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WO2009036108A1 - Procédés et compositions pour inhiber une fuite vasculaire - Google Patents

Procédés et compositions pour inhiber une fuite vasculaire Download PDF

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WO2009036108A1
WO2009036108A1 PCT/US2008/075921 US2008075921W WO2009036108A1 WO 2009036108 A1 WO2009036108 A1 WO 2009036108A1 US 2008075921 W US2008075921 W US 2008075921W WO 2009036108 A1 WO2009036108 A1 WO 2009036108A1
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fumagillol
tnp
derivative
block copolymer
acetyl
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Ofer Fainaru
Ofra Benny-Ratsaby
Judah Folkman
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Boston Childrens Hospital
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Boston Childrens Hospital
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/65—Tetracyclines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • 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

Definitions

  • the present invention relates generally to the treatment or prevention of diseases or disorders involving vascular leakage.
  • VEGF/VPF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • IL-8 interleukin- 8
  • TNP-470 O-(chloracetyl-carbamoyl) fumagillol
  • AGM- 1470 an analog of fumagillin
  • TNP-470 has the broadest anticancer spectrum of any known agent 4 ' 13 .
  • TNP-470 inhibited the growth of murine tumors up to 91%, human tumors up to 100% and metastatic tumors up to 100% in mice (reviewed in ref. 13 ).
  • mice were treated at the same optimal dose of 30 mg/kg subcutaneously every other day.
  • TNP-470 has shown evidence of antitumor activity when used as a single agent, with a number of objective responses reported with relapsed and refractory malignancies 14"16 .
  • TNP-470 is generally administered via injections, for example intramuscular or intravenous delivery systems or by a continuous intravenous infusion given every few days. TNP-470 is also typically administered at high doses for a specific period of time at infrequent time intervals, for example TNP-470 is administered at high doses as a chemotherapeutic. TNP- 470 is not generally administered orally.
  • the endothelial cell monolayer lining the vasculature forms a barrier that maintains the integrity of the blood fluid compartment, but permits passage of soluble factors and leukocytes in a regulated manner. Dysregulation of this process produces vascular leakage into underlying tissues, which accompanies the inflammation associated with pathological conditions involving edema 51"53 . Edema associated with vascular permeability also occurs in ischemic injury due to the secretion of vascular endothelial growth factor (VEGF) by hypoxic tissues, which increases tissue damage in animal models of stroke and myocardial infarction 54 ' 55 . Vascular permeability is characterized by altered cell-cell contacts and the appearance of paracellular pores between adjacent cells.
  • VEGF vascular endothelial growth factor
  • Integrity of the endothelial barrier is regulated in part by opposing roles of the actin cytoskeleton in which cortical F-actin stabilizes cell-cell contacts, whereas intracellular stress fibers exert tension to induce permeability 56 ' 57 .
  • Vascular permeability is a precisely regulated function that can contribute positively to immune responses and wound healing; however, leakage of fluid and immune cells into tissues can have serious and life-threatening consequences in a variety of diseases. Fluid accumulation in the lungs because of increased permeability of the pulmonary vasculature leading to respiratory insufficiency is a key element in acute respiratory distress syndrome 58 . Vascular leak after stroke or myocardial infarction due to the release of VEGF by hypoxic tissues substantially increases tissue injury after these events 54 ' 55 . Vascular leak and tissue edema contribute to organ failure in sepsis 59 .
  • vascular hyperpermeability has been implicated in numerous pathologies including vascular complications of diabetes, pulmonary hypertension and various edemas, and has been rendered responsible for decreasing efficacy of anti-cancer therapies due to loss of endogenous angiogenesis inhibitors into the urine.
  • diabetic retinopathy is a leading cause of blindness that affects approximately 25% of the estimated 16 million Americans with diabetes. It is believed that diabetic retinopathy is induced by hypoxia in the retina as a result of hyperglycemia. The degree of diabetic retinopathy is highly correlated with the duration of diabetes. There are two kinds of diabetic retinopathy.
  • the first, non-proliferative retinopathy is the earlier stage of the disease characterized by increased capillary permeability, microaneurysms, hemorrhages, exudates, and edema. Most visual loss during this stage is due to the fluid accumulating in the macula, the central area of the retina. This accumulation of fluid is called macular edema, and can cause temporary or permanent decreased vision.
  • the second category of diabetic retinopathy is called proliferative retinopathy and is characterized by abnormal new vessel formation, which grows on the vitreous surface or extends into the vitreous cavity. Neovascularization can be very damaging because it can cause bleeding in the eye, retinal scar tissue, diabetic retinal detachments, or glaucoma, any of which can cause decreased vision or blindness.
  • Non-proliferative retinopathy includes intensive insulin therapy to achieve normal glycemic levels in order to delay further progression of the disease, whereas the current treatment of proliferative retinopathy involves panretinal photocoagulation and vitrectomy.
  • the treatment of non-proliferative retinopathy while valid in theory, is mostly ineffective in practice because it usually requires considerable modification in the lifestyle of the patients, and many patients find it very difficult to maintain the near- normal glycemic levels for a time sufficient to slow and reverse the progression of the disease.
  • the current treatment of non-proliferative retinopathy only delays the progression of the disease and cannot be applied effectively to all patients who require it.
  • Another complication of diabetes, diabetic nephropathy is the dysfunction of the kidneys and the most common cause of end-stage renal disease in the USA. It is a vascular complication that affects the glomerular capillaries of the kidney and reduces the kidney's filtration ability. Nephropathy is first indicated by the appearance of hyperfiltration and then microalbuminuria. Heavy proteinuria and a progressive decline in renal function precede end- stage renal disease. It is believed that hyperglycemia causes glycosylation of glomerular proteins, which may be responsible for mesangial cell proliferation and matrix expansion and vascular endothelial damage. Typically before any signs of nephropathy appear, retinopathy has usually been diagnosed.
  • nephropathy can attenuate disease progression.
  • aggressive treatment including protein, sodium and phosphorus restriction diet, intensive glycemic control, ACE inhibitors (e.g., captopril) and/or nondihydropyridine calcium channel blockers (diltiazem and verapamil), C-peptide and somatostatin are also used.
  • the treatment regimen for early-stage nephropathy comprising dietary and glycemic restrictions is less effective in practice than in theory due to difficulties associated with patient compliance. Renal transplant is usually recommended to patients with end- stage renal disease due to diabetes. Survival rate at 5 years for patients receiving a transplant is about 60% compared with only 2% for those on dialysis. Renal allograft survival rate is greater than 85% at 2 years.
  • Nephrotic syndrome is a condition characterized by massive edema (fluid accumulation), heavy proteinuria (protein in the urine), hypoalbuminemia (low levels of protein in the blood), and susceptibility to infections. Nephrotic syndrome results from damage to the kidney's glomeruli. Glomeruli are tiny blood vessels that filter waste and excess water from the blood. The damaged glomeruli are characterized by hyperpermeability. Nephrotic syndrome can be caused by glomerulonephritis, diabetes mellitus, or amyloidosis. Presently, prevention of nephrotic syndrome relies on controlling these diseases.
  • ATIII Antithrombin III
  • vascular hyperpermeability has also been found to play a role in pathophysiology of nephrotic edema in human primary glomerulonephritis, such as idiopathic nephrotic syndrome (INS). It is believed that vascular hyperpermeability in nephrotic edema is related to the release of vascular permeability factor and other cytokines by immune cells. See Rostoker et al., Nephron 85:194-200 (2000).
  • Pulmonary hypertension is a rare blood vessel disorder of the lung in which the pressure in the pulmonary artery (the blood vessel that leads from the heart to the lungs) rises above normal levels and may become life threatening. Pulmonary hypertension has been historically chronic and incurable with a poor survival rate. Recent data indicate that the length of survival is continuing to improve, with some patients able to manage the disorder for 15 to 20 years or longer.
  • Pulmonary hypertension is caused by alveolar hypoxia, which results from localized inadequate ventilation of well-perfused alveoli or from a generalized decrease in alveolar ventilation. Treatment of pulmonary hypertension usually involves continuous use of oxygen. Pulmonary vasodilators (e.g., hydralazine, calcium blockers, nitrous oxide, prostacyclin) have not proven effective. Lung transplant is typically recommended to patients who do not respond to therapy.
  • Pulmonary vasodilators e.g., hydralazine, calcium blockers, nitrous oxide, prostacyclin
  • the present invention provides methods and compositions for inhibiting vascular leakage or inappropriate vascular permeability.
  • the methods and compositions are based in part on the finding that doxycycline has a synergistic effect with fumigillol derivatives for the inhibition of vascular permeability. Further, formulations of fumigillol derivatives for oral administration are described; these formulations can be administered orally with doxycycline to treat or prevent conditions characterized by or involving vascular permeability or leakage.
  • a fumagillol derivative is associated with a block copolymer comprising a hydrophobic moiety, wherein the hydrophobic moiety is part of a block copolymer comprising hydrophilic and hydrophobic moieties.
  • the block copolymer comprising hydrophilic and hydrophobic moieties forms a micelle.
  • the present invention relates to a method of inhibiting vascular leakage, the method comprising administering doxycycline and a composition comprising a formulation of a fumagillol derivative that inhibits vascular leakage, the formulation comprising the derivative associated with a block copolymer comprising a hydrophilic polymer moiety and a hydrophobic polymer moiety, the doxycycline and the composition comprising a fumigillol derivative administered in an amount effective to inhibit vascular leakage.
  • the block copolymer is a diblock copolymer.
  • the fumagillol derivative is associated with the hydrophobic moiety of such diblock copolymer.
  • the formulation comprises a micelle comprising said block copolymer associated with said fumagillol derivative.
  • the hydrophobic polymer moiety of said block copolymer is selected from the group consisting of poly(d,L- lactic acid), poly(L-lysine), poly(aspartic acid), poly(caprolactone) (PCL), and poly ⁇ ropylene oxide).
  • a hydrophobic polymer moiety is 1-15 kDa.
  • a hydrophobic polymer moiety is between 1-1OkDa, l-8kDa, l-5kDa, l-3kDa, 3-15kDa, 5-15kDa, 8-15kDa, 10-15kDa, 12-15kDa, 2-12kDa, 4- 1OkDa, 6-8kDa in size.
  • a hydrophobic polymer is approximately 2 kDa.
  • a hydrophobic moiety useful in the fumagillol derivative composition as disclosed herein is a poly(d,L-lactic acid) (PLA) polymer.
  • a hydrophilic polymer moiety useful in a block copolymer of the compositions disclosed herein is a poly(alkylene oxide) polymer.
  • the poly(alkylene oxide) polymer is a polyethene glycol (PEG) polymer, which is also referred to a poly(ethylene oxide) (PEO) or poly(oxythelene) in the art. Methoxy-PEG (mPEG) may be used.
  • a hydrophilic polymer moiety useful in the methods and composition disclosed herein is between 1-15 kDa.
  • a hydrophilic polymer moiety useful in the fumagillol derivative composition as disclosed is between 1-1OkDa, l-8kDa, l-5kDa, l-3kDa, 3-15kDa, 5-15kDa, 8- 15kDa, 10-15kDa, 12-15kDa, 2-12kDa, 4-1OkDa, 6-8kDa in size.
  • a hydrophilic polymer is approximately 2 kDa.
  • the fumagillol derivative composition as disclosed herein comprises a diblock copolymer comprising a PEG- PLA diblock copolymer having hydrophilic PEG and hydrophobic PLA moieties.
  • the fumagillol derivative composition as disclosed herein is formulated for oral administration.
  • a fumagillol derivative useful in the fumagillol derivative composition is the fumagillol derivative ( 3R,4S,5S,6R)-5-methoxy-4-( 2R,3R)-2-methyl-3-(3-methyl-2- butenyl)- oxiranyl)-l-oxaspiro(2,5)oct-6-yl(chloroacetyl) carbamate, also known as 6-0-(N- chloroacetylcarbamoyl) fumagillol or TNP-470.
  • fumagillol derivatives useful in the compositions as disclosed herein include but are not limited to, 6-O-(4- methoxyaniline) acetyl fumagillol; 6-0- (3,4, 5-trimethexyaniline)acetyl fumagillol; 6-O-(4- (N,N- dimethylethoxy) aniline) acetyl fumagillol; 6-O-(cyclopropylamino) acetyl fumagillol; 6- O-(cyclobutylamino)acetyl fumagillol; 4- ((cyclopropylamino)acetyl) oxy-2-(l,2-epoxy-l,5 20 dimethyl-4-hexenyl)-3- methoxy-l-chloromethyl-1 cyclohexanol; and 4- ((cyclobutylamino)acetyl) oxy-2-(l,2-epoxy-l,5 dimethyl-4-hexen
  • both the doxycycline and the formuation of a fumagillol derivative are administered orally.
  • the methods include the step, before the step of administering, of identifying a subject in need of inhibition of vascular leakage.
  • the methods described herein can treat or prevent vascular leakage that occurs in response to VEGF, bFGF, histamine, TNF or thrombin, among other agents.
  • the administering results in a synergistic decrease in vascular leakage relative to a decrease observed with doxycycline or the fumigillol derivative formulation administered alone.
  • a condition involving vascular leakage comprising administering doxycycline and a composition comprising an oral formulation of a fumagillol derivative that inhibits vascular leakage, wherein the fumagillol derivative is associated with a block copolymer comprising a hydrophilic polymer moiety and a hydrophobic polymer moiety, the doxycycline and the composition comprising an oral formulation of a fumagillol derivative administered in amounts effective to treat or prevent the condition involving vascular leakage.
  • the condition involving vascular leakage is selected from diabetes, non- proliferative diabetic retinopathy, diabetic nephropathy, nephrotic syndrome, glomerular vascular leakage, inflammation, Alzheimer's disease, ischemic injury, stroke, myocardial infarction, pulmonary hypertension, pulmonary edema, acute respiratory distress syndrome, sepsis, burn edema, tumor edema, brain tumor edema, IL-2 therapy- associated edema, cancer-associated proteinuria, trauma induced edema, including edema due to battlefield trauma, including, but not limited to pulmonary edema and vascular leak syndrome, and edema associated with allergic reactions.
  • kits for the treatment or prevention of a condition involving vascular leakage comprising doxycycline and a composition comprising an oral formulation of a fumagillol derivative that inhibits vascular leakage, wherein the fumagillol derivative is associated with a block copolymer comprising a hydrophilic polymer moiety and a hydrophobic polymer moiety, the kit further comprising packaging materials therefor.
  • composition comprising doxycycline and a fumagillol derivative that inhibits vascular leakage, wherein the fumagillol derivative is associated with a block copolymer comprising a hydrophilic polymer moiety and a hydrophobic polymer moiety and wherein the composition is formulated for oral administration.
  • kits comprising such a composition and packaging materials therefor.
  • Figure 1 shows NMR spectra for a block copolymer and a block copolymer conjugate with TMP-470. Differences in NMR pattern verify the binding of the ethylendiamine and the TNP470 to the polymer.
  • Figure 2 shows amine levels of mPEG-PLA-en polymer before and after TNP-470 binding.
  • TNBSA reaction was performed as described in the Examples; levels of 450nm absorption indicate the levels of free amines.
  • Figure 3 shows TNP-470 polymersomes' morphology and size.
  • (B) A typical DLS graph showing diameter distribution of TNP-470 polymersomes.
  • FIG. 4 shows inhibition of human umbilical vein epithelial cell (HUVEC) proliferation by TNP-470 polymersomes.
  • A Different concentrations of TNP-470 micelles (comprising between 62.5-100OnM of TNP-470 equivalent) showed a cytostatic effect on HUVECs as measured by WST-I (*p ⁇ 0.0005).
  • B HUVEC proliferation was not affected by polymeric micelles without a TNP-470. No significant differences in proliferation were detected, even with a double concentration of carrier than that of the TNP-470 polymersomes.
  • Figure 5 shows uptake of polymeric micelles labeled by 6-coumarin by HUVECs. Representative images taken after 20min, 4hr and 24hr of incubation with micelles.
  • A Overlay of a Green signal (micelles) and cell nuclei stained with DAPI (blue).
  • B Overlay of green and blue fluorescentic signals and transmission of light, x62 magnitude.
  • FIG. 6 shows the effect of TNP-470 polymersomes on tumor volume and mice weight.
  • A Tumor volume, measured during 14-18 days of daily oral administration of TNP- 470 polymeric micelles to C57/BL mice (*p ⁇ 0.05)
  • B Mice were weighted q.o.d, the results are an average weight of mice in each group.
  • Panel C presents photos that were taken on day 14, one mouse from each indicated group.
  • Figure 7 shows images taken by the Xenogen system, show the absorption of fluorescent polymeric micelles in the GI tract at different times post administration.
  • Figure 8 shows the results of a histological assay for intestinal absorption of orally administered polymeric micelles.
  • Figure 9 shows the effects of orally administered TNP-470 PEG/PLA micelles and doxycycline on tumor volumes of Lewis Lung Carcinoma tumors in mice.
  • Figure 10 shows the effects of TNP-470, doxycycline and the combination of TNP- 470 and doxycycline on tumor weights of Lewis Lung Carcinoma cells.
  • Figure 11 shows the effects of TNP-470, doxycycline and the combination of TNP- 470 and doxycycline on vascular permeability of tumor blood vessels as measured by Evans Blue leakage.
  • Figure 12 shows the effects of TNP-470, doxycycline and the combination of TNP- 470 and doxycycline on glomerular vascular permeability as measured by urinary Evans Blue leakage.
  • FIG. 13 shows the results of a balance beam assay for neurotoxicity of PEG-PLA- TNP-470 in mice. Mice were tested for foot-slip errors on the balance beam. Three groups were tested: mice that were given 1) oral PEG-PLA-TNP-470 (15 mg/kg eg. e.d) by gavage; 2) water by gavage; or 3) free TNP-470 by injection (30 mg/kg q.o.d.). Oral PEG-PLA-TNP-470 did not cause neurological symptoms when compared to water-treated control, whereas the free TNP-470 injections caused balance problems, suggesting adverse neurological side-effects.
  • Figure 14 shows further results regarding total proteinuria and total endostatinuria in tumor- induced and VEGF-induced glomerular vascular permeability.
  • the present invention provides methods and compositions for inhibiting vascular leakage.
  • the methods and compositions are based in part on the finding that doxycycline has a synergistic effect with fumigillol derivatives for the inhibition of vascular permeability. Further, formulations of fumigillol derivatives for oral administration are described; these formulations can be administered orally with doxycycline to treat or prevent conditions characterized by or involving vascular permeability or leakage.
  • the orally administrable fumigillol derivative composition comprises a fumagillol derivative associated with a block copolymer comprising a hydrophilic and a hydrophobic moiety.
  • the block copolymer comprising hydrophilic and hydrophobic moieties forms a micelle.
  • the present invention relates to methods of inhibiting vascular permeability, the methods comprising orally administering a composition comprising a fumagillol derivative formulated as disclosed herein.
  • TNP-470 has a short plasma half-life (a few minutes for the drug and less than 2 hr for the metabolites). It is desirable to administer TNP-470 to patients very often in doses that are limited by neurological toxicities. TNP-470 is also poorly soluble in water (1.9 mg/ml; see U.S. 5,536,623), and as such has low absorption and bioavailability if given orally. Therefore, modification of the formulation of fumagillol derivatives such as TNP-470 for oral administration in an oral dosage form is highly desirable.
  • fumagillol derivatives In order to administer fumagillol derivatives orally one must overcome several issues associated with the chemical properties of fumagillol derivatives such as their poor solubility in water, low absorption and poor bioavailability. Due to these properties, fumagillol derivatives are currently formulated for administration via injections (i.e. intramuscularly and intravenously). As demonstrated herein, the inventors have discovered a composition that formulates fumagillol derivatives such as TNP-470 for alternative routes of administration other than injections, by associating TNP-470 with a diblock copolymer, for example a diblock copolymer comprising PEG-PLA.
  • a diblock copolymer for example a diblock copolymer comprising PEG-PLA.
  • block copolymer- fumagillol derivative conjugates is a highly suitable formulation of fumagillol derivatives for oral administration.
  • the inventors demonstrate that TNP-470 was successfully conjugated to a modified PEG-PLA polymer through its amine, and formed nano-size polymersomes.
  • the inventors also demonstrate, using images taken by TEM, that spherical micelles were formed and size measurement with DLS showed a low range of size distribution around one hundred nanometers.
  • confocal microscopy images of fluorescently — labeled polymersomes the inventors demonstrate rapid uptake by Human Umbilical Vein Endothelial Cells (HUVECs), and when TNP-470 polymersomes were added, a significant inhibition of HUVEC proliferation was shown (as compared to no effect of the carrier itself).
  • mice In-vivo studies performed on mice showed a significant inhibition of subcutaneous Lewis Lung Carcinoma tumors with C57/B1 mice given a daily oral administration of TNP-470 micelles. A dose of 15mg/kg TNP-470 equivalent showed 63% inhibition without any weight loss to the mice. Additional in vivo studies demonstrate that TNP-470 polymersomes synergize with doxycycline to inhibit vascular permeability, both in the kidney and in tumors, and that induced by intradermal VEGF or histamine.
  • conjugating fumagillol derivatives such as TNP-470 to diblock copolymers provides an oral administration formulation and that such a fumagillol derivative-diblock copolymer conjugate, along with doxycycline, is useful to treat or prevent pathologies involving or characterized by vascular leakage or permeability.
  • X is -OH and Y is halogen, or X and Y are linked together to form a oxyrane ring
  • Rl represents hydrogen, hydroxyl, -CN; NO2, - CF3; formyl; C 1 -C 4 thioalkyl, acetamido; acetoxy; C 1 -C 6 alkyl, C 1 -C 4 aminoalkyl, C 1 -C 4 alkylaminoalkyl; C 1 -C 4 dialkylaminoalkyl; C 1 -C 6 alkyloxy, C 1 -C 6 aminoalkyloxy; C 1 -C 4 alkylaminoalkoyy, C 1 -C 4 dialkylaminoalkoxy, amino; C 1 -C 6 alkylamino; C 1 -C 4 dialkylamino; C1-C 4 dialkylamino; C1-C4 hyr
  • Fumagillol derivatives are disclosed in European Patent Application 0354787, 0357061, and 0415294 and Japanese patent application JP-A01-233275 and US Patent 5,290,807, which are incorporated herein in their entirety by reference. Fumagillol derivatives as described herein have, at a minimum, anti- angiogenic activity when tested, for example, in a HUVEC proliferation assay as known in the art and described herein below. It should be understood that the term “fumagillol derivative” refers to the derivatives of fumagillol of Formula I, whereas a “fumagillol derivative block copolymer conjugate” refers to such derivatives conjugated to a block copolymer.
  • angiogenesis refers to the development of blood vessels.
  • angiogenesis refers to the sprouting of new blood vessels from pre-existing blood vessels, characterized by endothelial cell proliferation and migration triggered by certain pathological conditions, such as the growth of solid tumors and metastasis.
  • anti-angiogenesis activity refers to an agent which inhibits or suppresses or reduces the rate of growth or creation of new blood vessels in the body in order to combat disease.
  • a compound or agent with anti-angiogenesis activity as used herein is an agent capable of inhibiting the formation of blood vessels.
  • vascular permeability and “vascular leakage” are used interchangeably herein.
  • a disease associated with vascular permeability for treatment with the present invention includes, but is not limited to, vascular complications of diabetes such as nonproliferative diabetic retinopathy and diabetic nephropathy, nephrotic syndrome, pulmonary hypertension, burn edema, tumor edema, brain tumor edema, IL-2 therapy-associated edema, glomerular vascular leakage, cancer-associated proteinuria and other edema-associated diseases or disorders, such as pulmonary edema and edema associated with delayed-type hypersensitivity allergic reactions and trauma associated edema.
  • diabetes such as nonproliferative diabetic retinopathy and diabetic nephropathy, nephrotic syndrome, pulmonary hypertension, burn edema, tumor edema, brain tumor edema, IL-2 therapy-associated edema, glomerular vascular leakage, cancer-associated proteinuria and other edema-associated diseases or disorders, such as pulmonary edema and edema
  • the term "retains anti-angiogenesis activity" means that a given fumagillol derivative has at least 50% of the anti-angiogenic activity of TNP-470 in a HUVEC assay of angiogenesis as described herein.
  • the method of the invention can be used to prevent the leakage of natural angiogenesis inhibitors from blood vessels.
  • the term "inhibit” or “inhibition” when used in reference to tumor growth or metastasis means the reduction or prevention of tumor growth and /or tumor metastasis in cancers. Inhibition includes slowing the rate of tumor growth and metastasis.
  • the tumor growth rate can be reduced by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150% or more compared to a control, untreated tumor of the same type.
  • Inhibition also means a reduction in the size of the tumor of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more compared to a control, untreated tumor of the same type.
  • the prevention of tumor growth and /or metastasis means no further increase in the size of the tumors from the time of start of treatment administration.
  • Prevention also means status quo of no new metastatic tumors detected (ie. no further spread of cancer) and/or no increased amount of tumor markers detected by methods known in the art.
  • vascular leakage or permeability are considered to be “inhibited” by a compound or treatment as the term is used herein when vascular leakage or permeability is reduced by at least 10% in a given vascular leakage or permeability assay and preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, up to and including complete inhibition, or 100%.
  • the Miles assay which uses intravenous injection of Evans blue dye (which binds to endogenous serum albumin) as a tracer can be used to assay macromolecular leakage from peripheral vessels after intradermal administration of test substances (Miles and Miles, 1952, J. Physiol. 118:684-686; Yano, et al., 2000, Clin. Cancer Res. 6:957-965).
  • nude mice and normal shaved mice are injected i.v. with 200 ⁇ l of 1% Evans blue dye (Sigma Chemical Co., St. Louis, MO). After 10 min, 50 ⁇ l of test agent or control samples is injected i.d. in both left and right flanks as well as at single or dual dorsal sites.
  • 50 ⁇ l of test agent or control samples is injected i.d. in both left and right flanks as well as at single or dual dorsal sites.
  • equally sized (8mm diameter) skin regions surrounding i.d. injection sites are removed 30 min after injection and placed in formamide (1 ml) at room temperature for 5 days, allowing for dye extraction. The A ⁇ io of samples is read, and extents of leakage are calculated by comparison with phosphate-buffered saline-treated controls.
  • Other in vivo assays for vascular leakage are described and reviewed by Ferrero, Methods MoI Med. 2004;98: 191-8, which is incorporated herein by reference
  • angiogenesis-mediated condition refers to diseases or disorders that are dependent on a rich blood supply and blood vessel proliferation for the disease pathological progression (eg. metastatic tumors) or diseases or disorders that are the direct result of aberrant blood vessel proliferation (e.g. diabetic retinopathy and hemangiomas).
  • Examples include abnormal vascular proliferation, ascites formation, psoriasis, age-related macular degeneration, thyroid hyperplasia, preclampsia, rheumatoid arthritis and osteo-arthritis, Alzheimer's disease, obesity, pleura effusion, atherosclerosis, endometriosis, diabetic/other retinopathies, ocular neovascularizations such as neovascular glauocoma and corneal neovascularization.
  • tumor refers to brain tumors including neuroblastoma, medulloblastoma, meningioma and glioblastoma; head and neck cancer, thyroid carcinoma, endocrine tumors, esophageal cancer, small cell and non- small cell lung cancer, colon cancer, rectal cancer, pancreatic cancer, gastric cancer, bladder cancer, hepatic cancer, malignant lymphoma, acute and chronic leukemia, Kaposi's sarcoma, glioma, hemangioma, osteosarcoma, soft tissue sarcoma, malignant melanoma, skin cancer, prostate cancer, breast carcinoma, choriocarcinoma, ovarian cancer, cervical cancer, uterine cancer and mesenchymal tumors among others.
  • tumor means a mass of transformed cells that are characterized, at least in part, by containing angiogenic vasculature.
  • the transformed cells are characterized by neoplastic uncontrolled cell multiplication which is rapid and continues even after the stimuli that initiated the new growth has ceased.
  • the term “tumor” is used broadly to include the tumor parenchymal cells as well as the supporting stroma, including the angiogenic blood vessels that infiltrate the tumor parenchymal cell mass.
  • a tumor generally is a malignant tumor, i.e., a cancer having the ability to metastasize (i.e. a metastatic tumor)
  • a tumor also can be nonmalignant (i.e. non-metastatic tumor).
  • Tumors are hallmarks of cancer, a neoplastic disease the natural course of which is fatal. Cancer cells exhibit the properties of invasion and metastasis and are highly anaplastic.
  • the term "metastases” or "metastatic tumor” refers to a secondary tumor that grows separately elsewhere in the body from the primary tumor and has arisen from detached, transported cells, wherein the primary tumor is a solid tumor.
  • the primary tumor refers to a tumor that originated in the location or organ in which it is present and did not metastasize to that location from another location.
  • a "malignant tumor” is one having the properties of invasion and metastasis and showing a high degree of anaplasia. Anaplasia is the reversion of cells to an immature or a less differentiated form, and it occurs in most malignant tumors.
  • copolymer also known as “heteropolymer” as used herein refers to a polymer derived from two (or more) monomeric species, as opposed to a homopolymer where only one monomer is used. Copolymerization refers to methods used to chemically synthesize a copolymer.
  • block copolymer refers to the polymer comprising more than one subunit (or oligomer) type, wherein the copolymer comprises regions of a polymer comprising one subunit type adjoined to a polymer region comprising a second subunit type, for example the term block copolymer refers to a copolymer comprised of two or more homopolymer subunits linked by covalent bonds. The union of the homopolymer subunits may require an intermediate non-repeating subunit, known as a junction block.
  • Block copolymers are made up of blocks of different polymerized monomers. Block copolymers are interesting because they can "microphase separate" to form periodic nanostructures.
  • Block copolymers are described in further detail in the section "Copolymers” herein below.
  • the term "diblock copolymer” as used herein refers to a block copolymer with two distinct blocks. A block copolymer with three distinct blocks is called a triblock copolymers. It is also possible to have tetrablocks, multiblocks, etc.
  • hydrophilic refers to a molecule or portion of a molecule that is typically charge-polarized and capable of hydrogen bonding, enabling it to dissolve more readily in water than in oil or other hydrophobic solvents. Hydrophilic molecules are also known as polar molecules and are molecules that readily absorb moisture, are hygroscopic, and have strong polar groups that readily interact with water. A “hydrophilic” polymer as the term is used herein, has a solubility in water of at least 100 mg/ml at 25°C.
  • hydrophobic refers molecules tend to be non-polar and prefer other neutral molecules and non-polar solvents. Hydrophobic molecules in water often cluster together. Water on hydrophobic surfaces will exhibit a high contact angle. Examples of hydrophobic molecules include the alkanes, oils, fats, and greasy substances in general. Hydrophobic materials are used for oil removal from water, the management of oil spills, and chemical separation processes to remove non-polar from polar compounds. Hydrophobic molecules are also known as non-polar molecules. Hydrophobic molecules do not readily absorb water or are adversely affected by water, e.g., as a hydrophobic colloid.
  • a "hydrophobic" polymer as the term is used herein has a solubility in water less than 10 mg/ml at 25°C, preferably less than 5 mg/ml, less than 1 mg/ml or lower.
  • hydrophobic drug refers to any organic or inorganic compound or substance having biological or pharmacological activity and adapted or used for a therapeutic purpose having a water solubility less than 10 mg/ml. Fumagillol derivatives tend to be hydrophobic drugs.
  • micelle refers to an arrangement of surfactant molecules (surfactants comprise a non-polar, lipophilic "tail” and a polar, hydrophilic "head”).
  • surfactants comprise a non-polar, lipophilic "tail” and a polar, hydrophilic "head”).
  • a micelle has the arrangement in aqueous solution in which the non-polar tails face inward and the polar heads face outward.
  • Micelles are typically colloid particles formed by an aggregation of small molecules and are usually microscopic particles suspended in some sort of liquid medium, e.g., water, and are between one nanometer and one micrometer in size.
  • a typical micelle in aqueous solution forms an aggregate with the hydrophilic "head” regions in contact with surrounding solvent, sequestering the hydrophobic tail regions in the micelle center.
  • This type of micelle is known as a normal phase micelle (oil-in- water micelle).
  • Inverse micelles have the headgroups at the centre with the tails extending out (water-in-oil micelle).
  • Micelles are approximately spherical in shape. Other phases, including shapes such as ellipsoids, cylinders, and bilayers are also possible.
  • the shape and size of a micelle is a function of the molecular geometry of its surfactant molecules and solution conditions such as surfactant concentration, temperature, pH, and ionic strength.
  • the process of forming micellae is known as micellisation.
  • terapéuticaally effective amount refers to an amount that is sufficient to effect a therapeutically or prophylactically significant reduction in a symptom associated with an angiogenesis-mediated condition or a vascular permeability-related condition when administered to a typical subject who has such a condition.
  • a therapeutically or prophylatically significant reduction in a symptom is, e.g. about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150% or more as compared to a control or non-treated subject.
  • the term "therapeutically effective amount” refers to the amount that is safe and sufficient to prevent or delay the development and further spread of metastases in cancer patients. The amount can also cure or cause the cancer to go into remission, slow the course of cancer progression, slow or inhibit tumor growth, slow or inhibit tumor metastasis, slow or inhibit the establishment of secondary tumors at metastatic sites, or inhibit the formation of new tumor metastasis.
  • treat or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow the development or spread of disease, including cancer and vascular permeability-related diseases or disorders.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already diagnosed with cancer as well as those likely to develop secondary tumors due to metastasis.
  • compositions or formulations are used interchangeably herein and refer to compositions or formulations that usually comprise an excipient, such as a pharmaceutically acceptable carrier that is conventional in the art and that is suitable for administration to mammals, and preferably humans or human cells.
  • excipient such as a pharmaceutically acceptable carrier that is conventional in the art and that is suitable for administration to mammals, and preferably humans or human cells.
  • Such compositions can be specifically formulated for administration via one or more of a number of routes, including but not limited to, oral, ocular and nasal administration and the like. Oral formulations are preferred.
  • polymeric drug delivery composition refers to the combination of drug, and block copolymer.
  • the term “medicament” refers to an agent that promotes the recovery from and/or alleviates a symptom of an angiogenesis-mediated or vascular permeability- mediated condition.
  • pharmaceutically acceptable carrier means any pharmaceutically acceptable means to mix and/or deliver a drug composition to a subject.
  • pharmaceutically acceptable carrier as used herein thus means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and compatible with administration to a subject, for example a human.
  • a diblock copolymer as described herein is a pharmaceutically acceptable carrier as the term is used herein.
  • Other pharmaceutically acceptable carriers can be used in combination with the block copolymer carriers as described herein.
  • polymer solution when used in reference to a block copolymer contained in such a solution, refers to water, i.e. aqueous, based composition having such block copolymer, or particularly to copolymer conjugates as described herein, such as a fumagillol derivative-PLA-PEG conjugate dissolved therein at a functional concentration.
  • Polymer solution includes all free flowing forms of the composition comprising the conjugated copolymers as described herein and water. Polymer solutions act to solubilize the drug in a form that is acceptable for administration at physiologically relevant temperatures (temperatures ⁇ 45°C).
  • aqueous solution includes water without additives, or aqueous solutions containing additives or excipients such as pH buffers, components for tonicity adjustment, antioxidants, preservatives, drug stabilizers, etc., as commonly used in the preparation of pharmaceutical formulations.
  • drug formulation refers to all combinations of drug with polymer, for example polymer solutions that are mixed with drug to form drug solutions, as well as mixtures of undissolved polymer with drug, i.e. polymeric drug delivery compositions, that are subsequently dissolved into an aqueous environment to form a drug solution.
  • administration refers to the presentation of formulations to humans and animals in effective amounts, and includes all routes for dosing or administering drugs, whether self- administered or administered by medical practitioners. Oral administration is preferred.
  • biodegradable as used herein means the block copolymer can chemically break down or degrade within the body to form nontoxic components.
  • the rate of degradation can be the same or different from the rate of drug release.
  • PLA refers to a polymer derived from the condensation of lactic acid or by the ring opening polymerization of lactide.
  • biodegradable polyesters refers to any biodegradable polyesters, which are preferably synthesized from monomers selected from the group consisting of D, L-lactide, D-lactide, L-lactide, D, L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid, ⁇ -caprolactone, ⁇ -hydroxy hexanoic acid, ⁇ -butyrolactone, ⁇ -hydroxy butyric acid, ⁇ -valerolactone, ⁇ -hydroxy valeric acid, hydroxybutyric acids, malic acid, and copolymers thereof.
  • the term "patient” refers to a human in need of the treatment to be administered.
  • the term "subject” and “individual” are used interchangeably herein, and refer to an animal, for example a human, to whom treatment, including prophylactic treatment, with a composition or compositions as described herein, is provided.
  • the term “mammal” is intended to encompass a singular “mammal” and plural “mammals,” and includes, but is not limited: to humans, primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras, food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and bears.
  • the mammal is a human subject.
  • the term “individual”, “subject”, and “patient” are used interchangeably
  • compositions, methods, and respective component(s) thereof are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.
  • consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • Fumagillol derivatives useful in the compositions and methods described herein retain anti-angiogenic activity, i.e., at least 50% of the anti-angiogenic activity of TNP-470, as measured in a HUVEC assay (see below). Numerous fumagillol derivatives meeting this criterion are known in the art. In one embodiment, suitable fumagillol derivatives for use in the compositions and formulations as disclosed herein are described in U. S Patent Application 5,290,807 which is incorporated herein in its entirety by reference.
  • suitable fumagillol derivatives for use in the compositions and formulations as disclosed herein are representative of general Formula II as disclosed in U.S. Patent Nos. 5,166,172; 5,290,807; 5,180,738 and 5,164,410, which are hereby incorporated by reference.
  • fumagillol derivatives for use in the compositions and formulations as disclosed herein are listed in International Patent Application WO03/027104 which is incorporated herein in its entirety by reference.
  • a fumagillol derivative is 6-O-(N-chloroacetylcarbamoyl) fumagillol, also known as TNP-470, a derivative of the fumagillol of Formula III of International Patent Application WO03/027104 shown below.
  • Formula III Formula III:
  • TNP- 470 6-O-(N-chloroacetylcarbamoyl) fumagillol
  • TNP-470 The synthesis of TNP-470 is disclosed in U.S. Patent No. 5,180,738 and 5,290,807 which are hereby incorporated herein in their entirety by reference.
  • Fumagillin has been disclosed to be an angiogenesis inhibitor in U.S. Patent No. 5,135,919 which is incorporated herein in its entirety by reference.
  • various fumagillol derivatives have been disclosed to be angiogenesis inhibitors in U.S. Patent Nos. 5,180,738; 5,164,410; 5,196, 406; 5,166,172; and 5,290,807 which are incorporated herein in their entirety by reference.
  • one fumagillol derivative ( 3R,4S,5S,6R)-5-methoxy-4- ( 2i?,Ji?)-2-methyl-3-(3-methyl-2- butenyl)-oxiranyl)-l-oxaspiro(2,5)oct-6-yl(chloroacetyl) carbamate, also known as 6-O-(N-chloroacetylcarbamoyl) fumagillol or TNP-470 (available from Takeda Chemical Industries, Ltd. of Japan) is a particularly potent anti-angiogenesis compound. Bhargava et al. review TNP-470 in Chapter 26 of Angiogenesis in Health and Disease, G. M. Rubanyi, ed., Marcel/Dekkker: 2000, pp. 387-406.
  • fumagillol derivatives include, for example, but not limited to, O-(3,4-dimethoxycinnamoyl)fumagillol; O-(4-methoxycinnamoyl) fumagillol; O-(3,4,5- trimethoxycinnamoyl)fumagillol; O-(4-Chlorocinnamoyl) fumagillol; 4-(3,4,5- trimethoxycinnamoyl)oxy-2-( 1 ,2-epoxy- 1 ,5-dimethyl-4- hexenyl)-3-methoxy- 1 -chloromethyl- 1-cyclohexanol; O-(4- trifluoromethylcinnamoyl)fumagillol; O-(4-nitrocinnamoyl)fumagillol; O-(3, 4-dimethoxy-6-nitrocinnamoyl)fumagillol; O
  • fumagillol derivatives include derivatives of the formula I, for example, but are not limited to: 1) 6-O-(4-methoxyaniline)acetyl fumagillol; 2) 6-O-(3,4,5- trimethoxyaniline) acetyl fumagillol; 3) 6-O-(2,4- dimethexyaniline) acetyl fumagillol; 4) 6-O- (3,4-dimethoxyaniline)acetyl fumagillol; 5) 6-O-(3,4-dimethoxy-6-nitroaniline)acetyl 5 fumagillol; 6) 6-O-(3,4-dimethexy-6-cyaneaniline)acetyl fumagillol; 7) 6-O-(4- allyloxyaniline) acetyl fumagillol; 8) 6-O-(4-(2-acetoxyethexy)aniline) acetyl fumagillol;
  • fumagillol derivatives useful in the methods and compositions as disclosed herein include, for example, but are not limited to 1) 6-O-(4- methoxyaniline) acetyl fumagillol; 2) 6-0- (3,4, 5-trimethexyaniline)acetyl fumagillol; 3) 6-O- (4-(N,N- dimethylethoxy) aniline) acetyl fumagillol; 4) 6-O-(cyclopropylamino) acetyl fumagillol; 5) 6-O-(cyclobutylamino)acetyl fumagillol; 6) 4- ((cyclopropylamino)acetyl) oxy-2- (l,2-epoxy-l,5 20 dimethyl-4-hexenyl)-3- methoxy-l-chloromethyl-1 cyclohexanol; and 7) 4- ((cyclobutylamino)acetyl) oxy-2- (l,2-ep
  • adjunct treatments with fumagillol derivatives can be performed.
  • use of the compostions as disclosed herein of the fumagillol derivative block copolymer conjugate in conjunction with other agents is encompassed in the present invention for treatment of angiogenesis-related diseases involving abnormally stimulated neovascularization, such as inflammatory diseases (rheumatism and psoriasis, among others), diabetic retinopathy and cancer.
  • a copolymer is a polymer comprising subunits of more than one type, i.e a copolymer can comprise subunits of A and subunits of B etc.
  • a copolymer is an alternating copolymer, for example, having a repeating structure comprising the different types of subunits, for example an alternating copolymer can have the formula: -A-B-A-B-A-B-A-B- A-B-, or -(-A-B-) n -.
  • fumagillol derivatives as disclosed herein are associated with a block copolymer to form a fumagillol derivative-block copolymer conjugate.
  • a block copolymer comprises subunits of more than one type, but instead of an alternating copolymer, a block copolymer comprises at a minimum, a block of subunits of one subunit type followed by a block of subunits of another subunit type.
  • a block copolymer comprising two subunits, for example A and B can have the formula of : -(A-A-) n -(B-B-) n - or -A-A-A-A-A-A-B-B-B-B-B-.
  • the number of the different subunits can be different, for example; A-A-A-A-B-B-B-B.
  • a diblock copolymer is a block copolymer comprising two different blocks of polymer subunits
  • a triblock copolymer is a block copolymer comprising three different blocks of polymer subunits
  • a tretrablock copolymer is a block copolymer comprising four different blocks of copolymer subunits, etc.
  • a block copolymer is a polymer comprising at least a first block comprising a polymer of hydrophobic monomers, and at least a second block comprising a polymer comprised of hydrophilic monomers.
  • a "block" copolymer differs from a non-block copolymer in that a block copolymer comprises blocks of polymer of one type (e.g. a block of hydrophilic monomers) that are joined to a block of polymer of another type (e.g. a block of hydrophobic monomers), as opposed to a non-block copolymer in which the different monomers are not joined together in blocks.
  • the block copolymer would have, e.g., the structure AAAAABBBBB.
  • a non-block copolymer of the same monomer subunits would have, for example, the structure ABAABABBABAABB or the structure ABABABABABAB, for example.
  • the blocks of a block copolymer as the term is used herein will have at least 5 monomers per block (i.e., for a block copolymer of A and B monomers, the A block will be at least 5 A monomers long, and the B block will be at least 5 B monomers long). In some embodiments the homopolymer blocks will be at least 10 monomers long, 15 monomers long, 20 monomers long or more.
  • a block copolymer as the term is used herein can have different block lengths (but each block will be at least 5 monomers long) - differences in block lengths can influence the ability of the block copolymer to form certain structures, e.g., micelles.
  • the monomers making up the hydrophilic block of a block copolymer can comprise different hydrophilic monomer subunits, and similarly, in some embodiments, monomers making up the hydrophobic block of a block copolymer can comprise different hydrophobic monomer subunits.
  • Diblock copolymers can be made using living polymerization techniques, such as atom transfer free radical polymerization (ATRP), reversible addition fragmentation chain transfer (RAFT), ring-opening metathesis polymerization (ROMP), and living cationic or living anionic polymerizations.
  • ATRP atom transfer free radical polymerization
  • RAFT reversible addition fragmentation chain transfer
  • REP ring-opening metathesis polymerization
  • living cationic or living anionic polymerizations such as atom transfer free radical polymerization (ATRP), reversible addition fragmentation chain transfer (RAFT), ring-opening metathesis polymerization (ROMP), and living cationic or living anionic polymerizations.
  • Block copolymers can "microphase separate” to form periodic nanostructures, for example, when one block is hydrophobic and the other hydrophilic. Microphase separation is a situation similar to that of oil and water. Oil and water don't mix together- they macrophase separate. If you have an "oil-like" first block and a “water-like” second block, the block copolymers undergo microphase separation. The blocks want to get as far from each other as possible, but they are covalently bonded, so they're not going to get very far.
  • the “oil” and “water” blocks form nanometer-sized structures, including micelles, which comprise essentially spherical arrangement with the hydrophilic blocks arrange to the outside of the sphere, in contact with an aqueous solution and the hydrophobic blocks forming an inner hydrophobic core.
  • Thermodynamic terms can describe how the different blocks interact.
  • the interaction parameter, "chi” gives an indication of how different, chemically, the two blocks are and whether or not they will microphase separate. Generally, if the product of chi and the molecular weight is large (greater than 10.5), the blocks will microphase separate. Conversely, if the product of chi and the molecular weight is too small (less than 10.5), the different blocks are able to mix, rather than microphase separate.
  • amphiphilic block copolymers are useful in the formulations and compositions of the present invention, for example as effective drug carriers that solubilize hydrophobic drugs into an aqueous environment.
  • amphiphilic block copolymers exhibiting self-association properties are disclosed in EP No. 0397 307 and EP0583955 and EP0552802, which are incorporated herein by reference.
  • useful biodegradable polyesters comprised by the hydrophobic block of copolymers described herein are, for example, biodegradable polyester oligomers or polymers synthesized from monomers selected from, e.g., D, L-lactide, D-lactide, L-lactide, D, L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid, ⁇ -caprolactone, ⁇ -hydroxy hexanoic acid, ⁇ -butyrolactone, ⁇ -hydroxy butyric acid, ⁇ -valerolactone, ⁇ -hydroxy valeric acid, hydroxybutyric acids, malic acid, and copolymers thereof.
  • monomers selected from, e.g., D, L-lactide, D-lactide, L-lactide, D, L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid, ⁇ -caprolactone, ⁇ -hydroxy hexanoic acid, ⁇
  • the biodegradable polyester is synthesized from monomers selected from the group consisting of D, L-lactide, D- lactide, L-lactide, D, L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid, ⁇ - caprolactone, ⁇ -hydroxy hexanoic acid, and copolymers thereof.
  • the biodegradable polyester is synthesized from monomers selected from the group consisting of D, L-lactide, D-lactide, L-lactide, D, L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid, and copolymers thereof.
  • the block copolymers comprise hydrophobic polyesters containing polyester bonds, for example but not limited to polylactic acid(PLA), polyglycolic acid(PGA), poly(D,L-lactic-co-glycolic acid)(PLGA), poly(caprolactone), poly(valerolactone), poly(hydroxybutyrate) and poly(hydroxy valerate).
  • the block copolymers comprise hydrophobic polymers, for example of said block copolymer is selected from the group consisting of poly(d,L-lactic acid), poly(L-lysine), poly(aspartic acid), poly(caprolactone) (PCL), poly(propylene oxide).
  • a hydrophobic polymer moiety is 1-15 kDa.
  • a hydrophobic polymer moiety is between 0.5-1OkDa, l-8kDa, l-5kDa, l-3kDa, 3-15kDa, 5-15kDa, 8-15kDa, 10- 15kDa, 12-15kDa, 2-12kDa, 4-1OkDa, 6-8kDa in size.
  • a hydrophobic polymer is approximately 2 kDa, for example 1.5kDa, 2kDa, or 2.5kDa.
  • a hydrophobic moiety useful in the composition as disclosed herein is a poly(d,L- lactic acid) (PLA) polymer, for example a poly(d,L-lactic acid) (PLA) polymer of IkDa (1000Da).
  • the copolymers comprise hydrophilic polymers, for example but not limited to polyethene glycol (PEG) polymer (which is also referred to a poly(ethylene oxide) (PEO) or poly(oxythelene) in the art).
  • a hydrophilic polymer is between 1-15 kDa.
  • a hydrophilic polymer moiety useful in the composition as disclosed is between 1-1OkDa, l-8kDa, l-5kDa, l-3kDa, 3-15kDa, 5-15kDa, 8-15kDa, 10- 15kDa, 12-15kDa, 2-12kDa, 4-1OkDa, 6-8kDa in size.
  • a hydrophilic polymer is approximately 2 kDa, for example 1.5kDa, 2kDa, or 2.5kDa.
  • a hydrophilic polymer is a poly(ethylene glycol) (PEG) polymer, for example a polyethylene glycol) (PEG) polymer of 2kDa (2000Da).
  • the copolymers comprise a diblock copolymer comprising a PEG-PLA diblock copolymer, where block copolymer comprises blocks of the hydrophilic PEG monomers and blocks of the hydrophobic monomer PLA.
  • hydrophilic blocks of a copolymer can be coupled to the hydrophobic blocks by covalent bonds, for example by ester or urethane links and the like.
  • Condensation polymerization and ring opening polymerization procedures may be utilized as may the coupling of a monofunctional hydrophilic block to either end of a difunctional hydrophobic block in the presence of coupling agents such as isocyanates.
  • coupling reactions may follow activation of functional groups with activating agents, such as carbonyl diimidazole, succinic anhydride, N-hydroxy succinimide and p-nitrophenyl chloroformate and the like.
  • hydrophilic blocks of a copolymer can comprise PEG or derivatized PEG monomers of an appropriate molecular weight.
  • PEG has particularly favorable biocompatibility, nontoxic properties, hydrophilicity, solubilization properties, and rapid clearance from a patient's body.
  • the hydrophobic blocks of a copolymer should also comprise biodegradable polyester momomers that are biodegradable and biocompatible.
  • biodegradable polyester momomers that are biodegradable and biocompatible.
  • the in vitro and in vivo degradation of hydrophobic, biodegradable polyester blocks of a copolymer are well understood and the degradation products are readily metabolized and/or eliminated from the patient's body.
  • Fumagillol derivatives such as TNP-470
  • block copolymers as disclosed herein.
  • One advantage of using the block copolymers as disclosed herein is that the fumagillol derivatives such as TNP-470 that have limited solubility or dispersibility in an aqueous or hydrophilic environment has enhanced solubility and/or dispersibility and can be administered via oral administration.
  • Step 1 Preparation of Block Copolymer.
  • a block copolymer containing a hydrophobic part having hydroxyl group at one end and a hydrophilic part at the other end is prepared by copolymerization of a biodegradable polyester polymer and a polyethylene glycol(PEG) polymer in the presence of stannous octate as a catalyst: The copolymerization is performed at 160-200. quadrature for 2-6 hours under a vacuum condition.
  • the polyester polymer includes polylactic acid(PLA), polyglycolic acid(PGA), poly(D,L-lactic-co-glycolic acid)(PLGA), poly(caprolactone), poly(valerolactone), poly(hydroxy butyrate) or poly(hydroxy valerate), and in some embodiments PLA and methoxypolyethyleneglycol is used as the polyethyleneglycol polymer.
  • Step 2 Binding of Linker by an Activation of Functional Groups of Block Copolymer.
  • the block copolymer is dissolved in an organic solvent and reacted with a linker at room temperature in the presence of pyridine and nitrogen:
  • the organic solvent includes, but without limitation, methylenechloride, and the linker includes p-nitrophenyl chloroformate, carbonyldiimidazole(CDI), N,N'-disuccinimidyl carbonate(DSC), or a mixture of these compounds, preferably p-nitrophenyl chloroformate.
  • the reaction is carried out for 2 to 6 hours, with a molar ratio of block copolyme ⁇ linke ⁇ pyridine ranging from 1:2:2 to 1:2:6.
  • Step 3 Preparation of a Conjugate of Drug and Biodegradable Polymer.
  • the linker- bound block copolymer is conjugated to a drug by covalent linkage to obtain a micelle monomer of a conjugate of drug and block copolymer, where the block copolymer obtained by reacting with hydrazine may be used:
  • the block copolymer reacted with hydrazine forms a micelle monomer by binding the linker to ketone group of a fumagillol derivative, while the block copolymer without hydrazine reaction forms a micelle monomer by binding the linker to amine group of a fumagillol derivative.
  • the fumagillol derivative is TNP-470.
  • anti-cancer agents such as, but without limitation, doxorubicin, adriamycin, cisplatin, taxol and 5-fluorouracil.
  • Step 4 Preparation of Sustained Release Micelle.
  • the micelle monomers prepared in Step 3 are dispersed in an aqueous solution to prepare sustained release micelles.
  • micelles are formed spontaneously by thermodynamic equilibrium.
  • Sustained release micelles thus prepared release a drug by way of hydrolysis and enzymatic action in vivo, and the released drug exerts the same effect as free drug does.
  • a fumagillol derivative for example TNP-470 is associated with a block copolymer.
  • association means that one entity is in physical association or contact with another.
  • a fumagillol derivative "associated with" a block copolymer can be either covalently or non-covalently joined to the block copolymer.
  • the association can be mediated by a linker moiety, particularly where the association is covalent.
  • association or “interaction” or “associated with” are used interchangeably herein and as used in reference to the association or interaction of a fumagillol derivative, e.g., TNP- 470 with a block copolymer, refers to any association between the fumagillol derivative with the block copolymer, for example a diblock copolymer comprising a hydrophilic polymer moiety and a hydrophobic polymer moiety, either by a direct linkage or an indirect linkage.
  • An indirect linkage includes an association between fumagillol derivative with the block copolymer, wherein said fumagillol derivative and block copolymer are attached via a linker moiety, e.g., they are not directly linked.
  • Linker moieties include, but are not limited to, chemical linker moieties.
  • a linker between the fumagillol derivative and the copolymer is formed by reacting the polymer and a linker selected e.g., from the group consisting of p-nitrophenyl chloroformate, carbonyldiimidazole(CDI), N,N'-disuccinimidyl carbonate(DSC), cis-aconitic anhydride, and a mixture of these compounds.
  • a linker selected e.g., from the group consisting of p-nitrophenyl chloroformate, carbonyldiimidazole(CDI), N,N'-disuccinimidyl carbonate(DSC), cis-aconitic anhydride, and a mixture of these compounds.
  • a direct linkage includes any linkage wherein a linker moiety is not required.
  • a direct linkage includes a chemical or a physical interaction wherein the two moieties, i.e. the fumagillol derivative and the block copolymer interact such that they are attracted to each other.
  • Examples of direct interactions include covalent interactions, non- covalent interactions, hydrophobic/hydrophilic, ionic (e.g., electrostatic, coulombic attraction, ion-dipole, charge-transfer), Van der Waals, or hydrogen bonding, and chemical bonding, including the formation of a covalent bond.
  • a fumagillol derivative such as TNP-470 and the block copolymer are not linked via a linker, e.g., they are directly linked.
  • fumagillol derivative, such as TNP-470 and the block copolymer are electrostatically associated with each other.
  • Doxycycline is a synthetic broad spectrum antibiotic derived from tetracycline. Doxycycline is active against a variety of bacteria, including Haemophilus influenzae, Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydia trachomatis, Neisseria gonorrhea, Rickettsia rickettsii, Bacillus anthracis (Anthrax), and Borrelia bergdorferi (infectious agent of Lyme disease), among others. Doxycycline kills bacteria by inhibiting protein synthesis by reversibly binding to the 30S subunit of bacterial ribosomes at the A site, thereby preventing the attachment of amionoacyl-tRNA.
  • Doxycycline is widely commercially available, and is sold under a variety of trade names, including Vibramycin (Pfizer), Doryx and VibroxTM, among others. Doxycycline is also used in the experimental or research setting to regulate transgenes under the control of a positive or negative tetracycline-responsive promoter element (the so-called “Tet-on” and “Tet-off ' systems). It should be understood that other tetracycline analogues or modified forms of doxycycline that inhibit vascular leakage can also be used in combination with fumagillol derivative compositions as described herein for the treatment of diseases or disorders involving or characterized by vascular permeability, including, but not limited to cancer. One of skill in the art can readily assess whether a given doxycycline or tetracycline analogue synergizes with a fumagillol derivative using assays such as those described and demonstrated herein.
  • compositions and administration are provided.
  • the fumagillol derivative in the formulations and compositions as disclosed herein is particularly useful in methods of treating angiogenesis-mediated and vascular permeability- related conditions in a mammal, for example a human.
  • Angiogenesis-mediated conditions herein also referred to as an "angiogenesis-dependent disease or disorder” can be selected from a group consisting of cancer, ascites formation, psoriasis, age-related macular degeneration, thyroid hyperplasia, preclampsia, rheumatoid arthritis and osteoarthritis, Alzheimer's disease, obesity, pleura effusion, atherosclerosis, endometriosis, diabetic/other retinopathies, neovascular glauocoma, age-related macular degeneration, hemangiomas, and corneal neovascularization.
  • an angiogenesis-mediated condition is cancer, where the rapidly dividing neoplastic cancer cells require an efficient blood supply to sustain their continual growth of the tumor.
  • cancer refers to any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites and also refers to the pathological condition characterized by such malignant neoplastic growths.
  • the blood vessels provide conduits to metastasize and spread elsewhere in the body. Upon arrival at the metastatic site, the cancer cells then work on establishing a new blood supply network.
  • Administration of fumagillol derivatives in the formulations and compositions as disclosed herein are useful to inhibit angiogenesis at the primary tumor site and secondary tumor site, embodiments of the invention serve to prevent and limit the progression of the disease. Any solid tumor that requires an efficient blood supply to keep growing is a candidate target.
  • candidates for the treatment described herein include carcinomas and sarcomas found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon/rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal marrow, tailbone, testicles, thyroid and uterus.
  • carcinomas include papilloma/carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma/adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma, lymphoma/leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinomas, basal cell carcinoma and sinonasal undifferentiated carcinoma.
  • sarcomas include soft tissue sarcoma such as alveolar soft part sarcoma, angiosarcoma, dermatofibro sarcoma, desmoid tumor, desmoplastic small round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangio sarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and chondrosarcoma.
  • an angiogenesis-mediated condition is age-related macular degeneration.
  • VEGF contributes to abnormal blood vessel growth from the choroid layer of the eye into the retina, similar to what occurs during the wet or neovascular form of age-related macular degeneration.
  • Macular degeneration often called AMD or ARMD (age-related macular degeneration)
  • AMD age-related macular degeneration
  • New blood vessels grow (neovascularization) beneath the retina and leak blood and fluid. This leakage causes permanent damage to light-sensitive retinal cells, which die off and create blind spots in central vision or the macula.
  • an angiogenesis-mediated condition is diabetic retinopathy- abnormal blood vessel growth associated with diabetic eye diseases.
  • diabetic retinopathy and retinopathy of prematurity (ROP) VEGF is released which promotes blood vessel formation. Released by the retina (light-sensitive tissue in back of the eye) when normal blood vessels are damaged by tiny blood clots due to diabetes, VEGF turns on its receptor, igniting a chain reaction that culminates in new blood vessel growth.
  • the backup blood vessels are faulty; they leak, bleed and encourage scar tissue that detaches the retina, resulting in severe loss of vision.
  • the subject in need of treatment can be a mammal, such as a dog or a cat, preferably a human.
  • an angiogenesis-mediated condition is rheumatoid arthritis.
  • Rheumatoid arthritis is characterized by synovial tissue swelling, leucocyte ingress and angiogenesis, or new blood vessel growth. The disease is thought to occur as an immunological response to an as yet unidentified antigen.
  • the expansion of the synovial lining of joints in rheumatoid arthritis (RA) and the subsequent invasion by the pannus of underlying cartilage and bone necessitate an increase in the vascular supply to the synovium, to cope with the increased requirement for oxygen and nutrients.
  • Angiogenesis is now recognised as a key event in the formation and maintenance of the pannus in RA (Paleolog, E. M., 2002).
  • a mononuclear infiltrate characterizes the synovial tissue along with a luxuriant vasculature.
  • Angiogenesis is integral to formation of the inflammatory pannus and without angiogenesis, leukocyte ingress could not occur (Koch, A. E., 2000). Disruption of the formation of new blood vessels would not only prevent delivery of nutrients to the inflammatory site, it could also reduce joint swelling due to the additional activity of VEGF, a potent proangiogenic factor in RA, as a vascular permeability factor.
  • an angiogenesis-mediated condition is Alzheimer's disease.
  • AD Alzheimer's disease
  • AD is the most common cause of dementia worldwide.
  • AD is characterized by an excessive cerebral amyloid deposition leading to degeneration of neurons and eventually to dementia.
  • the exact cause of AD is still unknown. It has been shown by epidemiological studies that long-term use of non-steroidal anti-inflammatory drugs, statins, histamine H2-receptor blockers, or calcium-channel blockers, all of which are cardiovascular drugs with an anti- angiogenic effects, seem to prevent Alzheimer's disease and/or influence the outcome of AD patients. Therefore, it has been speculated that in AD angiogenesis in the brain vasculature may play an important role in AD.
  • the brain endothelium secretes the precursor substrate for the beta-amyloid plaque and a neurotoxic peptide that selectively kills cortical neurons.
  • amyloid deposition in the vasculature leads to endothelial cell apoptosis and endothelial cell activation which leads to neovascularization.
  • Vessel formation could be blocked by the VEGF antagonist SU 4312 as well as by statins, indicating that anti-angiogenesis strategies can interfere with endothelial cell activation in AD (Schultheiss C, el. al., 2006; Grammas P., et. al., 1999) and can be used for preventing and/or treating AD.
  • an angiogenesis-mediated condition is obesity. It has been shown that the fumagillol derivative TNP-470 was able to prevent diet-induced and genetic obesity in mice (Ebba Brakenhielm et. al., Circulation Research, 2004;94:1579). TNP-470 reduced vascularity in the adipose tissue, thereby inhibiting the rate of growth of the adipose tissue and obesity development.
  • the angiogenesis-dependent disease or disorder is endometriosis. Excessive endometrial angiogenesis is proposed as an important mechanism in the pathogenesis of endometriosis (Healy, DL., et. al., 1998).
  • the endometrium of patients with endometriosis shows enhanced endothelial cell proliferation. Moreover there is an elevated expression of the cell adhesion molecule integrin vB3 in more blood vessels in the endometrium of women with endometriosis when compared with normal women. Strategies that inhibit angiogenesis can be used to treat endometriosis.
  • the method of treating an angiogenesis-mediated condition wherein the angiogenesis-mediated condition is cancer
  • the method as disclosed herein is applicable to all carcinomas and sarcomas.
  • the cancer is selected from the group consisting of papilloma/carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma/adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma, lymphoma/leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinomas, basal cell carcinoma, sinonasal undifferentiated carcinoma, soft tissue sarcoma such as alveolar soft part sarcoma, angiosarcoma, dermatofibro sarcoma
  • the fumagillol derivative in the formulations and compositions as disclosed herein is particularly useful in methods of inhibiting angiogenesis at a site of tumorigenesis in a mammal.
  • the fumagillol derivative in the formulations and compositions as disclosed herein administered at such sites prevents or inhibits blood vessel formation at the site thereby inhibiting the development and growth of the tumor.
  • Tumors which may be prevented or inhibited by preventing or inhibiting angiogenesis with the conjugate include but are not limited to melanoma, adenocarcinoma, sarcomas, thymoma, lymphoma, lung tumors, liver tumors, colon tumors, kidney tumors, non-Hodgkins lymphoma, Hodgkins lymphoma, leukemias, uterine tumors, breast tumors, prostate tumors, renal tumors, ovarian tumors, pancreatic tumors, brain tumors, testicular tumors, bone tumors, muscle tumors, tumors of the placenta, gastric tumors, metastases and the like.
  • compositions as disclosed herein comprising a fumagillol derivative can inhibit proliferation of blood vessel endothelial cells, thus having anti- angiogenesis effect or inhibition of angiogenesis. Accordingly, the compositions as disclosed herein comprising fumagillol derivatives can be used in methods for treating angiogenesis- mediated conditions, such as inhibiting growth and metastasis of cancer as well as treating other various diseases where inappropriate angiogenesis occurs or proliferation of blood vessel endothelial cells occur, for example but not limited to, inflammatory diseases, diabetic retinopathy, rheumatoid arthritis, psoriasis.
  • compositions comprising fumagillol derivatives as disclosed herein can be used as a cancer metastasis inhibitor or therapeutic agent against cancer, inflammatory diseases, diabetic retinopathy, rheumatoid arthritis, psoriasis and other retinopathies such as retinopathy of prematurity.
  • Doxycycline can increase the effectiveness of treatment with fumagillol derivatives for angiogenesis-related diseases or disorders.
  • the combination of doxycycline and fumagillol-derivative compositions as described herein also inhibits vascular permeability in a synergistic manner.
  • the combination of doxycycline and oral fumagillol-derivatives as described herein can treat or prevent diseases, disorders or conditions involving or characterized by vascular permeability.
  • Such diseases, disorders or conditions include, but are not limited to diabetes, non- proliferative diabetic retinopathy, diabetic nephropathy, nephrotic syndrome, glomerular vascular leakage, inflammation, Alzheimer' s disease, ischemic injury, stroke, myocardial infarction, pulmonary hypertension, pulmonary edema, acute respiratory distress syndrome, sepsis, burn edema, tumor edema, brain tumor edema, IL-2 therapy-associated edema, cancer-associated proteinuria, trauma associated edema and edema associated with allergic reactions.
  • compositions as described herein can be varied so as to obtain an amount of the active compound(s) which is effective to achieve the desired therapeutic response for a particular subject or patient.
  • the selected dosage level will depend upon the activity of the particular fumagillol derivative, the type of administration composition (i.e. tablet versus liquid oral administration), the severity of the condition being treated and the condition and prior medical history of the patient being treated.
  • the phrase "therapeutically effective amount" of a compound, e.g., a fumagillol derivative as described herein means a sufficient amount of the compound to treat disorders, at a reasonable benefit/risk ratio applicable to any medical treatment.
  • compositions and formulations as disclosed herein will be decided by the attending physician within the scope of sound medical judgment.
  • a "therapeutically effective amount" as the term is used herein need not eradicate a tumor or neoplasm. Rather, a therapeutically effective amount will slow the growth of a tumor or neoplasm relative to a rate without the therapeutic agent, for example the composition as disclosed herein comprising a fumagillol derivative-block copolymer conjugate. Thus, where tumor treatment is involved, it is preferred, but not required that the therapeutic agent actually kill the tumor.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific fumagillol derivative compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the compositions and formulations as disclosed herein which are employed; and like factors well known in the medical arts.
  • Efficacy of treatment can be judged by an ordinarily skilled practitioner. As disclosed in the Examples, efficacy can be assessed in animal models for tumor growth and diseases or disorders, involving or characterized by vascular permeability.
  • efficacy of treatment is evidenced when a reduction in a symptom of the cancer, for example a reduction in the size of the tumor or a slowing or cessation of the rate of growth of the tumor occurs earlier in treated, versus untreated animals.
  • efficacy in treatment of a disease or disorder involving or characterized by vascular permeability is evidenced by a reduction of at least 10% in an indicator of vascular permeability, e.g., edema, or by a secondary indicator such as a slowing or cessation of tumor growth.
  • an indicator of vascular permeability e.g., edema
  • secondary indicator such as a slowing or cessation of tumor growth.
  • the amount of each component to be administered also depends upon the frequency of administration, such as whether administration is once a day, twice a day, 3 times a day or 4 times a day, once a week; or several times a week, for example 2 or 3, or 4 times a week.
  • TNP-470 administration will be described in greater detail as a representative example of the administration procedures for all fumagillol derivatives in general.
  • TNP-470 6-O-(N- chloroacetylcarbamoy ⁇ fumagillol (TNP-470)
  • the following information may serve as a general guideline for administration.
  • the fumagillol-derivative formulations and compositions as disclosed herein are administered from once a day to several times a day, for example 2 times a day, three times a day, or four times a day.
  • the formulations and compositions as disclosed herein can be administered, for example three to five times a week, if it is to be plurally administered in a given week.
  • a suitable dose of the fumagillol derivative in the formulations and compositions as disclosed herein for a subject in need of treatment can be used according to conventionally used dose ranges of about 1 mg to about 2000 mg TNP-470 equivalent per kilogram of body weight.
  • conventional doses of fumagillol derivatives are about 0.1mg/kg to 40mg/kg body weight, preferably about 0.5mg/kg to 20 mg/kg body weight as disclosed in U.S. Patent 5,290,807.
  • a dose below the threshold used for chemotherapy can be used.
  • a suitable dose could be less than the conventionally used chemotherapeutic dose, for example, dose ranges of about l ⁇ g to lmg or O.l ⁇ g to 1 mg, or lmg to lOmg TNP-470 per kilogram of body weight can be used.
  • Reduced dosages of TNP-470, or other fumagillol derivatives can be used particularly where the fumagillol derivative is administered with doxycycline, which has synergistic effects with such derivatives in the treatment of tumors and diseases or conditions involving or characterized by vascular permeability. That is, the synergism between fumagillol derivatives and doxycycline permits the reduction of the dose of fumagillol derivative if so desired.
  • Dosages of doxycycline can be varied by the skilled clinician to achieve the desired degree of inhibition of vascular permeability or the desired therapeutic effect in combination with an oral fumagillol derivative composition.
  • Dosages can range, for example, from antimicrobial doses as large as 500 mg/day in adult humans, to sub-antimicrobial doses, e.g., 100-50 mg or less per day.
  • doses of doxycycline effective to treat diseases or disorders involving vascular permeability can be adjusted by the skilled clinician and range from, for example, 500 mg/day, 400 mg/day, 300 mg/day, 200 mg/day, 150 mg/day, 100 mg/day, 50 mg/day, 25 mg/day, 15 mg/day or less.
  • Doxycycline can be administered once a day or more frequently, if necessary, e.g., 2x/day, 3x/day, 4x per day or more. It can be convenient to co-ordinate the dosage interval of fumagillol derivative composition with that of doxycycline, such that both are administered together, but this is not necessary, nor should it be controlling if better results are seen with differing dose intervals.
  • TNP-470 if TNP-470 is administered once a week, it can be administered in an amount of from about 20 to about 200 mg/m /week; preferably in an amount of from about 40 to about 180 mg/m 2 /week; and most preferably in an amount of from about 135 to about 175 mg/m 2 /week. In some embodiments, if TNP-470 is administered daily, it may be administered in an amount of from about 1 to about 10 mg/m 2 /day; for example in an amount of from about 1.25 to about 5 mg/m 2 /day; or in an amount of from about 1 to about 3 mg/m 2 /day. For continuous administration, the component is usually administered for at least five consecutive days of the week.
  • the effective amount of a composition as disclosed herein comprising a fumagillol derivative can be determined using an anti-angiogenesis or vascular permeability assay as disclosed herein, and in some embodiments, the effective amount is less than the amount used as the conventionally effective dose.
  • compositions and formulations as disclosed herein are oral administration.
  • Solid dosage forms for oral administration include, for example but not limited to capsules, tablets, pills, powders and granules.
  • the compositions as disclosed herein may be mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as
  • compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the active components can also be in microencapsulated form, if appropriate, with one or more of the above- mentioned excipients.
  • the compound selected can be mixed with solid, powdered ingredients, such as lactose, saccharose, sorbitol, mannitol, starch, arnylopectin, cellulose derivatives, gelatin, or another suitable ingredient, as well as with disintegrating agents and lubricating agents such as magnesium stearate, calcium stearate, sodium stearyl fumarate and polyethylene glycol waxes.
  • solid, powdered ingredients such as lactose, saccharose, sorbitol, mannitol, starch, arnylopectin, cellulose derivatives, gelatin, or another suitable ingredient, as well as with disintegrating agents and lubricating agents such as magnesium stearate, calcium stearate, sodium stearyl fumarate and polyethylene glycol waxes.
  • disintegrating agents and lubricating agents such as magnesium stearate, calcium stearate, sodium stearyl fumarate and polyethylene glycol waxes.
  • compositions for topical e.g., oral mucosa, respiratory mucosa
  • oral administration can form solutions, suspensions, sustained-release formulations, or oral rinses.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and preservatives For examples of carriers, stabilizers and adjuvants, see University of the Sciences in Philadelphia (2005) Remington: The Science and Practice of Pharmacy with Facts and Comparisons, 21st Ed.
  • adjunct treatments To enhance the activity of anti-angiogenesis or anti-vascular permability treatments, use of adjunct treatments is contemplated.
  • fumagillin and fumagillol derivatives such as TNP-470 have been tested in conjunction with treatment with various other drugs to enhance efficacy for treatment of diseases induced by abnormally stimulated neovascularization, such as inflammatory diseases (rheumatism and psoriasis among others), diabetic retinopathy and cancer.
  • Soft gelatin capsules can be prepared with capsules containing a mixture of the active compound or compounds of the invention in vegetable oil, fat, or other suitable vehicle for soft gelatin capsules. Hard gelatin capsules can be prepared to contain the active compound.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active components, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents that are compatible with the maintenance of a micelle of a diblock copolymer as described herein. Liquid preparations for oral administration can also be prepared in the form of syrups or suspensions, e.g.
  • solutions or suspensions containing from 0.2% to 20% by weight of the active ingredients and the remainder consisting of sugar or sugar alcohols and a mixture of ethanol, water, glycerol, propylene glycol and polyethylene glycol provided that such solvent is compatible with maintaining the micelle form.
  • liquid preparations can contain coloring agents, flavoring agents, saccharin and carboxymethyl cellulose or other thickening agents.
  • Liquid preparations for oral administration can also be prepared in the form of a dry powder to be reconstituted with a suitable solvent prior to use. Oral formulations of fumagillol derivatives as described herein are quite stable when dried, making them well suited for the preparation of dry powder formulations that can be stored at, e.g., room temperature, without losing activity.
  • Such formulations can be re-constituted prior to use, or, alternatively, taken orally in dry form.
  • Preparations including dried oral fumagillol derivatives as described herein, mixed with an appropriate amount of dry doxycycline as described herein can provide an even more convenient dosage form that can be stored dry until use.
  • the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents
  • compositions and formulations as disclosed herein can be also administered via rectal or vaginal administration.
  • the compositions and formulations as disclosed herein can be in the form of suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active component.
  • Dosage units for rectal or vaginal administration can be prepared (i) in the form of suppositories which contain the active substance mixed with a neutral fat base; (ii) in the form of a gelatin rectal capsule which contains the active substance in a mixture with a vegetable oil, paraffin oil or other suitable vehicle for gelatin rectal capsules; (iii) in the form of a ready-made micro enema; or (iv) in the form of a dry micro enema formulation to be reconstituted in a suitable solvent just prior to administration.
  • the fumagillol-copolymer conjugates and doxycycline described herein can be used or administered in admixture or in combination with a gastric acid secretion-inhibitor and/or an antacid.
  • Gastric acid secretion inhibitors include, for example H 2 blockers (e.g. famotidine, cimetidine, ranitidine hydrochloride, etc.) and proton pump inhibitors (e.g. lansoprazole, omeprazole, etc.).
  • H 2 blockers e.g. famotidine, cimetidine, ranitidine hydrochloride, etc.
  • proton pump inhibitors e.g. lansoprazole, omeprazole, etc.
  • compounds which elevate the intragastric pH level such as magnesium carbonate, sodium hydrogen carbonate, magnesium hydroxide, magnesium oxide and magnesium hydroxide can be employed.
  • the oral dosage forms of the compositions and formulations as disclosed herein can be administered after the intragastric pH has been increased to alleviate the influence of gastric acid by the administration of a gastric acid secretion inhibitor and/or antacid.
  • compositions and formulations as disclosed herein can be in a form of enteric- coated preparation for oral administration comprising a fumagillol or fumagillol derivative block copolymer conjugate.
  • a fumagillol or fumagillol derivative containing core for coating with an enteric coating film can be prepared using an oleaginous base or by other known formulation methods without using an oleaginous base.
  • the compositions and formulations as disclosed herein in the form of the drug-containing core for coating with a coating agent may be, for example, tablets, pills and granules.
  • the excipient contained in the core is exemplified by saccharides, such as sucrose, lactose, mannitol and glucose, starch, crystalline cellulose and calcium phosphate.
  • Useful binders include polyvinyl alcohol, hydroxypropyl cellulose, macrogol, Pluronic F-68, gum arabic, gelatin and starch.
  • Useful disintegrants include carboxymethyl cellulose calcium (ECG505), crosslinked carboxymethylcellulose sodium (Ac-Di-SoI), polyvinylpyrrolidone and low- substituted hydroxypropyl cellulose (L-HPC).
  • Useful lubricants and antiflocculants include talc and magnesium stearate.
  • an enteric coating agent can be an enteric polymer which is substantially insoluble in the acidic pH and is at least partially soluble at weaker acidic pH through the basic pH range.
  • the range of acidic pH is about 0.5 to about 4.5, preferably about 1.0 to about 2.0.
  • the range of weaker acidic pH through basic pH is about 5.0 to about 9.0, preferably about 6.0 to about 7.5.
  • cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethyl acetate succinate (Shin-Etsu Chemicals), methacrylic copolymers (Rhon- Pharma, Eudragit® L-30D-55, L100-55, LlOO, SlOO, etc.), etc. can be mentioned as examples of the enteric coating agent. These materials are effective in terms of stability, even if they are directly used as enteric compositions.
  • micelles for example, spherical microparticles having particle diameters of about 0.01 ⁇ m to about lOOO ⁇ m are generated, or more preferably about 0.01 to about 5 ⁇ m (about IOnm to about 5,000 nm). This formation can be performed by the methods as disclosed in the Examples or as disclosed in Japanese Patent Application JP-A- 223533/1991.
  • micelles between 50 and 500nm are useful in the compositions as disclosed herein, for example about 50-100nm, 100-150nm, 150-200nm, 200-250nm, 250- 300nm, 300-350nm, 350-400nm, 450-500nm.
  • the micelles are about 130nm as disclosed in the examples.
  • the concentration or content of the fumagillol derivative in the composition administered to a subject can be appropriately selected according to the physicochemical properties of the composition.
  • the concentration can be about 0.0005 to about 30% (w/v) and preferably about 0.005 to about 25% (w/v).
  • the content is about 0.01 to about 90% (w/w) and preferably about 0.1 to about 50% (w/w).
  • additives such as a preservative (e.g. benzyl alcohol, ethyl alcohol, benzalkonium chloride, phenol, chlorobutanol, etc.), an antioxidant (e.g. butylhydroxyanisole, propyl gallate, ascorbyl palmitate, alpha- tocopherol, etc.), and a thickener (e.g. lecithin, hydroxypropylcellulose, aluminum stearate, etc.) can be used in the compositions and formulations as disclosed herein.
  • a preservative e.g. benzyl alcohol, ethyl alcohol, benzalkonium chloride, phenol, chlorobutanol, etc.
  • an antioxidant e.g. butylhydroxyanisole, propyl gallate, ascorbyl palmitate, alpha- tocopherol, etc.
  • a thickener e.g. lecithin, hydroxypropylcellulose, aluminum stearate, etc.
  • diblock copolymer conjugates as described generally need no further emulsifiers. Nonetheless, if necessary, one can use an additional emulsifier with the compositions and formulations as disclosed herein.
  • emulsifiers that might be used include pharmaceutically acceptable phospholipids and nonionic surfactants.
  • the emulsifiers can be used individually or in combinations of two or more.
  • the phospholipid includes naturally occurring phospholipids, e.g. egg yolk lecithin, soya lecithin, and their hydrogenation products, and synthetic phospholipids, e.g. phosphatidylcholine, phosphatidylethanolamine, etc.
  • the nonionic surfactant includes macro-molecular surfactants with molecular weights in the range of about 800 to about 20000, such as polyethylene-propylene copolymer, polyoxyethylene alkyl ethers, polyoxyethylene alkylarylethers, hydrogenated castor oil- polyoxyethylene derivatives, polyoxyethylene sorbitan derivatives, polyoxyethylene sorbitol derivatives, polyoxyethylene alkyl ether sulfate, and so on.
  • the proportion of the emulsifier is selected so that the concentration in a final administrable composition will be in the range of about 0.1 to about 10%, preferably about 0.5 to about 5%.
  • a stabilizer for further improving the stability of the compositions and formulations as disclosed herein such as an antioxidant or a chelating agent, an isotonizing agent for adjusting the osmolality, an auxiliary emulsifier for improving the emulsifying power, and/or an emulsion stabilizer for improving the stability of the emulsifying agent can be incorporated.
  • the isotonizing agent that can be used includes, for example, gylycerin, sugar alcohols, monosaccharides, disaccharides, amino acids, dextran, albumin, etc. These isotonizing agents can be used individually or in combination, with two or more.
  • An emulsion stabilizer that can be used which includes cholesterol, cholesterol esters, tocopherol, albumin, fatty acid amide derivatives, polysaccharides, polysaccharide fatty acid ester derivatives, etc.
  • compositions and formulations as disclosed herein can further comprise a viscogenic substance which can adhere to the digestive tract mucosa due to its viscosity expressed on exposure to water.
  • the examples of the viscogenic substance include, but are not particularly limited as long as it is pharmaceutically acceptable, such as polymers (e.g. polymers or copolymers of acrylic acids and their salts) and natural-occurring viscogenic substances (e.g. mucins, agar, gelatin, pectin, carrageenin, sodium alginate, locust bean gum, xanthan gum, tragacanth gum, arabic gum, chitosan, pullulan, waxy starch, sucralfate, curdlan, cellulose, and their derivatives).
  • polymers e.g. polymers or copolymers of acrylic acids and their salts
  • natural-occurring viscogenic substances e.g. mucins, agar, gelatin, pectin, carrageenin, sodium alginate
  • the additives conventionally used for preparing the oral compositions can be added.
  • the additives include excipients (e.g. lactose, corn starch, talc, crystalline cellulose, sugar powder, magnesium stearate, mannitol, light anhydrous silicic acid, magnesium carbonate, calcium carbonate, L-cysteine, etc.), binders (e.g. starch, sucrose, gelatin, arabic gum powder, methylcellulose, carboxymethylcellulose, carboxymethylcellulose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, pullulan, dextrin, etc.), disintegrators (e.g.
  • anionic surfactants e.g. sodium alkylsulfates etc.
  • nonionic surfactants e.g. polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene- castor oil derivatives, etc.
  • maltosyl-beta- cyclodextrin maltosyl-beta- cyclodextrin, maltosyl-beta- cyclodextrin-carboxylic acid, etc.
  • colorants corrigents, adsorbents, antiseptics, moistening agents, antistatic agents, disintegration retardants, and so on.
  • the proportion of these additives can be appropriately selected from the range that can keep the stability and absorption of the basis.
  • compositions and formulations as disclosed herein for oral administration of the present invention may also include flavoring agents.
  • flavoring agents include, for example, anise oil, lavender oil, lemon oil, orange essence, rose oil, powder green tea, bergamot oil, (alpha[litre]) borneol, Natural Peal Extract AH- 10, Sugar, bitter essence, pine flavor etc.
  • compositions and formulations as disclosed herein can exhibit potent pharmacological activity with low toxicity such that they are useful as a medicament for prevention and treatment for, interalia, angiogenesis-associated disease in mammals (e.g. mouse, rat, monkey, bovine, canine, human, etc.), said angiogenesis-associated disease including, for example, (1) inflammatory diseases such as rheumatoid arthritis, (2) diabetic retinopathy, and (3) benign and malignant tumors (e.g.
  • gastric cancer cancer of the esophagus, duodenal cancer, cancer of the tongue, pharyngeal cancer, brain tumors, neurilemoma, colorectal cancer, non- small-cell lung cancer, small cell carcinoma of the lung, hepatic carcinoma, renal cancer, cancer of the breast, biliary tract cancer, cancer of the pancreas, cancer of the prostate, cancer of the uterus, carcinoma of the uterine cervix, ovarian cancer, cancer of the urinary bladder, cancer of the skin, malignant melanoma, cancer of the thyroid, sarcomas of bone, hemangioma, hemangiofibroma, retinal sarcoma, cancer of the penis, solid tumors of childhood, Kaposi's sarcoma in AIDS, etc., inclusive of recurrencies and metastases to other organs).
  • compositions and formulations described herein can also treat or prevent diseases or disorders that involve or are characterized by vascular permeability. Tumors exhibit vascular permeability relative to non-tumor tissue, but the invention is not limited to the treatment of tumors. Additional diseases or disorders amenable to treatment to inhibit vascular permeability are discussed elsewhere infra.
  • compositions and formulations as disclosed herein are filled into capsule shells coated with an enteric coating agent as mentioned above for use as an enteric composition.
  • an enteric coating agent as mentioned above for use as an enteric composition.
  • the capsule shell for example, soft capsules (e.g. the product of R. P. Sealer) and hard gelatin capsules are used.
  • liquid or solid compositions and formulations as disclosed herein can be administered orally.
  • the liquid form it can be directly administered e.g., by drinking an elixir or suspension of the composition, or alternatively, into the digestive tract via a catheter or sonde for oral administration or administered in the usual manner in the unit dosage form of a hard capsule or a soft capsule.
  • the solid form it can be administered orally as powders, capsules, tablets, or the like in the usual manner. It can also be redispersed in a suitable dispersion medium and administered in a liquid form.
  • the oral dose of the fumagillol- derivative as disclosed herein is about 1 mg to about 3 g/day, preferably about 10 mg to about lg/day, as a fumagillol derivative.
  • the oral dose of the composition as disclosed herein is between the ranges of about 25 mg to about lg/day, and in some embodiments less than 25mg to lg/day, for example about 10 mg to about 0.5g/day, of a fumagillol derivative.
  • compositions and formulations as disclosed herein enhance stability and oral absorption properties of fumagillol derivatives and increase the pharmacological activity thereof, so that better assurance of therapeutic efficacy is achieved without the need for parenteral administration.
  • the dosage form of the compositions and formulations as disclosed herein is stable and exhibits remarkable inhibiting activity on tumor growth and metastasis based on its anti-angiogenesis and anti- vascular permeability activity in oral administration so that it can be used as clinically advantageous oral medicine.
  • Suspensions in addition to the active components, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • suspending agents as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • the present invention encompasses combination therapy in which the formulations and compositions as disclosed herein are used in combination with a chemotherapeutic agent such as Taxol, cyclophosphamide, cisplatin, gancyclovir and the like.
  • a chemotherapeutic agent such as Taxol, cyclophosphamide, cisplatin, gancyclovir and the like.
  • the chemotherapeutic agent may also be included within a micelle as described herein.
  • Such a therapy is particularly useful in situations in which the subject or patient to be treated has a large preexisting tumor mass which is well vascularized.
  • the chemotherapeutic agent serves to reduce the tumor mass and the conjugate prevents or inhibits neovascularization within or surrounding the tumor mass.
  • the chemotherapeutic agent may also be administered at lower doses than normally used and at such doses may act as an antiangiogenic agent or vascular permeability-inhibiting.
  • the second therapy can be administered to the subject before, during, after or as a combination thereof relative to the administration of the compositions as disclosed herein.
  • Anti-cancer therapies are well known in the art and are encompassed for use in the methods of the present invention.
  • Chemotherapy includes, but is not limited to an alkylating agent, mitotic inhibitor, antibiotic, or antimetabolite, anti-angliogenic agents etc.
  • the chemotherapy can comprise administration of CPT-Il, temozolomide, or a platin compound.
  • Radiotherapy can include, for example, x-ray irradiation, w- irradiation, ⁇ -irradiation, or microwaves.
  • chemotherapeutic agent or “chemotherapy agent” are used interchangeably herein and refers to an agent that can be used in the treatment of cancers and neoplasms, for example brain cancers and gliomas and that is capable of treating such a disorder.
  • a chemotherapeutic agent can be in the form of a prodrug which can be activated to a cytotoxic form.
  • Chemotherapeutic agents are commonly known by persons of ordinary skill in the art and are encompassed for use in the present invention.
  • chemotherapeutic drugs for the treatment of tumors and gliomas include, but are not limited to: temozolomide (Temodar), procarbazine (Matulane), and lomustine (CCNU).
  • Chemotherapy given intravenously includes vincristine (Oncovin or Vincasar PFS), cisplatin (Platinol), carmustine (BCNU, BiCNU), and carboplatin (Paraplatin), Mexotrexate (Rheumatrex or Trexall), irinotecan (CPT-Il); erlotinib; oxalipatin; anthracyclins- idarubicin and daunorubicin; doxorubicin; alkylating agents such as melphalan and chlorambucil; cis-platinum, methotrexate, and alkaloids such as vindesine and vinblastine.
  • the present invention encompasses combination therapy in which the formulations and compositions as disclosed herein are used in combination with, a cytostatic agent, anti-VEGF and/or p53 reactivation agent.
  • a cytostatic agent is any agent capable of inhibiting or suppressing cellular growth and multiplication. Examples of cytostatic agents used in the treatment of cancer are paclitaxel, 5-fluorouracil, 5-fluorouridine, mitomycin-C, doxorubicin, and zotarolimus.
  • Other cancer therapeutics include inhibitors of matrix metalloproteinases such as marimastat, growth factor antagonists, signal transduction inhibitors and protein kinase C inhibitors.
  • anti-VEGF agents include bevacizumab(AvastinTM), VEGF Trap, CP-547,632, AG13736, AG28262, SU5416, SUl 1248, SU6668, ZD-6474, ZD4190, CEP-7055, PKC 412, AEE788, AZD-2171, sorafenib, vatalanib, pegaptanib octasodium, IM862, DClOl, angiozyme, Sirna-027, caplostatin, neovastat, ranibizumab, thalidomide, and AGA- 1470, a synthetic analog of fumagillin (alternate names: Amebacilin, Fugillin, Fumadil B, Fumadil) (A. G. Scientific, catalog #F1028), an angio- inhibitory compound secreted by Asper
  • anti-VEGF agent refers to any compound or agent that produces a direct effect on the signaling pathways that promote growth, proliferation and survival of a cell by inhibiting the function of the VEGF protein, including inhibiting the function of VEGF receptor proteins.
  • agent or “compound” as used herein means any organic or inorganic molecule, including modified and unmodified nucleic acids such as antisense nucleic acids, RNAi agents such as siRNA or shRNA, peptides, peptidomimetics, receptors, ligands, and antibodies.
  • Preferred VEGF inhibitors include for example, AVASTIN® (bevacizumab), an anti-VEGF monoclonal antibody of Genentech, Inc.
  • VEGF Trap (Regeneron / Aventis). Additional VEGF inhibitors include CP-547,632 (3-(4-Bromo-2,6-difluoro- benzyloxy)-5-[3-(4-pyrrolidin 1-yl- butyl)-ureido]- isothiazole-4- carboxylic acid amide hydrochloride; Pfizer Inc.
  • VEGFR2- selective monoclonal antibody DClOl ImClone Systems, Inc.
  • angiozyme a synthetic ribozyme from Ribozyme (Boulder, Colorado) and Chiron (Emeryville, California)
  • Sirna-027 an siRNA-based VEGFRl inhibitor, Sirna Therapeutics, San Francisco, CA
  • Neovastat iEterna Zentaris Inc; Quebec City, CA
  • the present invention encompasses combination therapy in which the formulations and compositions as disclosed herein are used in combination or conjunction with therapeutics, physiotherapy and/or behavioral psychotherapy used in the treatment of rheumatoid arthritis, obesity, endometriosis, and Alzheimer's disease.
  • NSAIDS non-steroidal anti-inflammatory drugs
  • corticosteroids such as prednisone or cortisone
  • anti-malarial medications such as hydroxychloroquine
  • gold such as hydroxychloroquine
  • sulfasalazine gold
  • penicillamine a member of the group consisting of hydroxychloroquine
  • cyclophosphamide a member of the group consisting of hydroxychloroquine
  • cyclosporine a member of the group consisting of hydroxychloroquine
  • minocycline such as hydroxychloroquine
  • interleukin receptor antagonist such as interleukin receptor antagonist
  • the formulations and compositions as disclosed herein are particularly useful for administration in conjunction therapies used for treatment of diseases associated with vascular permeability, such as vascular complications of diabetes such as nonproliferative diabetic retinopathy and nephropathy, nephrotic syndrome, pulmonary hypertension, burn edema, tumor edema, brain tumor edema, IL-2 therapy-associated edema, trauma associated edema and other edema- associated diseases, as disclosed in International Application No: WO2003/086178 and U.S. Patent Applications US2005/0203013 and US2005/0112063 which are incorporated herein in their entirety by reference.
  • diseases associated with vascular permeability such as vascular complications of diabetes such as nonproliferative diabetic retinopathy and nephropathy, nephrotic syndrome, pulmonary hypertension, burn edema, tumor edema, brain tumor edema, IL-2 therapy-associated edema, trauma associated edema and other edema
  • the formulations and compositions as disclosed herein are particularly useful for administration in conjunction with IL-2 therapy, where the limiting factor of IL-2 therapy is IL- 2 therapy-associated edema as disclosed in International Application No: WO2003/086178 and U.S. Patent Applications US2005/0203013 and US2005/0112063.
  • Treatment for Alzhemier's disease include, but are not be limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), estrogen, steroids such as prednisone, vitamin E, menantine, donepezil, rivastigmine, tacrine, and galantamine.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • steroids such as prednisone, vitamin E, menantine, donepezil, rivastigmine, tacrine, and galantamine.
  • Treatment of endometrosis includes, but should not be construed as limited to, a combination oral contraceptives (estrogen plus a progestin), progestins (such as medroxyprogesterone, danazol (a synthetic hormone related to testosterone, gonadotropin- releasing hormone agonists (GnRH agonists such as buserelin, goserelin, leuprolide and nafarelin), and nonsteroidal anti-inflammatory drugs (NSAIDs) for pain control.
  • a combination oral contraceptives estrogen plus a progestin
  • progestins such as medroxyprogesterone, danazol (a synthetic hormone related to testosterone, gonadotropin- releasing hormone agonists (GnRH agonists such as buserelin, goserelin, leuprolide and nafarelin
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • Examples of treatment options for obesity include dieting and nutritional counseling, exercise regime, gastric-bypass surgery, and drugs such as a combination of fenfluramine and phentermine (often called fen-phen), orlistat, sibutramine , phentermine, benzphetamine, diethylpropion, mazindol, and phendimetrazine.
  • the formulations and compositions can comprise a plurality of micelles, wherein some micelles comprise fumagillol derivatives, and other micelles comprise other therapeutic agents, for example chemotherapeutic agents and antineoplastic agents or the doxycycline found herein to synergize with oral fumagillol derivative preparations.
  • formulations and pharmaceutical composition can, if desired, be presented in a suitable container (e.g., a pack or dispenser device), such as an FDA approved kit, which can contain one or more unit dosage forms.
  • a suitable container e.g., a pack or dispenser device
  • FDA approved kit which can contain one or more unit dosage forms.
  • the combination can be used to treat or prevent diseases or disorders characterized by or involving vascular leakage.
  • the combination provides improved treatment to reduce glomerular vascular leakage and cancer-associated proteinuria.
  • the combination can also be used to treat or prevent the development of pulmonary edema and ascites, and the edema associated with delayed-type hypersensitivity allergic reactions.
  • the combination treatment can also inhibit vascular leakage induced, for example, by VEGF and histamine. As shown in the Examples, the combination treatment reduced tumor vascular leakage and tumor growth relative to treatment with either agent alone and is thus useful for the treatment of tumors.
  • a given agent for example a fumagillol derivative
  • a fumagillol derivative is an anti-angiogenesis agent or can reduce or inhibit vascular permeability.
  • a composition or formulation comprising a fumagillol derivative block copolymer conjugate in accordance with the present invention can treat or prevent diseases associated with angiogenesis-mediated condition can be determined by its effect in the mouse model as shown in the Examples below.
  • a fumagillol derivative block copolymer as described herein will have anti- angiogenic activity (i.e., at least 50% of the anti-angiogenic activity of TNP-470) in a HUVEC assay as described in the Examples herein.
  • Another useful assay for determining if the compositions and formulations as disclosed herein have anti-angiogenesis activity is the CAM assay, which is frequently used to evaluate the effects of angiogenesis regulating factors because it is relatively easy and provides relatively rapid results.
  • An angiogenesis regulating factor useful in the methods and compositions described herein will modify the number of microvessels in the modified CAM assay described by Iruela-Arispe et al., 1999, Circulation 100: 1423-1431. The method is based on the vertical growth of new capillary vessels into a collagen gel pellet placed on the CAM.
  • the collagen gel is supplemented with an angiogenic factor such as FGF-2 (50 ng/gel) or VEGF (250 ng/gel) in the presence or absence of test agents.
  • an angiogenic factor such as FGF-2 (50 ng/gel) or VEGF (250 ng/gel) in the presence or absence of test agents.
  • the extent of the angiogenic response is measured using FITC-dextran (50 ⁇ g/mL) (Sigma) injected into the circulation of the CAM.
  • the degree of fluorescence intensity parallels variations in capillary density; the linearity of this correlation can be observed with a range of capillaries between 5 and 540. Morphometric analyses are performed, for example, by acquisition of images with a CCD camera.
  • Images are then analyzed by imported into an analysis package, e.g., NHImage 1.59, and measurements of fluorescence intensity are obtained as positive pixels.
  • Each data point is compared with its own positive and negative controls present in the same CAM and interpreted as a percentage of inhibition, considering the positive control to be 100% (VEGF or FGF-2 alone) and the negative control (vehicle alone) 0%.
  • Statistical evaluation of the data is performed to check whether groups differ significantly from random, e.g., by analysis of contingency with Yates' correction.
  • Additional angiogenesis assays are known in the art and can be used to evaluate fumagillol derivatives for use in the methods and compositions described herein. These include, for example, the corneal micropocket assay, hamster cheek pouch assay, the Matrigel assay and modifications thereof, and co-culture assays. Donovan et al. describe a comparison of three different in vitro assays developed to evaluate angiogenesis regulators in a human background (Donovan et al., 2001, Angiogenesis 4: 113-121, incorporated herein by reference).
  • the assays examined include: 1) a basic Matrigel assay in which low passage human endothelial cells (Human umbilical vein endothelial cells, HUVEC) are plated in wells coated with Matrigel (Becton Dickinson, Cedex, France) with or without angiogenesis regulator(s); 2) a similar Matrigel assay using "growth factor reduced” or GFR Matrigel; and 3) a co-culture assay in which primary human fibroblasts and HUVEC are co-cultured with or without additional angiogenesis regulator(s), the fibroblasts produce extracellular matrix and other factors that support HUVEC differentiation and tubule formation.
  • HUVEC Human umbilical vein endothelial cells
  • the co-culture assay provided microvessel networks that most closely resembled microvessel networks in vivo.
  • the basic Matrigel assay and the GFR Matrigel assay can also be used by one of skill in the art to evaluate whether a given fumagillol derivative is an angiogenesis-inhibiting agent as necessary for the methods and compositions described herein.
  • an in vitro angiogenesis assay kit is marketed by Chemicon (Millipore).
  • the Fibrin Gel In Vitro Angiogenesis Assay Kit is Chemicon Catalog No. ECM630.
  • angiogenesis assays are disclosed in International Application No: WO2003/086178 and U.S. Patent Applications US2005/0203013 and US2005/0112063, and involves assaying endothelial cells on a permeable substrate (e.g., a collagen coated inserts of "Transwells"), contacting the assay with a test compound (e.g., a fumagillol derivative block copolymer conjugate), treating the assay with a marker (e.g., FITC label) and a permeability- inducing agent (e.g.
  • a marker e.g., FITC label
  • a permeability- inducing agent e.g.
  • VEGF vascular endothelial growth factor
  • PAF platelet-activating factor
  • Compounds that are found to affect vascular permeability can be further tested for anti-tumor activity using existing methods.
  • the bioeffectiveness of fumagillol derivative block copolymer conjugate as an anti- angiogenic agent in a patient being treated with such fumagillol derivative block copolymer conjugate can be assessed by methods commonly known by person skilled in the art, for example, as disclosed in International Application No: WO2003/086178 and U.S.
  • composition or formulation as disclosed herein capable of preventing or treating nonproliferative diabetic retinopathy can be tested by in vitro studies of endothelial cell proliferation and in other models of diabetic retinopathy, such as Streptozotocin.
  • color Doppler imaging can be used to evaluate the action of a drug in ocular pathology (Valli et al., Ophthalmologica 209(13): 115-121 (1995)).
  • Color Doppler imaging is a recent advance in ulkasonography, allowing simultaneous two-dimension imaging of structures and the evaluation of blood flow. Accordingly, retinopathy can be analyzed using such technology.
  • compositions and formulations as disclosed herein are administered is dependent, in part, upon whether the treatment of a disease associated with vascular hyperpermeability, including non- proliferative retinopathy is prophylactic or therapeutic.
  • the manner in which compositions and formulations as disclosed herein are administered for treatment of retinopathy is dependent, in part, upon the cause of the retinopathy.
  • the compositions and formulations as disclosed herein can be administered preventatively as soon as the pre-diabetic retinopathy state is detected.
  • the present invention may be as defined, for example, in any one of the following numbered paragraphs.
  • a method of inhibiting vascular leakage comprising administering doxycycline and a composition comprising a formulation of a fumagillol derivative that inhibits vascular leakage, said formulation comprising said derivative associated with a block copolymer comprising a hydrophilic polymer moiety and a hydrophobic polymer moiety, said doxycycline and said composition comprising a fumigillol derivative administered in amounts effective to inhibit vascular leakage.
  • hydrophobic polymer moiety of said block copolymer is selected from the group consisting of poly(d,L-lactic acid), poly(L-lysine), poly(aspartic acid), poly(caprolactone) (PCL), poly ⁇ ropylene oxide).
  • hydrophobic moiety is a poly(d,L-lactic acid) (PLA) polymer.
  • hydrophilic polymer moiety of said block copolymer is a polyethene glycol (PEG) polymer.
  • hydrophilic moiety is a poly(ethylene glycol) (PEG) polymer.
  • said fumagillol derivative comprises a derivative selected from the group consisting of 6-O-(N-chloroacetylcarbamoyl) fumagillol (TNP-470), 6-O-(4-methoxyaniline)acetyl fumagillol; 6-0- (3,4, 5-trimethexyaniline)acetyl fumagillol; 6- 0-(4-(N 5 N- dimethylethoxy) aniline) acetyl fumagillol; 6-O-(cyclopropylamino) acetyl fumagillol; 6-O-(cyclobutylamino)acetyl fumagillol; 4- ((cyclopropylamino)acetyl) oxy-2- (l,2-epoxy-l,5 20 dimethyl-4-hexenyl)-3- methoxy-l-chloromethyl-1 cyclohexanol; and A- ((cyclobutylamin
  • said fumagillol derivative comprises 6-O-(N- chloroacetylcarbamoyl) fumagillol (TNP-470).
  • a method of treating or preventing a condition involving vascular leakage comprising administering doxycycline and a composition comprising an oral formulation of a fumagillol derivative that inhibits vascular leakage, wherein said fumagillol derivative is associated with a block copolymer comprising a hydrophilic polymer moiety and a hydrophobic polymer moiety, said doxycycline and said composition comprising an oral formulation of a fumagillol derivative administered in amounts effective to treat or prevent said condition involving vascular leakage.
  • a method of making a diblock copolymer micelle comprising a fumagillol derivative that has anti- angiogenic activity comprising conjugating said fumagillol derivative to a diblock copolymer comprising a hydrophilic polymer moiety and a hydrophobic polymer moiety and forming micelles of the resulting conjugate.
  • said fumagillol derivative comprises 6-O-(N- chloroacetylcarbamoyl) fumagillol (TNP-470).
  • a diblock copolymer micelle comprising a fumagillol derivative, said micelle produced by the method of paragraph 21.
  • said angiogenesis-mediated condition is selected from the group comprising cancer, metastatic tumors, psoriasis, age-related macular degeneration (AMD), thyroid hyperplasia, preclampsia, rheumatoid arthritis and osteoarthritis, Alzheimer's disease, obesity, pleural effusion, atherosclerosis, endometriosis, diabetic and other angiogenesis-mediated retinopathies, ocular neovascularization, and IL-2 therapy associated edema.
  • AMD age-related macular degeneration
  • TNP-470 (Takeda) was bound to a diblock polymer using two-step conjugation.
  • succinated mPEG-b-PLA-OOCCH 2 CH 2 COOH was dissolved in 10ml DMSO and reacted with ethyl(diethylaminopropyl) carbodiimide (EDC) and a catalyst N-hydroxysuccinimide (NHS)
  • EDC ethyl(diethylaminopropyl) carbodiimide
  • NHS catalyst N-hydroxysuccinimide
  • the same amount of succinated mPEG-b-PLA-OOCCH 2 CH 2 COOH is dissolved in 10ml MES buffer (pH 6) and the catalyst is N-hydroxysuccinimide (sulfo-NHS)).
  • the solution was fully dissolved and an excess of ethylendiamine was added while stirring and reacted at room temperature for 4 hr.
  • the polymer solution was then dialyzed (MWCO 1000, Spectra/Por Biotech Regenerated Cellulose JWR) and lyophilized.
  • the dry modified polymer was dissolved in 2OmI DMSO, and O.Sgr of TNP-470 dissolved in DMSO was added.
  • the polymeric micelles were prepared using dialysis methods.
  • the DMSO solution of the polymer-drug conjugate was dialyzed using regenerated cellulose dialysis bag (MWCO 2000, Spectra/Por Biotech Regenerated Cellulose, VWR) against double distilled water.
  • the polymersomes were self-assembled after the exchange of the organic solvent by water.
  • the polymeric micelles were lyophilized and stored at -2O 0 C in a dry environment until use.
  • NMR analysis In order to ensure the success of the conjugation, a Nuclear Magnetic Resonance (NMR) spectrometer analysis (NERCE/BEID, Harvard medical school) was performed for the different reaction steps: free drug, sample of polymer before and after the modification by ethylendiamine, as modified polymer after TNP-470 conjugation.
  • NMR Nuclear Magnetic Resonance
  • HUVEC proliferation assay HUVECs were exposed to different concentrations of polymeric micelles equivalent to 50-100OnM free TNP-470 (0.12-2.4 mg/ml micelles) and incubated in a serum — reduced medium for 48hr at 37 0 C. To omit the possible cytotoxic effect of the carrier, empty micelles were added to HUVECs at the same concentrations as the drug- micelles, and in a double maximum concentration (4.8mg/mi). A WST-I proliferation assay (Roche Diagnostics) was used. Cell viability was calculated as a percent of the absorbance of the formazan reading at 450 nm for experimental wells verses untreated cells. Data were derived from quadruplicate samples in two separate experiments.
  • the cells were washed twice with PBS and fixed with 4% parformaldehyde. After fixation, the cells were mounted using DAPI containing Vectashild (Vector), and the cells were imaged using Confocal microscopy.
  • TEM Transmission Electron Microscopy
  • TNP-470 polymeric micelles Size of TNP-470 polymeric micelles.
  • the average size and size distribution of polymeric micelles were determined by Dynamic Light Scatterer (DLS, DynaPro, Wyatt Technology). Four different samples of 2mg/ml TNP-470 polymersomes dispersed in double — distilled water were measured by 20 successive readings with DLS. Micelles' size was also measured after 24hr to evaluate their structural stability.
  • mice (Jackson Laboratories, Bar Harbor, MA) were injected subcutaneously with IxIO 6 Lewis Lung Carcinoma cells (LLC) at the back. When tumors reached a volume of 100 mm- the mice were divided into 5 groups, and treated every day for 12 days with TNP-470 polymeric micelles, administrated orally using a gavage needle.
  • LLC Lewis Lung Carcinoma cells
  • polymersomes Different doses of polymersomes were given daily: 15mg/kg (5 mice), 30mg/kg (3 mice) and 60mg/kg (1 mice) TNP-470 equivalent, respectively (5, 10, 20 mg polymeric drug per mouse, respectively).
  • the control groups were five mice that were given the same dose of polymersomes without the drug, and five mice that were given drinking water.
  • mice Every two days the weight of the mice was monitored and tumors dimensions were measured using a caliber. Tumor volume was calculated according to an ellipsoid formula.
  • Block copolymer micelles have been proposed for the delivery of hydrophobic drugs with low aqueous solubility (such as Paclitaxel).
  • the amphiphilic nature of diblock copolymers such as poly (lactide)-poly(ethylene glycol) (PLA-PEG), enables the formation of micelles in aqueous media.
  • the diblock copolymer In an aqueous environment, the diblock copolymer is self-assembled into a structure of hydrophobic core, formed by the association of the hydrophobic polymer and the drug, and a hydrophilic shell, formed by the hydrophilic polymer, commonly PEG.
  • Block copolymers as disclosed in U.S. Pat. No. 4,745,160 have been used form water insoluble, amphiphilic, non-crosslinked linear, branched or graft block copolymers having polyethylene glycol as the hydrophilic component and poly(D-, L-, or D, L-lactic acids) as the hydrophobic components.
  • Block copolymers as disclosed in U.S. Pat. No. 5,543,158 have been described to form nanoparticles or microparticles that are solid particles that are suspended in water that are formed from a water-insoluble block copolymer consisting essentially of poly(alkylene glycol) and poly(lactic acid). The molecular weight of the block copolymer is high and the copolymer is insoluble in water.
  • the biodegradable moieties of the copolymer are in the core of the nanoparticle or microparticle and the poly(alkylene glycol) moieties are on the surface of the nanoparticle or microparticle in an amount effective enough to decrease uptake of the nanoparticle or microparticle by the reticuloendothelial system.
  • Nanoparticles are prepared by dissolving the block copolymer and drug in an organic solvent, forming an oil and water emulsion by sonication or stirring, and collecting the nanoparticles containing the drug following precipitation. It does not provide for the solubilization of hydrophobic drugs.
  • the inventors demonstrate TNP-470 was successfully conjugated to a modified PEG- PLA polymer through its amine, and formed nano-size polymersomes.
  • the inventors demonstrate, using images taken by TEM, that spherical micelles were formed and size measurement with DLS showed a low range of size distribution around one hundred nanometers.
  • the inventors demonstrate rapid uptake by Human Umbilical Vein Endothelial Cells (HUVECs), and when TNP-470 polymersomes were added, a significant inhibition of HUVECs proliferation was shown (as compared to no effect of the carrier itself).
  • UUVECs Human Umbilical Vein Endothelial Cells
  • mice In-vivo studies done on mice showed a significant inhibition of subcutaneous Lewis Lung Carcinoma tumors with C57/BL mice given a daily oral administration of TNP470 micelles. A dose of 15mg/kg TNP-470 equivalent showed 63% inhibition without any weight loss to the mice.
  • NMR results from the different reaction steps are shown in Fig. 1.
  • the results show the binding of the ethylendiamine through the carboxic acid group at the original polymer, and the TNP-470 binding to the polymer through the amine.
  • TEM Transmission Electron Microscopy
  • TNP-470 polymeric micelles Size of TNP-470 polymeric micelles.
  • the average size and size distribution of polymersomes were determined by a Dynamic Light Scattering (DLS) instrument. To confirm that the structure of the micelle remained stable after preparation, the micelles' size was also measured after 24hr of incubation in aqueous medium at room temperature. The average diameter of micelles is presented in Table 1. .
  • Fig 3 B shows a typical graph of DLS analysis.
  • TNP-470 micelles TNP-470 micelles — day of preparation 1 day post preparation
  • HUVEC Proliferation The effect of different concentrations of TNP-470 polymersomes on the proliferation of HUVECs was evaluated using a WST-I reagent.
  • Fig. 3 shows the inhibition in proliferation after 48 hr of incubation.
  • Panel A shows the effect of TNP- 470 micelles
  • graph B shows the effect of micelles without drug on HUVECs.
  • a significant inhibition of HUVEC proliferation was detected from 0.3 mg/ml and up to 2.4 mg/ml micelles, which are 62.5nM — 100OnM TNP-470 equivalent (88% and 95% inhibition respectively).
  • the same concentrations of polymeric micelles without TNP-470, as well as double concentration (4.8mg/ml) showed no significant effect on HUVEC proliferation.
  • FIG. 5 shows representative images of cells incubated with the micelles for 20mm, 4 hr and 24 hr. After 20 mm micelles were already uptaken by the cells and located at the cytoplasm. After 2 hr, 4 hr, 7 hr and 24 hr the uptake increased and the micelles were detected as concentrated spots inside cell cytoplasm. Figure 5 shows representative cells incubated with micelles for 20mm, 4hr and 24 hr. [0247] In vivo results.
  • C57/BL mice bearing s.c LLC tumors showed inhibition in tumor growth when treated with PEG-PLA-TNP-470 micelles given orally. All doses showed a significant effect already after 14 days of daily treatment, as shown in figure 6A. The controlled groups had to be scarified on day 14, and the treatment continued up to day 18. The lowest dose (15mg/kg TNP-470 equivalent) showed a significant inhibition of tumor growth (63%) after 14 days (*p ⁇ O.05), without any weight loss to the mice, whereas higher doses of 30mg/kg and 60mg/kg TNP-470 equivalent showed 74%, 75% inhibition respectively, but also caused almost 20% weight loss (Fig. 6B).
  • mPEG-PLA-Rhodamine polymersomes (same polymer composition as mPEG-PLA-TNP-470) were given to a C57/B1 mouse orally by gavage. After 2 hr the mouse was killed and the intestine was sectioned. Confocal microscopy of the histological sections showed polymersomes absorbed into the vili of the small intestine. The micelles were taken up by columnar epithelium and found to penetrate this layer to the lamina intestinal, indicating oral absorption.
  • TNP-470 polymersomes or doxycycline After eight days of treatment, while the animals receiving TNP-470 polymersomes or doxycycline individually showed decreases in tumor volume by approximately 40% and 36%, respectively, relative to controls receiving no treatment (p ⁇ 0.05), the tumors of animals receiving TNP-470 polymersomes and doxycycline were inhibited by approximately 70% relative to non-treated controls (p ⁇ 0.005). Tumor weight was also further reduced by combination treatment relative to untreated controls (see Fig. 10). Thus, the combination of oral TNP-470 formulation and doxycycline has a profound effect on tumor growth.
  • Tumor vessel permeability was measured using Evans Blue dye in a Miles assay.
  • Figure 11 shows results of tumor vessel permeability normalized to tumor weight (OD620 nm/g tumor). Both TNP-470 polymersomes and doxycycline reduced tumor vessel permeability relative to untreated controls, and the combination further reduced permeability (p ⁇ 0.05). The combination is thus more effective not only in the inhibition of tumor growth, but also in the inhibition of vascular permeability.
  • TNP-470 inhibits murine cutaneous wound healing. / Surg Res 82, 268-74 (1999). 36. Whalen, C.T., Hanson, G.D., Putzer, K.J., Mayer, M.D. & Mulford, D.J. Assay of TNP-470 and its two major metabolites in human plasma by high-performance liquid chromatography- mas s spectrometry. / Chromatogr Sci 40, 214-8 (2002).

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Abstract

L'invention porte sur des dérivés de fumagillol et sur des compositions et des formulations de ceux-ci, et plus particulièrement sur des compositions et des formulations de dérivés du fumagillol, le dérivé du fumagillol étant associé à un copolymère à deux blocs. La présente invention porte également sur des compositions et des formulations comprenant des dérivés du fumagillol pour une administration orale ou une administration par l'intermédiaire de voix telles qu'une administration topique ou oculaire. La présente invention porte également sur des procédés pour traiter des états à médiation par angiogenèse par l'administration des compositions et formulations comprenant des dérivés du fumagillol comme décrit ici.
PCT/US2008/075921 2007-09-13 2008-09-11 Procédés et compositions pour inhiber une fuite vasculaire Ceased WO2009036108A1 (fr)

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WO2014152861A3 (fr) * 2013-03-14 2014-11-20 Zafgen, Inc. Méthodes de traitement de maladie rénale et d'autres troubles
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US9585909B2 (en) 2010-05-25 2017-03-07 Syndevrx, Inc. Polymer-conjugated MetAP2 inhibitors, and therapeutic methods of use thereof
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US10646463B2 (en) 2016-01-11 2020-05-12 Syndevrx, Inc. Treatment for tumors driven by metabolic dysfunction
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WO2003086178A2 (fr) * 2002-04-11 2003-10-23 Children's Medical Center Corporation Methodes permettant d'inhiber l'hyperpermeabilite vasculaire
WO2003086382A1 (fr) * 2002-04-11 2003-10-23 Children's Medical Center Corporation Conjugues de polymeres tnp-470 et utilisation de ceux-ci
WO2006084054A2 (fr) * 2005-02-02 2006-08-10 Children's Medical Center Corporation Procede de traitement de maladies angiogeniques

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WO2003086382A1 (fr) * 2002-04-11 2003-10-23 Children's Medical Center Corporation Conjugues de polymeres tnp-470 et utilisation de ceux-ci
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US10159692B2 (en) 2010-05-25 2018-12-25 Syndevrx, Inc. Polymer-conjugated MetAP2 inhibitors, and therapeutic methods of use thereof
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WO2014152861A3 (fr) * 2013-03-14 2014-11-20 Zafgen, Inc. Méthodes de traitement de maladie rénale et d'autres troubles
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EP3574922A1 (fr) * 2013-04-10 2019-12-04 Syndevrx, Inc. Inhibiteurs de fumagillol metap2 modifiés ou conjugués par un polymère destinés à être utilisés dans l'amélioration ou la restauration de la sensibilite de l'insuline
US10588904B2 (en) 2013-04-10 2020-03-17 Syndevrx, Inc. METAP2 inhibitors and methods of treating obesity
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US10646463B2 (en) 2016-01-11 2020-05-12 Syndevrx, Inc. Treatment for tumors driven by metabolic dysfunction
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