WO2002036111A1

WO2002036111A1 - New use of 2-(4-isobutylphenyl)propionic acid

Info

Publication number: WO2002036111A1
Application number: PCT/PL2001/000086
Authority: WO
Inventors: Marek Naruszewicz; Danuta Zapolska-Downar; Dariusz Majdas; Ryszard Harasym; Zdzislawa Miarczynska
Original assignee: Pabianickie Zaklady Farmaceutyczne Polfa
Priority date: 2000-10-31
Filing date: 2001-10-30
Publication date: 2002-05-10
Also published as: AU2002214422A1; PL343617A1

Abstract

The invention relates to the new use of 2-(4-isobutylphenyl)propionic acid (ibuprofen) for the preparation of a medicament for the treatment of hyperlipidaemia, elevating the level of high density cholesterol (HL) in the blood and/or prevention and treatment of atherosclerosis or cardiovascular diseases, associated with atherosclerosis, pharmaceutical composition for the treatment of hyperlipidaemia, elevating the level of high density cholesterol (HDL) level and/or prevention and treatment of atherosclerosis or cardiovascular diseases, associated with atherosclerosis, and a method of treatment of hyperlipidaemia, elevating the level of high density cholesterol (HDL) in the blood and/or prevention and treatment of atherosclerosis or cardiovascular diseases, associated with atherosclerosis.

Description

New use of 2- (4-isobutylphenyl) propionic acid

Field of invention

The present invention relates to the new use of 2- (4- isobutylphenyl) propionic acid (ibuprofen) for the preparation of a medicament for the treatment of hyperlipidaemia, elevating the level of high density cholesterol (HDL) in the blood and/or prevention and treatment of atherosclerosis or cardiovascular diseases, associated with atherosclerosis, pharmaceutical composition for the treatment of hyperlipidaemia, elevating the level of high density cholesterol (HDL) in the blood and/or prevention and treatment of atherosclerosis or cardiovascular diseases, associated with atherosclerosis, and a method of treatment of hyperlipidaemia, elevating the level of high density cholesterol (HDL) in the blood and/or prevention and treatment of atherosclerosis or cardiovascular diseases, associated with atherosclerosis. State of the art

Atherosclerosis is considered to be one of the major aetiological factors for cardiovascular diseases. According to the current theory, atherosclerosis is a chronic inflammatory process taking place within a wall of a blood vessel. Hyperlipidaemia is one of the essential risk factors for atherosclerosis and cardiovascular diseases associated with atherosclerosis. The important factor of the atherosclerosis development is atherogenic lipid profile, i.e. hyperlipidaemia with elevated LDL-cholesterol levels, elevated triglycerides levels and/or relatively lowered HDL- cholesterol levels. Besides lipid metabolism disorders, one of the main pathogenic factors is oxidative stress, said stress being the consequence of increased production of reactive oxygen species (ROS) . The source of ROS may be activated monocytes/macrophages . Oxidative stress may be caused by smoking cigarettes, one of the main risk factors for atherosclerosis. Like in other inflammatory processes, an early event in atherosclerosis is the adhesion of monocytes to endothelial cells, followed by transendothelial migration into the intima, where monocytes become the main source of foam cells in individuals with disorders of lipid metabolism. This process depends on both a disfunction of endothelial cells (expression of endothelial adhesive particles) and an activation of monocytes by chemotactic factors.

2- (4-Isobutylphenyl) propionic acid, known also under generic name ibuprofen, is a non-steroidal antiinflammatory drug (nsaid) , exhibiting antiinflammatory, analgesic and antipyretic activity. It is generally believed that these activities are linked with its inhibition of cyclooxygenase, a key enzyme in the arachidonic acid cascade.

Ibuprofen as a chemical compound as well as the processes for the preparation thereof have been described in US Patents No 3228831 and 3385886. Pharmacology and clinical activity of ibuprofen have been described for example in Am. J. Med., 77 (1A), 1-125 (1984).

It has now been surprisingly found that ibuprofen has the ability to reduce significantly blood triglycerides levels and increase blood high density (HDL) cholesterol levels and thus can be useful for treatment and prevention of hyperlipidaemia and atherosclerosis in susceptible individuals, as well as for treatment and prevention of cardiovascular diseases associated with atherosclerosis. Disclosure of the invention

In the first aspect the invention relates to the use of 2- (4-isobutylphenyl) propionic acid (ibuprofen) for the preparation of a medicament for the treatment of hyperlipidaemia, elevating the level of high density cholesterol (HDL) in the blood and/or prevention and treatment of atherosclerosis or cardiovascular diseases, associated with atherosclerosis.

In one of the preferred embodiments the invention relates to the use of ibuprofen the preparation of a medicament for lowering blood triglycerides level, especially in smokers .

In the next preferred embodiment of this aspect the invention relates to the use of ibuprofen for the preparation of a medicament for elevating the level of high density cholesterol (HDL) in the blood.

•In further embodiment of this aspect the invention relates to the use of ibuprofen for the preparation of a medicament for prevention and treatment of atherosclerosis. In yet further embodiment of this aspect the invention relates to the use of ibuprofen for the preparation of a medicament for prevention and treatment of coronary heart disease, especially acute coronary episodes, ischaemic heart disease and inhibiting vascular restenosis after coronary angioplasty.

In the second aspect the present invention relates to a pharmaceutical composition for the treatment of hyperlipidaemia, elevating the level of high density cholesterol (HDL) in the blood and/or prevention and treatment of atherosclerosis or cardiovascular diseases, associated with atherosclerosis, which comprises an effective amount of 2- (4-isobutylphenyl) propionic acid (ibuprofen) as an active ingredient, in combination with pharmaceutically acceptable carrier. In one of the preferred embodiments of this second aspect the invention relates to the composition as defined above for lowering the level of triglycerides in the blood, especially in smokers .

In the next preferred embodiment of this aspect the invention relates to the composition as defined above for elevating the level of high density cholesterol (HDL) in the blood.

In further embodiment of this aspect the invention relates to the composition as defined above for prevention and treatment of atherosclerosis.

In yet further embodiment of this aspect the invention relates to the composition as defined above for prevention and treatment of coronary heart disease, especially acute coronary episodes, ischaemic heart disease and inhibiting vascular restenosis after coronary angioplasty.

In a third aspect the present invention relates to a method of treatment of hyperlipidaemia, elevating the level of high density cholesterol (HDL) in the blood and/or prevention and treatment of atherosclerosis or cardiovascular diseases, associated with atherosclerosis, in mammals, especially man, which method comprises administering an effective amount of 2- (4-isobutylphenyl)propionic acid to said mammal in need of such treatment.

The method of the invention can be used in particular for lowering blood triglycerides level, especially in smokers .

The method of the invention can be particularly preferably used for elevating the level of high density cholesterol (HDL) in the blood. In a further preferred embodiment of this aspect the invention the method can be used for prevention and treatment of atherosclerosis.

In yet further embodiment of this aspect the invention relates to the method as defined above for prevention and treatment of coronary heart disease, especially acute coronary episodes, ischaemic heart disease and inhibiting vascular restenosis after coronary angioplasty. Brief description of Figures

Fig. 1 presents the adhesiveness of resting monocytes to non-stimulated (A) and TNF -activated endothelial cells before and after ibuprofen treatment in smokers and nons okers .

Fig. 2 presents the generation of superoxide radical O2- in both resting (A) and PMA-activated (B) monocytes before and after ibuprofen treatment in smokers and nonsmokers.

Fig. 3 presents the generation of H2O2 in resting (A) and PMA-activated (B) monocytes before and after ibuprofen treatment in smokers and nonsmokers. Detailed description of the invention The term „hyperlipidaemia" as used in the description and in the claims means the state of abnormally elevated level of lipids in the blood. The lipids, elevated levels of which may be treated according to the invention are especially total cholesterol, triglycerides and low-density lipoproteins (LDL) .

Thus the treatment and prevention of hyperlipidaemia will encompass lowering the level of cholesterol, triglycerides and low-density lipoproteins in the subject in the need of such treatment. ^"Treatment according to the method of the invention will also encompass elevating the level of high density cholesterol (HDL) .

The term „subject in the need of such treatment" means the subject in which hyperlipidemia has been found, including elevated level of triglycerides and/or lowered level of HDL cholesterol in the blood, as well as individuals at risk of having hyperlipidemia and/or atherosclerosis, especially subjects with a familial history of elevated blood lipids levels, peri- and post-menopausal females, females with surgically or chemically induced oestrogen deficiency, the aged individuals, the individuals with hyperglycaemia, diabetes, hypertension and obesity, smokers, individuals after myocardial infarct and/or coronary angioplasty, individuals after surgical by-pass procedure, as well as other individuals at risk of atherosclerosis developments for other reasons known for the person skilled in the art. Thus it will be appreciated by the person skilled in the art that prevention of atherosclerosis includes also elevating the level of HDL cholesterol in the blood.

The term ,,diseases associated with atherosclerosis" includes inter alia cardiovascular diseases, such as for example coronary heart disease, especially acute coronary episodes, ischaemic heart disease and vascular restenosis after coronary angioplasty.

The treatment according to the invention will include administration of ibuprofen both alone and as an adjunctive agent in combination with specific treatment of cardiovascular system.

In the method of the invention ibuprofen may be administered by any appropriate route, preferably by oral administration.

Ibuprofen preferably may be administered in the form of appropriate pharmaceutical formulation. Suitable form may be for example tablet or coated tablet, including parenterally coated tablet, sustained release tablet, soft or hard gelatine capsule, including sustained release capsule. Other suitable forms, especially in case of aged persons or persons having troubles with swallowing are solutions or syrups for oral administration, including also powders or granulates for ex tempore reconstitution in aqueous carrier. In the treatment and prevention according to the invention ibuprofen will be generally administered in the dosages for example from 20 mg to 1600 mg orally per day, in single or divided doses, generally one to four times a day. Generally however both the dosage regimen and the amount administered will be determined individually by the attending physician, depending on the needs and general state of individual patient. Exemplary single doses are 20 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 400 mg, 500 mg, 600 mg, 800 mg of ibuprofen, depending on the formulation and requirements of the medical treatment, exemplary dosage regimens being 20 mg to 800 mg 1 to 4 times per day, for example 200 mg 3 times per day, 400 mg 2 times per day, 400 to 800 mg once per day.

The invention will be described in more detail with reference to the folowing non-limiting Examples.

The meaning of abbreviations used is given below.

PBS - phosphate buffered saline PMA - phorbol myristate acetate

CRP - C-reactive protein

HUvΕC - human umbilical vein endothelial cells

TNF - tumor necrosis factor

IL-6 interleukina 6 Lp(a) - lipoproteina a

PBMC - peripheral blood mononuclear cells

The influence of ibuprofen on the plasma lipid profile and its effectioveness ^' as a protecting agent in atherosclerosis has been demonstrated in pharmacological tests in vivo and in vitro using freshly isolated monocytes from venous blood. Subjects tested

Tests were performed in a group of 9 smokers and 9 nonsmokers. None of the subjects had a history of hyperlipidemia, diabetes or family history of premature coronary artery disease. Venous blood was collected before and after a twoo-week course of ibuprofen (Polfa) 200 mg administered three times daily. Isolation of monocytes

25 ml of heparinised blood, diluted 1:1 with PBS was layered over 15 ml Ficoll-Paque and centrifuged (400 g for 40 min at 22°C) . The mixed mononuclear cell band was removed by aspiration, washed twice with PBS, divided into three aliquots and centrifuged. One aliquot was resuspended in RPMI-1640 media containing 20% autologous serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 iriM HEPES. The cell suspension was placed at 0,5 x 10^ monocytes per 16 mm multiwell dishes and incubated for 2 h at 37°C under humidified 5% CO2 in room air to allow the monocytes to adhere to the dish. The nonadherent cells were removed by washing twice with medium. The adherent cells were used to determine superoxide anion generation. The second aliquot was suspended in PBS prior to measurement of intracellular reactive specise (ROS) production. The third aliquot was suspended in medium 199 with 20 mM HEPES, counted and used in the adhesion assay. The mononuclear cell preparation consisted of approximately 30% monocytes and 70% lymphocytes.

Means and standard deviations were calculated on the basis the results of measurements and subjected to the unpaired Student's t-test between the groups or the paired test within each group. The level of significance was taken as p< 0,05. Example 1 Measurement of ibuprofen effect on lipid profile

The following parameters were measured to investigate the lipid profile: total cholesterol (Ch) , triglycerides (TG), low density cholesterol (ChLDL), high density cholesterol (ChHDL) , Apolipoprotein A (ApoAI), Apolipoprotein B (ApoB) ApoAI/ApoB ratio using enzymatic kits (CHOD-PAP, GPO-PA) from Boehringer Mannheim and Hitachi 902 autoanalyzer . HDL cholesterol was measured after precipitation of lipoprotein containing apoB with phosphotungstic acid in the presence of Mg2+, LDL-cholesterol after precipitaion of LDL with polyvinyl sulfate. Apo Al and apoB were determined by immunoturbidimetric method of the antigen-antibody reaction, on the basis of the test kits from Boehringer Mannheim and Hitachi 902 autoanalyzer.

It has been found (Table 1) that administration of ibuprofen resulted in significantly higher serum HDL cholesterol levels in both smokers (by 10%) and nonsmokers (by 15%) as well as in reduction of triglycerides level (by 14%) in smokers, having initial TG levels about twice higher comparing with nonsmokers. Example 2

Determination of systemic inflammatory response

Among inflammatory markers investigated by us were: Lp(a), fibrinogen, CRP and IL-6.

Lipoprotein a (Lp(a)) antigen in serum was measured with a turbidimetric immunoassay and commercially availabe Diapach kit (Dialab) . Plasma fibrinogen was determined using the method of Clauss and test kits from BioMerieux. Interleukin-6 was measured in serum with an ELISA kit (Boehringer Mannheim, Germany) . Serum C-reactive protein (CRP) concentration was measured by rate immunonephelometry on ARRAY-360 (Beckmann Instruments) .

It has been hown that ibuprofen significantly decreased plasma fibrinogen levels in smokers (p <0,01) and Lp(a) levels in nonsmokers (p <0,05). A tendency to reduce Lp ( ) , CRP and IL-6 in smokers has been noticed (Table 1) .

Table , The effect of ibuprofen on lipid parameters and markers of iriflammation

Smokers Nonsmokers

(mean ± SD) (mean + SD)

before after P before after P treatment treatment treatment treatment

Total cholesterol (mg/dl) 232.8 ± 37.4 230.4 ±34.8 0.522 197.0 ±17.4 193.1 ±23.9 0.508

Triglycerides (mg/dl) 153.7 ±87.5 132.2 ±68.5 0.039 90.4 ± 25.9 90.6 ±37.3 0.973

LDL cholesterol (mgdl) 142.6 ±38.6 141.2 ±36.2 0.637 95.5 ±24.6 87.1 ±20.6 0.054

HDL cholesterol (mg/dl) 53.6 ±11.4 59.1± 12.2 0.003 62.0 ± 13.8 71.6 ±13.5 0.0002

Apo Al (mg/dl) 143.0 ±17.9 142.0 ±14.7 d.769 145.9 ±18.9 153.0 ±19.2 0.051

Apo B (mg/dl) 110.5 ±22.6 106.9 ±19.0 0J96 9O.0±12.6 88.9 ±0.12.8 0.500

Apo AI/ApoB 1.3 ±0.3 1.3 ±0.3 0.367 1.6 ±0.3 1.7±0.3 0.005

Lp(a) (mgdl) 43.4 ±32.8 41 J ±29.2 0.279 ^■ 27.7 ±16.0 21.9 dr 13.5 0013

Fibrinogen (g/L) 3.4 ±0.5 3.0 ±0.6 0.005 2.6 ±0.6 2.4+0.6 0184

CRP (mgL) 2.4 ±0.8 2.0+1.0 0.197 1.5 ±0.6 1.3 ±0.3 0.245

IL-6 (pg/mJ) 2.0 + 2.9 IJ ±1.7 0.240 1.3+ 1.7 0.8 ± 1.2 0.446

Values before and after treatment were compared using paired Student's t test. HDL = high density lipoprotein- LDL = low density lipoprotein, CRP = C-reactive protein, IL-6 = interieυkm-6, Apo Al - apolipoprotein Al, Apo B = apolipoprotein B

Example 3

The effect of ibuprofen on adhesiveness of monocytes to endothelial cells.

The adhesiveness was measured by determining the amount of monocytes freshly isolated from peripheral blood of tested subjects which adhere to endothelial cells. The assay was were performed on endothelial cells isolated from human umbilical cords by collagenase digestion, both resting and activated with TNFα (disfunction of endothelial cells) . Second passage human umbilical vein endothelial cells

(HUVEC) were cultured in gelatin-coated 24-well plates. When confluent monolayers were formed, the medium was changed to medium 199 containing antibiotics, 10% FBS, 2 mM glutamine and 20 mM HEPES without ECGS. TNFα (100 U/ml) was added to some of the wells 12 h prior to the experiment. Next, HUVEC were washed with PBS and coincubated for 30 min with PBMC suspended in medium 199 with 20 mM HEPES to density of 1,5-2,0 x 10⁶/ml (0,5 ml per well). The PBMC suspension was withdrawn and the wells were washed twice with PBS to remove nonadherent cells. The endothelial cells with adherent mononuclear cells were detached by mild trypsinisation. The initial suspensions and the suspension from each well were counted three times. Cells detached from each well, consisiting of endothelial cells, monocytes and lymphocytes, were treated for 30 min at 4°C with saturating amounts of FITC-conjugated mouse ant-CD45 and PE-conjugated anti-CD14 monoclonal antibodies. Cells were next washed with FACS buffer, fixed in 1% paraformaldehyde and analysed (10000 cells per sample) by FACS (Becton Dickinson) . The proportion of monocytes in the suspension was established by measuring fluorescence I (FL I- CD45) and fluorescence II (FL II-CD14). The absolute number of monocytes adhering to endothelial cells was calculated in relation to the total number of cells obtained after trypsinisation. The results were expressed as percentage of monocytes added. It has been demonstrated (Fig. 1) that the adhesion of monocytes to both resting (A) and stimulated (B) endothelial cells was significantly higher in smokers than in nonsmokers. The administration of ibuprofen significantly reduced the adherence of monocytes to both resting (A) and stimulated (B) endothelial cells in both groups tested. Example 4 The assessment of the activation of monocytes

To assess the metabolic activation of monocytes intracellular production of superoxide anion 02^_ and hydrogen peroxide by resting and PMA-stimulated freshly isolated monocytes was investigated. These tests provide also a measure of oxdidative stress.

02^~ generation by monocytes was determined using an assay which measured the SOD-inhibitable reduction of cytochrome c (Johnston R.B., Measurements of O2- secreted by monocytes and macrophages, Methods in Enzymol. 1984; 105; 365-9) . Monocyte cultures were exposed for 1 h at 37°C to ferricytochrome c (80 μM) in Krebs-Ringer phosphate buffer with or without PMA (100 ng/ml) . A replicate assay was performed in the presence of SOD (40 μg/ml) to verify the contribution of O2- to the reduction of cytochrome c. Te reaction was stopped by transfer to an ice bath followed by centrifugation at 1200 g for 10 min. Absorbance was read at 550 nm. To determine nmol of O2^"" produced, the extinction coefficient E550 = 21,0 x 10³ m^~l cm^-1 was used. The conversion factor in the case of a standard light path of 1 cm and a reaction volume of 1 ml is 47,6. The SOD control value was subtracted to determine SOD-inhibitable nmol O2- produced. Results were expressed as nmol O2^~/lh/0,5 x 10⁶ monocytes .

It has been demonstrated (Fig. 2) that the levels of O2- generation by both resting (A) and activated (B) monocytes in smokers were significantly lowered after ibuprofen treatment in both smokers and nonsmokers group. Measurement of generation of reactive oxygen species ^H2°2 (cellular oxidation) was based on ROS-mediated conversion of non-fluorescent 2 ' , 1 " -dichlorofluorescein (DCFH) , loaded into cells as 2 ' , 1 ' -dichlorofluorescein diacetate, into fluorescent DCF reflecting enhanced oxidative stress (Bass D.A. et al . , Flow cytometric studies of oxidative product formation by neutrophils; A graded response to membrane stimulation. J. Immunol. 1983; 130:1910-17). In brief, freshly isolated PBMC were resuspended in PBS and incubated with 20 μM DCFH, with or without PMA (100 ng/ l) , for 30 min in the dark. Fluorescence intensity of the DCF fluorophore formed by peroxide oxidation or its non- fluorescent precursor was detected with a cytofluorimetric assay (FACScan, Becton Dickinson) . Monocytes were gated on the basis of FSC and SCC and the results were expressed as mean fluorescence intensity.

There were no significant differences between the groups tested in the H2O2 production by both resting and activated monocytes . Ibuprofen significantly reduced H2O2 production in both resting (A) and PMA-stimulated (B) monocytes in both smokers and nonsmokers groups tested (Fig. 3) .

The results obtained show that ibuprofen may be useful in the treatment of certain forms of atherosclerosis, especially in the light of its beneficial effect on HDL cholesterol and triglycerides levels, as well as in the treatment of cardiovascular diseases associated with atherosclerosis . Example of a pharmaceutical composition formulation

Coated tablet of the following composition (in mg) Ibuprofen 200

Colloidal silica 1

Lactose 65

Starch 50

Talc 7

Polyvidone 4,5

Magnesium stearate 2,5

Coating: saccharose, talc, acacia gum, cochineal red (E124), Carnauba wax, beewax

Claims

1. The use of 2- ( 4-isobutylphenyl) propionic acid for the preparation of a medicament for the treatment of hyperlipidaemia, elevating the level of high density cholesterol (HDL) in the blood and/or prevention and treatment of atherosclerosis or cardiovascular diseases, associated with atherosclerosis.

2. The use of claim 1, for the preparation of a medicament for lowering the level of triglycerides in the blood, especially in smokers.

3. The use of claim 1 for the preparation of a medicament for elevating the level of high density cholesterol (HDL) in the blood.

4. The use of claim 1 for the preparation of a medicament for prevention and treatment of atherosclerosis.

5. The use of claim 1 for the preparation of a medicament for prevention and treatment of coronary heart disease, especially acute coronary episodes, ischaemic heart disease and inhibiting vascular restenosis after coronary angioplasty.

6. A pharmaceutical composition for the treatment of hyperlipidaemia, elevating the level of high density cholesterol (HDL) in the blood and/or prevention and treatment of atherosclerosis or cardiovascular diseases, associated with atherosclerosis, comprising effective amount of 2- (4-isobutylphenyl) ropionic acid as an active ingredients, in combination with pharmaceutically acceptable carrier.

7. The composition of claim 6, for lowering the level of triglycerides in the blood, especially in smokers.

8. The composition of claim 6 for elevating the level of high density cholesterol (HDL) in the blood.

9. The composition of claim 6 for prevention and treatment of atherosclerosis.

10. The composition of claim 6 for prevention and treatment of coronary heart disease, especially acute coronary episodes, ischaemic heart disease and inhibiting vascular restenosis after coronary angioplasty.

11. A method of treatment of hyperlipidaemia, elevating blood high density cholesterol (HDL) level and/or prevention and treatment of atherosclerosis or cardiovascular diseases, associated with atherosclerosis, in mammals, especially men, which comprises administering an effective amount of 2- (4- isobutylphenyl) propionic acid to said mammal in need of such treatment .

12. The method of claim 11, for lowering the level of triglycerides in the blood, especially in smokers.

13. The method of claim 11, for prevention and treatment of atherosclerosis.

14. The method of claim 11, wherein said cardiovascular disease is coronary heart disease, especially acute coronary episode.

15. The method of claim 11, wherein said cardiovascular disease is ischaemic heart disease.

16. The method of claim 11, for elevating the level of high density cholesterol (HDL) in the blood.

17. The method of claim 11, for inhibiting vascular restenosis after coronary angioplasty.