WO2016010844A1 - Methods and compositions for the treatment of stroke - Google Patents

Methods and compositions for the treatment of stroke Download PDF

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Publication number
WO2016010844A1
WO2016010844A1 PCT/US2015/039900 US2015039900W WO2016010844A1 WO 2016010844 A1 WO2016010844 A1 WO 2016010844A1 US 2015039900 W US2015039900 W US 2015039900W WO 2016010844 A1 WO2016010844 A1 WO 2016010844A1
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Prior art keywords
peptide
angiotensin
administered
stroke
potassium channel
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PCT/US2015/039900
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French (fr)
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WO2016010844A4 (en
Inventor
Richard Franklin
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Tarix Pharmaceuticals Ltd
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Publication of WO2016010844A1 publication Critical patent/WO2016010844A1/en
Publication of WO2016010844A4 publication Critical patent/WO2016010844A4/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene

Definitions

  • the present invention is based, in part, on the surprising discovery that administration of at least one angiotensin(1-7) peptide in combination with at least one potassium channel blocker (e.g., dalfampridine), results in a synergistic improvement in one or more signs, symptoms, and/or complications of brain conditions including, but not limited to, stroke, vascular dementia, and/or traumatic brain injury.
  • the present invention provides, among other things, new options in the treatment of brain conditions that can be administered not only after the "golden hour” (traditionally recognized as the hour following an event such as a stroke or other brain trauma when treatment is thought to have maximum effect), but significantly after that time, even days or weeks after an event, and still achieve a significant improvement in a subject's condition.
  • the present invention provides methods of treating brain conditions including the steps of administering to a subject suffering from or who previously suffered from a brain condition at least one angiotensin (1-7) peptide, and administering to the subject at least one potassium channel blocker.
  • the at least one angiotensin (1-7) peptide and at least one potassium channel blocker are administered substantially simultaneously.
  • the at least one angiotensin (1-7) peptide is administered prior to admini stration of the at least one potassium channel b locker.
  • the at least one potassium channel blocker is administered prior to administration of the at least one angiotensin (1-7) peptide, in some embodiments, the at least one angiotensin (1-7) peptide and at least one potassium channel blocker are administered in overlapping regimen.
  • the presen t invention provides methods of treating brain conditions including the step of administering to a subject suffering from or who previously suffered from a brain condition, and was administered an angiotensin(1-7) peptide, a potassium channel blocker.
  • the present invention provides methods of treating brain conditions including the step of administering to a subject suffering from or who previously suffered from a brain condition, and was administered a potassium channel blocker, an angiotensin(1-7) peptide.
  • Various embodiments may be used to treat any of a vari ety of brain conditions.
  • a brain condition is selected from stroke, vascular dementia, and traumatic brain injury.
  • stroke is selected from ischemic stroke, hemorrhagic stroke, or a combination thereof.
  • Provided methods and compositions may be administered according to a variety of timepoints after development of a brain condition (e.g., a stroke).
  • a brain condition e.g., a stroke
  • at least one of an angiotensin (1-7) peptide and a potassium channel blocker are administered within 24 hours of the development of a brain condition (e.g., stroke).
  • at least one of an angiotensin (1-7) peptide and a potassium channel blocker are administered at least 24 hours after the development of a brain condition (e.g., stroke).
  • At least one of an angiotensin (1-7) peptide and a potassium channel blocker are administered at least 1 week after the development of a brain condition (e.g., stroke). In some embodiments, at least one of an angiotensin (1 -7) peptide and a potassium channel blocker are administered at least 4 weeks after the development of a brain condition (e.g., stroke).
  • any potassium channel blocker may be used in accordance with provided methods and compositions.
  • a potassium channel blocker is an FDA-approved potassium channel blocker.
  • a potassium channel blocker is selected from dalfampridine, amiodarone, dronedarone, dofetilide, sotalol, ibutilide, azimilide, bretylium, clofilium, E-4031 , nifekalant, tedisamil, sematilide, and combinations thereof.
  • a potassium channel blocker is administered at a dose other than an FDA-approved dose.
  • a potassium channel blocker is administered according to a dosing regimen other than an FDA-approved dosing regimen.
  • Various embodiments may be administered via any medically appropriate route.
  • an angiotensin (1-7) peptide and/or potassium channel blocker is administered via parenteral administration, rectal administration, oral administration, or a combination thereof.
  • parenteral administration is intravenous, subcutaneous, inhalation, intradermal, transdermal, and/or transmucosal administration.
  • an angiotensin (1-7) peptide is administered systemically.
  • an angiotensin (1-7) peptide and/or potassium channel blocker are administered at least once per day.
  • an angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-1,000 ug/kg/day. In some embodiments, an angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-500 ug/kg/day. In some embodiments, an angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-60 ug/kg/day.
  • angiotensin (1-7) peptides may be used in accordance with various embodiments.
  • the angiotensin (1-7) peptide comprises the naturally-occurring Angiotensin (1-7) amino acid sequence of Asp 1 -Arg 2 -Val 3 - Tyr 4 -Ile 5 -His 6 -Pro 7 (SEQ ID NO: 1).
  • the angiotensin (1-7) peptide is a functional equivalent of
  • the functional equivalent is a linear peptide.
  • the linear peptide comprises a sequence that includes at least four amino acids from the seven amino acids that appear in the naturally-occurring Angiotensin (1-7), wherein the at least four amino acids maintain their relative positions as they appear in the naturally- occurring Angiotensin (1-7).
  • the linear peptide contains 4-25 amino acids.
  • the linear peptide is a fragment of the naturally-occurring
  • the linear peptide contains amino acid substitutions, deletions and/or insertions in the naturally-occurring Angiotensin (1-7). In some embodiments, the linear peptide has an amino acid sequence of A
  • the linear peptide has an amino acid sequence of
  • an angiotensin (1-7) peptide comprises one or more chemical modifications to increase protease resistance, serum stability and/or bioavailability.
  • the one or more chemical modifications comprise pegylation.
  • the angiotensin (1-7) peptide is administered at an effective dose periodically at an administration interval such that at least one sign, symptom and/or feature of a brain condition is reduced in intensity, severity, duration, or frequency or has delayed onset.
  • the at least one sign, symptom and/or feature of a brain condition is selected from paralysis, memory loss, pain, seizure, dysphagia, aphasia, dysarthria, ataxia, depression, mood swings, and loss of vision.
  • an angiotensin(1-7) peptide is a non-peptidic angiotensin
  • a non-peptidic angiotensin (1 -7) receptor agonist is a 1 -(p-thienylbenzyl)imidazole.
  • the non-peptidic angiotensin (1-7) receptor agonist is a compound with the following structure:
  • FIG. 1 shows exemplary results from a forelimb placement test from rats exposed to one of: saline (vehicle control) or 50 ⁇ g/kgTXA302 (SEQ ID NO: 3), given subcutaneously for up to eight weeks beginning 28 days post tMCAO event, followed on day 1 13 (112 days after tMCAO event) by 2 mg/kg dalfampridine given orally twice per day for three days.
  • FIG. 2 shows exemplary results in a stepping from rats exposed to one of: saline
  • TXA302 (vehicle control) or 50 ⁇ g/kg TXA302 (SEQ ID NO: 3), given subcutaneously for up to eight weeks beginning 28 days post tMCAO event, followed on day 113 (112 days after tMCAO event) by 2 mg/kg dalfampridine given orally twice per day for three days.
  • FIG. 3 shows exemplary results from a body swing test (left turn-right turn) from rats exposed to one of: saline (vehicle control) or 50 ⁇ g/kg TXA302 (SEQ ID NO: 3), given subcutaneously for up to eight weeks beginning 28 days post tMCAO event, followed on day 1 13 (1 12 days after tMCAO event) by 2 mg/kg dalfampridine given orally twice per day for three days.
  • saline vehicle control
  • TXA302 SEQ ID NO: 3
  • FIG. 4 shows a graph of exemplary neuroscores from rats exposed to one of: saline (vehicle control) or 50 ⁇ g/kg TXA302 (SEQ ID NO: 3), given subcutaneously for up to eight weeks beginning 28 days post tMCAO event, followed on day 113 (112 days after tMCAO event) by 2 mg/kg dalfampridine given orally twice per day for three days.
  • saline vehicle control
  • TXA302 SEQ ID NO: 3
  • FIG. 5 shows a graph of exemplary results from a cylinder test of forelimb akinesia from rats exposed to one of: saline (vehicle control) or 50 ,ug/kg TXA302 (SEQ ID NO: 3), given subcutaneously for up to eight weeks beginning 28 days post tMCAO event.
  • FIG. 6 shows an example of the percent change of blood flow (left panel) and average vessel diameter (right panel) in rats exposed to one of: saline (vehicle control) or 50 ⁇ g/kg TXA302 (SEQ ID NO: 3), given subcutaneously for up to eight weeks beginning 28 days post tMCAO event, followed on day 113 (112 days after tMCAO event) by 2 mg/kg
  • FIG. 7 shows a selected view of Day 113 to Day 126 data from FIG. 1.
  • FIG. 7 shows the effect of Dalfampridine on animals receiving a vehicle control during phase 1 of the study.
  • FIG. 8 shows a selected view of Day 1 13 to Day 116 data from FIG. 1.
  • FIG. 8 shows the effect of Dalfampridine on animals receiving TXA302 (SEQ ID NO: 3) during phase 1 of the study.
  • FIG. 9 shows a selected view of Day 113 to Day 126 data from FIG. 2.
  • FIG. 9 shows the effect of Dalfampridine on animals receiving a vehicle control during phase 1 of the study.
  • FIG. 10 shows a selected view of Day 113 to Day 116 data from FIG. 2.
  • FIG. 10 shows the effect of Dalfampridine on animals receiving TXA302 (SEQ ID NO: 3) during phase 1 of the study.
  • FIG. 11 shows a selected view of Day 113 to Day 126 data from FIG. 3.
  • FIG. 1 1 shows the effect of Dalfampridine on animals receiving a vehicle control during phase 1 of the study.
  • FIG. 12 shows a selected view of Day 113 to Day 116 data from FIG. 3.
  • FIG. 12 shows the effect of Dalfampridine on animals receiving TXA302 (SEQ ID NO: 3) during phase 1 of the study.
  • FIG. 13 shows a selected view of Day 113 to Day 126 data from FIG. 4.
  • FIG. 13 shows the effect of Dalfampridine on animals receiving a vehicle control during phase 1 of the study.
  • FIG. 14 shows a selected view of Day 1 13 to Day 116 data from FIG. 4.
  • FIG. 14 shows the effect of Dalfampridine on animals receiving TXA302 (SEQ ID NO: 3) during phase 1 of the study.
  • 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.
  • 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.
  • biologically active refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism .
  • an agent that, when administered to an organism, has a biological effect on that organism is considered to be biologically active.
  • 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.
  • 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.
  • Brain Condition - as used herein, a "brain condition” is any disease, disorder or event that results in damage and/or dysfunction of at least a portion of a subject's brain.
  • Non- limiting examples of brain conditions include: stroke (both ischemic and hemorrhagic), vascular dementia, and traumatic brain injury.
  • Carrier or 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.
  • 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).
  • 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.
  • 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.
  • Dosing regimen is a set of unit doses (typically more than one) that are administered indi vidually to a subject, typically separated by periods of time.
  • a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
  • 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.
  • 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 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 may be 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.
  • “reduce,” or grammatical equivalents indicate values that are relative to a baseline measurement, 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.
  • in 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 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).
  • 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 m anufactured by the hand of man. Isolated substances and/or en titi es 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.
  • 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.
  • a substance is "pure” if it is substantially free of other components.
  • isolated cell refers to a cell not contained in a multi-cellular organism.
  • Polypeptide 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.
  • Protein 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.
  • 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., stroke or other brain condition).
  • risk is expressed as a percentage.
  • 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%.
  • risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples.
  • a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event (e.g., stroke or other brain condition).
  • a reference sample or group of reference samples are from individuals comparable to a particular individual.
  • relative risk is 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
  • 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).
  • Stability refers to the abili ty of the therapeutic agent to maintain its therapeutic efficacy (e.g., all or the majority of its intended biological activity and/or physiochemical in tegri ty) over extended periods of time.
  • the stabi lity 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).
  • 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.
  • 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).
  • Subject refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate).
  • a human includes pre- and post-natal forms.
  • 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.
  • 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 m an y biological an d chemical phenom en a.
  • 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.
  • an individual who is susceptible to a disease, disorder, condition, or event may be characterized by one or more of the following: (1) a genetic mutation associated wi th developmen t 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.
  • an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition.
  • an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
  • 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.
  • 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.
  • 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. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
  • the present invention provides, among other things, methods and compositions for treating brain conditions (e.g., stroke, including, but not limited to, ischemic and hemorrhagic stroke).
  • Provided methods of treating brain conditions include administering to a subject suffering from a brain condition an angiotensin (1-7) peptide, and administering a potassium channel blocker to the subject.
  • the combination of at least one angiotensin (1-7) peptide and at least one potassium channel blocker provides surprisingly positive results in the improvement of one or more si gns, symptoms and/or features of brain conditions.
  • angiotensin (1-7) peptides and potassium channel blockers to synergistically treat brain conditions (e.g., stroke) represents a new form of treatment for sufferers of brain conditions.
  • the beneficial effects of the combination of angiotensin (1 -7) peptides and potassium channel blockers is particularly surprising in light of the fact that such treatments may begin well after a stroke event, even days or weeks after the development of a brain condition, given dogma in the art that treatments administered outside the first several hours, particularly in the cases of stroke and/or traumatic brain injury, typically have limited effectiveness at best.
  • embodiments of the present invention provide entirely new treatment regimens for use by physicians in aiding stroke sufferers.
  • the brain is highly vulnerable to a disturbance in its oxygen supply. Anoxia and ischemia lasting only a few seconds can cause symptoms and if the condition persists for minutes, they can cause irreversible neuronal damage. Accordingly, stroke is a prominent cause of serious, long-term disability and a leading cause of death in the United States, with 500,000 new or recurrent cases occurring each year (see Kelly-Hayes et al., 1998, The American Heart Association Stroke Outcome Classification, Stroke, 29: 1274-1280). Stroke is also a significant burden on the medical industry, with total health costs for disability due to stroke being estimated at approximately $53 billion annually.
  • Ischemic stroke involves an obstruction in one or more blood vessels that supply blood to brain tissue, for example, occlusion resulting from atherosclerotic thrombii, or embolism.
  • Ischemic stroke (cerebral ischemia) represent approximately 88% of all strokes, making ischemic stroke one of the most common types of cerebrovascular injury. Ischemic conditions in the brain quickly lead to neuronal death, often leading to permanent sensorimotor deficits.
  • a hemorrhagic stroke is defined herein as the accumulation of blood anywhere within the cranial vault. Hemorrhagic strokes may result from many causes, including injury resulting from an expanding hematoma, which can disrupt or distort tissue.
  • a stroke may cause a wide variety of symptoms and/or complications. Without wishing to be held to a particular theory, it is thought that the specific signs, symptoms and/or complications, as well as their severity and duration, may be determined by where the stroke occurs in a brain as well as how severe it is. Because the brain controls or modulates nearly every system in the body, the range of potential signs, symptoms and/or complications is vast.
  • the signs, symptoms, and/or complications arising from a stroke include one or more of neurological impairmen t, cogni tive impairment, language impairment, emotional impairment (e.g., depression, anxiety), and motor impairment. According to various
  • exemplary, non-limiting signs, symptoms and/or complications of stroke include, but are not limited to: paralysis, memory loss, pain, seizure, dysphagia (difficulty swallowing), aphasia (loss of speech or language ability), dysarthria (difficulty articulating words), ataxia (lack of coordinated movements), depression, mood swings, and loss of vision.
  • paralysis is partial (e.g., limited to one muscle group, area of the body, and/or side of the body).
  • paralysis is substantially complete (e.g., affecting both sides of the body and most or all voluntary muscles below the neck).
  • exemplary, non-limiting assessment/rating scales include, but are not limited to: the National Institutes of Health Stroke Scale (NIHSS), the Canadian Neurological Scale, the Middle Cerebral Artery Neurological Score, Guy's Prognostic Score, the American Heart Association's Stroke Outcome Classification (AHA SOC), the Mini-Mental State Examination (MMSE), the Neurobehavioral Cognitive Status Examination (NCSE), the American Speech-Language-Hearing Association Functional Assessment of Communication Skills for Adults, the Boston Diagnostic Aphasia Examination, the Center for Epidemiologic Studies Depression (CES-D) Scale, the Geriatric Depression Scale, the Basic Activities of Daily Living (BADL), the Instrumental Activities of Daily Living
  • a major barrier in the treatment of both ischemic and hemorrhagic stroke is delivery of a therapeutic that will reach affected tissue. Given the effectiveness of the blood brain barrier, few compounds are capable of crossing into and affecting cerebral tissue.
  • angiotensin (1-7) peptides described below are able to cross the blood brain barrier without complex delivery systems such as modified stem cells or the like. Rather, in some embodiments, angiotensin (1-7) peptides may be delivered via intravenous or
  • rt-PA tissue plasminogen activator
  • thrombectomy e.g., suction thrombectomy
  • distal endovascular thrombectomy e.g., wherein the clot is physically seized and removed from the cerebral vessels.
  • angioplasty such as balloon angioplasty and/or placement of one or more stents are also common
  • Vascular dementia is the second most common form of dementia, behind
  • vascular dementia may result from problems with blood supply in the brain, such as those caused by ischemic or hemorrhagic stroke or from other causes that lead to development of lesions in the brain.
  • Other causes of vascular dementia include cerebral amyloid angiopathy, hypercholesterolemia, diabetes mellitus, or cardiovascular disease. Dementia resulting from one or more strokes is also known as “single-infarct dementia” or “multi-infarct dementia,” depending upon the root cause.
  • Treatment of vascular dementia has primarily focused on the prevention of further cerebrovascular lesions through use of antiplatelet drugs and life style changes (alteration of diet, cessation of smoking, etc).
  • Cholinesterase inhibitors such as galantamine have also been explored for use in this clinical scenario, but this type of treatment is concerned with
  • embodiments of the present invention represent a novel intravenous and subcutaneous therapy targeted to improving the underlying causes of the disease rather than management of symptoms or maximization of remaining tissue resources.
  • Traumatic brain injury a form of acquired brain injury, occurs when a sudden trauma causes damage to the brain. TBI can result when the head suddenly and violently hits an object (or vice versa), or when an object pierces the skull and enters brain
  • TBI Symptoms of a TBI can be mild, moderate, or severe, depending on the extent of the damage to the brain. A person with a mild TBI may remain conscious or may experience a loss of consciousness for a few seconds or minutes. Other symptoms of mild TBI include headache, confusion, lightheadedness, dizziness, blurred vision or tired eyes, ringing in the ears, bad taste in the mouth, fatigue or lethargy, a change in sleep patterns, behavioral or mood changes, and trouble with memory, concentration, attention, or thinking.
  • a person with a moderate or severe TBI may show these same symptoms, but may also have a headache that gets worse or does not go away, repeated vomiting or nausea, convulsions or seizures, an inability to awaken from sleep, di lation of one or both pupils of the eyes, slurred speech, weakness or numbness in the extremities, loss of coordination, and increased confusion, restlessness, or agitation.
  • Treatments for TBI focus primarily on preventing further injury or complications.
  • TBI TBI
  • Primary concerns in treating TBI include insuring proper oxygen supply to the brain and the rest of the body, maintaining adequate blood flow, and controlling blood pressure.
  • the primary treatment a TBI sufferer receives post-stabil ization is rehabilitation that involves individually tailored treatment programs in the areas of physical therapy, occupational therapy, speech/language therapy, psycho logy/ psychiatry, and social support.
  • Embodiments of the present invention provide a novel treatment for these patients.
  • angiotensin (1-7) peptide refers to both naturally- occurring Angiotensin (1-7) and any functional equivalent, analogue or derivative of naturally- occurring Angiotensin (1-7).
  • peptide and polypeptide are interchangeable terms and refer to two or more amino acids bound together by a peptide bond.
  • the terms “peptide” and “polypeptide” include both linear and cyclic peptide.
  • the terms “angiotensin ⁇ 1 -7)", “Angiotensin ⁇ 1 -7)", and “Ang-(l-7)” are used interchangeably.
  • Naturally-occurring Angiotensin (1-7) (also referred to as Ang-(1-7)) is a seven amino acid peptide shown below:
  • Angiotensinogen which is an ⁇ -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:
  • angiotensin may be formed by the action of various enzymes.
  • Angiotensin (1-7) is generated by action of Angiotensin-converting enzyme 2 (ACE 2).
  • ACE 2 Angiotensin-converting enzyme 2
  • Ang-(l-7) is an endogenous ligand for Mas receptors.
  • Mas receptors are G- protein coupled receptor containing seven transmembrane spanning regions.
  • angiotensin ⁇ 1 -7) receptor' encompasses the G Protein-Coupled Mas Receptors.
  • 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).
  • an angiotensin (1 -7) peptide suitable for the present invention is a functional equivalent of naturally-occurring Ang-(l-7).
  • 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-(1-7).
  • a functional equivalent of naturally-occurring Ang-(l-7) described herein has pro-angiogenic activity as determined using methods described herein or known in the art, or an activity such as nitric oxi de release, vasodil ation, improved endoth elial function, antidiuresis, or one of the other properties discussed herein, that positively impacts angiogenesis.
  • a functional equivalen t of natural ly-occurrin g Ang-(1-7) described herein can bind to or activate an angiotensin ⁇ 1-7) receptor (e.g., the G protein-coupled Mas receptor) as determined using various assays described herein or known in the art.
  • angiotensin ⁇ 1-7) receptor e.g., the G protein-coupled Mas receptor
  • a functional equivalent of Ang-(l -7) is also referred to as an angiotensin (1-7) analogue or derivative, or functional derivative.
  • a functional equivalent of angiotensin (1-7) shares amino acid sequence similarity to the naturally-occurring Ang-(l-7).
  • 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-(l-7).
  • a functional equivalent of Ang-(1-7) 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-(1-7). 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.
  • the WU-BLAST-2 software is used to determine amino acid sequence identity (Altschul et al, Methods in Enzymology 266, 460-480 (1996); http://l3last.wustL''edu/blast''README.html).
  • WU-BLAST-2 uses several search parameters, most of which are set to the default values.
  • HSP score (S) and HSP S2 parameters are dynamic values and are established by the program itself, depending upon the composition of the particular sequence, however, the m in imum values may be adjusted and are set as indicated above.
  • Ang-(1-7) is a fragment of the natural ly-occurring An g-(1 -7).
  • a functional equivalent, analogue or derivative of Ang-(1-7) contains amino acid substitutions, deletions and/or insertions in the naturally-occurring Ang-(1-7).
  • Ang-(1-7) functional equivalents, analogues or derivatives can be made by altering the amino acid sequences by substitutions, additions, and/or deletions.
  • 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.
  • the positively charged (basic) amino acids include arginine, lysine, and histidine.
  • the nonpolar (hydrophobic) amino acids include leucine, isoleucine, alanine, phenylalanine, valine, proline, tryptophane, 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.
  • An angiotensin ⁇ 1-7) peptide can be of any length.
  • an angiotensin ⁇ 1-7) 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, 4-1 1, 4-10, 4-9, 4-8, 4-7 amino acids).
  • the linear peptide contains 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.
  • an angiotensin ⁇ 1-7) peptide contains one or more modifications to increase protease resistance, serum stability and/or bioavailability.
  • suitable modifications are selected from pegylation, acetylation, glycosylation, biotinylation, substitution with D-amino acid and/or un-natural amino acid, and/or cyclization of the peptide.
  • amino acid in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain.
  • an amino acid has the general structure H 2 N-C(H)(R) --COOH.
  • an amino acid is a naturally-occurring amino acid.
  • an amino acid is a synthetic or un-natural amino acid (e.g., ⁇ , ⁇ -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.
  • synthetic or unnatural 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 half-life without adversely affecting its activity.
  • unconventional or un-natural amino acids include, but are not limited to, citrulline, ornithine, norleucine, norvaline, 4-(E)-butenyl-4(i?)-methyl-N-methylthreonine (MeBmt), N-methyl- leucine (MeLeu), aminoisobutyric acid, statine, and N-methyl-alanine (Me Ala).
  • Amino acids may participate in a disulfide bond.
  • 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.
  • angiotensin-( 1-7) peptides contain one or more L-amino acids, D-amino acids, and/or un-natural 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
  • 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 (cis and trans), - - 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.
  • paradigm polypeptide i.e., a polypeptide that has a biological or pharmacological activity
  • linkages such as (cis and trans), - - etc.
  • 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.
  • N-acyl derivatives of the amino terminal or of another free amino group examples 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 (fluorenylmethyi-0-CO-); (2) esters of the carboxy terminal or of another free carboxy or hy droxyl 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.
  • the acyl group may be an alkanoyl group (e.g
  • 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.
  • synthetic e.g., exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, classical solution synthesis, native-chemical ligation
  • recombinant techniques e.g., 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,
  • a resin e.g., benzhydrylamine resin, chloromethylated resin
  • 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
  • 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.
  • alpha amino protecting groups include acyl type protecting groups ⁇ e.g., trifluoroacetyl, formyl, acetyl), aliphatic urethane protecting groups ⁇ e.g., t-butyloxycarbonyl (BOC), cyclohexyloxycarbonyl), aromatic urethane type protecting groups (e.g., fluorenyl-9- methoxy-carbonyl (Fmoc), benzyloxycarbonyl (Cbz), Cbz derivatives) and alkyl type protecting groups ⁇ e.g., triphenyl methyl, benzyl).
  • acyl type protecting groups ⁇ e.g., trifluoroacetyl, formyl, acetyl
  • aliphatic urethane protecting groups e.g., t-butyloxycarbonyl (BOC), cyclohexyloxycarbonyl
  • aromatic urethane type protecting groups e.g., fluor
  • 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.
  • Ang-(1-7) peptides may be synthesized according to the FMOC protocol in an organic phase with protective groups.
  • the peptides are purified with a yield of 70% with high-pressure liquid chromatography (HPLC) on a CI 8 chromatography column and eluted with an acetonitrile gradient of 10-60%.
  • HPLC high-pressure liquid chromatography
  • 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).
  • 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 wi thin the same polypeptide.
  • 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 cl eavage.
  • exopeptidases whi ch represent the most promin en t 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)).
  • modified versions of 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.
  • D-amino acid of the same type may be used to generate more stable peptides.
  • 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.
  • 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)).
  • 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.
  • reverse L-pep tides 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.
  • 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 activi ty as the ori ginal L- amino acid peptide.
  • 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)).
  • 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.
  • non-naturally-occurring amino acids include ⁇ , ⁇ - disubstituted amino acids, " N -alkyl amino acids, C-a-methyl amino acids, ⁇ -amino acids, and ⁇ - methyl amino acids.
  • Amino acids analogs useful in the present invention may include, but are not limited to, ⁇ -alanine, norvaline, norleucine, 4-aminobutyric acid, orithine, hydroxyproline, 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.
  • 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)).
  • 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.
  • amino acid sequences of the peptides with N-terminal or C-terminal D-amino aci ds an d 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.
  • a functional equivalent, analogue or derivative of naturally-occurring Ang-(l -7) is a cyclic peptide.
  • 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.
  • Cyclic peptides as described herein may comprise residues of L-amino acids, D- amino acids, or any combination thereof.
  • Amino acids may be from natural or non-natural sources, provided that at least one amino group and at least one carboxyl group are present in the molecule; a- and ⁇ -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
  • N-terminal amino group e.g., acetylation or alkoxycarbonylation
  • side-chain modifications and/or substitutions e.g., methyl ation, benzylation, t-butylation, tosylation, alkoxycarbonylation, 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, ⁇ , ⁇ -tetramethylene cysteine, ⁇ , ⁇ -pentamethylene cysteine, ⁇ - mercaptopropionic acid, ⁇ , ⁇ -pentamethylene- ⁇ -mercaptopropionic acid, 2-mercaptobenzene, 2- mercaptoaniline, 2-mercaptoproline, ornithine, diaminobutyric acid, a-aminoadipic acid, m- aminomethylbenzoic acid and a, ⁇ -diam inopropioni c acid.
  • cyclization may be achieved by any of a variety of techniques well known in the art.
  • a bond may be generated between reactive amino acid side chains.
  • 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.
  • 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.
  • cyclization may be achieved by amide bond formation.
  • a peptide bond may be formed between terminal functional groups (i.e., the amino and carboxy termini of a linear peptide prior to cyclization).
  • the linear peptide comprises a D-amino acid.
  • 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.
  • Residues capable of forming a lactam bond include lysine, ornithine (Orn), a-amino adipic acid, m-aminomethylbenzoic acid, ⁇ , ⁇ -diaminopropionic acid, glutamate or aspartate. Methods for forming amide bonds are generally well known in the art.
  • carbodi imide-mediated lactam formation can be accomplished by reaction of the carboxylic acid with DCC, DIC, ED AC or DCCI, resul ting in the formation of an O-acylurea that can be reacted immediately with the free amino group to complete the cyclization.
  • cyclization can be performed using the azide method, in which a reactive azide intermediate is generated from an alkyl ester via a hydrazide.
  • cyclization can be accomplished using activated esters. The presence of electron withdrawing substituents on the alkoxy carbon of esters increases their susceptibility to aminolysis.
  • a thioether linkage may be formed between the side chain of a thiol-containing residue and an appropriately derivatized a-amino acid.
  • 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.
  • DCC carbodiimide coupling method
  • ED AC carbodiimide coupling method
  • any two thiol containing side-chains can be reacted with dibromoethane and diisopropylamine in DMF.
  • the invention provides linear angiotensin ⁇ 1-7) peptides.
  • the structure of naturally-occurring Ang-(1-7) is as follows:
  • peptides and peptide analogs of the invention can be generally represented by the following sequence:
  • Xaa 1 is any amino acid or a dicarboxylic acid. In certain embodiments, Xaa 1 is
  • Xaa 1 is a negatively-charged amino acid, such as Asp or Glu, typically Asp.
  • Xaa 2 is Arg, Lys, Ala, Cit (citrulline), Orn (ornithine), acetylated Ser, Sar, D-Arg and D-Lys.
  • Xaa is a positively-charged amino acid such as Arg or Lys, typically Arg.
  • Xaa 3 is Val, Ala, Leu, Nle (norleucine), IIe, Gly, Lys, Pro, HydroxyPro
  • Xaa is an aliphatic amino acid such as Val, Leu, IIe or Nle, typically Val or Nle.
  • Xaa 4 is Tyr, Tyr(P0 3 ), Thr, Ser, homoSer (homoserine), azaTyr (aza-a'-homo-L- tyrosine) or Ala.
  • Xaa 4 is a hydroxyl -substituted amino acid such as Tyr, Ser or Thr, typically Tyr.
  • Xaa 5 is IIe, Ala, Leu, norLeu, Val or Gly.
  • Xaa 5 is an aliphatic amino acid such as Val, Leu, IIe or Nle, typically IIe.
  • Xaa 6 is His, Arg or 6-NH 2 -Phe (6-aminophenylalaine). In certain embodiments,
  • Xaa 6 is a fully or partially positively-charged amino acid such as Arg or His.
  • Xaa 7 is Cys, Pro or Ala.
  • one or more of Xaa -Xaa is identical to the
  • Xaa 1 -Xaa 7 are identical to the corresponding amino acid in naturally-occurring Ang-(l-7).
  • all of Xaa 1 -Xaa 6 are identical to the corresponding amino acid in naturally-occurring Ang-(l-7).
  • Xaa 3 is Nle.
  • Xaa 3 is Nle, one or more of Xaa 1 -
  • Xaa 2 and Xaa 4-7 are optionally identical to the corresponding amino acid in naturally-occurring Ang-(1-7). In certain such embodiments, all but one or two of Xaa 1 -Xaa 2 and Xaa 4-7 are identical to the corresponding amino acid in naturally-occurring Ang-(l-7). In other words,
  • all of Xaa 1 -Xaa 2 and Xaa 4-7 ' are identical to the corresponding amino acid in naturally-occurring Ang-(1-7), resulting in the amino acid sequence: Asp 1 -Arg 2 -Nle 3 -Tyr 4 -Ile 5 - His 6 -Pro 7 (SEQ ID NO: 6).
  • the peptide has the amino acid sequence Asp -Arg ⁇ -Val " -
  • a linear angiotensin (1 -7) peptide as described herein is a peptide having a sequence of Asp 1 -Arg 2 -Val 3 -Tyr 4 -Ile 5 -His 6 -Pro 7 -Phe 8 -His 9 (SEQ ID NO: 22), which is identical to the sequence of Ang(l-9).
  • an angiotensin (1-7) peptide is a derivative of Ang (1-9).
  • 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.
  • a linear angiotensin (1-7) peptide is a peptide with an amino acid sequence of Ala 1 -Arg 2 -Val 3 -Tyr 4 -Ile 5 -His 6 -Pro 7 (SEQ ID NO: 23). Additional sequences derived from SEQ ID NO: 23 may be found in European Patent Application
  • the invention provides a cyclic angiotensin ⁇ 1 -7) (Ang-(1-7)) peptide analog comprising a linkage, such as between the side chains of amino acids
  • peptide analogs typically comprise 7 amino acid residues, but can also include a cleavable sequence.
  • 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 Asp 1 -Arg 2 -Val 3 -Ser 4 -Ile 5 -His 6 -Cys 7 (SEQ ID NO: 2), wherein a linkage is formed between Ser 4 and Cys''.
  • Another example of such an analog is Ala 1 -Arg 2 -Val 3 '-Ser 4 -Ile 5 -His 6 -Cys 7 (SEQ ID NO: 3), wherein a linkage is formed between Ser 4 and Cys 7
  • a thioether bridge is also referred to as a monosulfide bridge or, in the case of Ala-S- Ala, as a lanthionine bridge.
  • Thioether bridge-containing peptides can be formed by two amino acids having one of the following formulas:
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently -H, an alkyl (e.g.,
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are each independently -H or -CH 3 , such where all are -H.
  • the invention provides an Ang analog or derivative comprising a thioether bridge according to formula (I).
  • R 1 , R 2 , R J and R 4 are 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.
  • 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- penicill amine in position 7 (see, e.g., Gaisme, Trent and Spatola (2003) Biopolymers 71 , 534- 551).
  • 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.
  • 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.
  • the Ang analogs and derivati ves 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 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 Me-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.
  • Bioactivity of an analog or derivative 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, (1933) Gen. Pharmacol. 24:705- 713.
  • Ang analogs and derivatives where only the length of the peptide is varied include the following:
  • Ang-(1-7) (Asp 1 -Arg 2 VaP-Cyc 4 -Ile 5 His 6 Cyc 7 , SEQ ID NO: 7).
  • These analogs can have one of the thioether bridges shown in Formulae (I)-(I I I) as the Cyc 4"7 moiety, for example, where Cyc 4 and Cyc 7 are represented by Formula (I), such as where R'-R 4 are each -H or -CH 3 , typically -H.
  • the amino acids at positions 4 and 7 of the Cyc 4"7 analog are modified to allow introduction of the thioether-ring structures shown above.
  • 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.
  • 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.
  • an Ang analog or derivative of the invention has no intrinsic function but inhibits the effects of one or more naturally-occurring angiotensin compounds.
  • an Ang analog of the invention is represented by
  • Xaa 1 is any amino acid, but typically a negatively-charged amino acid such as Glu or Asp, more typically Asp.
  • Xaa 2 is a positivel y-charged amino acid such as Arg or Lys, typically Arg.
  • Xaa 3 is an aliphatic amino acid, such as Leu, IIe or Val, typically Val.
  • Cyc 4 forms a thioether bridge in conjunction with Cyc '.
  • Cyc 4 can be a D- stereoisomer and/or a L-stereoisomer, typically a D-stereoisomer.
  • Examples of Cyc 4 (taken with Cyc 7 ) are shown in Formulas (I), (II) and (III).
  • the R groups in Formulae (I), (II) and (III) are -H or -CH 3 , especially -H.
  • Xaa 5 is an aliphatic amino acid, such as Leu, IIe or Val, typically IIe.
  • Xaa 6 is His.
  • Cyc 7 forms a thioether bridge in conjunction with Cyc 4 , such as in Formula (I),
  • Cyc 7 can be a D-stereoisomer and/or a L-stereoisomer, typically a L-stereoisomer. Examples of Cyc 7 (taken with Cyc 4 ) are shown in Formulas (I), (II), (III) and (IV). Typically, the R groups in Formulae (I), (II),) and (III) and (IV) are -H or -CH 3 , especially -H. [0136] in certain embodiments, one or more of Xaa'-Xaa 6 (excluding Cyc 4 and Cyc 7 ) is identical to the corresponding amino acid in naturally-occurring Ang-(1-7).
  • all but one or two of Xaa'-Xaa 6 are identical to the corresponding amino acid in naturally-occurring Ang-(1-7). In other embodiments, all of Xaa'-Xaa 6 are identical to the corresponding amino acid in naturally-occurring Ang-(1-7).
  • Cyc 4 and Cyc 7 are independently selected from Abu (2- aminobutyric acid) and Ala (alanine), where Ala is present in at least one position.
  • cyclic analogs can have a thioether linkage formed by -Ala 4 -S-Ala 7 - (Formula (I), where R 1 -R 4 are each -H); -Ala 4 -S-Abu 7 - (Formula (I): R 1 -R 3 are -H and R 4 is -CH 3 ) or -Abu 4 -S-Ala 7 - (Formula (I): R 3 , R 3 and R 4 are -H and R 2 is -CH 3 ).
  • Specific examples of cyclic analogs comprise a -Abu 4 -S- Ala 7 - or -Ala 4 -S-Ala 7 - linkage.
  • the invention provides an Ang-(1-7) analog with a thioether-bridge between position 4 and position 7 having the amino acid sequence Asp -Arg - Val 3 -Abu 4 -Ile 5 -His 6 -Ala 7 (SEQ ID NO: 15) or the amino acid sequence Asp 3 -Arg 2 -Val 3 -Ala 4 - Ile 5 -His 6 -Ala 7 (SEQ ID NO: 16), which are represented by the following structural diagrams:
  • an Ang analog or derivative of the invention is represented by Formula (V):
  • Xaa , Xaa , Xaa , Xaa and Xaa are absent in certain embodiments.
  • (1) Xaa 10 is absent, (2) Xaa 9 and Xaa 10 are absent, (3) Xaa 8 , Xaa 9 and Xaa 10 are absent, (4) Xaa 1 is absent, (5) Xaa 3 and Xaa 10 are absent, (6) Xaa 1 , Xaa 9 and Xaa 10 are absent, (7) Xaa 1 , Xaa 8 , Xaa 9 and Xaa 10 are absent, (8) Xaa 1 and Xaa 2 are absent, (9) Xaa 1 , Xaa 2 and Xaa 30 are absent, (10) Xaa 1 , Xaa 2 , Xaa 9 and Xaa 10 are absent, or (11 ) Xaa 1 , Xaaa
  • Xaa 1 when present, is any amino acid, but typically a negatively charged amino acid such as Glu or Asp, more typically Asp.
  • Xaa 2 when present, is a positively charged amino acid such as Arg or Lys, typically Arg.
  • Nle 3 is norleucine.
  • Cyc 4 forms a thioether bridge in conjunction with Cyc 7 .
  • Cyc 4 can be a D- stereoisomer and/or a L-stereoisomer, typically a D-stereoisomer.
  • Examples of Cyc (taken with Cyc 7 ) are shown in Formulas (I), (II) and (III).
  • the R groups in Formulae (I), (II) and (I II) are H or CH 3 , especially -H.
  • Xaa 5 is an aliphatic amino acid, such as Leu, Nie, lie or Val, typically IIe.
  • Xaa 6 is His.
  • Cyc 7 forms a thioether bridge in conjunction with Cyc 4 , such as in Formula (I),
  • Cyc' can be a D-stereoisomer and/or a L-stereoisomer, typically a L-stereoisomer.
  • Examples of Cyc 7 (taken with Cyc 4 ) are shown in Formulas (I), (II) and (III).
  • the R groups in Formulae (I), (II) and (III) are -H or -CH 3 , especially -H.
  • Xaa 8 when present, is an amino acid other than Pro, typically Phe or IIe. In certain embodiments, IIe results in an inhibitor of Ang(l-8). In certain embodiments, Phe maintains the biological activity of Ang(l-8) or Ang(l-lO).
  • Xaa 9 when present, is His.
  • Xaa 10 when present, is an aliphatic residue, for example, IIe, Val or Leu, typically
  • one or more of Xaa'-Xaa 10 (excluding Nle 3 , Cyc 4 and
  • Cyc' is identical to the corresponding amino acid in naturally-occurring Ang (including Ang-(1- 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).
  • all but one or two of Xaa' -Xaa 10 are identical to the corresponding amino acid in naturally-occurring Ang.
  • all of Xaa 3 -Xaa 10 are identical to the corresponding amino aci d in naturally-occurring Ang.
  • Cyc 4 and Cyc 7 are independently selected from Abu (2- aminobutyric acid) and Ala (alanine), where Ala is present at at least one position.
  • cyclic analogs comprising a thioether linkage formed by -Ala 4 -S-Ala 7 - (Formula (I), where R'-R 4 are each -H); -Ala 4 -S-Abu 7 - (Formula (I): R '-R 3 are -H and R 4 is -CH 3 ) or -Abu 4 -S-Ala 7 - (Formula (I): R 1 , R 3 and R 4 are -H and R 2 is -CH 3 ).
  • Specific cyclic analogs comprise a -Abu 4 -S-Ala 7 - or -Ala 4 -S-Ala'- linkage.
  • the invention provides an Ang-(l-7) analog or derivative with a
  • the invention provides an Ang-(l-8) analog or derivative with a thioether-bridge between position 4 and position 7 having Ang-(l-8) antagonLstic activity, in particular an Ang(l-8) analog or derivative having the amino acid sequence Asp -Arg -Nle - Abu 4 -Ile 5 -His 6 -Ala 7 -Ile 8 (SEQ ID NO: 20), or the amino acid sequence Asp ⁇ Arg ⁇ Nle'-Ala 4 - Ile 5 -His 6 -Ala 7 -Ile 8 (SEQ ID NO: 21).
  • 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.
  • 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.
  • the present invention provides methods of treating brain conditions including administering to a subject who is suffering from or susceptible to one or more brain conditions an angiotensin (1 -7) receptor agonist.
  • angiotensin (1 -7) receptor agonist As used herein, the term
  • angiotensin-( 1 -7) receptor agonist is considered within the scope of the term “angiotensin (1-7) peptide” and encompasses any molecule that has a positive impact in a function of an
  • angiotensin ⁇ 1-7) receptor in particular, the G-protein coupled Mas receptor.
  • an angiotensin ⁇ 1-7) receptor agonist directly or indirectly enhances, strengthens, activates and/or increases an angiotensin ⁇ 1-7) receptor (i.e., the Mas receptor) activity.
  • an angiotensin ⁇ 1-7) receptor agonist directly interacts with an angiotensin ⁇ 1-7) receptor (i.e., 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.
  • the angiotensin (1-7) receptor agonist is a non-peptidic agonist.
  • an exemplary class of angiotensin- ⁇ 1 -7) receptor agonists are l -(p- thienylbenzyl)imidazoles. Examples of these non-peptide angiotensin ⁇ 1-7) receptor agonists are represented by Structural Formula (VI):
  • R 1 is halogen, hydroxyl, alkoxy wherein 1 to 6 carbon atoms are replaced by the heteroatoms O, S, or NH (preferably by substituted by a saturated cyclic ether such as tetrahydropyran or tetrahydrofuran,
  • alkylaryl or aryloxy that is unsubstituted or substituted by a substituent selected from halogen, and trifluoromethyl
  • R 4 is hydrogen, halogen (chloro, bromo, fluoro), or (C 1 -C 4 )-alkyl
  • X is oxygen or sulfur
  • Y is oxygen or -NH-;
  • R 5 is hydrogen, (C 1 -C 6 )-alkyl; or (C 1 -C 4 )-alkylaryl, where R 5 is hydrogen when Y is -NH-;
  • R 6 is (C 1 -C 5 )alkyl.
  • R is not halogen when R 2 is COOH or CO-0-(C 1 -C 4 )- alkyl.
  • an angiotensin-( 1-7) receptor agonist is AVE 0991, 5- formyl-4-methoxy-2-phenyl- 1 [ [4- [2-(ethylaminocarbonylsulfonamido)-5 -isobutyl-3 -thienyl] - phenyl]-methyl]-imidazole, which is represented by the following structure:
  • angiotensin -(1-7) receptor agoni sts are p- thienylbenzyl amides.
  • Examples of these non-peptide angiotensin-(l -7) receptor agonists are represented by Structural Formula (VII):
  • R 1 is (C 1 -C 5 )-alkyl that is unsubstituted or substituted by a radical chosen from MH 2 , halogen, O-(C 1 -C 3 >alkyl, CO-0-(C 1 -C 3 )-alkyl and C0 2 H, (C 3 -C 8 )-cycloalkyl, (C 1 -C 3 )-alkyl-(C 3 - C 8 )-cycloalkyl, (C6-C 10 )-aryl that is unsubstituted or substituted by a radical chosen from halogen and 0-(C 1 -C 3 )-alkyl, (C 1 -C 3 )-alkyl-(C6-C 10 )-aryl where the aryl radical is unsubstituted or substituted by a radical chosen from halogen and 0-(C]-C 3 )-alkyl, (C 1 -C 5 )-heteroaryl,
  • R is hydrogen, (C 1 -C6)-alkyl that is unsubstituted or substituted by a radical chosen from halogen and 0-(C 1 -C 3 )-alkyl, (C 3 -C 8 )-cycloalkyl, (C 1 -C 3 )-alkyl-(C 3 -C 8 )-cycioalkyl, (C 6 -C 10 )-aryl that is unsubstituted or substituted by a radical chosen from among halogen, 0-(C 1 -C 3 )-alkyi and CO-0-(C 1 -C 3 )-alkyl, or (C 1 -C 3 )-alkyl-(C 6 -C 10 )-aryl that is unsubstituted or substituted by a radical chosen from halogen and 0-(C 1 -C 3 )-aikyl;
  • R 3 is hydrogen, COOH, or COO-(C 1 -C 4 )-alkyl
  • R 4 is hydrogen, halogen; or (Ci-C 4 )-alkyl
  • R 5 is hydrogen or (C 1 -C 6 )-alkyl
  • R 6 is hydrogen, (C 1 -C6)-alkyl, (C 1 -C 3 )-alkyl-(C 3 -C 8 )-cycloalkyl, or (C 2 -C 6 )-alkenyl; and X is oxygen or NH.
  • angiotensin ⁇ 1 -7) receptor agonists are described in U.S.
  • angiotensin ⁇ 1-7) receptor agonists described above can be present as pharmaceutically acceptable salts.
  • 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 ⁇ , ⁇ '- dibenzylethylenediamine or ethylenediamine, or combinations thereof (e.g., a zinc tannate salt).
  • polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel and the like or with an organic cation formed from ⁇ , ⁇ '- dibenzylethylenediamine or ethylenediamine, or combinations thereof (e.g., a zinc tannate salt).
  • organic cation formed from ⁇ , ⁇ '- dibenzylethylenediamine or ethylenediamine, or combinations thereof (e.g., a zinc tannate salt).
  • the non-toxic, physiologically acceptable salts are preferred.
  • 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.
  • 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.
  • 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.
  • An aralkyl group is an alkyl group substituted by an aryl group.
  • Potassium channel blockers are a class of agents that interfere with conduction through one or more potassium channels.
  • potassium channel blockers are known to be effective cardiac agents, and are used as anti-arrhythmia agents.
  • Class III potassium channel blockers for example, are predominantly characterized in their effect on prolonging the repolarization of certain cardiac neurons and thus being useful for the treatment of particular cardiac abnormalities including ventricular tachycardia (VT) and-'or ventricular fibrillation (VP).
  • VT ventricular tachycardia
  • VP ventricular fibrillation
  • potassium channel blockers Any of a variety of potassium channel blockers are contemplated as within the scope of the present invention.
  • Non-limiting exemplary potassium channel blockers suitable for use in various embodiments include: dalfampridine, amiodarone, dronedarone, dofetilide, sotalol, ibutilide, azimilide, bretylium, clofilium, E-4031 , nifekalant, tedisamil, sematilide, and combinations thereof.
  • a potassium channel blocker may be at least one FDA- approved potassium channel blocker. In some embodiments, a potassium channel blocker is administered at a dose other than an FDA-approved dose. In some embodiments, a potassium channel blocker is administered according to a dosing regimen other than an FDA-approved dosing regimen. In some embodiments, a potassium channel blocker may not be an FDA- approved potassium channel blocker.
  • a potassium channel blocker may be 4-ammopyridine, also known as “fampridine” or “dalfampridine”.
  • Dalfampridine is a potassium channel blocker with the chemical formula C 5 H4N-NH2, that is thought to be selective for the Kvl family (also referred to as the "Shaker fam ily") of vo ltage activated potassium channels.
  • Dalfampridine has also been shown to have some beneficial effect in multiple sclerosis (MS). Without wishing to held to a particular theory, it is thought that dalfampridine may benefit MS sufferers by supporting robust action potential duration and amplitude.
  • an Ang (1-7) peptide and/or potassium channel blocker 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 or medicament (e.g., in the manufacture of a medicament for the treatment of the disease), as described herein.
  • 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)).
  • 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.
  • salt solutions e.g., NaCl
  • saline e.g., buffered saline
  • alcohols e.glycerol
  • ethanol glycerol
  • gum arabic vegetable oils
  • benzyl alcohols polyethylene glycols
  • gelatin carbohydrates such as lactose, amylose or starch
  • 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.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring and/or aromatic substances and the like
  • a water-soluble carrier suitable for intravenous administration is used.
  • composition or medicament 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.
  • composition or medicament can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings.
  • a composition for intravenous administration typically is a solution in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • 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.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water.
  • an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration .
  • An Ang (1-7) peptide and/or potassium channel blocker 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.
  • an Ang (1-7) peptide and/or potassium channel blocker as described herein is administered by any appropriate route.
  • an Ang (1-7) peptide and/or potassium channel blocker is administered subcutaneously.
  • subcutaneous tissue is defined as a layer of loose, irregular connective tissue
  • an Ang (1 -7) peptide and/or potassium channel blocker described herein is administered intravenously.
  • an Ang (1-7) peptide and/or potassium channel blocker described herein (or a composition or medicament containing an Ang (1-7) peptide and/or potassium channel blocker described herein) can be administered by inhalation, parenterally, intradermally, transdermally, rectally, or transmucosally.
  • an Ang (1 -7) peptide and/or potassium channel blocker is administered orally. More than one route can be used concurrently, if desired.
  • an Ang (1-7) peptide is administered in any non-intracerebroventricular manner.
  • 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 ischemic stroke).
  • 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 determined in accordance with standard clinical techniques.
  • 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, 1 1, 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.
  • 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, 1 1 , 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%.
  • 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.
  • an Ang (1-7) peptide and/or potassium channel blocker is administered at a therapeutically effective amount.
  • therapeutically effective amount is largely determined based on the total amount of the therapeutic agent contained in the pharmaceutical compositions of the present invention .
  • a therapeutically effective amount is largely determined based on the total amount of the therapeutic agent contained in the pharmaceutical compositions of the present invention .
  • 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).
  • appropriate doses or amounts to be administered may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • Ang (1 -7) peptide and/or potassium channel blocker may be present in varying amounts in various embodiments.
  • a therapeutically effective amount of an angiotensin (1-7) peptide may be an amount ranging from about 10-1000 mg (e.g., about 20 mg - 1,000 mg, 30 mg - 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, 100-1000 mg, 100-900 mg, 100-800 mg, 100-700 mg, 100-600 mg, 100-500 mg, 100-400 mg, 100-300 mg, 200-1000 mg, 200-900 mg, 200-800 mg, 200-700 mg, 200-600 mg, 200
  • an angiotensin (1-7) peptide and/or potassium channel blocker 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.
  • an angiotensin (1 -7) peptide and/or potassium channel blocker 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.
  • 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
  • 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 0.01 mg/kg weight to about 1
  • 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
  • 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
  • the angiotensin (1 -7) peptide and/or potassium channel blocker is administered at an effective dose ranging from about 1-1,000 ⁇ g/kg/day (e.g., ranging from about 1-900 ⁇ g/kg/day, 1-800 ⁇ g/kg/day, 1 -700 ⁇ g/kg/day, 1-600 ⁇ g/kg/day, 1-500 ⁇ g/kg/day, 1-400 ⁇ g/kg/day, 1-300 ⁇ g/kg/day, 1-200 ⁇ g/kg/day, 1-100 ⁇ g/kg/day, 1-90 ⁇ g/kg/day, 1-80 ⁇ g/kg/day, 1-70 ⁇ g/kg/day, 1-60 ⁇ g/kg/day, 1-50 ⁇ g/kg-''day, 1-40 ⁇ g/kg/day, 1- 30 ⁇ g/kg/day, 1 -20 ⁇ g/kg/day, 1-10 ⁇ g/kg/day .
  • an effective dose ranging from about 1-1,000 ⁇ g/kg/day (
  • the angiotensin (1 -7) peptide and/or potassium channel blocker is administered at an effective dose ranging from about 1-500 ⁇ g/kg/day. In some embodiments, the angiotensin (1-7) peptide and/or potassium channel blocker is administered at an effective dose ranging from about 1-100 ⁇ g/kg/day. In some embodiments, the angiotensin (1-7) peptide and/or potassium channel blocker is administered at an effective dose ranging from about 1-60 ⁇ g/kg/day.
  • the angiotensin (1- 7) peptide and/or potassium channel blocker 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 ug/kg/day.
  • the present invention provides several new dosing schedules not previously thought appropriate or beneficial in the treatment of stroke. In contrast to the established thinking regarding stroke treatment, where "time is brain,”
  • embodiments of the present invention allow for treatment to begin well after a stroke event.
  • several embodiments may include a dosing schedule wherein the first treatment of a patient with an Ang (1-7) peptide occurs more than 3 hours after the stroke event.
  • the first treatment of a patient with an Ang (1-7) peptide occurs more than 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90 hours, or 96 hours after the stroke event.
  • first treatment of a patient with an Ang (1-7) peptide occurs more th an 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days after a stroke event. In some embodiments, the first treatment of a patient with an Ang (1-7) peptide occurs more than 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks after the stroke event.
  • the administration of a potassium channel blocker may begin before, substantially at the same time as, or after administration of an angiotensin (1-7) peptide.
  • an Ang (1-7) peptide and/or potassium channel blocker will be used as a part of a combination therapy including at least one additional therapy or therapeutic. It is contemplated that any known therapy or therapeutic for the treatment of brain conditions ⁇ e.g., stroke) may be used with one or more Ang (1 -7) peptides and/or potassium channel blockers as disclosed herein.
  • Exemplary compounds that may be used with one or more Ang (1-7) peptides and/or potassium channel blockers include, but are not limited to, thrombolytic compounds, antioxidants or other reactive oxygen species agents, interferon beta- la ⁇ e.g., Avonex, Rebif, CinnoVex, ReciGen), interferon beta- 1 b (Betaseron), glatiramer acetate (Copaxone), mitoxantrone (Novantrone), natalizumab (Tysabri), fingolimod (Gilenya),
  • an Ang(l -7) peptide and/or potassium channel blocker may be administered prior to, concurrently with, or subsequent to one or more surgical procedures such as those outlined above ⁇ e.g.,
  • combination refers to agents or modalities that are administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics such that the subject is simultaneously exposed to both (or all) agents or modalities.
  • Each of the two or more agents or modalities may be administered according to a different schedule; it is not required that individual doses of different agents be administered at the same time, or in the same composition. Rather, so long as both (or more) agents are present in the subject's body simultaneously for some period of time, they are considered to be administered "in combination”.
  • kits or other articles of manufacture which contains an Ang (1 -7) peptide and/or a potassium channel blocker, or a formulation containing the same and provides instructions for its reconstitution (if lyophilized) and/or use.
  • Ki ts 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.
  • 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.
  • 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.
  • 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.
  • a container may contain a single dose of a stable formulation containing an Ang (1-7) peptide and/or a potassium channel blocker.
  • 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.
  • a container holding the formulation may be a multi-use vial, which al lows 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.,
  • kits or other articles of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • kits or other articl es of manufacture may include an in struction for self- administration.
  • angiotensin (1-7) peptides and the potassium channel blocker dalfampridine were used in a rat Transient Middle Cerebral Artery Occlusion (tMCAO) model, which is generally accepted model of ischemic stroke.
  • tMCAO Transient Middle Cerebral Artery Occlusion
  • TXA302 (SEQ ID NO: 3) dose volume was 0.5ml/kg for daily injections.
  • TXA302 was dissolved in PBS buffer to a concentration of 25 mg/ml (stock solution). For the working solution by daily injections TXA302 was dissolved in PBS to a concentration of 0.1 mg/ml for dose level 50 ⁇ g/kg.
  • Transient middle cerebral artery occlusion was performed according to the method described R. Schmid-Eisaesser et al. Briefly, the right CCA (Common Carotid Artery) was exposed through a midline neck incision and carefully dissected free from surrounding nerves and fascia - from its bifurcation to the base of the skull. The occipi tal artery branches of the ECA (External Carotid Artery) were then isolated, and these branches were dissected and coagulated. The EGA was dissected further distal ly and coagulated along with the terminal lingual and maxillary artery branches, which was then divided. The ICA (Internal Carotid Artery) was isolated and carefully separated from the adjacent vagus nerve, and the
  • pterygopalatine artery was ligated close to its origin with a 5-0 nylon suture (SMI, Belgium).
  • a 4-0 silk suture was tied loosely around the mobilized EC A stump, and a 4 cm length of 4- 0 monofi lament nylon suture (the tip of the suture was blun ted by using a fl ame, and the suture was coated with polylysine, prior to insertion) was inserted through the proximal ECA into the ICA and thence into the circle of Willis, effectively occluding the MCA.
  • the surgical wound was closed and the animals were returned to their cages to recover from anesthesia.
  • One hour and a half after occlusion rats were re-anesthetized, the monofilament was withdrawn to allow reperfusion, the surgical wound was closed and rats were returned to their cages.
  • mice were exposed to an angiotensin (1-7) peptide (TXA302) beginning 28 days after the stroke event and continuing for 56 days. Starting on Day 29, 28 days post-surgery, animals in Group 1 (saline control) and Group 2 (50 ⁇ /13 ⁇ 4 TXA302) received daily subcutaneous injection.
  • the first phase of this Example includes an end to the angiotensin (1-7) peptide treatment period at Day 85 followed by a four week washout period (ending on Day 113). Table 1 shows the group design used in this Example.
  • TXA302 enjoyed statistically significant improvements in performance within two weeks of treatment, as compared to vehicle control. Also, as shown in FIG. 1, these improvements continued through the duration of the study (phase one to Day 113 and phase two through Day 126) with trending toward increased performance over time.
  • Stepping Test (Pre-Operation, and Day 29, 43, 50, 57, 64, 71, 78, 85, 92, 99, 106 and 113)
  • FIG. 2 shows that administration of TXA302 provided a significant improvement in performance by day 43, as compared to vehicle control animals. As with the results of the limb placement test, FIG. 2 also shows a trend toward continuing improvement over time.
  • mNRS Rating Scale
  • each animal was also subjected to a well-established cylinder test for forelimb akinesia and exploratory behavior.
  • the cylinder test provides a way to evaluate a rat's spontaneous forelimb use to support the body against the wall of a cylinder enclosure.
  • the test takes advantage of the animal's innate drive to explore a novel environment by standing on its hind limbs and leaning toward enclosed walls.
  • rats were placed into a a transparent cylinder for 5 minutes and the number of wall contacts performed independently with the left and right forepaw were counted and noted to a maximum number of 20 wall contacts per animal per session. Only supporting contacts were counted (i.e., full appositions of the paws with open digits to the cylinder walls).
  • FIG. 5 shows that animals receiving TXA302 exhibited a significant increase in performance by Day 57 as compared to vehicle control animals and that this increased performance continued throughout the duration of the study.
  • each group of animals exhibited functional benefit from administration of dalfampridine, with animals having previously received TXA302 performing better than those having previously received vehicle only. Animals receiving dalfampridine after receiving TXA302 exhibited further increased performance benefit on all tests beyond that observed with TXA302 alone.
  • FIG. 7 shows, among other things, the effect of dalfampridine on animals that received only vehicle in phase one of the study. As expected, these animals saw an improvement in
  • FIG. 8 shows, among other things, the effects of dalfampridine on animals that received TXA302 (SEQ ID NO: 3) during phase one of the study as compared to control animals on days 1 13 and 116. While TXA302 exhibited dramatic improvements in performance, as described above, FIG. 8 shows that this improvement was further increased during dalfampridine administration and remained superior to controls, even when the control animal was also receiving dalfampridine.
  • FIG. 9 shows, among other things, the effect of dalfampridine on animals that received only vehicle in phase one of the study. As expected, these animals saw an improvement in performance during administration of dalfampridine, followed by a worsening of performance after cessation of dalfampridine substantially back to pre-dalfampridine levels.
  • FIG. 10 shows, among other things, the effects of dalfampridine on animals that received TXA302 (SEQ ID NO: 3) during phase one of the study as compared to control animals on days 113 and 116. While TXA302 exhibited dramatic improvements in performance, as described above, FIG. 10 shows that this improvement was further increased during dalfampridine administration and remained superior to controls, even when the control animal was also receiving dalfampridine.
  • FIG. 11 shows, among other things, the effect of dalfampridine on animals that received only vehicle in phase one of the study. As expected, these animals saw an improvement in
  • FIG. 12 shows, among other things, the effects of dalfampridine on animals that received TXA302 (SEQ ID NO: 3) during phase one of the study as compared to control animals on days 1 13 and 116. While TXA302 exhibited dramatic improvements in performance, as described above, FIG. 12 shows that this improvement was further increased during dalfampridine administration and remained superior to controls, even when the control animal was also receiving dalfampridine.
  • FIG. 13 shows, among other things, the effect of dalfampridine on animals that received only vehicle in phase one of the study. As expected, these animals saw an improvement in
  • FIG. 14 shows, among other things, the effects of dalfampridine on animals that received TXA302 (SEQ ID NO: 3) during phase one of the study as compared to control animals on days 1 13 and 116. While TXA302 exhibited dramatic improvements in performance, as described above, FIG. 14 shows that this improvement was further increased during dalfampridine administration and remained superior to controls, even when the control animal was also receiving dalfampridine.
  • FIG. 6 shows that animals receiving TXA302 showed statistically significant improvement in blood vessel diameter, as compared to vehicle control animals. Specifically, the right panel of FIG. 6 shows that animals receiving subcutaneously administered TXA302 followed by dalfampridine administration showed enhanced blood vessel diameter of approximately 25% larger than vehicle control animals.
  • FIG. 6 shows that by day 128, animals in the TXA302 treatment group shows a statistically significant improvement in blood flow as compared to vehicle control animals. Specifically, FIG. 6 (left panel) shows that animals receiving TXA302 followed by
  • dalfampridine administration showed enhanced blood flow ( ⁇ 45%) as compared to vehicle controls.
  • This Example shows, among other things, that administration of a combination of an angiotensin (1-7) peptide and a potassium channel blocker, even if administration begins well after a stroke event, results in synergistically improved outcomes after stroke.
  • these results represent the first time an angiotensin (1-7) peptide and a potassium channel blocker were shown to synergistically improve functional recovery after a stroke event.
  • angiotensin (1-7) peptides and potassium channel blockers represent a powerful and previously unknown treatment regimen for use in aiding patients who have suffered one or more brain conditions, including stroke events.

Abstract

The present invention provides, among other things, methods of treating brain conditions including the steps of administering to a subject suffering from or who previously suffered from a brain condition at least one angiotensin (1-7) peptide, and administering to the subject at least one potassium channel blocker. In some embodiments, the present invention provides methods of treating brain conditions including the step of administering to a subject suffering from or who previously suffered from a brain condition, and was administered an angiotensin(1-7) peptide, a potassium channel blocker. In some embodiments, the present invention provides methods of treating brain conditions including the step of administering to a subject suffering from or who previously suffered from a brain condition, and was administered a potassium channel blocker, an angiotensin(1-7) peptide.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF STROKE
BACKGROUND
[0001] Proper functioning of the central nervous system is essential in any animal.
Damage to the central nervous system, and particularly the brain, can have a wide range of physical, cognitive, and behavioral effects, including paralysis, dementia, disruption of proper motor function, and even death. As a result, timely administration of effective treatment after an injury to the brain is of critical importance. Unfortunately, several obstacles often make treatment difficult, including the blood brain barrier and even time itself. Due to these and other challenges, the development of effective mono- and/or combination therapies for treating brain conditions has proven extremely difficult.
SUMMARY OF THE INVENTION
[0002] The present invention is based, in part, on the surprising discovery that administration of at least one angiotensin(1-7) peptide in combination with at least one potassium channel blocker (e.g., dalfampridine), results in a synergistic improvement in one or more signs, symptoms, and/or complications of brain conditions including, but not limited to, stroke, vascular dementia, and/or traumatic brain injury. The present invention provides, among other things, new options in the treatment of brain conditions that can be administered not only after the "golden hour" (traditionally recognized as the hour following an event such as a stroke or other brain trauma when treatment is thought to have maximum effect), but significantly after that time, even days or weeks after an event, and still achieve a significant improvement in a subject's condition.
[0003] In some embodiments, the present invention provides methods of treating brain conditions including the steps of administering to a subject suffering from or who previously suffered from a brain condition at least one angiotensin (1-7) peptide, and administering to the subject at least one potassium channel blocker. In some embodiments, the at least one angiotensin (1-7) peptide and at least one potassium channel blocker are administered substantially simultaneously. In some embodiments, the at least one angiotensin (1-7) peptide is administered prior to admini stration of the at least one potassium channel b locker. In some embodiments, the at least one potassium channel blocker is administered prior to administration of the at least one angiotensin (1-7) peptide, in some embodiments, the at least one angiotensin (1-7) peptide and at least one potassium channel blocker are administered in overlapping regimen.
[0004] in some embodiments, the presen t invention provides methods of treating brain conditions including the step of administering to a subject suffering from or who previously suffered from a brain condition, and was administered an angiotensin(1-7) peptide, a potassium channel blocker.
[0005] In some embodiments, the present invention provides methods of treating brain conditions including the step of administering to a subject suffering from or who previously suffered from a brain condition, and was administered a potassium channel blocker, an angiotensin(1-7) peptide.
[0006] Various embodiments may be used to treat any of a vari ety of brain conditions.
In some embodiments, a brain condition is selected from stroke, vascular dementia, and traumatic brain injury. In some embodiments, stroke is selected from ischemic stroke, hemorrhagic stroke, or a combination thereof.
[0007] Provided methods and compositions, according to various embodiments, may be administered according to a variety of timepoints after development of a brain condition (e.g., a stroke). In some embodiments, at least one of an angiotensin (1-7) peptide and a potassium channel blocker are administered within 24 hours of the development of a brain condition (e.g., stroke). In some embodiments, at least one of an angiotensin (1-7) peptide and a potassium channel blocker are administered at least 24 hours after the development of a brain condition (e.g., stroke). In some embodiments, at least one of an angiotensin (1-7) peptide and a potassium channel blocker are administered at least 1 week after the development of a brain condition (e.g., stroke). In some embodiments, at least one of an angiotensin (1 -7) peptide and a potassium channel blocker are administered at least 4 weeks after the development of a brain condition (e.g., stroke). [0008] According to various embodiments, any potassium channel blocker may be used in accordance with provided methods and compositions. In some embodiments, a potassium channel blocker is an FDA-approved potassium channel blocker. In some embodiments, a potassium channel blocker is selected from dalfampridine, amiodarone, dronedarone, dofetilide, sotalol, ibutilide, azimilide, bretylium, clofilium, E-4031 , nifekalant, tedisamil, sematilide, and combinations thereof. In some embodiments, a potassium channel blocker is administered at a dose other than an FDA-approved dose. In some embodiments, a potassium channel blocker is administered according to a dosing regimen other than an FDA-approved dosing regimen.
[0009] Various embodiments may be administered via any medically appropriate route.
In some embodiments an angiotensin (1-7) peptide and/or potassium channel blocker is administered via parenteral administration, rectal administration, oral administration, or a combination thereof. In some embodiments, parenteral administration is intravenous, subcutaneous, inhalation, intradermal, transdermal, and/or transmucosal administration. In some embodiments, an angiotensin (1-7) peptide is administered systemically.
[0010] A variety of dosing regimen are contemplated as within the scope of various embodiments. In some embodiments, an angiotensin (1-7) peptide and/or potassium channel blocker are administered at least once per day.
[0011] Any of a variety of doses are contemplated as compatible with various embodiments. In some embodiments, an angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-1,000 ug/kg/day. In some embodiments, an angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-500 ug/kg/day. In some embodiments, an angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-60 ug/kg/day.
[0012] It is contemplated that various angiotensin (1-7) peptides may be used in accordance with various embodiments. In some embodiments, the angiotensin (1-7) peptide comprises the naturally-occurring Angiotensin (1-7) amino acid sequence of Asp1 -Arg2-Val3 - Tyr4-Ile5-His6-Pro7 (SEQ ID NO: 1). [0013] in some embodiments, the angiotensin (1-7) peptide is a functional equivalent of
SEQ ID NO: 1. In some embodiments, the functional equivalent is a linear peptide. In some embodiments, the linear peptide comprises a sequence that includes at least four amino acids from the seven amino acids that appear in the naturally-occurring Angiotensin (1-7), wherein the at least four amino acids maintain their relative positions as they appear in the naturally- occurring Angiotensin (1-7). In some embodiments, the linear peptide contains 4-25 amino acids. In some embodiments, the linear peptide is a fragment of the naturally-occurring
Angiotensin (1-7). In some embodiments, the linear peptide contains amino acid substitutions, deletions and/or insertions in the naturally-occurring Angiotensin (1-7). In some embodiments, the linear peptide has an amino acid sequence of A
Figure imgf000005_0003
Figure imgf000005_0004
. In some embodiments, the linear peptide has an amino acid sequence of
Figure imgf000005_0002
Figure imgf000005_0001
4 5 6 7
[0014] In some embodiments, an angiotensin (1-7) peptide comprises one or more chemical modifications to increase protease resistance, serum stability and/or bioavailability. In some embodiments, the one or more chemical modifications comprise pegylation.
[0015] In some embodiments, the angiotensin (1-7) peptide is administered at an effective dose periodically at an administration interval such that at least one sign, symptom and/or feature of a brain condition is reduced in intensity, severity, duration, or frequency or has delayed onset. In some embodiments, the at least one sign, symptom and/or feature of a brain condition is selected from paralysis, memory loss, pain, seizure, dysphagia, aphasia, dysarthria, ataxia, depression, mood swings, and loss of vision.
[0016] In some embodiments, an angiotensin(1-7) peptide is a non-peptidic angiotensin
(1 -7) receptor agonist. In some embodiments, a non-peptidic angiotensin (1 -7) receptor agonist is a 1 -(p-thienylbenzyl)imidazole. In some embodiments, the non-peptidic angiotensin (1-7) receptor agonist is a compound with the following structure:
Figure imgf000006_0001
or a pharmaceutically acceptable salt thereof.
[0017] 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
abo ut/ approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art.
[0018] 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.
BRIEF DESCRIPTION OF THE DRAWING
[0019] The Drawing, which is comprised of at least the following Figures, is for illustration purposes only, not for limitation.
[0020] FIG. 1 shows exemplary results from a forelimb placement test from rats exposed to one of: saline (vehicle control) or 50 μg/kgTXA302 (SEQ ID NO: 3), given subcutaneously for up to eight weeks beginning 28 days post tMCAO event, followed on day 1 13 (112 days after tMCAO event) by 2 mg/kg dalfampridine given orally twice per day for three days. [0021 ] FIG. 2 shows exemplary results in a stepping from rats exposed to one of: saline
(vehicle control) or 50 μg/kg TXA302 (SEQ ID NO: 3), given subcutaneously for up to eight weeks beginning 28 days post tMCAO event, followed on day 113 (112 days after tMCAO event) by 2 mg/kg dalfampridine given orally twice per day for three days.
[0022] FIG. 3 shows exemplary results from a body swing test (left turn-right turn) from rats exposed to one of: saline (vehicle control) or 50 μg/kg TXA302 (SEQ ID NO: 3), given subcutaneously for up to eight weeks beginning 28 days post tMCAO event, followed on day 1 13 (1 12 days after tMCAO event) by 2 mg/kg dalfampridine given orally twice per day for three days.
[0023] FIG. 4 shows a graph of exemplary neuroscores from rats exposed to one of: saline (vehicle control) or 50 μg/kg TXA302 (SEQ ID NO: 3), given subcutaneously for up to eight weeks beginning 28 days post tMCAO event, followed on day 113 (112 days after tMCAO event) by 2 mg/kg dalfampridine given orally twice per day for three days.
[0024] FIG. 5 shows a graph of exemplary results from a cylinder test of forelimb akinesia from rats exposed to one of: saline (vehicle control) or 50 ,ug/kg TXA302 (SEQ ID NO: 3), given subcutaneously for up to eight weeks beginning 28 days post tMCAO event.
[0025] FIG. 6 shows an example of the percent change of blood flow (left panel) and average vessel diameter (right panel) in rats exposed to one of: saline (vehicle control) or 50 μg/kg TXA302 (SEQ ID NO: 3), given subcutaneously for up to eight weeks beginning 28 days post tMCAO event, followed on day 113 (112 days after tMCAO event) by 2 mg/kg
dalfampridine given orally twice per day for three days.
[0026] FIG. 7 shows a selected view of Day 113 to Day 126 data from FIG. 1.
Specifically, FIG. 7 shows the effect of Dalfampridine on animals receiving a vehicle control during phase 1 of the study.
[0027] FIG. 8 shows a selected view of Day 1 13 to Day 116 data from FIG. 1.
Specifically, FIG. 8 shows the effect of Dalfampridine on animals receiving TXA302 (SEQ ID NO: 3) during phase 1 of the study. [0028] FIG. 9 shows a selected view of Day 113 to Day 126 data from FIG. 2.
Specifically, FIG. 9 shows the effect of Dalfampridine on animals receiving a vehicle control during phase 1 of the study.
[0029] FIG. 10 shows a selected view of Day 113 to Day 116 data from FIG. 2.
Specifically, FIG. 10 shows the effect of Dalfampridine on animals receiving TXA302 (SEQ ID NO: 3) during phase 1 of the study.
[0030] FIG. 11 shows a selected view of Day 113 to Day 126 data from FIG. 3.
Specifically, FIG. 1 1 shows the effect of Dalfampridine on animals receiving a vehicle control during phase 1 of the study.
[0031] FIG. 12 shows a selected view of Day 113 to Day 116 data from FIG. 3.
Specifically, FIG. 12 shows the effect of Dalfampridine on animals receiving TXA302 (SEQ ID NO: 3) during phase 1 of the study.
[0032] FIG. 13 shows a selected view of Day 113 to Day 126 data from FIG. 4.
Specifically, FIG. 13 shows the effect of Dalfampridine on animals receiving a vehicle control during phase 1 of the study.
[0033] FIG. 14 shows a selected view of Day 1 13 to Day 116 data from FIG. 4.
Specifically, FIG. 14 shows the effect of Dalfampridine on animals receiving TXA302 (SEQ ID NO: 3) during phase 1 of the study.
DEFINITIONS
[0034] 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.
[0035] 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.
[0036] 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%, 1 1%, 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).
[0037] 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.
[0038] Brain Condition - as used herein, a "brain condition" is any disease, disorder or event that results in damage and/or dysfunction of at least a portion of a subject's brain. Non- limiting examples of brain conditions include: stroke (both ischemic and hemorrhagic), vascular dementia, and traumatic brain injury.
[0039] 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. [0040] 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.
[0041] 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.
[0042] 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 indi vidually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent 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).
[0043] 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 may be 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.
[0044] Improve, increase, or reduce: As used herein, the terms "improve," "increase" or
"reduce," or grammatical equivalents, indicate values that are relative to a baseline measurement, 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.
[0045] In vitro: As used herein, the term "in 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.
[0046] In vivo: As used herein, the term "in 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).
[0047] 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 m anufactured by the hand of man. Isolated substances and/or en titi es 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. [0048] 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 defini tion of "risk."
[0049] 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.
[0050] 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.
[0051] 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., stroke or other brain condition). 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., stroke or other brain condition). In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, relative risk is 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
[0052] 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). [0053] Stability: As used herein, the term "stable" refers to the abili ty of the therapeutic agent to maintain its therapeutic efficacy (e.g., all or the majority of its intended biological activity and/or physiochemical in tegri ty) over extended periods of time. The stabi lity 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).
[0054] Subject: As used herein, the term "subject" refers to a human or any non-human 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.
[0055] 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 m an y biological an d chemical phenom en a.
[0056] 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.
[0057] 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, ischemic stroke) may be characterized by one or more of the following: (1) a genetic mutation associated wi th developmen t 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.
[0058] 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.
[0059] 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.
[0060] 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. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0061] The present invention provides, among other things, methods and compositions for treating brain conditions (e.g., stroke, including, but not limited to, ischemic and hemorrhagic stroke). Provided methods of treating brain conditions include administering to a subject suffering from a brain condition an angiotensin (1-7) peptide, and administering a potassium channel blocker to the subject. The combination of at least one angiotensin (1-7) peptide and at least one potassium channel blocker provides surprisingly positive results in the improvement of one or more si gns, symptoms and/or features of brain conditions.
[0062] The ability of angiotensin (1-7) peptides and potassium channel blockers to synergistically treat brain conditions (e.g., stroke) represents a new form of treatment for sufferers of brain conditions. The beneficial effects of the combination of angiotensin (1 -7) peptides and potassium channel blockers is particularly surprising in light of the fact that such treatments may begin well after a stroke event, even days or weeks after the development of a brain condition, given dogma in the art that treatments administered outside the first several hours, particularly in the cases of stroke and/or traumatic brain injury, typically have limited effectiveness at best. As a result, embodiments of the present invention provide entirely new treatment regimens for use by physicians in aiding stroke sufferers.
[0063] 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.
Brain Conditions
Stroke
[0064] The brain is highly vulnerable to a disturbance in its oxygen supply. Anoxia and ischemia lasting only a few seconds can cause symptoms and if the condition persists for minutes, they can cause irreversible neuronal damage. Accordingly, stroke is a prominent cause of serious, long-term disability and a leading cause of death in the United States, with 500,000 new or recurrent cases occurring each year (see Kelly-Hayes et al., 1998, The American Heart Association Stroke Outcome Classification, Stroke, 29: 1274-1280). Stroke is also a significant burden on the medical industry, with total health costs for disability due to stroke being estimated at approximately $53 billion annually.
[0065] There are two major types of stroke: ischemic and hemorrhagic. Ischemic stroke involves an obstruction in one or more blood vessels that supply blood to brain tissue, for example, occlusion resulting from atherosclerotic thrombii, or embolism. Ischemic stroke (cerebral ischemia) represent approximately 88% of all strokes, making ischemic stroke one of the most common types of cerebrovascular injury. Ischemic conditions in the brain quickly lead to neuronal death, often leading to permanent sensorimotor deficits. A hemorrhagic stroke is defined herein as the accumulation of blood anywhere within the cranial vault. Hemorrhagic strokes may result from many causes, including injury resulting from an expanding hematoma, which can disrupt or distort tissue.
[0066] A stroke may cause a wide variety of symptoms and/or complications. Without wishing to be held to a particular theory, it is thought that the specific signs, symptoms and/or complications, as well as their severity and duration, may be determined by where the stroke occurs in a brain as well as how severe it is. Because the brain controls or modulates nearly every system in the body, the range of potential signs, symptoms and/or complications is vast. In some embodiments, the signs, symptoms, and/or complications arising from a stroke include one or more of neurological impairmen t, cogni tive impairment, language impairment, emotional impairment (e.g., depression, anxiety), and motor impairment. According to various
embodiments, exemplary, non-limiting signs, symptoms and/or complications of stroke include, but are not limited to: paralysis, memory loss, pain, seizure, dysphagia (difficulty swallowing), aphasia (loss of speech or language ability), dysarthria (difficulty articulating words), ataxia (lack of coordinated movements), depression, mood swings, and loss of vision. In some embodiments, paralysis is partial (e.g., limited to one muscle group, area of the body, and/or side of the body). In some embodiments paralysis is substantially complete (e.g., affecting both sides of the body and most or all voluntary muscles below the neck). Those of skill in the art will be able to readily identify several additional signs, symptoms and/or complications of stroke, the treatment of each of which is contemplated as within the scope of the present invention. [0067] While it can be difficult to accurately catalog every sign, symptom or complication in order to determine an accurate clinical picture of a stroke sufferer, several impairment and rating scales exist to aid medical personnel in determining the proper
therapy(ies) for a patient as well as the effectiveness of therapy. Exemplary, non-limiting assessment/rating scales include, but are not limited to: the National Institutes of Health Stroke Scale (NIHSS), the Canadian Neurological Scale, the Middle Cerebral Artery Neurological Score, Guy's Prognostic Score, the American Heart Association's Stroke Outcome Classification (AHA SOC), the Mini-Mental State Examination (MMSE), the Neurobehavioral Cognitive Status Examination (NCSE), the American Speech-Language-Hearing Association Functional Assessment of Communication Skills for Adults, the Boston Diagnostic Aphasia Examination, the Center for Epidemiologic Studies Depression (CES-D) Scale, the Geriatric Depression Scale, the Basic Activities of Daily Living (BADL), the Instrumental Activities of Daily Living
(IADL), the Functional Independence Measure (FIM), and the Barthel Index. Those of skill in the art will recognize additional rating scales that may be of use according to some embodiments.
[0068] A major barrier in the treatment of both ischemic and hemorrhagic stroke is delivery of a therapeutic that will reach affected tissue. Given the effectiveness of the blood brain barrier, few compounds are capable of crossing into and affecting cerebral tissue.
Previously, delivery of compounds such as angiotensin (1-7), had to be made using
intracerebroventricular (ICV) delivery. Significantly, embodiments of the present invention, including the exemplary angiotensin (1-7) peptides described below, are able to cross the blood brain barrier without complex delivery systems such as modified stem cells or the like. Rather, in some embodiments, angiotensin (1-7) peptides may be delivered via intravenous or
subcutaneous routes.
[0069] Despite many medical advancements over the last several decades, the treatment of stroke remains very difficult. Without wishing to be held to a particular theory, factors contributing to the difficulty in treating stroke are thought to include: a) the difficulty in getting therapies to the brain due to the blood-brain-barrier, b) the difficulty in getting a patient treated within the limited window during which previously known therapies have been shown to be effective, and c) the heterogeneity of stroke manifestations which can lead to drastically differing signs, symptoms and/or complications and which may not manifest or become obvious until a significant period of time has passed since the stroke.
[0070] Despite the difficulties, several forms of therapy and/or treatment exist for stroke sufferers. One primary form of treatment for ischemic strokes is reperfusion therapy, wherein the clot or other obstruction is targeted for destruction. Recombinant tissue plasminogen activator (rt-PA) is one of the primary drugs used in this context, though it is known that rt-PA only has a limited time window of 4.5 hours or less during which it has a significant effect, with earlier treatment being correlated with better outcomes (see American Heart Association, Target: Stroke Time Lost is Brain Lost, Target: Stroke Campaign Manual, 2010, available at
www . strokeassociation.org/idc/ groups/heart- pubiic/@wcm/@hcm/@gwtg''documents/downloadable/ucm_308277.pdf; see also Singh et al., Endovascular treatment of acute ischemic stroke, 2013, J. Neurosci Rural Pract, 4(3): 298-303). Additional drugs used in reperfusion therapy include recombinant pro-urokinase, anticoagulants, antiplatelet therapies, and antihypertensives. In some cases, such as when an obstruction is severe enough, or when treatment with one or more drugs is either not appropriate or sufficient, a surgical endovascular procedure is often used. One primary type of surgical therapy is endovascular thrombectomy, in which the clot/thrombus is removed. Exemplary types of endovascular thrombectomy include, but are not limited to: proximal endovascular
thrombectomy (e.g., suction thrombectomy) and distal endovascular thrombectomy (e.g., wherein the clot is physically seized and removed from the cerebral vessels). Use of angioplasty, such as balloon angioplasty and/or placement of one or more stents are also common
interventional therapies used in certain stroke cases.
Vascular Dementia
[0071] Vascular dementia is the second most common form of dementia, behind
Alzheimer's Disease. Vascular dementia may result from problems with blood supply in the brain, such as those caused by ischemic or hemorrhagic stroke or from other causes that lead to development of lesions in the brain. Other causes of vascular dementia include cerebral amyloid angiopathy, hypercholesterolemia, diabetes mellitus, or cardiovascular disease. Dementia resulting from one or more strokes is also known as "single-infarct dementia" or "multi-infarct dementia," depending upon the root cause.
[0072] Treatment of vascular dementia has primarily focused on the prevention of further cerebrovascular lesions through use of antiplatelet drugs and life style changes (alteration of diet, cessation of smoking, etc). Cholinesterase inhibitors such as galantamine have also been explored for use in this clinical scenario, but this type of treatment is concerned with
maintenance of acetylcholine function in the brain, and not recovery or generation of an improved and sustained blood supply. Thus, embodiments of the present invention represent a novel intravenous and subcutaneous therapy targeted to improving the underlying causes of the disease rather than management of symptoms or maximization of remaining tissue resources.
Traumatic Brain Injury
[0073] Traumatic brain injury (TBI), a form of acquired brain injury, occurs when a sudden trauma causes damage to the brain. TBI can result when the head suddenly and violently hits an object (or vice versa), or when an object pierces the skull and enters brain
tissue. Symptoms of a TBI can be mild, moderate, or severe, depending on the extent of the damage to the brain. A person with a mild TBI may remain conscious or may experience a loss of consciousness for a few seconds or minutes. Other symptoms of mild TBI include headache, confusion, lightheadedness, dizziness, blurred vision or tired eyes, ringing in the ears, bad taste in the mouth, fatigue or lethargy, a change in sleep patterns, behavioral or mood changes, and trouble with memory, concentration, attention, or thinking. A person with a moderate or severe TBI may show these same symptoms, but may also have a headache that gets worse or does not go away, repeated vomiting or nausea, convulsions or seizures, an inability to awaken from sleep, di lation of one or both pupils of the eyes, slurred speech, weakness or numbness in the extremities, loss of coordination, and increased confusion, restlessness, or agitation.
[0074] Treatments for TBI focus primarily on preventing further injury or complications.
Primary concerns in treating TBI include insuring proper oxygen supply to the brain and the rest of the body, maintaining adequate blood flow, and controlling blood pressure. Often, the primary treatment a TBI sufferer receives post-stabil ization is rehabilitation that involves individually tailored treatment programs in the areas of physical therapy, occupational therapy, speech/language therapy, psycho logy/ psychiatry, and social support. Embodiments of the present invention provide a novel treatment for these patients.
Angiotensin (1-7) peptides
[0075] As used herein, the term "angiotensin (1-7) peptide" refers to both naturally- occurring Angiotensin (1-7) and any functional equivalent, analogue or derivative 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^ 1 -7)", "Angiotensin^ 1 -7)", and "Ang-(l-7)" are used interchangeably.
Naturally-occurring Angiotensin (1-7)
[0076] Naturally-occurring Angiotensin (1-7) (also referred to as Ang-(1-7)) is a seven amino acid peptide shown below:
Figure imgf000020_0002
It is part of the renin-angiotensin system and is converted from a precursor, also known as Angiotensinogen, which is an α-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:
Figure imgf000020_0001
[0077] 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).
[0078] Ang-(l-7) is an endogenous ligand for Mas receptors. Mas receptors are G- protein coupled receptor containing seven transmembrane spanning regions. As used herein, the term "angiotensin^ 1 -7) receptor' encompasses the G Protein-Coupled Mas Receptors. [0079] 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).
Functional equivalents, analogs or derivatives ofAng-(l-7)
[0080] 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-(1-7). For example, in some embodiments, a functional equivalent of naturally-occurring Ang-(l-7) described herein has pro-angiogenic activity as determined using methods described herein or known in the art, or an activity such as nitric oxi de release, vasodil ation, improved endoth elial function, antidiuresis, or one of the other properties discussed herein, that positively impacts angiogenesis. In some embodiments, a functional equivalen t of natural ly-occurrin g Ang-(1-7) described herein can bind to or activate an angiotensin^ 1-7) 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.
[0081] 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-(l-7).
[0082] In some embodiments, a functional equivalent of Ang-(1-7) 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-(1-7). 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 et al, Methods in Enzymology 266, 460-480 (1996); http://l3last.wustL''edu/blast''README.html). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adj ustable parameters are set with the following values: overlap span= 1 , overlap fraction^.125, word threshold (T)=l l. HSP score (S) and HSP S2 parameters are dynamic values and are established by the program itself, depending upon the composition of the particular sequence, however, the m in imum values may be adjusted and are set as indicated above.
[0083] In some embodiments, a functional equivalent, analogue or derivative of Ang-(1-
7) is a fragment of the natural ly-occurring An g-(1 -7). In some embodiments, a functional equivalent, analogue or derivative of Ang-(1-7) contains amino acid substitutions, deletions and/or insertions in the naturally-occurring Ang-(1-7). 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. 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, tryptophane, 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.
[0084] Examples of Ang-(1-7) functional equivalents, analogues and derivatives are described in the section entitled "Exemplary Angiotensin^ -7) Peptides" below.
[0085] An angiotensin^ 1-7) peptide can be of any length. In some embodiments, an angiotensin^ 1-7) 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, 4-1 1, 4-10, 4-9, 4-8, 4-7 amino acids). In some embodiments, the linear peptide contains 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.
[0086] In some embodiments, an angiotensin^ 1-7) 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 un-natural amino acid, and/or cyclization of the peptide.
[0087] 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., α,α-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 unnatural 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 half-life without adversely affecting its activity. Examples of unconventional or un-natural amino acids include, but are not limited to, citrulline, ornithine, norleucine, norvaline, 4-(E)-butenyl-4(i?)-methyl-N-methylthreonine (MeBmt), N-methyl- leucine (MeLeu), aminoisobutyric acid, statine, and N-methyl-alanine (Me Ala). 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.
[0088] In certain embodiments, angiotensin-( 1-7) peptides contain one or more L-amino acids, D-amino acids, and/or un-natural amino acids.
[0089] 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
Figure imgf000024_0002
(cis and trans), -
Figure imgf000024_0001
- 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. [0090] 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 (fluorenylmethyi-0-CO-); (2) esters of the carboxy terminal or of another free carboxy or hy droxyl 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.
[0091] 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,
hydroxym ethyl 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). [0092] 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 Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, N.Y., 2001.
[0093] 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-butyloxycarbonyl (BOC), cyclohexyloxycarbonyl), aromatic urethane type protecting groups (e.g., fluorenyl-9- methoxy-carbonyl (Fmoc), benzyloxycarbonyl (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.
[0094] Further, Ang-(1-7) 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 CI 8 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).
[0095] 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 wi thin the same polypeptide.
[0096] 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 cl eavage. Furthermore, exopeptidases, whi ch represent the most promin en t 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.
[0097] 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. I t is contemplated that reverse L-pep tides 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 activi ty as the ori ginal L- amino acid peptide.
[0098] 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 aUcyl group, consisting of a lower alkyl of from one to twen ty 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.
[0099] 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 α,α - disubstituted amino acids, "N -alkyl amino acids, C-a-methyl amino acids, β-amino acids, and β- methyl amino acids. Amino acids analogs useful in the present invention may include, but are not limited to, β-alanine, norvaline, norleucine, 4-aminobutyric acid, orithine, hydroxyproline, 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.
[0100] 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 aci ds an d 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
[0101] 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 derivati ve compounds of this invention.
[0102] Cyclic peptides as described herein may comprise residues of L-amino acids, D- amino acids, or any combination thereof. Amino acids may be from natural or non-natural sources, provided that at least one amino group and at least one carboxyl group are present in the molecule; a- and β-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 alkoxycarbonylation), with or without any of a wide variety of side-chain modifications and/or substitutions (e.g., methyl ation, benzylation, t-butylation, tosylation, alkoxycarbonylation, 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, β,β-tetramethylene cysteine, β,β-pentamethylene cysteine, β- mercaptopropionic acid, β,β-pentamethylene-β-mercaptopropionic acid, 2-mercaptobenzene, 2- mercaptoaniline, 2-mercaptoproline, ornithine, diaminobutyric acid, a-aminoadipic acid, m- aminomethylbenzoic acid and a, β-diam inopropioni c acid.
[0103] Following synthesis of a linear peptide, with or without N-acetylation and/or 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 K3Fe(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. 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. Residues capable of forming a lactam bond include lysine, ornithine (Orn), a-amino adipic acid, m-aminomethylbenzoic acid, α,β-diaminopropionic acid, glutamate or aspartate. Methods for forming amide bonds are generally well known in the art. Within one such method, carbodi imide-mediated lactam formation can be accomplished by reaction of the carboxylic acid with DCC, DIC, ED AC or DCCI, resul ting in the formation of an O-acylurea that can be reacted immediately with the free amino group to complete the cyclization. Alternatively, cyclization can be performed using the azide method, in which a reactive azide intermediate is generated from an alkyl ester via a hydrazide. 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 in DMF.
Exemplary Angiotensin- (1-7) Peptides
[0104] In certain aspects, the invention provides linear angiotensin^ 1-7) peptides. As discussed above, the structure of naturally-occurring Ang-(1-7) is as follows:
Figure imgf000031_0001
[0105] The peptides and peptide analogs of the invention can be generally represented by the following sequence:
Figure imgf000031_0002
or a pharmaceutically acceptable salt thereof.
[0106] Xaa1 is any amino acid or a dicarboxylic acid. In certain embodiments, Xaa1 is
Asp, Glu, Asn, Acpc ( 1 -aminocyclopentane carboxylic acid), Ala,
Figure imgf000031_0003
dimethylglycine), Pro, Bet (betaine, 1 -carboxy-N,N,N-trimethylmethanaminium hydroxide), Glu, Gly, Asp, Sar (sarcosine) or Sue (succinic acid). In certain such embodiments, Xaa1 is a negatively-charged amino acid, such as Asp or Glu, typically Asp. [0107] Xaa2 is Arg, Lys, Ala, Cit (citrulline), Orn (ornithine), acetylated Ser, Sar, D-Arg and D-Lys. In certain embodiments, Xaa is a positively-charged amino acid such as Arg or Lys, typically Arg.
[0108] Xaa3 is Val, Ala, Leu, Nle (norleucine), IIe, Gly, Lys, Pro, HydroxyPro
(hydroxyproline), Aib (2-aminoisobutyric acid), Acpc or Tyr. In certain embodiments, Xaa" is an aliphatic amino acid such as Val, Leu, IIe or Nle, typically Val or Nle.
[0109] Xaa4 is Tyr, Tyr(P03), Thr, Ser, homoSer (homoserine), azaTyr (aza-a'-homo-L- tyrosine) or Ala. In certain embodiments, Xaa4 is a hydroxyl -substituted amino acid such as Tyr, Ser or Thr, typically Tyr.
[0110] Xaa5 is IIe, Ala, Leu, norLeu, Val or Gly. In certain embodiments, Xaa5 is an aliphatic amino acid such as Val, Leu, IIe or Nle, typically IIe.
[0111] Xaa6 is His, Arg or 6-NH2-Phe (6-aminophenylalaine). In certain embodiments,
Xaa6 is a fully or partially positively-charged amino acid such as Arg or His.
[0112] Xaa7 is Cys, Pro or Ala.
[0113] In certain embodiments, one or more of Xaa -Xaa is identical to the
corresponding amino acid in naturally-occurring Ang-(1-7). In certain such embodiments, all but one or two of Xaa1 -Xaa7 are identical to the corresponding amino acid in naturally-occurring Ang-(l-7). In other embodiments, all of Xaa1 -Xaa6 are identical to the corresponding amino acid in naturally-occurring Ang-(l-7).
[0114] In certain embodiments, Xaa3 is Nle. When Xaa3 is Nle, one or more of Xaa1-
Xaa2 and Xaa4-7 are optionally identical to the corresponding amino acid in naturally-occurring Ang-(1-7). In certain such embodiments, all but one or two of Xaa1 -Xaa2 and Xaa4-7 are identical to the corresponding amino acid in naturally-occurring Ang-(l-7). In other
embodiments, all of Xaa1 -Xaa2 and Xaa4-7' are identical to the corresponding amino acid in naturally-occurring Ang-(1-7), resulting in the amino acid sequence: Asp1-Arg2-Nle3-Tyr4-Ile5- His6-Pro7 (SEQ ID NO: 6). [0115] in certain embodiments, the peptide has the amino acid sequence Asp -Arg~-Val" -
Ser4-Ile5-His6-Cys7 (SEQ ID NO: 2) or Ala1-Arg2Val3Ser3Ile5His6Cys7 (SEQ ID NO: 3).
[0116] 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 ID NO: 22), which is 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.
[0117] 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: 23). Additional sequences derived from SEQ ID NO: 23 may be found in European Patent Application
2,264,048, the disclosure of which is hereby incorporated by reference.
Exemplary Cyclic Angiotensin (1-7) Peptides
[0118] In certain aspects, the invention provides a cyclic angiotensin^ 1 -7) (Ang-(1-7)) peptide analog comprising a linkage, such as between the side chains of amino acids
corresponding to positions Tyr4 and Pro7 in 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 Asp 1 -Arg2-Val3-Ser4-Ile5-His6-Cys7 (SEQ ID NO: 2), wherein a linkage is formed between Ser4 and Cys''. Another example of such an analog is Ala1-Arg2-Val3'-Ser4-Ile5-His6-Cys7 (SEQ ID NO: 3), wherein a linkage is formed between Ser4 and Cys7
[0119] 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 bridge-containing peptides can be formed by two amino acids having one of the following formulas:
Figure imgf000034_0001
Figure imgf000034_0002
Formula (III)
[0120] In these formulae, R1, R2, R3, R4, R5 and R6 are independently -H, an alkyl (e.g.,
C1-C6 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, R5 and R6 are each independently -H or -CH3, such where all are -H.
[0121] In certain embodiments, the invention provides an Ang analog or derivative comprising a thioether bridge according to formula (I). Typically, R1, R2, RJ and R4 are 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- penicill amine in position 7 (see, e.g., Galande, Trent and Spatola (2003) Biopolymers 71 , 534- 551).
[0122] 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.
[0123] As discussed above, the Ang analogs and derivati ves 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 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 Me-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. Biological activity of an analog or derivative 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, (1933) Gen. Pharmacol. 24:705- 713.
[0124] Ang analogs and derivatives where only the length of the peptide is varied include the following:
[0125] a 4,7-cyclized analog designated [Cyc4-7] Ang-(1 -7), which is derived from natural
Ang-(1-7) (Asp1-Arg2VaP-Cyc4-Ile5His6Cyc7, SEQ ID NO: 7). a 4,7-cyclized analog designated [Nle3, Cyc4-7]Ang-(l-10), which is derived from natural Angiotensin I (Ang-(l-lO)) (Asp,-Arg2-Nle3-Cyc4-Ile5-His6-Cyc7-Phe8-His9-Leu!0, SEQ ID NO:
8);
a 4,7-cyclized analog designated [Nle3, Cyc JAng-O-S), which is derived from natural Angiotensin II (Ang-(l-8)) (Asp1-Arg2-Nle3-Cyc4-Ile5-His6-Cyc7-Phe8, SEQ ID NO: 9); 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: 10); a 4,7-cyclised analog designated [Nle3, Cyc^'JAng-Q-S), which is derived from natural Angiotensin IV (Ang-(3-8)) (Nle3-Cyc4-Ile5-His6-Cyc7-Phe8, SEQ ID NO: 11); a 4,7-cyclised analog designated [Nle3, Cyc4- 7]Ang-(1-7) derived from, natural Ang-(1 -7) (Asp1-Arg2-Nle3-Cyc4-ile5-His6-Cyc7, SEQ ID NO: 12); and a 4,7-cyclised analog designated [Nle3 , Cyc4-7 ]Ang-(l-9) derived from natural Ang-(l-9) (Asp1-Arg2-Nle3-Cyc4-Ile5-His6-Cyc7-Phe8-His9, SEQ ID NO: 13).
These analogs can have one of the thioether bridges shown in Formulae (I)-(I I I) as the Cyc4"7 moiety, for example, where Cyc4 and Cyc7 are represented by Formula (I), such as where R'-R4 are 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"7 analog 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-(1- 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):
[0128] Xaa1-Xaa2-Xaa3-Cyc4-Xaa5-Xaa6-Cyc7 (IV, SEQ ID NO: 14)
[0129] Xaa1 is any amino acid, but typically a negatively-charged amino acid such as Glu or Asp, more typically Asp.
[0130] Xaa2 is a positivel y-charged amino acid such as Arg or Lys, typically Arg.
[0131] Xaa3 is an aliphatic amino acid, such as Leu, IIe or Val, typically Val.
[0132] Cyc4 forms a thioether bridge in conjunction with Cyc '. Cyc4 can 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.
[0133] Xaa5 is an aliphatic amino acid, such as Leu, IIe or Val, typically IIe.
[0134] Xaa6 is His.
[0135] Cyc7 forms a thioether bridge in conjunction with Cyc4, such as in Formula (I),
(II) or (III). Cyc7 can 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. [0136] in certain embodiments, one or more of Xaa'-Xaa6 (excluding Cyc4 and Cyc7) is identical to the corresponding amino acid in naturally-occurring Ang-(1-7). In certain such embodiments, all but one or two of Xaa'-Xaa6 are identical to the corresponding amino acid in naturally-occurring Ang-(1-7). In other embodiments, all of Xaa'-Xaa6 are identical to the corresponding amino acid in naturally-occurring Ang-(1-7).
[0137] In certain embodiments, Cyc4 and Cyc7 are independently selected from Abu (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 R1-R4 are each -H); -Ala4-S-Abu7- (Formula (I): R1 -R3 are -H and R4 is -CH3) or -Abu4-S-Ala7- (Formula (I): R3 , R3 and R4 are -H and R2 is -CH3). Specific examples of cyclic analogs comprise a -Abu4-S- Ala7- or -Ala4-S-Ala7- linkage.
[0138] In certain embodiments, the invention provides an Ang-(1-7) analog with a thioether-bridge between position 4 and position 7 having the amino acid sequence Asp -Arg - Val3-Abu4-Ile5-His6-Ala7 (SEQ ID NO: 15) or the amino acid sequence Asp3-Arg2-Val3-Ala4- Ile5-His6-Ala7 (SEQ ID NO: 16), which are represented by the following structural diagrams:
Figure imgf000038_0001
Figure imgf000039_0001
[0139] In certain embodiments, an Ang analog or derivative of the invention is represented by Formula (V):
Xaa1Xaa2Nle3Cyc4Xaa5Xaa6Cyc7Xaa8Xaa9Xaa10 (V, SEQ ID NO: 17)
As discussed above, one or more of Xaa , Xaa , Xaa , Xaa and Xaa are absent in certain embodiments. For example, (1) Xaa10 is absent, (2) Xaa9 and Xaa10 are absent, (3) Xaa8, Xaa9 and Xaa10 are absent, (4) Xaa1 is absent, (5) Xaa3 and Xaa10 are absent, (6) Xaa1, Xaa9 and Xaa10 are absent, (7) Xaa1, Xaa8, Xaa9 and Xaa10 are absent, (8) Xaa1 and Xaa2 are absent, (9) Xaa1, Xaa2 and Xaa30 are absent, (10) Xaa1, Xaa2, Xaa9 and Xaa10 are absent, or (11 ) Xaa1, Xaa2, Xaa8, Xaa9 and Xaa10 are absent. For each of these embodiments, the remaining amino acids have the values described below.
[0140] Xaa1, when present, is any amino acid, but typically a negatively charged amino acid such as Glu or Asp, more typically Asp.
[0141] Xaa2, when present, is a positively charged amino acid such as Arg or Lys, typically Arg.
[0142] Nle3 is norleucine.
[0143] Cyc4 forms a thioether bridge in conjunction with Cyc7. Cyc4 can be a D- stereoisomer and/or a L-stereoisomer, typically a D-stereoisomer. Examples of Cyc (taken with Cyc7) are shown in Formulas (I), (II) and (III). Typically, the R groups in Formulae (I), (II) and (I II) are H or CH3, especially -H. [0144] Xaa5 is an aliphatic amino acid, such as Leu, Nie, lie or Val, typically IIe.
[0145] Xaa6 is His.
[0146] Cyc7 forms a thioether bridge in conjunction with Cyc4, such as in Formula (I),
(II) or (II I). Cyc' can 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.
[0147] Xaa8, when present, is an amino acid other than Pro, typically Phe or IIe. In certain embodiments, IIe results in an inhibitor of Ang(l-8). In certain embodiments, Phe maintains the biological activity of Ang(l-8) or Ang(l-lO).
[0148] Xaa9, when present, is His.
[0149] Xaa10, when present, is an aliphatic residue, for example, IIe, Val or Leu, typically
Leu.
[0150] In certain embodiments, one or more of Xaa'-Xaa10 (excluding Nle3, Cyc4 and
Cyc') is identical to the corresponding amino acid in naturally-occurring Ang (including Ang-(1- 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 Xaa3 -Xaa10 (for those present) are identical to the corresponding amino aci d in naturally-occurring Ang.
[0151] In certain embodiments, Cyc4 and Cyc7 are independently selected from Abu (2- aminobutyric acid) and Ala (alanine), where Ala is present at at least one position. Thus, encompassed are cyclic analogs comprising a thioether linkage formed by -Ala4-S-Ala7- (Formula (I), where R'-R4 are each -H); -Ala4-S-Abu7- (Formula (I): R '-R3 are -H and R4 is -CH3) or -Abu4-S-Ala7- (Formula (I): R1, R3 and R4 are -H and R2 is -CH3). Specific cyclic analogs comprise a -Abu4-S-Ala7- or -Ala4-S-Ala'- linkage.
[0152] In particular, the invention provides an Ang-(l-7) analog or derivative with a
1 2 thioether-bridge between position 4 and position 7 having the amino acid sequence Asp -Arg - Nle3-Abu4-lle5-His6-Ala7 (SEQ ID NO: 18) or the amino acid sequence Asp1-Arg2Nie3Ala4- Ile5-His6-Ala7 (SEQ ID NO: 19).
[0153] In another aspect, the invention provides an Ang-(l-8) analog or derivative with a thioether-bridge between position 4 and position 7 having Ang-(l-8) antagonLstic activity, in particular an Ang(l-8) analog or derivative having the amino acid sequence Asp -Arg -Nle - Abu4-Ile5-His6-Ala7-Ile8 (SEQ ID NO: 20), or the amino acid sequence Asp^Arg^Nle'-Ala4- Ile5-His6-Ala7-Ile8 (SEQ ID NO: 21).
[0154] 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 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 and isoindolyl.
Ang (1-7) Receptor Agonists
[0156] In some embodiments, the present invention provides methods of treating brain conditions including administering to a subject who is suffering from or susceptible to one or more brain conditions an angiotensin (1 -7) receptor agonist. As used herein, the term
"angiotensin-( 1 -7) receptor agonist" is considered within the scope of the term "angiotensin (1-7) peptide" and encompasses any molecule that has a positive impact in a function of an
angiotensin^ 1-7) 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 (i.e., the Mas receptor) activity. In some embodiments, an angiotensin^ 1-7) receptor agonist directly interacts with an angiotensin^ 1-7) receptor (i.e., 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-peptidic agonist.
[0157] An exemplary class of angiotensin-^ 1 -7) receptor agonists are l -(p- thienylbenzyl)imidazoles. Examples of these non-peptide angiotensin^ 1-7) receptor agonists are represented by Structural Formula (VI):
Figure imgf000042_0001
or pharmaceutically acceptable salts thereof, wherein:
R1 is halogen, hydroxyl,
Figure imgf000042_0003
alkoxy wherein 1 to 6 carbon atoms are replaced by the heteroatoms O, S, or NH (preferably by
Figure imgf000042_0004
substituted by a saturated cyclic ether such as tetrahydropyran or tetrahydrofuran,
Figure imgf000042_0006
alkylaryl, or aryloxy that is unsubstituted or substituted by a substituent selected from halogen,
Figure imgf000042_0005
and trifluoromethyl;
Figure imgf000042_0002
R4 is hydrogen, halogen (chloro, bromo, fluoro), or (C1-C4)-alkyl; X is oxygen or sulfur; Y is oxygen or -NH-;
R5 is hydrogen, (C1-C6)-alkyl; or (C1-C4)-alkylaryl, where R5 is hydrogen when Y is -NH-; and
R6is (C1-C5)alkyl.
[0158] In certain embodiments, R is not halogen when R2 is COOH or CO-0-(C1-C4)- alkyl.
[0159] in some embodiments, an angiotensin-( 1-7) receptor agonist is AVE 0991, 5- formyl-4-methoxy-2-phenyl- 1 [ [4- [2-(ethylaminocarbonylsulfonamido)-5 -isobutyl-3 -thienyl] - phenyl]-methyl]-imidazole, which is represented by the following structure:
Figure imgf000043_0001
[0160] Another exemplary class of angiotensin -(1-7) receptor agoni sts are p- thienylbenzyl amides. Examples of these non-peptide angiotensin-(l -7) receptor agonists are represented by Structural Formula (VII):
Figure imgf000044_0001
or a pharmaceutically acceptable salt thereof, wherein:
R1 is (C1-C5)-alkyl that is unsubstituted or substituted by a radical chosen from MH2, halogen, O-(C1-C3>alkyl, CO-0-(C1-C3)-alkyl and C02H, (C3-C8)-cycloalkyl, (C1-C3)-alkyl-(C3- C8)-cycloalkyl, (C6-C10)-aryl that is unsubstituted or substituted by a radical chosen from halogen and 0-(C1-C3)-alkyl, (C1-C3)-alkyl-(C6-C10)-aryl where the aryl radical is unsubstituted or substituted by a radical chosen from halogen and 0-(C]-C3)-alkyl, (C1-C5)-heteroaryl, or (d- C3>alkyl-(C 1 -C5)-heteroaryl ;
R is hydrogen, (C1-C6)-alkyl that is unsubstituted or substituted by a radical chosen from halogen and 0-(C1-C3)-alkyl, (C3-C8)-cycloalkyl, (C1-C3)-alkyl-(C3-C8)-cycioalkyl, (C6-C10)-aryl that is unsubstituted or substituted by a radical chosen from among halogen, 0-(C1-C3)-alkyi and CO-0-(C1-C3)-alkyl, or (C1-C3)-alkyl-(C6-C10)-aryl that is unsubstituted or substituted by a radical chosen from halogen and 0-(C1-C3)-aikyl;
R3 is hydrogen, COOH, or COO-(C1-C4)-alkyl;
R4 is hydrogen, halogen; or (Ci-C4)-alkyl;
R5 is hydrogen or (C1-C6)-alkyl;
R6 is hydrogen, (C1-C6)-alkyl, (C1-C3)-alkyl-(C3-C8)-cycloalkyl, or (C2-C6)-alkenyl; and X is oxygen or NH. [0161] Additional examples of angiotensin^ 1 -7) receptor agonists are described in U.S.
Patent No. 6,235,766, the contents of which are incorporated by reference herein.
[0162] Various angiotensin^ 1-7) 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 Ν,Ν'- dibenzylethylenediamine or ethylenediamine, or combinations thereof (e.g., a zinc tannate salt). The non-toxic, physiologically acceptable salts are preferred.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] An aralkyl group is an alkyl group substituted by an aryl group.
Potassium Channel Blockers
[0168] Potassium channel blockers are a class of agents that interfere with conduction through one or more potassium channels. In general potassium channel blockers are known to be effective cardiac agents, and are used as anti-arrhythmia agents. Class III potassium channel blockers, for example, are predominantly characterized in their effect on prolonging the repolarization of certain cardiac neurons and thus being useful for the treatment of particular cardiac abnormalities including ventricular tachycardia (VT) and-'or ventricular fibrillation (VP).
[0169] Any of a variety of potassium channel blockers are contemplated as within the scope of the present invention. Non-limiting exemplary potassium channel blockers suitable for use in various embodiments include: dalfampridine, amiodarone, dronedarone, dofetilide, sotalol, ibutilide, azimilide, bretylium, clofilium, E-4031 , nifekalant, tedisamil, sematilide, and combinations thereof.
[0170] In some embodiments, a potassium channel blocker may be at least one FDA- approved potassium channel blocker. In some embodiments, a potassium channel blocker is administered at a dose other than an FDA-approved dose. In some embodiments, a potassium channel blocker is administered according to a dosing regimen other than an FDA-approved dosing regimen. In some embodiments, a potassium channel blocker may not be an FDA- approved potassium channel blocker.
[0171] In some embodiments, a potassium channel blocker may be 4-ammopyridine, also known as "fampridine" or "dalfampridine". Dalfampridine is a potassium channel blocker with the chemical formula C5H4N-NH2, that is thought to be selective for the Kvl family (also referred to as the "Shaker fam ily") of vo ltage activated potassium channels. Dalfampridine has also been shown to have some beneficial effect in multiple sclerosis (MS). Without wishing to held to a particular theory, it is thought that dalfampridine may benefit MS sufferers by supporting robust action potential duration and amplitude.
Formulations and Dosing
[0172] In accordance wi th the methods of the invention, an Ang (1-7) peptide and/or potassium channel blocker 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 or medicament (e.g., in the manufacture of a medicament for the treatment of the disease), as described herein. 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)).
[0173] 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.
[0174] 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.
[0175] 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 a preferred embodiment, 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 .
[0176] An Ang (1-7) peptide and/or potassium channel blocker 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.
[0177] An Ang (1-7) peptide and/or potassium channel blocker as described herein (or a composition or medicament containing an Ang (1 -7) peptide and/or potassium channel blocker described herein) is administered by any appropriate route. In some embodiments, an Ang (1-7) peptide and/or potassium channel blocker 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 and/or potassium channel blocker described herein is administered intravenously. Alternatively, an Ang (1-7) peptide and/or potassium channel blocker described herein (or a composition or medicament containing an Ang (1-7) peptide and/or potassium channel blocker described herein) can be administered by inhalation, parenterally, intradermally, transdermally, rectally, or transmucosally. In some embodiments, an Ang (1 -7) peptide and/or potassium channel blocker is administered orally. More than one route can be used concurrently, if desired. In some embodiments, an Ang (1-7) peptide is administered in any non-intracerebroventricular manner.
[0178] 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 ischemic stroke).
[0179] 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 determined in 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, 1 1, 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, 1 1 , 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.
[0180] In various embodiments, an Ang (1-7) peptide and/or potassium channel blocker 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.
[0181 ] Therapeutically effecti ve dosage amounts of Ang (1 -7) peptide and/or potassium channel blocker, 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 10-1000 mg (e.g., about 20 mg - 1,000 mg, 30 mg - 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, 100-1000 mg, 100-900 mg, 100-800 mg, 100-700 mg, 100-600 mg, 100-500 mg, 100-400 mg, 100-300 mg, 200-1000 mg, 200-900 mg, 200-800 mg, 200-700 mg, 200-600 mg, 200-500 mg, 200-400 mg, 300-1000 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 - 700 mg, and 500 mg - 600 mg). In some embodiments, an angiotensin (1-7) peptide and/or potassium channel blocker 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 and/or potassium channel blocker 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. [0182] 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 0.001 mg/kg weight to 15 mg/kg weight, from about 0.001 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.
[0183] 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.
[0184] 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 0.01 1 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.
[0185] In some embodiments, the angiotensin (1 -7) peptide and/or potassium channel blocker is administered at an effective dose ranging from about 1-1,000 μg/kg/day (e.g., ranging from about 1-900 μg/kg/day, 1-800 μg/kg/day, 1 -700 μg/kg/day, 1-600 μg/kg/day, 1-500 μg/kg/day, 1-400 μg/kg/day, 1-300 μg/kg/day, 1-200 μg/kg/day, 1-100 μg/kg/day, 1-90 μg/kg/day, 1-80 μg/kg/day, 1-70 μg/kg/day, 1-60 μg/kg/day, 1-50 μg/kg-''day, 1-40 μg/kg/day, 1- 30 μg/kg/day, 1 -20 μg/kg/day, 1-10 μg/kg/day . In some embodiments, the angiotensin (1 -7) peptide and/or potassium channel blocker is administered at an effective dose ranging from about 1-500 μg/kg/day. In some embodiments, the angiotensin (1-7) peptide and/or potassium channel blocker is administered at an effective dose ranging from about 1-100 μg/kg/day. In some embodiments, the angiotensin (1-7) peptide and/or potassium channel blocker is administered at an effective dose ranging from about 1-60 μg/kg/day. In some embodiments, the angiotensin (1- 7) peptide and/or potassium channel blocker 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 ug/kg/day.
Dosing Schedules
[0186] According to various embodiments, the present invention provides several new dosing schedules not previously thought appropriate or beneficial in the treatment of stroke. In contrast to the established thinking regarding stroke treatment, where "time is brain,"
embodiments of the present invention allow for treatment to begin well after a stroke event. In particular, several embodiments may include a dosing schedule wherein the first treatment of a patient with an Ang (1-7) peptide occurs more than 3 hours after the stroke event. In some embodiments, the first treatment of a patient with an Ang (1-7) peptide occurs more than 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90 hours, or 96 hours after the stroke event. In some embodiments, first treatment of a patient with an Ang (1-7) peptide occurs more th an 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days after a stroke event. In some embodiments, the first treatment of a patient with an Ang (1-7) peptide occurs more than 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks after the stroke event.
[0187] According to various embodiments, the administration of a potassium channel blocker may begin before, substantially at the same time as, or after administration of an angiotensin (1-7) peptide.
Combination Therapies
[0188] In some embodiments, an Ang (1-7) peptide and/or potassium channel blocker will be used as a part of a combination therapy including at least one additional therapy or therapeutic. It is contemplated that any known therapy or therapeutic for the treatment of brain conditions {e.g., stroke) may be used with one or more Ang (1 -7) peptides and/or potassium channel blockers as disclosed herein. Exemplary compounds that may be used with one or more Ang (1-7) peptides and/or potassium channel blockers include, but are not limited to, thrombolytic compounds, antioxidants or other reactive oxygen species agents, interferon beta- la {e.g., Avonex, Rebif, CinnoVex, ReciGen), interferon beta- 1 b (Betaseron), glatiramer acetate (Copaxone), mitoxantrone (Novantrone), natalizumab (Tysabri), fingolimod (Gilenya),
Teriflunomide (Aubagio), aspirin, dipyridamole (Aggrenox), clopidogrel (Plavix) warfarin (Coumadin), one or more statins, or combinations thereof. In some embodiments, an Ang(l -7) peptide and/or potassium channel blocker may be administered prior to, concurrently with, or subsequent to one or more surgical procedures such as those outlined above {e.g.,
thrombectomy). [0189] The phrase "in combination", as used herein, refers to agents or modalities that are administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics such that the subject is simultaneously exposed to both (or all) agents or modalities. Each of the two or more agents or modalities may be administered according to a different schedule; it is not required that individual doses of different agents be administered at the same time, or in the same composition. Rather, so long as both (or more) agents are present in the subject's body simultaneously for some period of time, they are considered to be administered "in combination".
Kits
[0190] In some embodiments, the present invention further provides kits or other articles of manufacture which contains an Ang (1 -7) peptide and/or a potassium channel blocker, or a formulation containing the same and provides instructions for its reconstitution (if lyophilized) and/or use. Ki ts 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.
[0191] 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 Ang (1-7) peptide and/or a potassium channel blocker. 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 multi-use vial, which al lows 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, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, kits or other articl es of manufacture may include an in struction for self- administration.
EXAMPLES
Example 1 - Delayed Treatment of Stroke with TXA302 (SEQ ID NO: 3) and Dalfampridine
[0192] In this Example, angiotensin (1-7) peptides and the potassium channel blocker dalfampridine were used in a rat Transient Middle Cerebral Artery Occlusion (tMCAO) model was used, which is generally accepted model of ischemic stroke.
[0193] In this Example, a total of 30 Sprague Dawley rats were used, as two groups with
15 rats in each. Each rat was approximately 3 months old and weighed approximately 300 grams ± 20% at the initiation of the study.
[0194] Animal handling was performed according to the guidelines of the National
Institutes of Health (NIH) and the Association for the Assessment and Accreditation of
Laboratory Animal Care (AAALAC). Animals were fed ad libitum a commercial rodent diet, had free access to drinking water, and were housed under standard laboratory conditions with a 12 hour light/dark cycle.
[0195] In this Example, the day of the tMCAO procedure is defined as "Day 1 " of the study.
[0196] TXA302 (SEQ ID NO: 3) dose volume was 0.5ml/kg for daily injections.
TXA302 was dissolved in PBS buffer to a concentration of 25 mg/ml (stock solution). For the working solution by daily injections TXA302 was dissolved in PBS to a concentration of 0.1 mg/ml for dose level 50 μg/kg.
[0197] Transient middle cerebral artery occlusion was performed according to the method described R. Schmid-Eisaesser et al. Briefly, the right CCA (Common Carotid Artery) was exposed through a midline neck incision and carefully dissected free from surrounding nerves and fascia - from its bifurcation to the base of the skull. The occipi tal artery branches of the ECA (External Carotid Artery) were then isolated, and these branches were dissected and coagulated. The EGA was dissected further distal ly and coagulated along with the terminal lingual and maxillary artery branches, which was then divided. The ICA (Internal Carotid Artery) was isolated and carefully separated from the adjacent vagus nerve, and the
pterygopalatine artery was ligated close to its origin with a 5-0 nylon suture (SMI, Belgium). Next a 4-0 silk suture was tied loosely around the mobilized EC A stump, and a 4 cm length of 4- 0 monofi lament nylon suture (the tip of the suture was blun ted by using a fl ame, and the suture was coated with polylysine, prior to insertion) was inserted through the proximal ECA into the ICA and thence into the circle of Willis, effectively occluding the MCA. The surgical wound was closed and the animals were returned to their cages to recover from anesthesia. One hour and a half after occlusion rats were re-anesthetized, the monofilament was withdrawn to allow reperfusion, the surgical wound was closed and rats were returned to their cages.
[0198] Animals were subjected to the mNSS test (Neuroscore) at Day 2, 24 hours post reperfusion. Only animals with an overall score of≥ 10 were included in this Example.
[0199] Evaluation of the blood flow in the brain cortex and vessel constriction was carried out using a Flow-R Laser Doppler system, in which intracranial blood flow and vessel diameter (constriction/dilation) was monitored.
First Phase of Study - Treatment of Stroke Complications Wherein Treatment is Initiated 4 Weeks After Stroke Event
[0200] In the first ph ase of thi s Example, rats were exposed to an angiotensin (1-7) peptide (TXA302) beginning 28 days after the stroke event and continuing for 56 days. Starting on Day 29, 28 days post-surgery, animals in Group 1 (saline control) and Group 2 (50 μ§/1¾ TXA302) received daily subcutaneous injection. The first phase of this Example includes an end to the angiotensin (1-7) peptide treatment period at Day 85 followed by a four week washout period (ending on Day 113). Table 1 shows the group design used in this Example.
Table 1 Group Allocation
Figure imgf000058_0001
Limb Placement Test (Pre-Operation, and Day 29, 43, 50, 57, 64, 71, 78, 85, 92, 99, 106 and 113)
[0201] Animals were subjected to a limb placement test. The limb placing tests were divided into both forelimb and hindlimb tests. For the forelimb-placing test, the examiner held the rat close to a tabletop and scored the rat's ability to place the forelimb on the tabletop in response to whisker, visual, tactile, or proprioceptive stimulation . Similarly, for the hindlimb placing test, the examiner assessed the rat's ability to place the hindlimb on the tabletop in response to tactile and proprioceptive stimulation. Separate sub-scores are obtained for each mode of sensory input and added to give total scores (for the forelimb placing test: 0 = normal, 12 = maximally impaired; for the hindlimb placing test: 0 = normal; 6 = maximally impaired). Scores were given in half-point increments (see below).
Forelimb placing test (0-12);
Whisker placing (0-2);
Visual placing (forward(0-2), sideways (0-2))
Tactile placing (dorsal (0-2), lateral (0-2))
Proprioceptive placing (0-2) [0202] The results provided in FIG. 1 show that animals receiving treatment with
TXA302 enjoyed statistically significant improvements in performance within two weeks of treatment, as compared to vehicle control. Also, as shown in FIG. 1, these improvements continued through the duration of the study (phase one to Day 113 and phase two through Day 126) with trending toward increased performance over time.
Stepping Test (Pre-Operation, and Day 29, 43, 50, 57, 64, 71, 78, 85, 92, 99, 106 and 113)
[0203] Animals were tested for forelimb akinesia in a stepping test (ST). The animal was held by its hind limbs fixed with one hand and the forelimb not to be monitored with the other, while the unrestrained fore-paw touched the table. The number of adjusting steps were counted while the animal is moved sideways along the table surface (85 cm in approximately five seconds), in the forehand & backhand direction for both forelimbs.
[0204] FIG. 2 shows that administration of TXA302 provided a significant improvement in performance by day 43, as compared to vehicle control animals. As with the results of the limb placement test, FIG. 2 also shows a trend toward continuing improvement over time.
Body Swing Test (Pre-Operation, and Day 29, 43, 50, 57, 64, 71, 78, 85, 92, 99, 106 and 113)
[0205] Animals were also subjected to a body swing test. Each rat was held
approximately one inch from the base of its tail. It was then elevated to an inch above a surface of a table. The rat was held in the vertical axis, defined as no more than 10° to either the left or the right side. A swing was recorded whenever the rat moved its head out of the vertical axis to either side. Before attempting another swing, the rat had to return to the vertical position for the next swing to be counted. Twenty (20) total swings were counted. A normal rat typically has an equal number of swings to either side. Following focal ischemia, the rat tends to swing to the contralateral side (left side in this Example). Body swing scores were expressed as a percentage of rightward over total swings. [0206] The results of the body swing test are provided in FIG. 3, which shows that by day 50 a significant improvement in performance is enjoyed the TXA302 treatment group, with an apparently complete recovery in normal function appearing at Day 92 and continuing through at least Day 113. As with the limb placement and step tests above, performance appears to continue to improve over time until normal function is regained.
Neurological Scoring (Pre-Operation, and Day 2, 29, 43, 50, 57, 64, 71, 78, 85, 92, 99, 106 and 113)
[0207] Each animal was also subjected to a neuroscore test. The Modified Neurological
Rating Scale (mNRS), or Neuroscore, was performed before the operation, on Day 2 and on Day 15, Day 22, Day 29, Day 36, Day 43 and Day 50. The individual making the behavioral assessments was unaware of the drug/dose given (blind test). A total Neuroscore of 18 was possible, with higher scores correlating to worse functioning.
[0208] As shown in FIG. 4, by day 43, the TXA302 treatment group showed a statistically significant reduction in neuroscore as compared to vehicle control animals, and this effect was maintained throughout th e duration of the study .
Cylinder Test (Day 26, 57, 85, and 113)
[0209] Each animal was also subjected to a well-established cylinder test for forelimb akinesia and exploratory behavior. Briefly, the cylinder test provides a way to evaluate a rat's spontaneous forelimb use to support the body against the wall of a cylinder enclosure. The test takes advantage of the animal's innate drive to explore a novel environment by standing on its hind limbs and leaning toward enclosed walls.
[0210] To perform the test, rats were placed into a a transparent cylinder for 5 minutes and the number of wall contacts performed independently with the left and right forepaw were counted and noted to a maximum number of 20 wall contacts per animal per session. Only supporting contacts were counted (i.e., full appositions of the paws with open digits to the cylinder walls).
[0211] FIG. 5 shows that animals receiving TXA302 exhibited a significant increase in performance by Day 57 as compared to vehicle control animals and that this increased performance continued throughout the duration of the study.
Second Phase of Study - Treatment of Stroke Complications with A Potassium Channel Blocker After 10 Day Angiotensin (1-7) Peptide Washout Pperiod, and 112 Days After a Stroke Event
[0212] In the second phase of this Example, starting on Day 1 13 following a four week washout period after adm inistration of either vehicle or TXA302 was halted, every animal from both the vehicle control and TXA302 groups received 2 mg/kg dalfampridine twice per day for three days (equal doses 12 hours apart). The limb placement, step test, body swing test, and neuroscore tests were each performed at days 113, 1 14, 115, 1 16, and again at Day 126 and the results are shown in FIGS. 1, 2, 3 and 4, respectively. The second phase of this Example shows that administration of an angiotensin (1-7) peptide and a potassium channel blocker, even initiating treatment weeks after a stroke event, is able to provide a powerful synergistic benefit.
[0213] As shown in FIGS. 1-4, each group of animals exhibited functional benefit from administration of dalfampridine, with animals having previously received TXA302 performing better than those having previously received vehicle only. Animals receiving dalfampridine after receiving TXA302 exhibited further increased performance benefit on all tests beyond that observed with TXA302 alone.
[0214] The results of the limb placement test in phase two are also shown in FIGs 7 and
8. FIG. 7 shows, among other things, the effect of dalfampridine on animals that received only vehicle in phase one of the study. As expected, these animals saw an improvement in
performance during administration of dalfampridine, followed by a worsening of performance after cessation of dalfampridine substantially back to pre-dalfampridine levels. FIG. 8 shows, among other things, the effects of dalfampridine on animals that received TXA302 (SEQ ID NO: 3) during phase one of the study as compared to control animals on days 1 13 and 116. While TXA302 exhibited dramatic improvements in performance, as described above, FIG. 8 shows that this improvement was further increased during dalfampridine administration and remained superior to controls, even when the control animal was also receiving dalfampridine.
[0215] The results of the step test in phase two are also shown in FIGs 9 and 10. FIG. 9 shows, among other things, the effect of dalfampridine on animals that received only vehicle in phase one of the study. As expected, these animals saw an improvement in performance during administration of dalfampridine, followed by a worsening of performance after cessation of dalfampridine substantially back to pre-dalfampridine levels. FIG. 10 shows, among other things, the effects of dalfampridine on animals that received TXA302 (SEQ ID NO: 3) during phase one of the study as compared to control animals on days 113 and 116. While TXA302 exhibited dramatic improvements in performance, as described above, FIG. 10 shows that this improvement was further increased during dalfampridine administration and remained superior to controls, even when the control animal was also receiving dalfampridine.
[0216] The results of the body swing test in phase two are also shown in FIGs 11 and 12.
FIG. 11 shows, among other things, the effect of dalfampridine on animals that received only vehicle in phase one of the study. As expected, these animals saw an improvement in
performance during administration of dalfampridine, followed by a worsening of performance after cessation of dalfampridine substantially back to pre-dal fampridine levels. FIG. 12 shows, among other things, the effects of dalfampridine on animals that received TXA302 (SEQ ID NO: 3) during phase one of the study as compared to control animals on days 1 13 and 116. While TXA302 exhibited dramatic improvements in performance, as described above, FIG. 12 shows that this improvement was further increased during dalfampridine administration and remained superior to controls, even when the control animal was also receiving dalfampridine.
[0217] The results of the neuroscore test in phase two are also shown in FIGs 13 and 14.
FIG. 13 shows, among other things, the effect of dalfampridine on animals that received only vehicle in phase one of the study. As expected, these animals saw an improvement in
performance during administration of dalfampridine, followed by a worsening of performance after cessation of dalfampridine substantially back to pre-dalfampridine levels. FIG. 14 shows, among other things, the effects of dalfampridine on animals that received TXA302 (SEQ ID NO: 3) during phase one of the study as compared to control animals on days 1 13 and 116. While TXA302 exhibited dramatic improvements in performance, as described above, FIG. 14 shows that this improvement was further increased during dalfampridine administration and remained superior to controls, even when the control animal was also receiving dalfampridine.
Blood Vessel Diameter and Blood Flow Ratios
[0218] On day 128 (127 days after the stroke event), the blood vessel diameter of all animals was tested. FIG. 6 (right panel) shows that animals receiving TXA302 showed statistically significant improvement in blood vessel diameter, as compared to vehicle control animals. Specifically, the right panel of FIG. 6 shows that animals receiving subcutaneously administered TXA302 followed by dalfampridine administration showed enhanced blood vessel diameter of approximately 25% larger than vehicle control animals.
[0219] In addition to the measurement of blood vessel diameter, blood flow
measurements were also taken on Day 128 in both groups. Similar to vessel diameter above, FIG. 6 (left panel) shows that by day 128, animals in the TXA302 treatment group shows a statistically significant improvement in blood flow as compared to vehicle control animals. Specifically, FIG. 6 (left panel) shows that animals receiving TXA302 followed by
dalfampridine administration showed enhanced blood flow (~45%) as compared to vehicle controls.
[0220] This Example shows, among other things, that administration of a combination of an angiotensin (1-7) peptide and a potassium channel blocker, even if administration begins well after a stroke event, results in synergistically improved outcomes after stroke. To our knowledge, these results represent the first time an angiotensin (1-7) peptide and a potassium channel blocker were shown to synergistically improve functional recovery after a stroke event. Accordingly, angiotensin (1-7) peptides and potassium channel blockers represent a powerful and previously unknown treatment regimen for use in aiding patients who have suffered one or more brain conditions, including stroke events. EQUIVALENTS AND SCOPE
[0221] 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

CLAIMS We claim:
1. A method of treating a brain condition comprising
administering to a subject suffering from or who previously suffered from a brain condition at least one angiotensin (1-7) peptide; and
administering to the subject at least one potassium channel blocker.
2. The method of claim I, wherein the brain condition is selected from stroke, vascular dementia, and traumatic brain injury.
3. The method of claim 2, wherein the stroke is either ischemic stroke, hemorrhagic stroke, or a combination thereof.
2. The method of claim 3, wherein at least one of the angiotensin (1-7) peptide and potassium channel blocker are administered within 24 hours of a stroke.
3. The method of claim 3, wherein at least one of the angiotensin (1-7) peptide and potassium channel blocker are administered at least 24 hours after a stroke.
4. The method of claim 3, wherein at least one of the angiotensin (1-7) peptide and potassium channel blocker are administered at least 1 week after a stroke.
5. The method of claim 3, wherein at least one of the angiotensin (1-7) peptide and potassium channel blocker are administered at least 4 weeks after a stroke.
6. The method of any one of the above claims, wherein the potassium channel blocker is selected from: dalfampridine, amiodarone, dronedarone, dofetilide, sotalol, ibutilide, azimilide, bretylium, clofilium, E-4031, nifekalant, tedisamil, sematilide, and combinations thereof.
7. The method of claim 6, wherein the potassium channel blocker is administered at a dose other than an FDA-approved dose.
8. The method of any one of the above claims, wherein the angiotensin (1-7) peptide is administered via parenteral administration, rectal administration, oral administration, or a combination thereof
9. The method of any claim 8, wherein the parenteral administration is intravenous, subcutaneous, inhalation, intradermal, transdermal, and/or transmucosal administration.
10. The method of any one of the above claims, wherein the angiotensin (1-7) peptide is administered at least once per day.
11. The method of any one of the above claims, wherein the angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-1,000 ug/kg/day.
12. The method of any one of the above claims, wherein the angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-500 ug/kg/day.
13. The method of any one of the above claims, wherein the angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-60 ug/kg/day.
14. The method of any one of the above claims, wherein the angiotensin (1-7) peptide comprises the naturally-occurring Angiotensin (1-7) amino acid sequence of Asp -Arg -Val~ - Tyr4-Ile5-His6-Pro7 (SEQ ID NO: 1).
15. The method of any one of claims 1-13, wherein the angiotensin (1-7) peptide is a functional equivalent of SEQ ID NO: 1.
16. The method of claim 15, wherein the functional equivalent is a linear peptide.
17. The method of claim 16, wherein the linear peptide comprises a sequence that includes at least four amino acids from the seven amino acids that appear in the naturally-occurring
Angiotensin (1-7), wherein the at least four amino acids maintain their relative positions as they appear in the naturally-occurring Angiotensin (1-7).
18. The method of claim 16 or 17, wherein the linear peptide contains 4-25 amino acids.
19. The method of claim 16 or 17, wherein the linear peptide is a fragment of the naturally- occurring Angiotensin (1 -7).
20. The method of any one of claims 16-19, wherein the linear peptide contains amino acid substitutions, deletions and/or insertions in the naturally-occurring Angiotensin (1-7).
21. The method of any one of claims 16-19, wherein the linear peptide has an amino acid sequence of Aspi-Arg2-Val3-Ser4-Ile5-His6-Cys7 (SEQ ID NO: 2).
22. The method of any one of claims 16-19, wherein the linear peptide has an amino acid sequence of Ala'-Arg2-Val3-Ser4-Ile5-His6-Cys7 (SEQ ID NO: 3).
23. The method of any one of claims 1-22, wherein the angiotensin (1-7) peptide comprises one or more chemical modifications to increase protease resistance, serum stability and/or bioavailability.
24. The method of claim 23, wherein the one or more chemical modifications comprise pegylation.
25. The method of any one of the above claims, wherein the angiotensin (1-7) peptide is administered at an effective dose periodically at an administration interval such that at least one sign, symptom and/or feature of a brain condition is reduced in intensity, severity, duration, or frequency or has delayed onset.
26. The method of claim 25, wherein the at least one sign, symptom and/or feature of a brain condition is selected from paralysis, memory loss, pain, seizure, dysphagia, aphasia, dysarthria, ataxia, depression, mood swings, and loss of vision.
27. A method of treating a brain condition comprising
administering to a subject suffering from or who previously suffered from a brain condition, and was administered an angiotensin (1-7) peptide, a potassium channel blocker.
28. The method of claim 27, wherein the brain condition is selected from stroke, vascular dementia, and traumatic brain injury.
29. The method of claim 28, wherein the stroke is either ischemic stroke, hemorrhagic stroke, or a combination thereof.
30. The method of claim 29, wherein at least one of the angiotensin (1-7) peptide and potassium channel blocker are administered within 24 hours of a stroke.
31. The m ethod of claim 29, wherein at least one of th e angiotensin (1-7) peptide and potassium channel blocker are administered at least 24 hours after a stroke.
32. The method of claim 29, wherein at least one of the angiotensin (1-7) peptide and potassium channel blocker are administered at least 1 week after a stroke.
33. The method of claim 29, wherein at least one of the angiotensin (1-7) peptide and potassium channel blocker are administered at least 4 weeks after a stroke.
34. The method of any one of claims 27-33, wherein the potassium channel blocker is selected from: dalfampridine, amiodarone, droned arone, dofetilide, sotalol, ibutilide, azimilide, bretylium, clofilium, E-4031 , nifekalant, tedisamil, sematilide, and combinations thereof.
35. The method of any one of claims 27-33, wherein the potassium channel blocker is administered at a dose other than an FDA-approved dose.
36. A method of treating a brain condition comprising
administering to a subject suffering from or who previously suffered from a brain condition, and was administered a potassium channel blocker, an angiotensin (1-7) peptide.
37. The method of claim 36, wherein the brain condition is selected from stroke, vascular dementia, and traumatic brain injury.
38. The method of claim 37, wherein the stroke is either ischemic stroke, hemorrhagic stroke, or a combination thereof.
39. The method of claim 38, wherein at least one of the angiotensin (1-7) peptide and potassium channel blocker are administered within 24 hours of a stroke.
40. The method of claim 38, wherein at least one of the angiotensin (1-7) peptide and potassium channel blocker are administered at least 24 hours after a stroke.
41. The method of claim 38, wherein at least one of the angiotensin (1-7) peptide and potassium channel blocker are administered at least 1 week after a stroke.
42. The method of claim 38, wherein at least one of the angiotensin (1-7) peptide and potassium channel blocker are administered at least 4 weeks after a stroke.
43. The method of any one of claims 36-42, wherein the angiotensin (1-7) peptide is administered via parenteral administration, rectal administration, oral administration, or a combination thereof.
44. The method of any claim 43, wherein the paren teral admini stration i s in traveno us, subcutaneous, inhalation, intradermal, transdermal, and/or transmucosal administration.
45. The method of any one of claims 36-44, wherein the angiotensin (1-7) peptide is administered at least once per day.
46. The method of any one of claims 36-45, wherein the angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-1,000 ug/kg/day.
47. The method of any one of claims 36-45, wherein the angiotensin (1-7) peptide is administered at an effective dose ranging from about 50-500 ug/kg/day.
48. The method of any one of claims 36-45, wherein the angiotensin (1 -7) peptide is administered at an effective dose ranging from about 1-60 ug/kg/day.
49. The method of any one of claims 36-48, wherein the angiotensin (1-7) peptide comprises the naturally-occurring Angiotensin (1-7) amino acid sequence of Asp1-Arg2-Val"'-Tyr4-Ile5-His6- Pro7 (SEQ ID NO: 1).
50. The method of any one of claims 36-48, wherein the angiotensin (1-7) peptide is a functional equivalent of SEQ ID NO: 1.
51. The method of claim 50, wherein the functional equivalent is a linear peptide.
52. The method of claim 51 , wherein the linear peptide comprises a sequence that includes at least four amino acids from the seven amino acids that appear in the naturally-occurring Angiotensin (1-7), wherein the at least four amino acids maintain their relative positions as they appear in the naturally-occurring Angiotensin (1-7).
53. The method of claim 51 or 52, wherein the linear peptide contains 4-25 amino acids.
54. The method of claim 51 or 52, wherein the linear peptide is a fragment of the naturally- occurring Angiotensin (1-7).
55. The method of any one of claims 51-54, wherein the linear peptide contains amino acid substitutions, deletions and/or insertions in the naturally-occurring Angiotensin (1-7).
56. The method of any one of claims 51 -55, wherein the linear peptide has an amino acid sequence of Asp1-Arg2-Val3-Ser4-Ile5-His6-Cys7 (SEQ ID NO: 2).
57. The method of any one of claims 51-55, wherein the linear peptide has an amino acid sequence of Ala1-Arg2Val3Se4-Ile5His6Cys7 (SEQ ID NO: 3).
58. The method of any one of claims 36-57, wherein the angiotensin (1-7) peptide comprises one or more chemical modifications to increase protease resistance, serum stability and/or bioavailability.
59. The method of claim 58, wherein the one or more chemical modifications comprise pegylation.
60. The method of any one of claims 36-59, wherein the angiotensin (1-7) peptide is administered at an effective dose periodically at an administration interval such that at least one sign, symptom and/or feature of a brain condition is reduced in intensity, severity, duration, or frequency or has delayed onset.
61. The method of claim 60, wherein the at least one sign, symptom and/or feature of a brain condition is selected from paralysis, memory loss, pain, seizure, dysphagia, aphasia, dysarthria, ataxia, depression , mood swings, and loss of vision.
62. The method of any one of claims I, 27 and 36, wherein the at least one angiotensin (1-7) peptide is a non-peptidic angiotensin (1-7) receptor agonist.
63. The method of claim 62, wherein the non-peptidic angiotensin (1-7) receptor agonist is a compound with the following structure:
Figure imgf000072_0001
or a pharmaceutically acceptable salt thereof.
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