Inhibition of cellular adhesion

0001The invention relates to the use of a blackcurrant lipid in a dietetic or pharmaceutical composition with the aim of preventing the phenomena of adhesion responsible for certain thromboembolic, inflammatory and cancerous diseases.

0002We know that the polyunsaturated fatty acids of the ω3 and ω6 series have very important structural and functional roles. Polyunsaturated fatty acids can be designated by the number of carbon atoms, the number of double bonds and the number indicating the place of the first double bond counted from the methyl group which determines their metabolic family, noted ω. Thus in this nomenclature, dihomogammalinolenic acid (DHLA) is C20: 3ω6, eicosapentaenoic acid (EPA) is C20: 5ω3 and arachidonic acid (AA) is C20: 4ω6. The linoleic (LA, C18: 2ω6) and alphalinolenic (ALA, C18: 3ω3) fatty acids constitute the essential precursors of the other acids of the two families which are not synthesized by mammals. No metabolism allows one to pass from one family to another. The conversion of LA or ALA into the respective upper members of the two families is obtained by successive desaturation and elongation with relatively low yields.

0003Prostaglandins are a family of substances that have many biological effects. DHLA, AA and EPA are transformed under the action of cyclooxygenase, into prostaglandins of series 1 (PG1), 2 (PG2) and 3 (PG3), respectively. The PGs of series 1, 2 and 3 respectively comprise 1, 2 and 3 double bonds in their basic structure of fatty acids with 20 carbon atoms including a cyclopentene group. PG1 (from DHLA) has, among other properties, that of inhibiting the aggregation of blood platelets. On the other hand, the eicosanoids of series 2 (originating from AA), for example PG2, thromboxanes, for example PxA2 promote platelet aggregation. As for series 3 eicosanoids (from the EPA), their role is similar to that of series 1 eicosanoids.

0004It is assumed that the efficacy of EPA and DHLA in preventing thromboembolic cardiovascular disease is based on the favorable effects of PG3 and PG1 over the effects of PG2.

0005In addition, the lipoxygenase enzyme system leads to hydroxylated fatty acids and leukotrienes from the precursors AA and EPA. Recent studies indicate in particular that the compounds 13-hydroxy-octadecatrienoic acid (13-HODE) produced by the endothelial cells of the blood vessels and 12-hydroxy-eicosatetraenoic acid (12-HETE) produced by the platelets via the lipoxygenase play an important role in mediating the adhesion of cells to each other and consequently in the pathogenesis of thromboses, inflammatory diseases and the spread of cancer metastases. To simplify, adhesion would be influenced via the regulators 12-HETE and 13-HODE, positively by AA, precursor of 12-HETE and negatively by LA, leading to 13-HODE. DHLA is also unlikely to lipoxygenase.

0006It has been proposed, for example in the published French patent application No. 2,553,662, to add to a pharmaceutical composition or to a food product a mixture of a first fatty acid chosen from EPA or docosahexaenoic acid (DHA ) on the one hand and a second fatty acid chosen from DHLA, cis-linoleic acid, gammalinolenic acid (GLA) on the other hand, with the aim of preventing cardiovascular diseases. In this patent application, the fatty acids in question must have been isolated separately from natural fatty substances by iodization followed by saponification, extraction of the fatty acids by solvent, methylation thereof, separation of the methyl esters by chromatography and finally de-iodization. EPA, for example, is obtained this way from cod liver oil. The preparation of such a composition is particularly complicated.

0007In addition, the EPA is particularly unstable. Finally, some people cannot bear the reminiscences of bad tastes from fish oil, even deodorized or encapsulated.

0008EP-A-92076 relates to a lipid composition intended for oral, enteral or parenteral nutrition containing an oil supplying GLA in combination with a medium chain triglyceride and a long chain fatty acid facilitating the intestinal absorption of lipids. Depending on the prior art, a significant conversion of fatty acids to AA in the tissues should be expected.

0009We have found that it is possible by the use of a blackcurrant lipid to make available the EPA jointly with DHLA while depressing the bioavailability of AA without having the disadvantages associated with the ingestion of fish oil. , for example increased bleeding time which can cause bleeding. In this way, very interesting effects are obtained on the prevention of adhesion and aggregation of platelets, thromboses and the spread of cancer metastases.

0010The invention therefore relates to: the use of a blackcurrant lipid for the manufacture of a dietetic or pharmaceutical composition for promoting the bioavailability of dihomogammalinolenic and eicosapentaenoic acids compared to the bioavailability of arachidonic acid.

0011By blackcurrant lipid is meant according to the invention:<ul id="ul0001" list-style="dash"><li>blackcurrant seed oil (<u style="single">Ribes nigrum</u>), obtained by extraction from blackcurrant residues and refining, for example as indicated in European patent No 92,085 or European patent No 137,862,</li><li>a mixture of fatty acids from the hydrolysis or fractionation of blackcurrant seed oil, obtained for example according to European patent No 178,442 or according to European patent application No 271,747.</li><li>a pharmaceutically acceptable salt of the above fatty acids,</li><li>an oil obtained by reesterification of such a mixture of fatty acids with glycerol,</li><li>a mixture of the above lipids.</li></ul>

0012The blackcurrant lipid can be advantageously protected from oxidation by a fat-soluble antioxidant, for example ascorbyl palmitate, tocopherols, ascorbic acid in the presence of lecithin or a mixture of such antioxidants.

0013The dietary compositions may be in the form of emulsions, for example sauces, mayonnaises or margarines.

0014The pharmaceutical compositions can be in different forms suitable for the mode of administration, for example by the oral, enteral, rectal or parenteral route. One can for example prepare capsules, capsules, suppositories or syrups. In the case of enteral or parenteral administration, the compositions are in the form of physically or chemically stabilized, pyrogen-free and sterile solutions or emulsions.

0015The dose administered depends on the type and severity of the anomaly to be treated. It can be from 1 to 25 g of blackcurrant lipid and preferably from 2 to 5 g of blackcurrant oil per day in a single dose or preferably in 2 to 3 separate doses.

0016The examples below illustrate the invention. In these parts and percentages are by weight unless otherwise indicated.

Example 1

Incorporation of DHLA and EPA in tissues

0017Many pharmacological studies on the ability to modulate fatty acids in tissues through diet have mainly used the rat as an experimental model. It is known that in rats or mice, the ingestion of GLA leads to an increase in the incorporation of GLA in triglycerides or of DHLA in total lipids, but practically not in phospholipids. In humans, rabbits and guinea pigs, however, ingestion of GLA leads to the incorporation of DHLA into triglycerides and phospholipids in the blood, in the membrane of red blood cells and in all lipids. For this reason the guinea pig was preferred to the rat because, being much closer to humans with regard to the activity of desaturases, it better reflects the situation prevailing in humans. In addition, the liver was chosen as tissue since it constitutes the major site of fatty acid conversions.

1.1.

Experimental conditions

0018We form 3 groups of 7 male guinea pigs, weighing on average about 300 g which are placed in macrolon® cages. The groups are fed with semi-synthetic diets containing respectively blackcurrant seed oil (HPC), I, walnut oil (HN), II or lard (S), III ad libitum for 40 days . NHN is chosen as a comparison for HPC because it does not contain GLA and stearidonic (SA) acids, but more LA, so the total amounts of ω6 and ω3 acids and their ratio are comparable in the two oils.

0019All animals have free access to water containing 250 mg / l of ascorbic acid. After an overnight fast, the animals are anesthetized, the livers are removed, they are frozen quickly, they are finely ground and stored at -80 ° C until analysis. The compositions of the diet and of the lipids ingested are indicated in Tables 1 and 2 below.<tables id="tabl0001" num="0001"><img file="EP0374591B1_D0001.tif" /></tables><tables id="tabl0002" num="0002"><img file="EP0374591B1_D0002.tif" /></tables>

1.2.

Analysis of fatty acids

1.2.1.

0020The samples are prepared by homogenizing 1 g of frozen liver tissue in 20 ml of chloroform: methanol solvent mixture of ratio 2: 1 by volume and the total lipids (TL) are extracted therefrom after separation of the solids deposited by filtration, then elimination of the solvents .

1.2.2.

0021The TLs are separated into neutral lipids (NL) and polar lipids (PL) by passing over a column of silica gel. From the PL, the non-acid and acid fractions are separated on ion exchange columns filled with diethylaminoethyl-sepharose gel. The fraction of polar acid lipids is then desalted by passage over a column of silica gel.

1.2.3.

0022The NLs are separated into their various components by high performance thin layer chromatography (HPTLC) with glass plates coated with silicic acid. The plates are developed with a solvent mixture of petroleum ether: diethyl ether: acetic acid with a volume ratio 85: 15: 0.5, they are dried in air, they are sprayed with a solution of 0.005% of primulin in acetone (in weight / volume) and we visualize the bands corresponding to the different lipids with UV rays. The cholesterol esters (CE), triglycerides (TG) and free fatty acids (FFA) were identified by comparison with standard products and collected in glass tubes closed with Teflon-coated stoppers.

1.2.4.

0023The PLs are separated into their various components by HPTLC on silica gel plates impregnated with a 2% solution (weight / volume) of boric acid in absolute methanol and they are developed with a mixture of chloroform: methanol: triethylamine solvent: volume ratio water 30: 25: 34: 8. Acid phospholipids, phosphatidylserine (PS), phosphatidylinositol (PI), cardiolipin (CL) and non-acid phospholipids, phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were visualized as neutral lipids, identified in relation to standard products and collected as described above.

1.2.5.

0024The composition of TG, CE, FFA and PL is determined by gas chromatography of their methyl esters. The methyl esters are prepared by methylation in a known manner, then they are extracted with hexane. From the chromatograms obtained, the peaks of the compounds are identified by comparison with the chromatograms of standard products.

1.3.

Results

0025The results of lipid analysis are shown in Table 3 below:<tables id="tabl0003" num="0003"><img file="EP0374591B1_D0003.tif" /></tables><tables id="tabl0004" num="0004"><img file="EP0374591B1_D0004.tif" /></tables><tables id="tabl0005" num="0005"><img file="EP0374591B1_D0005.tif" /></tables>

0026The previous results show that the incorporation of GLA and DHLA is significantly greater in the case of the group fed with diet I, containing HPC.

0027There is no noticeable difference between the three groups with regard to the incorporation of AA. The result of these two observations is that the DHLA / AA ratio is significantly higher in the case of ingestion of HPC.

0028The incorporation of EPA is significantly higher in the case of ingestion of HPC than in the case of ingestion of NHN with regard to TG, CE, PC and PE. The same observation is true with respect to the ingestion of S with regard to TG and CE.

0029The unsaturation index shows that the guinea pig is capable of more than compensating for the differences in dietary fatty acid unsaturation.

0030In summary, the previous results demonstrate that the ingestion of HPC results in a significant incorporation of DHLA and higher DHLA / AA ratios in all the classes of hepatic lipids. In addition, the incorporation of EPA is greater for the HPC-based diet than for the NHN-based diet. It follows that the potential of PG1 and PG3 compared to PG2 is clearly demonstrated with regard to HPC.

Example 2

Influence of HPC ingestion on the fatty acid composition of plasma lipids in humans

0031A metabolic study is carried out in a healthy 32-year-old man to whom HPC concentrate capsules enriched with GLA (78%) and SA (16%) are administered for 6 weeks according to the table below:    days 1-20: 3 capsules / day, corresponding to approximately 1 g of GLA and 250 mg of SA / day.    days 21-26: 10 capsules / day, corresponding to approximately 3 g of GLA and 0.8 g of SA / day    days 27-37: interruption of administration due to influenza    days 38-42: 10 capsules / day, corresponding to approximately 3 g of GLA and 0.8 g of SA / day

0032The analysis of the fatty acids of the different classes of lipids in the blood serum is carried out by comparing with the control constituted by the average of a population receiving a placebo, by gas chromatography of the methyl esters.

0033The results are shown in Table 4 below.<tables id="tabl0006" num="0006"><img file="EP0374591B1_D0006.tif" /></tables>

0034The study of the previous results shows that the GLA ingested is incorporated into all classes of serum lipids and is rapidly metabolized to DHLA and to a lesser degree to AA. There is an increase in long chain fatty acids 3, in particular EPA, compared to the control, which seems to indicate that the SA is also incorporated.

0035We can conclude that the synthesis potential of PG1 and PG3 compared to PG2 is great when the diet is supplemented with HPC.

Example 3

Thrombosis and adhesion of blood platelets

3.1.

0036Study of the incorporation of fatty acids in the arterial walls. In this example, guinea pigs are fed diets containing different lipids for 6 weeks, then the fatty acid composition of the aortas of the animals is determined. The composition of the main fatty acids of the lipids of the diets is indicated in Table 5 and that of the fatty acids of the aortas in Table 6 below.<tables id="tabl0007" num="0007"><img file="EP0374591B1_D0007.tif" /></tables><tables id="tabl0008" num="0008"><img file="EP0374591B1_D0008.tif" /></tables>

0037There is a substantial increase in the rate of DHLA in the case of ingestion of HPC. This means that the potential for PG1 formation is increased in the blood vessels.

3.2.

0038Study of the influence of the diet on the biological functions of blood platelets. The capacity of guinea pig blood platelets marked with [³H] -adenine is measured to adhere to a thrombogenic surface constituted by discs covered with fibronectin after 4 weeks of ingestion of the diets of Example 3, Table 5. The number of plates adhered to the discs is counted. The results are shown in Table 7 below.<tables id="tabl0009" num="0009"><img file="EP0374591B1_D0009.tif" /></tables>

0039There is a decrease in adherence when ingesting HPC compared to all other diets, including one containing fish oil.

3.3.

Study of the thrombogenicity of the vascular wall and adhesion of platelets.

0040To study the influence of fatty acids in the diet on the thrombogenicity of blood vessels, groups of rabbits are fed the semi-synthetic HPC, HN and HP diets (Table 5, Example 3) and a commercial diet ("chow", VS). After 4 weeks on the diet, rabbits fed with HPC, HN and HP are injured in the carotid artery and then injected with [³H] -adenine-labeled blood platelets from donor rabbits fed the C diet. Measuring the in vivo accumulation of platelets on the injured artery tests the reactivity of the blood vessel. To discriminate the effect of the diet on the reactivity of the platelets from the effect on the reactivity of the blood vessel, platelets are isolated from the blood of rabbits fed on the 4 diets and their adhesion is measured ex vivo on discs covered with fibronectin.

0041The ability of blood vessels to synthesize 13-HODE and that of platelets to synthesize 12-HETE are also determined.

0042The results are shown in Tables 8 and 9 below:<tables id="tabl0010" num="0010"><img file="EP0374591B1_D0010.tif" /></tables>

0043It is found that in vivo the thrombogenicity of the blood vessel is the lowest with the HPC diet and that there is no difference between the HN and HP regimes. The ex vivo measurements of platelet adhesion show a reduced platelet reactivity with the HPC, HN and HP regimes compared with the C regime.<tables id="tabl0011" num="0011"><img file="EP0374591B1_D0011.tif" /></tables>

0044There is no difference in the synthesis of 12-HETE by the platelets of animals fed the HPC, HN and HP diets. On the other hand, the synthesis of 13-HODE by the vessels is greater with the HPC and HN regimes, which agrees with the greater content of linoleic acid in the blood vessels of these two groups and with the non-stick effect of 13-HODE. .

Example 4

Metastasis

4.1.

0045To study the influence of the diet on the proliferation and dissemination of metastases, rats were fed for 5 weeks with the diets of Example 3, Table 5, except that containing HN. A group of rats is fed a usual diet called "chow" (C). Tumor cells (Walker 256) are cultivated in a medium containing the radioactive nucleotide [¹²⁵ I] -uridine marking the tumor cells. The cells are then injected into the rats. The animals are sacrificed 24 h later and the radioactivity incorporated in the lungs is determined.

0046The results obtained are shown in Table 10 below.<tables id="tabl0012" num="0012"><img file="EP0374591B1_D0012.tif" /></tables>

0047It is found that HPC and HP produce the least incorporation of tumor cells. We can think that this effect is due to an interaction between the tumor cells and the endothelial cells of the internal walls of the blood vessels: there is less adhesion of the cells between them.

4.2.

0048A study is carried out in guinea pigs from the test of Example 3.2, but applied to animals to which tumor cells have previously been injected. The injection of tumor cells takes place after 4 weeks of ingestion of the diets and the animals are given diets for an additional 3 weeks. The capacity of blood vessels to synthesize 13-HODE is measured by incubating them with LA. The adhesion of the platelets to the discs is determined as in Example 3.2. The number of animals with lung lesions and the number of lesions per lung are also determined. The results are shown in Table 11 below.<tables id="tabl0013" num="0013"><img file="EP0374591B1_D0013.tif" /></tables>

0049It is found that, as in the case of normal animals (Example 3.2), the ingestion of HPC like that of HP results in a reduction in the adhesion of the platelets of animals into which tumor cells have been injected. The number of lungs with lesions is the lowest for HPC and HP, but the number of lesions per lung is significantly lower for HPC.

0050However, a histological study indicated that the lesions were of inflammatory and non-cancerous origin. The inflammation is therefore less for HPC.

0051The results of the analysis of 13-HODE show an increased presence of this anti-adhesion mediator when the animals are fed with PCH.

Example 5

Inflammation

0052Polynuclear leukocytes (PMN) play a decisive role in the inflammatory process and for this they need to leave the blood vessels to enter the peripheral tissues. One of the stages leading to this process is their prior adhesion to the internal wall of the blood vessels.

0053This adhesion was observed by studying the attraction between the PNMs isolated from guinea pigs fed with the diets of example 3, table 5 and monolayers of endothelial cells on discs. The results are shown in Table 12 below.<tables id="tabl0014" num="0014"><img file="EP0374591B1_D0014.tif" /></tables>

0054It is seen that ingestion of HPC results in a weak attraction of PMNs to endothelial cells.