salt thereof.

[0008] BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The following figures are for illustration purposes only and not for limitation.

[0010] FIG. 1A-1C shows graphs of percent split formation upon administration of angiotensin (1-7) in a human skin organ culture (HSOC), by either supplementation in the medium or injection. Split formation was measured and plotted relative to concentration of angiotensin (1-7), for example, from 0.01 pM to 1 pM or 100 pM. As a positive control, scFv to autoantibodies was used as a positive control. FIG. 1A shows percent split formation at various concentrations of angiotensin (1-7) from 0.01 pM to 100 pM supplemented in HSOC medium in HSOC without colored skin. FIG. IB shows percent split formation at various concentrations of angiotensin (1-7) from 0.01 pM to 100 pM injected in HSOC without colored skin. FIG. 1C shows percent split formation at various concentrations of angiotensin (1-7) from 0.01 pM to 1 pM injected in HSOC with only colored skin.

[0011] FIG. 2 shows histopathogic analysis of split formation in a human skin model. While an scFv positive control against desmoglein 1 and 3 autoantibodies, shows pronounced split formation in the microscopic images, an angiotensin (1-7) injected sample shows decreased split formation, indicative of amelioration of pemphigus and pemphigoid disease. Angiotensin (1-7) treatment resulted in a significant reduction in split formation.

[0012] FIG. 3 demonstrates the reduction of skin lesions in a preclinical model of a blistering skin disease by Angiotensin (1-7) treatment. Angiotensin (1-7) was administered daily from day 3 and the affected skin area was measured. Fig. 3A shows the reduction of affected skin area compared to the control over time and Fig. 3B the detailed results on the last treatment day.

[0013] DEFINITIONS

[0014] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g, a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and / or a clone.

[0015] Approximately or about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0016] Biologically active'. As used herein, the phrase “biologically active” refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. In particular embodiments, where a peptide is biologically active, a portion of that peptide that shares at least one biological activity of the peptide is typically referred to as a “biologically active” portion. In certain embodiments, a peptide has no intrinsic biological activity but that inhibits the effects of one or more naturally- occurring angiotensin compounds is considered to be biologically active.

[0017] Carrier or diluent. As used herein, the terms “carrier” and “diluent” refers to a pharmaceutically acceptable (e.g, safe and non-toxic for administration to a human) carrier or diluting substance useful for the preparation of a pharmaceutical formulation. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.

[0018] Complication'. As used herein, the term “complication” refers to an unfavorable evolution of a disease including the development of one or more signs, symptoms or, in some embodiments, even new pathological changes that manifest for a sustained period of time (e.g, weeks, months or years). In some embodiments, complication(s) may include a progression of a sign, symptom or other pathological change, for example, a minor memory loss growing worse over time, or a difficulty with one or more motor functions progressing to paralysis.

[0019] Dosage form: As used herein, the terms “dosage form” and “unit dosage form” refer to a physically discrete unit of a therapeutic agent for the patient to be treated. Each unit contains a predetermined quantity of active material calculated to produce the desired therapeutic effect. It will be understood, however, that the total dosage of the composition will be decided by the attending physician within the scope of sound medical judgment.

[0020] Dosing regimen: A “dosing regimen” (or “therapeutic regimen”), as that term is used herein, is a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent (e.g, an angiotensin (1-7) peptide) has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, the therapeutic agent is administered continuously over a predetermined period. In some embodiments, the therapeutic agent is administered once a day (QD) or twice a day (BID). Functional equivalent or derivative. As used herein, the term “functional equivalent” or “functional derivative” denotes, in the context of a functional derivative of an amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence. A functional derivative or equivalent may be a natural derivative or is prepared synthetically. Exemplary functional derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved. The substituting amino acid desirably has chemico-physical properties which are similar to that of the substituted amino acid. Desirable similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophilicity, and the like, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein. A “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.

[0021] In vitro'. As used herein, the term “ / ' / / vitro''" refers to events that occur in an artificial environment, e.g, in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism. In vivo'. As used herein, the term “z' / z vivo"" refers to events that occur within a multi- cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0022] Isolated'. As used herein, the term “isolated” refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated. In some embodiments, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure. As used herein, a substance is “pure” if it is substantially free of other components. As used herein, the term “isolated cell” refers to a cell not contained in a multi-cellular organism.

[0023] Prevent. As used herein, the term “prevent” or “prevention”, when used in connection with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder and / or condition. See the definition of “risk.”

[0024] Polypeptide'. The term “polypeptide” as used herein refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of ordinary skill in the art will understand that the term is not limited to lengthy chains and can refer to a minimal chain comprising two amino acids linked together via a peptide bond. As is known to those skilled in the art, polypeptides may be processed and / or modified.

[0025] Protein-. The term “protein” as used herein refers to one or more polypeptides that function as a discrete unit. If a single polypeptide is the discrete functioning unit and does not require permanent or temporary physical association with other polypeptides in order to form the discrete functioning unit, the terms “polypeptide” and “protein” may be used interchangeably. If the discrete functional unit is comprised of more than one polypeptide that physically associate with one another, the term “protein” refers to the multiple polypeptides that are physically coupled and function together as the discrete unit.

[0026] Risk: As will be understood from context, a “risk” of a disease, disorder, and / or condition comprises a likelihood that a particular individual will develop a disease, disorder, and / or condition (e.g., an autoimmune blistering skin disease, preferably pemphigus or pemphigoid). In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and / or event (e.g., an autoimmune blistering skin disease, preferably pemphigus or pemphigoid). In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, relative riskis 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore. Alternatively or additionally, risk can mean the presence of one of more risk factors for the autoimmune blistering skin disease, such as age of 40 years or older (or 50 years or older, or 60 years or older, having an ancestry associated with increased risk (such as Jewish, Indian, southeast European or Middle Easter, and / or being exposed to one or more trigger factors (e.g. drugs, thermal or electrical burns, surgical procedures, trauma, ultraviolet irradiation, radiotherapy, chemical preparations, transplants, or infections).

[0027] Sign: As used herein, the term “sign” refers to a departure from normal body function that indicates the presence of a disease or abnormality that is noticed by a person other than the patient (as opposed to a symptom, see below).

[0028] Stability: As used herein, the term “stable” refers to the ability of the therapeutic agent to maintain its therapeutic efficacy (e.g., all or the majority of its intended biological activity and / or physiochemical integrity) over extended periods of time. The stability of a therapeutic agent, and the capability of the pharmaceutical composition to maintain stability of such therapeutic agent, may be assessed over extended periods of time (e.g., for at least 1, 3, 6, 12, 18, 24, 30, 36 months or more). In certain embodiments, pharmaceutical compositions described herein have been formulated such that they are capable of stabilizing, or alternatively slowing or preventing the degradation, of one or more therapeutic agents formulated therewith. In the context of a formulation a stable formulation is one in which the therapeutic agent therein essentially retains its physical and / or chemical integrity and biological activity upon storage and during processes (such as freeze / thaw, mechanical mixing and lyophilization).

[0029] Subject. As used herein, the term “subject” refers to a human or any nonhuman animal (e.g, mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder. Substantially. As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0030] Suffering from'. An individual who is “suffering from” a disease, disorder, and / or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and / or condition.

[0031] Susceptible to An individual who is “susceptible to” a disease, disorder, and / or condition has not been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, condition, or event (for example, an autoimmune blistering skin disease, preferably pemphigus or pemphigoid disease) may be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and / or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and / or condition; (3) increased and / or decreased expression and / or activity of a protein associated with the disease, disorder, and / or condition; (4) habits and / or lifestyles associated with development of the disease, disorder, condition, and / or event (5) having undergone, planning to undergo, or requiring a transplant. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0032] Symptom: As used herein, the term “symptom” refers to a departure from normal body function that indicates the presence of a disease or abnormality that is noticed by the subject or patient.

[0033] Therapeutically effective amount. As used herein, the term “therapeutically effective amount” of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the symptom(s) of the disease, disorder, and / or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0034] Treating-. As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and / or reduce incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and / or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[0035] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0036] As used in this application, the terms “about” and “approximately” are used as equivalents. Any citations to publications, patents, or patent applications herein are incorporated by reference in their entirety. Any numerals used in this application with or without about / approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art.

[0037] Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.

[0038] Autoimmune blistering skin diseases, including pemphigus and pemphigoid, are rare autoimmune blistering diseases of the skin, often involving also the mucous membrane. The present invention provides, among other things, methods and compositions for treating an autoimmune blistering skin disease, preferably pemphigus, pemphigoid and related autoimmune blistering conditions.

[0039] Provided methods of treating an autoimmune blistering skin disease, preferably pemphigus or pemphigoid, include administering to a subject suffering from the autoimmune blistering skin disease an angiotensin (1-7) receptor agonist. In other words, the invention provides an angiotensin (1-7) receptor agonist for use in treating the autoimmune blistering skin disease. In yet other words, the invention provides the use of an angiotensin (1-7) receptor agonist in the manufacture of a medicament for treating the autoimmune blistering skin disease. The angiotensin (1-7) receptor agonist, wherever mentioned herein and unless indicated otherwise, is preferably an angiotensin (1-7) peptide, in particular angiotensin (1-7), a functional variant and / or a precursor thereof.

[0040] Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of “or” means “and / or” unless stated otherwise.

[0041] Autoimmune blistering skin disease Autoimmune blistering skin disease is a group of rare skin diseases. It occurs when the immune system (in particular autoantibodies) attack skin and sometimes also mucous membranes, i.e. the lining inside mouth, nose, and other parts of the body. This attack causes blisters (small pockets of body fluid within the upper layers of the skin) to form. The main groups of autoimmune blistering skin disease are pemphigus and pemphigoid, and those autoimmune blistering skin diseases that are not classified as pemphigus and pemphigoid are hereby referred to as autoimmune blistering conditions related to pemphigus and pemphigoid.

[0042] Pemphigus Pemphigus refers to blistering disorders caused by autoantibodies such as desmoglein 1 and desmoglein 3 that recognize components of the epidermis and lead to disruption of the intercellular junctions, loss of integrity and formation of blisters in the epidermis that break easily. In some embodiments, the pemphigus is one or more of pemphigus vulgaris, pemphigus foliaceus, IgA pemphigus, paraneoplastic pemphigus, pemphigus vegetans, drug-induced pemphigus, pemphigus erythematosus or Senear- Usher syndrome, endemic pemphigus or fogo selvagem, benign familial pemphigus or Hailey-Hailey disease, and combinations thereof. Preferably, the pemphigus is pemphigus vulgaris.

[0043] Pemphigus vulgaris: In some embodiments, the pemphigus is pemphigus vulgaris. In pemphigus vulgaris, blisters are formed in mouth and other mucous membranes causing red and white fluid-filled blisters or open sores, and blisters on skin near groin and legs.

[0044] Pemphigus foliaceus: In some embodiments, the pemphigus is pemphigus foliaceus. In pemphigus foliaceus, red to purple blisters develop on scalp, face, neck and back, but rarely mouth. The epidermis layer is affected and small blisters form in groups that break easily to form crusty lesions spreading over large areas of skin.

[0045] Pemphigus vegetans: In some embodiments, the pemphigus is pemphigus vegetans. Pemphigus vegetans forms thick lesions, typically in skin folds including groin and armpit regions.

[0046] Paraneoplastic pemphigus: In some embodiments, the pemphigus is paraneoplastic pemphigus. Paraneoplastic pemphigus is the rarest form of pemphigus in cancer patients that causes severe oral blisters.

[0047] Endemic pemphigus or fogo selvagem: In some embodiments, the pemphigus is endemic pemphigus or fogo selvagem. Endemic pemphigus is a form of pemphigus foliaceus that occurs most commonly in South and Central America, for example, Brazil.

[0048] IgA pemphigus: In some embodiments, the pemphigus is immunoglobulin A (IgA) pemphigus. IgA pemphigus causes lesions in the skin of trunk, upper and lower extremities, armpit and groin. It affects mucous membranes infrequently. IgA pemphigus manifests as blisters, pustules, erythema, erosions, and vegetating lesions. Flaccid vesicles and pustules form first, which then rupture to form erosions and crusted plaques, and the eruptions follow an annular pattern.

[0049] Benign familial pemphigus or Hailey-Hailey disease: In some embodiments, the pemphigus is benign familial pemphigus or Hailey-Hailey disease. Benign familial pemphigus is an inherited skin disorder that arises due to a mutation in a gene ATP2C1 that encodes SPCA1 (Secretory Pathway Calcium / manganese-ATPase), a calcium and manganese pump. Skin cells or keratinocytes are packed together by structures known as desmosomes that do not assemble in the absence of sufficient calcium as in benign familial pemphigus. Benign familial pemphigus starts as a painful crusted skin rash, typically in the armpit, groin, neck, under breasts and in the buttocks, in skin folds and creases, As the lesions get bigger, the center clears leaving a typical ring, and the lesions grow thicker which then causes maceration and painful cracks in the skin. It may sometimes involve mucous membranes or cause white bands on the fingernails and pits in the palms.

[0050] Drug-induced pemphigus: In some embodiments, the pemphigus is drug- induced pemphigus. Some medications, for example, antibiotics or blood pressure medication, might result in blistering, sometimes months later. Pemphigus erythematosus or Senear-Usher syndrome: In some embodiments, the pemphigus is pemphigus erythematosus or Senear-Usher syndrome. Pemphigus erythematosus sometimes presents with lupus and causes blisters on upper back, chest, cheeks and scalp that develop into red, scaly lesions.

[0051] In some embodiments, the pemphigus is a combination of one or more of pemphigus vulgaris, pemphigus foliaceus, IgA pemphigus, paraneoplastic pemphigus, pemphigus vegetans, drug-induced pemphigus, pemphigus erythematosus or Senear- Usher syndrome, endemic pemphigus or fogo selvagem, benign familial pemphigus or Hailey-Hailey disease, in any order and in any number of combinations. In some embodiments, the combination is concurrent. In some embodiments, the combination is sequential.

[0052] Pemphigoid

[0053] Pemphigoid causes blisters in a skin layer between the dermis and epidermis that do not break easily, sometimes having the appearance of hives or eczema. Autoantibodies, for example, anti-BPA2 and anti-BPAl, are directed against the basal layer of the epidermis and mucous membranes, causing skin cells to separate from one another and cause fluid accumulation between skin layers forming blisters sometimes covering a large area of skin. Pemphigoid diseases can be disintguished from pemphigus, by a negative Nikolsky sign, i.e. friction of non-lesional skin does not lead to intra epidermal disruption and visible erosion. In some embodiments, the pemphigoid is one or more of bullous pemphigoid (BP), mucous membrane pemphigoid (MMP, or cicatricial pemphigoid), pemphigoid gestationis, epidermolysis bullosa acquisita (EBA), Linear IgA disease, Anti-laminin gl / anti-p200 pemphigoid, Lichen planus pemphigoid, pemphigoid disease characterised by renal insufficiency, and combinations thereof. In some embodiments, which are preferred, the pemphigoid is bullous pemphigoid (BP). In some embodiments, the pemphigoid is a mucous membrane pemphigoid (MMP, or cicatricial pemphigoid). In some embodiments, the pemphigoid is pemphigoid gestationis. In some embodiments, the pemphigoid is a combination of one or more of the above-recited pemphigoids, in particular bullous pemphigoid (BP), mucous membrane pemphigoid (MMP, or cicatricial pemphigoid), or pemphigoid gestationis.

[0054] Bullous pemphigoid (BP)-. In some embodiments, the pemphigoid is a bullous pemphigoid. Bullous pemphigoid is a rare disease that causes large, fluid-filled blisters on the skin of the lower abdomen, upper thighs or armpits. Autoantibodies are targeted towards the fibers that connect epidermis to dermis layer of the skin. Bulbous pemphigoid is common in adults over 60. Bullous pemphigoid may be triggered by medications, e.g., etanercept, sulfasalazine, furosemide and penicillin, or light and radiation, e.g., ultraviolet light or radiation therapy, and medical conditions, e.g., psoriasis, lichen planus, diabetes, rheumatoid arthritis, ulcerative colitis and multiple sclerosis.

[0055] Mucous membrane pemphigoid (MMP) or cicatricial pemphigoid: In some embodiments, the pemphigoid is mucous membrane pemphigoid (MMP). Mucous Membrane pemphigoid is a group of rare chronic autoimmune disorders characterized by blistering lesions that affect the various mucous membranes of the body, for example, mucous membranes of the mouth and eyes. In some cases, blistering lesions also form on the skin, especially in the head and neck area. In addition, mucous membranes of the nose, throat, genitalia, and anus may be affected. As lesions heal, there may be scarring, which leads to serious complications affecting the eyes and throat.

[0056] Pemphigoid gestationis: In some embodiments, the pemphigoid is pemphigoid gestationis. Pemphigoid gestationis (previously called herpes gestationis) is a rare dermatosis of pregnancy. It is a bullous autoimmune, sub-epidermal dermatosis, clinically and pathogenetically similar to bullous pemphigoid.

[0057] Epidermolysis bullosa acquisita. In some embodiments, the pemphigoid is epidermolysis bullosa acquisita (EBA). EBA is characterized by autoantibodies against type VII collagen. Clinically, epidermolysis bullosa acquisita can either be the classic form or an inflammatory variant. The classic mechanobullous form presents with tense blisters and skin fragility preferentially localised to the extensor skin surfaces at trauma- prone areas. The inflammatory subtype can resemble bullous pemphigoid or raucous membrane pemphigoid.

[0058] Linear IgA disease: In some embodiments, the pemphigoid is Linear IgA disease. It is named for its main immunopathological feature, which is the linear binding of IgA at the dermal-epidermal junction. It is the most common pemphigoid disorder in children and has two peaks of onset- before the age of 5 years and after the age of 60 years. The major target antigen is BP 180, another is BP230. Anti-laminin gl / anti-p200 pemphigoid. In some embodiments, the pemphigoid is

[0059] Anti-laminin gl / anti-p200 pemphigoid. This disease clinically mimics bullous pemphigoid, although patients tend to be younger and concurrent psoriasis is seen in about a third of cases. Diagnosis is made by detection of autoantibodies against 200 kDa protein laminin yl (also referred to as p200 protein) of the dermal-epidermal junction. Lichen planus pemphigoides'. In some embodiments, the pemphigoid is Lichen planus pemphigoides. This disease always arises in conjunction with lichen planus. The main target antigen is BP180, another is BP230. It affects fairly young patients (mean age of onset 40-50 years), mainly arises on the limbs, and preferentially targets C- terminal epitopes in the immunodominant NC16A domain.

[0060] Pemphigoid disease characterised by renal insufficiency. In some embodiments, the pemphigoid is pemphigoid disease characterised by renal insufficiency. This disease is rare and characterized by autoantibodies against the a5 chain of type IV collagen. Symptoms

[0061] In some embodiments, the one or more symptoms of the autoimmune blistering skin disease, preferably pemphigus or pemphigoid disease, is / are selected from ulcers, blisters, eczema, hives, and combinations thereof. In some embodiments, the one or more symptoms the disease is ulcers. In some embodiments, the one or more symptoms of the disease is blisters. In some embodiments, the one or more symptoms of pemphigus or pemphigoid disease is eczema. In some embodiments, the one or more symptoms of the disease is hives.

[0062] In some embodiments, the ulcers or blisters are on skin or mucous membrane. In some embodiments, the ulcers are on skin. In some embodiments, the blisters are on skin. In some embodiments, the ulcers are on mucous membrane. In some embodiments, the blisters are on mucous membrane.

[0063] In some embodiments, the mucous membrane is of mouth, nose, throat, eyes, or genitals. In some embodiments, the mucous membrane is of mouth. In some embodiments, the mucous membrane is of nose. In some embodiments, the mucous membrane is of throat. In some embodiments, the mucous membrane is of eyes. In some embodiments, the mucous membrane is of genitals.

[0064] Administration

[0065] In some embodiments, the angiotensin (1-7) receptor agonist is administered at an effective dose periodically at an administration interval such that at least one symptom or feature of the autoimmune blistering skin disease, preferably pemphigus or pemphigoid disease is reduced in intensity, severity, duration, or frequency or has delayed onset relative to a control. In some embodiments, the angiotensin (1-7) receptor agonist is administered at an effective dose periodically at an administration interval such that at least one symptom or feature of the disease is reduced in intensity relative to a control. In some embodiments, the angiotensin (1-7) receptor agonist is administered at an effective dose periodically at an administration interval such that at least one symptom or feature of the disease is reduced in severity relative to a control. In some embodiments, the angiotensin (1-7) receptor agonist is administered at an effective dose periodically at an administration interval such that at least one symptom or feature the disease is reduced in duration relative to a control. In some embodiments, the angiotensin (1-7) receptor agonist is administered at an effective dose periodically at an administration interval such that at least one symptom or feature of the disease is reduced in frequency relative to a control. In some embodiments, the angiotensin (1-7) receptor agonist is administered at an effective dose periodically at an administration interval such that at least one symptom or feature of the disease has delayed onset relative to a control.

[0066] In some embodiments, the angiotensin (1-7) receptor agonist is administered at an effective dose periodically at an administration interval such that at least one symptom or feature of the autoimmune blistering skin disease, preferably pemphigus or pemphigoid disease is reduced in intensity, severity, duration, or frequency or has delayed onset.

[0067] In some embodiments, the angiotensin (1-7) receptor agonist is administered once per day. In some embodiments, the angiotensin (1-7) receptor agonist is administered once per week. In some embodiments, the angiotensin (1-7) receptor agonist is administered three times per month. In some embodiments, the angiotensin (1-7) receptor agonist is administered twice per month. In some embodiments, the angiotensin (1-7) receptor agonist is administered once per month.

[0068] In some embodiments, the administration is parenteral, rectal, oral, or a combination thereof. In some embodiments, the administration is parenteral. In some embodiments, the administration is rectal. In some embodiments, the administration is rectal. In some embodiments, the administration is oral. In some embodiments, the administration is a combination of one or more of parenteral, rectal, or oral. In some embodiments, the parenteral administration is intravenous, subcutaneous, inhalation, intradermal, transdermal, and / or transmucosal administration.

[0069] Subcutaneous administration is preferred, on particular for adults. Oral administration is also preferred, in particular for children. Angiotensin (1-7) peptides

[0070] In some aspects, provided herein is a method for treating an autoimmune blistering skin disease, preferably pemphigus or pemphigoid disease, comprising administering to a subject suffering from the disease, an angiotensin (1-7) peptide.

[0071] As used herein, the term “angiotensin (1-7) peptide” refers to both “naturally- occurring Angiotensin (1-7)” (also referred to as “Angiotensin (1-7)” or “Ang-(l-7)” herein) and any functional equivalent, analogue or derivative of naturally-occurring Angiotensin (1-7), preferably a functional variant and / or a precursor of naturally- occurring Angiotensin (1-7). As used herein, “peptide” and “polypeptide” are interchangeable terms and refer to two or more amino acids bound together by a peptide bond. As used herein, the terms “peptide” and “polypeptide” include both linear and cyclic peptide. The terms “angiotensin-(l-7)”, “Angiotensin (1-7)”, and “Ang-(l-7)” are used interchangeably.

[0072] Naturally -occurring Angiotensin ( 1-7)

[0073] Naturally-occurring Angiotensin (1-7) (also referred to as Ang-(l-7)) is a seven amino acid peptide: Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7(SEQ ID NO: 1). It is part of the renin-angiotensin system and is converted from a precursor, also known as Angiotensinogen, which is an a-2-globulin that is produced constitutively and released into the circulation mainly by the liver. Angiotensinogen is a member of the serpin family and also known as renin substrate. Human angiotensinogen is 452 amino acids long, but other species have angiotensinogen of varying sizes. Typically, the first 12 amino acids are the most important for angiotensin activity: Asp^Arg2- Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9-Leu10-Val11-Ile12(SEQ ID NO: 4), in particular the first 10 amino acids thereof.

[0074] Different types of angiotensin may be formed by the action of various enzymes. For example, Angiotensin (1-7) is generated by action of Angiotensin-converting enzyme 2 (ACE 2).

[0075] Angiotensin (1-7) is an endogenous ligand for Mas receptor (or G Protein- Coupled Mas Receptor, Uniprot identifier for human Mas receptor: P04201), for Mas- related G-protein coupled receptor member D (MrgD receptor, Uniprot identifier for human MrgD receptor: Q8TDS7), and, at high concentrations (e.g. 10'5M or higher) for the Angiotensin II receptor type 2 (AT2 receptor, Uniprot identifier for human AT2 receptor: P50052). Mas receptors are G-protein coupled receptor containing seven transmembrane spanning regions. As used herein, the term “angiotensin-(l-7) receptor” encompasses the above receptors, in particular the G Protein-Coupled Mas Receptors.

[0076] As used herein, the term “naturally-occurring Angiotensin (1-7)” includes any Angiotensin (1-7) peptide purified from natural sources and any recombinantly produced or chemically synthesized peptides that have an amino acid sequence identical to that of the naturally-occurring Angiotensin (1-7).

[0077] Functional equivalents, analogs or derivatives of Ang-(l-7) In some embodiments, an angiotensin (1-7) peptide suitable for the present invention is a functional equivalent of naturally-occurring Ang-(l-7). As used herein, a functional equivalent of naturally-occurring Ang-(l-7) refers to any peptide that shares amino acid sequence identity to the naturally-occurring Ang-(l-7) and retain substantially the same or similar activity as the naturally-occurring Ang-(l-7). For example, in some embodiments, a functional equivalent of naturally-occurring Ang- (1-7) described herein has pro-angiogenic activity as determined using methods described herein or known in the art, or an activity such as nitric oxide release, vasodilation, improved endothelial function, antidiuresis, or one of the other properties discussed herein, that positively impacts angiogenesis. In some embodiments, a functional equivalent of naturally-occurring Ang-(l-7) described herein can bind to or activate an angiotensin-(l -7) receptor (e.g. , the G protein-coupled Mas receptor) as determined using various assays described herein or known in the art. In some embodiments, a functional equivalent of Ang-(l-7) is also referred to as an angiotensin (1-7) analogue or derivative, or functional derivative. In some embodiments, a functional equivalent of Ang-(l-7) is a non-cyclic peptide.

[0078] Typically, a functional equivalent of angiotensin (1-7) shares amino acid sequence similarity to the naturally-occurring Ang-(l-7). In some embodiments, a functional equivalent of Ang-(l-7) according to the invention contains a sequence that includes at least 3 (e.g., at least 4, at least 5, at least 6, at least 7) amino acids from the seven amino acids that appear in the naturally- occurring Ang-(l-7), wherein the at least 3 (e.g., at least 4, at least 5, at least 6, or at least 7) amino acids maintain their relative positions and / or spacing as they appear in the naturally-occurring Ang- (1-7).

[0079] In some embodiments, a functional equivalent of Ang-(l-7) may encompass any peptide that contains a sequence at least 50% (e.g., at least 60%, 70%, 80%, or 90%) identical to the amino acid sequence of naturally-occurring Ang-(l-7). Percentage of amino acid sequence identity can be determined by alignment of amino acid sequences. Alignment of amino acid sequences can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Preferably, the WU-BLAST-2 software is used to determine amino acid sequence identity (Altschul etal. , Methods in Enzymology 266, 460-480 (1996); http: / / blast.wustl / edu / blast / README.html). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=l, overlap fraction=0.125, word threshold (T)= 11. HSP score (S) andHSP S2 parameters are dynamic values and are established by the program itself, depending upon the composition of the particular sequence, however, the minimum values may be adjusted and are set as indicated above.

[0080] In some embodiments, a functional equivalent, analogue or derivative of Ang- (1-7) is a fragment of the naturally-occurring Ang-(l-7). In some embodiments, a functional equivalent, analogue or derivative of Ang-(l-7) contains amino acid substitutions, deletions and / or insertions in the naturally-occurring Ang-(l-7). Ang- (1-7) functional equivalents, analogues or derivatives can be made by altering the amino acid sequences by substitutions, additions, and / or deletions. For example, one or more amino acid residues within the sequence of the naturally-occurring Ang-(l-7) (SEQ ID NO: 1) can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Substitution for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs (conservative substitution). For example, the positively charged (basic) amino acids include arginine, lysine, and histidine. The nonpolar (hydrophobic) amino acids include leucine, isoleucine, alanine, phenylalanine, valine, proline, tryptophan, and methionine. The uncharged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The negatively charged (acid) amino acids include glutamic acid and aspartic acid. The amino acid glycine may be included in either the nonpolar amino acid family or the uncharged (neutral) polar amino acid family. Substitutions made within a family of amino acids are generally understood to be conservative substitutions. For example, the amino acid sequence of a peptide inhibitor can be modified or substituted.

[0081] Examples of Ang-(l-7) functional equivalents, analogues and derivatives are described in the section entitled “Exemplary Angiotensin (1-7) Peptides” below.

[0082] An angiotensin-(l-7) peptide can be of any length. In some embodiments, an angiotensin-(l-7) peptide according to the present invention can contain, for example, from 4-25 amino acids (e.g, 4-20, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7 amino acids). In some embodiments, the linear peptide contains 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In some embodiments, an angiotensin-(l-7) peptide contains one or more modifications to increase protease resistance, serum stability and / or bioavailability. In some embodiments, suitable modifications are selected from pegylation, acetylation, glycosylation, biotinylation, substitution with D-amino acid and / or unnatural amino acid, and / or cyclization of the peptide.

[0083] As used herein, the term “amino acid,” in its broadest sense, refers to any compound and / or substance that can be incorporated into a polypeptide chain. In certain embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In certain embodiments, an amino acid is a naturally-occurring amino acid. In certain embodiments, an amino acid is a synthetic or un-natural amino acid (e.g, a,a- disubstituted amino acids, N-alkyl amino acids); in some embodiments, an amino acid is a d-amino acid; in certain embodiments, an amino acid is an 1-amino acid. “Standard amino acid” refers to any of the twenty standard amino acids commonly found in naturally occurring peptides including both 1- and d- amino acids which are both incorporated in peptides in nature. “Nonstandard” or “unconventional amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. As used herein, “synthetic or un-natural amino acid” encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and / or substitutions. Amino acids, including carboxy- and / or amino- terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, and / or substitution with other chemical groups that can change the peptide’ s circulating halflife without adversely affecting its activity. Examples of unconventional or un-natural amino acids include, but are not limited to, citrulline, ornithine, norleucine, norvaline, 4-(£)-butenyl-4(A)-methyl-N- methylthreonine (MeBmt), N-m ethyl -leucine (MeLeu), aminoisobutyric acid, statine, andN-methyl- alanine (MeAla). Amino acids may participate in a disulfide bond. The term “amino acid” is used interchangeably with “amino acid residue,” and may refer to a free amino acid and / or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

[0084] In some embodiments, angiotensin-(l-7) peptides contain one or more L- amino acids, D-amino acids, and / or un-natural amino acids.

[0085] In addition to peptides containing only naturally occurring amino acids, peptidomimetics or peptide analogs are also encompassed by the present invention. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. The non-peptide compounds are termed “peptide mimetics” or peptidomimetics (Fauchere et al., Infect. Immun. 54:283-287 (1986); Evans et al., J. Med. Chem. 30:1229-1239 (1987)). Peptide mimetics that are structurally related to therapeutically useful peptides and may be used to produce an equivalent or enhanced therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to the paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity) such as naturally- occurring receptor- binding polypeptides, but have one or more peptide linkages optionally replaced by linkages such as -CH2NH-, -CH2S-, -CH2-CH2-, -CH-CH- (cis and trans), -CH2SO-, -CH(OH)CH2-, -COCH2- etc., by methods well known in the art (Spatola, Peptide Backbone Modifications, Vega Data, 1(3):267 (1983); Spatola et al. Life Sci. 38: 1243-1249 (1986); Hudson et al. Int. J. Pept. Res. 14: 177- 185 (1979); and Weinstein. B., 1983, Chemistry and Biochemistry, of Amino Acids, Peptides and Proteins, Weinstein eds, Marcel Dekker, New-York,). Such peptide mimetics may have significant advantages over naturally-occurring polypeptides including more economical production, greater chemical stability, enhanced pharmacological properties (e.g, half-life, absorption, potency, efficiency, etc.), reduced antigenicity and others.

[0086] Ang-(l-7) peptides also include other types of peptide derivatives containing additional chemical moieties not normally part of the peptide, provided that the derivative retains the desired functional activity of the peptide. Examples of such derivatives include (1) N-acyl derivatives of the amino terminal or of another free amino group, wherein the acyl group may be an alkanoyl group (e.g. , acetyl, hexanoyl, octanoyl) an aroyl group (e.g, benzoyl) or a blocking group such as F-moc (fluorenylmethyl-O-CO-); (2) esters of the carboxy terminal or of another free carboxy or hydroxyl group; (3) amide of the carboxy-terminal or of another free carboxyl group produced by reaction with ammonia or with a suitable amine; (4) phosphorylated derivatives; (5) derivatives conjugated to an antibody or other biological ligand and other types of derivatives; and (6) derivatives conjugated to a polyethylene glycol (PEG) chain.

[0087] Ang-(l-7) peptides may be obtained by any method of peptide synthesis known to those skilled in the art, including synthetic (e.g, exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, classical solution synthesis, native-chemical ligation) and recombinant techniques. For example, the peptides or peptides derivatives can be obtained by solid phase peptide synthesis, which in brief, consist of coupling the carboxyl group of the C-terminal amino acid to a resin (e.g, benzhydrylamine resin, chloromethylated resin, hydroxymethyl resin) and successively adding N-alpha protected amino acids. The protecting groups may be any such groups known in the art. Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed. Such solid phase synthesis has been disclosed, for example, by Merrifield, J. Am. Chem. Soc. 85: 2149 (1964); Vale et al., Science 213: 1394-1397 (1981), in U.S. Patent Numbers 4, 305, 872 and 4,316, 891, Bodonsky et al. Chem. Ind. (London), 38: 1597 (1966); and Pietta and Marshall, Chem. Comm. 650 (1970) by techniques reviewed in Lubell et al. “Peptides” Science of Synthesis 21.11, Chemistry of Amides. Thieme, Stuttgart, 713-809 (2005). The coupling of amino acids to appropriate resins is also well known in the art and has been disclosed in U.S. Patent Number 4,244,946. (Reviewed in Houver-Weyl, Methods of Organic Chemistry. Vol E22a. Synthesis of Peptides and Peptidomimetics, Murray Goodman, Editor-in-Chief, Thieme. Stuttgart. New York 2002).

[0088] Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains. Generally, the procedures of cell cultures, infection, molecular biology methods and the like are common methods used in the art. Such standard techniques can be found in reference manuals such as, for example, Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001; and Sambrooket al., Molecular Cloning: A Laboratory Manual, 3rdedition, Cold Spring Harbor Laboratory Press, N.Y., 2001. During any process of the preparation of an Ang-(l-7) peptide, it may be desirable to protect sensitive reactive groups on any of the molecule concerned. This may be achieved by means of conventional protecting groups such as those described in Protective Groups In Organic Synthesis by T.W. Greene & P.G.M. Wuts, 1991, John Wiley and Sons, New-York; and Peptides: chemistry and Biology by Sewald and Jakubke, 2002, Wiley-VCH, Wheinheim p.142. For example, alpha amino protecting groups include acyl type protecting groups (e.g., trifluoroacetyl, formyl, acetyl), aliphatic urethane protecting groups (e.g, t-butyloxy carbonyl (BOC), cyclohexyl oxy carbonyl), aromatic urethane type protecting groups (e.g, fluorenyl-9- methoxy-carbonyl (Fmoc), benzyl oxy carbonyl (Cbz), Cbz derivatives) and alkyl type protecting groups (e.g, triphenyl methyl, benzyl). The amino acids side chain protecting groups include benzyl (for Thr and Ser), Cbz (Tyr, Thr, Ser, Arg, Lys), methyl ethyl, cyclohexyl (Asp, His), Boc (Arg, His, Cys) etc. The protecting groups may be removed at a convenient subsequent stage using methods known in the art.

[0089] Further, Ang-(l-7) peptides may be synthesized according to the FMOC protocol in an organic phase with protective groups. Desirably, the peptides are purified with a yield of 70% with high-pressure liquid chromatography (HPLC) on a C18 chromatography column and eluted with an acetonitrile gradient of 10-60%. The molecular weight of a peptide can be verified by mass spectrometry (reviewed in Fields, G.B. “Solid-Phase Peptide Synthesis” Methods in Enzymology. Vol. 289, Academic Press, 1997).

[0090] Alternatively, Ang-(l-7) peptides may be prepared in recombinant systems using, for example, polynucleotide sequences encoding the polypeptides. It is understood that a polypeptide may contain more than one of the above-described modifications within the same polypeptide.

[0091] While peptides may be effective in eliciting a biological activity in vitro their effectiveness in vivo might be reduced by the presence of proteases. Serum proteases have specific substrate requirements. The substrate must have both L-amino acids and peptide bonds for cleavage. Furthermore, exopeptidases, which represent the most prominent component of the protease activity in serum, usually act on the first peptide bond of the peptide and require a free N- terminus (Powell et al., Pharm. Res. 10: 1268-1273 (1993)). In light of this, it is often advantageous to use modified versions of peptides. The modified peptides retain the structural characteristics of the original L-amino acid peptides that confer the desired biological activity of Ang-(1- 7) but are advantageously not readily susceptible to cleavage by protease and / or exopeptidases.

[0092] Systematic substitution of one or more amino acids of a consensus sequence with D-amino acid of the same type (e.g, D-lysine in place of L-lysine) may be used to generate more stable peptides. Thus, a peptide derivative or peptidomimetic of the present invention may be all L, all D or mixed D, L peptide, in either forward or reverse order. The presence of an N-terminal or C-terminal D-amino acid increases the in vivo stability of a peptide since peptidases cannot utilize a D-amino acid as a substrate (Powell et al., Pharm. Res. 10: 1268-1273 (1993)). Reverse-D peptides are peptides containing D-amino acids, arranged in a reverse sequence relative to a peptide containing L-amino acids. Thus, the C-terminal residue of an L-amino acid peptide becomes N-terminal for the D-amino acid peptide, and so forth. Reverse D-peptides retain the same secondary conformation and therefore similar activity, as the L-amino acid peptides, but are more resistant to enzymatic degradation in vitro and in vivo, and thus can have greater therapeutic efficacy than the original peptide (Brady and Dodson, Nature 368:692-693 (1994); Jameson et al., Nature 368:744-746 (1994)). Similarly, a reverse-L peptide may be generated using standard methods where the C-terminus of the parent peptide becomes takes the place of the N-terminus of the reverse-L peptide. It is contemplated that reverse L-peptides of L-amino acid peptides that do not have significant secondary structure (e.g., short peptides) retain the same spacing and conformation of the side chains of the L-amino acid peptide and therefore often have the similar activity as the original L- amino acid peptide. Moreover, a reverse peptide may contain a combination of L- and D-amino acids. The spacing between amino acids and the conformation of the side chains may be retained resulting in similar activity as the original L-amino acid peptide.

[0093] Another effective approach to confer resistance to peptidases acting on the N- terminal or C-terminal residues of a peptide is to add chemical groups at the peptide termini, such that the modified peptide is no longer a substrate for the peptidase. One such chemical modification is glycosylation of the peptides at either or both termini. Certain chemical modifications, in particular N-terminal glycosylation, have been shown to increase the stability of peptides in human serum (Powell et al., Pharm. Res. 10: 1268-1273 (1993)). Other chemical modifications which enhance serum stability include, but are not limited to, the addition of an N- terminal alkyl group, consisting of a lower alkyl of from one to twenty carbons, such as an acetyl group, and / or the addition of a C-terminal amide or substituted amide group. In particular, the present invention includes modified peptides consisting of peptides bearing an N-terminal acetyl group and / or a C-terminal amide group.

[0094] Substitution of non-naturally-occurring amino acids for natural amino acids in a subsequence of the peptides can also confer resistance to proteolysis. Such a substitution can, for instance, confer resistance to proteolysis by exopeptidases acting on the N-terminus without affecting biological activity. Examples of non-naturally- occurring amino acids include a, a - disubstituted amino, N-alkyl amino acids, C-a- methyl amino acids, P-amino acids, and P-methyl amino acids. Amino acids analogs useful in the present invention may include, but are not limited to, P-alanine, norvaline, norleucine, 4-aminobutyric acid, orithine, hydroxy proline, sarcosine, citrulline, cysteic acid, cyclohexylalanine, 2-aminoisobutyric acid, 6-aminohexanoic acid, t-butylglycine, phenylglycine, o-phosphoserine, N-acetyl serine, N- formylmethionine, 3-methylhistidine and other unconventional amino acids. Furthermore, the synthesis of peptides with non-naturally-occurring amino acids is routine in the art.

[0095] In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods well known in the art (Rizo and Gierasch, Ann. Rev. Biochem. 61:387-418 (1992)). For example, constrained peptides may be generated by adding cysteine residues capable of forming disulfide bridges and, thereby, resulting in a cyclic peptide. Cyclic peptides can be constructed to have no free N- or C- termini. Accordingly, they are not susceptible to proteolysis by exopeptidases, although they may be susceptible to endopeptidases, which do not cleave at peptide termini. The amino acid sequences of the peptides with N-terminal or C-terminal D-amino acids and of the cyclic peptides are usually identical to the sequences of the peptides to which they correspond, except for the presence of N- terminal or C-terminal D-amino acid residue, or their circular structure, respectively. Cyclic Peptides

[0096] In some embodiments, a functional equivalent, analogue or derivative of naturally-occurring Ang-(l-7) is a cyclic peptide. As used herein, a cyclic peptide has an intramolecular covalent bond between two non-adjacent residues. The intramolecular bond may be a backbone to backbone, side-chain to backbone or side- chain to side-chain bond (i.e., terminal functional groups of a linear peptide and / or side-chain functional groups of a terminal or interior residue may be linked to achieve cyclization). Typical intramolecular bonds include disulfide, amide and thioether bonds. A variety of means for cyclizing polypeptides are well known in the art, as are many other modifications that can be made to such peptides. For a general discussion, see International Patent Publication Nos. WO 01 / 53331 and WO 98 / 02452, the contents of which are incorporated herein by reference. Such cyclic bonds and other modifications can also be applied to the cyclic peptides and derivative compounds of this invention. Cyclic peptides as described herein may comprise residues of L-amino acids,

[0097] D- amino acids, or any combination thereof. Amino acids may be from natural or nonnatural sources, provided that at least one amino group and at least one carboxyl group are present in the molecule; a- and P-amino acids are generally preferred. Cyclic peptides may also contain one or more rare amino acids (such as 4-hydroxyproline or hydroxylysine), organic acids or amides and / or derivatives of common amino acids, such as amino acids having the C-terminal carboxylate esterified (e.g, benzyl, methyl or ethyl ester) or amidated and / or having modifications of the N-terminal amino group (e.g. , acetylation or alkoxy carbonylation), with or without any of a wide variety of side-chain modifications and / or substitutions (e.g, methylation, benzylation, t- butylation, tosylation, alkoxy carbonylation, and the like). Suitable derivatives include amino acids having an N-acetyl group (such that the amino group that represents the N-terminus of the linear peptide prior to cyclization is acetylated) and / or a C-terminal amide group (i.e., the carboxy terminus of the linear peptide prior to cyclization is amidated). Residues other than common amino acids that may be present with a cyclic peptide include, but are not limited to, penicillamine, P,P-tetramethylene cysteine, P,P- pentamethylene cysteine, P -mercaptopropionic acid, P,P- pentamethylene-P-mercaptopropionic acid, 2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline, ornithine, di aminobutyric acid, a- aminoadipic acid, m- aminomethylbenzoic acid and a,P-diaminopropionicacid. Following synthesis of a linear peptide, with or without N-acetylation and / or

[0098] C-amidation, cyclization may be achieved by any of a variety of techniques well known in the art. Within one embodiment, a bond may be generated between reactive amino acid side chains. For example, a disulfide bridge may be formed from a linear peptide comprising two thiol-containing residues by oxidizing the peptide using any of a variety of methods. Within one such method, air oxidation of thiols can generate disulfide linkages over a period of several days using either basic or neutral aqueous media. The peptide is used in high dilution to minimize aggregation and intermolecular side reactions. Alternatively, strong oxidizing agents such as I2 and KsFe(CN)6 can be used to form disulfide linkages. Those of ordinary skill in the art will recognize that care must be taken not to oxidize the sensitive side chains of Met, Tyr, Trp or His. Within further embodiments, cyclization may be achieved by amide bond formation. For example, a peptide bond may be formed between terminal functional groups (i.e., the amino and carboxy termini of a linear peptide prior to cyclization). Within another such embodiment, the linear peptide comprises a D- amino acid.

[0099] Alternatively, cyclization may be accomplished by linking one terminus and a residue side chain or using two side chains, with or without an N-terminal acetyl group and / or a C-terminal amide.

[0100] Residues capable of forming a lactam bond include lysine, ornithine (Om), a- amino adipic acid, m-aminomethylbenzoic acid, a,P-diaminopropionic acid, glutamate or aspartate. Methods for forming amide bonds are generally well known in the art. Within one such method, carbodiimide-mediated lactam formation can be accomplished by reaction of the carboxylic acid with DCC, DIC, ED AC or DCCI, resulting in the formation of an O-acylurea that can be reacted immediately with the free amino group to complete the cyclization.

[0101] Alternatively, cyclization can be performed using the azide method, in which a reactive azide intermediate is generated from an alkyl ester via a hydrazide.

[0102] Alternatively, cyclization can be accomplished using activated esters. The presence of electron withdrawing substituents on the alkoxy carbon of esters increases their susceptibility to aminolysis. The high reactivity of esters of p-nitrophenol, N- hydroxy compounds and polyhalogenated phenols has made these "active esters" useful in the synthesis of amide bonds. Within a further embodiment, a thioether linkage may be formed between the side chain of a thiol -containing residue and an appropriately derivatized a-amino acid. By way of example, a lysine side chain can be coupled to bromoacetic acid through the carbodiimide coupling method (DCC, ED AC) and then reacted with the side chain of any of the thiol containing residues mentioned above to form a thioether linkage. In order to form dithioethers, any two thiol containing side-chains can be reacted with dibromoethane and diisopropylamine inDMF.

[0103] Exemplary Angiotensin-( 1-7) Peptides

[0104] In certain aspects, the invention provides non-cyclic (e.g, linear) angiotensin- (1-7) peptides. As discussed above, the structure of naturally-occurring Ang-(l-7) is asfollows: Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7(SEQ ID NO: 1)

[0105] The peptides and peptide analogs of the invention can be generally represented by the following sequence: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7, or a pharmaceutically acceptable salt thereof. Xaa1is any amino acid or a dicarboxylic acid. In certain embodiments, Xaa1is Asp, Glu, Asn, Acpc (1 -aminocyclopentane carboxylic acid), Ala, MeiGly (N,N-dimethylglycine), Pro, Bet (betaine, 1-carboxy- N,N,N-trimethylmethanaminium hydroxide), Glu, Gly, Asp, Sar (sarcosine) or Sue (succinic acid). In certain such embodiments, Xaa1is a negatively-charged amino acid, such as Asp or Glu, typically Asp. Xaa2is Arg, Lys, Ala, Cit (citrulline), Om (ornithine), acetylated Ser, Sar, D-Arg and D- Lys. In certain embodiments, Xaa2is a positively-charged amino acid such as Arg or Lys, typically Arg. Xaa3is Vai, Ala, Leu, Nle (norleucine), He, Gly, Lys, Pro, HydroxyPro (hydroxyproline), Aib (2- aminoisobutyric acid), Acpc or Tyr. In certain embodiments, Xaa3is an aliphatic amino acid such as Vai, Leu, He or Nle, typically Vai or Nle. Xaa4is Tyr, Tyr(POs), Thr, Ser, homoSer (homoserine), azaTyr (aza-od-homo-L- tyrosine) or Ala. In certain embodiments, Xaa4is a hydroxyl-substituted amino acid such as Tyr, Ser or Thr, typically Tyr. Xaa5is He, Ala, Leu, norLeu, Vai or Gly. In certain embodiments, Xaa5is an aliphatic amino acid such as Vai, Leu, He or Nle, typically He. Xaa6is His, Arg or 6-NH2-Phe (6-aminophenylalaine). In certain embodiments, Xaa6is a fully or partially positively-charged amino acid such as Arg or His. Xaa7is Cys, Pro or Ala.

[0106] In certain embodiments, one or more of Xaax-Xaa7is identical to the corresponding amino acid in naturally-occurring Ang-(l-7). In certain such embodiments, all but one or two of Xaa1- Xaa7are identical to the corresponding amino acid in naturally-occurring Ang-(l-7). In other embodiments, all of Xaax-Xaa6are identical to the corresponding amino acid in naturally-occurring Ang-(l-7).

[0107] In certain embodiments, Xaa3is Nle. When Xaa3is Nle, one or more of Xaa1- Xaa2and Xaa4'7are optionally identical to the corresponding amino acid in naturally- occurring Ang-(l-7). In certain such embodiments, all but one or two of Xaax-Xaa21 and Xaa4'7are identical to the corresponding amino acid in naturally-occurring Ang- (1-7). In other embodiments, all of Xaa1-Xaa2and Xaa4'7are identical to the corresponding amino acid in naturally-occurring Ang-(l-7), resulting in the amino acid sequence: Asp1-Arg2-Nle3-Tyr4-Ile5-His6-Pro7(SEQ ID NO: 5).

[0108] In certain embodiments, the peptide has the amino acid sequence Asp^Arg2- Val3- Ser4-Ile5-His6-Cys7(SEQ ID NO: 2) or Ala1-Arg2-Val3-Ser4-Ile5-His6-Cys7(SEQ ID NO: 3).

[0109] In some embodiments, a linear angiotensin (1-7) peptide as described herein is a peptide having a sequence of Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9(SEQ IDNO: 19), whichis identical to the sequence of Ang(l-9). In some embodiments, an angiotensin (1-7) peptide is a derivative of Ang (1-9). For exemplary Ang (1-9) peptides, including Ang(l-9) derivatives, see U.S. Patent Publication 2012 / 0172301, the disclosure of which is hereby incorporated by reference.

[0110] In some embodiments, a linear angiotensin (1-7) peptide is a peptide with an amino acid sequence of Ala1-Arg2-Val3-Tyr4-Ile5-His6-Pro7(SEQ ID NO: 20). Additional sequences derived from SEQ ID NO: 20 may be found in European Patent Application 2,264,048, the disclosure of which is hereby incorporated by reference.

[0111] Exemplary Cyclic Angiotensin (1-7) Peptides In certain aspects, the invention provides a cyclic angiotensin-(l-7) (Ang-(1-

[0112] 7)) peptide analog comprising a linkage, such as between the side chains of amino acids corresponding to positions Tyr4and Pro7in Ang. These peptide analogs typically comprise 7 amino acid residues, but can also include a cleavable sequence. As discussed in greater detail below, the invention includes fragments and analogs where one or more amino acids are substituted by another amino acid (including fragments). One example of such an analog is Asp1-Arg2-Val3-Ser4-Ile5-His6-Cys7(SEQ ID NO: 2), wherein a linkage is formed between Ser4and Cys7. Another example of such an analog is Ala1-Arg2-Val3-Ser4-Ile5-His6-Cys7(SEQ ID NO: 3), wherein a linkage is formed between Ser4and Cys7. In some embodiments, a cyclic angiotensin (1-7) peptide analog is a cyclic analog that does not have a sequence according to SEQ ID NO: 1. In some embodiments, a cyclic angiotensin (1-7) peptide analog is a cyclic analog that does not have a sequence according to SEQ ID NO: 2. In some embodiments, a cyclic angiotensin (1-7) peptide analog is a cyclic analog that does not have a sequence according to SEQ ID NO: 3. Although the following section describes aspects of the invention in terms of a thioether bond linking residues at the 4- and 7-positions, it should be understood that other linkages (as described above) could replace the thioether bridge and that other residues could be cyclized. A thioether bridge is also referred to as a monosulfide bridge or, in the case of Ala-S-Ala, as a lanthionine bridge. Thioether bridgecontaining peptides can be formed by two amino acids having one of the following formulas:

[0113] In these formulae, R1, R2, R3, R4, R5and R6are independently -H, an alkyl (e.g. , Ci-Ce alkyl, C1-C4 alkyl) or an aralkyl group, where the alkyl and aralkyl groups are optionally substituted with one or more halogen, -OH or -NRR’ groups (where R and R’ are independently -H or C1-C4 alkyl). In certain embodiments, R1, R2, R3, R4, R5and R6are each independently -H or -CH3, such where all are -H.

[0114] In certain embodiments, the invention provides an Ang analog or derivative comprising a thioether bridge according to formula (I). Typically, R1, R2, R3and R4are independently selected from -H and -CH3. Peptides comprising a thioether bridge according to formula (I) can be produced, for example, by lantibiotic enzymes or by sulfur extrusion of a disulfide. In one example, the disulfide from which the sulfur is extruded can be formed by D-cysteine in position 4 and L- cysteine in position 7 or by D-cysteine in position 4 and L-penicillamine in position 7 (see, e.g., Galande, Trent and Spatola (2003) Biopolymers 71, 534-551).

[0115] In other embodiments, the linkage of the two amino acids can be the bridges depicted in Formula (II) or Formula (III). Peptides comprising a thioether bridge according to Formula (II) can be made, for example, by sulfur extrusion of a disulfide formed by D-homocysteine in position 4 and L-cysteine in position 7. Similarly, peptides comprising a thioether bridge as in Formula (III) can be made, for example, by sulfur extrusion of a disulfide formed by D-cysteine in position 4 and L- homocysteine in position 7.

[0116] As discussed above, the Ang analogs and derivatives of the invention vary in length and amino acid composition. The Ang analogs and derivatives of the invention preferably have biological activity or are an inactive precursor molecule (inactive with regard to Ang-(l-7) function, the precursor may nevertheless have other function and be active in that regard) that can be proteolytically activated (such as how angiotensin(I), with 10 amino acids, is converted to active fragments by cleavage of 2 amino acids). The size of an Ang analog or derivative can vary but is typically between from about 5 to 10 amino acids, as long as the "core" pentameric segment comprising the 3-7 Nle-thioether-ring structure is encompassed. The amino acid sequence of an analog or derivative of the invention can vary, typically provided that it is biologically active or can become proteolytically activated. Generally, a precursor of Ang-(l-7) or a functional variant thereof is a molecule, preferably a peptide, which can be proteolytically activated; e.g. like Ang- (1-7) is generated by the cleavage of Angiotensin II by ACE2, by the cleavage of Angiotensin I by neprilysin (NEP), or by the cleavage of Ang-(l-9) by ACE / NEP). Thus, the precursor is a molecule that can be proteolytically activated to have the function of being a Mas and / or MrgD ligand, more specifically to have the functions described herein for the functional variant. The proteolytical activation, which preferably is a proteolytical cleavage, results in Ang-(f-7) or the functional variant thereof.

[0117] The precursor can be of any length. It may be 6-25 amino acids (e.g., 6-20, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7 amino acids) or preferably 7-25 amino acids (e.g., 7-20, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 7 amino acids) long, but more preferably it is from 8-25 amino acids (e.g., 8-20, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8- 9, 8 amino acids), even more preferably from 9-25 amino acids (e.g., 9-20, 9-15, 9-14, 9- 13, 9-12, 9-11, 9-10, 9 amino acids) or even more preferably 10-25 amino acids (e.g., 10- 20, 10-15, 10-14, 10-13, 10-12, 10-11, 10 amino acids) long, narrower ranges being preferred. In some embodiments, the precursor is 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long. In embodiments in which it is 6 or 7 amino acids long, it preferably has a deletion of the first two or the first amino acid position(s), respectively, corresponding to positions 1 and 2 of the heptamers described herein (e.g. SEQ ID NO: 1). In other words, the precursor preferably comprises amino acids downstream of the position corresponding to position 7 of the heptamers described herein, that can be cleaved off.

[0118] In a specific embodiment, the precursor is a peptide having a sequence of Asp1- Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9(SEQ ID NO: 19), which is the sequence of Ang-(l-9). The functional variant is a molecule, preferably a peptide, which is functional substantially as Ang-(l-7). It encompasses analogs and derivatives described herein, in particular those which are not precursors as defined above. All definitions and embodiments described herein for analogs and derivatives apply to functional variants, unless the context dictates otherwise. “Functional” means, in line with the characterization of Ang-(l-7), that the variant is capable of activating Mas receptor, MrgD receptor and / or AT2 receptor. Preferably, like Ang-(l-7), it may also have an activity such as nitric oxide release, vasodilation, improved endothelial function, antidiuresis, or one of the other properties discussed herein. The biological activity of a functional variant can be determined using methods known in the art, including radioligand binding studies, in vitro cell activation assays and in vivo experiments. See, for example, Godeny and Sayeski, (2006) Am. J. Physiol. Cell. Physiol. 291 :C 1297-1307; Sarr et al., Cardiovasc. Res. (2006) 71 :794-802; and Koziarz et al., (1993) Gen. Pharmacol. 24:705- 713. It should be understood that the functional variant (or the precursor) is one which has one or more functions of Ang- (1-7).

[0119] The functional variant can be of any length, preferably it is from 4-25 amino acids (e.g., 4-20, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7 amino acids), preferably from

[0120] 5-25 amino acids (e.g., 5-20, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7 amino acids), more preferably from 6-25 amino acids (e.g., 6-20, 6-15, 6-14, 6-13, 6-12, 6-11,

[0121] 6-10, 6-9, 6-8, 6-7 amino acids) or even more preferably 7-25 amino acids (e.g., 7-20, 7- 15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 7 amino acids) long, narrower ranges being preferred. In some embodiments, the functional variant is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long. In a preferred embodiment, the length of the functional variant is from 5 to 10 amino acids, preferably as long as the core pentameric segment comprising positions 4-7 of the heptamers described herein, e.g. SEQ ID NO: 1 or the 4-7 Nle-thioether-ring structure described below, is encompassed.

[0122] Typically, the functional variant shares amino acid sequence identity with Ang- (1-7). Preferably, the functional variant contains a sequence that includes at least 3 (e.g., at least 4, at least 5, at least 6, or at least 7) amino acids from the seven amino acids of Ang-(l-7) in the same order. The functional variant of Ang-(l-7) can also be defined to have a sequence at least 50% (e.g., at least 60%, 70%, 80%, 85%, or 90%) identical to the amino acid sequence of Ang-(l-7). The functional variant may contain amino acid substitutions, deletions and / or insertions in the sequence of Ang-(l-7). For example, one or more amino acid residues within Ang-(l-7) can be substituted by another amino acid. Preferred positions of Ang-(l-7) for substitution are 1, 4 and / or 7 (e.g. 1, 4, 7, 1+4, 1+7, 4+7 or 1+4+7). Substitution for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs (conservative substitution), as described elsewhere herein.

[0123] The functional variant may also comprise one or more substitutions with D-amino acids and / or non-natural (i.e. un-natural) amino acids, which are described in more detail elsewhere herein. The functional variant may also comprise substitution of non-naturally- occurring amino acids for natural amino acids, which are described in more detail elsewhere herein. The functional variant can be generally represented by the following sequence: Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7, which is described in more detail elsewhere herein. In a preferred embodiment, the functional variant is a non-cyclic peptide, i.e. a linear peptide. An example of such a linear peptide is SEQ ID NO: 20. Particular preferred examples are SEQ ID NO: 2 and SEQ ID NO: 3 (in linear but also in cyclic form). However, the functional variant may instead be a cyclic peptide, as described in more detail elsewhere herein. The functional variant, while typically a peptide, may also be a peptide mimetic, as described in more detail elsewhere herein. The functional variant does not comprise Ang-(l-8), i.e. amino acids 1-8 of SEQ ID NO: 4. Preferably, it also does not comprise amino acids 1-10, 1-11 and / or 1-12 of SEQ ID NO: 11.

[0124] Ang analogs and derivatives where only the length of the peptide is varied include the following: a 4,7-cyclized analog designated [Cyc4'7]Ang-(l-7), which is derived from natural Ang-(l-7) (Asp1-Arg2-Val3-Cyc4-Ile5-His6-Cyc7, SEQ ID NO: 6). a 4,7-cyclized analog designated [Nle3, Cyc4'7]Ang-(l-10), which is derived from natural Angiotensin I (Ang-(l-lO)) (Asp1-Arg2-Nle3-Cyc4-Ile5-His6-Cyc7-Phe8-His9- Leu10, SEQ ID NO: 7); a 4,7-cyclized analog designated [Nle3, Cyc4'7]Ang-(l-8), which is derived from natural Angiotensin II (Ang-(l-8)) (Asp1-Arg2-Nle3-Cyc4-Ile5-His6-Cyc7-Phe8, SEQ ID NO: 8); a 4,7-cyclised analog designated [Nle3, Cyc4'7]Ang-(2-8), which is derived from natural Angiotensin III (Ang-(2-8)) (Arg2-Nle3-Cyc4-Ile5-His6-Cyc7-Phe8, SEQ ID NO: 9); a 4,7-cyclised analog designated [Nle3, Cyc4'7]Ang-(3-8), which is derived from natural Angiotensin IV (Ang-(3-8)) (Nle3-Cyc4-Ile5-His6-Cyc7-Phe8, SEQ ID NO: 10); a 4,7-cyclised analog designated [Nle3, Cyc4'7]Ang-(l-7) derived from natural Ang- (1-7) (Asp1-Arg2-Nle3-Cyc4-Ile5-His6-Cyc7, SEQ ID NO: 11); and a4,7-cyclised analog designated [Nle3, Cyc4'7]Ang-(l-9) derived from natural Ang-(1- 9) (Asp1- Arg2-Nle3-Cyc4-Ile5-His6-Cyc7-Phe8-His9, SEQ ID NO: 12).

[0125] These analogs can have one of the thioether bridges shown in Formulae (I)-(III) as the Cyc4'7moiety, for example, where Cyc4and Cyc7are represented by Formula (I), such as where R'-p4are each -H or -CH3, typically -H.

[0126] As compared to the amino acid sequence of the natural angiotensin peptide, the amino acids at positions 4 and 7 of the Cyc4'7analog are modified to allow introduction of the thioether-ring structures shown above. In addition to the length of the Ang analogs, the amino acids at positions other than 3, 4 and 7 can be the same or different from the naturally-occurring peptide, typically provided that the analog retains a biological function. For analogs of inactive precursors, like [Cyc4'7]Ang-(l- 10), biological function refers to one or both of an analog’s susceptibility to angiotensin-converting enzymes that can cleave it to a biologically active fragment (e.g. Ang-(l-8) or Ang-(l-7)) or the biological activity of the fragment itself. In certain embodiments, an Ang analog or derivative of the invention has no intrinsic function but inhibits the effects of one or more naturally- occurring angiotensin compounds.

[0127] In certain embodiments, an Ang analog of the invention is represented by Formula (IV): Xaa1-Xaa2-Xaa3-Cyc4-Xaa5-Xaa6-Cyc7(IV).

[0128] Generally, Cyc herein refers to an amino acid position mediating cyclizisation. Xaa1is any amino acid, but typically a negatively-charged amino acid such as Glu or Asp, more typically Asp. Xaa2is a positively-charged amino acid such as Arg or Lys, typically Arg. Xaa3is an aliphatic amino acid, such as Leu, He or Vai, typically Vai. Cyc4forms a thioether bridge in conjunction with Cyc7. Cyc4can be a D-stereoisomer and / or a L-stereoisomer, typically a D-stereoisomer. Examples of Cyc4(taken with Cyc7) are shown in Formulas (I), (II) and (III). Typically, the R groups in Formulae (I), (II) and (III) are -H or -CH3, especially -H. Xaa5is an aliphatic amino acid, such as Leu, He or Vai, typically He. Xaa6is His. Cyc7forms a thioether bridge in conjunction with Cyc4, such as in Formula (I), (II) or (III). Cyc7can be a D- stereoisomer and / or a L-stereoisomer, typically a L-stereoisomer. Examples of Cyc7(taken with Cyc4) are shown in Formulas (I), (II), (III) and (IV). Typically, the R groups in Formulae (I), (II),) and (III) and (IV) are -H or -CH3, especially -H.

[0129] In certain embodiments, one or more of Xaa '-Xaa6(excluding Cyc4and Cyc7) is identical to the corresponding amino acid in naturally-occurring Ang-(l-7). In certain such embodiments, all but one or two of Xaa^Xaa6are identical to the corresponding amino acid in naturally-occurring Ang-(l-7). In other embodiments, all of Xaax-Xaa6are identical to the corresponding amino acid in naturally-occurring Ang-(l-7). In certain embodiments, Cyc4and Cyc7are independently selected from Abu

[0130] (2- aminobutyric acid) and Ala (alanine), where Ala is present in at least one position. Thus, cyclic analogs can have a thioether linkage formed by -Ala4-S-Ala7- (Formula (I), where R'-R4are each -H); - Ala4-S-Abu7- (Formula (I): R'-R3are -H and R4is - CH3) or -Abu4-S-Ala7- (Formula (I): R1, R3and R4are -H and R2is -CH3). Specific examples of cyclic analogs comprise a -Abu4-S-Ala7- or -Ala4-S-Ala7- linkage.

[0131] In certain embodiments, the invention provides an Ang-(l-7) analog with a thioether-b ridge between position 4 and position 7 having the amino acid sequence Asp1-Arg2-Val3- Abu4-Ile5-His6-Ala7(SEQ ID NO: 13) or the amino acid sequence Asp1-Arg2-Val3-Ala4-Ile5-His6-Ala7(SEQ ID NO: 14), which are represented by the following structural diagrams (specifically formulae):

[0132] In certain embodiments, an Ang analog or derivative of the invention is represented by Formula (V): Xaa1-Xaa2-Nle3-Cyc4-Xaa5-Xaa6-Cyc7-Xaa8-Xaa9- Xaa10(V).

[0133] As discussed above, one or more of Xaa1, Xaa2, Xaa8, Xaa9and Xaa10are absent in certain embodiments. For example, (1) Xaa10is absent, (2) Xaa9and Xaa10are absent, (3) Xaa8, Xaa9and Xaa10are absent, (4) Xaa1is absent, (5) Xaa1and Xaa10are absent, (6) Xaa1, Xaa9and Xaa10are absent, (7) Xaa1, Xaa8, Xaa9and Xaa10are absent, (8) Xaa1and Xaa2are absent, (9) Xaa1, Xaa2and Xaa10are absent, (10) Xaa1, Xaa2, Xaa9and Xaa10are absent, or (11) Xaa1, Xaa2, Xaa8, Xaa9and Xaa10are absent. For each of these embodiments, the remaining amino acids have the values described below.

[0134] Xaa1, when present, is any amino acid, but typically a negatively charged amino acid such as Glu or Asp, more typically Asp. Xaa2, when present, is a positively charged amino acid such as Arg or Lys, typically Arg. Nle3is norleucine. Cyc4forms a thioether bridge in conjunction with Cyc7. Cyc4can be a D- stereoisomer and / or a L- stereoisomer, typically a D-stereoisomer. Examples of Cyc4(taken with Cyc7) are shown in Formulas (I), (II) and (III). Typically, the R groups in Formulae (I), (II) and (III) are -H or -CH3, especially -H. Xaa5is an aliphatic amino acid, such as Leu, Nle, He or Vai, typically He. Xaa6is His. Cyc7forms a thioether bridge in conjunction with Cyc4, such as in Formula (I), (II) or (III). Cyc7can be a D-stereoisomer and / or a L-stereoisomer, typically a L-stereoisomer. Examples of Cyc7(taken with Cyc4) are shown in Formulas (I), (II) and (III). Typically, the R groups in Formulae (I), (II) and (III) are -H or -CH3, especially -H. Xaa8, when present, is an amino acid other than Pro, typically Phe or He. In certain embodiments, He results in an inhibitor of Ang(l-8). In certain embodiments, Phe maintains the biological activity of Ang(l-8) or Ang(l- 10). Xaa9, when present, is His.

[0135] Xaa10, when present, is an aliphatic residue, for example, He, Vai or Leu, typically Leu.

[0136] In certain embodiments, one or more of Xaa^Xaa10(excluding Nle3, Cyc4and Cyc7) is identical to the corresponding amino acid in naturally-occurring Ang (including Ang-(l-7), Ang(l-8), Ang(l-9), Ang(l-lO), Ang(2-7), Ang(2-8), Ang(2-9), Ang(2- 10), Ang(3-8), Ang(3-9) and Ang(3-10). In certain such embodiments, all but one or two of Xaa^Xaa10(for those present) are identical to the corresponding amino acid in naturally-occurring Ang. In other embodiments, all of Xaa^Xaa10(for those present) are identical to the corresponding amino acid in naturally-occurring Ang.

[0137] In certain embodiments, Cyc4and Cyc7are independently selected from Abu (2- aminobutyric acid) and Ala (alanine), where Ala is present at least one position. Thus, encompassed are cyclic analogs comprising a thioether linkage formed by - Ala4-S-Ala7- (Formula (I), where R'-R4are each -H); -Ala4-S-Abu7- (Formula (I): R1- R3are -H and R4is -CH3) or -Abu4-S-Ala7- (Formula (I): R1, R3and R4are -H and R2is -CH3). Specific cyclic analogs comprise a -Abu4-S-Ala7- or -Ala4-S-Ala7- linkage.

[0138] In particular, the invention provides an Ang-(l-7) analog or derivative with a thioether-b ridge between position 4 and position 7 having the amino acid sequence Asp'-Arg2-Nle3- Abu4-He5-His6-Ala7(SEQ ID NO: 15) or the amino acid sequence Asp1-Arg2-Nle3-Ala4-He5-His6-Ala7(SEQ ID NO: 16).

[0139] In another aspect, the invention provides an Ang-(l-8) analog or derivative with a thioether-b ridge between position 4 and position 7 having Ang-(l-8) antagonistic activity, in particular an Ang(l-8) analog or derivative having the amino acid sequence Asp1-Arg2-Nle3-Abu4-He5- His6-Ala7-He8(SEQ ID NO: 17), or the amino acid sequence Asp1-Arg2-Nle3-Ala4-He5-His6-Ala7-He8(SEQ ID NO: 18).

[0140] An alkyl group is a straight chained or branched non-aromatic hydrocarbon that is completely saturated. Typically, a straight chained or branched alkyl group has from 1 to about 20 carbon atoms, preferably from 1 to about 10. Examples of straight chained and branched alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n- butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl and octyl. A C1-C4 straight chained or branched alkyl group is also referred to as a "lower alkyl" group.

[0141] An aralkyl group is an alkyl group substituted by an aryl group. Aromatic (aryl) groups include carbocyclic aromatic groups such as phenyl, naphthyl, and anthracyl, and heteroaryl groups such as imidazolyl, thienyl, furyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl, pyrrolyl, pyrazinyl, thiazolyl, oxazolyl, and tetrazolyl. Aromatic groups also include fused polycyclic aromatic ring systems in which a carbocyclic aromatic ring or heteroaryl ring is fused to one or more other heteroaryl rings. Examples include benzothienyl, benzofuryl, indolyl, quinolinyl, benzothiazole, benzoxazole, benzimidazole, quinolinyl, isoquinolinyl andisoindolyl.

[0142] A ng (1-7) Receptor Agonists

[0143] In some embodiments, the present invention provides methods of treating the autoimmune blistering skin disease, preferably pemphigus or pemphigoid disease including administering to a subject who is suffering from the disease an angiotensin (1-7) receptor agonist. As used herein, the term “angiotensin-(l-7) receptor agonist” encompasses any molecule that has a positive impact in a function of an angiotensin- (1-7) receptor, e.g. Mas receptor and / or MrgD receptor, in particular the G-protein coupled Mas receptor. In some embodiments, an angiotensin- (1-7) receptor agonist directly or indirectly enhances, strengthens, activates and / or increases an angiotensin- (1-7) receptor (in particular the Mas receptor) activity. In some embodiments, an angiotensin- (1-7) receptor agonist directly interacts with an angiotensin-(l-7) receptor (in particular the Mas receptor). Such agonists can be peptidic or non- peptidic including, e.g., proteins, chemical compounds, small molecules, nucleic acids, antibodies, drugs, ligands, or other agents. In some embodiments, the angiotensin (1-7) receptor agonist is a non-pepti die agonist. An exemplary class of angiotensin-(l-7) receptor agonists are l-(p- thienylbenzyl)imidazoles. Examples of these non-peptide angiotensin-(l-7) receptor agonists are represented by Structural Formula (VI): or pharmaceutically acceptable salts thereof, wherein:

[0144] R1is halogen, hydroxyl, (Ci-C4)-alkoxy, (Ci-C8)-alkoxy wherein 1 to 6 carbon atoms are replaced by the heteroatoms O, S, or NH (preferably by O), (Ci-C4)-alkoxy substituted by a saturated cyclic ether such as tetrahydropyran or tetrahydrofuran, O- (Ci-C4)-alkenyl, O-(Ci-C4)-alkylaryl, or aryloxy that is unsubstituted or substituted by a substituent selected from halogen, (Ci-Cs)-alkyl, (Ci- Cs)-alkoxy and tri fluoromethyl;

[0145] R2is CHO, COOH, or (3) CO-O-(Ci-C4)-alkyl; R3is (Ci-C4)-alkyl or aryl; R4is hydrogen, halogen (chloro, bromo, fluoro), or (Ci-C4)-alkyl; X is oxygen or sulfur;

[0146] Y is oxygen or -NH-; R5is hydrogen, (Ci-C6)-alkyl; or (Ci-C4)-alkylaryl, where R5is hydrogen when Y is -NH-; and R6is (Ci-Cs)-alkyl.

[0147] In certain embodiments, R1is not halogen when R2is COOH or CO-O-(Ci- C4)-alkyl.

[0148] In some embodiments, an angiotensin-(l-7) receptor agonist is AVE 0991, 5- formyl-4-methoxy-2-phenyl-l[[4-[2-(ethylaminocarbonylsulfonamido)-5-isobutyl- 3-thienyl]-phenyl]- methyl]-imidazole, which is represented by the following structure: Another exemplary class of angiotensin-(l-7) receptor agonists are p- thienylbenzylamides. Examples of these non-peptide angiotensin-(l-7) receptor agonists are represented by Structural Formula (VII): or a pharmaceutically acceptable salt thereof, wherein:

[0149] R1is (Ci-Cs)-alkyl that is unsubstituted or substituted by a radical chosen from NH2, halogen, O-(Ci-C3)-alkyl, CO-O-(Ci-C3)-alkyl and CO2H, (C3-C8)-cycloalkyl, (Ci- C3)-alkyl-(C3-Cs)-cycloalkyl, (Ce-Cio)- aryl that is unsubstituted or substituted by a radical chosen from halogen and O-(Ci-C3)-alkyl, (Ci-C3)- alkyl-(C6-Cio)-aryl where the aryl radical is unsubstituted or substituted by a radical chosen from halogen and

[0150] O-(Ci-C3)-alkyl, (Ci-Cs)-heteroaryl, or (Ci-C3)-alkyl-(Ci-C5)-heteroaryl; R2is hydrogen, (Ci-C6)-alkyl that is unsubstituted or substituted by a radical chosen from halogen and O-(Ci-C3)-alkyl, (C3-Cs)-cycloalkyl, (Ci-C3)-alkyl-(C3-Cs)-cycloalkyl, (C6-Cio)-aryl that is unsubstituted or substituted by a radical chosen from among halogen, O-(Ci-C3)-alkyl andCO-O-(Ci-C3)-alkyl, or (Ci-C3)-alkyl-(C6-Cio)-aryl that is unsubstituted or substituted by a radical chosen from halogen and O-(Ci-C3)-alkyl; R3is hydrogen, COOH, or COO-(Ci-C4)-alkyl; R4is hydrogen, halogen; or (C1-C4)- alkyl; R5is hydrogen or (Ci-C6)-alkyl; R6is hydrogen, (Ci-C6)-alkyl, (Ci-C3)-alkyl- (C3-Cs)-cycloalkyl, or (C2-C6)-alkenyl; and X is oxygen orNH. Additional examples of angiotensin-(l-7) receptor agonists are described in

[0151] U.S. Patent No. 6,235,766, the contents of which are incorporated by reference herein.

[0152] Various angiotensin-(l-7) receptor agonists described above can be present as pharmaceutically acceptable salts. As used herein, “a pharmaceutically acceptable salt” refers to salts that retain the desired activity of the peptide or equivalent compound, but preferably do not detrimentally affect the activity of the peptide or other component of a system, which uses the peptide. Examples of such salts are acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like. Salts may also be formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, and the like. Salts formed from a cationic material may utilize the conjugate base of these inorganic and organic acids. Salts may also be formed with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel and the like or with an organic cation formed from N,N'- dibenzylethylenediamine or ethylenediamine, or combinations thereof (e.g., a zinc tannate salt).

[0153] The non-toxic, physiologically acceptable salts are preferred.

[0154] The salts can be formed by conventional means such as by reacting the free acid or free base forms of the product with one or more equivalents of the appropriate acid or base in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is then removed in vacuo or by freeze-drying, or by exchanging the cations of an existing salt for another cation on a suitable ion exchange resin. An alkyl group is a straight chained or branched non-aromatic hydrocarbon that is completely saturated. Typically, a straight chained or branched alkyl group has from 1 to about 20 carbon atoms, preferably from 1 to about 10. Examples of straight chained and branched alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n- butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl and octyl. A C1-C4 straight chained or branched alkyl group is also referred to as a “lower alkyl” group.

[0155] An alkenyl group is a straight chained or branched non-aromatic hydrocarbon that is includes one or more double bonds. Typically, a straight chained or branched alkenyl group has from 2 to about 20 carbon atoms, preferably from 2 to about 10. Examples of straight chained and branched alkenyl groups include ethenyl, n- propenyl, and n-butenyl.

[0156] Aromatic (aryl) groups include carbocyclic aromatic groups such as phenyl, naphthyl, and anthracyl, and heteroaryl groups such as imidazolyl, thienyl, furyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl, pyrrolyl, pyrazinyl, thiazolyl, oxazolyl, and tetrazolyl. Aromatic groups also include fused polycyclic aromatic ring systems in which a carbocyclic aromatic ring or heteroaryl ring is fused to one or more other heteroaryl rings. Examples include benzothienyl, benzofuryl, indolyl, quinolinyl, benzothiazole, benzoxazole, benzimidazole, quinolinyl, isoquinolinyl and isoindolyl.

[0157] An aralkyl group is an alkyl group substituted by an aryl group.

[0158] Formulations and Dosing

[0159] In accordance with the methods of the invention, an Ang (1-7) peptide or angiotensin (1-7) receptor agonist as described herein of the invention can be administered to a subject alone (e.g, as a purified peptide or compound), or as a component of a composition (in particular a pharmaceutical composition) or medicament (e.g, in the manufacture of a medicament for the treatment of the disease), as described herein or otherwise known in the art. The compositions can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition. The carrier and composition can be sterile. The formulation should suit the mode of administration, for example intravenous or subcutaneous administration. Methods of formulating compositions are known in the art (see, e.g, Remington's Pharmaceuticals Sciences, 17th Edition, Mack Publishing Co., (Alfonso R. Gennaro, editor) (1989)).

[0160] Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g, NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, sugars such as mannitol, sucrose, or others, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof. The pharmaceutical preparations can, if desired, be mixed with auxiliary agents (e.g, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring and / or aromatic substances and the like), which do not deleteriously react with the active compounds or interference with their activity. In a preferred embodiment, a water-soluble carrier suitable for intravenous administration is used.

[0161] The composition or medicament, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, sustained release formulation, or powder. The composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides.

[0162] The composition or medicament can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings. For example, in some embodiments, a composition for intravenous administration typically is a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose / water. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0163] An Ang (1-7) peptide or angiotensin (1-7) receptor agonist as described herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0164] An Ang (1-7) peptide or angiotensin (1-7) receptor agonist as described herein (or a composition or medicament containing an Ang (1-7) peptide or angiotensin (1- 7) receptor agonist described herein) is administered by any appropriate route. In some embodiments, an Ang (1-7) peptide or angiotensin (1-7) receptor agonist described herein is administered subcutaneously. As used herein, the term “subcutaneous tissue”, is defined as a layer of loose, irregular connective tissue immediately beneath the skin. For example, the subcutaneous administration may be performed by injecting a composition into areas including, but not limited to, thigh region, abdominal region, gluteal region, or scapular region. In some embodiments, an Ang (1-7) peptide or angiotensin (1-7) receptor agonist described herein is administered intravenously. Alternatively, an Ang (1-7) peptide or angiotensin (1-7) receptor agonist described herein (or a composition or medicament containing an Ang (1-7) peptide or angiotensin (1-7) receptor agonist described herein) can be administered by inhalation, parenterally, intradermally, transdermally, rectally, or transmucosally. In some embodiments, an Ang(l-7) peptide or angiotensin (1-7) receptor agonist is administered orally. More than one route can be used concurrently, if desired.

[0165] In some embodiments, a composition is administered in a therapeutically effective amount and / or according to a dosing regimen that is correlated with a particular desired outcome (e.g, with treating or reducing risk for an autoimmune blistering skin disease, preferably pemphigus or pemphigoid disease). Particular doses or amounts to be administered in accordance with the present invention may vary, for example, depending on the nature and / or extent of the desired outcome, on particulars of route and / or timing of administration, and / or on one or more characteristics (e.g., weight, age, personal history, genetic characteristic, lifestyle parameter, severity of cardiac defect and / or level of risk of cardiac defect, etc., or combinations thereof). Such doses or amounts can be determined by those of ordinary skill. In some embodiments, an appropriate dose or amount is determinedin accordance with standard clinical techniques. For example, in some embodiments, an appropriate dose or amount is a dose or amount sufficient to reduce a disease severity index score by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more. For example, in some embodiments, an appropriate dose or amount is a dose or amount sufficient to reduce a disease severity index score by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%. Alternatively or additionally, in some embodiments, an appropriate dose or amount is determined through use of one or more in vitro or in vivo assays to help identify desirable or optimal dosage ranges or amounts to be administered.

[0166] In various embodiments, an Ang (1-7) peptide or angiotensin (1-7) receptor agonist is administered at a therapeutically effective amount. As used herein, the term “therapeutically effective amount” is largely determined based on the total amount of the therapeutic agent contained in the pharmaceutical compositions of the present invention. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (e.g, treating, modulating, curing, preventing and / or ameliorating the underlying disease or condition). In some particular embodiments, appropriate doses or amounts to be administered may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0167] Therapeutically effective dosage amounts of angiotensin (1-7) peptides or angiotensin (1-7) receptor agonists, including derivatives, analogs, and / or salts may be present in varying amounts in various embodiments. For example, in some embodiments, a therapeutically effective amount of an angiotensin (1-7) peptide may be an amount ranging from about 10-1,000 mg (e.g., about 20 mg - 1,000 mg, 30 mg

[0168] - 1,000 mg, 40 mg - 1,000 mg, 50 mg - 1,000 mg, 60 mg - 1,000 mg, 70 mg - 1,000 mg, 80 mg - 1,000 mg, 90 mg - 1,000 mg, about 10-900 mg, 10-800 mg, 10-700 mg, 10-600 mg, 10-500 mg, 100-1,000 mg, 100-900 mg, 100-800 mg, 100-700 mg, 100-

[0169] 600 mg, 100-500 mg, 100-400 mg, 100-300 mg, 200-1,000 mg, 200-900 mg, 200- 800 mg, 200-700 mg, 200-600 mg, 200-500 mg, 200-400 mg, 300-1,000 mg, 300- 900 mg, 300-800 mg, 300-700 mg, 300-600 mg, 300-500 mg, 400 mg - 1,000 mg, 500 mg - 1,000 mg, 100 mg - 900 mg, 200 mg - 800 mg, 300 mg - 700 mg, 400 mg

[0170] - 700 mg, and 500 mg - 600 mg). In some embodiments, an angiotensin (1-7) peptide or angiotensin (1-7) receptor agonist is present in an amount of or greater than about 10 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg. In some embodiments, an angiotensin (1-7) peptide or angiotensin (1-7) receptor agonist is present in an amount of or less than about 1000 mg, 950 mg, 900 mg, 850 mg, 800 mg, 750 mg, 700 mg, 650 mg, 600 mg, 550 mg, 500 mg, 450 mg, 400 mg, 350 mg, 300 mg, 250 mg, 200 mg, 150 mg, or 100 mg. In some embodiments, the therapeutically effective amount described herein is provided in one dose. In some embodiments, the therapeutically effective amount described herein is provided in one day.

[0171] In other embodiments, a therapeutically effective dosage amount may be, for example, about 0.001 mg / kg weight to 500 mg / kg weight, e.g., from about 0.001 mg / kg weight to 400 mg / kg weight, from about 0.001 mg / kg weight to 300 mg / kg weight, from about 0.001 mg / kg weight to 200 mg / kg weight, from about 0.001 mg / kg weight to 100 mg / kg weight, from about 0.001 mg / kg weight to 90 mg / kg weight, from about 0.001 mg / kg weight to 80 mg / kg weight, from about 0.001 mg / kg weight to 70 mg / kg weight, from about 0.001 mg / kg weight to 60 mg / kg weight, from about 0.001 mg / kg weight to 50 mg / kg weight, from about 0.001 mg / kg weight to 40 mg / kg weight, from about 0.001 mg / kg weight to 30 mg / kg weight, from about 0.001 mg / kg weight to 25 mg / kg weight, from about 0.001 mg / kg weight to 20 mg / kg weight, from about O.OOl mg / kg weight to 15 mg / kg weight, from about O.OOl mg / kg weight to 10 mg / kg weight. In some embodiments, the therapeutically effective amount described herein is provided in one dose. In some embodiments, the therapeutically effective amount described herein is provided in one day.

[0172] In still other embodiments, a therapeutically effective dosage amount may be, for example, about 0.001 mg / kg weight to about 1 mg / kg weight, e.g. from about 0.001 mg / kg weight to about 0.9 mg / kg weight, from about 0.001 mg / kg weight to about 0.8 mg / kg weight, from about 0.001 mg / kg weight to about 0.8 mg / kg weight, from about 0.001 mg / kg weight to about 0.7 mg / kg weight, from about 0.001 mg / kg weight to about 0.6 mg / kg weight, from about 0.001 mg / kg weight to about 0.5 mg / kg weight, from about 0.01 mg / kg weight to about 1 mg / kg weight, from about 0.01 mg / kg weight to about 0.9 mg / kg weight, from about 0.01 mg / kg weight to about 0.8 mg / kg weight, from about 0.01 mg / kg weight to about 0.7 mg / kg weight, from about 0.01 mg / kg weight to about 0.6 mg / kg weight, from about 0.01 mg / kg weight to about 0.5 mg / kg weight, from about 0.02 mg / kg weight to about 1 mg / kg weight, from about 0.02 mg / kg weight to about 0.9 mg / kg weight, from about 0.02 mg / kg weight to about 0.8 mg / kg weight, from about 0.02 mg / kg weight to about 0.7 mg / kg weight, from about 0.02 mg / kg weight to about 0.6 mg / kg weight, from about 0.02 mg / kg weight to about 0.5 mg / kg weight, from about 0.03 mg / kg weight to about 1 mg / kg weight, from about 0.03 mg / kg weight to about 0.9 mg / kg weight, from about 0.03 mg / kg weight to about 0.8 mg / kg weight, from about 0.03 mg / kg weight to about 0.7 mg / kg weight, from about 0.03 mg / kg weight to about 0.6 mg / kg weight, from about 0.03 mg / kg weight to about 0.5 mg / kg weight, from about 0.04 mg / kg weight to about 1 mg / kg weight, from about 0.04 mg / kg weight to about 0.9 mg / kg weight, from about 0.04 mg / kg weight to about 0.8 mg / kg weight, from about 0.04 mg / kg weight to about 0.7 mg / kg weight, from about 0.04 mg / kg weight to about 0.6 mg / kg weight, from about 0.04 mg / kg weight to about 0.5 mg / kg weight, from about 0.05 mg / kg weight to about 1 mg / kg weight, from about 0.05 mg / kg weight to about 0.9 mg / kg weight, from about 0.05 mg / kg weight to about 0.8 mg / kg weight, from about 0.05 mg / kg weight to about 0.7 mg / kg weight, from about 0.05 mg / kg weight to about 0.6 mg / kg weight, from about 0.05 mg / kg weight to about 0.5 mg / kg weight. In some embodiments, the therapeutically effective amount described herein is provided in one dose. In some embodiments, the therapeutically effective amount described herein is provided in one day. In still other embodiments, a therapeutically effective dosage amount may be, for example, about 0.0001 mg / kg weight to 0.1 mg / kg weight, e.g. from about 0.0001 mg / kg weight to 0.09 mg / kg weight, from about 0.0001 mg / kg weight to 0.08 mg / kg weight, from about 0.0001 mg / kg weight to 0.07 mg / kg weight, from about 0.0001 mg / kg weight to 0.06 mg / kg weight, from about 0.0001 mg / kg weight to 0.05 mg / kg weight, from about 0.0001 mg / kg weight to about 0.04 mg / kg weight, from about 0.0001 mg / kg weight to 0.03 mg / kg weight, from about 0.0001 mg / kg weight to 0.02 mg / kg weight, from about 0.0001 mg / kg weight to 0.019 mg / kg weight, from about 0.0001 mg / kg weight to 0.018 mg / kg weight, from about 0.0001 mg / kg weight to 0.017 mg / kg weight, from about 0.0001 mg / kg weight to 0.016 mg / kg weight, from about 0.0001 mg / kg weight to 0.015 mg / kg weight, from about 0.0001 mg / kg weight to 0.014 mg / kg weight, from about 0.0001 mg / kg weight to 0.013 mg / kg weight, from about 0.0001 mg / kg weight to 0.012 mg / kg weight, from about 0.0001 mg / kg weight to 0.011 mg / kg weight, from about 0.0001 mg / kg weight to 0.01 mg / kg weight, from about 0.0001 mg / kg weight to 0.009 mg / kg weight, from about 0.0001 mg / kg weight to 0.008 mg / kg weight, from about 0.0001 mg / kg weight to 0.007 mg / kg weight, from about 0.0001 mg / kg weight to 0.006 mg / kg weight, from about 0.0001 mg / kg weight to 0.005 mg / kg weight, from about 0.0001 mg / kg weight to 0.004 mg / kg weight, from about 0.0001 mg / kg weight to 0.003 mg / kg weight, from about 0.0001 mg / kg weight to 0.002 mg / kg weight. In some embodiments, the therapeutically effective dose may be 0.0001 mg / kg weight, 0.0002 mg / kg weight, 0.0003 mg / kg weight, 0.0004 mg / kg weight, 0.0005 mg / kg weight, 0.0006 mg / kg weight, 0.0007 mg / kg weight, 0.0008 mg / kg weight, 0.0009 mg / kg weight, 0.001 mg / kg weight, 0.002 mg / kg weight, 0.003 mg / kg weight, 0.004 mg / kg weight, 0.005 mg / kg weight, 0.006 mg / kg weight, 0.007 mg / kg weight, 0.008 mg / kg weight, 0.009 mg / kg weight, 0.01 mg / kg weight, 0.02 mg / kg weight, 0.03 mg / kg weight, 0.04 mg / kg weight, 0.05 mg / kg weight, 0.06 mg / kg weight, 0.07 mg / kg weight, 0.08 mg / kg weight, 0.09 mg / kg weight, or 0.1 mg / kg weight. The effective dose for a particular individual can be varied (e.g., increased or decreased) over time, depending on the needs of the individual. In some embodiments, the angiotensin (1-7) peptide or angiotensin (1-7) receptor agonist is administered at an effective dose ranging from about 1-1,000 pg / kg / day (e.g, ranging from about 1-900 pg / kg / day, 1-800 pg / kg / day, 1-700 pg / kg / day, 1-600 pg / kg / day, 1-500 pg / kg / day, 1-400 pg / kg / day, 1-300 pg / kg / day, 1-200 pg / kg / day, 1- 100 pg / kg / day, 1-90 pg / kg / day, 1-80 pg / kg / day, 1-70 pg / kg / day, 1-60 pg / kg / day, 1- 50 pg / kg / day, 1-40 pg / kg / day, 1-30 pg / kg / day, 1-20 gg / kg / day, 1-10 gg / kg / day). In some embodiments, it is administered at an effective dose ranging from about 1-500 gg / kg / day. In some embodiments, the angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-100 gg / kg / day. In some embodiments, it is administered at an effective dose ranging from about 1-60 gg / kg / day. In some embodiments, it is administered at an effective dose selected from about 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000 gg / kg / day.

[0173] In some embodiments, the angiotensin (1-7) receptor agonist is administered at an effective dose ranging from about 1-1,000 gg / kg / day. In some embodiments, it is administered at an effective dose ranging from about 50-500 gg / kg / day. In some embodiments, it is administered at an effective dose ranging from about 1-60 gg / kg / day.

[0174] Combination Therapies

[0175] In some embodiments, an angiotensin (1-7) peptide or an angiotensin (1-7) receptor agonist will be used as a part of a combination therapy. In some embodiments, the angiotensin (1-7) peptide and / or receptor agonist may be administered prior to, concurrently with, or subsequent to one or more additional therapies. It is contemplated that any known therapy or therapeutic for the treatment of an autoimmune blistering skin disease, preferably pemphigus or pemphigoid disease may be used with one or more angiotensin (1-7) peptides and / or receptor agonists as disclosed herein. Exemplary therapies that may be used with one or more angiotensin (1-7) peptides or receptor agonists include, but are not limited to, medications that can help control pain and itching, medications that address complications such as sepsis (e.g., antibiotics), medications that reduce inflammation (e.g., corticosteroid), surgery to correct abnormal motion (e.g, surgery to correct fusing of finger or toes or abnormal bends in the j oints), surgery to improve the ability to eat a healthy diet (e.g., surgical dilation of the esophagus or placement of a feeding tube), skin grafts (e.g., OrCel composite cultured skin), gene therapy, bone marrow transplantation, protein replacement therapy, cell-based therapies, and / or combinations thereof, among others. Kits

[0176] In some embodiments, the present invention further provides kits or other articles of manufacture which contains an angiotensin (1-7) peptide, an angiotensin (1-7) receptor agonist or a formulation containing the same and provides instructions for its reconstitution (if lyophilized) and / or use. Kits or other articles of manufacture may include a container, a syringe, vial and any other articles, devices or equipment useful in administration (e.g., subcutaneous, by inhalation). Suitable containers include, for example, bottles, vials, syringes (e.g., pre-filled syringes), ampules, cartridges, reservoirs, or lyo-jects. The container may be formed from a variety of materials such as glass or plastic. In some embodiments, a container is a pre-filled syringe. Suitable pre-filled syringes include, but are not limited to, borosilicate glass syringes with baked silicone coating, borosilicate glass syringes with sprayed silicone, or plastic resin syringes without silicone.

[0177] Typically, the container may hold one or more formulations and a label on, or associated with, the container that may indicate directions for reconstitution and / or use. For example, the label may indicate that the formulation is reconstituted to concentrations as described above. The label may further indicate that the formulation is useful or intended for, for example, subcutaneous administration. In some embodiments, a container may contain a single dose of a stable formulation containing an angiotensin (1-7) peptide or angiotensin (1-7) receptor agonist. In various embodiments, a single dose of the stable formulation is present in a volume of less than about 15 ml, 10 ml, 5.0 ml, 4.0 ml, 3.5 ml, 3.0 ml, 2.5 ml, 2.0 ml, 1.5 ml, 1.0 ml, or 0.5 ml. Alternatively, a container holding the formulation may be a multiuse vial, which allows for repeat administrations (e.g, from 2-6 administrations) of the formulation. Kits or other articles of manufacture may further include a second container comprising a suitable diluent (e.g., BWFI, saline, buffered saline). Upon mixing of the diluent and the formulation, the final protein concentration in the reconstituted formulation will generally be at least 1 mg / ml (e.g., at least 5 mg / ml, at least 10 mg / ml, at least 20 mg / ml, at least 30 mg / ml, at least 40 mg / ml, at least 50 mg / ml, at least 75 mg / ml, at least 100 mg / ml). Kits or other articles of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fdters, needles, syringes, and package inserts with instructions for use. In some embodiments, kits or other articles of manufacture may include an instruction for self-administration. EXAMPLES

[0178] While certain compositions and methods of the present invention have been described with specificity in accordance with certain embodiments, the following example serves only to illustrate the compounds and methods of the invention, and it is not intended to limit the same. Example 1. Angiotensin (1-7) alleviates split formation in a human skin organ culture (HSOC) model of pemphigus and pemphigoid disease

[0179] A human skin organ culture (HSOC) model has been developed that reproduces the clinical phenotype of autoantibody-induced tissue pathology in pemphigus and pemphigoid disease. A recombinant single-chain variable fragment (scFv) targeting both desmoglein 1 (Dsg 1) and desmoglein 3 (Dsg3) is injected into pieces of human skin obtained surgically from abdomen or breast tissue, for example, to induce blistering. (Hartmann et al., Laboratory Animals, January 2023). The scFv injected alone is also referred to as a positive control herein for the model. As a negative control for the model, IVIG (intravenous immunoglobulins) were administered instead of the scFv. The human skin organ cultures were subsequently treated with different concentrations of angiotensin (1-7), either supplemented in the medium or injected. Angiotensin (1-7) was always administered in combination with the scFv. Samples were embedded in paraffin and cryomatrix, sectioned and immunostaining was performed on cryosections. Direct immunofluorescence was carried out for scFv. Immunostaining was quantified with ImageJ software on images obtained with identical microscope settings.

[0180] Split formation was measured and plotted in FIG. 1A-1C. relative to concentration of angiotensin (1-7), for example, from 0.01 pM to 1 pM or 100 pM. As a positive control, scFv to mimic autoantibodies was used. FIG. 1A shows percent split formation at various concentrations of angiotensin (1-7) from 0.01 pM to 100 pM supplemented in HSOC medium in HSOC without colored skin. The reduction in split formation was significant over control at all concentrations of angiotensin (1-7) when added to medium (p<0.0001, IVIG / scFV, IVIG / 0.01 pM, IVIG / 0.1 pM, IVIG / 1 pM, IVIG / 100 pM by Kruskal-Wallis test). FIG. IB shows percent split formation at various concentrations of angiotensin (1-7) from 0.01 pM to 100 pM injected in HSOC without colored skin. The reduction in split formation was significant over control at all concenrations of angiotensin (1-7) when injected (p<0.0001, IVIG / scFV, IVIG / 0.01 pM, IVIG / 0.1 pM, IVIG / 1 pM, IVIG / 100 pM by Kruskal-Wallis test). The reduction in split formation was significant over scFv at 0.1 pM concentration of angiotensin (1-7) when injected (p < 0.05, scFv / 0.1 uM by Mann-Whitney test).

[0181] Table 1. Percent Split formation upon Angiotensin (1-7) administration in HSOC (noncolored skin) FIG. 1C shows percent split formation at various concentrations of angiotensin (1-7) from 0.01 pM to 1 pM injected in HSOC with only colored skin. The reduction in split formation was significant over control in injected samples (p<0.001, IVIG / scFV, IVIG / 0.01 pM, IVIG / 0.1 pM, IVIG / 1 pM; p<0.01, scFV / 0.1 pM, scFV / 1 pM by Kruskal- Wallis test; p<0.05, scFV / 0.01 pM by Mann-Whitney test).

[0182] Table 2. Percent Split formation upon Angiotensin (1-7) injection in HSOC (colored skin)

[0183] FIG. 2 shows histopathogic analysis of split formation in the human skin organ culture model. While an scFv positive control against desmoglein 1 and 3 autoantibodies, shows pronounced split formation in the microscopic images, an angiotensin (1-7) injected sample shows decreased split formation, indicative of amelioration of an autoimmune blistering skin disease such as pemphigus and pemphigoid disease.

[0184] Overall, Angiotensin (1-7) treatment resulted in a significant reduction in split formation, indicative of treatment of an autoimmune blistering skin disease such as pemphigus and pemphigoid disease.

[0185] Example 2. Angiotensin (1-7) reduces skin lesions in a preclinical model of Epidermolysis Bullosa Acquisita.

[0186] The passive Epidermolysis Bullosa Acquisita (EBA) mouse model provides a robust and reproducible platform for investigating the immunopathogenesis of this antibody -mediated blistering disease of the skin and evaluating novel treatment strategies. The passive EBA mouse model is induced by the tail vein injection of anti -type VII collagen IgG antibodies, generated in rabbits by immunization against type VII collagen, into C57BL / 6 mice at day 0 and day 2. The daily subcutaneous injected treatment of the randomized mice with Angiotensin-(l-7) (lOOOpg / kg / day) or the equivalent amount of the control substance PBS starts at day 3 and is continued until day 11. The scoring as well as the blood collection is executed at day 4, 8 and 12, whereby the scoring at day 12 is completed with the organ removal. The scoring involves the evaluation of the general health and the area of affected skin in percent. As shown in Fig. 3A, administration of Angiotensin-(l-7) reduced the area of skin affected already from day 4 on, the first day of scoring. The difference to control became more pronounced over time. Fig. 3B shows the scoring on day 12 for individual mice (males and females) as well as mean values of the treatment groups. The reduction of affected skin area by administration of Angiotensin-(l-7) was highly significant (unpaired t test, p value = 0.0038).

[0187] EQUIVALENTS AND SCOPE

[0188] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims.

Claims

CLAIMS1. A method of treating an autoimmune blistering skin disease comprising administering an angiotensin (1-7) receptor agonist to a subject suffering from the autoimmune blistering skin disease.

2. The method of claim 1, wherein the administration is parenteral, rectal, oral, or a combination thereof, wherein the parenteral administration preferably is intravenous, subcutaneous, inhalation, intradermal, transdermal, and / or transmucosal administration.

3. The method of claim 1 or 2, wherein the autoimmune blistering skin disease is pemphigus, preferably selected from the group consisting of pemphigus vulgaris, pemphigus foliaceus, IgA pemphigus, paraneoplastic pemphigus, pemphigus vegetans, drug-induced pemphigus, pemphigus erythematosus (Senear-Usher syndrome), endemic pemphigus (fogo selvagem), benign familial pemphigus (Hailey-Hailey disease), and combinations thereof.

4. The method of claim 1 or 2, wherein the autoimmune blistering skin disease is pemphigoid, preferably selected from the group consisting of bullous pemphigoid (BP), mucous membrane pemphigoid (MMP, or cicatricial pemphigoid), pemphigoid gestationis, epidermolysis bullosa acquisita, linear IgA disease, anti-laminin gl / anti-p200 pemphigoid, lichen planus pemphigoides, pemphigoid disease characterised by renal insufficiency, and combinations thereof.

5. The method of any one of claims 1-4, wherein the angiotensin (1-7) receptor agonist is administered at an effective dose periodically at an administration interval such that at least one symptom or feature of the autoimmune blistering skin disease disease is reduced in intensity, severity, duration, or frequency or has delayed onset.

6. The method of claim 5, wherein the one or more symptoms of the autoimmune blistering skin disease is selected from ulcers, blisters, eczema, hives, and combinationsthereof, wherein the ulcers or blisters are preferably on skin or mucous membrane, and wherein the mucous membrane is preferably of mouth, nose, throat, eyes, or genitals.

7. The method of any one of claims 1-6, wherein the angiotensin (1-7) receptor agonist is administered once per day, once per week, three times per month, twice per month or once per month.

8. The method of any one of claims 1-7, wherein the angiotensin (1-7) receptor agonist is administered at an effective dose ranging from about 1-1,000 pg / kg / day, from about 50-500 pg / kg / day, or from about 1-60 pg / kg / day.

9. The method of any one of claims 1-8, wherein the angiotensin (1-7) receptor agonist is an angiotensin (1-7) peptide.

10. The method of claim 9, wherein the angiotensin (1-7) peptide is angiotensin (1- 7), a functional variant and / or a precursor thereof.

11. The method of claim 9, wherein angiotensin (1-7) has the amino acid sequence of Asp1-Arg2-Val3-Tyr4-Ile5- His6-Pro7(SEQ ID NO: 1).

12. The method of claim 10, wherein the functional variant of angiotensin (1-7) has the amino acid sequence of Asp1- Arg2-Val3-Ser4-Ile:’-His6-Cys7(SEQ ID NO: 2) or of Ala1- Arg2-Val3-Ser4-Ile5-His6-Cys7(SEQ ID NO: 3).

13. The method of any one of claims 9-12, wherein the angiotensin (1-7) peptide comprises one or more chemical modifications to increase protease resistance, serum stability and / or bioavailability, preferably, wherein the one or more chemical modifications comprise pegylation.

14. The method of any one of claims 1-8, wherein the angiotensin (1-7) receptor agonist is a non-peptidic angiotensin (1 -7) receptor agonist.

15. The method of claim 14, wherein the non-peptidic angiotensin (1-7) receptor agonist is a compound with the following structure:or a pharmaceutically acceptable salt thereof.

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