INDUCTION OF ARTERIOGENESIS

INDUCTION DE L'ARTERIOGENESE

English 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.

Claims

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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.

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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.


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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.


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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.


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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.

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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.

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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

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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.

Description

CA 02778735 2012-05-31
- 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.

CA 02778735 2012-05-31
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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.

CA 02778735 2012-05-31
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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.

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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

CA 02778735 2012-05-31
-
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,
23
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

CA 02778735 2012-05-31
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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

CA 02778735 2012-05-31
- 7 -
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

CA 02778735 2012-05-31
- 8 -
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,

CA 02778735 2012-05-31
_ 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

CA 02778735 2012-05-31
- 10 -
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

CA 02778735 2012-05-31
- 11
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

CA 02778735 2012-05-31
- 12 -
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

CA 02778735 2012-05-31
- 13 -
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

CA 02778735 2012-05-31
- 14 -
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.

CA 02778735 2012-05-31
- 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.

CA 02778735 2012-05-31
- 16
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

CA 02778735 2012-05-31
- 17 -
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

CA 02778735 2012-05-31
- 18
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

CA 02778735 2012-05-31
- 19 -
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

CA 02778735 2012-05-31
- 20 -
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.

CA 02778735 2012-05-31
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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

CA 02778735 2012-05-31
- 22 -
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\

CA 02778735 2012-05-31
- 23 -
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.

CA 02778735 2012-05-31
.
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.

CA 02778735 2012-05-31
- 25 -
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

CA 02778735 2012-05-31
- 26 -
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.

CA 02778735 2012-05-31
- 27 -
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,

CA 02778735 2012-05-31
- 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,

CA 02778735 2012-05-31
- 29 -
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.

CA 02778735 2012-05-31
- 30 -
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--();
0 096
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.

CA 02778735 2012-05-31
- 31
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

CA 02778735 2012-05-31
- 32 -
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 +

CA 02778735 2012-05-31
- 33 -
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
1111 NIG
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
5 DAYS
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
I 0
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

CA 02778735 2012-05-31
- 34 -
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
79
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).

CA 02778735 2012-05-31
- 35 -
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,

CA 02778735 2012-05-31
- 36 -
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

CA 02778735 2012-05-31
- 37
the intervention period) nod i oug-lerrn follow up investiolion monlli
after interi,enluon
period).

CA 02778735 2012-05-31
- 38 -
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

CA 02778735 2012-05-31
- 39 -
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

CA 02778735 2012-05-31
= 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

CA 02778735 2012-05-31
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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)

CA 02778735 2012-05-31
- 47 -
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,

CA 02778735 2012-05-31
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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

CA 02778735 2012-05-31
- 44 -
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

CA 02778735 2012-05-31
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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)

CA 02778735 2012-05-31
- 46 -
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
ASA 1 N
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.

CA 02778735 2012-05-31
- 47 -
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).

CA 02778735 2012-05-31
- 48 -
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

CA 02778735 2012-05-31
- 49 -
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
25 I 4-
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.
())

CA 02778735 2012-05-31
- 50 -
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

CA 02778735 2012-05-31
-51 -
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.

CA 02778735 2012-05-31
- 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.

CA 02778735 2012-05-31
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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

CA 02778735 2012-05-31
- 51 -
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

CA 02778735 2012-05-31
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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)

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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

CA 02778735 2012-05-31
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(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

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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

CA 02778735 2012-05-31
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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.

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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

CA 02778735 2012-05-31
-(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)

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(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

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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

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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

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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.

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( 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:
.

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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:
..____...
-1
Storage duration/temp.
[
GIN concentration 0 months = 6 months/25 'C 6 months/40U( =
0.397 itig 0.383 mg 0.355 nig
._ . -

CA 02778735 2012-05-31
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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

CA 02778735 2012-05-31
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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.

CA 02778735 2012-05-31
= 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
957
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

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'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.

CA 02778735 2012-05-31
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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

CA 02778735 2012-05-31
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.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.

CA 02778735 2012-05-31
- 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.
=

CA 02778735 2012-05-31
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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%

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(..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
40
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.