SE538144C2 - Use of dextran sulfate for inducing angiogenesis - Google Patents

Description

25 30 538 144 Traditional approaches in pro-angiogenic treatment include, among others, gene therapy, targeting genes of interest for ampli?cation or inhibition; protein therapy, which primarily manipulates angiogenic growth factors; and cell based therapies, which involve the implantation of speci?c cell types.

There are still serious, unsolved problems related to gene therapy. Difficulties include effective integration of the therapeutic genes into the genome of target cells, reducing the risk of an undesired immune response, potential toxicity, immunogenicity, in?ammatory responses, and oncogenesis related to the viral vectors used in implanting genes and the sheer complexity of the genetic basis of angiogenesis.

Pro-angiogenic protein therapy uses various growth factors, such as ?broblast growth factor (FGF) and vascular endothelial growth factor (VEGF), to promote angiogenesis. An obstacle of protein therapy is the mode of delivery. Oral, intravenous, intra-arterial, or intramuscular routes of protein administration are not always as effective, as the therapeutic protein may be metabolized or cleared before it can enter the target tissue. Cell based pro-angiogenic therapies are still in early stages of research, with many open questions regarding best cell types and dosages to use. lschemia is a restriction in blood supply to tissues, causing a shortage of oxygen and glucose needed for cellular metabolism. lschemia is generally caused by problems with blood vessels, with resultant damage to or dysfunction of tissue. lt also means local anemia and hypoxia in a given part of a body sometimes resulting from congestion, such as vasoconstriction, thrombosis or embolism.

Restoration of coronary blood ?ow after a period of prolonged lschemia often involve so-called reperfusion injury causing endothelial damage and an affected endothelium that takes on pro-coagulant and pro-in?ammatory phenotype. The reperfusion greatly accelerates ischemia-induced complement activation and deposition.

Dextran sulfate is a well-known complement inhibitor and has therefore been proposed to achieve cytoprotection of endothelium against reperfusion injury following lschemia.

Experimental Cell Research 215, 294-302 (1994) discloses that sulfated polysaccharides, such as heparin and dextran sulfate, can be used in vitro for collagen-induced vascular tube formation. However, in vivo experimental data indicated that the low molecular weight sulfated polysaccharide heparin (2.4 kDa) inhibited angiogenesis, Glycobiology 3, 567-573 (1993), Pathophysiology of Haemostasis and Thrombosis 23, 141-149 (1993). 538 144 U.S. patent no. 5,135,920 discloses that dextran sulfate with an average molecular weight of 500 000 Da is angiostatic, i.e. inhibits angiogenesis.

There is still room for improvements within the ?eld of angiogenesis in the art.

SUMMARY lt is a general objective to induce angiogenesis in a subject. lt is another objective to increase blood ?ow in a subject suffering from ischemia.

These and other objectives are met by embodiments as disclosed herein.

An aspect of the embodiments relates to dextran sulfate, or a pharmaceutically acceptable derivative thereof, having an average moiecuiar weight below 10 000 Da for inducing angiogenesis in a human subject suffering from a disease, disorder or medical condition causing ischemia in the human subject.

Another aspect of the embodiments relates to dextran sulfate, or a pharmaceutically acceptable derivative thereof, having an average moiecuiar weight below 10 000 Da for increasing blood flow in a subject suffering from ischemia.

A further aspect of the embodiments relates to dextran sulfate, or a pharmaceutically acceptable derivative thereof, having an average moiecuiar weight below 10 000 Da for vascularizing ischemic tissue in a subject.

Further aspect of the embodiments relates to dextran sulfate, or a pharmaceutically acceptable derivative thereof, having an average moiecuiar weight equal to or below 10 000 Da for in vitro or ex vivo use in increasing blood ?ow in a vascularized tissue and/or organ and/or for in vitro or ex vivo vascularizing a vascularized tissue and/or organ.

BRIEF DESCRIPTION OF THE DRAWINGS The embodiments, together with further objects and advantages thereof, may best be understood by making reference to the following description taken together with the accompanying drawings, in which: 538 144 Fig. 1 is a diagram illustrating mean body weight throughout the mouse critical limb ischemia model. Two- way ANOVA followed by Bonferroni post-hoc comparisons revealed no statistically significant differences between the groups.

Fig. 2 is a diagram illustrating mean blood ?ow by study group throughout the mouse critical limb ischemia model. Two-way ANOVA for repeated measures, followed by Bonferroni post-hoc test was performed.

Comparison of dextran sulfate treated groups ZM, 3M and 4M to control group 1M revealed statistically significant differences from days 14 and 21 through day 35 (p<0.001).

Fig. 3 is a diagram illustrating CD34 capillaries density of the groups through the critical limb ischemia study, following double staining with FlTC-labeled dextran (DA). DA bars representing functional capillaries. Statistical analysis performed using tvvo-way ANOVA followed by Bonferroni multiple comparisons.

Figs. 4A-4C illustrate CD34 capillaries density of the groups in the critical limb ischemia study (Fig. 4A - mouse from group 1 M vehicle control; Fig. 4B - mouse from group 3M dextran sulfate 30 mg/kg repeated; Fig. 4C - mouse from group 4M dextran sulfate 30 mg/kg single). The left diagrams illustrate CD34 staining and the right diagrams illustrate FlTC-labeled dextran staining.

Fig. 5 is a diagram illustrating capillaries density of the groups in the critical limb ischemia study of the endothelial marker smooth muscle actin (SMA). Statistical signi?cance according to tvvo-way ANOVA followed by Bonferroni multiple comparisons.

Fig. 6 compares blood flow measured in two mice with non-contact laser Doppler images 35 days after femoral artery ligation of the left hind limb. The lower panel has been vehicle treated (group 1M), the top panel has received treatment with dextran sulfate (group 3M).

Fig. 7 illustrates distribution of body weight in the groups used for the tMCAO rate-stroke study.

Fig. 8 illustrates Neuroscore by treatment group throughout the tMCAO rate-stroke study.

Fig. 9 illustrates stepping test by treatment group throughout the tMCAO rate-stroke study.

Fig. 10 illustrates forelimb placement test by treatment group throughout the tMCAO rate-stroke study. 10 15 20 25 30 538 144 Fig. 11 illustrates body swing test delta (left turn-right turn) by treatment group throughout the tMCAO rate-stroke study.

Fig. 12 illustrates cerebral blood ?ow ratio and percent change of vessels average diameter in both dextran sulfate groups as compared to vehicle control on day 29.

Fig. 13 illustrates SMA capillary density in rats treated by dextran sulfate 15 mg/kg daily as compared to vehicle control on day 30.

Fig. 14 illustrates GFAP area (area of positive cells in square microns per ><10 ?eld) in dextran sulfate treated group versus control vehicle group on day 30.

Figs. 15A and 15B illustrating the treatment effect on capillaries density for a rat from vehicle control group (Fig. 15A) and from dextran sulfate (15 mg/kg, daily) group (Fig. 15B).

All ?gures shows average + standard error of the mean DETAILED DESCRIPTION The present embodiments generally relate to angiogenesis, and in particular to the use of dextran sulfate for inducing angiogenesis in a subject.

The present embodiments are based on the discovery that dextran sulfate within a particular average molecular weight has angiogenesis inducing effect and blood ?ow increasing effect when administered to a subject, preferably a mammalian subject, and more preferably a human subject.

This effect of dextran sulfate of the embodiments was highly surprising in the light of the prior art disclosing that sulfated polysaccharides inhibited angiogenesis in vivo and that dextran sulfate with an average molecular weight of 500 000 Da is angiostatic, i.e. inhibits angiogenesis.

Experimental data as presented in herein in clear contrast shows that dextran sulfate of the embodiments has in vivo effects in inducing angiogenesis as seen by signi?cantly increasing mean blood ?ow, decreasing ischemia severity and increasing capillary density in an ischemia model and in a stroke model 10 15 20 25 30 538 144 Accordingly, an aspect of the embodiments relates to dextran sulfate, or a pharmaceutically acceptable derivative thereof, having an average molecular weight below 10 000 for use in inducing angiogenesis in a subject. ln the following, reference to (average) molecular weight and sulfur content of dextran sulfate applies also to any pharmaceutically acceptable derivative of dextran sulfate. Hence, the pharmaceutically acceptable derivative of dextran sulfate preferably has the average molecular weight and sulfur content as discussed in the following embodiments.

Dextran sulfate outside of the range of the embodiments are believed to have inferior or indeed no angiogenesis effect at all. For instance, heparin, another sulfated polysaccharide, with an average molecular weight of 2.4 kDa actually inhibited angiogenesis as did larger dextran sulfate molecules (Pathophysiology of Haemostasis and Thrombosis 23, 141-149 (1993); U.S. patent no. 5,135,920).

Furthermore, dextran sulfate of a molecular weight exceeding 10 000 Da generally has a lower effect vs. side effect profile as compared to dextran sulfate having a lower average molecular weight. This means that the maximum dose of dextran sulfate that can be safely administered to a subject is lower for larger dextran sulfate molecules (>10 000 Da) as compared to dextran sulfate molecules having an average molecular weight within the present range. As a consequence, such larger dextran sulfate molecules are less appropriate in clinical uses when the dextran sulfate is to be administered to subjects in vivo. ln addition, large dextran sulfate molecules have in fact the opposite effect as compared to dextran sulfate of the embodiments as is evidenced from U.S. patent no. 5,135,920.

Thus, there seems to be a very narrow range with regard to the average molecular weight of dextran sulfate within which dextran sulfate has angiogenesis effect when administered to a subject and that dextran sulfate molecules outside of the range of the embodiments have no or indeed angiogenesis inhibiting effect.

Dextran sulfate is a sulfated polysaccharide and in particular a sulfated glucan, i.e. polysaccharide made of many glucose molecules. Average molecularweight as de?ned herein indicates that individual sulfated polysaccharides may have a molecular weight different from this average molecular weight but that the average molecular weight represents the mean molecular weight of the sulfated polysaccharides. This further implies that there will be a natural distribution of molecularweights around this average molecular weight for a dextran sulfate sample. 10 15 20 25 30 538 144 Average molecular weight (MW) of dextran sulfate is typically determined using indirect methods such as gel exclusion/penetration chromatography, light scattering or viscosity. Determination of average molecular weight using such indirect methods will depend on a number of factors, including choice of column and eiuent, flow rate, caiibration procedures, etc. ln an embodiment, the dextran sulfate or the pharmaceuticaiiy acceptabie derivative thereof has an average molecular weight is within a range of 2 000 and 10 000 Da. ln another embodiment, the average molecular weight is within a range of 2 500 and 10 000 Da. ln a particular preferred embodiment, the average molecular weight is within a range of 3 000 to 10 000 Da. ln an optional, but preferred embodiment, less than 40 % of the dextran sulfate molecules have a molecular weight below 3 000 Da, preferably less than 35 %, such as less than 30 % or less than 25 % of the dextran sulfate molecules have a molecular weight below 3 000 Da. ln addition, or alternatively, less than 20 % of the dextran sulfate molecules have a molecular weight above 10 000 Da, preferably less than 15 %, such as less than 10 % or less than 5 % of the dextran sulfate molecules have a molecular weight above 10 000 Da. Thus, in a particular embodiment, the dextran sulfate has a substantially narrow molecular weight distribution around the average molecular weight. ln a particular embodiment, the average molecular weight of dextran sulfate, or the pharmaceuticaiiy acceptabie derivative thereof, is within a range of 3 500 and 9 500 Da, such as within a range of 3 500 and 8 000 Da. ln another particular embodiment, the average molecular weight of dextran sulfate, or the pharmaceuticaiiy acceptabie derivative thereof, is within a range of 4 500 and 7 500 Da. ln a further particular embodiment, the average molecular weight of dextran sulfate, or the pharmaceutically acceptabie derivative thereof, is within a range of 4 500 and 5 500 Da.

Thus, in a currently preferred embodiment the average molecular weight of dextran sulfate, or the pharmaceuticaiiy acceptabie derivative thereof, is preferably approximately 5 000 Da or at least substantially close to 5 000 Da, such as 5 000 i 500 Da, for instance 5 000 i 400 Da, preferably 5 000 i 300 Da or 5 000 i 200 Da, such as 5 000 i 100 Da. Hence, in an embodiment, the average molecular 538 144 weight of dextran sulfate, or the pharmaceutically acceptable derivative thereof, is 4.5 kDa, 4.6 kDa, 4.7 kDa, 4.8 kDa, 4.9 kDa, 5.0 kDa, 5.1 kDa, 5.2 kDa, 5.3 kDa, 5.4 kDa or 5.5 kDa. ln a particular embodiment, dextran sulfate, or the pharmaceutically acceptable derivative thereof, consists, on average, of about or slightly above 5 glucose units and has an average sulfate number per glucose unit of at least 2.0, such as of at least 2.5.

Dextran sulfate is a polyanionic derivate of dextran and contains sulfur. The average sulfur content for dextran sulfate of the embodiments is preferably 15 to 20 % and more preferably approximately 17 %, generally corresponding to about tvvo sulfate groups per glucosyl residue. ln a particular embodiment, the sulfur content of the dextran sulfate is preferably equal to or at least close to the maximum possible degree of sulfur content of the dextran molecules.

The dextran sulfate according to the embodiments can be provided as a pharmaceutically acceptable derivative of dextran sulfate. Such pharmaceutically acceptable derivatives include salts and solvates of dextran sulfate, e.g. a sodium or potassium salt.

Dextran sulfate, or the pharmaceutically acceptable derivative thereof, of the embodiments is preferably administered by injection to the subject and in particular by intravenous (i.v.) injection, subcutaneous (s.c.) injection or (i.p.) intraperitoneal injection, preferably i.v. or s.c. injection. Other parenteral administration routes that can be used include intramuscular and intraarticular injection. Injection of dextran sulfate, or the pharmaceutically acceptable derivative thereof, could alternatively, or in addition, take place directly in, for instance, an ischemic tissue or organ or other site in the subject body, at which angiogenesis and increased blood flow are to take place.

The dextran sulfate, or the pharmaceutically acceptable derivative thereof, of the embodiments is preferably formulated as an aqueous injection solution with a selected solvent or excipient. The solvent is advantageously an aqueous solvent and in particular a buffer solution. A non-limiting example of such a buffer solution is a citric acid buffer, such as citric acid monohydrate (CAM) buffer, or a phosphate buffer. For instance, dextran sulfate of the embodiments can be dissolved in saline, such as 0.9 % NaCl saline, and then optionally buffered with 75 mM CAM and adjusting the pH to about 5.9 using sodium hydroxide. Also non-buffered solutions are possible, including aqueous injection solutions, such as saline, i.e. NaCl (aq). Furthermore, other buffer systems than CAM could be used if a buffered solution are desired. 538 144 The embodiments are not limited to injections and other administration routes can alternatively be used including orally, nasally, bucally, rectally, dermally, tracheally, bronchially, or topically. The active compound, dextran sulfate, is then formulated with a suitable excipient or carrier that is selected based on the particular administration route.

Suitable dose ranges for the dextran sulfate of the embodiments may vary according to the size and weight of the subject, the condition for which the subject is treated, and other considerations. ln particular for human subjects, a possible dosage range could be from 1 pg/kg to 150 mg/kg of body weight, preferably from 10 pg/kg to 100 mg/kg of body weight. ln preferred embodiments, the dextran sulfate, or the pharmaceutically acceptable derivative thereof, is formulated to be administered at a dosage in a range from 0.05 to 30 mg/kg of body weight of the subject, preferably from 0.1 to 25 mg/kg of body weight of the subject, and more preferably from 0.1 to 15 mg/kg or from 0.1 to 10 mg/kg body weight of the subject.

Administration of dextran sulfate, or the pharmaceutically acceptable derivative thereof, of the embodiments is preferably initiated as soon as possible after injury or other condition causing ischemia or stroke in the subject or causing a medical condition that could be treated or at least alleviated by angiogenesis induction as triggered by the administration of dextran sulfate, or the pharmaceutically acceptable derivative thereof.

Administration of dextran sulfate, or the pharmaceutically acceptable derivative thereof, does not necessarily have to be limited to treatment of a present medical condition but could alternatively, or in addition, be used for prophylaxis. ln other words, dextran sulfate of the embodiments could be administered to a subject that will undergo a medical procedure, such as surgery, that may cause at local ischemia orother medical effect that could be treated, inhibited oralleviated by induction of angiogenesis and/or increasing blood ?ow.

The dextran sulfate of the embodiments can be administered at a single administration occasion, such as in the form of a single bolus injection. This bolus dose can be injected quite quickly to the patient but is advantageously infused over time so that the dextran sulfate solution is infused over a few minutes of time to the patient, such as during 5 to 10 minutes. 10 15 20 25 30 538 144 Alternatively, dextran sulfate of the embodiment can be administered at multiple, i.e. at least tvvo, occasions during a treatment period. The duration of such a treatment period is typically related to the endogenous time period of wound healing in different types and the type of insult. For more information of suitable treatment periods, reference can be made to Chapter 1 Overview of Wound Healing in Different Tissue Types, pages 3-40 of lndwelling Neural lmplants: Strategies for Contending with the ln Vivo Environment, ed. William M. Reichert, 2008 by Taylor & Francis Group, LLC (ISBN: 978-0-8493- 9362-4).

Generally, for acute diseases, such as causing acute ischemia in, for instance, stroke, myocardial infarction (MI), cell and organ transplantation, the duration of the treatment period could be a single administration but is preferably in the form of several administrations during a treatment period of, for instance, a week, a few weeks, or a month. Longer treatment periods up to three months or even a year can further improve healing and recovery.

For ischemic conditions of intermittent type, there can be an option to use the treatment as prophylaxis (prevention) or treatment directly after exacerbation of the disease. This type of administration protocol could be suitable for diseases such as multiple sclerosis (MS), amytrophic lateral sclerosis (ALS) and sickle cell disease. Treatment periods can be up to 1-3 months for treatment after exacerbation. For prophylaxis of the disease, optionally longer treatment periods can be used.

The induction of angiogenesis in a subject through administration of dextran sulfate, or the pharmaceutically acceptable derivative thereof, of the embodiments is preferably taking place in a human subject suffering from a disease, disorder or medical condition causing ischemia in the body of the human subject. lschemia is a restriction in blood supply to tissues, causing a shortage of oxygen and glucose needed for cellular metabolism. lschemia is generally caused by problems with blood vessels, with resultant damage to or dysfunction of tissue or organ. lt also means local anemia and hypoxia in a given part of a body sometimes resulting from congestion, such as vasoconstriction, thrombosis or embolism.

An effective treatment of ischemia or an effective approach in preventing or at least reducing the risk of suffering from ischemia is to induce angiogenesis. The angiogenesis causes an increase in blood flow in the relevant tissue and can thereby counteract any restriction in blood supply to the tissue caused by the disease, disorder or medical condition. 10 10 15 20 25 30 538 144 Non-Iimiting but illustrative examples of diseases, disorders or medical conditions that can cause ischemia include wound healing; peripheral ischemia, such as ischemia following transplantation of organs, tissues or cells, peripheral arterial disease, limb ischemia, Raynaud's syndrome, sickle cell disease, or thromboangiitis obliterans; coronary ischemia, such as caused by congestive heart failure or coronary arterial disease; ischemia in central nervous system, such as caused by traumatic brain injury, temporal arteritis, hypoxia caused by multiple sclerosis, stroke, amytrophic lateral sclerosis; or muscular dystrophic disease.

Wound healing generally involves four phases, typically denoted early phase, in?ammatory phase, proliferative phase and maturation and remodeling phase. Angiogenesis is one of the processes taking place during the proliferative phase. Administration of dextran sulfate, or the pharmaceutically acceptable derivative thereof, may promote the angiogenesis effect taking place as one of the sub-processes of wound healing. The process of angiogenesis occurs concurrently with ?broblast proliferation during wound healing when endothelial cells migrate to the area of the wound. Because the activity of ?broblasts and epithelial cells requires oxygen and nutrients, angiogenesis is imperative for other stages in wound healing, like epidermal and ?broblast migration.

Peripheral ischemia generally denotes ischemic states taking place in tissues and organs different from the heart (coronary ischemia) and the central nervous system (CNS ischemia). There can be various causes of peripheral ischemia. A typical example is transplantation of organs or tissue to a subject. The transplanted organ or tissue is then typically exposed to ischemia during the initial engraftment process taking place from the point of transplantation until new blood vessels have been formed around the transplanted organ or tissue. There is a high risk of damage to or dysfunction of the organ or tissue due to ischemia and hypoxia if sufficient blood supply is not established shortly after transplantation. Hence, induction of angiogenesis by dextran sulfate, or the pharmaceutically acceptable derivative thereof, according to the embodiments in connection with transplantation may signi?cantly reduce the risk of damage to or dysfunction of the transplanted organ or tissue due to ischemia and/or hypoxia.

Administration of dextran sulfate, or the pharmaceutically acceptable derivative thereof, according to the embodiments can be taking place prior to the transplantation in order to induce angiogenesis and provide increase blood ?ow at the site of transplantation prior to the actual transplantation event. ln such a case, the increase in blood flow induced by dextran sulfate of the embodiments may be sufficient to prevent or at least reduce ischemic damages to the transplanted organ or tissue. 11 538 144 Peripheral vascular disease (PVD), commonly referred to as peripheral artery disease (PAD) or peripheral artery occlusive disease (PAOD) or peripheral obliterative arteriopathy, refers to the obstruction of large arteries not within the coronary, aortic arch vasculature, or brain. PVD can result from atherosclerosis, in?ammatory processes leading to stenosis, an embolism, or thrombus formation. lt causes either acute or chronic ischemia. An efficient treatment of PVD is to restore blood ?ow by administration of dextran sulfate, or the pharmaceutically acceptable derivative thereof, according to the embodiments.

Limb ischemia, often referred to as acute limb ischemia, occurs when there is a sudden lack of blood ?ow to a limb. Acute limb ischemia is typically due to either an embolism or thrombosis. Thrombosis is usually caused by peripheral vascular disease (atherosclerotic disease that leads to blood vessel blockage), while an embolism can be due to air, trauma, fat, amniotic ?uid, or a tumor. Subjects suffering from limb ischemia would bene?t from administration of dextran sulfate, or the pharmaceutically acceptable derivative thereof, of the embodiments. ln medicine, Raynaud's phenomenon is excessively reduced blood ?ow in response to cold or emotional stress, causing discoloration of the fingers, toe, and occasionally other areas. Raynaud's phenomenon by itself is just a sign (hypoperfusion) accompanied by a symptom. When linked to pathogenesis, it can be part of Raynaud's disease (also known as primary Raynaud's phenomenon), where the cause is unknown, or part of Raynaud's syndrome (secondary Raynaud's phenomenon), which is a syndrome caused by a known primary disease, most commonly connective tissue disorders such as systemic lupus erythematosus. lt is a hyperactivation of the sympathetic nervous system causing extreme vasoconstriction of the peripheral blood vessels, leading to tissue hypoxia. Chronic, recurrent cases of Raynaud phenomenon can result in atrophy of the skin, subcutaneous tissues, and muscle, possibly causing ulceration and ischemic gangrene. Administration of dextran sulfate, or the pharmaceutically acceptable derivative thereof, of the embodiments can be an ef?cient means of reducing the risk of suffering from, treating or at least alleviating the symptoms of Raynaud's syndrome or disease.

Sickle-cell disease (SCD), or sickle-cell anaemia (SCA) or drepanocytosis, is a hereditary blood disorder, characterized by red blood cells that assume an abnormal, rigid, sickle shape. Sickling decreases the ?exibility of the red blood cells and results in a risk of an inadequate flow of blood to a part of the body. lnduction of angiogenesis using dextran sulfate, or the pharmaceutically acceptable derivative thereof, of the embodiments can be used to reduce the risk of developing peripheral ischemia in subjects suffering from SCD. 12 538 144 Thromboangiitis obliterans, also known as Buerger's disease or presenile gangrene, is a recurring progressive inflammation and thrombosis (clotting) of small and medium arteries and veins of the hands and feet. Thromboangiitis obliterans may therefore cause ischemia in the hands and feet due to restriction 5 of the blood ?ow these extremities. Administration of dextran sulfate, or the pharmaceutically acceptable derivative thereof, of the embodiments can be an ef?cient means to increase blood ?ow to the hands and feet.

Coronary ischemia is a medical term for not having enough blood through the coronary arteries. Coronary 10 ischemia is linked to heart disease as well as heart attacks. lt is also known as cardiac ischemia. Coronary arterial disease (CAD) occurs when fatty substances get stuck to the walls of coronary arteries, which narrows the arteries and constricts blood ?ow. This causes a lack of oxygen and blood to the heart, which can result in a myocardial infarction (heart attack). CAD causes constriction of arteries, which leads to a lack of blood ?owing through the arteries as well as oxygen, a process called atherosclerosis. 15 Atherosclerosis is the most common cause of coronary ischemia. increasing blood ?ow in the heart muscle through induction of angiogenesis triggered by administration of dextran sulfate, or the pharmaceutically acceptable derivative of the embodiments may be important to reduce the risk of or reduce the damage caused by coronary ischemia. Also thrombosis may be a cause to coronary ischemia. 20 lschemia in CNS may be due to various causes. For instance, traumatic brain injury may cause a blockage or restriction in blood ?ow to part of the brain. Such a restriction in blood ?ow may have severe consequences if hypoxia occurs in the brain. Increase in blood ?ow as caused by administration of dextran sulfate, or the pharmaceutically acceptable derivative thereof, of the embodiments can thereby be used to reduce the risk of permanent damages to the brain caused by ischemia following a traumatic 25 brain injury.

Temporal arteritis, also referred to as giant-cell arteritis (GCA), cranial arteritis or Horton disease, is an in?ammatory disease of blood vessels most commonly involving large and medium arteries of the head, predominantly the branches of the external carotid artery. lt is a form of vasculitis. induction of 30 angiogenesis and increase in blood ?ow as caused by administration of dextran sulfate, or the pharmaceutically acceptable derivative thereof, of the embodiments may be beneficial to subjects suffering from temporal arteritis. 13 10 15 20 25 30 538 144 Stroke, sometimes referred to as a cerebrovascular accident (CVA), cerebrovascular insult (CVI) or colloquially brain attack, is the loss of brain function due to a disturbance in the blood supply to the brain.

This disturbance is due to either ischemia or hemorrhage. ischemia is caused by either biockage of a blood vessel via thrombosis or arterial embolism, or by systemic hypoperfusion. Hemorrhagic stroke is caused by bleeding of blood vessels of the brain, either directly into the brain parenchyma or into the subarachnoid space surrounding brain tissue. Subject suffering from a stroke would benefit from a treatment that increases the blood ?ow to the brain to reduce the risk of damages caused by insuf?cient blood supply. As a consequence, administration of dextran sulfate, or the pharmaceutically acceptable derivative thereof, of the embodiments is advantageously given to subjects suffering from stroke.

Various neurological disorders may cause a restriction in blood supply to the CNS, such as part of the brain. For instance, Multiple Sclerosis lnternational2013, pages 1-6 (2013) discloses that early multiple sclerosis (MS) lesions are associated with hypoxia. Hence, subjects suffering from MS may bene?t from increased blood ?ow in order to treat or at least reduce or inhibit hypoxia associated with MS.

Amyotrophic lateral sclerosis (ALS), also referred to as motor neuron disease (MND) and Lou Gehrig's disease, is a neurodegenerative disease with various causes. lt is characterized by rapidly progressive weakness due to muscle atrophy and muscle spasticity, difficulty in speaking (dysarthria), swallowing (dysphagia), and breathing (dyspnea). Experiments have shown that ALS is associated with reduction in blood ?ow in, for instance, premotor frontal lobe regions, Acta Neurologica Scandinavia 116, pages 340- 344 (2007). lt is speculated that increased blood ?ow through induction of angiogenesis could be beneficial for subjects suffering from ALS. lnduction of angiogenesis according to the embodiments can further be used in connection with implantation of various medical devices, sensors, etc. where it may be advantageous to induce microcirculation towards or in connection with the implant.

Another aspect of the embodiments relates to a method for inducing angiogenesis in a subject. The method comprises administering dextran sulfate, or a pharmaceutically acceptable derivative thereof, having an average molecular weight below 10 000 Da to the subject.

A further aspect of the embodiments relates to use of dextran sulfate, or a pharmaceutically acceptable derivative thereof, having an average molecular weight below 10 000 Da for the manufacture of a medicament for inducing angiogenesis in a subject. 14 10 15 20 25 30 538 144 Yet another aspect of the embodiments relates to dextran sulfate, or a pharmaceutically acceptable derivative thereof, having an average molecular weight below 10 000 Da for use in increasing blood flow a subject suffering from ischemia.

A related aspect of the embodiments defines a method for increasing blood ?ow in a subject suffering from ischemia. The method comprises administering dextran sulfate, or a pharmaceutically acceptable derivative thereof, having an average molecular weight below 10 000 Da to the subject. Another related aspect of the embodiments defines use of dextran sulfate, or a pharmaceutically acceptable derivative thereof, having an average molecular weight below 10 000 Da for the manufacture of a medicament for increasing blood ?ow in a subject suffering from ischemia. ln a particular embodiment, dextran sulfate, or the pharmaceutically acceptable derivative thereof, is capable of increasing blood flow in an ischemic tissue or organ of the subject.

The tissue or organ can be a peripheral organ, the heart or CNS tissue, such as the brain, as discussed in the foregoing.

Still another aspect of the embodiments relates to dextran sulfate, or a pharmaceutically acceptable derivative thereof, having an average molecular weight below 10 000 Da for vascularizing ischemic tissue in a subject.

A related aspect of the embodiments de?nes a method for vascularizing ischemic tissue in a subject. The method comprises administering dextran sulfate, or a pharmaceutically acceptable derivative thereof, having an average molecular weight below 10 000 Da to the subject. Another related aspect of the embodiments de?nes use of dextran sulfate, or a pharmaceutically acceptable derivative thereof, having an average molecular weight below 10 000 Da for the manufacture of a medicament for vascularizing ischemic tissue in a subject.

The ischemic tissue could be a peripheral organ, the heart or CNS tissue, such as the brain, as discussed in the foregoing. 15 538 144 The subject is preferably a mammalian subject, more preferably a primate and in particular a human subject. The present embodiment can, however, be used also in veterinary applications. Non-Iimiting example of animal subjects include primate, cat, dog, pig, horse, mouse, rat.

The embodiments may also be applied to in vitro and/or ex vivo treatment of vascularized tissue and/or organs in order to induce angiogenesis in the vascularized tissue and/or organ, increase blood ?ow in the vascularized tissue and/or organ and/or vascularizing a vascularized tissue and/or organ. ln such a case, the dextran sulfate, or a pharmaceutically acceptable derivative thereof, can be added to the vascularized tissue and/or organ in various in vitro or ex vivo applications. For instance, dextran sulfate, or a pharmaceutically acceptable derivative thereof, can be added to a culture medium in which the vascularized tissue and/or organ is immersed or contacted in vitro. Alternatively, or in addition, the vascularized tissue and/or organ could be sprayed with a solution comprising dextran sulfate, or a pharmaceutically acceptable derivative thereof. Furthermore, if the vascularized tissue and/or organ is connected to an extracorporeal circulation pump or extracorporeal membrane oxygenation (ECMO) device, then dextran sulfate, or a pharmaceutically acceptable derivative thereof, could be added to the blood that is pumped through the vascularized tissue and/or organ.

EXPERIMENTS Peripheral artery disease - critical limb ischemia Peripheral artery disease (PAD) is a form of peripheral vascular disease (PVD) in which there are partial or total blockage of blood supply to a limb, usually the leg, leading to impaired blood flow and hypoxia in the tissue. When PAD advances it reaches the stage of critical limb ischemia (CLl) with skin ulcerations, gangrene and unavoidable amputations. Therapeutic angiogenesis emerged as a non-invasive means of promoting neovascularization in ischemic tissues. As disclosed in the present study systemic subcutaneous administration of dextran sulfate promote angiogenesis causing formation of small blood vessels and proliferation of endothelial cells. ln the present study a stable severe ischemia model (Journal of Experimental and Clinical Medicine 31, 128-132 (2006)) was applied to assess safety and ef?cacy of dextran sulfate on angiogenesis and functional outcome.

Materials Dextran sulfate with an average molecular weight within a range of 5-7 kDa was obtained from pK Chemicals A/S, Denmark. 16 538 144 An injection solution of dextran sulfate was prepared the day before start of the study. As vehicle, 0.9 % NaCl (saline) (Teva Pharmaceutical Industries Ltd) was used. The injection solution was prepared by adding the relevant volume of NaCl to the weighed compound to obtain a target concentration for administration (10 or 30 mg/kg body weight). The dextran sulfate was dissolved by vortexing or simply by turning the tube a few times. The solution was stored at 2-8°C over night for aggregates to stabilize.

The next day, the tube was vortexed and the solution was ?ltered through a 0.2 pm ?lter to obtain a sterile solution. Solutions were prepared at day 7, to use at days 8-21, and a second preparation was performed at day 21, to be used at days 22-35. The solution was stored at 2-8°C between application dates. ln total 60 male Balb/c mice, 9 weeks old, having an average body weight of 24.7 g at study initiation (day 0) were obtained from Harlan Laboratories, Israel. The minimal and maximal weight recorded in each group was within the range of 120 % of the group mean. Animals were handled according to the National Institute of Health (NlH) and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animals were housed in polysufone (PSU) cages (5/cage) measuring 42.5 >< 265.6 >< 18.5 cm, with stainless steel top grill having facilities for pelleted food and drinking water in glass- clear polycarbonate bottle; bedding: steam sterilized clean paddy husk (Harlan, Sani-chip, Cat#: 10688216) was used and bedding material was changed along with the cage at least twice a week.

Animals were fed ad libitum a commercial rodent diet (Teklad Certified Global 18 % Protein Diet cat #: 10688216). Animals had free access to autoclaved and acidi?ed drinking water (pH between 2.5 and 3.5) obtained from the municipality supply. Animals were housed under standard laboratory conditions, air conditioned and ?ltered (HEPA F6/6) with adequate fresh air supply (minimum 15 air changes/hour).

Animals were kept in a climate controlled environment. Temperatures range was 20-24°C and RH range was 30-70 % with 12 hours light and 12 hours dark cycle.

Surgical procedure On the day of surgery anesthesia was induced by 1.5 to 3.0 % isoflurane, 1.5 % N20 and 0.5 % Oz.

Under anesthesia, the mice were placed with ventral side up. A 0.5-1.0 cm incision was made in the skin in the inguinal area. The femoral artery was ligated proximally just after the distal part of the iliac artery and distally after its bifurcation with profound femoral artery with 6-0 silk thread, transected and excised between two ligatures. The wound was closed with 4-0 silk thread and the mice were allowed to recover.

Dextran sulfate treatment 17 538 144 On day 8, week 2 post-surgery, each animal in groups 2M and 3M were injected dextran sulfate solution s.c. three times a week. Animals in group 4M were injected s.c. once a week and group 1M received vehicle treatment (NaCI), see Table 1.

Table 1 - Group allocation Group Treatment Volume Route of administration 1M (n=15) Vehicle control 10 ml/kg 2M (n=15) Dextran sulfate 10 mg/kg 10 ml/kg s.c. repeated three times a week 3M (n=15) Dextran sulfate 30 mg/kg 10 ml/kg 4M (n=15) Dextran sulfate 30 mg/kg 10 ml/kg s.c. repeated once a week Body weigh measurements Body weight was measured on study day -1 prior to surgery and once a week thereafter. Between day 0 and day 7 a small reduction in mean body weight was observed in all animal groups 1M, 2M, 3M and 4M ranging from 1.1 g to 1.6 g, see Fig. 1. From day 14 onwards a gradual increase in weight was observed.

Accordingly, between day 0 and day 35 the mean increase in body weight ranged between 0.4 g to 1.7 g, see Table 2.

Table 2 - Mean change from baseline in body weight by study group Group Treatment Mean weight change from day 0 to day 35 (g) 1M Vehicle 3 times/week 0.4 2M Dextran sulfate 10 mg/kg 3 times/week 1.0 3M Dextran sulfate 30 mg/kg 3 times/week 1.7 4M Dextran sulfate 30 mg/kg 1 time/week 1.0 Blood flow measurements Blood flows in legs from both sides were measured with a non-contact laser Doppler before surgery on day -1 and on days: 1, 7, 14, 21, 28 and 35 post operation. Blood ?ow measurements was expressed as the ratio of the flow in the ischemic limb to that in the normal limb.

All animal groups 1M, 2M, 3M and 4M exhibited an increase in mean blood ?ow in the operated limb between day 1 and day 35, see Fig. 2. The mean blood ?ow increased from the baseline after surgery in the dextran sulfate treated group 2M (10 mg/kg 3 times/week) by 46.7 units; in the dextran sulfate treated group 3M (30 mg/kg 3 times/week) by 59.3 units and in the dextran sulfate treated group 4M (30 mg/kg 18 10 15 20 538 144 1 time/week) by 51.1 units compared to increase of 19.5 units in the vehicle control group 1M. This represents 2.2, 3.0 and 2.8 folds increase respectively in the mean blood flow due to dextran sulfate treatment, see Table 3 and Fig. 2.

Table 3 - Mean change in blood ?ow by study group Group Treatment Mean change in blood flow day 35 vs. day 1 1M Vehicle 3 times/week 19.5 2M Dextran sulfate 10 mg/kg 3 times/week 46.7 3M Dextran sulfate 30 mg/kg 3 times/week 59.3 4M Dextran sulfate 30 mg/kg 1 time/week 51 .1 Fig. 6 compares blood flow measured in a control mouse (group 1M) and a mouse treated with dextran sulfate according to group 3M. The ?gure shows non-contact laser Doppler images 35 days after femoral artery ligation of the left hind limb.

Macroscopic evaluation of ischemic severity Macroscopic evaluation of the ischemic limb was performed on day 7 and once a week thereafter by using morphological grades for necrotic area, see Table 4.

Table 4 - Morphological grades for necrotic area Grade 0 absence of necrosis Description 1 necrosis limiting to toe (toe loss) necrosis extending to a dorsum pedis (foot loss) necrosis extending to crus (knee loss) -ßOOIQ necrosis extending to a thigh (total hind-limb loss) The ischemic limb was macroscopically evaluated weekly from day 7 up to day 35 by using graded morphological scales for necrotic area, see Table 4. ln all animal groups treated with vehicle and dextran sulfate toe necrosis or foot amputation was found (graded from 1 to 2, see Table 6). Percent rates of foot amputation in each treatment group are displayed in Table 5 and Table 6. Foot amputation was found in vehicle treated control group 1M (15.4 %) and in dextran sulfate treated group 4M (30 mg/kg 1 time/week) (7.1 %). Toe necrosis rate in vehicle treated control group 1M was found to be 23.1 % animals. ln animal groups treated by dextran sulfate 2M and 3M, 35 days after HLl induction toe necrosis rate was 21.4 % 19 538 144 and 14.3 % respectively. ln animal group 4M treated by dextran sulfate no incidence of toe necrosis occurred (Table 6).

Table 5 - Incidence of mice with toe and limb necrosis on day 7 Group Incidence of mice with toe necrosis (%) Incidence of mice with limb amputation (%) 1 M 7.6 0.0 2i\/l 0.0 0.0 3M 0.0 0.0 4M 0.0 0.0 Table 6 - Incidence of mice with toe and limb necrosis on day 35 Group Incidence of mice with toe necrosis (%) Incidence of mice with limb amputation (%) 1 M 23.1 15.4 2M 21.4 0.0 3M 14.3 0.0 4M 0.0 7.1 In vivo assessment of limb function and ischemic damage Semi-quantitative assessment of impaired use of the ischemic limb was performed once a week post- 10 surgery using the following scale, see Table 7.

Table 7 - Assessments of limb function Grade Description 0 ?exing the toe to resist gentle traction of the tail 1 plantar ?exion 2 no dragging but no plantar ?exion 3 dragging of foot Limb function was graded as "Not applicab|e" in case of partial or full limb amputation. ln such case blood 15 ?ow measurements was not included in the statistical analysis. 20 538 144 ln parallel to the blood ?ow measurements and in comparison to the vehicle control group 1M, all dextran sulfate treated groups 2M, 3M and 4M exhibited better results in limb functional improvement, see Table 8 and Table 9 below. 5 Table 8 - lncidence of mice with limb function scores 0, 1, 2 and 3 on day 7 lncidence of mice lncidence of mice lncidence of mice lncidence of mice Group with limb function with limb function with limb function with limb function score 0 (%) score 1 (%) score 2 (%) score 3 (%) 1M 0.0 0.0 0.0 100.0 2M 0.0 0.0 21.4 79.6 3M 0.0 0.0 21.4 79.6 4M 0.0 0.0 7.1 92.9 Table 9 - lncidence of mice with limb function scores 0, 1, 2 and 3 on day 35 lncidence of mice lncidence of mice lncidence of mice lncidence of mice Group with limb function with limb function with limb function with limb function score 0 (%) score 1 (%) score 2 (%) score 3 (%) 1M 9.1 72.7 0.0 18.2 2M 50.0 42.9 7.1 0.0 3M 72.4 21.5 7.1 0.0 4M 69.2 30.8 0.0 0.0 lmmunohistochemistry and analysis of capillaries density 10 Mice were sacri?ced at study termination on day 36. Fluorescein isothiocyanate (FlTC) - dextran 500000 Da conjugate 10 mg/ml was injected i.v. in a dose of 200 ul per mouse 5 minutes before sacri?ce to all animals. Quadriceps muscle was dissected into coronary part. The muscle was ?xed in 2.5 % fresh paraformaldehyde (pH 7.4) for 24 hours and was then embedded in the paraf?n for smooth muscle actin (SMA) with mouse monoclonal antibodies (anti-SMA Ab-1, clone 1A4, 1:800, Thermo scienti?c) and 15 CD34 with anti CD34 (1 :200, Cedralene) immunostaining. Paraffin embedding was done according the standard embedding procedure.

Stained sections were evaluated and photographed by fluorescence microscope (E600; Nikon, Tokyo, Japan) equipped with plan ?uor objectives connected to a CCD camera (DMX1200F; Nikon). Under these 20 conditions Cy3 shows bright red ?uorescence: Ex (max): 543 nm; Em (max) 570 nm while ?uorescein 21 538 144 dextran shows intense green ?uorescence (Ex (max): 488 nm; Em (max): 530 nm). Digital images were collected and analyzed using Image Pro+ software. Four sections of muscle samples were taken from the same areas of eight animals from groups 1M, 3M and 4M. The area of blood vessels was measured.

Density was expressed as the mean number of capillaries per field of view. Total vessels represent all blood vessels in the measured area.

The number of CD34 positive capillaries was larger in dextran sulfate treated groups 3M and 4M compared to the control group 1M on day 35 of the study, see Figs. 3 and 4A-4C. CD34 positive staining is considered as an indication for small capillaries formation, and thus the obtained results support blood ?ow improvement observed in the animal groups treated with dextran sulfate. Dextran staining con?rmed that these capillaries are functioning and active. SMA staining revealed the same increase in capillaries formations as CD34 staining, see Fig. 5. lmpaired angiogenesis is one of the features of ischemic diseases. The most established target for therapeutic angiogenesis has been VEGF and its receptors. However, clinical trials to alleviate ischemia were disappointing, indicating the need for new therapeutic targets to treat ischemic diseases. ln the present study blood flow improvement in the mouse hind-limb ischemia model was examined in order to evaluate the ef?cacy of dextran sulfate. Repeated (three times a week) or (once a week) dextran sulfate administration at a dose of 30 mg/kg s.c. significantly restored blood perfusion compared to the vehicle treated control. On day 35, two and a half up to three folds higher blood ?ow perfusion values were observed in the dextran sulfate treated groups compared to the control group, with statistically significant effect starting fourteen days post treatment.

The collective data of the study confirmed the therapeutic efficacy of dextran sulfate given s.c. for the treatment of occlusive peripheral arterial disease in the Balb/c mouse animal model. Spontaneous amputations or toe necrosis rate also decreased in dextran sulfate treated animals compared to the control group. Dextran sulfate treatment improved limb functional restoration in all the drug treated animal group compared to the vehicle treated control. Dextran sulfate treatment did not cause any adverse effects in the treated animals. lmmunohistochemistry findings con?rmed the in vivo results. Collectively the data of this study con?rmed the therapeutic ef?cacy of dextran sulfate for the treatment of occlusive peripheral arterial disease in the mouse model. 22 10 15 20 25 30 538 144 Dextran sulfate treatment in mice with hind-Iimb ischemia resulted in a signi?cant and rapid recovery of blood ?ow, as measured by the laser Doppler and demonstrated also by decrease of limb ischemic severity and more rapid Iimb function improvement.

No adverse effect on general health was recorded in any of the groups. These data were confirmed by immunohistochemistry evaluation. The ?ndings re?ect changes in blood vessel morphology, i.e. capillaries density increase, and blood vessels angiogenesis.

Stroke The stroke tMCAO rat-model was used to evaluate the efficacy of dextran sulfate treatment. Rats were treated with dextran sulfate for 28 days via subcutaneous injections, starting at two hours after the surgical procedure, either at 30 mg/kg three times a week or at a daily dose of 15 mg/kg. During the study the neurological, motoric and somatosensory functions were monitored in a battery of behavioral tests.

Clear differences were demonstrated between the groups treated with dextran sulfate and the vehicle treated control group. Improvement in motor functions, as evaluated by Neuroscore, stepping test and body swing test, was demonstrated in both drug treated groups. Sensory motor functions also recovered following the dextran sulfate treatment. lt is likely that the effect of dextran sulfate treatments should be attributed to their angiogenic activity. This conclusion was supported by an increase in cerebral blood perfusion and Smooth Muscle Actin (SMA) positive capillaries density in the affected hemisphere.

Dextran sulfate treatment also reduced in?ammatory response compared to the vehicle treated control. ln view of these findings it may be concluded that dextran sulfate treatment clearly improved motor and somatosensory de?cits as well as cerebral blood perfusion and angiogenic activity in the rat stroke model.

Stroke is a prominent cause of serious, long-term disability and the third leading cause of death in the United States. Total health costs for disability due to stroke are estimated at 53.6 billion annually. lschemic strokes comprise over 88 % of all strokes, making them the most common type of cerebrovascular injury. lschemic conditions in the brain cause neuronal death, leading to permanent sensorimotor de?cits. lt is clear now that immediate treatment for stroke patients is often impossible in the clinical setting. Physicians need new treatment strategies for stroke treatments urgently. 23 10 15 20 25 30 538 144 Several animal models have been used to study cerebral ischemia in effort to understand its pathophysiology and to identify therapeutic strategies for minimizing the severity of ischemic damage.

Focal ischemia results in localized brain infarction and is induced by middle cerebral artery occlusion (MCAO) in the rat. lt has gained increasing acceptance as a model for hemispheric infarction in humans.

After MCAO a cortical and striatal infarct with temporal and spatial evolution occurs within the vascular territory supplied by the middle cerebral artery. ln the last decade growing evidence for behavioral assessment in the stroke animal studies have been collected. Functional improvement was found to be highly reliable as a measure for therapeutic ef?cacy.

One of the most promising innovative treatments for vascular complications in stroke is therapeutic angiogenesis, which emerged as a non-invasive mean for promoting neovascularization in ischemic tissues. ln this study the neuroprotective and rehabilitation potential of dextran sulfate was studied in transient MCAO rat-stroke model.

Materials Dextran sulfate with an average molecular weight within a range of 5-7 kDa was obtained from pK Chemicals A/S, Denmark.

Dextran sulfate was dissolved in 0.9 % NaCl (saline) (Teva Pharmaceutical Industries Ltd) to a concentration of 60 mg/ml for the three times a week injection and 30 mg/ml for the daily injection. The formulation is stable for one week. Animals received 0.5 ml/kg, equal to 30 and 15 mg/kg body weight. ln total 46 male SD rats having an average body weight of 342 at study initiation (day 0) were obtained from Harlan Laboratories, Israel. The minimal and maximal weight recorded in each group was within a range of :20 % of the group mean weight. Animals were handled according to guidelines of the National Institute of Health (NIH) and the Association forAssessment and Accreditation of Laboratory Animal Care (AAALAC). Animals were housed in polyethylene cages (5/cage) measuring 35 >< 30 >< 15 cm, with stainless steel top grill facilitating pelleted food and drinking water in plastic bottle; bedding: steam sterilized clean paddy husk (Harlan, Sani-chip cat#:2018SC+F) was used and bedding material was changed along with the cage at least twice a week. Animals were fed ad libitum a commercial rodent diet (Teklad Certi?ed Global 18 % Protein Diet cat #2 10688216). Animals had free access to autoclaved and acidified drinking water (pH between 2.5 and 3.5) obtained from the municipality supply. Animals were 24 10 15 20 25 30 538 144 housed under standard laboratory conditions, air-conditioned and ?ltered (HEPA F6/6) with adequate fresh air supply (Minimum 15 air changes/hour). Animals were kept in a climate-controlled environment.

Temperatures range was 20-24°C and RH range was 30-70 % with 12 hours light and 12 hours dark cycle.

Surgical procedure On the day of surgery anesthesia were induced with 4 % iso?urane in a mixture of 70 % N20 and 30 % Oz and maintained with 1.5-2 % iso?urane.

Transient middle cerebral artery occlusion was performed according to the method previously described in Stroke 29, 2162-2170 (1998). The right common carotid artery (CCA) was exposed through a midline neck incision and carefully dissected free from surrounding nerves and fascia -from its bifurcation to the base of the skull. The occipital artery branches of the external carotid artery (ECA) were then isolated, and these branches were dissected and coagulated. The ECA was dissected further distally and coagulated along with the terminal lingual and maxillary artery branches, which was then divided. The internal carotid artery (ICA) was isolated and carefully separated from the adjacent vagus nerve, and the pterygopalatine artery was ligated close to its origin with a 5-0 nylon suture (SMI, Belgium). Next a 4-0 silk suture was tied loosely around the mobilized ECA stump, and a 4 cm length of 4-0 mono?lament nylon suture (the tip of the suture was blunted by using a ?ame, and the suture was coated with polylysine, prior to insertion) was inserted through the proximal ECA into the ICA and thence into the circle of Willis, effectively occluding the MCA. The surgical wound was closed and the animals were returned to their cages to recover from anesthesia. Two hours after occlusion rats were re-anesthetized, monofilament was withdrawn to allow reperfusion, surgical wound was closed and rats were returned to their cages.

At one hour post occlusion, animals were subjected to neurological evaluation using the "Neuroscore for exclusion criteria". Only animals with an overall score of 2 10 were included in the study.

Dextran sulfate treatment Started two hours after occlusion (immediately after reperfusion), animals in groups 2M and 3M (dextran sulfate at doses of 30 mg/kg three times a week or 15 mg/kg daily) and animals in group 1M (vehicle control) were injected subcutaneously, see Table 10.

Table 10 - Group allocation 25 10 15 20 25 538 144 Group Treatment Dose Administration Treatment duration (days) 1 M (n=13) Vehicle 0 s.c. 3 times a 2M (n=15) Dextran sulfate 30 mg/kg week 28 3M (n=15) Dextran sulfate 15 mg/kg s.c daily Data Analysis Unless specified differently, all statistical analyses were performed using two-way ANOVA for repeated measures, followed by Bonferroni post-hoc comparison tests.

Body weights Throughout the study, no statistically significant differences in body weight were observed among the various treatment groups, see Fig. 7.

Neurological test score (Neuroscore) Evaluation: pre-operation, one hour after occlusion and on days 7, 14, 21 and 28 The Modi?ed Neurological Rating Scale (mNRS) was performed. The individual who made the behavioral assessments was unaware of the drug/dose given (blinded test). Neuroscore with total score 18 was performed according to Stroke 32, 1005-1011 (2001).

The Neuroscore included a set of clinical-neurological tests (composite of motor, sensory, re?ex and balance tests) that were used to assess the effect of the tested treatments. Neuroscore was graded on a scale of 0 to 18 (in which normal score is 0 and maximal de?cit score is represented by 18). As expected, in all groups of rats a sharp decline in neurological functions was observed two hours after tMCAO induction, with spontaneous improvement over time thereafter. Statistically signi?cant differences were exhibited in groups 2M treated with 30 mg/kg dextran sulfate three times per week and 3M treated with 15mg/kg dextran sulfate daily, as compared to the vehicle treated control, from the ?rst test on day 7 throughout the study up to day 28, see Fig. 8. No statistical difference was found between the two dosing schedules 2M and 3M.

Stepping Test Evaluation: pre-operation and on days 7, 14, 21 and 28 Animals were tested for forelimb akinesia using the stepping test. The animal was held with its hind limbs and one forelimb ?xed with one hand and the unrestrained fore-paw was drawn along the table. The 26 10 15 20 25 30 538 144 number of adjusting steps were counted while the animal was moved sideways along the table surface (85 cm in approximately ?ve seconds), in the forehand and backhand direction for both forelimbs.

Animals were tested for forelimb akinesia in the stepping test, commonly used for measurement of neuromuscular function, as an index for motoric function of the animal. Some improvement in motor function over time was observed in all the animals that were subjected to tMCAO, mostly as a result of spontaneous functional recovery. However, functional improvement in rats treated with dextran sulfate was more pronounced compared to vehicle treated controls. ln both treated groups of animals, group 2M and 3M, this improvement reached statistical signi?cance compared to control starting on the ?rst test on day 7 and continued to improve up to study termination on day 28, see Fig. 9. No statistical difference was found between the two dosing schedules 2M and 3M.

Forelimb Placing Evaluation: pre-operation and on days 7, 14, 21 and 28 For the forelimb-placing test, the examiner holds the rat close to a tabletop and scores the rat's ability to place the forelimb on the tabletop in response to whisker, visual, tactile, or proprioceptive stimulation.

Separate sub-scores were obtained for each mode of sensory input and added to give total scores (0 = normal, 12 = maximally impaired).

Forelimb placing test (0-12): Whisker placing (0-2); Visual placing (fon/vard (0-2), sideways (0-2)) Tactile placing (dorsal (0-2), lateral (0-2)) Proprioceptive placing (0-2).

For each subtest, animals were scored as followed: 0.0 = immediate response 0.5 = response within 2 seconds 1.0 = response of 2-3 seconds 1.5 = response of >3 seconds 2.0 = no response Forelimb placement test was used to assess somatosensory and sensory motor deficits. Similar to the other tests, some spontaneous improvement in sensory motor de?cits over time was observed in all animals that were subjected to tM CAO. However, all rats treated with dextran sulfate exhibited statistically 27 10 15 20 25 30 538 144 signi?cant improvement compared to vehicle control treatment, starting on day 14 and Iasting up to study termination on day 28, see Fig. 10. ln group 3M improvement in sensory motor de?cits reached statistical significance already at the ?rst testing on day 7.

Body swing test Evaluation: pre-operation and on days 7, 14, 21 and 28 The rat was held approximately one inch from the base of its tail. lt was then elevated to an inch above a surface of a table. The rat was held in the vertical axis, defined as no more than 10° to either the left or the right side. A swing was recorded whenever the rat moves its head out of the vertical axis to either side. Before attempting another swing, the rat was returned to the vertical position for the next swing to be counted. Twenty total swings were counted. A normal rat typically has an equal number of swings to either side. Following focal ischemia, the rat tends to swing to the contralateral side (left side in this case).

Body swing scores were expressed as a percentage of rightvvard over total swings. There was a spontaneous partial recovery of body swing scores (toward 50 %) during the first month after stroke.

Animals were tested for forelimb akinesia in the body swing test, commonly used for measurement of neuromuscular functions. Some spontaneous improvement in motor function over time was observed in all animals that were subjected to tMCAO. However, all rats treated with dextran sulfate exhibited statistically significant improvement compared to vehicle control treatment, starting on the ?rst test on day 7 and Iasting up to study termination on day 28, see Fig. 11. No statistical difference was found between the two dosing groups 2M and 3M.

Cerebral Blood Flow Assessment Evaluation: day 29 Evaluation of the blood ?ow on the brain cortex and vessel constriction was carried out using Flow-R Laser Doppler system, in which intracranial blood ?ow and vessels diameter (constriction/dilatation) was monitored. This was carried out on day 29 post stroke initiation. Doppler procedure was performed while the animals were under isoflurane anesthesia.

Animals were also examined for cerebral blood ?ow restoration at the damaged hemisphere on day 29.

Statistically signi?cant improvement in cerebral blood perfusion rate was observed in all animals that were subjected to tMCAO and treated with dextran sulfate (group 2M and 3M) versus control vehicle- treated group 1 M. Vessels diameter ratio also increased in dextran suflate treated animals versus control, see Figs. 12 and 13. 28 10 15 20 25 30 538 144 Samples Collection and Sacrifice On day 30 after MCAO, rats were anesthetized by ketamine/xylazine were transcardiacly perfused by buffered paraformaldehyde (PFA) 4 %. The brains were collected ?xed in 4 % buffered PFA for immunostaining and histological evaluation.

Two sections of brain samples were taken from the same areas of six animals from groups 1M and 3M.

Capillaries were counted under the microscope in a total of three random ?elds from each section. Density was expressed as the mean number of capillaries per ?eld of view. Treatment with dextran sulfate 15mg/kg daily increased the number of capillaries 30 days after stroke, as compared to the vehicle treated control group.

Improvement in the number of SMA capillaries with diameter less than 30 pm was observed in animals that were su bjected to tMCAO, and treated with dextran sulfate 15 mg/kg daily as compared to vehicle treated control group, as a result of angiogenic effect, see Fig. 14.

Mortality and clinical signs Eighteen rats died during the study. One rat died just after reperfusion before dosing, and seventeen rats within 10 hours after dosing (6 in group 1M, 6 in group 2M and 5 in group 3M). No adverse clinical signs unrelated to the model were observed in all the animal groups.

The stroke tMCAO rat-model is traditionally an accepted model for evaluating the neuroprotective and rehabilitation efficacy of drug treatments. This model was used in the present study to evaluate the efficacy of dextran sulfate treatment at two dosing schedules. Rats were treated with dextran sulfate for 28 days via subcutaneous injections, starting at tvvo hours after the surgical procedure, either at 30 mg/kg three times a week or at a daily dose of 15 mg/kg. During the study the neurological, motoric and somatosensory functions were monitored in a battery of behavioral tests.

As expected, some spontaneous recovery of neurological functions was observed during the 28 days follow-up after stroke induction. However, clear differences were demonstrated between the groups treated with dextran sulfate and the vehicle treated control group. No statistically signi?cant differences were noted however between the two dosing schedules. Improvement in motor functions, as evaluated by Neuroscore, stepping test and body swing test, was demonstrated in both drug treated groups (Figs. 8, 9 and 11). Sensory motor functions also recovered following the dextran sulfate treatment (Fig. 10). 29 10 15 538 144 The bene?cial effects were observed starting on the ?rst testing on day 7 of the treatment and continued to improve till study termination on day 28. The observed effects cannot be attributed to differences in general rats' health as all groups gained weight at the same rate with no significant differences between them (Fig. 7). ln addition, no observed differences in general clinical signs were noted. lt is likely that the effect of dextran sulfate treatments should be attributed to their angiogenic activity. This conclusion was supported by an increase in cerebral blood perfusion and SMA positive capillaries density in the affected hemisphere. Dextran sulfate treatment also reduced in?ammatory response compared to the vehicle treated control. ln view of these findings it may be concluded that dextran sulfate treatment clearly improved motor and somatosensory de?cits as well as cerebral blood perfusion and angiogenic activity in the rat stroke model.

The embodiments described above are to be understood as a few illustrative examples of the present invention. lt will be understood by those skilled in the art that various modi?cations, combinations and changes may be made to the embodiments without departing from the scope of the present invention. ln particular, different part solutions in the different embodiments can be combined in other con?gurations, where technically possible. The scope of the present invention is, however, de?ned by the appended claims. 30

Claims (17)

538 144 PATE NTKRAV

1. Dextransulfat, eller ett farmaceutiskt acceptabelt derivat darav, som har en medelmolekylvikt lika med eller under 10 000 Da for anvandning for att inducera angiogenes i en manniskopatient som lider av en sjukdom, rubbning eller medicinskt tillstand som orsakar ischemi i manniskopatientens kropp.

2. Dextransulfat, eller ett farmaceutiskt acceptabelt derivat darav, som har en medelmolekylvikt lika med eller under 10 000 Da for anvandning for att Oka blodflode i en patient som lider av ischemi.

3. Dextransulfat, eller det farmaceutiskt acceptabla derivatet darav, for anvandning enligt patentkrav 10 2, for anvandning for att Oka blodflode i en ischemisk vavnad eller organ hos patienten.

4.

5. Dextransulfat, eller ett farmaceutiskt acceptabelt derivat darav, som har en medelmolekylvikt lika med eller under 10 000 Da for anvandning for vaskularisering av ischemisk vavnad i en patient. 15 5.Dextransulfat, eller det farmaceutiskt acceptabla derivatet darav, for anvandning enligt nagot av patentkrav 1 till 4, van i medelmolekylvikten är inom ett intervall pa 2 000 och 10 000 Da.

6. Dextransulfat, eller det farmaceutiskt acceptabla derivatet darav, for anvandning enligt patentkrav 5, van i medelmolekylvikten är inom ett intervall pa 3 000 och 10 000 Da.

7. Dextransulfat, eller det farmaceutiskt acceptabla derivatet darav, for anvandning enligt patentkrav 6, van i medelmolekylvikten är inom ett intervall pa 3 500 och 9 500 Da.

8. Dextransulfat, eller det farmaceutiskt acceptabla derivatet darav, for anvandning enligt patentkrav 7, van i medelmolekylvikten är inom ett intervall pa 4 500 och 7 500 Da.

9. Dextransulfat, eller det farmaceutiskt acceptabla derivatet darav, for anvandning enligt patentkrav 8, van i medelmolekylvikten är inom ett intervall pa 4 500 och 5 500 Da.

10. Dextransulfat, eller det farmaceutiskt acceptabla derivatet darav, for anvandning enligt nagot av patentkrav 1 till 9, van i dextransulfatet, eller det farmaceutiskt acceptabla derivatet darav, har ett medelsvavelinnehall inom ett intervall fran 15 till 20 %. 538 144

11. Dextransulfat, eller det farmaceutiskt acceptabla derivatet darav, for anvandning enligt patentkrav 10, van i medelsvavelinnehallet är ungefar 17 %.

12. Dextransulfat, eller det farmaceutiskt acceptabla derivatet darav, for anvandning enligt nagot av patentkrav 1 till 11, van i dextransulfatet, eller det farmaceutiskt acceptabla derivatet darav, är formulerat som en vattenhaltig injektionslosning.

13. Dextransulfat, eller det farmaceutiskt acceptabla derivatet darav, for anvandning enligt nagot av patentkrav 1 till 11, van i dextransulfatet, eller det farmaceutiskt acceptabla derivatet darav, är formulerat for att administreras vid en dos inom ett intervall fran 0,05 till 30 mg/kg kroppsvikt hos patienten, foretradesvis fran 0,1 till 25 mg/kg kroppsvikt hos patienten, och annu hellre fran 0,1 till 15 mg/kg eller 0,1 till 10 mg/kg kroppsvikt hos patienten.

14. Dextransulfat, eller det farmaceutiskt acceptabla derivatet darav, for anvandning enligt nagot av patentkrav 2 till 13, van i patienten är en manniskopatient som lider av en sjukdom, rubbning eller medicinskt tillstand som orsakar ischemi i manniskopatientens kropp.

15. Dextransulfat, eller det farmaceutiskt acceptabla derivatet darav, for anvandning enligt patentkrav 1 eller 14, van i sjukdomen, rubbningen eller det medicinska tillstandet är valt fran en grupp som bestar av sarlakning; perifer ischemi, foretradesvis ischemi efter transplantation av organ, vavnad och/eller celler, perifer arteriell sjukdom, extremitetsischemi, Raynauds syndrom, sicklecellsjukdom eller tromboangiitis obliterans; koronarischemi, foretradesvis kronisk hjartsvikt eller en kranskarlssjukdom; ischemi i centrala nervsystemet, foretradesvis traumatisk hjarnskada, temporalisarterit, hypoxi orsakad av multipel skleros, stroke, amyotrofisk lateralskleros; muskeldystrofier.

16. Dextransulfat, eller det farmaceutiskt acceptabla derivatet darav, for anvandning enligt nagot av patentkrav 1 till 13, van i det farmaceutiskt acceptabla derivatet darav är ett salt av dextransulfat, foretradesvis eller natriumsalt av dextransulfat.

17. Dextransulfat, eller ett farmaceutiskt acceptabelt derivat darav, som har en medelmolekylvikt lika med eller under 10 000 Da for in vitro- eller ex vivo-anvandning for att 6ka blodflode i en vaskulariserad vavnad och/eller organ. som har en medelmolekylvikt lika )n vaskulariserad vavnad och/eller Body weight gain (g) 26, 26,0 25, 25,0 24, 24,0 23, 23,0 - 0 Time (days) —E9— Vehicle control --- TM-700 30mg/kg 3 times per week TM-700 30mg/kg once a week -TM-700 10mg/kg 3 times per week