WO2011038298A1 - Utilisation de dipyridamole dans l'ischémie chronique - Google Patents

Utilisation de dipyridamole dans l'ischémie chronique Download PDF

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Publication number
WO2011038298A1
WO2011038298A1 PCT/US2010/050305 US2010050305W WO2011038298A1 WO 2011038298 A1 WO2011038298 A1 WO 2011038298A1 US 2010050305 W US2010050305 W US 2010050305W WO 2011038298 A1 WO2011038298 A1 WO 2011038298A1
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dipyridamole
tissue
ischemia
ischemic
chronic
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PCT/US2010/050305
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English (en)
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Christopher G. Kevil
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Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • This invention relates to methods and compositions useful for the treatment of ischemia as well as chronic tissue ischemia, and more particularly to methods for o inducing new blood vessel growth in ischemic tissue.
  • Ischemia and especially chronic tissue ischemia can impair tissue function and result in tissue and organ damage.
  • chronic tissue ischemia in critical organ systems or body parts for example, heart, brain, kidneys, skin, limbs, or gastrointestinal5 tract, contributes significantly to human morbidity and mortality and there is a continuing need for therapeutic strategies that treat the ischemia and restore blood supply to affected regions.
  • the present invention is based, in part, on our discovery of compositions and
  • ischemia including chronic tissue ischemia such as that associated with a disorder, trauma or a congenital defect.
  • chronic tissue ischemia encompassed by the methods of the invention can stem from any of a wide range of medical conditions that result in the persistent or recurring restriction of blood supply to the tissue, for example, disorders such as peripheral artery disease, type 1 or type 2
  • chronic tissue ischemia can occur in a variety of tissue types including, for example, skeletal muscle, smooth muscle, cardiac muscle, neuronal tissue, skin, mesenchymal tissue, connective tissue, gastrointestinal tissue and bone.
  • this invention provides a method of treating ischemia in a 5 subject comprising administering to the subject a composition comprising a
  • dipyridamole or a pharmaceutically acceptable salt thereof, wherein dipyridamole is a primary drug in treating ischemia.
  • this invention provides a method of treating chronic tissue ischemia in a subject comprising administering to the subject a composition comprising a o therapeutically effective amount of dipyridamole or a pharmaceutically acceptable salt thereof.
  • dipyridamole is a primary drug in treating chronic tissue ischemia.
  • this invention provides a method of enhancing blood vessel growth in chronic tissue ischemia in a subject comprising administering to the5 subject a composition comprising a therapeutically effective amount of dipyridamole or a pharmaceutically acceptable salt thereof for a time sufficient to enhance blood vessel growth in the tissue.
  • dipyridamole is a primary drug in enhancing blood vessel growth in chronic tissue ischemia.
  • this invention provides a method of enhancing blood flow0 in chronic tissue ischemia in a subject comprising administering to the subject a
  • composition comprising a therapeutically effective amount of dipyridamole or a pharmaceutically acceptable salt thereof for a time sufficient to enhance blood flow in the tissue.
  • dipyridamole is a primary drug in enhancing blood vessel growth in chronic tissue ischemia.
  • the methods can be carried out by administering to a subject (e.g., a human patient) in need of treatment a pharmaceutically acceptable composition comprising dipyridamole or a pharmaceutically acceptable salt thereof.
  • a pharmaceutically acceptable composition comprising dipyridamole or a pharmaceutically acceptable salt thereof.
  • the dipyridamole or a pharmaceutically acceptable salt thereof can be formulated in various0 ways and can include pharmaceutically acceptable carriers. For ease of reading, we will not repeat the phrase "or a pharmaceutically acceptable salt thereof on every occasion. It is to be understood that where dipyridamole can be used, a pharmaceutically acceptable salt of the compound may also be used.
  • the invention features physiologically acceptable compositions of dipyridamole and methods by which the compositions can be administered to a patient 5 diagnosed as having, for example, a chronic tissue ischemic disorder.
  • these methods can include the steps of a) identifying a subject (e.g., a human patient) who is experiencing or is likely to experience a chronic tissue ischemic disorder; and b) providing to the subject a composition including dipyridamole for a time and in an amount sufficient to treat the ischemia, by e.g. stimulating blood vessel growth in the ischemic tissue.
  • the dipyridamole results in the formation of one or more blood
  • domesticated animals including, for example cats, dogs, horses, cows and other domesticated animals can also be treated.
  • the dipyridamole is administered for a time and in an amount sufficient to result in the growth of blood vessels in the ischemic tissue.
  • the new blood0 vessel growth may stem from any process that results in revascularization of the ischemic tissue, for example, angiogenesis, or arteriogenesis, or a combination of angiogenesis and arteriogenesis.
  • New blood vessel growth may be monitored over the course of treatment either directly, using, for example imaging techniques such as contrast angiography, contrast pulse sequence (CPS) ultrasound imaging for high-resolution perfusion,
  • CPS contrast pulse sequence
  • the nitrite may be administered until a symptom of chronic ischemia, e.g., intermittent claudication, claudication during rest, neuropathy, or defective tissue wound healing, improves.
  • the assessment of clinical benefit may entail comparison of the ischemic tissue with the corresponding non-ischemic tissue.
  • Choice of specific clinical endpoints may depend, in0 part, upon the nature of the underlying medical condition, e.g., cessation or amelioration of intermittent claudication may be useful for patients with peripheral artery disease or diabetes; healing of skin ulcers may be useful for patients with defective wound healing, and relief from gastrointestinal pain, diarrhea and constipation may be useful for patient suffering from bowel ischemia.
  • the amount of dipyridamole per dose can vary.
  • a subject can receive from about 0.05 mg/kg up to about 5000 mg/kg., e.g., about 0.05, 1, 5, 10, 25, 50, 5 100, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1250, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 mg/kg.
  • a subject can receive up to or up to about 165 mg/kg, 16.5 mg/kg, or 8.25 mg/kg.
  • dipyridamole in an amount such that the systemic concentration does not exceed 24.0 ⁇ (i.e., the dipyridamole is administered in a dose sufficient to produce o a systemic concentration of dipyridamole in the subject that does not exceed 24.0 ⁇ is preferred).
  • the dipyridamole can be administered in an amount such that the systemic concentration does not exceed 0.0005 ⁇ , 0.001 ⁇ , 0.002 ⁇ , 0.003 ⁇ , 0.004 ⁇ , 0.005 ⁇ , 0.01 ⁇ , 0.02 ⁇ , 0.03 ⁇ , 0.04 ⁇ , 0.05 ⁇ , 0.1 ⁇ , 0.15 ⁇ , 0.2 ⁇ , 0.25 ⁇ , 0.3 ⁇ , 0.35 ⁇ , 0.4 ⁇ , 0.45 ⁇ , 0.5 ⁇ , 0.55 ⁇ , 0.6 ⁇ , 1.2 ⁇ ,5 2.4 ⁇ , 3.6 ⁇ , 4.8 ⁇ , 6.0 ⁇ , 12.0 ⁇ , and 24.0 ⁇ .
  • exemplary dosages can produce a systemic concentration of nitrite in the subject of up to or up to about 2.4 ⁇ , 0.96 ⁇ , or 0.24 ⁇ .
  • the preferred therapeutic plasma levels for dipyridamole are in the range of about 0.8 ⁇ g/mL to about 1.5 ⁇ g/mL.
  • the frequency of treatment may also vary.
  • the subject can be treated one or more0 times per day (e.g., once, twice, three, four, five, or six times per day) or every so-many hours (e.g., about every 2, 4, 6, 8, 12, or 24 hours).
  • the time course of treatment may be of varying duration, e.g., for two, three, four, five, six, seven, eight, nine, ten or more days.
  • the treatment can be twice a day for three days, twice a day for seven days, twice a day for ten days.
  • Treatment cycles can be repeated at intervals, for example5 weekly, bimonthly or monthly, which are separated by periods in which no treatment is given.
  • the treatment can be a single treatment or can last as long as the life span of the subject (e.g., many years).
  • this invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of dipyridamole or a pharmaceutically0 acceptable salt thereof and a pharmaceutically acceptable carrier for treating chronic
  • this invention provides a pharmaceutical composition comprising a therapeutically effective amount of dipyridamole or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier for enhancing blood vessel growth in chronic tissue ischemia.
  • compositions can be administered to a subject in a variety of ways.
  • the compositions can be administered transdermally or injected (infused) intravenously, subcutaneously, sublingually, intracranially, intramuscularly,
  • compositions are also within the scope of the present invention.
  • the treatment regime can vary depending upon various factors o typically considered by one of ordinary skill in the art. These factors include the route of administration, the nature of the formulation, the nature of the patient's illness, the subject's size, weight, surface area, age, gender, other drugs being administered to the patient, and the judgment of the attending physician.
  • the compositions can be administered along with or in addition to other treatments for chronic tissue ischemia,5 e.g., drug therapy, immunotherapy, or surgery (e.g., aspirin therapy, statin therapy, or antihypertensive therapy).
  • sustained release formulations can be used.
  • drug depots or transdermal formulations would permit constant infusion of a therapeutically effective0 amount of dipyridamole systemically into the patient.
  • Disorders amenable to the methods of the invention can include any disorder that presents with chronic ischemia.
  • Conditions that result in chronic tissue ischemia due to a narrowing or blockage of an artery include but are not limited to, for example, atherosclerosis, arteriosclerosis, acute coronary syndrome, coronary artery5 disease (CAD), bowel ischemia and peripheral artery diseases.
  • chronic tissue ischemia that stems from a wound, e.g. a traumatic injury or a surgical procedure.
  • this invention provides a method for increasing PKA dependent eNOS phosphorylation with a concomitant increase in NO/nitrite in a patient0 which method comprises administering to said patient an effective amount of
  • FIGURES 1 A-C Panel A illustrates plasma levels of dipyridamole from an escalating dosing regimen of dipyridamole b.i.d. over 4 days.
  • Panel B reports plasma levels of dipyridamole from a single dosing regimen of 100 mg/kg dipyridamole b.i.d. over 4 days.
  • Panel C demonstrates a continuous dosing regimen of dipyridamole 200 mg/kg b.i.d. that achieves therapeutic plasma levels (0.8-1.2 ⁇ g/ml) by day 3.
  • n 10 mice per time point.
  • Panels B and C show day 7 CD31 and DAPI staining of ischemic tissue from acid water and dipyridamole treated mice, respectively.
  • Panels D and E illustrate day 7 CD31, Ki67, and DAPI staining of ischemic tissue from acid water and dipyridamole treated mice, respectively. Bar equals 100 ⁇ .
  • Panel F reports the vascular density in non-ischemic and ischemic tissue from acid water control or dipyridamole treated mice at day 7 per 500 ⁇ 2.
  • FIGURES 3 A-D Panels A and B show microfil vascular casting of the femoral artery and profunda femoris architecture at day 3 in acid water control non-ischemic and ischemic limbs, respectively.
  • Panels C and D illustrate microfil vascular casting of the femoral artery and profunda femoris architecture at day 3 in dipyridamole treated nonischemic and ischemic limbs respectively.
  • Arrows highlight microfil perfusion through adductor muscle collateral arteries depicting the results of an analysis of the effect of various doses of chronic sodium nitrate treatment on ischemic hind-limb blood flow.
  • FIGURES 4 A-D Panel A reports the number of branches from first order arteries at day 3 in both acid water control and dipyridamole non-ischemic and ischemic tissues.
  • Panel B shows the number of branches from first order arteries at day 5 in both acid water control and dipyridamole non-ischemic and ischemic tissues.
  • Panels C and D illustrate the distance between arterial branches in both acid water control and
  • mice per cohort.
  • Panel B illustrates the effect of dipyridamole therapy on vascular density in non-ischemic and ischemic tissue from wild o type control, wild type dipyridamole treated, and eNOS KO dipyridamole treated mice.
  • FIGURES 7 A-E shows that dipyridamole therapy significantly increases nonischemic and ischemic tissue nitrite levels at day 7 compared to acid water control.
  • FIGURE 8 depicts the results of a western blot performed for total eNOS and phospho-eNOS Serl l76 from vehicle control (C), dipyridamole treated (D), and
  • FIG. 9 Dipyridamole therapy restores diabetic ischemic hind limb blood flow o as measured by laser Doppler. Dipyridamole treatment (200 mg/kg) significantly
  • n 8 per cohort, *p ⁇ 0.05.
  • FIGS 10 A-D Dipyridamole therapy promotes ischemic angiogenesis in the diabetic hind limb.
  • Panels A and B illustrate CD31 (endothelial stain; red), Ki67
  • Panels C and D show CD31 (endothelial stain; red), Ki67 (proliferating cell stain; green), and DAPI (nuclear stain; blue) staining of non-ischemic and ischemic gastrocnemius tissue at day 7 from dipyridamole treated mice. Images are representative of 30 sections per tissue0 and treatment.
  • Figures 11 A-B Quantitative measurement of dipyridamole mediated vascular density and cell proliferation.
  • Panel A reports the quantitative measurement of vascular density (CD31:DAPI ratio) from images of ischemic (Isch) and non-ischemic (NI) vehicle control and dipyridamole treated diabetic mice at day 7.
  • Panel B shows the quantitative5 measurement of cell proliferation (Ki67:DAPI) ratio from images of Isch and NI vehicle control and dipyridamole treated diabetic mice at day 7.
  • n 20 per cohort, *p ⁇ 0.05.
  • Panel A illustrates a representative western blot of total eNOS between vehicle control and dipyridamole tissues compared to GAPDH.
  • Panel B shows a representative phospho-eNOS Serl 177 western blot between vehicle control and dipyridamole treatments compared to GAPDH.
  • Panel C reports western blot
  • FIG 14 A-B Dipyridamole therapy decreases diabetic tissue superoxide formation and protein oxidation.
  • Panel A reports tissue superoxide production in ischemic (Isch) and non-ischemic (NI) limbs of dipyridamole and vehicle control treated diabetic mice.
  • Peripheral Arterial Disease is a marker of systemic atherosclerosis which affects millions of people worldwide and is associated with significant morbidity and mortality (1-3).
  • Critical Limb Ischemia represents the end stage of PAD with severe obstruction of blood flow resulting in ischemic rest pain, ulcers, significant risk of limb loss, and is a source of significant socioeconomic costs.
  • therapeutic angiogenesis could have significant impact in the care of patients with PAD and CLI by preventing further tissue dysfunction.
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • eNOS endothelial nitric oxide synthase
  • NO nitric oxide
  • Various anti-platelet agents e.g. dipyridamole, clopidogrel, and others have shown some clinical efficacy in managing peripheral artery disease with the speculation that these agents could alter blood vessel growth and function (6, 7).
  • Nitric oxide and its metabolites have been implicated as key regulators of angiogenesis with several cellular mechanisms including but not limited to increased endothelial cell motility, increased endothelial cell proliferation and survival, and increased activity of signaling pathways implicated in angiogenesis (8, 9, 10).
  • NO donors bioconvertable metabolites (nitrite anion), and eNOS activators (e.g. statins or VEGFA) have been reported to promote angiogenesis and/or arteriogenesis in models of chronic hind limb ischemia (8, 11, 5).
  • pharmacologic potentiation of NO metabolic pathways could have significant potential for augmenting therapeutic angiogenesis during chronic ischemic tissue disorders.
  • Dipyridamole is a conventionally used anti-platelet agent primarily used for the secondary prevention of cerebrovascular disease. Dipyridamole has beneficial effects beyond platelet inhibition, including antithrombotic, anti-inflammatory, anti-proliferative, thrombolytic, and antioxidative properties. Dipyridamole has been shown to inhibit phosphodiesterases and potentiate the NO system. Moreover, dipyridamole being an adenosine uptake inhibitor increases local extracellular concentrations of adenosine which has been implicated in preconditioning and in stimulating VEGF production (12, 13, 14). However, the utility of dipyridamole for therapeutic angiogenesis has not been evaluated under chronic ischemic conditions.
  • This invention is directed to the discovery that dipyridamole is efficacious in ischemia-induced angiogenesis using the mouse hind-limb ischemia model.
  • dipyridamole rapidly restores ischemic tissue blood flow and stimulates angiogenesis through a Protein Kinase A (PKA) dependent eNOS pathway.
  • PKA Protein Kinase A
  • Dipyridamole is an antithrombotic agent, primarily used for secondary prevention of cerebrovascular disease in combination with aspirin (24). Although dipyridamole was originally intended as an anti-anginal agent, there has been no conclusive evidence to support its routine use in the care of patients with coronary artery disease (25).
  • dipyridamole could possibly enhance coronary collateral growth (31-33) and angiogenesis (34-36), yet the utility of dipyridamole as a therapeutic angiogenesis agent during chronic ischemia remains unknown.
  • the beneficial effects of the drug have been suggested to be secondary to its vasodilation or anti-platelet 5 functions with the use of dipyridamole for chronic tissue ischemia remaining untested.
  • This invention is directed to the discovery that dipyridamole hastens ischemic tissue reperfusion by augmenting ischemic tissue vascular density and collateral arteriolar function through a PKA dependent eNOS pathway.
  • dipyridamole therapy increases PKA dependent eNOS phosphorylation thereby increasing NO/nitrite o production to preserve endothelial cell density and proliferation.
  • dipyridamole enhanced utilization of the NO/nitrite endocrine system in non-ischemic tissue revealing a novel method of preconditioning which affects distant organ function.
  • this invention provides a method for increasing PKA dependent eNOS phosphorylation with a concomitant increase in NO/nitrite in a patient 5 which method comprises administering to said patient an effective amount of
  • ischemic tissue perfusion not only requires increased vascular density through angiogenesis but also involves changes in conduit vessel number or function through enhanced arteriogenesis activity. While dipyridamole is well known to0 stimulation vasodilation, the effect of dipyridamole on ischemic tissue arteriogenesis is less clear. Interestingly, dipyridamole therapy enhances collateral arteriolar branching and decreased the distance between branches in ischemic limbs from dipyridamole treated mice. Moreover, dipyridamole appeared to augment Microfil perfusion capability in both ischemic and non-ischemic limbs compared to vehicle control treated mice. These data5 suggest that dipyridamole treatment likely augments ischemic tissue blood flow by
  • Dipyridamole is well appreciated to inhibit adenosine uptake and adenosine deaminase, thus increasing the extracellular concentration of adenosine.
  • Adenosine classically modulates intracellular cAMP levels via activating adenylate cyclase activity0 and inhibiting phosphodiesterase activity, and has also been appreciated to promote
  • dipyridamole also exhibits phosphodiesterase inhibitory action toward PDE-5 (40), thus increasing cGMP levels that could play a role in ischemic angiogenesis involving a PKG dependent mechanism independent of NO/NOS activity as we have previously reported (16). Given the fact that dipyridamole induced augmentation of ischemic tissue vascular density is NO dependent and mediated by PKA, it is unlikely 5 that dipyridamole augmentation of intracellular cGMP plays a dominant role in this
  • dipyridamole induction of cGMP formation could contribute to ischemic tissue perfusion as both PKA inhibitor and NO scavenger treatments did not completely block the effects of dipyridamole on ischemic tissue blood flow.
  • dipyridamole therapy significantly increased systemic and tissue levels of nitrite compared to control mice. This observation is consistent with our discovery that augmenting tissue nitrite levels significantly enhances ischemic
  • dipyridamole0 is able to pharmacologically enhance the NO/nitrite endocrine system in non-target tissue that has significant effects on distant organs.
  • This invention is predicated, in part, on the finding that dipyridamole significantly enhances ischemia induced angiogenesis and rapidly restores tissue blood flow during0 chronic hind-limb ischemia through a PKA dependent NO pathway.
  • peripheral artery disease is a chronic arterial occlusive o disease of the extremities caused by atherosclerosis, characterized by high cardiovascular morbidity and mortality [46] .
  • Patients with type 2 diabetes mellitus (DM) are frequently afflicted with PAD and DM is associated with a 2-4 fold increase in the incidence of PAD compared to non-diabetics [47].
  • type 2 DM causes the amplification of the atherosclerotic process, endothelial cell dysfunction, glycosylation of extracellular5 matrix proteins and vascular denervation.
  • Therapeutic angiogenesis remains an attractive treatment modality for chronic ischemic disorders including PAD [49].
  • the establishment of patent blood vessel networks is a complex process that requires several factors and signaling pathways to stimulate vessel sprouting and remodeling of established vascular structures [50].
  • VEGF vascular endothelial growth factor
  • basic fibroblast growth factor vascular endothelial growth factor
  • bFGF vascular endothelial nitric oxide synthase
  • eNOS endothelial nitric oxide synthase
  • Dipyridamole is an antithrombotic and vasodilating agent effective in secondary prevention of cardiovascular disease [53]. Dipyridamole therapy could improve tissue perfusion through several mechanisms including antiplatelet effects, acting as a vasodilatory agent potentiating NO bioavailability, or possibly through its antioxidant properties via scavenging peroxyl radicals [54, 55]. Reports have suggested that dipyridamole could be effective in increasing capillary density in both a rat and rabbit heart following myocardial ischemia and augment collateral vessel growth via adenosine action in local ischemic tissues [56, 57].
  • dipyridamole augments ischemia-induced arteriogenesis through an endocrine nitric oxide-dependent pathway [55].
  • diabetes impairs both nitric oxide synthase activity and NO bioavailability leading to vascular dysfunction with concomitant enhanced oxidative stress [58-60]. Therefore, we tested the hypothesis that dipyridamole therapy is beneficial for diabetic chronic tissue ischemia and report here that
  • dipyridamole robustly augments ischemic tissue reperfusion and angiogenic activity in diabetic mice by decreasing oxidative stress and replenishing NO bioavailability. These data suggest that dipyridamole may be a useful agent for alleviating diabetic vascular dysfunction and tissue ischemia.
  • Dipyridamole has historically been used as an anti-platelet agent that is currently formulated with aspirin for hemostasis management after initial ischemic stroke, which confers significant protection against recurrent stroke [69] . Dipyridamole has also been suggested to augment coronary collateral perfusion and development which could augment cardiac function after ischemia/reperfusion injury [57, 70, 71]. Moreover, we have recently reported that dipyridamole therapy stimulates arteriogenesis during chronic hind limb ischemia involving the endocrine NO/nitrite system [55]. Despite these compelling observations, no studies have been performed directly examining the effect of dipyridamole on reperfusion of diabetic chronic hind limb ischemia. Here we
  • Diabetic vascular disease is known to encompass defective angiogenic responses during wound healing and tissue ischemia [72]. This defective angiogenesis response has been attributed to several pathophysiological mediators such as hyperglycemia, dyslipidemia, oxidative stress, and decreased NO bioavailability which all contributes to endothelial dysfunction [73].
  • dipyridamole therapy selectively enhanced vascular density in ischemic tissue in the diabetic setting demonstrating that endothelial cell dysfunction associated with the Db/Db murine model of type 2 diabetes could be 5 corrected.
  • dipyridamole preferentially increased endothelial cell proliferation in ischemic tissues along with a moderate but significant increase in cell proliferation in non-ischemic tissue.
  • Dipyridamole could be mitigating oxidative stress through several possible mechanisms including but not limited to direct anti-oxidant effects, decreased generation of reactive oxygen species from various sources, induction of different antioxidant enzymes or pathways, or a combination of these possibilities. While future studies are clearly needed to address these possibilities, our data are the first to identify dipyridamole0 reduction of oxidative stress as a primary protective mechanism of ischemia induced angiogenesis during diabetes.
  • Dipyridamole therapy not only preserved NO bioavailability and mitigated oxidative stress, but surprisingly affected cholesterol and blood glucose levels.
  • the effect on cholesterol is consistent with a previous report from Garcia-Fuentes et al. demonstrating that coconut-oil induced hypercholesterolemia was blunted with dipyridamole therapy; however, our study is the first to demonstrate this effect in a model of diabetes [81]. Few studies have revealed that dipyridamole therapy alters blood
  • dipyridamole effectively restores ischemic tissue reperfusion, angiogenic activity, and decreases oxidant stress in a diabetic model of femoral artery ligation emulating PAD.
  • dipyridamole has a5 good clinical safety profile along with well understood pharmacokinetics.
  • dipyridamole therapy for diabetic PAD represents an attractive clinical approach to stimulate ischemic angiogenesis and restore tissue blood flow.
  • dipyridamole therapy represents a viable approach to alleviating chronic peripheral tissue ischemia associated with type 2 diabetes.
  • dipyridamole treatment restores reperfusion of chronic hind limb ischemia in the murine B6.BKS-Lepr db/db diabetic model. Dipyridamole therapy quickly rectified ischemic hind limb blood flow to near pre-ligation levels within three days after starting therapy.
  • dipyridamole therapy significantly decreased tissue total nitric oxide metabolite levels (NOx) which were not associated with changes in eNOS expression or phosphorylation.
  • dipyridamole therapy significantly decreased ischemic tissue superoxide and protein carbonyl levels identifying a dominant antioxidant mechanistic response.
  • Dipyridamole therapy also moderately reduced diabetic hyperglycemia and attenuated dyslipidemia0 development over time.
  • compositions and methods for treatment of chronic tissue ischemia can be applied to, and are expected to benefit subjects having any of a variety of medical conditions that can give rise to chronic tissue ischemia.
  • the methods are based, inter alia, on the inventor' s discovery that
  • chronic tissue ischemia refers to a tissue condition comprising an ongoing restriction of blood supply to the tissue.
  • Chronic tissue ischemia is associated with a wide range of medical conditions that result in partial, substantially complete or complete reduction of blood flow to a body part or tissue comprising a body0 part and may be the result of disease, injury, or of an unknown cause, and may be
  • angiogenesis refers to the budding of new capillary branches from existing blood vessels.
  • anteriorgenesis refers to the growth of preexisting arteriolar connections into true collateral arteries.
  • systemic concentration refers to a concentration that is therapeutically effective, but has tolerable or no toxicity.
  • the term "therapeutically effective amount” refers to that amount that is sufficient to effect treatment, when administered to a subject in need of such treatment.
  • the therapeutically effective amount will vary depending upon the specific o activity of the therapeutic agent being used, the severity of the patient's disease state, and the age, physical condition, existence of other disease states, and nutritional status of the patient.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and5 absorption delaying agents and the like. The use of such media and agents for
  • compositions are well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the term "pharmaceutically acceptable salt” refers to salts of dipyridamole that have pharmacologically acceptable properties of dipyridamole.
  • Examples include, but are not limited to, pharmacologically acceptable reaction products of dipyridamole and inorganic or organic acids.
  • Suitable acids which are well known to those skilled in the art, include hydrochloric acid, tartaric acid, citric acid, and the like.5
  • Pharmaceutically acceptable salts can also be prepared from inorganic and organic bases.
  • Salts derived from inorganic bases include by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts.
  • the terms “treating,” “treatment” and the like are used herein to mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be0 prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect attributable to the disorder.
  • Treating also covers any treatment of a disorder in a subject, and includes: (a) preventing a disorder from occurring in a subject that may be predisposed to a disorder, 5 but may have not yet been diagnosed as having it; (b) inhibiting a disorder, i.e., arresting its development; or (c) relieving or ameliorating the disorder.
  • compositions and methods are intended to mean that the compositions and methods include the recited elements, but do not exclude others.
  • the term "primary drug” is intended to mean that the drug is o approved by the appropriate regulatory authority for the indication stated.
  • dipyridamole can be used in conjunction with other drugs, when referred to as a "primary drug," it is a drug of first choice in treating chronic tissue ischemia and is approved for the indication.
  • dipyridamole is used as the only drug in treating chronic tissue ischemia.
  • the patient is not treated with5 any other drug prior to treatment with dipyridamole for treating chronic tissue ischemia.
  • dipyridamole5 is commercially available.
  • the compounds of the invention can be formulated in accordance with their use.
  • the compounds can be formulated within compositions for application to cells in tissue culture or for administration to a patient.
  • any of the0 present compounds can be administered in the form of pharmaceutical compositions.
  • compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including 5 by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or
  • Methods for ocular delivery can include topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by balloon catheter or ophthalmic inserts surgically placed in the conjunctival sac.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular o injection or infusion; or intracranial, e.g., intrathecal or intraventricular administration.
  • Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump.
  • Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, and the like.
  • compositions which contain, as the active ingredient, one or more of the compounds described herein in combination with one or more pharmaceutically acceptable carriers.
  • the active ingredient is typically mixed with an excipient, diluted by an
  • excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container.
  • a carrier in the form of, for example, a capsule, sachet, paper, or other container.
  • the excipient serves as a diluent, it can be a solid, semisolid, or liquid material (e.g., normal saline), which acts as a vehicle, carrier or medium for the active ingredient.
  • compositions can be in the form of tablets, pills,5 powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10 % by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
  • the type of diluent can vary depending upon the intended route of administration. The resulting
  • compositions can include additional agents, such as preservatives.
  • the compounds may also be applied to a surface of a device (e.g., a catheter) or contained within a pump, patch, or other drug delivery device.
  • the therapeutic agents of the invention can be administered alone, or in a mixture, in the presence of a pharmaceutically acceptable excipient or carrier (e.g., physiological saline).
  • a pharmaceutically acceptable excipient or carrier e.g., physiological saline.
  • the excipient or carrier is selected on the basis of the mode and route of administration. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in
  • the active compound in preparing a formulation, can be milled to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it can be milled to a particle size of less than 200 o mesh. If the active compound is substantially water soluble, the particle size can be
  • excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium5 silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose.
  • the formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents.
  • the pharmaceutical compositions can be formulated so as0 to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
  • compositions can be formulated in a unit dosage form, each dosage containing, for example, from about 0.1 mg to about 1000 mg.
  • unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects5 and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition
  • the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
  • This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 1000 mg of the active ingredient of the present invention.
  • the tablets or pills of the present invention can be coated or otherwise
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, acetyl alcohol, and cellulose acetate.
  • liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein and/or known in the art.
  • the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
  • Compositions in can be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device can be attached to a face masks tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices which deliver the formulation in an appropriate manner.
  • composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration.
  • dosage e.g., dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration.
  • chemical characteristics e.g., hydrophobicity
  • route of administration e.g., the routes of administration.
  • the compounds of the invention can be provided in an aqueous
  • physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral adminstration.
  • sustained release formulations can be used.
  • drug depots or transdermal formulations would permit constant infusion of a therapeutically effective amount of dipyridamole systemically into the patient.
  • sustained release (“SR”) refers to formulations or dosage units of this invention that slowly and continuously release the o active ingradient, dipyridamole, over a period of several hours after administration.
  • this invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of dipyridamole or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier for treating chronic tissue ischemia.
  • this invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of dipyridamole or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier for enhancing blood vessel growth in chronic tissue ischemia.
  • the pharmaceutical composition is in a unit dosage form.0
  • the unit dosage form comprises from about 0.1 mg to about 1000 mg dipyridamole.
  • the pharmaceutical composition further comprises one or more active ingredients.
  • the one or more active ingredients are selected from the group consisting of antihypertensives, anti-diabetic agents, statins, anti-5 platelet agents, antibodies, immune suppressants, anti-inflammatory agents, antibiotics, and chemo therapeutics.
  • a patient who has chronic tissue ischemia is a candidate for treatment with the pharmaceutically0 acceptable compositions comprising dipyridamole described herein. Treatment can
  • compositions of the invention are administered for 5 a time and in an amount sufficient to result in the growth of new blood vessels in the ischemic tissue.
  • new blood vessel growth refers all phases of the process of blood vessel formation, including the initial signaling events, cellular recruitment of endothelial cells, the formation and enlargement o of new vessels and connection of new vessels with pre-existing vessels.
  • the new blood vessel growth may stem from any process that results in revascularization or
  • vasculogenesis typically is used to describe the embryonic development of blood vessels from angioblasts.
  • Angiogeneisis is generally understood to be a post-natal physiologic process required for wound healing.
  • Angiogenesis generally encompasses the formation of new capillaries or capillary branches by sprouting, budding and intussusception from pre-existent capillaries.
  • Arteriogenesis i.e., the growth of preexisting arteriolar connections into true collateral arteries, is generally understood to encompass the formation of mature arteries0 from pre-existent interconnecting arterioles after an arterial occlusion. It shares some features with angiogenesis, but the pathways leading to it can differ, as do the final results: arteriogenesis is potentially able to fully replace an occluded artery whereas angiogenesis typically cannot.
  • Capillaries are tubes formed by endothelial cells which are supported by vascular pericytes.
  • Arteries and veins are tubes that consist of multiple layers: the intima, which is composed of endothelial cells, pericytes, and a basement0 membrane; the media, which is composed principally of smooth muscle cells and their extracellular matrix; and, in the largest vessels, the adventitia, which is composed principally of fibroblasts and their extracellular matrix.
  • this invention provides a method of enhancing blood vessel growth in chronic tissue ischemia in a subject comprising administering to the subject a composition comprising a therapeutically effective amount of dipyridamole or a pharmaceutically acceptable salt thereof for a time sufficient to enhance blood vessel 5 growth in the tissue.
  • the blood vessel growth is enhanced via angiogenesis, arteriogenesis, or a combination thereof.
  • this invention provides a method of enhancing blood flow in chronic tissue ischemia in a subject comprising administering to the subject a composition comprising a therapeutically effective amount of dipyridamole or a o pharmaceutically acceptable salt thereof for a time sufficient to enhance blood flow in the tissue.
  • this invention provides a method of enhancing blood vessel growth in chronic tissue ischemia, wherein the chronic tissue ischemia is associated with a disorder, trauma, or a congenital defect.
  • the5 disorder is selected from the group consisting of cardiovascular disease, peripheral artery disease, arteriosclerosis, atherosclerotic cardiovascular disease, congestive heart failure, critical limb ischemic disease, acute coronary syndrome, intermittent claudication, type 1 and type 2 diabetes, skin ulcers, peripheral neuropathy, inflammatory bowel disease, ulcerative colitis, Crohn's disease, intestinal ischemia, and chronic mesenteric ischemia.0
  • the disorder is diabetes.
  • the disorder is type 2 diabetes.
  • this invention provides a method of enhancing blood vessel growth in chronic tissue ischemia, wherein the chronic tissue ischemia is associated with a trauma selected from the group consisting of wound, burn, laceration,5 contusion, bone fracture, infection, and surgical procedure.
  • this invention provides a method of enhancing blood vessel growth in chronic tissue ischemia, wherein the chronic tissue ischemia is associated with a, congenital defect selected from the group consisting of hernia, cardiac defect, and gastrointestinal defect.
  • this invention provides a method of enhancing blood vessel growth in chronic tissue ischemia, wherein the chronic tissue ischemia is associated with a narrowing or blockage of an artery.
  • the narrowing or blockage of an artery is due to atherosclerosis, arteriosclerosis, acute coronary syndrome, coronary artery disease, bowel ischemia and peripheral artery diseases.
  • this invention provides a method of enhancing blood vessel growth in chronic tissue ischemia, wherein the chronic tissue ischemia stems from medical conditions that result in restriction of blood supply to the tissue.
  • the tissue is selected from the group consisting of muscle, smooth muscle, skeletal muscle, cardiac muscle, neuronal tissue, skin, mesechymal tissue, connective tissue, gastrointestinal tissue, bone, epithelial tissue, loose connective tissue, and fibrous connective tissue.
  • the therapeutically effective amount of dipyridamole is in the range of about 0.05 mg/kg to about 5000 mg/kg per dose. In a further embodiment, the therapeutically effective amount of dipyridamole is in the range of about 8.25 mg/kg to about 165 mg/kg per dose.
  • this invention provides a method of enhancing blood vessel growth in chronic tissue ischemia, wherein the dipyridamole in the subject does not exceed a systemic concentration of 24.0 ⁇ . In a further embodiment, the dipyridamole in the subject does not exceed a systemic concentration of 2.4 ⁇ .
  • the preferred therapeutic plasma levels for dipyridamole are in the range of about 0.8 ⁇ g/mL to about 1.5 ⁇ g/mL.
  • this invention provides a method of enhancing blood vessel growth in chronic tissue ischemia, wherein the dipyridamole is administered topically, orally, intravenously, subcutaneously, sublingually, intracranically,
  • intramuscularly intraperitoneally, or intrapulmonarily.
  • this invention provides a method of enhancing blood vessel growth in chronic tissue ischemia, wherein the method is used in conjunction with a remedy for treating chronic tissue ischemia.
  • the remedy is selected from a drug therapy, a surgery, an anti-inflammatory agent, an antibody, exercise, or a lifestyle change.
  • Multiple signaling pathways contribute to new blood vessel growth. At the center of these pathways is hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor composed of a constitutively expressed HIF-1 ⁇ subunit and an oxygen-regulated HIF-1 a subunit.
  • HIF-1 hypoxia-inducible factor 1
  • the HIF-1 a subunit is continually synthesized and degraded within adequately perfused cells ; under hypoxic conditions, the degradation of HIF-1 a is inhibited, leading to its accumulation and dimerization with HIF-1 ⁇ , DNA binding, recruitment of coactivators and transcriptional activation of target genes.
  • hypoxia is a physiological stimulus that induces cells to produce angiogenic cytokines such as Vascular Endothelial Growth Factor (VEGF).
  • VEGF Vascular Endothelial Growth Factor
  • These secreted proteins bind to their cognate receptors (VEGFRs) on endothelial cells and activate signal transduction pathways that stimulate cells to undergo sprouting angiogenesis.
  • VEGF causes a massive signaling cascade in endothelial cells.
  • VEGF receptor-2 (VEGFR-2) starts a tyrosine kinase signaling cascade that stimulates the production of factors that variously stimulate vessel permeability (eNOS, producing NO), proliferation/survival (bFGF), migration (ICAMs/VCAMs/MMPs) and finally differentiation into mature blood vessels.
  • eNOS vessel permeability
  • bFGF proliferation/survival
  • ICMs/VCAMs/MMPs migration into mature blood vessels.
  • FGF growth factor
  • VEGFR and NRP-1 which can integrate survival signals
  • Angl and Tie2 which can stabilize vessels
  • PDGF BB-homodimer
  • PDGFR PDGFR
  • TGF- ⁇ endoglin and TGF- ⁇ receptors, which can increase extracellular matrix production
  • MCP- 1 Integrins ⁇ 3, ⁇ 5 and ⁇ 5 ⁇ 1 which can bind matrix macromolecules and proteinases
  • VE-cadherin and CD31 ephrin, which can determine formation of arteries or veins
  • plasminogen activators which can remodel extracellular matrix and release and activate growth factors
  • NOS and COX-2 AC133, which can regulate angioblast differentiation
  • Idl/Id3 which can regulate endothalial transdifferentiation.
  • angiogenic cytokines such as VEGF, PLGF and stromal-derived growth factor 1 (SDF-1) stimulate the mobilization and recruitment of a heterogeneous population of angiogenic cells from the bone marrow and other tissues to sites of angiogenesis and arteriogenesis.
  • Cell types that can participate in these responses are known as circulating angiogenic cells and include endothelial progenitor cells, myeloid, mesenchymal and hematopoietic progenitor cells.
  • Arteriogenesis seems to be triggered mainly by fluid shear stress, which is induced by the altered blood flow conditions after an arterial occlusion.
  • Arteriogenesis involves endothelial cell activation, basal membrane degradation, leukocyte invasion, proliferation of vascular cells, neointima formation, remodeling of the extracellular matrix and cytokine participation. More specifically, mechanical stresses cause endothelial cells to produce chemical facilitators that begin the process of increasing diameter.
  • An increase in shear stress causes an increase in the number of monocyte chemoattractant protein- 1 (MCP-1) molecules expressed on the surface of vessel walls as well as increased levels of TNF-a, bFGF, and MMP.
  • MCP-1 increases the tendency of monocytes to attach to the cell wall.
  • TNF-a provides an inflammatory environment for the cells to develop while bFGF helps induce mitosis in the endothelial cells.
  • MMPs remodel the space around the artery to provide room for the expansion of the new collateral artery.
  • Nitric oxide has been shown to positively regulate endothelial cell responses in both angiogenesis and arteriogenesis. NO increases the expression of various angiogenic factors, including VEGF, which, together with other mediators, increases NO levels via a positive feedback mechanism. In addition to stimulating the growth of nascent and immature blood vessels consisting of only fragile endothelial cells, NO recruits perivascular mural cells, which stabilize vessels and allow them to become fully functioning conduits. NO can also protect tissues against ischemic damage by slowing cellular respiration. NO has been shown to modulate several endothelial cell signaling pathways for example, Erkl/2 and PKC.
  • eNOS endothelial nitric oxide synthase
  • NO soluble guanylate cyclase
  • NO 3 nitrate
  • nitrite and nitrate are involved in regulating production of NO from NOS independent pathways.
  • Inorganic nitrite can undergo a one electron reduction back to NO through various mechanisms with oxygen-binding heme proteins (hemoglobin and myoglobin), deoxyhemoglobin, deoxymyoglobin, xanthine oxidoreductase, endothelial nitric oxide synthase, acidic disproportionation, and members of the mitochondrial electron transport chain, e.g., mitochondrial heme proteins all being potential electron donors.
  • the ability of nitrite to be reduced back to NO classifies it as a unique NO donor under biological conditions, e.g., tissue ischemia, in which many of these potential reducing agents are active.
  • NO interacts with several intracellular targets to form various NO-containing species including S-nitrosothiols, C- or N-S-nitroso compounds, and nitrosylheme adducts. Moreover, these nitroso-products may serve as a biological reservoir for NO, which can be liberated under certain conditions.
  • this invention provides a method of enhancing blood vessel growth in chronic tissue ischemia in a subject comprising administering to the subject a composition comprising a therapeutically effective amount of dipyridamole or a pharmaceutically acceptable salt thereof for a time sufficient to enhance blood vessel growth in the tissue, wherein the blood vessel growth is enhanced via anginogenesis through an endocrine Nitrite/NO Pathway.
  • the present methods for treating chronic tissue ischemia are carried out by administering dipyridamole for a time and in an amount sufficient to result in the growth of new blood vessels in the ischemic tissue.
  • compositions can vary depending on, for example, what is being administered, the state of the patient, and the manner of administration.
  • compositions can be administered to a patient suffering from chronic tissue ischemia in an amount sufficient to relieve or least partially relieve the symptoms of chronic tissue ischemia and its complications.
  • the dosage is likely to depend on such variables as the type and extent of progression of the chronic tissue ischemia, the severity of the chronic tissue ischemia, the age, weight and general condition of the particular patient, the relative biological efficacy of the composition selected, formulation of the excipient, the route of administration, and the judgment of the attending clinician.
  • Effective doses can be extrapolated from dose- response curves derived from in vitro or animal model test system.
  • An effective dose is a 5 dose that produces a desirable clinical outcome by, for example, improving a sign or symptom of chronic tissue ischemia or slowing its progression.
  • this invention provides a method of treating chronic tissue ischemia in a subject comprising administering to the subject a composition comprising a therapeutically effective amount of dipyridamole or a pharmaceutically acceptable salt o thereof, wherein the dipyridamole or the pharmaceutically acceptable salt is administered topically, orally, intravenously, subcutaneously, sublingually, intracranically,
  • intramuscularly intraperitoneally, or intrapulmonarily.
  • this invention provides a method of enhancing blood vessel growth in chronic tissue ischemia in a subject comprising administering to the5 subject a composition comprising a therapeutically effective amount of dipyridamole or a pharmaceutically acceptable salt thereof for a time sufficient to enhance blood vessel growth in the tissue, wherein the dipyridamole or the pharmaceutically acceptable salt is administered topically, orally, intravenously, subcutaneously, sublingually,
  • Method of the invention are applicable to any of a wide range of medical conditions which have as their underlying feature a persistent reduction of or partial or complete blockage of blood flow to a tissue or organ.
  • the methods are applicable to treatment of chronic tissue ischemia associated with a disorder, with a trauma or an5 environmental stress.
  • the reduction in blood flow to a tissue can be, for example, the result of a progressive blockage of an artery due to hardening and/or loss of elasticity due to an atheromatous plaque or the presence of a clot.
  • Reduction of blood flow to a tissue can also be the result of an environmental insult, for example, a traumatic injury or surgical procedure that interrupts the blood flow to a tissue or organ.
  • the methods are applicable to treatment of chronic tissue ischemia associated with a disorder, with a trauma or an5 environmental stress.
  • the reduction in blood flow to a tissue can be, for example, the result of a progressive blockage of an artery due to hardening and/or loss of elasticity due to an ather
  • this invention provides a method of treating ischemia in a subject comprising administering to the subject a composition comprising a
  • dipyridamole or a pharmaceutically acceptable salt thereof, wherein dipyridamole in the primary drug in treating ischemia.
  • this invention provides a method of treating chronic tissue ischemia in a subject comprising administering to the subject a composition comprising a therapeutically effective amount of dipyridamole or a pharmaceutically acceptable salt o thereof.
  • dipyridamole in the primary drug in treating chronic tissue ischemia include, for example, cardiovascular disease, peripheral artery disease, arteriosclerosis, atherosclerotic cardiovascular disease, critical limb ischemic disease, acute coronary syndrome, intermittent claudication, diabetes, including type 1 and type 2 diabetes, skin ulcers,5 peripheral neuropathy, inflammatory bowel disease, ulcerative colitis, Crohn's disease, intestinal ischemia, and chronic mesenteric ischemia.
  • the methods of the invention are also applicable to chronic tissue ischemia associated with a trauma, for example, a traumatic injury such as a wound, laceration, burn, contusion, bone fracture or chronic infection.
  • tissue injuries sustained as part of any0 surgical procedure for example, endarterectomy.
  • Procedures involving tissue or organ transplantation are within the scope of the invention. Examples include vascular bypass grafts, heart, liver, lung, pancreatic islet cell transplantation as well as transplantation of tissues generated ex vivo for implantation in a host.
  • the methods of the invention are also useful for treating a chronic ischemic condition brought about by exposure to an5 environmental insult, for example, chronic exposure to hypoxic conditions e.g., high altitude, or sustained aerobic exertion.
  • the chronic tissue ischemia is associated with a disorder, trauma, or a congenital defect.
  • the disorder is selected from the group consisting of cardiovascular disease, peripheral artery disease, arteriosclerosis,0 atherosclerotic cardiovascular disease, congestive heart failure, critical limb ischemic disease, acute coronary syndrome, intermittent claudication, type 1 and type 2 diabetes, skin ulcers, peripheral neuropathy, inflammatory bowel disease, ulcerative colitis, Crohn's disease, intestinal ischemia, and chronic mesenteric ischemia.
  • this invention provides a method of treating chronic tissue ischemia, wherein the chronic tissue ischemia is associated with a the trauma selected from the group consisting of wound, burn, laceration, contusion, bone fracture, infection, and surgical procedure.
  • this invention provides a method of treating chronic tissue ischemia, wherein the chronic tissue ischemia is associated with a congenital defect selected from the group consisting of hernia, cardiac defect, and gastrointestinal defect.
  • tissue types including, for example, muscle, smooth muscle, skeletal muscle, cardiac muscle, neuronal tissue, skin, mesechymal tissue, connective tissue, gastrointestinal tissue or bone.
  • Soft tissue such as epithelial tissue, e.g., simple squamous epithelia, stratified squamous epithelia, cuboidal epithelia, or columnar epithelia, loose connective tissue (also known as areolar connective tissue), fibrous connective tissue, such as tendons, which attach muscles to bone, and ligaments, which join bones together at the joints.
  • the methods of the invention can include the steps of identifying a subject (e.g., a human patient) who is experiencing or is likely to experience chronic tissue ischemia. Since chronic tissue ischemia can result from a wide range of medical conditions all of which have as their underlying feature a persistent reduction of or partial or complete blockage of blood flow to a tissue, the specific signs and symptoms will vary depending upon factor or factors responsible for the reduction of blood flow.
  • a subject e.g., a human patient
  • chronic tissue ischemia can result from a wide range of medical conditions all of which have as their underlying feature a persistent reduction of or partial or complete blockage of blood flow to a tissue, the specific signs and symptoms will vary depending upon factor or factors responsible for the reduction of blood flow.
  • peripheral artery disease a form of peripheral vascular disease in which there is partial or total blockage of an artery, usually due to atherosclerosis in a vessel or vessels leading to a leg or arm, can include intermittent claudication, that is, fatigue, cramping, and pain in the hip, buttock, thigh, knee, shin, or upper foot during exertion that goes away with rest, claudication during rest, numbness, tingling, or coldness in the lower legs or feet, neuropathy, or defective tissue wound healing.
  • PAD in the lower limb is often associated with diabetes, particularly type 2 diabetes.
  • Arm artery disease is usually not due to atherosclerosis but to other conditions such as an autoimmune disease, a blood clot, radiation therapy, Raynaud's disease, repetitive motion, and trauma. Common symptoms when the arm is in motion include discomfort, heaviness, tiredness, cramping and finger pain. PAD can be diagnosed by performing one or more diagnostic tests including, for example, an ankle brachial index (ABI) test, angiography, ultrasound, or MRI analysis.
  • ABSI ankle brachial index
  • Myocardial ischemia can have few or no symptoms, although typically, it is associated with a symptom such as angina, pain, fatigue elevated blood pressure.
  • Diagnostic tests for myocardial ischemia include: angiography, resting, exercise, or ambulatory electrocardiograms; scintigraphic studies (radioactive heart scans);
  • this invention provides a method of treating chronic tissue ischemia, wherein the chronic tissue ischemia is associated with a narrowing or blockage of an artery.
  • the narrowing or blockage of an artery is due to atherosclerosis, arteriosclerosis, acute coronary syndrome, coronary artery disease, bowel ischemia and peripheral artery diseases.
  • the chronic tissue ischemia stems from medical conditions that result in restriction of blood supply to the tissue.
  • the tissue is selected from the group consisting of muscle, smooth muscle, skeletal muscle, cardiac muscle, neuronal tissue, skin, mesechymal tissue, connective tissue, gastrointestinal tissue, bone, epithelial tissue, loose connective tissue, and fibrous connective tissue.
  • this invention provides a method of treating chronic tissue ischemia, wherein the therapeutically effective amount of dipyridamole is in the range of about 0.05 mg/kg to about 5000 mg/kg per dose.
  • the therapeutically effective amount of dipyridamole is in the range of about 0.05 mg/kg to about 5000 mg/kg per dose.
  • dipyridamole is in the range of about 8.25 mg/kg to about 165 mg/kg per dose.
  • this invention provides a method of treating chronic tissue ischemia, wherein the dipyridamole in the subject does not exceed a systemic
  • the dipyridamole in the subject does not exceed a systemic concentration of 2.4 ⁇ .
  • the preferred therapeutic plasma levels for dipyridamole are in the range of about 0.8 ⁇ g/mL to about 1.5 ⁇ g/mL.
  • the method of the invention can also be used in conjunction with other remedies known in the art that are used to treat chronic tissue ischemia including, drug therapy, surgery, anti-inflammatory agents, antibodies, exercise, or lifestyle changes. The choice of specific treatment may vary and will depend upon the severity of the chronic tissue 5 ischemia, the subject's general health and the judgment of the attending clinician.
  • compositions can also be formulated in combination with one or more additional active ingredients, which can include any pharmaceutical agent such antihypertensives, anti-diabetic agents, statins, anti-platelet agents (clopidogrel and cilostazol), antibodies, immune suppressants, anti-inflammatory agents, antibiotics, chemotherapeutics, and the o like.
  • additional active ingredients can include any pharmaceutical agent such antihypertensives, anti-diabetic agents, statins, anti-platelet agents (clopidogrel and cilostazol), antibodies, immune suppressants, anti-inflammatory agents, antibiotics, chemotherapeutics, and the o like.
  • this invention provides a method of treating chronic tissue ischemia, wherein the method is used in conjunction with a remedy for treating chronic tissue ischemia.
  • the remedy is selected from a drug therapy, a surgery, an anti-inflammatory agent, an antibody, exercise, or a lifestyle change.
  • Example 1 General methods using dupyridamole for treating chronic tissue ischemia
  • mice C57BL/6J mice weighing 20-25 grams were used for all studies. The mice were bred and housed at the Association for Assessment and Accreditation(SDf Laboratory Animal Care, International accredited Louisiana State University Health Science Center-Shreveport animal resource facility and maintained in accordance with the National Research Council's Guide for Care and Use of Laboratory Animals. All animal studies were approved by the institutional animal care and use committee and performed according to the US National Institutes of Health guidelines.
  • the Harvard eNOS _/ ⁇ mouseStrain (8 backcross generations) was breed and housed at our institution. Pharmaceutical grade dipyridamole was provided by from Boehringer-Ingelheim, Germany. Total and phospho-eNOS (Serine 1176) antibodies were purchased from Cell Signaling Technology.
  • the PKA inhibitor KT5720 was purchased from Calbiochem. All other reagents were obtained from Sigma Chemicals.
  • Plasma dipyridamole levels Blood samples were collected by retroorbital venepuncture into 7.5% EDTA anticoagulant solution. Specimens were centrifuged at 3,500 RPM for 15 minutes to obtain the plasma layer. Plasma levels of dipyridamole and its glucuronide were measured by HPLC with fluorescence detection (360 ex; 460 em) as previously reported (15).
  • Murine Chronic Hind-Limb Ischemia Model Chronic hind-limb ischemia was 5 induced in the C57BL/6J mice by ligating and transecting the left common femoral artery proximal to the origin of the profunda artery and its collateral branches as we have previously reported (11, 16). Mice were anesthetized with intraperitoneal injection of ketamine (100 mg/kg) and xylazine (8mg/kg). After measuring hind limb blood flows using laser tissue Doppler (see below), aseptic surgery was performed by a linear incision o at the left groin and exposing the left common femoral femoral artery which was
  • Murine Ischemia Arteriogenesis Model Ischemic arteriogenesis was evaluated as5 we have previously reported (11, 16). Briefly, acid water or dipyridamole treated mice were anesthetized and the femoral artery distal to the femoral profundus was ligated with 6-0 silk.
  • Laser Doppler Measurement of Tissue Blood Flow The Vasamedics Laserflo BPM2 deep tissue penetrating laser doppler device was used to measure hind limb blood flows. The tip of the laser probe was placed over the medial gastrocnemius muscles of0 both the hind limbs of the mice. Areas of blood vessels visible through the skin were avoided to ensure readings indicative of tissue blood flow in the muscle. Readings were recorded in ml of blood flow per 100 g tissue per min. Laser doppler blood flow readings were measured from the middle of the gastrocnemius muscle. Blood flow measurements were performed just prior to ligation, immediate post ligation and serially on days 1, 3, 5, 7, 14, and 21 post ligation. Percent blood flows were calculated as: (ischemic limb average blood flow /non-ischemic limb average blood flow) x 100.
  • Vascular Density Measurement Vascular density measurements were performed as we have previously reported (11, 16). The gastrocnemius muscles from the ischemic and nonischemic hind limbs were dissected and embedded in the OCT freezing medium, frozen and cut into 8 ⁇ sections. The slides were fixed at -20°C in 95% ethanol/5% glacial acetic acid for 1 hour. Slides were washed 3 to 4 times in cold PBS with 1% percent horse serum (5 minutes per wash) and blocked overnight with 5% horse serum in PBS at 4°C.
  • Tissue Nitrite Levels and eNOS western blotting Blood and tissue nitrite levels were measured using chemiluminescence techniques as we have previously reported (11, 16). Tissue specimens were homogenized with RIPA buffer containing protease and phosphatase inhibitors, protein lysates prepared and ran on SDS polyacrylamide gels. Phospho Serl 176 and total eNOS western blots were performed as previously reported (11, 18, 19).
  • KT5720 Inhibition ofPKA with KT5720: KT5720, a selective inhibitor of PKA was administered daily with dipyridamole to a separate cohort of mice to study the effects of dipyridamole with PKA inhibition.
  • KT5720 was used at a dose of 200 ⁇ g/kg after dissolving in 100% DMSO and administered intraperitoneally.
  • KT5720 i.p. treatments were began 2 hours post-ligation of the femoral artery inducing ischemia.
  • cPTIO a nitric oxide scavenger
  • dipyridamole Since dipyridamole has not been studied extensively in mice, it was necessary to establish a dosing regimen to achieve therapeutic plasma levels between 0.8-1.2 ⁇ g/ml in C57BL/6J mice. Dipyridamole was administered by gavage in a mixture of acid water (pH 2.0) plus 10% glycofurol twice daily (every 12 hours). Retroorbital sinus bleeds were performed to collect 500 ⁇ of whole blood to obtain plasma. Blood was collected 3 hours after the morning dose on the assigned day.
  • Figures 1A and B illustrate the first two 5 attempts to obtain reliable dipyridamole dosing in C57BL/6J mice. Panel A shows the results of a dose escalation study where dipyridamole was given b.i.d.
  • Panel B shows results from administration of o dipyridamole at a dose of 100 mg/kg b.i.d. Analysis of plasma revealed that the dosing regimen of 100 mg/kg b.i.d. achieved a steady state plasma dipyridamole concentration of 0.105 + 0.006 ⁇ g/ml that did not significantly change over time. Neither of the two dosing regimens achieved plasma therapeutic levels (0.8-1.2 ⁇ g/ml) necessary for study.
  • a third dosing regimen was performed using a dose of 200 mg/kg dipyridamole5 b.i.d. over a 4 day period.
  • Figure 1C illustrates that a 200 mg/kg b.i.d. dosing regimen quickly increased plasma dipyridamole levels to therapeutic levels of 0.925 + 0.14 ⁇ g/ml by day 3 which reached a plateau at day 4. Therefore, all subsequent experiments reporting the use of dipyridamole in the chronic hind limb ischemia model are dosed with 200 mg/kg dipyrdiamole b.i.d. three days prior to induction of hind limb ischemia and0 maintained on this dosing regimen throughout the remainder of the study.
  • FIG. 2 A Temporal serial measurements of the tissue blood flows by laser Doppler are shown in Figure 2 A. Dipyridamole treated mice quickly restored ischemic hind- limb5 blood flow back to baseline levels by day 5 post ligation which persisted through day 21 in contrast to acid- water control mice. Vascular density was evaluated by measuring the ratio of endothelial CD31 surface expression divided by DAPI nuclear counterstaining as we have previously reported in ischemic and non-ischemic tissues (11, 16).
  • Figures 2B and 2C shows representative immunofluorescent staining for CD31 (red) and DAPI0 (blue) nuclear counterstain in ischemic tissues at day 7 from acid water control and
  • Ischemic tissue from dipyridamole treated mice revealed a greater vascular density compared to ischemic tissue from acid water control mice as well as an increase over dipyridamole treated non-ischemic tissue.
  • Figure 2D and 2E show the amount of staining for Ki67 cell proliferation marker (green) in day 7 ischemic tissue of mice treated with either acid water or dipyridamole, respectively.
  • Dipyridamole treatment resulted in greater Ki67 staining of ischemic tissue with colocalization and adjacent staining of CD31.
  • Figure 2G demonstrates that dipyridamole treated ischemic tissue show a significantly greater proliferation index (ratio of
  • Ki67 DAPI staining
  • Figures 3A and 3B illustrate Microfil vascular casting at day 3 of acid water control non-ischemic and ischemic hind limbs, respectively.
  • Panels 3C and 3D show Microfil casting at day 3 of dipyridamole non-ischemic and ischemic hind limbs, respectively.
  • Dipyridamole treatment augmented collateral artery perfusion through the adductor muscle as indicated by Microfil contrast (arrows).
  • Figure 4 reports measurement of arterial branching and the distance between branches in both ischemic and non-ischemic limbs upon dipyridamole or vehicle treatment.
  • Dipyridamole therapy showed a moderate but insignificant increase in the number of arterial branches off of first order arteries day 3 (panel 4A). Conversely, dipyridamole therapy did significantly enhance branching at day 5 (panel 4B) compared to acid water vehicle control treatments. In addition, dipyridamole treatment did not alter the distance between arterial branches at day 3 (panel 4C), but did significantly decrease the distance between branch points at day 5 (panels 4D). These data suggest that dipyridamole therapy augments the ability of collateral perfusion in both non-ischemic and ischemic tissue and augments arterial branch number and decreases distances between branches.
  • dipyridamole can potentiate NO biological effects (20-22). Moreover, the potent effect of dipyridamole therapy suggests that its beneficial action likely involves key regulators of angiogenic activity (e.g. eNOS and NO). Therefore, whether the protective effects of dipyridamole therapy involved eNOS enzyme activity was examined next.
  • Figure 5A shows that dipyridamole therapy failed to restore ischemic hind limb blood flow in eNOS _/ ⁇ mice compared to wild type mice.
  • Figure 5B reports that dipyridamole restoration of ischemic tissue vascular density in 5 eNOS _/ ⁇ mice is significantly inhibited.
  • Figure 5C shows that dipyridamole induction of the proliferation index is significantly blunted in eNOS deficient mice.
  • Figure 6A shows that the PKA inhibitor KT5720 (200 ⁇ g/kg) significantly blocked dipyridamole enhancement of ischemic tissue blood flow.
  • Example 5 General methods using dipyridamole for treating chronic tissue ischemia associated with diabetes
  • B6.BKS-Lepr db/db diabetic mice were purchased from Jackson Laboratories (Bar Harbor, ME). 20 week old B6.BKS-Lepr db/db (Db/Db) diabetic mice were used for all experiments. Mice were housed at the Association for Assessment and 5 Accreditation of Laboratory Animal Care, International accredited Louisiana Health
  • Dipyridamole therapy and surgical model Dipyridamole was administered at a previously determined optimal dose of 200 mg/kg in Db/Db mice to obtain a steady state plasma therapeutic level between 0.8-1.2 ⁇ g/ml as we have reported [55, 61]. Unilateral chronic hind limb ischemia was induced by ligation, transection, and removal of the left common femoral artery proximal to the origin of the profunda artery and its collateral5 branches as we have previously reported [55, 61]. Dipyridamole or vehicle control (acid water with 10% glycofurol) therapy was administered by a gavage of either formulation (500 ⁇ volume) twice a day for the duration of the study.
  • Vascular Density Measurement Vascular density measurements were performed as we have previously reported [55, 61]. Briefly the gastrocnemius muscles from ischemic and non-ischemic hind limbs were removed, dissected, and embedded in OCT freezing medium. Frozen tissue blocks were cut into 5 ⁇ sections and slides prepared. A primary antibody against CD31 was added at a 1:200 dilution and incubated at 37°C for 1 hour. Slides were then washed and a Cy3 conjugated secondary antibody was added at a 1:250 dilution and incubated at room temperature for 1 hour. Slides were once again washed and mounted with coverslips using Vectashield DAPI.
  • a minimum of four slides per hind limb with three sections per slide were prepared for vascular density analysis. A minimum of two fields were acquired per section of muscle. Images were captured using a Hamamatsu digital camera in conjunction with a Nikon TE-2000 epifluorescence microscope (Nikon Corporation, Japan) at 200X magnification for CD31 and DAPI staining. Simple PCI software version 6.0 (Compix Inc., Sewickly, PA, USA) was used to determine the area CD31 and DAPI positive staining. Tissue vascular density was determined as the ratio between CD31 positive areas and DAPI positive regions.
  • Tissue NOx measurement and eNOS western blotting Tissue total NOx levels were measured using a chemiluminescent NO analyzer (GE Healthcare) as we have previously published [55, 61]. Briefly, vehicle control or dipyridamole treated mice were euthanized at day 7 and gastrocnemius non-ischemic and ischemic muscle tissue harvested and cut in a 5 mid-sagittal manner resulting in two proportional specimens.
  • Tissue superoxide and protein carbonyl measurements Tissue superoxide 5 production was measured using the hydroethidine (HE) HPLC method as previously published by Zielonka et al [62]. On day 7, Db/Db mice were injected i.p. with 300 ⁇ of HE (1 ⁇ g/ ⁇ l). HE injected animals were sacrificed one hour later and gastrocnemius muscle tissue from non-ischemic and ischemic hind limbs were isolated and cut in a mid- sagittal manner resulting in two equal portions of gastrocnemius tissue.
  • HE hydroethidine
  • Tissue proteins0 from one half were precipitated using acidified methanol and 2-OH-E + enriched using a micro-column preparation of Dowex 50WX-8 cation exchange resin and eluted with 10 N HC1.
  • 2-OH-E + product was then measured using fluorescence detection (ex: 490; em: 567) with a Shimadzu UFLC HPLC system.
  • 2-OH-E + superoxide production was normalized to total protein and reported as pmol/mg of protein.
  • Tissue protein carbonyls5 were measured from the remaining muscle half using a Protein Carbonyl ELISA from Cell Biolabs Inc (San Diego, CA) according to the manufacturer's instructions. Protein carbonyl formation was normalized to total protein and reported as nmols/mg of protein.
  • Example 2 Dipyridamole increases blood flow in a diabetic model of chronic hind limb ischemia Diabetes is one of the largest risk factors for development of PAD, which is further influenced age as well as sex, and is largely associated with type 2 diabetes [63, 64]. Therefore, we tested the efficacy of dipyridamole therapy in a type-2 diabetic murine model of PAD.
  • Figure 9 shows that dipyridamole treatment resulted in a rapid restoration of ischemic hind limb blood flow which was significantly increased by day 3 post ligation and maintained long term compared to vehicle control.
  • Dipyridamole increases vascular density in a model of chronic ischemia
  • Figure 10 illustrates vascular density (anti-CD31-red) and cell proliferation (anti-Ki67-green) staining of vehicle control treated non-ischemic and ischemic gastrocnemius muscle tissue ( Figures 10A and 10B) at day 7 post-ligation.
  • Figures IOC and 10D illustrate vascular density and cell proliferation staining of dipyridamole treated non-ischemic and ischemic gastrocnemius muscle, respectively.
  • Figure 11 reports quantitative immunohistochemical measurement of vascular density (CD31:DAPI ratio- Figure 11A) and cell proliferation (Ki67:DAPI- Figure 11B) indices confirming that dipyridamole therapy significantly and selectively increases ischemic limb vascular density concomitant with increased cell proliferation.
  • dipyridamole therapy slightly increased Ki67 staining of non-ischemic gastrocnemius tissue; however, this was not associated with a change in vascular density compared to vehicle control treated non-ischemic tissue.
  • FIG. 13A illustrates that dipyridamole treatment did not significantly alter the amount of total eNOS expression in ischemic versus non-ischemic tissue compared to vehicle control treatment.
  • Figure 13B shows eNOS Serl lW phosphorylation differences in ischemic and non-ischemic tissues from dipyridamole and vehicle control treatments. In both vehicle and dipyridamole treatments, eNOS phosphorylation was greater in non-ischemic versus ischemic tissue. Moreover, dipyridamole treatment did not significantly alter eNOS phosphorylation in ischemic tissue.
  • Panels 13C and 13D report western blot densitometry measurements for total and Serl 177 phospho-eNOS, respectively. These data demonstrate that dipyridamole therapy does not significantly alter eNOS expression or phosphorylation in diabetic muscle tissue.
  • FIG. 14A illustrates HPLC measurement of the superoxide specific 2-OH E + product in vehicle control and dipyridamole treated tissues. Superoxide generation was significantly increased in ischemic tissues of vehicle control treated diabetic mice compared to non-ischemic tissue. Importantly, dipyridamole therapy significantly decreased superoxide production in ischemic diabetic tissue compared to vehicle controls and also blunted superoxide production in non-ischemic tissue.
  • Figure 14A illustrates HPLC measurement of the superoxide specific 2-OH E + product in vehicle control and dipyridamole treated tissues. Superoxide generation was significantly increased in ischemic tissues of vehicle control treated diabetic mice compared to non-ischemic tissue. Importantly, dipyridamole therapy significantly decreased superoxide production in ischemic diabetic tissue compared to vehicle controls and also blunted superoxide production in non-ischemic tissue.
  • dipyridamole therapy significantly decreased diabetic tissue protein carbonyl levels in both ischemic and non-ischemic gastrocnemius tissue.
  • Type 2 diabetes associated cardiometabolic syndrome is characterized by both hyperglycemia as well as dyslipidemia (e.g. increased cholesterol and LDL levels and decreased HDL) which both contribute to vascular dysfunction.
  • dyslipidemia e.g. increased cholesterol and LDL levels and decreased HDL
  • triglyceride levels did not significantly change in either treatment group (dipyridamole or vehicle control, data not shown). Blood glucose in the dipyridamole treatment group was significantly decreased by day 7 post- ligation; however, the end glucose reading of 299.2 + 90.6 is still considered hyperglycemic. Interestingly, cholesterol and LDL levels in the5 dipyridamole treatment group did not vary over time; whereas vehicle control treated cholesterol and LDL levels progressively increased ( Figure 15).
  • Adair TH Growth regulation of the vascular system: an emerging role for5 adenosine. Am J Physiol Regul Integr Comp Physiol 2005, 289: R283-R296.
  • Isbell TS Sun CW, Wu LC, Teng X, Vitturi DA, Branch BG, Kevil CG,
  • ICM-1 Intercellular adhesion molecule- 1 (ICAM-1) regulates endothelial cell motility through a nitric oxide-dependent pathway. / Biol Chem 2004, 279: 19230-19238

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Abstract

La présente invention porte de manière générale sur la cicatrisation du corps humain à l'aide du dipyridamole. De plus, la présente invention porte sur des composés pour la cicatrisation des organes par angiogenèse, artériogenèse et normalisation de la fonction vasculaire à l'aide de dipyridamole. La présente invention porte sur une composition et sur un procédé d'utilisation de dipyridamole pour une angiogenèse, une artériogenèse thérapeutiques et une normalisation de la fonction vasculaire pour traiter un certain nombre de maladies, de lésions et d'autres affections telles qu'une maladie artérielle périphérique, une détérioration du tissu ischémique, une maladie intestinale inflammatoire, et d'autres par réparation facilitée du tissu. De plus, la présente invention porte sur une amélioration de la fonction organique à la suite d'une transplantation de divers organes tels que le cœur, le foie, le pancréas et l'intestin.
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EP3612178A4 (fr) * 2017-04-21 2021-01-13 Northeast Ohio Medical University Méthodes de traitement de l'insuffisance cardiaque
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