The present invention relates to a novel pharmaceutical composition and its use in particular within the framework of combating atherosclerosis.
Atherosclerosis is the first cause of mortality in the industrialized countries, with more than 20% of deaths (Murray J L, Lopez AD. Mortality by cause for eight regions of the world: global burden of disease study. Lancet 1997; 349: 1269-76). According to the WHO (1954), atherosclerosis is a variable combination of changes to the intima of the large and medium-calibre arteries involving the formation of an atheromatous plaque, i.e. a local accumulation of lipids, complex carbohydrates, blood and blood products, fibrous tissue and calcareous deposits; all accompanied by modifications of the media. This pathology manifests itself only after several years of sub-clinical evolution. Its prevention, even more than its treatment, therefore remains of the utmost importance.
The formation of the plaque is a succession of five stages (Tedgui A, Mallat Z. Formation de la plaque d'athérosclérose. Rev Prat 1999; 49: 2081-2086):
L-Arginine, a basic amino acid present in plant and animal proteins, is essential to the synthesis of tissue proteins. In different animal studies, when the diet is supplemented with L-Arginine (2% in the drinking water, a dose making it possible to double the plasmatic arginine concentration), it seems that the latter tends to have an antiatherogenic effect. This treatment also seems to have an effect on vascular reactivity. Böger et al. (Böger RH, Bode-Böger S M, Brandes R P, Phivthong-ngam L, Böhme M, Nafe R, et al. Dietary L-Arginine reduces the progression of atherosclerosis in cholesterol-fed rabbits. Circulation 1997; 96: 1282-1290), as well as Behr-Roussel et al. (Behr-Roussel D, Rupin A, Simonet S, Bonhomme E, Coumailleau S, Cordi A, et al. Effect of chronic treatment with the inducible nitric oxide synthase inhibitor N-iminoethyl-L-lysine or with L-arginine on progression of coronary and aortic atherosclerosis in hypercholesterolemic rabbits. Circulation 2000; 102: 1033-1038) demonstrate a stabilization of the atheromatous plaque area with an enriched diet over 12 to 16 weeks.
Statins are powerful cholesterol synthesis inhibitors. Their main indication is the reduction of plasmatic LDL-cholesterol in primary or secondary prevention of cardiac ischemic accidents (Vaughan C J, Murphy M B, Buckley B M. Statins do more than just lower cholesterol. Lancet 1996; 348: 1079-1082).
Statins inhibit HMG-CoA reductase by reversible competition with the substrate HMG-CoA, for the active site of the enzyme. They therefore lead to a reduction in the synthesis of mevalonate and its derivatives including cholesterol. Therefore, statins are indicated for the treatment of atherosclerosis.
However, at present, none of the treatments mentioned makes it possible to completely prevent and/or treat atherosclerosis.
An objective of the invention is therefore to provide a more effective method for the prevention and/or treatment of atherosclerosis.
The present invention follows in particular from the demonstration of an unexpected synergistic effect of the combination of a statin and L-Arginine within the framework of the prevention and/or treatment of atherosclerosis.
Thus, the present invention relates to the use,
in which R represents, —H,
or a pharmaceutically acceptable salt thereof, for the preparation of a medicament intended for the prevention or treatment of atherosclerosis, in particular of primary or secondary atherosclerosis, hypertension, diabetes, or neurodegenerative diseases, in particular Alzheimer's disease. When R represents —H, the compound of formula (I) corresponds to ornithine. When R represents
the compound of formula (I) corresponds to arginine. When R represents
the compound of formula (I) corresponds to citrulline.
These three amino acids possess a common metabolism in the organism.
Hypertension, diabetes and the neurodegenerative diseases, in particular Alzheimer's disease, have a vascular component linked to an endothelial dysfonction which can be compensated for by the synergistic combination of a compound of formula (I) and statin, in particular by the arginine-statin synergistic combination, according to the invention.
The expression “in combination” means that the statin and the compound of formula (I) are both present independently in the medicaments according to the invention, where they are in no event interlinked, either by means of weak bonds, such as electrostatic interactions, in order to form a salt for example, or by means of strong bonds, such as covalent bonds.
As is meant here, the word “statin” designates a compound belonging to the class of HMG-CoA reductase inhibitors. In particular, the term statin in no event corresponds to an amino acid derivative originating from pepstatin and having renin-inhibiting properties.
Moreover, advantageously, the compound of general formula (I), in particular arginine, is used as an adjuvant in order to increase the effects of the statins within the framework of the preparation of medicaments intended for the prevention or treatment of atherosclerosis, in particular of primary or secondary atherosclerosis, hypertension, diabetes, or neurodegenerative diseases, in particular Alzheimer's disease.
According to a preferred embodiment of the invention, the statin is chosen from the group comprising: atorvastatin, pravastatin, fluvastatin, rosuvastatin, lovastatin and simvastatin.
This list is not limitative, all the other statins known to a person skilled in the art can also be used according to the invention.
According to a particularly preferred embodiment of the invention, the statin is atorvastatin.
According to another preferred embodiment of the invention, the compound of formula (I) corresponds to the L-isomer.
According to yet another preferred embodiment of the invention, R represents
the compound of formula (I) then corresponding to arginine, in particular L-Arginine.
The invention relates more particularly to the use as defined above, of a statin, in particular atorvastatin, in a quantity suitable for the administration to an individual of a dose from about 5 mg/day to about 80 mg/day.
The invention also relates more particularly to the use as defined above, of a compound of formula (I), in particular L-Arginine, in a quantity suitable for the administration to an individual of a dose from about 1 g/day to about 30 g/day, in particular from about 5 g/day to about 30 g/day.
Advantageously doses of L-Arginine of less than 5 g/day are particularly suitable for children.
The invention also relates quite particularly to the use, as defined above, of L-Arginine, in a quantity suitable for the administration to an individual of a dose of at least about 10 g/day.
In a particular embodiment, the invention relates to the use, as defined above, of L-Arginine in a quantity suitable for the administration to an individual of a dose of about 0.15 g/kg/day, i.e. a dose of 0.15 g/day of L-Arginine per kg of bodyweight of the individual in question.
According to another particular embodiment of the use as defined above, the medicament is suitable for the administration to an individual of a single dose from about 5 mg to about 80 mg of statin and from about 5 g to about 30 g, in particular about 10 g, of L- Arginine.
The present invention also relates to products containing
in which R represents, —H,
or a pharmaceutically acceptable salt thereof, as combination products for simultaneous or separate use or use spread over time within the framework of the treatment of atherosclerosis, in particular of primary or secondary atherosclerosis, hypertension, diabetes, or neurodegenerative diseases, in particular Alzheimer's disease.
The expression “combination products” signifies that the statin and the compound of formula (I) are both present independently in the products according to the invention, where they are in no event interlinked, either by means of weak bonds, such as electrostatic interactions, in order to form a salt for example, or by means of strong bonds, such as covalent bonds.
According to a preferred embodiment of the invention, the statin is chosen from the group comprising: atorvastatin, pravastatin, fluvastatin, rosuvastatin, lovastatin and simvastatin.
According to a particularly preferred embodiment, the statin is atorvastatin.
According to another preferred embodiment of the invention, the compound of formula (I) corresponds to the L-isomer.
According to yet another preferred embodiment of the invention, R represents
the compound of formula (I) then corresponding to arginine, in particular L-Arginine.
According to a particularly preferred embodiment of the invention, said products are suitable for the administration to an individual of a dose from about 5 mg/day to about 80 mg/day of statin, in particular atorvastatin.
According to another particularly preferred embodiment of the invention, said products are suitable for the administration to an individual of a dose from about 1 g/day to about 30 g/day, in particular from about 5 g/day to about 30 g/day, of a compound of formula (I), in particular L-Arginine.
Advantageously doses of L-Arginine of less than 5 g/day are particularly suitable for children.
According to another particularly preferred embodiment of the invention, said products are suitable for the administration to an individual of a dose of L-Arginine of at least about 10 g/day.
In a particular embodiment, said products are suitable for the administration to an individual of a dose of L-Arginine of about 0.15 g/kg/day.
According to another particular embodiment of the products as defined above, the products are suitable for the administration to an individual of a single dose from about 5 mg to about 80 mg of statin and about 5 g to about 30 g, in particular about 10 g, of L-Arginine.
The present invention also relates to a pharmaceutical composition comprising as active ingredient:
in which R represents, —H,
or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable vehicle.
When R represents —H, the compound of formula (I) corresponds to ornithine.
When R represents
the compound of formula (I) corresponds to arginine.
When R represents
the compound of formula (I) corresponds to citrulline. These three amino acids possess a common metabolism in the organism.
As is meant here, the statin and the compound of formula (I) are both present independently in the pharmaceutical compositions according to the invention, where they are in no event interlinked, either by means of weak bonds, such as electrostatic interactions, in order to form a salt for example, or by means of strong bonds, such as covalent bonds.
According to a preferred embodiment of the invention, the statin is chosen from the group comprising: atorvastatin, pravastatin, fluvastatin, rosuvastatin, lovastatin and simvastatin.
This list is not limitative, all the other statins known to a person skilled in the art can also be used according to the invention.
According to a particularly preferred embodiment of the invention, the statin is atorvastatin.
According to another preferred embodiment of the invention, the compound of formula (I) corresponds to the L-isomer.
The natural amino acids found in the organism are essentially of L type.
According to yet another preferred embodiment of the invention, R represents
the compound of formula (I) then corresponding to arginine, in particular to L-Arginine.
According to another preferred embodiment of the invention, the pharmaceutical composition according to the invention is suitable for the administration to an individual of a dose from about 5 mg/day to about 80 mg/day of statin, in particular atorvastatin.
According to yet another preferred embodiment of the invention, the pharmaceutical composition according to the invention is suitable for the administration to an individual of a dose from about 1 g/day to about 30 g/day, in particular from about 5 g/day to about 30 g/day, of a compound of formula (I), in particular L-Arginine.
Advantageously doses of L-Arginine of less than 5 g/day are particularly suitable for children.
According to a particularly preferred embodiment, the pharmaceutical composition according to the invention is suitable for the administration to an individual of a dose of L-Arginine of at least about 10 g/day.
In a particular embodiment, the pharmaceutical composition as defined above is suitable for the administration to an individual of a dose of L-Arginine from about 0.15 g/kg/day.
According to another particular embodiment of the pharmaceutical compositions as defined above, the pharmaceutical compositions are suitable for the administration to an individual of a single dose from about 5 mg to about 80 mg of statin and from about 5 g to about 30 g, in particular about 10 g, of L-Arginine.
According to another preferred embodiment of the invention, the pharmaceutical composition according to the invention is suitable for administration by oral route.
According to yet another preferred embodiment of the invention, the pharmaceutical composition according to the invention is characterized in that it is presented in the form of a powder to be diluted, pills, sachets, tablets, capsules, or any other acceptable galenic form.
Twenty-four Watanabe rabbits (Centre de Production Animale, Olivet, France) which are hyperlipidaemic due to an hereditary LDL-receptor (LDL-R) deficiency, homozygous, and six weeks old, are used. Each animal is placed in an individual cage and receives a food ration of 200 g to 250 g per day. After acclimatization to the animal house for 7 days, four groups of six rabbits are formed:
The intake of L-Arginine in the enriched diet is such as to correspond to an L-Arginine intake of about 0.15 g/kg/day in humans.
The food intake is measured daily and the animals are weighed once a week. Plasma samples are taken every fifteen days from the marginal ear vein. The blood, collected in heparin tubes, is centrifuged (4° C., 2000 g, 15 minutes). Part of the plasma is deproteinized by a solution of sulphosalicylic acid (30%) then divided into aliquot fractions with a view to an assay of the amino acids. The remainder of the plasma is aliquoted into several fractions of about 200 μl, and stored at −80° C.
After eight weeks, a sampling of serum (about 1 ml) is carried out then the animals are anaesthetized with an injection of heparinized pentobarbital, and finally sacrificed.
The whole study is carried out in accordance with current regulations relating to animal experimentation.
2. Biochemical Analyses The lipid profile, i.e., cholesterol, triglycerides, HDL (High Density Lipoprotein) and LDL (Low Density Lipoprotein) cholesterol, is determined on a Hitachi 911 by the usual methods. The cholesterol is assayed according to an enzymatic colorimetric test using a cholesterol-esterase and a cholesterol-oxidase, followed by a reaction with a peroxidase. The latter reacts with hydrogen peroxide and releases a red derivative the stain intensity of which (measured between 550 and 700 nm) is directly proportional to the cholesterol concentration. The triglyceride assay is also based on this principle, using a lipase which releases glycerol, and a peroxidase which reacts with the hydrogen peroxide and forms a red compound. The HDL cholesterol is assayed by an enzymatic calorimetric test in two phases. The first selects the different lipid fractions by means of dextran sulphate. The second stage is similar to the cholesterol assay, involving a cholesterol-esterase and a cholesterol-oxidase modified by polyethylene glycol, and a peroxidase which releases a blue-violet derivative. The results are expressed in mmol/l. The kits necessary for all these assays are supplied by RANDOX (Montpellier, France). The LDLs are calculated using the Friedewald formula, from the total cholesterol, the HDL cholesterol, and the triglycerides (Friedewald et al. (1972) Clin. Chem. 18: 499-502).
The plasmatic arginine, ornithine and citrulline concentrations are determined on a JEOL (Tokyo, Japan) amino acid analyzer. The amino acids are separated by cation-exchange chromatography. On leaving the column, they are developed by a ninhydrin stain reaction. The reaction obtained is quantified by photometry at 570 and 440 nm. The results are expressed in μmol/l.
On the day of sacrifice, after anaesthesia, in the shortest possible time, the animal is opened along the whole length of the cervico-thoracic region. The aorta is incised as low as possible. Using curved scissors, the aorta is removed from the aortic arch as far as the level of the iliac bifurcation following the vertebral column. The aorta is first cleaned in physiological serum and the fatty tissues are removed from its external tunica. It is fixed for 20 minutes in a 2% paraformaldehyde-2% glutaraldehyde mixture then opened along its length. It is then again immersed in the same fixer for 24 hours, after which the aorta is rinsed in 70% ethanol, then stained with Soudan IV for 15 minutes. It is once again rinsed in 80% ethanol for 20 minutes then under running water for 1 hour (22). Then the aorta is photographed flat with a Nikon 995 digital camera. The images are analyzed by means of planimetry software (Scion Image) which makes it possible to determine the surface of the lesions. Finally the aorta is fixed in the same fixer with a view to microscopic study.
For optical microscopy, the aorta is cut into four segments:
Adjacent fragments are sampled for the electron microscopy.
The samples are progressively dehydrated in ethanol up to 100% ethanol then treated with paraffin. The sections thus impregnated with paraffin are included according to an oriented inclusion which makes it possible to have transversal sections. 5 μm sections are produced, which are stuck onto slides then stained with HES (haemalum/erythrosine/saffron).
4. Statistical Analyses
StatView software, version 5.0, was used for the statistical analysis of the data. All the values are given as the average+SEM. Comparison of the groups is carried out by a Kruskal-Wallis test. This test is a non-parametric version of variance analysis with a ranking factor. Comparison between two groups is carried out by a Mann-Whitney test, which is the non-parametric version of the t-test on independent series. Significant values for p<0.05 are considered. The significance of the correlations between quantitative variables is assessed by the Spearman test.
The treatment was carried out over 8 weeks (from T0 at the start of the treatment to T8).
1.1. Monitoring of the Animals: Weight and Food Intake
During the experimentation, the animals in the four groups have an identical growth curve (
The average food intake (
The measurement of the weights and daily food intake reflects the homogeneity of the animals with respect to these parameters whatever the group. At age 10 weeks (T4), their weight is comprised between 1.9 and 2.1 kg which corresponds to the data found in the literature (Murakami S, Kondo Y, Sakurai T, Kitajima H, Nagate T. Taurine suppresses development of atherosclerosis in Watanabe heritable hyperlipidemic (WHHL) rabbits. Atherosclerosis. 2002; 163: 79-87). The food intake increases as a function of age (from 97.6 to 139.5g/day for the control group for example) and remains relatively constant after T3.
1.2. The Lipid Profile
Treatments with atorvastatin and with atorvastatin plus arginine have a beneficial effect on the plasmatic cholesterol (
The plasmatic triglycerides concentration (
The HDL concentration (
Similarly, the LDL concentration (
On the one hand, the lipid profile is consistent with the literature: our animals at the start of the study (six weeks old) have cholesterolaemia between 20.4 mmol/l (control group) and 25.4 mmol/l (statin/arginine group), consistent with the work of Clubb et al. (Clubb F J, Cerny J L, Deferrari D A, Butler-Aucoin M M, Willerson J T, Buja L M. Development of atherosclerotic plaque with endothelial disruption in Watanabe heritable hyperlipidemic rabbit aortas. Cardiovasc Pathol 2001; 10: 1-11) which reports an average value of 21.9 +1.5 mmol/l. On the other hand, Dowell et al. (Dowell F J, Hamilton C A, Lindop G B, Reid J L. Development and progression of atherosclerosis in aorta from heterozygous and homozygous WHHL rabbits. Effects of simvastatin treatment. Arterioscler Thromb Vasc Biol. 1995; 15: 1152-60) have carried out a comparative study between heterozygous and homozygous Watanabe rabbits and have defined the homozygous rabbits as having a cholesterol level of more than 10 mmol/l, which is indeed the case with our animals.
Between T0 and T8, in the control group and the arginine group an increase in the plasmatic cholesterol concentration of 20% and 30% respectively is observed with age (
As regards the triglycerides, the same profile is observed for the control group and the arginine group: the triglycerides increase by more than half during the protocol (between T0 and T8). By contrast, the animals treated with statin (statin group and statin/arginine group) have a virtually constant concentration between T0 and T8, which implies a positive effect of the atorvastatin against hypertriglyceridaemia.
Arginine alone appears to have no appreciable effect on this lipid profile, which is consistent with numerous works (Böger R H, Bode-Böger S M, Brandes R P, Phivthong-ngam L, Böhme M, Nafe R, et al. Dietary L-Arginine reduces the progression of atherosclerosis in cholesterol-fed rabbits. Circulation 1997; 96: 1282-1290; Brandes R P, Brandes S, Böger R H, Bode-Böger S M, Mugge A. L-Arginine supplementation in hypercholesterolemic rabbits normalizes meukocyte adhesion to non-endothelial matrix. Life Sci 2000; 66: 1519-1524; Singer A H, Tsao P S, Wang B Y, Bloch D A, Cooke J P. Discordant effects of dietary L-arginine on vascular structure and reactivity in hypercholesterolemic rabbits. J Cardiovasc Pharmacol 1995; 25: 710-716).
1.3. Concentration of Arginine, Ornithine, and Citrulline
The plasmatic arginine concentration (
The ornithine profile (
The plasmatic citrulline concentration (
In general, the L-Arginine concentration (this parameter was assayed at times T0, T2, T4, T6 and T8, data not shown) doubled in the treated groups, i.e. the arginine group and the statin/arginine group.
3.1. Macroscopic Study of the Aortas
Evaluation of the surface area of the lesions (
If interest is confined to the distal half of the aorta, the results are very significant (
3.2. Microscopic Study of the Arterial Wall
Observation of the slides shows a smaller thickness of the lesions for the statin/arginine group compared with the statin group. The larger lesions are found in the two firsts sections (at the start and end of the aortic arch); on the last two sections (middle and end of the aorta), the lesions are less frequent. As regards their composition, the majority of lesions are more fibrous than cellular (comprising spumous and some polynuclear macrophages). There is no particular tendency as regards the composition of the lesions between the groups.
At macroscopic level, the lesion surface is unchanged in the statin group versus the control group, whereas numerous works are of the opposite opinion, in particular that of Kroon et al. (Aliev G, Burnstock G. Watanabe rabbits with heritable hypercholesterolaemia: a model of atherosclerosis. Histol Histopathol. 1998; 13: 797-817). After administration of pravastatin at a rate of 40 mg/kg/day for nine months, it shows a 53 to 80% reduction in the incidence of lesions. Similarly, Maeso et al. show a reduction in the size of the lesions in New Zealand White rabbits treated with atorvastatin, 2.5 mg/kg/day (Maeso R, Aragoncillo P et al. Effect of Atorvastatin on endothelium-dependant constrictor factors in dyslipidemic rabbits. Gen Pharma. 2000; 34: 263-272). Also, Böger (Böger R H, Bode-Böger SM, Brandes RP, Phivthong-ngam L, Böhme M, Nafe R, et al. Dietary L-Arginine reduces the progression of atherosclerosis in cholesterol-fed rabbits. Circulation 1997; 96: 1282-1290) has shown that lovastatin slowed down the plaque progression in New Zealand White rabbits.
Moreover, the arginine group has a lesion surface which is smaller than the control by a factor of 1.3 and the statin/arginine treatment shows the smallest lesion surface with 8.8% lesion compared with 13.7% for the control group.
Moreover, a significant inverse correlation between total lesion surface and arginine concentration is found throughout the protocol. This correlation is also found for ornithine.
Finally, at microscopic level, it appears that for the statin/arginine group, the lesions are less thick than for the other groups.
Number | Date | Country | Kind |
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04/04569 | Apr 2004 | FR | national |
The present application is a divisional of co-pending application Ser. No. 11/587,426, filed Oct. 25, 2006, which is a 371 National Stage of international application no. PCT/FR2005/001083, filed Apr. 29, 2005, which claims priority to French application no. 04/04569, filed Apr. 29, 2004. The entire contents of the above-referenced applications are hereby incorporated by reference in their entirety.
Number | Date | Country | |
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Parent | 11587426 | Jan 2007 | US |
Child | 12172362 | US |