EP1711201A2

EP1711201A2 - Use of low-dose erythropoietin for stimulating endothelial precursor cells, regenerating organs, and slowing down progression of end organ damages

Info

Publication number: EP1711201A2
Application number: EP05715203A
Authority: EP
Inventors: Ferdinand Hermann Bahlmann; Hermann Haller
Original assignee: Epoplus GmbH and Co KG
Current assignee: Epoplus GmbH and Co KG
Priority date: 2004-01-23
Filing date: 2005-01-22
Publication date: 2006-10-18
Also published as: EP2156843A2; AU2010200621A1; EA200702523A1; WO2005070450A3; US20070161552A1; EA200900874A1; NZ548697A; CN101601857A; WO2005070450A8; EA015350B1; BRPI0507048A; EP2156843A3; ZA200606515B; IL177017A0; DE102004004509B4; DE102004063927A1; UA86400C2; WO2005070450A2; DE102004004509A1; JP2007518769A

Abstract

The invention relates to the use of low-dose erythropoietin for stimulating physical mobilization, proliferation, and differentiation of endothelial precursor cells, stimulating vasculogenesis, treating diseases that are linked to a dysfunction of endothelial precursor cells, and producing pharmaceutical compositions used for the treatment of such diseases as well as pharmaceutical compositions containing erythropoietin and other appropriate agents for stimulating endothelial precursor cells.

Description

Use of low-dose erythropoietin to stimulate endothelial progenitor cells and to regenerate organs and slow the progression of end organ damage

description

The present invention relates to the use of, in particular low-dose, erythropoietin (EPO) alone or in combination with other chemical, thermal, mechanical and biological agents for stimulating the physiological mobilization, proliferation and differentiation of endothelial progenitor cells, for stimulating vasculogenesis, for the therapy of Diseases associated with dysfunction of endothelial progenitor cells and for the production of pharmaceutical compositions for the treatment of such diseases, as well as pharmaceutical compositions comprising erythropoietin and other suitable active substances for stimulating endothelial progenitor cells and for organ protection, organ regeneration, in particular vascular and tissue regeneration and slowing progression organ damage.

The present invention also relates to the use of erythropoietin, in particular in the low doses according to the invention, and / or suitable active ingredients for, preferably topical, use in the context of a cosmetic treatment, that is to say in the sense of “beauty care”, in particular of the human or animal body for the prophylaxis or reduction of wrinkles and fine lines, strengthening the connective tissue, for protecting such as for tightening the skin, especially the facial skin, against harmful environmental factors and as a make-up base. Furthermore, the topical use of erythropoietin according to the invention counteracts the development and development of age spots, refines the complexion, supports the skin's renewal process and accelerates hair growth.

The present invention also relates to the use of, preferably low-dose, erythropoietin, that is to say EPO, preferably metered as defined in the following paragraph “Dosage according to the invention by EPO”, suitable for producing a pharmaceutical composition and intended for application in adaptation to its natural circadian rhythm Human or animal body The endogenous erythropoietin production in humans has its acrophase (daily maximum) in the late afternoon, therefore the low-dose erythropoietin as defined above is preferably administered in the morning, in particular in the period from 6:00 to 10:00, around In this period, the EPO can be administered as a single dose or in multiple doses. This use as a single dose or in multiple doses is particularly preferred according to the invention beaten for all uses mentioned in accordance with the present teaching, in particular for the cosmetic and therapeutic treatment of the human and animal body or cell.

According to the invention, in a further embodiment, the use of erythropoietin provides for endothelial progenitor cells to be used at the same time as other cell therapy-employable to be administered with prior incubation with, preferably low-dose, erythropoietin in vitro and / or local or systemic application of, preferably low-dose, erythropoietin in vivo, in order to ensure the healing of the cell-therapeutic tissue cells with sufficient connection to the vascular system.

The invention therefore also relates to the use of, preferably low-dose, erythropoietin in vivo, preferably for an application in the morning in a period from 06:00 to 10:00, in the application of endothelial progenitor cells with at least one cell therapeutically usable cell population to improve healing the cell population that can be used for therapeutic purposes with sufficient connection to the vascular system. The invention also relates to the use of, preferably low-dose, erythropoietin in vitro for incubation with endothelial progenitor cells and at least one cell population which can be used for therapeutic purposes to improve the healing of the cell population which can be used for therapeutic purposes with sufficient connection to the vascular system.

The invention also relates to the use of erythropoietin, in particular in low doses, in particular for the production of a pharmaceutical composition or a kit for the prophylaxis or treatment of diseases or used in the context of transplantations or implantations, in sequential, sequential administration with at least one other chemical, thermal, mechanical or biological agent, in particular pharmacological active ingredient, to increase the number and function of endothelial progenitor cells and / or to regenerate on or to slow the progression of tissue damage.

The invention also relates to the use of erythropoietin, in particular for the production of a pharmaceutical composition or a kit, for the prophylaxis or treatment of diseases or used in the context of transplantations or implantations, in particular in low doses, for the simultaneous administration of erythropoietin and at least one other chemical, thermal, mechanical or biological agent to increase the number and function of endothelial progenitor cells and / or to regenerate or slow the progression of tissue damage.

The invention therefore relates to the preferably sequential, chronologically sequential or simultaneous administration of low-dose erythropoietin and, in a preferred embodiment, one or more other pharmacological active ingredients, for example VEGF; GM-CSF, M-CSF, thrombopoietin, SDF-1, SCF, NGF, PIGF, an HMG co-reductase inhibitor, an ACE inhibitor, an AT-1 inhibitor and a NO donor, so as to increase the number and To increase the function of endothelial progenitor cells and / or to bring about regeneration or slowing down of tissue damage. According to the invention, the sequence A) quantitative and qualitative optimization of stem cells and / or endothelial progenitor cells in the bone marrow or in tissue-derived "stem cell" niches B) mobilization of stem cells and / or endothelial progenitor cells from the bone marrow or other "stem cell" niches into the peripheral area Blood C) Quantitative and qualitative optimization of stem cells and / or endothelial progenitor cells in the peripheral Blood and / or ex vivo under selective culture conditions, preferably culture under hypoxic conditions with an oxygen content of 0.1% to 10% D) Homing of stem cells and / or endothelial progenitor cells to the site of the damage E) Adhesion and migration of stem cells and / or endothelial progenitor cells in the target tissue F) neovascularization can be influenced by endothelial progenitor cells.

The invention therefore also relates, in particular in vivo and in vitro, to the sequential, chronologically successive or simultaneous administration of low-dose erythropoietin according to the invention and optionally one or more other chemical, thermal, mechanical and biological agents, in order to increase the number and function of endothelial progenitor cells increase / or induce and regeneration or progression slowing of tissue damage, optionally and preferably in the above loading _^ written use in adaptation to the natural circadia- NEN rhythm of the body's EPO production, that is in an application form suitable for a dose in the period from 6:00 to 10:00 in the morning is suitable and determined.

The present invention also relates to the use of low-dose erythropoietin according to the invention for stimulating the physiological mobilization, proliferation and differentiation of endothelial progenitor cells, for stimulating vasculogenesis, for the therapy of diseases which are associated with dysfunction of endothelial progenitor cells, and for the production of pharmaceutical compositions for Treatment of such diseases and of pharmaceutical compositions containing erythropoietin and other suitable active substances for stimulation. include dothelial progenitor cells from or for patients with a) dysfunction of endothelial progenitor cells and b) with at least one cardiovascular risk factor such as hypertension, hypercholesterolemia, increased asymmetric dimethylarginine (ADMA) values, insulin resistance, hyperhomocysteinemia and c) at least one end organ damage such as Hypertrophy, micro-albuminuria, cognitive dysfunction, increase in intima media thickness in the carotid artery, proteinuria or a glomerular filtration rate (GFR) <80 ml / min, in particular 30, preferably 40 to 80 ml / min. The invention preferably relates to the use of low-dose EPO in the above-mentioned patient group defined in a) to c) in an embodiment which is suitable and intended to carry out the EPO application in a period from 6:00 to 10:00 in the morning ,

The vascular endothelium is a layer of cells that lines the blood vessels. The endothelium separates the blood from other vascular layers, whereby the endothelium is not only a passive barrier, but actively intervenes in the regulation of the vascular tone. Accordingly, an endothelium-dependent vasodilation is also spoken of. Due to its location, the endothelium is permanently exposed to hemodynamic stress and metabolic stress. In the case of pathogenic conditions, for example increased blood pressure, increased LDL values, increased values of the endogenous inhibitor of NO synthetase asymmetric dimethylarginine (ADMA), hypercholersterinemia, impaired kidney function with a glomerular filtration rate of 30, preferably 40 to 80 ml / min, insulin resistance or increased Blood sugar, there are often functional defects in the endothelium, which can then be morphologically ascertainable, such as the formation of atherosclerotic plaques and / or further end organ damage, such as left ventricular hypertrophy, microalbuminuria, proteinuria, neuropathies or microcirculation disorders can follow. A very early sign of a changed or reduced endothelial function or endothelial dysfunction is a reduction in endothelium-dependent vasodilation.

In coronary artery disease (CHD), but also in the presence of risk factors without CHD, such as hypertension, impaired kidney function, hyperlipoproteinemia, hyperhomocysteinemia, insulin resistance or diabetes, endothelial function defects manifest themselves in reduced production of NO (= EDRF) and increased endothelin Production. High plasma levels of endothelin lead to abnormal cell coalescence, inflammation, vascular growth and severe vasoconstriction. Endothelial dysfunction is also characterized by an increased production of adhesion molecules such as ICAM-1 and VCAM-1, as a result of which platelets and monocytes adhere to the endothelium to an increased extent. This leads to an increase in vascular tone. This creates an imbalance in a wide variety of systems, favoring vasoconstriction, adhesion, aggregation, coagulation, atherosclerosis and atherothrombosis. Even mental stress leads to measurable endothelial dysfunction that can last up to 4 hours.

Endothelial cells are also involved in the formation of new blood vessels. Blood vessel formation is important in a variety of processes, such as embryogenesis, the female reproductive cycle, wound healing, tumor growth and neovascularization of ischemic areas. Origin- Borrowed postnatal blood vessel formation, i.e. blood vessel formation after birth, was mainly attributed to angiogenic processes. Angiogenesis means the formation of new blood vessels through the sprouting of capillaries from an already existing vascular system. In angiogenesis, the basement membrane surrounding the blood vessels is first broken down using proteolytic enzymes and the extracellular matrix is fragmented in the perovascular space. The angiogenic stimuli released in the process cause already existing differentiated endothelial cells to migrate in the direction of the chemotactic stimulus, whereby they proliferate and are converted at the same time. The endothelial cells are then juxtaposed to form new vascular loops with a capillary-shaped lumen. Then the synthesis of a new basement membrane begins.

However, recent studies show that the formation of new blood vessels in the adult organism is based not only on angiogenesis, but also on vasculogenetic mechanisms. Vasculogenesis is understood to mean the formation of new vessels from endothelial progenitor cells that differentiate in situ. The dogma that vasculogenesis is restricted to embryogenesis has been refuted by the detection of endothelial progenitor cells (EPC) in the peripheral blood of healthy people and animals. Using animal models, it could be demonstrated that the endothelial progenitor cells derived from the bone marrow are actively involved in the neovascularization. It has also been shown that a specific CD34-positive subset of leukocytes that express endothelium-specific antigens is established in ischemic regions. In addition, in vitro endothelial progenitor cells (EPC) can be obtained from CD133 + and CD34 + cells. make a significant contribution to blood vessel formation in the adult organism (Asahara et al., Science, 275 (1997), 964-967; Crosby et al., Circ. Res., 87 (2000), 728-730; Gehling et al., Blood, 95 (2000), 3106-3112). Injection of isolated CD34 + cells or cultured endothelial progenitor cells has also been shown to accelerate blood flow restoration in diabetic mice (Schatteman et al., J. Clin. Invest, 106 (2000), 571-578) and neovascularization in vivo improved (Asahara et al., Circ. Res., 85 (1999), 221-228; Crosby et al., Circ. Res., 87 (2000), 728-730; Murohara et al., J. Clin. Invest ., 105 (2000), 1527-1536). In addition, it could be shown that neovascularization induced by CD34 + cells improves cardiac function (Kocher et al., Nat. Med., 7 (2001),. 430-436). In addition to CD34 + cells, CD34-negative mononuclear blood cells can also serve as a source for endothelial progenitor cells by appropriate transdifferentiation.

However, the mechanisms underlying the mobilization and differentiation of endothelial progenitor cells have not yet been fully elucidated. Molecular and cell biological studies suggest that various cytokines and angiogenic growth factors stimulate the mobilization of endothelial progenitor cells in the bone marrow. It is known, for example, that proangiogenic factors such as VEGF and GM-CSF can increase the number of endothelial progenitor cells (Asahara et al., EMBO J., 18 (1999), 3964-3972; Takahashi et al., Nat. Med., 5: 434-438 (1999). VEGF (Vascular Endothelial Growth Factor) is a protein that occurs in different isoforms and binds to the two tyrosine kinase receptors VEGF-R1 (flt-1) and VEGF-R2 (flk-1), for example on the surface growing endothelium cells occur (Wernert et al., Angew. Chemie, 21 (1999), 3432-3435). The activation of the VEGF receptors leads via the Ras-Raf-MAP kinase path to the expression of proteinases and specific integrins on the surface of endothelial cells or endothelial progenitor cells and finally to the initiation of the proliferation and migration of these cells in the direction of the angiogenic stimulus. GM-CSF (Granulocyte Macrophage Colony Stimulating Factor) is a cytokine that was previously known primarily for the stimulation of white blood cells including neutrophils, macrophages and eosinophils. PIGF (placenta growth factor) is known to stimulate mobilization of endothelial progenitor cells, but not their proliferation. From studies by Llevadot et al. (J. Clin. Invest, 108 (2001), 399-405) shows that HMG-CoA reductase inhibitors, especially statins, which are used as lipid-lowering drugs and reduce the morbidity and mortality of a coronary disease, are endothelial progenitor cells can mobilize. Dimmeier et al. (J. Clin. Invest., 108 (2001), 391-397) were also able to show that statins such as atorvastatin and simvastatin differentiated the differentiation of endothelial progenitor cells into mononuclear cells and CD34 + stem cells which were isolated from peripheral blood. significantly improve in vitro and in vivo. The treatment of mice with statins led to an increased number of differentiated endothelial progenitor cells, with statins having the same potent effect as VEGF.

The present invention is based on the technical problem of providing means and methods for improved stimulation of endothelial progenitor cells and for the therapy of diseases, in particular in connection with endothelial dysfunction Precursor cells are available as well as means and methods for the protection and regeneration of different tissues.

The present invention solves this technical problem by teaching, in particular low-dose, erythropoietin and / or its derivatives for stimulating the physiological mobilization of endothelial progenitor cells, the proliferation of endothelial progenitor cells, the differentiation of endothelial progenitor cells to endothelial cells and / or the migration of endothelial progenitor cells Use direction of an angiogenic or vasculogenic stimulus in a human or animal body. The stimulation of the mobilization and / or differentiation of endothelial progenitor cells according to the invention represents an important new therapeutic strategy for increasing postnatal neovascularization, in particular vasculogenesis, and for the treatment of diseases which are associated with dysfunction of endothelial progenitor cells and / or endothelial cells, and the Protection and regeneration of different tissues through harmful chemical, thermal, mechanical and biological agents.

The present invention also solves this technical problem through the teaching of using low-dose erythropoietin and / or its derivatives for the therapy of diseases or pathological conditions which are associated with dysfunction of endothelial progenitor cells and / or endothelial cells.

Furthermore, the present invention also solves this technical problem by teaching that low-dose erythropoietin and / or its derivatives for the protection and regeneration of different tissue types in the state of diseases or pathological conditions. to use those related to dysfunction of the specific tissue function.

The underlying technical problem is also solved by the sequential, temporally successive or simultaneous administration of low-dose erythropoietin and one or more other chemical, thermal, mechanical and biological agents.

The invention therefore relates in particular to the following embodiments A) to K) in each case alone and / or in combination:

A) Use of erythropoietin, preferably for the production of a pharmaceutical composition, for the prophylaxis or treatment of diseases, the erythropoietin and / or the pharmaceutical composition being suitable and intended for morning application to a human or animal body in a period of 6: 00 to 10:00.

B) Use of erythropoietin, preferably in combination with embodiment A), in particular for the production of a pharmaceutical composition for the prophylaxis or treatment of diseases, the pharmaceutical composition in its low dosage being suitable and intended for the prophylaxis or treatment of a human or animal Patients of a) at least one dysfunction of endothelial progenitor cells, b) at least one cardiovascular risk factor such as hypertension, hypercholestrinemic, insulin resistance, increased ADMA values or hyperhomocytemia and c) at least one end organ damage such as left ventricular hypertrophy, microalbuminuria, increased cognitive dima- tria -Media thickness in the carotid artery, proteinuria or has a glomerular filtration rate <80 ml / min, preferably 30 to 80 ml / min.

C) Use of erythropoietin, preferably in combination with embodiment A), B) or A) and B), for the cosmetic treatment of the human or animal body, in particular for the treatment of wrinkles, for strengthening the connective tissue, for protecting and tightening the Skin, to protect against damaging environmental effects, to treat age spots, to accelerate re-epithelialization, to accelerate hair growth and / or as a make-up base.

D) use of erythropoietin, preferably in combination with embodiment A), B) or A) and B), for producing a cosmetic preparation, in particular for topical application, for the cosmetic treatment of the human or animal body, in particular for treating wrinkles, to strengthen the connective tissue, to protect and tighten the skin, to protect against damaging environmental effects, to treat age spots, to accelerate re-epithelialization, to accelerate hair growth, and / or as a make-up base.

E) Use of erythropoietin, preferably in combination with one or more of the embodiments according to A), B), C) or D), and / or a mixture of endothelial progenitor cells with at least one cell therapy which can be used for cell therapy for the production of a pharmaceutical composition comprising E. rythropoietin and a mixture of endothelial progenitor cells with at least one cell therapeutically usable cell population for the regeneration of tissue or vessels in a human or animal body, the mixture having been brought into contact with erythropoietin in vitro before application.

F) Use of erythropoietin, preferably in combination with one or more of the embodiments according to A), B), C), D) or E), and / or a mixture of endothelial progenitor cells with at least one cell therapeutically usable cell population for producing a pharmaceutical composition , containing erythropoietin and / or a mixture of endothelial progenitor cells with at least one cell therapeutically usable cell population for the regeneration of tissues or vessels in a human or animal body, erythropoietin being administered to the animal or human body before, after or simultaneously with the application of the mixture ,

G) Use of erythropoietin, preferably in combination with one or more of the embodiments according to A) to F), and / or at least one chemical, thermal, mechanical or biological agent, in particular a pharmacological active ingredient, for the production of a pharmaceutical composition or a kit , containing erythropoietin and the at least one chemical, thermal, mechanical or biological agent for the prophylaxis or treatment of diseases, the pharmaceutical composition or the kit being suitable and intended for the sequential, sequential or simultaneous application of the erythropoietin with the at least one chemical , thermal, mechanical or biological agent. The invention therefore also relates to the use of erythropoietin in the manner referred to under G above, the mechanical agents being endoprostheses, preferably implant implants. tion body for the tooth, bone or ligament / tendon replacement. The invention also relates to the use of erythropoietin in the manner described above under G), the biological agents being solid organs such as the liver, kidney, heart, pancreas or skin. In this context, biological agents are also understood to mean hair implants. In a particularly preferred embodiment, the present invention therefore relates to the use of erythropoietin for the production of a pharmaceutical composition or a kit for systemic or local application to an implantation site of a biological agent of the aforementioned type or an endoprosthesis, in particular an implantation body such as a tooth, dentures , Dental implant, bone replacement, bone implant, for example hip joint prosthesis, ligament / tendon replacement, for example cruciate ligament, the erythropoietin being applied systemically or locally, for example a few weeks before the implantation of the biological or mechanical agent mentioned, that is to say for example the endoprosthesis the implantation and then the implantation. Another embodiment also provides for the implantation of the biological or mechanical agent mentioned, for example the endoprosthesis, at the same time as the use of erythropoietin. In a further embodiment, it is provided that the erythropoietin is carried out after the implantation of the endoprosthesis or mechanical or biological agent mentioned. According to these embodiments, the tissue or the body structure into which the implant, for example the tooth or the bone prosthesis is implanted, is mobilized or conditioned, and thus enables a considerably better and thus also Faster integration, for example the growth or ingrowth of the biological or mechanical agent, for example implant into the body structure.

H) Use of erythropoietin according to one or more of the embodiments according to A) to G), the pharmaceutical composition, when applied in the human or animal body, not leading to an increase in the hematocrit value, in particular by no more than 10% of the hematocrit value the application of erythropoietin.

I) Use of erythropoietin according to one or more of the embodiments according to A) to H) in a pharmaceutical composition, the erythropoietin in a low, non-erythropoietically effective dose being suitable and intended for the prophylaxis, treatments or therapies mentioned, in particular in one Dose of 0.001 lU / kg body weight / week to 90, in particular 50 lU / kg body weight / week is used.

K) Use of erythropoietin according to one or more of the embodiments according to A) to I), wherein the disease hypercholesterolemia, diabetes mellitus, insulin resistance, endothelium-mediated chronic inflammatory diseases, endotheliosis including reticuloendotheliosis, atherosclerosis, age-related cardiovascular disease, ischemic Diseases of the extremities, preeclampsia, Raynaud's disease, liver diseases such as hepatitis, cirrhosis, acute or chronic liver failure, bone and cartilage diseases or injuries, mucosal diseases or injuries, especially in the gastrointestinal tract, Crohn's disease, ulcerative colitis, pregnant shaft-induced hypertension, chronic or acute renal insufficiency, especially end-stage renal insufficiency, renal function restrictions with glomerular filtration rates of 30 to 80 ml / min, microalbuminuria, proteinuria, conditions with increased ADMA levels or wounds and secondary diseases thereof.

The invention also relates to the production of a kit containing erythropoietin, endothelial progenitor cells and at least one cell population which can be used for cell therapy, the erythropoietin preferably being present in low doses.

According to the invention it was surprisingly found that treatment with low-dose erythropoietin leads to the physiological mobilization of endothelial progenitor cells, the number of circulating endothelial progenitor cells being increased and their differentiation being induced. In addition, functional deficits of the endothelial progenitor cells occurring under certain pathological conditions are compensated for. According to the invention, it was possible to show that the number of circulating stem cells in patients with end stage chronic kidney disease is just as high as in healthy volunteers, but these have lost the ability to differentiate into endothelial cells via endothelial progenitor cells in these patients. In patients with chronic kidney disease, the number of cells that are adhesive and show an endothelial cell phenotype is significantly reduced compared to healthy volunteers (de Groot et al; Kidney Int. 2004; 66: 641-6). This functional drop in endothelial progenitor cells can be found with a moderate degree of renal function impairment with a glomerular filtration rate of 30, preferably 40, to 80 ml / min. According to the invention found that the number of circulating stem cells increases significantly by more than 50% both after treatment of these patients as well as healthy kidney patients and / or test subjects with low-dose erythropoietin according to the invention. In particular, the number of cells developing an endothelial phenotype increases dramatically. As demonstrated by a functional cell culture test, the low-dose erythropoietin treatment increases the ability of the endothelial precursor cell cells to adhere to the patients with chronic kidney disease with a glomerular filtration rate of 30, preferably 40 to 80 ml / min, for adhesion, in healthy subjects and / or patients by two to three times. The ability of differing endothelial progenitor cells or of endothelial cells for adhesion is one of the basic requirements for the formation of new tissues and / or vessels. In this way, erythropoietin can induce neovascularization, in particular vasculogenesis, in tissues or organs, for example in particular in kidneys, in which corresponding vasculogenic or angiogenic stimuli are released.

According to the invention, low-dose erythropoietin can be used to stimulate the physiological mobilization of endothelial progenitor cells, the proliferation of endothelial progenitor cells, the differentiation of endothelial progenitor cells to endothelial cells and / or for the migration of endothelial progenitor cells in the direction of a vasculogenic or angiogenic stimulus, in particular a human or a human body adult organism. According to the invention, low-dose erythropoietin can therefore advantageously be used to stimulate the formation of new vessels by vasculogenesis in tissues or organs who have pathological vascular changes. Due to the stimulation of endothelial progenitor cells by low-dose erythropoietin, the formation of endothelial tissue can also be induced. According to the invention, low-dose erythropoietin can therefore also be used for the treatment of diseases of the human or animal body which are associated with a dysfunction of endothelial progenitor cells and / or endothelial cells. Patient populations with such a dysfunction usually show cardiovascular risk factors such as hypertension, hypercholesterolemia, insulin resistance, hyperhomocysteinemia, increased ADMA levels, and end organ damage such as left ventricular hypertrophy, microalbuminuria, proteinuria or a glomerular filtration rate (preferably 40 GFR) , up to 80 ml / min.

The invention also relates to the use of low-dose erythropoietin for the protection and regeneration of tissue at risk of functioning due to the action of chemical, thermal, mechanical and biological agents. According to the invention, the topical application of low-dose erythropoietin also relates to the prophylaxis and reduction of existing wrinkles in the skin, in particular the facial skin, and the protection of the skin and the reduction of age spots. According to the invention, low-dose erythropoietin or a descendant can be used sequentially, sequentially in time or simultaneously with one or more other chemical, thermal, mechanical and biological agents. According to the invention, low-dose erythropoietin can be used therapeutically in adaptation to its circadian rhythm in order to achieve a maximum biological effect. Endothelial progenitor cells In a preferred embodiment, according to the invention, they are administered simultaneously with other cell populations that can be used in therapy, with prior incubation with low-dose erythropoietin in vitro and / or local and systemic application of low-dose erythropoietin in vivo, in order to heal the cell-therapeutic tissue cells with sufficient connection to the Ensure vascular system.

In connection with the present invention, “erythropoietin” or “EPO” is understood to mean a substance which controls the growth, differentiation and maturation of stem cells via erythroblasts to erythrocytes in correspondingly high doses.

Erythropoietin is a glycoprotein that has 166 amino acids, three glycosylation sites and a molecular weight of approximately 34,000 Da. As part of an EPO-induced differentiation of erythrocyte progenitor cells, globin synthesis is induced and the synthesis of the heme complex and the number of ferritin receptors are increased. This allows the cell to absorb more iron and synthesize functional hemoglobin. Hemoglobin binds oxygen in the mature erythrocytes. The erythrocytes and the hemoglobin they contain play a key role in the oxygen supply to the body. These processes are triggered by the interaction of EPO with a corresponding receptor on the cell surface of the erythrocyte progenitor cells (Graber and Krantz, Ann. Rev. Med. 29 (1978), 51-56). The term "erythropoietin" used here encompasses EPO of any origin, in particular human or animal EPO. The term used here covers not only the naturally occurring, that is to say wild-type forms of EPO, but also its derivatives, analogs, modifications, muteins, mutants or Other, as long as they show the biological effects of wild-type erythropoietin.

In connection with the present invention, “derivatives” are understood to mean functional equivalents or derivatives of erythropoietin which are obtained while maintaining the basic structure of erythropoietin by substitution of one or more atoms or molecular groups or residues, in particular by substitution of sugar chains such as ethylene glycol, and / or whose amino acid sequences differ from that of the naturally occurring human or animal erythropoietin protein in at least one position, but which essentially have a high degree of homology at the amino acid level and comparable biological activity. Derivatives of erythropoietin, such as can be used for example in the present invention, are known, inter alia, from WO 94/25055, EP 0 148 605 B1 or WO 95/05465.

"Homology" means in particular a sequence identity of at least 80%, preferably at least 85% and particularly preferably at least more than 90%, 95%, 97% and 99%. The term “homology” known to the person skilled in the art thus denotes the degree of relationship between two or more polypeptide molecules, which is determined by the agreement between the sequences. There can be a match both mean an identical match as well as a conservative amino acid exchange.

According to the invention, the term “derivative” also includes fusion proteins in which functional domains of another protein are present on the N-terminal part or on the C-terminal part. In one embodiment of the invention, this other protein can be, for example, GM-CSF, VEGF, PIGF, a statin or another factor which has a stimulating effect on endothelial progenitor cells. In a further embodiment of the invention, the other protein can also be a factor that has a stimulating effect on differentiated endothelial cells, for example angiogenin, VEGF (vascular endothelial growth factor) or bFGF (basic fibroblast growth factor). It is known from bFGF and VEGF that this growth factor exerts a strong mitogenic and chemotactic activity on endothelial cells.

The differences between an erythropoietin derivative and native erythropoietin may have arisen, for example, from mutations such as deletions, substitutions, insertions, additions, base changes and / or recombinations of the nucleotide sequences encoding the erythropoietin amino acid sequences. According to the invention, erythropoietin (EPO-) alpha, (EPO-) beta, Aranesp (darbepoetin alpha) or Gera (continuous erythropoietin receptor agonist) is preferably used. Of course, these can also be naturally occurring sequence variations, for example sequences from another organism or sequences that have been mutated naturally, or mutations that have been area of known agents, for example chemical agents and / or physical agents, have been specifically introduced into the nucleic acid sequences coding for erythropoietin. In connection with the invention, the term “derivative” therefore also includes mutated erythropoietin molecules, ie erythropoietin muteins.

According to the invention, peptide or protein analogs of erythropoietin can also be used. In connection with the present invention, the term “analogs” encompasses compounds which do not have an amino acid sequence identical to the erythropoietin amino acid sequence, but whose three-dimensional structure is very similar to that of erythropoietin and which therefore have a comparable biological activity. Erythropoietin analogs can be For example, these are compounds which contain the amino acid residues responsible for the binding of erythropoietin to its receptors in a suitable conformation and which can therefore mimic the essential surface properties of the erythropoietin binding region. Such compounds are described, for example, in Wrighton et al., Science, 273 ( 1996), 458. The EPO used according to the invention can be produced in various ways, for example by isolation from human urine or from the urine or plasma (including serum) from patients suffering from aplastic anemia nten (Miyake et al., JBC 252 (1977), 5558). Human EPO can, for example, also from tissue cultures of human kidney cancer cells (JA-OS 55790/1979), from human lymphoblast cells which have the ability to produce human EPO (JA-OS 40411/1982) and from a hybridoma culture obtained by cell fusion of a human cell line be won. EPO can also be produced by genetic engineering techniques by the desired protein is genetically produced by means of suitable DNA or RNA which codes for the corresponding amino acid sequence of the EPO, for example in a bacterium, a yeast, a plant, animal or human cell line. Such methods are described for example in EP 0 148 605 B2 or EP 0 205 564 B2 and EP 0411 678 B1.

The present invention relates in particular to the use of low-dose erythropoietin and / or derivatives thereof for stimulating the physiological mobilization of endothelial progenitor cells, the proliferation of endothelial progenitor cells, the differentiation of the endothelial progenitor cells to endothelial cells and / or for the migration of endothelial progenitor cells in the direction of a vascular or logical one angiogenic stimulus in a human or animal body, especially an adult organism.

The invention further relates to the sequential use of low-dose erythropoietin and at least one further suitable chemical, thermal, mechanical or biological agent or active substance, in particular pharmacological active substance, which increases the function and number of endothial progenitor cells, and the organ-protective and regenerative action of low-dose Erythropoietin increased.

Furthermore, the invention therefore preferably relates to the sequential, chronologically successive or simultaneous administration of low-dose erythropoietin and one or more other pharmacologically active substances, for example VEGF; GM-CSF, M-CSF, thrombopoietin, SCF, SDF-1, NGF, PIGF, an HMG co-reductase inhibitor, an ACE inhibitor, an AT-1 inhibitor and one NO donor in order to increase the number and function of endothelial progenitor cells and / or to bring about regeneration or slowing down of tissue damage. Sequence A) quantitative and qualitative optimization of stem cells and / or endothelial progenitor cells in the bone marrow or in tissue-derived "stem cell" niches B) mobilization of stem cells and / or endothelial progenitor cells from the bone marrow or other "stem cell" niches into the peripheral blood C) Quantitative and qualitative optimization of stem cells and / or endothelial progenitor cells in peripheral blood and / or ex vivo under selective culture conditions, preferably culture under hypoxic conditions with an oxygen content of 0.1% to 10% D) Homing of stem cells and / or endothelial progenitor cells at the site of the damage E) adhesion and migration of stem cells and / or endothelial progenitor cells into the target tissue F) neovascularization can be influenced by endothelial progenitor cells.

The present invention thus relates to the simultaneous as well as time-delayed application of endothelial progenitor cells and one or more cell therapeutically usable cell populations, in particular hepatocytes, myocytes, cardiomyocytes or islet cell transplants, after prior incubation with low-dose erythropoietin in vitro and / or local and systemic application of low dosed erythropoietin in vivo, which improves and accelerates the function, the healing as well as the vascularization and the connection to the bloodstream of the recipient of these cell therapeutically used cell populations. The present invention relates to the use of erythropoietin, in particular in a low dose, or suitable active ingredients for topical use in the sense of "beauty care", in particular prophylaxis or prompt reduction of wrinkles and fine lines, strengthening of the connective tissue, protection and tightening of the skin, in particular the facial skin , against harmful environmental factors and as a make-up base. Furthermore, the topical application of erythropoietin counteracts the development and development of age spots, refines the complexion and supports the renewal process of the skin, especially re-epithelialization. In addition, erythropoietin accelerates hair growth.

The present invention also relates to the use of low-dose erythropoietin suitable for the production of a pharmaceutical composition and intended for use in adapting to its circadian rhythm. Endogenous erythropoietin production has its acrophase (daily maximum) in the late afternoon, so low-dose erythropoietin should preferably be given in the morning, in particular between 6:00 and 10:00, in order to achieve a maximum biological, therapeutic or cosmetic effect.

The present invention relates to the use of low-dose erythropoietin for stimulating physiological mobilization, or / and for the proliferation and differentiation of endothelial progenitor cells, or / and for stimulating vasculogenesis, or / and for the therapy of diseases associated with dysfunction of endothelial progenitor cells are related, and / or for the production of pharmaceutical compositions for the treatment of such diseases and pharmaceutical compositions, which comprise erythropoietin and other suitable active substances for stimulating endothelial progenitor cells, in patients who a) at least one dysfunction of endothelial progenitor cells, and b) at least one cardiovascular risk factor such as hypertension, hypercholesterolemia, insulin resistance, hyperhomocysteinemia, increased ADMA levels and c) at least end organ damage such as left ventricular hypertrophy, microalbuminuria, cognitive dysfunction, increase in intimal media thickness in the carotid artery, proteinuria or a glomerular filtration rate (GFR) of less than 80 ml / min, in particular 30, preferably 40 to 80 ml / min exhibit.

In a preferred embodiment, the invention also relates to the sequential, chronologically successive or simultaneous administration of low-dose erythropoietin and one or more other chemical, thermal, mechanical and biological agents, so as to increase the number and function of endothelial progenitor cells and / or the regeneration or Slowing progression of tissue damage. The mechanical agents can be endoprostheses, preferably implant bodies, for example for the tooth, bone or ligament / tendon replacement. Furthermore, the biological agents can be solid organs, such as the liver, kidney, heart, pancreas or skin, or else hair implants. The invention therefore provides that EPO is used in particular in a low dose manner in order to allow, at the same time, subsequently or previously implanted mechanical agents, such as endoprostheses or biological agents, to grow or integrate better, faster and more efficiently into the surrounding body structure. The invention therefore also relates to the use of erythropoietin for the production of a pharmaceutical composition or Kits for improving, in particular for promoting and / or accelerating, the integration of biological agents or endoprostheses in surrounding body structures, in particular teeth, dentures, dental implants or other endoprostheses, such as bone replacements, bone implants, in particular hip joint prostheses or ligament / tendon replacements, such as cruciate ligaments , If necessary, provision can be made for the erythropoietin to be used together with cell populations suitable for cell therapy and / or endothelial progenitor cells. In the aforementioned use of erythropoietin for the production of a pharmaceutical composition or a kit for the improvement, in particular promotion and / or acceleration of the integration of biological or mechanical agents in target structures, in particular target tissue, target bone or target cartilage of a patient, can be provided in a further preferred embodiment be that the mechanical agents to be used are made, for example, of steel, ceramic, plastic or another matrix material. In addition, it can be provided that cell populations, osteoblasts, cells with osteogenic potential, platelets, blood cells or the like which are suitable as cell therapy are used in the use mentioned. In a further preferred embodiment it can be provided that in particular the mechanical agent to be used is contained in the pharmaceutical composition or the pharmaceutical kit together with organic adhesive, for example a fibrin adhesive.

In connection with the present invention, “endothelial progenitor cells” (endothelial progenitor cells; EPC) are understood to mean cells circulating in the bloodstream which have the ability to differentiate into endothelial cells Endothelial progenitor cells occurring in embryonic development are angioblasts. The endothelial progenitor cells occurring in the adult organism are angioblast-like cells which can be obtained from mononuclear cells, in particular CD34-CD14 + monocytes, and / or CD34 + stem cells which have been isolated from peripheral blood.

In connection with the present invention, “mobilization” or “physiological mobilization” means the process of activating stem cells and / or progenitor cells from the bone marrow or from alternative “stem cell” niches by means of growth factors, the stem cells or progenitor cells in the Blood circulation, especially in the peripheral blood.

In connection with the present invention, “proliferation” is understood to mean the ability of cells to enlarge and subsequently divide into two or more daughter cells. The EPO-mediated stimulation of endothelial progenitor cells thus relates in particular to the number and thus the division behavior of endothelial progenitor cells.

In connection with the present invention, “differentiation” of endothelial progenitor cells is understood to mean the development of mononuclear cells originating from the bone marrow or tissue-resistant niches via endothelial progenitor cells to endothelial cells. “Endothelial cells” are understood to mean the cells that make up the endothelium, that is to say the single-layered cell Lining vessels and serous cavities. Endothelial cells are characterized by the fact that they contain vascular active substances, release, for example, vasodilators such as EDRF (endothelial derived relaxing factor) or constricting substances such as endothelin, factors for inhibiting or activating blood coagulation and factors for regulating vascular permeability. Endothelial cells also synthesize components of the subendothelial connective tissue, in particular collagens of type IV and V, cell adhesion proteins such as laminin, fibronectin and thrombospondin, growth factors, for example for smooth muscle cells, and factors for the formation of new vessels.

In connection with the present invention, “migration” of the endothelial progenitor cells is understood to mean that the endothelial progenitor lines located in the bloodstream migrate in the direction of a vasculogenic or angiogenic stimulus and concentrate in the area of the vasculogenic or angiogenic stimulus. A “vasculogenic stimulus” means one chemical stimulus understood in a tissue or blood vessel of a human or animal body which acts specifically on endothelial progenitor cells and causes their migration to the location of the body from which the chemical stimulus originates. In this way, the process of vasculogenesis is induced by the vasculogenic stimulus. An "angiogenic stimulus" is understood to mean a chemical stimulus in a tissue or blood vessel of a human or animal body which acts specifically on differentiated endothelial cells and causes them to migrate to the location of the body from which the chemical stimulus originates. The angiogenic stimulus causes thus inducing angiogenesis.

In a further embodiment of the invention, the use of low-dose erythropoietin and / or derivatives thereof is used Increasing the adhesiveness of differentiating endothelial progenitor cells is planned. According to the invention, erythropoietin is used in particular to improve the ability of endothelial progenitor cells for adhesion, that is to say for cell-cell adhesion. The adhesion of differentiating endothelial progenitor cells or differentiated endothelial cells is one of the basic prerequisites for the formation of new vessels or a new endothelial tissue. Cell adhesion is mediated by protein molecules.

The present invention also relates to the use of low-dose erythropoietin for stimulating new vascularization, in particular the stimulation of vasculogenesis. In connection with the present invention, “vasculogenesis” is understood to mean the formation of new vessels from endothelial progenitor cells that differentiate in situ. According to the invention, the use of low-dose erythropoietin means that endothelial progenitor cells are increasingly involved in new vessel formation or local vascular formation The invention provides that the use of low-dose erythropoietin and / or its derivatives promotes the formation of new blood vessels and / or the replacement of damaged vascular areas by local formation of new blood vessels.

In a further embodiment of the invention, the use of low-dose erythropoietin and / or derivatives thereof is provided for the stimulation of endothelial progenitor cells for the formation of endothelial tissue. In a particularly preferred embodiment of the invention, the use of low-dose erythropoietin and / or derivatives thereof is provided for the therapy of disease states or diseases of the human or animal body which are associated with a dysfunction of endothelial progenitor cells or of secondary diseases thereof.

In connection with the present invention, “diseases”, “disease states” or “diseases” are understood to mean disturbances in life processes in organs or in the entire organism with the result of subjectively perceived or objectively ascertainable physical, mental or mental changes diseases which are associated with a dysfunction of endothelial progenitor cells, that is to say diseases which are either the result of such a dysfunction of these cells or which are mediated by these cells. Furthermore, according to the invention, "diseases", "disease states" or " Diseases "Disorders of life processes in organs or in the whole organism understood, which can be slowed down or slowed down in particular by the administration of low-dose erythropoietin or suitable active substances. “Subsequent diseases” are understood to be secondary diseases, that is to say a second disease that is added to a primary clinical picture.

In the context of the present invention, a "dysfunction" of endothelial progenitor cells is a disruption of essential cell functions such as metabolic performance, stimulus response, motility, division behavior or differentiation behavior. understood these cells. Dysfunction of endothelial progenitor cells can consist, for example, in that these cells do not proliferate or only inadequately. Since the use of erythropoietin stimulates the proliferation of endothelial progenitor cells, the deficient division behavior of both endothelial progenitor cells and already differentiated endothelial cells can be compensated and the number of endothelial progenitor cells or endothelial cells increased. Dysfunction of endothelial progenitor cells can consist, for example, of the impaired ability of these cells to differentiate into endothelial cells. The dysfunction of endothelial progenitor cells may also be due to their impaired ability to adhere and / or their ability to migrate towards an angiogenic or vasculogenic stimulus. Such dysfunctions of endothelial progenitor cells can lead, for example, to impairing or preventing the formation of new endothelial tissue and / or vasculogenesis. Dysfunction of endothelial progenitor cells can also be pathogenic, for example due to hypertension, hyperlipoproteinemia, increased ADMA blood levels, uremia or diabetes. The dysfunction of endothelial progenitor cells can result, for example, in a reduced production of NO (= EDRF) by NO synthases (NOS) from L-arginine, increased endothelin production and / or in an increased production of adhesion molecules such as ICAM-1 and VCAM- express.

According to the invention, the diseases associated with dysfunction of endothelial progenitor cells are, in particular, hypercholesterolemia, diabetes mellitus, insulin resistance, endothelium-mediated chronic inflammation. diseases such as vascular inflammation, endotheliosis including reticuloendotheliosis, atherosclerosis, age-related cardiovascular disease, ischemic diseases of the extremities, Raynaud's disease, preeclampsia, pregnancy-induced hypertension, chronic or acute renal insufficiency, in particular terminal renal insufficiency with renal insufficiency 30 / min, preferably 40 to 80 ml / min, microalbuminuria, proteinuria, increased ADMA levels, wound healing and secondary diseases thereof.

"Hypercholesterolemia" is characterized by increased concentrations of cholesterol in the blood. By far the most common form of primary hypercholesterolaemia is polygenic hypercholesterolaemia. Secondary hypercholesterolaemia often occurs in diabetes mellitus, nephrotic syndrome, hypothyroidism and liver diseases.

"Diabetes mellitus" is a combination of various forms of glucose metabolism disorders with different aetiology and symptoms. The AGE-RAGE system is particularly responsible for the development of vascular-related diabetic complications. AGEs (Advanced Glycation End Products) are the result of a series of complex reactions after long-term exposure of proteins or lipids to reducing sugars, for example glucose The formation of AGEs takes place during the normal aging process and to an increased extent in diabetes mellitus and Alzheimer's disease The binding of AGEs leads to oxidative stress, to activation of the transcription factor NF-κB and thus to a disruption of endothelial homeostasis. "Insulin resistance" is understood to mean the disturbed signal transmission in various body cells that ignore the physiological signaling cascade of insulin and so affected patients do not have a normal glucose metabolism.

"Endothelium-mediated chronic inflammatory diseases" are diseases or conditions of a human or animal body which are based on a defense reaction of the organism and its tissues to harmful stimuli, with certain signaling molecules changing the properties of endothelial cells so that, in conjunction with the activation of other cell types, leukocytes stick to the endothelial cells, eventually penetrate into the tissue and cause inflammation there. An example of an endothelium-mediated inflammation is leukocytic vasculitis. The transcription factor NF-B. plays a central role in the activation of an endothelium-mediated inflammatory event another system that leads to the development of endothelial cell-mediated chronic inflammation is the AGE-RAGE system.

“Endotheliosis” is understood to mean degenerative and proliferative endothelial changes in the non-thrombopenic purpura. “Reticuloendotheliosis” means diseases of the reticulohistiocytic system, such as reticulum, reticulosis, reticulohistiocytosis and Hand-Schüller-Christian disease.

“Raynaud's disease” is understood to mean ischemic states of the arteries of the fingers caused by vasoconstriction, that is to say vascular spasms. The primary Raynaud's disease is a purely functional disturbance of the small supplying vessels of the acra, during the secondary Rynaud's disease a other underlying disease, such as vascular inflammation.

“Preeclampsia is an endothelial and vascular disease of the maternal organism and appears to be the effect of endotheliotropic substances from the placenta. Preeclampsia is a multi-system disease that can lead to dysfunction of numerous organs and can manifest itself in a variety of symptoms. The circulatory disorders that are typical of the disease are the result of increased vascular resistance, which can vary locally. For pre-eclampsia, it is certain that endothelial dysfunction is a central component of the pathogenesis.

In the context of the present invention, “renal insufficiency” is understood to mean the limited ability of the kidneys to excrete substances that require urine, with the ability to regulate the electrolyte, water and acid-base balance to be lost in advanced stages. Terminal renal insufficiency is caused by a Breakdown of excretory and endocrine renal function characterized.

According to the invention, renal insufficiency can be acute renal insufficiency, which is also referred to as acute kidney failure, shock kidney or shock anuria. Acute renal insufficiency is characterized by a sudden partial or total loss of excretory kidney function as a result of mostly reversible kidney damage. The cause can be reduced perfusion due to hypovolemia, hypotension and dehydration as a result of blood loss (polytrauma, gastrointestinal or postpartum bleeding, major surgical interventions on the heart, vessels, abdomen or prostate ta), shock (myocardial infarction, embolism), severe infections (sepsis, peritonitis, cholecystitis), hemolysis (hemolytic-uraemic syndrome, paroxysmal hemoglobinuria, transfusion incident), myolysis (crush syndrome, rhabdomyolysis), myositis, burns Loss of water and electrolytes (massive vomiting, diarrhea, excessive sweating, hay, acute pancreatidis). Other causes can be nephrotoxins such as exogenous toxins, for example aniline, glycol compounds, methanol and the like, or endogenous toxins, for example myoglobin and oxalates. Other causes of acute renal insufficiency are kidney diseases, for example inflammatory nephropathies or rejection reactions after kidney transplantation. Acute renal failure can also be caused by urinary congestion as a result of urinary drainage problems. The treatment of acute renal insufficiency according to the invention with, preferably low-dose, erythropoietin leads, according to the invention, to preventing or at least reducing the progression of acute renal insufficiency.

According to the invention, renal failure can also be chronic renal failure. Chronic renal insufficiency is caused by vascular, glomerular and tubo-interstitial kidney diseases, infections and congenital or acquired structural defects. Causes of chronic renal failure include chronic glomerulopathy, chronic pyelonephritis, analgesic nephropathy, obstructive uropathy, and arteriosclerosis and arteriolosclerosis. Chronic renal failure ends in uremia. The treatment of chronic renal insufficiency according to the invention with low-dose erythropoietin leads, according to the invention, to a reduction in the progression of chronic renal insufficiency. In particular, the invention therefore relates to the use of preferably low-dose EPO for the manufacture of a medicament for preventing, reducing or slowing the damage to kidney tissue and / or for regenerating damaged kidney tissue in the case of acute or chronic renal failure.

In accordance with the invention, kidney function restriction means conditions in which the glomerular filtration rate has already fallen below 80 ml / min. The impairment of kidney function therefore refers to the early phase of glomerular, tubulointerstial and vascular kidney diseases. The treatment according to the invention of kidney function restrictions with low-dose erythropoietin leads, according to the invention, to a reduction in progression and to regeneration of the onset of kidney tissue and / or functional damage.

In the context of the present invention, “microalbuminuria” is understood to mean a clinical picture in which affected patients have an unphysiological excretion of albumin in the urine of more than 30 mg / 24 h. This increased excretion of albumin is and is an early sign of an onset of renal function deterioration Result of the first pathological remodeling processes in the kidney, accompanied by structural changes in the kidney architecture.

In connection with the present invention, “proteinuria” is understood to mean a clinical picture in which affected patients have an unphysiological excretion of proteins of more than 150 mg / 24 h. This increased excretion of protein Urine (> 150mg / 24h) is considered pathological and requires further medical clarification and therapy.

In connection with the present invention, “high ADMA values” is understood to mean a clinical picture in which the affected patients have an unphysiologically high ADMA blood concentration of more than 1.3 μmol / l. This increased ADMA concentration is associated with endothelial dysfunction and is a consequence metabolic dysfunctions in the breakdown or excretion processes of this molecule.

In connection with the present invention, “wound healing” is understood to mean the physiological processes for the regeneration of destroyed tissue or for closing a wound, in particular the new formation of connective tissue and capillaries. The wound healing can be a primary wound healing (sanatio per primam intentionem) , which is characterized by a quick and uncomplicated closure and extensive restitution ad integrum due to minimal formation of connective tissue between the well-perfused and possibly adapted wound edges of a clean wound. In the case of wounds with further apart, in particular bruised or necrotic wound edges or wound infections, secondary wound healing is delayed (Sanatio per second intentionem), in which the tissue defect is filled with granulation tissue and extensive scar tissue formation as a result of (a) bacterial inflammation e Epithelization from the edge completes wound healing. Wound healing is divided into three phases, namely the latency phase, the proliferation phase and the repair phase. The latency phase in turn, it is divided into the exudative phase with scab formation, especially in the first hours after the wound, and the resorptive phase with catabolic autolysis, which extends over a period of one to three days after the wound occurs.The proliferation phase is through an anabolic repair characterized by formation of collagen by fibroblasts and occurs on the fourth to seventh day after the wound has occurred. The repair phase, which is characterized by the transformation of the granulation tissue into a scar, begins on the eighth day after the wound has developed.

In the context of the present invention, a “wound” is understood to mean an interruption of the connection between body tissues with or without substance loss, which is caused by mechanical injury or physically-induced cell damage. For the purposes of the present invention, a wound is also referred to as a disease mechanical wounds, thermal wounds, chemical wounds, radiation-related wounds and illness-related wounds.Mechanical wounds are caused by external violence and occur primarily as cuts and stab wounds, bruises, lacerations, tears and abrasions, scratches and bites and bullet wounds. Thermal wounds are caused by the action of heat or cold. Chemical wounds are particularly caused by acid or alkali burns. Radiation-related wounds are caused, for example, by the action of actinic and ionizing radiation. Wounds caused by illness are particularly congestion-related wounds, traumatic wounds, diabetic wounds, etc. According to the invention, it is particularly provided that for wound healing low-dose erythropoietin is preferably applied topically or intravenously.

The present invention relates to the use of low-dose erythropoietin for the therapy of hypercholesterolemia, diabetes mellitus, insulin resistance, endothelium-mediated chronic inflammatory diseases, endotheliosis including reticuloendotheliosis, atherosclerosis, age-related cardiovascular diseases, ischemic diseases of the extremities, Raynaud disease diseases, preeclampsia Liver diseases such as hepatitis, cirrhosis of the liver, acute or chronic liver failure, bone and cartilage diseases or injuries, mucosal diseases or injuries, especially in the gastrointestinal tract, Crohn's disease, ulcerative colitis, pregnancy-induced hypertension, chronic or acute renal insufficiency, especially terminal renal insufficiency, particularly with terminal renal insufficiency, especially with renal insufficiency, renal insufficiency, especially with terminal renal insufficiency, especially with end-stage renal insufficiency Filtration rates <80 ml / min, in particular 30 to 80 ml / min, preferably 40 to 80 ml / min, microalbuminuria, proteinuria, increased ADMA levels, or he wounds and complications from it.

According to the invention, erythropoietin is administered to a patient in a therapeutically effective dose which is sufficient to cure or to prevent the state of a aforementioned disease, in particular a disease which is associated with a dysfunction of endothelial progenitor cells, the progression stop such an illness and / or relieve the symptoms of such an illness. The dose to be administered to a patient depends on many factors, such as the age, body weight and gender of the patient, the severity of the diseases, etc. "Dosing of EPO According to the Invention"

According to the invention, erythropoietin is preferably used in all uses, methods and compositions of the present teaching in small amounts, which are below the known amounts used for the therapy of renal anemia, in the sense of the present teaching under a low or low dose or dosage, in particular in vivo, ie per patient, EPO doses from 1 to 2000, preferably 20 to 2000, units (IU; International Units) / week, preferably doses from 20 to 1500 lU / week, in particular doses from 20 to 1000 lU / week, in particular doses from 20 to 950 IU / week, in particular doses from 20 to 900 lU / week, in particular doses from 20 to 850 lU / week, in particular doses from 20 to 800 lU / week, in particular doses from 20 to 750 IU / week, in particular doses of 20 to 700 IU / week, in particular doses of 20 to 650 IU / week, in particular doses of 20 to 600 IU / week, in particular doses of 20 to 550 IU / week, in particular doses of 20 bi s 500 lU / week, in particular doses from 20 to 450 lU / week, in particular doses from 20 to 400 lU / week, in particular doses from 20 to 350 IU / week, in particular doses from 20 to 300 lU / week, in particular doses of 20 up to 250 IU / week, in particular doses of 20 to 200 IU / week, in particular doses of 20 to 150 IU / week, depending on the severity of the disease and depending on the kidney function. According to the invention, doses of 1 to 450, preferably 1 to 9, lU / week are also to be used. All of the above doses provided according to the invention, for example from 1 to 2000 units (IU) / week and per patient, in particular for example from 500 to 2000 IU / week and per patient, are subpolycythemic doses, that is to say doses which do not lead to an increase in the hematocrit value, in particular compared to the hematocrit value before the treatment with EPO, do not lead to an increase in the hematocrit value of more than 10%, in particular 5%, preferably 2%. The subpolycythemic doses provided according to the invention correspond to weekly doses of approximately 1 to 90 units (IU) EPO / kg body weight, in particular 1 to 45, in particular 1 to 30 IU EPO / kg body weight, in particular 1 to 20 IU EPO / kg body weight, in particular 1 to 15 IU EPO / kg body weight, in particular 1 to 10 IU EPO / kg body weight, in particular 1 to 4 IU EPO / kg body weight, or a comparable weekly dose of Aranesp of 0.001 to 0.4 μg / kg body weight, 0.001 to 0.3 μg / kg body weight, 0.001 to 0.25 μg / kg body weight, 0.001 to 0.2 μg / kg body weight, 0.001 to 0.15 μg / kg body weight, 0.001 to 0.1 μg / kg body weight, 0.001 to 0.09 μg / kg body weight, 0.001 to 0.08 μg / kg body weight, 0.001 to 0.07 μg / kg body weight, 0.001 to 0.06 μg / kg body weight, 0.001 to 0.05 μg / kg body weight, 0.001 to 0.04 μg / kg body weight, 0.001 to 0.03 μg / kg body weight, 0.001 to 0.02 μg / kg body weight, 0.001 b is 0.01 μg / kg body weight, 0.001 to 0.009 μg / kg body weight, 0.001 to 0.008 μg / kg body weight, 0.001 to 0.007 μg / kg body weight, 0.001 to 0.006 μg / kg body weight, 0.001 to 0.005 μg / kg body weight, 0.001 up to 0.004 μg / kg body weight, 0.001 to 0.003 μg / kg body weight, 0.001 to 0.002 μg / kg body weight. Aranesp is a double PEG-ylated EPO.

According to the invention, erythropoietin is particularly preferred in all uses, methods and compositions of the present teaching in small amounts that are known to be below Amounts used for the therapy of renal anemia are used, EPO doses of 0.001 to 90, preferably 0.001 to 50, units being used in the sense of the present teaching under a low or low dose or dosage, in particular in vivo, ie per patient. IU; International Units) per kilogram of body weight and week, in particular doses from 0.05 to 45 lU / kg / week, in particular doses from 0.05 to 40 IU / kg / week, in particular doses from 0.05 to 35 lU / kg / Week, in particular doses from 0.05 to 33 lU / kg / week, in particular doses from 0.05 to 31 lU / kg / week, in particular doses from 0.05 to 29 lU / kg / week, in particular doses from 0 , 05 to 27 lU / kg / week, in particular doses from 0.05 to 25 lU / kg / week, in particular doses from 0.05 to 23 lU / kg / week, in particular doses from 0.05 to 21 lU / kg / week / Week, in particular doses from 0.05 to 20 lU / kg / week, in particular doses from 0.05 to 19 lU / kg / week, in particular doses from 0.05 to 17 lU / kg / week, in particular doses of 0.05 up to 15 lU / kg / week, in particular doses from 0.05 to 13 lU / kg / week, in particular doses from 0.05 to 11 lU / kg / week, in particular doses from 0.05 to 9 lU / kg / week, in particular doses from 0.05 to 7 IU / kg / week, in particular doses from 0.05 to 5 IU / kg / week, in particular doses from 0.05 to 3 IU / kg / week, in particular doses from 0.05 to 1 IU / kg / week depending on the severity of the disease and kidney function. According to the invention, doses of 0.001 to 20, preferably 0.05 to 10 lU / kg / week are also used. All of the above doses provided according to the invention, for example from 0.01 to 90 units (IU) / kg / week and per patient, in particular for example from 0.01 to 50 units IU / kg / week and per patient, are subpolycythemic doses, that is Doses which do not lead to an increase in the hematocrit value, in particular compared to the hematocrit value before treatment with EPO, do not lead to an increase in the hematocrit value of more than 10%, in particular 5%, preferably 2%. The subpolycythemic doses provided according to the invention correspond to weekly doses of approximately 0.001 to 90 units (IU) EPO / kg body weight, in particular 0.001 to 50, in particular 0.001 to 45 IU EPO / kg body weight, in particular 1 to 15 IU EPO / kg body weight, in particular 1 to 10 IU EPO / kg body weight, in particular 1 to 4 IU EPO / kg body weight, or a comparable weekly dose of Aranesp from 0.000005 to 0.45μg per kg body weight, 0.00025 to, 250 μg / kg body weight, 0.00025 to, 225 μg / kg body weight, 0.00025 to 0.2 μg / kg body weight, 0.00025 to 0.175 μg / kg body weight, 0.00025 to 0.165 μg / kg body weight, 0.00025 to 0.155 μg / kg body weight, 0, 00025 to 0.145 μg / kg body weight, 0.00025 to 0.135 μg / kg body weight, 0.00025 to 0.125 μg / kg body weight, 0.00025 to 0.115 μg / kg body weight, 0.00025 to 0.105 μg / kg body weight, 0, 00025 to 0.095 μg / kg body weight, 0.00025 to 0.085 μg / kg body weight icht, 0.00025 to 0.075 μg / kg body weight, 0.00025 to 0.065 μg / kg body weight, 0.00025 to 0.055 μg / kg body weight, 0.00025 to 0.055 μg / kg body weight, 0.00025 to 0.045 μg / kg Body weight, 0.00025 to 0.035 μg / kg body weight, 0.00025 to 0.025 μg / kg body weight, 0.00025 to 0.015 μg / kg body weight, 0.00025 to 0.005 μg / kg body weight. Aranesp is a double PEG-ylated EPO. The abovementioned low doses, for example the dose of 0.001 to 90 units / kg / week per patient, in particular for example from 0.001 to 50 units / kg / week and per patient, for the treatment according to the invention of diseases or disease states that are associated with dysfunction of endothelial progenitor cells is 90-150 lU / kg body weight / week (usually starting at 4000-8000 lU / week, compared to the starting dose usually used for the treatment of renal anemia). if the result of the therapy is unsatisfactory, however, also significantly higher) very low

Unless otherwise stated, the stated doses are single doses to be administered weekly, but can also be divided into several single doses in one week, that is to say they can be used as multiple doses.

A particularly preferred embodiment of the invention relates to the use of low-dose erythropoietin and / or its derivatives as defined in the above section “Dosage of EPO according to the invention” and / or derivatives thereof as an active ingredient for the production of a pharmaceutical composition or a medicament for the therapy of disease states or diseases with a dysfunction of endothelial progenitor cells.

According to the invention, an “active substance” is understood to mean an endogenous or foreign substance that influences specific functions of tissues, organs or organisms in a differentiated manner upon contact with target molecules or target cells or target tissues. According to the invention, erythropoietin as an active ingredient of the pharmaceutical composition according to the invention, upon contact with endothelial progenitor cells, influences their proliferation, differentiation and / or migration behavior in a human or animal organism in such a way that dys- functions of endothelial progenitor cells are balanced and the diseases arising from these dysfunctions can be effectively combated, alleviated or eliminated or these diseases can be effectively prevented, and that the use of low-dose erythropoietin can be used both for organ regeneration and to slow the progression of functional restrictions in different organs and organ systems leads.

In connection with the present invention, a “pharmaceutical composition” or a “medicament” is understood to mean a mixture used for diagnostic, therapeutic and / or prophylactic purposes, that is to say a mixture which promotes or restores the health of a human or animal body and which comprises at least one natural or comprises synthetically produced active ingredient that produces the therapeutic effect. The pharmaceutical composition can be both a solid and a liquid mixture. For example, a pharmaceutical composition comprising the active ingredient can contain one or more pharmaceutically acceptable components. In addition, the pharmaceutical composition may include additives commonly used in the art, for example stabilizers, manufacturing agents, release agents, disintegrants, emulsifiers or other substances commonly used to manufacture pharmaceutical compositions.

According to the invention, the use of erythropoietin in preferably low doses and / or a derivative thereof as an active ingredient for the manufacture of a medicament for the therapy of hypercholesterolemia, diabetes mellitus, insulin resistance, endo- thel-mediated chronic inflammatory diseases such as vascular inflammation, endotheliosis including reticuloendotheliosis, atherosclerosis, age-related cardiovascular disease, ischemic diseases of the extremities, Raynaud's disease, liver diseases such as hepatitis, cirrhosis of the liver, acute or chronic liver failure, bone or cartilage disorders, bone or cartilage disorders or injuries, particularly in the gastrointestinal tract, Crohn's disease, ulcerative colitis, preeclampsia, pregnancy-induced hypertension, acute or chronic renal failure, especially terminal renal failure, kidney function restrictions with glomerular filtration rates <80 ml / min, in particular 30, preferably 40 to 80 ml / min , Microalbuminuria, proteinuria, increased ADMA levels or wounds and secondary diseases thereof.

The pharmaceutical composition according to the invention can be suitable for topical as well as for systemic administration.

In a preferred embodiment of the invention it is provided that the pharmaceutical composition is used for parenteral, in particular intravenous, intramuscular, intracutaneous or subcutaneous administration. The drug containing erythropoietin is preferably in the form of an injection or infusion.

In a further use it is provided that the pharmaceutical composition containing erythropoietin is administered orally. For example, the drug containing erythropoietin is in a liquid dosage form such as a solution, suspension or emulsion, or a solid dosage form such as a tablet.

Another use provides that the pharmaceutical composition is suitable for pulmonary administration or for inhalation. The invention therefore provides that erythropoietin is administered directly to the patient's lungs in a therapeutically effective manner. This form of administration of erythropoietin enables a rapid delivery of an erythropoietin dose to a patient without the need for an injection. When erythropoietin is absorbed through the lungs, considerable amounts of erythropoietin can be released into the bloodstream via the lungs, which leads to increased amounts of erythropoietin in the bloodstream. In a preferred embodiment of the invention, the pharmaceutical composition to be absorbed via the lungs is an aqueous or non-aqueous solution or a dry powder. If the erythropoietin-containing medicament to be administered pulmonally is in the form of a dry powder, this preferably comprises particles containing erythropoietin, the particles having a diameter of less than 10 μm, so that the medicament can also reach distal regions of the patient's lungs. In a particularly preferred embodiment of the invention it is provided that the medicament to be administered pulmonally is in the form of an aerosol.

A particularly preferred embodiment of the invention relates to the use of erythropoietin for the production of a pharmaceutical composition for the therapy of diseases which are associated with a dysfunction of endothelial progenitor cells. Stand, wherein the pharmaceutical composition contains in addition to erythropoietin as an active ingredient at least one additional active ingredient for stimulating endothelial progenitor cells.

The further active ingredient is preferably an active ingredient which, in particular, stimulates the physiological mobilization of endothelial progenitor cells from the bone marrow or “other stem cells” niches. According to the invention, however, the further active ingredient can also be an active ingredient which in particular the division behavior However, according to the invention, there is also the possibility that the further active substance particularly stimulates the differentiation behavior and / or the migration behavior of endothelial progenitor cells. The further active substance that stimulates endothelial progenitor cells is particularly preferably VEGF, PIGF, GM - CSF, an HMG-CoA reductase inhibitor, in particular a statin such as simvastatin, mevastatin or atorvastatin, an ACE inhibitor such as enalapril, ramipril or trandolapril, an AT-1 blocker such as irbesartan, lorsartan or olmesaratan and / ora NO donor, especially L-arginine.

According to the invention, it is also provided that the at least one further active ingredient stimulates in particular differentiated endothelial cells, that is to say their proliferation and / or migration, but not endothelial progenitor cells. It is particularly preferably bFGF (basic fibroblast growth factor) or angiogenin.

A further embodiment of the invention relates to the use of erythropoietin and / or derivatives thereof as an active ingredient for producing a pharmaceutical composition for stimulating tion of endothelial progenitor cells, in particular to stimulate mobilization, proliferation, differentiation to endothelial cells and / or to migrate in the direction of a vasculogenic or angiogenic stimulus. According to the invention, the use of erythropoietin and / or its derivatives is furthermore provided as an active ingredient for producing a pharmaceutical composition for stimulating vasculogenesis and / or endothelial formation, in particular in the adult human or animal organism.

The present invention therefore also relates to pharmaceutical compositions for stimulating endothelial progenitor cells, in particular for stimulating their mobilization, proliferation, differentiation to endothelial cells and / or migration in the direction of a vasculogenic or angiogenic stimulus, for stimulating vasculogenesis and / or endothelial formation and for treating Diseases of the human or animal body associated with dysfunction of endothelial progenitor cells and / or endothelial cells. In particular, the present invention relates to pharmaceutical compositions or medicaments which comprise erythropoietin as an active ingredient and at least one further active ingredient for stimulating endothelial progenitor cells and / or differentiated endothelial cells. In a preferred embodiment, the present invention relates to pharmaceutical compositions containing erythropoietin and at least one further active ingredient selected from the group consisting of VEGF, PIGF, GM-CSF, an HMG-CoA reductase inhibitor, in particular a statin such as simvastatin, mevastatin or atorvastatin, an ACE inhibitor such as enalapril, ramipril or trandolapril, an AT-1 blocker such as irbesartan, lorsartan or olmesaratan, an NO donor, in particular L-arginine, bFGF and angiogenin. Another preferred embodiment of the invention relates to the use of erythropoietin for the production of a transplantable endothelial cell preparation. According to the invention, it is provided in particular that endothelial cells are produced in vitro by cultivating endothelial progenitor cells in the presence of erythropoietin and then transplanted into a recipient organism, in particular an organism suffering from a disease which is associated with a dysfunction of endothelial progenitor cells. For example, mononuclear cells (MNC) can be isolated from blood by means of density gradient centrifugation and cultured in suitable culture media in vitro. Methods for the isolation and in vitro cultivation of mononuclear cells are described, for example, in Asahara, Science, 275 (1997), 964-967; Dimmeier et al., J. Clin. Invest, 108 (2001), 391-397, and Llevadot et al., J. Clin. Invest., 108 (2001), 399-405. The mononuclear cells are then further cultivated in the presence of erythropoietin in order to stimulate the endothelial progenitor cells contained in the MNCs with regard to their proliferation and differentiation behavior and in particular to increase the number of differentiated adherent endothelial cells. According to the invention, it is also provided that the MNCs are cultivated in the presence of erythropoietin and at least one further substance which stimulates the proliferation and differentiation of endothelial progenitor cells. VEGF, PIGF, GM-CSF, an NO donor such as L-arginine, an ACE inhibitor such as Enapril, Ramipril or Trandolapril, an AT-1 blocker such as Irbesartan, Lorsartan or Olmesaratan or a are particularly preferred as a further substance HMG-CoA reductase inhibitor such as a statin, in particular simvastatin, mevastatin or atorvastatin, is used. In a further preferred application form of the invention, endothelial progenitor cells are administered simultaneously with other cell therapeutically usable cell populations, such as hepatocytes, myocytes, cardiomyocytes or islet cells, to corresponding patients with previous incubation with low-dose erythropoietin in vitro and / or local and systemic application of low-dose Erythropoietin in vivo, in order to ensure the healing of the cell-therapeutic tissue cells with sufficient connection to the vascular system.

Another preferred application of the invention also relates to the use of erythropoietin for the manufacture of a pharmaceutical composition or a kit for the sequential, sequential or simultaneous administration of low-dose erythropoietin and one or more other chemical, thermal, mechanical and biological agents to specific locations the body of a patient, for example to mobilize implantation target sites in order to increase the number and function of endothelial progenitor cells and / or to bring about the regeneration or slowing down of progression of tissue damage. The mechanical agents can be, for example, endoprostheses, preferably implant bodies, for example for the tooth, bone or ligament / tendon replacement. Furthermore, the biological agents can be solid organs, such as the liver, kidney, heart, pancreas or skin, or else hair implants. The invention therefore provides that EPO is used in particular in a low dose manner in order to better, faster and more efficiently, simultaneously, subsequently or previously implanted mechanical agents such as endoprostheses or biological agents, grow into the surrounding body structure. to have it integrated. The invention therefore also relates to the use of erythropoietin for the production of a pharmaceutical composition or a kit for the improvement, in particular for the promotion and / or acceleration, of the integration of biological agents or endoprostheses in surrounding body structures, in particular of teeth, dentures, dental implants or other endoprostheses , such as bone replacements, bone implants, in particular hip joint prostheses or ligament / tendon replacements, such as cruciate ligaments. If necessary, provision can be made for the erythropoietin to be used together with cell populations suitable for cell therapy and / or endothelial progenitor cells. In the aforementioned use of erythropoietin for the production of a pharmaceutical composition or a kit for the improvement, in particular promotion and / or acceleration of the integration of biological or mechanical agents in target structures, in particular target tissue, target bone or target cartilage of a patient, can be preferred in another Embodiment can be provided that the mechanical agents to be used, for example made of steel, ceramic, plastic or another material. In addition, it can be provided that cell populations, osteoblasts, cells with osteogenic potential, platelets, blood cells or the like which are suitable as cell therapy are used in the use mentioned. In a further preferred embodiment it can be provided that in particular the mechanical agent to be used is contained in the pharmaceutical composition or the pharmaceutical kit together with organic adhesive, for example a fibrin adhesive. In a further preferred embodiment of the invention, the use of erythropoietin or suitable active ingredients for topical use in the sense of "beauty care", in particular prophylaxis or prompt reduction of wrinkles and fine lines, strengthening of the connective tissue, protection and tightening of the skin, in particular the facial skin, against harmful environmental factors and as a make-up base The topical application of erythropoietin is intended to counteract the development and development of age spots, to refine the complexion, and to support the skin's renewal process, preferably through accelerated re-epithelialization but also hair growth.

In a further application form, the administration of low-dose erythropoietin is provided in adaptation to its circadian rhythm. Endogenous erythropoietin production has its acrophase (daily maximum) in the late afternoon, so low-dose erythropoietin should preferably be given in the morning, especially between 6:00 and 10:00, in order to achieve a maximum biological effect.

In a further preferred embodiment of the invention, the use of low-dose erythropoietin is provided for the pretreatment and / or further treatment of tissues or organs to be transplanted. The grafts are treated with low-dose erythropoietin before the transplant, preferably immediately before, in the donor organism. The recipient organism can also be treated with low-dose erythropoietin from the time of the transplant. Through this treatment of the organs to be transplanted or Tissue both directly before and after transplantation with erythropoietin is achieved according to the invention that new blood vessels quickly form in the transplant after a transplantation into a body due to the induced vasculogenesis and these newly formed blood vessels are quickly connected to the blood system of the recipient organism. The formation of endothelia is also quickly achieved in this way. The treatment of organ or tissue grafts with low-dose erythropoietin thus causes these systems to grow into the body more quickly, which considerably reduces the risk of rejection. In addition, organ regeneration is stimulated by the administration of low-dose erythropoietin.

In a further embodiment of the invention it is provided that the organ or tissue grafts are treated with low-dose erythropoietin in combination with at least one further factor which stimulates endothelial progenitor cells before transplantation. This factor is preferably a substance selected from the group consisting of VEGF, PIGF, GM-CSF, an HMG-CoA reductase inhibitor, for example a statin, in particular simvastatin, mevastatin or atorvastatin, an ACE inhibitor such as enalapril , Ramipril or trandolapril, an AT-1 blocker such as irbesartan, lorsartan or olmesaratan or an NO donor, in particular L-arginine. In a further embodiment, it is provided that the organ or tissue grafts are treated before transplantation in addition to erythropoietin with a further substance that stimulates the proliferation and migration of differentiated endothelial cells. This substance is particularly preferably angiogenin or bFGF. In a further embodiment it is provided that the pretreatment of the organ or tissue grafts with erythropoietin using isolated and possibly in vitro expanded endothelial progenitor cells.

Another particularly preferred embodiment of the invention provides that low-dose erythropoietin is used for the production of implantable or transplantable, cell-containing in vitro organs or tissues. According to the invention, it is particularly provided that the organ or tissue produced in vitro is treated with low-dose erythropoietin in vitro before the transplant or implantation in order to stimulate the endothelial progenitor cells present in the body of the recipient organism, in particular their physiological mobilization, migration, proliferation and differentiation , After the transplantation or implantation of the in vitro organ or tissue, the recipient organism is preferably treated further with low-dose erythropoietin in the doses according to the invention. By treating the in vitro organ or tissue before transplantation or implantation with erythropoietin and optionally aftertreatment of the recipient organism with erythropoietin, the invention achieves that in the in vitro organ or tissue system after transplantation or implantation into a body induced vasculogenesis quickly form new blood vessels and these newly formed blood vessels are quickly connected to the blood system of the recipient organism. The formation of endothelia and thus reendothelialization is also quickly achieved in this way. Treatment of the in vitro organ or tissue systems with low-dose erythropoietin thus causes these systems to grow into the body more quickly, thereby reducing the risk of rejection. ß is significantly reduced, and serves to protect the graft.

An “in vitro organ or tissue system” is understood to mean a transplantable or implantable cell-containing tissue or organ that is produced in vitro using defined cells and / or defined tissue and under defined culture conditions. Under an “implantable in vitro "Organ or tissue system" means a system which, in addition to cells, comprises foreign materials. A “transplantable in vitro organ or tissue system” is understood in particular to mean a cell-containing system which, in addition to cells, tissues or organs of the same or another individual, contains endogenous substances. In vitro organs or tissue are particularly characterized in that they largely correspond in their structure to the native organs or tissues to be replaced and can therefore take over the function of the replaced native organs or tissues in vivo.

In an embodiment according to the invention, it is provided that the in vitro organ or tissue systems are treated with erythropoietin in combination with at least one further factor which stimulates endothelial progenitor cells before transplantation or implantation. This factor is preferably one or more substances selected from the group consisting of VEGF, PIGF, GM-CSF, an HMG-CoA reductase inhibitor, in particular simvastatin, mevastatin or atorvastatin, an ACE inhibitor such as enalapril , Ramipril or trandolapril, an AT-1 blocker such as irbesartan, lorsartan or olmesaraatan, and an NO donor. In a further embodiment, seen that the in vitro organ or tissue systems are treated before transplantation or implantation in addition to erythropoietin with another substance that stimulates the proliferation and migration of differentiated endothelial cells. This substance is particularly preferably angiogenin or bFGF. In a further embodiment it is provided that the in vitro organ or tissue systems additionally contain isolated and possibly in vitro expanded endothelial progenitor cells.

Another preferred embodiment of the invention relates to the use of low-dose erythropoietin for the production of vascular prostheses or heart valves, the vascular prostheses or heart valves being coated with erythropoietin before insertion into a body, in particular a human body. By coating the vascular prostheses or heart valves with erythropoietin, endothelial progenitor cells in the body of the recipient organism are stimulated, in particular their mobilization from the bone marrow, their proliferation, their differentiation to endothelial cells and their migration to the vascular prostheses or heart valves used , After the vascular prosthesis or heart valves thus produced have been introduced into a body, the latter can be treated further with erythropoietin, in particular in the doses according to the invention. This results in the formation of endothelial layers on the vascular prostheses used and ingrowth into the affected areas of the body more quickly. In a preferred embodiment it is provided that for the coating of the vascular prostheses and heart valves additionally isolated and possibly in vitro expanded endothelial progenitor cells are used. The present invention also relates to a method for stimulating endothelial cell formation in vitro, comprising

a) the isolation of cell populations containing endothelial progenitor cells from blood by means of density gradient centrifugation,

b) culturing the isolated cell populations containing endothelial progenitor cells in cell culture medium, and

c) culturing the cell populations in the presence of low-dose erythropoietin.

According to the invention, the cell populations can be cultivated in the presence of a further substance which stimulates endothelial progenitor cells.

The present invention further relates to a method for the treatment of diseases associated with dysfunction of endothelial progenitor cells by administration of erythropoietin in a low dose as explained in the paragraph "Dosage according to the invention of EPO", alone or in combination with at least one other chemical, thermal, mechanical and biological agents to a patient with such a disease The method according to the invention is particularly suitable for the treatment of diseases of the human body such as hypercholesterolemia, diabetes mellitus, insulin resistance, endothelium-mediated chronic inflammatory diseases such as vascular inflammation, endotheliosis including reticuloendotheliosis, atherosclerosis, age-related cardiovascular disease, ischemic diseases of the extremities, Raynaud's disease, liver diseases such as hepatitis, liver cirrhosis, acute or chronic liver failure, bone and cartilage diseases or injuries, mucous membrane diseases or injuries, especially in the gastrointestinal tract, Crohn's disease, ulcerative colitis, preeclampsia, pregnancy-induced hypertension, acute or chronic renal insufficiency, especially terminal renal insufficiency, renal function impairments, <80 renal function impairments with glomerular filament disorders / <80 in particular 30 to 80, preferably 40 to 80 ml / min microalbuminuria, proteinuria, increased ADMA levels or wounds and secondary diseases are suitable.

In a preferred embodiment of the method according to the invention for the treatment of diseases which are related to dysfunction of endothelial progenitor cells, it is provided that the patient has at least one further active ingredient selected from the group consisting of VEGF, PIGF, GM-CSF, in addition to erythropoietin HMG-CoA reductase inhibitor and an NO donor is administered. The HMG-CoA reductase inhibitor administered is preferably a statin such as simvastatin, mevastatin or atorvastatin. The administered ACE inhibitor is an active ingredient such as enalapril, ramipril or trandolapril, and the administered AT-1 blocker is an active ingredient such as irbesartan, lorsartan or olmesaratan. The NO donor administered is preferably L-arginine.

In a further preferred embodiment of the method according to the invention for the treatment of diseases which are related to dysfunction of endothelial progenitor cells, it is provided that endothelial progenitor cells are isolated from the blood of a human organism, expanded and expanded in vitro using low-dose erythropoietin en- to differentiate dothelial cells and, after purification and isolation of the differentiated endothelial cells or the differentiating endothelial progenitor cells, to transplant them specifically into an area of the body, a tissue or an organ of a patient that is damaged due to the dysfunction of endothelial progenitor cells and / or endothelial cells to induce local endothelial regeneration there. In this way, a more targeted and faster treatment of the damaged body areas, tissues and / or organs of the patient is possible. This embodiment of the method according to the invention for the treatment of diseases which are associated with dysfunction of endothelial progenitor cells comprises the following steps:

a) isolation of cell populations containing endothelial progenitor cells from blood using density gradient centrifugation,

b) the cultivation of the cell populations containing endothelial progenitor cells in cell culture medium,

c) the cultivation of the cell populations containing endothelial progenitor cells in the presence of low-dose erythropoietin to stimulate the proliferation of endothelial progenitor cells and / or to differentiate them into endothelial cells,

d) the isolation and purification of the differentiated endothelial cells, and

e) the transplantation of the differentiated endothelial cells into a body with a disease which is associated with a dysfunction of endothelial progenitor cells. After the differentiated endothelial cells have been transplanted into a body, the latter can be treated with erythropoietin, in particular in the low doses provided according to the invention, that is to say in the doses defined in the section “Dosage according to the invention by EPO”, for example 1, preferably 0.001 to 90 lU / kg / week or 20 to 2000 lU / week.

According to the invention, the cell populations containing endothelial progenitor cells can be selected in vitro in the presence of at least one further active ingredient selected from the group consisting of VEGF, PIGF, GM-CSF, an HMG-CoA reductase inhibitor, an ACE inhibitor, an AT-1 blocker and one NO donor can be cultivated. The HMG-CoA reductase inhibitor used for the cultivation is preferably a statin such as Simvastatin, Mevastatin or Atorvastatin, the ACE inhibitors are substances such as Enalapril, Ramipril or Trandolapril and the AT-1 blocker is substances such as Irbsartan , Lorsartan or olmesaratan.

According to the invention, cell populations containing endothelial progenitor cells can be treated with a sequential, chronologically successive or simultaneous administration of low-dose erythropoietin and one or more other chemical, thermal, mechanical and biological agents, so as to increase the number and function of endothelial progenitor cells and / or regeneration or to slow the progression of tissue damage.

Another preferred embodiment of the invention relates to a method for the treatment of vascular diseases, comprising: a) isolation of cell populations containing endothelial progenitor cells from blood by means of density gradient centrifugation,

c) culturing the cell populations containing endothelial progenitor cells in the presence of erythropoietin to stimulate the proliferation of endothelial progenitor cells and / or to differentiate them into endothelial cells,

d) isolation and purification of the differentiated endothelial cells, and

e) the transplantation of the endothelial cells into a body with a vascular disease.

After the endothelial cells have been transplanted into the body with a vascular disease, the latter can be treated with erythropoietin, preferably in the low doses according to the invention, that is to say in the doses defined in the paragraph “Dosage according to the invention by EPO” of, for example, 0.001 to 90 units / kg / week or 20 IU / Week up to 2000 IU / week.

According to the invention, there is the possibility of the cell populations containing endothelial progenitor cells in the presence of at least one further active substance selected from the group consisting of VEGF, PIGF, GM-CSF, an ACE inhibitor, an AT-1 blocker and / or an HMG-CoA Cultivate reductase inhibitor. The ACE inhibitor used for cultivation is preferably substances such as enalapril, ramipril or trando- lapril, in the AT-1 blocker used for the cultivation for substances such as irbesartan, lorsartan or olmesaratan and in the HMG-CoA reductase inhibitor used in the cultivation for a statin such as simvastatin, mevastatin or atorvastatin.

According to the invention, cell populations containing endothelial progenitor cells can be treated with a sequential, chronologically successive or simultaneous administration of low-dose erythropoietin and one or more other chemical, thermal, mechanical and biological agents, in order to increase the number and function of endothelial progenitor cells and / or regeneration or to slow the progression of tissue damage. The mechanical agents can be endoprostheses, preferably implant bodies for the tooth, bone or ligament / tendon replacement. Furthermore, the biological agents can be solid organs, such as the liver, kidney, heart, pancreas or skin, or else hair implants. The invention therefore also provides that EPO is used, in particular, in a low-dose manner in order to allow, at the same time, subsequently or previously implanted mechanical agents such as endoprostheses or biological agents to grow or integrate better, faster and more efficiently into the surrounding body structure. The invention therefore also relates to the use of erythropoietin for the production of a pharmaceutical composition or a kit for the improvement, in particular for the promotion and / or acceleration, of the integration of biological agents or endoprostheses in surrounding body structures, in particular of teeth, dentures, dental implants or others Endoprostheses, such as bone replacements, bone implants, in particular right hip prostheses or ligament / tendon replacement, such as cruciate ligaments. In a preferred embodiment it can be provided that the erythropoietin is used together with cell populations suitable for cell therapy and / or endothelial progenitor cells. In the aforementioned use of erythropoietin for the production of a pharmaceutical composition or a kit for the improvement, in particular promotion and / or acceleration of the integration of biological or mechanical agents in target structures, in particular target tissue, target bone or target cartilage of a patient, can be provided in a further preferred embodiment be that the mechanical agents to be used are made, for example, of steel, ceramic, plastic or another material. In addition, it can be provided that cell populations, osteoblasts, cells with osteogenic potential, platelets, blood cells or the like which are suitable as cell therapy are used in the use mentioned. In a further preferred embodiment it can be provided that in particular the mechanical agent to be used together with organic adhesive, for example a fibrin adhesive, are contained in the pharmaceutical composition or the pharmaceutical kit.

The method according to the invention for the treatment of vascular diseases therefore provides for isolating endothelial progenitor cells from the blood of a human organism, expanding them in vitro using low-dose erythropoietin and differentiating them into endothelial cells and after purifying and isolating the differentiated endothelial cells or the differentiating endothelial cells Precursor cells then target these into a damaged blood vessel or an ischemic loading to transplant richly to induce local neovascularization. In this way, a more targeted and faster treatment of damaged blood vessels or ischemic tissue is possible. The method according to the invention for the treatment of vascular diseases is particularly suitable for the treatment of vascular diseases such as ischemia, in particular cerebral ischemia, ischemic diseases of the extremities, stroke, acute artery occlusion, arterial occlusive disease, Raynaud's disease and ergotism.

Further advantageous embodiments of the invention result from the subclaims.

The invention is illustrated by the following figures and examples.

Figure 1 shows the results of a FACS analysis of circulating CD34 + stem cells (cSC). (A-D): patient samples; (E-F): isotypic controls. cSC were identified from the additional expression of the CD34 marker (B and F), from the characteristic low to medium CD45 antigen expression (C and G) and from the characteristic light scattering properties (D and H). The absolute number of cSC was calculated per 100,000 mono- and lymphocytes.

FIG. 2 shows a quantitative determination of circulating stem cells using flow cytometry. The figure shows the time-dependent effect of an erythropoietin treatment using rhEPO (recombinant human erythropoietin) after 0, 2, 4, 6 and 8 weeks. n = 11, the values correspond to mean values +/- standard deviation. Medians represented as a line. *: p <0.01 compared to 2 weeks: Ψ: p <0.05 compared to 4 weeks, #: p <0.05 compared to 8 weeks.

FIG. 3 shows a quantitative determination of cultured endothelial progenitor cells (EPC). The figure shows that rhEPO treatment increases the relative number of EPCs. EPCs were isolated before the treatment of renal patients with rhEPO and 2, 4, 6 and 8 weeks after treatment of the patients with rhEPO and characterized by their adhesiveness and the two markers acLDL-Dil and UEA-1 FITC. n = 11, the values correspond to mean values +/- standard deviation. Medians represented as a line.

*: p <0.01 compared to the period before treatment; #: p <0.001 compared to the period before treatment.

FIG. 4 shows the quantitative determination of cultured endothelial progenitor cells (EPC). The figure shows that the absolute number of EPCs before starting rhEPO therapy is significantly reduced compared to healthy age and gender matched subjects. Patients with renal anemia therefore show a clear EPC dysfunction compared to control persons. This reduced number of functional EPCs is compensated for 8 weeks after the start of rhEPO therapy for renal anemia. EPCs were isolated before the treatment of renal patients with rhEPO and 2, 4, 6 and 8 weeks after treatment of the patients with rhEPO and characterized by their adhesiveness and the two markers acLDL-Dil and UEA-1 FITC. n =. 11 The 8-week course and the entire controls are shown as examples. The absolute values are shown on the one hand as individual values. Boxplots are also shown (90s / 75s / 50s / 25s and 10s percentiles and the mean). As healthy controls served as age- and gender-matched subjects, in whom EPCs were isolated and characterized analogously (n = 11).

Figure 5 shows the quantitative determination of cultured endothelial progenitor cells (EPC) in healthy young subjects. The figure shows that treatment with rhEPO treatment (30IU epoetin beta per kg body weight and week) increases the relative number of EPCs. EPCs were isolated before treatment with rhEPO and weekly after 1, 2, 3, 4, 5, 6 and 7 weeks after treatment with rhEPO and based on their adhesiveness and the two markers acLDL-Dil and UEA-1 FITC chara - ketrized. n = 4, the values correspond to mean values +/- standard deviation.

FIG. 6 shows a quantitative determination of cultured endothelial progenitor cells (EPCs). The representative photographs show that the absolute number of EPCs in uraemic patients is significantly reduced compared to healthy age- and sex-matched subjects (top row - in vivo). Patients with impaired kidney function therefore show a clear EPC dysfunction compared to control persons. If endothelial progenitor cells from a healthy subject are co-cultivated with serum from uraemic patients, the ability of the endothelial progenitor cells to differentiate (lower row - in vitro) decreases. A reduced kidney function with the resulting uremia leads to dysfunction of endothelial progenitor cells.

FIG. 7 shows a quantitative determination of cultured endothelial progenitor cells (EPCs) in 46 uremic patients with impaired kidney function compared to 46 age- and sex-matched subjects as boxplots (90s / 75s / 50s / 25s and 10s percentiles as well as the mean). The number of endothelial progenitor cells is significantly reduced in the uraemics compared to the healthy volunteers. Patients with impaired kidney function therefore show a clear EPC dysfunction compared to control persons.

Figure 8 shows the effect of erythropoietin on wound healing. The figure shows that when a standardized skin wound, which had been placed in a mouse using a tissue punch, was treated with erythropoietin, the wound was completely closed after seven to eight days, whereas when the wound was treated with physiological saline (saline), the wound only after was completely closed for thirteen to fourteen days. Treatment with erythropoietin or physiological saline started 7 days before the skin wound was placed. Recombinant human erythropoietin was administered once a week using a s.c. (subcutaneous) injection (0.1 mg / kg Aranesp) (group n = 5).

FIG. 9 shows that erythropoietin reduces the loss of kidney function after acute kidney failure (acute renal failure). Practice Dawley rats (250-300 g) were included in the study. The rats were anesthetized with ketamine (120 mg / kg) and rompun (10 mg / kg). One of the test groups received Aranesp 0.1 μg / kg body weight once on the day before induction of acute kidney failure. A group of experimental animals, each of which was injected with sc salt at the same time, served as a comparison. By placing an artery clamp on the right renal artery, the blood flow in the kidney interrupted for 45 minutes. During this time, a left side nephrectomy was performed. A sham operation was performed on another control group. The abdomen was opened, the left renal artery was freely prepared, but the blood supply was not interrupted and the contralateral right kidney was removed. All animals were anesthetized for 60 min and sacrificed 24 h after the operation. The 45-minute ischemia with subsequent reperfusion of the remaining right kidney led to a massive acute loss of kidney function in the animals treated with saline. This is reflected in a serum creatinine value which is 24 times 24 hours after the ischemia reperfusion than the value before the ischemia reperfusion (p <0.05). In contrast, the animals treated with the erythropoietin analog Aranesp showed only a four-fold increase in the serum creatinine values one day after induction of the ischemia-reperfusion damage. There was no increase in retention values in the left nephrectomized animals with sham operated right kidney. The figure shows the creatinine concentration in the serum of EPO-treated animals (IR + EPO), NaCI-treated animals (IR) and sham-operated animals (sham-OP) before ischemia-reperfusion (IR) injury and 24 hours after. The figure shows that the serum creatinine concentration in the Aranesp-treated animals is almost halved 24 hours after the ischemia-reperfusion injury compared to the control without (NaCI treatment).

FIG. 10 shows the Kaplan-Mayer survival curves of two test groups after induction of chronic renal failure, which were treated with either Aranesp or NaCl. 8 week old Spraque Dawley rats were included in the study. The rats were treated with ketamine (120 mg / kg) and rompun (10 mg / kg) anesthetized. The right kidney was removed from them on day 0, which was immediately fixed in formalin for histological examination. In the left kidney, the segment arteries that supply the upper and lower kidney poles were ligated. This leads to a kidney infarction in the corresponding kidney areas and only the middle third of the kidney remains functional. The rats were given Aranesp (0.1 μg / kg body weight) or NaCl sc once a week. The animals treated with the erythropoietin analog Aranesp have a significant survival advantage over the animals treated with saline (p = 0.027; log rank test).

FIGS. 11-18 show light microscopic kidney sections 6 weeks after induction of chronic kidney failure of two test groups, which were treated with either Aranesp or NaCl and whose Kaplan-Mayer survival curves are shown in FIG. 10.

FIG. 11 shows the histological changes in a Spraque-Dawley rat with chronic kidney failure after NaCl treatment once a week, starting immediately after induction of chronic kidney failure for a period of 6 weeks. Chronic renal failure results from the removal of the right kidney and the ligation of the segment arteries that supply the upper and lower kidney pole, the left kidney. The figure shows a medium-sized preglomerular artery with characteristic onion skin-like vascular wall proliferation in the case of severe hypertensive damage, a so-called malignant nephrosclerosis with endarteritis Fahr. FIG. 12 shows the histological changes in a Spraque-Dawley rat with chronic renal failure after NaCl treatment once a week, starting immediately after induction of chronic renal failure for a period of 6 weeks. Chronic renal failure results from the removal of the right kidney and the ligation of the segment arteries that supply the upper and lower kidney pole, the left kidney. The figure shows active focal segment mental glomerulosclerosis as a so-called proliferative FSGS (right glomerulum). The other glomerulum (left) shows ischemic collapse of the loop bundle. At the bottom of the picture is a small vessel with severe endothelial damage. The observed histological changes correspond to hypertensive organ damage or changes in the context of overload nephropathy after 5/6 nephrectomy.

FIG. 13 shows the histological changes in a Spraque-Dawley rat with chronic renal failure after NaCl treatment once a week, starting immediately after induction of chronic renal failure for a period of 6 weeks. Chronic renal failure results from the removal of the right kidney and the ligation of the segment arteries that supply the upper and lower kidney pole, the left kidney. The figure shows an almost complete sclerosis or destruction of a compensatory enlarged glomerulus with pronounced hyalinosis or fibrinoid necrosis of the associated afferent arterioles.

FIG. 14 shows the histological changes in a Spraque-Dawley rat with chronic renal insufficiency after every one time Weekly NaCl treatment starting immediately after induction of chronic renal failure for a period of 6 weeks. Chronic renal failure results from the removal of the right kidney and the ligation of the segment arteries that supply the upper and lower kidney pole, the left kidney. The figure shows a small preglomerular artery with characteristic onion-shell-like vascular wall proliferation and wall necrosis with severe hypertensive damage, a so-called malignant nephrosclerosis (see picture on the right). On the left you can see a normal (not yet damaged) arteriole.

FIG. 15 shows the histological changes in a Spraque-Dawley rat with chronic renal failure after Aranesp (EPO) treatment (0.1 mg / kg Aranesp) treatment once a week, starting immediately after induction of chronic renal failure for a period of 6 weeks. Chronic renal failure results from the removal of the right kidney and the ligation of the segment arteries that supply the upper and lower kidney pole, the left kidney. The figure shows a regular glomerulum with a delicate afferent vessel. No pathological finding can be found in the tubulointerstitium.

FIG. 16 shows the histological changes in a Spraque-Dawley rat with chronic renal insufficiency after Aranesp (EPO) treatment (0.1 mg / kg Aranesp) treatment once a week, beginning immediately after induction of chronic renal insufficiency for a period of 6 weeks. Chronic renal failure results from the removal of the right kidney and the ligation of the segment arteries that supply the upper and lower kidney pole, the left kidney. The figure shows a regular glomerulum with delicate afferent vessel (630x magnification). No pathological findings can be found in the tubo interstitium.

FIG. 17 shows the histological changes in a Spraque-Dawley rat with chronic kidney insufficiency after Aranesp (EPO) treatment (0.1 mg / kg Aranesp) treatment once every week, starting directly after induction of chronic renal failure for a period of 6 weeks , Chronic renal failure results from the removal of the right kidney and the ligation of the segment arteries that supply the upper and lower kidney pole, the left kidney. The figure shows a regular glomerulum with a delicate afferent vessel. No pathological finding can be found in the tubolointerstitium.

FIG. 18 shows the histological changes in a Spraque-Dawley rat with chronic renal failure after Aranesp (EPO) treatment (0.1 mg / kg Aranesp) treatment once a week, starting immediately after induction of chronic renal failure for a period of 6 weeks. Chronic renal failure results from the removal of the right kidney and the ligation of the segment arteries that supply the upper and lower kidney pole, the left kidney. The figure shows a regular glomerulum with a delicate afferent vessel (630x magnification). No pathological finding can be found in the tubolointerstitium.

Figure 19 shows the effect of EPO on the wound healing process. example 1

Effect of EPO in patients with renal anemia

The effect of erythropoietin in patients with renal anemia (Hb <10.5 g / dl) as a result of end-stage renal disease (premature renal failure; creatinine clearance <35 ml / min) was examined. 11 patients were treated with erythropoietin at weekly doses of 5000 IU rhE-PO (recombinant human erythropoietin) on average intravenously or subcutaneously over a period of at least 8 weeks. After erythropoietin treatment, the endothelial progenitor cells in the blood of the patients were examined over a period of 20 weeks, with endothelial progenitor cells being counted after 0, 2, 4, 6 and 8 weeks with regard to their number and also their differentiation status by means of flow cytometry and a culture test were analyzed.

Circulating peripheral blood stem cells (CPBSC) are a small cell population that express both the CD34 antigen and the CD45 antigen. On the basis of the ISHAGE guidelines, a test for determining the number of CPBSC by means of flow cytometry was developed (Sutherland et al., J. Hematother., 5 (1996), 213-226). Using this test, both the expression pattern of CD34 and CD45 cells and the morphology of the stem cells were determined. In this way, both the absolute number of CPBSC per μl and the percentage of CPBSC in the total number of leukocytes were determined.

Figure 1 shows the results of a FACS analysis of circulating CD34 + stem cells based on the ISHAGE guidelines. FIG. 2 shows the number of CD34 + stem cells determined by means of FACS analysis over a period of 8 weeks.

Cell culture test

Mononuclear peripheral blood cells (PBMCs) were isolated from human blood samples by density centrifugation via Ficoll according to the method described in Asahara, Science, 275 (1997), 964-967. The cells were plated on fibro-nectin on culture plates and kept in EC basal medium. EC basal medium consists of EBM-2 basal medium (from Clonetics) and EGM-2 quotas (hEGF; GA-1000 (gentamicin, amphotericin-B) FBS, VEGF, hFGF-B (w / heparin), R3-IGF- 1, Ascorbic Acid, Heparin). After 4 days of cultivation, non-adherent cells were removed by washing the plates. The remaining adherent cells were treated with trypsin and sown again. They were then cultivated for a further 3 days. Cells with endothelial phenotype were identified by positive staining for two different endothelial markers on day 7 after isolation. These are Dil-labeled low density acetylated lipoprotein (acLDL-Dil) and Ulex europaeus-Aglutinin-1 (UEA-1). The results of this investigation are shown in FIG. 3.

The results show that erythropoietin can mobilize endothelial progenitor cells and increase the number of circulating endothelial progenitor cells. Functional deficits that occur under certain pathological conditions such as renal anemia are compensated for. These results are shown in Figure 4

Flow cytometry was used to determine that in patients with end stage renal disease, the number of circulating CD34 + - Stem cells corresponds to the number of circulating CD34 + stem cells in the blood of healthy volunteers. After starting erythropoietin treatment, the number of CD34 + stem cells in the bloodstream increases significantly by more than 50%. Using the cell culture test, it was determined that after treatment with erythropoietin, the number of cells that develop an endothelial phenotype increases dramatically. In a functional cell culture test, the severely impaired ability of endothelial progenitor cells increased more than 3-fold.

Example 2

Improved wound healing through the systemic use of rhEPO

FVB / N mice were anesthetized by inhalation anesthesia with isofloran. The hair of the two hind legs was removed with the help of a depilatory lotion and disinfected with 70% alcohol. A skin wound was placed on the right flank of the mice using a sterile, 4 mm disposable biopsy tissue punch. The opposite side served as an internal control. Postoperative antibiotic shielding with penicillin G (20,000 units / kg) was performed once. Subcutaneous injections of recombinant human erythropoietin analog Aranesp (0.1 μg / kg body weight) were administered once a week over the entire study period over the entire study period. Treatment started seven days before the tissue punch was removed. The results are shown in Figure 8. They show that the administration of EPO significantly accelerates the wound healing process. Figure 19 shows the effect of erythropoietin on wound healing. The figure shows that when a standardized skin wound, which had been set in a mouse using a tissue punch, was treated with low-dose erythropoietin (20 IU EPO / kg / week), the wound was completely closed after seven to eight days, while when the wound was treated with physiological saline (saline) the wound was only completely closed after thirteen to fourteen days. Treatment of the test animals with high-dose erythropoietin (200IU EPO / kg / week) showed no acceleration in wound healing compared to the control group. Two of the experimental animals treated with the high dose of erythropoietin died during the observation period. Treatment with erythropoietin or physiological saline began after the skin wound was placed on the day of the operation. Recombinant human erythropoietin was administered once a week by sc (subcutaneous) injection (20 lU / kg EPO or 200 lU / kg EPO) (per group n = 5).

Example 3

Reduction of the progression of chronic renal failure through erythropoietin treatment

Eight week old Spraque-Dawley rats were anesthetized with ketamine (120 mg / kg) and Rompun (10 mg / kg). The right kidney was removed from the rats on day 0, which was fixed in formalin for histological examination immediately after removal. In the left kidney, the segment arteries that supply the upper and lower kidney poles were ligated. This leads to one Renal infarction of the corresponding kidney areas, whereby only the middle third of the kidney remains functional. The rats received the erythropoietin analog Aranesp once a week in a dose of 0.1 μg / kg body weight or injected subcutaneously (SC) as a control.

Figure 10 shows the Kaplan-Mayer survival curve of both test groups. The Aranesp-treated animals have a significantly improved survival compared to the saline-treated control animals.

FIGS. 15-18 show that after treatment with erythropoietin the kidney tissue shows no pathological changes, whereas after treatment with NaCl severe pathological changes are visible (see FIGS. 8-11). Further histological studies showed that a significantly higher vessel density (CD31) can be observed in animals treated with Aranesp than in animals treated with saline (data not shown).

Example 4

Reduction of the progression of acute renal failure

Spraque-Dawley rats with a body weight of 250 to 300 g were used for this investigation. One of the test groups received Aranesp once at the dose before induction of acute kidney failure in a dose of 0.1 μg / kg body weight. The rats were treated with ketamine (120 mg / kg body weight) and rompun (10 mg / kg) anesthetized. A group of experimental animals, each of which was injected with sc salt at the same time, served as a comparison. Blood flow in the kidney was stopped for 45 minutes by placing an artery clamp on the right renal artery. During this time, a left side nephrectomy was performed. A sham operation was performed on another control group. Here the abdomen was opened, the left renal artery was dissected, but the blood supply was not interrupted, and the contralateral right kidney was removed. All animals were anesthetized for 60 minutes and sacrificed 24 hours after surgery.

The 45-minute ischemia with subsequent reperfusion of the remaining right kidney led to a massive acute loss of kidney function in the saline-treated animals. This was expressed in a serum creatinine value that increased by a factor of 7 (p <0.05). In contrast, the animals treated with the erythropoietin analog Aranesp showed only a four-fold increase in the serum creatinine value one day after induction of the ischemia-reperfusion damage. There was no increase in retention values in the left nephrectonised animals with sham operated right kidney. The results are shown in Figure 9.

Example 5

Decreased ability to differentiate endothelial progenitor cells in patients with renal impairment The differentiation status of endothelial progenitor cells was analyzed in a culture test in 46 uraemic patients and 46 age- and sex-matched healthy control volunteers. It was surprisingly found that the number of endothelial progenitor cells in this differentiation assay in uremic patients was significantly reduced compared to the healthy controls (FIG. 7). If mononuclear cells from a healthy subject are isolated and cultivated in the presence of serum from a uraemic patient, the ability of the cells to differentiate into endothelial progenitor cells is reduced analogously (FIG. 6).

Example 6

Stimulation of the ability to differentiate endothelial progenitor cells in healthy volunteers

4 healthy young men were treated with 30IU epoetin beta per kilogram body weight once a week for 8 weeks. The ability of the endotheial progenitor cells to differentiate was determined in a culture assay before the subjects were treated with rhEPO and weekly after 1, 2, 3, 4, 5, 6 and 7 weeks after the subjects were treated with rhEPO on the basis of their adhesiveness and the two markers acLDL and UEA. A relative increase in EPCs of more than 50% was observed.

Claims

claims

1. Use of erythropoietin and / or derivatives for the production of a pharmaceutical composition containing a dose of 0.001 to 90 lU / kg body weight and week, preferably 0.05 to 50 lU / kg body weight / week for the prophylaxis or treatment of diseases, the erythropoietin in this dose is suitable and intended for the prophylaxis or treatment of a human or animal patient with a) at least one dysfunction of endothelial progenitor cells, b) at least one cardiovascular risk factor such as hypertension, hypercholestrinemia, increased asymmetric dimethylarginine (ADMA) values, increased insulin resistance or hyperhomocytenemia and c) at least one end organ damage such as left ventricular hypertrophy, microalbuminuria, cognitive dysfunction, increase in intimal media thickness in the carotid artery, proteinuria or a glomerular filtration rate <80 ml / min, preferably 30 to 80 ml / min.

2. Use of erythropoietin and / or derivatives for the production of a pharmaceutical composition containing a dose of 0.001 to 90 lU / kg body weight and week, preferably 0.05 to 50 lU / kg body weight / week, the erythropoietin being suitable and determined in this dose is for the cosmetic treatment of the human or animal body, in particular for the treatment of wrinkles, for strengthening the connective tissue, for protecting and tightening the skin, for protecting against damaging environmental influences, for treating age spots, for accelerating re-epithelialization, for accelerating hair growth and / or as a make-up pad.

3. Use of erythropoietin and / or derivatives for the production of a pharmaceutical composition containing a dose of 0.001 to 90 lU / kg body weight and week, preferably 0.05 to 50 lU / kg body weight / week for the production of a cosmetic preparation, in particular for topical application , the erythropoietin in this dose being suitable and intended for the cosmetic treatment of the human or animal body, in particular for the treatment of wrinkles, for strengthening the connective tissue, for protecting and tightening the skin, for protecting against damaging environmental effects, for the treatment of age spots , to accelerate re-epithelialization, to accelerate hair growth, and / or as a make-up pad.

4. Use of erythropoietin and / or derivatives for the production of a pharmaceutical composition containing a dose of 0.001 to 90 lU / kg body weight / week, preferably 0.05 to 50 lU / kg body weight / week and / or a mixture of endotelial precursor cells with at least a cell population which can be used for therapeutic purposes, the erythropoietin in this dose being suitable and intended for the regeneration of tissues and vessels in a human or animal body, and the mixture having been brought into contact with erythropoietin in vitro prior to application.

5. Use of erythropoietin and / or derivatives for the production of a pharmaceutical composition containing a dose of 0.001 to 90 lU / kg body weight and week, preferably 0.05 to 50 lU / kg body weight / week and / or a mixture of endotelial precursor cells with at least a cell population that can be used for therapeutic purposes, the erythropoietin in this dose is suitable and intended for the regeneration of tissues and vessels in a human or animal body, and the mixture being administered before, after or simultaneously with the application of the mixture

6. Use of erythropoietin and / or derivatives for the production of a pharmaceutical composition or a kit containing a dose of 0.001 to 90 lU / kg body weight / week, preferably 0.05 to 50 lU / kg body weight / week and / or at least one chemical, Thermal, mechanical or biological agent, in particular a pharmacological agent, for the production of a pharmaceutical composition or a kit containing erythropoietin in this dosage and the at least one chemical, thermal, mechanical or biological agent, for the prophylaxis or treatment of diseases, the Erythropoietin in this dose is suitable and is intended for the sequential, sequential or simultaneous application of erythropoietin with the at least one chemical, thermal, mechanical or biological agent.

7. Use of erythropoietin according to claim 6, wherein the mechanical agents are endoprostheses, preferably implant bodies for the tooth, bone or ligament / tendon replacement.

8. Use of erythropoietin according to claim 6, wherein the biological agents are solid organs, such as the liver, kidney, heart, pancreas, skin or hair implants.

9. Use of erythropoietin and / or derivatives for the production of a pharmaceutical composition containing a Dose of 0.001 to 90 lU / kg body weight and week, preferably 0.05 to 50 lU / kg body weight / week, the erythropoietin in this dose being suitable and intended for the prophylaxis or treatment of diseases, the disease being hypercholesterolemia, diabetes mellitus , Insulin resistance, endothelium-mediated chronic inflammatory diseases, endotheliosis including reticuloendotheliosis, atherosclerosis, age-related cardiovascular disease, ischemic diseases of the extremities, pre-eclampsia, Raynaud's disease, liver diseases such as hepatitis, cirrhosis of the liver, acute or chronic and liver failure, bone disorders Injuries, mucosal diseases or injuries, especially in the gastrointestinal tract, Crohn's disease, ulcerative colitis, pregnancy-induced hypertension, chronic or acute renal insufficiency, especially terminal renal insufficiency, kidney function restrictions with glomerular filter ation rates of 30 to 80 ml / min, microalbuminuria, proteinuria or wounds and complications thereof.

10. Use of erythropoietin and / or derivatives for the production of a pharmaceutical composition containing a dose of 0.001 to 90 lU / kg body weight / week, preferably 0.05 to 50 lU / kg body weight / week for the production of a kit containing erythropoietin, endothelial precursor cells and at least one cell population that can be used for therapeutic purposes, the erythropoietin preferably being present in low doses.

11. Use according to one of claims 1 to 10, wherein the pharmaceutical composition for stimulating the physiological mobilization of endothelial progenitor cells, the proliferation endothelial progenitor cells, which serves to differentiate endothelial progenitor cells to endothelial cells and / or to migrate endothelial progenitor cells in the direction of an angiogenic or vasculogenic stimulus.

12. Use according to claim 1 to 11, wherein the ability of differentiating endothelial progenitor cells for adhesion is increased.

13. Use according to claim 1 to 12, wherein the stimulation of endothelial progenitor cells leads to the formation of endothelial tissue.

14. Use according to one of claims 1 to 13, wherein the stimulation of the endothelial progenitor cells leads to the formation of new blood vessels.

15. Use of erythropoietin in a low dosage of 0.001 to 90 lU / kg body weight / week for the therapy of disease states or diseases of the human or animal body which are associated with dysfunction of endothelial progenitor cells.

16. Use according to claim 15, wherein the dysfunction of the endothelial progenitor cells consists in their impaired ability to proliferate, their impaired ability to differentiate into endothelial cells, their impaired ability to adhere and / or their impaired ability to migrate towards a vasculogenic or angiogenic stimulus.

17. Use according to claim 15 or 16, wherein the dysfunction of endothelial progenitor cells affects or prevents the formation of endothelial tissue and / or blood vessels.

18. Use according to one of claims 15 to 17, wherein the dysfunction of endothelial progenitor cells is pathogenic.

19. Use according to one of claims 15 to 18, wherein the disease states or diseases which are associated with dysfunction of endothelial progenitor cells are hypercholesterolemia, diabetes mellitus, insulin resistance, endothelium-mediated chronic inflammatory diseases, endotheliosis including reticuloendotheliosis, atherosclerosis, age-related atherosclerosis Cardiovascular disease, ischemic diseases of the extremities, pre-eclampsia, Raynaud's disease, liver diseases such as hepatitis, liver cirrhosis, acute or chronic liver failure, bone and cartilage diseases or injuries, mucosal diseases or injuries, especially in the gastrointestinal tract, Crohn's disease, colitis ulcerosa, pregnancy-induced hypertension, chronic or acute renal insufficiency, especially terminal renal insufficiency, renal function restrictions with glomerular filtration rates of 30 to 80 ml / min, microalbuminuria, prot auria, increased ADMA, or wounds and / or complications.

20. Use of erythropoietin in a low dose, in particular from 0.001 to 90 lU / kg body weight / week, for the therapy of hypercholesterolemia, diabetes mellitus, insulin resistance, endothelium-mediated chronic inflammatory diseases, endotheliosis including reticuloendotheliosis, atherosclerosis, age-related cardiovascular disease, ischemic diseases of the extremities, pre-eclampsia, Raynaud's disease, liver diseases such as hepatitis, liver cirrhosis, acute or chronic liver failure, bone and cartilage diseases or injuries, mucosal diseases or injuries, especially in the gastrointestinal tract, Crohn's disease, Ulcerative colitis, pregnancy-induced hypertension, chronic or acute renal insufficiency, in particular end-stage renal insufficiency, kidney function restrictions with glomerular filtration rates of 30 to 80 ml / min, microalbuminuria, proteinuria, increased ADMA values or wounds and / or secondary diseases thereof.

21. Use according to one of claims 1 to 20, wherein erythropoietin is administered per patient in a dose of 0.001 to 90 units / kg body weight / week.

22. Use according to claim 21, wherein erythropoietin is administered per patient in a dose of 0.05 to 50 units / week.

23. Use according to any one of claims 1 to 22, wherein the pharmaceutical composition is suitable for parenteral, in particular intravenous, intramuscular, intracutaneous or subcutaneous as well as topical administration.

24. Use according to claim 23, wherein the pharmaceutical composition is in the form of an injection or infusion.

25. Use according to any one of claims 1 to 22, wherein the pharmaceutical composition is suitable for pulmonary administration.

26. Use according to claim 25, wherein the pharmaceutical composition is in the form of an aqueous solution, non-aqueous solution or powder.

27. Use according to claim 25 or 26, wherein the pharmaceutical composition is in the form of an aerosol preparation.

28. Use according to any one of claims 1 to 22, wherein the pharmaceutical composition is suitable for oral administration.

29. Use according to claim 28, wherein the pharmaceutical composition is in the form of a solution, suspension, emulsion or tablet.

30. Use according to one of claims 1 to 29, wherein the pharmaceutical composition contains at least one further active ingredient for stimulating endothelial precursor cells.

31. Use according to claim 30, wherein the further active ingredient is VEGF, PIGF, GM-CSF, an ACE inhibitor, an AT-1 blocker, an HMG-CoA reductase inhibitor and / or an NO donor ,

32. Use according to claim 31, wherein the HMG-CoA reductase inhibitor is a statin such as Simvastatin, Mevastatin or Ator-vastatin, the ACE inhibitor is an active ingredient such as Enalapril, Ramipril or Trandolapril and / or the AT-1 blocker is an active ingredient such as Irbesartan, Lorsartan or Olmesaratan is.

33. Use of erythropoietin for the production of a transplantable endothelial cell preparation.

34. Use according to claim 33, wherein endothelial cells are produced in vitro by culturing endothelial progenitor cells in the presence of erythropoietin in a low dose, namely from 0.001 to 90 lU / kg / week.

35. Use according to claim 33 or 34, wherein the cultivation of the endothelial progenitor cells in the presence of at least one further active ingredient selected from the group consisting of VEGF, PIGF, GM-CSF, an ACE inhibitor such as enalapril, ramipril or trandolapril, an AT-1 Blockers such as irbesartan, lorsartan or olmesaratan, an HMG-CoA reductase inhibitor, in particular simvastatin, mevastatin or atorvastatin, and an NO donor, in particular L-arginine.

36. Use of erythropoietin in low doses, namely from 0.001 to 90 IU / kg / week, for the pretreatment and / or further treatment of tissue or organ transplants.

37. Use according to claim 36, wherein the pretreatment of the tissue or organ transplants is carried out using isolated endothelial progenitor cells.

38. Use of erythropoietin for the production of implantable or transplantable cell-containing in vitro organ or tissue systems, the in vitro organ or tissue systems being treated with erythropoietin before transplantation or implantation to induce vasculogenesis and / or endothelial cell formation.

39. Use according to claim 38, wherein the in vitro organ or tissue systems contain endothelial progenitor cells.

40. Use of erythropoietin for the production of vascular prostheses or heart valves, the vascular prostheses or heart valves being coated with erythropoietin.

41. Use according to claim 40, wherein the coating of the vascular prostheses or heart valves contains endothelial progenitor cells

42. Use according to any one of claims 1 to 41, wherein erythropoietin is human or animal erythropoietin.

43. Use according to claim 42, wherein erythropoietin is a derivative, an analog, a modification or a mutein of erythropoietin.

44. Use according to claim 42 or 43, wherein erythropoietin from human urine, the urine or plasma of patients suffering from apiastic anemia, tissue cultures of human kidney cancer cells, the ability to produce human erythropoietin-containing human lymphoblast cells or by cell fusion of a human or animal cell line hybridoma culture obtained is isolated.

45. Use according to claim 42 or 43, wherein erythropoietin is an erythropoietin produced by recombinant DNA techniques.

46. Pharmaceutical composition for stimulating endothelial progenitor cells, for stimulating the formation of endothelial tissue, for stimulating vasculogenesis and / or for treating diseases or disease states which are associated with dysfunction of endothelial progenitor cells, comprising erythropoietin and / or a derivative, an analog, a modification or a mutein thereof as an active ingredient and at least one further active ingredient selected from the group consisting of VEGF, PIGF, GM-CSF, an ACE inhibitor such as enalapril, ramipril or trandolapril, an AT-1 blocker such as irbesartan, lorsartan or olmesaratan, an HMG-CoA reductase inhibitor and an NO donor, preferably in a low dose, in particular from 0.001 to 90 lU / kg body weight / week.

47. Pharmaceutical composition for the prophylaxis and / or therapy of hypercholesterolemia, diabetes mellitus, insulin resistance, endothelium-mediated chronic inflammatory diseases, endotheliosis including reticuloendotheliosis, atherosclerosis, age-related cardiovascular disease, ischemic diseases of the extremities, pre-eclampsia, liver disease Hepatitis, cirrhosis of the liver, acute or chronic liver failure, bone and cartilage diseases or injuries, ligament and tendon diseases or injuries, mucosal diseases or injuries, especially in the gastrointestinal tract, Crohn's disease, ulcerative colitis, pregnancy-induced hypertension, chronic or acute renal insufficiency in particular terminal renal insufficiency, renal function restrictions with glomerular filtration rates of 30 to 80 ml / min, microalbuminuria, proteinuria, increased ADMA values or wounds and secondary diseases thereof, comprehensive- rythropoietin and / or a derivative, an analog, a modification or a mutein thereof as an active ingredient, preferably in a low dose, in particular from 1 to 90 lU / kg body weight / week.

48. Pharmaceutical composition according to claim 47, additionally comprising at least one further active ingredient selected from the group consisting of VEGF, PIGF, GM-CSF, an ACE inhibitor such as enalapril, ramipril or trandolapril, an AT-1 blocker such as irbesartan, lorsartan or olmesaratan , an HMG-CoA reductase inhibitor and an NO donor.

49. Pharmaceutical composition according to claim 46 or 437, wherein the HMG-CoA reductase inhibitor is a statin such as Simvastatin, Mevastatin or Atorvastatin, the ACE inhibitor is an active ingredient such as Enalapril, Ramipril or Trandolapril and / or the AT-1 blocker is an active substance such as irbesartan, lorsartan or olmesaratan.

50. The pharmaceutical composition according to claim 46 or 47, wherein the NO donor is L-arginine.

51. Use of erythropoietin according to claim 1 to 50, for the production of a pharmaceutical composition, for the prophylaxis or treatment of diseases, wherein the erythropoietin and / or the pharmaceutical composition is suitable and intended for morning application to a human or animal body in one Period from 6:00 to 10:00.

52. Kit containing erythropoietin, endothelial progenitor cells and at least one cell therapy that can be used for cell therapy, the erythropoietin preferably being present in low doses.

53. Use of erythropoietin and / or derivatives for the production of a pharmaceutical composition or a kit containing a dose of 0.001 to 90 lU / kg body weight and week, preferably 0.05 to 50 lU / kg body weight / week for the prophylaxis or treatment of diseases of the human or animal body, the erythropoietin being suitable and determined in the low dose mentioned, the integration of a mechanical or biological agent, in particular an endoprosthesis, in particular an implant, for example a dental implant, denture, a bone implant, a bone replacement, in particular a joint prosthesis , a ligament / tendon replacement, such as of the cruciate ligaments or a solid organ in the implant or the endoprosthesis surrounding body structures to improve, in particular to promote and / or accelerate.

54. Use according to claim 53, wherein the pharmaceutical preparation or the kit additionally contains a cell therapeutic, in particular endothelial progenitor cells and / or other cell therapy usable cell population for the regeneration of tissues and vessels.

55. Use according to one of claims 53 or 54, wherein the endoprosthesis is made of steel, ceramic, plastic or another matrix material.

56. Kit containing erythropoietin in a dose of 0.001 to 90 lU / kg body weight / week, preferably 0.05 to 50 lU / kg body weight / week, an endoprosthesis and, if appropriate, a cell theory therapeutic agent, preferably endothelial progenitor cells or other cell population that can be used for therapeutic purposes.