CA1151068A - Fatty acid and derivatives thereof for use in treatment of prophylaxis of thrombo-embolic conditions - Google Patents

Abstract

ABSTRACT

(A11-Z?-5,8,11,14,17-cicosapentaenoic acid administration to a mammal, including man, for treat-ment or prophylaxis of thrombo-embolic conditions, for increasing bleeding time, for disintegrating or dispersing already formed thrombi or platelet clumps for modifying or controlling adherence of blood platelets to damaged tissue, for rendering blood platelets less readily aggregatable and/or more readily disaggregatable from one another, and for adding to blood in extra-corporeal circulation.
Pharmaceutical fcrmulations containing (a11-Z)-5,8,11,14,17-eicosapentaenoic acid or a pharmaceuti-cally acceptable salt, ester or amide thereof.

Description

1151S3~8

- 2 -Fatty Acid and Derivatives Thereof in Treatment or Prophylaxis of Thrcmbo,Emholic Cbnditions.

m e present inventian relates to the treatment or prophylaxis of th~ h~1mb~1ic cQnditions.
AlthDugh it is known that nEmy substances can affert platelet aggregation, it cannot be pr~A;cted from a knowledge of the effect of a particular suk-stance an aggregatian of platelets in vitro, w*~her or not the substan oe will have an inhibitory or sti~latory (ar neutral) effect thrombus formatiQn - in vivo. m is is largely because it is not kncwn what initiates formation of a thrombus or embolus in, for example, a stroke or n~x~mdial infarction. As an example of this unpredictability, Aspirin* i5 a good inhibitor of platelet aggregation in vitro and in vivo, kut it is not an anti-thromkotic agent, in particular it cannot disp~rse a preformed thrombus.
M. J. Silver, J. B, Smith, et al., (Prostaglandins Dec. 1973, VQ1.4~ No. 6, pages 863 tD 875) shr~ed that many oompounds can influence in vitro the platelet aggregating effects produced ~y the essent;~l dietary oomponent ararhi~onic acid (5,8,1l,14-eioosa-tetraenoic acid, alternatively C20:4; n-6 acid, i.e. a fatty acid containIng 20 carbcn atoms having 4 r~rbDn-to, carbon c~s- double bonds, the one at the highest *trademark ~' 115~ i8 numbered p~sition ~eing at a position 6 bonds from the end of the m~lecule re~Dte from the carboxyl group, and n bPing the number of carbon atoms in the straight chain). These in vitrD tests in human citrated platelet rich plasma cannot be unambig~ously related to _ vivo behaviour in the thromkus formation-prone nEunnal, including man. M. J. Silver et al.
foNnd in their tests that the platelet aggregation induced by arachidonic acid, as sodium arachiodonate, can be inhibited by many materials including adeno-sine; ~ -naphthol; non-steroidal, anti-inflammatory agents such as indomethacin, sodium salicylate and aspirin; human aIbu~in; unsaturated fatty acids, such as 11, 14, 17-eicosatrienoic acid, 8,l1,14~
eicosatrienoic acid (dihomo-y-linolenic acid, DHIA), 5,8,11,14,17-e;rnsapentaenoic acid, 5,8,11,14-eicosa-tetraynoic acid, and 4,7,10,13,16,19_docosahexaenoic acid. They also found that the platelet aggregation induced by collagen an~ a second wave of platelet aggregation induced by adenosine diphosFhate (ADP) could be inhibited by ~ -naphthol, Aspirin,* 8,11,14-eicosatrienoic acid, 5,8,11,14,1~-eicosapentaenoic acid and human ~ nin. Silver et aI. further found that Yarious fatty acids their own did n~t induce platelet aggre~ation. The acids they mntioned were 8,11,14-eioDsatrien~ic acid; 11,14,17~eicosatrienoic *trademark ~, - 1~510~8 - 4 - Xl55 acid; 5,P"11,14,17-cicosapentaenoic acid, 5,8,11,14~
eicosatetraynoic acidS 4,7,10,13,16,19-docosahexaenoic acid; linolenic acid; linoleic acid; oleic acid;
arachidic acid; stearic acid; and decanoic acid.
It will be apprcciated tha~ many of the com-pounds found by Silver et al. to be anti-aggregatory are unsuitable for use in therapy. For examplc, adenosine is rapid]y absorbed by cells and so would not be available in the body for long enough to be of value. ~ naphthol is toxic as it is a phenolic compound. Albumin is unsuitable because it would put an undesirable load on the kidney and give rise to glomerulant damage in the kidney. As non-steroidal anti-inflammatory agents frequently give rise to gastric lesions, they should preferably be avoided in any therapy requiring long term prophylactic oral administration as is often desirable in cardio-vascular treatments.
Silver et al. appear to conclude that arachidonic acid has an important place in hemostasis and throm-bosis, and that its effects can be inhibited in vitro hy various compounds, particularly albumin. They suggeste(l that albumin may be an important controlling factor in hcmostasis and that the ability of albumin to bind ara(hido3lic aci~ in circulatin~ hlood migh~
b~ thc wa~; it inllibits the cffects of arachidon;c ilS1~68 acid. Th~y further suggeste~l that the net binding capacity of albumin for arachidonic acid may depend on, ~or e~amJ)lc, ~he availability of binding sites an~ competition between arachidonic acid, other ~atty acids and other classes of substallces for those sites. Presumably, thercfore, the more competing substances there are available; particularly other fatty ac.ids, the more free arachidonic acid there would be and the more likely platelet aggregation would be, and thereore if these phenomena were to be related, the more likely thrombus formation would be.
This suggests that other fatty acids should be removed from the diet.
Attempts have been made to investigate in man the effects of various fatty acids on diseases involving thrombus ormation, but no clcar conclusion has cmerged.
For example, the Norwegian Vegetable Oil Experiment of 1965-66 was carried out before the work of Silver et al and was reported by H. Natvig, Chr. F.
Borch~revink, et al in Scand. J. Clin. Lab. Invest.
22, Su~pl. 105, 1-20, ~1968). The study compared the effects on human mortali~y ratcs caused by various corvnary hear~. diseases, includi.ng myoc.lrdial in:far~tion, of two dicts, O]lC contai.nin~ sunflo~er sec~ oil (al)out 63o of linoleic acjd) and the othe.

11510~i8 - G - X15~

cont~ ing linseed oil (about 55~ OI linolenic acid);
10 ml. nf either oil b~ing take7lper day. The group taking the lnore highly unsaturatcd linolenic acid l~as found t:o be mGre at risk than the g3'0Up taking S t]le linoleic acid.
Linoleic acid, and, in rats, eicosapentaenoic and docosahexaenoic acids are kno~l to decrease blood plasma cholesterol levels, which are bclieved to be connected with atherosclerosis. Atherosclerosis is often found in persons who have suffered from a myo-cardial infarct. However, there appears to be no causal relationship, because Robertson (Lancet, (19S9), i, 44) found that in Jamaica, although extensive atherosclerosis is regularly found in the native pop-lS ulation at necropsy, it is very seldom associatedwith secondary thrombi or with myocardial infarction.
Further, myocardial infarcts can occur in the absence of highly developed atherosclerosis.
Yet another possible dietary factor that has been suggested (P.B. ~crnoff, A.L. Willis, K.J. Stone, J.A. Davis and G.P. McNicol, British Med. J., 1977, 2, 1441-1444) as helping to inhibit thrombosis is ~HLA.
Dl1LA is a biosynthetic precursor o~ prostaglandin E
(PGEl), wl~ich is 2 powerful inhibitor of platelet functio3l, ?nd was said to be Ittrac~ive as an anti-throrllbotic agent. It was found Lhat theré w~s ? ~5 1151~68 7 XlSS

hopc~, a rise (mean 55~ ) in production of the dcs-irablc PGEl but in six men out of the eight tested there was also a rise (mean 33~) in production of the undesirable prcstaglandin E2 (PG~2). Furthermore S these results were not clearly dose related. There was also a lo~ering of heparin-neutralising activity of plasma, and this activity has been found to be high in thrombotic states. However, the authors did not know the extent to which heparin neutralising activity reflects basic pathological mechanisms, and so its relationship with thrombosis was unclear.
The authors of the paper speculated that "Perhaps small doses of DHLA may be equally if not more effec-tive than major dietary manipulations in preventing and treating these conditions" i.e. atherosclerosis and coronary heart disease. However, the author of an editorial in the same edition of the Journal (pages 1437 and 1438) was more cautious and thought that "Trial~ of agents and regimens that modify the platelet prostaglandin mechanisms must be carried out before we can tell whether the results obtained by McNicol and his colleagues have any clinical appli-ca~ion". The reasons for his caution lay in the ignorance that exists of the Jnechanisms involve~ in vi~ in throml~otic situations, ~hen investigative tests hav~ only ~ecn carrie~l out on shed blood.

1~510f~8 ~ X155 This at least partially attractive work witn DHLA thrcws some doubt on the frequently quoted vie~
that wlsatllr2ted fatty acids in the diet are morc beneficial than their more saturated analogues, especially as the even less saturated linoleic and linolenic acids can be metabolised to DHLA. This doubt is strengthened by the -fact that arachidonic acid which is undesirable (see Silver et al and - - Kernoff et al above) is even more unsaturated tfour carbon-carbon cis-double bonds) than DHLA (three cis-carbon-carbon double bonds).
We have now surprisingly found that among the many fatty acids (all Z)-5,8,11,14,17-eicosapentaenoic acid or its salts, esters or amides can be used to treat effectively, or provide effective prophylaxis against, thrombo embolic conditions, hereinafter referred to simply as thrombosis. Examples of con-ditions where our indings may be useful are in the treatment or prophylaxis of cardiovascular disease mediated by the formation of a thrombus or thrombi, for example myocardial in~arction, stroke , or deep v~in thrombosis during surgicai ope~rations.
We have found that ~all 7)-~,8,11,14,17-eicosa-~- pen~aencic acid (hereinaf~er referred to simply as eicosapentaenoic acid, also ~no~n as icosapentacnoic acid) ~ n injccted intraveilollsly into rabbits incre;~se~i ~l,cir blce~illg time, ~hus den~ollstratillg a dccrease in the telldency of the blood to produce thrombi or adhere to damaged tissue, thus enabling one to modify and/or control ~ound-healing.
When infuscd into rabbit lung, eicosapentaenoic acid gives rise to a substance which has a po~Jerful anti-aggregatory action on blood platelets, ~ ,icosapentaenoic acid also has the un'usual and important ability to disperse or disin~egrate already formed thrombi or platelet clumps. For example, blood from an anaesthetised rabbit was allowed to drip over a continuously weighed collagen strip taken from the Achilles tendon of another rabbit. As the blood flowed over the strip, platelets and other cells adhered to it to form a thrombus until there was no further ~ain in weight of the strip. The b-lood was returned to the first rabbit under gravity. When eicosapentae-noic acid was infused into the blood passing over the loaded strip a decrease in weight was observed, showing that at least part of th~ aggregated platelets and other cells had been disaggregated from the loaded strip.
This a~ility o~ eicosapentaenoic ~cid to bring about dispersion or disaggregation of thrombus is important in the treatment of thrombosis" and also in its pro-phylactic treatment. ~'hen a thrombus is being formedin an artery (or vein) there is a reduction in thc blood flow (which flow would be completely stoppc~ if llS10t~8 - 10 - XiSS

the vcssel were to become completely occluded).
This rcduction in blood flow brings about ischae~nia, which ~roduces pain. The reduccd blood ~low can, however, carry therapeutic materials to the site of thrombus formation. In ~ivo with eicosapentaenoic acid the residual blood flow and any blood flow in the col]ateral circulation can carry the acid to the site of thrombus formation where the eicosapentaenoic acid and its metabolites can disaggregate the thrombus and restorefull blood flow. Accordingly, this invention also provides a method of restoring full blood flow in a partly occluded blood vessel by administering eicosapentaenoic acid. The adminis-tration can also be used prophylactically to help to keep blood vessels clear.
We have also found that human platelets, whe~
pre-incubated with eicosapentaenoic acid and then incubated with arachidonic acid and stimulated with ADP, aggregate less readily ~han when the pre-incubation is carried ou~ with arachidonic acid.
This suggested to us that, if human platele~s could be 'primed' with eicosapentaeroic acid, they would be ].e5s susceptible to A~DP stimulation and so less liable to form thrombi.
The Applic-Jn~s believc, althou~h they do no~
wish ~o be bound by ~his belief, th2t n _ ivo the 115~068 - 1l - X155 eicosapenta~lloic acid, in contrast to arachidonic acid, not only itself has an anti-aggregatory e~ect on blood platelets but its metabolites, presumab]y prostaglandinsof the a-17 series, also have an anti-aggregatory effect on the plate]ets, or at worst a reversible aggregating effect, whereas many of the metabolites of arachidonic acid, such as PGH2 and TXA2, have an irreversible aggregating e~fect on platelets. This net anti-aggregating profile for eicosapentaenoic acid is, the Applicants believe, responsible for its surprisingly beneficial properties.
The dose of eicosapentaenoic acid needed for therapeutic or prophylactic effect will vary with the route of administration and the nature of the condition being treated, but will generally be at least 1 gram (g), prefcrably from 1.5 to 7.5 g,especially 2 to 6 g ~or example 5 g per day. This is the dose for an a~erage 70 g man and the dose ~or other men or animals will generally vary pro-rata according to th~ir weight, i.e. about 20 to l00 mg~kg.
Eicosapentaenoic acid may be added to extra-cor-poreally circulating blood to prevent, substantially or comple~ely, aggregation of biood platclets induced by contact with the ~lachine or with other non-tissue materials.
Eico~apentaenoic acid is lino~l to be prescnt in oystels and other sca oods, in cod liver oil ~nd in il5~0~8 other oils, e.g. menhaden oil, rrom ~hich it may be extractcd by mcthods kno~n in the art or described in thc literature. The eicosapentaenoic acid may also be s3~nthesised by conventional methods of S synthetic organic chemistry. The rou~e chosen will depend on the availability of suitable starting materials, and on the relative costs of the various routes available to provide eicosapentaenoic acid of the right quality for human medical or veterinary use. Care should be taken in extractive and pre-parative processes to avoid, or keep low, the isomer-isation of cis-double bonds to ~rans- double bonds.
The amounts of eicosapentaenoic acid in naturally occurring or readily extractable materials, such as cod 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. Furthermore, 2S cod liver oil ~and other fish oils) is rich in vitamin A (at least 850 international units (I.U.) per gram) and vitamin D (at least 85 I.U. per gram) administering enough cod liver oil to give the nec~ssary amowlt of eicosa-pentaenoic ~cid would administer amounts of these vitamins great~y ex~ceedillg the recom,nended daily dose for hulnalls and Tlould 1ead to hyper-vitaminosis. Tne 115~068 - 13 - XlS~

rccommelldcd daily dose is 5000 I.IJ. for vitamin A
and 400 I.U. for vitamin D in humans. In the U.S.A.
the Food and Drugs Administra~ion has laid down that the daily in~ake of vitamin A should not exceed 10,000 I.U. and of vitamin D should not exceed 400 I.U. ~nowlts above this require a doctor's pres-cription.
Therefore to avoid complications, which may arise through the recipient receiving vitamin doses for other medicinal reasons, or at his or her own instigation, a formulation is preferably provided which comprises eicosapentaenoic acid, or a pharma-ceutically acceptable salt, ester or amide thereof, and a pharmaceutically acceptable carrier, the formulation being substantially free of vitamins.
Because of the complex and to some extent uncer-tain e~fects of acids less unsaturated than eicosa-pentaenoic acid a formulation is preferably provided comprising eicosapentaenoic acid, or a pharmaceutically acceptable salt, ester, or amide thereof~ and a pharmaceutically acceptable carrier, ~he formulation being substantially frce of other, less wlsaturated acids, or their salts, esters or a~TIides. ln eicosapentaenoic acid obtaiIled ~rom natllral sources, such as fish ~ils, there is usua31y a pro~ortion of (all-Z) 7,10,l3,l6,19-~13cosal)entae]loic - 14 Xl55 acid (hereinafter rc~errcd to as docosapentaenoic acid) and/or of (all-Z)4,7~10,13,16,19-docosahcxacnoic acid (hereillaftcr rcferred to as docosa~lexaenoic acid) (as such or as thei.r derivatives i.e.tl~ir esters, salt:s or amides). lt is not necessary to try to remove these equally or more unsaturated acids (or their deriva-tives~, because they behave in a way simi;ar to eicosa-pentaenoic acid, but are less active.
The excessive calorie intake mentioned above, if, for example, cod liver oil or menhaden oil were used as the source of the eicosapentaenoic acid, may be substantially overcome, although some control of calorie intake in the remainder of the diet may still be necessary, by administering a formulation cGm-prising eicosapentaenoic acid, or a pharmaceuticallyacceptable salt, ester or amide thereof, and a pharmaceutically acceptable carrier, at least 50~, e.g. greater than 56~, by weight of the latty acid content of the formulation being provided by eicosa-pentaenoi.c acid. However, if the eicosapentaenoicacid is to be administered without modification of - the recipient's diet, the acid ~and any ~ocosapent-a.cnoic aci.d or docosahexaenoic acid) should represent at least 90~0, prefcrably at leasl: 95~ or all, by ~eight of thc- atty acid content of ~he adminis~ere~
material.

115~068 - ]5 - ~lSS

Arachidonic acid should precrably be absent or at most should be no more than 5~0 of the fatty acid content. For example a prefeIred quality of eicosap~ntacnoic acid comprises at least 90~ of S the acid, abou~ 2o Of each of arachidonic and dihomo-~-linolenic acids, the balance being docosahexaenoic, docosapentaenoic, palmitic or oleic acids; and other pharmaceutically acceptable fatty acids. If vitamins are present, as they may be, they should preferably not be present in amounts that would lead to their recommended daily intake being exceeded.
Pormulations used according to the invention should also be free of saturated fatty acids and their salts, esters or amides. Preferably the formulations should be free of unsaponifiable materials.
By administering the eicosapentaenoic acid as at least 50%, preferably at least 90~, of the fatty acid content, it should be possible to avoid sub-stantial alteration of the diet of the recipient, excep~ perhaps to reduce slightly the calorific content of the diet to allow for the extra calories from the eicosapcntaenoic acid (and other fatty acids). Howc-ver, if preferred, it may be possible to adn~.inister the eicosapentaenoic acid by replacing, s~ buttcr and/or ordin~ry margarine by a special mar~.~rille, e.g. of thc emulsion type, formulclted so il510~

that in normal usage the recipient l~ould receive the required amount of the eicosapentaenoic acid.
Cooki~g oils and fats may also be similarly formulated to con~ain the eicosapentaenoic acid.
The eicosapentaenoic acid (and o~her acids) need not be used as the acid itself but may be used as its pharmaceutically acceptable salts, esters or amides (which would 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 ester being the triglyceride or ethyl ester, but the methyl ester could perhaps also be used. The alcohol used to esterify the acid should preferably be non-polymeric and should preferably contain no more than three hydroxyl groups in the molecule. Further, the ester used is preferably not the cholesteryl ester as this would lead to some cholesterol being liberated which may lead to an increase in the serum cholesterol level. For the 3ame reason the formulations of the - 16a -present invention are preferably free of cholesterol, as such or as a derivative thereof convertible to cholesterol in the body of the recipient. The preferred salts are the sodium or potassium salts or any other pharmaceutically acceptable solid salt, aR these are more suitable for making into tablets. Tablets may comprise a pharmaceutically acceptable solid derivative, e.g., a salt, of eicosapentaenoic acid.
As eicosapentaenoic acid is highly unsaturated, 1151~t;8 - 17 - X~.55 it an~ its derivatives are readily o~idisable and formulations containi.ng them should preferably also contain anti~oxidants, such as butylated hydroxy toluene, butylated hydroxy anisole, propyl gallate~
a pharmaceutically acccptable quinone and ~-toco pherol. Some anti-oxidants may also contribute to the anti-thrombo-embolic effect.
Although it is preferred to administer the eic~s-apentaenoic acid (or its salts, esters or amides) (active compound) orally as this is a convenient route for routine administration, the active compound may be administered by any route by which it may be successfully absorbed, e.g. paren~erally ~i.e. sub-cutaneously, intramuscularly or intravenous~y), rectally or, in the case of women, vaginally.
While it is possible for the active compound to be administercd as a raw chemical or as a simple mixture of components, it is preferable to present it as a pharmaceutical for~ulation. The formulations, both for veterinar)~ and for human medical use, of the present invention comprise the active compound a~
above defined, together with one or more acceptable carriers therefor and optionally other therapeutic ingledicn~s. The carrier(s) must be 'acceptable' in the sen-;c of beir;g ccmpati.ble with the other i.ngredi.ent.c o~ ~he formula.tion and not dcleteriolls to 1151~i8 ~ X155 the rccipicnt thereof. Formulations which contain eicosapentaelloic acid itself are prefer~bly non-~4ucnus. Unit doses, e.g. tablets or capsules of ~ formulation generally contain from 0.25 to 1.0 g, e.g. 0.5 ~ of the active compound.
Generally three doses would be administered per day.
Formulations which may be used include those suitable for oral rectal, vaginal, or parenteral (including subcutaneous, intramuscular and intra-renous) administration As eicosapentaenoic acid itself is a liquidand tends to be unpala~able, it is preferably adrninistered per orally in a capsule, for example one of soft gelatin, so that the eicosapentaenoic acid is not tasted. The capsule would generally be of a size to permit the required dose of eicosa-pentaenoic acid to be administrable in one, two or three capsules at each dose taking and so a capsule would be generally about 0.5 ml in size. Another way of disguising the taste of the acid is to formulate it as an emulsion to be taken orally. The acid could also be formulated to be spontaneously emulsifiable on being taken orally or on l~eing diluted before ~dministration. An emulsion could also be of the mu]tiple type; e.g. the acid ~ou3cl be made into lAn oil-in-water er,lulsicn ~lit~ a pl~al~mace~ltica]ly - l9 - Xl55 acceptable surface active agent and then this emulsion could be cmulsified in another oil, e.g. arachis oil.
Alternatively, the acid could be similarly formulated into a water-in-oil emuls on and then this emulsion S itsel emulsified in water. The various types of emulsion could be presented as an oral gel or as a stiff emulsion, such as an emulsion margarine.
Other methods of disguising the taste are to absorb the acid onto a carrier or carriers such as kaolin, chalk, calcium phosphate, calcium sulphate, starch, a micro-crystalline cellulose,or methyl or other modified cellulose. The resulting powder could be sold as such or flavoured, and perhaps made into 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.
As for the salts, e.g. the sodium or potassium salts, ~hese also tend to be unpalatable and tablets containing them, and representing for example 0.5 g of acid, should pre~erably be coa~cd e.g. by film or sugar. Other mcthods of oral administratio~, e.g.
cache~ or lozengc, may also be used in appropriate circumstances. The estcrs or amidcs may be formu lated as for the acid or the salts, depending on whether they are liquid or solid, respectively.

1~51068 If desired an oral form~lation can be presented as a sustainad release formulation, for example as beads or micro-capsules in a capsule.
A formulation for intr.rlJKliLlr administration could be in the form of an e~ulsion. A formulation for intravenous injection could be in the form of a nixtNre that would spontaneously emulsify upon injection.
~or rectal administration the acid or derivative oould be fc~miLated into a suppository in a trigly-ceride base, e.g. cocoa butter, a Witepsol* or Suppocire*
or placed in a soft gelatin suppository capsule.
The formLla~;~nc may conveniently be presented in unit dosage form and may be pre$ared ky any of the methods well kncwn in the art of pharmacy. All methods incl~de the step of bringing into asscciation the active compound with the carrier which constitutes one or more accessory ingredients. In general the formLlations are prepared by uniformly an~ inti~ately bringing into association the active ccn~x~md w~th liquid carriers ~r finely divi,ded solid carriers or koth, and then, if necessary, shaping the prDduct into the desired formulation. In the present specification and claims the term "carrier" includes one which is sui~hle for administration to a recipient and substantially encloses the active *trademark D
.

compound e.g. the body of a capsule or tlle coating on a coated tab].et.
To improve the effectiveness of the cicosapent-aenoic acid, the formulation may also include a phosphodiesterase inhibitor, such as theophylline or dipyridamole.
Accordingly, the present invention.provides:-(a) (all -Z)-5,8,11,14,17-eicosapentaenoic acid, or a pharmaceutically acceptable salt, ester or amide thereof, for use in the treatment or prophy-laxis of a thrombo-embolic condition;
(b) a formulation comprising (all -Z)-5,8,11,14,17-eicosapentaenoic acid or a pharmaceutically acceptable salt, ester or amide thereof, and a pharmaceutically acceptable carrier, at least 50~ of the fatty acid content of the formulation being provided by (all-Z)-5,8,11,14,17-eicosapentaenoic acid (i.e. as such or as a derivative);
(c) a formulation comprising (all -Z)-5~8,11,14,17-eicosapentaenoic acid, or a pha.rmaceuticallyaccepta~le salt, ester or amide thereof, and a phar-. maceutically acceptable carrier, the formulation hcing substantially free of ~Titamins;
~d) a ormulatioll comprising (all -Z)-5,8,11,14,17-: 25 ei.cosapellt.. lenoic acid, or a pharmaceutic21]y acccpt-able salt~ es~el or amide thcrcof, and a.

~15~6i8 pharmaceutically acceptable carrier, the formulation being substantially free of other, less unsaturated acids (i.e. as such or as their derivatives);
(e) a method of preparing a pharmaceutical form-5 ulation according to (b), ~c~ or (d);
(f) a margarine, butter, cooking oil or fat formu-lation inc].uding tall -Z)-5,8,11,14,17-eicosapentae-noic acid or a salt, ester or amide thereof in an amount to provide at least 3~ by weight of the eicosapentaenoic acid (i.e. as such or as a derivative);
(g) a method for the treatment or prophylaxis of a thrombo-embolic condition in a mammal inc.luding man, which comprises administering a therapeutic or prophylactic anti-thrombo-embolic amount of (all -Z)-5,8,11,14,17-eicosapentaenoic acid or a pharmaceuti-cally acceptable sa].t, ester or amide thereof;
th) a method according to (g) using a formulation according to any one of ~b), (c) and (d).
(i) a method of increasing the bleeding time of a mammal, including man, which comprises administering an effective amount o~ (all-Z)-5,8,11,14,17-eicosa-pentaenoic acid, or a salt, es~er or amide thereof;
~j~ a method of dispersing or disintegrating an alread~- .ormed thrGm~us or platelet clump in a mammal illCl~ding lllall, Wl'i.CIl COmpl`iseS ad;lliiliStra~iOil of an ,.

115:10G8 effective amount of (all-Z)-5,8,11,14,].7-eicosa-pentaenoic acid or a salt, ester or amide thereof;
(~) a metllod. of modifying alld/or con.trolling adherence o blood platelets to damaged tissue in a mammal~ includi.ng man, whi.ch comprises admin-istering an effective amount of (all-Z)-5,8,31,14,17-eicosapentaenoic acid or a salt, ester or amide thereof;
(1) a method of rendering blood platelets in a mammal, including man, less readily aggregatable and/or more readily disaggregatable from one another by administering an e~fective a~.ount of (all-Z)-5,8~ 14,17-eicosapentaenoic acid or a salt~ ester or amide thereof;
~5 (m) a me~hocl according to any one of methods (i) to tl) using a formulation according to any one of (b), (c) and (d);
tn) a method of extra-corporeally circulating blood to ~d from a mammal, including man, which includes ac~ninister~ng to the blood, i.ntra- or extra-corporeally, an amount effective to prcvent, sub-stantially or completely a~gregation of blood platelets, of (all-Z)-5,8,11,14,17-eicosapentaenoic aci.d or a salt, ester or amide thereof; and (ol a ll\etl-lod of restoring or maintaillin~ full blood f3.o~ in a part3.y occluded bloocl vesse]. in a n~;l,nmal, ~lSl~
~ 24 -including man, which oorprises administering eicosa-pentaenoic acid or a salt, ester, or amide thereof.
The present inventian is illustrated by the follownng EXamples.

E~E 1 Blood from human volunteers wh~ had not taken aspirin for the previous tw~ weeks ~as collected from an ante-cubital vein in sodium citrate (O.llM), 1 p~rt of citrate to 9 p3rts of blood. Plasma was sep~rated from the blood by centrifugation at 160 g (5 minutes) as a p~atelet rich plasma (PRP).
Studies on platelets were preformed with ara-chidonic acid ~), eicosapentaenoic ~acid (EPA) prepared as potassium salts (see Sc W r, K., Moncada, S., Ubatuba, F.B., And Vane, J. R., Eur. J. Pharmac., 1978, ~J 103) and with A~P or thrombin in a coagulation apparatus e.g. 'Fibromate'* ~Bie & Eernsted CbFenhagen, Denmark).
Aggregation was reoorded both turbidime*rir~lly and nephelcmetrically in a cylindrical cuvette, cantaim ng 300~1 of PRP at 37 & and st;rred magneti-cally at 800 rpm; alternatively a Payton dual channel aggregometer was used with 50ql1 PRP.
In oDntrast to M , EPA did not induce aggregation in human PRP at concentrations of EPA (1.33, 2.66 and *trademark `' ~C~' ~, , llSlOG8 75 _ X155 5.~ m~l) about 4 or more times greatcr than M (0.33, 0.66 and 1.3 m~l). At lower concentrations in the range of fro~n n. ol to 0.5 mM EPA somewhat inhibited platelet aggrcgation incluced by AD~ (2~M) in the human PRP.
The anti-aggregating effect of EPA (0.065 mM), however, was not due to its conversion by platelet cyclo-oxygenase because the anti-aggregating effect was present with aspirin-treated platelets which did not respond to AA (0.065 mM) but we~e aggregated by thrombin (0.04-0.4 U/ml), and the anti-aggregating effect was also present in first phase aggregation induced by ADP (2 to 5~M) in aspirin-treated platelets.

_ AMPLE 2 Vascular tissue (thorafic and abdominal aorta) . . .
was obtained from freshly killed rats. Approximately 100 mg of tissue was chopped and washed once in ice cold Tris buffer (.O.OS M, pH 7.5~. After testing its ability to inhibit thrombin-induced platelet - aggregation when added to the pla~celet cuvette, the tissue was washed several times in 10 ml of ice cold Tris buffer to remove blood and adhcring platcle~s. The tissuc was tl~ell quickly frozen to -~0~ crushed to a coarse pc~wder all~ r~-suspendcd in 11510~8 - 26 - Xl55 five volumcs of Tris buffer. This suspension of vascula-r tissuc was kept on icc during the experi-ments and used for incubation studies.
~lood was obtained from the ante-cubital vein of human volunteers that had taken aspirin (1.5 g per day) for the 3 days before blood sampling.
Washed human platelets werc obtained from this blood as described by Vargaftig, B.B., Tranier, Y., and Chignard, M., (Prostaglandins, 1974, 8, 133).
Aggregation tests were carried out as in Example 1.
To see if the suspension of vascular tissue could synthesise any material having an anti-aggre-gatory effect Oll human platelets, platelets were obtained as described above from volunteers who had taken aspirin, so that their platelets could not produce prostaglandin endoperoxides that could be utilised by the vascular tissue to make anti-aggregatory material. Moreover, washed platelets were used to avoid any possibility of the vascular tissue utili~ing any AA in the plasma. Under these conditions anti-aggrega~ing activity could be formed by the vasc~lar tissue only from elldogenous or exogenously added precursors.
The initial suspension or ~ascular tissue (10 to 50~1~ described abovc inhibited aggregation induced by thrc!mbil) ~0.09 to 0.4 U/ml). lllis inh;l)itory .~

11510~

activi.ty was abolished by repeated washing (5 to 20 times) of the tissue by centrifuging (30 seconds in an l,p~cndorf ccntrifuge), pouring off the supernatant and resuspcnding in fresh buffer (0.5 ml).
The gcneral levcl of inhibitory activity against primary phase aggregation induced by ADP (2 to 5~M) or aggregation induced by thrombin (0.04 to 0.4 U/ml) could be restored by adding washed vascular tissue and EPA to the washed platelets from aspirin-treated volunteers. The generation of anti-aggregating activity was prevented by the pretreatment of the washed vascular tissue with indomethacin (5 to lO~g/
ml). Thus, the vessel wall cyclo-oxygenase could utilise EPA to form anti-aggregating activity.
The anti-aggregating activity formed might have been due to displacement of endogenous AA by EPA
and not to 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 so DHLA doe~ not displace AA.

The Ef_e t of Eico.sapentacnoic Acid on Bleeding Time In the Rabbit Four male New Zealand white rab~ s (Kanc,l) ~51068 - 28 - Xl55 weighing 2.0 to 2.5 kg were anaesthetized with sod;.um pentobarbitone (40 mg/kg). 'rhe margillal ear vei.n was cannulated for infusions (0.1 ml/min) of eicosapentaenoic acid. The potassium salt of eicosapentaenoic acid (95~ pure and containing about 2~ AA and 2~ DHLA, balance C-18 fatty acids) was dissolved 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 S minutes before and contir.uously during the measurement of bleeding time.
The internal surface of the ear without the can-nula was carefully shaved. The ear was transillumi-nated so that blood vessels were clearly visible.
Cuts, approximately 0.4 cm long and deep enough to : cause an upwelling of blood within 15 seconds, were made with a new scalpel blade in an area free of visible blood vessels and in a direction parallel to the nearest blood vessel. The cut was gently 2Q blotted every 15 seconds with filter paper (Whatman No.l).
Bleeding time was measured to the nearest lS
seconds from the time of incision until dots of blood were no longer visible on the filter paper. If there was a plasma exudate ~rom thc cut, the end point was considered as the time wherl the exudate 115~068 no longcr had a rcddish tinge. When bleeding time was longer than 10 minutes, the cut was then blotted every 30 seconds. The blecdillg time at each dose was a mcan of 3 estimations.
Two rabbits were pretrcated with aspirin 100 mg/
kg l.V. injection 4 hours before the experiment.
Two rabbits were given 0.5 ml Tris pH 7.5 in 4 ml saline in the same way to act as controls. The results obtained are set out in Tables 1 and 2.
Table 1 Controls i.e. no aspirin Dose Bleeding Time*
g/kg/min Rabbit 1 Rabbit 2 0 3.5 3.0 16.0 100 19.816.5 200 **23.0 ilS10~8 Tablt~ 2 Pretreated with aspirin 100 mg/kg i.v. injection 4 hours before test begun _ Dose . Bleeding Time*
~Ig/kg/mln _ _ 0 Rabb t 3 Rabb;t 4 . 100 7.3 6.3 200 4.5 **7.5 * Mean of 3 estimations ** Rate of infusion 0.2 ml/min Accordingly when treated with aspirin, the rabbits showed little or no increase in bleeding time.
A rabbit treated with 75~ prue EPA ga~e similar results after allowing from the lower purity of the acid.

EY.AMPLE 4 Conversion o. Eicosapentaenoic Acid in the Circulation of the ~G~.
Jntravenous infusion of eicosapentaenoic acid (0.2 to 2 m~ kg 1 mill 1) caused systemi.c ~nd pulmonary hypotension in chloralosc an~esthetized dogs~ Blood-- 31 - XlS5 bathed iso]atcd strips of bovine coronary artery and rabbit coeliac artery are known to be relaxed by the powerfully anti-aggregatory n-aterial PGI2 (S to 10 ng/ml). When treated with antagonists of catecholamilles and angiotensin II, these bio-assay tissues, bathed in arterial blood relaxed during infusion of eicosapentaenoic acid (0.6 to 2 mg kg lmin 1, 2 dogs), by an amount equivalent to about 10 to 20 ng/ml PGI2 at the highest rates.
In one of these dogs after administration of indo-methacin ~5 mg/kg), subsequent infusion of eicosa-pentaenoic acid (2 mg kg lmin 1 for 10 min) still caused hypotension but did not release any detect-able activity in the bioassay tissues~
, _, ~
Disaggregating efect of Eicosapentaenoic Acid in the Rabbit Rabbits (2-3 kg) were anaesthetized with pento-barbitone sodium 30 mg/kg and heparinized ~2000 U/kg).
A carotid artery was dissected and blood was exterior-ized and delivered with a roller pump to superfuse a strip of collagen from the Achilles tendon of a diffcrent rabbit. As the blood flowed over the tendon s~rip, the strip increased in lveight over a perlod of 35 min up to a miximum of ~rom 1~0 to 200mg.

1151Vti8 - 32 - ~ X155 Therea~er any decrease in weight was due to p]atclet disaggregation.
Eicosapentaenoic acid infused intravenously (50-500llg/kg/min) into five ra~bits induced small disaggregating e-ffects (approxilllately 20 mg). This effec~ of eicosapentaenoic acid could be inhibited by pre-treating the rabbits with aspirin (150 mg/kg).

EXA~PLE 6 A soft gelatin capsule to contain about 0.5 ml was sterilised and then filled with a composition con-taining more than 90~ of EPA, about 2% AA, about 2~o DHLA with the balance including paimitic and oleic acids.
The capsule was then sealed.
The capsule used may be transparent or coloured, and may also be of the hard gelatin type or made of polymethyl methacrylate for example.

A tablet formulation comprised:
Sodium eicosapentaenoa~e 281 mg St~rch 62 mg Lactose 250 mg Poly~7inyl pyrrolidone 3.5 mg Magnesium S~cclra~e 3.5 mg - 1~51068 Butylated hydro.~y toluene 2 ppm TOTAL 600 mg The tablet was coa,ted with sugal~ al~hough other coating agents could be used.

The formulation described in Example 7 in untab],etted powder form may be used to fill hard gelatin capsules with 600 mg of the formulation.

-About 250 g of a conventional soft margarine formulation was thoroughly mixed with 8 g of eicosapentaenoic acid until a smooth consistency was reached.

Male New Zealand rabbits (2-2.5 kg) were given aspirin (10 or 100 mg/kg i.v.). A control group received only the liquid vehiclc used for dissolving the aspirin. Two to four hours later the animals were anaesthetised Wit}l pentobarbitone and cutaneous bleeding time was measured as described in Example 3, befol-e and during the infusioll of EPA ~potassium salt, 95~ ~ure as usc~l in Example 3) at cliferent rates 11510~8 (1,50,200 or ~00 llg jkg/min). Duplicate or tripli-cate measurcments were done for each condition. The results ob~ained are set out in Table 3 below.
Aspirin (10 mg/kg produced a small but significant (p ~0.0001) incrcasc in bleeding time, the average of triplicate determination in five rabbits was 489 + 27 s (mean ~ s.e.m.) compared to the controls 278 + 48 s. The value of 288 + 11 s in the animals treated with a large dose (100 mg/kg) of aspirin was not significantly higher than ~he control.
In the group receiving no aspirin, EPA (1 ~g/kg/
min) prolonged bleeding time by mGre than 100~ (p~
0.00001). A further increase in bleeding time is observed at higher rates of infusion and a plateau value of about 1000 sec is attained at the rate of 50 ~g/kg/min.
In the animals treated with a,spirin (10 or 100 mg/kg) EPA failed to produce a significant modification in the bleeding time.

115~0~i8 l`ablc 3 ~ - EPA ~g/kg/min Pretreatmcllt - - _ = 50 20~ 400 None 278 + 48 6~2 + 69 36] + 69 951 + 87 1020 + 89 n = 8 n = 5 n = 5 n = 4 n = 4 ASA 10 mg/kg 489 l 27 607 + 97 678 + 115 839 + 160 693 + 114 n = 5 n - 5 n = 5 n = 5 n = 4 ASA 100 mg/kg 288 + 11 309 + 13 327 + 82 428 + 86 364 + 26 n = S n = 3 n = 3 n = 4 n = 3 Mean bleeding time in seconds ~ s.e.m.
ASA = Aspirin Using the method of Example 3~ the effects were ob-served of various fatty acids on the aggregation induced by 11~, 9~-epoxymethano-15-hydroxyprosta -5,13-dienoic acid (an analogue of PGH2) ~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 analogue was added; the amount of fatty acid used was about 1.5 mM. The amounts OI inhibition obtained six minutes after addition of PGH2 analogue are set out in 15. Table 4.

- ~151068 Ta~le 4 __ _ Aci~l ~ Inhibition Eicosapentaelloic acid lO0 (all-~)-9,12,15-octadecatrienoic acid 40 (all-Z)-9,12-octadecadienoic acid 55 (all-Z)-6,9,12-octadecatrienoic acid . 63 Z-9-octadecaenoic acid 68 Control 0 Eicosapentaenoic acid also exhibited 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-octa-decatrienoic acid and ~all-Z~-6,9,12-octadecatrienoic acid, which are 2,3-dinor analogues of the 11,14,17-eicosatrienoic acid and 8,11,14-eicosatrienoic acid used in the paper by Silver et al referred to above, were not very effective as anti-aggregation agents as compared to eicosapentaenoic acid.

Claims (24)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A formulation comprising (all-Z)-5,8,11,14,17-eicosapentaenoic acid, or a pharmaceutically acceptable salt, ester or amide thereof, and a pharmaceutically acceptable carrier; at least 50 by weight of the fatty acid content of the formu-lation being provided by (all -Z)-5,8,11,14,17-eicosapentaenoic acid or the derivative thereof.

2. A formulation according to claim 1 in which at least 90% by weight of the fatty acid content of the formulation is provided by (all-Z)-5,8,11,14,17-eicosapentaenoic acid or a derivative thereof.

3. A formulation according to claim 1 or 2 which includes in its fatty acid content about 2% of arachidonic acid, about 2% of dihomo-.gamma.-linolenic acid, with the balance being pharmaceutically acceptable and comprising docosahexaenoic, docosa-pentaenoic, palmitic, oleic acid or other fatty acids, or derivatives thereof.

4. A formulation comprising (all-Z)-5,8,11,14,17-eicosapentaenoic acid, or a pharmaceutically acceptable salt, ester or amide thereof, and a pharmaceutically acceptable carrier, the formulation being substantially free of other, less unsaturated acids or derivatives thereof.

5. A formulation according to claim 1 or 2, sub-stantially free of other, less unsaturated acids or derivatives thereof.

6. A formulation comprising (all-Z)-5,8,11,14,17-eicosapentaenoic acid, or a pharmaceutically acceptable salt, ester or amide thereof, and a pharmaceutically acceptable carrier, the formulation being substantially free of vitamins.

7. A formulation according to claim 1, 2 or 4, substantially free of vitamins.

8. A formulation according to claim 1, 4 or 6, in which the eicosapentaenoic acid is present in the form of its sodium or potassium salt.

9. A formulation according to claim 1, 4 or 6, in which the eicosapentaenoic acid is present as its triglyceride or ethyl ester.

10. A formulation according to claim 1, 2 or 6, in which eicosapentaenoic acid itself is used.

11. A formulation according to claim 1, 4 or 6, including an antioxidant.

12. A formulation according to claim 1, 4 or 6, including a flavouring agent.

13. A formulation according to claim 1, 4 or 6, in which the carrier is or includes a capsule enclosing the remainder of the formulation.

14. A formulation according to claim 1, 4 or 6, in which the eicosapentaenoic acid, salt, ester or amide forms a disperse phase in the carrier which is a liquid.

15. A formulation according to claim 1, 4 or 6, in a capsule in which the-eicosapentaenoic acid, salt, ester or amide forms a disperse phase in the carrier which is a liquid.

16. A formulation according to claim 1, 4 or 6, in tablet form, the carrier being solid.

17. A formulation according to claim 1, 4 or 6, in unit dosage form.

18. A formulation according to claim 1, 4 or 6, in unit dosage form containing 0.25 to 1.0 g of eico-sapentaenoic acid, as such or as a salt, ester or amide.

19. A formulation according to claim 1, 4 or 6, substantially free of saturated fatty acids or their salts, esters or amides.

20. A tablet comprising a solid derivative of (all-Z)-5,8,11,14,17-eicosapentaenoic acid in an amount effective for the treatment or prophylaxis of thrombo-embolic conditions, together with at least one tableting excipient.

21. A tablet according to claim 20, containing 0.25 to 1.0 g of said acid.

22. A method of preparing a formulation according to claim 1, 4 or 6, comprising bringing the components into operative association with one another.

23. A margarine, butter, cooking oil or fat formulation including (all-Z)-5,8,11,14,17-eicosapentaenoic acid or a salt, ester or amide thereof in an amount to provide at least 3% by weight of eicosapentaenoic acid, in the form of the acid itself or a derivative thereof.

24. A margarine formulation according to claim 23, in the form of an emulsion margarine.