US20020115603A1 - Methods and compositions for the treatment of peripheral artery disease - Google Patents

Methods and compositions for the treatment of peripheral artery disease Download PDF

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US20020115603A1
US20020115603A1 US09/886,856 US88685601A US2002115603A1 US 20020115603 A1 US20020115603 A1 US 20020115603A1 US 88685601 A US88685601 A US 88685601A US 2002115603 A1 US2002115603 A1 US 2002115603A1
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fgf
therapeutically effective
effective amount
patient
mutein
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Martha Whitehouse
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Novartis Vaccines and Diagnostics Inc
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Chiron Corp
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Priority to US09/886,856 priority Critical patent/US20020115603A1/en
Priority to DE60132343T priority patent/DE60132343T2/de
Priority to AU2001268680A priority patent/AU2001268680A1/en
Priority to EP07024542A priority patent/EP1894570A3/fr
Priority to AT01946663T priority patent/ATE383168T1/de
Priority to CA002414020A priority patent/CA2414020A1/fr
Priority to EP01946663A priority patent/EP1324766B1/fr
Priority to JP2002504301A priority patent/JP2004501164A/ja
Priority to PCT/US2001/019978 priority patent/WO2001098346A2/fr
Assigned to CHIRON CORPORATION reassignment CHIRON CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WHITEHOUSE, MARTHA J.
Publication of US20020115603A1 publication Critical patent/US20020115603A1/en
Priority to US10/845,911 priority patent/US7186407B2/en
Priority to US11/671,382 priority patent/US7541337B2/en
Assigned to NOVARTIS VACCINES AND DIAGNOSTICS, INC. reassignment NOVARTIS VACCINES AND DIAGNOSTICS, INC. MERGER (SEE DOCUMENT FOR DETAILS). Assignors: CHIRON CORPORATION, NOVARTIS BIOTECH PARTNERSHIP, INC.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention relates to methods and pharmaceutical compositions for treating peripheral artery disease, particularly the administration of compositions that contain recombinant fibroblast growth factor-2 (rFGF-2).
  • rFGF-2 fibroblast growth factor-2
  • Coronary artery disease (CAD) and peripheral artery disease (PAD) are conditions characterized by insufficient blood flow, usually secondary to atherosclerosis.
  • CAD coronary artery disease
  • PAD peripheral artery disease
  • CAD cerebral artery disease
  • CAD cerebral artery disease
  • PAD peripheral artery disease
  • Identification and management of risk factors are important in the medical management of both CAD and PAD.
  • Pharmacologic management of risk factors may include anti-hypertensives, lipid-lowering agents, and hypoglycemic agents; smoking cessation, diet, and exercise are often prescribed with variable compliance.
  • Pharmacologic management aimed at reduction of symptoms of ischemia often includes vasodilators, anti-anginal, and anti-platelet therapy.
  • Mechanical revascularization by percutaneous angioplasty (with or without a stent) and direct surgical reconstruction improve blood flow and reduce symptoms.
  • restenosis after angioplasty and progression of disease may limit the duration of the benefit.
  • PAD afflicts approximately 11 million patients in the United States. Approximately one third of these patients experience intermittent claudication (discomfort, pain, fatigue, or heaviness in the leg muscles that consistently is brought on by the same amount of muscular activity and relieved by rest). Claudication is similar to angina and represents ischemic muscle pain that may be localized to the hip, buttock, thigh, or calf. It occurs predictably with the same amount of physical stress. Atherosclerosis is systemic, but often one lower limb is more affected than the other. Patients may develop critical limb ischemia, with rest pain, non-healing ulcers, and/or gangrene. Rest pain occurs when blood supply is inadequate to meet the basic nutritional requirements at rest and typically localizes in the toes or foot of the affected limb.
  • compositions and methods for treating peripheral artery disease (PAD) in a patient are provided.
  • Pharmaceutical compositions comprising a therapeutically effective amount of fibroblast growth factor, such as FGF-2, and a pharmaceutically acceptable carrier are provided.
  • Such compositions when administered in accordance with the methods of the invention provide effective treatment for PAD patients including those suffering intermittent claudication associated with this disease.
  • Such compositions may also be administered to PAD patients to prevent progression of critical limb ischemia to amputation.
  • the methods of the invention comprise administering pharmaceutical compositions comprising a therapeutically effective amount of a growth factor, such as FGF-2, as an intra-arterial infusion (IA), intravenous infusion (IV), intramuscular injection (IM), or subcutaneous injection (SC).
  • a growth factor such as FGF-2
  • IA intra-arterial infusion
  • IV intravenous infusion
  • IM intramuscular injection
  • SC subcutaneous injection
  • a single-dose administration of FGF-2 is efficacious for the treatment of PAD.
  • Therapeutic benefits may be obtained with multiple doses without compromising safety.
  • Administration of FGF-2 improves peak walking time in patients with PAD for at least 90 days after FGF-2 administration.
  • FGF-2 can be used to treat patients suffering from critical limb ischemia including those with resting pain with and without non-healing ulcers. Additionally, FGF-2 can be used to treat PAD patients suffering from critical limb ischemia.
  • the FGF-containing composition of the invention can be administered as adjuncts to vascular
  • FIG. 1 sets forth the DNA sequence (SEQ ID NO:1) encoding fibroblast growth factor-2 (FGF-2) having the amino acid sequence set forth in FIG. 2; this FGF-2 is of bovine origin.
  • FGF-2 fibroblast growth factor-2
  • SEQ ID NO:2 The translated amino acid sequence (SEQ ID NO:2) is also shown.
  • FIG. 2 sets forth the amino acid sequence (SEQ ID NO: 2) for the 146 amino acid residue bovine FGF-2 encoded by the DNA sequence set forth in FIG. 1.
  • FIG. 3 sets forth the DNA sequence (SEQ ID NO:3) encoding the translated amino acid sequence (SEQ ID NO:4) for the 146 amino acid residue FGF-2 of human origin.
  • FIG. 4 sets forth the DNA sequence (SEQ ID NO:5) encoding the translated amino acid sequence (SEQ ID NO:6) for the 155 amino acid residue FGF-2 of bovine origin.
  • FIG. 5 sets forth the DNA sequence (SEQ ID NO:7) encoding the translated amino acid sequence (SEQ ID NO:8) for the 155 amino acid residue FGF-2 of human origin.
  • FIG. 6 shows the relative change in peak walking time (PWT) at day 90 with administration of rFGF-2 in patients in a phase II clinical study.
  • PWT peak walking time
  • three patient groups were assessed: a group administered a placebo on both days 1 and 30; a group administered a single dose of rFGF-2 (30 ⁇ g/kg) on day 1 and a placebo on day 30; and a group administered a dose of rFGF-2 (30 ⁇ g/kg) on both days 1 and 30.
  • the mean and standard error are indicated for the measured PWT in each of these groups.
  • the ANOVA analysis excluded patients with missing data and revascularized patients.
  • the ANOVA of Ranks test included patients with missing data and revascularized patients by assigning the lowest rank.
  • Pairwise comparison indicated a p value of 0.026 between the single dose and placebo groups and a p value of 0.45 between the double dose and placebo group.
  • the figure provides the primary efficacy analysis of the clinical trial, which specified the use of log-transformed data. This is considered appropriate statistical management of data when the results have skewness or kertosis such as is often seen in treadmill tests.
  • FIG. 7 shows absolute change in PWT at days 90 and 180 for the patient groups receiving placebo, single-dose rFGF-2, or double-dose rFGF-2.
  • PWT at baseline is subtracted from the PWT at day 90 and the differences are summed for each group and a mean determined; the data are analyzed by an analysis of variance (ANOVA).
  • ANOVA analysis of variance
  • FIG. 8 shows the percent absolute change in PWT in the three patient groups shown at day 90 and day 180. The percent change in PWT averaged across the two rFGF-2 groups is also shown (designated Any FGF).
  • FIG. 9 shows the measured ABI (ankle brachial index) for the three patient groups of the phase II clinical study.
  • a baseline measurement, a day-90 measurement, and the corresponding change between the baseline and day-90 measurement are indicated.
  • the mean change in ABI is also shown for the three patient groups.
  • the ABI is described in An Office Based Approach to the Diagnosis and Treatment of Peripheral Arterial Disease (2000) Society of Vascular Medicine and Biology (Medical Communications Media, Inc., Wrightstown, Pa.) herein incorporated by reference. Subjects having an ABI>1.2 at baseline are excluded from the analysis.
  • FIG. 10 shows the results of the WIQ severity of claudication for the three patient groups in the phase II clinical study at day 90 and day 180. Values represent the percentage of patients in each group indicating an improvement, no change, or worsening of this condition.
  • FIG. 11 shows the severity scores at baseline, day 90, and day 180 for distance, speed, and stair climbing for each group.
  • the figure demonstrates that the results for the single-dose group were better than the results for the placebo group for WIQ distance, speed, and stair climbing.
  • the figure is shown with a scale where higher scores are better.
  • FIG. 12 depicts the physical summary scores from the short form 36 (SF-36). A change of 1 point is associated with an increased lifespan of 2 years. The change scores in the figure indicate an improvement in the single-dose group versus the placebo group by greater than 2 points at day 90.
  • FIG. 13 summarizes the results of the study.
  • FIG. 14 shows the measured ABI (ankle-brachial index) for the three patient groups of the phase II clinical study, when subjects having an ABI>1.2 at anytime (i.e., baseline, day 90, and/or day 180) are excluded from the analysis.
  • a baseline measurement, a day-90 measurement, and the corresponding change between the baseline and day-90 measurement are indicated.
  • the mean change in ABI is also shown for the three patient groups.
  • FIG. 17 shows a scatter plot of PWT90 versus PWTB plus an unrestricted spline regression curve for the placebo ( ⁇ ), single-dose ( ⁇ ), and double-dose ( ⁇ ) groups.
  • FIG. 18 shows the same scatter plot from FIG. 16 plus curves representing regression model 2 described in Table 15 as applied to the placebo ( ⁇ ), single-dose ( ⁇ ), and double-dose ( ⁇ ) groups.
  • FIG. 19 shows the scatter plot of PWT180 versus PWTB plus an unrestricted spline regression curve for the placebo ( ⁇ ), single-dose ( ⁇ ), and double-dose ( ⁇ ) groups.
  • FIG. 20 shows the effect of single administration by intra-arterial infusion (IA) or intramuscular injection (IM) and 14-day continuous intra-arterial infusion on total hindlimb blood flow in a rat bilateral PAD model.
  • IA intra-arterial infusion
  • IM intramuscular injection
  • PBS Phosphate-buffered solution
  • PDA peripheral artery disease
  • angiogenesis angiogenic growth factors that promote the formation of new blood vessels from preexisting ones
  • endothelial cells leave their resting state and start to digest the underlying basement membrane followed by proliferation, migration, and finally formation of a hollow tube (Gerwins et al. (2000) Crit. Rev. Oncol. Hematol. 34(3):185-194).
  • Fibroblast growth factors bind to cell surface receptors that are ligand-stimulatable tyrosine kinases.
  • VEGF vascular endothelial growth factor
  • FGF FGF
  • PDGF vascular endothelial growth factor
  • compositions and methods for treating PAD in a patient are provided.
  • the compositions and methods are useful in the treatment and prevention of claudication and critical limb ischemia due to PAD.
  • critical limb isehemia is used for all patients with chronic ischemic rest pain, ulcers, or gangrene attributable to objectively proven arterial occlusive disease.
  • critical limb ischemia implies chronicity and is to be distinguished from acute limb ischemia.
  • acute limb ischemia is intended any sudden decrease or worsening in limb perfusion causing a threat to extremity viability. See, J. Vasc. Surg. 31:S135, S168, herein incorporated by reference.
  • the methods of the invention utilize angiogenic agents, such as angiogenic members, of the fibroblast growth factor (FGF) family, including preferably FGF-1, FGF-2, FGF-4, FGF-5, FGF-18, and most preferably FGF-2. It is recognized that all angiogenic growth factors herein described may be recombinant molecules. Also, it is recognized that compositions of the invention may comprise one or more fibroblast growth factors as angiogenic agents as well as biologically active variants thereof. Variants of an FGF sequence include, but are not limited to, angiogenically active fragments, analogues, and derivatives.
  • fragment is intended a polypeptide consisting of only a part of the intact FGF sequence and structure, and can be a C-terminal deletion, N-terminal deletion, or both.
  • analogues is intended analogues of either the angiogenic agent FGF or fragment thereof that comprise a native FGF sequence and structure having one or more amino acid substitutions, insertions, or deletions.
  • Peptides having one or more peptoids (peptide mimics) and muteins, or mutated forms of the angiogenic agent are also encompassed by the term analogue.
  • derivatives any suitable modification of the angiogenic agent, fragments of the angiogenic agent, or their respective analogues, such as glycosylation, phosphorylation, or other addition of foreign moieties, so long as the angiogenic activity is retained.
  • Methods for making fragments, analogues, and derivatives are available in the art. See generally U.S. Pat. Nos. 4,738,921, 5,158,875, and 5,077,276; International Publication Nos. WO 85/0083 1, WO 92/04363, WO 87/01038, and WO 89/05822; and European Patent Nos. EP 135094, EP 123228, and EP 128733; herein incorporated by reference.
  • Such variants should retain angiogenic activities and thus be “angiogenically active.”
  • the variants may be measured for angiogenic activity using standard bioassays.
  • Representative assays include known radioreceptor assays using placental membranes (see, e.g., U.S. Pat. No. 5,324,639; Hall et al. (1974) J. Clin. Endocrinol. and Metab. 39:973-976; and Marshall et al. (1974) J. Clin. Endocrinol. and Metab. 39:283-292).
  • Additional assays include mitogenic activity as determined in an in vitro assay of endothelial cell proliferation.
  • This activity is preferably determined in a human umbilical vein endothelial (HUVE) cell-based assay, as described, for example, in any of the following publications: Gospodarowicz et al. (1989) Proc. Natl. Acad. Sci. USA 87:7311-7315; Ferrara and Henzel (1989) Biochem. Biophys. Res. Commun. 161:851-858; Conn et al. (1990) Proc. Natl. Acad. Sci. USA 87:1323-1327; Soker et al. (1998) Cell 92:735-745; Waltenberger et al. (1994) J. Biol. Chem.
  • a further biological activity is involvement in angiogenesis and/or vascular remodeling, which can be tested, for example, in the corneal pocket angiogenesis assay as described in Connolly et al. (1989) J. Clin. Invest. 84:1470-1478 and Lobb et al.
  • the variant has at least the same activity as the native molecule.
  • Fibroblast growth factor-2 (FGF-2), including recombinantly produced forms (rFGF-2), is a potent mitogen and angiogenic agent that has utility for treatment of coronary artery disease (angina) and peripheral artery disease (claudication).
  • FGF-2 is normally made in many body tissues and is involved in the body's response to certain ischemic conditions, the body's own supply of FGF-2 may not be sufficient to circumvent the complications of atherosclerosis and arterial insufficiency/ischemia.
  • compositions and methods of the invention can be used to treat PAD patients, even those suffering a wide spectrum of related clinical ailments, including but not limited to coronary artery disease (CAD), myocardial infarctions, stroke, diabetes, dyslipidemias, hypertension, and patients who have had surgical or catheter-based revascularizations.
  • Fibroblast growth factors particularly FGF-2, can be used to treat PAD patients suffering from claudication, including those having critical limb ischemia.
  • Critical limb ischemia when left untreated, can progress to acute limb ischemia and ultimately necessitate amputation of the limb.
  • the methods of the invention can be used to prevent acute limb ischemia.
  • the FGF-containing compositions of the invention are administered intra-arterially (IA), intravenously (IV), intramuscularly (IM), subcutaneously (SC), transmurally, and the like to a patient in need thereof.
  • transmural administration is intended localized delivery of the composition into the blood vessel or body lumen wall including neointimal, intimal, medial, advential, and periviascular spaces, particularly adjacent to the target site.
  • target site is intended the area surrounding or immediately surrounding the blood supply into the extremities, e.g, legs.
  • Intra-arterial administration involves delivery of the FGF-containing composition into at least one artery.
  • the infusion is typically divided into several arteries in the legs, e.g., the left and right common femoral arteries, but is sometimes administered into a single artery.
  • the infusion can be administered for about 1 minute, 1 to 5 minutes, 10 to 20 minutes, or 20 to 30 minutes into each artery in both legs.
  • the infusion can be repeated from time to time to achieve or sustain the predicted benefit.
  • the timing for repeat administration is based on the patient's response as measured by symptoms and hemodynamic measures.
  • a therapeutically effective dose or amount of FGF, such as FGF-2, that is to be given as an infusion can be divided into two doses, and a single dose administered into each leg of a patient undergoing treatment. In this manner, the total dose is delivered such that the angiogenic agent is presented to both legs of the patient.
  • a therapeutically effective dose or amount of FGF as defined elsewhere herein is administered via IA infusion using a bilateral delivery method such that the procedure can be completed with a single puncture.
  • FGF therapeutically effective amount or total dose of FGF, such as rFGF-2
  • one-half of the therapeutically effective amount or total dose of FGF is infused into the common femoral artery of the first leg, followed by guiding the catheter over the bifurcation of the aorta to the contralateral iliac artery and common femoral artery and then infusing the remainder of the total dose into the femoral artery of the second leg.
  • the rate of each infusion, one into each leg, is about 1 mL/per minute over about a 10-minute period, with a short interruption between the first and second infusion.
  • the second infusion generally begins within about 1 hour of the first infusion, but can begin up to 2, 3, or 4 hours after the first infusion.
  • the second infusion begins within about 30 minutes, more preferably within about 20 minutes, even more preferably within about 10 minutes, still more preferably within about 5 minutes of the completion of the first infusion.
  • Each infusion can take less than about 10 minutes, such as 3, 4, or 5 minutes, so long as the FGF is not administered as a bolus.
  • the therapeutically effective dose or amount of FGF can be divided between the two legs of the patient such that unequal portions of the total dose are delivered to each leg, for example, one-third to one leg, and two-thirds to the other leg.
  • the advantage of the bilateral delivery method is that the two infusions, one into each leg, can be accomplished with a single puncture to the subject.
  • the sight of the puncture is preferably at groin level.
  • a brachial approach may be used if deemed preferable by the treating physician. With this procedure, the catheter can be guided more distally, such as in the area just above the knee, as long as the obstruction to blood flow remains distal to the point of infusion.
  • the therapeutically effective amount of FGF can be delivered by direct IA puncture into each common femoral artery.
  • FGF vascular endothelial growth factor
  • one-half of the dose of FGF is administered into one common femoral artery, while the other half of the dose of FGF is administered into the other common femoral artery.
  • Direct IA puncture can be advantageous in that it avoids the catheterization procedure required with bilateral delivery, but it necessitates two punctures when the therapeutically effective dose is to be divided and infused into both legs.
  • each infusion is delivered at a rate of about 1 mL per minute over about a 10-minute period, with a short interruption between the first and second infusion.
  • the second infusion generally begins within about 1 hour of the first infusion, but can begin up to 2, 3, or 4 hours after the first infusion.
  • the second infusion begins within about 30 minutes, more preferably within about 20 minutes, even more preferably within about 10 minutes, still more preferably within about 5 minutes of the completion of the first infusion.
  • Each infusion can take less than about 10 minutes, such as 3, 4, or 5 minutes, so long as the FGF is not administered as a bolus.
  • the therapeutically effective dose or amount of FGF such as rFGF-2, can be divided between the two legs of the patient such that unequal portions of the total dose are delivered to each leg.
  • Delivery of the FGF-containing compositions in accordance with the methods of the invention may be accomplished through a variety of known intravascular drug delivery systems.
  • Such delivery systems include intravascular catheter delivery systems.
  • a variety of catheter systems useful for the direct transmural infusion of angiogenic growth factors into the blood vessel are well known in the art.
  • any of a variety of diagnostic or therapeutic type catheters could be used.
  • balloon catheters can be used. Balloon catheters having expandable distal ends capable of engaging the inner wall of a blood vessel and infusing an angiogenic growth factor directly therein are well described in the patent literature. See, for example, U.S. Pat. Nos.
  • Direct intramuscular (IM) injections can be used to administer the angiogenic agents of the invention.
  • the agents for injection can include the FGF protein or angiogenically active fragments of the protein as well as the gene or plasmid encoding the angiogenically active FGF protein or fragment.
  • Injections are administered to the affected limb(s), in the thigh or calf, in the vicinity of existing vessels, near collateral flow vessels or conduit vessels such as arteries and arterials.
  • the therapeutically effective dose of angiogenic agent is administered as a single injection, or can be divided and administered as multiple injections.
  • the therapeutically effective amount or dose is delivered as 1 to about 20 injections, 1 to about 15 injections, more preferably 1 to about 10 injections.
  • a single dose of angiogenic agent can be administered intramuscularly, and repeated as needed based on symptoms and/or hemodynamic measures.
  • Local delivery such as with IM injection can provide the added benefit of administering lower doses of the angiogenic agent.
  • Example 4 and the copending application entitled “Dose of an Angiogenic Factor and Method of Administering to Improve Myocardial Blood Flow,” filed Aug. 11, 2000 and assigned U.S. patent application Ser. No. 09/637,471, based on U.S. provisional application no. 60/148,746, filed Aug. 13, 1999, both of which herein incorporated by reference.
  • IM injection(s) The advantage to IM injection(s) is that it is less likely to result in hypotension, is more likely to have a longer half-life in the ischemic area, is less invasive, and therefore, can be repeated more frequently than the IA infusion.
  • An IA infusion or an IM injection(s) could be “boosted” by an IM injection(s) every 1-2 months as warranted by clinical symptoms.
  • Recombinant FGF-2 releases nitric oxide, a potent vasodilator, aggressive fluid management prior to (proactively) and during the infusion is critical to patient's safety.
  • Administration of IV fluids e.g., 500-1000 mL of normal saline
  • administration of boluses of IV fluids e.g., 200 mL normal saline
  • decreases of systolic blood pressure e.g., ⁇ 90 mm Hg
  • compositions comprising fibroblast growth factor can be administered to a patient with peripheral artery disease, including those with claudication, in conjunction with vascular or mechanical bypass surgery or angioplasty.
  • the FGF including but not limited to FGF-2, can be administered with and without a stent during surgery.
  • the FGF may thus be administered as an adjunct to vascular surgery involving mechanical bypass and angioplasty.
  • compositions of the invention provide a safe and therapeutically effective amount of fibroblast growth factor to improve blood flow.
  • safe and therapeutically effective amount is intended an amount of a fibroblast growth factor such as FGF-2, or angiogenically active variant or fragment thereof, that when administered in accordance with the invention is free from major complications that cannot be medically managed, and that provides for objective improvement in patients having symptoms of PAD. It is recognized that the therapeutically effective amount may vary from patient to patient depending upon age, weight, severity of symptoms, general health, physical condition, and the like. Other factors include the mode of administration and the respective amount of FGF included in the pharmaceutical composition.
  • a therapeutically effective amount of an angiogenic agent of the invention is about 0.1 ⁇ g/kg to about 100 ⁇ g/kg, preferably about 0.20 ⁇ g/kg to about 75 ⁇ g/kg, more preferably about 0.4 ⁇ g/kg to about 50 ⁇ g/kg, even more preferably about 0.50 ⁇ g/kg to about 35 ⁇ g/kg, more preferably still about 1.0 ⁇ g/kg to about 30 ⁇ g/kg based on actual body weight.
  • the angiogenic agent is FGF-2
  • a therapeutically effective amount of FGF-2 is about 0.
  • compositions and methods of the invention are useful for treating or preventing PAD and symptoms associated with PAD, including claudication and critical limb ischemia.
  • desired therapeutic responses include increased exercise capacity, improvement in ankle-brachial index, reduction in body pain and claudication.
  • desired therapeutic responses include resolution of unremitting rest pain that is not controllable by analgesic, healing of ulcers, and prevention of gangrene and amputation.
  • Methods for monitoring efficacy of administration of FGF, particularly FGF-2, for treatment of PAD are well known in the art. See, for example, methods for monitoring increased blood flow into affected limbs, including, but not limited to, Doppler ultrasound, plethysmography (Macdonald (1994) J. Vas. Tech. 18:241-248), and magnetic resonance spectroscopy, ankle-brachial or toe systolic pressure index at rest and after a period of exercise, and increased collateral vessel density using angiography.
  • Clinical indicators of efficacy include total treadmill walk time (i.e., peak walking time, PWT) and time to onset of claudication; and patient quality of life questionnaires.
  • the FGF-containing pharmaceutical compositions of the invention will be delivered for a time sufficient to achieve the desired physiological effect, i.e., angiogenesis, and/or restoration of endothelial cell function and the promotion of collateral blood vessels.
  • the compositions may be administered as a single bolus, or multiple injections.
  • the angiogenic factor will be delivered as an infusion over a period of time. It is recognized that any means for administration are encompassed including sustained-release formulations, plasmids, or genes, as well as other routes of administration.
  • the total amount of time may vary depending on the delivery rate and drug concentration in the composition being delivered.
  • the time of administration may vary from 1 second to about 24 hours, more usually from about 1 minute to about 6 hours, specifically from about 5 minutes to about 30 minutes.
  • a single intra-arterial dose administration is efficacious in the treatment of PAD.
  • FGF-containing compositions provide the patient with a safe and therapeutically efficacious treatment for PAD that lasts at least 1 month, 2 months, generally 3 months, 4 months, 6 months, and, in some cases, more than 6 months before a further treatment is needed.
  • the angiogenic agent such as FGF-2, can be administered once or twice per day about every week, preferably every month or more preferably every other month, even more preferably every 3 months, even more preferably every 4 months, and even more preferably still about every 6 months.
  • fibroblast growth factors and related molecules are able to restore endothelial cell function and to promote endothelial and/or smooth muscle cell proliferation.
  • the fibroblast growth factors (FGF) are a family of at least twenty-three structurally related polypeptides (named FGF-1 to FGF-23) that are characterized by a high degree of affinity for proteoglycans, such as heparin.
  • the various FGF molecules range in size from 15 to at least 32.5 kDa, and exhibit a broad range of biological activities in normal and malignant conditions including nerve cell adhesion and differentiation (Schubert et al. (1987) J. Cell. Biol. 104:635-643); wound healing (U.S. Pat. No.
  • the FGF family is a family of pluripotent growth factors that stimulate to varying extents fibroblasts, smooth muscle cells, epithelial cells, endothelial cells, myocytes, and neuronal cells. FGF-like polypeptides are also contemplated for use in the compositions and methods of the present invention.
  • FGF-like polypeptides that bind FGF receptor 1, particularly receptor 1-C, bind to heparin-like molecules, and have angiogenic activity.
  • heparin-like molecule is intended heparin, proteoglycans, and other polyanionic compounds that bind FGF, that dimerize FGF, and that facilitate receptor activation.
  • FGF-2 is designated as well as variants and fragments thereof, which are known in the art. For example, see U.S. Pat. Nos.
  • the FGF can be from any animal species including, but not limited to, avian, canine, bovine, porcine, equine, and human.
  • the FGF is from a mammalian species, preferably bovine or human in the case of FGF-2.
  • the FGF may be in the native, recombinantly produced, or chemically synthesized forms as outlined below.
  • the FGF is FGF-2, it may be the 146 amino acid form, the 153-155 amino acid form, or a mixture thereof depending upon the method of recombinant production. See U.S. Pat. No. 5,143,829, herein incorporated by reference.
  • angiogenically active muteins of the FGF-2 molecule can be used. See, for example, U.S. Pat. Nos. 5,859,208 and 5,852,177, herein incorporated by reference.
  • Biologically active variants of the FGF polypeptide of interest are also encompassed by the methods of the present invention.
  • such variants include fragments, analogues, and derivatives.
  • Such variants should retain angiogenic activities and thus be “angiogenically active” as measured using standard bioassays noted above.
  • Variants of the native FGF used in the compositions and methods of the invention will generally have at least 70%, preferably at least 80%, more preferably about 90% to 95% or more, and most preferably about 98% or more amino acid sequence identity to the amino acid sequence of the reference FGF molecule.
  • sequence identity is intended the same amino acid residues are found within the variant and the reference FGF molecule when a specified, contiguous segment of the amino acid sequence of the variant is aligned and compared to the amino acid sequence of the reference FGF molecule, which serves as the basis for comparison.
  • an angiogenically active variant thereof will generally have at least 70%, preferably at least 80%, more preferably about 90% to 95% or more, most preferably about 98% or more, sequence identify to the full-length amino acid sequence set forth in FIG. 3 (SEQ ID NO:3).
  • FGF receptor-binding peptides can be used as described in, for example, WO98/21237 or U.S. application Ser. No. 09/407,687, filed Sep. 28, 1999, herein incorporated by reference.
  • a polypeptide that is a biologically active variant of a reference polypeptide molecule of interest may differ from the reference molecule by as few as 1-15 amino acids, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • the percentage sequence identity between two amino acid sequences is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the segment undergoing comparison to the reference molecule, and multiplying the result by 100 to yield the percentage of sequence identity.
  • the contiguous segment of the amino acid sequence of the variant polypeptide may have additional amino acid residues or deleted amino acid residues with respect to the amino acid sequence of the reference polypeptide molecule.
  • the contiguous segment used for comparison to the reference amino acid sequence will comprise at least twenty (20) contiguous amino acid residues, and may be 30, 40, 50, 100, or more residues. Corrections for increased sequence identity associated with inclusion of gaps in the variant's amino acid sequence can be made by assigning gap penalties. Methods of sequence alignment are well known in the art for both amino acid sequences and for the nucleotide sequences encoding amino acid sequences.
  • the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm.
  • a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is utilized in the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences.
  • Another preferred, nonlimiting example of a mathematical algorithm for use in comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc.
  • Gapped BLAST can be utilized as described in Altschul et al.
  • PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the ALIGN program Dayhoff (1978) in Atlas of Protein Sequence and Structure 5:Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.) and programs in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wisconsin), for example, the GAP program, where default parameters of the programs are utilized.
  • FGF polypeptide variants provide substantial guidance regarding the preparation and use of FGF polypeptide variants.
  • one of skill in the art can readily determine which modifications to the native nucleotide or amino acid sequence will result in a variant that is suitable for use as a therapeutically active component of a pharmaceutical composition of the present invention for use in the methods of the invention directed to treatment of patients having peripheral artery disease.
  • Fibroblast growth factors such as FGF-2 are formulated into pharmaceutical compositions for use in the methods of the invention.
  • a pharmaceutically acceptable carrier may be used in combination with the angiogenic agent such as FGF-2 and other components in the pharmaceutical composition.
  • pharmaceutically acceptable carrier is intended a carrier or diluent that is conventionally used in the art to facilitate the storage, administration, and/or the desired effect of the therapeutic ingredients.
  • a carrier may also reduce any undesirable side effects of the angiogenic agent, i.e., FGF or variant thereof.
  • a suitable carrier should be stable, i.e., incapable of reacting with other ingredients in the formulation. It should not produce significant local or systemic adverse effect in recipients at the dosages and concentrations employed for therapy.
  • Suitable carriers for this invention are those conventionally used large stable macromolecules such as albumin, gelatin, collagen, polysaccharide, monosaccarides, polyvinylpyrrolidone, polylactic acid, polyglycolic acid, polymeric amino acids, fixed oils, ethyl oleate, liposomes, glucose, sucrose, lactose, mannose, dextrose, dextran, cellulose, mannitol, sorbitol, polyethylene glycol (PEG), heparin alginate, and the like.
  • Slow-release carriers such as hyaluronic acid, may also be suitable.
  • Stabilizers such as trehalose, thioglycerol, and dithiothreitol (DTT) may also be added. See, for example, copending U.S. Application Serial No. 60/229,238, entitled “Stabilized FGF Formulations Containing Reducing Agents,” herein incorporated by reference.
  • FGF formulations comprising DTT as described in this application are defined herein as “stabilized FGF-DTT formulations and include stabilized FGF-2-DTT formatting.”
  • Other acceptable components in the composition include, but are not limited to, buffers that enhance isotonicity such as water, saline, phosphate, citrate, succinate, acetic acid, and other organic acids or their salts.
  • the angiogenic agents of the invention may be administered using a patch for slow release.
  • Such formulation may include DMSO.
  • Preferred pharmaceutical compositions may incorporate buffers having reduced local pain and irritation resulting from injection.
  • buffers include, but are not limited to, low phosphate buffers and succinate buffers.
  • the pharmaceutical composition may additionally comprise a solubilizing compound that is capable of enhancing the solubility of an angiogenic agent or variant.
  • the pharmaceutical composition comprising the angiogenic agent FGF or angiogenically active variant thereof should be formulated in a unit dosage and in an injectable or infusible form such as solution, suspension, or emulsion. It can also be in the form of lyophilized powder, which can be converted into solution, suspension, or emulsion before administration.
  • the pharmaceutical composition may be sterilized by membrane filtration, which also removes aggregates, and stored in unit-dose or multi-dose containers such as sealed vials or ampules.
  • compositions of the present invention can also be formulated in a sustained-release form to prolong the presence of the pharmaceutically active agent in the treated patient, generally for longer than one day.
  • sustained-release formulation Many methods of preparation of a sustained-release formulation are known in the art and are disclosed in Remington 's Pharmaceutical Sciences (18 th ed.; Mack Pub. Co.: Eaton, Pa., 1990), herein incorporated by reference.
  • the agent can be entrapped in semipermeable matrices of solid hydrophobic polymers.
  • the matrices can be shaped into films or microcapsules.
  • Such matrices include, but are not limited to, polyesters, copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al. (1983) Biopolymers 22:547-556), poly-actides (U.S. Pat. No. 3,773,919 and EP 58,481), polyactate polyglycolate (PLGA), hydrogels (see, for example, Langer et al. (1981) J. Biomed. Mater. Res. 15:167-277; Langer (1982) Chem. Tech.
  • microcapsules can also include hydroxymethyl cellulose or gelatin-microcapsules and poly-methylmethacylate microcapsules prepared by coacervation techniques or by interfacial polymerization. Microparticles such as heparin alginate beads may also be used.
  • microemulsions or colloidal drug delivery systems such as liposomes and albumin microspheres, may also be used See a Remington 's Pharmaceutical Sciences ( 18 th ed.; Mack Pub. Co.: Eaton, Pa., 1990).
  • FGF-2 of FIG. 2 also known as basic FGF (bFGF)
  • bFGF basic FGF
  • human FGF-2 of FIG. 3 SEQ ID NO:4
  • angiogenically active fragment or mutein thereof can be utilized in the practice of the invention.
  • the nucleotide sequence encoding bovine FGF-2 is set forth in FIG. 1 (SEQ ID NO: 1).
  • the nucleotide sequence encoding human FGF-2 is set forth in FIG. 3 (SEQ ID NO:3). See also, U.S. Pat. No. 5,604,293, herein incorporated by reference.
  • the dose of FGF-2 that is predicted to result in clinical benefit to a patient whose exercise capacity is limited by claudication associated with PAD ranges from about 0.1 ⁇ g/kg to about 100 ⁇ g/kg of the FGF-2, preferably about 0.20 ⁇ g/kg to about 75 ⁇ g/kg, more preferably about 0.4 ⁇ g,/kg to about 50 ⁇ g/kg, even more preferably about 0.50 ⁇ g/kg to about 35 ⁇ g/kg, more preferably still about 1.0 ⁇ g/kg to about 30 ⁇ g/kg, and most likely from 0.3 to 3.5 mg as a standard dose.
  • the therapeutically effective dose of FGF-2 is about 0.1 ⁇ g/kg to about 1 ⁇ g/kg, about 1 ⁇ g/kg to 3 ⁇ g/kg, about 3 ⁇ g/kg to about 5 ⁇ g/kg, about 5 ⁇ g/kg to about 7 ⁇ g/kg, about 7 ⁇ g/kg to about 8 ⁇ g/kg, about 8 Ag/kg to about 9 ⁇ g/kg, about 9 ⁇ g/kg to about 9.9 ⁇ g/kg, such as about 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, or 9.9 ⁇ g/kg, up to about ⁇ g/kg, about 10 ⁇ g/kg to about 15 ⁇ g/kg, about 15 ⁇ g/kg to about 20 ⁇ g/kg, about 20 ⁇ g/kg to about 30 ⁇ g/kg, about 30 ⁇ g/kg to about 40 ⁇ g/kg, about 40 ⁇ g/kg, about 40 ⁇ g/kg, about 40 ⁇ g/kg, about 40
  • the standard dose to be administered in accordance with the methods of the present invention ranges from about 4.0 ⁇ g to about 7.2 mg, such as about 4.0 ⁇ g to about 0.3 mg, preferably from about 0.3 mg to about 1.0 mg, even more preferably from about 1.0 mg to about 2.0 mg, more preferably still from about 2.0 mg to about 2.5 mg, from about 2.5 mg to 3.5 mg, from about 3.5 mg to about 4.5 mg, from about 4.5 mg to about 5.5 mg, from about 5.5 mg to about 6.5 mg, up to about 7.2 mg.
  • the standard dose is a sufficient amount of FGF-2 to accommodate dosing any one of the majority of human PAD patients, ranging from the smallest patient (e.g., 40 kg) at the lowest dosage (about 0.1 ⁇ g/kg) through the larger patients (e.g., 150 kg) at higher dosages (about 48 ⁇ g/kg for this embodiment).
  • the standard dose ranges from about 0.2 mg to about 3.0 mg, from about 0.5 mg to about 2.5 mg, preferably about 2.1 mg, depending upon the route and mode of administration.
  • the standard dose to be administered in accordance with the methods of the present invention ranges from about 7.0 jig to about 0.7 mg, about 8 ⁇ g to about 0.6 mg, about 9 ⁇ g to about 0.5 mg, about 0.1 mg to about 0.4 mg, preferably about 0.21 mg for a 70 kg patient.
  • the standard dose for a 70 kg patient ranges from about 7.0 ⁇ g to about 0.7 mg, including 8 ⁇ g, 9 ⁇ g, 0.1 mg, 0.2 mg. 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.65 mg, up to about 0.7 mg.
  • FGF-2 is a glycosaminoglycan- (e.g., heparin) binding protein and the presence of a glycosaminoglycan (also known as a “proteoglycan” or a “mucopolysaccharide”) optimizes activity and area under the curve (AUC), the dosages of FGF-2 of the present invention may be administered within 20 to 30 minutes of an intravenous (IV) administration of a glycosaminoglycan, such as a heparin.
  • IV intravenous
  • Various fractionated and unfractionated heparins, proteoglycans, and sulfated mucopolysaccharides such as chondroitin sulfate can be used in the practice of the invention.
  • Low molecular weight heparins ( ⁇ 10,000 d) and unfractionated (i.e., high molecular weight) heparins (>10,000 d) can be used in the practice of the invention. These molecules can be administered together with the rFGF-2 or within 20 to 30 minutes of administration of the rFGF-2. Heparin is suitably dosed at 20-80 units/kg, and preferably at 40 units/kg.
  • the unit dose contains a sufficient amount of FGF-2 ranging from about 0.1 ⁇ g/kg to about 80 ⁇ g/kg. More typically, the systemic unit dose comprises 0.3 mg to 3.5 mg of the FGF-2 of FIG. 2 (SEQ ID NO:2) or the FGF-2 of FIG. 3 (SEQ ID NO:4), or an angiogenically active fragment or mutein thereof. Dosages for local delivery comprising about 0.01 ⁇ g to about 500 ⁇ g up to about 3 mg may be used. When administered locally as with IM injections, the dose may be the same as, one-tenth of, or one-hundredth of the dose administered intra-arterially.
  • the unit dose is typically provided in solution or reconstituted lyophilized form containing the above-referenced amount of FGF-2 and an effective amount of one or more pharmaceutically acceptable buffers, stabilizers, and/or other excipients as described elsewhere herein.
  • FGF-2 having the amino acid sequence of FIG. 2 (SEQ ID NO:2) is made as described in U.S. Pat. No. 5,155,214, entitled “Basic Fibroblast Growth Factor,” which issued on Oct. 13, 1992, and which is incorporated herein by reference in its entirety.
  • SEQ ID NO: 1 a DNA of FIG. 1 (SEQ ID NO: 1), which encodes a bFGF (hereinafter “FGF-2”) of FIG.
  • a cloning vector such as pBR322, pMB9, Col E 1, pCRI, RP4 or ⁇ -phage
  • the cloning vector is used to transform either a eukaryotic or prokaryotic cell, wherein the transformed cell expresses the FGF-2.
  • the host cell is a yeast cell, such as Saccharomyces cerevisiae .
  • the resulting full length FGF-2 that is expressed has 146 amino acids in accordance with FIG. 2 (SEQ ID NO:2).
  • SEQ ID NO:2 has four cysteines, i.e., at residue positions 25, 69, 87 and 92, there are no internal disulfide linkages. ['214 at col. 6, lines 59-61.] However, in the event that cross-linking occurred under oxidative conditions, it would likely occur between the residues at positions 25 and 69.
  • the 146-residue mammalian FGF-2 of FIG. 2 (SEQ ID NO:2), which is of bovine origin, like the corresponding 146-residue human FGF-2 of FIG. 3 (SEQ ID NO:4) is initially synthesized in vivo as a polypeptide having 155 amino acids (Abraham et al. (1986) EMBO J. 5(10):2523-2528; FIG. 4 (SEQ ID NO:6) of bovine origin; FIG. 5 (SEQ ID NO:8) of human origin).
  • the 146-residue FGF-2 molecules When compared to the full-length 155-residue FGF-2 molecules, the 146-residue FGF-2 molecules lack the first nine amino acid residues, Met-Ala-Ala-Gly-Ser-Ile-Thr-Thr-Leu (SEQ ID NO:9), at the N-terminus of the corresponding full-length bovine and human 155-residue FGF-2 molecules (FIG. 4 (SEQ ID NO:6) and FIG. 5 (SEQ ID NO:8), respectively).
  • the 155-residueFGF-2 of human or bovine origin, and biologically active variants thereof, can also be used in the compositions and methods of the present invention in the manner described for the bovine and human 146-residue FGF-2 molecules.
  • the 155-residue form may exist as 153-155 residues, or mixtures thereof, depending upon the method of recombinant protein production.
  • the mammalian FGF-2 of FIG. 2 (SEQ ID NO:2) differs from human FGF-2 of FIG. 3 (SEQ ID NO:4) in two residue positions.
  • the recombinant FGF-2 employed in the present compositions and methods was purified to pharmaceutical quality (90% or greater purity by weight of total proteins, preferably 92% or greater purity, more preferably 95% or greater purity, preferably substantially pure, that is about 98% purity by weight of total proteins) using the techniques described in detail in U.S. Pat. No. 4,956,455, entitled “Bovine Fibroblast Growth Factor,” which issued on Sep. 11, 1990 and which is incorporated herein by reference in its entirety.
  • the first two steps employed in the purification of the recombinant FGF-2 used in a unit dose of a pharmaceutical composition of the invention are “conventional ion-exchange and HPLC purification steps as described previously. “[U.S. Pat. No.
  • the third step which the '455 patent refers to as the “key purification step” ['455 at col. 7, lines 5-6]
  • the third step is heparin-SEPHAROSE(® affinity chromatography, wherein the strong heparin binding affinity of the FGF-2 is utilized to achieve several thousand-fold purification when eluting at approximately 1.4 M and 1.95 M NaCl ['455 at col. 9, lines 20-25].
  • Polypeptide homogeneity may be confirmed by reverse-phase high pressure liquid chromatography (RP-HPLC). Buffer exchange was achieved by SEPHADEX® G-25(M) gel filtration chromatography.
  • the therapeutically active agent in the unit dose of the present invention also comprises an angiogenically active fragment” of the FGF-2 of FIG. 2 (SEQ ID NO:2).
  • angiogenically active fragment of the FGF-2 of FIG. 2 SEQ ID NO:2
  • angiogenically active fragment of the FGF-2 of FIG. 2 SEQ ID NO:2
  • angiogenically active fragment also applies to human FGF-2 of FIG. 3 (SEQ ID NO:4).
  • An “angiogenically active fragment” of the FGF-2 of FIG. 4 (SEQ ID NO:6) or FIG. 5 (SEQ ID NO:8) is a fragment of FGF-2 that has about 80% of the 155 residues of FIG. 4 (SEQ ID NO:6) or FIG. 5 (SEQ ID NO:8), respectively.
  • the FGF-2 fragment should have two cell binding sites and at least one of the two heparin binding sites.
  • the two putative cell binding sites of the analogous 146-residue human FGF-2 (hFGF-2; SEQ ID NO:4) occur at about residue positions 36-39 and about 77-81 thereof. See Yoshida et al. (1987) Proc. Natl. Aca. Sci. USA 84:7305-7309, at FIG. 3.
  • the two putative heparin binding sites of hFGF-2 occur at about residue positions 18-22 and 107-111 thereof. See Yoshida (1987), at FIG. 3.
  • the two putative cell binding sites occur at about residue positions 45-48 and about 86-90 thereof, and the two putative heparin binding sites occur at about residue positions 27-31 and about 116-120 thereof.
  • the two putative cell binding sites are at about residue positions 45-48 and about 86-90, and the two putative heparin binding sites are at about residue positions 27-31 and about 116-120 of the 155-residue bovine FGF-2 (FIG. 4; SEQ ID NO:6).
  • N-terminal truncations of the FGF-2 of FIG. 2 do not eliminate its angiogenic activity in cows.
  • the art discloses several naturally occurring and biologically active fragments of the FGF-2 that have N-terminal truncations relative to the FGF-2 of FIG. 2 (SEQ ID NO:2).
  • An active and truncated bFGF-2 having residues 12-146 of FIG. 2 was found in bovine liver and another active and truncated bFGF-2, having residues 16-146 of FIG. 2 (SEQ ID NO:2) was found in the bovine kidney, adrenal glands, and testes.
  • the angiogenically active fragments of a mammalian FGF typically encompass those terminally truncated fragments of an FGF-2 that have at least residues that correspond to residues 30-110 of the FGF-2 of FIG. 2 (SEQ ID NO:2); more typically, at least residues that correspond to residues 18-146 of the FGF-2 of FIG. 2 (SEQ ID NO:2).
  • hybrid FGF molecules may be constructed comprising peptides from different native sequences as well as combinations of native and synthetic sequences. Again, the hybrid molecules will retain the ability to bind with FGF receptors.
  • the unit dose of the present invention also comprises an “angiogenically active mutein” of the FGF-2 of FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:4), FIG. 4 (SEQ ID NO:6), or FIG. 5 (SEQ ID NO:8).
  • angiogenically active mutein is intended a mutated form of the FGF-2 of FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:4), FIG. 4 (SEQ ID NO:6), or FIG. 5 (SEQ ID NO:8) that structurally retains at least 80%, preferably 90%, of the 146 residues of the FGF-2 sequence shown in FIG.
  • the mutations are “conservative substitutions” using L-amino acids, wherein one amino acid is replaced by another biologically similar amino acid.
  • conservative substitutions include the substitution of one hydrophobic residue such as Ile, Val, Leu, Pro, or Gly for another, or the substitution of one polar residue for another, such as between Arg and Lys, between Glu and Asp, or between Gln and Asn, and the like.
  • the charged amino acids are considered interchangeable with one another.
  • Suitable substitutions include the substitution of serine for one or both of the cysteines at residue positions 87 and 92, which are not involved in disulfide formation.
  • substitutions include any substitution wherein at least one constituent cysteine is replaced by another amino acid so that the mutein has greater stability under acidic conditions, see for example U.S. Pat. No. 5,852,177 which is herein incorporated by reference.
  • One such substitution is the replacement of cysteine residues with neutral amino acids such as for example: glycine, valine, alanine, leucine, isoleucine, tyrosine, phenylalanine, histidine, tryptophan, serine, threonine, and methionine (U.S. Pat. No. 5,852,177).
  • substitutions are introduced at the FGF-2 N-terminus, which is not associated with angiogenic activity. However, as discussed above, conservative substitutions are suitable for introduction throughout the molecule.
  • FIG. 1 SEQ ID NO:1
  • FIG. 3 SEQ ID NO:3
  • FIG. 4 SEQ ID NO:5
  • FIG. 5 SEQ ID NO:7
  • an FGF-2 polypeptide mutein or fragment of a mutein having angiogenic activity for use within the unit dose, compositions, and methods of the present invention.
  • an angiogenically active mutein of the FGF-2 of FIG. 2 SEQ ID NO:2
  • FIG. 3 SEQ ID NO:4
  • FIG. 4 SEQ ID NO:6
  • FIG. 5 (SEQ ID NO:8), one uses standard techniques for site-directed mutagenesis, as known in the art and/or as taught in Gilman et al. (1979) Gene 8:81 or Roberts et al. (1987) Nature 328:73 1, to introduce one or more point mutations into the cDNA of FIG. 1 (SEQ ID NO: 1), FIG. 3 (SEQ ID NO:3) FIG. 4 (SEQ ID NO:5), or FIG. 5 (SEQ ID NO:7) that encodes the FGF-2 of FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:4), FIG. 4 (SEQ ID NO:6), or FIG. 5 (SEQ ID NO:8), respectively.
  • compositions of the invention comprise an angiogenically effective dose of a mammalian FGF-2 of FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:4), FIG. 4 (SEQ ID NO:6), FIG. 5 (SEQ ID NO:8) or an angiogenically active fragment or mutein thereof, and a pharmaceutically acceptable carrier.
  • the safe and angiogenically effective dose of the pharmaceutical composition of the present invention is in a form and a size suitable for administration to a human patient and comprises (i) 1.0 ⁇ g/kg to 30.0 ⁇ g/kg of an FGF-2 of FIG.
  • the safe and angiogenically effective dose comprises about 0.1 ⁇ g/kg to about 1 ⁇ g/kg, about 1 ⁇ g/kg to 3 ⁇ g/kg, about 3 ⁇ g/kg to about 5 ⁇ g/kg, about 5 ⁇ g/kg to about 7 ⁇ g/kg, about 7 ⁇ g/kg to about 8 ⁇ g/kg, about 8 ⁇ g/kg to about 9 ⁇ g/kg, about 9 ⁇ g/kg to about 9.9 ⁇ g/kg, such as about 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, or 9.9 ⁇ g/kg, up to about 10 ⁇ g/kg, about 10 ⁇ g/kg to about 15 ⁇ g/kg, about 15 ⁇ g/kg to about 20 ⁇ g/kg, about 20 ⁇ g/kg to about 30 ⁇ g/kg, about 30 ⁇ g/kg, about 30 ⁇ g/kg, about 30 ⁇ g/kg, about 30 ⁇ g/kg, about 30 ⁇ g/kg,
  • a typical pharmaceutical composition comprises 0.1 mg/ml to 10 mg/ml, more typically 0.3 mg/ml to 0.5 mg/ml, of FGF-2, more particularly recombinant FGF-2 (rFGF-2), having the sequence set forth in FIG. 2 (SEQ ID NO:2), or in FIG. 3 (SEQ ID NO:4), or an angiogenically active fragment or mutein thereof, 10 mM thioglycerol, 135 mM NaCl, 10 mM Na citrate, and 1 mM EDTA, pH 5.0.
  • a suitable diluent or flushing agent for the above-described composition is any of the above-described carriers.
  • the diluent is the carrier solution itself comprising 10 mM thioglycerol, 135 mM NaCl, 10 mM Na citrate, and 1 mM EDTA, pH 5.0.
  • the rFGF-2 of FIG. 2 (SEQ ID NO:2) or an angiogenically active fragment or mutein thereof is unstable for long periods of time in liquid form.
  • the pharmaceutical composition of the present invention comprising an effective amount of rFGF-2 or an angiogenically fragment or mutein thereof, in a pharmaceutically acceptable aqueous carrier should be stored frozen at ⁇ 60° C. Thawed, the solution is stable for 1 month at refrigerated conditions.
  • a typical unit dose would comprise about 5-10 ml of the above described composition having 1.5-8 mg of FGF-2 of FIG. 2 (SEQ ID NO:2), or FIG. 3 (SEQ ID NO:4).
  • the pharmaceutical composition comprises a unit dose of FGF-2 of FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:4), or an angiogenically active fragment or mutein thereof in lyophilized (freeze-dried) form.
  • the unit dose of FGF-2 would be capable of being stored at room temperature for substantially longer than 6 months without loss of therapeutic effectiveness. Lyophilization is accomplished by the rapid freeze drying under reduced pressure of a plurality of vials, each containing a unit dose of the FGF-2 of the present invention therein. Lyophilizers, which perform the above described lyophilization, are commercially available and readily operable by those skilled in the art.
  • the lyophilized product Prior to administration to a patient, the lyophilized product is reconstituted to a known concentration, preferably in its own vial, with an appropriate sterile aqueous diluent, typically 0.9% (or less) sterile saline solution, or a compatible sterile buffer, or even sterile deionized water. See, for example, copending U.S. Application Serial No. 60/229,238, entitled “Stabilized FGF Formulations containing Reducing Agents,” herein incorporated by reference.
  • a single dose comprising from 0.2 ⁇ g/kg to 36 ⁇ g/kg of the FGF-2 of FIG. 2 (SEQ ID NO:2), the FGF-2 of FIG.
  • an angiogenically active fragment or mutein thereof is withdrawn from the vial as reconstituted product for administration to the patient.
  • an average 70 kg man that is being dosed at 24 ⁇ g/kg would have a sufficient volume of the reconstituted product withdrawn from the vial to receive an infusion of (70 kg x 24 ⁇ g/kg) 1680 ⁇ g (i.e., 1.680 mg).
  • the pharmaceutical composition in solution form is generally administered by infusing the unit dose substantially continuously over a period of about 10 to about 30 minutes, although it is recognized that the composition may be administered over a longer period of time.
  • a portion e.g., one half
  • portions of the unit dose may be administered to a plurality of vessels until the entire unit dose has been administered. After administration, the catheter is withdrawn using conventional protocols known in the art.
  • Signs of angiogenesis and a therapeutic benefit are seen as early as two weeks to one month following the FGF-2 administration.
  • FGF-2 The recombinantly produced FGF-2 (rFGF-2) having the sequence shown in FIG. 2 (SEQ ID NO:2) was formulated as a unit dose and pharmaceutical composition. The various formulations are described below.
  • the rFGF-2 unit dose was provided as a liquid in 3 cc type I glass vials with a laminated gray butyl rubber stopper and red flip-off overseal.
  • the rFGF-2 unit dose contained 1.2 ml of 0.3 mg/ml rFGF-2 of FIG. 2 (SEQ ID NO:2) in 10 mM sodium citrate, 10 mM monothioglycerol, 1 mM disodium dihydrate EDTA (molecular weight 372.2), 135 mM sodium chloride, pH 5.0.
  • each vial (and unit dose) contained 0.36 mg rFGF-2.
  • the vials containing the unit dose in liquid form were stored at 2° to 8° C.
  • the diluent was supplied in 5 cc type I glass vials with a laminated gray butyl rubber stopper and red flip-off overseal.
  • the rFGF-2 diluent contains 10 mM sodium citrate, 10 mM monothioglycerol, 135 mM sodium chloride, pH 5.0.
  • Each vial contained 5.2 ml of rFGF-2 diluent solution that was stored at 2 to 8 C.
  • Such agents may also be administered to prevent progression of critical limb ischemia to amputation.
  • the rFGF-2 pharmaceutical composition that was infused was prepared by diluting the rFGF-2 unit dose with the rFGF diluent. In order to keep the EDTA concentration below the limit of 100 ⁇ g/ml, the total infusion volume was increased up to 40 ml when proportionately higher absolute amounts of FGF-2 were administered to patients.
  • Peripheral artery disease as defined by resting anklebrachial index (ABI) less than 0.9, is a common condition afflicting about 15% of adults greater than 55 years of age. About 33% of these individuals are symptomatic with claudication; about 25% will progress. With worsening blood flow limitation, the spectrum of PAD runs from mild to moderate to severe claudication, followed by limb-threatening ischemia, initially characterized by rest pain, then poor wound healing, and impending or overt gangrene.
  • ABSI resting anklebrachial index
  • phase II trial was undertaken to assess the efficacy of intra-arterial administration of rFGF-2 on exercise capacity in patients with intermittent claudication due to infra-inguinal PAD.
  • the phase II PAD trial was a multicenter, randomized, double-blind, placebo-controlled, regimen finding study of rFGF-2 to evaluate the safety, pharmocokinetics, and efficacy by intra-arterial (IA) infusion over 20 minutes in PAD subjects with moderate to severe intermittent claudication.
  • IA intra-arterial
  • Major selection criteria for inclusion of a patient in the trial were age greater than 40 years, exercise limited by claudication, index ankle brachial index (ABI) of less than 0.8 at rest, patent femoral inflow, medically stable for greater than 4 months, and informed consent.
  • Major selection criteria for exclusion of a patient from the trial were evidence of malignancy (according to ACS guidelines), creatinine greater than 2.0 mg/dL, urine protein greater than or equal to 2+or greater than 300 mg/day, proliferative retinopathy, and/or other conditions impacting safety or compliance.
  • 190 patients participated in the phase II PAD trial. Baseline characteristics of the patient population are shown in Tables 1-3. TABLE 1 Baseline characteristics of the phase II PAD clinical trial patient population.
  • Placebo SINGLE DOUBLE Any FGF Number of Subjects 63 66 61 127 Median Age (yrs) 67 65 68 67 Male 73% 71% 82% 76% Female 27% 29% 18% 24% ABI at Rest (index) 0.55 0.57 0.55 0.56 PWT at Baseline 5.32 5.15 5.81 5.48 COT at Baseline 1.97 2.03 2.20 2.13 Current Smoker 38% 24% 21% 23% Past Smoker 43% 59% 61% 60% None Smoker 19% 17% 18% 17% Structured Exercise 56% 50% 49% 50%
  • the rFGF-2 was administered by intra-arterial (IA) infusion over 20 minutes divided between two legs on days 1 and 30.
  • the dose administered was 30 ⁇ g/kg of rFGF-2.
  • the trial patients were divided into three groups: placebo; single dose (rFGF-2 on day 1); and double dose (rFGF-2 on days 1 and 30).
  • the primary endpoint used in the study was a change in peak walking time (PWT) at day 90 on a Gardner graded exercise protocol.
  • Recombinant FGF-2 (rFGF-2) was formulated in a solution containing 0.3 mg/ml rFGF-2, 10 mM sodium citrate, 0.3 mM EDTA, 10 mM thioglycerol, 135 mM sodium chloride, pH 5.0. Each 5 ml vial contained 3.7 ml of clear colorless solution (1. 1 mg rFGF-2 per vial). Vials containing rFGF-2 were labeled “rFGF-2” and supplied frozen. Drug product was thawed at room temperature prior to preparation of dose; detailed instruction for pharmacists were provided in study manuals. Thawed, undiluted active drug product could be stored refrigerated at 2-8° C. for 30 days.
  • Drug product was diluted with placebo (diluent) and filtered before administration.
  • the filter was sterile, non-pyrogenic, and low protein binding. Filtration of the drug product through a 0.22 micron syringe filter (e.g., Millipore, Millex-GV, #SLGVR25LS or equivalent) would remove particle with no resultant loss in strength or potency. Thawed, undiluted drug product was used within 8 hours.
  • a 0.22 micron syringe filter e.g., Millipore, Millex-GV, #SLGVR25LS or equivalent
  • Placebo (diulent) was supplied as a clear, colorless solution indistinguishable from the drug product. It contained 10 mM sodium citrate, 0.3 mM EDTA, 10 mM thioglycerol, 135 mM sodium chloride, pH 5.0. Vials containing diluent were labeled “placebo,” supplied in a liquid state, and stored refrigerated at 2-8° C.
  • FIG. 7 shows absolute change in PWT at days 90 and 180 for the patient groups receiving placebo, single-dose rFGF-2, or double-dose rFGF-2.
  • PWT at baseline is subtracted from the PWT at day 90 and the differences are summed for each group and a mean determined.
  • the data are analyzed by an analysis of variance (ANOVA).
  • FIG. 8 shows the percent absolute change in PWT in the three patient groups shown at day 90 and day 180. The percent change in PWT averaged across the two rFGF-2 groups is also shown (designated Any FGF).
  • FIG. 9 shows the measured ABI (ankle brachial index) for the three patient groups of the phase II clinical study.
  • a baseline measurement, a day-90 measurement, and the corresponding change between the baseline and day-90 measurement are indicated.
  • the mean change in ABI is also shown for the three patient groups.
  • the ABI is described in An Office Based Approach to the Diagnosis and Treatment of Peripheral Arterial Disease (2000) Society of Vascular Medicine and Biology (Medical Communications Media, Inc., Wrightstown, Pa.).
  • the anklebrachial index is the ratio of the systolic pressure in the foot to the systolic pressure in the arm as measured by a Doppler ultrasound device.
  • the normal ABI is 1.
  • An ABI less than 0.9 is considered diagnostic of PAD.
  • the mean ABI of the target population enrolled in the trial was .56 in the index leg at rest.
  • the index leg is the leg with the lower ABI.
  • FIG. 9 shows the mean ABI (top panel) at baseline, day 90, and day 180 for each group.
  • the bottom panel shows the mean change in ABI at day 90 and at day 180 for each group. There is a positive directional change in the treatment groups compared to placebo.
  • FIG. 10 represents the WIQ severity of claudication at days 90 and 180 for single- and double-dose groups relative to the placebo group.
  • the bar values represent the percentage of patients in each group who improved, stayed the same, or became worse in each group. At day 90, greater than 50% of the patients in the treatment groups were improved whereas less than 40% of the placebo patients were improved. At day 180 this apparent treatment benefit is lost.
  • FIG. 12 depicts the physical summary scores from the short form 36 (SF-36).
  • the SF-36 is a general validated quality of life instrument consisting of 36 questions.
  • the SF-36 has 12 domains, which can be collapsed into two summary scores, physical and mental.
  • a change of 1 point is associated with an increased lifespan of 2 years.
  • the change scores in the figure indicate an improvement in the single-dose group versus the placebo group by greater than 2 points at day 90.
  • the analysis plan provided for the tracking of treatment response in three pre-specified subgroups of the phase II clinical trial patient population: diabetes (type I or II, yes vs no), smoking (current vs non-current, which included those individuals who had smoked in the past or had never smoked), and median age ( ⁇ 68 years vs >68 years).
  • diabetes type I or II, yes vs no
  • smoking current vs non-current, which included those individuals who had smoked in the past or had never smoked
  • median age ⁇ 68 years vs >68 years.
  • the initial analysis plan pre-specified that subjects having a baseline ankle brachial index (ABI)>1.2 (consistent with non-compressible artery) be excluded from the analysis (see FIG. 9).
  • ABSI ankle brachial index
  • subjects having an ABI>1.2 at anytime i.e., baseline, day 90, and/or day 180
  • this post-hoc analysis indicates that both the single-dose and double-dose groups had a statistically significant improvement in ABI compared to the placebo group at day 90. This significance was not apparent at day 180, though the trend persisted for both the single-dose and double-dose groups.
  • the overall P-value for the primary efficacy analysis was 0.075 (ANOVA).
  • the post-hoc responder analysis shows a higher percentage of responders in the single-dose group and a greater magnitude of effect in the single-dose group. In addition, it suggests that the treatment effect persists at 180 days. The strongest predictor of response at 180 days is the response at 90 days.
  • FIG. 15 shows a hypothetical plot of PWT90 versus PWTB when absolute change score is assumed to be the correct variable.
  • FIG. 16 shows a hypothetical plot of PWT90 versus PWTB when relative change score is assumed correct.
  • the original analysis plan used absolute change score as the analysis variable, because there was not strong initial guidance from PIs/consultants to use relative score, and the absolute change score was used in the analysis of a related study directed to treatment of coronary artery disease (CAD). There was interest in a potential for a combined indication of FGF for CAD and PAD, which would be facilitated by consistent use of the same analysis variable.
  • FIGS. 17 - 19 The results of the post-hoc regression analysis using the models described in Tables 15 and 16 are shown in FIGS. 17 - 19 .
  • FIG. 17 shows a scatter plot of PWT90 versus PWTB plus an unrestricted spline regression curve for each treatment group.
  • use of absolute change score or relative change score as the analysis variable does not appear to be fully consistent with the observed data.
  • FIG. 18 shows the same scatter plot plus curves representing regression model 2 described below.
  • Regression models provide a more flexible method for assessing the change in PWT at day 90 and adjusting for baseline PWT.
  • the curved shape of the scatter plot suggests that a regression model that produces curves will better represent or fit the study data.
  • regression models would have PWT90 as the analysis variable, and predictor variables that include PWTB and (PWTB) 2 .
  • Other baseline variables such as smoking status can also be included in the regression model, if they are important confounders.
  • Model 1 for PWT90 includes only the predictor variable PWTB but is more flexible than using the absolute change score because model 1 can have any slope (i.e., model 1 does not require the slope to be 1.0). Model 1 better fits the data than either the absolute or relative change scores.
  • Model 2 for PWT90 includes both PWTB and (PWTB) 2 as predictor variables, and accommodates the curve shape of the scatter plot (see FIG. 18). Model 2 appears to provide a better fit to the study data than model 1, as seen by:
  • Model 3 for PWT90 includes smoking status (at baseline), PWTB, and (PWTB) 2 , and adjusts for current smoking status. TABLE 15 Regression models for PWT90. All regression models are adjusted for Center (Site). 1 2 3 Variables Trt. Single Trt. Single Trt. Single in Model Trt. Double Trt. Double Trt.
  • the single-dose group had a statistically significant improvement in PWT at day 90, with pairwise p-values of 0.032, 0.027, and 0.015 (for models 1, 2, and 3, respectively).
  • the single-dose group had an average increase in PWT over the placebo group of 69.8, 71.7, and 79.3 seconds.
  • the double-dose group had a trend or statistically significant improvement in PWT at day 90, with pairwise p-values of 0.096, 0.0678, and 0.037.
  • the double-dose group had an average increase in PWT over the placebo group of 56.7, 61.8, and 71.5 seconds; and thus the double-dose group is more similar to the single-dose group in increased PWT than to the placebo group.
  • FIG. 19 shows the scatter plot of PWT180 versus PWTB plus an unrestricted spline regression curve for each treatment group.
  • the shape of the data also do not support a slope of 1 or an intercept of 0.0.
  • the shape of the day 180 PWT data are only slightly curved.
  • Table 16 shows the three regression models for PWT180 as the analysis variable and adjusted for PWTB, and other baseline subject characteristics. All three models indicate similar results, with the single-dose group having a 22- to 26-second benefit over the placebo group, and the double-dose group apparently not different than the placebo group. TABLE 16 Regression models for PWT180. All regression models are adjusted for Center (Site). 1 2 3 Variables Trt. Single Trt. Single Trt. Single in Model Trt. Double Trt. Double Trt.
  • This study defined an effective dose, route, and regimen for treatment of PAD with rFGF-2.
  • Administering a double-dose of rFGF-2 was not better than administering a single-dose of FGF-2.
  • the magnitude of benefit in PWT was greater than 1 minute, with the duration of benefit observed at both 3 and 6 months.
  • about 40% of the patients receiving the single-dose treatment experienced an increase in PWT of greater than 2 minutes at both day 90 and day 180, compared with only about 22% of patients receiving placebo and about 26% of patients receiving a double dose of rFGF-2.
  • a phase III, multicenter (up to 50 sites), double-blind, placebo-controlled, dose-optimization study is conducted.
  • the primary objective of this trial is to evaluate safety and efficacy of an intra-arterial (IA) infusion of 3.0 ⁇ g/kg or 30.0 ⁇ g/kg rFGF-2 versus placebo in peripheral artery disease (PAD) subjects with moderate to severe claudication.
  • IA intra-arterial
  • PID peripheral artery disease
  • the trial enrolls 450 subjects (150 per arm) with moderate to severe claudication limiting exercise.
  • Inclusion criteria and exclusion criteria are shown in Table 17. Sample size may be adjusted based on DSMB evaluation of variability of peak walking time at 90 days after 225 subjects are enrolled. TABLE 17 Synopsis of Phase III Clinical Trial.
  • the study drug, rFGF-2 having the sequence shown in FIG. 2 is contained at 0.35 or 3.5 mg/mL in a lyophilized powder, to be reconstituted with normal saline; it is formulated in 10 mM sodium citrate, 1 mM EDTA, 10 mM dithiothreitol (DTT), 4% glycine, 1% glucose at pH 6.0.
  • Treatment consists of infusion of 20 mL at 1 mL/min of placebo, 3.0 ⁇ g/kg or 30.0 ⁇ g/kg rFGF-2, divided equally between two legs, via the common femoral artery.
  • Patients are monitored for acute safety variables including systolic hypotension associated with IA infusion and any evidence of allergic reactions, as well as frequency and severity of adverse events, changes in laboratory parameters (especially urine protein), evidence of retinal toxicity, and evidence of seroconversion (antibody formation).
  • DSMB will review SAEs and abnormal laboratory tests during enrollment.
  • Primary efficacy variable is change from baseline in peak walking time (PWT) at 90 days as measured by Gardner graded exercise test time, adjusted for baseline PWT, smoking status, and center. Secondary efficacy is established based on the following parameters:
  • PCSS physical component summary score
  • Data are analyzed with an intent to treat analysis using ANOVA of Ranks with last value after baseline carried forward for missing data, or lowest rank for subjects without a post-baseline PWT assessment, adjusting for baseline PWT, smoking status, and center.
  • Bilateral ligation of the femoral artery is designed to establish peripheral arterial insufficiency without impairing resting muscle blood flow.
  • the high blood flow reserve of muscle is markedly reduced while residual muscle blood flow is sufficient to support resting blood flow needs; e.g., compare (Yang et al. (1990) J. Appl. Physiol 69:1353-1359; Yang and Terjung(1993) J. Appl, Physiol. 75(1):452-457; Mackie and Terjung (1983) Am. J. Physiol. 245:H265-H275).
  • FGF-2 delivery was initiated/achieved at the time of femoral artery ligation by: a) a 14-day continuous intra-arterial infusion; b) a single intramuscular injection; or c) a single intra-arterial injection as follows.
  • Six of the rats received FGF-2 at a dose of 5 ⁇ g/kg/day for 14 days for a total of 70 ⁇ g/kg; the other two received vehicle alone (PBS).
  • PBS vehicle alone
  • a final volume of 0.435 mL of pump solution for each rat was calculated based on the initial rat weight of approximately 325 g.
  • a second group of rats received vehicle or FGF-2 with a single intra-arterial injection. This injection was given over the span of 10 minutes, into the femoral artery of one hindlimb, upstream of the point of ligation. The volume to be injected was 0.35 mL.
  • Six rats received 1.5 ⁇ g/kg FGF-2 total dose; six received 15 ⁇ g/kg FGF-2; six received 30 ⁇ g/kg FGF-2; and two received vehicle alone.
  • a third group of rats received a single intramuscular injection. This injection was split between two sites in the medial hamstring on one hindlimb, in the region of collateral formation. The volume to be injected was 100 ⁇ L per site, for a total volume of 0.2 mL. Six rats received 0. 15 ⁇ g/kg FGF-2 total dose; six received 1.5 ⁇ g/kg FGF-2; six received 15 ⁇ g/kg FGF-2; and four received vehicle alone.
  • Muscle blood flow was determined in a blinded manner, utilizing radiolabeled microspheres during treadmill running, as used extensively (Yang and Terjung(1993) J. Appl, Physiol. 75(1):452-457; Mackie and Terjung (1983) Am. J. Physiol. 245:H265-H275; Mathien and Terjung (1986) Am. J. Physiol. 245:H1050-H1059; Mathien and Terjung (1990) Am J. Physiol. 258:H759-H765; Yang et al. (1990) J. Appl. Physiol 69:1353-1359; Yang et al. (1995) Circ. Res. 76.448-456; Yang et al.
  • Microspheres (15 ⁇ m diameter), labeled with 85 Sr or 141 Ce ( ⁇ 10 mCi/g), were obtained commercially (NEN, Boston) in a suspension of 10% dextran containing 0.05% Tween 80. A well-mixed suspension of microspheres was carefully infused into the arch of the aorta, followed by a saline flush, over a 15-20 second period. Direct comparisons of injection sites (left ventricle versus aortic arch) gave the same blood flows to the kidneys and hindlimb muscles.
  • T tissue and RBS is the reference blood sample.
  • Surgical preparation for in vivo blood flow determination was a modification of that described by Laughlin et al. (1982) J. Appl. Physiol 52:1629-1635. Animals were anesthetized with ketamine/ACE-promazine (100 mg/0.5 mg per kg) and a catheter was inserted into the right carotid artery to the arch of the aorta for later infusion of the microspheres. A catheter (PE 50 tapered) was also placed into the caudal artery for the withdrawal of blood sample. Both catheters were filled with saline containing heparin (100 IU/ml), led under the skin, and exteriorized at the back of the neck.
  • Muscles (Greene (1963) Anatomy of the Rat, New York Hafner Pub. Co.) include: biceps femoris, semitendinosus, semimembranosus, caudofemoralis, adductor group, gluteus group, tensor fascias latae, quadriceps group, soleus, plantaris, gastrocnemius, tibialis anterior, extensor digitorurm longus, deep lateral and posterior crural muscles. The tibia, fibula, femur and foot are also weighed and counted.
  • Intramuscular administration of rFGF-2 increased blood flow in a dose-dependent manner (FIG. 20). When administered intra-arterially, the 15 ⁇ g/kg rFGF-2 dose was just as efficacious as the 30 ⁇ g/kg dose. Continuous infusion over a 14-day period did not provide a significantly different efficacy relative to the single IA infusion or single IM injection mode of administration.

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WO2001098346A3 (fr) 2003-05-01
US7541337B2 (en) 2009-06-02
WO2001098346A2 (fr) 2001-12-27
US7186407B2 (en) 2007-03-06
US20040209817A1 (en) 2004-10-21
AU2001268680A1 (en) 2002-01-02
JP2004501164A (ja) 2004-01-15
EP1324766A2 (fr) 2003-07-09
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