CA2778735A1 - Induction of arteriogenesis - Google Patents

Abstract

The present invention inter alia relates to a method of promoting collateral circulation comprising the step or exposing a subject to a therapeutically effective amount of an NO donor wherein the therapeutically effective amount of the NO donor promotes arteriogenesis sufficient to augment collateral circulation in a physiological or pathological condition.

Description

- I -Inducting' or Arteriagenesis The present invention relates to methods of treating or preventing an arterial instil tic ieney by the administration of a NO (nitric Nide) Llonor, Cardiovascular diseases as well as other diseases involving a cardiovascular and. more specifically, arterial insufficiency have an enormous economic importance. hi icrinany.
for example, about 280000 patients stilThr every year from a cardiac intrcl,Vv :11)01.1i (15000 patients die_ one important reason for a cardiovascular disease is tire or complete Occlusion of arterial vessels resulting in a reduced simply of oxyLren rind nutrients of the iisRue stimlied by the arterial vessel.
Angina pectoris, the chest pain, is a clinical syndrome reflecting inadequate (is vgcli flir myocardial metabolic demands with resultant ischemia and is g,enerally canseil by obstruction (stenosis) or spasm of coronary arteries, Arteriogenesis is a process in which already pre-existing small arteriolar coilaicrals can develop to full functional conductance arteries which bypass the sire 4.l. an itrterial occlusion an4.1/or compensate blood flow to isehemic territories supplir.-7d by artery. Consequently, arteriogcnesis is a highly effective endogenous mechanism lorIliç
maintintince anti regeneration of the blood flow atter an acute or chronic occilNiN ç i.:10:111 in an arterial vessel ln this ease the collaterals can function us natural bypasses.
Al/CflOgClleSiti is n process distinct crom ariv,iorcricsis er ncovascularization, µvho.c a de novo fibrillation of arterial vessels occur (Fitischmann and Schaper, Journal of Pathology 2000, 190:338-i42).
lo Nitroglycerin (glyceryl trinitrate) is used since deer-Wes as a vasochlatating agent in cardiovascular diseases as coronary artery disease (CAD, also ischemic heart disea,;is or coronary artery disease), which is the leading cause of death and lisrih,iriv\Nor IcINNide Nit rolOyceriii has Neil solcly used to treat the symplon1S of these diseases e angina pectoris duc to its vasodilating effect on veins and arteries, resultinglil t reduced 3!) workload and energy consumption of thc heart (by decreasing preload and altilload) kiti well as i:l11 if ICI-CiAWLI myocardial oxygen supply (by dilating the collar:tr.,. arterics) Fliese symptoms include chest pain, pressure, discomfort, or dyspnea. 1-lowcyci, 1111100) [ ill lias not bcen used for curing the underlying disease or improving its ptog,nosis.

- 2 -Consequently, nitroglycerin has been and is primarily used for the acute relief or prophylaxis of angina pectoris attacks, the most common symptom of CAD
In the art, it has becri described that nitroglycerin is not able to induce anplopenesis (neovaseularisation) or arteriogencsis in a setting where this substance ha:, been administered continuously (I lopkins et al. Journal of Vascular Surgery 27:886 8O..4 Troidl et al. Journal of Cardiovascular Pharmacology 55; l i trio CO I
(.1)).
There is a need for providing agents thr promoting, collateral circulation.
hi a first aspect, the present invention relates to a method of treatinp or preventing. an amerial insufficieney, wherein an NO donor is administered in an intermining manner to a subject in an amount effective for the induction of arteriogenesis.
ln the context of the present invention, it has been surprisingly Ibund that NO donors are effective in the induction of artcriogenesis even if they are not 2kiniinis1ercd constant IN hut in a manner where platUita levels are only elevated FOr i sl tor. I ,iine (see the cx.tniptv Section.). Consequently, the present invention provides effective agents for the promotion of collateral circulation. Based on the finding that NO donors are capable of inducing arteriogenesis, the present invention now provides all VIICC11Ve 1001 for pro, eit..ny ;Intl treating an arterial insufficiency.
According to the present invention, the term "treatment- or "prevention"
ittcaw; that not only symptoms of' the, disease are relieved but that also the disease itself is Ire:lied oi prevenicd. In a preferred embodiment, the kalif "treatment" Mean,: mirth-min)/ the p1oV1Itisis of said disease, According to the invention, the term "arterial insufficiency" refers to any insufficient blood 10 or oxygen supply or any other insufficient supply of a tissue which is provided h an artery. This insufficient supply can he overt:a:mg: by the methods and uses 1.)1. the piesent invention wherein an NO donor is used to increase the supply of a given tissue lie Liricrial insuffieieriey may occur both during physical rust or during an exercise In a preferred embodiment of the present invention, the arterial insiifliciene is due to instill-161ml oxygen or blood supply of a tissue supplied by the artery Or1 bypass 01 ?thunt during physical rest ra. exercise.

- 3 -According to a further preferred embodiment, the arterial insufficiency is due to an increased demand of oxygen or blood now of a tissue supplied by the artery or A bypass or shunt.
This itiereascd demand of oxyE,en or blood now can have several reasons ineluclinw but not limited to increased sport or physieal activity, and increased mental activily or a disease requiring an increased demand of oxygen or blood flow.
According, to a clinker prete I
rra. ciii...)or.,ment, the arterial inStlifieiericy k eharauteriiLd hy iu a partial (stenosis) or complete occlusion of an arterial vessel. I I
.11 Lie come !st ol thL present invention, the term -partial occlusion" is equivalent to a stenosis.
The partial or complete ocolusion of an arterial vessel is a well-known phenomenon. 11 can have various reasons including but not limited to deposition of material in the blood 15 vessels (including non-reyascularisable stenoses), compression from external tissue or next to the vessel or a dysfunction of the endothelium or the veSSCI resulting in a pariidoXIC
vasoconstriciimi during exercise.
In a preferred erribOdiment. the arterial insufficiency is due lo the deposition of mate' 20 the blood vessels.
The deposition of materials in the blood vessels is a well-known phenomenon re-ii limy et,.
in atlieroselcrosis.
25 III a further preferred embodiment, the arterial insufficiency is due to an emeinal in1crnal compression of an artery.
An internal vompression of art artery may be clue to an illSO to ci tumor putting pressure on the artery. lurthermore, this includes a vasospastieal constriciion oldie artery 30 as e.g. in Prinzrnetal'S angina. In addition, this also includes the paradosie vasoconstriction which e.t . sometimes occur in an endothelial dysfunction.
An external compression may be dile to an accident or any external have which can put pressure on an artery.
3.5 In u further preferred embodiment, the arterial insufficiency is a vascular disease.

- 4 -Accordion to a further preferred embodiment, the arterial insufficiency is a disease selected from the nroup consisting of atherosclerosis, an ischcmic disease and a Radler chronic arterial disease.
In a further preferred embodiment, the arterial insufficiency is a coronary arterial In a preferred embodiment, the coronary insufficiency is an atherocelerotie coronary arterial insufficiency. in particular coronary artery disease (eon:only head 4.1i!;1: ;Ise or 1(1 ischemic heart disease). stable angina pectoris, unstable angina pecioris..
ischemia or chronic myocardial ischernia, tetite coronary syndrome, or myocardial infarct (heart attack or ischernic myocardial infarct).
In a further preferred embodiment, the coronary insufficiency is a non-atherw,clerolie, in particular coronary microyascular disease or small vessel diseasc, Prini.mettil-14 angina and cardiac syndrome X, In a further preferred embodiment. the arterial insufficiency is a cerebral ariorial In a prefcrred ernhoilimerit, the cerebral arterial iusullieicricy is an atherot;clerolie Lerebra I
arterial insufficieney, in particular cerebral ischemia, prc-stroke, transient ischemic attack (m1111 StWke), stroke., vascular dementia, ischem ic disease. or isc hem le cerchrr ?vase II disease.
The eercbral arterial insufficiency may also be isehemic mierovascular brain vessel vascular deMentia, Subcortical arteriosclerotic encenbaloptithy diSeaSe), Alzheimer's disease, or Parkinson's disease.
SO In o preferred embodiment, thc arterial insufficiency is ii peripheral oriel In a preferred embodiment, the peripheral arterial insufficiency is an atliero,;c1crotie peripheral arterial insufficiency, in particular peripheral vascular disease (peripheral artery disease (PAD) or peripheral artery oeolusive disease (PAOD)).
In a preferred enthodinicrit, the peripheral arterial insufficiency is an rion-athero,,cicrotie peripheral arterial insufficiency, in particular 1(aynaird's syndrome (vasovasinatic).
thrombangi iris obl derails, endangitis obliterans or liticrecr's disease (recurring progressive -jnflaiiììntioii rtir.1 thrombosis (clotting) of small and medium arteries and veins of the hands and feet), vascular inflammatory disease (vasculitis), dialiciie ischemia. dhihetk-neuropaihy and compartment syndromes, liìa furthei preferred embodiment, thc arterial insufficiency may bc an intestinal artecuil insufficieney, in particular an atherosclerotic intestinal arterial insullicicney, in paiticular ischemie latwel disease, mesenteric ischeinia, or mesenicric infarction.
In a further preferred embodiment, Ilic arterial insufficiency may bc an urog.canal Ariel ial insufficiency, in particular an athcroselerotie urogenital artelial iiisulïic ienc y iiipaiticidar erectile dysfunction, renal artery disease, renal ischemia, or renal infarction.
In a further preferred embodiment, the arterial insufficiency may he a ners,.:il insufficiency, in particular finnitus, Is Furthermore, the arterial insufficiency may he in the context of scliµroderma sclerosis).
In a preferred embodiment, the arterial insufficiency is a central retinal artery insufficiency, in particular un atherosclerotic central retinal artery iiisillficiunc\ in particular ocular arterial insufficiency, In a further preferred embodiment, the arterial insufficiency is characterized by an Likctice of an encloitielial dysfunction, The endothelial dysfunction is a well-knovvii systemic pathological sum: ol the endothelium and can be broadly defined as an inthalanec between vasorlitating and vasoconstricting substances produced hy or acting on dic endothelium.
In a further prcicTred embodiment, the uteri& insufficiency is a cltronii.
arterial insufficiency. In the context of the present invention, the term --chronic arterial insufficiency- means that the course of the arterial iniiffic ienc s chronic and (Men proe,redient, AcCOrCiing to u further preferred embodiment, the chronic arterial insufficiency includes endothelial dysfunction, atherosclerosis, coronary artery disQase (o)rOnary heart disease or iselietnic heart disease), stûble angina pectoris, coronary microvascular disease or small vessel disease, Primintetars angina and cardiac -;yri4.-Iroilie X. vascular derncraia.. ischeinic brain disease, or ischeinic cerebrovascular disease, iselietnie rnicrovaseillar brain disease, small vessel vascular dementia, I
su.)cortical atherosclerotic encephalopathy s disease). AlLticimer's disease, Parkinson's disease, peripheral vascular disease wei ipheral artery disease (PAD) or peripheral oricry occlusive disease (PA01)1.
Ihrombangiitis oblitcrans, endangitis obliterans or 1-itie.q!er's disease, vascular inflianinalory disease (vasealitis), diabetic isehemia, diabetic neuroputhy, ischemic bowel discisk:.. el cud,:
dysfunction, renal artery disease, tinnittis, and seleroderma (systemic sclerosis) According to the invention, the term NO donor" refers to either ton...r. it ;
c CY\ Ilk: itself or simp any molecule which is capable to release NO alter having been administert.scl to a 1 ;ec,.
Preferably, the NO donor is nilric oxide, sodium nitroprusside, nitroglyeei tillyceryl trinitrate), isosorbide mononarate, isosorbide dinitrale, pentaerythritol teiranilralc (P1- IN).
molsidom in, amyl nitrite or nicorandil.
.
In a preferred embodiment., the NO donors may. be selected frorn 1; 11 ;11g:
AnorgrIpic:
nitric oxide nitrite nitrate Organic nitrates:
GIN (glyeeryl trinitrate: nitroglycerin) PI-:TN letranitrate) ISDN (isosorbitIc dinilnite) ISMN (iSOsorbide mononitra(r) Nicorandil Oraanie nitrites:
IAN (istamiy1 nitrite: amyl nitrite) (isotally1 nitrite) N-nitroso compounds:
N-Nitrusamincs;
1)ephostatin NDMA
derivates of N-methyl-N-nitrosol.1112a N-llydroxy-Nitrosamines:
Dopastin Cupferron Alanosine N-NitrosimineS
N-Diazeniumdiolates (NON( Htc):
l 0 sperm ine NONOate DEA-NONOate DE'IA-NONOate S-Nitrosothiols:
S-nitroso-N-aectylnenicillaminc (SNAP) mcial-NO-coinulexes:
Iron complexes:
Nitroprussidi: (sodium nitroprusside) Dinitrosyl-iron complexes Iron-Sulfur Cluster Nitrosyls (as e_g. Roussin's Red Salt, Roussin's Black Sall. Rutisiii-s Red Ester) Ruthenium complexes NO releils.ing.heterocycles:
Ileterocyclic N-oxides:
Euroxans Mesoionic l leterocyeles Sydnonimines Os e.g. mo Is idom ine, J insidom iiìc (SIN- l ), ciclo,dclontinc. pi rs idoni ine.
marsidornine) Mesoionic oxatriazoles .35 iiianidinc ind N-hydroxvalianidincs:
.-aruinine L-lioinuargininc N-hydroxy -1 ine N-hydroxy-Lhomoargtnine ()tiler;
A lky I C-nitruso compounds 1., Aryl C-nitroso compounds Oximes N-hydroxyureas ln a preferred embodiment, llie NO donor is ;:tn organic nitrate with a 11.1%c:cm1 backbone.
In a further preferred embodiment, the NO donor is selected from Hu...group c n,iltiili of nitroglycerin Wlyceryl ttini(rate), glyccro1-1,2-dinitrate (1,2.-(_i1)N) tind il ccrol 1,3-rhnitrate (1,3-0I)N), glyeerol-l-nitratc (1-CMN) and glyccrol-7-nitrate (2-CMN).
l5 In a particular preferred embodiment, the NO donor is nitroglycerin.
Preferably, the NO donor is a short acting NO donor, Accordinp. io illy invention.. the term 'shout acting NO donor- refers either to NO itself or to an NO donor kkhiCh.
NO
shortly, with a short halt' life time of less then e.g. 45, 'i() or preferably l ininiite,õ altcr having been administered to a subject. 1=;xam1ies of. short ;:teting No donor, are nitroglycerin (g1yceryl trinitrate.), amyl nitrite and sodium nitroprusside.
Nitroglycerin is an especially ',referred exanyle of sucli a short acting NO
donoi in an embodiment, the Nt) donor used in the voritent of the present invention is N
which according to one aspect of the invent ion Is a solid pharmaceutical preparation fur oromucosal or oral administration characterized in that it contains between PAY., and 2 weight% g,lyceryl trinitrate (( i l'N), at least one diluent, out: carrier material. and il leasi one substance that reduces the volatility or GTN, whereby this substance 41 non-volatil,...
ester stabilizer whose melting point is nut higher than 60 'C.
=l'he invention further relates to a method of treating or preventing an arlenal insult-1u ioney_ wherein an NO donor is administered in an intermitting manner to LI subject in iii amount effective tor the induction a arIcriogenesis, and wherein the NO doff!' is GIN
m A ,=;014.1 pharmaceutical preparation (Or oromucosal or oral administration cliaricteritµ.'il in that it contains an abst:prbatc comprising bctwecti 0.05 and 2 weight% GIN and a iton-%okitile ester stabiliei on a carrier material, _ 9 _ In the context. 4if' the present inve n...on, the terms "Ci IN" or "nitroglycerin are S imultaneously.
In an embodiment, the present invention exploits the surprising 11111.14)g that highly ;table., non-liquid preparations of GIN can be manufactured using a novel process 1.111creiii 6 IN
is combined with non ..volatile carboxylic acid esters. Those esters which are surtahlc tbr this purpose arc those with a melting point of 60'e or less rind which can be luplid or Fr:Ali:1W a pasty or semi-solicl consistency at ambient temperatures ranging froin ahont I 5"C
to about 2f) C. As described herein, it has now been discovered that a high IN
stabilized.
non-liquid preparation of CIIN rt.'sults when Ci IN, phlermatized in a suitable diluent to form a GIN concentrate, is then contacted with a suitable carrier material in a GIN-containing slurry which is then (or contemporaneously) admixed with a -.unable stabilizer in accordance with the teachings provided hcrcin. The resulting absorbate is highly stable. The resulting absorbate caii be ill !he form ()I a powilci or granules. The absorbate (.7all also be pressed to thrill a tablet type or comp,,,,iio, Without wishing to be bound by theory, the stabilizer entraps the GIN on and/or within the carrier material thereby preventing volatilization or escape of GIN from the non-liquid ).11.)sorba1e.
I h.:nee the invention results in highly prolonged shell life and improved Aability as:
compared with conventional GIN preparations, including Cl-rN in a diluent cie,ionririly used for phlegmatizatiou purrivws.
The present invention is a significant advanocinent in the preparation ;mil clinical availability of stabilized medicines with a prolonged slier fife whose aci,võ
incred,õi is.
by its nature, volatile and unstable such as but not limited to GIN. The irnesent invention has broad-reaching implications tor medicinal chemistry and formularies licretotOre unava i la ble.
Certain preferred preparations contain between 0.1 and 1 weight% glyT),1 triniirate The non-volatile ester stabilizer can he solid oi semi-solid at a teinperatuie of 20 in ceiiitii prereeted embodiments while the non-volatile ester stabilizer can be liquid iit others. hi preferred embodiments, ihe non-volatile ester stabilizer is scleeted From thL
p.roup consisting of: 111011Q- and diglyceridesõ polyethoxylated glycerides, esters of het ie avid. L)-alpha tocopheryl polyethylene glycol 1000 succinate and solid triglycericies, and1 111.Ntures cif any one of these substances. The non-volatile ester stabilizer COO be thet1 TN a concentration o' 0.2 to 10 weight based on the total weight of the plepaiiiiion According to the present invention. the GTN, diluent and stabilizer form a horiweiwous Preparation in some embodiments. In currently preferred embodiments, the mass ratio of the non-volatile ester stabilizer to GIN is 1:)etween 2 and 40; and the mass ratio of the diluent to non-voliiile csicr stabilizer is between I alld 9.5. In some proton-N:1 embodillle111.ti, tbe carrier material is selected fmnt the group consisting ol: magnesium aluminomelasilicatc, dibasic calcium phosphate, isoinall arid mixtures of am one ot the foregoing.
According to the present invention, the above. described sulk! pharmaceutical preparalion can further include ul least one excipient suitable for sub];
algual administration. which is selected from the group consisting of: water-soluble mono-, di-, and polysaccharides. as well as their alcohols. In currently preferred embodiments, the excipient suitable Ibr sublinetial administration is selected from the g,rolip consisting of:
fructose, plucose, lac.tose, maltose, inaltitol, mannitol, surhitol, sucrose. trehalose, and NV
ill1/1 and mixtures of any uric of the foregoing. In particularly preferred embodiments, I he e \cipient suitable for sublingual administration is xylitol and/or ism-milt al concentrations of 1-K-tweerr and Q5 weight%. In even more preferred embodiments. the excipicnt for 1.5 sublingual administration is isomalt, 1\111101 is contained at concentralions of tietweell 70 and 95 weight%, based on the total weiL.111 of the preparation.
In yet other embodiments of the solid pharmaceutical preparation, the piepnirnion Further comprises at least 0.0 I to :3.0 wcieht% old flavoring agent.
In a preferred embodiment., the NO donors may be selected from ihe 14 11 i In one plan:Wady pieferred embodiment, the solid pharmaceutical picpaiai ion tor oromticosal or oral administration contains an absorbate comprising heixveen (..) 05 and 2 weight%) CNN and a non-volalile ester stabilizer on a carrier material.
According to the teachings or the present invention. any of the lim=roinp.
pharmaceutical preparation can be in the form of a free-flowing powder (..n-live-flowing granules. 'Ihey can be packaged as a single dose in the form Of n stick pack oi sachet.
10 In another aspect, the present invention provides a process fOr the mainikieture el. a Pharmaceutic:al preparation with the active solistrince glyeeryl trinitrate tor oroniticosAl m-oral administration characterized in that it contains between 0.61 and 7 wcight''.,) 1.yr_cryi trinitron: r(iTIN), thc process comprising the steps cif: n) preparing a mixture conipribong itl Olat..7 carrier material selected [Tom the group consisting of: magnesium 15 aluminometasilicate, dibasic calcium phosphaie, fructose, glucose, isoinalt, lactose, maltose, inallitol, mannitol, sorbitol, sucrose, lrehnlosc, xylitol arid mixtures of any one ul thc foregoing; b) preparing. a (i.IN solution comprising 01 leas( one non-volatile stabilizer whose melting point is not higher than 60 "C; c) adding in a step wise lab,hirin lliç

GTN solution to the carrier material; and d) mixing until the active substance has been homogeneously distributed, optionally followed by a drying step.
In an embodiment, the preparation used in the context of the present invention mar be produced hy n process for the manufacture of a solid pharmaceutical preparation with the active substance GTN for orotnueosal or oral administration eharacterii0,1 in tlmt ul contains between 0.05 and 2 11/1, GTN, the process comprising the of: a) preparing H GTN solution comprising plileginahzect G IN and at least one non olinile ester stabilizer; b) adding in a stepwise mariner the GIN solution fOrmed in ,teri to a earlier material; c) optionally addine, further excipients; d) mixing until Mc active substance has been homogeneously distributed, optionally followed hy a drying step.
In other embodiment, the preparation used in the context of the pieseut illµention Iiirther be produced hy a process frit the manufacture of a SOIld phainiacentival preparation with the active substance GIN lin- owniucosal or oral administration Qliziractcnifil 111 lhzil it contains between 0.01 mid 2 weight % (ITN, the pioecss eomprising the sieps of a) providing GTN admixed with al least one non-volatile ester stabilizer: b) addinu, iii stepvvise manner die GIN -stabilizer admixture of step a) to a carrier malciial. c) optionally adding further excipients; end d) mixing until llie active substance has been homogeneously distributcd, optionally followed by a drying step.
For purposes of clarification, and in no manner intended to be limiting, thc ll)llciWiliLt definition of terms used herein is provided:
Diluent is a substance which permits phlegmatization of a volatile substance su1/4.11 3'5 (1 IN
and penniksa li preixiration of a liquid concentrate. As also described elsewheie suilnble diluents inelticleõror example (but not limited 10), medium chain iiiglyecrid(.=s (N1(.."1') (e.g,, Cr,.:), propylene glycol and ethanol. Fot example, when refeiciice herein is mode to a GTN concentrate, it is GIN phlegmatizeel in a suitable dil nen. stieh 0 example, MCI.
Stabilizer is a substance which increases the shibility of a volatile substance such GIN
beyond that exhibited by thesuo I
stance in a niere diluent. As also deNeriheit elsewhere herein, suitable stabilizers include hut are not limited to non-volatile carhiroõ lie tcit1 estk:rs.
.3 5 Generally speaking and as descrilwd elsewhere herein, suitable stabillicrs can he sclected from a group 01' carboxylic acid esters with similar polarity as IN and which rimy Ire liquid, solid or semi-solid at ambient temperatures but licpicfy at about 60 For pin poses of the present invention, MCI sueli as, for example, medium chain triglyeerides aeeordinc LO the fitIrOpean Pharmacopoeia are not contemplated as a stabiliier within the leachinp,s of thr present invention.
Carrier material is a 000-liquid substance which renders a composition acemiliiir to the present invention as a powder or a granule. As also described clscwheri..µ
herein. ;unable carrier materials include but are not limited to :water soluble carbohydrines ;mil respective alcohols such as, but not limited to, isomalt which has a porows istrileiiire and inorganic compounds with porous structures such as, but not limited to, anhy(lrokr,: dibasic calcium phosphate and magnesium alumiimmetasilieinc, Or mixtures ot any one of Ilic foreeping.
Absorbate as used hercin means a composition comprising an admixture of at least an active ingredient such as GTN in a phlegmatized form with at least one carrier material and a siìihil iiçr. or purposes of the present invention, the ITlaSS ratio between dilliout und slrthilizer in one currently preferred embodiment is 19:5; in certain ;Ale' pielerred embodiments, the ratio is 19;10. In yet other preferred embodiments the diluent mass ratio is 19:2, I 9:3,19:4,19:6,19:7,19:8,199,19:12,19:15, and 1,1.
The teachings of the present invention have inter ;Ilia resulted uí i the ,airpriing and unexpected finding that thc free-flowing absorbote with its at least 10-101ii 12,roater surface area as compared with a conventional compressed tabletized form of i'IN can uuiiuijuiiitc or prevent volatilization and/oi evaporation of GTN, even though the (.iTN in them): has a greater opportunity to escape due to lhe absorbate's extensive surface! area.
One of skill in the art would not have predicted this based on the state of the art heron: the present invention.
Consequently, the present relates to (he use of a pharmaceutical preparation which is stable during storage, in the tbrin of a free-flowing IIMV(1(.1 or granules., which, in addition to at least one non-liquid carrier substance and optional additional excipients, eumprises at least one stabilizer substanee, which significantly reduces the volatility of GTN and is selected from the group kir non-volatile eL,ters whose Incltillt; point is not higher than GO "C. Without remaining hound by ihrs theca> it is assumed that esters with a polarity very close to that of (JUN surround the GIN molecules on the inner surface of the carrier material and prevent volatilization of the -is Stabilizers, which are solid, semi-solid or pasty at room temperature, are especially well suited as exemplified below. Again, without remaining bound by this theoly, ii is currently thought that the absorbed solutions which form theI I t mosor.7a,c, 01 COMpletdy gOlidify following proptrration, are especially effective at trapping and thereby preventing the GIN from evaporating. For example, vii.teri a porous carrier 16, used. the GIN becomes encapsulated in the pores of the carrier -i ilre Stabilizer substance solidifies in the pores. The pharmaceutical preparation as described in the 'Present invention is suitable for filling in individual packages, such as stick packs, capsules oi sachcIs.lr example. In the case of stick packs, particularly preferred materials and cold-Trunnions are described in (1) International Patent Application PCT/EY2012/000802, ihe entire contents of which is herein incorporated by reference and (2) German Patent Application No 1..)1.. 10 2011 012 491.8 filed on February 25, 2011. Ace()rding to DE 10 2011 017>
.191.8 (e.g.
example 5) the prclerred materials for packaging, the GIN coritairaav prvimrarion W according to the invention are Composite films which contain a layer compi Ishii!, a co-polymer of acrylonitrile units and one or rnore other monomers (AN-copolynicrs) on the surface lacing the pliarmuveutical preparation. In the case of slick packs, most prcIerred are aluminium composite films containing a layer made of aerylonitrile-methy laciy late copolymer or impact-modified acrylonitrile-methylaerylate copolymer on the side, liieli is in contact willi the pharmaceutical composition. The pharmaceutical preparations rind methods as described in the present invention can also be used, however.
produer ion of' other solid pharmaceutical Ruins, e.g. tablets. mini-tablets Or pellet!,;.
A particularly significant advamage of one aspect of the prescin Invention 1-.
that prOduVliOn of pharmaceutical preparations of volatile, unstable ingredients can Ile carried out without the use of volatile anti flammable solvents, enabling the preparations to be manufactured without the use of energy-intensive drying steps and elaborate solvent recovery processes. However, the present invention also 17.onteniplates production or pharnia.ecutical preparations of volatile ingredients such as GIN
plilegirnaizecl in ethanol.
Within the framework of the tests exemplified below, upon which the invention was discovered, it was unexpectedly discovered that the volatility of Cil-N is significainly reduced under both standard storage conditions as \veil as uncle, stress condition thtotigh the use of a non-volatile ester stabilizer whose melting point is not higher than about 60 C. ror the purposes of the invention non-volatile means that the stahili/er preferably has a boiling point above or at about 200 "C' (measured at normal ambient pressure.). The prelerivtI maximum melting point of 60 c'C results from the kt-i that the GIN is also heated to this temperature during the absorbate production proces,-;
temperatures should be avoided due to stability issues of the GIN. A more oieferred mcItinu point is at or about 0 to 50 C, fill even trwre preferred melting.
pow' is or aboul 20 to 45 "C. tInd a most preferred melting point is at or about 30 to 410 Furthermore, ancl very importantly, the use Of the ester stabilizer described herein docs not compromihe the disinlegration properties of the gran u les, the release of the active substance. ku- its absorption into the body. the processes of the presciil invention result in a clinic:111V
advantageous composition, which induces a rapid absorption of the active NIIIi;.111(`C vhen cutoiriirily adminislen;r1 via the oral mucosa and a resulting rapid IL:dm:lion in the symptoms of the condition in life-threatening, einergencieS SUCh as all attack of angina pectoris.
As described earlier, the fin:sent invention's ("ITN component is pro %/Edxc. .41 ,. 11 .je _WM of a phlegrnatized (iTN concentrate; in a preferred embodiment, the dili,ciul For qieh a concentrate is MeT. Due to its explosive properties, GTN intended for pharmaceutical purposes is phIcgtnalized by the manufacturer, which reduces the risk posed by the hazardous properties. The tnattix used for plilegmatization can be in liquid midfor powder form. For example, GIN is commercially available as a 5 ,so.1 u.ion in Mc: l . such us Miu,ly0W 812, s.is a ;.4 solution in propylene glycol, as it 10% concentrate in lilt:lose triturate or a 2.2.5% dilution in glucose, Miglyollt) 812 is a prepara t.on iõromprising ii faltY
acid fraction or a maximum of about 7..% cupruie acid (060), about 50-80%
capry lie acid (Cc). about 70-50% capric acid (( '10.0), ft maximum of about MI laiiric and a maximum of. about 1% myristic acid (CH.0) in keeping with the art¨reeogilized standards sel by the European Pharmacopoeia. If these GTN concentrates are used dirc(.=tly lOr the preparation of the absorbate according to the invention, then die cli l iiciil LISOLI Jo,-phlegmatization is also contained in the finished product. According to the present invention, a preferred ratio of di luent:stabili4er is between about 1 and N.
more pi elerably between about 1.2 and 5,0, even inure preferably between about 1 5 and 10:
iind most preferably between about 1.9 and 3.8. In other equally (mend embodiments of the present invention. GIN can be plilegmatized in a volatile solvent such as but not limited to ethanol; in such instances, little or no diluent is present in the finished product.
The stabilized GIN-eorilaining composition according to the invention comprises an absorbate comprising concentrations of at or about 0.2 to 10 weight % of ihe non-volatile ester stabilizer having it inching point not higher than or at about 60 'C.':
stabiliici concentrations in certain preferred embodiments include 0.5, 1.0, 1.5,2Ø
2,5, i O. 1Ø S.O.
10 6.0, 7.0 or 8.0 weight %. It is also contemplated to useini xtures Of LIR: stabili/ing esters described. Ill title/I cases, the quantities refer to the total fur these substani..e... the < ;TN
concentration iii the final absorbate composition is within a range of at or abillit () 0'7", to 2 weight%; in certain preferred embodiments CiTN concentrations include 0.10.
0.15. 0 70.
0.25, 0.30, 0.35, 0/10, 0.50, 0.60, 0.70, 0_80, 1.0 or 1.5 weight o. Unless othei wisc siatcd, 15 nil weight percentages refer to the total composition. The rnaSs ratio of Mtn Iverts¶ l N is within the range 0f at or about 2 to 40; one preferred embodiment includes a ratio of :1 to 20, whilc: a ratio of 5, 10, and 15 is particularly preferred in other embodiments.

- I S -As earlier explained, in a preferred embodiment of the. i atvention the lion-volaiik ester stabilizer whose melting point is not higher than or at about 60 sclecicd from the group of liquid, solid, semi-solid or pasty substances at room lemperature. In pail iµularly preferred embodiments of the inveniion, the stabilizer is chosen from a group of ,aibmiinces that result in a homogeneous solution when admixed with the phlegmatized i1N
concentrate. Especially prefei red stabilizer substances arc solid or 131`,t).
ill loom temperature, and include but are not limited to triglyeerides, diglyeeridc-i, and mortoglycerides; polyethoxylated triglycerides, diglyeericies, and inoitoglyccrides. eqers of lactic. acid; and !Minim iocopheryl polyethylene glycol MOO succinate 1 P( ì).
As proposed earlier, it is suspeeled that the absorbates, which partially or completely solidity following preparation, are especially effective at preventing the ur-N from evaporating.
In the ease of stabilizersI 1 sea:exc. :rom the group consisting of triglycerides.
triglycerides include, for example. hard fat in accordance with 1..18P/N1', 11 la is.
commercially available as Gelueiren" 13/01 from (.iattetbss,= (Sahli Prics1 ('edex. 1 ranee).
In the case of niono- and diglycerides include, for example, glycerol inonooleate. which is, c.g., commercially available as Cithrollt) (iMO HP from Croda 1 tNetietal., (.iermany), glycerol inonocaPrylocaprate in aCCOldfince with ihe European Pliarrnaµopeia (Ph. Eur.), sold for example under the commercial mime (_'apmulTM MCM I .P b Abitec (Janesville, USA), or mono- Lind diglycericles in accordance with USP/N1 =
Polyethox.s'latect glycerides include for example olcoyl inacrog,01-6.glycei ides in accordance with 1.ISP!Nf, whieh are, e.g., commercially available as Labra11141) 191/CS from Ciattetbsie. In other .
embodiments. preferred stabilizers are selected lioni the group consisting of r.,;ter.s ol look:
acid including, for example. eetyl laciatc ni.tl myristyl lactate_ which arc..
commercially available as Crodainorrm CI. and CrodarnolT" MI_ resp, from Croda imbl I
(Nettetal, (iermany).
The absorbate composition SIS llSed in the present invention also contains at least oric pharmaceutically suitable carrier material charaelerized by a larg,e inner siirintc tea 10 capable of absorbing, for example, oily liquids. Preferred carrier materials of that kind include, bin are not limited to, maglic.sitan aluminometasilicate in accordance with USP/NF, one example of which is commercially available as Nensiiin4n tl..S2 from Fuji Chemical Industry (Japan), aiihydrous dibasic calcium phosphate in accordanct=
with USP/NF, one example of which is commercially available as Fitiicaliii* from Fuji Chemical Industry (Japan), isornalt according to the European l'harimicopoeiti onc e.xample of which is commercially available as Galen IQ' from Iir%/120-Palatinit GmbH
(Maiiiikiin, Germany) or mixtures of any one of thc .foregoing types of carrier minerals.

The absorbale composition as used in the present invention can ;Ilso contain other pharmaceutically acceptable exeipients whieh support sublingual tel .ease active sAihstancc. an(' are selected (but not limited lo) from among the group watei-soliible mono-, di-, anil polysaL,clii:trides, as well as thcir rilixhols. This excipient is selected especially from the: group including but not limited to fructose, glucose, isonia11..
sorbitol, .,;iicroseõ trelialow, :And xylitol and/or die)coil In certain prelCrred embodiments, these excipients arc present at a total concentiation or al or about 70 to 95 weight v/o. In the case or niiNdurcs the concenlralion ()I each individual substance is at or about 20 to 95 weight % whereby in certain oilier pick:I:red in c iti bod i Me nts concentrations include 25, 30, 35, 10, /15, 50, 55, 60, 6.5, 7'5õ 80_ or weight Vo. In certain embodiments, isomalt is especially preferred. If can scrvc both its a carrier material when a dillient for the active ingredient such as OTN is an oily i-iolution and can also serve a.s i=tdditional bulk material.
In addition, the ahsorl:inte preparation as used in the present invention can contain other excipients, such as flavoring agents. Flavoring agents ale 'Ned especially iii the case or preparations for oral or st,171ilify,tial alministration in order to i die rtnixc actienhincc among patients. In certain preferred embodiment according to the invenlion., they are used al concentrations or at or about 0.01 to 3_0 weight %, whereby Ilic osp...cially preferred concentrations in certain other embodiments include at or about 0 I, 0.5, 1.
1.5, 2 or 2.5 weieht A currently preferred liwinula comprises:
Contents Quantity J gl CO'N (5Vo) ill diluent !VIC'T 79,3.5 cilycerol intmoemprylocaprate Ph. Eur. =19.81 .Anhydrous dibasic calcium phosphate [00.80 isomtalt 1800.00 __ Total 2000.00 TN comentration 0.2%
2$ Another currently preferred formula eompriscs:
Contents Qua' t I ity I gl GTN (5%) in diluent M(' 10.0 Solid Triglyeerides 20.0 _Anhydrous dibasic calcium phosphate /19.9 lsomalt 880. I
=
Pepperm int flavoring agent 10.0 Total 1000.00 GIN concentration 0.2%
Yet allot tic:r currently pretrred formula comprises:
Contents Quantity gl Ci l'N (5%) in diluent MCI 40.0 Oleoy1macto2,01-6-glycerides 200 Anhydrous dibasic calcium phosphate .s0.0 'soma It Kg0.0 . _ flavorint.; .4.!ent 10 0 "fotal 1000.00 GTN concentration =
And, another currently preferred formula con:irises:
Contents Quantity 1.g1 GIN (5%) in diluent MCI 40.0 Sol irl Trie,lyer.Ticlvs 10.0 Cilyµxrol inunoAprylocapratc Ph, ur. 10.0 AnhYdrOLIS (MIMIC efilei Jim phosphnio= 500 lsoinalt 880.0 Peppermint flavoring agent 10.0 1000.00 GIN concentration 0.2%
And, yet another currently prefeyed_formula comprisq;._ _ Coittc.Ints Quantity 1.14I
GIN (5%) in diluent MCI' 40.0 maerou,o1-61,1yceridcs 10.0 Triglycerìdes 10.0 Anhydrous dibasic calcium phosphate 50.0 lsomalt 880.0 Peppermint flavoring agent 10.0 'I'otal 1000.0 _ . .
(13"1"N concentration 0.1%
õ
And, another currently preferred formula compricec: .
Conic:As Ottani ity I gl , _ GIN (5%) in diluent propylene glycol 1.Ø

Glycerol monocaprylocaprate Ph. Eur. 1.0 __ Anhydrous dibasic calcium phirsphate 5.0 I:WM[1R 89,0 Peppermint flavoring agent Total 100.00 _ concentration 0.2%
And, yet another current.ly preferred formula compriscA:
C'ontenis Quantity Iv GIN (5%) in diltroril propylene elycor Oleoylmnerogo1-6-glyeericles 2.0 Anhydrous dibasic calcium phosphate = 5.0 Isomalt 88 0 I)pperinint flavor;
dig rimttrit 1.0 100.0 (TN concentration 0.2%
And, inothcr currently preferred formula eornprise;!;: =
Contents GIN 5 % in diluent ethanol 4.0 Glycerol monooleale ________________________ 4.0 Anhydrous dibasic calcirmt phosphate Isornalt 89.8 Peppermint flavoring agcni _________________ 1.0 'I'otal 103 8 GIN concentration after evaporation of ethanol 0 2%
And, yet another currently preferred formula comprises!
Contents Qw-111111Y 1111 0.2 Oleoyl macrogol-o=slyeeridcs 4.0 Anhydrous dibasic calcium phosphate $.0 Isorrialt 89.8 .Peppermint flavoring agent 1.0 100.0 GM concentration 0.2% ________________________________ 1 The inelhOd as described in the present invention comprises the mixture of a concentiate of a volatile chemical, such a.s for example a GIN concentrate in which GIN is solubilited in a suitable diluent with a. non-volatile ester stabilizerf waose melting point is not linther than about 60 C until a homogeneous solution results ill certain em.-kodiments, stabilizing esters not already in liquid form at room ternperaturc arc heated to il or about lO'C (Move theit melting point and then mixed with the N I
. ¨ coneciitiate as described above at that temperature. This intermediate solution is then admixed %Nitll carrier material which is powdered or granulated and mixed mechanically until a homogeneouS, free-flowine, powder or granulate absorbate is formed. For purposes ()I the iC.ì present invention, free-flowing powder or granulate absorbate means an absorbate whiel) clues not have a wet or oily or sticky consistency or is not a liquid. In another embodiment.
the GTN concentrate can first be combined with a carrier material capable ()I
absorbing especially laree quantities of oil - up to I 00% al its oil weight, for example - and thLii the other components siielu as the stabilizer can be added. Thus in certain criditaliments iit which the active substance now contained in a carrier material is mi \ed with the non-volatile ester stabilizer whose melting point is not higher than 60 "C., it is possible to dispense entirely with the use of volatile and flammable solvents dui ine the prialuetion process. 'flits enables the production of a preferred embodiment of the absorb:0c according to the invention vvithoul energy-intensive drying steps and elaborate solvent recovery 20 processes. Thus, one preferred embodiment of the production process of the present invention is espevially suitable on an industrial scale. This is a sienificant advancement with industrial benefits heretofore unavailable.
However, as described earlier, other embodiments of the production process as described 25 in the present invention contemplate the use of G'I'N concentrates comprising a volatile solvent such as ethanol. Even in the cast: of' this particular production process, a herctolore unavailable stabilized free-flowing absorhate can still 1)0 successfully manufactured without any disadvantages or compromises in the resulting allsorbate. In the omhodimen( of the production methocl in which a GIN concentrate in a vt.)latilc diluent is 30 used, the concentrate is absorbed by kl solid carrier material and contemporancoudy or In ll second step the liquid or liquefied stabilizing ester is added. The restillinr_.) ;Itirry. is dried, ['or example at a temperature of 30 'C. Then the other excipients are added arid mised marl a homogeneous free flownie, powder or granulate absorbate is formed.
i5 In yet another embodiment of the production method as described in the present invciition, GIN can be diluted under careful attention of the explosion risk directly in tlw stabilizing ester. This mixture is then absorbed hy a solid carrier material. Then thc other excipienis ale Hilded and mixed until a homogeneous free flowing powder or granulate abNorbate is formed, This process is most suitable when the stabilimr is a liquid.
In any of the proposed production methods described herein, a sieving, OF
thsaggregating S step can be employed in order to ameliorate the flowing behavior of the powder rub:lure.
lri yet another embodiment of the production method as described in the present invention, the method Can differ from the above in that first a CiTN concentrate is tibsorbed I)) a solid carrier material and tiCCOrld the liquid or liquclied stabilizing, ester is added. Then the other io excipients are added and mixed until a homogenous free flowing, powder or gianulate ubsorbate is formed. This process is especially effective if stabilizer iind 1....11-N in diluent cannot be mixed homogeneously e.g. solid triglyeerides as stabilizers and GIN
in diluent propylene glycol.
15 According to the invention, the NO donor is administered in an amount capable of inducing, arieriogenesis. The skilled person will appreciate that this amount will depend on the subject to which the NO donor is administered. Generally., f a11101101 lo he administered may be between 0.1 and mg per day, but this can vary due to the weight of the subject, its hemodynamic response lo the NO donor and/or the severity of the disease.
In a preferred embodiment, the amount of the NO donor is applied in a dosage rtr O..' up io 0.8 mg (0.2, 0.3, 0.4, 0.6, 0.8) for at least l- up to maximal 11-timc5 d maximal daily dosage of 3.2 mg. These numbers especially ipply in eases where the NO
donor is n itroglycer According to the invention, the 'um "administration of an NO donor" means that a given dosage of the NO donor is administered. Depending on the way of adminisu awn, iht.=
skilled person will appreciate that the administration may take some time. In a preferred embodiment, the NO donor is administered in .on-ri of a spray, chewable capsulc mho table as, inhalahle aerosol or powder, granules, powder or a tablet, which means thu (lie administration may he completed within seconds. However, the!It... "I
.t.ninI.!,..rat.oti or ihe Nt) donor may also take long,er, e.g. if the NO donor is administered to the put ic tit kv wit) of infusion or by ointment or patch, Modes of administration of the NO donor aft blither discussed below.
Furthermore, according to the invention, the NO donor is administered in a manner capable of inducing arteriogenesis.

As shown in the examples, the invenlors of the present invention have surprisingly found out dial an NO donor is capable of inducing arterioneriesis when administered in an intermitting manner.
According to the invention, the term "intermitting manner- means that the NO
donor is administered in a way thar its plasma levels are Only elevated in a short-term manner after the administration of the NO donor but then again decline, This ean hç
rellievcd for example if the N() donor is a short acting NO donor as delined ziboµe and the i4lininistration of the short acting NO donor is followed hy a time period without administration and then the NO donor is again administered to the silbjeer,-ur...) ri ermore..
this way of administration avoids that the subject is developing tolerances aellinst the effect of NO.
However, it is equally possible that the N() donor is a long acting NO donor, in ihr, case, however, in order to achieve the decline ill plasma levels, care has lo be taken Mat Ile!
administration of the long acting NO donor is only shortly and that ihe plasma levet,:
obtained are noi too high.
In a preferred embodiment, the plasma levels of the NO donor ate elevated for not more that 180, [20, or 60 minutes, or for not more than 50, 40, 313, 15, I or 5 minutes.
Furthermore, this also implies that the NO donor can be a.dministcred in chronival manner, i.e. without taking account of disease developments implying an acute treatment with the NO donor, Furthermore, it also implies that a therapy plan elm be established without taking account of disease developments implying an acute in:anneal with tho NO
donor.
In thc context of the present invention, the NO donol is Mier aria administered lo induce arteriogenesis. This implies that the NO donor can also be administered zit Dino poirns or time periods where there is no necd 11õIr viisodilation and sucli u n:lief of sinpronil..: tikc pain relicf'.
thk is in contrast to past applicalions where the NO donor, e.g.
nitroglycerin, has bi.M1 lltied 10 achieve a relief or neille (i.e. immediate) prevention of the ,,,ymptoins oi a corresponding disease. Ihese .symptoms for example include pain and/ol dyspirea in the 15 case Or 4 cardiovascular disease. and the relief or ;wolv prevention of the symptom,: was achieved by vasodilation and resulting pain antlfor dyspnea relief. However, the purpose of the administration of the 'NO donor was, as discussed above, nor the treatment 01 llic underlying disease, heetiuse it was well known that the diseases cannot he liealvd by vasodilation or pain relief.
The identification cif an NO donor as a pro-artoriogenie agent., therclOre, also makes it possible that the NO donor is administered at time points or tilite periods %here thcie is 110 need for such a relief of symptoms like pain relief. In a further preferred 1/4:111htithiliont, the NO donor can also be diriiriislered in cases where there are no curre sponding, symptoms likc dyspnea or pain or in eases where such sympioms are not to be expected.
in 111 the context of the prest.Ant invention, the lam "intermittemly" also MetilisIliit iii N() dOnOr, in particular the short acting NO donor, is not administered continuously_ tor example by means of long term intravenous infusion or with the help of an implanted pimp which constantly delivers the N(..) donor to the stilljeet. Rather, this tei in also means that there is an interval between two administrations of the NO donor, and that the NO
donor is given several times, e.g at least 1, 2, 3,1, 5, 6, g, 9, 12 or 1 6 times a day As the skilled person will appreciate, one administration of die NO donor may include an administration in one or more dosau,c fOrms, e.g. tablets or hubs (puffs) in ease of a spray.
For example, one administration may include the administration or two iableis or one to three hubs (purrs).
As to the schedule of administration, the skilled person will appreciate thin Melt: aii; many ways to achieve this intermitting administration. For example, it is possible to administer the NO donor at least once a day and at least on one clay a week for al least two weeks.
However, it is equally possible lo administer the NO donor for only one week if the NO
donor is administered several times during this week.
Preferable, the NO donor is administered once, twice ur three times a day, wherein even More preferred the time period between two administrations of the NO donor is al leasi 4 hours, in Inirticular 8 hours, in partivular at lento 10 hours or 12 hours Although possible, it is not necessary that the time periods between two adminktrat ions of the N(_) donor are the same_ Rather, it is preferred lhat these time periods differ, depending on the individual adminisiralion schedule.
In a preferred embodiment, the NO donor is administered al least on one day ii week.
However, the NO donor way also he adminis,crew I ,1 on 2, 3, 4, 5, 6 or 7 days a 1\ LA\

especially preferred embodiment, the NO donor is administered at least on 3 or 4 days a week..
Accordine, to the invention, it is possible to administer the NO donor tor e.1 petiod of several weeks or months. This is particularly pieferrecl in ordei to induce arteriogenesis efficiently, although also a shorter administration of one of two weeks is possible.
In 3 preferred embodiment, the NO donor is administered ibr 2 to 8 weeks. It is equally preferred to administer the NO donor ibr 3 to 6, 3 to 8, 3 to 10 or /I to 8, 4 to 10 or 4 lo to weeks. These numbers are only examples and may vary depending, on the individual schedule atilt. subject.
In a preferred embodiment, the NO donor is taken at least once a week lor al least 8 weeks, in particular for at least I 2 wecks, In a further preferred embodiment, the NO donor is taken not longei than b. ti or 12 months. However, it is also possible to take the NO donor for 2, 3 Or even more years Furthermore, it is also possible that the NO donor is administered for decades or- even through the whole life of the subject.
to In the context of such long-term administrations, it is preferred that the NO
donor is administered once or twice a week or at least once or twice a week.
It has been described previously that an exogerious sliiminlion :if pulsatile she,ir lial'ON irp 25 an individual rimy rcstilt in arteriogenesiis. Furthermore. it has Iwo, deseriht.-d how the pulsatile shear forecs can be measured (wO2010/072,1 I6.
Consequently, in a preferred embodiment, the NO donor is rultninistered in conjunclion with an exogenous stimulation of the pulsatile shear forces in the artery.
With respect lti said embodiment of the invention, the NO donor should be4 m.rnin.N11.-tcd a way that it is active in the body of the subject when the exogenous stimulation is applied.
In this context, active means that either the .NO release is not yet terminated or OW NO
released From the NO donor is still present and active. Depending on the specific ) donor to be used, its physiological halftime in the subject and its Formulation. the Wed person will be capable of determining when the NC) donor has to be administeied kr Mc subject in order to ensure that it is active upon the exnenotis stimulation.

.
In the case of nitroglycerin, the halftime and its persistenve in the body of the subject has been intensively studied, sec c.g. Armstrong el al. Circulation 59-585-588 (1979) or Armstrong et al. Circulation 62:160-166 (19X0) (e.g. after intravenous i..)r sublingual application, where 11 is 7 to 5 minutes).
In general, the halftime of nitroglycerin is 2 to 5;
inmates.
In a preferred embodiment, the NO donor is administered in the time pet kid of 30 minutes before the onset of the exogenous stimulation until 30 minutes alter the terminal ion of the exogenous stimulation In another embodiment, the NO donor is administered in the lime pet mil of 30.
prelerably to 10 minutes befbre the onset of the exogenous stimulation until .50, prcli.rdbly 1 to 10 minutes atter the termination of the exogenous stimulation.
Is More preferably, the NO donor is administered in the time period of 15 minutes. preterably

5 in Mules, more preferably 2mittL i t es minutes before the exogenous stimulation until ,50, preferably 15, more preferably 5 minutes after the onset of the exogenous stimulation.
In a further preferred embodiment, the NO donor is administered once a day.
five times week for 6 weeks 2-5 minutes before the exogenous stimulation.
The exogenous stirmantion of the pulsatile shear forces may be iteltieved by tiny known way. This includes a stimulation with the help of medicaments like medicNiments which increase the blood pressure.
In a preferred embodiment, said stimulation is achieved by physical exercise or the application of an endogenous fork...e to the arterial vessel.
According to the invention, the term "physical cxetretsc" Means :111), training Of tlit. tiLihjecl.
including but not limited to training in exercise rooms. jogging, walking,, nordic won, log.
swirnming, dartciii,cycling and hiking. '1'lle skilled person will appreciate that any exercise will be helpful in the context of the invention, provided that it is poi footled in conjunction with the administration of the NO donor. Preterably, the !eon -pllystet.d eXenzitit.:" does not include unsupervised, unproscribed routine movements Rs.-eaSlIal wa lk lug or house work.

As discussed above. it has been found ill llic context of the present invention that an NO
doiwr is capable of inducing, arteriotwricsis. This enables not only the Ireatmcni ()I an already existing disease. Rather, hi the context of the present invention, it is also possible to prevent the disease. Consequently, in a preirreci embodiment of the present invention, the method aims at the prevention of said arterial insufficiency.
As shown in the emimple section, in the context of the picsent invention. it has bolt possible to reduce the intarel size in case of an already existing occlusion.
Hirt]
.lormore, has been possible to reduee arrhythmias in the subjects. Consequently. in a pkµteilei.1 embodiment of the present invention, the method results in a reduction of the mraict in reduced arthyilimias or in a decreased ST segment elevation.
The NC) donor can be administered in any suitable way so that it eon be incoupoiated into the subject. This includes an oral, parenteral or intravenous administration a.; well as the injection of the NO clonor into the body oí the subject, but also an administratuin to a mucous membrane or the subject.
Consequently, in a preferred embodiment of the prescint invention. the NO
donor is adm in isitered lingually, sublingually, in ha lat ivc ly, buca I ly, Iransinncosn11). or oromucosally.
In case or a lingual, sublingual or oromucosal administration, it is prefcrred that the Ni donor, pretirably nitroglycerin, is administered with the help of a viay.u chewable eapstile or in the form of all' powder or granules or even by an inhalatot device, from which the N() donor can be easily inhaled and adsorbed. It is equally ritelerrcil that the NO
donor is administered in the form (Willi inflatable gas, aerosol or powdet.
Preferably, the administration of the NO donor is a non topical administration.. i c.. that the NO donor is not administered to the skin of the subject, 1 11 1,1 I
0 t0111("NI of the present invention, thc term -skin" excludes mucous membranes attic subject The NO donor can be formulated in any suitable way tipt the above mentioned administration modes. Such formulations are known to the perS4311 Skillai in the art and include the formulation in suitable buffers, in a as, aerosol, as iìililcls, pOWLia or granules.
In a preferred embodiment, rhc NO donor is formulated ill ;=1 way that ;illows a last release of the NO donor From the formulation, This includes e.g. formulations which do not hold back the NO donor for a longer time period, bid which release the NO donor 1\
1111111 e 45, 30 01 15, 10, 5 minutes or 1 minute or even in some seconds, e.g. seconds.
Through the invention, it is preferred that the suhlect lo which the NO donor is applied is a human subject In a further aspect, the present invention also relates to nn NO donor for use in a motliod for the prevention or treatincid of au arterial inSulficieney, wherein iie Nl/
donor is administered in an airionin and manner effective for the induction of nricriogene,ii,,.
All features and preferred embodiments discussed above for ihe method of treating or preventing an arterial i .nsu.,..e.eney also apply to the NO donor tbi use according lo this aspect of the invention.
In another aspect, the present invention also relates to a method of the suppreY,ion of neg,ative effects associated with any treatment of an arterial iiisliffieiertey which l ann-ateriogenic or inhibiting artcriogenesis, comprisingaLin -1 f austering to a subject mibiected to said Ireatment an NO donor in on amount and manner effective for the indmtion ot.
arterioc,criesis.
In a preferrecl embodiment, said treatment is an acetyl salicv lie acid (ASA,), glyeoproichillbIlla antagonists, or etanercept (soluble tumor necrosis factor1 I
a.paii rot opror) treatment.
1t is known in the art that ASA is an inhibitor of arieriou,encsis (Singer CI
, Vasa 2006, I 74-177). Consequently, the ASA treatment of eardiovascular diseases, although belly, a standard therapy. has significant side effects and ilisadvaiilages. In the c4,nicN.1 or the present invention, it has been found that NO donors ore capable of overeormiv,2, the gCllitive effeciN wisociated with an ASA treatment (see example sec(ion). Based on Most!
the inventors conclude that also the negative side effects associated wah olhot medications like glycoproleinl Mina antagonists or etanereept treatment can also he d I
led.
Furthermore, the present invention also relates 14) an NO donor for use in n method of Me suppression of negative effects associated with any treatment of an fillet iil imairlicioncv whjçh is anti-ateriogenic or inhibiting arteriogetiesis, wherein the NO donor is administered to a subject subjected to said treatment in an amount and mariner . l'ti.lic !Or Ihe induction of arteriogenesis.

In a preferred eintimliment. said treatment is ail acetyl salicylic acid (ASA), glycoproteinlIbIlla antagonists, or etanercept (soluble turnor necrosis factor alpha receptor) trcatinent.
All features and pret'erred embodiments discussed above for the method of treating of preventing an arterial insulTiciency also apply to the method for the suppression Lii.
negative effects according to this aspect of the invention or 10 said NO donor for Ilse according to this aspect of the invention.
In a further aspect, the present invention also relates to a method 11.ir the prevention or.
treatment of a cardiac arrhythmia, wherein an NO donor .s i 1 acniiiiisicral to a =411-1JeL.1 in an amount and manlier effective for the treatment of said cardiac arrhythmia. 1 urthermoie. ilic present invention also relates to an NO donor for use in a method fi..)t the prevention or treatment of a cardiac arrhythmia, wherein the N) donor is administered to a sublet:1 in un amount mid manner effective .tor the treatuiCnt of said cardiac arrhythmia.
In the context of the present invention, the inventors have found that No donors ;ire capable to prevent and treat arrhythmias (see the eXample section).
All features and embodiments defined above vvith respect to the N(.) donot and its formulation and administration also apply to this tnethod or NO oi donor Ibr ue acct it mg to the invention.
The present invention also relates to a method of promoting collateral circulation comprising the step of. exposing a subject lo a therapeutically effective amount ol oil NO
donor wherein the therapeutically effective amount of the N(.-.) donor pommies artcriogenesis sufficient to augment collateral circulation in a physiological or palliolopreol condition.
The term collateral circulation describes the circulation or blood thronvli collateral vessels. These vessels are small arterioles, which are part of o timi( interconnects perfusion territories of arterial branches. In the ease that the main artery itself is not capal)le of sufficiently supplying a tissue, e.g. due to an arterial oeclmioii. these collateral vessels arc recruited and can develcip to large conductance arteries, to bypass the site of an arterial occlusion and/or to compensate blood flow to ischemic territories Supplied by the or insufficient artery. in Ihe context or the present invernion., the promotion (4c:01181er-1d cirvulation occurs via arteriOgencsis, - 2g -According to the invention, the term "physiological condition" denotes imy condition of the subject. which ì noi related to any disease.
Aceordinu to the invention, tlie term "pathological condition-- denoies tiiy condition orltic subject which is related to a disvase.
Preferably, the subject suffers from an arterial insufficiency.
All lealures and preferred etnboditnents discussed above for the method of treating or preventing an arterial insufficiency also apply to the method of- promoting collateral circulat With respect to the aspects defined above where the NO donor k aditiiiiistQred in a manner sufficient to induce arterioone.,;is this manner is preli;rably zin intermitting planner as defined above.
1'he invention is further described hy the allached figures and examples, whit.li are intended to illustrate, but !lotto thrift the i aivention, Short 1)eseription of the Figures Figure I: Course of the ST segment elevation per beat after 17P0 (-final occlusion to induce infarct) of 5- and 10-days-control-groups. ECG graph in middle L1.11.2y iiiiiir=ites 5 DAYS RIP PBS, ri-X: 0.10,1 0.016 111V; ECCi graph in black indicates 5 DAYS
SI IAM
PBS, n=8: 0.134 0.034 niV; ECG graph in lighi grey indicates 10 DAYS RIP
l;us II-7:
0.055 1. 0.033 InV; ECG graph in dark grey indicates 10 DAYS SHAM P1.3S, rv=
i'1).1 24 0.019 inV.
ECU was recorded 91D minutes after EPO. Course of the. ST segment clevatiou per beat at first X minutes revealed no differences between 5- and 10-days sham-groniN
ancl 5-days-RIP-group. Only in the 10-days RIP-group a lower S'I' segment elevation was obseived.
Figure 2: ST segment elevation or 5- and 10-days-control-groups. Column shows SI.
segTrient elevation of 5 DAYS SHAM PBS group: column 2 shows ST segment dc,vation of 5 DAYS RIP PBS group; column 3 shows ST segment elevation or I() DAYS 51 )AM
PBS group:, column 4 shows ST segment elevation of 10 DAYS RIP PBS groiip deviation is indicated in error bars; asterisk indicates significant compared to 11) DAYS
SHAM PBS (nomirifil p value < 0,025); double asterisk indium.: !significant compared to 10 DAYS RIP PBS (nom ina 1 p value < 0.025), Diagram shows mean of ST segment elevation maximum pe' youp. Ariel 5 days there as tio significant difference found between RIP and SHAM. After 10 days in rho RIP r roup ST segment elevation maximum was significantly lower eompared to sham (1') anti RIP control (**) (*nominal p-value < 0.025).
Figure 3: Course of the ST segment elevation per beat after FPO (module I:
Sham Operation without the RIP). E( X.) p,raptt in black indicates 5 DAYS SHAM PBS.
n=8:
0.134 1. 0.031 InV: LeCi graph in light grey indicates 5 DAYS SHAM NTG.11.--7:
() 12.1 0.058 iriV; ECG graph in middle grey indicates 5 DAYS Si IAM NT( i-Pl.....AC113('). ii-6:
1).131 0.043 inV.
The course or the ST segment elevation per heal a ner FPO revealed no diiteiences between sham Ciffil.rol arid treated groups after 5 days.

Figure 4: ST segment elevation (module 1: Sham operation without the KIP).
Column I
shows 5 DAYS SI IAM 111-3S; column 2 shows 5 DAYS SHAM NT(]-Placebo; coiumn shows 5 DAYS SHAM NIG; standard deviation is indicated hy errt.ir bars.
No difICrcnce in ST segment elevation maximum was found between sham control and treated groups.
Figure 5: Course of the ST segment elevation pr beat after ITO (module 7'=
intermittcni (NT(])). graph in light grey indicates 5 DAYS RIP PBS. n -8.
ltP1 0,016 inV; ECG graph in middle grey indicates 5 DAYS NT(1-111.ACLI:i0_ n--();

0.061 itiV; FCC,. graph in black indicates 5 DAYS RIP NTG, n=7: 0.052 + 0.010 iììV
Compared to control treatment with PBS or NTG-Placebo a lower ST ch:vation course was detected alter NT(] treatment days atter RIP.
In the NT(] eroop ("5 DAYS R11" NTG¨) ST segment elevation is significantly decreased compared to the PBS group. There is no significance between the PBS and N
5 PLACEBO-group.
Figure 6: ST segment elevation (module 2: NO intermittent (NI G)). Column l 11(1lNs DAYS RIP PBS-, ci.iluttin 2 shows DAYS NTG-PLACE130; column i shows 5 DAYS
RIP NTCi; standard deviation is indicated hy error bars, asterisk indicates sirniticaiii decrease of ST segment elevation comparc,d to PBS group (nominal p-valuc 0,01?).
Alto- treatment with NIG, thc ST segment elevation maximum was significantly decreased compared to PBS and NIVI-Plaecho treatment 5 days alter RIP
(*nominal p-value 0.017).
Figure 7: Course of the ST segment elevation per beat idler 171'0 ; NO
continuous (ISDN retard)). ECG graph in light grey indicates 5 DAYS RIP 1.13S, 0.104 0.016 mV; EC(] graph in middle grcy indicates 5 DAYS ISDN-PLACEB0, ii 7:
0.110 'IL 0.069 inV; ECG graph in black indicates 5 DAYS HIP ISDN. 0.062 0.027 m V.
Compared to coorot treatment with PBS or ISDN-Placebo a lower ST serincia cleyation course was detected alter ISI7N treatment 5 days atter RIP.
ST segment elevation in the ISDN group ("5 DAYS RIP ISDN") is decreased compared to the PBS group hin there is nu significance ati WCII as between the PBS and ISDN
PLACE130-group.

Figure 8; ST segment elevation (module 3: NO comititions (ISDN retard)).
t.'oluinn I
shows 5 DAYS R.IP PBS; column 2 shows 5 DAYS RIP ISDN-PLACIIIi(); column 3 Shows 5 DAYS RIP ISDN; standard deviation is indicated by emit has.
After treatment with 1S1)N, the ST segment elevation maximum was noii-signitiedinly decreased compared to PBS and ISDN-Placebo treatment 5 days aller RIP (nominal p-value < 0.017).
Figure 9: Course of the ST segment elevation per beat alter 11'0 (module 17 NO
=
intermittent plus ASA). FUG graph in light grey indicates 5 DAYS RIP PBS. it-8: 0 0,1 1 0.016 mV, ECC.i graph in middle p,rey indicates 5 DAYS RIP ASA + PBS, ri 7 tr 0.098 mV; ECCi graph in dark grey indicates 5 DAYS RIP ASA l N P111(114) n-.6.
0.144+ 0.091 mV; ECG graph in black indicates 5 DAYS RIP NI(.; ASA, tr-/ 0M88 +
0.07 1 inV.
ls Ireatincnt with NIG I ASA was compared to with PBS-I-ASA, NIG-PlacebolASA and PBS. In general, all curves overlay at the same range.
ST segment elevation in the group treated with PBS and ASA is higher compared to the PBS control group, but there is mi significance as well as between the ASA
N l'Ci-PLACTBO-group. In the ASA + NICi-group ST .st:grric:iit I:it:Nation is decreased compared to the group treated with ASA and PBS.
Figure 10: ST segment elevation (module 4: NO intermittent plus ASA). 'oliimit 1 shows 5 DAYS RIP PRS: column 2 shows 5 DAYS RIP PBS+ASS; column 3 shows 5 DAYS
KIP N'Ri-PLACE1-30; column 4 sillows 5 DAYS RIP NIG-PLACM)+ASS, column 5 shows 5 DAYS RIP NI'Ci; column 6 s;hows 5 DAYS RIP NTG.i ASS: standard deviAnon is indicated by error bars.
Treatment with NTG+ASA WUS compared to PIIS+ASA, NICI-PlaccbulA SA and PBS.
Furthermore, all ASA groups (PBS+ASA, NTO-I'lacebo+ASA, N'I(J 1 ASA) 1+...re compared to their controls (PBS, NIG-Placebo, NI(J). No significant dilierenees were detected.
Figure I ArrhythmiaN
during FPO (module 1: Sham Operation (without tho RIP)) Numbers ols columns are given in consecinive order of thr I.:Annus in group !Vb. Column l shows 5 DAYS SHAM PBS; column 2 shows 5 DAYS SIIAM N.Hr-PLACkliO;
column 3 shows 5 DAYS SHAM NTG.
In iiCCOrdanee v ith Lown clrissi íiciI iun. all shan't groups were predt..miiiiantl scaled into grade I
In the "5 DAYS SHAM PBS" group 87.5% of the rats have class 1Vh aiillytlimitkt and 12.5% class IVa. In the "5 DAYS SHAM NT(i-PLACEBO" group 1.43. i% have IVb arrhythmias and 16.7% class IVa and in the -5 DAYS Si IAM .NTG" group 145.7"ti have IVb ;:trrhythmitts and 14."P/n class 111a arrhythinias.
Figure 12: A rrhythmias during FPO (module 2: NO intermittent (NTG)). Numbers of columns are given in consecutive order of the columns in g MUT) I VI). Cohami I .illokk.s 5 DAYS RIP PliS; Column 2 shows 5 DAYS KIP NT(J-PLACE130: Column 3 shows 5 DAYS Rlp While arrhythmias in both control groups, PBS and NITI-Placebo, were predominantly sealed into grade IVa, the NIci treated group was more often scaled mitt r.radc 0 In die "5 DAYS RIP PI3S" group, 75.0% the rats have class IVb arrhythinias, 17 5%
IVa and 12.5% class 0. Regarding the "5 DAYS RIP NT(-PLACT.110" croup, (4.;
or the rats showed elass IVb arrhythmias, 16.7% I V a and 16.7% class 111b arrh)thinias Interestincly, the "5 DAYS RIP NIG" group shows 42.9% class !VI) arrlo,thmias and 57. I% class 0 arrhythm Figure 13: Arrhythmias during FPO (morlide 3: NO continuous (ISDN retaid)).
Numbers or columns arc given i .11 consecutive order of the columns in group IVb. Column 1 shows 5 DAYS RIP PBS; eolumn 2 shows 5 DAYS RIP ISDN-PLACEBO; column 3 shows 5 DA S RIP ISDN.
Irl all groups, arrhythmias were similarly more often scaled into gradc I Va.
In the ".5 DAYS ISDN-PLACIA30" croup, 57.1'4) of the rats have class 1 V h arth)itinitas, 14.3% class IVa and 28.6% class íllb. Mc ='5 DAYS RIP ISDN" group shows fess sovcre arrhythm iur with 57.1% class IVb, 28.6 c,',/0 class IV?, and 14.3% class 0 arrhydnuitri Figure 14: Arrhythmias during F.PO (module 41: NO intermittent plus ASA).
Numbers 0i.
columns are given in consecutive order or the columns in group IVb. Column I
shows Ai DAYS RIP ASS + PBS; column 2 shows 5 DAYS RIP ASS + NIG-PLACA-111J: toluirm shows 5 DAYS RIP ASS +

Arrhythmins were similarly scaled more into grade Wa iii 111 groups.
In the "5 DAYS RIP ASS + PBS" group, in llic group treated µ4'ith AS N
PLACEBO and in dic -5 DAYS RIP ASS + group 83.3%
of the rats posses class IVb arrhythmias nndI 6.7% elasti I Ia.
Figure 15: VPB-Score. Column 1 shows SHAM PBS; Column 2 shows 511.AM N
Placebo; Column 3 shows SHAM NFU; Column ,1 shows RIP PHs; column 5 shows RIP
NIC1-Placebo; Column 6 shows RIP N FG; Column 7 shows RIP PBS; Column 8 shows RIP ISDN-CC; Column 9 shows RIP ISDN; Column 10 shows RIP PBS + ASS. Column 11 shows RIP NICi-CC ASS; Column 12 shows I(IPNTG + ASS.
The VI3P score shows the percentage of each Lown grade of every group. The Sham groups have higher VI-IP-scores, Compared to the group with an ischemic plott.lei.il (con(rol group, treated with PBS), more rats show severe arrhythrnias. The 0:eminent %k reveals reduced arrhythinias. and consequently a lower: VPB-Score. The VP1i-SLoreiit groups treated with ASA alum: or NICI + ASA is 14lier compared 10 the controls (trea(ed with PBS).
Regarding the pereentagc.; or each Lown grade of every group, n VRI) Nuork.
call be ascertained. The more animals show ti higher grade, the higher is the v1-11 Figure 16: Infarct size of 5-days- and I 0-days-control-groups. Column I shows SHAM PBS, ri-8: 13.36 5.22%; column 2 shows 5 DAYS RIP PBS. n-8: 11,05 5,12%; column 3 shows 10 DAYS SF1AM PBS, n-7: 13.7 I - 0.06%; column 1 5ho' s DAYS RIP PliS, TI6: 6.57 3.26%;
standard deviation is indicaled hy error bars:. i,isterisk indicates significant compared 10 10 DAYS SI IAM PBS (nominal p-ya I tic 0 013).
After an ischernic protocol of 5 days then: is no significantly smaller a-IL-act size measurable, but after a RIP of 10 days the intruded area is significantly deLrcased (nominal p-value < 0.013).
Attu 90 minutes of LAD occlusion and 20 minutes teperinsion, Mind siie anal red, The -10 DA YS RIP PliS- group has a significantly smaller farm cl ;irea t'ont pared to the "10 DAYS SHAM PBS" Brom). There is no significance between both 5 DAYS r.roup Figure 17: Inthret size (module 1: Sham Operation (without the RIP)). Column I
,õ;litlws S
DAYS SHAM PBS, n-8: 13,36 . 5.22mV; column 2 shows 5 DAYS SI !Aryl N

P1 ACEB0,1:16: 11.21 + 5.79 rriV; column 3 shows 5 DAYS SHAM NIG, n T 14.09 5.18 mV; standard deviation is indicated by error bars.
The infarct size shows no difference between the SHAM groups.
There is no significance between the three SI IAM..groups, Figure 18: Infarct size (module 2: NO intermittent (NIG)). Column I !.;how.; S
DAYS RIP
PBS, ii-8; 11.05 J.. 5.12%; column 2 shows 5 DAYS NIG-PLACEBO: 11-6, 9.80 F 6 mV; column 3 shows 5 DAYS RfP i, 11-7:
3.61 .1- 2.08%: standard deviation is indicated by error bars, asterisk indicates signilleant L:ompared to 5 DAYS
RIP PBS
(nominal p-value < 0.017).
Hie infarct size is siguilicautly smaller after treatment with NIG compared lo controls (treated with PI-IS) (nominal p-value < 0.017).
Compared to the "5 DAYS RIP PBS", a significantly smaller infarct arcaI
.8 008ervuri 111 the "5 DAN'S IMP NIG" group. There is no signiticanco between llw PBS imd N
PLACEBO-group.
Figure 19: Inrarel sitc (module 3: NO continuous (ISDN retard)). Colwyn' 1 Si DAYS RIP PBS, n-8; 11.05 5.12%; column 2 shows 5 DAYS ISDN PLALF.11(), n-6.
9.97i 3.6S %; column 3 shows 5 DAYS RIP ISDN, n-7; 7,59 .4.indard deviation is indicated by error bars.
The infarct size after treatment with 1SDN is smaller compared to controls (treated with IBS or ISDN-Placebo), but there is no significance.
'Hie infarct size in the ISDN group ("5 DAYS RIP ISDN") is smaller compared to ilic 11.3S
group, as well as the ISDN-Pl...A.CEBO-group.
Figure 20: Infarct size (module 4: NO intermittent plus ASS). Column l shows 3 IMYS
RIP PBS, u-8; 11.05 .1 5,12%; Column 2 shows 5 DAYS RIP ASS PBS, ti¨b: 12.51 +.
3.05%; Column 3 shows 5 DAYS NIG-PLACEBO: 11-6; 9.80 6.79 "4: Column .1 shows DAYS RIP NTO-1)1 ACEBO i ASS, n=6: 13.92 + 1.71%; C011.111111 shows DAYS
RIP NIG, n-7: 11.05 5_12%; Column 6 shows 5 DAYS RIP NIG + ASS. n 6.. 13.00 1-3.82%;standard deviation is indicated by error bars. asterisk indicates sienilWani compared to 5 DAYS RIP NIG (nominal p-yalue 0.017).
The infarct size after treatment xvith NIG plus ASS is significantly increased compnred to the trealrnent with NIG alone (nominal p-valuc . 0.017).

The infarct size in the c,rotip treated with ASA (-5 DAYS ASS PBS-) is minimally increased compared to the PBS control group, is well as the ASS .1 NTG-PLA(.1;130-group. There is no difference between the ASS i NTG-group and the r.rottp treated with ASS and PBS. However, the infarct area in the NTG group is signiticandy compared to the ASA + NTO group.
Figure 21: TTC-staining,. The pictures 5howti slices of three levels. Int-au:tn.! l issue Aains pale-white salve they lack the enzymes with which the ITC.' Naos_ flitis the iirc=as oI
necrosis $trc clearly discernible and quantifiable.
1(i Figure 22: Collateral diameters of Ft..01 (module I. Sham Operation (without the RIP)).
Column 1 shows 5 DAYS SHAM PBS, n=3: R2.7 3,7 pill; VOIURIll 2 ShOWS YS
SHAM NTC;-PLACT:14(..), n-3.. 89.6 um I 10.6 um: column 3 shows 5 DAYS Si IAM
NTG, n-3: 86.8 i 9.0 t,trn; standard deviation is indicated by error bars IS l'here is no growth or collaterals and no differences measurable between the SHAM
groups.
There is no significance between the three SHAM-groups, Figure 23: Collateral diameters of ROI (module 2: NC) intermittent (NT(i)).
Column I
20 shows 5 DAYS RIP PBS, n=3: 129,8 6,9 pm; column 2 shows 5 DAYS RIP NIG
PLACEBO: n-3; 127.0 1 12.1 tttn; column 3 shcivvs 5 DAYS RIP NIG, ii . 158 I
0.2 gm; standard deviation is indicated by error bars, asterisk indicates significant compared to 5 DAYS RIP 'NT( i (nominal p-value 0.033).
Diameters of collaterals are signilicantly increased by treatment with N
compared to 25 controls (treated with PBS or N'IT3-Pkicebo) (nominal p-value <
0.(13.1).
Compared to the "5 DA YS RIP PBS", the diameters of the col laterals iii tin:
It()I in Mc "5 DAYS RIP NTG" group are sig,nificantly ineretised. There is no (filleiroce behAecri the p135 and NTO-PLACEBO-group.
30 Figure 2,1: Collateral diameicrs oi" ROI (module 3; N() continuous (ISDN
retard)). Column shows 5 DAYS KIP PBS, n-3: 129.8 6,9 p.tni, column 2 shows 5 DAYS ISDN-PLACEBO, n-3: 133,0 l 11.5 um; column 3 shows 5 DA y S RIP ISDN, Ir. 1: 1,18.2 / 1.3 ilm; standard deviation is indicated by ell or bars, No ditTercriccs are measurable in the diameter of collaterals alter treatment with ISDN oi 1SDN-Placebo.
The diameters of the collalerals in the ISDN group ("5 DAYS RIP ISDN") are enhanced compared to the PBS group, as well as compared to the ISDN-PI .A(14.11() group.
Figure 25: Collateral diameter of ROI (module 4: NO intermittent plus ASA).
Column 1 shows 5 DAYS 11.111 PBS, 11-3; 129.8 6.9 mm, column 2 shows 5 DAYS RIP PBS
.4- ASS, n=3; 1(Y2.5 'LE 8.0 um; column 3 shows 5 DAYS RIP Ni't i-PI At.'17;130: n [2/ 0 12.1 i.tm: column 4 shows 5 DAYS NIG-PLACEBO + ASS, n-3: 97.1 8.01 um. column 5 shows 5 DAYS KIP NIG, n-3: 158.4 9.2 1.tm: column 6 shows 5 DAYS RIP ASS
124..1 + 5.6 priy, standaid deviation is indicated hy error hors, one asterisk indicates significant compared to 5 DAYS RIP PBS (nominal p-valne < (1.0;)):, klutible asterisk indicate significant compared to 5 DAYS ItIP ASS .1 NIG (nominal 0.039).
Diameters of collaterals arc significantly smaller after treatment with A.SA
compared to controls (treated with PiiS). Aii addilional treatment with NT(.; abolished the inhibiting effect of ASS. N1'(-treatment alone shows significantly increased(. 11.ametei compared to controls (treated with PBS) (nominal p-value 0.039).
The diameters in die group treated with PliS and ASS ore signilleantiv smaller Lompared to the PBS control group as well as the ASS + NT(i-PLACEBO-group. but there no siiiitivance, In the ASA l vro -group diameter are 1 .nereascd compared to the group treated with PBS and ASA.
Figure 26: MicroCT imaging of the "ItOl": (A) "5DAYS SIIAM (1-1) "5DAYS
SHAM NIG"; (C) "5DAYS RIP ISDN'', (D) "5DAYS RIP PBS", (E) "!!,DAY!-, it ii) NIG"; (F)"5DAYS RIP ASS I PBS; (Co -5DAYS RIP ASS l NIG", tin: pictures show the growth of the collateral diameter in the region of interest t-sv the ischemie treated with PBS (D), NTG (E), or ISDN ((,') compared to SIIAM =
treated with PBS (A) or NI(.i (8). Inhibition of collateral growth by treanneni with ASA
(F) is partially abolished by additiolikil treatment with NTC (Ci).
Figure 27: Study Flow Chart. Duration of the baseline period is estimated to he approximately 7 weeks. Duration Of the intervention period will be six weeks, 1 he follow up period will include all immediate investigation (one day up to maximal three days aftet the intervention period) nod i oug-lerrn follow up investiolion monlli after interi,enluon period).

Example 1 Pre-Clinical Study 1. INTRODUCTION
One important mechanism of arteriogenesis ; I 1;
.s .11c.tic..on oí shear sttess -tcross recruited collateral arteries.
NO plays a fundamental rule in this scenario, since it regulates the yasotlilatory capability of the artery as well as therapeutic proliferation aspects on the smooth muscle cells of collateral arteries.
15 Here we evaltiaied the effects of Nitrolingual Am Spray ((i. Pohl-Boskamp Co.K(i, Holiodockstedt. Germany; l.S_ American brand name Nitrolinguar Puinp,iprily) in a unique non-myocardial infarct arleriogcnesis mOdel. Collateral growth in Ihis motto' is induced via repetitive occlusion of the left anterior descending coronary arter (I At)).
Intaret sia in these animals was measured as the endpoint al the end of the ewerimeni.
20 Thus, no interference between myocardial infarction and arieriogonesis has weaken the experiment. Moreover we evaluated the effect of acetyl salicylic acid (ASA) in this model of repetitive coronary occlusion as a possible inhibitor cif arteriogericsis.
We el/nth:lied whether a concomitant application of NC) (intermittent use ()I' ni(rog15.cerin) may compensate for this neg,ativc efIcel or ASA.
2. MATERIALS AND ME'llIODS
1.1. Animal Preparation Male Sprague-Dawley rats (300 g body weight at study start: ¨ 182) are ivied for experiments. For surgery (day 0), rats are promedicated (ketainine 50 mg/1W
phis sylmeine 11 mg/MI intraperitoneal) and intimated. Oral intubation (14-0 polyethylene inhint:,) is done under direct observation of the vocal cords with tin oloseope. General anesthesia is introduced and ma ha:lined by isonurane inhalation ( l .0% to 2.0%, with I
00ck, 35 Body temperature is controlled at 37 'V by an electric heating table.
Suigery is performed using aseptic technique. The animal is initially placzed uii iis dorsal side arid eiliancotis clips are Fixed. With a BioAmp differential amplifier coupled to a Poworl.i.th data ai.gliciaiori system (AD Ins(ruments) F.C.:(i parameters (heart rate) are monitored and recorded during surgery. The heart is exposed by left thoracotomy. A mini-pneumatic snare oc.chidet (we the Mini-Pneumatic Snare Oeclilder section for details) is implanted around the mid to proximal let) anterior descending coronary artery (I .A1)). Confirmation that the ocu hider is functional, i.e., producing myocardial ischemia, i deltmnined initially by observation of blanching and hypokincsis of the left ventricle (LV) arid by obsei vat ion of the electrocardiogram (ST elevation) during inflation. Rats are randomly divided into 4 therapeutic moduls:
Module 1; Sham Operation Module 2: NO intermittent (nitroglycerin) Module 3; NO continuous (retard preparation of isosorhide tiiniirate) Module 4: NO intermittent plus ASA
Alter instrumentation and measurements, the elicst is closed under positive.
end -expiratory pressure, and the thoracic cavity is evacuated of air. The occluders an:
tunneled subcutaneously arid exteriorized between The scapulae. These catheters are proteeted b} a stainless steel spring coil connected to i ring, that s secured subcutaneously between thc scapulae. Alter the surgery, analgesic (buprenorphine 0.05 mg/ki!, S(:) and antibiotic (cnrofloxacin 10 ing/kg SC) are administered. Rats are observed in a rocoverv 1:0:2k: tor 7 hours; and then transterred to the animal can: facility vvherc they are continuous!), monitored by technicians. For 3 clays after the surgery, huprcnorphioe (0.5 Ing/Itg S( ) is taken for pain. On the third clay alter the surgery (day 3), ischemie protocol is started. Alter 5 resp. 10 days (only in module lA and 213) of the experimental protocol (day 3 n.Np day 1.3), the rats are anesthetized, and the chest is opened by mitl thortieotomy.
In the olio o-C1' group, the hearts are immediately excised. For the final infarct size detection the I Al) vill be permanently occluded (linal permanent occlusion, F1'0) and infarct Ate NN, ill nicam.ircd via TTC: staining.
1.2. Mini-Pneumatic Snare Oceluder for Rat Heart A mint-pneumatic snare occluder is used consisting of a mini-balloon, sheath tubing, suture, and catheter. The balloon (7 rom long) is made of soft latex nit,mbiline and is sufficiently pliable to give negligible physical force on the coronary vessels during balloon donation. The balloon is mounted within an umbrella sheath (3.2 or 4.1i Hun in diameter.
$5 12 rnm in length; protects the balloon from lihrous infiltration).
Pirilenc (5.--0) ipat,sed around the 1.A1) and attached to the sheath, securing the oecluder to the heart, No (hal iScheinia is produced hy balloon inflation. Inflation volume is small (O..' ro 0.2 nil, air), 1)111 occlusion occurs by 2 physieal actions: "crimping" the LAD toward = 40 -upward/outside and compressing the I,AI) by Ilic inflated balloon/sheath. I h I
.11..00ri is connected to a catheter (PE-50) that is. exteriorized. Balloon inflation and deflation lire controlled from outside the rat cage.
1.3. Measurements of ECG Parameters In ail four modules (I-4) we will at the beginning (day 3) and the end (day 8 resp clay 13) of the experimental protocol (RIP) perform Ihe coronary occlusion for 40 seconds (equivalent to an occlusion in the, RIP; see page 6) and measure ECG
examine the heart rate and ST elevation.
1.4. Coronary Microvascular imaging With Micro-CT
In addition we propose to use Micro-CI as a further endpoint to One group of rats (3 rats of each group in each module; total oll(i rats) is prepated lot cot unary vascular visualization via micro-CT. The coronary circulation is filled with contrast medium (yellow microlil) by modification of the methodology for micio-CT study in the rats_ The viscosity of the contrast medium enables filling up to coronary uloriolar with no or minimal filling, of capillaries. The excised heart is immediately cannot:lied by an aortic cannula, and coronary circulation is perrused rettogyndely ni min lit!, A perInsate (25 'V lo 27 C saline with 2% procaine) is used to avoid myocArdiol iitutabolic contraction and maximally dilate the coronary vasculattire. Polyethylene tubine is inserted into the I.V via a left appendage through the mitral valve to unload the I...V. Warmed contrast medium (42 'C) is injected at a pressure of 85 rnirrH1. kir 1 minutes while perfusion pressure is monitored. The heart is cooled hy immersion into cold saline (0 to 4 C) until the (yellow microfil) solidified. !lien, the head is rctuoved and lised in ,I%
paraformaldchyde solution (4 "( ) overnight. Whole hearts are uscd for micro-CT imaging of coronary collateral growth. The coronary vasculattire is visualiAed with micro CT, In brief, the whole heart is scanned in 3o I' increments around 360n about its apex-to-base longitudinal axis. The spatial resolution selected in the present study has ail 18418418 ut) voxel size to focus on the site ot collateral vessels and 10 minimize die signals from smaller vessels. Finally, ( ' l liia are reconstructed as 31) images. I he main purpose ol these images is to establish the presence or absence of aderial-arterial anastomolic connections. Collateral vessels.
i.e,, arterial--15 arterial anastottiolie connections, are counted by independent observers for the groups.
1.5. Experimental firotocnI

The repetitive ischemia protocol (R1P) is introduced 1)y automatised inflation of the oculinik:r using the following proincol: 40 seconds of occliAion every 20 1111111110'. for 7- hours 20 minutes, followed by a period of "rest- (deflation) for 5 hours -10 minutes. .1-11i5 8-hour set is repeated .3 times a day for 5 resp. 10 days (only in module IA
and 2A) The 1,A1) is 0i:eluded automatically. by remote inflation o, deflation through the catheter hi sham rats (see module 1), Ihe balloon is implanted, but RIP is not applied.
Rats under RI
protocol arc randomly divided into the thrce modules 2, anti -.1.
1.6. Infarct Size 1)citction O
Iniart.:1 size will be detected by ITC staining after final permanent occlusion. A fief resp.
l0 days (only in module IA and 2A) of the experimental protocol, the occluder r; inflated permanently for 90 minutes. Infarct size will be measureLl by Tit staining (11-10,'group), Therefore rats are anaesthesized and undergo again the ECG recording, lo the 15 occlusion (S' I elevation) and to calculate li:C( i parameters and the numbers of In animals without collaterals, coronary occlusion C211.1SCS deterioraiion of systemic hemodynamics and arrhyllanias. including premature ventricular contractions, µ,entrictilar tachycardia, and ventricular fibrillation; in animals with well developed collatcrals. no such adverse effects are noted.
The chest is opened by mid thoracotomy. The heart is immediately exciscd ;.tite.1!.ectiout,1 from ape"; to base in 2-nim-thick transverse slices parallel to the itirioventricalar Slices are incubated with 0.0k) mo1/1 suction) phospliale buffer coniainimg I
0"0 if ipli<liy1 tetraiolium chloride (TTC) and 8(!fo dextral.] Iltr 20 rnin. at 37'('. Slices tile ['Red in I
forimildc.itydo and then photographed with a digital camera mounted on ci stereomicroseope. The infarcterl size is quantified using a coinpulcrized planmetric program (Adobe Phutoshop). 'Mc MI-art:led area is indentified as the 'ITC-negative tissue and is expressed as a percentage of the area of the left ventricle (I,V).
:to 1.7. Details Regarding Testing Compounds ASA Merck Chemicals NO intermittent (NTG) nitroglycerin solution-. Nil 110.0".
Spray, G. Pohl-Roskamp c'S K(1.
lionenlockstedt, Germany NO continuous (ISI)N retard) isosorbide dinitrate retard pellets;
Nitrosorbonli) retard; G. l'ohl-Boskamp (..imbl I
Az Co. KG, Hohenlockstedt. ( tormaii) Carrier Compound fOr NO intermittent (NT(3-Placebo) placebo solution of Nitrolingual akin" Spray.
Pohl-Boskarrip GmbH & cii KG, Holtentockstedt, Germany NO continuous ('.itrrier Compound (ISDN-Placebo) nontral pelletsol Nitrosorbon': rciard G Pohl Boskamp GmbH & Co. KG. I lolientoukstedt, Ciermany Control buffer PBS (phosphate buffered saline) LH. Route, Tirnepuint and Concentration of Delivery to Aniinal Ali medication (ASA and NTG arid ISON retard) is given uplront to a followirm ok.clusion time oldie device. The control buffer (PBS) is given in the same way prior to the two occ I ilSions.
NO intermittent (NTG) A. new test solution is prepared every morning at eight o'clock. The solution is taken from the vials via syringes.
NO intermittent (NTG) is given twice a day with a time interval of !I hours Due 10 (he chronic instrumentation of the rats and lo avoid further stross, NTG p.iven via buccal application. 50 pl of the daily prcpared test1 ti sour_on eUn1iiiuiiij.i 3.3 nitroglycerin (equivalent to n human dose. 010.8 mg) is administered per applkalion in module 1, 2 and 4, ihe tiiiic point of application is directly upfront to balloon intlation at 9 a.m. and 5 p.m., thus with rmiKilnal effects on recruited collateral arteries This concentration is taken from the above mentioned retieiion vials right before administration.
Carrier compound solution is served as a stock solution for the preparation of the test solution.
Carrier compound for NO intermittent ("NIG-Placebo) Carrier compound is administered in a way identical to NO inicrmittent.
NO contintions (ISDN retard) The medication for prolonged NO delivery (retard preparation isosorbide dinitiate acting nitrate ISDN) is delivered as retarded pellets 1x per day, For the retard preparation ISDN a dosage of 2.6 mg ISI)N/ral is chosen.
Therefore I
pellets are suspended in 0.5 ml drinking water and are applied via gavage at 9 a.m every morning (equivalent of a human dose of 2mWkg/liW).
NO continuous Carrier Compound (ISDN-Piaccbo) Carrier compound is administered in a way identical to NO continuous.
No intermittent talus ASA (acetylsalicylic acid) F.:very morning at 9.30 a.m. 2.22 mg ASA per rat is given dissolved in 0.'.) nil drinking water via gavage directly into the stomach.
The ASA concentration of 2.22 me, ASA per rat (6.31 ii/k) correlates with the human dosage of 100 mg/day.
.5 1_9. AnittuilS and Groups 10 rats per groups (FPO- final permanent (reclusion to induce infarcts) Group d: 3 additional animals are treated with the same medications and lnzation .,cheme like the corresporidinu groups a, h and c, btit without 1,130. These 9 EIlliMaIS pet Modulc an:
uscrl for micro C:f images.
Module 1: Sham Operation (without the A. Control buffer (phosphate buffered saline PBS) with functional ITO
for infaict site detection 11-20 1. n=10: "5 DAYS 511AM P1.5S"
2. n-10 "10 DAYS SI IAM PBS"
13. Carrier compound without NO plus functional FPO for infarct size deleci ion 11-10: "5 DAYS SHAM .NTG-PLACEBO-C. NTG with functional ITO for inlliret size detection ri-10: "5 DAYS SI IAM NITG-D. A 1.) ri-3 A2.) n=3 B) n=3 C) n=3 for micro C.1 images n=12 total: n=52 Module 2: NC) intermittent:
A. intermittent control buffer with functional FPO for infarct size detection n-20 1. n10; "5 DAYS 1<11' PBS"
2. n=10: "1() 1)A YS RIP PIS' B. intermittent Carricr compound plus functional FPO for inlarct size derkm ion n10: "5 DAYS RIP NTG-PLACEBO"
C. Intermittent N1'ì with functional FPO for infarct S17C detection n-10: "5 DAYS RIP NTG"
D. A I .) n-3 A2.) n===1 R) 11-3 C) n=3 for micro CT intaues n-12 total:
Module 3: NO continuous:
A. Continuous Control buficr (drinking writer) with functii.ma I fP() for in tarc detection (11-= 10); "5 DAYS RIP DW"
B. Continuous Carrier compound pins functional FPO Cor infarct size detection 1.1,111: "5 DAYS RIP ISDN-PLACI7:130"
C. C'ontinnous NO functional FPO for infarct sit: dcieetion n=10: "5 DAYS RIP ISDN"
D. A.) n=3 B.) ii-3 C.) ii-3for total: n-(39) Module 4: NO intermittent plus ASA:
A. InIermittent Control buffer plus ASA with functional FPO for infarci size (filet:non 0: "3 DAYS RIP Pliti+A SA"
B. Intel:1111MM NO Carrier compound plus ASA plus functional FPO íin inforcl -;ize detection n-1 0: "5 C)AYS R IP NTG-PLACLBO l ASA"
C. Inlenni0ent NTG plw,µ. ASA runclionul 11)0 for ifllrc1 size dtcctioii ii-10: "5 DAYS KIP NTG+ASA"
D. A.) 11-3 13.) n=3 C.) n=-3 for micro (.7.1' images total: ii-39 3. RESULTS
3.1 Final Permanent Occlusion I.Al) occlusion allowed a prospective study of the function of collateral vcsst.61s. Such vessels can protect myocardial tissue at risk of isehernia ater calumny( occlusion, At the end of the RMI protocol we performed the permanent 1.A1) occlusion in one subgroup of all groups and measured 1C:Ci parameters to examine Si wginent elevation in and ventricular arrhythmias. After 90 minutes of permanent occlusion e determined the intareted arca.
3.2 ECG Analysis Is Fleetrocardiographic manifestations of isehernia initiated by LAD
occlusion are less pronounced when collateral vessels are present.
3.3. ST Segment Elevation During LAD occlusion there is an inverse correlation between the inagninidc of ST
segment elevation and the extent of the collateral supply.
Collateral function is an important determinant of die direction of ST segment respoip;c ischemia during acute coronary occlusion. Reversible ST segment elevation doting acute LAD occlusion is related to inadequate collateral arterial function. In patient:, with reversible ST segment depression, cur-unary collalcral function appears to be better and, as a consequence, shows less isehemia results, During a 90 minutes Occlusion the ST segment elevation in the "10 DAYS SI IAM
PBS- is sie,nifienntly higher compared to the "10 DAYS RIP PBS" group DAYS SHAM. n-7:
0.039 inVy., 10 DAYS RIP, ri-7: 0.055 1 0.033 mV). In coniriv:t, S
lst.T,riielit elevation in the "S) DAYS SHAM PHS- is similar to the "5 DAYS RIP PBS-1_2.10iip (I
DAYS SHAM, n-8: 0.134 0.034 inV; 5 DAYS RIP. n=8; 0.104 0.010 ItiV1(1.11t.s. 1 and 2).
Module 1: Sham Operation (without the RIP) There is no significance betAveen the three SHAM-groups (5 DAYS SI1AM PBS ii-8:
0.1:M .1- 0.034 mV; 5 DAYS SHAM NTG-PLACIAl0, ti-==6: 0.131 0.04 ; inV 5 DAYS
SHAM NTG, n-7: 0.124 0.058 rnV) (Figs. 3 and 4).
Module Z: NO intermittent (NTG) In the NIG group (-5 DAYS RIP NT(jì") ST elevation is significantly decreased compared to the PBS group (5 DAYS RIP PBS, n-8: 0.104 0.016 triV; 5 DAYS RIP
NT(, 0.052 0.030 inV), There is no significance between the P135 and NTG-PI AO:BO-group (5 DAYS NTG-PLACEBO: n-6; 0.096 0 061 ItiV) (,l is. 5 kind

6), Module 3: NO continuous JISDN retard) ST segment elevation in the ISDN group ("5 DAYS RIP ISDN") is dcerezised compared to the PBS group (5 DAYS RIP PBS, n-8; 0.104 0.016 HIV-, 5 DAYS RIP ISDN, n--7;
0.062 0,027 rtiV), but there is no significance as well as between the PBS and ISDN-PLACEBO-group (5 DAYS ISI)N-PI .A( 1-140, n=7: 0.110 L 0.069 inV) (Figs, 7 Module 4: NO intermittent olus ASA
ST segment elevation in the group treated with Pl35 and ASA is higher compared to the PBS control group (5 DAYS RIP ASA + PBS, n-7: 0.138 i 0.098 rnV; 5 DAYS RIP
Pl3S, n-8; 0.104 (1.016 mV), hut there is no significance ie.; vvc11 us belWeell the PLACA ,I30-group (5 DA YS RIP ASA + NTG-PLACE1-10, ti-6; 0.144 L 0.091 m V).
In the ASA + ST
elevation is decreased compared lo the group treated with ASA :Ind PBS (5 DAYS RIP N'IG + ASA, ii-7: 0.088 0.07 I mV) (Figs. V and 10).
3,4, Ventricular Arrhythmias The importance of ventricular premature beats (VPBs) results twin Owir pothIe ntiSOC iatiOn with an increased risk for cardiac sudden death, V PHs were stratified ding to the LOY4)11 classification. A high LOW/1 grade has hen shown to piedict mortality alter i;ieute myocardial infarction.

Grade 0: 110 ventricular eetopic beats Grade I: OCCaSional, isolated VPB
Grade 11: frequent VPH I/min or 30/h) Grade III: multiform VI'13 (a) VPB
(b) Bigenimus Cirade IV: repetitive VPB
(o) Couplets (b) Salvos Grade V: Fmrty VPB
Module 1: Sham 0..pitration (wi(hout the RIP) lïi the "5 DAYS SI IAM PHS- group 87.5% a the rats hav l:is lVt, arlivtinnids and 12.5% class I Va. In the "5 DAYS SHAM NTG-PLA(.-.Tlit 3" group 83.3 , havclVb arrhythmias and 16.7% class IVa and in the "5 DAYS SHAM NIG" 85.7').i., have 1Vb arrhythmias and 14.3% class Illaarro Itirmas yta õ. ig, 111 -Module 2; NO intermittent (NTG1 In the "5 DAYS RIP PBS" group, 75.0% of the raN have class [VI) arrhydnnias, 1Va and 12.5% class 0. Regarding the "5 DAYS RIP NIG-131,ACIAl()" group.
06.1".4, el the rats showed class IVb arrhythinias, 16.7% IVa and 16.7% class 1Ilb Interestingly, Ott: -5 DA.YS RIP vrc- group shows 42.9% class IVh arrh),1hnth.h and 51. I u/rk c kiss arrhythmias (rig, 12), 31) Module 3: NO continuous (ISDN retard) In the "5 DAYS 1SDN-PLAC131.30" group, 57.1% (of the rats have class IVI-) arc F1, ilitnias, 14.,Vf/i) class IVa and 28.6% class 111b. The "5 DAYS RIP ISDN- group show;
less :se).ere arrhyllimias with 57.1% class 1Vb, 28.6 % elasK IVa and 11.3% class 0 arrhyllimi:ps (Fig.
13).

Module 4: NO intermittent plus ASA
In the "5 DAYS ItIP ASA t PBS" group, in the groin) trc.!ated with ASS -F. NTG-PLACER() and in the "5 DAYS KIP ASS + NICF grrnip 83.3% a the rats Posses class 'VII rrhythmias and 16.7% class Illa.
Rogarding the percentttge of each Low!, efrido of every group, a VII' icore can be ascertained. The non: animal show a higher gradeõ the higher is the VI3l1 ,;core I S) Figure I 5: VPB-Score PK-group Score .Module 1 SHAM PBS _______________________________ 5.88 SHAM NTG-PLACEBO
SI IAM NTG 5.77 Module 2 RIP PHS 5.10 RIP NTG-PLACEBO 4_84 RIP N-Ri 3,60 Module 3 ____________________________ RIP PBS 5.10 RIP ISDN-PLACEBO 5.29 KIP ISI)N 4.57 Module 4 RIP ASA .t PBS 5.50 RIP ASA + NTG-PLACEBO 5.50 RIP ASA + NTG 5.50 Table I: VP13-Sore 3.5. Infarct Size After 90 minutes of LAD occlusion and 20 minutes reperfusion, ittrarct size was amity/v(1 The "10 DAYS RIP PBS" group has a significantly smaller infarct area compar,...d to the -10 DAYS SHAM PBS" group (10 DAYS KIP PBS, ri-6: 6.5/ + 3.201?==,_ 10 DAYS
SHAM PBS, n=7: 13,71 6.06%). There is no sietiificance between both 5 DAYS
ttroups (5 DAYS 51.1AM PBS, 13.36 + 5,22%; 5 DAYS RIP PBS, n=8; 11.0 + 5 12",,i) (lig.
16).
Module 1: Sham Operation (without the RIP) There is no significance between the three SHAM-volips (5 DAYS SIIAM PBS. n 1136 .1.. 5,22 my-, 5 DAYS SI IAM NTG-PLACI-=140, 11-6v 14.21 -f= 5.79 mV: 5 I
S
51IAM N'1(i, n-7: 14.09 + 3.18 mV) (Fig,. 17).
f5 Module 2: NO intermittent (NTG1 Compared to the "5 DAYS RIP PBS", a significantly smaller infarct (111N1 is observed in the "5 DAYS RIP NTG" group (5 DAYS RIP PBS, n=8: 11.05 5.17%; 5 DAYS RIP
NT(J, n--=7: 3.61 + 2.08%). There is no significance between the PBS ttid N
PI ,ACEBO-group (5 DAYS NTCi-PLACEBO! n=6; 9_80 6.79 in V) (Fig. 18).
Module 3: NO continuous (ISDN retard 'rho inrarc1 size in Ihe ISDN group ("5 DAYS RIP ISDN") is smaller compared to the PBS
group (5 DAYS RIP PBS, n=8: 11.05 1 5.12%; 5 DAYS RIP ISDN. rt=7: 7,59 +
as well LIN the ISDN= PEA( 1-1-10-group (3 DAYS ISDN-P1,ACEBO, 11-6; 9,9 /
.()'") (Fill,.19), Module 4: NO intermittent _plus ASA
The infarct size in the group treated with ASA c.5 DAYS ASA + PBS") is minimally increased compared to the PBS control group (5 DAYS RIP ASA .1. PBS., 11¨(: l 3.05%; 5 DAYS RIP P1.3S, 11-8; 11.05 1 5.12'), as well as the ASA + N IKI-PLACI..130=
group (5 DAYS RIP ASA 1 N-rci=pl,Acy.Bo, ii-6: 13.92 1.71%) There is no difictcnce between the ASA + NIG-group and the g,roup treated with ASA iiiiJ PBS (1 ie.
()) I lowever, the infarct area in the NICi group is significantly smaller compared to the A.SA
+ NTG group (5 DAYS RIP NT(3, nr=7: 11.05 i .12%; 5 DAYS RIP NT(.1 ASS. 11=6i 13.001 3.82%).
3_6. Coronary Mierovascular Imaging With Micru-CT
C:ollateral m.er.cs arc pre-existent vessels running parallel to vi major arterv. in case the major artery is occluded, even for a short period of time (40 sec during this RIP), collaterals assume the blood supply. As a result, et-dish:Jai arteries in this area (R01. region of interest) start to grow in length (clearly visible, by the cork screw pattern) :mil most notably in their diameter. So we measured the diameter of the collaterals in the R( Module I: Sham Operation twithout the RIP) Therc is no signilicance between the three SHAM-p.n.:nips (5 DAYS SI IAIVI
PBS. n 3:
82.7 . 3.7 pm; 5 DAYS SHAM NIG-PLACEBO, ri-3: 89.6 pm l Ill.() pin, 5 DAYS
SHAM N'F(J, 86.8 + 9.0 pm) (Figs. 22 and 26), Module 2: NO intermittent oirc) Compared to the "5 1..LAYS RIP PBS", the diameters of the coil:tier-firs in the Rol in dic -5 DAYS RIP NIG" group are significantly increased (5 DAYS RIP PBS. 11 1: I '."9 8 6.9 pm; 5 DAYS RIP NTG, 158.4 9.2 pm). There is no differenec berween the PBS
and NTG-PLACEBO-group (5 DAYS NTC-PLACE130: n=3; 121.0 12.1 11111) (Figs. 23 and 26).
Module.3: NO ennfir* s (ISDN retard) *
The diameter of the collateral in 1Iw ISDN group (-5 DAYS 1:IP ISDN-) :ire enhoncod compared to the PBS group (5 DAYS RIP PBS, n=3: 129.8 6.9 prir; 5 DAYS MI' ISDN.
11-3: 148.2 11,3 jun), Hs well as compared to the ISDN-PIACI:410 group (", DAYS
1,;5 ISDN-PLACEBO, n-3: 133.0 11.51.1111) (Figs, 24 and 2(,).
Module. 4; NO intermittent phis ASA

The diameter in the group treated vvith PBS and ASA are smaller compared 14) Me PBS
control group (5 DAYS RIP PBS + ASA, n=3: 102.'5 + 8.0 gm; 5 DAYS RIP
129.8 6.9 um), but there is lin significance as well as the ASA + NI( i-11 .AC1...130 gyoup (5 DAYS N('G-PLACIA30 + ASA, n-3: 97.1 4:: 8,61 tin). In lli ASA i NH.; group diameter are increased compared io the group treated with PBS and AS.A (5 DAYS
KIP
ASA + NTG, 111.4 7'7 5.( pm) (Figs. 25 and 26).
to 4. ( :tinclusion We examined the groups -to DAYS SIIAM PBS" and "5 DAYS SHAM Pl..tS". each without a RIP (repo!
.selem.e protocol) and the group l 0 DAYS
R.1P P135" and "5 DAYS RIP PIIS", each with a RIP of five arid len days.
Ivleasuremcni Of infarct volume aller a 90 minute permanent I .Al) occlusion (I:1'0. !Mal permanent occlusion) revealed significantly St/Willi:kr infarcled aleas ill the 10 IAA ys Rip group than in -I() DAYS SHAM" group. In contrast, rifler a RIP 4)1 live das.
no differences became apparent in the SI IAM and RIP group.
Moreover, we u.s4,14.1 ECG parameters for examinations and evaluation lOr the first time. We found the maximal ST elevation after FPC) of the 1.AI) showed no crucial diflerences between "5 DAYS RIP PBS" and SI IA M groups, yet However. after 10 days SI
elevations vvere significantly decreased in the RIP group.
Aside from ST elevation measurement during FPO, we were able to analvze and e\
allude arrhythmias in differentiated way, Based on these novel insights into the chaincieri/atior, of rat RNA! Model. we lir use a 5 day RIP in case of an expected stimidalion ot artcriogenesis, degree of ST
elevation enhancement and the infarct volume tiller a 10 day RIP can be obtained with [no-arterlOgenie substances within a 5 day RIP, yet, This provides additional parameters bein, able to approve our results of infarct volume ineasurerricut.
The intermittent application of NTO solution (iwlet:, daily on buccal iniicosa) doeioased serious arrhythmias of the rat heart (luring FPO compared to the coffin)!
rroill) Additionally, infarct volume is decrea.scd by more than 50% atter 90 minutes IT(_) compared to the control grim!). This reduction in infarct size is not even ohiainable with controls set to a 10 clays RIP. Furthermore, a treatment with NEU solution attenuated 5'1' elevation (luring FPO. On the basis of thc per analyses, significantly enlarged eollate.ral arteries were measurable.

- 3, -The treattnent of the rats with ISDN retard (once daily intragastrally)id!30 led to decreases in s-r elevation during ITO, less urrhythrmas and reduced infarct v1iiiiie.
Ilowcycr. these improvements of infirrcl parameters are less diSlinct compared with NI(.
treatment, Moreover, they clic] lint show any signiriCance.
Compared to controls, the treatment with ASA showed MI impairment of f:CCi parameters ffirl an increase of laurel volumes due to tinpaircd collateral growth. .1 he.,,e negative effects cif ASA on trt.t.=riogenesis are already known. interestingly, they can he partly abolished throur.li an additional N troniment (twice daily on buccal nuIeo.,l) I luis, collateral diamclers were enlarged in the ROI Fuld Ff.'0 parameters were enhanced NeverIlic,:less, infarct volumes after FP() showed no reduction.
The SHAM groups did not differ among each other.
Further on, there were no differences measured between the Placebo group:3 and their corresponding control groups.
In conclusion, the presented results indicate that an intermittent treatment with NI(_ solution decreases the size of an experimentally induced myocardial infarct.
In addition., effects on cardiac rhythm may ameliorate. These insirlits are of outstandinv, relevanci, lot clinical aspects.

Example 2 Clinical Study This study aims to investig,aie the elreets of a supervised,pA I
ys.c,titi-eontrollcd stand:trail:A.1 exercise proram for the symptomatic treatment, functional inipiovenient and All augmentation of the arteriogenic cApAcity in patients with chronic stable CAD.
1 Study DeSign 1.1 1 Imotheses and Study Arms 1.1.1 Hypotheses Active physician-controlled exercise training with intermittent :ipplicAtion of IN
is supet ior to Active physician-contrOlIed exercise trainiq, l limo (A+) > (A-) I I Passive physician-controllcd exercise trainine. (( 'ardioAcccr) with intermittent application of GIN is superior to passive physicion-c)utrollcd exercise training without GIN.
(P+) (["-) Ill Conservative CAD therapy with intermittent application of (AIN is superior it) conservative CAI) therapy without (C I ) > (C .) 1.1.2 Study Arms A+ Active physician-emilrolled exercise training with intermittent application of (AIN
A- Active physician-controlled exercise iraining Passive physician-controlled exercise. training (CardioAcculg) with iniri niittciil application of GIN
P- Passive physician-controlled exercise training (CardioAccelk) C i Conservative CAD therapy with intermittent application olC.i C'- Conservative CAD therapy Patients may use Ci'IN in case of angina pecloris, however will he supplied with an additional study (STN fur (he study use.
Active physician-controlled exercise training with intermittent application of CTN.
Best medical therapy ,tot.l usual care as detailed in the current guidelines (Al IA. 1 S(') fOi the care for patients with chronic stable angina. Daily (Mon-Fri) physical exercise intervals (treadmill) of 30 min 1 W/kg bw, following risk stratification and individual calculation arid adjustment of training intensity as detailed in the current FAUPli guidelines, [oral of six weeks. (J11NI use for thc treatment of angina episodes is permitted. In addition. ;TN
0.1 mg is administered 2-5 min before the onset of exercise.
Active physician-controlled exercise training_ Bost medical therapy1 am. usual care íi detailed in the current guidelines (Al IA, LtiC) tbr the care for patients.:
with chronic stable angina. Daily (Mon-Fri) physical exercise intervals (treadmill) of 30 min bw).
t011owing risk stratification and individual calculation and adjtudinciii of trainine. intensity as detailed in the current FLACPR guidelines, for a total of six weeks. IN
use tOr the treatment of angina episodes is permitted.
Passive physician-controlled exercise training (CardioAccee) with intermitivii1 aPPlicalion a CTN. Hest medical therapy and usual care as detailed in the current guidelines (Al IA, FS() for the care for patients with chronic stable angina.
Daily (Mon-rii) CartlioAccee') treatment intervals of one hour per day l'ur a total of six weeks, as delailed (Arora KR, Cli)It, TM, Jain I), Fleishman 0, Crawford Mc Kiernan Nest KW. The multicenter study of enhanced external counterptilsni ion (MI.JST-1.T.C1'): effect of EE.CP cm excrcise-induced myocardial ischemia and ane,inal episodes. t Ain Coll Carcliol. 1999 Jun;33(7): 1833-10). GTN use for the treatment of .:ing,ina episodk.÷, permitted, in addition, G-FN 0.4 mg is administered 7-5 min betbre the onset of Q.\ etc ise.
(..l TN use for the treatment of angina episodes is perinittecl.
Passive physician-controlled exercise training (CardioAcecla). Hest medical therapy and usual care as detailed in the current guidelines (Al lA, [S(') .I.Or the eare for patients with chronic stable angina. Daily (Moit-Fri) CardioAceel treatment intervubs of 011Q hour lO per day for a total of six weeks, as detailed (Arora et al., supra). GTN
use for the treatment of angina episodes is permitted.
Conservative CAI) therapy with intermittent application of CTN.
Hest medical therapy mid usual care as detailed in the current guidelines (Al A, l..S( ') tor 15 the care tbr patients with chronic stable ane,ina.. GTN use for the treatment of Linginti episodes is permitted. lit addition, GTN 0.4 mo is administered once daily, preferably before the onset via voluntary activity of (laity life, Conservative CAD therapy. Best medical therapy and usual care as detailed in !h.., current 20 eilidelines (A1-1A, ESC) for the care for patients with chronic stable angina. & IN use for the treatmentit I
'arigina episodes is permitted.
1.2 Clinical Trial Design ?5 1.2.1 Clinical Trial Design - general The study is designed as a = prospective 3() = random i7.ed = Multicenter (German Sitc, US-Sire) - 'D6 -clinical trial, to evaluate glyceryl trinitrate (Nitrolinguan effects on Li.xereise ekipaehy, the proposed pathophysiological mechanism being fin induction of pro-arter ingirnic Meets.
1.2.2 Study Em_po.ms Primary Changes in functional exercise capacity, as m urec.
ins I
on visit 3 by peak voloole of oxygen uplialce (V02 max) and maximum oxygen uptake al anaerobic threshold (Ví) , inaN A ) From baseline in a standardized exercise treadmill lest (sETT).
Secondary (7lianges in 0 ) Time to exercise-induced ischemia as incasured by time to a > I -nun S.V-segmeni depression in a standardized exercise treadmill test (sETT), (2) the hemodynaMie responses to the sETT, as gUanlined by ilic rale-pressure prodiiel (R PP) I , which is defined as I
1.re systolic blood pressure Oirm tho healt rate (bprii), 1 !cart rate, blood pressure, and ST segment trends are electronically ineamired at the J-point I 60 ms, (3) the number or angirru episodes per day, (4) cscrcisc duration on sETT, (5) Relative Peak Slope Index (RPSI), (4) Doppler-dLniyed maximal systolic accelcria ion IACCmaxj, (5) CCS and NY! IA functional status, (6) Mike Treadmill Score, Thc Rate-pressure r.iroducl (RPP) 15 a sensitive index of myocardial oxygen consumption (rriV02).
Patients are cdtegorized by the rate pressure product (RPP) that existed al the. lime ot mayirourn ST deptession, tri the dbsence of ST dr!pression, the maximum RPP is recorded.
? The [Juke treadmill score calculates risk; it equals the exercise time in minutes minus (S times the ST-sewrivent deviation, during or after exerc*e, in millimeters) minus (4 timo5 the anquid which has a value of "0" if there is 110 angina, "1" if angina occurs, and "2' if nngin,-1 is tile reason for stopping the test). Among outpatients with suspected CAD, the two thirds of pationl.: with

(7) Incidence of cardiovascular events during the treatment pliiisc and (X) same as primary endpoint, but one month aftor intervention period.
1.2.3 Patients Eligible patients must tie clinically stable, receiving beforç enrolment an antianginal iil CAD therapy thin is in Rill iiiceordance with the current ESC/A1 IA guidelines lor ihe treatment of chronic stable CAD.
Prohibited tuedicai . long-acting nitrates = Sildenafil etc, = Anti-intlarnmatory compounds (other Ilan aspirin) such LIS steroids or claiiereciii inclusion Criteria:
Age > 18 yr;
Documented evidence of stable coronary artery disease by either positive niicic,ar exorcise stress testing, angiographically documented coronary -itenosis or histOry tif documented ST-elevation or myocardial infarction 7o SCOrCl5 indicating low risk had a tour-year survival rate of 99% (averatle anniial mortality ialv 0.25"/0), and the u/u who had scores indicating high rdc had a four -ye-1r survival rate of /9% (avelacie mortality tato The score works well tor both inpinients and outpatients., and preliminary data sucpent that the score workS egUally well for men Hid women [Gibbons et 6[;., 2003..AHA/ACC
Guldellnel Exclusion Criteria:
Nitrate intolerance or intolerance to any component of the study medication.
Medication that poses a risk of pharmacologically interacting with G-TN.
Acute coronary syndrome or unstable -angina <6 weeks prior.
Left main stenosis of PCI or CABG
- months prior.
Coronary angiography woc...!kli prior, Congestive heart failure/ EF of ,-.4.300/0.
Valvular heart disease or myocarditis.
Uncontrolled hypertension with blood pressure valut4!-;> 180 / 1.00 mmHg Severe symptomatic PAD, varicosis, deep vein thrombc.)si!-; (current or in documented medical history), phlebitis or ulcer.
Coagulation disorder or therapeutic anticoagulation.
Cardiac arrhythmlas that interfere with ECP triggering.
ECG characteristics that would invalidate ST segment monitoring: baseline ST
segment depres!jon, pacemaker-dependent rhythms, QRS duration >0.12 s, arrhythmias other than sinus arrhythmia.
FEV1 < 1.51.
Current participation in a cardiac exercise rehabilitation program.
Randomization Euro111.:(.1 patictits are randomized in a ratio to receive/undergo either activc Cann Accellc therapy or usual care. i.e. a contininnion oí' the baseline.' 114.2111Men1 in accordance wilh current guidelines. Within Iliese groups, rialients are randomized in a I :1 ration lo enher a " 1(-3-rN" or a "4.1TN" group to receive glyeiõaid trinitrate caller addition to their standard medical ion, or not.
L2.4 Study Planning, Conduction anti Management The trial is planned by Arieriogenesis Network Art.Nei.
Study management1111 W... .)0 covered by Arteriogenes is Network Art.Nei.
c/o Campus 'reel-moll:Ties Freiburg Cimb1-1 Technolomy Trataer of the University of Freiburg CEO: Prof. Dr. Bernhard Arriolds Stefan-Meier Sinitic 8, 79104 Freiburg ((ìermany) Phone: +49 (0)761 203 4990 Facsimile! +49 (0)761 203 1992 Sponsor of the trial is CTF, reporting structures and reporting schemes will be detailed tiller the participaiilig centers have been assigned.
Research Sites participating eenlers: to be determined contact in case of questions, dif:sern Minion of info 2s contact 111 eat;e of adverse everii. dissemination of info 1.2.5 Study Flow Chart and Protocol 10 The Study Flow Chart is givcn in Figure 27.

1.16 Treatment Assignment Randomization will be done at thc conducting centers via envelopes.
Stratification will be done according, to age-groups, gcrider and morbidity Study visits are conducted by an investigator.
Study centers in advance aNsiga blinded investigators that arc unaware 01 the randomization, t.i.ral who carry out the medical examinations and testing at Baseline and First Follow-up, At each study visit, patients are instructed It) 1111 in a short standarditcd qualib, of life assessment form (SF-363).
1 5 Patients assigned to the C+ / C- groups are contacted on a regular basis by study personnel to help control potential bias effects as these subjects do not have as regular contacts with study personnel aS do the CarclioAccutl. or exercise groups, 1_2.7 Study Visits Visit I: Eligibility Screening (day I) = Medical history, including previous interventions, physical exam = Enrol r nem yin Visit 2: Baseline Visit (until day 11 (1-3 clays)) = Detailed medical history and physical exam, including assessment of number of angina episodes per day, CCS and NYFIA stains and assessment ol. voluntary physical activity, = Treadmill testing on a standard, calibrated treadmill egitipilicat with Cardiopulmonary testing capability (modified Naughton protocol).
http.//vvww rand.org/healtn/suivays_tools/rnos/mos_eare_36item.htmi -(1 -functional exercise eapacily (VC)2 max and VO1 max AT), time to exercise-induced ischemir as measured by time to a >1-mm ST-segment depression, rate -pressure product (RPP), heart rate, blood pressure, anti ST segment trends electronically measured at the J-point i 0 ms, exercise duration, DUKE treadmill score, continuous monitoring of vital signs *mei. 12-lead ECG and V07, µvith Vo2max defined as VO2 at maximum level of exercise the, individual patient is nble to achieve (respiratory ratio >I, anaerobic threshold) = Relative Peak Slope Index (RI'S!) = Doppler-derived maximal systolic acceleration I Aci'maxi = Randomization Interim Visits (non-scheduicd) Patients are advised to contact the study center at any time regarding theii medical condition. Patients are scheduled to return for their first follow-up visits at (, weelo. alkr IS randomization.
Visit 3: Short-term follow-up (1-3 days after intervention_period) = Medical history and physical exam, including assessment or niimbei iiigiritt episodes per clay, CCS arid NYI IA status and assessment of voluntar) plwsieal activity.
= Treadmill testing on a standard, calibrated treadmill equipment with cardiopulmonary testing capability (modified Naughton protocol)' functional exercise capacity (V02 rnax and VO, max Al), (MR' (0 exercise induced ischernin as measured by time to a >l-mrn depresion, rate presmire product (RPP), heart rate, blood pressure, and S.1' segment trends electroniLally measured at the J-point + 60 Ins. exercise duration, DUKE treadmill score.
continuous monitoring of. vital signs Mel. 1.2-Icad ECG and VO:,, with 1.701ilux dclined as VO2 at maximum level or ex.ereise the individual patient is able 10 achieve (respiratory ratio >1, anaerobic threshold) = Relative Peak Slope Inclex (RPSI) = DOppler-derived maximal systolic, acceleration [MY:max l ib Incidence of cardiova.seular events during the treatment phase Visit 4: Lonw,:term follow-np (1 month ;iller intervention period) - tr -(The rational of this study point is to evaluate the long term effect of the study medication after the intervention period).
= Medical history and physical exam, including assessment. ()I' number of angina episodes per day, CCS and NYI-IA status and assessment of voluntary physical activity.
= Treadmill testing on a standard, cal i br ate(' treadmill equipment with cardiopulmonary testing capabi lily (modified Naughton protocol):
functional exercise capacity (VOz max and VO, max AT), lime to exercise-Induced in ischemia as measured by time to a >I .mm ST-segment 4.14;presNion, product (kW), heart rale. blood pressure, and ST septum( trends clecnonically measured at the .1-1ioint + 60 ms, exercise duration, DUKE treadmill score, continuous monitoring of vital Aigns incl. 12-lead ECG and V02, with VO,max defined as VQ: at maximum level of exercise the individual patient is able to achieve (respiratory ratio >1, nnaerobie threshold) = Relative Peak Slope Iiirlex (ItP,S1) = Doppler-derived maximal systolic: acceleration IA( C'iriax]
1.2.8 Statistical Considerations The main efficacy parameter is functional exercise capacity, as meastued by peak N.oluine of oxygen uptake (V02 max) and maximum oxygen uptake at anaerobic threshold (V02 max AI) in a standardized exercise treadmill test (sETT). We assume no difference at baseline bui significantly higher values in the GIN groups nt t011ow-up.
Statistic-al Methods There are two major sourees or- variance to be considered in this trial: ( iTN
ticatnient effects and effects of active training/passive training/conservative therapy.
Accordingly data will be analysed iit i two-way ANOVA. Any therapy effects not rehired to G FN will be reported in a descriptive way without inference statislic.
For sevondary parameters parnineliic or non-paramclric tests will be applied as appropriate.
Sample Si/c / Power To establish the necessary sample size I'm the proposed two-way-ANOVA. e itiI
the following assumptions (based on literature revic!w and iniernol data.):
,;la(istical power-80%, standard deviation for outcome ineasure=10 of mean, eiThet siie (golip difference in change between ci-rN yes/no) ¨ 5% of mean. Power was established in a Monte Carlo simulation based on 10000 repeats per sample size over a range of n per group Crum 30 to 60 patients, This simulation established a minimum sample site of subjects per group, to allow for potential drop-outs we propose to include 50 subjects per group, resulting in a total sample size 01'300 patients, 1.3 Ethical anti Legal "%spec%
The investigators plan and conduct any esperiments involving humans. including identifiable samples taken from humans and identifiable data, iii compliance \V Ilh (a) the Declaration ol' Helsinki (Ethical Principles for Medical Research Involvinv Subjects) concluded by the World Medical Association (WMA) iii June 1964, as last revised:
(h) the ICII Harmonised Tripartite Guideline: Guideline for Good Clinical Practice international Conference on Htlfmonisation of eelmical Requirements for ReOstrat icin ot=
Pharmaceuticals For Human Iise (ICIIE6, 1 May l 996) as well as ) ipp1icabte German regulations (e.g. Arzfi(:imittelgeset7) in their current forms, as well as applicable FDA regulations (e.g. Guidance for Sponsors, Inycstir.ators.
Informed Consent Elements, 21 CFR 50.25(e).
5. List of Abbreviations Doppler.derived maximal systolic acceleration Art.Net.: Network SubeOnlractors of CFI' CAD: Coronary Artery Discase CarcliciAecµe; personalized counterpulsation therapy CCS: Canadian Class Society (Angina classificatio)) CIF: Campus Tcelinologies Freiburg, FSS: fluid shear stress CITN: glyceryl trinitrate ballon pump NY1.1A: New York l Icart. Msociation RPSI: Relative Pcuk Slope index sEIT: a standurclii.cd exercise treadmill icsi SMC: vascular smooth muscle cell VO., max: peak volume of tmygen uptake VO-, max AT: maximum oxygen uptake at anaerobic threshold Comparative Examples The tbllowing Exatnples illustrate inter alin the production of' various lormulaiions according to the invention without reducing the scope Ellie invention to these Examples.
Comparative Example l Commercially available G'I'N tablets each µvitli a total weight of 35 me.,, cunt111111T, 0 mg CITN and lactose monohydrate, glycerol monostearate, pre agglutinated starch, calcium stearate and colloidal silicon dioxide as cxeipients. were packaged individually in Film slick packs and stored at 40 'CI 75% ref. humidity For ..ffet! 111011thS.
Comparative Example 2 ......._..
Contents Quantity in diluent lactose monohydratc !WA 0.60 Polyethylene glycol 400 0.36 isomalt Xylitol 12.01 Silicon dioxide. (Aeroperlq(i) 0.25 Total 30.03 (AN concentration 0 70%
-Me active substance triturate was mixed well with the liquid polyethylene 0 col; the other substances were added in the sequence listed ;:ibove and mixed 200-mg port ionii or the free-flowing powder were tilled in stiek packs and stored at 40 u('/75% rci.
humidity lbr three months.
Fxatriple 3 .,Contents Quainily GIN (5%) in diluent MCI 1.033 'Friethyl citrate 1.0 ;2 !wino It 16 513 Xylitol 6 248 Silicon dioxide (Aeroperh1,) 0.207 'Iota!
CiTN concentration = 0.20/ %
Triethyl citrate was mixed with (i'lts.1 phk-!ginttt ized iii i diluent of medium liiiii to trivIyeerides (MCT). The solution was mixed well with the isomalt. Then xylitol and finally silicon dioxide were added and mixing was continued. 200-mg portions of the free-flowing powder we rc tilled in slick packs and stored al 40 "(.775% rel.
humidity lor ihiec months.
The GIN concentration was quantified after production and at various points (intuit!.
storage using FIN ,( tiiilysis. The individual doses were dissolved in a suitable solvent to pertbrm the analysis, 'file (His! was deteet(:(.1 USilig (.1V-VIS detector al a wavelength of 225 rim.

( concentration following sioragc at 40 T/75% rel. humidity Product 0 months 2 weeks 1 month 3 months according to Comp. 0.391 mg 0.079 mg, 0.065 mg, example 1 Comp. 0.407 Mg n.c. 0.305 mg 0.245 my.
example 2 Lxample 3 0 415 mg ii.. n.c. 0.394 rni2 * The test was terminated alter ono month because mote than 80% or Ihe had :ilready been lost.
nut conducted This initial comparative test proves Mai neither the commercially available tablet. DM
powder mixture with the substances contained in a conventional tablei, nor a preparation with polyethylene g,lycol is suitably stable in a stick pack. Theon. I
y iiceoptablc level oî
storage stability was achieved with the addition of the stabilizing ester according lo lr invention logether with a (iTN concentrate phlegmatized in MCT as a liquid.
Example 4 Contents Quantity le,1 OTN (5%) in MCT 00 = FP( I .00 Magnesium alum inornems icaie 2.50 Isomalt +1.51 .1 Mal 50.00 GYN concentration 0 20%
TPcis was melted ni 50 C and mixed µvith CITN concentrate in a diluent of MC
I. While t5 still warm, the mixture was blended well with the magnesium alum inuniciasilicale. Hien isomall was added and mixing was continued. 200-mg portions of die tree-flowing powder were filled in stick packs and stored at 25 C/60% rel. humidity. The (ITN
concentration MIS quantified immediately after production and at various points during storap.e disclosed under Example 3. lIiv results are presented ; II 1 .11 õre 2o..ow.ng table:
.

Storage. duration/temp. 0 munihs 3 months/25 "C. 6 montlisi)., 'I.' . __ .
OTN concentration 0/100 mg 0.398 nig 0.392 mg 1 Example 5 -Contents Quantity [g.1 (.1IN (5%) in diluent MCT
Glycerol monocaprylocaprate Ph. Fur, 0.52 Magnesium aluml momelasilicate I .'.)0 Isoinalt 'Total 50.03 GTN concentration 0..20'.!.o ___1 Thc glycerol inonocaprylocaprnic was melted at /10 C and mixed with a ( i'IN
cl.mixiitrillc in MCT diluent. While still warm, thc mixture was blended well with the itiagnesiiim aluminometasilicate. Then isomalt was added iiiit.1 mixing was continued. 211)-inr, porl kills of the free-flow ine powder were tilled in stick packs and sti..ired i1 10 0(yht,/0 ro. humidity, and at 25 u(2/60% rel. humidity. -I'he (TN concentration was Ettimit Hied iinniediaiel .ilier production and at various points (hiring storage as disclosed tinder Example 3. Mc results arc presented in Ihe following table:
..____...

Storage duration/temp.
[
GIN concentration 0 months = 6 months/25 'C 6 months/40U( =
0.397 itig 0.383 mg 0.355 nig ._ . -Example (i Contents Quantity lg1 GTN (5%) in diluent MCT
Glycerol trionocaprylocapraie Ph, Hit. 19.85 Anhydrous dibasic calcium phosphate 100.80 Isomalt 1800.00 Tothl 2000.00 GIN concentration The glycerol monocaprylocaprate was incited in 40 'C .trid mixed with a Ci IN
t:onecntrate in ti WIlk still warm. the mixture was blcuded well with the anhydi oth dibasic calcium phosphale. Then isomalt was added and mixing was continued.
200-me, portions dale free-flowing powder were filled in stick packs and stored at 40 '075" rel.
humidity and at 25 uc/60910 rel. humidity, The GIN concentration was quantified immediately liner production anti at various points during storage as disclosed undet Example 3. Thu results are presented in ihe following Storage duration 0 momhs 3 months Ci months GTN concentration at 40 C 0.380 rag 0.380 mg 0 383 nig concentration at 25 C 0.380 mg 0.380 mg 0.379 mg Comparative example 3 =-=
C011telltR Quantity [g]
GIN (5%) in diluent MCT I .193 Anhydrous dibasic ealcioin phosphate I 513 19.80 = ¨
7.50 Total 30.000 _GIN concentration CITN concentrate in MC r diluent was blended well with the anhydrous dibasic calcium phosphate. Then isornalt and xylitol were added and [nixing was continued. 200-mg portions of the l'rce flowing powder were filled i,i stia pdcks and stored in 40 "(:./75.0 rel.
humidiiy and at 25 C/60% rel. humidity. The ( iTN concentration was quantified immediately after production and at various points durin storage tts disclosed under Example 3. The results arc presented in the following table:
Storage duration 0 months =3 months GIN concentration at /10 C 0.410 mg 0.340 mg GTN concentration at 25 C 0.410 mg 0.363 mg, A comparison between the data from Example 6 according to the invention and the non stabilized preparation according to comparative Example 5 reveals that a clinically significant average loss of 17% and 11% occurred vvithout stabilization alter a stolagt:
period of three months at 40 9C and 25 C, respectively. A product having this decree of susceptibility to deter ioration and loss of active inercri '01 is not suitable cominert.,ially lot sale as n pharmaceutical. In sharp contrast. the content of the iiciivc substance in 1.xaniple 6 remained nearly constant for six months when in the presence of a stabiliier in accordance with the present .nveni The t011owing Examples illustrate tiirther the benefit of stabilifed formulas according to the invention. Additionally., the 1011owing Examples demonstrate the henclits 4110N:tures of the stabilizers as contemplated by the present invention. Importantly, the 1011owing Examples illustrate stabilization obtained at even more elevated temperaiiiroN., i e . 50 C.

= lo Example 7:
(lc:Intents Quantity [g]
GIN (5%) in diluent M(.1. 43.) (.ilycerol monoeaprylocaprate Ph. Fur. 16.6 TPGS 16.6 Magnesium aluminometasilic441U 54.9 Peppermint flavoring, agent 11.0 Total 1100.00 (_3.I'N concentration 0 ?gfo The glycerol monocaprylocaprate and TPGS were melted at 50 'V. and mixed with IN
concentrate MCT diluent. While si ill warm, the mixture was blended well will, the magnesium aluminometasiiicate, Then isoluali ;aid peppermint flavoring agent welt, added and mixing was continued. 200-mg portions of the live-flowing powder were lined in stick packs and sloted al 50 C and at 40 '(-175% rel, humidity. l'he GIN
concentrinion quantifled immediately after production and at various points during, storage kis disclosed under kxample io Fxample :.ontents Quail( ily (iTN (5%) in diluent MCI 40.0 Solid ..lriglyeerides 20.0 Anhydrous dibasic calcium phosphate .19.9 I SOM a It 880. I
Peppermint flavoring agent 10.0 ' I 'ota 1 1000.00 GIN concentration 'I'he solid triglyeericles were melted at 50 "C zind mixed with i'l'N
concentrate in Mt.
diluent. While still warm, the mixture was blended well with the anhydrous dibasic calcium phosphate. Then isomalt was added in port irms Followed by the peppermint flavoring agent. and mixing was continued. 200-mg portions of the free-llowing powder were filled in stick packs and stored it 50 'C and at 40 "075% rel, humidity.
i he (J
Concentration was quantified immediately alter production and at various points dining, storage as disclosed under Example '1.
Example ):
Contents: Quantity IJ
(..31-N (5%) in diluent MCT 40 0 Solid Triglycerides 10_0 Glycerol monocaprylocaprate Ph. Fur, 10.0 Anhydrous dibasic calcium phosphate 50.0 IsomaIt 880.0 Peppermint flavoring agent l 0,0 Total I 000.00 (VIN concentration .._._.....
in The solid triglyeerides and glycerol monocaprylocaprate were melted at Mt (' and Mixed with a CITN concentrate in MCT diluent. While still warm, the mixture was hie I .1 vcll with the anhydrous dibasic enlcium phosphate. Then isomalt was added in portions followed by the peppermint fla.voring agent and mixing WEIS confirmed. 200 mg portions of the free-flowing Powder were filled in stick packs and stored at 10 `)C ind at /it) rel. humidity. The (SIN concentration was inamii Fla immediately atter production and at various points during stonige as disclosed under 1.L.xample 3.

bsamplc 10:
Contents = Quantit) GTN (5%) in diluent MCI 10.0 Oleoylmaeroe,O1-6-glyi.",eridos 20.0' Anhydrous dibasic calcium phosphate 50.0 Isomalt 880.0 Peppermint flavoring agent 10.0 Total 1000.00 GTN concentration IQ%
The olcoyl inacrogo1 6-glycerides were mixed with GTN in MCI'. Ihe mixture was blended well with the anhydrous dibasic cale inn) phosphate. Then isoman was dcidcil in portions followed by the peppermint flavoring agent and mixing was columned 700-mg portions of the free-flowing powder were filled in stick packs and stored at 50 and il 40 C./75% rel. humidity. Tile GIN concentration was quantified immediately after produetion and al various points during storage as disclosed under Pxample 3.
Example. I I:
Contents Qiiantit). 11411 GTNI (5%) in diluent MCT 10,0 (llycerol moriooleate 20 0 Anhydrous dibasic calcium phosphate 50.0 Isornalt 880.0 Peppermint fl3vOring agent = 10.0 Total i 000.00 comontration .1'he glycerol monooleate was melted at 50 C and mixcd with a GTN concentrate in MC
diluent. the mixturc was blended well with the anhydrous dibasic calcium phosphate.
Then isomalt was added in porlious followed by the peppermint irlavoimp, arcui and mixing was continued. 200-mg portions or fret.7-liowing powder werc lilted in slid.
packs and stored at 50 C and at 40 wC/75% rel. humidity. The CiTN
concentration quantified immediately after production and at various points during storat,te n. di,,Llosed under Example 3.
The results rrom the storage tests wilh the preparations rrom Examples 7 11 nre pre-:k=nlet.:1 in the following table:
_Storage duration at 50 c'C 0 months I month 2 monIlis Example 7 0.404 mg 0.380 mg 0.369 iii.
ample 8 0.394 mg 0.390 mg. 0.384 mg Example 9 0,391 mg 0.395 0.389 mg Example 10 0.394 mg 0.392 mg 0.388 mg F.xamplc 11 0.390 mg 0.376 mg 0,377 mg Taken together the results unambiguously show that loss of GIN is significant reduced through the addition of a non-volatile ester stabilizer as compared with Ihe stabili/ei-nee composition trom comparative F.NiluipieS 4 and 5.

- 74 -1-;xample l2;
Contents Quantity 1141 CiIN (5%) in diluent MCT 40.0 Olcoyl macrogo1-6-glycerides 10.0 'Iriglyeerides 10.0 Anhydrous dibasic calcium phosphate 50 0 Isomalt 880.0 Peppermint Flavoring agent 10.0 Total 1000.0 GTN concentration Oleoyl macrogol-6-glycerides and triglyeerides were mixed with CiIN
plileiïintiivd ui Mit 'rho mixture was blended well with the anhydrous dibasic calcium phosphate. Ilictt isomalt was added in portions .o..f 11 owed hy the peppermint flavoring agent titt.1 mixing 1.3,as continued. 200-mg, portions of the free-flowing powder were filled iil stick packs :a id stored at 50 Ç and at 40 'Crb% rel. humidity.
Example 13:
Contents Quantity fgl ( ;TN (Wn) in diluent MCI. 40 0 Glyµ'ierol monooleate 10.0 Mono- Lind Diglycerides 10.0 Anhydrous dibasic, ealeium phosphate 30.0 lsoundl 8K0.0 Peppermint flavorine. nem 10.0 Total 1000.0 GTN concentration 0 Glycerol monooleate and mono- anti ftlyeerides (type (õieleol) were melted al 50 and mixed with a GIN concentrate in a MCI diluent. The mixture WM INCH µk anhydrous dibasic calcium phosphate. Then isomalt was added in portions followed hytlic peppermint flavoring agent and inixin, WIN continued. .200-mg portion of the tree-flowing i5 powder were filled in stick packs and stored at 50 C and at 40 C/75%
rel.
=

Example 14:
Contents Quantity Igi GIN (5%) in diluent MCI 40.1 Myristyl lactate 20.0 Aiihydrous dibasic calcium phosphate 50,0 lsomalt X70 9 Peppermint flavoring tgent 10 I
Total 1000.1 (TN concentration 0.2%, Myristyl lamite was [nixed with a GTN concentrate in MC 'l. diluent. Thc..-mixture 1.1 its blended well with thc anhydrous ditnitiic caid0M 1)h0Sphate..lhen irriiriliwm;
added in portions followed bv the peppemiint flavoring agent and mixing was continued.
200-im!, portions or the ftee-flowing powder were [Bled ill stick packs inct stored al 50 "C jiil /10 c'C/75% rel. humidity.
to Example 15:
Contents Quantity ig.l (5%) in diluent propylene glycol 4.0 Glycerol MOTIOcitprylucaprate Fur. 1,0 Anhydrous dihn.µiic caleium phosphate 5.0 Isornalt 89.0 ...........
Peppermint flavoring agent l .0 1 'out I 100.00 GTN concentration 0.2%

(..iTN concentrate in propylene glycol diluent was blended well with the i.iiihydrous dibasiC
calcium phosphate. In a separate vessel the glycerol monocaprylocaprate was inclicd al 40 C and added to the absorbed diluted isoinall was added in porriolix followed by the peppeimini flavoring agent and mixing was continued. 200. ma portions. of the froe-nowine, powder were filled in stick packs and stored at 50 ^C and at rel. humidity.
Example 16:
Contents =Quantity 11;1 CiIN (5%) in diluent propylene glycol Oleoylmacrogo1-6-glyeerideS
Anhydrous dibasic calcium phosphate = 5 0 Isoinalt t48,0 Peppermint flavoring agent 1.0 100.0 GIN concentration (ITN concentrate in propylene g,lyeol diluent was blended well with the ;nth) drons dibasic calcium phosphate, oleoy I macrogo1-6-glycericles were added to the absorbed dinned i IN.
Then isomalt was added in portions followed by the peppermint flavoring agent and mixing was continued. 200-mg portions of the free-llowing powder were tilled in stick frocks Hrid stored at 50 QC and at 40 3C/75% rel. humidity.
It is expected that the 6'1'N-containing formulations of Examples 12-16 wall-Tani demonstrate the advantages of the inclusion ()la slribil izer in the above described tilN
absorbate compositions even when held at stressful storage temperatures such 0 As described elsewhere herein, the preparatiolis exemplified above according to the invention can be supplied as a single dose iii the !Wm of a stick pack, a capsule in r siielnd.
In addition, it is possible to manufacture tablets, mini-tablets or pellets for oionnicosal or sublingual administration from ti stabiliced powders or granules, as necessary. FolIovt ing the addition of other tillers, disintegranis, glidants, binders, and Inbriearn6 using roulirh:
and customary protocols.

Claims

Claims 1. A method of treating or preventing an arterial insufficiency, wherein an NO donor is administered in an intermitting manner to a subject in an amount effective for the induction of arteriogenesis.
2. The method of claim 1,. wherein the arterial insufficiency is due to insufficient oxygen or blood supply of a tissue supplied by the artery or a bypass or shunt during physical rest or exercise.
3. The method of any of claims 1 or 2, wherein the arterial insufficiency is due to an increased demand of oxygen or blood flow of a tissue supplied by the artery or a bypass or shunt 4. The method of any of claims 1 to 3, wherein the arterial insufficiency is characterized by a partial or complete occlusion of an arterial vessel.
5. The method of any of claims 1 to 4, wherein the arterial insufficiency is due to the deposition of material in the blood vessels.
6. The method of any of claims 1 to 5, wherein the arterial insufficiency is due to an external or internal compression of an artery.
7. The method of any of claims 1 to 6, wherein the arterial insufficiency is a vascular disease.
8. The method of any of claims 1 to 6, wherein the arterial insufficiency is a disease selected from the group consisting of atherosclerosis, an ischemic disease and a further chronic arterial disease.
9. The method of any of claims 1 to 6, wherein the arterial insufficiency is a visionary arterial insufficiency.
10. The method of any of claims 1 to 6, wherein the arterial insufficiency is a cerebral arterial insufficiency.

11. The method of any of claims 1 to 6, wherein the arterial insufficiency is a peripheral arterial insufficiency.
12. The method of any of claims 1 to 6, wherein the arterial insufficiency is an intestinal arterial insufficiency.
13. The method of any of claims 1 to 6, wherein the arterial insufficiency is an urogenital arterial insufficiency.
14, The method of any of claims 1 to 6 the arterial insufficiency is a nerval arteial insufficiency.
15. The method of any of claims 1 to 6, wherein the arterial insufficiency is in the context of scleroderma.
16. The method of any of claims 1 to 6, wherein the arterial insufficiency is a central retinal artery insufficiency.
17. The method of any of claims 1 to 16, wherein the arterial insufficiency characterized by an absence of an endothelial dysfunction.
18. The method of any of claims 1 to 17, wherein the NO donor is nitric oxide, sodium nitroprusside, nitroglycerin (glyceryl trinitrate), isosorbide mononitrate, isosorbide dinitrate, pentaerythritol tetranitrate (PETN), molsidomin, amyl nitrite or nicorandil, 19. The method of any of claims 1 to 6, wherein the NO donor is a short acting NO
donor.
20. The method any of claims 1 to 19, wherein the NO donor is Nitroglycerin.
21. The method of any of claims 1 to 19, wherein the NO
donor is Nitroglycerin.
at least on one day a week for at least two weeks.
22. The method of any of claims 1 to 21, wherein the NO donor is administerd fin a period of several weeks or months.
23. The method of any a claims 1 to 22, wherein the NO donor is administered ill conjunction with an exogenous stimulation of the pulsatile shear forces in the artery.

24. The method of claim 23, wherein the NO donor is administered in the time period of 30 minutes before the onset of the exogenous stimulation until 30 minutes after the termination of the exogenous stimulation.
25. The method of claim 24, wherein the NO donor is administered in the time period of 15 minutes before the exogenous stimulation until 30 minutes after the onset of the exogenous stimulation.
26. The method of any of claims 23 to 25, wherein said stimulation is achieved by physical exercise or the application of an endogenous force to the arterial vessel.
27. The method of any of claim 1 to 26, wherein the method aims at the prevention of said arterial insufficiency.
28. The method of any of claims 1 to 27, wherein the NO donor is administered lingually, sublingually, inhalatively, bucally, transmucosally or oromucosally.
29. An NO donor for use in a method for the prevention or treatment of an arterial insufficiency, wherein the NO donor is administered in an intermitting manner in an amount effective for the induction of arteriogenesis.
30. The NO donor for use according lo claim 29, with the features as defined in any of claims 2 to 28.
31. A method of the suppression of negative effects associated with any treatment of an arterial insufficiency which is anti-anteriogenic or inhibiting arteriogenesis, comprising administering to a subject subjected to said treatment an NO donor in an amount and manner effective for the induction of arteriogenesis.
12. An NO donor for use in a method of the suppression of negative with any treatment of an arterial insufficiency which is anti-anteriogenic or inhibiting arteriogenesis, wherein the NO donor is administered to a subject subjected to said treatment in an amount and manner effective for the induction of arteriogenesis 33. The NO donor for use according to claim 32 or the method of claim 31, with the features as defined in any of claims 2 to 28.

34. A method for the prevention or treatment or a cardiac arrhythmia, wherein an NO
donor is administered to n subject in an amount and manner effective l'or the treatment of said cardiac arrhythmia.
35. The method of claim 34, with the features as defined in any of claims 11.t to 7'S
36. An NO donor for use in a method for the prevention or treatment or a cardiac arrhythmia, wherein the NO donor is administered to a subject in an amount and mannerNiasese effective for the treatment of said cardiac arrhythmia.
37, The NO donor for use according to claim 36, with the features as &lined in an of claims 18 to 28.
38. A method of promoting collateral circulation comprising the step of exposing a subject to a therapeutically effective amount of an NO donor wherein the therapeutically effective amount of the NO donor promotes arteriogenesis sufficient to augment collateral circulation in a physiological or pathological condition.
39. The method of claim 38, wherein the subject suffers from an arterial insufficiency 40. The method of claim 19, with the features as defined in any of claims 2 to 28.
41. A method of treating or preventing an arterial insufficiency, wherein an NO donor is administered in an intermitting manner to a subject in all amount effective for the induction of arteriogenesis, and wherein the NO donor is glyceryl trinitrate in a solid pharmaceutical preparation for oromucosal or oral actin administration characterized in that it contains between 0.05 and 2 weight% glyeeryl trinilrate (GTN), al leas( one diluent, one carrier material and al least one substance that reduces the volatility of GTN, whereby this substance is non-volatile ester stabilizer whose melting point is not higher than 60 °C.
42. The method of claim 41, wherein the arterial insufficiency is dire to insufficient oxygen or blood supply of a tissue supplied by the artery or a bypass or shunt during physical rest or exercise.
43, The method of any of Claims 41 or 42, wherein the arterial insufficiency is due to an increased demand or oxygen or blood flow of a tissue supplied by the artery or a by pass or shunt.

44. The method of any of claims 41 to 43, wherein the arterial insufficiency is characterized by a partial or complete occlusion of an arterial vessel.
45. The method of any of claims 41 to 44, wherein the arterial insufficiency is due to the deposition of material in the blood vessels.
46. The method of any of claims 41 to 45, wherein the arterial insufficiency is due to an external or internal compression of an artery.
47. The method of any of claims 41 to 46, wherein the arterial insufficiency is a vascular disease.
48. The method of any of claims 41 to 46, wherein the arterial insufficiency is a disease selected from the group consisting of atherosclerosis, an ischemic disease and a further chronic arterial disease.
49. The method of any of claims 41 to 46, wherein the arterial insufficiency is a coronary arterial insufficiency.
50. The method of any of claims 41 to 46, wherein the arterial insufficiency is a cerebral arterial insufficiency.
51. The method of any or claims 41 to 46, wherein the arterial insufficiency is a peripheral arterial insufficiency.
52. The method of any of claims 41 to 46, wherein the arterial insufficiency is an intestinal arterial insufficiency.
53. The method of any of claims 41 to 46, wherein the arterial insufficiency is an urogenital arterial insufficiency.
54. The method of any of claims 41 to 46, wherein the arterial insufficiency is a nerval arterial insufficiency.
55. The method of any of claims 41 to 46, wherein the arterial insufficiency is in the context or scleroderma.

56. The method of any of claims 41 to 16, wherein the arterial insufficiency is a central retinal artery insufficiency.
57. The method of any of claims 41 to 56, wherein the arterial insufficiency characterized by an absence of fan endothelial dysfunction.
58. The method of any of claims 41 to 57, wherein the preparation contains between 0. 1 and 1 weight% glyceryl trinitrate.
59. The method of any of claims 41 to 58, wherein the non-volatile ester is solid or semi solid at a temperature of 20 °C.
60. The method of any of claims 41 to 59, wherein the non-volatile ester stabilizer is selected from the group consisting of: mono- and diglycerides, polyethoxylated glycerides, esters of lactic acid, D-alpha tocopheryl polyethylene glycol 1000 succinate and solid triglycerides, and mixtures of any one of these substances.
61, The method of any of claims 41 to 60, wherein the preparation contains the non-volatile ester stabilizer at a concentration of 0.2 to 10 weight%, based on the total weight of the preparation.
62. The method of any of claims 41 to 61, wherein GTN, diluent and stabilizer form a homogeneous solution.
63. The method of any of claims 41 to 62, wherein the mass ratio of the non-volatile ester stabilizer to GTN between 2 and 40.
64. The method of any of claims 11 to 63, wherein the mass ratio of diluent to the non-volatile ester stabilizer is between 1 and 9.5.
65. The method of any of claims 41 to 64, wherein the carrier material is selected from the group consisting or: magnesium aluminometasilicate, dibasic calcium phosphate, isomalt and mixtures of any one of the foregoing.
66. The method of any of claims 41 to 65, wherein the preparation further includes at least one excipient suitable for sublingual administration, which is selected from the group consisting of: water-soluble mono-, di-. and polysaccharides, as well as their alcohols.

67. The method of any of claims 11 to 66, wherein the excipient suitable for sublingual administration is selected from the group consisting of: fructose, glucose, isomalt, lactose, maltose, maltilol, mannitol, sorbitol, sucrose, trehalose, an xylitol and mixtures of any one of the foregoing.
68. The method any of claims 41 to 67, wherein the excipient suitable for sublingual administration to be included at minimum is xylitol and/or isomalt at concentrations of between 20 and 95 weight%.
69. The method of any of claims 41 to 68, wherein the excipient suitable lot sublingual administration is isomalt which is contained at concentrations of between 70 and 95 weight%, based on the total weight of the preparation.
70. The method of any of claims 41 to 69, wherein the preparation is in the form of a free-flowing powder or free-flowing granules.
71. The method of any of claims 41 to 70, wherein the preparation is packaged as a single dose in the form of a stick pack or sachet.
72. The method of any of claims 41 to 71, wherein the preparation further comprises at least 0.01 to 3.0 weight% of a flavoring agent.
73. A method of treating or preventing an arterial insufficiency, wherein an NO donor is administered in an intermitting manner to a subject in an amount effective for the induction or arteriogenesis, and wherein the NO donor is GTN in a solid pharmaceutical preparation for oromucosal or oral administration characterized in that it contains an absorbate comprising between 0.05 and 2 weight% GTN and a non-volatile ester stabilizer on a carrier material.
74. The method of claim 73, with the features as defined in any of claim 12 to 57 75. The method of any of claims 41 to 74, wherein the NO donor at least once a day and at least on one day a week for at least two weeks.
76. The method of any of claims 41 to 75, wherein the NO donor administered for a period of several weeks or months.

77. The method of any of claims 41 to 76, wherein the NO donor is administered in conjunction with an exogenous stimulation of the pulsatile shear threes in the artery.
78. The method or claim 77, wherein the NO donor is administered in the time period of 30 minutes before the onset of the exogenous stimulation until 10 minutes alter the termination of the exogenous stimulation.
79. The method of claim 78, wherein the NO donor is administered in the time period of 15 minutes before the exogenous stimulation until 30 minutes after the onset of the exogenous stimulation.
80. The method of any of claims 77 to 79, wherein said stimulation is achieved by physical exercise or the application of an endogenous three to the arterial vessel.
81. The method of any of claim 41 to 80, wherein the method aims at the prevention of said arterial insufficiency.
82. The method of any of claims 41 to 81, wherein the NO donor is administered lingually, sublingually, bucally, or transmucosally, 81 An NO donor for use in a method for the prevention or treatment of an arterial insufficiency, wherein the NO donor is administered in an intermitting manner in an amount effective for the induction of arteriogenesis. and wherein the NO donor is as defined in any of claims 41 or 73.
84. The NO donor for use according to claim 83, with the features as defined in any of claims 42 to 82.
85. A method of the suppression or negative effects associated with any treatment of an arterial insufficiency which is anti-anteriogenic or inhibiting arteriogenesis, comprising administering to a subject subjected to said treatment an NO donor in an amount and manlier effective for the induction of arteriogenesis, and wherein the NO
donor is as defined in any of claims 41 or 73.
H. An NO donor for use in a method of the suppression of negative effects associated with any treatment of an arterial insufficiency which is anti-anteriogenic or inhibiting arteriogenesis, wherein the NO donor is administered to a subject subjected to said treatment in an amount and manner effective for the induction of arteriogenesis and wherein the NO donor is as defined in any of claims 41 or 73.
87. The NO donor for use according to claim 86 or the method according to claim 85.
with the features as defined in any of claims 42 to 82.
88. A method for the prevention or treatment of a cardiac arrhythmia, wherein an NO
donor is administered to a subject in an amount and manner effective for the treatment of said cardiac arrhythmia, and wherein the NO donor is as defined in any of claims 41 or 73.
89. The method of claim 88, with the features as defined in any of claims 58 to 82.
90. An NO donor for use in a method for the prevention or treatment of a cardiac arrhythmia, wherein the NO donor is administered to a subject in an amount and manner effective for the treatment of said cardiac arrhythmia. and wherein the NO
donor is as defined in any of claims 41 or 73.
91. The NO donor for use according to claim 90, with the features as defined in any of claims 58 to 82.
92. A method of promoting collateral circulation comprising the step of exposing a subject to a therapeutically effective amount of an NO donor wherein the therapeutically effective amount of the NO donor promotes arteriogenesis sufficient to augment collateral circulation in a physiological or pathological condition and wherein the NO
donor is as defined in any of claims 11 or 73.
93. The method of claim 92, wherein the subject suffers from an arterial insufficiency.
91. The method of claim 93, with the features as defined in any of claims 47 to 80.