CA2390285A1 - Non-mitogenic fgf-2 protects against ischemia and/or reperfusion injury - Google Patents

Abstract

Fibroblast growth factor-2 (FGF-2) is acutely cardioprotective towards non-ischemic as well as ischemic myocardium. The potent mitogenic activity of FGF-2, however, may pose limitations to some of its clinical applications. In this study we examined whether a recombinant FGF-2 mutant (S117A) that is no longer mitogenic retains cardioprotective properties. Administration of S117A FGF-2 after 30 min of ischemia and during reperfusion of the ex vivo perfused rat heart resulted in significant protection (comparable to that of wild type FGF-2) against myocardial contractile dysfunction. In an in vivo study, rat myocardial infarction was induced by irreversible left coronary ligation; S117A FGF-2, or saline, were administered by direct intramyocardial injection into the ischemic left front ventricular wall. One day later: infarct size (assessed histologically), and plasma cTnT levels (assessed by Western blotting) were significantly decreased in the S117A FGF-2-, compared to the saline- treated control group, by 32.2% (P<0.01) or 28.5 % (P<0.01), respectively;systolic pressure, rates of contraction and relaxation and developed pressure, assessed in the Langendorff mode, were significantly increased in the S117A FGF-2 group compared to saline-treated group. One week after infarction, echocardiography showed significantly improved contractile function (ejection fraction, fractional shortening), in the S117A FGF-2 or wild type FGF-2 treated hearts compared to the saline treated group. At 6 weeks post infarction, however, S117 FGF-2-treated hearts had similar scar size and contractile function (systolic pressure, developed pressure, rates of contraction and relaxation, ejection fraction, fractional shortening) to the saline-treated group, while wild type FGF-2-treated hearts continued to display significantly improved contractility and reduced scar size.
The wild type-, but not S117-, FGF-2-treated group had significantly increased microvessel density at or near the scar area. It is concluded that acute cardioprotection by FGF-2 against ischemia and/or reperfusion -induced contractile dysfunction and tissue damage is independent of its mitogenic/ angiogenic activity.
Thus the non-mitogenic, non-angiogenic S117 FGF-2 may serve as an agent of secondary injury prevention during early (one week) management of myocardial infarction and re-establishment of blood flow. Long-term protection of underperfused myocardium is likely to require increased vessel formation, and the mitogenic/angiogenic activity of wild type FGF-2.

Description

v . ~ Y I~~ b ' a! I II ~ I

r Non-mitogenic FGF-2 protects against ischemia and/or reperfusion injury FIELD OF THE INVENTION
The present invention relates generally to the field of pharmaceutical compounds and medical treatments. More specifically, the present invention is related to a mutant fibroblast growth factor (FGF-2) which is non-angiogenic but protects against ischemia and/or reperfusion injury.
BACKGROUND OF THE INVENTION
Our recently published work (22) demonstrated that the polypeptide FGF-2 may represent a promising, potent new treatment to reduce ongoing damage during development of myocardial infarction ('heart attack'). FGF-2, administered acutely, directly onto the ischemic myocardium shortly after surgically induced occlusion, or during the re-establishment of flow, after global ischemia of the ex vivo heart (22), conferred significant acute as well as long-term protection from ischemia-induced myocardial injury and contractile dysfunction. Several studies have used FGF-2 in animal models, as well as in small open-label clinical trials, as a long-term, angiogenic agent, to promote formation of new blood vessels after infarction (for a summary of these see reference (54) and reported beneficial effects [23-25], although a recent double-blind, randomized, controlled clinical trial (55) did not show significant improvement in perfusion or exercise tolerance in FGF-2-treated patients.
These studies have nevertheless established that FGF-2 is well tolerated overall.
FGF-2 is a pluripotent protein and a potent mitogen. The ability to promote proliferation is central to the ability of FGF-2 to promote angiogenesis. As a mitogen, however, FGF-2 may pose risk to patients with a cancer history, or at risk i ~~ n~ i ~i s a for cancer; or at those at risk for retinopathy. Flugelman et al. [26]
suggested that FGF-2 might play a role in plaque instability, which mechanism could be at least partly explained by the mitogenic and chemotactic effects of FGF-2 on the broad range of cells resident within plaques. Several other experimental studies have also demonstrated that prolonged exposure to high local levels of FGF family of peptides can cause hemangioma-like tumors and vascular malformations [27,28], and can increase neointimal development [29,30]. The very same property therefore that FGF-2 has been used and tested for (angiogenesis) may be the property that actually limits or precludes its use.
While the beneficial or potentially beneficial effects of FGF-2 for the heart have been considered to be derived from its angiogenic properties (collateral vessel formation and improved perfusion), it is perhaps not as well appreciated that FGF-2 has direct, acutely protective effects on cardiac myocytes, making it a very promising agent of secondary injury prevention. It is not known if the ability of FGF-2 to protect cells from injury directly is in any way dependent on it mitogenic/angiogenic potential .It would thus be important to examine whether mutations that decrease FGF-2 mitogenicity retain its ability to be cardioprotective.
Recently, Bouche and coworkers demonstrated that a single mutation on FGF-2 significantly reduced its ability to stimulate proliferation of fibroblastic or endothelial cells; this mutation however did not alter the ability of the molecule to have specific effects on cell differentiation (57): the mutation did not influence the ability of FGF-2 to promote neurite outgrowth in PC12 cells, or the epithelial-mesenchymal transformation in bladder caarcinoma cells. These results suggested i i i ~i to us that other end-points associated with FGF-2 acting on cells, such as protection from injury, may be separable from mitogenicity. We therefore decided to examine the hypothesis that the above mentioned FGF-2 mutant retained the cardioprotective properties of the intact molecule. Our results, outlined below, have demonstrated that indeed, non-mitogenic FGF-2 is acutely cardioprotective for the ischemic myocardium (with or without reperfusion).
SUMMARY OF THE INVENTION
According to a first aspect of the invention, there is provided a pharmaceutical composition comprising purred non-angiogenic FGF-2.
According to second aspect of the invention, there is provided a method of promoting recovery of cardiac function in a patient following myocardial infarction comprising administering an effective amount of purified non-angiogenic FGF-2 to said patient.
According to a third aspect of the invention, there is provided a method of treating ischemia or reperfusion injury comprising administering an effective amount of purified non-angiogenic FGF-2 to a patient at risk of developing or suffering from ischemia or reperfusion injury.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Effect of wt.FGF-2 and S117A FGF-2 on proliferation of neonatal rat myocytes. Rat neonatal myocytes were incubated respectively with low serum (0.5%), high serum (5%), low serum plus wt.FGF-2 (final conc. 10 nglml) or low serum plus S117A FGF-2 (final conc. 10 ng/ml) for 24 hours. 3H-TdR was added to every well of cells at 1 NCi/well. At 24 hours later, 3H-TdR incorporation into the i, ni s t myocytes was determined. Compared with low serum control, ** P<0.01; Compared with high serum control, # P<0.05, ## P<0.01. n=4 in each group.
Fi ure 2: Effect of S117A FGF-2 on the extent of myocardial injury after MI: MI was produced by permanent ligation of the left coronary artery and normal saline (100N1), wt.FGF-2 or S117A FGF-2 (2 Ng in 100u1 saline) were administered intramyocardially by injection into three sites at the lower half of the left front ventricular wall, within 10 minutes from ligation. At 24 hours or 6 weeks later, hearts were harvested for determination of infarct size using TTC
staining.
Blood samples were collected for measurement of plasma cTnT levels. (A) Relative infarct size at 24 hours post MI. (B) Relative scar size at 6 weeks post surgery. (C) Plasma cTnT levels at 24 hours post MI. Compared with saline control, **P<0.01.
n=6-8 in all groups.
Fib: Effect of S117A FGF-2 on cardiac contractile function at 24 h and 6 weeks post MI. After surgery with or without treatment, hearts were harvested for Langendorff perfusion to determine cardiac mechanical function. (A), (B) and (C) show respectively Systolic Pressure, tdP/dtmax and Developed Pressure among all groups at 24 hours post MI. (D), (E) and (F) show, respectively, Systolic Pressure, tdP/dtmax and Developed Pressure among all groups 6 weeks post MI. Compared with saline control, ** P<0.01. n=6 in all groups.\
Figure 4: Effect of S117A FGF-2 administered during reperfusion on cardiac functional recovery from ischemia and/or reperfusion injury. Ex vlvo hearts were perfused in the Langendorff mode. All hearts were submitted to 20 min equilibration, 30 min global ischemia and 60 min reperFusion. At the beginning of n i ~i t reperfusion, normal saline (10m1) supplemented or not with wt.FGF-2 or S117A
(10 microg) were introduced directly into the perfusion buffer; perfusion continued for 60 min. (A), (B) and (C) show respectively Systolic Pressure, tdP/dtmax and Developed Pressure among all groups. Compared with saline control, **P<0.01.
n=7 5 in all groups.
Fi ure 5: Effect of S117A FGF-2 on angiogenesis post MI: 6 weeks after surgery, rat heart 1, 2 and 6 weeks after coronary ligation and intramyocardial injections was processed for cryosectioning and simultaneous immunolocalization of a-sm-actin and Von Willebrand Factor (vWF) or phosphorylated histone 3 (pH3) using monoclonal anti-a-sm-actin, polyclonal anti-vWF and polyclonal anti-pH3 antibodies to evaluate angiogenesis. Staining without the primary antibodies was used to control for non-specific fluorescence. Vessels were defined as round or ellipse structures with a central lumen lined by staining positively to a-sm-actin and vWF. The number of positively stained microvessels was counted in 40 random fields from at least three independent blocks at x100 magnification. Compared with saline control, *P<0.05.
Figure 6. Amino acid sequence of wild-type FGF-2.
Figure 7. Amino acid sequence of FGF-2 S117A.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the i ~ ~~~ a ~~ i ~i i t present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.
DEFINITIONS
As used herein, "angiogenic" refers to promoting the formation of new blood vessels.
As used herein, "effective amount" refers to the administration of an amount of a given compound that achieves the desired effect.
As used herein, "purified" does not require absolute purity but is instead intended as a relative definition. For example, purification of starting material or natural material to at least one order of magnitude, preferably two or three orders of magnitude is expressly contemplated as falling within the definition of "purified".
As used herein, the term "treating" in its various grammatical forms refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease state, disease progression, disease causitive agent or other abnormal condition.
As used herein, "cardioprotective" refers to the protection of cardiac myocites.
As used herein, "mitogenic" refers to the promotion of cellular proliferation.
As used herein, "FGF-2" refers to fibroblast growth factor-2, the amino acid sequence of which is shown in Figure 6.
As used herein, "non-mitogenic FGF-2" or "non-angiogenic FGF-2"
refers to an FGF-2 mutant which is non-angiogenic and non-mitogenic. As will be i ~ni v appreciated by one of skill in the art, these terms are used herein interchangeably in some contexts. An exemplary example of a non-angiogenic FGF-2 mutant is S117A
FGF-2, the amino acid sequence of which is shown in Figure 7, although other suitable non-angiogenic FGF-2 mutants may also be used within the invention.
As used herein, "bioactive fragment" refers to a fragment of non-angiogenic FGF-2 peptide which retains cardioprotective activity.
Described herein is the use of non-angiogenic FGF-2 or a bioactive fragment thereof for treating or preventing a number of diseases or conditions, including but by no means limited to myocardial infarction, ischemia, reperfusion injury, dystrophies, skin wounds, blood vessel injuries, ischemic heart disease, arteriosclerosis and the like.
As discussed herein, FGF-2 is acutely cardioprotective towards non-ischemic as well as ischemic myocardium. The potent mitogenic and angiogenic activity of FGF-2, however, may pose limitations to some of its clinical applications (in cancer patients, or in patients suffering from ischemic heart disease and arteriosclerosis). In this study we examined whether a recombinant FGF-2 mutant (S117A) that is no longer mitogenic retains cardioprotective properties.
Administration of S117A FGF-2 after 30 min of ischemia and during reperfusion of the ex vivo perfused rat heart resulted in significant protection (comparable to that of wild type FGF-2) against myocardial contractile dysfunction. In an in vivo study, rat myocardial infarction was induced by irreversible left coronary ligation;

2, or saline, were administered by direct intramyocardial injection into the ischemic left front ventricular wall. One day later: infarct size (assessed histologically), and i~,, ni a i plasma cTnT levels (assessed by Western blotting) were significantly decreased in the S117A FGF-2-, compared to the saline- treated control group, by 32.2%
(P<0.01 ) or 28.5 % (P<0.01 ), respectively;systolic pressure, rates of contraction and relaxation and developed pressure, assessed in the Langendorff mode, were significantly increased in the S117A FGF-2 group compared to saline-treated group.
One week after infarction, echocardiography showed significantly improved contractile function (ejection fraction, fractional shortening), in the S117A
FGF-2 or wild type FGF-2 treated hearts compared to the saline treated group. At 6 weeks post infarction, however, S117 FGF-2-treated hearts had similar scar size and contractile function (systolic pressure, developed pressure, rates of contraction and relaxation, ejection fraction, fractional shortening) to the saline-treated group, while wild type FGF-2-treated hearts continued to display significantly improved contractility and reduced scar size. The wild type-, but not S117-, FGF-2-treated group had significantly increased microvessel density at or near the scar area. It is concluded that acute cardioprotection by FGF-2 against ischemia and/or reperfusion -induced contractile dysfunction and tissue damage is independent of its mitogenic/
angiogenic activity. Thus the non-mitogenic, non-angiogenic S117 FGF-2 may serve as an agent of secondary injury prevention during early (one week) management of myocardial infarction and re-establishment of blood flow. Long-term protection of underperfused myocardium is likely to require increased vessel formation, and the mitogenic/angiogenic activity of wild type FGF-2.
It is of note that methods of producing and purifying wild-type FGF2 are well known in the art and are applicable for the purification and/or isolation of i ~ ~ i i ~i i non-angiogenic FGF-2 or a bioactive fragment thereof and subsequent use thereof in the preparation of pharmaceutical compositions, as described below. See for example PCT WO 00/13701, which is incorporated herein by reference.
As will be appreciated by one of skill in the art, non-angiogenic FGF-2 or a bioactive fragment thereof, may be administered to a patient in need of said treatment systemically, intra-arterially or intravenously (ie simply inject into circulation). In other embodiments, implants coated with non-angiogenic FGF-2 or a bioactive fragment thereof may be placed onto or within vessels, near the heart, or into other organs.
In yet other embodiments, the non-angiogenic FGF-2 may be administered via direct intramyocardial injection (during surgery or transthoracically or tarnsendocardially) or using any type of catheterization, including the 'stilleto' catheter. See, for example, Bao et al., 2001, Intramyocardial delivery of FGF2 in combination with radio frequency transmyocardial revascularization, Catheter Cardiovasc Interv. 2001 Ju1;53(3):429-34, which is incorporated herein by reference.
In yet other embodiments, the non-angiogenic FGF-2 may be used as an agent in gene therapy, that is, instead of administering the peptide, a gene therapy vector carrying a nucleic acid molecule encoding a non-angiogenic FGF-peptide or bioactive fragment thereof is administered.
It is of note that other methods of administering FGF-2 are well known in the art. See for example, Bush et al., Pharmacokinetics and pharmacodynamics of recombinant FGF-2 in a phase I trial in coronary artery disease, J Clin Pharmacol.
2001 Apr;41 (4):378-85 and Post et aL,Therapeutic angiogenesis in cardiology using i, ~~ i m i protein formulations, Cardiovasc Res. 2001 Feb 16;49(3):522-31, both of which are incorporated herein by reference.
In yet other embodiments, non-angiogenic FGF-2 or a bioactive fragment thereof may be added as an additive to cardioplegic solutions during open 5 heart surgery and/or heart transplantation: It is of note that this would protect heart viability.
It is further of note that non-angiogenic FGF-2 or a bioactive fragment thereof may be used to treat any kind of 'degenerative' disease in any tissue, including skeletal muscle, for example, dystrophies, and brain. In other words, non-10 angiogenic FGF-2 or a bioactive fragment thereof may be used to treat any kind of ischemic disease in any tissue, as well as used to promote skin wound healing or prevent or treat blood vessel injury, which untreated may lead to arteriosclerosis or ischemic heart disease. Because the mutant would not be expected to promote the proliferation of any of the cells involved in the atherosclerotic or restenotic lesion, it would acutely decrease on-going vessel injury and thus decrease the 'repair' inflammatory response.
In some embodiments, non-angiogenic FGF-2 or a bioactive fragment thereof may be combined with other compounds or compositions known in the art such that the non-angiogenic FGF-2 or a bioactive fragment thereof is a pharmaceutical composition in the form of, for example, a pill, tablet, liquid, film or coating using means known in the art and as discussed below.
It is of note that suitable dosages of FGF-2 are well known in the art, for example, PCT WO 00/13701, which is incorporated herein by reference. In i ; i i Ei addition, given that non-angiogenic FGF-2 will not have the same potential side effects as wild-type FGF-2, higher dosages may in fact be used in some instances.
In some embodiments, the dosage may be about 0.2 wg/kg to 48 ~g/kg of the subject. As will be apparent to one knowledgeable in the art, the total dosage will vary according to the weight, health and circumstances of the individual.
In some embodiments, the above-described pharmaceutical composition at concentrations or dosages discussed above may be combined with a pharmaceutically or pharmacologically acceptable carrier, excipient or diluent, either biodegradable or non-biodegradable. Exemplary examples of carriers include, but are by no means limited to, for example, polyethylene-vinyl acetate), copolymers of lactic acid and glycolic acid, poly(lactic acid), gelatin, collagen matrices, polysaccharides, poly(D,L lactide), poly(malic acid), poly(caprolactone), celluloses, albumin, starch, casein, dextran, polyesters, ethanol, mathacrylate, polyurethane, polyethylene, vinyl polymers, glycols, mixtures thereof and the like. Standard excipients include gelatin, casein, lecithin, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene stearates, colloidol silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethycellulose phthalate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, i r i ~i polyvinylpyrrolidone, sugars and starches. See, for example, Reminaton: The Science and Practice of Pharmacy, 1995, Gennaro ed.
As will be apparent to one knowledgeable in the art, specific carriers and carrier combinations known in the art may be selected based on their properties and release characteristics in view of the intended use. Specifically, the carrier may be pH-sensitive, thermo-sensitive, thermo-gelling, arranged for sustained release or a quick burst. In some embodiments, carriers of different classes may be used in combination for multiple effects, for example, a quick burst followed by sustained release.
In other embodiments, the above-described pharmaceutical composition at concentrations or dosages described above may be encapsulated for delivery. Specifically, the pharmaceutical composition may be encapsulated in biodegradable microspheres, microcapsules, microparticles, or nanospheres. The delivery vehicles may be composed of, for example, hyaluronic acid, polyethylene glycol, poly(lactic acid), gelatin, poly(E-caprolactone), or a poly(lactic-glycolic) acid polymer. Combinations may also be used, as, for example, gelatin nanospheres may be coated with a polymer of poly(lactic-glycolic) acid. As will be apparent to one knowledgeable in the art, these and other suitable delivery vehicles may be prepared according to protocols known in the art and utilized for delivery of the.
Alternatively, the delivery vehicle may be suspended in saline and used as a nanospray for aerosol dispersion onto an area of interest. Furthermore, the delivery vehicle may be dispersed in a gel or paste, thereby forming a nanopaste for coating a tissue or tissue portion.

i ~ i ~i It is of note that the compositions as described above may be combined with permeation enhancers known in the art for improving delivery.
Examples of permeation enhancers include, but are by no means limited to those compounds described in U.S. Pat. Nos. 3,472,931; 3,527,864; 3,896,238;
3,903,256;
3,952,099; 4,046,886; 4,130,643; 4,130,667; 4,299,826; 4,335,115; 4,343,798;
4,379,454; 4,405,616; 4,746,515; 4,788,062; 4,820,720; 4,863,738; 4,863,970;
and 5,378,730; British Pat. No. 1,011,949; and Idson, "1975, J. Pharm. Sci. 64:901-924.
It is further of note that non-angiogenic FGF-2 or a bioactive fragment thereof may be combined with other treatments or pharmaceuticals known in the art which are routinely coadministered with wild type FGF-2. In addition and as discussed below, it is also of note that non-angiogenic FGF-2 may be combined or administered with wild type FGF-2 as part of a treatment regimen.
Myocardial infarction or a heart attack is the rapid development of myocardial necrosis caused by a critical imbalance between the oxygen supply and demand of the myocardium. This usually results in an acute reduction of blood supply to a portion of the myocardium. As discussed herein, non-angiogenic FGF-is cardioprotective in that it protects cardiac myocites from further damage and prevents loss of contractile function. Thus, administration of non-angiogenic or a bioactive fragment thereof to a patient at risk of having or having suffered a myocardial infarction will accomplish at least one of the following: will not induce cellular proliferation, promote recovery of cardiac function, reduce organ damage (present, ongoing and caused by treatment), and improve outcome.

i,,,, i i Gi Ischemia is the interruption of blood flow to a tissue region or organ.
Administration of non-angiogenic FGF-2 or a bioactive fragment thereof to a patient at risk of developing ischemia or being treated for ischemia will accomplish at least one of the following: reduce organ or tissue injury, protect against contractile disfunction, will not induce cellular proliferation and prevent reperfusion injury, thereby allowing the ischemia to be repaired by traditional means known in the art without the associated side-effects.
Reperfusion injury occurs when blood flow is restored to an area in which blood flow has been interrupted. Administration of non-angiogenic FGF-2 to a patient at risk of developing reperfusion injury would accomplish at least one of the following: protect the tissue or organ from contractile disfunction, reduce organ injury and improve outcome.
As used herein, °dystrophies" refers to any of a number of known neuromuscular disorders. Examples include but are by no means limited to muscular dystrophy, Duchenne muscular dystrophy, ALS and the like. Administration of non-angiogenic FGF-2 or a bioactive fragment to a patient suffering from or at risk of developing a dystrophy will accomplish at least one of the following: protect tissues and/or muscles from ongoing injury, protect cells from loss of contractile function, eliminate side effects caused by wild-type FGF-2 such as tumors and vascular malformations.
Non-angiogenic FGF-2 or a bioactive fragment thereof is also an effective treatment for skin wounds and the like, as the pharmaceutical composition will protect the local cells from further injury but will not induce cellular proliferation i ~ i i ci which would lead to scar formation. It is of note that in these embodiments, the non-angiogenic FGF-2 or bioactive fragment may be combined with a permeation enhancer and may be arranged to be applied topically, that is, on the skin surface, as discussed above.
5 Non-angiogenic FGF-2 or a bioactive fragment thereof is also an effective treatment for blood vessel injuries and the like, as the pharmaceutical composition will accomplish at least one of the following: protect against loss of contractile function, protect against further injury, and will not induce cellular proliferation, thereby reducing the risk of narrowing of the blood vessel.
10 As discussed below, non-angiogenic FGF-2 or a bioactive fragment thereof will also be an effective treatment for atherosclerosis as administration of the pharmaceutical compound to an individual suffering from or at risk of developing atherosclerosis will prevent plaque instability and will not induce smooth muscle cell proliferation, as discussed below.

15 The invention provides kits for carrying out the methods of the invention.
Accordingly, a variety of kits are provided. The kits may be used for any one or more of the following (and, accordingly, may contain instructions for any one or more of the following) uses: treating ischemia, myocardial infarction, reperfusion injury, dystrophies, skin wounds or blood vessel injury in an individual; preventing undesired proliferation of cells in an individual at risk of ischemia, myocardial infarction, reperfusion injury, dystrophies, skin wounds or blood vessel injury; preventing one or more symptoms of ischemia, myocardial infarction, reperfusion injury, dystrophies, skin wounds or blood vessel injury or the like in an individual at risk of ischemia, myocardial infarction, nm i reperfusion injury, dystrophies, skin wounds or blood vessel injury ; reducing severity one or more symptoms of ischemia, myocardial infarction, reperfusion injury, dystrophies, skin wounds or blood vessel injury in an individual; reducing recurrence of one or more symptoms of ischemia, myocardial infarction, reperfusion injury, dystrophies, skin wounds or blood vessel injury in an individual; and delaying development of ischemia, myocardial infarction, reperfusion injury, dystrophies, skin wounds or blood vessel injury in an individual.
It is of note that the individual may be a human patient.
The kits of the invention comprise one or more containers comprising a non-angiogenic FGF-2 or a bioactive fragment thereof, a suitable excipient as described herein and a set of instructions, generally written instructions although electronic storage media (e.g., magnetic diskette or optical disk) containing instructions are also acceptable, relating to the use and dosage of the non-angiogenic FGF-2 or bioactive fragment thereof for the intended treatment. The instructions included with the kit generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers of the non-angiogenic FGF-2 or bioactive fragment thereof may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
The invention will now be described by way of examples; however, the invention is not in any way limited by the examples and the examples are intended for illustrative purposes.
EXAMPLE 1. Materials Recombinant rat wild type low molecular weight ('lo') FGF-2 was i n i produced and purified from Escherichia coli bacteria according to previously published procedures [31,32]. Recombinant human mutant FGF-2(S117A) was produced in E. coli as described [33]. Briefly, pGEX FGF-2 S117A bacteria was subcultured in TB medium (final carbencilin concentration 10~,g/ml) at 37°C under shaking (240rpm) to obtain a OD600=0.4-0.6, then IPTG was added to final concentration 0.4mM. 4 hrs later, bacteria pellets were harvested by centrifuge at 4°C, 5000g, 10min. The extracted supernatant of the bacteria was gone through Glutathione-Sepharose 4B (Pharmacia Biotech), and the GST carrier was removed from fusion proteins by cleavage with 25 units of thrombin (Pharmacia Biotech) in 500.1 PBS. The eluted S117A FGF-2 was further purified through Heparine Sepharose CL-6B (Pharmacia Biotech) with gradient concentration of NaCI
solutions (1, 2 and 3M). 12% SDS-PAGE was performed to check the purity and quantity of this protein.
EXAMPLE 2. Myocardial infarction and intracardiac injections:
MI was produced by permanent ligation of the left coronary artery using previously reported method [34, 35]. In brief, rats were anesthetized with 2-2.5% isofluorane inhalation. The chest was opened by a left-size thoracotomy at the level of the fourth rib, the pericardium was incised and the left coronary artery was ligated with a silk suture. Within 10 minutes of coronary ligation, normal saline (100 p1), wt.FGF or S117A FGF-2 (2 Ng in 100 w1 normal saline) was injected into three sites at the lower half of the (ischemic, akinetic) left front ventricular wall. The chest was then evacuated and closed. Animals were placed in an incubated chamber and allowed to recover for 24-48 h (as required). Coronary ligation resulted in 30%

n '~ ~~ ,n i ~i mortality, similar in all groups. At various time points after coronary ligation (1, 3, and weeks), animals were anesthetized with a ketamine:xylazine cocktail (90mg/kg:
10mg/kg, i.p.) and echocardiography was performed as described below. Blood samples were collected from the carotid artery and centrifuged to obtain plasma.
5 Rats were euthanized by decapitation at the sixth week, and hearts were harvested for determination of hemodynamic function or infarct size (n=6-8).
EXAMPLE 3. Determination of infarct size Extent of myocardial infarction was estimated as described [36].
Briefly, the heart was traversely cut into slices (2 mm vertical length) and incubated with 1 % 2,3,5-triphenyltetrazolium chloride (TTC) at 37°C for 10 min.
Infarct tissue appears pale because of absence of critical tissue factors necessary to interact with TTC to form the dye. Viable tissue stains red because of formation of formazan dye in the cell. The extent of infarction was estimated as the ratio of the area that remained unstained by TTC over the total ventricular area, in all slices, using Sigma ScanPro photographic analysis software. As rats do not possess cardiac collateral circulation, the infarcted area is expected to be very similar to area at risk.
EXAMPLE 4. Echocardiography [37,39]
Transthoracic echocardiography was perfiormed on rat at 1, 2, 3, 4, 5 and 6 weeks after coronary ligation and intracardiac injections of FGF-2s using the Agilent SONOS 5500(Agilent Technologies, Andover, MA, USA) and a 12-MHz linear array transducer. Short axis two-dimensional views of the left ventricle at the papillary muscle level were used to obtain M-mode targeted recordings. The following echocardiographic parameters were measured in each heart using the i i i ~i leading edge method of the American Society of Echocardiography [38]: Left ventricle(LV) anterior wall thickness in end-diastole (LVAWd) and in end-systole (LVAWs), LV posterior wall thickness in end-diastole (LVPWd) and in end-systole (LVPWs), LV internal chamber dimension in end-diastole (LVIDd) and in end-systole (LVIDs). LV fractional shortening (FS%) was calculated as (LVIDd-LVIDs)/LVIDd x100%. Relative wall thickness (RWT) was calculated as (LVAWd+LVPWd)/LVIDd.
EXAMPLE 5. Measurement of hemodynamic functions After rats were euthanized, their hearts were isolated and arrested in immediately in cold buffer. Langendorff mode was employed to measure myocardial mechanical functions 24 hours and six weeks after coronary ligation and intracardiac injections, as described before [22]. In vitro effect of S117A FGF-2 on cardiac injury induced by global ischemia was also observed on Langendorrf model. All hearts from three groups, control, S117A FGF-2 and wt.FGF-2, were subjected to 20 min equilibration, 30 min ischemia and 60 min reperfusion. 10 ml PBS, 10Ng S117A
FGF-2 or 'lo' FGF-2 (both in 10 ml PBS) was perfused respectively with perfusate for 5 min immediately reperfusion. Developed pressure (DP), end diastolic pressure (EDP), and maximal rate of contraction/relaxation were used to assess myocardial mechanical functions.
EXAMPLE 6. Determination of plasma troponin T (TnT) levels Concentration of rat plasma cardiac TnT (cTnT) from each group was determined by western blotting, as reported previously [22]. Western bands were estimated by densitometry (Bio-RAD Model GS-670, Bio-RAD Laboratories, Inc., Hercules, CA,USA) and standardized as percent of control.

i;;~ ;ni s i EXAMPLE 7. Cardiac myocytes culture Media, sera and antibiotics were purchased from GIBCO/BRL.
Myocyte culture were obstained from neonatal rat hearts (1-2 days old), as described before [40]. All cells were devided into four groups (four wells for each):
5 low serum (0.5%), high serum (5%), wt.FGF-2 and S117A FGF-2 groups. In later two groups, wt.FGF-2 or S117A FGF-2 was added respectively to the cells at at final concentration of 10 ng/ml after the cells were ttreated with low serum (0.5%) medium for 24 hours. 24 hours later, 3H-TdR was added to every well of cells at 1 NCi/well. Another 24 hours later, 3H-TdR incorporation into the myocytes was 10 determined and used as assessment of changes of myocytes proliferation.
EXAMPLE 8. Immunofluorescence Rat heart 1, 2 and 6 weeks after coronary ligation and intramyocardial injections was processed for cryosectioning and simultaneous immunolocalization of a-sm-actin and Von Willebrand Factor (vWF) or phosphorylated histone 3 (pH3) 15 using monoclonal anti-a-sm-actin, polyclonal anti-vWF (sigma) and polyclonal anti-pH3 (Upstate Biotechnology) antibodies to evaluate angiogenesis [41,42].
Staining without the primary antibodies was used to control for non-specific fluorescence.
Vessels were defined as round or ellipse structures with a central lumen lined by staining positively to a-sm-actin and vWF. The number of positively stained 20 microvessels was counted in 40 random fields from at least three independent blocks at x100 magnification.

i ~ ii n i . 21 EXAMPLE 9. Statitical analysis All values were reported as meantSD. One way ANOVA with post-hoc testing was used for significant examination among all groups. P<0.05 was considered statistically significant.
EXAMPLE 10. Effect of S117A FGF-2 on proliferation of neonatal rat myocytes As shown in Fig 1, after incubation with 10 ng/ml wt.FGF-2, 3H-TdR
incorporation into myocytes was increased by 86.8% (P<0.01 ) compared to low serum control. In S117A FGF-2 treated cells, 3H-TdR incorporation was not significantly different from that in low serum incubated cells {915211097 vs 72011717 cpm, P>0.05), but significantly lower {32.0% or 25.0% lower) than that treated with 'lo' FGF-2 or high serum {P<0.01 ). These data indicate that both 'lo' FGF-2 and high serum could strongly stimulate proliferation of neonatal rat myocytes, while S117A FGF-2 did not show such a biological activity.
EXAMPLE 11. Effect of S117A FGF-2 on myocardial injury after MI
Within 10 min of coronary ligation, normal saline, wt.FGF-2 or S117A
FGF-2 was introduced directly into ischemic left ventricle. 24 hours or six weeks later after MI, animals were sacrificed, hearts removed and degree of myocardial damage assessed using TTC staining, followed by morphometric analysis. Infusion of either wt.FGF-2 or S117A FGF-2 resulted in significantly smaller infarct area compared to saline-injected controls 24 hours post coronary ligation {P<0.01 ). To further assess myocardial damage 24 hours after MI, we examined cardiac TnT levels in serum.
Relative plasma TnT levels were found decreased by 37.7% or 28.5% in wt.FGF-2 or S117A FGF-2 treated rats compared to saline injected controls {P<0.01 ).

i , i ~i , 22 But six weeks post MI, there was an apparent difference of effect between wt.FGF-2 and S117A FGF-2 on scar size. While infarct size in wt.FGF-2-treated animals was still significantly smaller than that in control group, area of infarcts in S117A FGF-2-injected rats was indistinguishable from those infused with saline (P>0.05). Results are shown in Fig 2A,2B and 2C.
EXAMPLE 12. Effect of S117A FGF-2 on myocardial function after MI: ex vivo study Echocardiography Echocardiography conducted on live anesthetized animals was used to continuously assess contractile properties of the infracted hearts from each group.
As shown in Table 1, 1 week after MI, EF and FS in wt.FGF-2 or S117A FGF-2 injected hearts were significantly higher than those in saline injected controls (P<0.05 or 0.01 ). 2 weeks after MI, these two parameters were indistinguishable between S117A FGF-2 and saline groups, although they were still spectacularly higher in wt.FGF-2 treated rats compared to saline injected ones (P<0.01 ). At weeks, cardiac dimensions seemed improved in the wtFGF-2-group (but not the S117 group) compared to the saline group.
EXAMPLE 13. Isolated hearts Cardiac mechanical functions were also evaluated at two time points after coronary ligation and intramyocardial injections of FGF-2s or saline. As seen in Fig 3A,B and C, 24 hours post surgery, systolic pressure in both wt.FGF-2 and S117A FGF-2 treated hearts was increased by 74.1 % and 80.2% (P<0.01 ), respectively, compared to saline treated controls. tdp/dtmax were also significantly improved (P<0.01 ). At different level of preload (0-7.5mmHg), wt.FGF-2 or i ni o FGF-2 injected hearts displayed significantly higher developed pressures than saline injected hearts (P<0.01 ). 6 weeks after surgery (Fid 3D, E and F), these parameters in wt.FGF-2 infused hearts still remained significantly improved (P<0.01 ) while in S117A FGF-2 infused hearts, they did not show significant changes compared to saline administered controls (P>0.05).
EXAMPLE 14. Effect of S117A FGF-2 on myocardial function: in vitro study Rat hearts were collected from normal animals and hanged on Langendorff device. All hearts were submitted to 20 min equilibration, 30 min global ischemia and 60 min reperfusion. At beginning of reperfusion, normal saline (10m1), wt.FGF-2 or S117A (10Ng in 10 ml normal saline) was perfused for 5 min with perfusate. As expressed in Fig 4A, B and C, systolic pressure in wt.FGF-2 or FGF-2 group was 48.8% and 38.7% higher than that in saline group (P<0.01 ).
tdp/dtmax were also significantly improved in these two groups compared to control group (P<0.01 ). Under different preload, wt.FGF-2 or S117A FGF-2 perfused hearts displayed significantly higher developed pressures than saline perfused ones (P<0.01 ).
EXAMPLE 15. Effect of S117A FGF-2 on angiogenesis post MI
Immunofluorescence showed that six weeks after coronary ligation, microvessel density in 'lo' FGF-2 treated hearts was 52.9% or 67.5% higher than those in S117A FGF-2 or saline injected hearts (P<0.05). There was no significant difference of microvessel density between S117A FGF-2 and saline infused hearts (P>0.05). This shows that S117 FGF-2 is not angiogenic, while wild type FGF-2 (as expected) is. It also shows that single adminsitration of wtFGF-2 to the heart can L ~~ p, I VI i induce angiogenesis, and resulting benefits, several weeks later.
EXAMPLE 16. DISCUSSION
S117 FGF-2 administered intramyocardially protects ischemic myocytes from ongoing injury, and protects from loss of contractile function, during the development of myocardial infarction resulting from irreversible coronary occlusion, to an extent similar to the wild type FGF-2. Cardioprotection from FGF-2 is detected up to one week from occlusion.
S117, administered during reperfusion after 30 minutes of global ischemia, protects the ex vivo heart from contractile dysfunction resulting from ischemia-reperfusion injury, to an extent similar to that of wild type FGF-2 The protective effects of S117, unlike wild type FGF-2, are no longer detectable at 2-6 weeks post myocardial infarction.
S117, unlike wild type FGF-2, did not stimulate new blood vessel growth in the infarcted myocardium; this likely explains lack of long-term protection Most importantly, an FGF-2 mutant that is not angiogenic (and thus poses no risk for large groups of patients) is as efficacious as the wild type protein in protecting heart from injury, for up to one week. This 'short-term' protection is all that would be required for patients being treated for myocardial infarction, since re-establishment of circulation is achieved during this time window by standard means (thrombolytics and angioplasty). Administration of S117FGF-2 as an adjunct treatment together with thrombolytics and angioplasty would significantly reduce injury (present, ongoing and 'reperfusion' induced) to the heart and result in improved outcome. Since acute coronary occlusion is a major killer in the Western ~ i ,I, 1~ I 11 i world, a significant number of patients would benefit from such treatment.
Results of acute cardioprotection by wt.FGF-2 in this paper are consistent with our previous work and those by other investigators. We once reported [22] that local single-dose wt.FGF-2 administration shortly after the onset of 5 ischemia confers protection from acute and chronic cardiac dysfunction and damage. This cardioprotective effect by wt.FGF-2 is identical when used before or after reperfusion. Other experiments on mouse [43), rat [20,21 ], rabbit [18,19], canine [10-17] and pig [4-9] showed that wt.FGF-2, administered systemically or locally (intracoronary, intramyocardium, perivascular delivery), in the form of protein 10 formulations or by means of gene transfer, has a significant cardioprotective role against myocardial ischemic injury. Even FGF-1, a factor from the same family of growth factors as FGF-2, was also reported to exert a cardioprotection against myocardial injury induced by ischemia [44, 45).
Due to obvious cardioprotection by FGF-2, this growth factor has been 15 used in clinical trials, and beneficial effects on patients with occlusive artery disease were reported by a couple of investigators [46-50]. For examples, Laham et al.
reported that local perivascular delivery of FGF-2 in patients undergoing coronary bypass surgery resulted in significant improvement in blood perfusion of ischemic myocardium and a reduction of the target ischemic zone [48]. Pecher et al.
found 20 that left ventricular ejection fraction was apparently increased and clinical appearance had a pronounced improvement in CHD patients with intramyocardial injection of FGF-2 [50]. It is suggested basically that the mechanism of these beneficial influences induced by FGF-2 mainly relates to to its mitogenic activity to d I 41 I

stimulate formation of new vessels. But the capacity of this angiogenic agent to cause cellular proliferation could have some additional serious side effects.
It has been demonstrated experimentally that prolonged exposure of skeletal muscle or myocardium to high local levels of FGF family peptides can cause hemangiomalike tumors and vascular malformations [51], although even in the animal experimental studies demonstrating tumor development, there has been no evidence of malignant transformation. Furthermore, FGFs has been found to be present in atherosclerotic plaques [52]. Under various experimental circumstances their administration increases neointimal smooth muscle cell proliferation and neointimal mass [43, 53], and this might play a role in plaque instability [26], which is an important cause for occurrence of complications in CHD patients. Therefore, it would be a good strategy to construct a novel, non-mitogenic FGF-2 while its cardioprotective role can be reserved.
S117A FGF-2, a novel FGF-2 mutant that does not show proliferative stimulation of neonatal rat myocytes, but can still be cardioprotective against myocardial ischemic injury in in vivo and in vitro experimental studies.
Direct intracardiac injection of S117A FGF-2 caused a significant improvement of cardiac mechanical functions and a reduction of both infarct size and plasma cTnT
level, which suggest a smaller injury induced by ischemia compared to saline injected controls. !n vitro study also showed a significantly better recovery of cardiac functions in ischemia/reperfusion hearts perfused with this FGF-2 mutant immediately after reperfusion. These data indicate that non-mitogenic S117A
FGF-2, like native FGF-2, can exert a strongly beneficial effect against ischemic injury.

i i ~i Our in vivo studies were conducted on a model of irreversible coronary occlusion, ie, a significant portion of the left ventricle was rendered permanently ischemic, without any attempts to re-establish blood flow. This likely explains why the beneficial effects of non-angiogenic S117 FGF-2 were no longer tenable after one week, while the protection by wild type FGF-2 (which induced angiogenesis) was evident at least up to 6 weeks. As the ultrasound results indicated, 2-6 weeks post MI, cardiac function of S117-treated animals was not improved over saline-treated controls. One can predict that re-establishment of blood flow in the treated hearts, assuming that it happens within a one week from occlusion, would have enabled long-term protection. In addition, and based on our ex vivo reperfusion data, on can predict that introduction of S117FGF-2 during the re-establishment of blood flow (through catheterization for example) would be of benefit, by reducing 'lethal' reperfusion injury that occurs in addition to ischemic damage.
While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.

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Claims (7)

1. A pharmaceutical composition comprising purified non-angiogenic FGF-2.

2. The pharmaceutical composition according to claim 1 wherein the non-angiogenic FGF-2 is FGF-2 S117A.

3. A method of promoting recovery of cardiac function in a patient following myocardial infarction comprising administering an effective amount of purified non-angiogenic FGF-2 to said patient.

4. The method according to claim 3 wherein the non-angiogenic FGF-2 is FGF-2 S117A.

5. The method according to claim 3 wherein the patient is at risk of developing at least one cancer.

6. A method of treating ischemia or reperfusion injury comprising administering an effective amount of purified non-angiogenic FGF-2 to a patient at risk of developing or suffering from ischemia or reperfusion injury.

7. The method according to claim 6 wherein the non-angiogenic