Methods of use of fibroblast growth factor, vascular endothelial growth factor and related proteins in the treatment of acute and chronic heart disease Number:7,091,179 from the United States Patent and Trademark Office (PTO) owispatent Disclosed herein is a rational, multi-tier approach to the administration of growth factor proteins in the treatment of heart disease. Also disclosed is a method to evaluate the effectiveness of the administration of growth factor proteins comprising the clinical assay of CPK-MB levels in a patient undergoing treatment with growth factor proteins. In addition, there is disclosed a method for treatment of heart disease comprising administration of a therapeutically effective amount of a growth factor protein by oral inhalation therapy.<H endothelial, fibroblast, Methods, of, use, , 7091179.html, Patent, pto, 7091179

Title: Methods of use of fibroblast growth factor, vascular endothelial growth factor and related proteins in the treatment of acute and chronic heart disease

Abstract: Disclosed herein is a rational, multi-tier approach to the administration of growth factor proteins in the treatment of heart disease. Also disclosed is a method to evaluate the effectiveness of the administration of growth factor proteins comprising the clinical assay of CPK-MB levels in a patient undergoing treatment with growth factor proteins. In addition, there is disclosed a method for treatment of heart disease comprising administration of a therapeutically effective amount of a growth factor protein by oral inhalation therapy.

Patent Number: 7,091,179 Issued on 08/15/2006 to Franco

Inventors: Franco; Wayne P. (Rocky Hill, CT)
Appl. No.: 10/731,197

Filed: December 9, 2003

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
09828330	Apr., 2001	6759386
60195624	Apr., 2000

Current U.S. Class: 514/2 ; 514/12; 514/8; 530/300

Current International Class: A61K 38/18 (20060101); A61K 38/00 (20060101)

Field of Search: 424/9.1 514/2,8,12,14 530/300,324,399

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4296100	October 1981	Franco
4409237	October 1983	Cairns et al.
5006343	April 1991	Benson et al.
5254330	October 1993	Ganderton et al.
5607918	March 1997	Eriksson et al.
5915378	June 1999	Lloyd et al.
5925012	July 1999	Murphy-Chutorian et al.
6436902	August 2002	Backstrom
6620784	September 2003	Ferrara et al.

Foreign Patent Documents


WO 98/49300	Nov., 1998	WO
WO 00/40086	Jul., 2000	WO

Other References

Sellke et al. (Jun. 1998) "Therapeutic Angiogenesis With Basic Fibroblast Growth Factor: Technique and Early Results." Ann Throac Surg 65(6): 1540-1544. cited by examiner .
Bikfalvi et al. (1997) "Biological Roles of Fibroblast Growth Factor-2." Endocrine Reviews 18(1): 26-45. cited by examiner .
Spallarossa et al. (Aug. 15, 1999) "Evaluation of Growth Hormone Administration in Patients With Chronic Heart Failure Secondary to Coronary Artery Disease." The American Journal of Cardiology 84(4): 430-433. cited by examiner .
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Vassenelli et al. (Dec. 1987) "Comparison of Different Pharmacological Interventions on Enzymatic Parameters During Acute Myocardial Infarction." Clinical Biochemistry 20(6): 441-447. cited by examiner .
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Primary Examiner: Romeo; David S.
Assistant Examiner: Gamett; Daniel C.
Attorney, Agent or Firm: Ernest D. Buff & Associates, LLC Buff; Ernest D. Pierson; Theodore J.

Parent Case Text

REFERENCE TO RELETED APPLICATIONS

This application is a division of U.S. patent application Ser. No. 09/828,330, filed Apr. 6, 2001, now U.S. Pat. No. 6,759,386, claims priority under Title 35, U.S.C. .sctn.119(e) of U.S. Provisional Application No. 60/195,624, Filed Apr. 6, 2000.

Claims

What is claimed is:

1. A method for monitoring the clinical effectiveness of the administration of a formulation comprising one or more therapeutic growth factor proteins in the treatment of coronary artery disease, the method comprising the steps of: a. selecting a patient displaying symptoms of coronary artery disease; b. administering at least one dose of an effective amount of a first therapeutic growth factor protein formulation comprising a growth factor protein selected from the group consisting of FGF-1, FGF-2, VEGF-B, and mixtures thereof by inhalation therapy; c. obtaining a sample of a biological fluid from the patient displaying symptoms of coronary artery disease; d. performing an assay of the biological fluid to determine an amount of CPK-MB present in the fluid; e. determining, based on monitoring the amount of CPK-MB present in the fluid, whether an additional dose of a therapeutic growth factor protein formulation is necessary; f. depending on the results of the step e), administering one or more additional doses of a second growth factor protein formulation comprising a growth factor protein being selected from the group consisting of FGF-1, FGF-2, VEGF-B, and mixtures thereof; and g. repeating steps c) through f) until the assayed levels of CPK-MB in the biological fluid indicates the clinical effectiveness of the administration of the pharmaceutical formulation and amelioration of the symptoms of coronary artery disease in the patient, or until there is a contraindication to continued treatment.

2. The method of claim 1 wherein the growth factor protein formulation is a dry powder formulation.

3. The method of claim 1 wherein the growth factor protein formulation is a liquid aerosol formulation.

4. The method of claim 1, wherein the symptoms of coronary artery disease are brought on by a condition selected from the group consisting of myocardial infarct, unstable angina, an acute anginal attack and reperfusion injury.

5. The method of claim 4, wherein the reperfusion injury is induced by a procedure selected from the group consisting of thrombolytic therapy, bypass surgery and angioplasty.

6. A method for monitoring the clinical effectiveness of the administration of a potentially therapeutic pharmaceutical formulation selected from the group consisting of FGF-1, FGF-2, VEGF-B, and mixtures thereof, in the treatment of chronic coronary artery disease, the method comprising the steps of: a. selecting a patient displaying symptoms of chronic coronary artery disease; b. administering at least one dose of an effective amount of a first therapeutic growth factor protein formulation comprising a growth factor protein selected from the group consisting of FGF-1, FGF-2, VEGF-B, and mixtures thereof by inhalation therapy; c. monitoring one or more clinical indicators of chronic coronary artery disease; d. determining, based on monitoring the one or more clinical indicators of chronic coronary artery disease, whether an additional dose of a therapeutic growth factor protein formulation is necessary; e. depending on the results of the step e), administering one or more additional doses of a second growth factor protein formulation comprising a growth factor protein being selected from the group consisting of FGF-1, FGF-2, VEGF-B, and mixtures thereof; and f. repeating steps c) through f) until there is a clinical indication of amelioration of the symptoms of chronic coronary artery disease in the patient, or until there is a contraindication to continued treatment.

7. The method of claim 6 wherein the growth factor protein formulation is a dry powder formulation.

8. The method of claim 6 wherein the growth factor protein formulation is a liquid aerosol formulation.

Description

FIELD OF THE INVENTION

The present invention relates generally to strategies and methods for the treatment of chronic and acute heart disease through the delivery of one or more related protein growth factors such as fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF).

BACKGROUND OF THE INVENTION

Chronic myocardial ischemia is the leading cardiac illness affecting the general population in the Western world. Since the occurrence of angina symptoms or objective physiological manifestations of myocardial ischemia signifies a mismatch between myocardial oxygen demand and the available coronary blood flow, the goal of therapy is to restore this balance. This can be achieved either by attempting to prevent further disease progression through modification of risk factors, or by more aggressive modes of therapy such as reducing the myocardial oxygen demand (i.e. reducing the heart rate, myocardial contractility or blood pressure) by using anti-anginal medications, or by restoring the blood supply by means of mechanical interventions such as percutaneous transluminal angioplasty or its variants, or coronary artery bypass surgery, coronary angioplasty (PTCA) or bypass surgery (CABG). When CABG is selected as the treatment option, its success may be limited by the inability to provide complete revascularization in those patients in whom the artery that supplies a viable but underperfused myocardial territory is not graftable because of diffuse disease, calcifications, or small size. Complete revascularization cannot be achieved in up to 37% of patients undergoing CABG. This number is probably much lower today. However, patients who undergo complete revascularization have improved 5-year survival and angina-free survival compared with patients who have incomplete revascularization. Therefore, an adjunctive treatment strategy is warranted in patients undergoing CABG if complete revascularization is not possible. Percutaneous catheter-based revascularization is often precluded secondary to the same attributes that made the myocardial territory ungraftable: diffuse disease and small or calcified vessels.

The field of angiogenesis research was initiated 30 years ago by a hypothesis that tumors are angiogenesis-dependent. Folkman, J. "Tumor angiogenesis: therapeutic implications." N. Engl. J. Med. 285: 1182 1186 (1971). Shortly thereafter, in the early 1970's, it became possible to passage vascular endothelial cells in vitro for the first time. Bioassays for angiogenesis were developed subsequently through that decade. The early 1980's saw the purification of the first angiogenic factors. Clinical applications of angiogenesis research are being pursued along three general lines: 1) prognostic markers in cancer patients; 2) anti-angiogenic therapy (in cancer treatment); and 3) angiogenic therapy (treatment of heart disease).

In discussing the field of angiogenesis, it is important to differentiate 3 different processes that contribute to the growth of new vessels. Vasculogenesis is the primary process responsible for the growth of new vasculature during embryonic development, and it may play an as yet undefined role in mature adult tissues. Arteriogenesis refers to the appearance of new arteries possessing fully developed tunica media, while true angiogenesis describes the growth of collateral-like vessels lacking the development of media. In the case of coronary circulation, arteriogenesis is usually taken to mean new, angiographically visible epicardial vessels while angiogenesis refers to thin-walled intramyocardial collaterals.

Occlusion of coronary arteries is often associated with development of collateral circulation in patients with atherosclerosis. Although the existence of collateral circulation in such patients is associated with improved clinical outcomes, the net effect is rarely adequate to compensate fully for the flow lost to occlusion of native epicardial coronary arteries. A number of growth factors have been associated with myocardial and peripheral limb ischemia, particularly basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (FGF-1), and vascular endothelial growth factor (VEGF), which have been shown to induce functionally significant angiogenesis in animal models of myocardial and limb ischemia. These promising preclinical results have rapidly lead to the study of these growth factors in patients with chronic myocardial ischemia using intracoronary (IC), intravenous (i.v.), and local delivery (myocardial injection).

Therapeutic myocardial angiogenesis is a novel approach to the treatment of myocardial ischemia based on the use of proangiogenic growth factors to induce the growth and development of new blood vessels to supply the myocardium at risk. Angiogenesis is a complex process involving endothelial and smooth muscle cell proliferation and migration, formation of new capillaries, and extracellular matrix turnover. Various heparin-binding growth factors, including basic fibroblast growth factor (FGF-2), acidic fibroblast growth factor, and vascular endothelial growth factor (VEGF) induce angiogenesis in chronic myocardial ischemia. Given the typically long time course of new collateral vessel development, most attempts to stimulate myocardial angiogenesis have used methods of prolonged growth factor delivery, including gene therapy, continuous infusions, repeated injections, or sustained release polymers. However, some of these options are not feasible or practical in patients with ischemic heart disease, making single dose administration, if effective, a potentially superior strategy in these patients.

Angiogenesis is a complex process that involves endothelial cell migration and proliferation, extracellular matrix breakdown, attraction of pericytes and macrophages, smooth muscle cell proliferation and migration, formation and "sealing" of new vascular structures, and deposition of new matrix. A number of growth factors, including the fibroblast growth factors (FGF) and vascular endothelial growth factors (VEGF) are integrally involved in the angiogenic response in ischemic conditions and in certain pathological states. The availability of these factors has led to studies, which have demonstrated a therapeutic benefit in various animal models of acute and chronic myocardial ischemia. In particular, basic fibroblast growth factor is an attractive candidate as an agent for therapeutic angiogenesis.

The therapeutic goal of attempting to ameliorate chronic ischemic conditions through revascularization by administration of various protein growth factors is feasible only due to the chronic nature of the condition and the resulting long-term time scale for treatment. In acute clinical situations, such as myocardial infarct, or therapeutic procedures likely to lead to reperfusion injury, the luxury of long time scales for revascularation is not available. However, the administration, via various routes, of growth factors such as FGF has been demonstrated to be effective in reducing the effects of myocardial infarct within a time frame that precludes a therapeutic contribution from the angiogenic function of these proteins. See, for example, my earlier U.S. Pat. No. 4,296,100, the disclosure of which is hereby incorporated specifically by reference. Thus, by a mechanism yet to be elucidated, protein growth factors such as FGF and VEGF and related proteins are capable of demonstrating a therapeutic utility in situations involving acute damage to the heart.

SUMMARY OF THE INVENTION

In a first embodiment, the present invention provides a method for the systematic, multi-tiered treatment of heart disease by delivery of therapeutic growth factor proteins comprising the steps of a.) selecting a patient displaying symptoms of heart disease; b.) administering at least one dose of an effective amount of a first therapeutic growth factor protein formulation by oral inhalation; c.) monitoring levels of CPK-MB in the patient; d.) determining whether administration of the growth factor protein formulation was effective in treating the symptoms of heart disease in the patient; e.) administering one or more additional doses of a second growth factor protein formulation by a method of delivery more invasive than delivery by oral inhalation; and f.) repeating steps c.) through e.) until there is a clinical indication of amelioration of the symptoms of heart disease in the patient, or until there is a contraindication to continued treatment. Preferably, the protein formulation comprises a growth factor protein selected from the group consisting of FGF-1, FGF-2, VEGF, and mixtures thereof. In one aspect of this embodiment, the method of the invention contemplates application where the symptoms of heart disease are acute. These acute symptoms of heart disease can be brought on by a condition selected from the group consisting of myocardial infarct, unstable angina, an acute anginal attack, and reperfusion injury. Furthermore, the reperfusion injury is induced by a procedure selected from the group consisting of thrombolytic therapy, bypass surgery and angioplasty. Alternatively, the method of the present invention contemplates applicatin where the symptoms of heart disease are chronic.

In an alternative embodiment, the method of the present invention encompasses the administration of therapeutic amounts of a growth factor protein formulation in the treatment of heart disease by delivering the protein formulation by inhalation therapy. Preferably, in the practice of the present invention, the protein formulation is a dry powder formulation. Alternatively, the protein formulation is a liquid aerosol formulation.

In another embodiment, the present invention provides a method for monitoring clinical effectiveness of administration of a growth factor protein formulation in the treatment of heart disease, the method comprising the steps of obtaining a sample of a biological fluid from a patient displaying symptoms of heart disease; performing an assay of the biological fluid to determine an amount of CPK-MB present in the fluid; administering a therapeutic amount of a growth factor protein formulation to the patient; and repeating the last two steps until the assayed amount of CPK-MB in the biological fluid has decreased by an amount sufficient to indicate the clinical effectiveness of the administration of the growth factor protein formulation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the lung, indicating a mechanism of delivery of aerosol drug particles through the lung and into the bloodstream.

FIG. 2 is an illustration of the results of measured regional wall thickening in the LAD (normal) and LCX (collateral-dependent) distribution.

FIG. 3 is an illustration of, at top, MRI perfusion images of the left ventricle and, at bottom, the ischemic zone extent in all groups of test animals.

FIG. 4 is an illustration of histopathological sections from the LCX distribution demonstrating an increased number of capillaries in all treatment groups.

DETAILED DESCRIPTION OF THE INVENTION

Recent advances in growth factor therapy for the treatment of ischemic disease of the heart and peripheral vasculature offer hope of a novel treatment strategy that is based on generation of new blood supply in the diseased heart. Members of the fibroblast growth factor family, vascular endothelial growth factor family, and several other molecules have all been shown to result in functionally significant angiogenesis in animal models of acute and chronic myocardial and peripheral limb ischemia. The promising preclinical data have propelled the use of these angiogenic growth factors in clinical studies of ischemic heart and peripheral vascular disease. These growth factors are presumed to induce neovascularization by stimulating endothelial and smooth muscle cell proliferation and migration, dissolving the extracellular matrix, attracting pericytes and macrophages, and finally forming and "sealing" new vascular structures with deposition of new matrix.

In the surgical treatment of acute ischemic conditions, approximately 500 000 PTCA and 375 000 CABG procedures are performed annually in the United States. However, a significant number of patients are suboptimal candidates for CABG or PTCA or do not achieve complete revascularization with these procedures. These patients would likely benefit from additional measures to achieve enhanced revascularization, and therapeutic angiogenesis may serve this role. Several studies have demonstrated that chronic administration of FGF-2 results in significant myocardial angiogenesis in animal models of myocardial ischemia and infarction. However, because of the protracted time course required for new collateral vessel development, many attempts to stimulate myocardial angiogenesis have used methods of prolonged growth factor delivery, including gene therapy, continuous infusions, repeated injections, and sustained release polymers. Many of these therapeutic strategies, particularly those requiring repeated access or major surgical intervention, are impractical, or less than ideal, from a clinical standpoint. The pericardial space offers potentially unique advantages in convenience, safety, and efficacy as a cardiovascular drug depot site for the administration of proangiogenic growth factors.

In investigating the effects of a single intrapericardial injection of increasing FGF-2 doses in a porcine model of chronic myocardial ischemia, separate saline and heparin control arms were used to address the potential angiogenic effects of heparin alone or in combination with FGF-2. However, no significant differences were found between the heparin (at the dose used) and saline arms in any of the measured parameters. Intrapericardial FGF-2, on the other hand, resulted in an improvement in left-to-left angiographic collaterals, occluded LCX coronary artery blood flow, LCX (ischemic territory) myocardial perfusion, and LCX regional wall function as measured by MRI. Improvements in ischemic territory regional wall function and myocardial perfusion were positively correlated with FGF-2 dose, with near normalization of wall function and perfusion in the 2 mg FGF-2 group. Qualitative histopathologic examination showed increased myocardial vascularity in FGF-2-treated animals without any adverse findings.

In considering growth factor-induced neovascularization, it is important to distinguish intramyocardial collateral developmentfrom formation of epicardial collaterals (neoarteriogenesis). The process of intramyocardial collateral development (angiogenesis) is characterized by appearance of thin-walled vessels with poorly developed tunica media generally under 200 .mu.m in diameter and by an increase in the number of true capillaries (<20 .mu.m in diameter containing only a single endothelial layer), whereas the neoarteriogenesis is characterized by development of larger vessels (>200 .mu.m in diameter) with well developed tunica media and adventitia that usually form close to the site of the occlusion of a major epicardial coronary artery (bridging collaterals) or extend from one coronary artery to the other. The distinction between these two groups of newly formed vessels is important not only from the point of view of their location but also because stimuli for their development appear to be quite different and because they may exhibit different physiological properties. It is unclear whether intrapericardially administered FGF-2 exerts its beneficial effects on myocardial revascularization by acting on the epicardial surface (where it is in greatest concentration) to induce collateralization around sites of occlusion in the epicardially situated major coronary arteries, or whether it diffuses into the myocardium and myocardial microcirculation to induce angiogenesis at a more microscopic level, or both. However, the demonstrated effectiveness of the low-dose (30 .mu.g) intrapericardial FGF-2 suggests that the presence of FGF-2 on the epicardial surface may play a key role in inducing functionally significant angiogenesis.

Fibroblast Growth Factors

Acidic fibroblast growth factor (aFGF), also referred to as FGF-1, is a monomeric, acidic protein of approximately 18 kDa. It shares about 55% homology with the basic protein FGF-2. Both are prototypes for the FGF family members and their three dimensional structure are known.

Basic fibroblast growth factor (bFGF), also referred to as FGF-2, is a 16.5 Kd 146 amino acid protein that belongs to the FGF family, which now comprises more than 22 structurally related polypeptides. One of the key differences between the various FGFs is the presence or absence of the leader sequence required for conventional peptide secretion (absent in FGF-1 and FGF-2). Another difference is the varied affinity for the different isoforms of FGF receptors. As for most heparin-binding growth factors, bFGF binds with high affinity to cellular heparin sulfates and, with even higher affinity, to its own tyrosine kinase receptors (FGF receptors 1 and 2). The ability of bFGF to bind cell surface and matrix heparin sulfates serves both to prolong its effective tissue half-life and to facilitate its binding to the high affinity receptors. While bFGF is present in the normal myocardium, its expression is stimulated by hypoxia and hemodynamic stress.

FGF-2 is a pluripotent mitogen capable of stimulating migration and proliferation of a variety of cell types including fibroblasts, macrophages, smooth muscle and endothelial cells. In addition to these mitogenic properties, FGF-2 can stimulate endothelial production of various proteases, including plasminogen activator and matrix metalloproteinases, induce significant vasodilation through stimulation of nitric oxide release and promote chemotaxis. FGF-2 is present in the normal myocardium and its expression is potentiated by hypoxia or hemodynamic stress. Because of its heparmn-binding properties, FGF-2 binds avidly (Kd 10.sup.-9 M) to endothelial cell surface heparin sulfates. This interaction serves to prolong effective tissue half-life of the FGF-2 protein, facilitates its binding to its high-affinity receptors and plays a key role in stimulation of cell proliferation and migration. bFGF also possesses a plethora of other biological effects such as the ability to stimulate NO release, to synthesize various proteases, including plasminogen activator and matrix metalloproteinases, and to induce chemotaxis. Homozygous deletion of the bFGF gene is associated with decreased vascular smooth muscle contractility, low blood pressure and thrombocytosis. One interesting aspect of bFGF is its biological synergy with VEGF. Thus, a combination of bFGF and VEGF is far more potent than bFGF alone in inducing angiogenesis in vitro and in vivo. Furthermore, bFGF induces VEGF expression in smooth muscle and endothelial cells.

Despite significant levels of bFGF in normal tissues, the growth factor does not appear to be biologically active, as suggested by the lack of on-going angiogenesis. While the precise explanation for this lack of activity of the endogenous bFGF is uncertain, contributing factors probably include very low levels of expression of FGF receptors 1 and 2 and syndecan-4, another transmembrane protein involved in bFGF-dependent signaling. In addition, endogenous bFGF may be sequestered in the extracellular matrix by binding to heparin sulfate-carrying proteoglycan percelan and, thus, be unavailable to bind to its signaling receptors.

Vascular Endothelial Growth Factor

Similar to bFGF, vascular endothelial growth factor (VEGF) transcripts are detected in all cardiac tissues. VEGF and the expression of its receptors in the heart are induced 7-fold by hypoxia/ischemia. The unique feature of VEGF was thought to be the narrow spectrum of activity, presumed to be confined to endothelial cells because of the restricted expression of its receptors. However, recent studies suggest VEGF receptor expression is more widespread and includes monocytes and some smooth muscle cells. Furthermore, VEGF is capable of inducing bFGF expression, thereby further increasing its biological spectrum of activity. VEGF is a potent and specific mitogen for vascular endothelial cells that is capable of stimulating angiogenesis during embryonic development and tumor formation. The VEGF family of structurally related growth factors has five mammalian members, VEGF, VEGF-B, VEGF-C, VEGF-D, and placenta growth factor (PIGF), all encoded by separate genes. Stacker, S. A. and Achen, M. G. "The vascular endothelial growth factor (VEGF) family: signaling for vascular development." Growth Factors 17: 1 11 (1999).

Fibroblast Growth Factor (FGF), in its human and bovine basic forms, and in its human acidic form, has been used successfully to treat ischemic heart disease, including chronic angina, by stimulating angiogenesis (the growth of new blood vessels). Vascular Endothelial Growth Factor (VEGF) has also been used to treat ischemic heart disease by stimulating angiogenesis. FGF has been demonstrated to show a reduction in acute myocardial infarct size after treatment. Treatment of myocardial infarct (MI) requires acute intervention by health care professionals, whereas angiogenesis may take at least several days to occur to a sufficient extent to demonstrate any clinical benefit in the affected patient. It is the purpose of this invention to utilize a form of FGF and/or VEGF, or other related growth factor proteins, to bring immediate relief from MI, unstable angina, or an anginal attack and then, utilizing the same or an alternate delivery system, to promote angiogenesis for the relief of subacute or more chronic symptoms.

Angiogenesis begins when blood-starved areas of the heart send out receptor signals. The purpose of this invention is achieved by administration to the affected patient of an effective amount of a form of FGF and/or VEGF via inhalation delivery techniques. Inhalation treatment with FGF and/or VEGF for the management of coronary artery disease should be successful because the lung is one of the least blood or oxygen starved organs. The FGF or VEGF would end up on the left atrium of the heart and from there travel to the coronary arteries where it would be most useful. The inhalation of FGF and/or VEGF into the lungs could be used for the treatment of MI, unstable angina, or an anginal attack. This delivery system could also be used before, during, and/or after thrombolytic therapy (such as administration of recombinant tissue plasminogen activator) to help alleviate ischemic or reperfusion injury.

After successful treatment of acute myocardial infarct or acute ischemia via the methods of the present invention, angiogenesis may also occur. If it does not occur within two or three weeks, then the inhalation therapy could be repeated or the FGF and/or VEGF could be given through a catheter into the coronary arteries or by direct injection in the left atrium, or ventricular myocardium via a limited thoracotomy. For the treatment of acute myocardial infarct (with or without thrombolytic therapy), unstable angina or an anginal attack, the least invasive method would be preferred. Besides inhalation into the lungs, other available methods of delivery could be sublingual, intranasal, or IV utilizing one of the forms of FGF and/or VEGF. If the least invasive approaches are not successful in the treatment of acute myocardial infarct or acute ischemic, then alternate deliver systems should be explored. As clinically indicated, the FGF and/or VEGF could be given through a catheter into the coronary arteries or by direct injection into the left atrium or ventricular myocardium via a limited thoracotomy. To assess the efficacy of VEGF or FGF in the treatment of acute myocardial infarct or unstable angina, you could follow levels of creatine phosphokinase-myocardial band (CPK-MB) isoenzymes. Even a minimal elevation above normal range would be considered significant. With treatment, the rise in the level should be less when compared to placebo.

Alternative methods of delivery for treatment of coronary artery disease should also be considered. FGF and/or VEGF could be administered directly into the myocardium during transmyocardial laser revascularization, into the coronary arteries during angioplasty, or by injection into the coronary arteries, myocardium or bypass grafts during coronary bypass surgery. When injected into the myocardium, slow release forms of FGF or VFGF should be considered. It might also be possible to inject FGF and/or VFGF into the myocardium via a catheter passed during cardiac catheterization.

To promote angiogenesis for the relief of chronic angina or ischemia the least invasive method would be preferred. The inhalation of FGF and/or VEGF into the lungs could be used to achieve this goal. Besides inhalation into the lungs, other available methods of delivery could be sublingual, intranasal, or IV utilizing one of the forms of FGF and/or VEGF. If the least invasive approaches are not successful in promoting angiogenesis, then alternate delivery systems should be explored. As clinically indicated the FGF and/or VEGF could be given through a catheter into the coronary arteries or by direct injection into the left atrium or ventricular myocardium via a limited thoracotomy.

Methods of Delivery of Growth Factors

Despite promising preclinical data, the progression of angiogenic growth factor therapy to the clinical trials stage awaits a practical delivery strategy. This requirement essentially eliminates all forms of prolonged or frequent repetitive intracoronary infusions. Local perivascular delivery is more easily adaptable to clinical trials, but it requires open-chest surgery. One such form of delivery is heparin alginate capsules that provides prolonged (4 to 5 weeks) first-order kinetics release of the growth factor from the polymer. The capsules are easily implanted and do not provoke an inflammatory response. One potential advantage of perivascular delivery is the absence of the endothelial barrier and the absence of the rapid washout that is typical with intravascular administration.

The pericardial space may potentially serve as a drug delivery reservoir that might be used to deliver therapeutic agents to the heart. Chronic intrapericardial FGF-2 delivery in a rabbit model of angiotensin II-induced cardiac hypertrophy resulted in a localized myocardial angiogenic response. A single intrapericardial injection of FGF-2 with or without heparin resulted in localized angiogenesis and myocardial salvage in a canine model of myocardial infarction. Moreover, the concentration of FGF-2 and VEGF in the pericardial fluid of patients with unstable angina has been documented to be higherthan that in patients with nonischemic heart disease, suggesting that increases in the levels of proangiogenic growth factors inthe pericardial space may reflect an endogenous and, indeed, physiological response to myocardial ischemia and injury. Accordingly, the pericardium may serve as a useful reservoir for proangiogenic growth factor administration in patients with coronary disease.

An alternative approach to perivascular administration of bFGF involves intrapericardial delivery of the growth factor. A major advantage of this approach is that it can be accomplished via a catheter, obviating the need for open-chest surgery. However, the clinical application of intrapericardial delivery is limited to a small number of patients currently being enrolled in coronary angiogenesis trials because of the high prevalence (80 to 90%) of prior coronary artery bypass surgery in this group of patients.

The feasibility of short duration intracoronary or intravenous infusions and endomyocardial injections has also been tested in animal models. Intravenous infusions are appealing because of their practicality, low cost and applicability to a broad group of patients. Furthermore, treatment can be easily repeated and may not require any special facilities. The downside includes systemic exposure to a growth factor and the potential for adverse effects such as NO-mediated hypotension.

Intracoronary infusions are easily carried out in any cardiac catheterization laboratory and are also applicable in most patients with coronary disease. However, the need for left heart catheterization limits this approach to a single session or, at most, infrequent repetitions. While somewhat more "local" than intravenous infusions, intracoronary infusions are also likely to result in systemic exposure to the growth factor and may precipitate systemic hypotension. A variation on the same theme is transvascular intracoronary administration with a local delivery catheter. This approach, while it is potentially feasible, remains experimental at this time, and it is still associated with significant systemic recirculation.

Detailed evaluation of tracer-labelled growth factor uptake and retention in the myocardium, and its systemic distribution following intracoronary and intravenous infusions, demonstrated that both forms of delivery are associated with relatively low uptake in the target (ischemic) area of the heart. Thus, at 1 hour after injection, 0.9% of the injected bFGF was found to be present in the ischemic myocardium following intracoronary administration and 0.26% following intravenous administration. Perhaps more importantly, 24 hours later, very small amounts of the growth factor remained in the myocardium (0.05% for intracoronary and 0.04% for intravenous administration).

Intramyocardial delivery of growth factors is the least evaluated form of therapy at this time. The appeal of this mode of delivery includes the possibility of targeting the desired areas of the heart, which is likely to provide higher efficiency of delivery and prolonged tissue retention. The drawbacks are its invasive nature, and requirements for highly specialized equipment and a high skill level for the operator. Furthermore, no conclusive data regarding the physiological efficacy of this mode of administration are available to date.

The pharmacokinetics and tissue distribution of protein growth factors administered by various techniques have not been clearly defined. Although an i.v. delivery strategy is very appealing in terms of technical safety, ease of administration, and lack of need for cardiac catheterization, it is unclear whether i.v. delivered growth factors achieve therapeutic myocardial concentrations without untoward systemic effects. In addition, IC infusions may not result in more significant myocardial deposition and retention with the added invasiveness of the delivery technique. The relevance of tissue distribution becomes apparent when one considers the potential systemic toxicity of these agents in terms of hemodynamic effects, recirculation, and organ deposition, with the potential to induce pathologic angiogenesis and tumor genesis.

Pulmonary Routes of Administration Pulmonary delivery of potentially therapeutic agents provides a direct route to the circulation, with a minimum of discomfort and pain, and is a cost-effective alternative in comparison to the more invasive routes of delivery typically utilized for administration of FGF, VEGF, and related proteins. Traditionally, noninvasive delivery systems do not work for macromolecules; pills or tablets enter the stomach, where enzymes and hydrochloric acid rapidly degrade the protein or peptide. The oral administration of proteins and peptides is under investigation, but no satisfactory system is commercially available yet. No acceptable transdermal delivery systems have been found because of proteins' size constraints or inherent physical properties that prohibit these large molecules from crossing the diverse layers of the skin without the addition of irritating enhancers.

The biology of the lung makes it a favorable environment for noninvasive drug delivery (see FIG. 1). Studies have shown that most large-molecule agents are absorbed naturally by the lungs, and once absorbed in the deep lung, they pass readily into the bloodstream without the need for enhancers used by other noninvasive routes. Patton, J. S. Adv. Drug Delivery Rev. 1996, 19, 3. On inhalation, air passes through the trachea, which branches more than 17 times into successively smaller tubes that constitute the bronchial network, eventually reaching the grapelike clusters of tiny air sacs known as alveoli. Each breath of air is distributed deep into the lung tissue, to the alveolar epithelium, the surface area of which measures .sup..about.100 m.sup.2 in adults--roughly equivalent to the surface area of a standard singles tennis court. This large area is made up of about half a billion alveoli, from which oxygen passes into the bloodstream via an extensive capillary network.

The potentially most significant barrier to the delivery of compounds via the lungs is the tightly packed, single-cell-thick layer known as the pulmonary epithelium. In the lungs, the epithelium of the airway is very different from that of the alveolus. Thick, ciliated, mucus-covered cells line the surface of the airway, but the epithelial cell layer thins out as it reaches deeper into the lungs, until reaching the tightly packed alveolar epithelium. Most researchers believe that protein absorption occurs in the alveoli, where the body absorbs peptides and proteins into the bloodstream by a natural process known as transcytosis.

Logically, there is no reason to expect safety problems related to the inhalation of a substance to be any different from those associated with the injection of the same amount of the substance. A growing quantity of safety data indicates that inhaling proteins can be safe for patients with healthy or diseased lungs. The safety of therapeutic inhalation is further supported by the existence of more than 20 small-molecule and one large-protein drug inhalation products approved by the U.S. Food and Drug Administration (FDA); this group of therapeutic inhalants contains 13 different excipients.

Most aerosol systems today deliver a total amount of <100 .mu.g of drug per puff to the deep lung; this amount is too low to enable timely delivery of many macromolecules if the required dose is in the milligram doses. Traditional inhalation systems have been designed primarily to deliver some of the most potent drugs in use today, the bronchodilators and bronchosteroids to treat asthma. Both types of compounds are bioactive in the lung at 5 20 .mu.g per dose. In contrast, many peptide and protein compounds need to be delivered to the deep lung at much larger doses of 2 20 mg. Adjei, A. L.; Gupta, P. K. Inhalation Delivery of Therapeutic Peptides and Proteins; Marcel Dekker: New York, 1997.

Bioavailability After the aerosolized drug reaches the deep lung, it must be absorbed with high enough bioavailability to make the system practical. As early as 1925, insulin inhalation for the treatment of diabetes was shown to work in humans, but the bioavailability was low (<3%). More recently, several inhalation studies comparing insulin administration by aerosol inhalation (using cumbersome devices) and by subcutaneous injection for the reproducibility of dosing have shown that the variability in glucose response to the two methods was equivalent. Bioavailability in more recent studies with aerosol insulin was up to 25%, supporting the use of such a method of administration. Laube, B. L.; Georgopolos, A.; Adams, G. K. J. Am. Med. Assoc. 1993, 269, 2106. Insulin administered by oral inhalation effectively normalized diabetic patients' plasma glucose levels without adverse effects. Numerous patents have issued, directed to methods, formulations and devices for the oral administration of insulin via inhalation therapy. See, for example, U.S. Pat. Nos. %,952,008; 5,858,968; and 5,915,378, the disclosures of which are hereby incorporated specifically by reference.

Bioavailability studies in humans of the aerosol administration of lutenizing hormone-releasing hormone (LHRH), a decapeptide, and its analogues also have demonstrated that appropriate bioactive systemic levels can be achieved to treat conditions such as endometriosis and prostate cancer. Thus, using delivery and formulation technology available today, as would be recognized by one of skill in the appropriate art, it will be possible to deliver an effective amount of FGF and/or VEGF, and related growth factor proteins, in the treatment of chronic and acute heart disease.

The mechanism of macromolecule absorption in the deep lung is thought to occur via normal physiological processes that can deliver active compounds with relatively high bioavailability without requiring the addition of penetration enhancers. LHRH analogues (used in treating osteoporosis), composed of 10 amino acids, can reach 95% bioavailability; however, interferon-.alpha.(used in treating hepatitis B and C), composed of 165 amino acids, attains 29% bioavailability. Some smaller peptides such as glucagon (29 amino acids) and somatostatin (28 amino acids) reach 1% bioavailability. The degree of bioavailability is thought to depend on the peptide or protein susceptibility to certain hydrolytic enzymes in the lung.

How a macromolecular drug is formulated also affects its delivery to the deep lung. Many macromolecules are formulated as dry powders because they are more stable as solids than as liquids. Compared with liquid aerosol particles, which are mostly water (97%), dry powder aerosol particles can carry 50 100% of the drug. In general, more puffs would be necessary to deliver the equivalent amount of drug to the alveolar epithelium from a liquid aerosol device. Liquid formulations also carry the risk of microbial growth; the risk of lung infections due to bacterial and fungal contaminants is greatly reduced with dry powder systems. By greatly lowering the possibility of microbial contamination, dry powder systems offer a safer technology.

In the liquid state, individual protein or peptide molecules are extremely mobile. When water is removed, macromolecules usually pack together in an amorphous state, unlike the highly ordered packing that occurs in crystallization. When water is removed from proteins, the protein molecules remain mobile and chemical stability stays low in the initial amorphous powder that forms. When a critical amount of water has been removed, a kind of molecular gridlock occurs, producing a greatly increased chemical stability called the "amorphous glass state." In this state, previously mobile molecules slow down drastically. As long as the glass transition temperature of the powder is higher than any environmental temperatures that may occur during normal human use, the powder will remain in a glass state.

Systematic, Multi-Tiered Approach to the Use of Growth Factor Proteins in the Treatment of Acute and Chronic Heart Disease.

Of the various treatment modalities currently in use or under investigation for the delivery of therapeutically effective doses of various growth factor proteins, a wide range of levels of invasiveness are involved. Obviously, intravenous administration is among the least invasive, but questions remain as to the ultimate delivery of the proteins to physiological sites at therapeutically effective levels. Next most invasive is intracoronary infusion through catheters. Although requiring surgical intervention, the insertion and manipulation of catheters has seen increasingly widespread use in the treatment of the symptoms of heart disease and a number of other clinical conditions. However, for most, if not all, cardiac patients, there is a very low level of toleration of such catheterizations procedures, so that the possibility of repeated deliver of growth factor proteins is extremely limited.

Next on the relative scale of invasiveness is intrapericardial injection of growth factors. Although requiring more substantive surgical procedures, this technique can be utilized in conjunction with other surgical procedures such as coronary artery bypass surgery and would, thus, not constitute an additional traumatic burden on the patent. Alternatively, relatively minor incisions can be made in the chest wall to permit direct interpericardial delivery. Again, due to the invasive nature of the procedures utilized in this manner of delivery, the realistic possibility of repeated administration via this route is very low.

At the most invasive end of the spectrum is direct myocardial injection of FGF and related proteins. This, of course, requires open heart surgery to achieve access to the delivery site. As such, this approach is feasible only when used in conjunction with surgical intervention for other purposes, such CABG. Again, the major drawback here is that there is very little practical opportunity for repeated delivery of the therapeutic protein.

At the opposite end of the invasiveness spectrum, intra-pulmonary inhalation therapy, preferably via dry powder formulations, offers significant advantages over previous delivery strategies. As discussed above, formulation and delivery technology has reached a state where a number of therapeutic macromolecules, including insulin, can now be delivered consistently, and at clinically effective levels via inhalation therapy. An added advantage arising from the non-invasive nature of inhalation therapy is that it is particularly attractive in the treatment of chronic heart conditions that require repeated dosing over longer time intervals. The non-invasive nature of the therapy also proves to be of significant advantage in the treatment of acute heart conditions such as the onset of a myocardial infarct. For patients known to be at risk for such a cardiac event, it will be possible to carry a relatively compact dry powder inhalation device so that at the onset of symptoms, the patient can self-administer a dose of growth factor that may prove to be significantly effective in reducing the damage induced by the MI, and may eventually prove to constitute the difference between life and death.

Recognizing the scope of therapies potentially available in the treatment of both acute and chronic heart disease, it is therefore an aspect of the present invention to provide a systematic, multi-tiered therapeutic approach to the administration of FGF, VEGF and related growth factor proteins. This approach must, of necessity, recognize the relative invasiveness of different treatment modalities, and the likelihood of repeated recourse to such treatment procedures.

In implementing the rational, multi-tiered therapeutic approach of the present invention, it is recognized that differing approaches need be taken with respect to chronic and acute conditions. In the case of chronic conditions, the initial tier of therapeutic treatment is the administration of therapeutic levels of FGF (acidic or basic), VEGF, or related growth factor proteins, either individually or in combination, via dry powder inhalation therapy. Ideally, this therapy should be utilized as soon as possible after the onset of acute symptoms. For this form of delivery, repeated doses can be administered, at levels and at dosage ranges as set forth in the examples below.

Upon appropriate monitoring of the clinical effectiveness of the initial tier of therapy, as disclosed herein, the health practitioner can assess the advisability of proceeding to the next tier of interventional therapy. As described above, the next most invasive level of therapy would entail the intracoronary delivery, via catheter, of therapeutic doses of one or more of the growth factor proteins. As alluded to above, in the acute stage of, heart disease, the health practitioner does not have the option of a great deal of time in which to assess the success of alternative treatment options. Thus, the ability to assess, short-term, the efficacy of a particular treatment is essential to formulating the overall therapeutic strategy. The methods of the present invention, disclosed below, for assessing on a short term basis the effectiveness of growth factor protein treatment are essential to the rational, multi-tiered approach to the treatment of heart disease disclosed and claimed herein.

Upon assessment that the clinical effectiveness of intracoronary delivery of the protein growth factor has not met the desired therapeutic goal, the health care provider must consider options involving far more invasive surgical intervention. Among these would be the intra-pericardial injection of FGF, VEGF, and/or other related protein growth factors. If the health care provider has reached the point in assessment of therapeutic options where coronary angioplasty (PTCA) or bypass surgery (CABG) is mandated, then the delivery of one or more growth factor proteins becomes feasible. At this level of therapy, for patients whose condition does not require PTCA or CABG, but whose response to previous levels of therapy has not been adequate, an alternative option is to utilize a limited thoracotomy for intrapericardial delivery of the therapeutic protein(s).

At a final level of therapeutic intervention, FGF or other protein may be delivered by direct injection into the myocardium during transmyocardial laser revascularization, or during coronary bypass surgery. At this level of treatment, it is also possible to implant slow-release beads comprising the therapeutic protein for both long- and short-term benefit.

An additional aspect of the treatment of acute symptomatic conditions is that, unlike uncontrollable incidents arising from unstable angina, acute anginal attacks, or onset of myocardial infarct, certain therapeutic procedure have the potential to create symptoms that can be alleviated through administration of FGF, VEGF, and/or related proteins. Specifically, reperfusion injury can occur during any procedure when blood flow is temporarily curtailed or restricted, upon reinstitution of full blood flow. Examples would be in the course of thrombolytic therapy (such as the administration of recombinant tissue plasminogen activator), as well as in bypass surgery and angioplasty. As the data included herein demonstrate, the extent of reperfusion injury that can result in such situations can be ameliorated through administration of FGF, VEGF, and/or related proteins prior to reinstatement of full blood flow. Thus, the rational, multi-tier therapeutic approach for the treatment of acute conditions of the present invention can be modified to include the administration of the appropriate growth factor protein or mixtures thereof prior to initiation of the procedure raising the risk of reperfusion injury.

The rational, multi-tier approach to treatment of heart disease with FGF, VEGF and/or related growth factor proteins can be adapted to treatments for chronic, as opposed to acute, conditions. The initial tier, as with acute conditions, is based on delivery of the therapeutic proteins via inhalation therapy, preferably using dry powder formulations. Thus, for patients exhibiting the symptoms of chronic ischemic disease, initial treatment involves inhalation therapy with a therapeutically effective amount and formulation of FGF, VEGF, and/or related proteins according to a dose level and dosing regimen as set forth in the Examples below. Due to the long-term nature of such conditions, the progress from less invasive to more invasive treatment modalities does not need to progress on a shortened time scale as is the case for treatment of acute conditions. Thus, multiple administrations of the protein(s) via inhalation therapy are possible, preferably accompanied by clinical evaluation of the effectiveness of previous treatments. In this fashion, dose levels and/or dose schedules can be adjusted based upon the results of periodic clinical evaluation of the presence of markers such as CPK-MB, as disclosed more fully below.

If the clinical evaluations do not reveal sufficient progress in amelioration of symptoms associated with the disease state, then the health care provider can move to the next tier, or level, of treatment, moving further along the spectrum of increasing invasiveness. Thus, the next tier would involve intracoronary perfusion via catheter. After a period of monitoring of the therapeutic effectiveness of the intracoronary perfusion, the health care provider can assess whether it will b necessary to move to the next, more invasive, tier of treatment. Assuming that the patient's condition has not responded to treatment to date, then it is likely that the health care provider will be forced to consider more invasive surgical treatments such as bypass surgery or coronary angioplasty. If clinical conditions dictate such an escalation of therapy, then the next tier of therapy, interpericordial injection of the growth factor protein(s) can be implemented in conjunction with the surgery. Alternatively, for patients whose condition does not warrant, or cannot support, angioplasty or bypass surgery, a limited thoracotomy may be used to achieve interpericordial delivery of the protein(s).

If symptoms or clinical testing do not evidence sufficient progress in treatment, then the health care provider may elect to move therapy to the highest tier of invasiveness. Thus, intermyocardial delivery of FGF, VEGF and/or related proteins may be achieved in conjunction with surgical procedures.

Integral with the rational, multi-tier approach of the methods of the present invention, is the use of a rapid, easily accomplished clinical evaluation procedure designed to provide the health care provider with an indication of the efficacy of growth factor therapy. Accordingly, the present invention provides an assay technique that satisfies this need. As one of skill in the relevant art would recognize, the criteria used to diagnose myocardial infarction (MI) can be of critical importance clinically. The most widely accepted diagnostic criteria for MI are those of the World Health Organization, first proposed over 20 years ago. These criteria require the presence of at least 2 of the following 3 criteria: (1) a history of ischemic-type chest discomfort; (2) evolutionary changes on serial electrocardiograms; and (3) a rise and fall in serum cardiac enzymes. Joint International Society and Federation of Cardiology/World Health Organization Task Force "Nomenclature and criteria for diagnosis of ischemic heart disease," Circulation 59: 707 709 (1979). Of importance here the use of creatine kinase (CK) and the more myocardium specific MB isoenzyme, CK-MB, as markers for MI. Wagner, G. S. "Optimal use of serum enzyme levels in the diagnosis of acute myocardial infarction," Arch Intern Med 140: 33 38 (1982). Data compiled in conjunction with the large, multicenter Platelet Glycoprotein Ilb/illa in Unstable Angina: Receptor Suppression Using Integrilin Therapy (PURSUIT) trial suggest that small CK-MB elevations represent clinically important evidence of myocardial necrosis and should be considered sufficient cardiac-marker criteria for a diagnosis of MI in patients with acute coronary syndromes. Alexander, J. H., et al., "Association Between Minor Elevations of Creatine Kinase-MB Level and Mortality in Patients With Acute Coronary Syndromes Without ST-Segment Elevation," JAMA 283: 347 353 (2000).

The conclusion to be drawn from this data is that monitoring of the level of CK-MB can provide useful information for the clinical practitioner is assessing the short-term efficacy of various levels of treatment with FGF, VEGF and/or related growth factor proteins. Thus, the method of the present invention contemplates implementation of a rational, multi-tier therapeutic treatment strategy for administrat