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  Pharmaceutical Patents  

 

Title:  Protection of cardiac myocardium
United States Patent: 
7,504,379
Issued: 
March 17, 2009

Inventors:
 Edelberg; Jay (New York, NY), Xaymardan; Munira (Toronto, CA)
Assignee:
  Cornell Research Foundation, Inc. (Ithaca, NY)
Appl. No.:
 11/129,076
Filed:
 May 13, 2005


 

Web Seminars -- Pharm/Biotech/etc.


Abstract

The invention provides compositions and methods for protecting vascular tissues from injury that occurs, for example, during occlusion of one or more arteries. In some embodiments, the injury is myocardial infarction. The compositions of the invention include combinations of platelet derived growth factor, vascular endothelial growth factor, and angiopoietin-2.

Description of the Invention

SUMMARY OF THE INVENTION

The invention provides compositions and methods for protecting vascular tissue in a patient from damage, for example, during occlusion of an artery. In some embodiments, the compositions and methods are used to prevent or treat tissue injuries resulting from myocardial infarction.

The methods of the invention involve administering to the patient a composition comprising a therapeutically effective amount of platelet-derived growth factor, angiopoietin-2, and vascular endothelial growth factor. The composition can also include a pharmaceutically acceptable carrier.

In one embodiment, the invention provides a method of promoting vascular health in a patient by administering to the patient a therapeutically effective amount of platelet derived growth factor, angiopoietin-2, and vascular endothelial growth factor.

In another embodiment, the invention provides a method of protecting cardiac tissue in a patient from damage during myocardial infarction by administering to the patient a therapeutically effective amount of platelet derived growth factor, angiopoietin-2, and vascular endothelial growth factor.

In yet another embodiment, the invention provides a method of improving survival of transplanted tissue in a patient by administering to the patient a therapeutically effective amount of platelet derived growth factor, angiopoietin-2, and vascular endothelial growth factor.

Administration can be parenteral, as by intravascular, intravenous, intraarterial, intraperitoneal, intraventricular infusion, stent, infusion catheter, balloon catheter, bolus injection, topical administration as by direct application to tissue surfaces during surgery, or oral. Alternatively, administration can be directly into the heart or into the vasculature of the patient.

The platelet-derived growth factor employed can, for example, be platelet derived growth factor AB, platelet derived growth factor A, platelet derived growth factor B, or mixtures thereof. In some embodiments, the platelet-derived growth factor can have a sequence comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or a mixture thereof. The angiopoietin-2 can, for example, have a sequence comprising SEQ ID NO:7, SEQ ID NO:8, or a mixture thereof. The vascular endothelial growth factor can, for example, have a sequence comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or a mixture thereof.

The size of necrosis, vascular injury or myocardial infarction in the patient can be reduced by such compositions and methods of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides pharmaceutical compositions comprising an effective amount of platelet-derived growth factor (PDGF), angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) that are effective for preventing damage to cardiac tissues as a result of vascular conditions or diseases, for example, those conditions and diseases that can lead to occlusion of an artery. Such administration is given before, during or as soon as possible after arterial occlusion.

Vascular Diseases

The vascular diseases treated by the present invention are vascular diseases of mammals. The word mammal means any mammal. Some examples of mammals include, for example, pet animals, such as dogs and cats; farm animals, such as pigs, cattle, sheep, and goats; laboratory animals, such as mice and rats; primates, such as monkeys, apes, and chimpanzees; and humans.

According to the invention, endothelial cells within normal vascular tissues change as they grow older, exhibiting reduced angiogenesis and losing their ability to communicate with other cells by secreting signaling agents. These changes can lead to a diminished capacity for blood vessel formation, a reduction in blood flow to the associated organ or system, and an inability to recover from injuries or diseases that can block or adversely affect blood vessels.

Many pathological conditions can lead to vascular diseases that can block blood vessels such as atherosclerosis, preeclampsia, peripheral vascular disease, erectile dysfunction, cancers, renal failure, heart disease, and stroke. Such conditions are associated with alterations in the normal vascular condition of the affected tissues and/or systems.

Accordingly, this invention relates to methods for treating endothelial dysfunction, such as a vascular condition or a circulatory condition associated with loss, injury or disruption of the vasculature within an anatomical site or system. The term "vascular condition" or "vascular disease" refers to a state of vascular tissue where blood flow is, or can become, impaired or even blocked.

Examples of vascular conditions or vascular disease to which the methods of the invention apply are those in which the vasculature of the affected tissue or system is injured, senescent, or otherwise altered in some way, so that blood flow to the tissue or system is reduced or is in danger of being reduced. Vascular, circulatory or hypoxic conditions to which the methods of the invention apply are those associated with, but not limited to, myocardial infarction, maternal hypoxia (e.g., placental hypoxia, preeclampsia), abnormal pregnancy, peripheral vascular disease (e.g., arteriosclerosis), transplant accelerated arteriosclerosis, deep vein thrombosis, erectile dysfunction, cancers, renal failure, stroke, heart disease, sleep apnea, hypoxia during sleep, female sexual dysfunction, fetal hypoxia, respiratory disorders due to smoking, anemia, hypovolemia, vascular or circulatory conditions which increase risk of metastasis or tumor progression, hemorrhage, hypertension, diabetes, vasculopathologies, disorders due to surgery (e.g., per-surgical hypoxia, post-operative hypoxia), Raynaud's disease, endothelial dysfunction, regional perfusion deficits (e.g., limb, gut, renal ischemia), stroke, thrombosis, frost bite, decubitus ulcers, asphyxiation, poisoning (e.g., carbon monoxide, heavy metal), altitude sickness, pulmonary hypertension, sudden infant death syndrome (SIDS), asthma, chronic obstructive pulmonary disease (COPD), congenital circulatory abnormalities (e.g., Tetralogy of Fallot) and erythroblastosis (blue baby syndrome). In particular embodiments, the invention provides methods of treating loss of circulation or endothelial dysfunction in a mammal such as a human patient.

Thus, the invention is directed to methods of promoting vascular health by preventing or treating diseases or conditions like those listed above. In some embodiments, the disease or condition is stroke, atherosclerosis, acute coronary syndromes including unstable angina, thrombosis and myocardial infarction, plaque rupture, both primary and secondary (in-stent) restenosis in coronary or peripheral arteries, transplantation-induced sclerosis, peripheral limb disease, intermittent claudication and diabetic complications (including ischemic heart disease, peripheral artery disease, congestive heart failure, retinopathy, neuropathy and nephropathy), or thrombosis.

In some embodiments, the vascular condition or vascular disease arises from injured or damaged myocardium. As used herein injured or damaged myocardium refers to myocardial cells that have been exposed to ischemic conditions. These ischemic conditions may be caused by a myocardial infarction, transplant injury, heart surgery or cardiovascular disease. The lack of oxygen causes the death of the cells in the surrounding area, leaving an infarct that can eventually scar.

Preferably, injured or damaged myocardium is treated with the methods and compositions of the invention before damage occurs (e.g. when damage is suspected of occurring or a possibility exists of potential vascular injury), during injury or as quickly as possible after injury or damage occurs. Hence, the methods and compositions of the invention are advantageously employed on tissues that may be experiencing, are experiencing, have just experienced or are in danger of ischemia, myocardial infarction, heart attack or loss of blood flow. The methods and compositions of the invention are also advantageously employed on recently damaged vascular tissues (e.g. myocardium) and on not so recently damaged vascular tissues (e.g. myocardium).

As used herein "recently damaged vascular tissues" refers to vascular tissues (or myocardium) that has been damaged within zero to one day of treatment being started. In a preferred embodiment, the tissue (or myocardium) has been damaged within zero to twelve hours of the start of treatment. In a further preferred embodiment, the tissue or myocardium has been damaged within 6 hours of the start of treatment.

The methods and compositions of the invention can be used to prevent or to treat vascular conditions such as those listed above. These methods involve administering an effective amount of a combination of platelet-derived growth factor (PDGF), angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF). Such an effective amount is effective when it reduces the extent of damage due to vascular injury, vascular occlusion or myocardial infarction.

Platelet-Derived Growth Factor

Naturally occurring, platelet-derived growth factor ("PDGF") is a disulfide-bonded dimer having two polypeptide chains, namely the "A" and "B" chains, with the A chain being approximately 60% homologous to the B chain. Naturally occurring PDGF is found in three dimeric forms, namely PDGF-AB heterodimer, PDGF-BB homodimer, or PDGF-AA homodimer. Hannink et al., Mol. Cell. Biol., 6, 1304-1314 (1986). PDGF-AB has been identified as the predominant naturally occurring form. However, some data indicates that the PDGF-BB homodimer may be effective for wound healing. Each monomeric subunit of the biologically active dimer, irrespective of whether it is an A chain monomer or a B chain monomer, contains eight cysteine residues. Some of these cysteine residues form interchain disulfide bonds that hold the dimer together. As used herein, the term PDGF means any PDGF polypeptide or protein, including PDGF A, PDGF B, PDGF AB, PDGF BB, and PDGF AA.

The A polypeptide of human PDGF can be any mammalian PDGF A polypeptide including, for example, human, mouse, rat, rabbit, goat, bovine, horse, sheep and any other mammalian PDGF A polypeptide. The following sequence is one example of an amino acid sequence of a human PDGF A polypeptide (SEQ ID NO:1) -- see Original Patent.

The following sequence is an example of a mouse PDGF A sequence (SEQ ID NO:2) -- see Original Patent.

Other sequences for PDGF A can readily be obtained by one of skill in the art, for example, from the GenBank database of sequences. Variability in these and other sequences is permitted so long as the PDGF A polypeptide can dimerize with PDGF B and/or function in cell-to-cell communication.

The PDGF B polypeptide found in human platelets has been identified as a 109 amino acid cleavage product (PDGF-B.sub.109) of a 241 amino acid precursor polypeptide Johnsson et al., EMBO Journal, 3(5), 921-928 (1984). An example of a human sequence for the PDGF B polypeptide is provided below (SEQ ID NO:3) -- see Original Patent.

The following sequence is an example of a mouse PDGF B sequence (SEQ ID NO:4) -- see Original Patent.

A 109 amino acid PDGF B polypeptide is believed to be the mature form of PDGF in humans and constitutes a cleavage product of the PDGF-B precursor protein. Homology with the precursor protein begins at amino acid 82 of the 241 amino acid precursor protein and continues for 109 amino acids yielding, for example, a polypeptide with the following sequence (SEQ ID NO:5) -- see Original Patent.

Another form of PDGF-B (PDGF-B.sub.119), corresponds to the first 119 amino acids of the PDGF-B precursor protein (SEQ ID NO:6) -- see Original Patent.

This PDGF-B.sub.119 form has also been identified as a major cleavage product of the precursor protein when the entire gene is expressed in a transfected mammalian host. See U.S. Pat. No. 5,149,792.

Human platelet-derived growth factor is believed to be the major mitogenic growth factor in serum for connective tissue cells. PDGF can positively affect mitogenesis in arterial smooth muscle cells, fibroblast cells lines, and glial cells. Deuel et al., J. Biol. Chem., 256(17), 8896-8899 (1981). See also, e.g., Heldin et al., J. Cell Physiol., 105, 235 (1980) (brain glial cells); Raines and Ross, J. Biol. Chem., 257, 5154 (1982) (monkey arterial smooth muscle cells). PDGF is also believed to be a chemoattractant for fibroblasts, smooth muscle cells, monocytes, and granulocytes.

Angiopoietin-2

An angiogenic factor, which was originally called TIE-2 ligand-1 (TL1), but which is also referred to as angiopoietin-1 (Ang1) is required for normal vascular development in the mouse. Angiopoietin-2 (also called TIE-2 ligand-2, TL2) is believed to be a naturally occurring antagonist for Ang1 and the angiopoietin-2 receptor. A description of the cloning and sequencing of TL1 (Ang1) and TL2 (Ang2) as well as for methods of making and uses thereof is provided in PCT International Publication No. WO 96/11269 published Apr. 18, 1996, PCT International Publication No. WO 96/31598 published Oct. 10, 1996 and S. Davis, et al., Cell 87: 1161-1169 (1996) each of which is hereby incorporated by reference.

One example of a sequence of a human angiopoietin-2 precursor polypeptide is as follows (SEQ ID NO:7) -- see Original Patent.

Another example of a sequence of a human angiopoietin-2 precursor polypeptide is as follows (SEQ ID NO:8) -- see Original Patent.

Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) (Ferrara et al., Endo. Rev., 13: 18-32 [1992]) is a potent angiogenic factor that acts via the endothelial cell-specific receptor tyrosine kinases fins-like tyrosine kinase (Flt1) and fetal liver kinase (Flk1) (also designated KDR). Shibuya et al., Oncogene, 5: 519-524 (1990); Devries et al., Science, 255: 989-991 (1992); Quinn et al., Proc. Natl. Acad. Sci. USA, 90: 7533-7537 (1993); Millauer et al., Cell, 72: 835-846 (1993); Matthews et al., Proc. Natl. Acad. Sci. USA, 88: 9026-9030 (1991); Terman et al., Biochem. Biophys. Res. Commun., 187: 1579-1586 (1992); Terman et al., Oncogene, 6: 1677-1683 (1991); Oelrichs et al., Oncogene, 8: 11-18 (1993). These two VEGF receptors and a third orphan receptor, Flt4 (Pajusola et al., Cancer Res., 52: 5738-5743 [1992]; Galland et al., Oncogene, 8: 1233-1240 [1993]; Finnerty et al., Oncogene, 8: 2293-2298 [1993]) constitute a subfamily of class III receptor tyrosine kinases that contain seven extracellular immunoglobulin-like domains and a split intracellular tyrosine kinase domain. Mustonen and Alitalo, J. Cell. Biol., 129: 895-898 (1995). See also WO 94/10202 published May 11, 1994 and PCT/US93/00586 filed Jan. 22, 1993 (Avraham et al.). These three receptors have 31-36% amino acid identity in their extracellular ligand-binding domains.

VEGF is a homodimeric, cysteine-rich protein that can occur in at least four forms due to alternative splicing of its mRNA. Ferrara et al., Endo. Rev., 13: 18-32 [1992]). One example of a VEGF polypeptide that may be used in the invention is a polypeptide of the following sequence (SEQ ID NO:9) -- see Original Patent.

Another example of a VEGF polypeptide that may be used in the invention is vascular endothelial growth factor B precursor, which has the following sequence (SEQ ID NO:10) -- see Original Patent.

Another example of a VEGF polypeptide that may be used in the invention is vascular endothelial growth factor C preproprotein, which has the following sequence (SEQ ID NO:11) -- see Original Patent.

Another example of a VEGF polypeptide that may be used in the invention is vascular endothelial growth factor D precursor, which has the following sequence (SEQ ID NO:12) -- see Original Patent.

As recognized by one of skill in the art, each of the PDGF, angiopoietin-2 and VEGF polypeptides from different mammalian species have similar amino acid sequences. According to the invention any PDGF, angiopoietin-2 and VEGF polypeptide from any mammalian species can be utilized in the practice of the invention so long as the PDGF, angiopoietin-2 and VEGF polypeptide can stimulate endothelial cells to promote angiogenesis.

Compositions

The polypeptides of the invention, including their salts, are preferably administered so as to achieve a reduction in at least one symptom associated with a myocardial infarction, or a decrease in the degree or amount of tissue injury associated with myocardial infarction or heart attack.

To achieve the desired effect(s), the polypeptides or variants thereof, may be administered as single or divided dosages, for example, of at least about 0.01 mg/kg to about 500 to 750 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg, at least about 0.1 mg/kg to about 100 to 300 mg/kg or at least about 1 mg/kg to about 50 to 100 mg/kg of body weight, although other dosages may provide beneficial results. The amount administered will vary depending on various factors including, but not limited to, the polypeptides chosen, the disease, the weight, the physical condition, the health, the age of the mammal, whether prevention or treatment is to be achieved, and if the peptide is chemically modified. Such factors can be readily determined by the clinician employing animal models or other test systems that are available in the art.

Administration of the combination of therapeutic agents in accordance with the present invention may be in a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the polypeptides of the invention may be essentially continuous over a pre-selected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.

To prepare the composition, polypeptides are synthesized or otherwise obtained, purified as necessary or desired and then lyophilized and stabilized. A given polypeptide can then be adjusted to the appropriate concentration, and optionally combined with other agents. The absolute weight of a given polypeptide included in a unit dose can vary widely. For example, about 0.01 to about 2 g, or about 0.1 to about 500 mg, of the combination of polypeptides of the invention. Alternatively, the unit dosage can vary from about 0.01 g to about 50 g, from about 0.01 g to about 35 g, from about 0.1 g to about 25 g, from about 0.5 g to about 12 g, from about 0.5 g to about 8 g, from about 0.5 g to about 4 g, or from about 0.5 g to about 2 g.

Daily doses of the polypeptide combination of the invention can vary as well. Such daily doses can range, for example, from about 0.1 g/day to about 50 g/day, from about 0.1 g/day to about 25 g/day, from about 0.1 g/day to about 12 g/day, from about 0.5 g/day to about 8 g/day, from about 0.5 g/day to about 4 g/day, and from about 0.5 g/day to about 2 g/day.

Thus, one or more suitable unit dosage forms comprising the therapeutic polypeptides of the invention can be administered by a variety of routes including oral, parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), rectal, dermal, transdermal, intrathoracic, intrapulmonary and intranasal (respiratory) routes. The therapeutic polypeptides may also be formulated for sustained release (for example, using microencapsulation, see WO 94/07529, and U.S. Pat. No. 4,962,091). The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to the pharmaceutical arts. Such methods may include the step of mixing the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

When the therapeutic polypeptides of the invention are prepared for oral administration, they are generally combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. For oral administration, the polypeptides may be present as a powder, a granular formulation, a solution, a suspension, an emulsion or in a natural or synthetic polymer or resin for ingestion of the active ingredients from a chewing gum. The active polypeptides may also be presented as a bolus, electuary or paste. Orally administered therapeutic polypeptides of the invention can also be formulated for sustained release, e.g., the polypeptides can be coated, micro-encapsulated, or otherwise placed within a sustained delivery device. The total active ingredients in such formulations comprise from 0.1 to 99.9% by weight of the formulation.

By "pharmaceutically acceptable" it is meant a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.

Pharmaceutical formulations containing the therapeutic polypeptides of the invention can be prepared by procedures known in the art using well-known and readily available ingredients. For example, the polypeptide can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, solutions, suspensions, powders, aerosols and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include buffers, as well as fillers and extenders such as starch, cellulose, sugars, mannitol, and silicic derivatives. Binding agents can also be included such as carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone. Moisturizing agents can be included such as glycerol, disintegrating agents such as calcium carbonate and sodium bicarbonate. Agents for retarding dissolution can also be included such as paraffin. Resorption accelerators such as quaternary ammonium compounds can also be included. Surface-active agents such as cetyl alcohol and glycerol monostearate can be included. Adsorptive carriers such as kaolin and bentonite can be added. Lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols can also be included. Preservatives may also be added. The compositions of the invention can also contain thickening agents such as cellulose and/or cellulose derivatives. They may also contain gums such as xanthan, guar or carbo gum or gum arabic, or alternatively polyethylene glycols, bentones and montmorillonites, and the like.

For example, tablets or caplets containing the polypeptides of the invention can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate. Caplets and tablets can also include inactive ingredients such as cellulose, pre-gelatinized starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, zinc stearate, and the like. Hard or soft gelatin capsules containing at least one polypeptide of the invention can contain inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil. Moreover, enteric-coated caplets or tablets containing one or more polypeptides of the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum.

The therapeutic polypeptides of the invention can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous, intraperitoneal or intravenous routes. The pharmaceutical formulations of the therapeutic polypeptides of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension or salve.

Thus, the therapeutic polypeptides may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion containers or in multi-dose containers. As noted above, preservatives can be added to help maintain the shelve life of the dosage form. The active polypeptides and other ingredients may form suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active polypeptides and other ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

These formulations can contain pharmaceutically acceptable carriers, vehicles and adjuvants that are well known in the art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name "Dowanol," polyglycols and polyethylene glycols, C.sub.1-C.sub.4 alkyl esters of short-chain acids, ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the name "Miglyol," isopropyl myristate, animal, mineral and vegetable oils and polysiloxanes.

It is possible to add, if necessary, an adjuvant chosen from antioxidants, surfactants, other preservatives, film forming, keratolytic or comedolytic agents, perfumes, flavorings and colorings. Antioxidants such as t-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene and .alpha.-tocopherol and its derivatives can be added.

Also contemplated are combination products that include one or more polypeptides of the present invention and one or more other anti-microbial agents. For example, a variety of antibiotics can be included in the pharmaceutical compositions of the invention, such as aminoglycosides (e.g., streptomycin, gentamicin, sisomicin, tobramycin and amicacin), ansamycins (e.g. rifamycin), antimycotics (e.g. polyenes and benzofuran derivatives), .beta.-lactams (e.g. penicillins and cephalosporins), chloramphenical (including thiamphenol and azidamphenicol), linosamides (lincomycin, clindamycin), macrolides (erythromycin, oleandomycin, spiramycin), polymyxins, bacitracins, tyrothycin, capreomycin, vancomycin, tetracyclines (including oxytetracycline, minocycline, doxycycline), phosphomycin and fusidic acid.

Additionally, the polypeptides are well suited to formulation as sustained release dosage forms and the like. The formulations can be so constituted that they release the active polypeptide, for example, in a particular part of the intestinal or respiratory tract, possibly over a period of time. Coatings, envelopes, and protective matrices may be made, for example, from polymeric substances, such as polylactide-glycolates, liposomes, microemulsions, microparticles, nanoparticles, or waxes. These coatings, envelopes, and protective matrices are useful to coat indwelling devices, e.g., stents, catheters, peritoneal dialysis tubing, draining devices and the like.

For topical administration, the therapeutic agents may be formulated as is known in the art for direct application to a target area. Forms chiefly conditioned for topical application take the form, for example, of creams, milks, gels, dispersion or microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, aerosol formulations (e.g., sprays or foams), soaps, detergents, lotions or cakes of soap. Other conventional forms for this purpose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols. Thus, the therapeutic polypeptides of the invention can be delivered via patches or bandages for dermal administration. Alternatively, the polypeptide can be formulated to be part of an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer. For long-term applications it might be desirable to use microporous and/or breathable backing laminates, so hydration or maceration of the skin can be minimized. The backing layer can be any appropriate thickness that will provide the desired protective and support functions. A suitable thickness will generally be from about 10 to about 200 microns.

Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. The active polypeptides can also be delivered via iontophoresis, e.g., as disclosed in U.S. Pat. Nos. 4,140,122; 4,383,529; or 4,051,842. The percent by weight of a therapeutic agent of the invention present in a topical formulation will depend on various factors, but generally will be from 0.01% to 95% of the total weight of the formulation, and typically 0.1-85% by weight.

Drops, such as eye drops or nose drops, may be formulated with one or more of the therapeutic polypeptides in an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, or via a plastic bottle adapted to deliver liquid contents dropwise, via a specially shaped closure.

The therapeutic polypeptide may further be formulated for topical administration in the mouth or throat. For example, the active ingredients may be formulated as a lozenge further comprising a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the composition of the present invention in a suitable liquid carrier.

The pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are available in the art. Examples of such substances include normal saline solutions such as physiologically buffered saline solutions and water. Specific non-limiting examples of the carriers and/or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions such as phosphate buffered saline solutions pH 7.0-8.0.

The polypeptides of the invention can also be administered to the respiratory tract. Thus, the present invention also provides aerosol pharmaceutical formulations and dosage forms for use in the methods of the invention. In general, such dosage forms comprise an amount of at least one of the agents of the invention effective to treat or prevent the clinical symptoms of a specific infection, indication or disease. Any statistically significant attenuation of one or more symptoms of an infection, indication or disease that has been treated pursuant to the method of the present invention is considered to be a treatment of such infection, indication or disease within the scope of the invention.

Alternatively, for administration by inhalation or insufflation, the composition may take the form of a dry powder, for example, a powder mix of the therapeutic agent and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator, insufflator, or a metered-dose inhaler (see, for example, the pressurized metered dose inhaler (MDI) and the dry powder inhaler disclosed in Newinan, S. P. in Aerosols and the Lung, Clarke, S. W. and Davia, D. eds., pp. 197-224, Butterworths, London, England, 1984).

Therapeutic polypeptides of the present invention can also be administered in an aqueous solution when administered in an aerosol or inhaled form. Thus, other aerosol pharmaceutical formulations may comprise, for example, a physiologically acceptable buffered saline solution containing between about 0.1 mg/ml and about 100 mg/ml of one or more of the polypeptides of the present invention specific for the indication or disease to be treated. Dry aerosol in the form of finely divided solid polypeptide that are not dissolved or suspended in a liquid are also useful in the practice of the present invention. Polypeptides of the present invention may be formulated as dusting powders and comprise finely divided particles having an average particle size of between about 1 and 5 .mu.m, alternatively between 2 and 3 .mu.m. Finely divided particles may be prepared by pulverization and screen filtration using techniques well known in the art. The particles may be administered by inhaling a predetermined quantity of the finely divided material, which can be in the form of a powder. It will be appreciated that the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating the particular infection, indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units. Moreover, the effective amount may be achieved using less than the dose in the dosage form, either individually, or in a series of administrations.

For administration to the upper (nasal) or lower respiratory tract by inhalation, the therapeutic polypeptides of the invention are conveniently delivered from a nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Nebulizers include, but are not limited to, those described in U.S. Pat. Nos. 4,624,251; 3,703,173; 3,561,444; and 4,635,627. Aerosol delivery systems of the type disclosed herein are available from numerous commercial sources including Fisons Corporation (Bedford, Mass.), Schering Corp. (Kenilworth, N.J.) and American Pharmoseal Co., (Valencia, Calif.). For intra-nasal administration, the therapeutic agent may also be administered via nose drops, a liquid spray, such as via a plastic bottle atomizer or metered-dose inhaler. Typical of atomizers are the Mistometer (Wintrop) and the Medihaler (Riker).

Furthermore, the active ingredients may also be used in combination with other therapeutic agents, for example, pain relievers, anti-inflammatory agents, antihistamines, bronchodilators and the like, whether for the conditions described or some other condition.

The present invention further pertains to a packaged pharmaceutical composition for controlling microbial infections such as a kit or other container. The kit or container holds a therapeutically effective amount of a pharmaceutical composition for controlling cardiac damage and instructions for using the pharmaceutical composition for reducing the effects of myocardial infarction. The pharmaceutical composition includes at least one polypeptide of the present invention, in a therapeutically effective amount such that the effects of myocardial infarction are minimized.
 

Claim 1 of 32 Claims

1. A method of increasing survival of blood vessels by reducing apoptosis of blood vessel cells during or after vascular injury in a patient consisting of administering to the patient a therapeutically effective amount of platelet derived growth factor, angiopoietin-2, and vascular endothelial growth factor, and thereby increasing survival of blood vessels by reducing apoptosis of blood vessel cells in the patient during or after vascular injury.

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