RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63/310,921, filed on February 16, 2022. The entire teachings of the above application are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present disclosure relates to methods, uses, and kits for treating impaired blood flow/perfusion in a subject.

BACKGROUND

[0003] Severely impaired blood flow impairs tissue perfusion, resulting in organ dysfunction from poor oxygen delivery, poor nutritional delivery, poor clearance of toxic metabolic byproducts, poor dissemination of locally produced molecular distress signals, and poor delivery of salutary progenitor and inflammatory cells that would be mobilized by these distress proteins to repair ischemic tissue. Vascular pathologies contributing to impaired blood flow include atherosclerosis, medial sclerosis, fibromuscular dysplasia, vasculitis, thrombosis, and embolization. In the brain, ischemic events result in stroke and vascular dementia. In the heart, ischemia can cause angina, heart attack, and/or congestive heart failure. In the legs, ischemia results in claudication and/or chronic limb threatening ischemia (CLI). In the lungs, ischemia from embolization or vasculitis causes poor oxygen and carbon dioxide exchange, as well as pulmonary hypertension and right heart failure. Mesenteric ischemia causes malnutrition. Renal ischemia leads to hypertension and renal failure. Spinal ischemia leads to motor and/or sensory dysfunction. Penile ischemia results in erectile dysfunction. Retinal ischemia causes loss of vision. Scalp ischemia causes hair loss. Auditory canal ischemia causes deafness. Venous thrombosis causes systemic or portal venous hypertension, swelling, and ulceration. Organ failure is the final result of impaired circulation.

[0004] Fibrinolysis and neovascularization are natural biologic processes that are not optimized when ischemic organ dysfunction becomes evident.

[0005] Fibrinolysis is an enzymatic process that begins when clot first appears and leads to clot dissolution. It is naturally self-limiting; an anti-fibrinolytic cascade is activated as soon as fibrinolysis starts. This balance is necessary to prevent hemorrhage. In large tissue beds, the rate this balance is restored is quicker than the rate of clot lysis. Residual thrombus is further degraded by inflammatory cells if they have access to the clot. Unfortunately, in vascular disease, the long segments of residual organized clot between atherosclerotic plaque lesions do not resolve. Pharmacologic fibrinolysis is possible, usually by infusion of thrombolytics by catheter. However, risk of hemorrhage limits such potent infusions to 48 to 72 hours. Complete and permanent clearance of all clot in extensive tissue beds is usually not feasible within this interval.

[0006] Neovascularization preserves tissue perfusion through growth of collateral arteries

(arteriogenesis); growth of capillaries, arterioles and venules (angiogenesis); and growth of lymphatics (lymphangiogenesis). Endothelial shear stress initiates arteriogenesis, while hypoxia stimulates angiogenesis. Natural neovascularization processes are often sufficient to maintain tissue perfusion through growth in diameter, length, and number of collateral vessels. However, when the disease burden is high, for example when there is multi-level arterial occlusive disease that attenuates shear stress or the patient has co-morbidities (e.g. age, diabetes) that affect availability and function of salutary reparative cells, the process is insufficient to restore tissue perfusion. Certainly, as the lack of perfusion advances, the ischemic environment itself impairs normal biological reparative processes.

[0007] Current interventions to treat severely impaired blood flow include bypassing around the blockages or opening the occluded vasculature directly with endarterectomy or using balloons, stents, atherectomy devices, and lasers, or administering potent thrombolytic agents. Invasive revascularization (surgery- or catheter-based) is typically offered to prevent amputation in CLI cases. Thrombolytics and catheter procedures are used for treatment of stroke. Despite significant procedural risk, variable durability, and high cost, these approaches remain the standard of care. Immediate risks of these interventions include acute failure, bleeding, infection, intimal hyperplasia, and injury to other organs. Long term risks include re-thrombosis at the intervention site, blood-surface incompatibility, progression of disease at or above or below the intervention, aneurysmal degeneration, bleeding, poor graft incorporation, and infection. Complex revisions are frequently needed, with progressively shorter durability and at increased risk. Costs can be staggering.

[0008] Thus, new, safe and durable strategies to treat or avoid vascular pathologies are needed. For example, compositions and methods for treatment of arterial occlusive disease and chronic limb-threatening ischemia are needed which improve perfusion by stimulating fibrinolysis safely and restoring natural neovascularization to reduce the need for invasive revascularization and amputation. New, safe, and durable strategies are also required for treatment of other forms of severely impaired blood flow, such as thromboembolic stroke.

SUMMARY

[0009] Provided herein are methods of treating impaired perfusion in a subject comprising administering to the subject an extended delivery regimen of a pharmaceutical composition comprising a blood cell growth factor. The impaired perfusion may be a result of vascular occlusion and/or vascular stenosis in the subject, or it may be a result of atherosclerosis, medial calcific sclerosis, fibromuscular dysplasia, vasculitis, embolism, thrombosis, and/or intimal hyperplasia.

[0010] An extended delivery regimen as described herein is effective to stimulate in the subject one or more of: an increase in plasma and serum concentration of plasmin and Fibrin Degradation Products (FDP); effectors of neovascularization; progenitor cell division; mobilization of progenitor cells into circulation; and/or production of granulocytes and/or monocytes. The methods described herein may stimulate fibrinolysis and/or neovascularization in the subject.

[0011] In some embodiments of the methods provided, the blood cell growth factor is a granulocyte colony-stimulating factor (G-CSF), a granulocyte-macrophage colonystimulating factor (GM-CSF), a macrophage colony-stimulating factor (M-CSF), a combination thereof, a functional equivalent thereof, a derivative thereof, or a biosimilar thereof.

[0012] The pharmaceutical composition for use in the methods may be filgrastim, tbo- filgrastim, filgrastim-sndz, filgrastim-aafi, Accofil, balugrastim, biograstim, efbemalenograstim alfa, Grastofil, Hexal, lenograstim, lipegfilgrastim, Pelgraz, ratiograstim, sargramostin, Tevagrastim, Emgrast, Fegrast, filgrastim (Lupin), Grafeel, Neukine, Religrast, a combination thereof, a functional equivalent thereof, a derivative thereof, or a biosimilar thereof. In some embodiments, the pharmaceutical composition is a long-acting pharmaceutical composition such as, e.g., pegfilgrastim, pegfilgrastim-bmez, pegfilgrastim- cbqv, pegfilgrastim-jmbd, eflapegrastim, TX-01 (a filgrastim biosimilar in development by Tanvex BioPharma Inc. (San Diego, CA)), a combination thereof, a functional equivalent thereof, a derivative thereof, or a biosimilar thereof.

[0013] The extended delivery regimen described may comprise administering a dosage of the pharmaceutical composition to the subject 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or 15 times over the course of about 1 month. [0014] In some embodiments, the extended delivery regimen comprises a dosage of the pharmaceutical composition administered to the subject 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, or about 40 times over the course of about 2 months, about 3 months, about 4 months, or over 4 months.

[0015] In some embodiments, the dosage of the pharmaceutical composition is administered to the subject daily, about once every 2 days, about once every 3 days, about once every 4 days, about once every 5 days, about once every 6 days, about once every 7 days, about once every 8 days, about once every 9 days, about once every 10 days, about once every 11 days, about once every 12 days, about once every 13 days, about once every 14 days, about once every 15 days, about once every 16 days, about once every 17 days, about once every 18 days, about once every 19 days, about once every 20 days, about once every 21 days, about once every 22 days, about once every 23 days, about once every 24 days, about once every 25 days, about once every 26 days, about once every 27 days, about once every 28 days, about once every 29 days, about once every 30 days, about once every 45 days, about once every 60 days, about once every 90 days, or about once every 120 days.

[0016] The dosage of the pharmaceutical composition may be administered to the subject once daily, twice daily, or three times daily.

[0017] The extended delivery regimen may be discontinuous, in that administration of the pharmaceutical composition to the subject occurs for a period of time; is stopped for a period of time; and is then re-initiated. For example, the period of time in which the administration occurs or is stopped may be about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, or longer than 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, or longer than 1 year.

[0018] In some embodiments, the dosage of the pharmaceutical composition comprises a weight-based dose of about 1 mcg/kg, about 2 mcg/kg, about 3 mcg/kg, about 4 mcg/kg, about 5 mcg/kg, about 6 mcg/kg, about 7 mcg/kg, about 8 mcg/kg, about 9 mcg/kg, about 10 mcg/kg, about 11 mcg/kg, about 12 mcg/kg, about 13 mcg/kg, about 14 mcg/kg, or about 15 mcg/kg of the blood cell growth factor. Alternatively, the dosage of the pharmaceutical composition may comprise a fixed dose of about 100 mcg, about 200 mcg, about 300 mcg, about 400 mcg, about 500 mcg, about 600 mcg, about 700 mcg, about 800 mcg, about 900 mcg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, or about 15 mg of the blood cell growth factor.

[0019] The methods may entail subcutaneously administering, intravenously administering, intraperitoneally administering, intramuscularly administering, topically administering, parenterally administering, or administering the pharmaceutical composition using an on-body injector.

[0020] In one embodiment, the pharmaceutical composition is filgrastim and the extended delivery regimen comprises administering to the subject about 1 to about 10 fixed dosages of about 550 mcg to about 960 mcg of the pharmaceutical composition about once every 3 days for about 2 weeks to about one month.

[0021] In one embodiment, the pharmaceutical composition is filgrastim and the extended delivery regimen comprises administering to the subject about 1 to about 10 weight-based dosages of about 6 mcg/kg to about 11 mcg/kg of the pharmaceutical composition about once every 3 days for about 2 weeks to about one month.

[0022] The impaired perfusion in the subject may be a symptom of vascular occlusion or vascular stenosis resulting from one or more of atherosclerosis, medial calcific sclerosis, fibromuscular dysplasia, vasculitis, embolism, thrombosis, and intimal hyperplasia. The impaired perfusion may be a symptom of arterial disease or chronic venous disease.

[0023] The arterial disease may be ischemic cardiomyopathy, vascular dementia, lacunar infarcts, bland (non-hemorrhagic) infarct, cerebral vasculitis, ischemic retinopathy, pulmonary hypertension, pulmonary embolism, upper or lower extremity chronic limbthreatening ischemia, upper or lower extremity claudication, hypothenar hammer syndrome, vascular impotence, vascular alopecia, vascular deafness, chronic mesenteric ischemia, chronic renal artery disease, and/or post-infection micro thrombosis, or chronic residual ischemia after vasculitis (inflammation) resolves of Takayasu’s arteritis, Kawasaki’s arteritis, ischemic blindness caused by temporal arteritis, marijuana induced vasculitis, and/or thromboangiitis obliterans (Buerger’s disease).

[0024] The chronic venous disease may be chronic deep vein thrombosis (DVT) induced venous hypertension, phlegmasia (acute DVT) alba dolens, phlegmasia cerulea dolens, effort thrombosis (upper arm) (“Paget Shroeder disease”), chronic mesenteric venous disease, portal venous hypertension, and/or DVT induced pelvic congestion. [0025] In some embodiments, the impaired perfusion is a symptom of peripheral artery disease and presents in one or more limb of the subject. In such embodiments, the methods may further comprise using compression on the one or more limb of the subject.

[0026] For example, compression may be useful where the impaired perfusion presents as claudication or chronic limb-threatening ischemia in the subject.

[0027] The applied compression may be mechanical compression or pneumatic compression. The compression may be peristaltic or pulsatile.

[0028] In some embodiments, compression is applied to the one or more limb of the subject at least 1 hour daily, at least 2 hours daily, at least 3 hours daily, at least 4 hours daily, or at least 5 hours daily. Compression may be applied in at least 1 session a day, at least 2 sessions a day, at least 3 sessions a day, at least 4 sessions a day, or at least 5 sessions a day. Compression may be applied 1 day per week, 2 days per week, 3 days per week, 4 days per week, 5 days per week, 6 days per week, or 7 days per week. Compression may be applied for at least: about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, or longer than 12 months. In some embodiments, compression is applied until symptoms of the subject resolve.

[0029] Applying compression may comprise contacting the one or more limb of the subject with a limb compression device.

[0030] In one embodiment, where the subject’s impaired perfusion involves chronic limbthreatening ischemia and the subject has no revascularization treatment options (sometimes referred to as “no-option CLI"), the extended delivery regimen may comprise administering 6 mcg/kg to 11 mcg/kg subcutaneously every 3 days for one month and the applying compression may comprise contacting the affected limb of the subject with a limb compression device until symptoms resolve or the affected limb requires amputation.

[0031] The limb compression device may comprise at least 1 inflatable compression sleeve, at least 2 inflatable compression sleeves, at least 3 inflatable compression sleeves, at least 4 inflatable compression sleeves, at least 5 inflatable compression sleeves, at least 6 inflatable compression sleeves, at least 7 inflatable compression sleeves, or at least 8 inflatable compression sleeves, wherein pressure is delivered to one or more inflatable bladders in one or more of the inflatable compression sleeves. In some embodiments, the one or more inflatable bladders are inflated to a maximum pressure of about 60 mm/Hg, about 70 mm/Hg, about 80 mm/Hg, about 90 mm/Hg, about 100 mm/Hg, about 110 mm/Hg, about 120 mm/Hg, about 130 mm/Hg, about 140 mm/Hg, or about 150 mm/Hg; inflated to the maximum pressure in 0.5 seconds or less, 0.4 seconds or less, 0.3 seconds or less, 0.2 seconds or less, or in about 0.25 seconds; deflated to a minimum pressure of about 10 mm/Hg; and deflated to the minimum pressure in 0.5 seconds or less, 0.4 seconds or less, 0.3 seconds or less, 0.2 seconds or less, or in about 0.25 seconds. For example, the one or more inflatable bladders may be held inflated for about 3 seconds and deflated for about 17 seconds for a total cycle time of about 20 seconds.

[0032] In some embodiments, applying compression may comprise contacting the one or more limbs of the subject with the limb compression device in sessions of at least 10 minutes; at least 20 minutes; at least 30 minutes; at least 1 hour; at least 2 hours; at least 3 hours; or at least about 4 hours.

[0033] Some embodiments of the methods may further comprise inflating one or more of the inflatable compression sleeves to a foot, an ankle, then a calf of the subject in sequence, or to a calf, an ankle, then a foot of the subject in sequence.

[0034] In some embodiments, the methods further comprise stopping for a period of time one or both of the administering of the pharmaceutical composition and the applying compression. The period of time of the stopping may be about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, or longer than 1 year.

[0035] Some embodiments of the methods provided entail treating peripheral artery disease presenting in one or more limb of a subject by identifying a subject as suitable for treatment of the peripheral artery disease using an extended delivery regimen of a pharmaceutical composition comprising a blood cell growth factor; and administering to the subject the extended delivery regimen of the pharmaceutical composition comprising the blood cell growth factor. Treatment of peripheral artery disease in one or more limb of a subject in this fashion may further entail applying compression to the one or more limb of the subject as described herein. The applying compression force may further entail using ultrasound shockwave therapy applied with a transducer and a coupling material such as ultrasonic gel if being effected with a manual transducer, or in a water or saline bath if effected in a tub. The compression force may also be augmented or effected by an external vibrating surface under the feet or by an external vibrating unit akin to a commercially available massage device.

[0036] For example, the identifying a subject may comprise identifying a subject as having claudication or chronic limb-threatening ischemia, wherein the subject: has claudication or chronic limb-threatening ischemia that cannot be treated with compression; refuses or disfavors treatment with compression; cannot tolerate treatment using compression; and/or has failed to improve with treatment using compression or for whom continued treatment using compression will fail to improve the peripheral artery disease. [0037] In some embodiments, identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen comprises identifying the subject as having persistent or increased pain or pain upon exertion, coldness, claudication, ulceration, or gangrene in the affected one or more limb of the subject.

[0038] The affected one or more limb of the subject may be a toe, foot, ankle, calf, or portion thereof. In such cases, the identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen may comprise identifying the subject as having (i) hemodynamics showing an ankle-brachial index (ABI) measurement of less than 0.5, less than 0.49, less than 0.48, less than 0.47, less than 0.46, or less than 0.45, (ii) anon-pulsatile or monophasic ankle or pedal Doppler waveform, and/or (iii) anon- pulsatile or monophasic pedal (transmetatarsal or toe) Doppler or photoplethysmography (PPG) waveform.

[0039] In some embodiments, the subject may have undergone a toe or forefoot amputation and the wound is closed with the skin sutured. In such cases, identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen comprises identifying the subject as having toe pressure of 30 mmHg or less in a non-diabetic subject or 50 mmHg or less in a diabetic subject.

[0040] In some embodiments, the subject may have undergone a toe or forefoot amputation and the wound is left open. In such cases, identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen may comprise identifying the subject as having an ankle-brachial Index (ABI) measurement of less than 0.5, or ankle or pedal non-pulsatile or monophasic waveforms after undergoing limb compression for about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or more than 8 weeks.

[0041] In some embodiments, identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen may comprise identifying the subject as having an absence of granulation in an open wound in the affected one or more limb of the subject after undergoing limb compression period for about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or more than 8 weeks. [0042] In some embodiments, identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen may comprise identifying the subject as having forefoot ischemic rest pain with preserved tissue turgor and no muscle atrophy.

[0043] In some embodiments, identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen may comprise identifying the subject as having advanced CLI with severe ischemic rest pain and loss of tissue turgor, and atrophy of muscle mass.

[0044] In some embodiments, identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen may comprise identifying the subject as having (i) tissue loss due to ulceration or gangrene and (ii) an ABI measurement of less than 0.5 or non-pulsatile or monophasic ankle or pedal waveforms.

[0045] The methods of treating peripheral artery disease presenting in one or more limb of a subject may further comprise applying compression to the one or more limb as described herein.

[0046] Also provided herein are uses of a blood cell growth factor in the manufacture of a medicament for treating impaired perfusion in a subject. The blood cell growth factor for use in the manufacture of a medicament for treating impaired perfusion may comprise one or more of: a granulocyte colony-stimulating factor (G-CSF), a granulocyte-macrophage colony-stimulating factor (GM-CSF), a macrophage colony-stimulating factor (M-CSF), filgrastim, tbo-filgrastim, filgrastim-sndz, filgrastim-aafi, Accofil, balugrastim, biograstim, efbemalenograstim alfa, Grastofil, Hexal, lenograstim, lipegfilgrastim, Pelgraz, ratiograstim, sargramostin, Tevagrastim, Emgrast, Fegrast, filgrastim (Lupin), Grafeel, Neukine, Religrast, pegfilgrastim, pegfilgrastim-bmez, pegfilgrastim-cbqv, pegfilgrastim-jmbd, eflapegrastim, or TX-01 (a filgrastim biosimilar in development by Tanvex BioPharma Inc. (San Diego, CA)), a combination thereof, a functional equivalent thereof, a derivative thereof, or a biosimilar thereof.

[0047] A dosage of the medicament may be effective to stimulate in the subject one or more of: an increase in serum and plasma concentration of plasmin; effectors of neovascularization; progenitor cell division; mobilization of progenitor cells into circulation; and/or production of granulocytes and/or monocytes.

[0048] Also provided herein are kits for treating impaired perfusion in a subject. The kits may comprise a pharmaceutical composition comprising a granulocyte colony-stimulating factor (G-CSF), a granulocyte-macrophage colony-stimulating factor (GM-CSF), a macrophage colony-stimulating factor (M-CSF), filgrastim, tbo-filgrastim, filgrastim-sndz, filgrastim-aafi, Accofil, balugrastim, biograstim, efbemalenograstim alfa, Grastofil, Hexal, lenograstim, lipegfilgrastim, Pelgraz, ratiograstim, sargramostin, Tevagrastim, Emgrast, Fegrast, filgrastim (Lupin), Grafeel, Neukine, Religrast, pegfilgrastim, pegfilgrastim-bmez, pegfilgrastim-cbqv, pegfilgrastim-jmbd, eflapegrastim, or TX-01 (a filgrastim biosimilar in development by Tanvex BioPharma Inc. (San Diego, CA)), a combination thereof, a functional equivalent thereof, a derivative thereof, or a biosimilar thereof in a dose effective to stimulate in the subject one or more of: an increase in serum or plasma concentration of plasmin; effectors of neovascularization; progenitor cell division; mobilization of progenitor cells into circulation; and/or production of granulocytes and monocytes. Some embodiments of the kit include a compression device for compression of vasculature comprising a mechanical pump or a pneumatic pump attached to an inflatable compression sleeve. The kits may further comprise instructions for use of the pharmaceutical composition and instructions for use of the compression device, where applicable. The kits may further comprise a device for administering the pharmaceutical composition.

[0049] Also provided herein are methods of administering a fixed dose of a blood cell growth factor to a subject in need thereof. The methods may comprise administering to the subject an extended delivery regimen of a pharmaceutical composition comprising a blood cell growth factor by determining the weight of the subject and administering to the subject a fixed dose of about 450 mcg, about 550 mcg, about 640 mcg, about 780 mcg, or about 900 mcg of the blood cell growth factor based on the weight of the subject in accordance with Table 14 herein. In some embodiments, the methods comprise determining the weight of the subject and administering to the subject a fixed dose of about 450 mcg, about 550 mcg, about 640 mcg, about 780 mcg, or about 900 mcg of filgrastim based on the weight of the subject in accordance with Table 12 herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1A - Figure ID depict ankle-brachial index (ABI) measurements (Figure 1A and Figure IB) and proteomic and biochemical analysis (Figure 1C and Figure ID) for the patient discussed in Example 12 and Example 13.

[0051] Figure 2A - Figure 2L are fluoroscopic images and ABI data for the patient discussed in Example 27.

[0052] Figure 3A - Figure 3G provide an ABI line graph and fluoroscopic images for the patient discussed in Example 16. [0053] Figure 4A - Figure 4E are photographs and fluoroscopic images for the patient discussed in Example 17 A.

[0054] Figure 5 A - Figure 5C are bar graphs showing serum nitrite levels (mM) in patients being treated either with filgrastim and limb compression or with limb compression alone discussed in Example 26.

[0055] Figure 6A - Figure 6D are bar graphs showing serum and plasma concentration of plasmin and Fibrin Degradation Products in patients being treated with filgrastim and limb compression or limb compression alone discussed in Example 31.

[0056] Figure 7A - Figure 7D are angiograms for the patient discussed in Example 10.

DETAILED DESCRIPTION

I. INTRODUCTION

[0057] Described herein are methods to treat impaired perfusion by increasing the flow/passage of fluids to, from, and/or through tissue, increasing arterial inflow to tissue, and/or increasing venous drainage of fluids from tissue. This process is accomplished by stimulating innate fibrinolysis to dissolve chronic thrombus and innate neovascularization to grow the diameter, number, and length of collateral vessels present in the tissue, or promote maturation of new arteries, arterioles, veins, venules, and capillaries.

[0058] A diminished number and function of circulating reparative progenitor cells impairs neovascularization, particularly in elderly dysvascular patients. Rauscher FM, et al., (2003) Circulation 108:457-63; Hill JM, et al., (2003) N Engl J Med 348:593-600; Heiss C, et al., (2005) J AmColl Cardiol 45: 1441-8. Diabetes, heart failure, and renal insufficiency compound this deficit. Vasa M, et al., (2001) Circ Res 89:El-7; Loomans CJ, et al., (2004) Diabetes 53:195-9; Tepper OM, et al., (2002) Circulation 106:2781-6. The rate of presentation of the progenitor cells to the ischemic tissue is diminished by impaired arterial inflow. Therefore, neovascularization is particularly disadvantaged in the population most in need: patients with vascular disease, for example those with chronic limb-threatening ischemia (CLI).

[0059] The BASIL trial (J Vase Surg 2010; 51) highlights the yearly amputation-free survival rates of surgery and angioplasty for CLI: 80% at one year follow-up, 65% at two years, 55% at three years, 45% at four years, and 40% at five years follow-up. Patients spend 5-6 weeks being treated outside the home in the first year, and 2-3 weeks per year thereafter. Hospitalization, intervention, revisions, perioperative studies, wound complications, and rehabilitation all contribute to escalating costs and adversely impact quality of life, especially when these interventions fail. Insufficient growth of new arteries (arteriogenesis), arterioles, capillaries, and venules (angiogenesis) and insufficient fibrinolysis are responsible for the progressive severity of chronic limb-threatening ischemia resulting from vascular occlusive diseases.

[0060] Endothelial shear stress initiates collateral artery growth (in length, diameter, and number). Multi-level arterial occlusive disease in CLI attenuates this stimulus. Without this shear stress stimulus, the endothelial cells lining small collateral arteries are not “activated”, and thus cannot initiate arteriogenesis. Endothelial activation includes the upregulation of nitric oxide synthase, the enzyme responsible for endothelial induced arterial dilatation and loosening of the gap junctions between endothelial cells. Endothelial cells grow as a monolayer and do not proliferate until contact inhibition is relieved. Endothelial activation also causes the expression of proteins and surface adhesion molecules that attract and capture circulating monocytes and progenitor cells that would orchestrate arteriogenesis. Moreover, without sufficient inflow of oxygenated nutritive blood flow into the ischemic tissue, and without efficient clearance of metabolic waste products from the ischemic tissue, the environment for neovascularization deteriorates. Further, protein distress signals (e.g., stromal cell-derived factor 1 (SDF-1)) released from ischemic tissue do not disseminate effectively. These proteins would normally recruit pro-arteriogenic reparative cells. Even if recruited, these cells may not make it back to their target as flow to the ischemic tissue is impaired and the cells travel unimpeded paths elsewhere and are captured.

[0061] Angiogenesis is also impaired by this trafficking problem, even though its stimulus, hypoxia, is present. These hemodynamic challenges can be addressed with external compression, for example with an external pump worn on the limb, or sound waves applied through the skin or bone (in the case of stroke) or by vibration. The frequency and amplitude of vascular deformation may be modulated by external mechanical energy to restore kinetic energy to the decelerating blood and to optimize the endothelial shear stress stimulus. This will improve inflow of oxygenated nutritive blood, clearance of waste products of metabolism, and trafficking of elements needed to promote neovascularization. In the lower extremities, compression can also induce fissuring within the thrombus allowing fibrinolysis to occur over a greater area and in less time.

[0062] Transduction of an external force applied by a programmed pneumatic compression pump into a physiological effect was examined in methods disclosed herein at the protein and cellular levels. Intermittent compression increases shear stress across the endothelium, causing it to increase production of monocyte chemoattractant protein- 1 (MCP- 1), a protein which attracts salutary cells to the activated endothelium, as well as serum nitric oxide produced as result of increased nitric oxide synthase activity. Further, endothelial cell membrane surface adhesion molecules such as platelet endothelial cell adhesion molecule (PECAM-1), vascular cell adhesion molecule (VC AM), junctional adhesion molecule (JAM), and intercellular adhesion molecule (ICAM) all increase.

[0063] The proteins analyzed in examples herein are involved in different aspects of vessel growth: endothelial tube formation, media cell growth, hormonal regulators, matrix degradation, and inflammation. Additionally, previously occluded named vessels became visible on follow up angiograms, consistent with fibrinolysis. Levels of serum and plasma concentration of plasmin and of Fibrin Degradation Products (FDP) were measured as an index of fibrinolysis.

[0064] The results provided herein demonstrate that an environment favoring fibrinolysis and neovascularization is created by the methods described herein.

[0065] Angiographic and diagnostic duplex ultrasound imaging of subjects treated with methods described herein show occluded arterial segments and slow transit time through the ischemic limb before treatment. Following therapy, angiogram and duplex ultrasound imaging confirmed recanalization of previously occluded arterial segments, along with improved ankle pressures and toe and ankle doppler derived waveforms. The contrast transit time was also much faster. Most importantly, plasmin and fibrin degradation products (from clot breakdown) increased.

[0066] Angiographic and diagnostic duplex ultrasound imaging caudal to multi-level arterial occlusive disease also show severely attenuated luminal arterial flow before treatment. This translates to diminished endothelial shear stress, which is required to activate the endothelium so collateral arterial growth (arteriogenesis) can ensue. This stimulus is delivered by using external limb compression. Providing shear stress activates the endothelium, the first step in stimulating collateral growth (arteriogenesis). Evidence for endothelial cell activation include increased Monocyte Chemoatactic Protein (MCP-1) production, endothelial adhesion molecule expression, and nitric oxide synthase upregulation (as measured by increased serum nitrite and nitrate). These were measured before and after administration of intermittent compression to the limb, providing biologic evidence of endothelial activation.

[0067] Circulating monocytes and progenitor cells contribute significantly to arteriogenesis. Attracted by MCP-1, they attach to activated endothelial surface adhesion molecules and are then dragged sub-endothelial to orchestrate arterial growth. In vascular disease, circulating progenitor cells are decreased in number and diminished in function. Granulocyte colony stimulating factor (G-CSF) was used to increase the number of these cells in the circulation.

[0068] CLI clinical trials using either compression alone for variable months or granulocyte colony stimulating factor (G-CSF) alone daily for a week or less or up to 10 days showed limited benefit in achieving limb salvage. Treatment failures observed in these trials are attributed to limitations of monotherapy. Specifically, during the G-CSF trials, the impact of the hemodynamic failure was not addressed. In the limb compression studies, the progenitor cell deficits and the impediment of chronic thrombus were not addressed. Treatment failures observed in these trials are further attributed to the limited duration of G- CSF administration.

[0069] The biochemical and cytometry data support hemodynamic, clinical, and angiographic evidence that fibrinolysis and neovascularization are promoted by an approach that combines use of G-CSF and external compression when applied as described herein. Amputation-free survival was observed to increase in patients with failed previous interventions and with severe ischemia presenting with ischemic forefoot rest pain, nonhealing wounds and/or gangrene.

[0070] Compression devices may be used to effect limb compression. In some cases, limb compression is programmable or programmed. Examples of suitable limb compression devices include the ArtAssist® pneumatic compression device (ACI Medical, Inc, San Marcos, CA), an intermittent programmable pneumatic compression device used to increase arterial blood flow in patients with chronic limb-threatening ischemia. Louridas G, et al., (2002) Int Angiol 21:28-35; van Bemmelen PS, et al., (2001) Arch Surg 136:1280-5, discussion 1286; Montori VM, et al., (2002); Int Angiol 21:360-6; Kavros SJ, (2008) J Vase Surg 47:543-9; the contents of each of which are incorporated by reference herein in their entireties. Sequential venous emptying of calf, ankle, and foot pneumatic compression cuffs or inflatable compression sleeves increases the arteriovenous gradient locally, which thereby lowers the resistance across the capillary network and increases blood flow into the ischemic tissue. It is envisioned herein that the rapid inflation causes the endothelium to release nitric oxide, a powerful vasodilator (Liu K, et al., (1999) J Orthop Res 17:415-20; Guzman RJ, et al., (1997) Surgeryl22:273-9; discussion 279-80) and stimulator of arteriogenesis (the formation and widening of medium-sized blood vessels); Unthank JL, et al., (1996) Circ Res 79:1015-23; incorporated herein by reference in their entireties. The association of limb compression with arteriogenesis has been inferred from angiography (van Bemmelen P, et al., (2003) Ann Vase Surg 17:224-8), sustained improved arterial hemodynamics, and increased skin perfusion. Eze AR, et al., (1996) Am J Surg 172:130-4; discussion 135; incorporated by reference in their entireties. However, limb compression is associated with variable outcomes, particularly in elderly patients with diabetes, renal failure, and/or congestive heart failure. This may be due to the reduced number and function of circulating reparative progenitor cells in these populations, and the inability to clear chronic fibrin cross-linked thrombus.

[0071] Examples herein describe the clinical course of 75 patients treated with an intermittent pneumatic limb compression device or with a granulocyte colony stimulating factor (G-CSF) and an intermittent pneumatic limb compression device, including 2 patients treated with G-CSF and limb compression after compression alone failed to improve the patient’s condition. Six additional patients treated with filgrastim and limb compression participated in a confirmatory study of cytometry and proteins involved in fibrinolysis and neovascularization. G-CSF is a cytokine growth factor that dramatically boosts the circulating number of reparative progenitor (stem) cells (Bolwell B, et al., (1997) Bone Marrow Transplant 19:215-9; Haas R, Murea S. (1995) Cytokines Mol Ther 1:249-70. Gazitt Y. (2002) Curr Opin Hematol 9:190-8) needed for neovascularization. Further, G-CSF induces endothelial cells to proliferate, migrate, and stimulate repair of mechanically wounded endothelial monolayers. Bussolino F, et al., (1991) J Clin Invest 87:986-95; Bussolino F, et a., (1993) Int J Clin Lab Res 23:8-12. G-CSF-assisted neovascularization strategies have been reported clinically. Kudo FA, et al., (2003) Int Angiol 22: 344-8; Kawamura A, et al., (2006) J Artif Organs 9:226-33; Huang P, et al., (2005) Diabetes Care 28:2155-60; incorporated by reference herein. It is envisioned herein that G-CSF will potentiate the arteriogenesis observed with limb compression use, and the angiogenesis stimulated by the hypoxia, by providing a needed boost in the depleted circulating stem cell pool while also increasing plasma and serum concentration of plasmin to stimulate fibrinolysis as reported in the Examples.

[0072] Previously, neovascularization was attempted by delivering large numbers of progenitor cells directly into ischemic tissue in a single session, either by multiple intramuscular injections or by arterial embolization of the cells, most of which are distributed quickly throughout the body with only a percentage arriving in the ischemic tissue (due to the multi-level occlusive disease), and a smaller percentage possibly engrafting. The approach described herein avoids the costly and invasive hospital-based bone marrow harvest, ex vivo cell processing, and cell reinjection required in these previously described neovascularization strategies, with improved efficacy.

[0073] The approach described herein overcomes the problems of non-uniform progenitor cell distribution after direct injection into the ischemic calf musculature, and limited survival of these cells. An alternative approach, single-session intra-arterial injection, has the potential to provide a more uniform progenitor cell distribution. However, significant “pass through” in lower resistance arteries results in cell sequestration in other organs. Healing of a dehisced trans metatarsal amputation wound, or of an ankle wound with an exposed Achilles’ tendon, would be challenging even in the setting of good arterial perfusion. That healing was observed in examples herein in the setting of severe ischemia demonstrates efficacy. The healing was correlated with hemodynamic and angiographic evidence of neovascularization. This occurred despite severe comorbidities including coronary artery disease, renal insufficiency, diabetes, and congestive heart failure. When successful, traditional revascularization (surgery, catheter technology) produces immediate improvement in blood flow but it is significantly less durable compared to neovascularization and fibrinolysis obtained by methods herein, and it is not always successful. These methods result in gradual but more durable improved blood flow, wound healing, and symptom abatement over time (weeks and months), and at lower risk and cost.

[0074] Neovascularization in chronic limb-threatening ischemia (CLI) fails for numerous reasons: poor biochemical environment, hemodynamic failure, progenitor cell deficiency, clot and plaque obstruction, and poor immunity. The poor biochemical environment relates to poor delivery of oxygenated nutritive blood flow resulting in poor energy use as inefficient anaerobic glycolysis replaces more efficient oxidative phosphorylation pathways (e.g., Kreb’s cycle). The local tissue acidosis and adenosine triphosphate (energy currency) production failure result in protein and enzyme denaturation and cell membrane permeability leading to apoptosis. Synthetic regenerative pathways become impaired. A goal of methods herein is to restore the healthy biological environment so biochemical and cellular processes conducive to fibrinolysis and neovascularization are restored. Arteriogenesis is stimulated by endothelial shear stress in the arteries. However, multi-level occlusive diseases attenuate endothelial shear stress and thus arteriogenesis. Multi-level occlusive disease also impairs inflow of oxygenated nutritive blood, clearance of waste products of metabolism, dissemination of protein signals from the ischemic tissue, and arrival of pro-angiogenic cells to the ischemic tissue. Progenitor cells participate in angiogenesis and arteriogenesis and are observed to be deficient in number and in function in CLI patients. Further, these patients have poor immunity.

[0075] To promote neovascularization, the subject in methods provided herein is administered a growth factor such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or a macrophage colonystimulating factor (M-CSF). Examples of growth factors suitable for use in the methods provided herein include: filgrastim, tbo-filgrastim, filgrastim-sndz, filgrastim-aafi, Accofil, balugrastim, biograstim, efbemalenograstim alfa, Grastofil, Hexal, lenograstim, lipegfilgrastim, Pelgraz, ratiograstim, sargramostin, Tevagrastim, Emgrast, Fegrast, filgrastim (Lupin), Grafeel, Neukine, Religrast, combinations thereof, functional equivalents thereof, derivatives thereof, or biosimilars thereof, or an agent that safely promotes CSF production in the body. Additional examples of growth factors suitable for use in the methods provided herein include factors that have been modified or adapted to be “long-acting” growth factors, such as pegfilgrastim, pegfilgrastim-bmez, pegfilgrastim-cbqv, pegfilgrastim-jmbd, eflapegrastim, and TX-01 (a filgrastim biosimilar in development by Tanvex BioPharma Inc. (San Diego, CA)). The growth factors stimulate progenitor cell division and mobilize the progenitor cells into circulation from stem cell niches. Further, the growth factor promotes pro-angiogenic cytokines for endothelial activation, endothelial proliferation, vascular wall assembly, extracellular matrix (ECM) degradation and tissue growth, and metabolic breakdown of obstructive fibrin cross linked thrombus. The proteins involved are monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF), placental growth factor (PIGF), hepatocyte growth factors (HGF), fibroblast growth factor-a (FGF-a), fibroblast growth factor-P (FGF-P), platelet derived growth factor AA (PDGF-AA), platelet derived growth factor BB (PDGF-BB), platelet derived growth factor AB (PDGF-AB), agiopoietin-1, hepatocyte growth factor, interleukin-6, interleukin-8, membrane metalloproteinase (MMP) (1, 2, 3 and 9), insulin growth factor- 1, hepatocyte growth factor, TGF-[3. The growth factor further induces fibrinolysis by increasing serum and plasma concentration of plasmin and fibrin degradation products. Additionally, the growth factors treat impaired immunity by increasing macrophage activity and increasing population of neutrophils, monocytes and tumor necrosis factor-a (TNF-a).

[0076] In response to shear stress, vascular endothelial cells produce MCP-1, attracting effectors of arteriogenesis. VEGFA promotes vascular permeability, angiogenesis, vasculogenesis, vascular endothelial cell growth, and cell migration and inhibits apoptosis. HGF plays a central role in angiogenesis and tissue regeneration. FGF-P promotes proliferation and differentiation of vascular endothelial cells, smooth muscle cells and fibroblasts. TNF-a activates signal transduction pathways including nuclear factor-kb, jun N- terminal kinase, p38, extracellular signal regulated kinase, and phosphoinositide 3-kinase. GM-CSF stimulates stem cells to produce granulocytes and monocytes. Monocytes exit the circulation and migrate into tissue and mature to macrophages. TGF-J31 controls cell growth, cell proliferation, cell differentiation and apoptosis. MMP-9 degrades extra cellular matrix, which is extremely important in angiogenesis and neovascularization. The metalloproteinase activity results in physical space into which the endothelial tube can grow. PIGF belongs to the VEGF family and is a key molecule in angiogenesis and vasculogenesis. PDGF regulates cell growth and division and is a potent mitogen for cells of mesenchymal origin including smooth muscle cells that participate in neovascularization. PDGF is central to the growth and maturation of the vessel supporting structure (the media). Without the media the endothelial tubes would rupture. The media also provides the elasticity to absorb some of the systolic energy from the blood flow to offset the systolic force on the fragile endothelium. Ang-1 is critical for vessel maturation, particularly of the media, adhesion, migration, and survival. IL- 6 is an anti-inflammatory myokine that stimulates growth of vascular SMC in a PDGF- dependent manner. IGF-1 regulates migration of human vascular endothelial cells and angiogenesis. Plasmin is a fibrinolytic enzyme. FDP as used herein means fibrin degradation products arising from the plasmin effect on chronic thrombus.

[0077] The attenuated shear stress stimulus needed for arteriogenesis in chronic limbthreatening ischemia (CLI) can be restored with limb compression. Limb compression also increases inflow of oxygenated nutritive blood, clears waste products of metabolism, and enhances the traffic of elements needed for neovascularization. The circulating progenitor cell (CPC) population is also depressed in CLI.

[0078] In methods and examples herein, the compression of the limb is affected by a sleeve covering a portion of the limb or alternatively the entire limb. The sleeve contains a bladder, which is filled intermittently by a pump with either air or a fluid, or alternatively contains small electromagnets that attract toward each other in response to a pulsed current. The compression upstroke delivers a rapid force to the underlying limb sufficient to activate the vascular endothelium in the limb vasculature. This causes endothelial "activation" by exerting the mechanical force transduced by the endothelium into a biochemical response. The biochemical response includes expression of MCP-1, increase of nitric oxide synthase (NOS) activity, and expression of cell membrane adhesion molecules (e.g., PCAM, VCAM, JAM). In examples herein the levels of MCP-1, serum nitrate and nitrite were measured in venous blood before and after use of a pneumatic pump. MCP-1 is a homing signal for circulating monocytes and vascular progenitor cells and was observed to be elevated after compression. NOS results in release of a potent vasodilator which results in endothelial cell separation and ablation of contact inhibition to mitosis. Nitric oxide is rapidly broken down to serum nitrate and nitrate which were measured and observed to be elevated in patients after compression. The PECAM-1 adhesion molecule was measured by cytometry (CD31+ cells) and was observed to increase following compression. Endothelial activation was observed to initiate arteriogenesis. The adherent captured progenitor cells and monocytes were dragged sub-endothelial and their phenotype changed closer to a macrophage. At that point proteins were elicited that caused the endothelium to divide (e.g., VEGF A, FGF, PLGF), the underlying media to grow (e.g., PDGF-AA and BB, FGF), and the underlying matrix to be dissolved to allow for vessel expansion (e.g., MMP-9).

[0079] Embodiments of the methods described herein use a compression protocol in which a pneumatic pump was used to effect compression of the vasculature by an inflatable compression sleeve or cuff. The pump exerted a pressure of 120 mmHg in 0.3 seconds, which inflated the sleeve and effected compression of the limb. The pressure was held for 1 second for each inflation and the sleeve was inflated every 20 seconds. The device was worn in a seated position for 3 hours on both limbs simultaneously or where the subject had no contralateral leg, on the leg being treated.

II. INDICATIONS

[0080] The methods described herein can be advantageous in the treatment of various vascular pathologies in a subject. The neovascularization and fibrinolysis that result from the methods described herein are advantageous in the treatment of conditions caused by reduced or occluded blood flow (i.e., impaired perfusion). Examples of vascular pathologies treatable by the methods described herein are described below.

[0081] Impaired perfusion or impaired blood flow in a subject is caused by vascular occlusion and/or vascular stenosis. Such occlusion or stenosis may result from one or more of atherosclerosis (arterial), medial calcific sclerosis (arterial), fibromuscular dysplasia (arterial), vasculitis (inflammation) (arterial and venous), embolism (arterial and venous), thrombosis (arterial and venous), and intimal hyperplasia (arterial and venous) (a physiologic healing response to injury to the blood vessel wall). [0082] Impaired perfusion may be categorized by the major organ or body system affected. For example, impaired blood flow may be an arterial system disease or a venous system disease.

[0083] Examples of arterial system diseases which may be treated using the methods described herein include coronary artery disease (e.g., ischemic cardiomyopathy); cerebrovascular disease [e.g., vascular dementia, lacunar infarcts, bland (non-hemorrhagic) infarct, cerebral vasculitis, or ischemic retinopathy]; pulmonary disease (e.g., pulmonary hypertension, pulmonary embolism, or vasculitis post-resolution of inflammation); and peripheral artery disease (e.g., affecting arteries outside the heart not in brain or lungs) [(such as upper and lower extremity chronic limb-threatening ischemia, upper and lower extremity claudication, hypothenar hammer syndrome, vascular impotence, vascular alopecia, vascular deafness, chronic mesenteric ischemia, chronic renal artery disease, and/or post-infection micro thrombosis, or chronic residual ischemia after vasculitis (inflammation) resolves such as Takayasu’s arteritis, Kawasaki’s arteritis, ischemic blindness caused by temporal arteritis, marijuana induced vasculitis, and/or thromboangiitis obliterans (Buerger’s disease)].

[0084] Examples of venous system diseases which may be treated using the methods described herein include chronic venous disease such as chronic deep vein thrombosis (DVT) induced venous hypertension, phlegmasia (acute DVT) alba dolens, phlegmasia cerulea dolens, effort thrombosis (upper arm) (“Paget Shroeder disease”), chronic mesenteric venous disease, portal venous hypertension, and DVT induced pelvic congestion.

[0085] Examples of vasculitis which may be treated using the methods described herein include disorders resulting in small vessel thrombosis such as following infection with SARS-CoV-2 or disease resulting therefrom (COVID-19), or a disorder from a noxious agent such as marijuana or tobacco induced thromboangiitis obliterans (Buerger’s disease), or a disorder from other inflammatory diseases. The small vessel occlusive disease resulting from these conditions is addressed after the vasculitis inciting cause is removed and the inflammation resolves.

[0086] In some embodiments, the methods described herein are useful to treat or ameliorate consequences of ischemia following thromboembolic stroke, heart attack, and peripheral artery disease including vascular dementia, paralysis, congestive heart failure, angina, claudication, mesenteric ischemia, renal failure, vasculitis, impotence, embolism, blindness, loss of hearing, loss of hair, recurrent sepsis, venous thrombosis, pulmonary embolism, pulmonary hypertension, venous ulceration, effort thrombosis, mesenteric venous thrombosis, varicose veins, pelvic congestion syndrome, portal hypertension, disabling venous hypertension, and/or phlegmasia.

[0087] In some embodiments, the methods provided are useful to treat a chronic arterial condition in a subject, such as upper limb-threatening ischemia or lower limb-threatening ischemia (collectively, “chronic limb-threatening ischemia” or “chronic limb ischemia,” “CLI”), claudication, coronary artery disease, thromboembolic stroke, lacunar brain infarct, pulmonary arterial hypertension from thrombosis, vasculogenic impotence, atherosclerosis, medial arterial sclerosis, vasculitis, impaired circulation resulting from embolization, forefoot ischemic rest pain, non-healing foot ulcer, non-healing foot wound, gangrene, angina from coronary artery disease, intestinal angina from chronic mesenteric arterial disease, vasculogenic hearing impairment, vasculogenic alopecia, and thromboembolic retinal pathology.

[0088] In some embodiments, the methods provided herein promote venogenesis in the subject. Promotion of venogenesis can be useful in the treatment of a subject suffering from a venous condition such as venous hypertension, venous thrombosis, disabling severe limb swelling, bleeding from hypertensive varicose veins, and venous stasis ulceration at the ankles.

III. COMPOSITIONS

[0089] An aspect of the present disclosure provides methods, uses, and kits for treating a subject having impaired perfusion by administering a pharmaceutical composition and, when necessary due to severity of disease and remoteness from the pumping action of the heart, applying compression. The pharmaceutical composition for use in the methods described herein may be any pharmaceutical composition that stimulates progenitor cell division, mobilizes progenitor cells into circulation, produces granulocytes and monocytes, and/or increases serum or plasma concentration of plasmin and fibrin degradation products or effectors of neovascularization in a subject. In some embodiments, the pharmaceutical composition for use in the methods described herein stimulates progenitor cell division. In some embodiments, the pharmaceutical composition for use in the methods described herein mobilizes progenitor cells into circulation. In some embodiments, the pharmaceutical composition for use in the methods described herein produces granulocytes and monocytes in the subject. In some embodiments, the pharmaceutical composition for use in the methods described herein increases serum or plasma concentration of plasmin. In some embodiments, the pharmaceutical composition for use in the methods described herein stimulates increased production of effectors of neovascularization.

[0090] The pharmaceutical composition may comprise a granulocyte colony-stimulating factor (G-CSF), a granulocyte-macrophage colony-stimulating factor (GM-CSF), a macrophage colony-stimulating factor (M-CSF). Examples of growth factors suitable for use in the methods provided herein include: filgrastim, tbo-filgrastim, filgrastim-sndz, filgrastim- aafi, Accofil, balugrastim, biograstim, efbemalenograstim alfa, Grastofil, Hexal, lenograstim, lipegfilgrastim, Pelgraz, ratiograstim, sargramostin, Tevagrastim, Emgrast, Fegrast, filgrastim (Lupin), Grafeel, Neukine, Religrast, combinations thereof, functional equivalents thereof, derivatives thereof, or biosimilars thereof, or an agent that safely promotes CSF production in the body. Additional examples of growth factors suitable for use in the methods provided herein include factors that have been modified or adapted to be “long-acting” growth factors, such as pegfilgrastim, pegfilgrastim-bmez, pegfilgrastim-cbqv, pegfilgrastim-jmbd, eflapegrastim, and TX-01(a filgrastim biosimilar in development by Tanvex BioPharma Inc. (San Diego, CA)).

[0091] In some embodiments, the pharmaceutical composition is compounded as an injectable formulation for subcutaneous (SQ) administration to the subject. In related embodiments, the pharmaceutical composition is formulated sufficiently pure for administration to a human subject. In certain embodiments, these compositions optionally further include one or more additional therapeutic agents. In certain embodiments, the additional therapeutic agent induces natural production of a colony stimulating factor in the patient. In certain embodiments, the additional therapeutic agent or agents are selected from the group consisting of growth hormone, anabolic steroids, and growth factors, keratinocyte growth factor (KGF), fibroblast growth factor (FGF), insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), epidermal growth factor (EGF), platelet derived growth factor (PDGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), heparin-binding EGF (HBEGF), and thrombospondins.

[0092] In some embodiments, the additional therapeutic agent is a compound, composition, biological or the like that potentiates, stabilizes or synergizes or even substitutes for the ability of the pharmaceutical composition (e.g., filgrastim) to induce neovascularization and/or fibrinolysis. Also included are therapeutic agents that may beneficially or conveniently be provided at the same time as the pharmaceutical composition (e.g., filgrastim), such as agents used to treat the same, a concurrent or a related symptom, condition or disease. In some embodiments, the drug may include without limitation anti- tumor, antiviral, antibacterial, anti-mycobacterial, anti-fungal, or anti-apoptotic agents. Drugs that are included in the compositions of the disclosure are well known in the art. See for example, Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman, et al., eds., McGraw-Hill, 2001, the contents of which are herein incorporated by reference herein.

[0093] As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences Ed. by Gennaro, Mack Publishing, Easton, PA, 2006 provides various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as glucose and sucrose; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil, and soybean oil; glycols such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, preservatives and antioxidants that can also be present in the composition, according to the judgment of the formulator.

IV. DOSE

[0094] Treatment of vascular pathology by methods provided herein may involve administering a granulocyte colony-stimulating factor (G-CSF), a granulocyte-macrophage colony-stimulating factor (GM-CSF), a macrophage colony-stimulating factor (M-CSF), or administering to a subject a pharmaceutical composition comprising such a growth factor and applying compression to effect neovascularization and/or fibrinolysis. Examples of growth factors suitable for use in the methods provided herein include: filgrastim, tbo-filgrastim, filgrastim-sndz, filgrastim-aafi, Accofil, balugrastim, biograstim, efbemalenograstim alfa, Grastofil, Hexal, lenograstim, lipegfilgrastim, Pelgraz, ratiograstim, sargramostin, Tevagrastim, Emgrast, Fegrast, filgrastim (Lupin), Grafeel, Neukine, Religrast, combinations thereof, functional equivalents thereof, derivatives thereof, or biosimilars thereof, or an agent that safely promotes CSF production in the body. Additional examples of growth factors suitable for use in the methods provided herein include factors that have been modified or adapted to be “long-acting” growth factors, such as pegfilgrastim, pegfilgrastim-bmez, pegfilgrastim-cbqv, pegfilgrastim-jmbd, eflapegrastim, and TX-01 (a filgrastim biosimilar in development by Tanvex BioPharma Inc. (San Diego, CA)).

[0095] The pharmaceutical compositions, according to the method of the present disclosure, may be administered using any amount and any route of administration effective for effecting neovascularization and/or fibrinolysis. In some embodiments, the pharmaceutical composition is administered to the subject in a dose effective to stimulate progenitor cell division, to mobilize progenitor cells into circulation, and/or to produce granulocytes and monocytes in the subject. In some embodiments, the pharmaceutical composition is administered to the subject in a dose effective to stimulate upregulation of plasmin and effectors of neovascularization.

[0096] The individual physician may modulate an extended duration regimen in view of the response of the patient treated to the therapy, including dose, dosing interval, and duration of administration. Dosage and administration of the pharmaceutical composition are adjusted to provide sufficient levels of the active agent(s) therein or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, e.g., intermediate or advanced stage of the chronic illness; age, weight, and gender of the patient; diet, time and frequency of administration; route of administration; drug combinations; reaction sensitivities; and tolerance/response to therapy. Short-acting pharmaceutical compositions might be administered hourly, every 2 to 4 hours, every 6 to 8 hours, or every 12 hours, daily, every other day, every 3 to 4 days, or every 5 to 6 days depending on half-life and clearance rate of the particular composition. Long-acting pharmaceutical compositions might be administered every week, once every 2 weeks, once every 3 weeks, or once a month depending on half-life and clearance rate of the particular composition.

[0097] The G-CSF, GM-CSF, M-CSF, filgrastim, tbo-filgrastim, filgrastim-sndz, filgrastim-aafi, Accofil, balugrastim, biograstim, efbemalenograstim alfa, Grastofil, Hexal, lenograstim, lipegfilgrastim, Pelgraz, ratiograstim, sargramostin, Tevagrastim, Emgrast, Fegrast, filgrastim (Lupin), Grafeel, Neukine, Religrast, pegfilgrastim, pegfilgrastim-bmez, pegfilgrastim-cbqv, pegfilgrastim-jmbd, eflapegrastim, and TX-01 (a filgrastim biosimilar in development by Tanvex BioPharma Inc. (San Diego, CA)) or combination thereof may be administered in a dose of at least 1 mcg/kg; at least 5 mcg/kg; or at least 10 mcg/kg. In an embodiment of the method, the pharmaceutical composition is administered in a dose of at least 500 mg; at least 600 mg; at least 700 mg; at least 800 mg; at least 900 mg; or at least 1,000 mg. In an embodiment of the method, the pharmaceutical composition is administered according to a dosage regimen selected from: once, at least once per day, every second day, every third day, every fourth day, every fifth day, every sixth day, weekly, fortnightly, and monthly. In an embodiment of the method, 10 doses of the pharmaceutical composition are administered every third day for about a month. In an embodiment of the method, 5 doses of the pharmaceutical composition are administered every third day for about two weeks.

[0098] The pharmaceutical compositions of the disclosure are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression “dosage unit form” as used herein refers to a physically discrete unit of active agent in a pharmaceutical composition appropriate for the patient to be treated. It will be understood, however, that dosing of the compositions of the present disclosure may be adjusted by the attending physician within the scope of sound medical judgment.

V. ADMINISTRATION

[0099] As formulated with an appropriate pharmaceutically acceptable carrier in a desired dosage, the pharmaceutical composition provided herein is administered to subjects topically such as intravenous, sub-cutaneous, ocularly (as by solutions, ointments, or drops), nasally, bucally, orally, rectally, parenterally, intracistemally, intravaginally, or intraperitoneally.

[0100] Liquid dosage forms for ocular, oral, or other systemic administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active agent(s), the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the ocular, oral, or other systemically delivered compositions can also include adjuvants such as wetting agents and emulsifying and suspending agents.

[0101] Dosage forms for topical or transdermal administration of an inventive pharmaceutical composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active agent is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. For example, ocular or cutaneous routes of administration are achieved with aqueous drops, a mist, an emulsion, or a cream. Administration may be therapeutic, or it may be prophylactic. The disclosure includes ophthalmological devices, surgical devices, audiological devices or products, which contain disclosed compositions (e.g., gauze bandages or strips), and methods of making or using such devices or products. These devices may be coated with, impregnated with, bonded to or otherwise treated with a composition as described herein.

[0102] Transdermal patches have the added advantage of providing controlled delivery of the active ingredients to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0103] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. In order to prolong the effect of an active agent, it is often desirable to slow the absorption of the agent from subcutaneous or intramuscular injection. Delayed absorption of a parenterally administered active agent may be accomplished by dissolving or suspending the agent in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the agent in biodegradable polymers such as polylactide-poly glycolide. Depending upon the ratio of active agent to polymer and the nature of the particular polymer employed, the rate of active agent release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the agent in liposomes or microemulsions, which are compatible with body tissues.

[0104] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the active agent(s) of this disclosure with suitable nonirritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active agent(s).

[0105] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active agent is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, acetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof.

[0106] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active agent(s) may be admixed with at least one inert diluent such as sucrose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active agent(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. VI. PATIENT IDENTIFICATION

[0107] Provided herein are methods of identifying subjects suitable for treatment using the methods described herein. In some embodiments, subjects are identified as patients suitable for treatment based on presence of one or more of the following indications: vascular disease, arterial occlusive disease, chronic limb-threatening ischemia, Fontaine Class lib ischemia, Fontaine Class III ischemia, Fontaine Class IV ischemia, no-option chronic limb ischemia, and stroke. In some embodiments, the subjects are identified as patients suitable for treatment using the methods described herein where they have undergone previously failed interventions for revascularization, including, for example, surgical- or catheter-based revascularization. In some embodiments, the patients identified for treatment using the methods described herein suffer from no-option chronic limb ischemia and have given consent for amputation of the affected limb.

[0108] Subjects may be identified as patients suitable for treatment using the methods described herein by applying certain exclusion criteria. For example, subjects may be excluded from treatment using the methods described herein where: the subject presents with acute limb ischemia requiring emergency treatment; the subject presents with non- salvageable extremity (e.g., foot) (e.g., extensive gangrene, advanced infection, rigor mortis, knee/hip flexion contracture, post-stroke paralysis, and hemiparesis); severe carotid stenosis; sepsis proximal to forefoot; the subject presents with untreated hypercoagulability disorder, sickle cell anemia, or myeloproliferative disorder; the subject is on dialysis or has a sustained elevated creatinine level (> 3.5, > 3.6, > 3.7, > 3.8, > 3.9, or > 4 mg/dl); the subject presents with severe dementia, is bed-ridden, or is likely to show non-compliance; the subject cannot tolerate compression as defined herein; the subject has a body mass index greater than 34; the subject presents with severe venous insufficiency causing painful severe swelling, swelling causing disability, venous stasis ulceration and dermatitis; the subject presents with uncorrected significant aorto-iliac, common femoral, and/or profunda femoral artery disease; the subject presents with active cancer; the subject is allergic to a pharmaceutical composition described herein; the subject presents with uncorrected symptomatic coronary artery disease; the subject has a history of lymphoma or leukemia; and/or allergy to filgrastim or other blood cell growth factor.

[0109] For example, some embodiments may entail treating peripheral artery disease presenting in one or more limbs of a subject by identifying a subject as suitable for treatment of the peripheral artery disease using an extended delivery regimen of a pharmaceutical composition comprising a blood cell growth factor; and administering to the subject the extended delivery regimen of the pharmaceutical composition comprising the blood cell growth factor. Treatment of peripheral artery disease in one or more limb of a subject in this fashion may further entail applying compression to the one or more limb of the subject as described herein.

[0110] Identifying a subject may comprise identifying a subject as having claudication or chronic limb-threatening ischemia. A subject who has claudication or chronic limbthreatening ischemia that cannot be treated with compression may be suitable for treatment with the methods described herein. A subject who refuses or disfavors treatment with compression may be suitable for treatment with the methods described herein. A subject who cannot tolerate treatment using compression may be suitable for treatment with the methods described herein. A subject who has failed to improve with treatment using compression or for whom continued treatment using compression will fail to improve the peripheral artery disease may be suitable for treatment with the methods described herein.

[OHl] In some embodiments, identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen comprises identifying the subject as having persistent or increased pain or pain upon exertion, coldness, claudication, ulceration, or gangrene in the affected one or more limb of the subject.

[0112] The affected one or more limb of the subject may be a toe, foot, ankle, calf, or portion thereof. In such cases, the identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen may comprise identifying the subject as having hemodynamics showing an ankle-brachial index (AB I) measurement of less than 0.5, less than 0.49, less than 0.48, less than 0.47, less than 0.46, or less than 0.45, or having a monophasic or non-pulsatile ankle Doppler waveform or having a monophasic or non-pulsatile Doppler or photoplethysmographic (PPG) toe or transmetatarsal (“pedal”) waveforms if the toes cannot be evaluated due to gangrene or previous amputation. In some embodiments, identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen may comprise identifying the subject as having hemodynamics showing non-pulsatile waveforms (i.e., no detectable flow). It will be understood that waveforms may be determined by measurement not only at the ankle but may also or alternatively be determined by measurement at the transmetatarsal level or at the toes. [0113] In some embodiments, the subject may have undergone a toe amputation and the wound is closed with the skin sutured. In such cases, identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen comprises identifying the subject as having toe pressure of 30 mmHg or less in a non-diabetic subject or 50 mmHg or less in a diabetic subject after undergoing limb compression for about 5 days. [0114] In some embodiments, the subject may have undergone a toe amputation and the wound is left open. In such cases, identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen may comprise identifying the subject as having an ABI measurement of less than 0.5, or having non-pulsatile or monophasic ankle or pedal waveforms when ABI is not accurate as a result of medial calcinosis or severe advanced atherosclerosis. In some embodiments, identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen may comprise identifying the subject as having an absence of granulation in an open wound in the affected one or more limb of the subject after undergoing limb compression period for about 5 days.

[0115] In some embodiments, identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen may comprise identifying the subject as having early onset rest pain with preserved tissue turgor and no muscle atrophy. [0116] In some embodiments, identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen may comprise identifying the subject as having advanced CLI with severe ischemic rest pain and loss of tissue turgor, atrophy of muscle mass, and dry cool skin.

[0117] In some embodiments, identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen may comprise identifying the subject as having tissue loss due to ulceration or gangrene and an ABI measurement of less than 0.5 or non-pulsatile or monophasic waveforms. It will be understood that ABI is not always an accurate measure of hemodynamic status in all subjects. For example, a subject may present with incompressible or relatively incompressible arteries. Thus, in some embodiments, identifying the subject as suitable for treatment of the peripheral artery disease using the extended delivery regimen may comprise identifying the subject as having non- pulsatile or monophasic ankle or pedal waveforms.

[0118] In the foregoing methods of treating peripheral artery disease presenting in one or more limb of a subject, the method may further comprise applying compression to the one or more limb as described herein. VII. DEFINITIONS

[0119] The term “impaired perfusion” or “impaired blood flow” as used herein shall mean reduced flow and/or passage of fluids to, from, and/or through tissue of a subject. Impaired perfusion may include decreased arterial inflow to tissue and/or decreased venous or any combination thereof. Impaired perfusion may be due to decreased diameter, length, and/or number of arteries, and veins. Impaired perfusion may be due to fibrin cross-linked thrombus in arteries and/or veins, or may be due to impaired break-down or digestion of fibrin crosslinked thrombus. Impaired perfusion may be visible with the naked eye in macroscopic vessels either in the operating room or on contrast studies such as angiogram (catheter, MR, or CT) or nuclear tracer studies (e.g., technetium blood perfusion scan). Impaired perfusion may be visible in microscopic vessels (arterioles, venules, capillaries) by microscope. Impaired perfusion may be visible in tissues as observed by decreased perfusion (capillary refill time, oxygen tension, hemodynamic measurement, and spectral imaging), decreased tissue healing, and/or increased swelling. Evidence of impaired perfusion includes pain, decreased wound healing, poor ambulation distance, and/or reduced tissue function. For example, tissue function may be assessed in the brain by cognitive and neurological assessment; in the heart by cardiac function (on echocardiogram or stress test) and/or angina; in the lungs by reduced oxygenation and CO2 clearance and/or increased right ventricular pressures; in the kidneys by reduced glomerular function; in the mesentery by post prandial pain, decreased absorption, weight loss, and/or atrophy; in the skin as decreased turgor, sweating, and/or hair loss; in the auditory system as decreased hearing; in the visual system as impaired vision; in the venous system as blood clot causing increased swelling; in the lymphatic system as increased swelling; and in the limbs as pain and slowed healing of dysvascular wounds and poor hemodynamics.

[0120] The term “extended delivery regimen” as used herein shall mean delivery of a pharmaceutical composition or other treatment regimen over an extended period of time or delivery of a pharmaceutical composition with prolonged half-life and/or enhanced absorption and bioavailability. An extended delivery regimen may include administering to a subject a dosage of a pharmaceutical composition (which may be a long-acting or a shortacting formulation) 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times over the course of about 1 month. In some embodiments, an extended delivery regimen includes administering a dosage to a subject 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times over the course of about 2 months, about 3 months, about 4 months, or over 4 months. Dosages of a pharmaceutical composition may be administered to a subject daily, about once every 2 days, about once every 3 days, about once every 4 days, about once every 5 days, about once every 6 days, about once every 7 days, about once every 8 days, about once every 9 days, about once every 10 days, about once every 11 days, about once every 12 days, about once every 13 days, about once every 14 days, about once every 15 days, about once every 16 days, about once every 17 days, about once every 18 days, about once every 19 days, about once every 20 days, about once every 21 days, about once every 22 days, about once every 23 days, about once every 24 days, about once every 25 days, about once every 26 days, about once every 27 days, about once every 28 days, about once every 29 days, or about once every 30 days, consistent with an extended delivery regimen of the pharmaceutical composition. Also consistent with an extended delivery regimen are once daily, twice daily, or three times daily administrations of the dosages of the pharmaceutical composition to the subject. Examples of extended delivery regimens include discontinuous administrations, for example, where the pharmaceutical composition is administered for a period of time (e.g., about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, or longer than 1 year); stopped for a period of time; and then re-initiated.

[0121] The term “blood cell growth factor” or “growth factor” as used herein shall mean factors that stimulate production or activation of blood cells. Blood cell growth factors include factors that stimulate progenitor cell division, mobilization of progenitor cells into circulation, production of granulocytes and/or monocyte, and/or increase in serum plasmin concentration in a subject. Blood cell growth factors include white blood cell growth factors, red blood cell growth factors, and platelet growth factors. In some embodiments, blood cell growth factors stimulate neutrophil production, mobilization, or activation. Examples of blood cell growth factors include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colonystimulating factor (M-CSF), combinations thereof, functional equivalents thereof, derivatives thereof, and/or biosimilars thereof. In some embodiments, a blood cell growth factor may be filgrastim, tbo-filgrastim, filgrastim-sndz, filgrastim-aafi, Accofil, balugrastim, biograstim, efbemalenograstim alfa, Grastofil, Hexal, lenograstim, lipegfilgrastim, Pelgraz, ratiograstim, sargramostin, Tevagrastim, Emgrast, Fegrast, filgrastim (Lupin), Grafeel, Neukine, Religrast, pegfilgrastim, pegfilgrastim-bmez, pegfilgrastim-cbqv, pegfilgrastim-jmbd, eflapegrastim, and TX-01 (a filgrastim biosimilar in development by Tanvex BioPharma Inc. (San Diego, CA)), combinations thereof, functional equivalents thereof, derivatives thereof, and/or biosimilars thereof.

[0122] The term “neovascularization” as used herein shall mean the formation of new blood vessels in the form of functional vascular and micro-vascular networks, which are capable of perfusion, by red blood cells. Further, neovascularization is used herein to characterize the growth in length and diameter of vessels already present, and of new vessels. These vascular and micro-vascular networks serve as collateral circulation in response to local poor perfusion or ischemia, for example, as a result of blockage of a major vessel. [0123] The term “revascularization” as used herein shall mean a process in which the blood circulation of an organ or area is restored by surgical intervention. Standard methods include bypass surgery using implantable devices such as tubular prosthetic or biologic (human or animal) grafts that re-route blood around the occluded vessels. Alternatively, catheters are used to cut through or simply dilate the blockage or to place stents to expand a new flow lumen within the blocked area.

[0124] The term “fibrinolysis” as used herein shall mean a process in which fibrin crosslinked blood clots are digested or prevented from growing or stabilizing.

[0125] The term “thrombolysis” as used herein shall mean a process in which a pharmaceutical composition (i.e., thrombolytic agent) acts directly on thrombus to induce fibrinolysis of clots in arteries or veins. Thrombolytics include tissue plasminogen activator (tPA), streptokinase, urokinase [Eminase (anistreplase). Retavase (reteplase), Streptase (kabikinase)] and are administered in acute conditions to dissolve clots for example after heart attack, ischemic stroke, acute limb ischemia, venous thrombosis, or pulmonary embolism. Thrombolytics have high potency and risk of bleeding; therefore, thrombolytics are typically not administered for more than 72 hours. The side effects of thrombolytics include death, intracranial hemorrhage, hemorrhage anywhere, nausea, headache, dizziness, low blood pressure, mild fever, bleeding from wounds or gums, rash, and itching.

[0126] The term “compression” as used herein shall refer to application of a mechanical force to tissue to produce a biological effect. Compression may be created by, e.g., mechanical or pneumatic means. Compression may be peristaltic or pulsatile in nature. In some instances, compression includes application of a mechanical force perpendicular to the flow of blood intermittently compressing and relaxing so as to result in increasing flow of blood across the endothelium of a subject. The increase in blood flow may be sufficient to induce endothelial activation. Compression includes application of a force to squeeze or put pressure on a region of tissue, an organ, or a limb of a subject. A force applied may be a direct force applied by physical contact to the tissue or may be an indirect force applied to the tissue by propagation through, e.g., a tissue, a material, or other substrate. Compression may result in a decrease in volume of a tissue or substance to which the force is applied. Compression may be intermittent, e.g., consisting of sequential periods of compression followed by release of compression. In methods, kits, examples, and claims herein, compression may be affected by contacting tissue of a subject with a compression device, such as by placing an inflatable compression sleeve of such a device on a limb or on a portion of a limb of a subject. The inflatable compression sleeve may contain a bladder, which is filled intermittently by a pump with either gas or a fluid, or alternatively may contain small electromagnets that attract toward each other in response to a pulsed current. The subject may wear an inflatable compression sleeve of a compression device on any limb, such as a foot, an ankle, or a calf. In some embodiments, a subject may wear more than one inflatable compression sleeve on more than one limb. The compression upstroke delivers a rapid force to the underlying limb sufficient to activate the vascular endothelium in the limb vasculature. Compression may include a process by which the abnormal hemodynamics resulting in arterial or venous dysfunction are managed using an external device programmed to generate sufficient endothelial shear stress to activate the endothelium and promote vessel growth. Such a device may be a programmed compression device. The device may be a pneumatic limb compression device (“PLC device”). In general, compression increases inflow of oxygenated nutritive blood flow, promotes clearance of toxic metabolic byproducts, delivery of distress proteins from the ischemic tissue into the circulation, and delivery of proteins and cells in response to these distress proteins back into the tissue. In regions of the body where compression is not feasible (e.g. brain, chest, head, abdomen), proximity to the heart may be sufficient to supply the kinetic energy needed to generate sufficient shear stress. In some embodiments, compression may be performed using methods and or apparatus as described in U.S. Patent Application Publication No. 20160175184, the contents of which are incorporated herein by reference in their entirety.

[0127] The term “endothelial activation” as used herein shall mean a process by which the endothelium is stimulated to produce nitric oxide (NO) by up-regulation of nitric oxide synthase, to express surface adhesion molecules to capture circulating salutary endothelial and mesenchymal progenitor cells and monocytes, and to produce MCP-1 which acts as a homing signal to attract these cells to the endothelium. The result of endothelial activation is that these mobilized circulatory cells attach to the endothelial membrane adhesion molecules and are then dragged sub-endothelial where these cells undergo phenotypic change into a macrophage-like cell that produces the proteins that promote proliferation of endothelial cells in the tunica intima (e.g., VEGF, FGF), vascular smooth muscle cells in the tunica media (e.g., PDGF, Angiopoietin-1), and enzymes required to digest the underlying matrix in the media (e.g., MMP activity precedes endothelial tube expansion). The NO elicited by the activated endothelium dilates the vessel and decreases contact inhibition between endothelial cells (which grow in a monolayer) so they can start to divide and spread across the widened lumen.

[0128] The term “subject” as used herein shall mean any individual organism, including any mammal such as a human, a non-human primate, a dog, a cat, or a rodent that is screened for or selected for treatment using the methods described herein or used for clinical testing of the methods described herein. A “patient” may be a subject, and vice versa. The terms “patient” and “subject” are used herein interchangeably.

EXAMPLES

[0129] The following examples, embodiments, and claims are illustrative of the foregoing detailed description of the inventive subject matter and are not meant to be further limiting. The details of one or more embodiments are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are now described. Other features, objects and advantages of the invention will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. 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 this invention belongs. In the case of conflict, the present description will control.

[0130] The present invention is further illustrated by the following non-limiting examples.

Example 1. Chronic limb-threatening ischemia (CLI) patients

[0131] The Fontaine classification system is a method of grading clinical presentation of subjects with peripheral artery disease. Fontaine Stage lib, claudication, is characterized by pain upon exertion; Fontaine Stage III chronic limb-threatening ischemia is characterized by rest pain, and Fontaine Stage IV chronic limb-threatening ischemia is characterized by ischemic ulcers or gangrene (see Fontaine R, Kim M, Kieny R., Helv Chir Acta. 1954;21(5- 6):499-533; Hardman RL, Jazaeri O, Yi J, Smith M, Gupta R. Semin Intervent Radiol. 2014 Dec;31(4): 378-88)). 62 patients suffering from atherosclerosis resulting in Fontaine Stage lib, Ill, and/or IV chronic limb-threatening ischemia were treated with limb compression. Each was provided with a limb compression device to be used at home 3 hours a day in seated position with inflatable compression sleeves on both legs. Average age of these 62 patients was 70; 68% were male and 32% female; and 10% were Fontaine Class lib, 24% Fontaine Class III, and 76% Fontaine Class IV. Almost all were on multiple medications: statins (74%); aspirin (79%); Angiotensin-receptor blockers (18%); ACE inhibitors (45%); Clopidogrel (44%); Cilostazol (13%); warfarin (24%); beta-blockers (68%).

[0132] Most suffered from multiple co-morbidities and risk factors. 97% suffered from hypertension, 74% suffered from hyperlipidemia, 67% were diabetic (44% insulin dependent), 26% on dialysis, 42% had coronary artery disease, 84% were current or former smokers, average hemoglobin Ale and creatinine levels were 7% and 2.0 respectively.

[0133] In addition, 15 patients suffering from chronic limb-threatening ischemia Fontaine Stage III or Fontaine Stage IV in a lower extremity were treated with a combination of filgrastim and limb compression. Of these 15 patients, CLI in 13 patients resulted primarily from atherosclerosis; CLI in 2 resulted from vasculitis. All 15 were so-called “no option” CLI patients, i.e. patients not surgically reconstructable as result of inadequate bypass conduits, unfavorable anatomy, or poor operative risk for whom amputation is the only alternative and for whom amputation was standard of care. Two of the 15 were previously treated with limb compression alone. One was treated for CLI in an upper extremity after recovering from lower extremity CLI. Each was administered an extended duration regimen of filgrastim intravenously, via subcutaneous injection (SQ) in the lower abdomen with filgrastim drawn from vials, or from pre-filled syringes. Doses were 10 micrograms per kilogram body weight (mcg/kg) when delivered intravenously, or 600 mcg, 780mcg or 960 mcg filgrastim when delivered (SQ), or 780 mcg when delivered from pre-filled syringes. Doses were administered every 3 days for 5, 7 or 10 doses. The extended duration regimen also comprised discontinuous administration of 10 doses every 3 days where there was an interval between the fifth dose and the sixth dose of from 10 days to four months. The extended duration regimen for one patient was 7 doses every 3 days as a result of being unable to tolerate limb compression. Each patient was provided a limb compression device with inflatable compression sleeves for both legs to be used at home 3 hours a day in seated position. Two patients lacked contralateral limbs and therefore used the device on the treatment leg only. One patient declined to use the limb compression device on the contralateral limb. One patient was treated with filgrastim-sndz. The six additional CLI patients whose clinical course was not assessed participated in a confirmatory study of cytometry, nitrite, and proteins involved in fibrinolysis and neovascularization and were treated with 600 mcg, 780 mcg or 960 mcg filgrastim every 3 days for a month, total of 10 doses, and limb compression.

[0134] Of the 62 patients receiving limb compression alone, 19 consented to have blood drawn to measure cytometry, serum nitrite, and proteins involved in fibrinolysis and neovascularization. Of the 15 patients receiving filgrastim and limb compression and followed clinically, 8 consented to have blood drawn to measure cytometry, serum nitrite, and certain proteins involved in fibrinolysis and neovascularization. In addition, 6 CLI patients whose clinical course was not assessed participated in a confirmatory study of cytometry, nitrite, and proteins involved in fibrinolysis and neovascularization and were treated with 600 mcg, 780 mcg or 960 mcg filgrastim every 3 days for a month and limb compression.

[0135] Most patients receiving limb compression alone, and patients that consented to have blood drawn for testing, suffered from multiple co-morbidities and risk factors and took multiple medications, as detailed in Table 1.

[0136] Average age of the 14 patients who received filgrastim and were followed clinically was 67; 50% were male and 50% female; 8% were Fontaine Class III and 92% Fontaine Class IV. Most suffered from multiple co-morbidities and risk factors; 92% suffered from hypertension, 58% suffered from hyperlipidemia, 42% were diabetic (none on dialysis, 25% insulin dependent), 42% had coronary artery disease, 42% were current or former smokers, average hemoglobin Ale and creatinine levels were 7% and 1.2 respectively. Almost all were on multiple medications: statins (100%); aspirin 67(%); Angiotensinreceptor blockers (33%); ACE inhibitors (42%); Clopidogrel (58%); Cilostazol (0%); warfarin (17%); beta-blockers (50%). A comparison of demographics, co-morbidities, risk factors, medications, and symptoms of patients treated with limb compression and the patients treated with filgrastim and limb compression whose blood was assayed is contained in Table 1.

[0137] Demographics, co-morbidities, medications, risk factors, and symptoms of 7 patients treated with filgrastim and limb compression not included in Table 1 were comparable. Table 1. Patient demographics, co-morbidities, risk factors, medications, and symptoms (Numbers in parentheses are standard deviation from the mean) [0138] The majority of patients also had histories of significant failed revascularizations, including bypass procedures to circumvent and stents to open blocked blood vessels. Patients treated with filgrastim and compression had significantly more failed procedures than patients treated with compression alone. One patient who failed limb compression and then began filgrastim treatment alone had 14 procedures, skewing results to some extent. See Table 2. (Filgrastim and compression numbers and percent do not include the 2 patients who had previously failed compression alone, the 6 patients who participated in the confirmatory study and patient 15 treated with filgrastim-sndz.)

Table 2. Previous failed re-vascularization procedures.

[0139] To support clinical and hemodynamic results of both treatments, blood samples for biochemical analysis were drawn from patients in each group to measure and compare biomarkers of fibrinolysis, endothelial activation, and neovascularization. Of the 62 patients treated with compression alone, 19 consented to have blood drawn and assayed. (Of the 19, 2 deteriorated rapidly after the first draw and dropped out.) Of the 15 patients treated with filgrastim and compression, 8 consented to have blood drawn. One patient was in both groups. A third group of 6 patients treated with filgrastim and limb compression also had blood drawn and assayed.

Example 2: Extended delivery regimen and limb compression to induce fibrinolysis and neovascularization

[0140] Fibrinolysis and neovascularization as described herein may be induced by performing a method according to the following exemplary description. Inclusion criteria for patients to be considered for treatment with this “extended delivery regimen” and limb compression include, for example, “no-option” or “non-reconstructable” CLI patients who are unsuitable for revascularization interventions such as percutaneous transluminal angioplasty (PTA) or bypass graft surgery (see, e.g., Rumenapf G, Morbach S. Int J Low Extrem Wounds. 2014;13:378-389, the contents of which are incorporated by reference herein in their entirety). In some cases, this includes patients with prior failed invasive revascularization interventions.

[0141] The pharmaceutical composition to be administered in the extended delivery regimen described herein may be filgrastim, tbo-filgrastim, filgrastim-sndz, filgrastim-aafi, Accofil, balugrastim, biograstim, efbemalenograstim alfa, Grastofil, Hexal, lenograstim, lipegfilgrastim, Pelgraz, ratiograstim, sargramostin, Tevagrastim, Emgrast, Fegrast, filgrastim (Lupin), Grafeel, Neukine, Religrast, combinations thereof, functional equivalents thereof, derivatives thereof, or biosimilars thereof, or an agent that safely promotes CSF production in the body. Additional examples of growth factors suitable for use in the methods provided herein include factors that have been modified or adapted to be “long-acting” growth factors, such as pegfilgrastim, pegfilgrastim-bmez, pegfilgrastim-cbqv, pegfilgrastim-jmbd, eflapegrastim, and TX-01 (a filgrastim biosimilar in development by Tanvex BioPharma Inc. (San Diego, CA)). Alternatives can be substituted, including any G-CSF (granulocyte colonystimulating factor), GM-CSF (granulocyte-macrophage colony-stimulating factor), M-CSF (macrophage colony-stimulating factor), or equivalents.

[0142] Filgrastim (Amgen Inc., Thousand Oaks, CA) is indicated for neutropenia resulting from chemotherapy and for mobilization of hematopoietic progenitor cells into the peripheral blood for collection by leukapheresis. It is now packaged as 300 microgram (mcg) and 480 mcg single use vials and 300 mcg and 480 mcg single use pre-filled syringes in dispensing packs of 10. The recommended dose for mobilization of peripheral blood progenitor cells is 10 mcg/kg (e.g., a 132 lb/60 kg patient requires 600 mcg or two 300 mcg vials or pre-filled syringes) a day SC for at least 4 days. Patients administered filgrastim from vials drawn into syringes and injected SQ required multiple vials. To obtain a dose of 600 mcg requires 2 300 mcg vials, 780 mcg requires 1 300 mcg and 1 480 mcg vial, and 960 mcg requires 2480 mcg vials). When contents of a vial are drawn into a syringe to be injected into a patient, some filgrastim drug product invariably remains in the vial after drawing into the syringe.

Similarly, some filgrastim drug product invariably remains in a syringe after administration in the patient.

[0143] In some embodiments, administration results in dose/kg that is not 10 mcg/kg, the dose/kg will vary depending on patient weight, i.e., patients do not receive 10 mcg/kg unless they happen to be the appropriate weight (i.e. 60 kg for 600 mcg; 96 kg for 960 mcg). Depending on how a provider wishes to manage inventory, if a full vial or syringe is used, there will be a range of patient weights, and effective doses, actually delivered. An example using one possible combination of vials and resulting range of patient weights for that combination where 10 mcg/kg desired as the maximum effective dose is provided in Table 3. As patient weight within a band increases, the effective dose/kg decreases until the next weight range is reached. The fewer the combinations, the wider the range of possible effective weights. Table 3. Example of possible filgrastim vial combinations and resulting patient body weight range

[0144] The 15 patients receiving filgrastim and limb compression that were assessed clinically were administered filgrastim in accordance with Table 4A. Patients 1 and 2 were administered filgrastim intravenously. Patients 3 to 14 were administered filgrastim drawn from vials. Patient 15 was administered filgrastim-sndz supplied in pre-filled syringes.

Table 4A. Filgrastim dosage administered per patient and effective body weight dose

[0145] The 6 patients receiving filgrastim and limb compression as part of a corroborating study and not assessed clinically were administered filgrastim in accordance with Table 4B. Table 4B. Filgrastim dosage administered per patient and effective body weight dose

[0146] Long-acting G-CSF formulations such as pegfilgrastim (Neulasta®) and biosimilars are administered according to a single fixed dose for all patient weights, e.g., 6 mg in a single use pre-filled syringe for all non-pediatric patient weights.

[0147] Limb compression is performed using a compression device, such as the ArtAssist® pneumatic compression device (ACI Medical, Inc, San Marcos, CA) 3 hours a day until improvement stops.

Example 3: Limb compression

[0148] Patients were treated at their homes with limb compression using a compression device: the ArtAssist® Arterial Assist Device® (ACI Medical, Inc, San Marcos, CA). This pneumatic compression device was to be used in the seated position for 3 hours daily (e.g., a one-hour session, 3 times daily) with inflatable compression cuffs on both legs. The sequential rapid inflation (0.3 sec to 120 mmHg) of three leg sleeves/cuffs (calf, ankle and foot) provided shear stress stimulus and facilitated flow of oxygenated nutritive blood. The pressure was held in each of the sleeves/cuffs for one second followed by rapid deflation. The inflation/deflation cycles were separated by a delay of up to 20 seconds, such that 3 cycles occur per minute. Patients were instructed to use the device at home on both legs in the seated position for a minimum of 3 hours a day. Patient compliance was generally good.

Example 4: Clinical summaries-patient 1 (limb compression; filgrastim + limb compression

[0149] Patient 1, a 56-year-old Fontaine Stage IV male, presented with dehiscence of a healed transmetatarsal amputation (TMA) following thrombosis of a femoral to peroneal artery bypass graft constructed a year earlier with in situ ipsilateral saphenous vein. The graft developed diffuse intimal hyperplasia (IH). Graft thrombosis occurred during balloon angioplasty of the significant stenoses caused by the intimal hyperplasia. The original transmetatarsal amputation was done for forefoot gangrene. When the bypass graft thrombosed, the foot became severely ischemic, with ankle- brachial index (ABI) dropping to 0 mmHg, ankle waveforms becoming non-pulsatile, and transcutaneous oxygen tension (TcP02) on the foot dorsum dropping to 3 mmHg. Patient had a moderate ankle extension deformity.

[0150] Vascular history includes bilateral carotid endarterectomy and coronary artery revascularization. Patient was human immunodeficiency virus (HIV) positive and on antiretroviral therapy, which was thought to contribute to the diffuse IH. Viral counts were undetectable. Outflow embolization and occlusion of the bypass graft arose during angioplasty of the IH stenoses. Subsequent progressive necrosis of the TMA wound led to complete dehiscence and gangrene of the edges. The amputated metatarsal bones were partially exposed. HIV physician granted clearance to use filgrastim. Standard of care is below knee amputation.

[0151] Patient began an extended delivery regimen of 10 doses 10 mcg/kg filgrastim administered intravenously in hospital every 3 days and 3 hours limb compression.

[0152] The patient’s wound granulated slowly. At 6 months, ABI increased to 0.43, ankle pulsatility returned. The foot dorsum TcPO2 increased to 24 mmHg. The TMA wound was slowly granulating in and the distance between the edges was shortening. At 8 months, ABI increased further to 0.64, doppler waveform amplitude increased, and the open dehisced TMA wound healed fully. A dehisced TMA wound does not normally heal following graft thrombosis with ABI originally zero. Angiogram at 1 year showed the “corkscrew” collateral growth characteristic of arteriogenesis. Importantly, previous segmental arterial occlusion appeared to have recanalized. Contrast transit time was brisk compared to the pre-treatment angiogram.

[0153] When the ABI rose to 0.43 (still moderate to severe perfusion impairment), a podiatrist performed an Achilles tendon release to treat the chronic ankle extension deformity. The 2 cm incision over the released tendon became infected, with calcaneal osteomyelitis diagnosed despite wound care and IV antibiotics. Since a partial calcinectomy after TMA would leave the patient with a dysfunctional foot, a below knee amputation was performed to control the infection. Patient survived amputation free for 367 days.

Example 5: Clinical summaries-patient 2A (limb compression; filgrastim + limb compression)

[0154] Patient 2, a 44-y ear-old Fontaine Stage IV female, presented with severe ischemic forefoot rest pain, non-healing large posterior ankle ulcer exposing the Achilles tendon, and another large non-healing ulcer on the foot dorsum. Ipsilateral external iliac artery stenoses were treated with balloon angioplasty and stenting. Two previous femoral to tibial artery bypass grafts had failed within a month of surgery. Medical history included insulin dependent diabetes, renal insufficiency, congestive heart failure, morbid obesity, and coronary revascularization. ABI on the treatment limb was 0.4, TcPO2 was 23 mmHg on the skin overlying the medial calcaneus and 1 mmHg on the foot dorsum. There was no metatarsal level or digital pulsatility. Angiogram showed superficial femoral, popliteal, and tibial artery occlusive disease. Standard of care is below knee amputation.

[0155] Patient began an extended delivery regimen in the hospital of 10 mcg/kg filgrastim intravenously every 3 days, total 10 doses, and limb compression.

[0156] The wound base granulated slowly, anterior much quicker than posterior. Four months after treatment, ABI was 0.5; At 5 months, the anterior foot ulcer had healed. At 12 months, TcPO2 increased to 46 mmHg (medial calcaneus) and 35 mmHg (foot dorsum). Metatarsal level pulsatility improved, but no toe pulsatility.

Example 6: Clinical summaries-patient 2B (limb compression; filgrastim + limb compression)

[0157] Patient returned to clinic one year later as Fontaine Stage IV with a cyanotic 5 ^th toe after blunt trauma. Given no toe pulsatility, this injury would ordinarily not heal without revascularization. ABI had increased to 0.7 and there was pulsatility in the mid-foot.

Angiogram before the second course of filgrastim was compared to the pre-treatment (first course of filgrastim) angiogram from the year before. It showed corkscrew collateral growth. Segmental recanalization of previous occluded segmental arterial occlusive disease was observed. Contrast transit time was brisk, and much improved compared to the previous year. Despite the documented hemodynamic and anatomic improvement, patient was still deemed high risk for a third revascularization.

[0158] Patient began a second extended delivery regimen of 10 mcg/kg filgrastim every 3 days total 10 doses.

[0159] Seven months after the second course of filgrastim ABI increased further to 1.0 and pulsatility returned to all 5 toes. The Achilles tendon wound, though still open, was granulating slowly. The large ulcer over the exposed Achilles’ tendon eventually closed over 5 years. Patient has remained ambulatory with healed ulcers and amputation free for 13 years as of 2021.

Example 7: Clinical summaries-patient 3 (limb compression; filgrastim + limb compression)

[0160] Patient 3, a 65-year-old Fontaine Stage IV female, presented with severe ischemic forefoot rest pain, a non-healing dorsal wound ulcer at the site of an attempt at retrograde catheter recanalization through the dorsalis pedis artery, and gangrene of toes 1 and 2. Two femoral distal bypass grafts had failed acutely, followed by failed angioplasty and stent placement in the popliteal artery.

[0161] Two hypercoagulable disorders were diagnosed: anticardiolipin antibody (lupus anticoagulant) and thrombocytosis. ABI was 0, TcPO2 was 2mmHg. No bypass targets or conduit were identified. Angiogram showed occlusion of the superficial femoral and the popliteal arteries, an occluded popliteal artery stent, and three vessel tibial artery occlusions. Standard of care is below knee amputation.

[0162] Patient began an extended delivery regimen of 600 mcg filgrastim (8.7mcg/kg based on patient weight) subcutaneously every 3 days, total 10 doses, and limb compression. [0163] In the first month, ischemic rest pain improved. At 3 months, angiogram showed improvement in the number of blood vessels surrounding the knee and corkscrew collateral formation extending down to the foot; named vessels became apparent. Over 8 months, wounds healed, and gangrene sloughed. Toe pulsatility returned and remained stable, at which time patient discontinued use of compression. At 8 years ABI was 0.65.

[0164] Patient remained amputation free 11 years before dying of chronic renal failure.

Example 8: Clinical summaries-patient 4 (limb compression; filgrastim + limb compression)

[0165] Patient 4, a 68-year-old Fontaine Stage III morbidly obese insulin dependent diabetic female tobacco user, presented with ischemic rest pain and short distance calf claudication after 2 failed femoral popliteal bypass grafts and significant infra-geniculate soft tissue scarring from previous surgeries. ABI was 0.3; toe pressure 0.17. Dorsalis pedis waveform was monophasic. Patient stopped tobacco use for 2 weeks as condition of treatment. Standard of care is below knee amputation.

[0166] Patient began an extended delivery regimen of 960 mcg filgrastim (8.6mcg/kg based on patient weight) every 3 days. A home visit revealed that the vials taken home were stored improperly and therefore likely ineffective, an observation confirmed by low neutrophil and white blood cell counts from blood drawn the day after the fifth dose was taken. About 6 weeks later second course of 960 mcg filgrastim every 3 days was begun, total 10 doses, but with an 18-day gap between the 5th dose and the 6th dose.

[0167] After completing a second course of filgrastim, symptoms improved, with claudication occurring at 2 blocks rather than less half a block. ABI increased to 0.68; toe pressure increased to 0.43. Dorsalis pedis waveform became biphasic. [0168] Six months later patient’s walker was caught in a doorway and the metal bottom impaled her midfoot dorsum. She also reported resuming tobacco use. To manage the significant soft tissue injury, a below knee amputation was performed.

[0169] Patient remained amputation free 413 days before management of the soft tissue injury resulting from trauma to the foot necessitated amputation.

Example 9: Clinical summaries-patient 5 (limb compression; filgrastim + limb compression)

[0170] Patient 5, a 70-year-old Fontaine Stage IV male, was referred by a hematologistoncologist with diagnosis of blue toe syndrome without an embolic source after a hypercoagulable work-up and a 2 month history of forefoot rest pain, toe cyanosis, and deep venous thrombosis. Referring vascular surgeon deemed patient unsuitable for invasive revascularization due to extensive evidence of pedal and distal tibial artery occlusive disease. Standard of care is below knee amputation.

[0171] Limb compression is contraindicated in the setting of acute deep venous thrombosis. A vena cava temporary filter was placed to protect from pulmonary embolism so compression could be used.

[0172] Patient began an extended delivery regimen of 600 mcg filgrastim every 3 days total 10 doses and started limb compression with a gap of 20 days between the 5th and 6th doses.

[0173] Skin on the toes looked better perfused and rest pain was reported to improve within 2 weeks. However, in the next 2 weeks toes looked more ischemic and patient reported no symptomatic benefit. The day after the 10th dose of filgrastim the patient complained of feeling lethargic. CBC revealed a drop in the hemoglobin count of 2 points compared to when treatment started. Stool guaiac was positive for occult blood. Upper endoscopy and CT scan led to diagnosis of Stage 4 stomach adenocarcinoma.

[0174] Patient was started on Folinic acid (leucovorin), Fluorouracil (5-FU), and Oxaliplatin (El oxatin) but the tumor did not respond. He was then placed on Taxol and Cyramza (anti-angiogenic) and tumor receded to where it became undetected. Meanwhile, both legs became ischemic, worse on treatment limb. Gangrene progressed to involve the whole forefoot, necessitating below knee amputation. Patient had below knee amputation at 86 days and ultimately died from brisk tumor recurrence one year after tumor became undetectable. Had the cancer been known, patient would not have been treated with a growth factor. Example 10: Clinical summaries-patient 6 (limb compression; filgrastim + limb compression)

[0175] Patient 6, a 75-year-old Fontaine Stage IV insulin dependent diabetic male, presented with an infection under previously dry gangrene of toes 1 and 2, occluded posterior tibial and peroneal arteries, previous bypass graft, and after laser recanalization resulting in acute thrombosis of dorsalis pedis artery shortly before enrollment. There were no bypass targets. ABI was 0.55 with monophasic DP Doppler waveforms and biphasic above-ankle PT Doppler waveforms. Severe medial sclerosis and incompressible extremity arteries limited accuracy and utility of hemodynamic measurements. Figure 7A and Figure 7B are angiograms of distal calf and foot before treatment. Contralateral limb was previously amputated below the knee. Patient could ambulate with prosthesis. Recent thrombotic complications suggest endogenous activation of the fibrinolytic system. Standard of care is below knee amputation.

[0176] Patient refused consent to below knee amputation of remaining leg. Infected toes were amputated acutely, the wound thoroughly irrigated and debrided, and opposing tissue planes loosely approximated. Antibiotics were given IV. A week later the wound looked unhealthy, was reopened and underwent further debridement.

[0177] Patient began an extended delivery regimen of 960 mcg filgrastim every 3 days, total 10 doses, with a gap of 11 days between the 5th and 6th doses, and limb compression. [0178] At 3 months PT ABI was 0.62, DP Doppler waveforms became biphasic. PT Doppler waveforms also improved. Angiography showing recurrent stenosis of the popliteal artery where previous angioplasty had been performed, yet the wound eventually healed. At 9 months operative angiogram with hand injection of contrast through 4F catheter in femoral artery shows recanalization and neovascularization; cork-screw collaterals around the ankle and into the foot. Improved contrast transit were observed. Figure 7C and Figure 7D. Open toe amputations healed.

[0179] Patient remained ambulatory with prosthesis on contra-lateral limb and amputation free for 12 years, at which point patient was lost to follow up.

Example 11: Clinical summaries-patient 7 (limb compression; filgrastim + limb compression)

[0180] Patient 7, a 71-year-old Fontaine Stage IV male, presented with forefoot gangrene following 2 failed femoral to tibial artery bypasses, failed laser recanalization and atherectomy, failed popliteal and tibial artery angioplasty, a 2 pack per day smoking history, altered sensorium from alcoholism, vascular dementia, hypertension, and hyperlipidemia. Contralateral limb previously amputated above the knee after a similar course.

[0181] Living in VA assisted living facility, patient used his remaining leg to transfer to and from a wheelchair and care for himself. Forefoot had poor perfusion. He developed forefoot dry gangrene from recurrent injury. ABI was 0.28, too low to heal a transmetatarsal amputation unless revascularized for what would have been the third time in this leg. However, no conduit or suitable target was identified to achieve a durable outcome. He underwent TMA with primary closure. Standard of care is below knee amputation.

[0182] After TMA, patient began an extended delivery regimen of 600 mcg filgrastim (8.3mcg/kg based on patient weight) every 3 days for 5 doses as transfer to a VA nursing home precluded further treatment. Patient was sent to in-patient rehab for strengthening, protecting the wound, limb compression, and preventing tobacco use. After 2 months, ABI increased to 0.53 and closed TMA wound edges were observed to heal. Limb compression stopped, patient became sedentary, mostly sleeping. Use of foot to stabilize himself in a wheelchair caused recurrent trauma to the TMA resulting in a dry eschar developing over the traumatized TMA and requiring below knee amputation 123 days after beginning treatment.

Example 12: Clinical summaries-patient 8A (limb compression; filgrastim + limb compression)

[0183] Patient 8, a 67-year-old Fontaine III male, presented with thrombosed left femoral distal polytetrafluoroethylene (PTFE) graft, 14 previous vascular interventions (7 in each leg), left calf claudication at less than one half block, ischemic rest pain in the toes, and dependent rubor with pallor on elevation (signs of poor blood flow). Patient had a history of hypertension, diabetes mellitus type II, high cholesterol, congestive heart failure, cerebrovascular accident, deep vein thrombosis, chronic obstructive pulmonary disease, chronic renal insufficiency, dye allergy, gout, and glaucoma. Tobacco use had stopped 3 months earlier.

[0184] Two months of limb compression yielded no symptomatic benefit or improvement in blood flow. In the symptomatic left leg, ABI was 0.17 at the dorsalis pedis (DP); 0.12 at the posterior tibia (PT); Toe Brachial Index (TBI) was 0; all critically low. In the non- symptomatic contralateral right leg, DP ABI was 0.38 and PT ABI 0.49; right TBI was also 0. Standard of care is below knee amputation.

[0185] Patient began an extended delivery regimen of 600 mcg filgrastim every 3 days and limb compression, which patient reported doing 6 hours a day. After the 5th dose rest pain abated and patient stopped administration. Pain free walking distance improved dramatically and blood flow showed significant improvement in both legs.

[0186] Before treatment, CD34+ circulating progenitor cell counts were 9 per 10,000 cells and VEGFR2+ endothelial progenitor cell counts were 2 per 10,000 cells. One day after the 5th filgrastim dose, CD34+ and VEGFR2+ increased to 24 and 5 cells per 10,000 respectively. Figures 1C and ID tabulate proteomic and biochemical data for this patient with filgrastim and limb compression compared to limb compression alone. Increases in plasmin and fibrin degradation products (FDP) are in Table 5.

Table 5. Comparison of increase in plasmin and FDP between limb compression alone and filgrastim and limb compression in patient 8

[0187] These data show the pro-fibrinolytic effect of filgrastim and compression relative to compression alone.

Example 13: Clinical summaries-patient 8B (limb compression; filgrastim + limb compression)

[0188] Patient 8 returned to clinic 3 months later and presented as Fontaine Stage IV in severe pain with blunt injury to first toe. The toe was cyanotic. Toe pressure was 0. Patient began another extended delivery regimen of 600 mcg filgrastim every 3 days, total 5 doses, and renewed compression. Over several months, toe pressures gradually improved, the wound healed, and pain resolved.

[0189] Five months later, left DP ABI increased to 0.55 and left PT ABI increased to 0.52. Pulsatility was observed in the left great toe for the first time. In the contra-lateral limb (right) DP and PT ABI increased to 0.78 and 0.69 respectively. Right great toe TBI increased to 0.31. See Figure 1A and Figure IB. Over 12 months, posterior tibial ABI (PT, square points), dorsalis pedis ABI (DP, diamonds), and toe brachial index (TBI) (triangles) in the right and left foot all progressed. Pain free ambulation distance increased from less than one block pretreatment to nearly 14 blocks of slow walking and 6 blocks of rapid walking.

[0190] Five years later, patient’s left iliofemoral prosthetic bypass graft placed over 10 years earlier thrombosed. A new iliofemoral graft was placed during which 4 previous femoral grafts had to be explanted. Patient developed a lymphocele that became infected, necessitating removal of the new graft and placement of a cryopreserved graft, which also thrombosed several weeks later. Patient was amputation free for 5 years until the iatrogenic infection and thrombosis of subsequent graft required above the knee amputation, a common result of this complication.

Example 14: Clinical summaries-patient 9 (limb compression; filgrastim + limb compression)

[0191] Patient 9, a 63-year-old Fontaine Stage IV female, presented with acute lower leg ischemia and gangrene of foot and a medical history that included diabetes, obesity, and tobacco use. The superficial and popliteal arteries were chronically occluded and the profunda artery was acutely ligated (occluded) after her third mycotic aneurysm rupture in 3 weeks. Two femoral popliteal bypass grafts (one prosthetic, one autogenous) had ruptured from mycotic infection a week apart before this. To prevent further potentially fatal hemorrhage, the grafts were explanted, and the superficial femoral artery ligated. A profunda thrombectomy and patch angioplasty was performed to improve blood flow so a below knee amputation would heel. That vascular patch ruptured from the same necrotizing mycotic infection. The profunda and common femoral artery were then ligated. The patient was treated with antibiotics IV while her leg demarcated and the open groin/thigh wounds from rupture managed. The foot was not salvageable and allowed to demarcate. Necrosis and gangrene progressed from the foot to the distal third of the calf. Recent thrombotic complications suggest endogenous activation of the fibrinolytic system. Standard of care is high above knee amputation.

[0192] Patient began an extended delivery regimen of 600 mcg filgrastim every 3 days, stopping after 7 doses because of inability to tolerate limb compression.

[0193] Despite ligation of the femoral and profunda arteries and explantation of the graft, the distal thigh and upper calf progressively warmed, suggesting improved blood flow. Due to the calf and foot necrosis, limb compression was limited to the upper calf. Patient did not want to continue compression after the seventh dose due to discomfort. By then warmth of upper calf had perceptibly improved, implying improved blood flow. The high above knee amputation was no longer required. A below knee amputation was performed, with successful healing, saving the knee.

Example 15: Clinical summaries-patient 10 (limb compression; filgrastim + limb compression)

[0194] Patient 10, a 79-y ear-old Fontaine Stage III female presented with severe foot pain after a second femoral tibial bypass graft occluded in her right leg. DP and PT ABI in the treatment limb were 0.16 and 0 respectively (critical); right TBI was 0. Standard angiogram with a power injector and full-strength contrast showed occlusion of grafts and native femoral/popliteal and tibial arteries. Collateral flow was not detected. A non-traditional nitroglycerine angiogram with power injector and full-strength contrast was used to dilate small collateral arteries that had formed over the years. These collaterals were small, plentiful, and readily observed. The foot turned red and warm. The nitroglycerine effect was brief; circulation reverted back to baseline. The contralateral (non-symptomatic) limb DP and PT ABI were 0.8 and 0 respectively, left toe TBI was 0.35. Standard of care is below knee amputation.

[0195] Patient began an extended delivery regimen of 600 mcg filgrastim (10.2 mcg/kg based on patient weight) every 3 days stopping after 5 doses when rest pain resolved.

[0196] Several weeks later, patient was seated in a chair when a 6 mm varix in the treated foot instep was traumatized. EMS placed a pressure dressing on the midfoot to control bleeding. It was left on until the following day (>24 hours). The varix thrombosed. Full thickness skin necrosis corresponding to the circumferential dressing was observed in her instep and midfoot. Patient presented now as Fontaine Stage IV. She weighed only 45 kg with paucity of soft tissue in both feet (“skin on bone”). An urgent third operative bypass by a different surgical team was attempted and immediately failed. A fourth bypass was attempted (by another surgical team) and also eventually failed. No specific hypercoagulability was identified. As the devitalized skin sloughed the tarsal bones and midfoot tendons became exposed. Standard of care is below knee amputation.

[0197] Patient began a second extended delivery regimen of 600 mcg filgrastim every 3 days to re-establish blood flow to the skin on the foot.

[0198] Despite all the failed interventions, 9 months after the first treatment, DP and PT ABI in the treatment limb increased to 0.39 and 0.3 respectively, waveforms improved, and contralateral limb ABI DP and PT increased to 1.06 and 0.91 respectively. An intra-operative angiogram with hand injection of 50% dilute contrast and C-arm imaging demonstrated a rich network of collateral arteries in both calf and foot. Contrast transit was brisk, and the collateral network was ample and clearly visualized. No nitroglycerine or power injector was needed to make them visible, and only 10 ml of half strength contrast was hand injected from the groin. Notwithstanding the improved blood flow, the exposed hind foot bone and tendons necessitated amputation. Example 16: Clinical summaries-patient 11 (limb compression; filgrastim + limb compression)

[0199] Patient 11, a 77-year-old Fontaine Stage III male, presented with increasing ischemic forefoot rest pain. A previous femoral to below knee popliteal bypass had failed shortly after being placed. Left DP and PT ABIs were 0.34 and 0.38 respectively; left great toe TBI was 0.2. Diffuse infra-geniculate arterial occlusive disease was not conducive to further revascularization.

[0200] Patient used compression dutifully for three years, but symptoms progressed to ischemic forefoot rest pain with no change in ABI. Figure 3A shows a plot of the stable but low ABI over this period. Toward the end of this period pain increased and patient began to feel worse. Standard of care is below knee amputation.

[0201] Patient began an extended delivery regimen of 600 mcg filgrastim (7.9mcg/kg based on patient weight) every 3 days and limb compression. Filgrastim administration was stopped after the 5th dose after ischemic forefoot rest pain abated (patient declined to take the next 5 doses), demonstrating effect of an extended delivery regimen of filgrastim compared to limb compression alone. Over the next 6 weeks ambulation distance increased to 2 blocks. Left DP and PT ABI rose to 0.50 and 0.46 respectively; left great toe TBI increased to 0.38. Follow up angiogram revealed large, corkscrew and straight collaterals, demonstrating fibrinolysis and neovascularization. The mature vascular pattern of collateral growth and fibrinolysis mimic a bypass graft from the groin to the ankle. Figures 3B - 3G. Patient remained amputation free for 2 years as of last follow up.

Example 17A: Clinical summaries-patient 12 (filgrastim + limb compression)

[0202] Patient 12, a 36-year-old Fontaine Stage III female athlete, presented with vasculitis resulting from exposure to what at the time was an unknown agent, leading to acute occlusion of her right superficial femoral and popliteal arteries and severe rest pain. Intraarterial thrombolysis led to thrombosis of the profunda femoral artery. Three emergency bypass surgeries failed. Standard of care when patient presented was high above knee amputation. Standard of care is below knee amputation.

[0203] Patient began an extended delivery regimen of 600 mcg filgrastim every 3 days and daily limb compression. The patient was observed to improve and the first toe gangrene (Figure 4A) sloughed (Figure 4B) and the patient was able to walk increasingly long distances. CT angiogram after two months (Figure 4C - Figure 4E) shows the right calf artery filled with contrast before the normal left calf (Figure 4C, note contrast in right calf before left calf) despite occluded superficial femoral, profunda femoral, and popliteal arteries (Figure 4D, note right superficial femoral profundal femoral and popliteal artery occlusions). Remarkably, Figure 4E axial CTA images show the tibial vessels on the right larger than the tibial vessels on the normal left leg. (CTA resolution was inadequate to pick up the many collaterals that likely developed to bridge the thigh to the calf to account for contrast reaching the right calf first). Angiogram of lower right extremity 3 years later showed rich collateralization. Patient progressed for 4 years and was able to climb stairs and hike; she retained the limb 10 years later.

Example 17B: Clinical summaries-Thromboembolic stroke-patient 12 (filgrastim) [0204] Patient 12 was re-hospitalized 5 years later and presented as Fontaine Stage IV with left arm intra-arterial thrombosis and gangrene of fingertips. She had resumed smoking tobacco and marijuana and was suspected to have recurrent marijuana associated vasculitis. Importantly the right leg did not deteriorate. Catheter directed left upper extremity thrombolysis failed, during which patient got worse. Two subsequent emergent revascularizations (bypasses) failed immediately. Catheter directed thrombolysis was reattempted during which patient sustained a left cerebral infarct in the middle and posterior circulation distribution resulting in thromboembolic posterior cerebrovascular stroke. The left arm remained ischemic and digit gangrene progressed. Following the stroke, patient was placed in neuro intensive care. The right upper and lower arms had flaccid paralysis. Intraventricular drainage was performed to prevent herniation. Metabolic brain activity was pharmacologically lowered (induced coma). Patient seemed moribund and was placed on a ventilator. MRI showed infarcts in brain tissue perfused by the middle and posterior circulation. Catheter directed intracerebral thrombectomy was attempted. Hyperbaric oxygen therapy was started. CT scan showed evolution of a cerebellar infarct. Consideration of removing life support was initiated, but postponed due to EEG activity.

[0205] Patient began an extended delivery regimen of 600 mcg filgrastim every 3 days after patient’s father inquired about and requested treatment with filgrastim that had been effective previously with the lower limb ischemia. After 2 weeks subject became oriented, was moved to a neuro rehab facility and gradually recovered speech, cognition, memory, and vision.

Example 17C. Clinical summaries-CLI upper extremity-patient 12 (filgrastim + limb compression)

[0206] Flaccid paralysis in right arm and leg and left arm weakness and ischemia and progressive gangrene of fingertips of left hand remained. Standard of care is to allow ischemic area of the fingers to demarcate. [0207] Extended delivery regimen of 600 mcg filgrastim every 3 days was repeated and daily compression of left upper extremity begun to address left hand ischemia. Gangrene eventually sloughed from fingertips, and patient regained function of her right hand and leg and became ambulatory. (Motor function remains not fully normal in either limb (rate both 4 out of 5).

Example 18: Clinical summaries-patient 13 (filgrastim + limb compression)

[0208] Patient 13, a 40-y ear-old Fontaine Stage IV female referred with a diagnosis of thromboangiitis obliterans (Buerger’s vasculitis), presented with progressive enlarging ischemic ulceration on the foot dorsum, gangrene, and severe rest pain. Patient was directed to stop smoking. She returned 2 months later after auto-amputation of the fifth toe and hyperbaric oxygen, smoking cessation, and frequent wound care failed to stop gangrene progression (70% percent of the foot dorsum was now ulcerated). Cellulitis was treated with IV antibiotics and debridement to remove the gangrenous tissue. Toe pressures were not measurable. Angiogram showed SFA and popliteal atherosclerosis and distal tibial and plantar arch arterial occlusive disease with diminutive cork-screw collaterals consistent with Buerger’s disease, confirming thromboangiitis obliterans. Patient was ambulatory at short distances despite previous toe amputations in both feet. Symptoms and ulcer continued to worsen. ABI was 0.7. The end of the acute inflammatory phase was measured, for example, by C reactive protein or erythrocyte sedimentation rate, Standard of care is below knee amputation.

[0209] Patient began an extended delivery regimen of 600 mcg filgrastim every 3 days, total 10 doses, and limb compression.

[0210] After 5 months, healthy granulation formed and completely covered the wound. At 10 months, ABI was measured as 1.13. By 18 months, foot wounds were healed completely. At 3 years, angiogram showed rich collateral flow into the foot. No named arteries were visible from the distal third of the calf distally. Another toe was amputated after a traumatic injury in 2020. Patient remained amputation free as of last follow up in 2021, 9 years later. Example 19: Clinical summaries-patient 14 (filgrastim + limb compression) [0211] Patient 14, a 26-year-old Fontaine Stage III male, presented with severe ischemic rest pain and short distance calf and thigh claudication as result of recurrent occlusion of multiple bypass grafts from and including common iliac artery to infra-geniculate arterial circulation. The profunda artery was also occluded. Past medical history included juvenile onset diabetes, morbid obesity, and hypercoagulability. ABI was 0. Angiogram showed multi-level arterial occlusive disease from the common iliac artery origin to the lower calf where the posterior tibial reconstituted and perfused the plantar artery. Standard is care high above knee amputation.

[0212] Patient began an extended delivery regimen of 960 mcg filgrastim every 3 days, total 10 doses, and limb compression.

[0213] After one month, rest pain resolved and patient could walk one block. Improvement continued after patient entered a weight loss program and exercised, reaching 4 blocks of pain free walking before onset of thigh and calf claudication. Patient remained amputation free for 4.5 years before lost to follow up.

Example 20: Clinical summaries-patient 15 (filgrastim-sndz + limb compression)

[0214] Patient 15, a 74-year-old Fontaine Stage III male, presented with bilateral calf claudication (left>right), multiple prior endovascular procedures on both legs including left superficial femoral artery (SFA) stenting and angioplasty and right SFA stenting, tibioperoneal recanalization, left forefoot rest pain, left foot numbness, and inability to sleep resulting from two recent failed left SFA drug coated balloon angioplasties. Stents were occluded. Standard of care is below knee amputation.

[0215] Patient began an extended delivery regimen of 780 mcg filgrastim-sndz (Zarxio™, Sandoz Inc. Princeton, NJ) using pre-filled syringes every 3 days for a total of 10 doses.

[0216] After 6 weeks, pain resolved and patient was able to walk increasing distances. At 9 months patient showed progressive improvement. Patient remained amputation free 6 years later.

Example 21: CLI treated with filgrastim + limb compression - Amputation Free Survival

[0217] Standard of care for all 15 no option patients clinically assessed and treated with filgrastim/filgrastim-sndz and limb compression was amputation. In other words, but for the extended delivery regimen, all would have lost limbs. One that did lose a li b to a. below knee amputation due to a gangrenous foot nevertheless avoided a high above knee amputation. (Patient had had her femoral vessels ligated in the groin for a mycotic rupture.) Iambs were lost for various causes including foot sepsis, trauma, malignancy, non- compliance, tight pressure bandage around instep and forefoot induced gangrene following bleeding from a ruptured foot varix, and mycotic infection of an iliac bypass graft 4 years after the procedure. The remainder retain limbs 5 to 12 years later. These durable outcomes contrast with invasive revascularization and standard of care major amputation, both associated with diminished life expectancy. Example 22: CLI treated with filgrastim + limb compression-Blood flow and ABI results

[0218] A total of 15 patients were treated with a combination of an extended delivery regimen of filgrastim every 3 days and directed to use limb compression at home 3 hours a day until symptoms and wounds improved, limb-threatening ischemia resolved, or the limb was lost. Of the 15, 2 had used limb compression unsuccessfully before treatment with filgrastim. Follow-up ranged from 6 months to 12 years.

[0219] Ankle-brachial index and forefoot perfusion measured by photoplethysmography were observed to increase significantly. Toe pulsatility was observed in patients who had no toe pulsatility before treatment. Neovascularization and fibrinolysis were observed on followup angiogram. Ischemic wounds healed and forefoot rest pain resolved. These observations were supported by biochemical assays and cytometry.

[0220] Patients treated with filgrastim and limb compression observed a 49% increase in ABI compared to a 9% increase in ABI in patients treated with limb compression alone. See Table 6.

Table 6: Comparison of Increases in Ankle-Brachial Index (ABI)

Example 23: Neovascularization and fibrinolysis - measurement of effectors

[0221] Neovascularization and fibrinolysis are natural processes that can be both observed in angiograms of affected limbs and measured by increases in their effectors. To measure relevant cytometry, ELISA assays of relevant proteins, and serum nitrite levels, blood was drawn from patients treated with limb compression alone and with extended duration regimens of filgrastim and limb compression. In all cases, blood was drawn before and after 2 hours of limb compression. Two samples were drawn for each timepoint. On days blood was drawn, patients were instructed to administer an additional hour of limb compression at home to bring the total to 3 hours a day.

[0222] For patients administered limb compression only, blood was drawn on day 1 (N=19) and on day 30 (N=17; 2 patients did not complete the study). For patients administered filgrastim and limb compression (N=14), blood was drawn on day 1 before any filgrastim was administered, on the day after the 5th dose, and on the day after the 10th dose. One patient had blood drawn after dose 7. [0223] After 10 doses, serum and plasma levels of plasmin were increased 2,000% (p< 0004); serum and plasma levels of fibrin degradation products were observed to increase over 500% (0.004); and serum levels of VEGFA (p 0.01), Hepatocyte Growth Factor (p 0.0001), PDGF-AA (p 0.05) and PDGF-BB (p 0.03), angiopoietin-1 (p 0.02), and MMP-9 (p< 0.00001) were observed to increase from 268% to 984% compared to the levels before treatment. PLGF, Insulin GF-1, TNF, IL-6, and TGFb were also observed to increase. Serum levels of nitrite were observed to increase 268% (p=0.003)

[0224] After the tenth dose of filgrastim cell counts of CD34+, VEGFR2+, and PECAM-1 (CD31+) increased by 83% ±57%, 93% ±59%, and 17% ±15% respectively (p< 03 or less). These data indicate that neovascularization and fibrinolysis was effected to a greater extent in patients treated with filgrastim and limb compression compared to patients treated with limb compression only.

Example 24. CLI treated with filgrastim + limb compression - Cytometry results [0225] Levels of circulating CD34±, VEGFR2±, and PECAM-1 (CD31±) cells were measured using flow cytometry in patients receiving an extended delivery regimen of filgrastim every 3 days. From Day 1 before initial filgrastim administration to the day after the tenth dose of filgrastim, cell counts of CD34±, VEGFR2±, and PECAM-1 (CD31±) increased by 83% ±57%, 93% ±59%, and 17% ±15% respectively (p< 03). Absolute neutrophil counts were observed to increase 664% ±251% and absolute monocyte counts were observed to increase 134% ±251% after the fifth dose of filgrastim. Percent increase is the average of the percent changes for each patient (each patient is their own control).

Example 25. CLI treated with filgrastim + limb compression; limb compression alone - ELISA results

[0226] Endothelial activation, neovascularization, and fibrinolysis can be measured at the biochemical level by measuring their effectors. For example, MCP-1 for endothelial activation, VEGFA for neovascularization, and plasmin for fibrinolysis.

[0227] Blood was drawn from patients treated with an extended duration regimen of filgrastim and limb compression as described in Example 21. Table 7 contains serum concentration measured by ELISA of proteins associated with different aspects of fibrinolysis and neovascularization before and after 2 hours of limb compression on days 1, one day after the fifth dose, and one day after a tenth dose. There were statistically significant increases in concentration of MCP-1, a marker of the endothelial activation needed to promote arteriogenesis on all 3 days. The increased MCP-1 levels support the hypothesis that limb compression increases the endothelial shear stress needed to activate the endothelium to promote arteriogenesis. On none of the 3 days did 2 hours of limb compression result in statistically significant increases in concentration of the other proteins, indicating limb compression had no impact on these markers.

[0228] ELISA identified statistically significant increases (p<0.01) in serum concentrations of multiple proteins from Day 1 to one day after the 5th dose and from Day 1 to one day after the 10th dose. Serum concentration increases of these proteins from before filgrastim administration on day 1 to the days after doses 5 and 10 are in Table 7. Percent increase is the average of the percent changes calculated for each patient (patient being their own control). From Day 1 to the day after the 5th dose and from Day 1 to the day after the 10th dose markers for neovascularization, including VEGFA, Angiopoietin-1 (day 5 doses only), PDGF-AA, PDGF-BB, MMP-9, TNF, and TGFb increased significantly (p< 05). Markers for fibrinolysis, plasmin and Fibrin Degradation Products (FDP), also increased significantly. Table 7. Change in protein expression after 5 and 10 doses of filgrastim. Paired T-Tests using the concentrations were used to derive P values.

[0229] The decrease in serum concentration of MCP-1 from day 1 to one day after the 5th dose and one day after the 10th dose is not statistically significant. MCP-1 serum concentration was, however, shown to increase after 2 hours of limb compression (15%±21%, p=.05 day after the 5th dose; 21%±20%, p=.05 day after the 10th dose).

[0230] Limb compression alone was evaluated in 19 patients treated 3 hours a day from blood drawn before and after 2 hours of limb compression on day 1 (N=19) and on day 30 (N=17) (2 patients lost limbs and did not complete the study). Concentration is average concentration for all patients. A P value less than 0.05 is considered significant. There was a statistically significant increase from t=0 to t=2 hours on day 1 for MCP-1 (mean increase 19%; p=0.006) and IL-6 (mean increase 21%; P=0.03) but for no other protein. There were no statistically significant differences in serum concentration of any other of these proteins from t=0 to t=2 hours of limb compression on day 30. Nor were there statistically significant increases in serum concentration of any of these proteins from Day 1 to Day 30 with limb compression alone. Table 8 shows proteomic data (ELISA) before and after limb compression.

Table 8. Change in serum concentration of proteins of patients treated with limb compression alone

[0231] These data indicate that filgrastim and limb compression increases concentration of proteins associated with of endothelial activation, neovascularization, and fibrinolysis and that filgrastim is primarily responsible for the increase in serum concentration in proteins involved in neovascularization and fibrinolysis. [0232] The increase in serum concentration of MCP-1 (along with the increased serum nitrite activity) supports the hypothesis that limb compression promotes endothelial activation, likely by increasing endothelial shear stress.

[0233] Comparing ELISA concentrations of proteins at all data points before any filgrastim administration (Day 1 and Day 30 for patients undergoing compression alone; Day 1 t=0 for patients receiving filgrastim after limb compression on Day 1) and concentrations one day after the fifth and tenth doses of filgrastim demonstrates the significant impact of an extended duration regimen of filgrastim on proteins involved in fibrinolysis and neovascularization. See Table 9. Table 9. Summary comparison (unpaired group analysis) of P values are derived from unpaired T Test of ELISA data. N is the number of ELISA results.

Example 26. CLI treated with filgrastim + limb compression - Serum Nitrite levels [0234] Endothelial activation, a promoter of arteriogenesis, can be measured by nitric oxide synthase (NOS) activity. NOS cannot be measured in tissues without biopsy, but increased NOS activity increases nitric oxide (NO) which breaks down quickly (due to its short half-life) to nitrite and nitrate, which can be measured. We measured serum nitrite, a stable end-product of NO, using a quantitative commercially available nitrate/nitrite colorimetric assay kit according to manufacturer’s protocol.

[0235] For patients treated with filgrastim and limb compression, concentration of serum nitrite (nM/ml) increased relative to Day 1 (t=0, before limb compression and filgrastim) 72% + 37% (p<0.001) one day after the fifth dose and 268+159% (p=0.003) one day after the tenth dose of filgrastim. See Table 7. Concentration of serum nitrite also increased from before to after two hours of limb compression on the day after the 5th dose (30%+27%) and the day after the 10th dose 9%+5% (p=0.01). Serum nitrite concentration for each patient treated with filgrastim and limb compression on Day 1 and the day after the 5th dose and the day after the 10th dose are in Figure 5C.

[0236] For patients treated with limb compression alone, concentration of serum nitrite increased from before to after two hours of limb compression 28%+28%, (p<0.0001) on Day 1 and 7%+6% (p<0.0001) on day 30. Concentration of serum nitrite also increased over 30 days of 3 hours daily limb compression, 169%+158% (p<0.001). See Table 8. Serum nitrite concentration for each patient treated with limb compression alone on Days 1 and 30 before and after 2 hours of limb compression are in Figures 5 A and 5B respectively.

[0237] These data demonstrate that while serum nitrite increased significantly after treatment with both limb compression alone and with filgrastim and limb compression, the percentage increase in serum nitrite concentration in patients treated with filgrastim and limb compression was considerably higher than the percentage increase in serum nitrite concentration in patients treated with limb compression alone.

Example 27. Neovascularization (filgrastim + limb compression)

[0238] An extended delivery regimen of filgrastim and limb compression was observed to promote neovascularization and fibrinolysis. This was evidenced by patient angiograms that specifically show the growth of corkscrew collaterals and the recanalization of previously occluded arterial segments. For example, a 79-year-old female presented with severe foot pain after a second femoral tibial bypass graft occluded in her right leg. DP and PT ABI in the treatment limb were 0.16 and 0 respectively (critical); right toe brachial index was 0. Traditional angiogram showed occlusion of grafts and native femoral/popliteal and tibial arteries. Collateral flow was not detected. A non-traditional nitroglycerine angiogram with power injector and full-strength contrast was used to dilate small collateral arteries not visible on the initial angiogram. These collaterals were small, plentiful, and readily observed. The foot turned red and warm. The nitroglycerine effect lasted only a few minutes. Six months after treatment began an intraoperative angiogram with hand injection of 50% dilute contrast and C-arm imaging (and no nitroglycerine) demonstrated a rich network of collateral arteries in the right thigh, calf, and foot. Contrast transit was brisk. The ephemeral collateral network displayed briefly before treatment was now readily visible. This finding confirmed stable, durable, collateral growth.

[0239] Traditional angiograms and non-traditional nitroglycerine angiograms (NTG) of the ischemic limb were taken before treatment. Intraoperative angiograms with hand injection of 50% dilute contrast and C-arm imaging were taken 6 months after treatment started. The initial traditional angiogram shows occluded superficial femoral, popliteal, and pedal arteries and small collaterals around the knee (Figure 2A, thigh; Figure 2B, above and below knee; and Figure 2C, calf and ankle).

[0240] To demonstrate the effect of treatment, nitroglycerine angiograms before treatment and intraoperative angiograms with hand injection 6 months later are shown side by side in Figures 2D to 2L. Figure 2D is NTG thigh angiogram before treatment; Figure 2E is angiogram with hand-injection of 10 ml 50% dilute contrast at 6 months. Figure 2F is NTG angiogram of knee before treatment; Figure 2G is angiogram with hand-injection of 10 ml 50% dilute contrast at 6 months. Figure 2H is NTG angiogram of the calf/ankle/foot before treatment; Figure 21 is angiogram with hand-injection of 10 ml 50% dilute contrast of the distal calf ankle/foot at 6 months. Figure 2J is angiogram with hand-injection of 10 ml 50% dilute contrast of the plantar arch on the lateral foot at 6 months showing visible blood flow into the foot where none was apparent on nitroglycerine angiogram before treatment. In sum, following treatment, a technique typically less sensitive than both traditional and nitroglycerine angiogram needed a smaller volume of lower contrast without vasodilation to make visible vessels that previously could not be seen with much stronger techniques.

Example 28. Fibrinolysis (filgrastim + limb compression)

[0241] An extended delivery regimen of 5 or 10 doses of 600 mcg or 960 mcg of filgrastim every 3 days and limb compression 3 hours a day was observed to increase serum and plasma concentrations of plasmin, the enzyme that breaks down fibrinogen, and Fibrin Degradation Products (FDP), the proteins released when fibrinogen breaks down. This is consistent with the recanalization of previously occluded arterial segments observed on follow up angiography. Serum and plasma concentration of plasmin and FDP from Day 1 to the day after the 5th dose and from Day 1 to the day after the 10th dose are contained in Table 10.

Table 10. Increase in concentration of plasma and FDP relative to baseline (N = number of assays)

[0242] Two hours of limb compression did not significantly change the serum concentration of plasmin and FDP before and after limb compression on Day 1 or on Day 30 or from Day 1 to Day 30. See Table 11. Table 11. Increase in serum concentration (ng/ml) of plasma and FDP relative to baseline with compression alone (N = number of assays)

[0243] When all data points are included, the difference in concentration of plasmin and FDP in subjects with no exposure to filgrastim and subjects one day after they have received a fifth or tenth dose of filgrastim is highly significant (P<0.001 ). Mean measure of nanograms per milliliter and standard deviation are in Table 12. P values are derived from unpaired t-test. N=number of results.

Table 12. Summary comparison (unpaired group analysis) of concentration of plasmin and FDP with no filgrastim and one day after 5 or 10 doses of filgrastim.

Example 29. Vasculitis resulting in chronic limb-threatening ischemia

[0244] Vasculitis is inflammation of blood vessels that occurs when the body's immune system attacks blood vessels improperly. It can be caused by infection, a medicine, or another disease and can affect arteries, veins, and capillaries. The cause is often unknown. Buerger’s disease (thromboangiitis obliterans) is one form of vasculitis often caused by tobacco use affecting the extremities. Marijuana is also known to cause vasculitis. They and other forms of vasculitis can be characterized as having an acute inflammatory phase and a chronic residual ischemic phase. Inflammation can often be reduced by halting use of the inciting inflammatory agent, e.g. tobacco or marijuana. Once the acute inflammatory phase resolves, its consequences, damaged and/or occluded vessels, remain. The chronic residual ischemic phase has no widely accepted intervention. The involved vessels are too small and numerous to access directly with catheter lysis; moreover, catheter directed thrombolysis from the larger proximal vessels into these tissue beds would require an extended period outside the acceptable safe window (resulting in life threatening hemorrhage). Typically, an inflammatory agent such as G-CSF would not be used to treat CLI caused by an inflammatory process (e.g., Buerger's disease). However, the present inventors now contemplate the use of an inflammatory agent such as G-CSF, GM-CSF (granulocytemacrophage colony-stimulating factor), M-CSF (macrophage colony-stimulating factor), filgrastim, tbo-filgrastim, filgrastim-sndz, filgrastim-aafi, Accofil, balugrastim, biograstim, efbemalenograstim alfa, Grastofil, Hexal, lenograstim, lipegfilgrastim, Pelgraz, ratiograstim, sargramostin, Tevagrastim, Emgrast, Fegrast, filgrastim (Lupin), Grafeel, Neukine, Religrast, pegfilgrastim, pegfilgrastim-bmez, pegfilgrastim-cbqv, pegfilgrastim-jmbd, eflapegrastim, CHS-1701 (a filgrastim biosimilar developed by Udenyca), and/or TX-01 (a filgrastim biosimilar in development by Tanvex BioPharma Inc. (San Diego, CA)), or equivalents to treat the chronic residual ischemic phase left after the acute inflammatory phase resolves, including in Buerger’s disease. In some embodiments, the acute inflammatory phase of vasculitis, including, e.g., the inflammatory phase of Buerger's disease, is monitored. Once the inflammatory phase is over as measured, for example, by C reactive protein or erythrocyte sedimentation rate, an extended delivery regimen and compression as described herein is administered to treat the occluded arteries and veins. Neovascularization and fibrinolysis reopen occluded vessels and accelerate development of new collaterals to improve blood flow to alleviate the chronic residual ischemia following the acute inflammatory phase of vasculitis, including Buerger’s disease.

Example 30. Stroke - filgrastim

[0245] Stroke (including “thromboembolic,” “ischemic,” or “thrombotic” stroke) results from occluded vasculature in the brain. The present inventors now contemplate the use of an inflammatory agent (i.e., a blood cell growth factor) such as G-CSF, GM-CSF (granulocytemacrophage colony-stimulating factor), M-CSF (macrophage colony-stimulating factor), filgrastim, tbo-filgrastim, filgrastim-sndz, filgrastim-aafi, Accofil, balugrastim, biograstim, efbemalenograstim alfa, Grastofil, Hexal, lenograstim, lipegfilgrastim, Pelgraz, ratiograstim, sargramostin, Tevagrastim, Emgrast, Fegrast, filgrastim (Lupin), Grafeel, Neukine, Religrast, pegfilgrastim, pegfilgrastim-bmez, pegfilgrastim-cbqv, pegfilgrastim-jmbd, eflapegrastim, CHS-1701 , and/or TX-01 (a filgrastim biosimilar in development by Tanvex BioPharma Inc. (San Diego, CA)), or equivalents for the treatment of stroke. In some embodiments, fibrinolysis reopens occluded vessels in the brain to improve blood flow and alleviate stroke symptoms; neovascularization may also create new vessels.

[0246] Patients presenting with stroke, including thromboembolic stroke, may be administered filgrastim at a dose of about 10 mcg/kg or about 600 mcg, 780 mcg, or 960 mcg every third day for at least 2 doses. In some embodiments, patients are administered 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses, or at least 10 doses of filgrastim or other pharmaceutical compositions described herein. Where the stroke patient is treated with a long-acting blood cell growth factor such as pegfilgrastim, pegfilgrastim-bmez, pegfilgrastim-cbqv, pegfilgrastim-jmbd, eflapegrastim, CHS-1701, TX-01, a combination thereof, a functional equivalent thereof, a derivative thereof, or a biosimilar thereof, the long- acting blood cell growth factor may only need to be administered to the patient in a single dose to treat the stroke by inducing fibrinolysis and/or neovascularization.

Example 31. Stimulation of fibrinolysis

[0247] Fibrinolysis entails the dissolution of chronic thrombus by enzymatic degradation of fibrin. In 14 patients undergoing an extended delivery regimen and limb compression as described herein, Enzyme Linked Immunosorbent Assay (ELISA) revealed increased serum and plasma concentration of plasmin and fibrin degradation products (FDP) over the course of the extended delivery regimen. The effect of an extended duration regimen of filgrastim on the serum (N=8) and plasma (N=6) concentration of plasmin on the day after the 5 ^th and 10 ^th doses of filgrastim relative to Day 1 (at T=0) can be seen in Figures 6A and 6B. The effect of an extended duration regimen of filgrastim on the serum and plasma concentration of FDP on the day after the 5 ^th and 10 ^th doses of filgrastim relative to Day 1 (at T=0) can be seen in Figures 6C and 6D. For patient 1406, the last assay was the day after dose 7, as patient discontinued administration.

[0248] The three patients marked with blue * (P008, 1403, and 1406) represent patients with post intervention thrombotic complications shortly before treatment. Relative to other patients, serum and plasma concentration of plasmin and FDP are significantly elevated on Day 1 and do not increase appreciably after 5 or 10 doses. All of these patients had recent thromboses suggests endogenous activation of the fibrinolytic system. Further, the modest change in the plasmin and FDP levels in these patients after beginning treatment relative to their high baselines, combined with the significant increases in plasma and FDP levels in the other patients on the day after 5 or 10 doses to approximately the magnitude of the patients with recent thromboses on Day 1 support the notion that filgrastim promotes fibrinolysis at a physiologic level over the course of treatment.

[0249] It is now contemplated that G-CSF, GM-CSF (granulocyte-macrophage colonystimulating factor), M-CSF (macrophage colony-stimulating factor), filgrastim, tbo- filgrastim, filgrastim-sndz, filgrastim-aafi, Accofil, balugrastim, biograstim, efbemalenograstim alfa, Grastofil, Hexal, lenograstim, lipegfilgrastim, Pelgraz, ratiograstim, sargramostin, Tevagrastim, Emgrast, Fegrast, filgrastim (Lupin), Grafeel, Neukine, Religrast, pegfilgrastim, pegfilgrastim-bmez, pegfilgrastim-cbqv, pegfilgrastim-jmbd, eflapegrastim, CHS-1701, and/or TX-01 (a filgrastim biosimilar in development by Tanvex BioPharma Inc. (San Diego, CA)), or equivalents elevate plasmin and may stimulate upregulation of fibrinolysis. Angiogram revealed recanalization of previously occluded segments of tibial and pedal arteries and increased contrast flow through the tissues, demonstrating fibrinolysis. Furthermore, ELISA measures of serum and plasma concentration of plasmin and Fibrin Degradation Products increased.

Example 32. Extended duration regimen of filgrastim induces fibrinolysis

[0250] Blood was drawn from chronic limb-threatening ischemia patients (N=14) treated with extended duration regimens of filgrastim and limb compression 3 hours a day every 3 days according to Example 3 before and immediately after 2 hours of observed limb compression on day 1 and on the day after the 5th dose (N=12) and the day after the 10th dose (N= 12) of filgrastim. (Not all patients provided blood after the 5th dose or completed 10 doses.) During the course of or following treatment, no hemorrhagic complications were observed.

[0251] Proteins associated with Fibrinolysis: Compared to Day 1, concentration of plasmin increased l,600%±2,603% (p<0.002) the day after the 5th dose and 2,231%±2,013% (p<0.004) the day after the 10th dose. FDP concentration increased 554%±475% (p=0.006) the day after the 5th dose and 555%±442% (p=0.004) the day after the 10th dose. This > 10- fold increase in plasmin and the > 5-fold increase in FDP after filgrastim administration in 14 patients were not associated with hemorrhage and were limb compression independent, as concentration did not increase from before limb compression (t=0) to after 2 hours of limb compression.

[0252] Proteins associated with Neovascularization: The serum concentrations of VEGF- A, HGF and MMP-9 increased significantly the day after both a fifth and a tenth dose of G- CSF (i.e., filgrastim) as compared to Day 1. These findings were limb compression independent as concentration did not increase from before limb compression (t=0) to after 2 hours of limb compression and were not observed in patients treated with limb compression alone and reflected a significant (p<0.05) elevation of Platelet Derived Growth Factor AA and BB (PDGF AA and BB), Tumor Necrosis Factor (TNF), and TGFb on one day after the 5th dose and one day after the 10th dose relative to Day 1. The increase in the serum concentration of Angiopoietin 1 reached significance (p=0.05) one day after the 5th dose. [0253] Influence of limb compression in CLI patients receiving Filgrastim: Serum concentrations of proteins involved in fibrinolysis and neovascularization were not influenced by 2 hours of supervised limb compression. After 2 hours of supervised limb compression, the serum concentration of MCP-1 increased 15%±21% (p=0.05) on day 14, and 21%±20% (p=0.05) on 29. Serum nitrite increased 169%±158% (p<0.001) and 313±168% (p<0.001) on days 14 and 29 compared to Day 1. Lastly, 2 hours of limb compression led to an increase in serum nitrite of 28%±28% (p<0.001) on Day 1, and 7%±4% (p<0.001) on the day after the 10th dose.

[0254] Cytometry (counts per 10,000 cells): Cytometry confirmed the significant percentage increase in CD34+ progenitor and VEGFR2+ endothelial progenitor cells one day after the fifth and tenth doses of filgrastim. The number of circulating CD34+ progenitor cells was 83%±57% (p<0.004) and the number of VEGFR2+ endothelial progenitor cells was 93%±59% (p<0.002) higher measured one day after the tenth dose of filgrastim compared to Day 1. The differential blood cell count also shows the significant percentage increase in white blood cell count (WBC) and neutrophils the day after a fifth dose of filgrastim. Cell blood count of neutrophils increased a mean of 6-fold as compared to baseline (day 1) when measured 1 day after 5 doses of filgrastim. Measurements of CD31+, CD34+, VEGFR2+, WBC, neutrophils, lymphocytes, and monocytes are contained in Table 13.

Table 13. Circulatory cells in CLI patients treated with filgrastim and limb compression after

5 and 10 doses of filgrastim.

[0255] Serum nitrite before and after 2 hours of limb compression: Endothelial activation includes increased nitric oxide synthase activity, yielding nitric oxide (NO), a promoter of arteriogenesis. Serum Nitrite (mM) increased significantly after 2 hours of limb compression with the ArtAssist® pneumatic compression device on day 1 and on day 29 in both the limb compression alone and in the Filgrastim + limb compression groups. Additionally, at 29 days, serum nitrite concentration increased 170%±160% (p<0.001) at 29 days in the limb compression alone group and 313±168% (p<0.001) in the Filgrastim + limb compression group. Percent change was calculated as the average of the percent changes calculated for each patient (patient being his/her own control). Example 33. Contralateral Limb Ankle-Brachial Index, Toe Brachial Index, and Waveforms Before and After Treatment

[0256] Contralateral limbs were not a treatment focus. However, certain patients experienced meaningful increases in ABI, TBI, and/or waveforms in their contralateral limbs after treatment with filgrastim and limb compression. See Table 14. These data demonstrate that treatment with filgrastim and limb compression as described herein has potential for systemic improvement in blood flow.

Table 14. Changes in contralateral ABI, TBI, and waveforms before and after treatment with filgrastim and limb compression

Example 34. Multi-tier dosing regimen

[0257] Filgrastim is available in 300 mcg and/or 480 mcg single use vials and syringes and dispensed in packs of 10. Patients 1 and 2 were administered 10 mcg/kg filgrastim intravenously; patients 3 to 14 were administered 600 mcg (2 300 mcg vials) or 960 mcg (2 480 mcg vials) filgrastim drawn from vials into syringes, the contents of which were then injected SQ in the lower abdomen; patient 15 received 780 mcg filgrastim (1 300 mcg prefilled syringe and 1 480 mcg pre-filled syringe). Patient weights ranged from 59 kg (130 lbs) to 115 kg (253 lbs), and the actual effective dose administered to these 15 patients ranged from 6.6 mcg/kg to 10.9 mcg/kg. The mean dose actually administered was 8.7 mcg/kg (arithmetic midpoint). See Table 4A.

[0258] A fixed dose for a range of patient weights is more clinically and operationally convenient than a dose that must be adjusted according to patient weight. A fixed dose eliminates the need to calculate dosage for each patient, avoids the need to draw less than the full vial from a single use vial or inject less than a full single use syringe, and reduces opportunity for error. Furthermore, a fixed dose more readily lends itself to a larger single pre-filled vial or syringe per administration, avoiding the need to mix and match sizes depending on patient weight. Only one syringe is needed. A fixed dose in a vial or pre-filled syringe can reduce inventory, needle sticks, and waste. [0259] A patient weight range of 59 kg to 115 kg is too broad for a single fixed dose to deliver a patient dosage of between 6.6 mcg/kg to 10.9 mcg/kg actually dosed to these patients. However, a tier of different doses would allow for a single vial or syringe to accommodate a range of patient weights with no patient receiving more or less than the desired dose.

[0260] Examples of 2- and 3-tiered dose/weight ranges is shown in Table 15.

Table 15. Multi-tiered fixed dose blood cell growth factor (e.g., filgrastim) dosing regimen

[0261] A fixed dose for a range of patient weights according to the dosing regimen provided in Table 15 is more clinically and operationally convenient, requiring only one syringe per administration rather than 2 or 3 that must be mixed and matched depending on patient weight. Patient weight ranges and dose size can be adjusted, or additional tiers created, to address desired populations or for other reasons. Equivalents and Scope

[0262] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[0263] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

[0264] It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of’ is thus also encompassed and disclosed.

[0265] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. [0266] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0267] It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects. [0268] All references cited herein, whether in print, electronic, computer readable storage media or other form, are expressly incorporated by reference in their entirety, including but not limited to, abstracts, articles, journals, publications, texts, treatises, internet web sites, databases, patents, and patent publications. [0269] While the present invention has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the invention.