WO2017141185A1 - Procédés et compositions de traitement l'athérosclérose - Google Patents

Procédés et compositions de traitement l'athérosclérose Download PDF

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WO2017141185A1
WO2017141185A1 PCT/IB2017/050876 IB2017050876W WO2017141185A1 WO 2017141185 A1 WO2017141185 A1 WO 2017141185A1 IB 2017050876 W IB2017050876 W IB 2017050876W WO 2017141185 A1 WO2017141185 A1 WO 2017141185A1
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increased levels
subject
levels
hgf
angiogenic
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PCT/IB2017/050876
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Nestor Raul GONZALEZ
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The Regents Of The University Of California
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the present invention is generally related to the field of medicine and methods and compositions for treating atherosclerosis.
  • Atherosclerosis is a systemic disease process in which fatty deposits, inflammation, cells, and scar tissue build up within the walls of arteries, is the underlying cause of the majority of clinical cardiovascular events.
  • Atherosclerosis can develop in large and small arteries supplying a variety of end-organs, including the heart, brain, kidneys, and extremities.
  • the current disclosure further relates to methods and compositions for treating or ameliorating atherosclerosis or AD in a subject.
  • Cardiovascular disease is the leading global cause of death, accounting for 17.3 million deaths per year, a number that is expected to grow to more than 23.6 million by 2030. Nearly 787,000 people in the U.S. died from heart disease, stroke and other cardiovascular diseases in 2011. That's about one of every three deaths in America. About 2,150 Americans die each day from these diseases, one every 40 seconds. About 85.6 million Americans are living with some form of cardiovascular disease or the after-effects of stroke. Heart disease is the No. 1 cause of death in the world and the leading cause of death in the United States, killing over 375,000 Americans a year. Heart disease accounts for 1 in 7 deaths in the U.S. 1
  • Intra-cranial atherosclerosis is the most common cause of stroke worldwide. It accounts for at least 10% of all strokes in the United States and as much as 33% to 67% of stroke in countries with predominantly Asian, Hispanic, and Black populations. ICAS carries a worse prognosis than other stroke etiologies with an annual rate of recurrent stroke and death of 15% despite intensive medical management, and as high as 25% in certain populations. [0006] Therefore, there is a continuing need for methods and compositions for treating, ameliorating or preventing atherosclerotic disease and stroke.
  • the current disclosure provides methods and compositions for treating atherosclerosis, and is based on inhibiting anti-angiogenesis in vivo. Aspects relate to a method for treating atherosclerosis in a subject comprising: administering a surgical treatment to a subject determined to have one or more of: increased levels of HGF, decreased levels of VEGF165a, increased levels of VEGFRl, increased levels of endostatin, increased levels of angiostatin, and/or increased levels of TSP-2.
  • the method further comprises administering a non-surgical therapeutic to a subject determined to not have one or more of increased levels of HGF, decreased levels of VEGF165a, increased levels of VEGFRl, increased levels of endostatin, increased levels of angiostatin, and/or increased levels of TSP-2.
  • the subject is determined to have or is one that has all of: increased levels of HGF, decreased levels of VEGF165a, increased levels of VEGFRl, increased levels of endostatin, increased levels of angiostatin, and increased levels of TSP-2.
  • the subject is determined to have or is one that has all of: increased levels of HGF, decreased levels of VEGF 165a, increased levels of VEGFRl , increased levels of endostatin, and increased levels of angiostatin.
  • the subject is determined to have or is one that has all of: increased levels of HGF, increased levels of VEGFRl, increased levels of endostatin, increased levels of angiostatin and increased levels of TSP-2.
  • the subject is determined to have at least 1, 2, 3, 4, 5, or 6 of: increased levels of HGF, decreased levels of VEGF165a, increased levels of VEGFRl, increased levels of endostatin, increased levels of angiostatin, and/or increased levels of TSP- 2.
  • the atherosclerosis is intracranial atherosclerosis.
  • Atherosclerosis is a medical condition characterized by the hardening and narrowing of the arteries.
  • the increased HGF comprises an increase in an HGF polypeptide wherein the polypeptide is a fragment of the HGF protein.
  • the HGF fragment is an anti-angiogenic factor.
  • the fragment is a K1, K2, K3, or K4 fragment.
  • the fragment is a K1 fragment.
  • the fragment is a K2 fragment.
  • the fragment is a K3 fragment.
  • the fragment is a NK4 fragment.
  • the non-surgical treatment comprises one or more of an ACE inhibitor, an angiotension II receptor blocker, an antiarrhythmic, an antiplatelet, aspirin, a beta blocker, a calcium channel blocker, a clot buster, digoxin, a diuretic, a nitrate, or a blood thinner.
  • the surgical treatment comprises encephaloduroarteriosynangiosis, stent placement, angioplasty, heart bypass, valvuloplasty, enhanced external counterpulsation, ablation, pacemaker placement, implantation of a cardioverter defibrillator, lead extraction, left ventricular assist device placement, or heart transplant.
  • the method further comprises determining the levels of at least two angiogenic factors in the subject. In some embodiments, the determining is done by reviewing or receiving a report of results of angiogenic factor levels in a patient sample. In some embodiments, the determining is done by measuring the angiogenic factor level in a patient's sample. The measuring may be done by a method known in the art or described herein. In some embodiments, the patient is one that has had angiogenic factors measured in a sample from the patient by a method known in the art or described herein. In some embodiments, the patient's sample has been tested according to a method described herein or according to an assay described herein.
  • the angiogenic factor are two or more of vascular endothelial growth factor (VEGF) isoforms 165a (VEGF 165a), VEGF 165b, VEGF-D, VEGF receptor (VEGFR)1, VEGFR2, VEGFR3, platelet-derived growth factor (PDGF)-AA, PDGFF-BB, fibroblast growth factor (FGF) b, hepatocyte growth factor (HGF), K1, K2, NK3, K4, heparin-binding epidermal growth factor (HB-EGF), transforming growth factor (TGF) ⁇ , TGF ⁇ 2, bone morphogenic protein (BMP)-2, BMP-9, stromal cell derived factor (SDF)-l, interleukin (IL) 4, thrombospondin (TSP)-l, TSP-2, endostatin, and angiostatin.
  • VEGF vascular endothelial growth factor
  • VEGF 165a vascular endothelial growth factor
  • VEGFR
  • the method further comprises comparing the level of the angiogenic factor to a control.
  • the control is the level of an angiogenic factor in a normal patient.
  • the control is the level of an angiogenic factor in a diseased patient (i.e. one that has atherosclerosis).
  • the control is a cut-off value.
  • the cut-off value may be at least, at most, or exactly 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 12, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900,
  • the method further comprises performing a statistical analysis of the levels of the angiogenic factors.
  • the statistical analysis is principal component analysis.
  • the increase or decrease in levels are assessed by a principal component analysis.
  • the method further comprises comparing the level to a cut-off value. In some embodiments, an increased level is above a cut-off value and a decreased level is below a cut-off value.
  • the subject is a human patient.
  • the subject may be a mammal, such as a human, or a common laboratory experimental animal, such as a rat, mouse, cat, dog, rabbit, donkey, horse, or pig.
  • the method further comprises administration of an anti- angiogenic blocking agent.
  • the anti-angiogenic blocking agent is administered prior to surgery.
  • the anti-angiogenic blocking agent may be administered at least, at most, or exactly, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 days or hours (or any range derivable therein) before or after surgery.
  • the anti-angiogenic blocking agent is administered locally to an atherosclerotic region during surgery.
  • the anti-angiogenic blocking agent is GMI-1070, a proteinase activated receptor (PAR) - 1 agonist, meziothrombin, granzyme A, activated protein C receptor - endothelial protein C receptor (APC-EPCR), kalikrein (KLK) 4,KLK5, KLK6, matrix metalloproteinase (MMP1), proatherocytin, pen C13; PAR-4 antagonists, tcY- H 2 ; heparan sulfate proteoglycans, analogues of heparin sulfate proteoglycans, aprotinin, or combinations or derivatives thereof.
  • PAR proteinase activated receptor
  • APC-EPCR activated protein C receptor - endothelial protein C receptor
  • KLK kalikrein
  • KLK5 kalikrein
  • KLK6 matrix metalloproteinase
  • proatherocytin pen C13
  • the angiogenic factor is a circulating blood factor.
  • the levels of the at least two angiogenic factors are or were determined from a blood sample from the subject.
  • the sample may be a blood sample or other patient sample.
  • the patient sample is a urine, fecal, DNA, RNA, tissue, serum, whole blood, or plasma sample.
  • the anti-angiogenic blocking agent is GMI-1070, aproteinase activated receptor (PAR) - 1 agonist, meziothrombin, granzyme A, activated protein C receptor - endothelial protein C receptor (APC-EPCR), kalikrein (KLK) 4,KLK5, KLK6, matrix metalloproteinase (MMP1), proatherocytin, pen C13; PAR-4 antagonists, tcY- H 2 ; heparan sulfate proteoglycans, analogues of heparin sulfate proteoglycans, aprotinin, or combinations thereof.
  • PAR proteinase activated receptor
  • APC-EPCR activated protein C receptor - endothelial protein C receptor
  • KLK kalikrein
  • KLK5 kalikrein
  • KLK6 matrix metalloproteinase
  • proatherocytin pen C13
  • PAR-4 antagonists
  • the subject has undergone previous treatment for atherosclerosis. In further embodiments, the subject has not yet undergone a treatment for atherosclerosis. In some embodiments, the subject was a poor or non-responder to the previous treatment. In some embodiments, the previous treatment was a non-surgical treatment described herein.
  • the atherosclerosis is intracranial.
  • the method further comprises administering a surgical treatment to the subject.
  • the agent may be administered in a time-frame described previously or in the description that follows.
  • an assay system comprising agents for detecting proteins or protein levels, wherein the proteins comprise one or more of VEGF165a, VEGF165b, VEGF-D, VEGFR1, VEGFR2, VEGFR3, PDGF-AA, PDGFF-BB, FGFb, HGF, K1, K2, K3, K4, HB-EGF, TGF ⁇ , TGF ⁇ 2, BMP-2, BMP-9, SDF-1, IL 4, TSP-1, TSP-2, endostatin, and angiostatin.
  • the proteins comprise one or more of VEGF165a, VEGF165b, VEGF-D, VEGFR1, VEGFR2, VEGFR3, PDGF-AA, PDGFF-BB, FGFb, HGF, K1, K2, K3, K4, HB-EGF, TGF ⁇ , TGF ⁇ 2, BMP-2, BMP-9, SDF-1, IL 4, TSP-1, TSP-2, endostatin, and angiostatin.
  • the assay system consists of agents for detecting VEGF165a, VEGF165b, VEGF-D, VEGFR1, VEGFR2, VEGFR3, PDGF-AA, PDGFF-BB, FGFb, HGF, K1, K2, K3, K4, HB-EGF, TGF ⁇ , TGF ⁇ 2, BMP-2, BMP-9, SDF-1, IL 4, TSP-1, TSP-2, endostatin, and angiostatin.
  • the assay system consists of agents for detecting VEGF165a, VEGF165b, VEGF-D, VEGFRl, VEGFR2, VEGFR3, PDGF-AA, PDGFF-BB, FGFb, HGF, K1, K2, K3, K4, HB- EGF, TGF ⁇ , TGF ⁇ 2, BMP-2, BMP-9, SDF-1, IL 4, TSP-1, TSP-2, endostatin, angiostatin, and a control.
  • the control may be one or multiple detecting agents to determine a normalized or base-line expression level.
  • the proteins consist of HGF, VEGF165a, VEGFRl, endostatin, and angiostatin.
  • the proteins consist of HGF, VEGF165a, VEGFRl , endostatin, angiostatin, and a control. In some embodiments, the proteins consist of HGF, VEGF 1.65a, VEGFRl , endostatin, angiostatin, and TSP-2. In some embodiments, the proteins consist of HGF, VEGF 165a, VEGFRl, endostatin, angiostatin, TSP-2 and a control. In some embodiments, the proteins consist of HGF, VEGFRI, endostatin, angiostatin, and TSP-2. In some embodiments, the proteins consist of HGF, VEGFRI, endostatin, angiostatin, TSP-2 and a control.
  • the agent for detecting HGF comprises an agent that detects a fragment of the HGF protein.
  • the fragment is NKl, NK2, NK3, or NK4 fragment.
  • the assay system further comprises one or more of a buffer, a detectable label, or a solid support. In some embodiments, the assay system comprises one or more elements described throughout the disclosure.
  • the agent for detecting the angiogenic factors is a polypeptide.
  • the polypeptide is an antibody.
  • the agent is labeled with a detectable label.
  • a further aspect relates to a method for predicting a subject's response to atherosclerosis medical treatment comprising: determining the levels of at least two angiogenic factors in the subject; wherein the levels were or are determined using an assay system described herein; and predicting that the subject will respond poorly if the subject is determined to have one or more of increased levels of HGF, decreased levels of VEGF165a, increased levels of VEGFRI , increased levels of endostatin, increased levels of angiostatin, and/or increased levels of TSP-2.
  • a further aspect relates to a method for predicting a subject's response to atherosclerosis medical treatment comprising: determining the levels of at least two angiogenic factors in the subject; wherein the levels were or are determined using an assay system described herein; and predicting that the subject will respond poorly if the subject is determined to have one or more of increased levels of HGF, increased levels of VEGFRI, increased levels of endostatin, increased levels of angiostatin, and/or increased levels of TSP- [0027]
  • a further aspect relates to a method for predicting a subject's response to atherosclerosis medical treatment comprising: determining the levels of at least two angiogenic factors in the subject; wherein the levels were or are determined using an assay system described herein; and predicting that the subject will respond poorly if the subject is determined to have one or more of increased levels of HGF, decreased levels of VEGF165a, increased levels of VEGFRI, increased levels of endostatin, and/or increased levels of angiostatin.
  • the method further comprises administering a non-surgical therapeutic if the subject does not have one or more of increased levels of HGF, decreased levels of VEGF165a, increased levels of VEGFRl, increased levels of endostatin, increased levels of angiostatin, and/or increased levels of TSP-2.
  • the method further comprises administering a non-surgical therapeutic if the subject does not have one or more of increased
  • FIG. 1 Further aspects relate to a method for treating atherosclerosis in a subject comprising: determining the levels of at least two angiogenic factors in the subject; wherein the levels were or are determined using an assay system described herein; administering a surgical treatment if the subject is determined to have one or more of increased levels of HGF, increased levels of VEGFRl, increased levels of endostatin, increased levels of angiostatin, and/or increased levels of TSP-2.
  • the method further comprises administering a non-surgical therapeutic if the subject does not have one or more of increased levels of HGF, increased levels of VEGFRl, increased levels of endostatin, increased levels of angiostatin, and/or increased levels of TSP-2.
  • Further aspects relate to a method for predicting a subject's response to atherosclerosis medical treatment comprising: determining the levels of at least two angiogenic factors in the subject; predicting that the subject will respond poorly if the subject is determined to have one or more of increased levels of HGF, decreased levels of VEGF 165a, increased levels of VEGFRl, increased levels of endostatin, increased levels of angiostatin, and/or increased levels of TSP-2, In some embodiments, the subject is predicted to respond poorly when the subject is determined to have: increased levels of HGF, decreased levels of VEGF 165a, increased levels of VEGFRl, increased levels of endostatin, increased levels of angiostatin and increased elvels of TSP-2.
  • the subject is predicted to respond poorly when the subject is determined to have: increased levels of HGF, decreased levels of VEGF165a, increased levels of VEGFRl, increased levels of endostatin, and increased levels of angiostatin. In some embodiments, the subject is predicted to respond poorly when the subject is determined to have: increased levels of HGF, increased levels of VEGFRl , increased levels of endostatin, increased levels of angiostatin and increased elveis of TSP-2. In some embodiments, the subject has been diagnosed with atherosclerosis. In some embodiments, the subject has not received any treatment for atherosclerosis. In some embodiments, the subject has not responded poorly to any medical treatment, such as a nonsurgical medical treatment.
  • the atherosclerosis is intracranial atherosclerosis.
  • increased HGF comprises an increase in an HGF polypeptide and wherein the polypeptide is a fragment of the HGF protein.
  • the fragment is I , NK.2, N 3, or NK.4 factor.
  • the medical treatment is non-surgical.
  • the method further comprises administration of surgical treatment if the subject is predicted to respond poorly to medical treatment.
  • the surgical treatment comprises encephaloduroarteriosynangiosis, stent placement, angioplasty, heart bypass, valvuloplasty, enhanced external counterpulsation, ablation, pacemaker placement, implantation of a cardioverter defibrillator, lead extraction, left ventricular assist device placement, or heart transplant.
  • the subject is predicted to respond to non-surgical medical treatment when the subject does not have one or more of increased levels of HGF, decreased levels of VEGF165a, increased levels of VEGFRl, increased levels of endostatin, and/or increased levels of angiostatin.
  • the subject is predicted to respond to non-surgical medical treatment when the subject does not have one or more of increased levels of HGF, decreased levels of VEGF165a, increased levels of VEGFRl, increased levels of endostatin, increased levels of angiostatin, and/or increased levels of TSP-2. In some embodiments, the subject is predicted to respond to non-surgical medical treatment when the subject does not have one or more of increased levels of HGF, increased levels of VEGFRl, increased levels of endostatin, increased levels of angiostatin, and/or increased levels of TSP- 2. In some embodiments, the method further comprises administration of a non-surgical treatment.
  • the non-surgical treatment comprises one or more of an ACE inhibitor, an angiotension II receptor blocker, an antiarrhythmic, an antiplatelet, aspirin, a beta blocker, a calcium channel blocker, a clot buster, digoxin, a diuretic, a nitrate, or a blood thinner.
  • the at least two angiogenic factors comprise two or more of VEGF165a, VEGF165b, VEGF-D, VEGFRl, VEGFR2, VEGFR3, PDGF-AA, PDGFF-BB, FGFb, HGF, K1, K2, K3, K4, HB-EGF, TGF ⁇ , TGF ⁇ 2, BMP-2, BMP-9, SDF-1, IL 4, TSP-1, TSP-2, endostatin, and angiostatin.
  • Further aspects relate to the use of a surgical treatment for the treatment of atherosclerosis in a subject determined to have one or more of: increased levels of HGF, decreased levels of VEGF 165a, increased levels of VEGFRl, increased levels of endostatin, increased levels of angiostatin, and/or increased levels of TSP-2. Further aspects relate to the use of a surgical treatment for the treatment of atherosclerosis in a subject determined to have one or more of: increased levels of HGF, increased levels of VEGFRl , increased levels of endostatin, increased levels of angiostatin, and/or increased levels of TSP-2.
  • Further aspects relate to the use of a non-surgical treatment for the treatment of atherosclerosis in a subject determined not to have one or more of: increased levels of HGF, decreased levels of VEGF165a, increased levels of VEGFRl , increased levels of endostatin, and/or increased levels of angiostatin. Further aspects relate to the use of a non-surgical treatment for the treatment of atherosclerosis in a subject determined not to have one or more of: increased levels of HGF, decreased levels of VEGF165a, increased levels of VEGFRl, increased levels of endostatin, increased levels of angiostatin, and/or increased levels of TSP- 2.
  • Further aspects relate to the use of an anti-angiogenic blocking agent for treating atherosclerosis in a subject in need thereof. Further aspects relate to the use of of an anti- angiogenic blocking agent in the preparation of a medicament for treating atherosclerosis.
  • Further method aspects relate to a method of assessing level of response of a subject to a clinical management regimen of Intracranial atherosclerotic stroke (ICAS) or stroke, comprising: measuring circulating blood level of pro-angiogenic factor (PAGF) in the subject prior to the start of the clinical management regimen (PAGF L 0 ), measuring circulating blood level of anti-angiogenic factor (AAGF) in the subject prior to the start of the clinical management regimen (AAGF L 0 ), measuring PAGF circulating blood level in the subject at a time point of or after a clinical management regimen (PAGF Lt), measuring AAGF circulating blood level in the subject at a time point of clinical management regimen (AAGF L t ), comparing PAGF L t with PAGF Lo and AAGF L t with AAGF L 0 , and designating the subject as responding to the clinical management regimen if the PAGF L t is higher than PAGF LO or as lacking response to the clinical management regimen if the PAGF L 0 is higher than PAGF
  • the PAGF is selected from the group consisting of BMP2, BMP9, PDGF-AA, PDGF-BB, HGF, FGF2, KGF, HB-EGF, IL4, TPO, TGF-beta 1, TGF-beta 2, VEGF, VEGFD and SDF-1 alpha
  • the AAGF is selected from the group consisting of endostatin, angiostatin, sVEGFRl, thrombospondin-1 and thrombospondin-2.
  • the subject is a human patient.
  • a method of treating or ameliorating Intracranial atherosclerotic stroke (ICAS) or stroke in a subject comprising (a) subjecting the subject to an encephaloduroarteriosynangiosis (EDAS) procedure, (b) administering to the subject a neovascularization inducer, or a combination of (a) or (b).
  • EDAS encephaloduroarteriosynangiosis
  • the neovascularization inducer comprises an angiogenic factor.
  • the neovascularization inducer is selected from the group consisting of BMP2, BMP9, PDGF-AA, PDGF-BB, HGF, FGF2, KGF, HB- EGF, IL4, TPO, TGF-beta 1, TGF-beta 2 , VEGF, VEGFD, SDF-1 alpha, or a combination thereof.
  • the neovascularization inducer comprises monocytes.
  • the neovascularization inducer are monocytes that are obtained from blood of the subject and cultured in a culture medium prior to use.
  • the method comprises (a) subjecting the subject to the encephaloduroarteriosynangiosis (EDAS) procedure, and (b) administering to the subject the neovascularization inducer locally at the site of the EDAS, wherein the neovascularization inducer comprises BMP2, BMP9, PDGF-AA, PDGF- BB, HGF, FGF2, KGF, HB-EGF, IL4, TPO, TGF-beta 1, TGF-beta 2 , VEGF, VEGFD, SDF- 1 alpha, or a combination thereof.
  • EDAS encephaloduroarteriosynangiosis
  • the administering comprises administering to the subject a DNA construct encoding the neovascularization inducer agent.
  • the subject is a human being.
  • a composition which composition comprising a neovascularization inducer in an effective amount to induce cerebrovascularization in a subject having Intracranial atherosclerotic stroke (ICAS) or stroke.
  • the neovascularization inducer comprises BMP2, BMP9, PDGF-AA, PDGF-BB, HGF, FGF2, KGF, HB-EGF, IL4, TPO, TGF-beta 1, TGF-beta 2 , VEGF, VEGFD, SDF- 1 alpha, or a combination thereof.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the neovascularization inducer comprises monocytes.
  • the neovascularization inducer are monocytes that are obtained from blood of the subject and cultured in a culture medium prior to use.
  • a method of fabricating a composition comprising providing a neovascularization inducer in an effective amount to induce cerebrovascularization in a subject having Intracranial atherosclerotic stroke (ICAS) or stroke, and forming the composition.
  • IAS Intracranial atherosclerotic stroke
  • the neovascularization inducer is BMP2, BMP9, PDGF-AA, PDGF-BB, HGF, FGF2, KGF, HB-EGF, IL4, TPO, TGF-beta 1, TGF- beta 2 , VEGF, VEGFD, SDF- 1 alpha, or a combination thereof.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the neovascularization inducer comprises monocytes.
  • the neovascularization inducer are monocytes that are obtained from blood of the subject and cultured in a culture medium prior to use.
  • FIG. 1 shows Graphic representation of Angiogenic Profiles (AP) selected by Principal Component Analysis (PCA) AP-4 was significantly associated to failure of medical management and poor mRS at 6 months follow up independently of age and baseline mRS. HGF and several anti angiogenic factors are the dominant components of AP-4.
  • PCA Principal Component Analysis
  • FIG. 2A-B shows a simulation using the ordinal logistic model predictor profiler for 5,000 runs with uniform random values, depicting the probability of mRS 0 to 2 (a) and 3 to 5 (b) with a range of possible AP4s.
  • the shadow areas represent the confidence of prediction.
  • FIG. 3 shows the relative levels of angiogenic factors.
  • FIG. 4 shows the VEGF-165A Ratios in ICAS treatment.
  • the histogram showing mean VEGFA-165a/b ratios over time for the IMM and EDAS group. While an initial elevation of the ratio in week 1 immediately after surgery (red arrow) could be attributed to the operative insult, elevations in the IMM group in months 3 and 6 (green arrow) may indicate a protective effect.
  • ICAS intracranial arterial stenosis
  • medical pharmacological, surgical or endovascular - e.g., intravascular manipulations or implants
  • ICAS can be produced by multiple etiologies including intracranial atherosclerosis, inflammatory process, degenerative diseases, moyamoya disease or undefined arteriopaties.
  • neovascularization refers to the formation of functional microvascular networks with red blood cell perfusion.
  • Neovascularization differs from angiogenesis in that angiogenesis is only a component of the neovascularirzation, mainly characterized by the protrusion and outgrowth of capillary buds and sprouts from pre-existing blood vessels. See, e.g., Martin Rucker, et al., Elsevier: Biomaterials 27(2006) pp 5027-5038.
  • the term “neovascularization” is used interchangeably with the term “cerebrovascularization” or “revascularization”, which refers to neovascularization within the intracranial space or area.
  • the terms “neovascularization,” “cerebrovascularization,” and “vascularization” are used interchangeably.
  • the term “neovascularization inducing agent” refers any agent that is effective to stimulate or induce or otherwise promote cerebrovascularization or revascularization.
  • the term neovascularization is also referred to as "collateral formation.”
  • the term "agent” refers to neovascularization inducing agent in an effective amount for promoting neovascularization (e.g., cerebrovascularization).
  • An example of the agent is a VEGF such as VEGF-D.
  • the term also encompasses a PEGylated agent bearing a short alkyl chain, a short polymer chain, a short poly(amino acid) chain, or acyl group such as methyl or ethyl or acetyl, for example. More detailed discussion of neovascularization inducing agent is described below.
  • the term “agent” is also referred to from time to time as "bioactive agent", “compound”, “chemical”, “chemical compound”, peptide, polypeptide, or protein.
  • PAGF pro-angiogenic growth factor
  • AAGF anti-angiogenic growth factor or agent
  • neovascularization inducing agent inducing statistically significant result of neovascularization under clinical conditions.
  • terapéuticaally effective amount is an amount of an agent that is sufficient to produce a statistically significant, measurable change of a condition using the agent disclosed herein as compared with the condition in the repaired tissue without using the agent. Such effective amounts can be gauged in clinical trials as well as animal studies. [0065] Such a statistically significant, measurable, and positive change of a condition in repaired tissue using the agent disclosed herein as compared with the condition without using the agent is referred to as being an "improved condition.”
  • safe and effective amount refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this disclosure.
  • the specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.
  • derivative refers to neovascularization inducer agent which have been chemically modified, for example by ubiquitination, labeling, pegylation (derivatization with polyethylene glycol) or addition of other molecules.
  • a molecule is also a "derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties can improve the molecule's solubility, absorption, biological half-life, etc.
  • the moieties can alternatively decrease the toxicity of the molecule, or eliminate or attenuate an undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences, 18th edition, A. R.
  • a “derivative" polypeptide or peptide is one that is modified, for example, by glycosylation, pegylation, phosphorylation, sulfation, reduction/alkylation, acylation, chemical coupling, or mild formalin treatment.
  • a derivative may also be modified to contain a detectable label, either directly or indirectly, including, but not limited to, a radioisotope, fluorescent, and enzyme label.
  • the term "functional" when used in conjunction with “derivative” or “variant” refers to a compound or agent which possess a biological activity that is substantially similar to a biological activity of the neovascularization inducer compound or agent of which it is a derivative or variant.
  • substantially similar in this context is meant that at least 50% of the relevant or desired biological activity of a corresponding neovascularization inducer compound or agent is retained, e.g., preferably the variant retains at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100% or even higher (i.e., the variant or derivative has greater activity than the original neovascularization inducer compound or agent), e.g., at least 1 10%, at least 120%, or more compared to a measurable activity of the neovascularization inducer compound or agent.
  • compositions, methods, and respective component(s) thereof are used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.
  • the term "consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%), more preferably up to 5%, and more preferably still up to 1%> of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about” meaning within an acceptable error range for the particular value should be assumed.
  • active fragment or variant is meant a fragment that is 100% identical to a contiguous portion of the peptide, polypeptide or protein, or a variant that is at least 90%, preferably 95% identical to a fragment up to and including the full length peptide, polypeptide
  • a variant for example, may include conservative amino acid substitutions, as defined in the art, or nonconservative substitutions, providing that at least e.g. 10%), 25%), 50%), 75% or 90% of the activity of the original peptide, polypeptide or protein is retained.
  • conservative amino acid substitutions as defined in the art, or nonconservative substitutions, providing that at least e.g. 10%), 25%), 50%), 75% or 90% of the activity of the original peptide, polypeptide or protein is retained.
  • the terms "peptide”, “polypeptide” or “protein” are used interchangeably herein, although typically they refer to peptide sequences of varying sizes.
  • polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) or single base mutations in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population with a propensity for a disease state, that is susceptibility versus resistance.
  • SNPs single nucleotide polymorphisms
  • Derivative polynucleotides include nucleic acids subjected to chemical modification, for example, replacement of hydrogen by an alkyl, acyl, or amino group.
  • Derivatives e.g., derivative oligonucleotides, may comprise non-naturally-occurring portions, such as altered sugar moieties or inter-sugar linkages. Exemplary among these are phosphorothioate and other sulfur containing species which are known in the art.
  • Derivative nucleic acids may also contain labels, including radionucleotides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, substrates, cofactors, inhibitors, magnetic particles, and the like.
  • expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules, siRNA, ribozymes, and the like.
  • Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.
  • nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA).
  • non-translated sequences e.g., introns
  • the information by which a protein is encoded is specified by the use of codons.
  • amino acid sequence is encoded by the nucleic acid using the "universal" genetic code.
  • heterologous in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form.
  • a heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.
  • sample is used herein in its broadest sense.
  • a sample comprising polynucleotides, polypeptides, peptides, antibodies and the like may comprise a bodily fluid; a soluble fraction of a cell preparation, or media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; and the like.
  • patient refers to a mammalian subject to be treated.
  • subject refers to a human
  • methods of the disclosure find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • ameliorated or “treatment” refers to a symptom which is approaches a normalized value (for example a value obtained in a healthy patient or individual), e.g., is less than 50% different from a normalized value, preferably is less than about 25% different from a normalized value, more preferably, is less than 10% different from a normalized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests.
  • a normalized value for example a value obtained in a healthy patient or individual
  • a normalized value for example a value obtained in a healthy patient or individual
  • Angiogenic factors may be pro- or anti-angiogenic factors, unless specified otherwise. The description of certain angiogenic factors is provided below.
  • Endostatin is a 20-kDa C-terminal fragment derived from the proteolytic cleavage of collagen XVIII.
  • Collagen XVIII is quite a ubiquitous molecule.
  • perlecan and agrin is one of the major proteoglycans of basal membranes and the adjacent connective tissue.
  • Endostatin' s antiangiogenic function involves inhibition of endothelial cell proliferation and migration. This is achieved by multiple mechanisms that include binding with the integrin ⁇ 5 ⁇ 1 to activate the Src-kinase pathway, which downregulates the activity of RhoA GTPase and inhibits signaling pathways mediated by small kinases of the Ras and Raf families.
  • Endostatin also inhibits angiogenesis by inhibiting the actions of VEGF and FGF by competing for binding to either glypican or their high-affinity receptors. Endostatin potently inhibits endothelial cell migration via down regulation of c-myc expression and inhibition of MAPK (p38 and INK). Several reports have also indicated that endostatin induces endothelial cell apoptosis by abrogation of cyclin Dl expression, which produces arrest of the cell cycle in Gl, and by induction of autophagy, by modulating Beclinl and ⁇ - catenin levels.
  • Angiostatin is a 38-kDa fragment derived from the proteolytic cleavage of plasmin that contains the first four kringle domains of plasminogen. Among these individual kringles, Kl to K3 are the most potent inhibitors of endothelial cell growth. The complete mechanisms by which angiostatin produces antiangiogenesis are not totally understood. However, in general the preservation of the kringle structure is essential for the maintenance of the anti angiogenic effects. There are reports of possible receptors of angiostatin (ATPase, angiomoietin), as well as indications that angiostatin may arrest the endothelial cell cycle at G2.
  • Endothelial apoptosis has also been linked to the antiangiogenic activity of angiostatin. Due to the nonoverlapping nature of the inhibitory pathways, endostatin and angiostatin may act synergistically. This ability has been confirmed in vitro by isobolographic analysis of treatment of endothelial cells with a combination of angiostatin and endostatin, which resulted in synergistic inhibition. Applicant's study demonstrated that endostatin and angiostatin vary together in ICAS patients and they were important components of the anti- angiogenic profile associated with to failure of medical management and poor functional status. In addition, angiostatin and endostatin constituted the main components of the resolved angiogenic profile for angiographic neovascularization, which has a negative correlation with the formation of new vessels after surgery.
  • VEGFR-1 C.
  • the soluble VEGFR-1 is an approximately 100-kDa molecule encoded by the sFlt- 1 (VEGFR-1) gene on chromosome 13ql2.
  • VEGFR-1 is produced by different human cells including endothelium, vascular smooth muscle, monocytes, placental trophoblasts, corneal epithelial cells, and proximal tubular cells of the renal epithelium.
  • the soluble VEGFR-1 has antiangiogenic effects by either ligating circulating members of the VEGF family (VEGFA 165a, VEGF-D, VEGF-C, PDGFAA, PDGFBB) or by forming heterodimers with monomer VEGFRs in the cell surface, creating decoy surface receptors for these molecules.
  • VEGFR-1 displayed high covariance with endostatin and angiostatin, and the VEGFR-1 PAC coefficient was comparable to the endostatin and higher than the one for angiostatin in the anti-angiogenic profile, AP4.
  • AP4 angiostatin in the anti-angiogenic profile
  • HGF is a heterodimer with two subunits (a: 69KDa and ⁇ : 34KDa) encoded by the HGF gene on chromosome 7q21.1.
  • HGF is secreted by cells of mesenchymal origin as a pro- HGF inactive precursor that is bound to heparin-like proteoglycans within the extracellular matrix of most tissues.
  • Pro-HGF is cleaved into the mature ⁇ heterodimer by different extracellular proteases including HGF activator, urokinase-type plasminogen activator, Factors XI and XII, matriptase and hepsin.
  • HGF by activation of its Met receptor (RTK receptor tyrosine kinase) acts as a pleiotropic factor that stimulates multiple biological processes, such as cell proliferation, survival, motility, differentiation and morphogenesis during embryogenesis, organ regeneration, and tumor invasiveness.
  • Met receptor RTK receptor tyrosine kinase
  • the a subunit of the HGF is constituted by a N-terminal hairpin and subsequent four kringle domains.
  • SAMMPRIS the presence of good collaterals seemed to influence the stroke risk: 0/66 (0%) in the medical arm and 0/51 (0%) in the stent arm had vessel territory stroke when collaterals were complete (Liebeskind DS. Stroke. 2003;34:2279-2284; Wei L, et al. Stroke. 2001;32:2179-2184; and Liebeskind et al . ISC 2012; February 103 New La).
  • Arteriogenesis is the outgrowth of a mature collateral arterioles upon stenosis or occlusion of a major conductance artery driven by hemodynamic factors such as an increase in stretch and/or fluid shear stress (FSS) on endothelial cells.
  • FSS fluid shear stress
  • Encephaloduroarteriosynangiosis is a form of indirect revascularization initially employed for the treatment of pediatric moyamoya disease.”
  • branches of the external carotid artery ECA
  • STA superficial temporal artery
  • MMA middle meningeal artery
  • STA superficial temporal artery
  • MMA middle meningeal artery
  • Prior trials by our clinical laboratory have demonstrated that EDAS produces vascular connections between the ECA branches and the intracranial circulation in adults with moyamoya 12 and more recently that EDAS also induces formation of new vessels in patients with ICAS averting stroke. In these patients, EDAS has successfully reduced transient ischemic attacks, prevented stroke, and demonstrated angiographic neovascularization with improvement in cerebral perfusion. 13
  • Figure 1 shows baseline and 5 months post EDAS surgical angiogram of the external carotid artery. Notice the enlargement of the STA (white arrow) and the MMA (black arrow), as well as multiple anastomosis between these vessels and the middle cerebral artery branches (arrowheads) and cerebral cortical blush.
  • FIG. 3 illustrates the mechanisms of vessel formation.
  • Such vessel formation mechanisms are reported to involve one or more of the following agents: monocyte chemoattractant protein- 1 (MCP-1), bone morphogenetic proteins (BMP) 2 and 9, platelet derived growth factor (PDGF), intercellular adhesion molecule- 1 (ICAM-1), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), heparin binding epidermal growth factor (HB-EGF or FIBEGF), interleukin 4 (IL4), thrombopoietin (TPO), transforming growth factor (TGF)-P, and/or stromal cell derived factor
  • Indirect revascularization techniques such as encephaloduroarteriosynangiosis (EDAS) were initially used for the treatment of moyamoya disease in children.
  • EDAS encephaloduroarteriosynangiosis
  • This technique offers several advantages: (1) it obviates the need for temporary occlusion of cerebral vessels, which is inherent to bypass, (2) it avoids hyperperfusion since a direct anastomosis with immediate increase in blood flow is not established, and (3) it is technically less demanding than bypass surgery.
  • Applicants have demonstrated that in adults with moyamoya disease, EDAS produces robust revascularization with a quantifiable increase in collateral circulation that develops gradually, avoiding the potential problems of abrupt flow restoration with direct bypass and stenting.
  • Applicants introduced the use of EDAS for its treatment.
  • Embodiments of methods described herein comprise administration of a surgical procedure.
  • the surgical procedure may be, for example, a surgical procedure known in the art or described herein or selected from encephaloduroarteriosynangiosis, stent placement, angioplasty, heart bypass, valvuloplasty, enhanced external counterpulsation, ablation, pacemaker placement, implantation of a cardioverter defibrillator, lead extraction, left ventricular assist device placement, or heart transplant. IV. Additional Methods
  • a method of assessing level of response of a subject to a clinical management regimen of Intracranial atherosclerotic stroke (ICAS) or stroke comprising: measuring circulating blood level of pro-angiogenic factor (PAGF) in the subject prior to the start of the clinical management regimen (PAGF L 0 ), measuring circulating blood level of anti-angiogenic factor (AAGF) in the subject prior to the start of the clinical management regimen (AAGF L 0 ), measuring PAGF circulating blood level in the subject at a time point of or after a clinical management regimen (PAGF L t ), measuring AAGF circulating blood level in the subject at a time point of clinical management regimen (AAGF Lt), comparing PAGF L t with PAGF L 0 and AAGF L t with AAGF L 0 , and designating the subject as responding to the clinical management regimen if the PAGF L t is higher than PAGF L0 or as lacking response to the clinical management regimen if the PAGF L
  • the PAGF is selected from the group consisting of BMP2, BMP9, PDGF-AA, PDGF-BB, HGF, FGF2, KGF, HB-EGF, JL4, TPO, TGF-beta 1, TGF-beta 2, VEGF, VEGFD and SDF-1 alpha
  • the AAGF is selected from the group consisting of endostatin, angiostatin, sVEGFRl, thrombospondin-1 and thrombospondin-2.
  • the subject is a human patient.
  • a method of treating or ameliorating Intracranial atherosclerotic stroke (ICAS) or stroke in a subject comprising (a) subjecting the subject to an encephaloduroarteriosynangiosis (EDAS) procedure, (b) administering to the subject a neovascularization inducer, or a combination of (a) or (b).
  • EDAS encephaloduroarteriosynangiosis
  • the neovascularization inducer comprises an angiogenic factor.
  • the neovascularization inducer is selected from the group consisting of BMP2, BMP9, PDGF-AA, PDGF-BB, HGF, FGF2, KGF, HBEGF, IL4, TPO, TGF-beta 1, TGF-beta 2 , VEGF, VEGFD, SDF-1 alpha, or a combination thereof.
  • the neovascularization inducer comprises monocytes.
  • the neovascularization inducer are monocytes that are obtained from blood of the subject and cultured in a culture medium prior to use.
  • the method comprises (a) subjecting the subject to the encephaloduroarteriosynangiosis (EDAS) procedure, and (b) administering to the subject the neovascularization inducer locally at the site of the EDAS, wherein the neovascularization inducer comprises BMP2, BMP9, PDGF-AA, PDGF- BB, HGF, FGF2, KGF, HB-EGF, IL4, TPO, TGF-beta 1, TGF-beta 2 , VEGF, VEGFD, SDF- 1 alpha, or a combination thereof.
  • EDAS encephaloduroarteriosynangiosis
  • the administering comprises administering to the subject a DNA construct encoding the neovascularization inducer agent.
  • the subject is a human being.
  • Embodiments of the disclosure relate to assays and products for the diagnosis and treatment of atherosclerosis.
  • the levels of angiogenic factors can be determined by measuring the levels of polypeptides encoded by these genes in a patient sample.
  • Methods suitable for this purpose include, but are not limited to, immunoassays such as ELISA, RIA, FACS, dot blot, Western Blot, immunohistochemistry, and antibody-based radioimaging. Protocols for carrying out these immunoassays are well known in the art. Other methods such as 2-dimensional SDS-polyacrylamide gel electrophoresis can also be used. These procedures may be used to recognize any of the polypeptides encoded by the angiogenic factors described herein.
  • One example of a method suitable for detecting the levels of target proteins in samples is an immunoassay such as an ELISA.
  • an immunoassay such as an ELISA.
  • antibodies or other molecules capable of specifically binding to the target proteins i.e. angiogenic factors
  • a selected surface exhibiting protein affinity such as wells in a polystyrene or polyvinylchloride microtiter plate.
  • patient samples to be tested are added to the wells. After binding and washing to remove non-specifically bound immunocomplexes, the bound antigen(s) can be detected.
  • Detection can be achieved by the addition of a second antibody which is specific for the target proteins and is linked to a detectable label.
  • Detection may also be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • a second antibody followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • cells in the peripheral blood samples can be lysed using various methods known in the art. Proper extraction procedures can be used to separate the target proteins from potentially interfering substances.
  • protein-containing samples from the patient are immobilized onto the well surface and then contacted with the antibodies. After binding and washing to remove non-specifically bound immunocomplexes, the bound antigen is detected. Where the initial antibodies are linked to a detectable label, the immunocomplexes can be detected directly. The immunocomplexes can also be detected using a second antibody that has binding affinity for the first antibody, with the second antibody being linked to a detectable label.
  • Another typical immunoassay involves the use of antibody competition in the detection.
  • the target proteins are immobilized on the well surface.
  • the labeled antibodies are added to the well, allowed to bind to the target proteins, and detected by means of their labels.
  • the amount of the target proteins in an unknown sample is then determined by mixing the sample with the labeled antibodies before or during incubation with coated wells. The presence of the target proteins in the unknown sample acts to reduce the amount of antibody available for binding to the well and thus reduces the ultimate signal.
  • Different assay formats can have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immunocomplexes. For instance, in coating a plate with either antigen or antibody, the wells of the plate can be incubated with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate are then washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated” with a nonspecific protein that is antigenically neutral with regard to the test samples. Examples of these nonspecific proteins include bovine serum albumin (BSA), casein and solutions of milk powder.
  • BSA bovine serum albumin
  • the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
  • a secondary or tertiary detection means can also be used. After binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the control and/or clinical or biological sample to be tested under conditions effective to allow immunocomplex (antigen/antibody) formation.
  • These conditions may include, for example, diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween and incubating the antibodies and antigens at room temperature for about 1 to 4 hours or at 49°C overnight. Detection of the immunocomplex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.
  • solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween
  • BGG bovine gamma globulin
  • PBS phosphate buffered saline
  • the contacted surface can be washed so as to remove non-complexed material.
  • the surface may be washed with a solution such as PBS/Tween, or borate buffer.
  • a solution such as PBS/Tween, or borate buffer.
  • the second or third antibody can have an associated label to allow detection.
  • the label is an enzyme that generates color development upon incubating with an appropriate chromogenic substrate.
  • a urease e.g., glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS- Tween).
  • the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azido-di-(3-ethyl)-benzhiazoline-6- sulfonic acid (ABTS) and hydrogen peroxide, in the case of peroxidase as the enzyme label.
  • a chromogenic substrate such as urea and bromocresol purple or 2,2'-azido-di-(3-ethyl)-benzhiazoline-6- sulfonic acid (ABTS) and hydrogen peroxide, in the case of peroxidase as the enzyme label.
  • Quantitation can be achieved by measuring the degree of color generation, e.g., using a spectrophotometer.
  • Suitable labels may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • the labels can be suitable for small scale detection or more suitable for high-throughput screening.
  • suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or it may be quantified.
  • a response that is simply detected generally comprises a response whose existence merely is confirmed
  • a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property.
  • the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.
  • luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence. Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6.sup.th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferases.
  • fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue.TM., and Texas Red.
  • suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6.sup.th ed.).
  • the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker.
  • Suitable functional groups including, but not are limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule.
  • the choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.
  • Attachment of the fluorescent label may be either directly to the cellular component or compound or alternatively, can by via a linker.
  • Suitable binding pairs for use in indirectly linking the fluorescent label to the intermediate include, but are not limited to, antigens/antibodies, e.g., rhodamine/anti-rhodamine, biotin/avidin and biotin/strepavidin.
  • RIA radioimmunoassay
  • An example of RIA is based on the competition between radiolabeled-polypeptides and unlabeled polypeptides for binding to a limited quantity of antibodies.
  • Suitable radiolabels include, but are not limited to, I 125 .
  • a fixed concentration of I 125 -labeled polypeptide is incubated with a series of dilution of an antibody specific to the polypeptide. When the unlabeled polypeptide is added to the system, the amount of the I 125 -polypeptide that binds to the antibody is decreased.
  • a standard curve can therefore be constructed to represent the amount of antibody-bound I 125 -polypeptide as a function of the concentration of the unlabeled polypeptide. From this standard curve, the concentration of the polypeptide in unknown samples can be determined.
  • Various protocols for conducting RIA to measure the levels of polypeptides in breast cancer cell samples are well known in the art.
  • Suitable antibodies as binding agents in the methods described herein can include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, Fab fragments, and fragments produced by a Fab expression library.
  • Antibodies can be labeled with one or more detectable moieties to allow for detection of antibody-antigen complexes.
  • the detectable moieties can include compositions detectable by spectroscopic, enzymatic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical or chemical means.
  • the detectable moieties include, but are not limited to, radioisotopes, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers such as fluorescent markers and dyes, magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors, and the like.
  • Protein array technology is discussed in detail in Pandey and Mann (2000) and MacBeath and Schreiber (2000), each of which is herein specifically incorporated by reference. These arrays typically contain thousands of different proteins or antibodies spotted onto glass slides or immobilized in tiny wells and allow one to examine the biochemical activities and binding profiles of a large number of proteins at once. To examine protein interactions with such an array, a labeled protein is incubated with each of the target proteins immobilized on the slide, and then one determines which of the many proteins the labeled molecule binds. In certain embodiments such technology can be used to quantitate a number of proteins in a sample, such as a breast cancer biomarker proteins.
  • protein chips has some similarities to DNA chips, such as the use of a glass or plastic surface dotted with an array of molecules. These molecules can be DNA or antibodies that are designed to capture proteins. Defined quantities of proteins are immobilized on each spot, while retaining some activity of the protein. With fluorescent markers or other methods of detection revealing the spots that have captured these proteins, protein microarrays are being used as powerful tools in high-throughput proteomics and drug discovery.
  • the earliest and best-known protein chip is the ProteinChip by Ciphergen Biosystems Inc. (Fremont, Calif).
  • the ProteinChip is based on the surface-enhanced laser desorption and ionization (SELDI) process.
  • Known proteins are analyzed using functional assays that are on the chip.
  • chip surfaces can contain enzymes, receptor proteins, or antibodies that enable researchers to conduct protein-protein interaction studies, ligand binding studies, or immunoassays.
  • the ProteinChip system detects proteins ranging from small peptides of less than 1000 Da up to proteins of 300 kDa and calculates the mass based on time-of-flight (TOF).
  • TOF time-of-flight
  • the ProteinChip biomarker system is the first protein biochip-based system that enables biomarker pattern recognition analysis to be done. This system allows researchers to address important clinical questions by investigating the proteome from a range of crude clinical samples (i.e., laser capture microdissected cells, biopsies, tissue, urine, and serum). The system also utilizes biomarker pattern software that automates pattern recognition-based statistical analysis methods to correlate protein expression patterns from clinical samples with disease phenotypes.
  • the levels of polypeptides in samples can be determined by detecting the biological activities associated with the polypeptides. If a biological function/activity of a polypeptide is known, suitable in vitro bioassays can be designed to evaluate the biological function/activity, thereby determining the amount of the polypeptide in the sample. VI. Compositions
  • composition which composition comprising a neovascularization inducer in an effective amount to induce cerebrovascularization in a subject having Intracranial atherosclerotic stroke (ICAS) or stroke.
  • IFS Intracranial atherosclerotic stroke
  • the neovascularization inducer comprises BMP2, BMP9, PDGF-AA, PDGF-BB, HGF, FGF2, KGF, HB-EGF, IL4, TPO, TGF-beta 1, TGF-beta 2 , VEGF, VEGFD, SDF-1 alpha, or a combination thereof.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the neovascularization inducer comprises monocytes.
  • the neovascularization inducer are monocytes that are obtained from blood of the subject and cultured in a culture medium prior to use.
  • the pharmaceutical composition described herein may be administered to a subject in need of treatment by a variety of routes of administration, including orally and parenterally, (e.g., intravenously, subcutaneously or intramedullary), intranasally, as a suppository or using a "flash" formulation, i.e., allowing the medication to dissolve in the mouth without the need to use water, topically, intradermally, subcutaneously and/or administration via mucosal routes in liquid or solid form.
  • the pharmaceutical composition can be formulated into a variety of dosage forms, e.g., extract, pills, tablets, microparticles, capsules, oral liquid.
  • the term "compound” is used interchangeably with "agent” or "molecule.”
  • compositions may also be included as part of the pharmaceutical composition pharmaceutically compatible binding agents, and/or adjuvant materials.
  • the active materials can also be mixed with other active materials including antibiotics, antifungals, other virucidals and immunostimulants which do not impair the desired action and/or supplement the desired action.
  • the mode of administration of the pharmaceutical composition described herein is oral.
  • Oral compositions generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets.
  • the aforesaid compounds or agents may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. Some variation in dosage will necessarily occur, however, depending on the condition of the subject being treated.
  • These preparations should produce a serum concentration of active ingredient of from about 0.01 nM to 1,000,000 nM, e.g., from about 0.2 to 40 .mu.M.
  • a preferred concentration range is from 0.2 to 20 .mu.M and most preferably about 1 to 10 .mu.M.
  • concentration of active ingredient in the drug composition itself depends on bioavailability of the drug and other factors known to those of skill in the art.
  • the mode of administration of the pharmaceutical compositions described herein is topical or mucosal administration.
  • a specifically preferred mode of mucosal administration is administration via female genital tract.
  • Another preferred mode of mucosal administration is rectal administration.
  • polymeric and/or non-polymeric materials can be used as adjuvants for enhancing mucoadhesiveness of the pharmaceutical composition disclosed herein.
  • the polymeric material suitable as adjuvants can be natural or synthetic polymers.
  • Representative natural polymers include, for example, starch, chitosan, collagen, sugar, gelatin, pectin, alginate, karya gum, methylcellulose, carboxymethylcellulose, methylethylcellulose, and hydroxypropylcellulose.
  • Representative synthetic polymers include, for example, poly(acrylic acid), tragacanth, poly(methyl vinylether-co-maleic anhydride), poly(ethylene oxide), carbopol, poly(vinyl pyrrolidine), poly(ethylene glycol), poly(vinyl alcohol), poly(hydroxyethylmethylacrylate), and polycarbophil.
  • Other bioadhesive materials available in the art of drug formulation can also be used (see, for example, Bioadhesion— Possibilities and Future Trends, Gurny and Junginger, eds., 1990).
  • dosage values also varies with the specific severity of the disease condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted to the individual need and the professional judgment of the person administering or supervising the administration of the aforesaid compositions. It is to be further understood that the concentration ranges set forth herein are exemplary only and they do not limit the scope or practice of the disclosure.
  • the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.
  • the formulation may contain the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, corn starch and the like; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; and a sweetening agent such as sucrose or saccharin or flavoring agent such as peppermint, methyl salicylate, or orange flavoring may be added.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, corn starch and the like
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or saccharin or flavoring agent such as pepper
  • dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings.
  • tablets or pills may be coated with sugar, shellac, or other enteric coating agents.
  • Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
  • the solutions or suspensions may also include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methylparabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as ethylenediaminetetraacetic acid
  • compositions can be prepared as formulations with pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carriers Preferred are those carriers that will protect the active compound or agent against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatable polymers can be used, such as polyanhydrides, polygly colic acid, collagen, and polylactic acid. Methods for preparation of such formulations can be readily performed by one skilled in the art.
  • Liposomal suspensions are also preferred as pharmaceutically acceptable carriers.
  • Methods for encapsulation or incorporation of compounds into liposomes are described by Cozzani, I; Jori, G.; Bertoloni, G.; Milanesi, C; Sicuro, T. Chem. Biol. Interact. 53, 131-143 (1985) and by Jori, G.; Tomio, L.; Reddi, E.; Rossi, E. Br. J. Cancer 48, 307-309 (1983), for example.
  • These may also be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.
  • liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound is then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.
  • appropriate lipid(s) such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol
  • the pharmaceutical composition described herein may be administered in single (e.g., once daily) or multiple doses or via constant infusion.
  • the compounds may also be administered alone or in combination with pharmaceutically acceptable carriers, vehicles or diluents, in either single or multiple doses.
  • Suitable pharmaceutical carriers, vehicles and diluents include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents.
  • the pharmaceutical compositions formed by combining the compounds of this disclosure and the pharmaceutically acceptable carriers, vehicles or diluents are then readily administered in a variety of dosage forms such as tablets, powders, lozenges, syrups, injectable solutions and the like.
  • These pharmaceutical compositions can, if desired, contain additional ingredients such as flavorings, binders, excipients and the like according to a specific dosage form.
  • tablets containing various excipients such as sodium citrate, calcium carbonate and/or calcium phosphate may be employed along with various disintegrants such as starch, alginic acid and/or certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and/or acacia.
  • various lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tabletting purposes.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules. Preferred materials for this include lactose or milk sugar and high molecular weight polyethylene glycols.
  • the active pharmaceutical agent therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin and/or combinations thereof.
  • solutions of the compounds of this disclosure in sesame or peanut oil, aqueous propylene glycol, or in sterile aqueous solutions may be employed.
  • Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • the sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art.
  • the compounds of the disclosure are conveniently delivered in the form of a solution or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurized container or nebulizer may contain a solution or suspension of a compound of this disclosure.
  • Capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound or compounds of the disclosure and a suitable powder base such as lactose or starch.
  • the pharmaceutical composition provided herein can also be used with another pharmaceutically active agent effective for a disease such as neurodisorders, cardiovascular disorders, tumors, AIDS, depression, and/or type-1 and type-2 diabetes.
  • additional agents can be, for example, antiviral agent, antibiotics, anti-depression agent, anti-cancer agents, immunosuppressant, anti-fungal, and a combination thereof.
  • composition described herein can be formulated alone or together with the other agent in a single dosage form or in a separate dosage form.
  • Methods of preparing various pharmaceutical formulations with a certain amount of active ingredient are known, or will be apparent in light of this disclosure, to those skilled in this art.
  • For examples of methods of preparing pharmaceutical formulations see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19th Edition (1995).
  • the composition of disclosure can be formulated into a scaffold.
  • a scaffold can include a carrier, which can be biodegradable, such as degradable by enzymatic or hydrolytic mechanisms.
  • carriers include, but are not limited to synthetic absorbable polymers such as such as but not limited to poly(.alpha.- hydroxy acids) such as poly (L-lactide) (PLLA), poly (D, L-lactide) (PDLLA), polyglycolide (PGA), poly (lactide-co-glycolide (PLGA), poly (-caprolactone), poly (trimethylene carbonate), poly (p-dioxanone), poly (-caprolactone-co-glycolide), poly (glycolide-co- trimethylene carbonate) poly (D, L-lactide-co-trimethylene carbonate), polyarylates, polyhydroxybutyrate (PUB), polyanhydrides, poly (anhydride-co-imide), propylene-co-
  • PLLA polyglycolide
  • carriers include cellulosic polymers such as, but not limited to alkylcellulose, hydroxyalkylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, carboxymethylcellulose, and their cationic salts.
  • carrier include synthetic and natural bioceramics such as, but not limited to calcium carbonates, calcium phosphates, apatites, bioactive glass materials, and coral-derived apatites.
  • the carrier may further be coated by compositions, including bioglass and or apatites derived from sol-gel techniques, or from immersion techniques such as, but not limited to simulated body fluids with calcium and phosphate concentrations ranging from about 1.5 to 7-fold the natural serum concentration and adjusted by various means to solutions with pH range of about 2.8-7.8 at temperature from about 15-65 degrees C.
  • compositions including bioglass and or apatites derived from sol-gel techniques, or from immersion techniques such as, but not limited to simulated body fluids with calcium and phosphate concentrations ranging from about 1.5 to 7-fold the natural serum concentration and adjusted by various means to solutions with pH range of about 2.8-7.8 at temperature from about 15-65 degrees C.
  • Other examples of carriers include collagen (e.g. Collastat, Helistat collagen sponges), hyaluronan, fibrin, chitosan, alginate, and gelatin, or a mixture thereof.
  • the carrier may include heparin-binding agents; including but not limited to heparin-like polymers e.g. dextran sulfate, chondroitin sulfate, heparin sulfate, fucan, alginate, or their derivatives; and peptide fragments with amino acid modifications to
  • the scaffold may be in the form of a liquid, solid or gel.
  • the scaffold can be a carrier that is in the form of a flowable gel.
  • the gel may be selected so as to be injectable, such as via a syringe at the site where bone formation is desired.
  • the gel may be a chemical gel which may be a chemical gel formed by primary bonds, and controlled by pH, ionic groups, and/or solvent concentration.
  • the gel may also be a physical gel which may be formed by secondary bonds and controlled by temperature and viscosity.
  • gels include, but are not limited to, pluronics, gelatin, hyaluronan, collagen, polylactide-polyethylene glycol solutions and conjugates, chitosan, chitosan & b-glycerophosphate (BST-gel), alginates, agarose, hydroxypropyl cellulose, methyl cellulose, polyethylene oxide, polylactides/glycolides in N-methyl-2- pyrrolidone. See for example, Anatomical Record (2001), 263(4), 342-349, the teachings of which are incorporated herein by reference.
  • the carrier may be photopolymerizable, such as by electromagnetic radiation with wavelength of at least about 250 nm.
  • Photopolymerizable polymers include polyethylene (PEG) acrylate derivatives, PEG methacrylate derivatives, propylene fumarate-co-ethylene glycol, polyvinyl alcohol derivatives, PEG-co-poly(-hydroxy acid) diacrylate macromers, and modified polysaccharides such as hyaluronic acid derivatives and dextran methacrylate.
  • PEG polyethylene
  • PEG methacrylate derivatives propylene fumarate-co-ethylene glycol
  • polyvinyl alcohol derivatives polyvinyl alcohol derivatives
  • PEG-co-poly(-hydroxy acid) diacrylate macromers PEG-co-poly(-hydroxy acid) diacrylate macromers
  • modified polysaccharides such as hyaluronic acid derivatives and dextran methacrylate.
  • the scaffold may include a carrier that is temperature sensitive.
  • a carrier that is temperature sensitive. Examples include carriers made from N-isopropylacryl amide (NiPAM), or modified NiPAM with lowered lower critical solution temperature (LCST) and enhanced peptide (e.g. NELLl) binding by incorporation of ethyl methacrylate and N-acryloxysuccinimide; or alkyl methacrylates such as butylmethacrylate, hexylmethacrylate and dodecylmethacrylate (PCT Int. Appl. WO/2001070288; U.S. Pat. No. 5, 124,151, the teachings of which are incorporated herein by reference).
  • the carrier may have a surface that is decorated and/or immobilized with cell adhesion molecules, adhesion peptides, and adhesion peptide analogs which may promote cell-matrix attachment via receptor mediated mechanisms, and/or molecular moieties which may promote adhesion via non-receptor
  • mediated mechanisms binding such as, but not limited to polycationic polyamino- acid- peptides (e.g. poly-lysine), polyanionic polyamino-acid-peptides, Mefp-class adhesive molecules and other DOPA-rich peptides (e.g. poly-lysine-DOPA), polysaccharides, and proteoglycans.
  • polycationic polyamino- acid- peptides e.g. poly-lysine
  • polyanionic polyamino-acid-peptides e.g. poly-lysine
  • Mefp-class adhesive molecules e.g. poly-class adhesive molecules
  • DOPA-rich peptides e.g. poly-lysine-DOPA
  • the carrier may be comprised of sequestering agents such as, but not limited to, collagen, gelatin, hyaluronic acid, alginate, poly(ethylene glycol), alkylcellulose (including hydroxyalkylcellulose), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl- methylcellulose, and carboxymethylcellulose, blood, fibrin, polyoxyethylene oxide, calcium sulfate hemihydrate, apatites, carboxyvinyl polymer, and poly(vinyl alcohol). See for example, U.S. Pat. No. 6,620,406, herein incorporated by reference.
  • sequestering agents such as, but not limited to, collagen, gelatin, hyaluronic acid, alginate, poly(ethylene glycol), alkylcellulose (including hydroxyalkylcellulose), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl- methylcellulose, and
  • the carrier may include buffering agents such as, but not limited to glycine, glutamic acid hydrochloride, sodium chloride, guanidine, heparin, glutamic acid hydrochloride, acetic acid, succinic acid, polysorbate, dextran sulfate, sucrose, and amino acids. See for example, U.S. Pat. No. 5,385,887, herein incorporated by reference.
  • the carrier may include a combination of materials such as those listed above.
  • the carrier may be a PLGA/collagen carrier membrane.
  • the scaffold can be an implant of the various embodiments described herein.
  • the composition according to this disclosure may be contained within a time release tablet.
  • a bioactive agent described herein can be formulated with an acceptable carrier to form a pharmacological composition.
  • Acceptable carriers can contain a physiologically acceptable compound that acts, for example, to stabilize the composition or to increase or decrease the absorption of the agent.
  • Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the anti-mitotic agents, or excipients or other stabilizers and/or buffers.
  • physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms.
  • Various preservatives are well known and include, for example, phenol and ascorbic acid.
  • phenol and ascorbic acid include, for example, phenol and ascorbic acid.
  • the composition can have a dosage of about 1 ng to about 500 mg, for example, about 10 ng, 20 ng, 50 ng, 100 ng, 200 ng, 500 ng, 1 micro gram, about 10 micro gram, about 50 micro gram, about 100 micro gram, about 200 micro gram, about 500 micro gram, or about 1 mg (or any range derivable therein).
  • Embodiments of the composition can be administered in a variety of unit dosage forms depending upon the method of administration.
  • unit dosage forms suitable may include powder, tablets, pills, capsules.
  • a method of fabricating a composition comprising providing an anti-angiogenic agent in a therapeutically effective amount and forming the composition.
  • the anti-angiogenic agent is GMI-1070, aproteinase activated receptor (PAR) - 1 agonist, meziothrombin, granzyme A, activated protein C receptor - endothelial protein C receptor (APC-EPCR), kalikrein (KLK) 4,KLK5, KLK6, matrix metalloproteinase (MMP1), proatherocytin, pen C13; PAR-4 antagonists, tcY- H 2 ; heparan sulfate proteoglycans, analogues of heparin sulfate proteoglycans, aprotinin, or combinations thereof.
  • PAR aproteinase activated receptor
  • APC-EPCR activated protein C receptor - endothelial protein C receptor
  • KLK kalikrein
  • KLK5 kalikrein
  • KLK6 matrix metalloproteinase
  • proatherocytin pen C13
  • a method of fabricating a composition comprising providing a neovascularization inducer in an effective amount to induce cerebrovascularization in a subject having Intracranial atherosclerotic stroke (ICAS) or stroke, and forming the composition.
  • IAS Intracranial atherosclerotic stroke
  • the neovascularization inducer is BMP2, BMP9, PDGF-AA, PDGF-BB, HGF, FGF2, KGF, HB-EGF, JL4, TPO, TGF-beta 1, TGF- beta 2 , VEGF, VEGFD, SDF-1 alpha, or a combination thereof.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the neovascularization inducer comprises monocytes.
  • the neovascularization inducer are monocytes that are obtained from blood of the subject and cultured in a culture medium prior to use.
  • step (1) through step (3) reactions are performed and repeated until the desired peptide is obtained: (1) a free-amine is reacted with carboxyl terminus using carbodiimide chemistry, (2) the amino acid sequence is purified, and (3) the protecting group, e.g., the FMOC protecting group, is removed under mildly acidic conditions to yield a free amine.
  • the peptide can then be cleaved from the resin to yield a free standing peptide or peptide mimetics.
  • any of the disclosed therapies described herein may be used in any particular combination.
  • the compositions and related methods may also be used in combination with the administration of traditional therapies.
  • the therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
  • the other agents and/or a proteins or polynucleotides are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the therapeutic composition would still be able to exert an advantageously combined effect on the subject.
  • compositions of the disclosure administered to a patient/subject will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the composition. It is expected that the treatment cycles would be repeated as necessary. It is also contemplated that various standard therapies, such as hydration, may be applied in combination with the described therapy.
  • ICAS Intracranial atherosclerosis
  • TIA transient ischemic attack
  • Surgical management The EDAS technique has been described in detail in prior publications. 14 Patients eligible for surgery continued intensive medical management with no interruption. The EDAS surgeries were performed at the Ronald Reagan Hospital at UCLA.
  • Angiogenic factor measurements Blood samples were collected at each visit. Blood was extracted in EDTA tubes, serum was immediately separated by centrifugation at 3500rpm for 15 minutes at 4°C, and stored in 2 cc cryovials at -80°C.
  • VEGF vascular endothelial growth factor
  • VEGF-D vascular endothelial growth factor
  • VEGFR soluble VEGF receptor
  • PDGF platelet-derived growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • FIB-EGF heparin-binding epidermal growth factor
  • TGF transforming growth factor
  • TGF ⁇ TGF ⁇ 2
  • BMP-9 stromal cell derived factor
  • IL interleukin
  • TSP thrombospondin
  • Descriptive statistics were computed to summarize demographic and angiogenic factor data.
  • Principal component analysis was applied to the angiogenic factors at each time point to reduce data dimensionality and to derive the equations for composite indexes that were denominated as angiogenic profiles (APs). As no p values are computed during the creation of the APs, this methodology does not increase the false discover ⁇ ' rate, while capturing as much of the variability in the original variables as possible.
  • APs with eigenvalues higher or equal to 1.0 were included in the inferential analysis. This threshold was selected as the components with eigenvalues above 1 accounted for 75% of the total variance.
  • a stepwise logistic regression model was built using the APs as predictor variables. Then, the significantly associated APs were incorporated in a multivariable logistic regression including the relevant clinical predictor variables: age, gender, previous stroke, hypertension, hypercholesterolemia, diabetes, and obesity (defined as a BMI > 30). The fitness of each model was evaluated using R square and AUCs of the corresponding ROCs. Odds ratios were calculated for each variable from the regression parameters with its corresponding 95% confidence intervals.
  • mRS modified Rankin Score
  • mRS modified Rankin Score
  • a "neovascularization index” was defined as the Perren grades plus an additional point if evidence of collaterals in the ASITN grades were observed in cerebral angiography at 6 months after surgery.
  • a linear regression model was built using stepwise regression with minimum BIC stopping rule and backward direction for the outcome variable "neovascularization index,” including as predictor variables: age, diabetes, smoking history, and the baseline APs. If more than one AP was associated with a specific response, the coefficients of the regression were used to resolve the final angiogenic profile associated with the outcome variable.
  • Table 3 depicts the medians and interquartile range (IQR) for the baseline angiogenic factor measurements for each group. Transformed angiogenic factor values were used to conduct the principal component analysis. There were 7 APs with eigenvalues higher than 1. They were named consecutively API to AP7 in order of decreasing eigenvalue. The details of the coefficients for each transformed angiogenic factor value are provided in the Table 4 and a graphic representation of the composition of the APs is depicted in Figure 1.
  • IQR interquartile range
  • the multivariable logistic regression model parameter results and odds ratios are described in table 6.
  • the AP4 is characterized by high levels of HGF, lower levels of the proangiogenic isoform VEGF165a, and high levels of the antiangiogenic factors VEGFR1, Endostatin and Angiostatin.
  • Applicants built a multivariable ordinal logistic regression model to evaluate the association of the APs to functional outcomes at 6 months from enrollment in terms of modified Rankin Score (mRS).
  • mRS modified Rankin Score
  • a prediction profiler was constructed using the ordinal model with 5,000 simulation runs of random factor settings to facilitate the interpretation of the association between AP4 and the outcome 6-months mRS.
  • Figure 2 shows the graphical query of the factor AP4 in the multivariate model as predictor variable for the calculated probabilities of the 6-months mRS.
  • Angiogenesis has been extensively studied in peripheral vascular disease and myocardial ischemia. 15 ' 16 However, limited investigations have been conducted evaluating the role of angiogenesis in stroke and intracranial atherosclerosis. The pioneering works of Arenillas 17 and Navarro-Sobrino 18 ' 19 have shown that angiogenesis and the balance of pro and antiangiogenic factors may have an influence in acute stroke and in the evolution of intracranial atherosclerosis. Arenillas et al. performed a study on 40 patients with ICAS with a single time measurement of VEGF and endostatin.
  • Our results show a strong association between circulating anti angiogenic profile AP4 and failure to medical management, poor functional status, and reduced neovascularization.
  • the anti angiogenic profile was a stronger predictor of failure than other clinical factors, including a history of previous stroke, diabetes, and age.
  • the anti angiogenic profile was also a predictor of poor functional status independent of baseline mRS and age.
  • digital subtraction angiographic evaluation of patients that received an indirect revascularization with EDAS showed a negative correlation between neovascularization and the antiangiogenic factors endostatin and angiostatin.
  • PCA provides insights into patterns and relationships in the data. 20 This approach allowed us to overcome, to some extent, the current limitations in the biological understanding of all the interactions and roles of the proangiogenic and antiangiogenic factors in response to cerebral ischemia in ICAS.
  • AP4 was the most antiangiogenic, based on its PCA coefficients. AP4 was found to be statistically significant with regards to failure to medical management and outcomes at 6 months.
  • this prospective cohort study of patients with severe ICAS identified a circulating angiogenic profile, characterized by a high correlation between factors HGF, endostatin, angiostatin, and VEGFR-1, which was independently and significantly associated with failure to medical management and poor functional status at 6 months. It also showed a profile dominated by endostatin and angiostatin as significant negative predictor of neovascularization after EDAS surgery. This angiogenic profile exhibited the highest contribution of antiangiogenic factors compared to the other profiles that describe the dataset.
  • Setting 5 Describe the setting, locations, and relevant dates, including periods of recruitment, exposure, follow-up, and data collection
  • Participants 6 (a) Give the eligibility criteria, and the sources and methods of selection of participants. Describe methods of follow-up
  • Variables 7 Clearly define all outcomes, exposures, predictors, potential confounders, and effect modifiers. Give diagnostic criteria, if applicable
  • Data sources/ 8* For each variable of interest, give sources of data and measurement details of methods of assessment (measurement). Describe comparability of assessment methods if there is more than one group
  • Bias 9 Describe any efforts to address potential sources of bias
  • Quantitative variables 1 1 Explain how quantitative variables were handled in the analyses. If applicable, describe which groupings were chosen and why
  • Descriptive data 14* (a) Give characteristics of study participants (eg.
  • Limitations 19 Discuss limitations of the study, taking into account sources of potential bias or imprecision. Discuss both direction and magnitude of any potential bias
  • Funding 22 Give the source of funding and the role of the funders for the present study and, if applicable, for the original study on which the present article is based
  • intracranial artery diagnosed by 2. Any hemorrhagic infarct within 14 days before angio, TCD, MRA, or CTA. enrollment or any other intracranial hemorrhage
  • VEGFD (pg/mi) 1016.1 (703.5) 868.6 (619.0)
  • PDGFAA (pg/ml) 88.4 (182.1) 56.3 (155.0)
  • PDGFBB (pg/ml) 84.0 (149.9) 101.4 (178.0)
  • FGFb (pg/ml) 1.4 (1.5) 0.4 (1.4)
  • HGF (pg/ml) 401.9 (833.6) 216.2 (1 19.5)
  • HBEGF (pg/ml) 1.0 (1.1) 1.4 (1.5)
  • VEGFR1 (pg/ml) 178.1 (156.1) 114.4 (71.5)
  • VEGFR2 (pg/ml) 13334.4 (3724.0) 14594.8 (4727.9)
  • VEGFR3 (pg/ml) 397595.0 (140122.0)
  • TSP1 (pg/ml) 102431.1 (12876.1) 97226.8 (35094.8)
  • TSP2 (pg/ml) 29983.4 (26279.6) 36365.0 (25849.3)
  • Angiostatin (ng/ml) 766.6 (334.5) 624.0 (213.4)
  • Table 4 (Can be supplementary table 1)
  • Tr TGFB 1 0.360 -0.005 -0.047 -0.023 -0.020 -0.010 -0.049
  • Tr TGFB2 0.509 -0.161 0.035 -0.039 -0.037 0.104 -0.058
  • Tr BMP9 0.070 0.044 0.438 -0.141 0.077 -0.189 -0.058
  • Tr TSP2 0.166 0.254 0.582 0.202 -0.521 -0.136 0.189
  • Ischemic stroke is the leading cause of adult disability and the second cause of all death worldwide.
  • IAS intracranial atherosclerosis
  • EDAS Encephaloduro-arteriosynangiosis
  • EMS Encephalomyosynangiosis
  • EMS a similar form of indirect revascularization, uses the temporals muscle rather than the superficial temporal artery.
  • EDAS is promising as a treatment for ICAS, the angiogenic mechanisms, which cause vessel growth are poorly understood.
  • Applicants present the development of a reproducible animal model of ischemia and synangiosis, and evaluated the development of neovascularization on the surgical side.
  • the muscle is reattached to the bone with 2 10-O nylon sutures ensuring close approximation of the surfaces of the muscle and the brain.
  • the field is then irrigated and the wound is closed with interrupted stitches. All animals postoperative care and euthanasia were performed according to the Guidelines of the NIH and the Chancellors Animal Research Committee at UCLA. After euthanasia mice were perfused transcardially with PBS followed by 4% paraformaldehyde. Tissues were paraffin-embedded after decalcification and section in sequential coronal ⁇ slides. Specimens underwent H&E staining along with immunofluorescence studies for markers of cellular proliferation (Ki67), endothelial cell (CD-31) and nuclei (DAPI).
  • VEGFA165a/b ratio Mean age was 61.8 ⁇ 12.3, and 53% were females.
  • Intracranial atherosclerosis is one of the most common causes of stroke worldwide. It accounts for at least 10% of all strokes in the United States and as much as 67%) in countries with predominantly Asians, Hispanics, and Blacks, which constitute the maj ority of the world population.
  • ICAS carries a worse prognosis than other stroke etiologies, with an annual rate of recurrent stroke and death of 15%> despite intensive medical management, and as high as 35% in certain populations.
  • the objective of this study was to identify circulating angiogenic factor profiles associated to success or failure of intensive medical treatment in ICAS patients.
  • vascular endothelial growth factor isoforms 165a and 165b
  • VEGF-D vascular endothelial growth factor
  • VEGFR soluble VEGF receptor
  • PDGF platelet-derived growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • HGF heparin-binding epidermal growth factor
  • TGF transforming growth factor
  • BMP bone morphogenic protein
  • SDF stromal cell derived factor
  • IL interleukin
  • TSP thrombospondin
  • TSP-2 endostatin
  • endostatin endothelial growth factor
  • angiostatin angiostatin.
  • levels of angiogenic factors were determined by plasma multiplex sandwich ELISA.
  • Kuba K, Matsumoto K, Date K, Shimura H, Tanaka M, Nakamura T. Hgf/nk4, a four- kringle antagonist of hepatocyte growth factor, is an angiogenesis inhibitor that suppresses tumor growth and metastasis in mice. Cancer Res. 2000;60:6737-6743

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Abstract

La présente invention concerne des procédés et des compositions de traitement de l'athérosclérose, et est basée sur l'inhibition anti-angiogenèse in vivo. Des aspects de l'invention concernent un procédé de traitement de l'athérosclérose chez un sujet, comprenant : l'administration d'un traitement chirurgical à un sujet dont il a été déterminé qu'il présentait ou moins l'un de : taux accrus de HGF, taux réduits de VEGF165a, taux accrus de VEGFR1, taux accrus d'endostatine, et/ou taux accrus d'angiostatine.
PCT/IB2017/050876 2016-02-16 2017-02-16 Procédés et compositions de traitement l'athérosclérose WO2017141185A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108864274A (zh) * 2018-07-27 2018-11-23 新乡医学院 一种无纯化标签的血管内皮细胞生长因子VEGF165b的纯化方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090175791A1 (en) * 2007-11-09 2009-07-09 Peregrine Pharmaceuticals, Inc. Anti-VEGF Antibody Compositions and Methods

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090175791A1 (en) * 2007-11-09 2009-07-09 Peregrine Pharmaceuticals, Inc. Anti-VEGF Antibody Compositions and Methods

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
G ONZALEZ ET AL.: "Intracranial arterial stenoses: current viewpoints, novel approaches, and surgical perspectives", NEUROSURG REV., vol. 36, no. 2, April 2013 (2013-04-01), pages 175 - 185 *
GONZALEZ ET AL.: "Abstract TMP15: Circulating Antiangiogenic Profile Predicts Lower Degrees of Neovascularization After Indirect Revascularization With Encephaloduroarteriosynangiosis (EDAS", PATIENTS WITH INTRACRANIAL ATHEROSCLEROSIS (ICAS, vol. 47, no. 1, February 2016 (2016-02-01), Retrieved from the Internet <URL:http://stroke.ahajournals.org/content/47/Suppl_1/ATMP15.short> [retrieved on 20170707] *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108864274A (zh) * 2018-07-27 2018-11-23 新乡医学院 一种无纯化标签的血管内皮细胞生长因子VEGF165b的纯化方法

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