Title of Invention

A PHARMACEUTICAL COMPOSITION FOR STIMULATING ENDOTHELIAL PRECURSOR CELLS

Abstract The present invention relates to the use of low-dosage erythropoietin for stimulation of physiological mobilization, proliferation and differentiation of endothelial progenitor cells, for stimulation of vasculogenesis, for therapy of diseases related to a dysfunction of endothelial progenitor cells, and for production of pharmaceutical compositions for treatment of such diseases as well as pharmaceutical compositions that include erythropoietin and other suitable active ingredients for stimulation of endothelial progenitor cells.
Full Text FfORM 2
THE PATENT ACT 1970 (39 of 1970)
&
The Patents Rules, 2003 COMPLETE SPECIFICATION (See Section 10, and rule 13)
1. TITLE OF INVENTION
USE OF LOW DOSE ERYTHROPOIETIN FOR STIMULATING ENDOTHELIAL PRECURSOR CELLS, REGENERATING PRGANS, AND SLOWING DOWN PROGRESSION OF END ORGAN DAMAGES
2. APPLICANT(S)
a) Name : EPOPLUS GMBH & CO. KG
b) Nationality : GERMAN Company
c) Address : FEODOR-LYNEN^STRASSE 5,
30625 HANNOVER, GERMANY
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is| to be performed : -

Description
The present invention relates to the use of erythropoietin (EPO) especially in low doses, alone or in combination with other chemical, thermal, mechanical and biological agents, for stimulation of physiological mobilization, proliferation and differentiation of endothelial progenitor cells, for stimulation of vasculogenesis, for therapy of diseases related to a dysfunction of endothelial progenitor cells, and for production of pharmaceutical compositions for treatment of!such diseases as well as pharmaceutical compositions that include erythropoietin and other suitable active ingredients for stimulation of endothelial progenitor cells as well as for organ protection for organ regeneration, especially formation of new vessels and tissues, and for slowing the progression for organ damage. The present invention also relates to use of erythropoietin, especially in the inventive low doses, and/or suitable actibe ingredients for application, preferably topical, in cosmetic treatment, and therefore in the sense of "beauty care' for the human or animal body, especially for prevention or reduction of creases and wrinkles, for strengthening of the Connective tissue, for protection and tightening of the skin, especially facial skin, against harmful environmental factors, and as make up foundation. Furthermore, the inventive topical use of erythropoietin counteracts the formation and further development of age spots, refines the skin texture, supports the skin rejuvenation process and accelerates hair growth.
The present invention also relates to the use of erythropoietih, preferably in low doses, or in other words EPG. preferably dosed as defined in the section entitled "Inventive dosing of EPO" hereinafter, for production of a pharmaceutical composition that is suitable and designed for application in a manner adapted to the natural cipcadian rhythm of EPO In the human or animal body. In humans, endogenous erythropoietin production has its aero phase (daily maximum)) in the rate afternoon, and so the inventive administration of the low-dosage erythropoietin as defined in the foregoing preferably rakes place in the morning especially in the period from 6:00 to 10:00 a.m. in order in this way to achieve a maximum biological and therefore also therapeutic effect. Within this time period, the EPO can be administered as a single dose or as multiple doses According to the invention, this use as a single dose or as multiple doses is proposed particularly preferably for all uses cited according to the present teaching, especially for cosmetic and therapeutic treatment of the human and animal body or cells.
According to the invention, it is provided in a further embodirtierit of the use of erythropoietin that endothelial progenitor cells will be applied simultaneously with other cell population usable for cell therapy, after prior incubation with low-dosage erythropoietin in vitro, preferably in low doses, or local as well as systemic application of erythropoietin In ViVO, preferably in low doses according to the invention, in order in this way to ensure that the tissue cells usejable! for cell therapy settlee With sufficient binding to the vascular system.
The invention therefore also relates to the use of erythropoietin in ViVO, preferably in low doses, preferably for morning application in a period from 06:00 to 10:00 a.m, during application of endothelial progenitor cells having at least one cell population usable for cell therapy, In order to improve the settling, with sufficient binding to the vascular system of the cell population usable for cell therapy, The invention also relates to the use of erythropoietin in vitro, preferably in low doses for incubation with endothelial progenitor cells and at least one cell population usable for cell therapy in order to improve the settling, with sufficient binding to the vascular system of the cell population usable for cell therapy.
The invention also relates to the use of erythropoietin especially in low doses, especially in low doses, especially for production of a pharmaceutical composition or of a kit for prevention or implantatin, in sequential, timed successive administration with at least one other chemical, thermal, mechanical or biological agent especially a ph;Jrnldcological active ingredient, fur increasing the rlurnuer and function of endotlwli:-\l progenitor" cells and/or for regeneration or slowinp me progrt-lssion of tissue damage.
Tht- Irlvelltion also telates to Hie use of use of erythropoietin, especially for production of a pharmaUHJtlcal composition or of a kit. for prevention or treatment of diseases or for use during trcm|:;pBntation or implantation, especially in low dosWs, for sinlultan80us administration of erythropoietin and at least one other chemical, thermal, mechanical or biological agent, for increasing the number and function of endothelial progenitor cells and/or tor regeneration or slowing the progression of tissue damage. The invention ttlerefore relates to the preferably sequential, timed successive or simultaneous administration of low dosage eryHII'opoietln plu~, irl a preferred embodiment, one ur rTiore cjthu pharmacological active ingredients, such 8S VEC3F: GM-CSF. MCSF, Hlrombopoietin, SDF-1, SCF, NG~, PIGF, an HMG coreductase inhibitor an ACE inhibitor, an AT 1 inhibitor and an NO donor in order in this way to mcrSase the nurntx?r and function of endothelial progenitor ceils anrl/or te bring about regsneratiurl or slowing of the proCjresslurl ()f tis->ue darruqe. Ir! this connection, the IrKention acc>JrrJing to the invention IS 10 influence the follol/virlg sequurlce: 1\) qUcHltitativc and qualitative optimization of stem cfdls and/or, -ndothelial progenitor cells in hone malT()\N or" In specific tissue niches for stem cells: B) fnobillzCition olstcr-n l:eil<: endothelial prodonitor cells from bolle marrow or other cell niches irlto peripheral blood: c quantitative and qualitative cjptlmization of stem progenitor in blood ex vivo under selective culture conditions preferably cultures hypoxic vjiui an oxygen concerltratlon to d homing the damage site e adhesion ami migration into target tissue: f neovascularization by cells.> The Invention therefore also relates to the sequential, timed successive or simultaneous administration of erythropoietin, in low doses according to the invention especially in vivo and in vitro, and if necessary also of one Dr more chemical, thermal, mechanical or biological agents, in order in this way to Irlcrease the number and functionfof endothelial progenitor cells and/or to bring about regeneration ur slowing of the progression of tissue dama9l'. an opti(Jm~1 and preferable use being as described hereinabove, in a mariner adapted to the natural circadian rhythm of endogenous EPO production oi) in pther words In an application fOrTT! that !S suitable and designed for administration in the period from 6:011 to1 0:00 a.m.
The; present Irlvuntion dbo relates to trie use of erythropoietin in low doses according to the invention, tor stimulation of pllysiological mobilization, proliferation and d.tfer'entldtiof) of cnduUlelial proUftO'rtitor eelb, tor stimulation tA 'laSCtdnl]t;rlesls tor thd21PV of rJK'.UciSP-rO.I.-.ttc'dtod
dysfunction uf endothelial progenitor cells, and for production of pnannClceutical compositions fur treatment of such diseases as well as pharmaceutical compositions that include erythropoietin arlu other suitable active ingredients for stimulation of endothelial progE:enitor cells, of or for patients wltl, a) a dysfunction of endothelial progenitor cells and b) with at least one cardiovascular risk factor such as

hypertension, Hypercholesterolemia, elevated asymmetric dtaethylarginille (ADMA) levels, insulin resistance, hyperhomocysteinernia and c) at least one end-organ damage, such as left ventricular hypertrophy, microalbuminuria, cognitive dysfunction, increased thickness of the intima media in the carotid artery, proteinuria or a glomerular filtration rate (GFR) of <: ml especially preferably to mllmin hie invention relates the aforesaid use of low-dosage epg in patient groups defined a c an embodiment that is suitable and designed for undertaking application epo periucj from vascular endothelium layer cells lines blood vessels trie keeps separate tram other layers however not fth d passive barrier but it participates actively re vascu f tone this capability also refem as endutthlun-l di vasod lation. because its location exposed fit all times hemodynzjmic metabolic stre>. In paHIOgeniq condition:-,, such as high blood pressure, elevated IDL levels 8loVat:;(j levul:.~ of asymmetric dirr1ettlylal'ginlnr~, which
is ttle endugenous inhibitor of NO synthetase, hypercholesterolemia restricted renal function with a glomerular filtration rate of 2>0,
preferably 40 to 80 ml/min, insulation resistance or high blood glucose. Were frequently develop functional detects of the endothelium,
potentially followed by morphologically detectable damage, sucji 4 forma110n of aterosclerotic plaques and/or further end-organ
damage, such as left ventricular hypertrophy,
microalbuminuna, proteinuria, neuropathies or microcirculation ilnpiirments, A very early sign of altered or reduced endothelial function
or endothelial dysfunction is a decline of endothelium-dependentl vasodilation, In the case at coronary heart disease, or even when risk
factors arc present without coronary Heart disease, especially hypertension, restricted renal function,
hyperlipoproteinemia, hyperrlOmocysteinemia, insulin resistance or diabetes, endothelial function defects are ITICHilfested in decreased production of NO (:..;; EDRF) and Irlcreasej Endothelin production. High plasma levels ot endothelin lead to abnormal coalescence ot cells, inflammatlorl. vascular tumors and severe vascular stenoses Endothelial function impairments are additionally (:;haracterlzed bV increased productiorl of adhesion molecuic;s such as ICAM laid VCAfvll, whereby thrombocytGs and mCinocyte::; adhere to an Iflcreased degree to th8 endothelium T fl is results in increased VSsolpnia. Thus a disequlliUrturn t8voring vasoconstriction adhesion, ag\J1eg;jtion. cuagulatioll. attlerosclerosi-, and atheruthrorntJosis develop::; ifl thF: 11"lost dlvurs(;syc:;tpms Even niental stress leads to mea::;urable endothelial dysfunction, which can persist tor a— long a~o 4 hours. Endothelial cells also pGrticlpate in the formation of new blood vessels. Blood vessel formation is important in a large number" ot processes, such as embryogen8sis, the female reproductive cycle, wound healing, tumor growth and neovascularization of ischemic regions. Originally, postnatal blood vessel formation, or Ir1 other words blood vessel formation after birth, was attributed mainly to angiogenic processes. By angiogenesis there will be understood the development of new blood vessels by sprouting of capillaries tram a preexisting vascular system During angiogenesis, the basement mejnbrane surrounding Hie blood vessels is first destroyed by means of proteolytic enzymes, and the exlracellular matrix in the perivascuar space is fragmented. The anglogerw; stITnUII released thereby cause already existing differentiated endothelial cells to migrate toward the chemotactic stimulus, during Milch process they simultaneously proliferate and are transformed. New vascular loops with a capillary type lumen are then formed by accretion of endothelial cells Thereafter synthesis of a rle\N l)asement rnenlbrane begins
riecent Investigations however, shew! thai t;,e formation ot newj blbod vess81~': in thp adult organism depend.:; flol only 011 ariglogc'rk-sis but also on v8::;e;ulogenic mechanisms. By vdsculogenesis there is ImderstuorJ formation of new vessels fron, Pfldottldlial proqenHor coils undergoing clHerentiation In ::itu The; bt,7it:f tlld.t va ~culog(;n8;"iis,"- cunflned to emUIY(Ju(?nesi~ was refuted by the detection of endothelial prug(;nitor cells (EPC) in peripheral blood of rjearthy humans and anlrnah. By uSjng animal models, it was proved that the endothelial progenitor cells derived from bone marrow particibate actively in neovascularization It was also shown that a specific C034-positive subgroup of leukocytes expresslflg endothelium-speciflc antigens becomes established in Ischemic regions. In addition, endothelial progenitor cells (EPC) that contribute significantly to the formation of blood vessels in the adult organism can be obtained in vitro from CD133+ and CD34+ cells (Asahara et a/., Science, 275 (1997), 964-967; Crosby et a/., Cite. Res., 87 (2000), 728-730; Gehling et al.. B/ood, 95 (2000), 31063112) It Was also shown that trJJection of isolated CD34+ cells or cultivated endothelial progenitor cells accelerates restoration of Wood flow in diabetic mice (Schatteman jet al.. J. Clin. Invest.. 106 (2000),571-578) and improves .leovascularization In vivo (Asahara et al.. Circ. Res' 85 (1999) 221 et al., Nat. Med .. 7 (2001). 4:30436). Besides C034+ cells, C034-negetive mononuclear blood cells can al~o be used as a Source of endothelial progenitor cells by appropriate tr ansrJ.fierentiatiorS.
However, the mechanisms underlying mobllHd.tion and dif1i:.Jfr;"!T/tlat|ofllof endothelial progenitor cells have not yet bc.'ofl tully expld.jned. MolC;CUIr3r hiological and cytolJloloyical
investigations indicate that various cytokines and angiogenic growHI factors -lave stirTIUIating effects on mobilization of endothelial progenitor cells in bone marrow. For example, it IS known thai proanglogenlc factors such as VEGF and GMCSF can Increase the number of endothelial progenitor cells (Asahara et al., EMBO, J., 18 (1999), 3964-3972; Takahashi et al., Nat. Med., 5 (1999), 434438) VEGF (vascular endothelial growth factor) IS a protein that occurs in various Ispfmms and that binds to the two tyrosine kinase receptors VEC3F-R1 (flt-1) and VEGF-R2 (flk-1), which occur, for example, on H-JI8 surface of growing endothelial cells (Wemert et al., Angew. Chemie, 21 (1999), 3432-3435). Activation of VEGF receptors leads via the Ras-RafMAP kinase pathway to expression of proteinases and specific integnns on the surface of endothelial cells or endothelial progenitor cells, and finally to initiation of proliferation and migration (If these cells toward the angiogenic stimulus. GM CSf-granulocyte-macrophage colony-stimulating factor) is a cytokine n-l21t heretofore was known mainly for stimulation (jf white blood corpuscles. Including neutrophils, macrophages and eosinophils. PIGF (placental grov,1h factor) is known tC\ stimulate the mobilization of endothelial progenitor celis but :1ot proliferation thereof. From investigations by LleViidot et al. (.J. Clin. Invest.. 108 (7001), 399-405). jj fc)llows HW.t HMQ~-Co,A, reductase inhibitors, especially statins, which are usod as lifJlcJ-ln\N(;rinq m8Jicam8nts and which reduce the morbirJity and rrjortality of coronary disease, are able to rflOUill/8 t';l-ldothclial progenitor- cells. Oirnmeler et al. (J Clin. Il\vt':-,t 1i)2, (~)()CJ1}. 391307) wti|e able tu :3how fuitrici that
Statins such as atorvastatin and simvastatin significantly improve the; difrrreititiation, in vitlo and In vivo, of endothelial progenitor
3

pancreas or skin 1 n this cOrtncictlor1 f:,:-,ir :fi;):ints !, „\~)c L'el understuud :is L1 iuluglcal d. implantation -)upp(Jr1 fOI a t()uth, tooth rcpl:1cCtTH:'r\t, toottl irrlplant, bon(; replaccrTlcnt. bone inlplant, for e;xarnple hip jOint prosthcsl::.J, IMarneflt/tGndorl repl::\cemc:r1tco" tOP example crJicia e ligament. WllCICill the erythropoicotin is dppllod sy~;temically or locally prior to irnplC3ntatlorl of Hie said biological or mechanical Sgent. or in other word-;, for example, tilt- enduprosthesis, tor example some Wt?oks prior to implantation, dllt';l' which implantation is perforrried In a further embodirricnt. It is also provided that Implantation of the said biological or mechanical agent such as the endoprosthesis, be undertaken simultaneously with the use of erythropoietin, III a further embodiment, it is pruvided that the erythropoietin r)e administered after implantation of the said elldoprosthe.:;ih or ot the mechanical or biological agent. According to thc;.(-; embodiments, the tissue or the) body structure in wllll;h the implant such as a tooth or bone prosthesis will be implanted is mobilized or conditioned, H IIS 8nd.bling COiiSidcfd.bly bGtter and tllUS faster integration at the blologb';i or n!c::ch8nical agent, suci I as an implant, lor examfJIe |iY' ;ro7l!h onto or into Ule body structure HI The; LLi; d er{Hf;)pOletlr\ 8ccol'ding to one or1 mow uf the 8!l1hocJ:mt/[!L :ICc(Jrding to A to (31, \tJhnrC'lfi the prlc,T!Tidu;;utcaicll:pUS!!tl)n doe::; [1(,j l(;iJd tu any H1crt=-,JSC oj the tldn2toc.Jli durifl'd applicatlurl in ttw human Ofjanijinal bo.Jv. e~p(:'C!;j,lly n(.1\ nwre tllarl 1 UOI(~ ot ilK: value at tliC hUITIatOu:l [.Hlor to application uf the urythropoietln.
I) Tile use of c["V\hropoictin according to (JriC; 01 more: of ernbodimd:?nls dccordir1\:j to A.\ 10 r!) HI 0 pl'lcurnaceutlcal composition, whuC'ln H\l? erythropuictin i-,) r.uitable and deskjnc;u tur the said prevention, treatment or the'rapy. in a Imv du:;;e thdt cannot activate elythropoic:si~; f;:'c.;pccially In a dose of OOU1 lU/kQ of body weight per week, up to 90. especially 50 lUikg of body weight per week \ I he in\iCr~lii)!1 alsu rci3tc:~; 1n the pmductiofl of a kit contalni!\rj N;rtl irOpolctltl endoH\8llal progenltur cells dnd at lc.<:i crjl pnpulutlch usablr.: fur ceil trlerapy. wflerein the p l pre: in low dcrtje> According to the: lrlvclltlc..H1. it has SIJrpri~;jngly bU;11 found that treatment v.llth oosage 8tyHHopoietir1 ICdd~J to prlyslologic;:.j I motJill! atlOrl uf (";ndoUlelial progenitor1 cells, wherelrl the nurnhcr oj circulating endothelial progenitor c(;,lls is lncrc;a::;ed and cJittererl nation thereof is inducpcL In addition, functlUflcii cioflck.'": ot the endothelial progtfnllor' cc.'lls that occur under certain pathological condition-; drc compensated, According to the invention, It has beer! :,hOWrl Mat the number uf circulating stem cells in patient-, with chronic renal disorder in the terminal stage is just as nig-'1 as in riealthy subjects, [Jut in these patients they have lo~)t the ability to differentiate to endothelial cells via endothelial progenitor ^ells;, Thus the number of cells capable; of adhesion and exnibiting an endoHlelial cell phenotype is distinctly reduced ir) patients with chrpnic mnal dhordor compared wittl hc;alH\y subjects (de Groot et al., Kujncy Int. 200{1:66:641~6) This furlctional decline of endothelial progenitor cells carl already be seen In moderatE? restriction -Jf tile rer\al tWIctlon with ,~ glomerular tiltrdtion rate of :-30, preferably 40 to 80 ml/min, According to the invention, it has now been tound that Hie number of circul:;J.tin~J stern cells JrlcreasiC—' sicJrl ritly by rnore thar! 50^, after treatment ..nitll ICN-c.Ja.-'age; cV/ttlropoietlr1 according to the Invention, not only in tr H:-'0 I.,; pC1tier.t:~ but also in patients and/or -;ul)jccts with hC21iHIY -xjr-'efs In particular. thE.' number of ceils t!1at develop df! c:nrjuthe!la! prIE-'notype Increases dram8.tlcally Ar-:, VI'd::; linmon::.{r,:;rc:cJ by mf;ans of a functional cell culture tC';st. the impairRcj adl18:;IGn ability of the endothelial progenitor cpils in Uatiefil': \lvitl! chronic renal disorder, characteriled by" (Jl()rl\i-!li-" 1111 'j'lnn rl:-irf:~ uf:-;{) preferably 40 to 80 ml/mirl,
is incrc-ased bV ,J l-\c!or of three by tl 18 IC!w-du:.:;a~Je erythropiietm trQ3tm—nt Ir1 subjects andl ur patterlb witll healthy kidneys. It i~; irlcr-a:)ed l)y a factor of two to three;, 1 rie mJhE.sion ShilttV 01 endothelial progenitor1 cells undcrCJ()in~j dtfferC'ritltJtion and of endothelial cells is one of the basic prerequisite:) fOI' the formation of naw tl:-;~oues ancJ/or vessels. In this way erythropoietin is able to induce neovascularization, especially vasculogenesis. in tisSUt':s or organs, of whicjl kidneys are a particular exafT)plL~. in wtlich corrGsponding vasculogenic or angiogenic stimuli are released.
According to HH- invention, low-dosage etythropol8tin can be used to stimulate prlysiological mobilization of endothelial progenitor cells, pr oliferatioll of endothelial progenitor cells. dlfferentla~ion of endothelial progenitor cells to endothelial l;eils and; or migr ation of endothelial progenitor1 cells tuward a 78sculDgerw; ur angiuljonic s1 itnuli JS In a hurndh or 3111 rllal b,„dy e:S;.H::'clally an adult organism. A.ccording 10 tho WJOntlorj, tOVv-dusa~-e erythropoietin car! thewfure be cniploYfC;d advantageously to stimulate the formation of new vessels 17 v:';:;CLItogenesi~; if 1 tissues or org3fls in which
i Vlisculdr ,;hanges afe pre:-,cnt In 3ddlt!OI I th8 rT:iii:U'i tndutflid <:111 s.o.uc can disc be iflducfyj fv the :: cit endothelial prosjenlto uy lowcjqsage:: ijylrlrup according to invention lowdo::. crylhlu letr. c8n therefore al:-.. he employed trf::dl di::: ot the: human or arwnal body that arp h i a ay: f u ic: n r doth.d p ito ci c i. jatleflt jpulfc.ugh hillnsj> (1 rly-;jun(;ti()H usually have c:cHdlova::,cular risk factors such; a;\ tft'PE-rtc'ri'Olurl, llypuchole:-tc:rolernia, insulin resistance, HYP0r-1()rl)Ocy::;teinClllia. olevdtl":d AOMA levels, and end- iCJk|~1\1 dal1k\gc' :;.;uch a::; Inti ventricular hypertrophy. nlicroalbumlrllHia. prCJte;inU\ld or 3 glomerular filtration ICIte l(Jr:nl Oil 30 pruferably 40 to 80 ml/min.
II1\-, inverrtion also relates to the use ot lowdosag6 erythropolE=!tin for protection and regeneration of tissue whu-)e function has been joopardized by the action of chemical, tholmal, mechanical or biological agents According to the Invention topical application of low-

dosage erythropoietin also relates to prevention and reduction of already existinq wrir Ikle5 of the skin, especially of the facial skin, to protection of tll(~ skin and to reduction of age spots, Accordinq tu M indention such use of 10wdOSdge er)'1IHUIJoictM (if nf a cJeriv3tive can take place sequentially. In timed succe::,;c:n Of xilTlultaJleously with one or more otl i,~r cherni(:81 0101 maio 'nechanicai or biological agenb. According to tile Invention. iO'N-dusage erythropoietin can be therafleufccallv use:i ili a rVCInncr adapted to its Orcadian rhythm if CHcJf:or 1,\ thiS :.C:iY i(, achieve a maXltTlldrn bloioQIC81 ("1(6"j in.:, pi crrc'd -rnbodlnler(t accon.Jirlg to the Inventiorl erickthnll::;! prU':Jc;n!tcr cells are applied simult(}neou~:]ly 'NiH1 ml18r cei1, plipuiakirls uSd.bii:0 lur ceil therapy. artt~1 prior inCU()8tion toilri IC'N-du-~agc: erytr IrCJpoietin in vitro and/or ; I
ia:> .I i:\': 5v:;terrllC .Ipplicallon oj luw d0SCi\.jE::;
i:-ryrHIK'Jpotet:r' iii VI/(., .II ol, Ji..:r il\ thi—) \Nlfy tu ensure that the j
tissue cell::; uS(1 blc tor eel! HWI'dPY Sf ;ttlE- with sufficient Dlndlfkj to the va:;cUlar -:;ystcnl
in connection wiH1 till' present invention, there will be LHIderstodd by "erythropc)ictin" or 'ErG" a substancS that in appropriately hig—1
do~;age contmls Hie gro\Nth, differentiation and maturation ot stem eels via erylhroblasts to erythrocytes
Ery1hropoiE;tin is a glycoprotein -laving 166 amino acids, three glycosylation sites and a molecular weight of about 34.000 Da. During
EPO-induced differentiation of erythrocyte progenitor c,-lls, globirj synHwsis is induced, synthesis of the heme complex is augmented
and the number of ferritin receptors is increased. The! el)y Hie cell (can take up mare iron and synthesize functional hemoglobin. In mature
er)1hrocytes. hemoglobin1 binds oxygen, Thus the erythrocytes J and the hemoglobin contained therein playa key role in supplyinC)
oXV;Jen to the organism. T~lose prCJcesses are initiated throupn the interaction of EPa with an appropratf? receptor on V:e cell suriace
ot thE! erythrocyte progenitor cells (Graber and Krantz. Ann. Rev rW 2C1 (1(j18).51'Jf))
Thp jprrll "~rythr()pniptir" 11;;f,J IIH8 IrlcltJdes fl-'O of (c\j(-;,jy Wnjlri. espf:,cldlJy hum;;n ! ,-;'iirTial fPO. Tile terril u::,i:'U ilere
r::;riCOiTipa:;;[;e:: riot ur:ly jh; flaturiJIly occurring, or In uttler jlJorp--; 'Nllc.i-type form:-, l)f [f-JO l)ut also its duiv3tl\fes, ;:Hidlu~l~',
fTlurJ.flcallufl::: rrlutems, mutants or OH1EHS. as lonq 11-) thr.:y r :xhlfoit the biological effect-:; of wlldtyp(~ c; eyt h! C![JI.' :(,;1 ir i
In Connection with the pre::;ent invention, there will bt,' I Jrl rstood by "derivatives" functional equivalents or dCIIVaAivfO of erythropoietin
(hat. while retaining the [.1asic cryHHopoietin structure, ar(~ obtained by substitution of one or rnHf atoms or molecular groups or
residues, especially bV -ubstitution ot sugar chains -;uch as etrjylenE? -tycol, anctor wl10se amino acid sequencB-; differ from that of
the rlaturaily occurring 11UrT1an or anirTIGII etythropoietin protejn in at least one position but essentially have a high degree of
flOrllOlogy at the amino acid level and cornfJarable biological ditivity, Erythropoietin derivatives such as can be employed, for example,
In the present invention are known from WO94250S5, EP 01486p5 B1 or WO 95/05465, among ottlor
sources. l
"Honlology" means especially a sequence identity of at least 80~\,. preferably at least 85',{, and particularly preferably at least more than 90C:',~, 95'-.-, 9Tcj and 9f:J%, Tlw l"Ufl f"H)rnology" known ty thi"; persorl skilled in the art t!IU„; refers to the degree of relationsrlip between 1:\'10 nr moro polypeptide molecules, This IS determined by tr18 agreement befweerl f1„" sequence's SUch agreement can m(;an (tflHIel :(Jentical Ciljreement ur else a conservative exc!i:'nqrj- U1 3r!1:nt) -c!d;:,
Ac(;0rdinq tu the Invention the t(:;rm "de'livatlvF also inc:IIJ(h")s tuf ;iur I proteins It I which functional domains of another r HuteM ;:Jre prci.':.;ult un thF: N tcrrnin2\1 pd.rt or Oil H)8 C T(;rmlf)~\ p;.in llljonij ic'l r Ii.ii.'diri IRni ot nIL: ltlvHltic)j-1 tlli::;
other r")l'otc;irl may be, for example, GM-CSF, VEGF, PIG!, /jl s:t..-lllf1 or ('I 11 ot.l H„;r factor that has a stimulating effect Drl el](J(-thc:lid.1 pmgerntur cells In a tuilhf;f embodiment of the iftvanllUfl rho oHIer protein may also be a factor that hils a stilTJ-JiiJtlflg eHect on differentiated endothelial cells, tor eXilmpie anglO(jeniti VEGF (vascular endothelldl growth factor) or- bfGF (basic fibroblast gro\>vtfl factor). Regarding bFC3f dnd VEGF, ft is known that these groUrth factors exert a strong mitogenic and chemotactic activity on endothelial cells
1lle ditierences between an erythropoietin derivative and na'tive' erythropoietin may arise, for example, through mutations such as
deletions, substitutions, insertions, addition's, base exchanges and/or" recombinations of the rlucleotld('-; sequences coding for the
erythropoietin anllno aCid ::-equences, According to the invention, (EPG-) alpha.
Lf-'O bE:\td AranBSp (darbepoetin slfa) or CEHA f
Inuou::; erytrlropoietin receptor antagonist) al'(;~
preferably used as erythropoietin, Obviously such
d!fteref1c8,) C3.li also be naturally occurring s.eqUGI1CE:! va^atkns ::;l](;h 33 sequences from another orqarilsrrt cJf :-;l;i;uelC- r c;rvd!( ic;tin, using common me2J.ns known in tjjie; art
:)uen (i:: c~"cmicdl agent-; and/or pfjysical a~:,wnt-;, Irl corinHci IlYn "lith llle invention, therefore, the term "deriviltive" di:,c, ir cluck':: nllJtatccJ eryl 1lropo!,~tin molecule-:,, or- ir1 UUIHI Nelras (,'lrytf |r(Jpoiclif. mutcins,
Acc()ldmg tu the;: invf.?ntion, peptide Of proteifl analogs of (JryrJi fupcwdif i may also be usc'cJ. in connection with the present iih/l:fltion, the term "an[.1log-;" illclude-; compounds that do not hnve any amino acid sequenc8 identical to the ery11lfopoii311rl anW10 acid sequcnl.e but -lave a tllrGedimensional structure greatly resf:?rnbiing llldt of erytiropoli2urr, so that they have cum parable biological activity. Erythropoietin analogs rTlay be, for example, compound-, that contain, in a suitable corlformatiorl, the amino acid residues responsible for binding of erythropoietin to its receptors and that are therefon? ablE;; to simulate the essential surface properties of the erythropoietin binding region. Cumpounds of thi- type are ufescribed, for example, in Wrighton et aL, Science, 273 (199C) 458 The EPO used according to the invention can be pmduced in various wW for example by isolation tram human urine or tram the urine Of plasnIGi. (including serum) of patil:;nts suffering fwm aplastic anenil2jMiyake et al.. J,8C 252 (18//), 5558). f\S an example, human" ErO can aiso be obtained frum tissue cultures of human renal cancer ceil] (JA Unexamined Application 55790/1979), from -1Umar lymphoblast cells, which r13Vr.: tr,c; dbility to pruduce rluman EPO (,JA. Uiiex, ;m nf..;cj Appllcdtiof ,jU41119,3;),. and from a hybridornJ. culteJrt; obta:rit:cJ by ceil fusion of::; "Unld.l cc;lliine [PO can dl:,o bE:' ;Jic::JuCed by metrlOd,- of gerle technology US1I1U ,'iuit2ble D-'-;\ or nfUA codlriSj fer the.' 2J.pfHopnatu amino aCid ')c'quencc of If O to prouupe tne de~"r8d proteifl by gCfletic m)~Jlm(;rirq. fur example in a bacterium, in
6

a yeast, or in a pldfJI. (mi'lidl Cit hUrJkJH..- i Lrlt; ~:-\J\~,h rltethods arc!
descrllxU for example, in EP 0148()05 [!,? or IP 07055641:3? and IF U'4116?K 81 The present lnvc::ntiofl relates in pariicul8r to tile use of low dOS;;1.ge c:rytll!OpOif;;lt1rl and/or derivatives thereot fol stnTlul2tiori of pilysiologlcal rTloblllzation of endothelial progenitor cells proliferation of endothelial progenitor cells, differerHiation of endoHlelial progenitor cells to endothelial cells and/or migration of endothelial progenitor cells toward a vas(;uloge~1 Ic or angiogenic stimulus Wl a human or anlnlal body, especially an adult organism. The Invention also relates to sequential use ot low dosage erythropoietin and at least one turHler suitable chemical, thermal, mecharllcal or biological aldent or active ingredient, especially a pharmacological active Ingredient, that increases tl18 function and number of endothelial progE;nitor cEdis alld also potentiates thE effect ot low dosa~Je er„ 1 rllOpoletin a::, rGgards organ protection and rcgeneratlurl. FurthemlUfc. the invention ttH~retore [fdates pll-;fe»ably to :sequf;ntlQ!, tlmf.d successive' or simult;:Hlrc~UUS arlmirw,:;tration CJt lel,'v-dt>:',J.gc' erythropoietin plus uneJr more; other pll;>HiT:8Cuioqlcai actlU8 in~W_)(jlf::!nt,-:, suerl as VEUF-GMC-3F. f/1-CSF. tfllur-nbopoletin. scr. SDF1. NOI" FICF. an HfACi ,-,yeUiJc1asf::" Inhibiter. Wl ACE it It libitor. Wl AT 1 intlllJItCH ,me! \:HI NO dOrior. in order in this way in incred-;e Mil" hlHnlJd and furlc;on of endothelial progenltur cells
(-jllG,'U:\~)lil'll._~;:.iL")l )ut rt-::D8nc;rJtic'n d~!C\\Irl(j (jt thl) rrogrc-;sior1 uf ti:,sue dama-Jt. In H II- connection, the
interltion dccc)f"(iing to Hie inventilHI i~) to influence the followll1~J sequence: A) quarltitativl' and qualitative optlfnlzati and/or1 endothelial pro-Jenitor cells in bone marTOvV cr in specific tissue tllche:- for stern cell::.;; 8) tllubillz;ation of stem cell;:; and/or
endoth('li;a1 progenitor cells tmm bone marrow or other "stem cell" niches into peripheral blood: C) quantitative and qualitative
optlrTI l/ation of stem cells arid/or endothelial progenitor cells in peripheral blood and/or ex vivo under selective culture conditions,
preferably cultums under hypoxic conditions wiHI an oxygen conceitration of 0,1% to 10%; D) MOrning 01 stem cells and/or endothelial
progenitor cells to the darrlage site, E) adhesion and migration at stem cells and/or endothelial proqenltor cells Into the target tissue: F)
neovascularization by endothelial progenitor cells.
The presont invention therefore relatEo's to application, simultaneously or at different times Df endothelial progenitor ceil-; and 0[IE-: or
more cell populations usable for cell therapy especially hepatocytes, myocytes. cardiomyocytt~<: dr l:sland transplants after prior> incubation with low-dosage er~(thr()poiotln if I vitro and/or local as well 8S systemic appiicatlor \ ot lOIN.dosage prythropolE-:tin 111
VIVO, thus ilnprov!n~; and ;:1cc8leratirHj the Junction, settling. 7;iscjjlarvciticin cHid connection to tile Uloorl circulation of the reCiPient of
H\(:,sp coil puplllatlOrlS uSf:d for cell therapy.
I iif:' pr(;~ cnt lfl'vC;[iticln relates to lilt; 11:-)(' of erythropoietin,; :T:cc^:~{:IY' li:,';. :]r :;Ult;'Jbk- activi-; inCJI'C'diprl !t> tor
topical appllciHiur: !r) the: sem.u of "beauty cafe", 8;;;;pecially tor preveritiCi[l or !imc.:l'y n;;dlJction ot Cle2i--;es and wrinkles,
stncmuthc:ninq uf the connnctivc tl~-)ue. protecliun Gnd tiqlltf:,ningjj IIFJ— :~kin. r=::;pCCii1lly facial skin. agai!1-t harmful envirunmo'lUi
[aciol::0. [me! 3S rnakeLJp tUU[ldation. F urthermon:.! the topiC:ol use of erythropoietin cuunteracb the formatiun and further
development of age spots, refine;-; the sklr, texture: arid support^ thp skin rejuvenation process, especially r-el)!th(:li;jli/2:ition. In
addition, erythropoietin
accelerJtoshairgro'wttl.
The present inveiltl(J!l also relates to the use of IO\-v-dosage eryHHopoietirl for production of a pharmaceutical composition tfiat is
suitable and designed tOf application in a manner adapted to tho circadiafl endogenous i'hyttim of erytliropoietJII. Endogenous
erythropoietin production lidS its aero phase (J:;,ily trt3.xil1 lurTi) in tie late dfternoon. and so the adrilinistt at I c..;f \ rJt n ie luw dosage
erythropoietin prefGrably tak'.:'s place \r the rT:oming, especially lietwecn b.00 and 10:00 8 ril. ir" rdet in tr11S way to BChl8Ve a
maximurTI
tJ'ulogical trlc;rdp('JUtlC Of cosmetic effect.
Th -le,;'::1 W!\l(';!'!lur: relates to the use ot !v.'v-(hsaq(l)
CT/HHUpOicti:- 1„;r >,tirl1ulatlon d physioioglCd.i rnnhillzaticHI 6W](mkr pi:)! .r.3.tie,r* eclb,) 1 ':00 i fer stirn ulatlon uf va,:culoqc:1 Hc)~;IS and/or Tor therapy 0+ di,-e8ses rcdated tu (] dystunctlcHI of c:ndotlibli,"1 r-'logc-r)!Wr
cells, and/rH for product:ull ot pl":2,;l))~r.A,j:IC;,~i c:"r_:!0n:: to: :r{_- iliflrJ ,~ljC'h cii'A>;1': (->( ;"1ncl
of pharmaceutical cc>rlH()sition::; that Irlcludc erythropoietin and other ::;u!talJle Mctlve ingredients h)1 stimulation uf enuothelial
plogeritor c(:,;113. In patlAnts with a) a dYc,function of endothelial prUwrtltor c<:>lls, ahu b) at Inast ono
cardiovascular risk factor such as Hyperierwion.
\\ypercholestcrotcmi::'l. insulin resi-tance,
hyperhomocysteinl>miii elevated ADM/1, 18v81s and c) at least! onb end-or~an damage such as lott ventricular hypertrophy,
microdlbuminurl3, cogrlltive dysfunction, increased thickness uf the intima media in the carotid artery, proteinuria ur EJ glomerular
filtration rate (C.FH) of less than 80 rnl/min, especidlly 30 preferably 40 to 80 ml/min
In a preterrE:cJ (-mt)udiment the inventi()fi ak;;o relates to soquentlal nmed successive or simultaneous administration of low-do~:;agl'
erythropoietin as well as or18 or more: other chemical. therrri81. mechanical and biological agents In order in Hils "NaV tu increase thE:
!1l1rr:t,\Ar ahd functlorl uf endothelial progenitor cells a.ndor to br ng about rege:nerdlion or SIC/Nil1q of the progrer-SIOrl of tISSIW
danlMgo Suetl mechariicztl 3':Jerl1s can be endoprusthe::;;es preferably irrlpl?mtatluf SUppuri-.; for teeth, bor\u-o or llqamentAendon
rep!dcer\:,:;ri(' Furtllslmore, the biukAJIcal 3gf;l\1S can be ,:;clid c,rr|j;2ii'::-,dd' i):'i liver, -IcJnE'-Y::;, I,c,c1ri p::.ulcr\';3S or skin, or everi
hair irnpldnts 'The; invention therefore provides that F.:PU t';;-.fJ!X,ld y 1:'11 o \lv (Josos vMIII be uc.;cd -;() that n\b.:h;lnicai d.u°"L Ocjch
as 8ncJopro';;ttH3ses or biological agents ir!lpl;:JntC:"l.l :„;imultwlc;mis y, -;ubs(:?qu(;ntly Or u,.:t()i,:i1 11 )'.1 ';;"1": u: (Y;J ,-)1 bt: if
il„';cFdted [icttOI, f;:-,:;tc:r and more cHic'el illy In1<: the surroundlncj structure irwerltiun thetefme also r to u uf> crythmpol8tin for production uf a phannaceutical compo-illOn 01' bt a. kit for Improving, especially for promote) or accl'lorating,
integration uf biological agents or endoprostheses Into surrounding tody structures, especially uf teoth. tooth replacements, tooth
implants or other endoprostheses, sudl (IS bone replacE:;ment—, bW implants, especially II ip JOint prostheses or ligamenVtencJon
replacements, suer) as cruciate ligaments. In this connection, it can be p ovided if necessary that Hie erythropoietin will be used togeth8r
witrl cell populations suitable for cell therapy and/or endoUleliai progenitor cells. In the aforesaid use of eryHHopOletin for production ot a
pildrmaceutical composition or 01 a kit for improving, especially for Promoting or accPIMating, integration of biological or mechanical
agents into tar-)et structure—, especially target tissue, tcuget bones or target cartilage 01 a patient it can be provided in a fur1fiE::1
pref()rred embodlrrtl:-'nt that Hw mechanical agents to be used \Nill be made, for examc-ic, of steEd, cemmic, plastic or anotrter matrix
material, Irf addition, it Cd.f I t)e provided that o:::,teoblasts. cedis pdfing osteogeft ur,t(-nti;J! thromtJocytes, blood c(;lls or s:m\[rt
7

aCJcnts Cdrl be IJ:':'C"cj a~, u?!1 PUfJuld.tlurlS suiU;\blt' f(;r (bt';l| therd.py 'rl W, rJi"(;2,c.ni d.ppllCiltlUII In a furH'ler prcterTeu embc)cflmc:nL it car" U~ IJfOvided ttl21t titt-; rr)(::!cr lanical aqc.'ill pi particular to br? u::,ed , In connection 'Nith the present invention, Hiel\:; will he ur1l.ici(SiO(yj by "proliferation' H\e ability of cells to become; 'JlqOI and subsequently divide into two or more dauljhter Geils, The FPOri(,E;dl2ited stimulation ot endothelial prow.;nito! ceil.c thus relates in particular to the} r lllrliber -Hili thu:,c the diYldrf-U CL)';;"j!UI C endijthelizd progenitor cells
if) connuCI:uri',Vlt\: HiP present lnvc;nilon trlelC; will be urtdc;r)(joo" by "cJlffetT;ntiallUrt' of endoHlelldl proqenitur cells tile oevclo[Jrrll,l:: (")11l1Ufl()nuclc6r cell;) oli-Jindtlllg from the h'OH":' it1 r1-' ••.;' ::|jr:cH tis::uc: ni I:
progenitor cells into endothelial cells. By "endothelial ceils'-' therein be understood the cells that form the endothelium, or in other words the monolayer cellular lining of vessels and serous cavities. Ejidothelial cells are characterized in that they release vasoactive substances, for example vasodilating substances such as EDRF (endothelial derived relaxing factor) or constricting substances such as endothelin, factors for inhibition or activation of blood clotting arW factors for regulation of ascular permeability. Endothelial cells also synthesize components of the subendotheliai connective tissue, especially type IV and V col lagens, 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, there will be understood by 'migration' of endothelial progenitor cells the fact that the endothelial progenitor cells present in the bloodstream migrate towar J a vasculogenic or angiogenic stimulus and become concentrated in the region of the vasculogenic or angiogenic stimulus. By "vasculogenic stimulus" there will be understood a chemical stimulus in a tissue or blood vessel of a human or animal body that acts specifically on endothelial progenitor cells and brings about migration thereof to that site in the body from which the chemical stimulus originates- In ths way, the vascutogenesis process is induced by the vasculogenic stimulus. By "angiogenic stimulus" there will be understood a chemical stimulus in a tissue or blood vessel of a human or animal body that acts specifically on differentiated endothelial cells and brings atiout migration thereof to that site in the body from which the chemical stimulus originates. In this way, induction of angiogenesis is induced by the angiogenic stimulus.
In a further embodiment of the invention, there is provided the use of low-dosage erythropoietin and/or derivatives thereof for increasing the adhesion ability of endothelial progenitor cells undergoing differentiation. According to the invention erythropoietin is used in particular for improving the adhesion ability or in other words the cell-to-ce)! adhesion of endothelial progenitor cells, The adhesion of endothelial progenitor cells undergoing differentiation or differentiated endothelial cells is one of the basic prerequisites for the formation of new vessels or of new endothelial tissue. Cell adhesion is mediated by protein molecules.
The present invention also relates to the use of low-dosage ejytnropoietin for stimulation of the formation of new vessels, especially stimulation of vasculogenesis. In connection with the present invention, there will be understood by "vasculogenesis" the formation of new vessels from endothelial progenitor cells undergoing differentiation in situ.
According to the invention, therefore, it is ensured by the use of low-dosage erythropoietin that endothelial progenitor cells can participate to an increased degree in formation of new vessels or in focal formation of new vessels to restore damaged vascular regions. According to the invention, therefore, it is provided that the use of low-dosage erythropoietin and/or its derivatives will promote formation of new blood vessels and/or replacement of damaged vascular regions through local formation of new blood vessels, In a further embodiment of the invention, there is provided the use of low-dosage erythropoietin and/or derivatives thereof for stimulation of endothelial progenitor cells for formation of endothelial tissue.
In a particularly preferred embodiment of the invention, there is provided the use of low-dosage erythropoietin and/or derivatives thereof
for the therapy of pathological states or diseases of the hum^n or animal body associated with a dysfunction of endothelial progenitor
cells, or of sequelae thereof. •
fn connection with the present invention, there will be understood py ""diseases", "pathological states" or "disorders" impairments of vital
processes in organs or in the entire organism, resulting in subjectively experienced or objectively detectable physical, emotional or mental
changes. According to the invention, these diseases are associated in particular with a dysfunction of endothelial progenitor cells, or in
other words diseases that either are the result of such a dysfunction of these cells or are mediated by these cells. Also according to the
present invention, there will be understood by "diseases", "patljolqgical states" or "disorders" impairments of vital processes in organs or
in the entire organism that can be arrested or in particular slowed in their progression by administration of low-dosage erythropoietin or
suitable active ingredients. By "sequelae" there will be understood secondary diseases, or in other words a second disorder occurring in
addition to a primary clinical condition. |
In connection with the present invention, there will be underetckxj by "dysfunction" of endothelial progenitor cells an impairment of essential cell functions such as metabolic activities, response iff stimuli, motility, dividing behavior or differentiation behavior of these cells. A dysfunction of endothelial progenitor ceifs may mean, for e>c&mple, that these cells proliferate not at all or only inadequately. Since the proliferation of endothetial progenitor ceils is stimulated by the use of erythropoietin, the deficient dividing behavior both of endothelial progenitor cells and of already differentiated endothelial cells c^n thereby be compensated and the number of endothelial progenitor cells or endothelial cells increased. Dysfunction of endothelial projjennor cells may consist, for example, of impaired ability of these ceils to differentiate to endotheiial cells. A dysfunction of endothelial progenitor cells may also be caused by their impaired adhesion ability and/or
8

their impaired ability to migrate toward an angiogenic or vasculdjerjic stimulus. Such dysfunctions of endothelial progenitor cells may lead,
for example, to impairment or prevention of the formation of new endothelial tissue and/or of vasculogenesis. A dysfunction of endothelial
progenitor cells may also have a pathogenic cause, for example due to hypertension, hyperlipoproteinemia, elevated ADMA
blood levels, uremia or diabetes. The dysfunction of endothelial progenitor cells may be manifested, for example, by reduced production of
NO (=EDRF) by NO synthases (NOS) from L-arginine, increased endothelin production and/or increased production of adhesion
molecules such as ICAM-1 and VCAIvM. \.
According to the invention, the diseases associated witti a dysfunction of endothelial progenitor cells are in particular
hypercholesterolemia, diabetes meltitus, insulin resistance, enWielium-mediated chronic inflammatory disorders such as vascular
inflammations, endotheliosis including retfculoendotheliosisjj atherosclerosis, age-related ardiovascular disease, ischemic
disorders of the extremities, Raynaud's disease, preedamnsia pregnancy- induced hypertension, chronic or acute renal failure,
especially terminal renal failure, renal function restrictions with glomerular filtration rates of 30 to 80 ml/min, preferably 40 to 80 ml/min,
microaJbuminuria, proteinuria, elevated ADMA levels, wound healing and sequelae thereof.
"Hypercholesterolemia" is characterized by elevated concentrations of cholesterol in the blood. By far the most frequent form of primary
hypercholesterolemia is polygenic hypercholesterolemia. Sejjomiary hypercholesterolemia frequently occurs in diabetes mellitus,
nephrotic syndrome, hypothyroidism and hepatic disorders. i j
"Diabetes mellitus" encompasses various forms of glucose metabolism impairments having different etiologies and symptoms. In
particular, the AGE-RAGE system is responsible for the deveppment of diabetic complications related to vascular systems. AGEs
(advanced glycation end products) are formed by a series of complex reactions following prolonged 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 mellits and Alzheimer's disease. Binding of AGEs leads! \o oxidative stress, activation of the NF-K-B transcription factor and thus
an impairment of endothelial homeostasis. j
By "insulin resistance" there will be understood impaired signal jtensmission in various body cells, which ignore the physiological signal
cascade of insulation. Affected patients therefore lack normal glucose metabolism.
"Endothelium-mediated chronic inflammatory disorders" are disorders or conditions of a human or animal body that are caused by a
defense response of the organism and its tissues to harmful stimuli, wherein certain signal molecules alter the properties of endothelial
cells, with the result that, in interaction with the activation of oner cell types, leukocytes remain adhering to endothelial cells, finally
penetrating into the tissue and causing inflammation therein. One example of endothelium-mediated inflammation is leukocytic vasculitis.
A central role in activation of an endothelium-mediated inflammatory event is played by the NF-KB transcription factor. Another system
leading to the development of endotheliaf cell-mediated chronic inflammations is the AGE- RAGE system.
By "endotheliosis" there will be understood degenerative and proliferative endothelial changes during non-thrombopenic purpura. By
"reticuloendotheliosis" there will be understood diseases of the reticulohistiocytrc system, such as reticulum, reticulosis,
reticulohistiocytosis and Hand-SchuHer-Chfistian disease. i
By "Raynaud's disease" there will be understood episodically occuning ischemic states caused by vasoconstriction, or in other words
vascular spasms, usually in the arteries of the fingers. Primary Raynaud's disease is a purely functional impairment of the small vessels
supplying the distal parts of the extremities, whereas secondary Raynaud's disease accompanies another disease such as vascular
inflammation, h
"Preeclampsia" is an endothelial and vascular disease of the aternal organism, apparently caused by endotheliotropic substances from
the placenta. Preeclampsia is a multisystem disorder that may lead to functional impairments of numerous organs and be manifested by
diverse symptoms. The circulatory impairments typical of the disorder result from increased vascular resistance, which can vary locally in
severity. For preeclampsia it has been confirmed that an endothelial dysfunction is the central component of the pathogenesis.
In connection with the present invention, by "renal failure" thera will be understood the restricted ability of the kidneys to excrete
substances normally contained in the urine. In advanced stages, thsi ability to regulate the electrolyte, water and acid-base balance is also
lost, Terminal renal failure is characterized by collapse of the excrelpry and endocrine function of the kidneys.
According to the invention, renal failure may be acute renal failure, which is also referred to as acute renal insufficiency, shock kidney or
shock aneuria. Acute renaf failure is characterized by sudden partial or total toss of the excretory function of the kidneys as a result of
kidney damage that is usually reversible. The causes may be hypoperfusion due to hypovolemia, hypotension and dehydration resulting
from blood losses (polytrauma, gastrointestinal or postpartum bleeding, major surgical procedures on the heart, vessels, abdomen or
prostate), shock (myocardial infarction, embolism), serious infections (sepsis, peritonitis, cholecystitis), hemolysis (hemolytic-uremic
syndrome.paroxysmal hemoglobinuria, transfusion reaction), myolysis (crush syndrome, rhabdomyolysis, myositis, bums), water and
electrolyte losses (massive vomiting, diarrhea, excessive sweating, fleus, acute pancreatitis). Further
causes may be nephrotoxins such as exogenous toxins, for example aniline, gtycol compounds, methanol and the like, or endogenous
toxins, for example myoglobin and oxalates, Further causes of |jacute renal failure are renal disorders, for example inflammatory
nephropatrn'es or rejection reactions following kidney transplantation. Acute renal failure may also be caused by urinary retention
following obstruction of the urine flow. The inventive treatment of acuje renal failure with erythropoietin, preferably in low doses, leads
according to he invention to prevention or at (east diminution of the pnjgression of acute renal failure, According to the invention, renal
failure may also be chronic renat failure. Causes of chronic renal failure are vascular, glomerular and tubulointerstitial kidney disorders,
infections and congenital or acquired structural defects. Causes of chronic renal failure include chronic glomerulop athy, chronic
pyelonephritis, analgesic nephropathy, obstructive uropathy, arteriosclerosis and arteriolosclerosis. The terminal stage of chronic renal
failure is uremia. The inventive treatment of chronic renal failure With low-dosage erythropoietin leads according to the invention to
diminution of the progression of chronic renal failure.
In particular, the invention therefore relates to the use of EPO, preferjably in low doses, for production of a drug for prevention, diminution
or slowing of the damage to kidney tissue and/or for regeneration of imaged kidney tissue in cases of acute or chronic renal failure.
According to the invention, there will be understood by renal funcflon restriction conditions in which the glomerular filtration rate has
already slowed to less than 80 ml/min. Renal function restriction thetefdre relates to the early phase of glomerular, tubuointerstjtJal and
vascular kidney disorders. The inventive treatment of renal function restrictions with low-dosage erythropoietin leads according to the
invention to diminution of the progression or to regeneration of the beginning kidney tissue and/or function damage.

In connection with the present invention, there will be understood byHmitroalbuminuria" a clinical picture in which affected patients exhibit unphysiological excretion of albumin in the urine in excess of 30 mg per 24 hours. This increased albumin excretion is an early sign of the beginning of renal function deterioration, and is a consequence [pf jthe first pathological transformation processes in the kidneys, accompanied by structural alterations of the kidney architecture.
In connection with the present invention, there will be understood) by "proteinuria" a clinical picture in which affected patients exhibit unphysiological excretion of proteins in the urine in excess of 150 rrjjj per 24 hours. This increased protein excretion via the urine (> 150 mg per 24 hours) is considered to be pathological, requiring further medical investigation and therapy.
in connection with the present invention, there will be understood by if high ADMA levels" a clinical picture in which affected patients exhibit an unphysiologically high ADMA blood concentration En excess of 1.3 pmoi/!. This elevated ADMA concentration is associated with an endothelial dysfunction and is a consequence of metabolic dysfunctions in the processes of degradation and excretion of this molecule.
In connection with the present invention, there will be understood! by "wound healing" the physiological processes for regeneration of destroyed tissue and for closing a wound, especially formation of r»sw connective tissue and capillaries. Wound healing may be primary wound healing (first intention healing), which in the case of a dear wound is characterized by rapid and complication-free closure and largely complete recovery, resulting from minimal formation of new pwective tissue between the wound edges, which have a good blood supply and have been approximated if necessary. In the case of wounds with wound edges that are further apart, especially crushed or necrotic wound edges, and of wound infections, delayed secondarVjwound healing (second intention healing) takes place. In such cases the tissue defect becomes filled with granulation tissue as a result of (a)bacterial inflammation, and scar tissue is formed more extensively. Epithelialization starting from the edge represents the completion of wound healing. Such wound and it takes place on the fourth to seventh day after the wound occurred. The repair phase begins jjn (he eighth day after the wound occurred, and is characterized by transformation of the granulation tissue into a scar.
In connection with the present invention, there will be understood by a "wound" a break in the continuity of body tissues with or without loss of substance, caused by mechanical injury or physically related cell damage. Within the meaning of the present invention, a wound is also considered to be a disease. Types of wound are mechanical wounds, thermal wounds, chemical wounds, radiation- related wounds and disease-related wounds- Mechanical wounds are caused by external violence and occur in particular as cut and stab wounds, crushing, lacerating, tearing and abrading wounds, scratch and bite wounds and projectile wounds. Thermal wounds are caused by exposure to heat or cold. Chemical wounds are caused in particular by burning with acids or alkalis. Radiation-related wounds are caused, for example* by exposure to actinic and ionizing radiation. Wounjfc occurring in relation to disease are in particular congestion-related wounds, traumatic wounds, diabetic wounds etc According to the invention, it is provided in particular that low-dosage erythropoietin will be administered fo,r wound healing, preferably topically or intravenously.
The present invention relates to the use of low-dosage erythropoietin for the therapy of hypercholesterolemia, diabetes mellitus, insulin resistance, endothelium-mediated chronic inflammatory disorders, endothetiosis including reticuloendotheliosis, atherosclerosis, age-related cardiovascular disorders, ischemic disorders of the extremities, preeclampsia, Raynaud's disease, hepatic disorders such as hepatitis, cirrhosis of the liver, acute or chronic liver failure, bone and cartilage disorders or lesions, mucous membrane disorders or lesions, especially in the gastrointestinal tract. Crohn's disease, ulcerative colitis, pregnancy-induced hypertension, chronic or acute renal failure, especially terminal renal faifure, renal function restrictions vflth glomerular filtration rates of "Inventive dosing of EPO"
According to the invention, it is preferred for ail uses, methods aiid Compositions of the present teaching that erythropoietin be used in small quantities, smaller than the quantities known to be used for the treatment of renal anemia. Within the meaning of the present teaching, there will be understood by a small or low dose or dosage, especially in vivo, or in other words per patient. EPO doses of 1 to 2000, preferably 20 to 2000 units (IU; international units)/week, preferably doses of 20 to 1500 lU/week, especially doses of 20 to 1000 lU/week, especially doses of 20 to 950 iU/week, especially doses of 20 to 900 Ill/week, especially doses of 20 to 850 lU/week, especially doses of 20 to 800 lU/week, especially doses of 20 to 750 lU/we^k, especially doses of 20 to 700 lU/week, especially doses of 20 to 650 lU/week, especially doses of 20 to 600 lU/week, especially dosesj iof 20 to 550 IU/week, especially doses of 20 to 500 IU/week, especially doses of 20 to 450 tU/week, especially doses of 20 to 400 tU/weSk. Especially doses of 20 to 350 lU/week, especially doses of 20 to 300 (U/week, especially doses of 20 to 250 lU/week, especially dosesj of 20 to 200 lU/week, especially doses of 20 to 150 lU/week, according to the severity of the disorder and depending on renal function) According to the invention, it is also provided that doses of 1 to 450, preferably 1 to 9 lU/week will be used. All of the foregoing dos^s provided according to the invention, for example of 1 to 2000 units (IU)/week per patient, especially, for example, of 500 to 2000 lUWek per patient, are subpolycythemic doses, or in other words doses that do not lead to an increase of the hematocrit, and in particular] do not lead to an increase of more than 10%, especialty 5%, preferably 2% in the hematocrit compared with the hematocrit prior to the tr ^0

weight. Aranesp is a doubly PEGylated EPO. According to thei invention, it is particularly preferred for all uses, methods and compositions of the present teaching that erythropoietin be used in small qualities, smaller than the quantities known to be used for the treatment of renal anemia. Within the meaning of the present teaching, the^e will be understood by a small or low dose or dosage, especially in vivo, or in other words per patient, EPO doses of 0.001 to 90, preferably 0.001 to 50 units (1U; international units) per kilogram of body weight per week, especially doses of 0.05 to 45 lU/kg/week, espe&iafly doses of 0.05 to 40 lU/kg/week, especially doses of 0.05 to 35 lU/kg/week, especially doses of 0.05 to 33 lU/kg/week, especially doses of 0.05 to 31 lU/kg/week, especially doses of 0.05 to 29 lU/kg/week, especially doses of 0.05 to 27 ID/kg/week, especially doses of 0-05 to 25 lU/kg/week, especially doses of 0.05 to 23 lU/kg/week, especially doses of 0.05 to 21 lU/kg/week, especially doses of 0-05 to 20 lU/kg/week, especially doses of 0.05 to 19 fU/kg/week, especially doses of 0.05 to 17 lU/kgrweek, especially doses of 0.05 to 15
lU/kg/week, especially doses of 0.05 to 13 lU/kg/week, especially doses of 0.05 to 11 lU/kg/week, especially doses of 0.05 to 9 lU/kg/week, especially doses of 0-05 to 7 iU/kg/week, especially|doses of 0.05 to 5 lU/kg/week, especially doses of 0.05 to 3 lU/kg/week, especially doses of 0.05 to 1 lU/kg/week, according to the severity of the disorder and depending on renal function. According to the invention, it Is also provided that doses of 0.001 to 20, preferabw 0J05 to 10 iU/kg/week will be used. All of the foregoing doses provided according to the invention, for example of 0.01 to 90 units (IU)/kg/week per patient, especially, for example, of 0.01 to 50 lU/kg/week per patient, are subpolycythemic doses, or in other words doses that dp not lead to an increase of the hematocrit, and in particular do not lead to an increase of more than 10%, especially 5%, preferably 2% in the hematocrit compared with the hematocrit prior to the treatment with EPO. The subpolycythemic doses provided according to thejjinvention correspond to weekly doses of about 0.001 to 90 units (IU) of EPO/kg of body weight, especiaify 0,001 to 50, especially 0.001 Ito 45 IU of EPO/kg of body weight, especially 1 to -15 1U of EPO/kg of body weight, especially 1 to 10 IU of EPO/kg of body weight, especially 1 to 4 IU of EPO/kg of body weight, or a comparable weekly dose of Aranesp of 0.000005 to 0-45 ug per kilogram of body weight, 0.00025 to 0.250 u,g/kg of body weight, 0.00025 to 0.225 ug/kg of body weight, 0.00025 to 0.2 fig/kg of body weight 0.00025 to 0.175 udjkg of body weight. 0-00025 to 0.165 ug/kg of body weight, 0.00025 To 0.155 ug/kg of body weight, 0.00025 to 0.145 fig/kg of body wept, 0-00025 to 0.135 ug/kg of body weight, 0.00025 to 0.125 ug/kg of body weight, 0,00025 to 0.115 u.g/kg of body weight, 0.00025 to 0.105 ug/kg of body weight, 0.00025 to 0.095 ug/kg of body weight, 0.00025 to 0.085 ug/kg of body weight, 0.00025 to 0.075 "ig/kg o(|botiy weight, 0.00025 to 0.065 ug/kg of body weight, 0.00025 to 0.055 ug/kg of body weight, 0.00025 to 0.045 ug/kg of body weight, 0.010025 to 0.035 p-g/kg of body weight, 0.00025 to 0.025 "g/kg of body weight, 0.00025 to 0.015 pg/kg of body weight, 0.00025 to 0.005 tig/kg of body weight. Aranesp is a doubly PEGylated EPO. Compared with the initial dose of 90 to 150 lU/kg of body weight per week (beginning with 4000 to 8000 lU/week as a rule, but even much higher if the result of therapy is not satisfactory) usually used for therapy ol renal anemia, the small doses cited above - for example the dose of 0.001 to 90 units/kg/week per patient, and especially, for example, of 0.001 to 50 units/kg/week per patient, as provided according to the invention for the treatment of diseases or pathological states associated with dysfunction of endothelial progenitor cells - are extremely low.
Unless otherwise specified, the cited dosages are one-time doses to be administered weekly, although they can also be divided into several individual doses in a week, or in other words administered by multiple dosing.
A particularly preferred embodiment of the invention relates to the use of low-dosage erythropoietin and/or its derivatives as defined in the foregoing section entitled "Inventive dosing of EPO" as active ingreqierjt for production of a pharmaceutical composition or as a drug for the therapy of pathological conditions or diseases associated with a dysfunction of endothelial progenitor cells. According to the invention, there will be understood by "active ingredient" an endogenous or exogenous substance that, on contact with target molecules or target cells or target tissues, influences specific functions of tissues, organs or organisms in differentiated manner. According to the invention, therefore, if is provided that erythropoietin, as active ingredient of the inventive pharmaceutical composition, upon contact with endothelial progenitor cells, will influence the proliferation, differentiation and/or migration behavior thereof in a human or animal organism in such a way that dysfunctions of endothetial progenitor cells can be compensated and the diseases occurring as a. consequence of these dysfunctions can be effectively controlled, alleviated or eliminated, or these diseases can be effectively prevented. It is also provided that the use of fow-dosage erythropoietin will lead both to organ regeneration and to slowing of the progression of functional restrictions in different organs and organ {Systems.
tn connection with the present invention, there will be understood by "pharmaceutical composition" or "drug" a mixture used for diagnostic,
therapeutic and/or preventive purposes, or in other words a mixture th^t promotes or restores the health of a human or animal body, which
mixture includes at least one natural or synthetically produced Wye ingredient that brings about the therapeutic effect. The
pharmaceutical composition may be either a soiid or a liquid mixture. For example, a pharmaceutical composition that includes the active
ingredient may contain one or more pharmaceutJcally tolerable components. The pharmaceutical composition may additionally include
additives normally used in the art, for example stabilizers, finishing agents, release agents, disintegrants, emulsifiers or other substances
normally used for production of pharmaceutical compositions- !
According to the invention, there is provided In particular the use of erythropoietin, preferably in low doses, and/or a derivative thereof as
active ingredient for producing a drug for the therapy of hypercholestorotemia, diabetes mellitus, insulin resistance, endothelium-
mediated chronic inflammatory disorders such as vascular inflammafbns, endotheliosis including reticuloendotheliosis, atherosclerosis,
age-related cardiovascular disease, ischemic disorders of the extremities, Raynaud's disease, hepatic disorders such as hepatitis,
cirrhosis of the liver, acute or chronic liver failure, bone and cartilage d sorters or lesions, mucous membrane disorders or lesions,
especially in he gastrointestinal tract, Crohn's disease, ulcerative coliti^preedampsia, pregnancy-induced hypertension, chronic or acute
renal failure, especially terminal renal failure, renaf function restrictions with glomerular filtration rates of preferably 40 to 80 ml/min, microalbuminuria, proteinuria, elevated ADMA levels or wounds and sequelae thereof.
The inventive pharmaceutical composition may be suitable both for topical and for systemic administration.
In a preferred embodiment of the invention, it is provided that the pharmaceutical composition will DO used for parenteral, especially
intravenous, intramuscular, intracutaneous or subcutaneous administration. Preferably the erythropoietin- containing drug has the form of
an injection or infusion. jj
In a further use, it is provided that the erythropoietin- containing pharmaceutical composition will be administered orally. For example, the
erythropoietin-containing drug is administered in a liquid presentation sucji as a solution, suspension or emulsion, or a solid presentation
such as a tablet. I
11

In a further use, it is provided that the pharmaceutical composition wi | bd suitable for pulmonary administration or for inhalation. According to the invention, therefore, it is provided that erythropoietin will be administered in therapeutically effective manner directly to the lungs of the patient. This form of administration of erythropoietin permits rapt) delivery of an erythropoietin dose to a patient without the need to perform an injection. By absorption of erythropoietin through the lun is, considerable quantities of erythropoietin can be delivered via the lungs to the bloodstream, leading to elevated erythropoietin concentrations in the bloodstream. In a preferred embodiment of the invention, the pharmaceutical composition to be absorbed through the lungs is an aqueous or nonaqueous solution or a dry powder. When the erythropoietin-containing drug to be administered by the! pulmonary route is in the form of a dry powder, the said powder preferably comprises erythropoietin-containing particles, wherein the particles have a diameter of smaller than 10 urn, thus enabling the drug to reach even distal regions of the patient's lungs- In a particularly preferred embodiment of the invention, it is provided that the drug to be administered by the pulmonary route will be in the form of an aerosol.
A particularly preferred embodiment of the invention relates to the use of erythropoietin for production of a pharmaceutical composition for therapy of diseases associated with a dysfunction of endothelial progenitor cells, wherein the pharmaceutical composition contains not only erythropoietin as active ingredient but also at least one further additional active ingredient for stimulation of endotheliaf progenitor cells.
The further active ingredient is preferably an active ingredient that in particular stimulates the physiological mobilization of endothelial progenitor cells from bone marrow or "other stem cell" niches. Accoijlinj) to the invention, however, the further active ingredient may also be an active ingredient that in particular stimulates the dividing behayiorfor m other words the proliferation, of endothelial progenitor cells. According to the invention, however, the possibility also exists that the further active ingredient wilt stimulate in particular the differentiation behavior and/or the migration behavior of endothelial progenitor cells. Particularly preferably, the further active ingredient that stimulates endothelial progenitor ceils is VEGP, PIGF, GM-CSF, an HMG-CoA reductase inhibitor, especially a statin such as simvastatin, mevastatin or atorvastatin, an ACE inhibitor such as enalapnl, ramibril or trandolapril, an AT-1 blocker such as irbesartan, lorsartan or olmesaratan, and/or an 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 endotheHal cells, or in other words the proliferation and/or migration thereof, but not endothelial progenitor cells. Particularly preferably, it will be bFGF (basic fibroblast growth factor) or angiogenic A further embodiment of the invention relates to the use of erythropc ietin and/or derivatives thereof as active ingredient for production of a pharmaceutical composition for stimulation of endothelial progenitor cells, especially for stimulation of mobilization, proliferation, differentiation to ©ndothelial cells and/or migration toward a vasculogenic or angiogenic stimulus. According to the invention, it is further provided that erythropofetin and/or its derivatives will be used as active ingredient for production of a pharmaceutical composition for stimulation of vasculogenesis and/or endotnelium formation, especially in the adult human or animal organism. The present invention therefore also relates to pharmaceutical compositions for stimulation of endothelial progenitor cells, especially for stimulation of mobilization, proliferation, differentiation thereof to endothelfal cells and/or migration toward a vasculogenic or angiogenic stimulus, for stimulation of vasculogenesis and/or endotnelium formation and for treatm ent of diseases of the human or animal body that are associated with a dysfunction of endothelial progenitor cells and/or endothelial cells. In particular, the present invention relates to pharmaceutical compositions or drugs that contain erythropoietin as active ingredient and at feast one further active ingredient for stimulation of endothelial progenitor cells and/or differentiated endothelial cells. In a preferred embodiment, the present invention relates to pharmaceutical compositions containing erythropoietin land at least one further active ingredient selected from the group comprising VEGF, PIGF, GM-CSF, an HMG-CoA reductase inhibitor, especially a statin such as simvastatin, mevastatin or atorvastatin, an ACE inhibitor such as enalapnl, ramipril or trandolapril, an AT-1 blocker such as irbesartan, lorsartan or olmesarqtan, an NO donor, especially L-arginine, bFGF and angiogenic A further preferred embodiment of the invention relates to the use of erythropoietin for production of a transplantable endothelial cell preparation. According to the invention, it is provided in particular in this embodiment that endothelial cells will be produced in vitro by cultivation of endothelial progenitor ceils in the presence of erythropoietin and will then be transplanted into a recipient organism, especially an organism suffering prom a disease associated with a
dysfunction of endotheii ai progenitor cells. For example, mononuclear celts (MNC) can be isolated from blood by density gradient centrifugation and cultivated in suitable culture media in vitro. Methods for isolation and in vitro cultivation of mononuclear cells are described, for example, in Asahara, Science, 275 (1997), 964-967; laimriieier et al, J. Clin. Invest, 108 (?001), 391-397 and Llevadot et af., J. Clin. Invest, 108 (2001) 399405- The mononuclear cells are flien further cultivated in the presence of erythropoietin, in order to stimulate the proliferation and differentiation
behavior of the endothelial progenitor cells contained in the MNCs, and especially to increase the number of differentiated adherent endothelial cells. According to the invention, it is also provided tha| tiwj MNCs will be cultivated in the presence of erythropoietin and at least one further substance that stimulates the proliferation and differentiation of endothelial progenitor cells. Particularly preferably, there is used as the further substance VEGF, PIQF, GM-CSF, an NO doflior such as L-arginine, an ACE inhibitor such as enalapril, ramipril or trandolapril, an AT-1 blocker such as irbesartan, lorsartan or olmesaratan, or an HMG-CoA reductase inhibitor such as a statin, in particular simvastatin, mevastatin oratorvastatin.
In a further preferred embodiment of the invention, endothelial progenitor cells are applied to corresponding patients simultaneously with other cell populations usable for cell therapy, such as hepatoses, myocytes, cardiomyocytes or island cells, after prior incubation with tow-dosage erythropoietin in vrtro and/or local as well as systemic application of low-dosage erythropoietin in vivo, in order in this way to ensure that the tissue cells usable for cell therapy settle with sufficient binding to the vascular system.
A further preferred embodiment of the invention also relates to the use of erythropoietin for production of a pharmaceutical composition or
of a kit for sequential, timed successive or simultaneous administration of low-dosage erythropoietin as well as one or more other
chemical, |j
prior to insertion into a body, especially a human body. By such coaling of the vascular prostheses or heart valves with erythropoietin, it is ensured that endothelial progenitor celis in the body of the recipient Wanism will be stimulated. In particular, their mobilization from bone marrow, their proliferation, their differentiation to endothelial cells aridIheir migration to the inserted vascular prostheses or heart valves will be stimulated. After the vascular prosthesis or heart valves prcjjuckl in this way have been introduced into a body, such a body can be treated further with erythropoietin, especially in the inventive IUS. Thereby endothelial layers form more rapidly on the inserted vascular prostheses, and growth into the relevant area of the body takes place more rapidly. In a preferred embodiment, it is provided that

isolated endothelial progenitor cells, which have been expanded^ in I vitro if necessary, are additionally used for coating the vascular prostheses and heart valves.
The present invention also relates to a method for stimulation pf Endothelial cell formation in vitro, comprising a) isolation of cell populations containing endothelial progenitor cells from blood by m|ans of density gradient centritugation, b) cultivation of the isolated cell populations comprising endothelial progenitor cells in eel) culture iridium, and c) cultivation of the cell populations in the presence of low-dosage erythropoietin.
According to the invention, cultivation of the cell populations cah take place in the presence of a further substance that stimulates endothelial progenitor cells.
The present invention further relates to a method for treatment of diseases associated with a dysfunction of endothelial progenitor cells, wherein erythropoietin, in a small dose such as explained in the seiion entitled "Inventive dosing of EPCA alone or in combination with at least one other chemical, thermal, mechanical and biological agent, is administered to a patient with such a disease. The inventive method is suitable in particular for treating diseases of the human body such as hypercholesterolemia, diabetes mellitus. insulin resistance, endothelium-mediated chronic inflammatory disorders such as | vascular inflammations, endotheliosis including reticuloendothetiosis, atherosclerosis, age-related cardiovascular disorder, ischemic disorders of the extremities, Raynaud's disease, hepatic disorders such as hepatitis, cirrhosis of the liver, acute or chronic iiver failure, bone and cartilage disorders or lesions, mucous membrane disorders or lesions, especially in the gastrointestinal tract, Crohn's disease, ulcerative colitis, preeclampsia, pregnancy-induced hypertension, acute or chronic renal failure, especially terminal renal failure, renal function restrictions with glomerular filtration rates of In a further preferred embodiment of the inventive method for reaiment of diseases associated with a dysfunction of endothelial
progenitor cells, it is provided that endothelial progenitor cells will be isolated from the blood of a human organism, expanded in vitro using
low-dosage erythropoietin and differentiated to endothelial ceils, after which the differentiated endothelial cells or the endothelial
progenitor cells undergoing differentiation will be purified and isolated, then transplanted selectively 'nto a patients body region, tissue or
organ that has been damaged because of the dysfunction of endothelial progenitor cells and/or endothelial cells, in order to induce local
formation of new endothelium therein- In this way the damaged body regions, tissues and/or organs of the patient can be treated more
selectively and rapidly. This embodiment of the inventive method foil treatment of diseases associated with a dysfunction of endothelial
progenitor ceils comprises the following steps: a) isolation of ceil populations containing endothelial progenitor cells from blood by means
of density gradient centritugation, b) cultivation of the cell populations containing endothelial progenitor celts in cell culture medium, c)
cultivation of the cell populations containing endothelial progenitor cells in the presence of low-dosage erythropoietin in order to stimulate
proliferation of endothelial progenitor cells and/or differentiation thereof to endothelial cells, d) isolation and purification of the differentiated
endothelial cells, and e) transplantation of the differentiated endothelial cells into a body with a disease associated with a dysfunction of
endotheliat progenitor cells. j
After transplantation of the differentiated endothelial cells into a body, such a body can be treated further with erythropoietin, especially in the low doses provided according to the invention, or in other wordsilhe doses defined in the section entitled "Inventive dosing of EPO", for example of 1 and preferably 0.001 to 90 lU/kg/week or of 20 to 2000 lU/week.
According to the invention, the cell populations containing endothelial progenitor cells can be cultivated in vitro in the presence of at least one further active ingredient selected from the group comprising VEGF, PIGF, GM-CSF, an HMG- CoA reductase inhibitor, an ACE inhibitor, an AT-1 blocker and an NO donor. Preferably the HMG-CoA reductase inhibitor used for cultivation will be a statin such as simvastatin, mevastatin or atorvastatin, the ACE inhibitors will be substances such as enalapril, ramipril or trandolapril, and the AT-1 blocKer wif) be substances such as irbesartan, lorsartan or olmesaratan.
According to the invention, cell populations containing endotheliat progenitor cells can be treated with sequential, timed successive or
simultaneous administration of low- dosage erythropoietin as well as one or more other chemical, thermal, mechanical or biological
agents, in order in this way to increase the number and function of endothelial progenitor cells and/or to bring about regeneration or
slowing of the progression of tissue damage, A further preferred embojliment of the invention relates to a method for treatment of vascular
disorders, comprising: a) isolation of cell populations containing endothelial progenitor cells from blood by means of density gradient
centrifugation, b) cultivation of the ceil populations containing endothelial progenitor celhs in cell culture medium,
c) cultivation of the cell populations containing endothelial progenitbr cells \v\ Ahe presence o1 erythropoietin in order to stimulate
proliferation of endothelial progenitor cells and/or differentiation thereof to endothelial cells, d) isolation and purification of the
differentiated endothelial cells, and e) transplantation of the endothelial cells Into a body with a vascular disorder, After transplantation of
the endothelial cells into the body with a vascular disorder, such a body can be further treated with erythropoietin, especially in the tow
doses according to the invention, or in other words the doses defined in the section entitled "Inventive dosing of EPO", for example of
0.001 to 90 units/kg/week or of 20 lU/week to 2000 lU/week. j
According to the invention, it is possible to cultivate the cell populations containing endothefial progenitor cells in the presence of at least one further active ingredient selected from the group comprising VEGF PIGF, GM-CSF, an ACE inhibitor, an A"M blocker and/or an HMG-CoA reductase inhibitor. Preferably the ACE inhibitor used for olivaition will be substances such as enalapril, ramipril ortrandoiapril, and the AT-1 blocker used for cultivation will be substances such as rbesartan, lorsartan or otmesaratan, and the HMG-CoA reductase inhibitor used for cultivation will be a statin such as simvastatin, mevastatin or atorvastatin. According to the invention, cell populations containing endothelial progenitor cells can be treated with sequentia timed successive or simultaneous administration of low- dosage ©rythropoietin as well as one or more other chemical, thermal, mechan pal or biological agents, in order in this way to increase the number and function of endothelial progenitor cetis and/or to bring about regeneration or slowing of the progression of tissue damage. Such mechanical agents can be endoprostheses, preferably implantatiojji supports for teeth, bones or ligament/tendon replacements.
13

Furthermore, the biological agents can be solid organs such as liverjkidheys, heart, pancreas or skin, or even hair implants. The invention therefore provides that EPO, especially in low doses, will be used so that mechanical agents such as endoprostheses or biological agents implanted simultaneously, subsequently or beforehand can grow or be integrated better, faster and more efficiently into the surrounding body structure. The invention therefore also relates to the use of erythropoietin for production of a pharmaceutical composition or of a kit for improving, especially for promoting and/oif accelerating, integration of biological agents or endoprostheses into surrounding body structures, especially of teeth, tooth replacements tooth implants or other endoprostheses, such as bone replacements, bone implants, especially hip joint prostheses or ligament/tendon replacements, such as cruciate ligaments. In a preferred embodiment, it can then be provided that the erythropoietin will be used together Jith cell populations suitable for cell therapy and/or endothelial progenitor cells. In the aforesaid use of erythropoietin for production of a pharmaceutical composition or of a kit for improving, especially for promoting and/or accelerating, integration of biological or mecnan cal agents into target structures, especially target tissue, target bones or target cartilage of a patient, it can be provided in a further preferred embodiment 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 osteoblasts, cells having osteogenic potential, thrombocytes, blood cells or similar agents can be used in the present application as cell populations suitable for cell therapy. In a further preferred embodiment, it can be provided that the mechanical agent in particular to be used will be contained in the pharmaceutical composition or in the pharmaceutical kit together with organic adhesive, such as a fibrin glue. The inventive method for treatment of vascular disorders thus provides that endothelial progenitor cells wilt be isolated from the blood of a human organism, expanded in vitro using low-dosage erythropoietin and differentiated to endothefial cells, after which the differentiated endothelial cells or the endothelial progenitor ceils undergoing differentiation will be purified and isolated, then transplanted selectively into a damaged blood vessel or an ischemic region, in order to induce local neovascularization therein. In this way damaged blood vessels or ischemic tissues can be treated more selectively and rapidly. The inventive method for treatment of vascular disorders is suitable in particular for treatment of vascular disorders such as ischemia, especially cerebral ischemia, ischemic disorders of the extremities, stroke, acute arterial occlusion, arterial occlusive disease, Raynaud's disease and ergotism, Further advantageous embodiments of the invention are specified in the dependent claims. The invention will be explained in more detail on the basis of the figures and examples hereinafter. Fig. 1 shows the results of a FACS analysis of circulating CD34+ stem cells (cSC). (A-D): patients' samples; (E-F): isotype controls. cSC were identified by means of the additional expression of the CD34 marker (B and F), by means of the characteristic tow to moderate CD45 antigen expression (C and G) and by means of the characteristic ijghlj scattering properties (D and H). The absolute cSC number was calculated per 100,000 monocytes and lymphocytes.
Fig. 2 shows a quantitative assay of circulating stem cells by means ui nuw cytometry. The figure shows the time- dependent effect of 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 are shown by lines.
*: p shown is the course over 9 week$ and all the controls. The absolute values are shown on the one hand as individual values, in addition, box plots are presented (90th/75th/50th/25th and 10th percentiles as well as the mean value). Subjects of matched age and sex in whom EPCs were isolated and characterized analogously (n -11) served as healthy control.
Fig. 5 shows the quantitative assay of cultivated endothelial progMor cells (EPC) in healthy young subjects. The figure shows that treatment with rhEPO (30IU of epoetin beta per kg of body weight m week) increases the relative number of EPCs. EPCs were isolated before the treatment of the subjects with rhEPO as well as weekW at 1, 2, 3, 4, 5, 6 and 7 weeks after treatment of the patients with rhEPO. And were characterized by means of their adhesion ability and the two markers acLDL-Dil and UEA-1 FITC. n A 4, the values correspond to mean values ± standard deviation.
Fig. 6 shows a quantitative assay of cultivated endothelial progenitoi icells (EPC). The representative photographs show that the absolute number of EPCs in uremic patients is significantly reduced comparod with healthy subjects of matched age and sex (top row = in vivo). Patients with restricted renal function therefore exhibit distinct EPC dysfunction compared with control subjects. If endothelial progenitor cells of a healthy subject are cocultivated with serum of uremic patiehtsl the differentiation ability of his or her endothelial progenitor cells is reduced (bottom row = in vitro). Thus restricted renaf function] wiljh uremia derived therefrom leads to dysfunction of endothelial progenitor cells.
Fig. 7 shows a quantitative assay of cultivated endothelial progenitor cells (EPC) in 46 uremic patients with restricted renal function versus 46 subjects of matched age and sex, presented in the form of box frfojs (90th/75th/50th/25th and 10th percentiles as well as the mean value). The number of endothelial progenitor cells in the uremic patients is significantly reduced compared with the healthy subjects. Patients with restricted renal function therefore exhibit distinct EPC disfunction compared with control subjects, Fig. 8 shows the effect of erythropoietin on wound healing. The figun£ snows that, when a standardized skin wound inflicted on mice using a tissue punch was treated with erythropoietin, it already closed completely after seven to eight days. In contrast, when, the wound was treated with physiological salt solution (saline), it did not closej completely until after thirteen to fourteen days. Treatment with erythropoietin or physiological salt solution began 7 days before M skin wound was inflicted. Recombinant human erythropoietin was administered one time per week by s.c. (subcutaneous) injection (0.1 W/kg Aranesp) (n = 5 in each group),
1|4

Fig. 9 shows that erythropoietin reduces the toss of renal function after aWe renal failure (acute renal insufficiency). Sprague Dawley rats
(250 to 300 g) were included in the study. The rats were anesthetized with ketamine (120 mg/kg) and Rompun (10 mg/kg). One of the
experimental groups received 0.1 p-g of Aranesp per kg of body weiht one time on the day before induction of the acute renal failure. For
comparison, there was used a group of experimental animals, each of which was given an s.c.injection of saline at the same time. By
application of an arterial clamp to the right renal arteries, the blood ftow into the kidney was interrupted for 45 minutes. During this period,
a left nephrectomy was performed. A sham operation was performed on a further control group. In this procedure, the abdomen was
opened to expose the left renal artery, but the blood supply was Unt interrupted and the contralateral Right kidney was removed. All
animals were anesthetized for 60 min and killed 24 h after the operation. In the animals treated with saline, the 45-minute ischemia with
subsequent reperfusion of the remaining right kidney led to massive acute loss of renal function. This is reflected by the fact that the
serum creatinine level 24 h after ischemia and reperfusion was 7 timps higher than the level before ischemia and reperfusion (p contrast, the animals treated with the erythropoietin analog Aranesp exhibited only a four-fold increase in the serum creatinine levels one
day after induction of damage by ischemia and reperfusion. No increase in retention levels was found in the animals subjected to left
nephrectomy and a sham operation on the right kidney. The figure shows the creatinine concentration in the serum of EPO-treated
animals (IR+EPO), Nad-treated animals (IR) and sham-operated anjjnaK (sham OP) before ischemia-reperfusion (IR) injury and 24 hours
thereafter. It is evident from the figure that the serum creatinEne concentration 24 hours after ischemia-reperfusion injury is almost halved
in the animals treated with Aranesp compared with the control without Aranesp (NaCI treatment).
Fig. 10 shows the Kaplan-Mayer survival curves of two experimental groups treated either with Aranesp or NaCI after induction uf chrome
renal failure. 8-week old Sprague Dawley rats were included in the study. The rats were anesthetized with ketamine (120 mg/kg) and
Rompun (10 mg/kg). Their right kidney was removed on day 0 anq was immediatefy fixed in formalin for histological examination. The
segmental arteries supplying the upper and lower renal poles of the left kidney were ligated. Thereby renal infarction occurred in the
corresponding kidney areas, and only the middle third of the kidney remained functional. One time per week, the rats received Aranesp
(0.1 M-9*9 A body weight) or NaCi by s.c. injection. The animals treated with the erythropoietin analog Aranesp exhibited a significant
survival advantage compared with the animals treated with saline (pffO-027; log rank test).
For the two experimental groups that were treated either with Aranesp or NaCI and whose Kaplan-Mayer survival curves are illustrated in
Fig. 10, Figs. 11 to 18 show optical microscopic kidney sections 6 weeks after induction of chronic renal failure.
Fig. 11 shows the histological changes in a Sprague-Dawley rat with chronic renal failure after NaCI treatment one time per week for a
period of 6 weeks, beginning immediately after induction of chronic renal failure. The chronic renal failure was caused by removal of the
right kidney and ligation of the segmental arteries supplying the upper and lower renal poles of the left kidney. The figure shows a
medium-sized preglomerular artery with characteristic onionskin-like vessel wall proliferation associated with severe hypertensive
damage, known as Fahr's malignant nephrosclerosis with endarteritis.
Fig. 12 shows the histological changes in a Sprague-Dawley rat with chronic renal failure after NaCI treatment one time per week for a
period of 6 weeks, beginning immediately after induction of chronic renal failure. The chronic renal failure was caused by removal of the
right kidney and ligation of the segmental arteries supplying the upper and tower renal poles of the left kidney. The figure shows florid
focal-segmental glomerulosclerosis, known as proliferative FSGS (right glomerulus). The other glomerulus (left) exhibits ischemia collapse
of the loop convolution. A small vessel with severe endothelial damage is visible in the lower part of the photograph- The observed
histological changes correspond to hypertensive organ damage or changes associated with overload nephropathy following 5/6
nephrectomy.
Fig. 13 shows the histological changes in a Sprague-Dawiey rat with chronic renal failure after NaCI treatment one time per week for a
period of 6 weeks, beginning immediately after induction of chronic renal failure. The chronic renal failure was caused by removal of the
right kidney and figation of the segmental arteries supplying the upper and lower renal poles of the left kidney. The figure shows almost
complete sclerosis or destruction of a glomerulus with compensatory enlargement and pronounced hyalinosis or fibrinoid necrosis of the
associated afferent arterioles.
Fig, 14 shows the histological changes in a Sprague-Dawley rat with chronic renal failure after NaCI treatment one time per week for a
period of 6 weeks, beginning immediately after induction of chronic renal failure. The chronic rena! failure was caused by removal of the
right kidney and ligation of the segmental arteries supplying the upper and lower renal poles of the left kidney. The figure shows a small
preglomerular artery with characteristic onionskin-like vessel wail j proliferation and wall necrosis associated with severe hypertensive
damage, known as malignant nephrosclerosis (see right photographj). A normal (and as yet) undamaged arteriole is visibte on the left.
Fig. 15 shows the histological changes in a Sprague-Dawiey rat with chronic renal failure after Aranesp (EPO) treatment (0.1 mg of
Aranesp per kg) one time per week for a period of 6 weeks, begi^nifjig Immediately after induction of chronic renal failure. The chronic
renal failure was caused by
removal of the right kidney and ligation of the segmental arteries ^jipdlying the upper and lower renal poles of the left kidney. The figure
shows a normal glomerulus with delicate afferent vessel. No pathological signs were observed in the tubutointerstitium.
Fig. 16 shows the histological changes in a Sprague-Dawley rarJMtti chronic rena! failure after Aranesp (EPO) treatment (0.1 mg of
Aranesp per kg) one time per week for a period of 6 weeks, begjnning immediately after induction of chronic renal failure. The chronic
renal failure was caused by
removal of the right kidney and ligation of the segmental arteries supplying the upper and lower renal poles of the left kidney. The figure
shows a normal glomerulus with delicate afferent vessel (630X magnification). No pathological signs were observed in the
tubulointerstitium.
Fig. 17 shows the histological changes in a Sprague-Dawley ratiwijh chronic renal failure after Aranesp (EPO) treatment (0.1 mg of
Aranesp per kg) one time per week tor a period of 6 weeks, beg r^nijig immediately after induction of chronic renal failure. The chronic
renal failure was caused by
removal of the right kidney and ligation of the segmental arteries flying the upper and lower renal poles of the left kidney. The figure
shows a normal glomerulus with delicate afferent vessel. No pathological signs were observed in the tubulointerstilium.
Fig. 18 shows the histological changes in a Sprague-Dawley rat with chronic renal failure after Aranesp (EPO) treatment (0.1 mg of
Aranesp per kg) one time per week for a period of 6 weeks, begjhnijig immediately after induction of chronic renal failure. The chronic
renal failure was caused by
15

removal of the right kidney and ligation of the segmentat arteries supplying the upper and lower renal poles of the left kidney. The figure
shows a normal glomerulus with delicate afferent vessel (63p magnification). No pathological signs were observed in the
tubulointerstitium.
Fig. 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 renal disease in the terminal stage (preterminal renal failure; creatin me clearance intravenously or subcutaneously with erythropoietin in weekly doses; averaging 5000IU of rhEPO (recombinant human erythropoietin) tor
a period of at least 8 weeks. After erythropoietin treatment, the endothelial progenitor cells in the blood of the patients were investigated
over a period of 20 weeks, the endothelial progenitor cells being] analyzed with regard to number and differentiation status by flow
cytometry and a culture test after 0,2,4,6 and 8 weeks.
Circulating peripheral blood stem cells (CPBSC) represent a small population of cells that express both the CD34 antigen and the CD45
antigen. A test based on the ISHAGE guidelines has been developed to determine the number of CPBSC by flow cytometry (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 pPBSC per JJ and the content of CPBSC as a percentage of the
total leukocyte count were determined.
Fig. 1 shows the results of an FACS analysis of circulating CD34+ stJBm bells on the basis of the ISHAGE buidelines.
Fig. 2 shows the number of CD34+ stem cells measured by FACS aijijalyjsis over a period of 8 weeks.
Cell culture test
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficolj density centrifugation from human b cod samples in accordance with
the method described in Asahara, Science, 275 (1997). 964-967. Tne cells were plated out on culture plates with fibronectin and
maintained in EC basal medium. EC basal medium consists of EBM-2 basal medium (Clonetics Co.) an i EGM-2 Quots (hEGF; GA-100
(gentamicin, amphotericin-B) FBS, VEGF, hFGF-B (w/heparin), R3-IGP-1, ascorbic acid, hepain). After 4 days of cultivation,
nonadherent cells were removed by washing the plates. The remaining adherent cells were treated with trypsin and plated out once again.
Thereafter they were cultivated for a further 3 days. Cells with th# endothelial phenotype were identiled by positive staining for two
different endothelial markers on day 7 after isolation. These
are DiMabeled acetylated low density lipoprotein (acLDL-Dit) and Ul$x europaeus aglutinin-1 (UEA-1). Th|e results of this investigation are
presented in Fig. 3. |
The results show that erythropoietin is able to mobilize endothelial proc enitor cells and to increase the itiumber of circulating endothelial
progenitor cells. In the process, functional deficits that occur in certain pathological states such as renal anemia are compensated. These
results are j
presented in Fig. 4, By means of flow cytometry it was found that thelhurnber of circulating CD34+ stem a ills in patients with renal disease
in the terminal stage corresponds to the number of circulating CD34+ stem cells in the blood of healthy subjects. After the erythropoietin
treatment is started, the number of CD34+ stem cells in the bloodstream increases significantly by more than 50%. By using the cell
culture assay, it was determined that, after treatment with erythropoietin, the number of cells that develop an endothelial phenotype
increases dramatically. In one functional cell culture test, the greatly impaired ability of endothelial progen tor cells increased by a factor of
greater than 3.
Example 2
Improved wound heating through systemic use of rhEPO FVB/N mice were anesthetized by inhalation anesthesia with isoflorane. The fur
on the two rear limbs was removed using a depilatory lotion and disinfected with 70% alcohol. A sterile 4 mm disposable biopsy tissue
punch was used to inflict a skin wound on the right flank of each of the mice. The opposite side served a s internal control. Postoperative
antibiotic cover with penicillin G (20,000 units/kg) was administered one time. Throughout the entire period of investigation, subcutaneous
injections of the recombinant human erythropoietin analog Aranesp (0,1 ug/kg of body weight) were applied one time per week throughout
the entire study period. The treatment began seven days before removal of the tissue punch. The results are presented in Fig. 8. They
show that administration of EPO considerably accelerates the woundlpealing process. Fig. 19 shows the 6 ffect of erythropoietin on wound
healing. The figure shows that, when a standardized skin wound inflicted on mice using a tissue punch was treated with low-dosage
erythropoietin (20 ID EPO/kg/week), it already closed completely aftejf seven to eight days. In contrast, when the wound was treated with
physiological salt solution (saline), it did not close completely until after thirteen to fourteen days. Ir the case of treatment of the
experimental animals with high-dosage erythropoietin (200 IU BPO/kg/week), no acceleration of wound healing could be observed
by comparison with the control group. Two of the experimental;animals treated with high-dosage erythropoietin died during the
observation period. The treatment with erythropoietin or physiological salj solution began on the day of the operation, after the skin wound
was inflicted. Recombinant human erythropoietin was administered erne lime per week by s.c. (subcutaneous) injection (20 lU/kg EPO or
200IU/kgEPO)(n = 5ineachgroup). ' I
Example 3
Reduction in the progression of chronic renal failure through erifthilopoietin treatment Ekjht-week-clld Sprague-Dawley rats were
anesthetized with Ketamine (120 mg/kg) and Rompun (10 mg/kg). Thfiir right kidney was removed on day 0 and was immediately fixed in
formalin for histological examination. The segmental arteries supplymg the upper and lower renal poles of the left kidney were ligated.
Thereby renal infarction occurred in the corresponding kidney areas, ghd only the middle third of the kidnej' remained functional. One time
per week, the rats received the erythropoietin analog Aranesp in a dtjse of 0.1 M.g/kg of body weight or h aCI by s.c. injection for control
purposes.
Fig. 10 shows the Kaplan-Mayer survival curves for both experimental grpups. The animals treated with Aitanesp have distinctly improved
survival compared with the control animals treated with saline.
Figs. 15 to 18 show that the renal tissue exhibits no pathological changed after treatment with erythropoietic whereas severe pathological
changes are visible after treatment with NaCI (compare with Figs. 8 to[fl1). Further
histotogical investigations revealed that a distinctly greater vessel density (CD31) can be observed in animals treated with Aranesp than in
animals treated with saline (data not shown). '
16

Example 4
Reduction in the progression of acute renal failure Sprague-Dawley rats with a body weight of 250 to 300 g were used for this investigation. One of the experimental groups received Aranesp in a dose of 0.1 ,ag/kg of body wsight one time on the day before induction of acute renal failure. The rats were anesthetized with ketamine (120 mg/kg of body weight) and Rompun (10 mg/kg). For comparison, there was used a group of experimental animals, eeioh af which was given an s.c. injection of saline at the same time. By application of an arterial clamp to the right renal artery, the blood flow into the kidney was interrupted for 45 minutes. During this period, a left nephrectomy was performed. A sham operation was performed on a further control group. In this procedure, the abdomen was opened to expose the left renal artery, but the blood supply was not interrupted and the contralateral right kkJn|ey was removed. All animals were anesthetized for 60 min and killed 24 h after the operation.
En the animals treated with saline, the 45-minute ischemia with subsequent reperfusion of the remaining right kidney led to massive acute loss of renal function. This is reflected by the fact that the serum creatinine level increased by a factor of 7 (p Reduced differentiation ability of endothelial progenitor cells in patients with restricted renal function The differentiation status of endothelial progenitor cells was analyzed by a culture test in 46 uremic patients as well as 46 healthy o jntrol subjects of matched age and sex. It was surprisingly found that the number of endothelial progenitor cells in this differentiation assa/ is significantly reduced in uremic patients compared with the healthy controls (Fig. 7). If mononuclear cells of a healthy subject are isolate d and cultivated in the presence of serum of a uremic patient, the ability of these cells to differentiate to endothelial progenitor cells is reduqed analogously (Fig. 6). Example 6
Stimulation of the differentiation ability of endothelial progenitor celts in healthy subjects Four healthy ydung males were treated with 30IU of epoetin beta per kilogram of body weight one time per week for a period of 8 weeks. The differentiation ability of their endothelial progenitor cells was determined in a culture assay, based on their adhesion ability and the two markers acLDL and UEA, before treatment of the subjects with rhEPO as well as weekly at 1,2,3,4,5,6 and 7 weeks after treatment of the subje rts with rhEPO. A relative increase of greater than 50% was observed in the EPCs.
17

WE CLAIM:
1. The use of erythropoietin and/or derivatives for production df a pharmaceutical composition corjtaining a dose of 1 to 90 lU/kg of
body weight per week, preferably 1 to 45 lU/kg of body weight per week, for prevention or trsatment of diseases; wherein the
erythropoietin in this dose is suitable and designed for prevention or treatment of a human or anim al patient exhibiting a) at least one
i dysfunction of endothelial progenitor cells, b) at least one cardiovascular risk factor such as hypertension, hyperc-
hoiesterolemia, elevated asymmetric dimethylarginine (ADM|) levels, increased insulin resistance or hyperhomocysteinemia
and c) at least one end-organ damage, namely left I', ventricular hypertrophy, micros ibuminuria, cognitive dysfunction,
increased thickness of the intima media in the carotid artery, proteinuria or a glomemlar filtration ra :e of 30 to 80 ml/min.
2. The use of erythropoietin and/or derivatives for production o|f a pharmaceutical composition con aining a dose of 1 to 90 lU/kg of body weight per week, preferably 1 to 45 lU/kg of body weight per week, wherein the erythropoietin in this dose is suitable and designed for cosmetic treatment of the human or animal body, especially for treatment ol v Tinkles, for strengthening of the connective tissue, tor protection and tightening of the skin, for protection against harmful environmental effects, tor treatment of age spots, tor acceleration of reepithelialization, for acceleration of hair growth and/or as makeup found ation.
3. The use of erythropoietin and/or derivatives for production ol a pharmaceutical composition containing a dose of 1 to 90 lU/kg of body weight per week, preferably 1 to 45 lU/kg of body weight per week, for production of a cosmetic preparation, especially for topical application, wherein the erythropoietin in this dose is suitable and designed for cosmetic t'eatment of the human or animal body, especially for treatment of wrinkles, for strengthening df the connective tissue, for protection and tightening of the skin, for protection against harmful environmental effects, for treatment of age spots, for acceleration of reepithelialization, for acceleration of hair growth and/or as makeup foundation.
4. The use of erythropoietin and/or derivatives for production of a pharmaceutical composition containing a dose of 1 to 90 Ill/kg of body weight per week, preferably 1 to 45 lU/kg of body weight per week, and/or a mixture of endotf eliaf progenitor cells with at least one cell population usable for cell therapy, wherein the erythroiioietin in this dose is suitable and de signed for regeneration of tissues or vessels in a human or animal body, and wherein the mixture has been brought into contact wji'th erythropoietin in vitro prior to application.
5. The use of erythropoietin and/or derivatives for production of a pharmaceutical composition containing a dose of 1 to 90 !U/kg of body weight per week, preferably 1 to 45 lU/kg of body weight per week, and/or a mixture of endoth slial progenitor cells with at least one cell population usable for cell therapy, wherein the erythropoietin in this dose is suitable and des igned for regeneration of tissues or vessels in a human or animal body, and wherein the mixturi [ssc: erythropoietin] is administere i before, after or simultaneously with application of the mixture.
6. The use of erythropoietin and/or derivatives for production of a pharmaceutical composition or of a kit containing a dose of 1 to 90 !U/kg of body weight per week, preferably 1 to 45 lU/kg of body weight per week, and/or at least onj chemical, thermal, mechanical or biological agent, especially a pharmacological active ingredient, for production of a pharmaceutical composition or of a kit containing erythropoietin in this dosage and the at least one chemical, thermal, mechanical or bic logical agent, for prevention or treatment of diseases, wherein the erythropoietin in this dose is suitable and designed for sequential, timed successive or simultaneous application of the erythropoietin with the at least one chemical, thermal, mechanical or I >iological agent.
7. The u&e of erylhropoietin according to claim 6, wherein the mecpanjical agents are endoprostheses, preferably implantation supports for teeth, bones or ligament/tendon replacements.
8. The use of erythropoietin according to claim 6, wherein the biological I agents are solid organs such as! liver, kidneys, heart, pancreas, skin or hair implants.
9. The use of erythropoietin and/or derivatives for production of a pnarmaceutical composition containing a dose of 1 to 90 Ill/kg of body weight per week, preferably 1 to 45 lU/kg of body weigh! per week, wherein the erythropoiein in this dose is suitable and designed for prevention or treatment of diseases, wherein the disease is hepatic disorders such as hepatitis, cirrhosis of the liver, acute or chronic liver failure, bone and cartilage disorders or fusions, mucous membrane disordes or lesions, especially in the
18

gastrointestinal tract, Crohn's disease, ulcerative colitis, irerial function restrictions with glomerular filtration rates of 30 to 80 ml/min, microalbuminuria, proteinuria, or wounds and sequelae thereof.
10. The use of erythropoietin and/or derivatives for production of a pharmaceutical compositor containing a dose of 1 to 90 tU/kg of body weight per week, preferably 1 to 45 lU/kg of body weight per week, for production of a hit containing erythropoietin, endothelial progenitor cells and at least one cell population usable for cell therapy, wherein the erythropoietin is preferably present in low dosage.
11. The use according to one of claims 1 to 10. wherein the pharmaceutical composition is used for stimulation of physiological mobilization of endothelial progenitor cells, proliferation of; endothelial progenitor cells, differen iation of endothelial progenitor cells to endothelial ceils and/or migration of endothelial progenitor cells toward a vasculogenic or an jiogenic stimulus.
12. The use according to claims 1 to 11, wherein the adh^sijjn ability of endotheUa! progenitor cells undergoing differentiation is increased.
13. The use according to claims 1 to 12, wherein the stimulation of endothelial progenitor cells leads to formation of endothelial tissue.
14. The use according to one of claims 1 to 13, wherein the stimulation of endothelial progenitor pells leads to formation of new'blood vessels.
15. The use of erythropoietin in a low dosage of 1 to 90 Ill/kg of body weight per week for the theripy of pathological states or diseases of the human or animal body associated with a dysfunction of endothelial progenitor cells, ard wherein the pathological states or diseases associated with a dysfunction of endothelial progenitor cells are hepatic disorders such as hepatitis, cirrhosis of the liver, acute or chronic liver failure, bone and cartilage disorders or lesions, mucous membrane disorders or lesions, especially in the gastrointestinal tract, Crohn's disease, ulcerative colitis, renal function restrictions with glomerular filtration rates of 30 to 60 ml/min, microalbuminuria, proteinuria, elevated ADMA levels ur wounds and sequelae thereof.
16. The use according to claim 15, wherein the dysfunction of the endothelial progenitor cells consists of their impaired proliferation ability, their impaired ability to differentiate to endothelia) cells, their impaired adhesion ability and/or their impaired ability to migrate toward a vascuiogenic or angiogenic stimulus.
17. The use according to claim 15 or 16, wherein the dysfunction of endothelial progenitor cells impairs or prevents the formation of endothelial tissue and/or blood vessels.
18. The use according to one of claims 15 to 17, wherein the dysfunction of endothelial progenitor cells has a pathogenic cause.
19. The use of erythropoietin in a low dose, especially of 1 to 90 lU/kg of body weight per week, for the therapy of hepatic disorders such as hepatitis, cirrhosis of the liver, acute or chronic liver failure!, bone and cartilage disorders or les ions, mucous membrane disorders or lesions, especially in the gastrointestinal tract, Crohn's disease, ulcerative colitis, renal fuiction restrictions with glomerular filtration rates of 30 to 80 ml/min, microalbuminuria, proteinuria, elevated ADMA levels or wounds md/or sequelae thereof.
20. The use according to one of claims 1 to 19, wherein erythropoietin is administered to each patient in a dose of 1 to 90 units/kg of body weight per week.
21. The use according to claim 20, wherein erythropoietin is admir»istjpred to each patient in a dose of' to 45 units/week.
22. The use according to one of claims 1 to 21, wherein the pharmaceutical composition is suitable for parenteral, especially intravenous, intramuscular, intracutaneous or subcutaneous as well as topical administration.
23. The use according to claim 22, wherein the pharmaceutical composition has the form of an injectiorj or infusion.
24. The use according to one of claims 1 to 21, wherein the pharmaceutical composition is suitable for | mlmonary administration.
25. The use according to claim 24, wherein the pharmaceutical composition has the form of an aqueou s solution, a nonaqueous solution or a powder.
26. The use according to claim 24 or 25, wherein the pharmaceutical domposition has the form of an ae osol preparation.
27. The use according to one of claims 1 to 21, wherein the pharmaceutical composition is suitable for o ral administration.
28. The use according to claim 27, wherein the pharmaceutical coitlposition has the form of a solution, s uspension, emuision or tablet.
29. The use according to one of claims 1 to 28, wherein the pharmaceutical composition contains at least one further active ingredient for stimulation of endothelial progenitor cells.
19

30. The use according to claim 29, wherein the further active ingn$dbnt is VE6F, PIGF, GM-CSF, an ACE inhibitor, an AT-1 blocker, an HMG-CoA reductase inhibitor and/or an NO donor.
31. The use according to claim 30, wherein the HMG-CoA reductase! inhibitor is a statin such as simvastatin, mevastatin or atorvastatin, the ACE inhibitor is an active ingredient such as enaiapril, ramiprjt or trandoiapril and/or the AT-11 locker is an active ingredient such as irbesartan, lorsartan or olmesaratan.
32. The use of erythropoietin tor production of a transplantable endothelial preparation.
33. The use according to claim 32, wherein endothelial celis are produced in vitro by cultivation of [endothelial progenitor cells in the presence of erythropoietin in a small dose, namely of 0.001 to 90 lU/kg/week.
34. The use according to claim 32 or 33, wherein the cultivation of tine endotheiial progenitor cells takiss place in the presence of at least one further active ingredient selected from the group comprising VEQF, PIGF, GM-CSF, an ACE inhibitor such as enataprif, ramipril or trandoiapril, an AT-1 blocker such as irbesartan, lorsartan or olmesaratan, an HMG-CoA reductase inhibitor, especially simvastatin, mevastatin or atorvastatin, and an NO donor, especially L- arginine.
35. The use according to one of claims 1 to 34, wherein erythroppiet n iA human or animal erythropoietin.
36. The use according to claim 35, wherein erythropoietin is a derivative, an analog, a modification or a mutein of erythropoietin.
37. The use according to claim 35 or 36, wherein erythropoietin t> isolated from human urine, from the urine or plasma of patients suffering from aplastic anemia, from tissue cultures of hum^in renal cancer ceils, from human tymphoblast cells, which have the ability to produce human erythropoietin, or from a hybridoma culture obtained by cell fusion of a hi iman or animal cell tine.
38. The use according to claim 35 or 36, wherein erythropoietin is an erythropoietin produced by means of DNA recombination techniques.
39. A pharmaceutical composition for stimulation of endothelial progenitor cells, for stimulation of the formation of endothelial tissue, for stimulation of vasculogenesis and/or for treatment of diseases or pathological slates associated with a dysfunction of endothelial progenitor cells, comprising erythropoietin and/or a derivative, £in analog, a modification or a mu:ein thereof as the active ingredient as well as at least one further active ingredient selected from tho group comprising VEGF, P1GF, GM-CSF, an ACE inhibitor such as enalaprii, ramipril or trandoiapril, an AT-1 blocker such as irbesartan, lorsartan or olmesaratan, aji HMG-CoA reductase inhibitor and an NO donor, preferably in a low dose, especially of 1 to 90 lU/kg of body weight per week.
40. A pharmaceutical composition tor prevention and/or therapy of hepatic disorders such as hepatitis, cirrhosis of the liver, acute or chronic liver failure, bone and cartilage disorders or lesions, ligoment and tendon disorders or le sions, mucous membrane disorders or lesions, especially in the gastrointestinal tract, Crohn's disease, ulcerative coiitis, renal function restrictions with glomerular filtration rates of 30 to 80 ml/min, microalbuminuria, proteinuria elevated ADMA levels or wouncs and sequelae thereof, comprising erythropoietin and/or a derivative, an analog, a modification or a mutein thereof as the active ingredient, preferably in a small dose, especially of 1 to 90 lU/kg of body weight per week.
41. A pharmaceutical composition according to claim 40, additionally including at least one further active ingredient selected from the group comprising VEGF, PIGF, GM-CSF, an ACE inhibitor such as enalaprii, ramipril or tra idolapril, an AT-1 blocker such as irbesartan, lorsartan or olmesaratan, an HMQ-CoA reductase inhibitor and an NO donor.
42. A pharmaceutical composition according to claim 39 or 40, wherein the HMG-CoA reductase inh ibitor is a statin such as simvastatin, mevastatin or atorvastatin, the ACE inhibitor is an active ingredient such as enalaprii, ramipril or trandoiapril and/or the AT-1 blocker is an active ingredient such as irbesartan, torsartan oroimesaratan.
43. A pharmaceutical composition according to claim 39 or 41, Wherein the NO donor is L-arginine.
44. The use of erythropoietin according to claims 1 to 43 for production of a pharmaceutical composition for prevention or treatment of diseases, wherein the erythropoietin and/or the pharmaceutical composition is suitable and d 45. A kit containing eiythropoietin, endothelial progenitor ceils ajid at least one cell population disable for cell therapy, wherein the erythropoietin is preferably present in low dosage.
£0

46. The use of efythropoietin and/or derivatives for production of a pharmaceutical composition or of b kit containing a dose of 1 to 90 lU/kg of body weight per week, preferably 1 to 45 lU/kg of body weight per week, for prevention or treatment of diseases of the human or animal body, wherein the ©rythropoietin in the sra low dose is suitable and designed for improving, especially tor promoting and/or accelerating, the integration of a mechanical or biological agent, especially in endoprosthesis, especially an implant, for example a tooth implant, a tooth replacement, a! bone implant, a bone replacemen, especially a Joint prosthesis, a ligament/tendon replacement, such as the cruciate ligaments, or a solid organ into the implant or ft e body structures surrounding the endoprosthesis.
47. The use according to claim 46, wherein the pharmaceutical preparation or the kit additionally contains a cell therapeutic, especially endothelial progenitor ceils and/or other cell populations usable for cell therapy tor regeneration of issues and vessels.
48. The use according to one of claims 46 or 47, wherein the endoprosthesis is made of steel, ceramic, plastic or another matrix material.
49. A kit containing erythropoietin in a dose of 1 to 90 lU/kg of body weight per week, preferably 1 to 15 lU/kg of body weight per week, an endoprosthesis and if necessary a cell therapeutic, preferably endothelial progenitor cells or ot ler celt populations usable for cell therapy.
50. The use of erythropoietin and/or derivatives for production of a pharmaceutical composition containing a dose of 1 to 90 lU/kg of body weight per week, wherein the erythropoietin in this dose is suitable and designed for prevention or treatment of insulin resistance.
51. The use of erythropoietin and/or derivatives according to one of claims 15 to 18 for production of a pharmaceutical composition containing a dose of 1 to 90 ID/kg of body weight per week, wherein the erythropoietin in this tee is suitable and designed for prevention or treatment of insulin resistance.

52. The use of erythropoietin in a smalt dose of 1 to 90 lU/kg of body weight per week for therapy of i lsulin resistance. A kit containing erythropoietin, endothelial progenitor celts and at least one cell population usable for cell therapy, wheiein the erythropoietin is preferably present in low dosage.
53. The use of erythropoietin and/or derivatives for production of a pharmaceutical composition or of la kit containing a dose of 0.001 to 90 lU/kg of body weight per week, preferably 0.05 to 50 lU/kg of body weight per week, for prevention or treatment of diseases of the human or animal body, wherein the erythropoietin in the said low dose is suitable and designed for improving, especially for promoting and/or accelerating, the integration of a mechanical or biological agent, especially an endoprosthesis, especially an implant, for example a tooth implant, a tooth replacement, a bone .mplant, a bone replacement, especially a joint prosthesis, a ligament/tendonreplacement, such as the cruciate ligaments, or a solid organ into the implant or the body structures surrounding the endoprosthesis.
54. The use according to claim 53, wherein the pharmaceutical preparation or the kit additionally contains a cell therapeutic, especially endothetial progenitor cells and/or other cell populations usable for bell therapy for regeneration of tissues and vessels.
55. The use according to one of claims 53 or 54, wherein the endoprosthesis is made of steel, ceramic], plastic or another matrix material,
56. A kit containing erythropoietin in a dose of 0.001 to 90 lU/kg of body weight per week, preferably 0.05 to 50 ID/kg of body weight per week, an endoprosthesis and if necessary a cell therapeutic, prefeiably endothelial progenitor cells or ether cell populations usable for cell therapy. I
Dated this 21st day of July, 2006.

^

Abstract
The present invention relates to the use of low-dosage erythropoietin for stimulation of physiological mobilization, proliferation and differentiation of endothelial progenitor cells, for stimulation of vasculogenesis, for therapy of diseases related to a dysfunction of endothelial progenitor cells, and for production of pharmaceutical compositions for treatment of such disc ases as well as pharmaceutical compositions that include erythropoietin and other suitable active ingredients for stimulation of endothelial progenitor cells.
To
The Controller of Patents
The Patent Office
Mumbai
22

Documents:

873-mumnp-2006-abstract(10-10-2007).doc

873-mumnp-2006-abstract(10-10-2007).pdf

873-mumnp-2006-abstract.pdf

873-mumnp-2006-abstract.rtf

873-mumnp-2006-cancelled pages(10-10-2007).pdf

873-mumnp-2006-claims(granted)(10-10-2007).doc

873-mumnp-2006-claims(granted)(10-10-2007).pdf

873-mumnp-2006-claims.pdf

873-mumnp-2006-claims.rtf

873-mumnp-2006-correspondence(01-01-2009).pdf

873-MUMNP-2006-CORRESPONDENCE(26-3-2010).pdf

873-mumnp-2006-correspondence(ipo)-(20-01-2009).pdf

873-mumnp-2006-correspondence-others.pdf

873-mumnp-2006-correspondence-received.pdf

873-mumnp-2006-description (complete).pdf

873-mumnp-2006-drawing(10-10-2007).pdf

873-mumnp-2006-drawings.pdf

873-mumnp-2006-form 1(21-07-2006).pdf

873-mumnp-2006-form 18(21-07-2006).pdf

873-mumnp-2006-form 2(granted)-(10-10-2007).doc

873-mumnp-2006-form 2(granted)-(10-10-2007).pdf

873-mumnp-2006-form 5(17-07-2006).pdf

873-mumnp-2006-form-1.pdf

873-mumnp-2006-form-2.doc

873-mumnp-2006-form-2.pdf

873-mumnp-2006-form-26.pdf

873-mumnp-2006-form-3.pdf

873-mumnp-2006-form-5.pdf

873-mumnp-2006-power of attorney(01-01-2009).pdf

abstract1.jpg


Patent Number 227826
Indian Patent Application Number 873/MUMNP/2006
PG Journal Number 10/2009
Publication Date 06-Mar-2009
Grant Date 20-Jan-2009
Date of Filing 21-Jul-2006
Name of Patentee EPOPLUS GMBH & CO., KG.
Applicant Address FEODOR-LYNEN-STRASSE 5, 30625 HANNOVER,
Inventors:
# Inventor's Name Inventor's Address
1 BAHLMANN FERDINAND HERMANN KLEINE DUWELSTRASSE 21, 30717 HANNOVER,
2 HALLER HERMANN AN DER TRIFT 8D, 30559 HANNOVER,
PCT International Classification Number A61K38/18
PCT International Application Number PCT/EP2005/000618
PCT International Filing date 2005-01-22
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 102004004 509.7 2004-01-23 Germany