UTILISATION DU 3-CARBOXY-N-ETHYL-N,N-DIMETHYLPROPAN-1-AMINIUM OU D'UN SEL PHARMACEUTIQUEMENT ACCEPTABLE POUR LE TRAITEMENT DE L'ATHEROSCLEROSE

USE OF 3-CARBOXY-N-ETHYL-N,N-DIMETHYLPROPAN-1-AMINIUM OR A PHARMACEUTICALLY ACCEPTABLE SALT THEREOF IN THE TREATMENT OF ATHEROSCLEROSIS

Abrégé français

L'invention concerne l'utilisation du 3-carboxy-N-éthyl-N,N-diméthylpropan-1-aminium et de ses sels pharmaceutiquement acceptables : le fumarate hydrogéné du 3-carboxy-N-éthyl-N,N-diméthylpropan-1-aminium et le phosphate hydrogéné du 3-carboxy-N-éthyl-N,N-diméthylpropan-1-aminium, pour la prévention et le traitement de l'athérosclérose.

Abrégé anglais

Use of 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium and its pharmaceutically acceptable salts: 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium hydrogen fumarate and 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium dihydrogen phosphate, in the prevention and treatment atherosclerosis.

Revendications

10
Claims
1. 3-Carboxy-N-ethyl-N,N-dimethylpropan-1-aminium or a pharmaceutically
acceptable salt thereof for use in the prevention and treatment of
atherosclerosis.
2. Use according to claim 1, wherein pharmaceutically acceptable salt is 3-
carboxy-N-ethyl-N,N-dimethylpropan-1-aminium hydrogen fumarate.
3. Use according to claim 1, wherein pharmaceutically acceptable salt is 3-
carboxy-N-ethyl-N,N-dimethylpropan-1-aminium dihydrogen phosphate .

Description

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Use of 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium or a pharmaceutically
acceptable salt thereof in the treatment of atherosclerosis
Technical Field
The present invention relates to 3-carboxy-N-ethyl-N,N-dimethylpropan-1-
aminium
or a pharmaceutically acceptable salt thereof for use in the prevention and
treatment of atherosclerosis. Examples of pharmaceutically acceptable salts of
3-
carboxy-N-ethyl-N,N-dimethylpropan-1-aminium are: 3-carboxy-N-ethyl-N,N-
dimethylpropan-1-aminium hydrogen fumarate and 3-carboxy-N-ethyl-N,N-
dimethylpropan-1-aminium dihydrogen phosphate.
1.0 Background Art
Atherosclerosis is a complex chronic inflammatory process of the vascular wall
that progresses over decades. It is characterized by the accumulation of
oxidized
low-density lipoproteins (LDL), increased cell death and hypertrophic
degeneration
of the arterial wall, causing narrowing of the inner diameter of the vessel
and, thus,
impairing blood flow. It can occur in any area of the body, but is most
important
when it develops in the blood vessels of the heart or brain. The narrowing is
due to
the formation of plaques (raised patches) in the inner lining of the arteries.
These
plaques consist of oxidized LDL, decaying muscle cells, fibrous tissue, clumps
of
blood platelets, cholesterol, macrophages, T-lymphocytes and sometimes
calcium.
Atherosclerotic lesions commonly develop in regions of turbulent blood flow
and
are found most often in people with elevated cholesterol concentrations. The

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number and thickness of plaques increases with age, causing loss of the smooth
lining of the blood vessels and encouraging the formation of thrombi (blood
clots).
Sometimes fragments of thrombi break off and form emboli, which travel through
the bloodstream and block smaller vessels, thus, causing ischemic damage of
the
tissues.
Atherosclerosis and its clinical manifestations are a major cause of morbidity
and
mortality in the modern society. Atherosclerotic heart disease, involving the
coronary arteries (coronary heart disease), is the most common cause of death,
accounting for one-third of all deaths. Atherosclerotic interference with
blood
supply to the brain (stroke) is the third most common cause of death after
cancer.
Vascular insufficiency is another clinical manifestation of atherosclerosis
which
causes a great deal of serious illness by reducing the flow of blood in other
major
arteries, such as to the kidneys, legs, and intestines.
Unfortunately, atherosclerosis produces no symptoms until the damage to the
arteries is severe enough to restrict blood flow. Restriction of blood flow to
the
heart muscle due to atherosclerosis can cause angina pectoris or a myocardial
infarction (a heart attack). Restriction of blood flow to the muscles of the
legs
induces intermittent claudication (pain in the legs that occurs during
exercise and
is relieved by rest). Narrowing of the arteries supplying blood to the brain
may
cause transient ischemic attacks (symptoms and signs of a stroke lasting less
than
24 hours) and episodes of dizziness, or ultimately, to a stroke itself.

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3-Carboxy-N,N,N-trimethylpropan-1-aminium (GBB) is known mostly as a bio-
precursor of carnitine, a key molecule in the regulation of myocardial energy
metabolism , was primarily characterised as a toxic substance, which
accelerates
respiration, causes salivation and lacrimation, pupil dilation,
vasoconstriction and
heart stop in diastole LINNEWEH, W. Gamma- Butyrobetain, Crotonbetain und
Carnitin im tierischen Stoffwechsel. Hoppe-Seylers Zeitschrift fur
physiologische
Chemie. 1929, vol.181 , p.42-53. At the same time, in later papers it has been
shown that administration of 3-carboxy-/V/V,N-trimethylpropan-1-aminium does
not induce any toxic effects as it is extremely low toxic (LD50 7000 mg/kg,
s.c.)
3.0 ROTZSCH, W. lber die Toxizitat des Carnitins und einiger verwandter
Stoffe. Acta
biol. med. germ. 1959, vol.3, p.28-36.
The combination of 3-carboxy-N,N,N-trimethylpropan-1-aminium with 3-(2,2,2-
trimethylhydrazinium)propionate dihydrate in the treatment of atherosclerosis
was
presented in WO 2010/149654 A (GRINDEKS JSC) 29.10.2010. Nevertheless the
effect of 3-carboxy-/V,N,N-trimethylpropan-1-aminium, where it is used alone,
for
the treatment of atherosclerosis is not reported.
A new derivate of 3-carboxy-N,N,N-trimethylpropan-1-aminium, 3-carboxy-N-ethyl-
/V,N-dimethylpropan-1-aminium, as a new compound with cardioprotective
activity
was disclosed in WO 2011/048201 A (GRINDEKS JSC) 28.04.2011.
Apolipoprotein E knockout (ApoE-/-) mice are frequently used experimental
model
of the atherosclerosis for the assessment of anti-atherosclerotic activity of
tested
substances.

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Disclosure of the invention
This invention is directed to treating atherosclerosis by decreasing the total
area
and volume of atherosclerotic lesions.
The lesion progression inhibition is achieved by treatment with 3-carboxy-N-
ethyl-
N,N-dimethylpropan-1-aminiumor its pharmaceutically acceptable salts: 3-
carboxy-
N-ethyl-N,N-dimethylpropan-1-aminium hydrogen fumarate) or 3-carboxy-N-ethyl-
N,N-dimethylpropan-1-aminium dihydrogen phosphate, possibly through alteration
of lipid and cholesterol metabolism.
io A therapeutically effective amount of 3-carboxy-N-ethyl-N,N-
dimethylpropan-1-
aminium or its pharmaceutically acceptable salt is about 0.01 to 500
mg/kg/day,
preferably 0.1 to 100 mg/kg/day.
It was surprisingly and unexpectedy found that 3-carboxy-N-ethyl-N,N-
dimethylpropan-1-aminium and its pharmaceutically acceptable salts: 3-carboxy-
N-ethyl-N,N-dimethylpropan-1-aminium hydrogen fumara te and 3-carboxy-N-ethyl-
N,N-dimethylpropan-1-aminium dihydrogen phosphate posses pronounced anti-
atherosclerotic effect.
The anti-atherosclerotic activity of 3-carboxy-N-ethyl-N,N-dimethylpropan-1-
aminium, 3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium-hydrogen fumarate or
3-carboxy-N-ethyl-N,N-dimethylpropan-1-aminium dihydrogen phosphate can be
determined by assessing the effect of these compounds on the portion of the
aortic surface covered by atherosclerotic lesions.

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3-Carboxy-N-ethyl-/V,N-dimethylpropan-1-aminium can be used in pharmaceutical
preparations containing the compound, or a pharmaceutically acceptable salt
thereof, in combination with a pharmaceutically acceptable carrier or diluent.
Suitable pharmaceutically acceptable carriers include inert solid fillers or
diluents
5 and sterile aqueous or organic solutions. Thus, for oral administration
the
compounds can be combined with a suitable solid or liquid carrier or diluent
to
form capsules, tablets, powders, syrups, solutions, suspensions and the like.
The
pharmaceutical compositions may, if desired, contain additional components
such
as flavorants, sweeteners, excipients and the like. For parenteral
administration
io the compounds can be combined with sterile aqueous or organic media to
form
injectable solutions or suspensions. The injectable solutions can then be
administered intravenously, intraperitoneally, subcutaneously, or
intramuscularly.
Anti-atherosclerotic activity
Female ApoE-/- mice weighing 18 - 20 g were maintained on a 12 h dark/12 h
light
cycle in air-conditioned rooms (22.5 0.5 C, 50 5% humidity) with unlimited
access to food and water.
Mice were adapted to local conditions for one week before the beginning of the
study. At the age of 8 weeks, mice were randomly assigned to five equally
sized
groups (n = 10). To induce experimental atherosclerosis (atherosclerotic
lesions in
the aorta), animals of all groups were fed with WESTERN RD (P) diet (Cat
82316)

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from Special Diets Services (Great Britain) for 4 months. During these 4
months,
mice from different experimental groups received following treatment:
1. Control group ¨ drinking water;
2. 3-Carboxy-/VAN-trimethylpropan-1-aminium group ¨ 3-carboxy-
/VAN-trimethylpropan-1-aminium 10mg/kg in drinking water;
3. 3-Carboxy-N-ethyl-/V,N-dimethylpropan-1-aminium group ¨ 3-carboxy-
N-ethyl-/V,N-dimethylpropan-1-aminium 10 mg/kg in drinking water;
4. 3-Carboxy-N-ethyl-/V,N-dimethylpropan-1-aminium hydrogen fumarate
group ¨ 3-carboxy-N-ethyl-/V,N-dimethylpropan-1-aminium hydrogen
fumarate 17.5 mg/kg in drinking water;
5. 3-Carboxy-N-ethyl-/V,N-dimethylpropan-1-aminium dihydrogen
phosphate group ¨ 3-carboxy-N-ethyl-/V,N-dimethylpropan-1-aminium
dihydrogen phosphate 16.8 mg/kg in drinking water.
The doses of the tested substances were adjusted to be equimolar with 10 mg/kg
dose of 3-carboxy-N-ethyl-/V,N-dimethylpropan-1-aminium.
The dosing of the test compounds was confirmed by measuring the consumption
of drinking water every 2 days and adjusting the concentration of supplemented
substances.
After 4 months, mice were injected intraperitoneally (i.p.) with 1,000 Ul of
heparin
ip. and sacrificed under anesthesia (sodium pentobarbital, 50 mg/kg i.p.).
The size of atherosclerotic lesions was determined in whole aorta. The aortas
from
arch to bifurcation were cleaned from surrounding tissues, cut out and fixed
in 4%

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formaldehyde. Afterwards whole aorta was longitudinally opened, pinned onto
silicone plates and stained for lipids with Sudan IV. Images of the aorta were
captured using a digital camera and the total area of the lesion was
calculated
using Image-Pro Plus 6.3 software. The extent of atherosclerosis was expressed
as the percentage of the aortic surface covered by lesions compared to the
total
aortic surface.
All analyses were performed by an observer blinded to the treatment group.
After 4-month exposure to Western RD diet, the apoE-/- mice developed marked
atherosclerotic lesions. Analysis of Sudan IV stained aortas showed that area
of
atherosclerotic lesions in the control group averaged 14-16% of the total
aortic
surface. Four-month administration of 3-carboxy-N-ethyl-/V,N-dimethylpropan-1-
aminium at the dose of 10 mg/kg, 3-carboxy-N-ethyl-/V,N-dimethylpropan-1-
aminium hydrogen fumarate at the dose of 17.5 mg/kg, 3-carboxy-N-ethyl-/V,N-
dimethylpropan-1-aminium dihydrogen phosphate at the dose of 16.8 mg/kg (the
latter two equimolar to 10mg/kg of 3-carboxy-N-ethyl-/V,N-dimethylpropan-1-
aminium) induced a statistically significant reduction in the size of
atherosclerotic
lesions. 3-Carboxy-/V/V,N-trimethylpropan-1-aminium (10 mg/kg) had no effect
on
the size of atherosclerotic lesions.
Effects of 4-month treatment with 3-carboxy-/V/V,N-trimethylpropan-1-aminium,
3-
carboxy-N-ethyl-/V,N-dimethylpropan-1-aminium, 3-carboxy-N-ethyl-/V,N-
dimethylpropan-1-aminium hydrogen fumarate and 3-carboxy-N-ethyl-/V,N-

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dimethylpropan-1-aminium dihydrogen phosphate on the area of atherosclerotic
lesions in aorta of apoE-/- mice are presented in Table 1.
Table 1
Effects of 3-carboxy-/V/V,N-trimethylpropan-1-aminium, 3-carboxy-N-ethyl-/V,N-
dimethylpropan-1-aminium, 3-carboxy-N-ethyl-/V,N-dimethylpropan-1-aminium
hydrogen fumarate and 3-carboxy-N-ethyl-/V,N-dimethylpropan-1-aminium
dihydrogen phosphate on the area of atherosclerotic lesions
Treatment group Area of atherosclerotic lesions (%)
Control 100.0 10.7
3-Carboxy-NAN-trimethylpropan-1-
96.0 10.6
aminium, 10 mg/kg
3-Carboxy-N-ethyl-/V,N-
43.6 8.6"
dimethylpropan-1-aminium, 10 mg/kg
3-Carboxy-N-ethyl-/V,N-
dimethylpropan-1-aminium hydrogen 47.9 5.7"
fumarate, 17.5 mg/kg
3-Carboxy-N-ethyl-/V,N-
dimethylpropan-1-aminium dihydrogen 64.4 7.5"
phosphate, 16.8 mg/kg

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Values are given as mean SEM. Statistical analysis was performed by
Student's
t-test; *p<0.05 versus control group and #p<0.05 versus 3-carboxy-NAN-
trimethylpropan-1-aminium group; n=10.
The results are presented as a percentage relative to the control group which
was
assigned a value of 100%.
Results presented in Table 1 show that 3-carboxy-N-ethyl-NN-dimethylpropan-1-
aminium, 3-carboxy-N-ethyl-NN-dimethylpropan-1-aminium hydrogen fumarate
and 3-carboxy-N-ethyl-NN-dimethylpropan-1-aminium dihydrogen phosphate had
a protective effect on formation of atherosclerotic lesions in aorta of apoE-/-
mice.
3-Carboxy-NAN-trimethylpropan-1-aminium, in contrary, was not effective.