DE2831507A1 - FORMULATIONS CONTAINING (ALL-Z) -5,8,11,14,17-EICOSAPENTAIC ACID AND / OR THEIR DERIVATIVES AND METHOD OF PREPARATION - Google Patents

Description

DR. BERG DJPL.-TNG. STAPF DIPL.-ING. SCHWABE DR. DR. SANDMAIR

PATENTANWÄLTE Postfach 860245 ■ 8000 Munich 86 2 O 3 I 5 G

<f

Dr. Berg Dipl.-Ing. SUpf and Partner, POBox 860245, 8000 Munich 86 '

Your reference Our reference 29 320 Mauerkircherstraße 45 June 8th 1978 Vour ref. Our ref. ^ 8000 MUNICH 80 Lawyer file number: 29 320

TUE WELLCOME FOUNDATION LIMITED L ο η d ο n ₃ NW 1 / Great Britain

(Alle-Z) -5j8jll _J l4jl7-eicosapentaenoic acid and / or its derivatives containing formulations, as well as methods for

their manufacture

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• (089) 988272 telegrams: Bank accounts: Hypo-Bank Munich 4410122850

988273 BERGSTAPFPAXENT Munich (BLZ 700200 U) Swift Code: HYPO DE MM

988274 TELEX: '. Bayet Vereinsbank Munich 453100 (bank code 70020270) 98 3310 0524560 BERG d Postscheck Munich 65343-808 (bank code 700 100 80)

The present invention relates to (all-Z) -5,8-, 11,14,17-eicosapentaenoic acid and / or formulations containing their derivatives, as well as “processes for their preparation.

Although it is known that many substances inhibit platelet aggregation can influence, it is not possible from a knowledge of the special effect of a substance predict the aggregation of platelets, whether the substance has an inhibiting or stimulating (or neutral) effect on thrombus formation in vivo will or not. This is mainly because it is not known what causes a thrombus or embolus to form for example, strokes or myocardial infarction initiated. As an example that a prediction is not possible, It should be noted that aspirin is a good inhibitor of platelet aggregation in vitro and in vivo is that, however, it is not an anti-thrombotic agent, and in particular cannot dissolve a pre-formed thrombus.

M-.J. Silver, J.B. Smith, et al., (Prostaglandins Dec. 1973, Vol. 4, No. 6, pages 863 to 875) showed in vitro that many Compounds that can influence the platelet aggregating effects caused by the diet component arachidonic acid (5j8, ll, 14-eicosatetraenoic acid, optionally also C20: '4; n-6 acid, i.e. a fatty acid with 20 carbon atoms that

Contains 4 double bonds, the one in the position with the highest number being in a position 6 bonds away from the end of the molecule opposite the carboxyl group, and η denotes the number of carbon atoms in the straight chain) . These in vitro studies on human, citrated, platelet-rich plasma cannot be transferred unequivocally to the in vivo behavior of a mammal that is prone to thrombus formation, including humans. MJ Silver et al. found in their investigations that the platelet aggregation induced by arachidonic acid, as sodium arachidonate, can be inhibited by many materials such as including adenosine, 3-naphthol, non-steroidal anti-inflammatory agents such as indomethacin, sodium salicylate and aspirin, unsaturated fatty acids such as 11,14,17-eicosatrienoic acid, 8,11,14-eicosatrienoic acid (dihomo-γ-linolenic acid = DHLA), 5,8,11,14,17-eicosapentaenoic acid, 5, 8,11,14-eicosatetrainic acid, 4.7 , 10,13,16,19-docosahexaenoic acid and human albumin. They also found that the collagen-induced platelet aggregation and a second wave of platelet aggregation induced by adenosine diphosphate (ADP) were caused by β-naphthol, aspirin, 8,11,14-eicosatetraenoic acid, 5,8,11,14,17-eicosapentaenoic acid and human albumin could be inhibited. Silver et al. found about it

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In addition, numerous fatty acids cause platelet aggregation by themselves do not induce. The acids these researchers mentioned were 8,11,14-eicosatrienoic acid, 11,14,17 " Eicosatrienoic acid, 5,8,11,14,17-eicosapentaenoic acid, 5,8,11,14-eicosatetrainic acid, 4,7,10,13,16,19-docosahexaenoic acid, linolenic acid, Linoleic acid, oleic acid, arachidic acid, stearic acid and capric acid (deanic acid).

Silver et al. seem to conclude that arachidonic acid is a has an important place in hemostasis and thrombosis, and that their effects in vitro through various compounds, in particular by albumin. They suggest that albumin is an important control factor in hemostasis and that albumin's ability to bind arachidonic acid in circulating blood may be the way on which it inhibits the effects of arachidonic acid. she also indicated that this binding capacity, for example on the availability of the binding sites and the competition between arachidonic acid and other fatty acids and other substance classes. It can therefore be assumed that the more free arachidonic acid is available and the more likely platelet aggregation occurs, the more competing substances are available, and consequently when these phenomena are related to each other stand, a thrombus formation is all the more likely.

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Various attempts have been made to study the effects of various fatty acids on human diseases including thrombus formation, but without giving clear results.

For example, the "Norwegian Vegetable Oil Experiment 1965-66" was used prior to the work of Silver et al. carried out and about it by H. Natvig, Chr. P. Borchgrevink et al. in scand. J. Clin. Lab. Invest. 22, Suppl. 105, 1-20, (1968). The study compares the effects of two diets on human mortality rates caused by various coronary heart diseases, including myocardial infarction, one diet containing sunflower oil (about 63 % linoleic acid) and the other one containing linseed oil (about 55% linolenic acid) and 10 ml of each Oils per day were taken. It was found that the group that consumed the more unsaturated linolenic acid had a greater risk than the group that received the linoleic acid.

Linoleic acid and, in rats, eicosapentaenoic and docosahexaenoic acids are known to reduce blood plasma cholesterol levels that is believed to be associated with atherosclerosis. Atherosclerosis is often found in people who have had a myocardial infarction. However, there does not seem to be any

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sale relationship because Robertson [Lancet, (1959) j i, 44] found that atherosclerosis is very rarely accompanied by secondary thrombi or myocardial infarction, although it occurs in of the indigenous population is usually found extensive after autopsy. Furthermore, myocardial infarction can occur in the absence of advanced atherosclerosis.

Yet another possible dietary factor that has been suggested to aid in the inhibition of thrombosis is DHLA (Kernoff PB, Willis AL, Stone KJ, Davis JA and McNicol GP, British Med. J., 1977, 2, 1441-1444). DHLA is a biosynthetic precursor of prostaglandin E ^ (PGE ₁ ) which is a potent inhibitor of platelet function and is said to be attractive as an anti-thrombotic agent. It has been found that there is, as hoped, an increase (55 % average) in the production of the desired PGE. However, in six of the eight men examined there was also an increase (on average 33 %) in the formation of the undesired prostaglandin Ep (PGEp). In addition, these results were not clearly dose-related. There was also a decrease in the heparin-neutralizing activity of the plasma, and this activity was found to be high in thrombotic conditions. However, the authors knew nothing about

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the degree to which the heparin-neutralizing activity fundamental pathological mechanisms reflected, and therefore their relationship to thrombosis was unclear.

The authors of the paper theorized that "perhaps small doses of DHLA are equivalent to, if not greater, effectiveness than major dietary manipulations for the prevention and treatment of these conditions "i.e., atherosclerosis and coronary artery disease. However, the author of an editorial was in the same issue of the journal (pp 1437 and 1438) more cautiously and assumed that "experiments with agents and applications that use the platelet-prostaglandin mechanism modify, must be done before we can make a statement as to whether the McNicol and his colleagues obtained results have some clinical application. "The reasons for his caution lie in the inadequate knowledge of the specified in vivo mechanism in thrombotic conditions, if research studies have only been carried out on blood samples.

This at least partially attractive work with DHLA expresses some doubts about the frequently cited view suggest that highly unsaturated fatty acids are more beneficial in the diet than their more saturated analogues, especially as

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the even less saturated linoleic and linolenic acids in the Metabolism can be transferred to DHLA. This doubt is supported by the fact that arachidonic acid, the is undesirable (cf. the work by Silver et al. and Kernoff et al. discussed above) even more unsaturated is (4 carbon-carbon double bonds) as DHLA (3 carbon-carbon double bonds).

It has now surprisingly been found that among the many fatty acids (all-Z) -5 _J 8311, 14 ₃ 17-eicosapentaenoic acid or its salts, esters or amides for the effective treatment or for the effective prophylaxis against thromboembolic conditions, the following for the sake of simplicity are referred to as thrombosis sake, can be used. Examples of conditions where this may be useful for the treatment or prophylaxis of a cardiovascular disease caused by the formation of a thrombus or thrombi are, for example, myocardial infarction, strokes, or thromboses of deep veins in the course of surgical procedures.

It was found that (alle-Zj-Sjßj11,14,17-eicosapentaenoic acid (hereinafter referred to simply as eicosapentaenoic acid) when injected intravenously in rabbits increases bleeding time, indicating a decrease in the tendency of the blood to develop Formation of thrombi or to stick to damaged tissue

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proves. Eicosapentaenoic acid releases when infused into the rabbit's lungs an increase to a substance that has a potent anti-aggregation effect on platelets. Eicosapentaenoic acid also has the unusual and important ability to dissolve thrombi that have already formed.

For example, blood from an anesthetized rabbit was passed through a continuously weighed collagen strip, that came from the Achilles tendon of another rabbit, drip. When the blood flowed over the strip, platelets adhered and other cells thrombus thereon until no further weight gain of the strip occurred. The blood was returned to the first rabbit under gravity. If eicosapentaenoic acid is in the over the loaded strip of guided blood was infused, a weight loss was observed indicating that at least some of the aggregated platelets and other cells from the loaded strip were dissolved. This ability eicosapentaenoic acid to dissolve a thrombus is for the treatment of thrombosis, and also for its prophylaxis, important.

It has also been found that human platelets, when preincubated with eicosapentaenoic acid and subsequently incubated with arachidonic acid and stimulated with ADP wor-

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the goods aggregate less easily than if the preincubation with arachidonic acid. This suggests that human platelets are resistant to ADP stimulation are less sensitive and therefore less prone to the formation of thrombi if they are treated with eicosapentaenoic acid could be "loaded".

The dose of eicosapentaenoic acid required for therapeutic or prophylactic effect will vary with the route of administration and the nature of the condition being treated, but will usually be at least Ig, preferably from 1.5 to 3 g per day. This is the dose for a human weighing an average of 70 kg and the dose for other people or animals will vary according to the proportion of their weight: ¹ ^ n, ie about 20 to 40 mg / kg.

It is known that eicosapentaenoic acid is found in cod liver oil and others oils, e.g. in menhaden oil, from which they are known to the person skilled in the art or described in the literature Procedure can be extracted. The eicosapentaenoic acid can also by conventional methods of the synthetic organic chemistry can be synthesized. The route chosen will depend on the availability of the appropriate starting materials depend. In practice, the route used will depend on the relative cost of the various routes available

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Manufacture of eicosapentaenoic acid of the right quality for human medicine! See or veterinary use depend .

The amounts contained in naturally occurring or easily extractable materials such as cod liver oil or menhaden oil are such that it would not be possible to administer the desired amount of eicosapentaenoic acid without simultaneously administering too many calories in the form of other fatty acids. In addition, since cod liver oil (and other fish oils) is rich in vitamin A [at least 850 international units (IU) per gram] and vitamin D (at least 85 IU per gram), administration of an amount containing the necessary amount of eicosapentaenoic acid would also do so at the same time Add amounts of these vitamins that the recommended daily allowance for humans would be significantly exceeded and hypervitaminosis would occur. The recommended daily intake in humans is 5000 IU for vitamin A and 400 IU for vitamin D. In the USA, the Food and Drugs Administration has determined that the daily intake of vitamin A should not exceed ^{10,000 IU and vitamin D 1 JOO IU.} Exceeding amounts require a doctor's prescription.

In order to avoid complications that arise as a result

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it can be assumed that the recipient is taking vitamin doses for other medical reasons or of his own accord, a formulation is preferably prepared that contains eicosapentaenoic acid, or a pharmaceutically acceptable salt, Ester or amide thereof, and a pharmaceutically acceptable one Contains carrier that is essentially free of vitamins.

Because of the complex and somewhat uncertain effects of other unsaturated acids besides eicosapentaenoic acid, a formulation is preferably created that contains eicosapentaenoic acid, or a pharmaceutically acceptable salt, ester or amide thereof, and a pharmaceutically acceptable one Contains carrier, the formulation essentially is free from other unsaturated acids, or their salts, esters or x ^ iiden.

The above-mentioned excessive calorie intake when using, for example, cod liver oil or menhaden oil as a source of the Eieosapentaenoic acid can be avoided for the most part, although some control of caloric intake in the rest The diet will usually still be necessary by having one Formulation that creates eicosapentaenoic acid, or a pharmaceutical one compatible salt, ester or amide thereof, and a pharmaceutically acceptable carrier, with at least 50 percent by weight of the fatty acid content of the formula

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lation are eicosapentaenoic acid. However, if the eicosapentaenoic acid is to be administered without substantial modification of the patient's diet, the acid should constitute at least 90%, preferably at least 95%, or all of the fatty acid content of the administered material. Arachidonic acid should preferably not be present, or at most not be more than 5 % of the fatty acid content. For example, a suitable grade of eicosapentaenoic acid contains at least 90 % of the acid, about 2 % each of arachidonic and dihomo-γ-linolenic acids, and the balance of palmitic and oleic acids, and other pharmaceutically acceptable materials. If vitamins are present, when this may be the case, they should preferably not be present in such amounts as to exceed their recommended daily intake.

Formulations used according to the invention should furthermore also be free from saturated fatty acids and their salts, esters or amides. Preferably the formulations are also free of unsaponifiable materials.

If eicosapentaenoic acid is administered in an amount of at least 90 % of the fatty acid content, it should be possible to avoid a significant change in the patient's diet, perhaps with the exception of a minor reduction.

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tion of the calorie content of the diet to be reduced by the eicosapentaenoic acid to compensate for the extra calories that are consumed. However, if preferred, it may be possible to use the eicosapentaenoic acid as a substitute for, for example, butter and / or ordinary margarine in the form of a special margarine, e.g. of the emulsion type, so formulated is that with normal use the patient ingests the required amount of eicosapentaenoic acid. Also edible oils and fats can similarly be formulated to contain the eicosapentaenoic acid.

The eicosapentaenoic acid need not be used as the acid itself but they can be in the form of their pharmaceutically acceptable salts, esters or amides (which are used as acid equivalents definitely knows) be taken. It can be ester or Amides that. can be converted into the acid in vivo, and "other pharmaceutically acceptable products are used, the preferred ester being the ethyl ester, however, the methyl ester may also be used. Of the the ester used is preferably not the cholesteryl ester, as this leads to the release of some cholesterol, which in turn leads to an increase in serum cholesterol levels can lead. The preferred salts are the sodium or potassium salts or any other pharmaceutically acceptable salts solid salts, as these are suitable for processing into tablet

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th are suitable. Accordingly, the present invention relates also a tablet containing a pharmaceutically acceptable, contains solid derivative of (all-Z) -5,8,11,14,17-eicosapentaenoic acid.

Because eicosapentaenoic acid is highly unsaturated, and they themselves and Their derivatives, as well as formulations which contain these compounds, are easily oxidizable, should formulations containing these compounds also contain anti-oxidants such as butylated hydroxytoluene, butylated Hydroxyanisole, propyl gallate, a pharmaceutically acceptable quinone and α-tocopherol. Any anti-oxidants but can also contribute to the anti-thrombotic-embolic Make an impact.

Although it is preferred to administer the eicosapentaenoic acid (or its salts, esters or amides) (active compound) orally, however, since this is a convenient route for routine administration, the active compound can be on any any other route in which it can be successfully absorbed, e.g., parenterally (i.e. subcutaneously, intramuscularly or intravenously), rectally, or in women also vaginally.

Although it is possible to use the active compound as such or

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To be administered as a simple mixture of components, it is intended to be administered as a pharmaceutical formulation. The formulations according to the invention, both for veterinary and for human medicine, contain the active compound as defined above, together with one or more compatible carriers therefor and optionally together with other therapeutic ingredients. The carrier or carriers must be "compatible" in the sense that they are compatible with the other ingredients of the formulation and not harmful or even toxic to the patient. Formulations which contain the eicosapentaenoic acid itself are preferably non-aqueous. Unit doses of a formulation, e.g. tablets or capsules, usually contain between 0.25 and 1.0 g, e.g. 0.5 g ₃ , of active compound. C "are administered three doses per day livable.

Formulations include those suitable for oral, rectal, vaginal or parenteral (including subcutaneous, intramuscular and intravenous) administration.

Since eicosapentaenoic acid itself is a liquid and too tends to have an unpleasant taste, it is preferably per os administered in a capsule, for example in a capsule made of soft gelatin, so that the eicosapentaenoic acid does not taste good. The capsule will usually be that size

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have that it is made possible that required for administration Dose of eicosapentaenoic acid in one or two capsules for each dose, and accordingly will a capsule usually about 0.5 ml in size. Another way of masking the taste of the acid is there in formulating it as an emulsion to be taken orally. The acid can also be formulated so that it is spontaneously emulsifiable upon oral ingestion or it can be diluted prior to administration. An emulsion could also be of the multiple type, for example by using the acid using a compatible surfactant is converted into an oil-in-water emulsion, and then this emulsion could emulsified in another oil, e.g. arachis oil. Alternatively, the acid can be poured into a water tank in a similar way. Oil emulsion transferred and then this emulsion itself again be emulsified in water. The various types of emulsion can be used as an oral gel or as a stiff emulsion, for example as emulsion margarine. Other methods of masking the taste are to that the acid is on one or more carriers, such as kaolin, chalk, calcium phosphate, calcium sulfate, Starch, a microcrystalline cellulose or a methyl or otherwise modified cellulose. The received Powder can be used as such or after adding flavor

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substances are sold and perhaps made into tablets or capsules, each tablet or capsule for example contains about 0.5 g of eicosapentaenoic acid as such or in the form of a solid derivative. The tablets can with a film or coated with sugar.

Since the salts of eicosapentaenoic acid, for example the Sodium or potassium salts, and tablets containing these salts with a content of, for example, 0.5 g of acid tend to taste unpleasant, they should preferably be coated e.g. with a film or with sugar. Other formulations for oral administration, e.g., cachets or Lozengen (lozenge lozenges) can also be used in appropriate cases. The esters or amides can like be formulated in the case of the acid or the salts, depending on whether they are liquid or solid.

If desired, an oral formulation can also be used as a formulation be presented with uniform release, for example in the form of beads or microcapsules in a Capsule.

A formulation for intramuscular administration can be found in In the form of an emulsion. A formulation for intravenous injection may be in the form of a mixture which

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spontaneously emulsified upon injection.

For rectal administration, the acid or a derivative thereof can be used as a suppository with a triglyceride base, e.g., cocoa butter WITEPSOL (suppository mass from Chemische Werke Witten GmbH) or SUPPOCIRE formulated or in a suppository capsule be placed from soft gelatin.

The formulations may suitably be in the form of a unit dose and by any method known to those skilled in the art of pharmacy getting produced. All methods include the step of bringing into association the active compound with the carrier, the consists of one or more accompanying components. Usually the formulations are made by using the active compound evenly and intimately with liquid carriers, or finely divided solid carriers, or with both, brings together, and then, if necessary, shaped the product into the desired formulation. In the In the present specification and claims, the term "carrier" includes one suitable for administration to is suitable for a patient and essentially includes the active compound, e.g., the capsule body or the coating on a coated tablet.

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Accordingly, the present invention provides

(a) (all-Z) -5j8, ll ₃ l4 _J 17-eicosapentaenoic acidj or a pharmaceutically acceptable salt, ester or amide thereof for use in the treatment or prophylaxis of thromboembolic conditions;

(b) a formulation containing (all-Z) -5 ₅ 8 ₃ ll, 14 ₃ 17-eicosapentaenoic acid, or a pharmaceutically acceptable salt ₃ ester or amide thereof, and a pharmaceutically acceptable carrier, with at least 50 percent by weight of the fatty acid content of the formulation (all-Z) -5,8,11 ₃ 1 ¹ J _{3 are} 17-eicosapentaenoic acid (ie, as such or as a derivative);

(c) a formulation containing (all-Z) -5 ₃ 8, ll, 14,17-eicosapentaenoic acid, or a pharmaceutically acceptable salt, EsL iv or amide thereof, and a pharmaceutically acceptable carrier, the formulation being essentially free of Vitamins is;

(d) a formulation comprising (all-Z) -5,8, ll ₃ l4,17-eicosapentaenoic acid, or a pharmaceutically acceptable Saiz ₃ ester or amide thereof, and a pharmaceutically acceptable carrier, wherein the formulation is substantially free of other unsaturated acids (ie as such or in the form of their derivatives);

(e) Process for the preparation of a pharmaceutical formulation according to (b), (c) or (d);

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(f) a margarine, butter, edible oil or fat formulation containing (all-Z) -5,8, ll, 14,17-eicosapentaenoic acid or a salt ₃ ester or amide thereof, in such an amount that these at least 3 percent by weight eicosapentaenoic acid (ie, as such or in the form of a derivative);

(g) a method for the treatment or prophylaxis of a thromboembolic condition in a mammal, including of the human receiving the administration of a therapeutic or prophylactic anti-thrombo-embolic Amount of (all-Z) ~ 5,8,11,14,17-eicosapentaenoic acid, or a pharmaceutically acceptable salt, ester or amide thereof; and

(h) Method according to (g) using a formulation according to (b), (c) or (d).

The present invention is illustrated in more detail by the following examples.

example 1

Blood was drawn from human volunteers who had not taken aspirin in the previous two weeks taken from a vein in front of the elbow in sodium citrate (0.11 molar), using 1 part citrate for 9 parts blood became. The plasma was separated from the blood by centrifugal

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yaw at 160 g (5 minutes) as platelet-rich plasma (PRP> won.

Investigations on the platelets were made with arachidonic acid (AA) ₃ eicosapentaenoic acid (EPA) ₃ as potassium salts (cf. Schrör, K., Moncada, S., Ubatuba, PB and Vane, JR, Eur. J. Pharmac, 1978, jj7_, 103) and with ADP or thrombin in a coagulation apparatus, eg "Pibromate" (Bie & Bernsted, Copenhagen, Denmark).

The aggregation was registered both turbidimetrically and nephelometrically in a cylindrical cuvette which contained 300 μl PRP at 37 ° C. and was stirred magnetically at 800 rpm; optionally a "Payton dual channel aggregometer" with 500 μΐ PL. ⁵ used.

In contrast to AA, EPA induced aggregation in human PRP at concentrations of EPA (1.33, 2.66 and 5.3 mmol) that were about four times or more the concentrations of AA (0.33, 0.66 and 1 , 3 mmol) were not. At lower concentrations, ranging from 0.01 to 0.5 mmol, EPA somewhat inhibited the ADP (2 μmol) induced platelet aggregation in the human PRP.

The anti-aggregating effect of EPA (0.065 mmol) was not

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due to its conversion by platelet cyclooxygenase, since the anti-aggregating effect in aspirin-treated platelets that did not respond to AA (0.065 mmol), however, aggregated by thrombin (0.04-0.4 U / ml) was present, and the anti-aggregating effect was present was also present in the first phase aggregation induced by ADP (2 to 5 µmoles) in aspirin-treated platelets is induced.

Example 2

Vascular tissue (thoracic and abdominal aorta). Approximately 100 mg of tissue was dissected and once in ice cold Tris buffer (0.05 mol, PjT 7.5). After examining his Ability to inhibit thrombin-induced platelet aggregation after addition to the platelet cuvette, the tissue was soaked several times in 10 ml of ice-cold Tris buffer washed to remove blood and adhering platelets. The tissue was then rapidly frozen to -60 ° C, too 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.

The blood was drawn from the vein in front of the elbow

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Human volunteers received during 3 days before who had taken aspirin (1.5 g per day). This blood became washed human platelets obtained as described by Vargaftig, B.B., Tränier, Y. and Chignardj M. (Prostaglandins, 197 4 ".8, 133) has been. The aggregation tests were carried out as in Example 1.

To see if the vascular tissue suspension is any material with anti-aggregatory effects on human To synthesize platelets, platelets, as described above, were obtained from volunteers who received aspirin had taken so that their platelets had no prostaglandin endoperoxides could form, which would have from the vascular tissue for the heat division of anti-aggregatory material can be used. In addition, washed platelets were used to avoid any possibility of that the vascular tissue utilizes any AA in the plasma. Under these conditions it could be anti-aggregating Activity can only be formed by the vascular tissue from endogenous or exogenously added precursors.

The initial suspension of the vascular tissue as described above (10 to 'jO μΐ) inhibited thrombin-induced (0.0 * 1 to 0j4 U / ml) aggregation. This inhibiting activity

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This was done by washing the tissue repeatedly (5 to 20 times) by centrifugation (30 seconds in an Eppendorff centrifuge), pouring off the supernatant liquid and resuspending it in fresh buffer (O ₃ 5 ml). The primary inhibitory activity against induced by ADP (2 to 5 micromoles) primary phase or aggregation by thrombin ₍₅ 0 04 to 0.4 U / ml) induced aggregation could by adding washed vascular tissue and EPA to the washed platelets from aspirin treated volunteers are restored. The development of anti-aggregating activity was prevented by pretreating the washed vascular tissue with indomethacin (5 to 10 μg / ml). As a result, the vessel wall cyclooxygenase could use the EPA to develop anti-aggregating activity.

The development of the anti-aggregating activity can result from the displacement of the endogenous AA by EPA and not by direct use of EPA. However, the same concentrations of vascular tissue washed with resulted Incubated DHLA does not lead to the formation of anti-aggregating Material.

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Example J>

The effect of eicosapentaenoic acid on bleeding time when Rabbits

Four male New Zealand white rabbits (ranch) weighing from 2.0 to 2.5 kg were given sodium pentobarbitone
(40 mg / kg) anesthetized. A cannula was inserted into the marginal ear vein for infusions of eicosapentaenoic acid (0.1 ml / min). The potassium salt of eicosapentaenoic acid
(95 $ purity with a content of about 2 % AA and 2 %
DHLA, remainder CLg fatty acids) was dissolved in 50 mmol Tris-HCl buffer with a P ₁ value of 8.0 and protected from light
kept on ice. Infusions of either the Tris vehicle or the eicosapentaenoic acid were performed 5 minutes before and continuously during the measurement of the bleeding time. The inner surface of the ear was carefully shaved without the cannula area. The ear was x-rayed so that the blood vessels were clearly visible. There were cuts with
a new scalpel blade in one of the visible blood vessels. vascular free area and made in a direction parallel to the next blood vessel that is approximately 0.4 cm long and
were deep enough to cause blood to swell within 15 seconds. The section was gently wiped every 15 seconds with filter paper (Whatman No. 1).

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As a bleeding tent, the time was measured from the time of the incision to the nearest 15 seconds when blood stains were no longer visible on the filter paper. If a plasma exudate emerged from the incision , the end point was considered to be the time at which the exudate no longer had a reddish color. If the bleeding time was longer than 10 minutes, the cut was then wiped every 30 seconds. The bleeding time at each dose was an average of 3 determinations.

Two rabbits received an injection of aspirin 4 hours prior to the experiment of 100 mg / kg i.v. Two rabbits serving as control animals received 0.5 ml Tris with a p ^ value of 7.5 in 4 ml of saline in the same way. The results obtained are set out in Tables I and II.

Table I.

Control animals did not receive aspirin

Bleeding time *

EPA dose (min)

(yg / kg / min) rabbit 1 rabbit 2

0 3.5 3.0

50 16.0

100 19.8 16.5

200 23.00 **

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Table II

Pretreated with aspirin injection of 100 mk / kg i.V. 4 hours before the start of the experiment.

Bleeding time *

EPA dose (min)

(yg / kg / min) rabbits 3 rabbits 4

0 5.3 4.7

100 7.3 6.3

200 4.5 7.5 **

* Average of 3 determinations ** ■ Infusion rate 0.2 ml / min.

As a result, the aspirin treated rabbits showed little or no increase in bleeding time.

A rabbit treated with 75 # purity EPA gave similar results when. took into account the lower purity of the acid. _a

Example 4 Conversion of eicosapentaenoic acid in the dog's circulation .

Intravenous infusion of eicosapentaenoic acid (0.2 to

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2 mg kg min) causes systemic and pulmonary hypotension in dogs anesthetized by chloralosis. Blood bathed isolated strips of bovine coronary artery and rabbit peritoneal artery were passed through the highly potent anti-aggregatory material PGI (5 to 10 ng / ml) relaxes. When treated with antagonists of C at echo land.-nen and angiotensin II, these relaxed in arterial blood bathed tissue from the living animal during the infusion of

- 1-1 eicosapentaenoic acid (0.6 to 2 mg kg min, 2 dogs), equivalent to about 10 to 20 ng / ml PGIp at the highest rates. In one of these dogs, after the administration of indomethacin (5 mg / kg), caused a subsequent infusion

-1 -1 of eicosapentaenoic acid (2 mg kg min for 10 minutes) nor hypotension, but did not release any detectable activity in the tissues of the living animal.

Example 5 Disaggregating Effect of Eicosapentaenoic Acid in Rabbits

Rabbits (2 to 3 kg) were anesthetized with 30 mg / kg sodium pentobarbitone and heparinized (2000 o / kg). A big Carotid artery was dissected out. Blood is drawn and a pump is used to impregnate a strip of collagen promoted from the Achilles tendon of another rabbit. When the blood flows over the tendon strip, it increases

909848/0481 - / 30-

Weight of the strip for a period of 35 minutes to a maximum in the range of 180 to 200 mg. Thereafter was any weight loss due to platelet dissolution.

Eicosapentaenoic acid infused intravenously in five rabbits (50 to 500 µg / kg / min) induced minor dissolving effects (approximately 20 mg). This effect of eicosapentaenoic acid could be achieved by pretreating the rabbit with aspirin (150 mg / kg) can be inhibited.

Example 6

A soft gelatin capsule for a content of about 0.5 ml was sterilized and then filled with a preparation which contained more air 90 % EPA, about 2 % AA, about 2 % DHLA and the remainder palmitic and oleic acid. The capsule was then sealed.

The capsule used can be transparent or colored and it can also be of the hard gelatin type or for example be made from polymethyl methacrylate.

Example 7

A tablet formulation consisted of the following ingredients:

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- / 31 -

Sodium eicosapentaenoate 281 mg

Strength 62 mg

Lactose 250 mg

Polyvinylpyrrolidone 3 ₃ 5 rag

Magnesium te ar at 3 j 5 mg

Butylated hydroxytoluene 2 ppm

Total: 600 mg

The tablet was coated with sugar ₅ although other coating agents could also have been used.

Example 8

The formulation described in Example 7 can be used in untabletted Powder form can be used for filling hard gelatin capsules, using 600 mg of the formulation.

Example 9

About 25O g of a conventional soft margarine formulation was thoroughly mixed with 8 g of eicosapentaenoic acid, was until a smooth consistency is achieved.

Example 10

Male New Zealand rabbits (2 to 2 ₅ 5 kg) were given intravenous aspirin (10 or 100 mg / kg). A control group received only that to dissolve the aspirin

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used carrier liquid. 2 to 4 hours later the animals anesthetized with pentobarbitone and the skin bleeding time as described in Example 3, measured before and during the infusion of EPA (potassium salt, 95 # purity, as in Bei game 3 used) at different speeds. (1, 50, 200 or 400 yg / kg / min). There were two for each condition multiple or triple measurements performed. The results obtained are set out in Table III below. Aspirin (10 mg / kg) produced a small but significant (p <0, .0001) Increase in bleeding time, the mean of the triplicate determination in five rabbits being 489 ± 27 s (Mean ± s.e.m.) was in comparison to the control values of 278 + 48 s. The value of 288 ± 11 s in the with animals treated with a large aspirin dose (100 mg / kg) was not significantly higher than the control experiment.

In the non-aspirin group, EPA (1 µg / kg / min) prolonged bleeding time by more than 100 % (p <0.00001). A further increase in bleeding time was observed at higher infusion rates and a plateau value of approximately 1000 s was reached at the rate of 50 yg / kg / min.

In the animals treated with aspirin (10 or 100 mg / kg) EPA failed to achieve a significant modification in bleeding time.

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Table III

Pretreatment

No

ASA (10 mg / kg) ASA (100 mg / kg)

EPA (yg / kg / mirv)

200

± 48 642 ± 69 861 ± 69 96I ± 87 1020 ± 89

n = 8 n = 5 n = 5 n = 4 n = 4

± 27 607 ± 97 678 ± 115 839 ± 160 693 ± 114

n = 5 n = 5 n = 5 n = 5 n = 4

± 11 309 ± 13 327 ± 82 428 ± 86 364 ± 26

n = 5 n = 3 n = 3 n = 4 n = 3

ASA = aspirin

Mean bleeding time in seconds ± s.e.m.

Claims (1)

DR. BERG DIPL.-fNG. STAPF DIPL.-ING. SCHWABE DR. DR. SANDMAIR PATENTANWÄLTE PO Box 860245 8000 Munich 86 £ O 3 I ζ) Q Or. Berg Dipl.-Ing. Stapf and Partner, P.O.Box 860245, 8000 Munich 86 ' Your reference Our reference 29 320 Mauerkircherstraße 45 July 8th 19 ' Your ref. Our ref. 8000 MUNICH 80 Lawyer file number: 29 520 Claims 1. Formulation containing (all-Z) -5.8, Hjl ^ lT taenic acid, or a pharmaceutically acceptable salt, ester or amide thereof, and a pharmaceutically acceptable carrier, where at least 50 percent by weight of the fatty acid content of the formulation (all-Z) -5,8, ll, l4,17-eicosapentaenoic acid or a Are a derivative of it. 2. Formulation according to claim 1, characterized in that at least 90 percent by weight of the Fatty acid content of the formulation (all-Z) -5,8, ll, 14,17-eicosapentaenoic acid or a derivative thereof. 3 Formulation according to Claim 2, characterized in that χ / κ 909840/0481 * (089) 98 82 72 Telegrams: Bank accounts: Hypo-Bank Munich 4410122850 988273 BERGSTAPFPATENT Munich (BLZ 70020011) Swift Code: HYPO DE MM 988274 TELEX: Bayet Vereinsbank Munich 453100 (BLZ 70020270) 983310 0524560 BERG d Postscheck Munich 65343-808 (BLZ 70010080) ORIGINAL INSPECTED indicates that at least 95 percent by weight of the Fatty acid content of the formulation (all-Z) -5,8, ll, l4,17-eicosapentaenoic acid or a derivative thereof. 4. Formulation according to claim 1, characterized in that that the total fatty acid content, or essentially the total fatty acid content (all-Z) -5,8,11,14,17-eicosapentaenoic acid or a derivative thereof. 5. Formulation according to one of claims 1 to 3, characterized in that in the fatty acid content of the formulation about 2% arachidonic acid, about 2 % dihomo-γ-linolenic acid, and the remainder palmitic and oleic acids, and other pharmaceutically acceptable fatty acids or derivatives thereof. 6. Formulation containing (all-Z) -5,8,11,14,17-eicosapentaenoic acid, or a pharmaceutically acceptable salt, ester or amide thereof, and a pharmaceutically acceptable one Carriers, the formulation being essentially free of other unsaturated acids or derivatives thereof. 7. Formulation according to one of claims 1 to 3j, characterized in that it is substantially is free from other unsaturated acids or derivatives thereof. 909848/0481 8. Formulation j containing (all-Z) -5,8, ll, 14,17-eicosapentaenoic acid, or a pharmaceutically acceptable salt, ester or amide thereof, and a pharmaceutically acceptable one A carrier, the formulation being substantially free of vitamins. 9. Formulation according to one of Claims 1 to 7, characterized in that it is essentially free of vitamins. 10. Formulation according to one of claims 1 to 9 j, characterized in that the eicosapentaenoic acid is in the form of its sodium or potassium salt. 11. Formula tion according to one of claims 1 to 9 j, characterized in that the eieosapentaenoic acid is in the form of their ethyl ester. 12. Formulation according to one of claims 1 to 9, characterized in that the eicosapentaenoic acid is used as such. 13. Formulation according to one of claims 1 to 12, characterized in that it is an antioxidant contains. 909848/0481 lh. Formulation according to one of Claims 1 to 13, characterized in that it contains a flavoring agent. 15. Formulation according to one of claims 1 to 1H, characterized in that the carrier is a solid. 16. Formulation according to claim 15, characterized in that that the carrier is a capsule or comprises a capsule containing the remainder of the formulation. 17. Formulation according to one of claims 1 to 14, characterized in that the carrier is a Liquid is. 18. Formulation according to claim 17 , characterized in that the eicosapentaenoic acid, its salt, ester or amide forms a disperse phase in the carrier liquid. 19. Formulation according to one of claims 17 and 18, characterized in that it is in capsule form is present. 909848/0481 20. Formulation according to one of claims 1 to 15, characterized in that it is in tablet form is present. 21. Formulation according to one of claims 1 to 20, characterized in that it is in a appropriate form for oral, parenteral, rectal, vaginal or intrapulmonary administration. 22. Formulation according to one of claims 1 to 21, characterized in that it is in the form a unit dose. 23. A formulation according to claim 22, characterized ge kennze ⁴ seframe that it contains 0.25 to 1.0 g of eicosapentaenoic acid. 24. A method for producing a formulation according to any one of claims 1 to 23, characterized in that that one brings the components together in a usable preparation. 25 margarine, butter, edible oil or fat formulation, characterized in that it (all-Z) -5,8,11,14,17-eicosapentaenoic acid, or a salt, ester or 909848/0481 Amide of the same in such an amount that this at least 3 percent by weight eicosapentaenoic acid in the form of the Acid itself or a derivative thereof. 26. Margarine formulation according to claim 25, characterized in that it is in the form of an emulsion margarine. 27. Formulation according to one of the preceding claims, characterized in that it essentially is free from saturated fatty acids or their salts, esters or amides. 28. Use of (all-Z) -5,8,11,14,17-eicosapentaenoic acid or a pharmaceutically acceptable salt, ester or amide thereof for the treatment or prophylaxis of one thrombo-embolic condition. 29. Anti-thrombo-embolic agent containing (all-Z) -5,8,11,14,17-eicosapentaenoic acid or a pharmaceutically acceptable salt, ester or amide thereof. 30. Formulation according to one of claims 1 to 24 or 27 for use in the treatment or prophylaxis of a thrombo-monobolic State. 909848/0481 31. Anti-thrombo-embolic agent, characterized in that it is a formulation according to one of claims 1 to 2k or 27. 32. Tablet, characterized in that it contains a pharmaceutically acceptable solid derivative of (all-Z) -5,8,11, lh , 17-eicosapentaenoic acid. 909848/0481