USOO9572817B2

(12) United States Patent (10) Patent No.: US 9,572,817 B2 Sieveking et al. (45) Date of Patent: Feb. 21, 2017

(54) USE OF ANDROGENS FOR VASCULAR Zampetaki et al. Vascular repair by endothelial progenitor cells. REGENERATION AND ENDOTHELLAL Cardiovascular Research (2008) 78, 413-421.* REPAIR Ray et al. Sex Steroids and Stem Cell Function. Mol. Med. (2008) 14 (7-8):493-501.* Kawamoto et al. Therapeutic Potential of Ex Vivo Expanded (75) Inventors: Daniel Peter Sieveking, Summer Hill Endothelial Progenitor Cells for Myocardial Ischemia. Circulation. (AU); Martin Kean Chong Ng. 2001; 103:634-637.* Mosman (AU) Foresta et al. Androgens stimulate endothelial progenitor cells through an androgen receptor-mediated pathway. Clinical Endocri (73) Assignee: THE HEART RESEARCH nology (2008) 68, 284-289.* INSTITUTE LTD., Newtown (AU) Zhao et al. Autologous endothelial progenitor cells transplantation promoting endothelial recovery in mice. Transplant International 20 (*) Notice: Subject to any disclaimer, the term of this (2007) 712-721.* patent is extended or adjusted under 35 Kang et al. Effect of Oral Administration of Testosterone on U.S.C. 154(b) by 961 days. Brachial Arterial Vasoreactivity in Men With Coronary Artery Disease. The American Journal of Cardiology vol. 89 Apr. 1, 2002. (21) Appl. No.: 13/002,382 869-864. Englsih et al. Low-Dose Transdermal Testosterone Therapy (22) PCT Filed: Jul. 4, 2008 Improves Angina Threshold in Men With Chronic Stable Angina A Randomized, Double-Blind, Placebo-Controlled Study. Circulation. (86). PCT No.: PCT/AU2O08/OOO993 2000; 102:1906-1911.* Mulligan et al. Prevalence of hypogonadism in males aged at least S 371 (c)(1), 45 years: the HIM study. IntJ Clin Pract, Jul. 2006, 60, 7,762-769.* (2), (4) Date: Oct. 17, 2011 Buckle et al., “Hyperglycaemia Inhibits. Thioredoxin-Medicated Angiogenesis: Implications for Impairment of Neovascularisation (87) PCT Pub. No.: WO2010/000011 in Diabetes Mellitus.” Heart, Lung and Circulation, Elsevier, 2007. PCT Pub. Date: Jan. 7, 2010 pp. S211-S217, vol. 16. Chu, K. et al., “Circulating Endothelial Progenitor Cells as a New (65) Prior Publication Data Marker of endothelial Dysfunction or Repair in Acute Stroke.” Stroke, 2008, pp. 1441-1447, vol. 39. US 2012/OO39851 A1 Feb. 16, 2012 Dunn, L. et al., “Gene Silencing of Thioredoxin Interacting Protein Dramatically Rescues Diabetes-Related Impairment of (51) Int. Cl. Angiogenesis in Vitro and in Vivo.' Heart, Lung and Circulation, A6 IK3I/56 (2006.01) Elsevier, 2009, pp. S243, vol. 18. A6 IK3I/568 (2006.01) Dunn, L. et al., “Rescue of Diabetes-Related Impairment of A6 IK3I/5685 (2006.01) Angiogenesis by Gene Silencing of Thioredoxin-Interacting Pro (52) U.S. Cl. tein.” Heat Lung and Circulation, Elsevier, 2008, S3-S4, vol. 17. CPC ...... A61 K3I/56 (2013.01); A61K 3 1/568 Dunn, L. et al., “Type 1 Diabetes Mellitus is Associated with (2013.01); A61 K3I/5685 (2013.01) Impairment of Late Outgrowth Endothelial Progenitor Cell Func (58) Field of Classification Search tion.” Heart, Lung and Circulation, Elsevier, 2008, pp. S138, vol. CPC ... A61K 3 1/56; A61K 3 1/568: A61K 31/5685 17. See application file for complete search history. Marumo, T. et al., “Aldosterone Impairs Bone Marrow-Derived Progenitor Cell Formation.” Hypertension, 2006, pp. 490-496, vol. (56) References Cited 48. (Continued) U.S. PATENT DOCUMENTS 6,017,924. A * 1/2000 Edwards et al...... 514,292 6,583,129 B1* 6/2003 Mazer et al...... 514,167 Primary Examiner — Taeyoon Kim 2002fOO65273 A1 5, 2002 Chein (74) Attorney, Agent, or Firm — Fenwick & West LLP 2003. O1994.64 A1 10, 2003 Itescu 2005/0233992 A1 10, 2005 Itescu FOREIGN PATENT DOCUMENTS (57) ABSTRACT EP 1210951 B1 2, 2005 The present invention relates to the field of therapy and WO WO O3,O2O329 A1 3, 2003 WO WO 2006/078853 A2 T 2006 prophylaxis of vascular tissue damage and/or complications WO WO 2007/035451 A2 3, 2007 and/or disease of vascular tissue by vascular regeneration WO WO 2007/149548 A2 12/2007 and endothelium repair. The inventors have found that administration of an androgen receptor agonist alleviates OTHER PUBLICATIONS one or more adverse vascular diseases and/or vascular complications thereof or adverse effects of androgen defi Rosano et al. Acute Anti-Ischemic Effect of Testosterone in Men ciency, with implications for prophylactic and therapeutic With Coronary Artery Disease. Circulation. 1999;99:1666-1670.* interventions. Webb et al. Effects of Testosterone on Coronary Vasomotor Regu lation in Men With Coronary Heart Disease. Circulation. 1999; 100:1690-1696.* 15 Claims, 12 Drawing Sheets US 9,572.817 B2 Page 2

(56) References Cited

OTHER PUBLICATIONS PCT International Search Report and Written Opinion, PCT Appli cation No. PCT/AU2008/000993, Aug. 14, 2009, 9 pages. Schulze, P.C. et al., “Hyperglycemia Promotes Oxidative Stress Through Inhibition of Thioredoxin Function by Thiordexin-Inter acting Protein.” Journal of Biological Chemistry, 2004, pp. 30369 30374, vol. 279, No. 29. Werner, G.S. et al., “Direct Assessment of Coronary Steal and Associated Changes of Collateral Hemodynamics in Chronic Total Coronary Occlusions.” Circulation, Jul. 23, 2002, pp. 435-440, vol. 106. * cited by examiner U.S. Patent Feb. 21, 2017 Sheet 1 of 12 US 9,572,817 B2

Fig la

Effect of DHT on EC Migration (Scratch)

rer

50 U.S. Patent Feb. 21, 2017 Sheet 2 of 12 US 9,572,817 B2

Fig 1b Effect of DHT on Female EC Migration (Scratch Assay)

U.S. Patent Feb. 21, 2017 Sheet 3 of 12 US 9,572,817 B2

Fig 2a

Effect of DHT on Male EC Proliferation

140

8 O

60

4. O

2 O U.S. Patent Feb. 21, 2017 Sheet 4 of 12 US 9,572,817 B2

Fig 2b

Effect of C or Ferae EC Proliferation

30 U.S. Patent Feb. 21, 2017 Sheet S of 12 US 9,572,817 B2

Fig 3a

Effect of DHT or The FOrator

BO 160 4 120 100.

50

k o so so w * w N s SS' 3. s sw S s U.S. Patent Feb. 21, 2017 Sheet 6 of 12 US 9,572,817 B2

Fig 3b Effect of DHT on Female EC Tubulogenesis (Matrigel)

U.S. Patent Feb. 21, 2017 Sheet 7 of 12 US 9,572,817 B2

Fig. 4

U.S. Patent Feb. 21, 2017 Sheet 8 of 12 US 9,572,817 B2

1.3 1.2 -- Control 1.1 rur-Control 4 OHT indu-Castrate 10 -- Casata + DHT 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 D.0 Pre-Op Post-Op d3 d5 d7 d9 11 14 U.S. Patent Feb. 21, 2017 Sheet 9 of 12 US 9,572,817 B2

Fig 5b

2.50 -- Contro 2.25 -- Contro + DHT -- Castrate 2.00 i-- Castrate + DHT

1.75 150 1.25 1.00 0.75 0.50 0.25 0.00 Preop d3 d5 7 d9 d11 14 U.S. Patent Feb. 21, 2017 Sheet 10 of 12 US 9,572,817 B2

Fig 5c

AA 2.00 --Control and Control+DHT 1.75 avan Castrate

-- Castake + OHT

25 O10..50 7 O0.5 0.25 HP = 0.015 0.00 Preop d3 d5 d7 d9 d11 d4 U.S. Patent Feb. 21, 2017 Sheet 11 of 12 US 9,572,817 B2

Fig 6a

Spleen Angiogenic Cells

Non-schaemic lschaemic

U.S. Patent Feb. 21, 2017 Sheet 12 of 12 US 9,572,817 B2

Fig 6b

Bone Marrow Angiogenic Cells

Non-schaemic 80 ischaemic 70 60

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US 9,572,817 B2 1. 2 USE OF ANDROGENS FOR VASCULAR become narrow, blood flow to the heart can slow down or REGENERATION AND ENDOTHELIAL stop, causing chest pain, shortness of breath, heart attack, REPAIR and other symptoms. Recent clinical research has included investigation of the FIELD OF THE INVENTION therapeutic benefit of mobilising endothelial progenitor cells (EPCs). Hill et al., “Outcomes and Risks of Granulocyte The present invention relates to the field of therapy and Colony-Stimulating Factor in Patients with Coronary Heart prophylaxis of vascular tissue damage and/or complications Disease'. Journal of the American College of Cardiology, and/or disease of vascular tissue by vascular regeneration Vol. 46, No. 9 (2005) investigated vascular progenitor cells 10 for the potential for cardiovascular repair following injury. and endothelial repair. Hill et al., found that although investigators using the BACKGROUND OF THE INVENTION hind-limb ischaemia model and carotid injury model and a small clinical trial had suggested favourable effects of a Angiogenesis/Vasculogenesis cytokine mobilization approach, no progenitor cell mobili 15 zation was observed. Hill et al., concluded that administra tion of granulocyte colony-stimulating factor to CAD Angiogenesis and vasculogenesis are important processes patients mobilizes CD34+/CD133+ progenitor cells into the in animal development. A large body of evidence indicates circulation, however no objective evidence was obtained that angiogenesis and vasculogenesis play a critical role in that there was any cardiac benefit. physiological processes such as wound healing and tissue Further research in the area of angiogenesis and cardio vascularisation in response to injury and ischaemia, and the vascular regeneration is essential to the treatment of isch enhancement of collateral circulation. The development of emic heart disease. With economic development and the coronary collateral circulation in response to coronary associated decline in communicable diseases globally, car ischaemia ameliorates adverse outcomes of ischemic heart diovascular disease and in particular ischemic heart disease disease, such as acute myocardial infarction, angina and 25 is now the leading cause of mortality worldwide and is heart failure. Myocardial viability after acute myocardial expected to remain so well into the twenty first century. infarction correlates with the extent of collateral blood flow Coronary Vascular Surgery with the affected vascular segment. Therapeutic neovascu Both Surgical revascularization (coronary artery bypass larisation is therefore considered as a very promising grafting) and percutaneous coronary intervention (PCI) are approach to prevent the serious clinical consequences (heart 30 established treatment options for coronary heart disease. failure, gangrene, neuronal disability) of abnormal tissue However, a major drawback of PCI is damage to the remodelling in patients suffering from cardiovascular isch endothelium caused by the stent. emic disease. Expansion of a stent in a target artery induces local injury The mechanisms for ischaemia-induced collateral forma of the vessel wall, primarily from the disruption of the tion, however, are poorly understood. 35 endothelial lining (the endothelium is a layer of flat cells Heart Failure lining the closed internal spaces of the body Such as the Heart failure, characterized by a severe deficiency in inside of blood vessels and lymphatic vessels and the heart). Ventricular pump function, is a major public health problem. Following the injury, the reparatory mechanisms are acti It arises through a number of intrinsic factors and extrinsic vated leading to recovery of the endothelial coverage over factors. An example of one such extrinsic factor includes 40 the stent struts. myocyte loss that is unmatched by myocyte replacement, Disruption of the endothelium causes the activation and frequently the end result of cardiovascular disease. Replace adherence of platelets and recruitment of the monocytes and ment and/or regeneration of functional cardiac muscle after leukocytes. The time that elapses between the endothelial an ischemic insult to the heart is thought possible by disruption caused by the expanded stent and full coverage of stimulating proliferation of endogenous mature cardiomyo 45 the struts with new endothelial cells carries the highest risk cytes or resident cardiac stem cells, by implanting exog of in-stent thrombosis. At the same time the initial events of enous donor-derived or allogeneic cells, or by Supplement in-stent restenosis also occur, primarily migration and pro ing the angiogenic mechanisms that occur naturally using liferation of smooth muscle cells. The key event in rebuild defined growth factors or vessel forming cells (Dimmeler, S. ing the endothelial layer over the stent struts is the recruit etal “Unchain my heart: the scientific foundations of cardiac 50 ment of circulating endothelial progenitor cells (EPC), their repair”J Clin Invest. Vol. 115, No. 3, 2005; and Orlic, D. et adherence and attachment to the Surface of the stent and al “Mobilized bone marrow cells repair the infarcted heart, vascular wall between the struts. The full coverage of the improving function and survival. Proc Natl AcadSci USA. prothrombotic metal struts with new endothelial cells Vol. 98, No. 18, 2001). reduces the initially high risk of thrombosis. In addition to the heart, other end-organ targets of car 55 Limb Ischaemia diovascular disease include the kidney and the brain. The Critical limb ischaemia, the most severe form of periph endothelial layer is a key regulator of blood flow to these eral vascular disease, leads to over 160,000 major limb organs. As such, maintenance of the endothelium is thought amputations per year. Therapeutic options are limited and necessary for the prevention of end-organ effects such as largely ineffective for the most severe patients. Vascular kidney disease and stroke. 60 regeneration is one potential option to repair and regenerate Coronary Artery Disease and Ischemic Heart Disease the ischemic tissues of these patients to improve the blood The disease process underlying most ischemic heart dis flow in the affected areas. ease is atherosclerosis of the coronary arteries (coronary Any discussion of documents, acts, materials, devices, heart disease). Atherosclerosis is a disease whereby plaque articles or the like which has been included in the present builds up on the inside of arteries. This fatty material 65 specification is solely for the purpose of providing a context thickens, hardens and may eventually block the artery. This for the present invention. It is not to be taken as an admission makes it harder for blood to flow. If the coronary arteries that any or all of these matters form part of the prior art base US 9,572,817 B2 3 4 or were common general knowledge in the field relevant to regeneration. As used herein vascular regeneration is to be the present invention as it existed before the priority date of understood in its broadest sense to also include other repair each claim of this application. processes including endothelial cell migration, proliferation and mobilisation. SUMMARY OF THE INVENTION The term “endothelial progenitor cell' or “EPC” shall be construed in its broadest context to mean a cell that, when 1. Background functional or normal, is capable of proliferating to form blood vessels e.g., in the formation of a vascular network or in vascular repair, Such as by angiogenesis and/or neovas In work leading to the present invention, the inventors cularisation. EPCs may be functional, non-functional, or sought to investigate the role of androgens (and their with 10 have impaired function (for example with respect to this drawal) in the maintenance of vascular homeostatic and proliferative ability and developmental capability), and non regenerative processes. The inventors sought to investigate functional EPCS or EPCs having impaired function may be the role of androgens in vascular disease and injury and in identified readily (for example by their aberrant morphology particular on the cardiovascular response to ischaemia and and impaired proliferative ability). vascular injury. 15 The term “endothelial cell or “EC as used herein is to The inventors made the following hypothesis: be construed in its broadest sense to refer to a cell capable (i) androgens have an effect on the rate and extent of of forming the endothelium. EPCs are understood t have an vascular regeneration including neovascularisation and ability to differentiate into endothelial cells. angiogenesis, The inventors studied the effects of androgens on three (ii) androgens effect the rate and extent of endothelial key vascular repair (angiogenic) processes: Vascular tubu progenitor cell (EPC) mobilisation and function, and logenesis, endothelial cell migration, and endothelial cell (iii) there are gender dependent differences in the androgenic proliferation. The investigations were conducted in both in regulation of vascular genesis. vitro and in vivo settings. The inventors hypothesised that their findings would have In Summary, the present inventors have demonstrated that implications for understanding of the repair of the vascular 25 androgens promote angiogenesis and neovascularisation system and its end organs (such as for example the heart or processes and this has important implications for Subjects kidneys) in response to aging, disease and injury and in with impaired vascular function or vascular disease and/or vascular complications thereof, and that administration of an particular the repair and regeneration of the cardiovascular androgen has utility in preventing one or more vascular system. diseases and/or vascular complications thereof from arising The inventors hypothesised that their findings would have 30 or developing and/or reducing the severity of vascular important implications for understanding of vascular effects diseases and/or vascular complications thereof and/or of the age-related decline in circulating androgens and enhancing Vascular repair e.g., by promoting, inducing or gender differences in outcomes following coronary disease enhancing vascular repair through tubulogenesis, angiogen or complications as a result thereof. esis and neovascularisation processes. The inventors further hypothesised that their findings 35 As exemplified herein, the inventors have shown that in would have important implications for understanding of vitro androgen administration induced dose-dependent male androgen deficiency and androgen therapy particularly older EC migration and a dose-related increase in EC prolifera subjects. The inventors theorized that knowledge of the tion. Chronic exposure to androgens also increased tubule androgen effects on vascular regeneration and EPC function formation. will provide a better understanding of gender differences in 40 As exemplified herein, the inventors demonstrated in vivo vascular diseases and vascular injury and will guide efforts that male castration markedly decreased in vivo Vasculari to exploit the wider therapeutic benefit of androgens in sation processes and furthermore that androgen replacement vascular therapies. reversed castration-effects on angiogenesis. As used herein the terms to “promote vascular repair or As exemplified herein, the inventors demonstrated in vivo "enhance vascular repair shall be construed in their broad 45 that male castration markedly inhibited the rate of recovery est context to include the initiation or enhancement or from hind limb ischaemia. Androgen replacement not only improvement of any process that enhances the ability of a reversed castration-effects, but also accelerated recovery in cell to contribute to vascular repair and/or induces, initiates, hind limb ischaemia. These results were also mirrored in derepresses or otherwise enhances vascular repair for clinical evaluations of ischemic damage and motor function. example by augmenting a cells capacity for vascular repair 50 These findings Suggest that repair and/or regeneration of and/or angiogenesis and/or neovascularisation. damaged (or aged) endothelium can be augmented by andro As used herein, the term “angiogenesis' shall be taken to gens. These findings Support the hypothesis that androgens mean a process by which new blood vessels are formed participate in promotion of vascular repair and has particular whether from extant blood vessels or not and/or the enlarge implications for cardiovascular regeneration and the role of ment of pre-existing blood vessels. 55 androgen replacement in Subjects deficient of androgens, As used herein, the term “neovascularization' shall be especially men. taken to mean a development of new blood vessels in tissues Taken together, these findings by the inventors have not possessing extant blood vessels. Neovascularization in indicated that administration of an androgen receptoragonist adults is thought to be a mixed phenomenon of angiogenesis alleviates one or more adverse vascular diseases and/or and vasculogenesis. Vasculogenesis is understood to be the 60 vascular complications thereof or adverse effects of andro differentiation of endothelial progenitor cells or stem cells or gen deficiency, with implications for prophylactic and thera cells capable of adopting an endothelial lineage to endothe peutic interventions. lial cells (ECs) and Subsequent formation of primary vas cular plexus. 2. Specific Embodiments Angiogenesis and neovascularisation are understood to be 65 important processes of vascular regeneration and may col The scope of the invention will be apparent from the lectively or alternatively be referred to herein as vascular claims as filed with the application that follow the examples. US 9,572,817 B2 5 6 The claims as filed with the application are hereby incor vascular repair The cell is functional with respect to vascular porated into the description. The scope of the invention will repair so as to be capable of participating in one or more also be apparent from the following description of specific vascular repair processes, such as: 1) direct and/or indirect embodiments. participation in angiogenesis, vasculogenesis and/or neovas The present invention relates to methods of therapy which cularization; 2) direct and/or indirect participation in promote vascular repair and regeneration. The present endothelial repair and regeneration and; 3) direct and/or invention also provides a use of an androgen receptor indirect participation in end organ recovery and repair agonist in the preparation of a medicament for conditions following ischaemia Such as myocardial preservation and/or which benefit from promoting vascular repair and regenera regeneration following myocardial infarction. The cell can tion. 10 The methods are particularly useful for example: be functional by virtue of being functional in nature, or by to promote angiogenesis and neovascularisation in a Sub virtue of having been genetically-modified or genetically ject in need thereof repaired. to treat and prevent diseases and ailments involving Examples of cells include for example endothelial pro angiogenesis, such as cardiovascular disease, myocar 15 genitor cells, outgrowth endothelial cells, endothelial colony dial or cerebral infarctions, mesenteric or limb ischae forming cells, bone marrow mononuclear cells, hemopoietic mia, wounds and vascular occlusion or Stenosis stem cells, erythroid progenitors and colon forming units, to accelerate wound healing, or the vascularisation of a mesenchymal stem cells, cardiac stem cells, embryonic stem skin graft, musculocutaneous flap or other Surgically cells, any stem or progenitor cell capable of adopting a transplanted tissue, or to enhance the healing of Surgi vascular cell lineage, pluripotent or progenitor cells induced cally created anastomosis, and from differentiated cells, side population cells, satellite cells, to promote endothelial recovery after injury for example myoblasts, CD 34 positive and/or CD 133 positive and/or following angioplasty. KDR positive cells, Stro bright and/or VCAM-1 positive According to the present invention, Vascular repair is cells, differentiated cells including endothelial cells, fibro enhanced by virtue of the therapeutic composition enhanc 25 blasts, monocytes, myocytes. etc. ing the ability of one or more cells that participate directly In another aspect the present invention provides a method or indirectly in vascular repair and renewal to mobilise of promoting vascular repair in a Subject in need thereof, the and/or to migrate, proliferate and form tubules or otherwise method comprising administering to a cell in Vivo or ex participate in vascular repair and/or angiogenic and/or neo Vivo, an amount of a composition effective to activate, vascularisation and/or arteriogenic mechanisms in a subject. 30 upregulate or stimulate or otherwise augment expression of The methods of the invention are particularly relevant to an androgen receptor, thereby enhancing the ability of the subjects with low levels of circulating androgens and to cell to participate in vascular repair. aging Subjects. It will be understood that expression of an androgen In a first aspect the present invention provides a method receptor in a cell can be augmented in vivo or ex vivo. EX of promoting vascular repair in a Subject in need thereof, the 35 Vivo augmentation of the expression of an androgen receptor method comprising administering to the Subject a composi in a cell will require the administration of the cell to the tion comprising a therapeutically effective amount of an subject. It will be understood that the augmented cell can androgen receptor agonist, wherein said administration optionally be co-administered with an androgen receptor results in promoting vascular repair in the Subject. agonist and/or a cell treated with an androgen receptor In one embodiment, the invention additionally or alter 40 agonist. nately includes one or both of (i) treating a cell to render it In another aspect the invention provides a method of capable of participating in vascular repair or enhancing the treatment or prophylaxis of one or more vascular diseases ability of the cell to participate in vascular repair; and (ii) and/or vascular complications thereof in a Subject in need administering the cell alone or together with an androgen thereof or in a subject at risk of developing Such complica receptor agonist to the Subject. 45 tions, said method comprising administering to the Subject a Accordingly in another aspect the present invention pro therapeutically effective amount of an androgen receptor vides a method of promoting vascular repair in a subject in agonist, wherein said administration results in promoting need thereof, the method comprising administering to the vascular repair in the Subject. Subject a therapeutically effective amount of a composition Accordingly this aspect of the invention additionally or comprising a cell treated to render it capable of participating 50 alternately includes one or both of (i) treating a cell to render in vascular repair or enhancing the ability of the cell to it capable of participating in vascular repair or enhancing the participate in vascular repair, wherein said administration ability of the cell to participate in vascular repair; and (ii) results in promoting vascular repair in the Subject. administering the cell alone or together with an androgen In another embodiment the method further involves iso receptor agonist to the Subject. lating a cell capable of participating in vascular repair from 55 Accordingly in another aspect the present invention pro a Subject. vides a method of treatment or prophylaxis of one or more According to any of the preceding embodiments the vascular diseases and/or vascular complications thereof in a composition for administration can also include one or more Subject in need thereof or in a Subject at risk of developing additional cells. The one or more additional cells can be Such complications, said method comprising administering treated or untreated cells. 60 to the subject a therapeutically effective amount of a com Accordingly, the invention further includes one or both of position comprising a cell treated to render it capable of (i) treating an additional cell to render it capable of partici participating in vascular repair or enhancing the ability of pating in vascular repair or enhancing the ability of the cell the cell to participate in vascular repair, wherein said admin to participate in vascular repair, and (ii) administering the istration results in promoting vascular repair in the Subject. additional cell to the subject. 65 In another embodiment the method further involves iso The cell according to any of the forgoing embodiment can lating a cell capable of participating in vascular repair from be for example any cell that is capable of being involved in a Subject. US 9,572,817 B2 7 8 According to any of the preceding embodiments the developing one or more vascular diseases and/or vascular composition for administration can also include one or more complications thereof and/or a subject in need of vascular additional cells. The one or more additional cells can be repair or enhanced vascular repair treated or untreated cells. (ii) obtaining an amount of a composition comprising an Accordingly, the invention further includes one or both of 5 androgen receptor agonist and/or a cell treated with an (i) treating an additional cell to render it capable of partici androgen receptor agonist and/or a cell treated to augment pating in vascular repair or enhancing the ability of the cell the expression of an androgen receptor, and to participate in vascular repair, and (ii) administering the (iii) administering the composition to the identified subject additional cell to the subject. in a therapeutic amount. The cell according to any of the forgoing embodiment can 10 be for example any cell that is capable of being involved in In an alternate embodiment the method provides alterna vascular repair The cell is functional with respect to vascular tively or in addition to step (iii), a step of recommending repair so as to be capable of participating in one or more administration of a composition comprising an androgen vascular repair processes, such as: 1) direct and/or indirect receptor agonist. participation in angiogenesis, vasculogenesis and/or neovas 15 In another aspect the invention provides a method of cularization; 2) direct and/or indirect participation in treatment or prophylaxis comprising: endothelial repair and regeneration and; 3) direct and/or (i) identifying a Subject having one or more vascular dis indirect participation in end organ recovery and repair eases and/or vascular complications thereof or is at risk of following ischaemia Such as myocardial preservation and/or developing one or more vascular diseases and/or vascular regeneration following myocardial infarction. The cell can complications thereof and/or a subject in need of vascular be functional by virtue of being functional in nature, or by repair or enhanced vascular repair virtue of having been genetically-modified or genetically (ii) obtaining an amount of a composition comprising an repaired. androgen receptor agonist and/or a cell treated with an Examples of cells include for example endothelial pro androgen receptor agonist and/or a cell treated to augment genitor cells, outgrowth endothelial cells, endothelial colony 25 the expression of an androgen receptor, and forming cells, bone marrow mononuclear cells, hemopoietic (iii) formulating the composition at (ii) with a Suitable stem cells, erythroid progenitors and colon forming units, carrier or excipient wherein said composition is in an mesenchymal stem cells, cardiac stem cells, embryonic stem amount to alleviate or prevent one or more vascular diseases cells, any stem or progenitor cell capable of adopting a and/or vascular complications thereof according to any vascular cell lineage, pluripotent or progenitor cells induced 30 embodiment herein in a subject in need thereof; and from differentiated cells, side population cells, satellite cells, (iv) administering said formulation to said Subject. myoblasts, CD 34 positive and/or CD 133 positive and/or In an alternate embodiment the method provides alterna KDR positive cells, Stro bright and/or VCAM-1 positive tively or in addition to step (iv), a step of recommending cells, differentiated cells including endothelial cells, fibro administration of a composition comprising an androgen blasts, monocytes, myocytes. etc 35 receptor agonist. Examples of complications, such as conditions or diseases In a particularly preferred embodiment, the method of the that would benefit from said treatment include but are not invention comprises repeated administration of a formula limited to any condition associated with a narrowed or tion comprising an androgen receptor agonist. obstructed blood vessel, for example, narrowing of an artery, The present invention also provides a method for identi vein, or of a capillary system. Specific examples of Such 40 fying an androgen receptor agonist, Suitable for use in a conditions or disease include but are not limited to coronary formulation for the treatment or prophylaxis of one or occlusive disease, carotid occlusive disease, arterial occlu vascular diseases and/or vascular complications thereof and/ sive disease, peripheral arterial disease, atherosclerosis, or for promoting vascular repair or enhancing vascular myointimal hyperplasia (for example due to vascular Sur repair, said method comprising gery or balloon angioplasty or vascular stenting), throm 45 (i) Subjecting a cell to an amount of a candidate androgen boangiitis obliterans, thrombotic disorders, vasculitis, and receptor agonist in the presence and optionally in the the like. absence of an androgen receptor antagonist and Examples of conditions or diseases that can be prevented (ii) detecting a parameter indicative of vascular repair and using the methods of the invention include, but are not (iii) selecting the candidate agonist that enhances vascular necessarily limited to, heart attack (myocardial infarction) or 50 repair. other vascular death, stroke, death or loss of limbs associ Parameters that are indicative of vascular repair include ated with decreased blood flow, and the like. for example tubulogenesis, endothelial cell migration and Other forms of therapeutic methods include, but are not endothelial cell migration. necessarily limited to, the use of an androgen to accelerate In one embodiment, the parameter indicative of vascular healing of wounds or ulcers; to improve the vascularisation 55 repair is determined in an endothelial cell for example of skin grafts or reattached limbs so as to preserve their HUVEC or HCAEC, or a co-culture of fibroblasts and/ECs, function and viability; to improve the healing of Surgical or an EPC. anastomoses (for example, as in re-connecting portions of In one embodiment the method of identification involves the bowel after gastrointestinal Surgery.) (i) Subjecting a cell to an amount of a candidate agonist in In one embodiment of the invention the method relates to 60 the presence and optionally in the absence of an androgen treating a Subject that is androgen deficient. In one embodi receptor antagonist and ment the Subject is a male. (ii) detecting migration of the cell, and The present also provides a method of treatment or (iii) selecting a candidate agonist that enhances cell migra prophylaxis of a subject in need thereof, said method com tion. prising: 65 In one embodiment the cell is in a monolayer. (i) identifying a subject having one or more vascular dis In a different embodiment the method of identifying an eases and/or vascular complications thereof or is at risk of androgen receptor agonist comprises US 9,572,817 B2 9 10 (i) Subjecting a cell to an amount of a candidate agonist in Each embodiment describing a composition or compound the presence and optionally in the absence of an androgen or its use shall be taken to apply mutatis mutandis to a receptor antagonist and formulation comprising the composition or compound or its (ii) detecting cell numbers, and use unless the context requires otherwise or specifically (iii) selecting a candidate agonist that enhances cell num stated otherwise. bers. Those skilled in the art will appreciate that the invention In another embodiment the method of identifying an described herein is susceptible to variations and modifica androgen receptor agonist comprises tions other than those specifically described. It is to be (i) Subjecting a cell to an amount of a candidate agonist in understood that the invention includes all such variations the presence and optically in the absence of an androgen 10 and modifications. The invention also includes all of the receptor agonist steps, features compositions and compounds referred to or (ii) detecting vascular network formation indicated in this specification, individually or collectively, (iii) selecting a candidate agonist that enhances vascular and any and all combinations or any, two or more of said network formation. steps or features. 15 The present invention is not to be limited in scope by the In one embodiment the cell is in a co-culture of cells. specific embodiments described herein, which are intended Co-cultures can be assessed for example using immunohis for the purpose of exemplification only. Functionally tochemistry for EC specific markers and image analysis equivalent products, compositions and methods are clearly software. within the scope of the invention, as described herein. It is to be understood that all of the embodiments relating to the methods of treatment or prophylaxis and the methods BRIEF DESCRIPTION OF THE DRAWINGS promoting vascular repair in a Subject in need thereof as described herein are applicable and relevant to this aspect. FIG. 1a. is a bar chart of effects of DHT on male EC The present invention clearly extends to the direct product migration. of any method of identification or isolation of a compound 25 FIG. 1b. is a bar chart of effects of DHT on female EC or other composition of matter described herein. migration. It is to be understood that any compound of the present FIG. 2d. is a bar chart of effects of DHT on male EC invention, identified or isolated compound or other compo proliferation. sition of matter in Substantially pure form, that is a com FIG. 2b. is a bar chart of effects of DHT on female EC pound or composition free from contaminants that might 30 proliferation. cause adverse side effects or contraindications or antagonise FIG. 3a. is a bar chart of effects of DHT on male EC the activity of the active compound or other composition of tubulogenesis. matter, can be formulated into a medicament Suitable for FIG. 3b. is a bar chart of effects of DHT on female EC treatment according to the present invention. tubulogenesis. Accordingly, in one example, the present invention fur 35 FIG. 4. depicts effects of Castration and DHT Treatment ther provides for the use of a compound or other composi on Angiogenesis in Male Mice. Castrated and sham-cas tion of matter as described according to any embodiment trated mice with and without DHT treatment were implanted herein, for the therapy or prophylaxis of one or more with matrigel plugs. After 14 days, plugs were excised and vascular diseases and/or vascular complications thereof in immunohistochemically stained for the endothelial cell spe the manufacture of a medicament for the treatment of 40 cific antigen von Willebrand Factor (VWF). vascular diseases and/or vascular complications thereof and/ FIG. 5a. depicts effect of Castration and DHT treatment or promotion of vascular repair. on the recovery of mice Subjected to femoral artery ligation. In a preferred embodiment the compound or composition Limb perfusion ratio. For each group of mice, the mean of matter is an androgen receptor agonist and/or a cell (SEM) ratio of perfusion in the ischemic-non-ischemic treated with an androgen receptor agonist and/or a cell 45 limb at the indicated times was determined by LDPI. Dif treated to augment ferences between groups were assessed by 2-way ANOVA As used herein the term "derived from shall be taken to with Bonferroni correction (brackets at right); *P<0.05, indicate that a specified integer may be obtained from a **P<0.01, ***P<0.001 comparisons between castrate and particular source albeit not necessarily directly from that castrate+DHT at individual time point. SOUC. 50 FIG. 5b. depicts effect of Castration and DHT treatment Throughout this specification, unless the context requires on the recovery of mice Subjected to femoral artery ligation. otherwise, the word “comprise', or variations such as “com Tissue damage score. Mice were scored as follows: 0, no prises' or “comprising, will be understood to imply the tissue damage; 1 mild discoloration; 2, moderate discolou inclusion of a stated Step or element or integer or group of ration: 3, Tissue loss/necrosis; 4, loss of foot or more. The steps or elements or integers but not the exclusion of any 55 mean score (SEM) was determined at each time point. other step or element or integer or group of elements or Differences between groups were assessed by 2-way integers. ANOVA with Bonferroni correction (brackets at right), Throughout this specification, unless specifically stated *P<0.05, comparisons between castrate and castrate--DHT otherwise or the context requires otherwise, reference to a at individual time point. single step, composition of matter, group of steps or group 60 FIG. 5c. depicts effects of Castration and DHT treatment of compositions of matter shall be taken to encompass one on the recovery of mice Subjected to femoral artery ligation. and a plurality (i.e. one or more) of those steps, composi Foot Movement Score. Mice were scored as follows: 0, tions of matter, groups of steps or group of compositions of normal response (plantar/toe flexion in response to tail matter. traction); 1, plantar but not toe flexion; 2, no plantar or toe Each embodiment described herein is to be applied muta 65 flexion; 3, dragging of foot. The mean score (SEM) was tis mutandis to each and every other embodiment unless determined at each time point. Differences between groups specifically stated otherwise. were assessed by 2-way ANOVA with Bonferronicorrection US 9,572,817 B2 11 12 (brackets at right), **P<0.01, ***P<0.001 comparisons Agonists can also be described as being androgenic. As between castrate and castrate+DHT at individual time point. exemplified herein the androgen receptor agonist is most FIG. 6a. is a bar chart of effects of Castration and DHT preferably an androgen. Treatment on Mobilization of Spleen Angiogenic cells. Naturally circulating androgenic steroids that are particu MNCs were isolated from the spleens of mice and cultured larly useful in this invention are testosterone and 5C.-dihy in endothelial growth medium, and the mean (SEM) num drotestosterone (DHT) and any derivatives thereof. In one ber (per x200 field) of angiogenic cells was determined. embodiment the androgen receptor agonist is an ester *P<0.05 compared with respective control. derivative. FIG. 6b. is a bar chart of effects of Castration and DHT Other androgenic steroids that may demonstrate pro Treatment on Mobilization of Bone Marrow Angiogenic 10 angiogenic effects include, for example, boldenone, flu cells. MNCs were isolated from the bone marrow of mice oxymesterone, mestanolone, mesterolone, methandros and cultured in endothelial growth medium, and the mean tenolone, 17-methyltestosterone, 17C.-methyl-testosterone (SEM) number (per x200 field) of angiogenic cells was 3-cyclopentyl enol ether, norethandrolone, normethandrone, determined. P<0.05 compared with respective control. 15 Oxandrolone, oxymetholone, prasterone, Stanlolone, stano Zolol, and testosterone. DETAILED DESCRIPTION OF THE Other pro-angiogenic agents also include for example INVENTION anabolic steroids such as androisoxazole, bolasterone, clostebol, ethylestrenol, formyldienolone, 4-hydroxy-19 Androgen Receptor nortestosterone, methenolone, methyltrienolone, nan drolone, okymesterone, quinbolone, Stenbolone, trenbolone. The principal action of androgen is to regulate gene Physiological androgens, also include for example, expression through the androgen receptor, which belongs to Androstenedione, 4-hydroxy-Androstenedione, 11B-hy the superfamily of nuclear receptors. Nuclear receptors are droxyandrostenedione, Androstanediol, Androsterone, Epi ligand-inducible transcription factors that mediate the sig 25 androsterone, Adrenosterone, Dehydroepiandrosterone, nals of a broad variety of fat-soluble hormones, including Dehydroepiandrosterone Sulphate Testosterone, Epitestos the steroid and vitamin D3 hormones, thyroid hormones terone, 5C.-dihydrotestosterone, 5 B-dihydrotestosterone, retinoids he androgen receptor is activated by binding of 11 B-hydroxytestosterone and 11-ketotestosterone androgenic hormones testosterone and dihydrotestosterone. Additionally, non-steroidal androgen receptor agonists The androgen receptor can modulate gene expression 30 are also likely to exert pro-angiogenic effects. Non-steroidal directly by interacting with specific elements in the regula agonists are described for example in US 2008/0057068. tory regions of target genes or indirectly by activating Age Related Decline in Androgens—Androgen Deficiency various growth factor signalling pathways. In one embodiment of the invention the subject has low Androgens can act as chemical messengers in a wide levels of circulating androgens. In an alternate embodiment 35 the Subject has androgen levels within a normal range. range of species and target tissues to produce both slow According to Wald etal (2006) “Testosterone replacement genomic responses, and rapid non-genomic responses. therapy for older men”, Journal of Andrology, Vol. 27, No. Although it is clear that genomic responses to androgens are 2, there is no agreement on the definition of hypogonadism mediated by the formation of a complex of the hormone and in older men, and therefore a combination of clinical Science its cognate steroid-hormone nuclear receptor, new evidence 40 and testosterone measurements is usually used as a tool to indicates that rapid responses are mediated by a variety of determine whether testosterone replacement therapy is indi receptor types associated with the plasma membrane or its cated. The normal range of testosterone levels in young caveolae components, potentially including a membrane males is suggested to be valid for older men as well. The associated nuclear receptor. measurement of total blood testosterone is currently consid Androgen Receptor (AR) is differentially expressed on 45 ered the most appropriate test to determine whether an older male and female cells. Furthermore, sex Steroids may act in patient is hypogonadal or not. Some investigators have a gender-specific manner, with greater effects of estrogens Suggested that a total testosterone level of 200 mg/dL can and/or androgens in male or female cells (McCrohon JA, et appropriately be use as a cut-off value, below which an al. “Androgen receptor expression is greater in macrophages individual should be considered hypogonadal, regardless of from male than from female donors. A sex difference with 50 age. However, the presence of hypogonadism is uncertain in implications for atherogenesis.” Circulation 2000 Vol. 101 patients whose total testosterone levels are in the border line No. 3). As such, in light of the ability of androgens to range, between 200 and 300 mg/dL. Others consider indi promote vascular repair, modulation of AR expression in viduals whose total testosterone levels are less than 300 various tissues may augment said repair. mg/dL to be hypogonadal. Various methods for the upregulation of AR expression 55 As used herein the term “androgen deficiency” is to be will be known to those skilled in the art. These may include, understood in its broadest sense to include any decline in prolonging the half life of AR, prevention of mechanisms of circulating androgens in a subject. A decline in circulating the degradation of AR, or overexpression of AR by trans androgens can be as a result of age or disease or trauma. It fection of cells or tissues with cDNA encoding AR (Lee, D. will be understood that according to the present invention a et al. “Expression and Degradation of Androgen Receptor: 60 subject that is said to be androgen deficient can be fully or Mechanism and Clinical Implication' The Journal of Clini partially deficient and that a level of androgens that consti cal Endocrinology & Metabolism Vol. 88, No. 9) tutes what is meant by partially deficient is subjective and Androgen Receptor Agonists dependent on the individual and their circumstances. The As used herein the term "androgen receptor agonist' person skilled in the relevant art is capable of determining refers to any natural or synthetic compound, that binds to, 65 whether a subject is androgen deficient or not and whether and/or has an affinity for, and activates an androgen receptor. the methods of the present invention are likely to be useful. Androgen receptor agonists include full and partial agonists. Dosage Rates US 9,572,817 B2 13 14 It will be understood that a person skilled is capable of VCAM-1 positive cells, differentiated cells including appropriately determining a dosage rate of an androgen endothelial cells, fibroblasts, monocytes, myocytes. etc receptor agonist for the methods of administration as In one preferred embodiment a cell is of cardiovascular described herein. Dosage rates are dependent upon the route lineage or is capable of differentiating into a cell of said of administration and the vasculature, tissue or organ to be lineage. targeted and treated. Cells can be used in combination. For example the In the context of androgen deficiency, assuming that methods of the invention can involve infusion of different testosterone is being administered, a dosage regimen may haematopoietic stem cell populations and ex vivo expanded include applying a dose of testosterone to the Subject in an and treated endothelial progenitor cells. 10 Cells (such as for example EPCs) can be isolated from a amount sufficient for the testosterone to reach the blood healthy Subject and may be saved for use at a later date, and stream of the Subject so as to achieve a serum concentration typically such cells are frozen under conditions that retains within a range between about 300 mg testosterone per dL their viability. It will be appreciated that the cells may be serum to about 1000 mg testosterone per dL serum within at obtained and enriched (expanded if necessary) before vas least about 24 hours of dosing of the androgen. 15 cular complications arise or are apparent in a subject, and Supraphysiological testosterone is considered to be 40 kept for immediate administration when necessary. nM. Trials conducted by others have involved weekly intra Alternatively, an cell is isolated from a subject in need of muscular administration of 250 mg testosterone enanthate, treatment. believed to affect Supraphysiological concentrations of tes A cell that has a capacity for repair can be isolated and/or tosterone. Supraphysiological dosing regimens for testoster treated, transformed and/or transfected prior to administra one enanthate involve weekly intramuscular injections rang tion to a Subject. Methods for isolating and/or administering ing from 200-600 mg. such cells will be apparent to the skilled artisan and/or According to in vitro experiments performed by the described herein. inventors, the highest dose of DHT which is a more potent For example, a natural source of EPCs includes bone androgen than testosterone used was 400 nM. A number of 25 marrow (e.g., with and without previous bleeding), periph studies have administered DHT for androgen therapy. Typi eral blood (e.g., with and without enhancement from mar cally, administration is via a transdermal gel with doses row), and peripheral blood mononuclear cells. Other sources ranging from 70-250 mg. are not to be excluded. Administration of DHT however can result in the hor Cells may be isolated from such a source by any suitable monal changes of increased plasma DHT together with 30 method, typically involving cell fractionation and concen negative feedback Suppression of the pituitary-testicular axis tration. (plasma total and free T, LH, FSH) see Ly L. P. et al. “A A cell may be treated or rendered capable of participating double-blind, placebo-controlled, randomized clinical trial in repair by being Subjected to, contacted with, grown or of transdermal dihydrotestosterone gel on muscular strength, otherwise cultured in the presence of an androgen receptor mobility, and quality of life in older men with partial 35 agonist. An androgen receptor agonist can for example be androgen deficiency.” J Clin Endocrinol Metab. 2001; added to a growth medium for a cell. Alternatively a cell can 86:40784.088. Site specific or localised administration of be treated in the presence of a composition capable of DHT may be recommended. activating, up regulating or otherwise augmenting the Cells expression of an androgen receptor in said cell. In vitro The present invention encompasses cells in vivo and ex 40 treatment of cells are provided for example in Example 1. Vivo capable of participating in vascular repair, enhancing The scope of the invention is understood to include all vascular repair or preventing or reducing vascular disease or methods of treatment or stimulation of a cell. complication(s) thereof. The present invention encompasses Pharmaceutical Compositions the stimulation or treatment of cells in vivo or ex vivo to As used herein, pharmaceutical composition” means a participate in migration, proliferation or tubulogenesis and/ 45 “therapeutically effective amount of the active ingredient, or to facilitate other cells to perform these processes. that is, the androgen receptor agonist, together with a The cell is functional with respect to vascular repair so as pharmaceutically acceptable carrier or diluent. A “therapeu to be capable of participating in one or more vascular repair tically effective amount’ as used herein refers to that amount processes, such as: 1) direct and/or indirect participation in which provides a therapeutic effect for a given condition and angiogenesis, vasculogenesis and/or neovascularization; 2) 50 administration regimen. direct and/or indirect participation in endothelial repair and As used herein, the term “administering refers to bring regeneration and; 3) direct and/or indirect participation in ing a cell and/or Subject in contact with a compound of the end organ recovery and repair following ischaemia Such as present invention. Administration is in Vivo, i.e. in cells or myocardial preservation and/or regeneration following myo tissues of living organisms, for example humans or ex vivo. cardial infarction. The cell can be functional by virtue of 55 In one embodiment, the present invention encompasses being functional in nature, or by virtue of having been administering the compounds and compositions of the pres genetically-modified or genetically repaired. ent invention to a Subject. Examples of cells include for example endothelial pro Administrations genitor cells, outgrowth endothelial cells, endothelial colony Pharmaceutical compositions containing the compounds forming cells, bone marrow mononuclear cells, haematopoi 60 of this invention can be administered to a subject by any etic stem cells, erythroid progenitors and colon forming method known to a person skilled in the art, such as orally, units, mesenchymal stem cells, cardiac stem cells, embry parenterally, intravascularly, paracancerally, transmucosally, onic stem cells, any stem or progenitor cell capable of transdermally, intramuscularly, intranasally, intravenously, adopting a vascular cell lineage, pluripotent or progenitor intradermally, Subcutaneously, Sublingually, intraperitone cells induced from differentiated cells, side population cells, 65 aly, intraventricularly, intracranially, intravaginally, by inha satellite cells, myoblasts, CD 34 positive and/or CD 133 lation, rectally, intratumorally, or by any means in which the positive and/or KDR positive cells, Stro bright and/or androgen receptor agonist can be delivered to tissue (e.g., US 9,572,817 B2 15 16 needle or catheter). Alternatively, topical administration nadal men 4 to 8 hours after application, and these levels may be desired for application to mucosal cells, for skin or gradually decrease over the next 24 hours Non-genital ocular application. Another method of administration is via patches are known to provide midnormal serum levels 8 to aspiration or aerosol formulation. Wald et al., (2006) (ibid) 12 hours after the application of 2 nongenital patches. describes various routes of administration and dosage Transdermal testosterone gel (1% testosterone in a hydroal regimes. It is to be understood that Wald et al., relates to coholic gel is also available. The skin serves as a reservoir acute treatment and not vascular repair as described herein, for the sustained release of testosterone into the systemic and that the person skilled in the art such as for example a circulation, allowing a single application of this formulation physician will know how to formulate, prescribe or admin to provide continuous transdermal delivery of testosterone ister an appropriate dosage of the compound for the methods 10 for 24 hours, producing circulating testosterone levels that of the present invention. approximate the normal levels (eg 300-1000 ng/dL) (Waldet In one embodiment, the pharmaceutical compositions are al., ibid). administered orally, and are thus formulated in a form Further, in another embodiment, the pharmaceutical com Suitable for oral administration, that is as a solid or a liquid positions are administered as a Suppository, for example a preparation. Suitable solid oral formulations include tablets, 15 rectal Suppository or a urethral Suppository. capsules, pills, granules, pellets, powders, and the like. Further, in another embodiment, the pharmaceutical com Suitable liquid oral formulations include Solutions, Suspen positions are administered by Subcutaneous implantation of sions, dispersions, emulsions, oils and the like. In one a pellet. embodiment of the present invention, the androgen receptor In a further embodiment, the pellet provides for controlled agonist is formulated in a capsule, for example a gelatine release of a compound over a period of time. A single capsule. Oral testosterone undecanoate Supplementation has intramuscular dose of a biodegradable testosterone micro been used in androgen therapy. In another embodiment the sphere formulation can provide normal testosterone levels in agonist can be formulated as Sublingual formulations such as hypogonadal men for up to 11 weeks (see Wald et al., 2006). a cyclodextrin-complexed testosterone Sublingual formula Formulations tion. 25 As used herein “pharmaceutically acceptable carriers or Further, in another embodiment, the pharmaceutical com diluents' are well known to those skilled in the art. The positions are administered by intravenous, intraarterial, or carrier or diluent may be a solid carrier or diluent for solid intramuscular injection of a liquid preparation. Suitable formulations, a liquid carrier or diluent for liquid formula liquid formulations include solutions, Suspensions, disper tions, or mixtures thereof. sions, emulsions, oils and the like. In one embodiment, the 30 Solid carriers/diluents include, but are not limited to, a pharmaceutical compositions are administered intrave gum, a starch (e.g. corn starch, pregeletanized starch), a nously, and are thus formulated in a form suitable for Sugar (e.g., lactose, mannitol, Sucrose, dextrose), a cellulosic intravenous administration. In another embodiment, the material (e.g. microcrystalline cellulose), an acrylate (e.g. pharmaceutical compositions are administered intraarteri polymethylacrylate), calcium carbonate, magnesium oxide, ally, and are thus formulated in a form suitable for intraar 35 talc, or mixtures thereof. terial administration. In another embodiment, the pharma Suitable excipients and carriers may be, according to ceutical compositions are administered intramuscularly, and embodiments of the invention, solid or liquid and the type is are thus formulated in a form suitable for intramuscular generally chosen based on the type of administration being administration. used. Liposomes may also be used to deliver the composi Intramuscular injection of long-acting esters, such as 40 tion. Examples of suitable solid carriers include lactose, enanthate, is a traditional form of testosterone. Testosterone Sucrose, gelatin and agar. Oral dosage forms may contain cypionate and testosterone proprionate are also considered Suitable binders, lubricants, diluents, disintegrating agents, here. Testosterone enanthate and cypionate have longer a coloring agents, flavoring agents, flow-inducing agents, and longer action and have the same pharmacokinetic profile. melting agents. Liquid dosage forms may contain, for They reach peak serum testosterone levels 24 hours after 45 example, Suitable solvents, preservatives, emulsifying administration and decline gradually within 2 weeks. agents, Suspending agents, diluents, Sweeteners, thickeners, In another embodiment an intramuscular dose of a bio and melting agents. Parenteral and intravenous forms should degradable testosterone microsphere can be used (see Wald also include minerals and other materials to make them et al., (2006)) compatible with the type of injection or delivery system Further, in another embodiment, the pharmaceutical com 50 chosen. Of course, other excipients may also be used. positions are administered topically to body Surfaces, and For liquid formulations, pharmaceutically acceptable car are thus formulated in a form suitable for topical adminis riers may be aqueous or non-aqueous Solutions, Suspensions, tration. Suitable topical formulations include gels, oint emulsions or oils. Examples of non-aqueous solvents are ments, creams, lotions, drops and the like. For topical propylene glycol, polyethylene glycol, and injectable administration, the compounds of this invention or their 55 organic esters such as ethyl oleate. Aqueous carriers include physiologically tolerated derivatives such as salts, esters, water, alcoholic/aqueous solutions, cyclodextrins, emulsions N-oxides, and the like are prepared and applied as Solutions, or Suspensions, including saline and buffered media. Suspensions, or emulsions in a physiologically acceptable Examples of oils are those of petroleum, animal, vegetable, diluent with or without a pharmaceutical carrier. or synthetic origin, for example, peanut oil, soybean oil, Two types of transdermal patches are available, Scrotal 60 mineral oil, olive oil, Sunflower oil, and fish-liver oil. and nongenital and these are characterised by favourable Parenteral vehicles (for subcutaneous, intravenous, pharmacokinetic behaviour and have proven to be an effec intraarterial, or intramuscular injection) include Sodium tive mode of delivery (Findlay J C, Place V. Snyder P J. chloride Solution, Ringer's dextrose, dextrose and sodium “Treatment of primary hypogonadism in men by the trans chloride, lactated Ringer's and fixed oils. Intravenous dermal administration of testosterone. J Clin Endocrinol 65 vehicles include fluid and nutrient replenishers, electrolyte Metab. 1989: 68:369-373). Daily use of the scrotal patch can replenishers such as those based on Ringer's dextrose, and produce midnormal serum testosterone levels in hypogo the like. Examples are sterile liquids Such as water and oils, US 9,572,817 B2 17 18 with or without the addition of a surfactant and other administration, the compounds of this invention or their pharmaceutically acceptable adjuvants. In general, water, physiologically tolerated derivatives Such as salts, esters, saline, aqueous dextrose and related Sugar Solutions, and N-oxides, and the like are converted into a solution, Sus glycols such as propylene glycols or polyethylene glycol are pension, or emulsion, if desired with the Substances custom preferred liquid carriers, particularly for injectable solutions. 5 ary and Suitable for this purpose, for example, solubilises or Examples of oils are those of petroleum, animal, vegetable, other. or synthetic origin, for example, peanut oil, soybean oil, Disease Therapy mineral oil, olive oil, Sunflower oil, and fish-liver oil. Where appropriate disease therapy includes preventing, In addition, the compositions may further comprise bind treating, Suppressing, inhibiting or reducing the risk of ers (e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellu 10 lose, guar gum, hydroxypropyl cellulose, hydroxypropyl reducing the incidence of ameliorating symptoms of delay methyl cellulose, povidone), disintegrating agents (e.g. ing progression of, or diminishing pathogenesis of a disease. cornstarch, potato starch, alginic acid, silicon dioxide, cros In some embodiments, the Subject is human, while in carmellose Sodium, crospovidone, guar gum, sodium starch other embodiments, the Subject is non-human. In some glycolate), buffers (e.g., Tris-HCl, acetate, phosphate) of 15 embodiments, the Subject is mammalian. In one embodi various pH and ionic strength, additives such as albumin or ment, the Subject is simian, bovine, feline, canine, Ovine, gelatin to prevent absorption to Surfaces, detergents (e.g., porcine, equine, or murine. Tween 20, Tween 80, Pluronic F68, bile acid salts), protease In one embodiment, the subject is male, while in another inhibitors, Surfactants (e.g. sodium lauryl Sulfate), perme embodiment, the subject is female. In one preferred embodi ation enhancers, solubilizing agents (e.g., cremophor, glyc ment the Subject is an older male. erol, polyethylene glycerol, benzlkonium chloride, benzyl As used herein, the terms “prevention' and “treatment' benzoate, cyclodextrins, Sobitan esters, Stearic acids), anti shall not be taken to require an absolute i.e., 100% abroga oxidants (e.g., ascorbic acid, Sodium metabisulfite, butylated tion of impaired vascular function or vascular complication, hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose, or an absolute i.e., 100% prevention of the development of hydroxypropylmethyl cellulose), Viscosity increasing agents 25 a vascular complication in a subject having risk factors (e.g. carbomer, colloidal silicon dioxide, ethyl cellulose, therefor, and it is Sufficient that there is a significant reduc guar gum), Sweetners (e.g. aspartame, citric acid), preser tion in the adverse vascular effect(s) using the method of the Vatives (e.g., Thimerosal, benzyl alcohol, parabens), color present invention compared to the absence of prophylaxis or ing agents, lubricants (e.g. Stearic acid, magnesium Stearate, therapy in accordance with the present invention. polyethylene glycol, Sodium lauryl Sulfate), flow-aids (e.g. 30 colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, Similarly, the term “alleviating or “alleviate' as used triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl throughout this specification shall not be taken to require cellulose, sodium lauryl Sulfate), polymer coatings (e.g., abrogation of impaired vascular function or vascular com poloxamers or poloxamines), coating and film forming plication in a Subject that is more than a significant effect agents (e.g. ethyl cellulose, acrylates, polymethacrylates), 35 compared to the absence of treatment in accordance with the and/or adjuvants. present invention. In one embodiment, the pharmaceutical compositions Similarly, the terms “inhibit”, “enhance”, “repress', provided herein are controlled release compositions, i.e. “delay”, “enhance”, “induce”, “activate” and “promote' as compositions in which the compound of this invention is used throughout this specification shall not be taken to released over a period of time after administration. Con 40 require any particular quantitative change, merely a modi trolled or Sustained release compositions include formula fied level and/or activity and/or expression, or modified tion in lipophilic depots (e.g. fatty acids, waxes, oils). In timing thereof, that is significant compared to the absence of another embodiment, the composition is an immediate treatment in accordance with the present invention. release composition, i.e. a composition in which all of the compound is released immediately after administration. 45 Examples of the Invention In yet another embodiment, the pharmaceutical compo sition can be delivered in a controlled release system. In yet Example 1 another embodiment, a controlled release system can be placed in proximity to the therapeutic target, i.e., the heart, In Vitro thus requiring only a fraction of the systemic dose (see, e.g., 50 Goodson, in Medical Applications of Controlled Release, Description of In Vitro Methods: supra, vol. 2, pp. 116-138 (1984). Other controlled release The effect of the non-aromatizable androgen dihydrotes systems are discussed in the review by Langer (Science tosterone was assessed on three key angiogenic processes: 249:1627-1633 (1990). endothelial cell (EC) migration, EC proliferation and EC The preparation of pharmaceutical compositions which 55 tubulogenesis. Cells were treated with 0, 4, 40 or 400 nM contain an active component is well understood in the art, dihydrotestosterone (DHT, a potent androgen) with and for example by mixing, granulating, or tablet-forming pro without the androgen-receptor antagonist hydroxyflutamide cesses. The active therapeutic ingredient is often mixed with (HF). excipients which are pharmaceutically acceptable and com (i) EC Migration patible with the active ingredient. For oral administration, 60 EC migration was assessed via a scratch assay and boyden the compounds of this invention or their physiologically chamber assay. Human Umbilical Endothelial Cells (HU tolerated derivatives Such as salts, esters, N-oxides, and the VEC) monolayers were grown in the presence of varying like are mixed with additives customary for this purpose, doses of DHT+Hydroxyflutamide (HF), and either scratched Such as vehicles, stabilizers, or inert diluents, and converted or placed on top of permeable membranes. EC migration by customary methods into Suitable forms for administra 65 across the culture Surface or through the membranes was tion, Such as tablets, coated tablets, hard or soft gelatin assessed at 24 h and 6 h respectively via light and fluores capsules, aqueous, alcoholic or oily Solutions. For parenteral cence microscopy respectively. US 9,572,817 B2 19 20 (ii) EC Proliferation (i) Matrigel Plug Implantation Model EC proliferation was assessed using two methods: direct Commercially available Matrigel (BD biosciences) with counting of cell numbers and the MTS Assay. HUVECs out Phenol Red was supplemented with bfGF (100 ng/mL) were seeded at a density of 1x104 cells/ml (12 well plates and implanted subcutaneously (250 (1) in male mice. After for counting, 96 well plates for MTS assay) in endothelial a period of fourteen days, Matrigel plugs were removed growth media in the presence of varying doses from the mice and assessed for vascularization using stan DHT+Hydroxyflutamide (HF). After 48 h, cells were either dard immunohistochemical techniques. This model and harvested from 12 well plates and counted or incubated with method is described in detail in Passaniti A, et al. A simple, MTS reagent in the 96 well plates with absorbance measured quantitative method for assessing angiogenesis and antian 10 giogenic agents using reconstituted basement membrane, Subsequently at 490 nm measured. heparin, and fibroblast growth factor. Lab Invest. 1992: (iii) EC Tubulogenesis 67:519-528. Vascular network formation by HUVECs was assessed (ii) Hindlimb Ischaemia Model using a previously described co-culture assay of fibroblasts Complete femoral artery excision from the left hind limb and ECs (Sieveking, D. et al J Am Coll Cardiol 2008: 15 of male mice was performed using an operative procedure 51:660-8). Co-cultures were grown in the presence of developed in the laboratory of the late Dr. Jeffrey Isner, DHT+Hydroxyflutamide (HF). After 72 h, co-cultures were which was modified to include disruption of the subcutane assessed using immunohistochemistry for EC specific mark ous branch in the medial thigh. Perfusion of the hindlimbs ers and image analysis Software. was measured by laser Doppler perfusion imaging (LDPI; Description of In Vitro Results: Moor Instruments) which utilises a near-infrared laser diode (i) Endothelial Cell (EC) Migration to measure Subcutaneous blood flow as a function of light DHT administration induced dose-dependent male EC scattering by moving red blood cells (Doppler shift). This migration after 24 h (138+7%, 280+23% and 306+32% vs. technique allows for repeated, non-invasive, quantitative 100% control value for DHT at 4, 40, and 400 nM, respec measurements of tissue perfusion that are expressed as the tively, P<0.01, ANOVA). Addition of the androgen receptor 25 ratio of signal in the ischemic and contralateral non-isch antagonist HF abrogated DHT mediated EC migration emic hind limb. Perioperative LDPI provides a method of (96+6% and 94+6% vs. 100% control value for 40 nM demonstrating Successful Surgical outcome (i.e. presence of DHT+HF and 400 nM+HF respectively, P>0.05) (FIG. 1a). critical limb ischaemia). The serial changes in perfusion that DHT did not augment EC migration in female donor cells are observed following femoral artery excision and revas (90+5%, 96+5% and 93+8% vs. 100% control value for 30 cularization have been shown to correlate with changes in DHT at 4, 40, and 400 nM, respectively, P>0.05, ANOVA) vessel density as determined by histological analysis (FIG. 1b). (Couffinhal T, et al. Mouse model of angiogenesis. Am J (ii) EC Proliferation Pathol. 1998: 152: 1667-1679.). DHT induced a dose-related increase in proliferation after In addition to the quantitative analysis of perfusion in the 48 h (112.8%, 118+5% and 127+4% vs. 100% control value 35 foot by LDPI, the animals were clinically evaluated and for DHT at 4, 40, and 400 nM, respectively, P-0.001, scored for physical evidence of ischemic tissue damage and ANOVA). Addition of the androgen receptor antagonist HF impairment of motor function. abrogated DHT mediated EC proliferation (94+4% and (iii) Angiogenic Cell Mobilization 93.3% vs. 100% control value for 40 nM DHT+HF and 400 Mononuclear cells were harvested from the spleens and nM+HF respectively, Pid-0.05) (FIG. 2a). DHT did not aug 40 bone marrow of all animals and assessed via cell culture and ment EC proliferation in female donor cells (92+5%, 92+3% flow cytometry. Cells were and stained with fluorescein and 89-6% vs. 100% control value for DHT at 4, 40, and isothiocyanate-lectin and/or 1,1(-dioctadecyl-3,3,3(.3(-te 400 nM, respectively, P>0.05, ANOVA) (FIG. 2b). tramethylindocarbocyanine (DiD)-labelled, acetylated low (iii) EC Tubulogenesis density lipoprotein (Vasa M. et al. Number and migratory Chronic exposure to DHT also increased tubule formation 45 activity of circulating endothelial progenitor cells inversely (115+7%, 137+5% and 157+6% vs. 100% control value for correlate with risk factors for coronary artery disease. Circ DHT at 4, 40, and 400 nM, respectively, P<0.001, ANOVA). Res. 2001: 89:e1-e7). Addition of the androgen receptor antagonist HF abrogated The animal models are both well established for the in DHT mediated EC tubulogenesis (94+4% and 93+3% vs. Vivo assessment of angiogenesis in the presence (Hindlimb 100% control value for 40 nM DHT+HF and 400 nM+HF 50 Ischaemia) or absence (Matrigel plug) of an ischemic stimu respectively, P>0.05) (FIG. 3a). DHT did not augment EC lus. tubulogenesis in female donor cells (92+3%, 90+3% and Detailed Description of In Vivo Results: 92-3% vs. 100% control value for DHT at 4, 40, and 400 (i) Matrigel Plug Implantation Model nM, respectively, P>0.05, ANOVA) (FIG. 3b). Male castration markedly decreased in vivo Vasculariza 55 tion of Matrigel plugs (4.8+0.2x10-3 vs. 1.3+0.1x10-3 (m2 Example 2 VWF+ cells/(m2 matrigel for control vs. castrated mice, P<0.0001). Androgen replacement reversed castration-ef In Vivo fects on angiogenesis (4.3+0.3x10-3 (m2 v WF+ cells/(m2, P<0.001 vs. castrated mice)(FIG. 4). Description of Mouse Models Used 60 (ii) Hindlimb Ischaemia Model Male castration markedly inhibited the rate of recovery Animals from hindlimb ischaemia (Laser Doppler Perfusion Index Male C57B 16/J mice were either castrated or sham (LDPI) ischemic/non-ischemic ratio after 11 days, control: castrated after reaching sexual maturity (6 weeks). After a 0.54+0.04, castrate: 0.39+0.04, P-0.05). Androgen replace period of 10 days, 50% of both castrated and sham-castrated 65 ment not only reversed castration-effects, but also acceler mice received a 1 cm Subcutaneous Silastic implant contain ated recovery in hindlimb ischaemia (LDPI after 7 days, ing DHT. control: 0.39+0.03, castrate+DHT: 0.6+0.06, P<0.01) (FIG. US 9,572,817 B2 21 22 5a). These results were also mirrored in clinical evaluations requiring medical or Surgical treatment or have chronic of ischemic damage and motor function (FIGS. 5b and c medical diseases or medications likely to interfere with safe respectively). participation in androgen therapy. Full medical history These findings Suggest that androgens participate in including cardiovascular risk factors and clinical examina modulation of cardiovascular regeneration with particular tion will be undertaken on all patients. implications for the role of androgen replacement in men. Blood Collection, Biochemical Markers, Hormone Assays (iii) Angiogenic Cell Mobilization and Monocyte Isolation: To test the hypothesis that the observed differences in Fasting morning blood samples will be obtained at weeks: angiogenic responses were associated with differences in 0, 1, 6 and 14 of the trial. Serum T. DHT and oestradiol will mobilization of angiogenic cells, MNCs were isolated from 10 be measured at 0, 6 and 14 weeks by standard methods. In mice used in both animal models outlined above. MNCs, addition lipid profile, fibrinolytic markers, prostatic specific were harvested from both the spleen, which serves as a antigen and full blood count including haemoglobin will be reservoir of circulating cells, and the bone marrow. Castra measured by the Royal Prince Hospital Pathology. Serum tion of male mice markedly decreased angiogenic cells EPO and VEGF will be measured by ELISA. For EPC detected in the spleens of mice, both in the presence (22+1.7 15 studies a 20 ml blood sample will be collected and buffered vs. 16+0.8 Angiogenic cells/x200 field for control vs. cas with sodium citrate. Mononuclear cells will be isolated with trated mice, P-0.05) and absence (15.4+1 vs. 10.3+0.9 the use of a Ficoll density gradient (Biocol, Biochrom) Angiogenic cells/x200 field for control vs. castrated mice, according to standard protocols. P<0.05) of an ischemic stimulus (FIG. 6a). In the absence of Quantitation of EPCs: ischaemia DHT treatment reversed castration effects on EPCs and early haematopoietic stem cells will be quan angiogenic cell mobilisation (10+0.9 Angiogenic cells/x200 titated by FACS analysis (Beckman Coulter FC 500 at HRI) field for castrate+DHT P>0.05 vs. control). Indeed, in the for CD34+/KDR+ and CD34+/CD133+ cells, respectively. presence of ischaemia, not only was angiogenic cell mobi Mononuclear cells will be resuspended in 100 "1 of a FACS lization normalized, but augmented (26t1.3 Angiogenic buffer containing phosphate-buffered saline, 0.1% bovine cells/x200 field for castrate+DHT P-0.05 vs. respective 25 albumin, and aprotinin (20"1/mL). Immunofluorescent cell control). In the bone marrow, castration dramatically staining will be performed with the use of the fluorescent reduced the numbers of angiogenic cells in the presence of conjugated antibodies to CD34-fluorescein isothiocyanate ischaemia, a finding which was reversed by DHT treatment (FITC) (Becton Dickinson) in combination with KDR (Control: 60+2.4, Castrate: 26+1.7, castrate+DHT: 36+3 (R&D Systems) or CD133-phycoerythrin (Miltenyi). For the Angiogenic cells/x200 field, P-0.001 for castrate and cas 30 identification of KDR-- cells, indirect immunolabeling will trate--DHT vs. control, PK0.05 for castrate vs. castrate-- be performed with the use of a biotinylated goat mononu DHT) (FIG. 6b). clear antibody against the extracellular domain of human Together, these in vivo findings suggest that androgens KDR (R&D Systems). IgG2a-FITC-PE antibody (Becton participate in modulation of cardiovascular regeneration Dickinson) will be used as a negative control. Cell fluores with particular implications for the role of androgen replace 35 cence will measured immediately after staining. ment in men. To assess the reproducibility of the measurements, two separate blood samples will be obtained, on day 0 from 5 Example 3 subjects. The intraclass correlation between the two probes will be computed. Human Studies 40 EPC Colony Forming Unit (CFU) Assay: In endothelial basal medium (CellSystems) with supple Effects of Exogenous Androgen Administration on ments, 1x107 mononuclear cells will be seeded on human Endothelial Progenitor Cell Mobilisation and fibronectin-coated plates (Sigma-Aldrich). After 48 hours, Function in Men 1x106 nonadherent cells will be transferred into new 45 fibronectin-coated wells to avoid contamination with mature Rationale: Androgens are known to stimulate erythropoi endothelial cells and nonprogenitor cells. After seven days, esis and haematopoietic stem cell proliferation by direct endothelial CFUs in at least 3 wells will be counted by two effects and also via stimulation of EPO production. Recent observers blinded to the subject’s treatment. Confirmation of evidence suggests that EPO is a potent stimulus for EPC endothelial-cell lineage will be performed by immunostain mobilisation and ischaemia-induced angiogenesis. We will 50 ing for KDR and CD31. evaluate the effects of androgen replacement in on EPC Reverse Transcription Polymerase Chain Reaction (RT mobilisation and function in elderly men with partial andro PCR): gen deficiency. EPC expression of AR, VEGF, VEGF receptors (1, 2 and Prospective Evaluation of Androgen Effects on EPC Mobi VEGF 1651 and 2) will be assessed by RT-PCR (11). lization and Function in a Randomised Placebo-Controlled 55 Effects of DHT and T on EPC Proliferation and EPC Trial Involving Elderly Men with Partial Androgen Defi mediated Tubulogenesis in vitro: will be evaluated using the ciency. EC proliferation and fibroblast co-culture assays as outlined Patient Recruitment: in 1.3 and 1.4. Healthy, ambulatory men over the age->60 years with Statistical Analysis: partial androgen deficiency (n=40), defined as the presence 60 We calculated that for men receiving ART, 40 participants on 2 occasions of low serum total testosterone (<15 nmol/L) would be needed to detect a 30% increase in EPCs in a will be randomised to 3 months treatment with intramuscu randomised study with 80% power and 5% significance. lar testosterone injection (100 mg fortnightly, Sustanon, Anticipated Results and Further Studies: Organon Laboratories) or placebo. The patients will be Given that T stimulates haematopoietic stem cell prolif enrolled via an ongoing collaboration with AI2 and the 65 eration, we anticipate that T replacement will stimulate both Andrology Clinic at Concord Hospital, Sydney. Men will be EPC and early haematopoietic progenitor cell mobilisation. excluded from the study if they have prostatic disease Functional assays will assess the in vitro effects of andro US 9,572,817 B2 23 24 gens on EPC proliferation and tube formation. Future stud 9. The method of claim 1, comprising administering to the ies may evaluate these effects of testosterone in the context Subject a plurality of dosages of the composition. of men with CAD, by evaluating both EPC mobilisation and 10. The method of claim 1, comprising administering the myocardial perfusion endpoints. composition to the Subject daily, weekly or fortnightly. It will be appreciated by persons skilled in the art that 11. The method of claim 1, comprising detecting the numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without presence of one or more indicator of vascular repair in the departing from the Scope of the invention as broadly subject after administration of the composition to the sub described. The present embodiments are, therefore, to be ject. considered in all respects as illustrative and not restrictive. 12. The method of claim 11, wherein the one or more The invention claimed is: 10 indicator of vascular repair is one or more of the group 1. A method of promoting or enhancing vascular repair in consisting of tubulogenesis, endothelial cell migration, and a male Subject who is Suffering from a vascular blockage, endothelial cell proliferation. wherein the method comprises administering to the Subject 13. The method of claim 1, comprising selecting a subject a composition comprising a therapeutically effective amount of an androgen receptor agonist so that the Subject is 15 who is suffering from critical limb ischaemia or throm chronically exposed to the androgen receptor agonist, and boangiitis obliterans as a Subject Suitable for administration wherein the composition is administered in an amount of the composition. Sufficient to stimulate angiogenesis, neovascularization and/ 14. A method of promoting or enhancing vascular repair or vascular regeneration in the Subject to thereby promote or in a male Subject who is suffering from critical limb ischae enhance vascular repair in the Subject, wherein the vascular mia, wherein the method comprises administering to the blockage is a result of critical limb ischaemia or throm Subject a composition comprising a therapeutically effective boangiitis obliterans. amount of an androgen receptor agonist so that the Subject 2. The method of claim 1, wherein the androgen receptor is chronically exposed to the androgen receptor agonist, and agonist is an androgen. wherein the composition is administered in an amount 3. The method of claim 1, wherein the androgen receptor 25 Sufficient to stimulate angiogenesis, neovascularization and/ agonist is a steroidal androgen. or vascular regeneration to thereby promote or enhance 4. The method of claim 1, wherein the androgen receptor vascular repair in the Subject. agonist is testosterone. 15. A method of promoting or enhancing vascular repair 5. The method of claim 1, wherein the subject is androgen in a male Subject who is suffering from thromboangiitis deficient. 30 obliterans, wherein the method comprises administering to 6. The method of claim 1, wherein the subject is not the Subject a composition comprising a therapeutically androgen deficient. effective amount of an androgen receptor agonist so that the 7. The method of claim 1, wherein the androgen receptor Subject is chronically exposed to the androgen receptor agonist is dihydrotestosterone (DHT). agonist, and wherein the composition is administered in an 8. The method of claim 1, wherein the androgen receptor 35 amount Sufficient to stimulate angiogenesis, neovasculariza agonist is dehydroepiandrosterone (DHEA), androsterone, tion and/or vascular regeneration to thereby promote or epitestosterone, androstenedione, dehydroepiandrosterone, enhance vascular repair in the Subject. or androstanediol. k k k k k