Note: Descriptions are shown in the official language in which they were submitted.
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
X155
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
1151068
- 9 - X155
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
- 12 - X155
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
1151068
- 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~
- 16 - X155
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 -
1151068
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
1151068
- 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
- 20 -
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
.
1151068
- 21 - X155
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
- 22 - X155
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
- 23 - X155
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~
- 27 - X155
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.
AMPLE 3
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
- 29 - X155
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
- 30 - X155
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-
1151068
- 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~
,
EXAMPLE 5
_, ~
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.
LXAMPLE 7
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
33 X155
Butylated hydro.~y toluene 2 ppm
TOTAL 600 mg
The tablet was coa,ted with sugal~ al~hough
other coating agents could be used.
EXAMPLE 8
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.
EXAMPLE 9
-
About 250 g of a conventional soft margarine
formulation was thoroughly mixed with 8 g of
eicosapentaenoic acid until a smooth consistency was
reached.
EXAMPLE 10
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
- 34 - X155
(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
X155
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
EXAMPLE 11
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
- 36 - X155
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.
