SE448678B - PHARMACEUTICAL PREPARATION CONSISTING ESSENTIAL OF EICOSAPENTAIC ACID OR DERIVATIVE THEREOF - Google Patents

Description

Arachidonic acid, such as sodium arachidonate, can be inhibited by many materials including adenosine; B-naphthol; non-steroidal anti-inflammatory drugs such as indomethacin, sodium salicylate and aspirin; human albumin; unsaturated fatty acids, such as 11,14,17-eicosatrienoic acid, 8,11,14-eicosatrienoic acid (dihomo-Y-linolenic acid, DHLA), 5,8,11,14,17-eicosapentaenoic acid, 5,8,11,14-eicosatetraynic acid acid and 4,7,10,13,16,19-docosahexaenoic acid. They also found that platelet aggregation induced by collagen and a second wave of platelet aggregation induced by adenosine diphosphate (ADP) could be inhibited by β-naphthol, aspirin, 8, 11, 14-eicosatrienoic acid, S, 8, 11, 14, 17-echo- sapentaenoic acid and human albumin. Silver et al further found that different fatty acids alone did not induce platelet aggregation. The acids they mentioned were 8,11,14-eicosatrienoic acid; 11,14,17-eicosatrienoic acid; 5,8,11,14,17,-eicosapentaenoic acid, 5,8,11,14-eicosatetraenoic acid; 4,7,10,11,13,16,19,9-docosahexaenoic acid; linolenic acid; linoleic acid; oleic acid; arachidonic acid; stearic acid; and decanoic acid. ' It should be understood that many of the compounds that Silver et al found to be anti-aggregating are unsuitable for use in therapy. For example, adenosine is rapidly absorbed by cells and thus would not be available in the body long enough to be of any value.

B-naphthol is toxic because it is a phenolic compound.

Albumin is inappropriate because it would put an unwanted burden on the kidneys and cause glomerulus damage in the kidneys. Because non-steroidal anti-inflammatory drugs often cause gastric lesions, they should preferably be avoided in any therapy that requires prolonged prophylactic oral administration, which is often desirable in cardiovascular treatments.

Silver et al seem to conclude that arachidonic acid has an important place in hemostasis and thrombosis and that its effects can be inhibited in vitro by various ////////// 1 (NR gšjhlxfv 10 15 20 25 30 35 443 eva They suggested that albumin may be an important monitoring factor in hemostasis and that the ability of albumin to bind. Arachidonic acid in circulating blood could be the way in which it inhibits the effects of arachidonic acid. They further suggested that the albumin's net binding capacity for arachidonic acid may be due to, for example, the availability of binding sites and competition between arachidonic acid, other fatty acids and other classes of substances for sites.In addition, it is likely that rancid substances that are available, especially other these binding- the more competitive fatty acids, the more free arachidonic acid would there is and the more likely platelet aggregation would be, and, if these phenomena were related to each other, the more likely le thrombus formation be. This suggests that other fatty acids should be removed from the diet.

Attempts have been made to investigate in humans the effects of various fatty acids on diseases that cause thrombus formation, but no clear conclusion has emerged.

For example, the Norwegian experiment with vegetable oil was performed in 1965-66 before the work of Silver et al and it was reported by H. Natvig, Chr. F.— Borchgrevink, et al in Scand. J. Clin. Lab. Invest. go, Suppl. 105, l-20, (1968). The study compared the effects on human mortality, caused by various coronary heart diseases including myocardial infarction, of two diets, one of which contained sunflower seed oil (about 63% linoleic acid) and the other contained linseed oil (about 55% linolenic acid), with 10 ml of linoleic acid. of each oil was ingested per day. The group that ingested the more unsaturated linolenic acid was found to be more at risk than the group that ingested the linoleic acid.

Linoleic acid, and, in rats, eicosapentaenoic acid and docosahexaenoic acid are known to reduce blood plasma cholesterol levels, which are thought to be associated with atherosclerosis.

Atherosclerosis is often detected in people who have suffered from a myocardial infarction. However, there does not appear to be a causal relationship, as Robertson (Lancet (1959), i, 44) found that in Jamaica, although widespread atherosclerosis was frequently detected in the native population at necropsy, it is very rarely associated with secondary thrombi or myocardial infarction. infarction. In addition, myocardial infarctions can occur in the absence of severely developed atherosclerosis. Yet another possible dietary factor that has been suggested (P. B. Kernoff, A. L. Willis, K. J. Stone, J. A. Davis and G. P. McNicol, British Ned. J., 1977, 3, 1441-1444) to help inhibit thrombosis is DHLA.

DHLA is a biosynthetic precursor of prostaglandin E1 (PGE1), a potent inhibitor of platelet function, and was said to be attractive as an antithrombotic agent. It was found that there was, as expected, an increase (average 55%) in the production of the desirable PGE1, but in 6 men out of 8 tested there was also an increase (average 3%) in the production of the undesirable prostaglandin E2 (PGE2). In addition, these results were not clearly dose-related. There was also a decrease in heparin-neutralizing activity of plasma, and this activity has been found to be high in thrombotic conditions.

However, the authors did not know the extent to which heparin-neutralizing activity affects basic pathological mechanisms, and therefore its association with thrombosis was unclear.

The authors of the article speculated that "perhaps small doses of DHLA may be equally if not more effective than greater dietary manipulation in the prevention or treatment of these conditions" ie atherosclerosis and coronary heart disease. The author of an editorial article 10 15 20 25 30 35 448 678 S kel in the same edition of British Med. J. (pp. 1437 and 1438) was, however, more cautious, thinking that "Studies of agents and diets that modify platelet prostaglandin mechanisms must be performed before we can say whether the results obtained by McNicol and his colleagues have any clinical application". The reason for his caution lies in the ignorance that exists about the mechanisms involved in vivo in thrombotic conditions, when exploratory tests have only been performed on shed blood. d This at least partially attractive work with DHLA casts some doubt on the often held view that unsaturated fatty acids in the diet are healthier than their more saturated analogs, especially as the even less saturated linoleic and linolenic acids can be metabolized to DHLA. This doubt is reinforced by the fact that arachidonic acid, which is undesirable (see Silvér et al and Kernoff et al above), is even more unsaturated (four carbon-carbon cis double bonds) than DHLA (three carbon-carbon cis double bonds).

It has now surprisingly been found that among the many fatty acids (all Z) -5,8,1l, 14,17-eicosapentaenoic acid or its salts, esters or amides can be used for effective treatment, or effective prophylaxis, of thromboembolic conditions, which hereafter simply called thrombosis. Examples of conditions where the present findings may be useful are in the treatment or prophylaxis of cardiovascular diseases mediated by the formation of a thrombus or thrombi, for example myocardial infarction, stroke, or deep venous thrombosis during surgical operations.

It has been found that (all Z) -5,8,11,14,17,7-eicosapentaenoic acid (hereinafter simply referred to as eicosapentaeno- also known as acid, icosapentaenoic acid), when injected intravenously into rabbits, increases their bleeding time, thus. ..f 10 15 20 25 30 35 448 678 6 showing a reduction in the blood's tendency to produce thrombi or adhere to damaged tissue, whereby it is possible to modify and / or regulate wound healing. When eicosapentaenoic acid is infused into rabbit lung, it gives rise to a substance that has a strong anti-aggregating effect on platelets.

Eicosapentaenoic acid also has the unusual and important ability to dissolve or disintegrate already formed thrombi or platelet clots. For example, blood from an anesthetized rabbit was allowed to drip over a continuously weighed collagen strip taken from another rabbit's Achilles tendon. As blood flowed across the strip, platelets and other cells adhered to it, to form a thrombus, until no further weight gain of the strip appeared. The blood was returned to it, the first rabbit under gravity. When eicosapentaenoic acid was infused into the blood that passed over the loaded strip, a decrease in weight was observed, indicating that at least some of the aggregating platelets and other cells had disaggregated from the loaded strip. The ability of this eicosapentaenoic acid to cause dissolution or disaggregation of thrombi is important in the treatment of thrombosis, and also in its prophylactic treatment. When a thrombus forms in an artery (or vein), there is a reduction in blood flow (which flow would stop completely if the vessel were completely closed). This reduction in blood flow causes ischemia, which produces pain.

However, the reduced blood flow may carry therapeutic materials to the thrombus site. With eicosapentaenoic acid in vivo, the residual blood flow and each blood flow in the collateral circulation can carry the acid to the thrombus site, where eicosapentaenoic acid and its metabolites can disaggregate the thrombus and restore full blood flow. Accordingly, administration of a pharmaceutical formulation according to the present invention enables full blood flow to be restored in a partially occluded blood vessel. The administration can also be used prophylactically to help keep the blood vessels open.

It has also been found that human platelets, when preincubated with eicosapentaenoic acid and then incubated with arachidonic acid and stimulated with ADP, aggregate less rapidly than when preincubated with arachidonic acid. This is thought to suggest that if human platelets could be "charged" with eicosapentaenoic acid, they would be less susceptible to ADP stimulation and thus less likely to form thrombi.

It is now believed, although not to be bound by this belief, that eicosapentaenoic acid, in contrast to arachidonic acid, not only alone has an anti-aggregating effect on platelets in vivo, but also its metabolites, probably A-type prostaglandins. 17, has an anti-aggregating effect on platelets, or in the worst case a reversible aggregating effect, whereas many of the arachidonic acid metabolites, such as PGH2 and TXA2, have an irreversible aggregating effect on platelets. It is now believed that this net anti-aggregating profile for eicosapentaenoic acid is responsible for its surprisingly beneficial properties. The dose of eicosapentaenoic acid required for therapeutic or prophylactic effect varies with the mode of administration and the nature of the condition being treated, but is generally at least 1 g, preferably from 1.5 to 7.5 g, especially 2 to 6 g, e.g. 5 g per day.

This is the dose for an average 70 kg person and the dose for other people or animals generally varies proportionally according to their weights, ie about 20 to 100 mg / kg.

Eicoasapentaenoic acid can be added to extracorporeal circulating blood to prevent, substantially or completely, aggregation of platelets induced by contact with the machine or with other non-tissue materials.

The presence of eicosapentaenoic acid is known in oysters and other seafood, in fish liver oil and in other oils, such as menhaden oil, from which it can be extracted by methods known in the art or described in the literature. Eicosapentaenoic acid can also be synthesized by conventional methods in synthetic organic chemistry. The route chosen depends on the availability of suitable starting materials, and on the relative costs of the various routes available to provide the right quality eicosapentaenoic acid for human or veterinary use.

Caution should be exercised in extractive and preparative procedures to avoid, or keep low, the isomerization of cis double bonds to trans double bonds. The amounts of eicosapentaenoic acid in naturally occurring or easily extractable materials, such as fish liver oil or menhaden oil, are such that it would not be possible to obtain the desired amount of eicosapentaenoic acid by administering them without also administering too many calories in the form of other fatty acids. Since cod liver oil (and other fish oils) are rich in vitamin A (at least 850 international units (IU) per gram) and vitamin D (at least 85 IU per gram), adequate administration of cod liver oil to provide the necessary amount of eicosapentaenoic acid would also give amounts of these vitamins that greatly exceed the recommended daily doses for humans and would lead to hyper-vitaminosis.

The recommended daily dose is 5000 I.U. for vitamin A and 400 I.E. for vitamin D for humans.

In the United States, the Food and Drug Administration has established that daily intake of vitamin A should not exceed io ooo LE. and vitamin n should not exceed 4oo I.E. Quantities above this require a doctor's prescription.

To avoid complications which may arise from the recipient receiving vitamin doses for other medical reasons, or from his or her own voluntary ingestion, a preparation comprising eicosapentaenoic acid, or a pharmaceutically acceptable salt, ester or amide thereof, and a pharmaceutically acceptable carrier, the formulation being substantially free of vitamins.

Depending on the complex and to some extent various effects of acids which are less unsaturated than eicosapentaenoic acid, a formulation comprising eicosapentaenoic acid, or a pharmaceutically acceptable salt, an ester or an amide thereof, and a pharmaceutically acceptable carrier are preferably provided. is substantially free of other, less unsaturated acids, or their salts, esters or amides.

In eicosapentaenoic acid obtained from natural sources, such as fish oils, there is usually a proportion of (all-Z) 7, 10, 13, 16, 19-docosapentaenoic acid (hereinafter referred to as docopentanoic acid) and / or of (all-Z) 4, 7, 10, 13, 16, 19- -docosahexaenoic acid (hereinafter referred to as docosahexaenoic acid) (as such or as their derivatives, ie their esters, salts or amides). It is not necessary to try to remove these equal or more unsaturated acids (or their derivatives), because they behave in the same way as eicosapentaenoic acid, but they are less active.

The above-mentioned high caloric intake, if for example fish liver oil or menhaden oil is used as the source of the eicosapentaenoic acid, can mainly be avoided, although some control of the caloric intake in the rest of the diet may still be necessary, by administering a preparation comprising eicosapentaenoic acid, or a pharmaceutically acceptable salt, ester or amide thereof, and a pharmaceutically acceptable carrier, wherein at least 50% by weight, for example more than 56% by weight, of the fatty acid content of the formulation is provided by eicosapentaenoic acid.

However, if eicosapentaenoic acid is to be administered without modification of the recipient's diet, the acid (or any docosapentaenoic acid or docosahexaenoic acid) should represent at least 90% by weight, preferably at least 95% by weight or all of the fatty acid content of the administered material.

Arachidonic acid should preferably be absent or present to a maximum of 5% by weight of the fatty acid content. For example, a preferred grade of eicosapentaenoic acid comprises at least 90% by weight of the acid, about 2% by weight of each arachidonic acid and dihomo-Y-linolenic acid, the remainder being docosahexaenoic acid, docosaentaenoic acid, palmitic acid or oleic acid, and other pharmaceutically acceptable acids. - tabla fatty acids. If vitamins are present, as they may be, they should preferably not be present in amounts which would lead to the recommended daily intake of it being exceeded.

Formulations used in accordance with the present invention should also be free from saturated fatty acids and their salts, esters or amides. Preferably, the preparations should be free of unsaponifiable material.

By administering eicosapentaenoic acid as at least 50% by weight, preferably at least 90% by weight of the fatty acid content, it should be possible to avoid significant changes in the diet of the recipient, with the exception of any slight reduction in the caloric content of the diet to include the extra calories. from eicosapentaenoic acid (or other fatty acids). If preferred, however, it may be possible to administer eicosapentaenoic acid by substituting, say, butter: and / or plain margarine with a special margarine, eg of the emulsion type, prepared so that in normal use the recipient would receive the required amount of eicosapentaenoic acid. Cooking oils and fats can also be prepared in a similar way to contain the eicosapentaenoic acid. Eicosapthenic acid (and other acids) need not be used as the acid itself but can be used as its pharmaceutically acceptable salts, esters or amides (which would then be measured as their acid equivalents). Esters or amides which can be converted in vivo to the acid and other pharmaceutically acceptable products may be used, the preferred esters being triglyceride or ethyl ester, but the methyl ester could optionally also be used. The alcohol used to esterify the acid should preferably be non-polymeric and should preferably contain at most three hydroxyl groups in the molecule. In addition, the ester used is preferably not cholesterol ester, as this would lead to the release of some cholesterol, which could lead to an increase in serum cholesterol levels. For the same reason, the formulations of the present invention are preferably free of cholesterol, as such or as a derivative thereof which is convertible to cholesterol in the body of the recipient. The preferred salts are sodium or potassium salts or any pharmaceutically acceptable solid salt, as these are more suitable for preparation into tablets.

Tablets may comprise a pharmaceutically acceptable solid derivative, for example a salt, of eicosapentaenoic acid.

Since eicosapentaenoic acid is highly unsaturated, it and its derivatives are readily oxidizable, and formulations containing them should preferably also contain antioxidants, such as butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate, a pharmaceutically acceptable quinone and α-tocopherol. Certain anti-oxidants may also contribute to the antithrombo-embolic effect. Although it is preferred to administer eicosapentaenoic acid (or its salts, esters or amides), (active compound) orally as this is a convenient route of routine administration, the active compound may be administered by any means. ways in which it can be successfully absorbed, eg parenterally (ie subcutaneously, intramuscularly or intravenously), rectally or, women, vaginally. While it is possible to administer the active compound as a raw chemical or as a simple mixture of components, it is preferable to present it in the form of a pharmaceutical preparation. For both veterinary and human use, the formulations of the present invention comprise the active compound, as defined above, together with one or more acceptable carriers therefor and optionally other therapeutic ingredients. The carrier or carriers must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Formulations containing the eicosapentaenoic acid itself are preferably non-aqueous. Unit doses, for example tablets or capsules of a preparation, usually contain from 0.25 to 1.0 g, for example 0.5 g of the active compound. Generally, 3 doses can be administered per day.

Such formulations which may be used include those suitable for oral, rectal, vaginal or parenteral administration including subcutaneous, intravenous administration. intramuscular or Since the eicosapentaenoic acid itself is a liquid and tends to be distasteful, it is preferably administered orally in a capsule, for example one of soft gelatin, so that the eicosapentaenoic acid is not tasted.

The capsule should generally be of a size that allows the required dose of eicosapentaenoic acid to be administered. dispensable in 1, 2 or 3 capsules at each dose, and thus the capsule should be approximately 0.5 ml in size. Another way to mask the taste of the acid is to prepare it as an emulsion to be taken orally. The acid could also be formulated to be spontaneously emulsifiable when taken orally or when diluted for administration.

An emulsion could also be of the multiple type; for example, the acid could be reconstituted into an oil-in-water emulsion with a pharmaceutically acceptable surfactant and then this emulsion could be emulsified in another oil, eg peanut oil. Alternatively, the acid could be similarly prepared into a water-in-oil emulsion and then this emulsion types of emulsions could be presented as an oral in turn am fl give ß in water. The various gels or as a rigid emulsion, such as an emulsion margarine. Other ways to mask the taste are to absorb the acid on one or more carriers, such as kaolin, chalk, calcium phosphate, calcium sulfate, starch, a microcrystalline cellulose, or methyl or other modified cellulose. The resulting powder could be sold as such or flavored, and optionally in tablets or capsules, each tablet or capsule containing, for example, about 0.5 g of eicosapentaenoic acid as such or in the form of a solid derivative. Tablets could be film or sugar coated.

Regarding the salts, ie the sodium or potassium salts, these also tend to be distasteful, and tablets which contain them, and represent, for example, 0.5 g of acid, should preferably be coated with, for example, film or sugar. Other methods of oral administration, such as capsules or lozenges, may also be used in appropriate circumstances. The esters or amides can be prepared as the acids or salts, depending on whether they are liquids or solids. If desired, an oral formulation may be presented as a sustained release formulation, for example as beads or microcapsules in a capsule.

A preparation for intramuscular administration could be in the form of an emulsion. A preparation for intravenous injection could be in the form of a mixture which would spontaneously emulsify upon injection.

For rectal administration, the acid or derivative could be formulated into a suppository in a triglyceride base, such as cocoa butter, a Witepsol or Suppocire, or placed in a soft gelatin suppository capsule.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active compound into association with the carrier, which consists of one or more additive ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired formulation. In the present description and claims, the term "carrier" includes one which is suitable for administration to a recipient and substantially encloses the active compound, for example, the body of a capsule or the coating on a coated tablet.

To improve the efficacy of eicosapentaenoic acid, the formulation may also include a phosphodiesterase inhibitor, such as theophylline or dipyridamole.

Accordingly, the present invention relates to: A pharmaceutical preparation consisting essentially of (all-Z) -5,8,11,14,17-eicosapentaenoic acid or a pharmaceutically acceptable salt, preferably sodium or potassium salt, thereof or a pharmaceutically acceptable ester , preferably triglyceride or ethyl ester, or amide thereof, and a pharmaceutically acceptable carrier and optionally an antioxidant and optionally a flavoring agent, wherein at least 50% by weight, preferably 90% by weight, of the fatty acid content in the formulation consists of (all-Z) -5,8,11,14,17,7-eicosapentaenoic acid or pharmaceutically acceptable esters, salts and amides thereof and wherein the formulation is substantially free of vitamins and / or other, less unsaturated acids and their derivatives and saturated fatty acids and their salts, esters and amides.

The pharmaceutical preparation may be presented in unit dosage form, such as in capsule or tablet form. The pharmaceutical preparation can be used to treat a thromboembolic disorder or disease in a mammal.

The present invention is illustrated by the following examples. 'EYCEIMPEL l. Blood from volunteers who had not taken aspirin for the previous two weeks was collected from an antecubital vein in sodium citrate (0.1 M), 1 part citrate to 9 parts blood. Plasma was separated from the blood by centrifugation at 160 g (5 minutes) as a platelet-rich plasma (PRP).

Platelet studies were performed with arachidonic acid (AA), eicosapentaenoic acid (EPA) is prepared as potassium salts (see Schrör, K., Moncada, S., Ubatuba, FB, and Vane, JR, Eur. J. Pharmac., 1978 , A1, 103) and with ADP or thrombin in a coagulation apparatus, eg "Fibromate" (Bie & Bernsted, Copenhagen, Denmark).

Aggregation was recorded both turbidimetrically and nephelometrically in a cylindrical cuvette containing 300 μl of PRP at 37 ° C and magnetically stirred at 800 rpm; alternatively, a Payton two-channel aggregator with 500 μl PRP was used.

In contrast to AA, EPA did not induce aggregation in human PRP at concentrations of EPA (1.3, 2.66, and 5.3 mM) about 4 or more times greater than AA (44, 678, 168). 0.33, 0.66 and 1.3 mM). At lower concentrations in the range of 0.01 to 0.5 mM, EPA inhibited some platelet aggregation induced by ADP (2 PM) in human PRP.

However, the anti-aggregating effect of EPA (0.065 mM) was not due to its conversion by platelet cyclooxygenase, as the anti-aggregating effect occurred with aspirin-treated platelets which were not affected by AA (0.065 mM) but which were aggregated. of thrombin (0.04-0.4 units / ml), and the anti-aggregating effect also occurred in the first phase aggregation induced by ADP (2 to 5 Pm) in aspirin-treated platelets.

EXAMPLE 2 Vascular tissue (thoracic and abdominal aorta) was obtained from recently killed rats. Approximately 100 mg of tissue was chopped and washed in ice-cold Iris buffer (0.05 M, pH 7.5). After testing its ability to inhibit thrombin-induced platelet aggregation when added to the platelet cuvette, the tissue was washed several times in 10 ml of ice-cold Tris buffer to remove blood and adherent platelets. The tissue was then rapidly frozen to -60 ° C, crushed to a coarse powder and resuspended in 5 volumes of Tris buffer. This suspension of vascular tissue was kept on ice during the experiments and used for incubation studies.

Blood was obtained from the antecubital vein of volunteers who had taken aspirin (1.5 g per day) for 3 days before blood sampling. Washed human platelets were obtained from this blood as described by Vargaftig, B.B., Tranier, Y., and Chignard, M., (Prostaglandins, 1974, §, 133). Aggregation tests were performed as in Example 1. To see if the suspension of vascular tissue could be synthesized to an anti-aggregating effect on human platelets, the platelet was obtained as described above. had taken aspirin so that their platelets could not produce prostaglandin endoperoxides which could be used by the vascular tissue to produce anti-aggregating material. In addition, washed platelets were used to avoid any possibility for the vascular tissue to utilize any AA in the plasma.

Under these conditions, anti-aggregating activity could be generated by the vascular tissue only from endogenous or exogenously delivered precursors.

Initial suspension of vascular tissue (10 to 50 μl as described above inhibited thrombin-induced aggregation (0.04 to 0.4 units / ml) This inhibitory activity was destroyed by repeated washing (5 to 20 times) of the tissue by centrifugation. (30 in an Eppendorff centrifuge), decantation of the supernatant and resuspension in fresh buffer (0.5 ml) Ä The general level of inhibitory activity against primary phase aggregation induced by ADP (2 to 5 μM) or aggregation induced by thrombin ( 0.04 to 0.4 units / ml) could be restored by adding washed vascular tissue and EPA to the washed platelets of aspirin-treated volunteers The generation of anti-aggregating activity was prevented by the pretreatment of the washed vascular tissue with indomethacin (5 to 10 pg / ml) Thus, vessel wall cyclooxygenase could utilize EPA to form anti-aggregating activity.

The anti-aggregating activity formed could have been due to the reduction of endogenous AA by EPA and not to the direct utilization of EPA. However, the same concentrations of DHLA incubated with washed vascular tissue did not lead to the formation of anti-aggregating material and thus DHLA does not displace AA. few: 10 15 20 25 30 35 448 678 18 EXAMPLE 3 Four white New Zealand male rabbits (Ranchk weighing 2.0 to 2.5 kg, anesthetized with sodium pentobarbitone (40 mg / kg). The marginal ear vein was cannulated for infusions (0, 1 ml / min) of the eicosapentaenoic acid The potassium salt of eicosapentaenoic acid with a purity of 95% and which contained about 2% AA and 2% DHLA, the rest C-18 fatty acids) was tested in 50 mM Tris-HCl buffer pH 8.0, kept on ice and shielded from light. Infusions of either the Tris vehicle or eicosapentaenoic acid were made 5 minutes before and continuously during the measurement of the bleeding time. The inner surface of the ear, which was without a cannula, was carefully shaved. the ear was illuminated so that the blood vessels were clearly visible. Incisions, which were about 0.4 cm long and deep enough to cause swelling of blood within l5 s, were made with a scapula, i. an area that was free of visible blood vessels and in a direction parallel to the nearest blood vessel. The section was gently dried every 15 seconds with filter paper (Whatman No. 1).

The bleeding time was measured for the next 15 s from the time of incision until blood stains were no longer visible on the filter paper. If there was a plasma excretion from the section, the end point was considered to be the time when the 1m excretion no longer had a reddish color. When the bleeding time was longer than 10 minutes, the incision was wiped every thirty seconds. The bleeding time for each dose was an average of 3 determinations.

Two rabbits were pretreated with aspirin 100 mg / kg intravenous injection 4 hours before the experiment. Two rabbits were given 0.5 ml of Tris pH 7.5 in 4 ml of physiological saline in the same manner to act as controls.

The results obtained are shown in Tables 1 and 2.

TABLE l 448 678 19 Controls, ie no aspirin EPA dose Bleeding time pq / kq / min min Rabbit l Rabbit 2 å i i o 3.5 § 3.0 É 56 I 5 16.0 § loo 1 9, s f 16, 5; zoo {; ** 23.0 l à TABLE 2 Pretreated with aspirin 100 mg / kg i.v. injection 4 hours before the start of the test EPA Dose Bleeding time X ng / kg / min min Rabbit 3 Rabbit 4 O f I 10 0,, zoo, x *, x XX Average of 3 determinations Infusion rate 0.2 ml / min 10 15 20 25 30 35 448 678 20 From the tables it appears that when the rabbits were treated with aspirin they showed little or no increase in bleeding time. u A rabbit treated with EPA of 75% purity gave similar results if one considers the lower purity of the acid.

EXAMPLE 4 9my§2êliQ9_§2_sië9§a2sn§ase§xr§_i_sa_h2a§§_ël9êsir: culture Intravenous infusion of eicosapentaenoic acid (0.2 to 2 mg kg_l min_1) caused systemic and pulmo ~ nallgçohaßiaï in dogs. Blood bath insulated strips of oxcoronary artery and rabbit coeliacus partner are known to be relaxed by the strong anti-aggregating material PGI2 (50 to 10 ng / ml).

When these bioassay tissues; treated with catecholamine and angiotensin II antagonists, bathed in arterial blood, they relaxed under infusion of eicosapentaenoic acid (0.6 to 2 mg kg'l 1 corresponded to about 10 to 20 ng / ml PGI2 at the highest rates. min-, 2 dogs), in an amount which, after administration of indomethacin (5 mg / kg) in one of these dogs, subsequently infused eicosapentaenoic acid (2 mg kg-lmin_l for 10 min) still caused hypotension, but did not release any detectable activity in the bioassay tissues. .

EXAMPLE 5 9i§a¶¶rererau§e -__ sššeëë_ë! Fäë2ëaea§§aee§2§§_i_ëaaia Rabbits (2-3 kg) were anesthetized with pentobarbitone sodium 30 mg / kg and treated with heparin (2000 units / kg).

A carotid artery was cut open and blood was drawn and delivered by means of a pump to pour a strip of collagen from another rabbit's Achilles tendon. As blood flowed over the tendon strip, the weight of the strip increased over a period of 35 minutes up to a maximum of from 180 to 200 mg. Thereafter, each decrease in weight was due to platelet disaggregation. Eicosapentaenoic acid, which was infused intravenously (50-500 pg / kg / min) in five rabbits induced small disaggregating effects (about 20 mg). This effect of eicosapentaenoic acid could be inhibited by pretreatment of the rabbits with aspirin (150 mg / kg).

EXAMPLE 6 A soft gelatin capsule containing about 0.5 ml was sterilized and then filled with a composition containing more than 90% of EPA, about 2% AA and about 2% DHLA, the remainder including palmitic and oleic acids.

The capsule was then sealed.

The capsule used may be transparent or colored, and may also be of the hard gelatin type or made of polymethyl methacrylate, EXAMPLE 7 A tablet formulation comprising: Sodium mycosapentaenoate 281 mg Starch 62 mg Lactose 250 mg Polyvinylpyrrolidone 3.5 mg Magnesium stearate 3.5 mg Butylated ppm TOTAL 600 mg The tablet was coated with sugar, although other coating agents could be used.

EXAMPLE 8 The formulation described in Example 7 in unbleached powder form may be used to fill hard gelatin capsules with 600 mg of the formulation.

EXAMPLE 9 Approximately 250 g of a conventional soft margarine preparation was carefully mixed with 8 g of eicosapentaenoic acid until a uniform consistency was achieved. EXAMPLE 10 Aspirin (10 or 100 mg / kg iv) was given to New Zealand male rabbits (2-2.5 kg). One control group received only the liquid vehicle used to dissolve the aspirin. Two to four hours later, the animals were anesthetized with pentobarbitone and cutaneous bleeding time was measured, as described in Example 3, before and during the infusion of EPA (potassium salt, 95% purity used in Example 3) at different rates (1, 50, 200 or 400 Pg / kg / min). Double or triple measurements were made for each condition. The results obtained are shown in Table 3. Aspirin (10 mg / kg produced a small but significant (p- <0.000l) increase in bleeding time, and the mean of triplicate determinations in five rabbits was 489 É 27 s (mean É sem) compared to the controls 278 É 48 s. The value 288 Éll si of the animals treated with a large dose (100 mg / kg) of aspirin was not significantly higher than the control.

In the group that did not receive any aspirin, EPA (1 pg / kg / min) prolonged the bleeding time by more than 100% (p 4 0.0000l). A further increase in bleeding time was observed at higher infusion rates and a plateau value of about 1000 s was achieved at the rate of SO Fg / kg / min. In animals treated with aspirin (10 or 100 mg / kg), EPA did not cause a significant change in bleeding time. 448 678 23 = fl uHmm <n .awm H uwwnaxwm H ø fi uwmc fl øwm fi n m fifl uß fl zwaonww MHC flflfl m M flfl NUCH m fifi ww H www mm H www Nm M ßwm mä M mom HH M www m1 \ wa oo fi Mu mH fl mm Hu mfl m m fifl M wßw »QH ßow» NM www mx \ wa od V flfi w flfl mHC MNC .. WHC mw wo ~ oH ßw H Hwm mm M Hmm mm H Nwm ww H wßw = wmcH cow oo ~ om HQ wc fifl wamswnuwm cHE \ vx \ mQ dmm m .HAMm / NH. EXAMPLE 11 Using the method of Example 3, the effects of various fatty acids on the aggregation induced by 11α, 9α-epoxymethano-15-hydroxyprosta-5,13-dienoic acid (an analog of PGH2) were observed (Upjohn ) of aspirin-treated platelets in human PRP.

In each test, the fatty acid was incubated with the platelets for about 6 minutes before the PGH2 analog was added; the amount of fatty acid used was about 1.5 mM. The amounts of inhibition obtained 6 minutes after the addition of the PGH2 analog are shown in Table 4.

TABLE 4 Acid% inhibition Eicosapentaenoic acid 100 (all-Z) -9,12,15-octadecatrienoic acid 40 (all-Z) -9,12-octadecadienoic acid 55 (all-Z) -6,9,12-octadecatrienoic acid 63 Z-9 octadecenoic acid 68 Control O Eicosapentaenoic acid also showed 100% inhibition at a concentration of 1.0 mM and about 95% inhibition at a concentration of 0.5 mM.

It should be noted that (all-Z) -9,12,15-octadecatrienoic acid and (all-Z) -6,9,12-octadecatrienoic acid, which are 2,3-dinor analogs of 11, 14, 17- eicosatrienoic acid and 8,11,14-eicosatrienoic acid, which were used in the above work by Silver et al, were not very effective as anti-aggregating agents compared to eicosapentaenoic acid. m)

Claims (10)

10 15 20 25 30 448 678 25 PATENT REQUIREMENTS A pharmaceutical formulation, characterized in that it consists essentially of (all-Z) -5,8,1l, 14,17- -eicosapentaenoic acid or a pharmaceutically acceptable salt, preferably sodium or potassium salt thereof, or a pharmaceutically acceptable ester, preferably triglyceride. ethyl ester, or amide thereof, and a pharmaceutically acceptable carrier and optionally an antioxidant and optionally a flavoring agent, wherein at least 50% by weight, preferably 90% by weight, of the fatty acid content of the preparation is (all ~ Z) -5 , 8, 11, 14, 17, 17-Eicosapentaenoic acid or pharmaceutically acceptable esters, salts and amides thereof and wherein the preparation is substantially free of vitamins and / or other, less unsaturated acids and their derivatives and saturated fatty acids and their salts, esters and amides . Pharmaceutical preparation according to Claim 1, characterized in that it consists essentially of (a1-Z) -5,5,8,11,14,17-eicosapentaenoic acid or a pharmaceutically acceptable salt, preferably sodium or potassium. salt, thereof, a pharmaceutically acceptable ester, preferably triglyceride or ethyl ester, or amide thereof, and a pharmaceutically acceptable carrier, an antioxidant and a flavoring agent, the preparation being substantially free of other, less unsaturated acids and their derivatives and saturated fatty acids and their salts, esters and amides. Pharmaceutical preparation according to Claim 1, characterized in that it consists essentially of (all-Z) -5,5,8,11,14,17-eicosapentaenoic acid or a pharmaceutically acceptable salt, preferably sodium or potassium. salt, thereof, a pharmaceutically acceptable ester, preferably triglyceride or ethyl ester, or amide thereof, and a pharmaceutically acceptable carrier, an antioxidant and a flavoring agent, the preparation being substantially free from vitamins and saturated fatty acids as well as their salts, esters and amides. Pharmaceutical preparation according to any one of the preceding claims, characterized in that it is in the form of a capsule. Pharmaceutical formulation according to any one of claims 1 to 3, characterized in that the eicosapentaenoic acid, salt, ester or amide forms a dispersed phase in the carrier, which is a liquid, the formulation being enclosed in a capsule. IN Pharmaceutical preparation according to one of Claims 1 to 3, characterized in that it is in tablet form, the carrier being a solid. Pharmaceutical preparation according to any one of the preceding claims, characterized in that it is in unit dosage form. Pharmaceutical preparation according to Claim 7, characterized in that it contains 0.25 to 1.0 g of eicosapentaenoic acid, as such or as a salt, an ester or an amide. _ Pharmaceutical preparation according to one of Claims 1 to 3, characterized in that it is in tablet form and in that it consists essentially of a solid ester, a solid salt or a solid amide of (a1l-Z) - -5.8 , 11,14; 17-eicosapentaenoic acid. A pharmaceutical preparation according to any one of claims 1-9, for use in the treatment of a thromboembolic disorder or disease in a mammal.