Inhibition of cell adhesion for reducing cancer cell metastases

EXAMPLES

The invention is illustrated by the following Examples in which parts and percentages are by weight, unless otherwise stated.

EXAMPLE 1

Incorporation of DHLA and EPA in the tissues

Many pharmacological studies on the ability to modulate the fatty acids of the tissues by diet have used primarily rats as the 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 the total lipids, but not the phospholipids. By contrast, in human beings, rabbits and guinea pigs, the ingestion of GLA leads to the incorporation of DHLA in the triglycerides and the phospholipids of the blood, in the membrane of the red corpuscles and in all the lipids. For this reason, guinea pigs have been preferred to rats because, being much closer to man in regard to desaturase activity, they reflect better the situation prevailing in human beings. Also, the liver was chosen as the tissue because it represents the major site of fatty acid conversions.

1.1 Experimental Conditions

3 Groups of 7 male guinea pigs each weighing approximately 300 g are formed and are placed in cages of MACROLON. The groups are fed on semi-synthetic diets respectively containing blackcurrant seed oil (HPC), I, walnut oil (WO), II, or lard (L), III, "ad libitum" for 40 days. The WO is selected for comparison with the HPC because it does not contain the acids GLA and stearidonic acid (SA), but more LA, so that the total quantities of .omega.6 and .omega.3 and their ratios are comparable in the two oils.

All the animals have free access to water containing 250 mg/1 ascorbic acid. After one night without food, the animals are anaesthetized, their livers are removed and rapidly frozen, finely ground and then stored at -80.degree. C. lipids ingested are shown in Tables 1 and 2 below.

TABLE 1 ______________________________________ Composition of the base diet: Ingredient % ______________________________________ Casein 30 Starch 20 Sucrose 10 Glucose 3.8 Cellulose 15 Mineral salts 6 Vitamins 2.2 Potassium acetate 2.5 Magnesium oxide 0.5 Lipid 10 ______________________________________

TABLE 2 ______________________________________ Fatty acid composition of the lipids of the diets (in mol-%) Fatty acids HPC WO L ______________________________________ C14:0 -- -- 2.7 C16:0 7.4 8.6 32.4 C16:1 -- -- 3.4 C17:0 -- -- 0.5 C17:1 -- -- 0.3 C18:0 0.8 1.9 14.2 C18:1.omega.9 10.4 20.8 36.7 C18:2.omega.6 48.1 57.7 8.4 C18:3.omega.6 17.1 -- -- C18:3.omega.3 12.7 11.0 0.7 C18:4.omega.3 2.6 -- -- C20:1.omega.9 0.6 -- 0.7 C20:2.omega.6 0.3 -- -- Total .omega.6's 65.5 57.7 8.4 Total .omega.3's 15.3 11.0 0.7 .omega.6:.omega.3 ratio 4.3 5.2 12.0 Unsaturation index.sup.1 207.6 169.2 59.8 ______________________________________ Legend .sup.1 The unsaturation index is the sum of the products a .times. b wher a is the molar percentage of each unsaturated fatty acid and b is the number of double bonds of that particular fatty acid.

1.2 Analysis of the Fatty Acids

1.2.1

The samples are prepared by homogenizing 1 g frozen hepatic tissue in 20 ml of a solvent mixture of chloroform and methanol (ratio 2:1 by volume) and the total lipids (TLS) are extracted after separation of the precipitated solids by filtration, followed by elimination of the solvents.

1.2.2

The TLS are separated into neutral lipids (NLS) and polar lipids (PLS) by passage through a column of silica gel. The non-acidic and acidic fractions are separated from the PLS in ion exchange columns filled with diethylaminoethyl sepharose gel. The fraction of acidic polar lipids is then freed from salts by passage through a column of silica gel.

1.2.3

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

1.2.4

The PLS are separated into their various components by HPTLC on silica gel plates impregnated with a 2% (weight/volume) solution of boric acid in absolute methanol. The plates are developed with a solvent mixture of chloroform, methanol, triethylamine and water in a ratio by volume of 30:25:34:8. The acidic phospholipids phosphatidyl serine (PS), phosphatidyl inositol (PI), cardiolipine (CL), and the non-acidic phospholipids phosphatidyl ethanolamine (PE) and phosphatidyl choline (PC) were visualized in the same way as the neutral lipids, identified by comparison with the standard products and collected as described above.

1.2.5.

The composition of the TGS, CES, FFAS and PLS is determined by gas-phase chromatography of their methylesters. The methylesters are prepared by methylation in known manner and are then extracted with hexane. The peaks of the compounds in the chromatograms obtained are identified by comparison with the chromatograms of standard products.

1.3 Results

The results of analysis of the lipids are shown in Table 3 below:

TABLE 3 __________________________________________________________________________ TGS CES FFAS CL PI PS PC PE __________________________________________________________________________ Composition of the fatty acids in the lipid classes of the liver of guinea pigs fed on diet I (mol-%) C18:2.omega.6 47.1 33.0 25.7 68.9 21.1 25.3 31.6 19.9 C18:3.omega.6 (GLA) 8.1 9.1 3.6 1.5 3.2 4.0 4.5 2.7 .omega.6 C20:3.omega.6 (DHLA) 2.3 7.0 1.4 2.1 7.5 3.6 2.7 2.3 C20:4.omega.6 (AA) 1.2 3.6 1.8 3.0 11.3 5.6 7.0 18.2 C18:3.omega.3 9.2 5.2 4.8 5.5 1.5 1.4 2.2 1.9 C18:4.omega.3 0.6 0.3 0.4 -- 0.1 -- 0.1 -- .omega.3 C20:5.omega.3 (EPA) -- 0.2 -- -- -- -- 0.1 0.2 C22:5.omega.3 0.1 0.1 0.2 -- 0.3 1.1 0.3 1.2 C22:6.omega.3 0.2 -- 0.7 0.2 0.3 2.2 0.7 3.5 Ratio 1.9 1.9 0.9 0.7 0.7 0.7 0.4 0.1 C20:3.omega.6 (DHLA) C20:4.omega.6 (AA) Unsaturation 178 172 116 190 137 132 136 172 index __________________________________________________________________________ Composition of the fatty acids in the lipid classes of the liver of guinea pigs fed on diet II (mol-%) C18:2.omega.6 53.8 47.2 23.7 69.4 25.1 34.0 41.3 27.0 C18:3.omega.6 (GLA) 0.6 0.4 0.7 -- -- 0.1 -- 0.4 .omega.6 C20:3.omega.6 (DHLA) 0.4 1.0 0.3 0.2 1.9 0.5 0.2 0.4 C20:4.omega.6 (AA) 0.8 2.3 2.0 2.5 14.8 4.7 4.6 14.4 C18:3.omega.3 9.1 6.5 4.5 4.0 1.0 1.5 2.2 1.8 C18:4.omega.3 -- -- 0.1 -- -- -- -- -- .omega.3 C20:5.omega.3 (EPA) -- -- -- -- 0.1 -- -- 0.1 C22:5.omega.3 0.3 -- 0.1 0.2 0.3 0.7 0.3 1.2 C22:6.omega.3 0.2 -- 0.9 0.6 0.4 1.8 0.9 4.5 Ratio 0.5 0.5 0.2 0.1 0.1 0.1 <0.1 <0.1 C20:3.omega.6 (DHLA) C20:4.omega.6 (AA) Unsaturation 162 150 96 179 133 121 126 167 index __________________________________________________________________________ Composition of the fatty acids in the lipid classes of the liver of guinea pigs fed on diet III (mol-%) C18:2.omega.6 22.4 25.5 17.7 60.4 13.7 21.1 27.5 16.0 C18:3.omega.6 (GLA) 0.2 0.1 0.6 -- 0.1 0.1 -- -- .omega.6 C20:3.omega.6 (DHLA) 0.2 0.2 0.2 0.2 2.8 0.8 0.4 0.3 C20:4.omega.6 (AA) 0.7 1.7 2.7 3.5 12.7 6.7 5.2 17.5 C18:3.omega.3 1.5 0.8 2.1 0.5 0.1 0.5 0.6 0.3 C18:4.omega.3 0.1 -- 0.2 -- -- -- -- -- .omega.3 C20:5.omega.3 (EPA) 0.1 -- 0.2 -- 0.4 0.2 0.3 0.7 C22:5.omega.3 0.6 0.2 0.3 0.3 0.8 1.5 0.7 2.1 C22:6.omega.3 0.7 0.3 1.6 1.9 1.1 4.1 2.5 10.7 Ratio 0.3 0.1 0.2 0.1 0.3 0.1 0.1 <0.1 C20:3.omega.6 (DHLA) C20:4.omega.6 (AA) Unsaturation 104 104 91 170 124 129 119 196 index __________________________________________________________________________

The above results show that the incorporation of GLA and DHLA is considerably greater in the case of the group fed on diet I containing HPC.

There is no significant difference between the three groups in regard to the incorporation of AA. The result of these two observations is that the DHLA:AA ratio is considerably higher in the case of the ingestion of HPC.

The incorporation of EPA is considerably greater in the case of the ingestion of HPC than in the case of the ingestion of WO in regard to the TGS, CES, PCS and PES. The same observation is true for the ingestion of L in regard to the TGS and CES.

The unsaturation index shows that guinea pigs are capable of largely compensating the difference in unsaturation of the fatty acids of the diets.

In short, the above results show that the ingestion of HPC results in the substantial incorporation of DHLA and in higher DHLA/AA ratios in all the classes of hepatic lipids. In addition, the incorporation of EPA is greater for the regime based on HPC than for the regime based on WO. It follows from this that the potential of the PG1S and PG3S by comparison with the PG2S is clearly demonstrated in regard to HPC.

EXAMPLE 2

Influence of the Ingestion of HPC on the Fatty Acid Composition of the Plasma Lipids in Human Beings

A metabolic study is conducted on a 32-year-old man in good health to whom capsules of HPC concentrate enriched with GLA (78%) and SA (16%) are administered for 6 weeks in accordance with the following schedule:

______________________________________ Days 1-20: 3 capsules/day, corresponding to approxi- mately 1 g GLA and 250 mg SA/day Days 21-26: 10 capsules/day corresponding to approxi- mately 3 g GLA and 0.8 g SA/day Days 27-37: administration interrupted because of influenza Days 38-42: 10 capsules/day, corresponding to approx. 3 g GLA and 0.8 g SA/day ______________________________________

Analysis of the fatty acids of the various classes of blood serum lipids is carried out by gas chromatography of the methylesters in comparison with the control consisting of the mean of a group receiving a placebo.

The results are shown in Table 4 below.

TABLE 4 __________________________________________________________________________ Fatty acid composition of the various classes of serum lipids in mol-% of the HPC concentrate and the control PLS FFAS TGS CES HPC HPC HPC HPC Fatty acids Concentrate Control Concentrate Control Concentrate Control Concentrate Control __________________________________________________________________________ C20:3.omega.6 (DHLA) 8.35 3.83 0.93 0.48 1.60 0.38 1.97 0.88 C20:4.omega.6 (AA) 16.30 14.92 1.61 1.29 3.12 1.49 11.75 9.68 C20:3.omega.6 0.51 0.26 0.58 0.02 0.51 0.25 0.17 0.09 C20:4.omega.6 C20:5.omega.3 (EPA) 0.94 0.68 -- -- 0.58 0.27 0.91 0.65 C22:5.omega.3 1.52 1.28 0.87 0.19 0.90 0.50 0.18 -- C22:6.omega.3 2.52 2.76 0.57 0.15 0.36 0.19 0.45 0.43 __________________________________________________________________________

Evaluation of the above results shows that the GLA ingested is incorporated in all the classes of serum lipids and is rapidly metabolized into DHLA and, to a lesser extent, into AA. An increase is observed in the long-chain .omega.3 fatty acids, particularly EPA, in relation to the control, which seems to suggest that SA is also incorporated.

It may be concluded that the synthesis potential of the PG1S and PG3S is high by comparison with the PG2S when the regime is supplemented with HPC.

EXAMPLE 3

Thrombosis and Adhesion of Blood Platelets

3.1

Study of the incorporation of fatty acids in the arterial walls.

In this Example, guinea pigs are fed on diets containing various lipids over a period of 6 weeks, after which the fatty acid composition of the aortas of the animals is determined. The composition of the principal fatty acids of the lipids of the diets is shown in Table 5 and that of the fatty acids of the aortas in Table 6 below.

TABLE 5 ______________________________________ Composition of the principal fatty acids (in mol-%) in the diets containing the lipids HPC WO L FO ______________________________________ C14:0 0.1 0.1 1.8 3.0 C16:0 7.0 7.8 26.4 24.9 C16:1 0.1 0.1 2.1 3.3 C18:0 1.4 2.1 15.4 12.8 C18:1.omega.9 11.3 17.3 37.3 34.8 C18:2.omega.6 46.0 58.5 15.4 11.9 C18:3.omega.6 16.8 -- -- 0.1 C18:3.omega.3 14.3 13.9 1.1 2.0 C18:4.omega.3 3.0 -- 0.2 0.4 C20:5.omega.3 -- -- 0.1 1.7 C22:6.omega.3 -- -- 0.2 3.2 Total .omega.6's 63.0 58.5 12.6 12.7 Total .omega.3's 17.3 13.8 1.8 7.3 .omega.6:.omega.3 ratio 3.6 4.2 8.7 1.8 ______________________________________ Legend: FO = oil containing 20% fish oil, 70% lard and 10% walnut oil L = lipid containing 90% lard and 10% grapeseed oil

TABLE 6 ______________________________________ Composition of the principal fatty acids (in mol-%) of the aortas of guinea pigs fed on the diets containing the lipids HPC WO L FO ______________________________________ C18:1.omega.9 20.46 27.60 43.19 41.76 C18:2.omega.6 30.58 32.95 6.51 5.97 C18:3.omega.6 4.47 0.33 0.12 0.03 C18:3.omega.3 6.07 4.10 0.71 0.64 C20:3.omega.6 (DHLA) 1.43 0.13 0.10 0.05 C20:4.omega.6 0.97 0.62 0.91 0.91 C20:5.omega.3 -- -- -- 0.01 ______________________________________

A substantial increase is observed in the level of DHLA where HPC is ingested. This means that the formation potential of the PG1S is increased in the blood vessels.

3.2

Study of the influence of diet on the biological functions of the blood platelets.

The capacity of the blood platelets of guinea pigs marked with [.sup.3 H]-adenine to adhere to a thrombogenic surface in the form of discs coated with fibronectin is measured after 4 weeks on the diets of Example 3, Table 5. The number of platelets which have adhered to the discs is counted. The results are shown in Table 7 below.

TABLE 7 ______________________________________ Diet containing the lipids HPC WO L FO ______________________________________ Number of platelets 1.9 2.3 4.0 2.3 which have adhered to the discs/cm.sup.2 .times. 10.sup.5 ______________________________________

Where HPC is ingested, there is a reduction in adhesion compared with all the other diets, including that containing fish oil.

3.3.

Study of the thrombogenicity of the vessel wall and adhesion of the platelets,

To study the influence of the fatty acids of the regime on the thrombogenicity of the blood vessels, groups of rabbits are fed with the semi-synthetic diets HPC, WO and FO (Table 5, Example 3) and a commercial diet ("chow", C). After 4 weeks of the regime, a lesion is made in the carotid artery of the rabbits fed with HPC, WO and FO. The rabbits are then injected with blood platelets marked with [hu 3H]-adenine from donor rabbits fed on diet C. "In vivo" measurement of the accumulation of the platelets on the wounded artery tests the reactivity of the blood vessel. To discriminate the effect of the regime on the reactivity of the platelets from its effect on the reactivity of the blood vessel, platelets are isolated from the blood of rabbits fed on the 4 diets and their adhesion to discs coated with fibronectin is measured "ex vivo".

The capacity of the blood vessels to synthesize 13-HODE and the capacity of the platelets to synthesize 12-HETE is also determined.

The results are shown in Tables 8 and 9 below.

TABLE 8 ______________________________________ Number of platelets Diet containing the Commercial which have adhered to lipids diet the discs/cm.sup.2 .times. 10.sup.5 HPC WO FO C ______________________________________ In vivo 36 52 48 69 Ex vivo 15 18 9 50 ______________________________________

It can be seen that the thrombogenicity of the blood vessel "in vivo" is lower with the HPC regime and that there is no difference between the WO and FO regimes. The "ex vivo" measurements of the adhesion of the platelets show a reduced platelet reactivity with the HPC,WO and FO regimes compared with the C regime.

TABLE 9 ______________________________________ Diet containing the Commercial lipids diet HPC WO FO C ______________________________________ Metabolites of 30.2 27.0 4.9 12.6 the lipoxygenase 13-HODE (ng/cm.sup.2 of vessel) 12-HETE (ng/cm.sup.2 2.7 2.4 1.2 9.3 platelets) ______________________________________ Legend: ng = nanogram

No difference is observed in the synthesis of 12-HETE by the platelets of the animals fed on the HPC, WO and FO diets. By contrast, the synthesis of 13-HODE by the vessels is greater with the HPC and WO regimes which is consistent with the higher content of linoleic acid in the blood vessels of these two groups and with the anti-adhesive effect of 13-HODE.

EXAMPLE 4

Metastases

4.1

To study the influence of diet on the proliferation and dissemination of metastases, rats are fed on the diets of Example 3, Table 5, except for that containing WO, over a period of 5 weeks.

One group of rats is fed on a standard so-called "chow" diet (C). Tumour cells (Walker 256) are cultured in a medium containing the radioactive nucleotide [.sup.125 I]-uridine marking the tumour cells. The rats are then injected with the cells. The animals are killed 24 hours later and the radioactivity incorporated in the lungs is determined.

The results are shown in Table 10 below.

TABLE 10 ______________________________________ Diet containing the Commercial lipids diet HPC L FO C ______________________________________ Number of tumour 3.6 13.1 1.0 8.5 cells .times. 10.sup.4 ______________________________________

It can be seen that HPC and FO produce the lowest incorporation of tumour cells. It may be assumed that this effect is attributable to an interaction between the tumour cells and the endothelial cells of the inner walls of the blood vessels: there is less inter-cell adhesion.

4.2

A study is conducted in guinea pigs on the basis of the test of Example 3.2, but applied to animals injected beforehand with tumour cells.

The animals are injected with the tumour cells after 4 weeks' ingestion of the diets and are kept on the diets for another three weeks. The capacity of the 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 lesions of the lungs and the number of lesions per lung are also determined. The results are shown in Table 11 below.

TABLE 11 ______________________________________ Diet containing the lipids HPC WO L FO ______________________________________ Number of platelets which 3.6 6.6 7.1 4.1 have adhered to the discs/ cm.sup.2 .times. 10.sup.5 % of lungs with 33 40 55 33 lesions/total Number of lesions 8.9 9.8 21 22 per lung Mol-% of 13-HODE in the 9.4 8.6 5.5 2.9 inner wall of the blood vessels ______________________________________

It can be seen that, as in the case of the normal animals (Example 3.2), the ingestion of HPC, like that of FO, results in a reduction in the adhesion of the platelets in the animals injected with tumour cells. The number of lungs bearing lesions is lowest for HPC and FO although the number of lesions per lung is distinctly lower for HPC.

However, a histological study has shown that the lesions were of inflammatory origin and non-cancerous. Accordingly, there is less inflammation with HPC.

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

EXAMPLE 5

Inflammation

The polynuclear leucocytes (PMNS) play a decisive part in the inflammatory process and, to that end, they have to leave the blood vessels to enter the peripheral tissues. One of the steps leading to this process is their preliminary adhesion to the inner wall of the blood vessels.

This adhesion is observed by studying the attraction between the PNMS isolated from guinea pigs fed on the diets of Example 3, Table 5 and monolayers of endothelial cells on the discs. The results are shown in Table 12 below.

TABLE 12 ______________________________________ Diet containing the lipids HPC WO L FO ______________________________________ Number of PMNS per disc 1391 1938 1310 2193 ______________________________________

It can be seen that the ingestion of HPC results in a weak attraction of the PMNS to the endothelial cells.