CA2217258A1 - Triglycerides - Google Patents

Abstract

Triglycerides with two or three different fatty acids chosen from the twelve essential fatty acids, oleic acid and other fatty acids containing 8 to 26 carbon atoms, useful in nutrition and in medicine.

Description

CA 022172~8 1997-10-02 TRIGLYCERIDES

Field of Invention The invention relates to triglycPri~le R~k~- .,u-ld The Ps~ 1 fatty acids (EFAs) consist of a series of twelve co.ll~oullds tr~tetl in Table 1 below. T.inolçic acid (LA) the parent conlpoulld of the n-6 series of EFAs, and alpha-linole-nic acid (ALA) the parent compound of the n-3 series, are usually the main dietary EFAs. The parent co~ ou,lds are metabolizedby the sequence of reactions shown in Table 1. In 4~ tive terms, as judged by their levels in cell membranes and in other lipid fractions, dihomo-gamma-linolenic acid (DGLA) and ~r~chidonic acid (AA) are the main EFA metabolites of the n-6 series, while eicos~pPnt~PnQic acid (EPA) and doco~hPY~Pnc ic acid (DHA) are themain metabolites of the n-3 series. DGLA, AA, EPA and DHA are illlpol~lt con~tituPnt~ of most of the lipids in the body. As well as being illl~Ul~t in thPm~Plves they can also give rise to a wide range of oxygenated derivatives, the eicosanoids, including the prost~gl~ntlin~, leukotrienes and other compounds.
The elong~ti~n reactions shown in Table 1, in which 2 carbon atoms are added to the chain, tend to be rapid, whereas the cle~tl-r~tion reactions in which an extra double bond is introduced tend to be very slow. Thus for example gamma-linolPnic acid (GLA) is rapidly converted t~ DGLA while stP~ridonic acid is readily collverted to 20:4n-3 and so these pairs of compounds are equivalent in dietary terms. However, DGLA is only slowly converted to AA. The reactions are not normally reversible nor, in man, are n-3 and n-6 series acids inter-convertible.

W O 96/31457 PCT/GB96J'~2 The table is as follows:-n-6 n-3 18:2 delta-9,12 18:3 delta-9,12,15 (linoleic acid) ¦ (alpha-linol~nic acid) delta-6 desaturase 18:3 delta-6,9,12 1, 18:4 delta-6,9,12,15 (gamma-linolenic acid) l (ste~ri~onic acid) elongation 20:3 delta-8,11,14 J, 20:4delta-8,11,14,17 (dihomo-gamma-linolenic acid) delta-5 ~ s~t~ ce 20:4 delta-5,8,11,14 1, 20:5 delta-5,8,11,14,17 (~rhirlQnic acid) ¦ ('~icoC~r~nt~noic acid') elongation 22:4 delta-7,10,13,16 ~l 22:5 delta-7,10,13,16,19 (adrenic acid) delta-4 desaturase 22:5 delta-4,7,10,13,16 ~l 22:6 delta-4,7,10,13,16,19 ('doco~hPY~ noic acid') The acids, which in nature are of the all-cis configuration, are system~ti~lly named as derivatives of the collcs~onding oct~der~noic, eiro~noic or ~ococ~noic acids, e.g. delta-9, 12-oct~-ler~ n~ic acid or delta-4,7,10,13,16, l9-doco~hPY~noic acid, but nnm~ric~l decign~tinns such as, col.c~Llondingly~

W 096/31457 PCT/GB~G/OC8~X

18:2 n-6 or 22:6 n-3 are convenient. Tniti~lc, for example, EPA for the 20:5 n-3 acid C_icosa~nt~P-nOiC acid) or DHA for the 22:6 n-3 acid (_oco~hPy~pnoic _cid), are also used but do not serve when n-3 and n-6 acids of the same chain length and degree of lm~tll~tion exist as for ~Y~mple with the 22:5 acids. Trivial names in more or less common use in the n-6 series are as shown. Of the n-3 series only 18:3 n-3 has a commonly used trivial name, alpha-linolPnic acid, though the name ste~riclonic acid is coming into use for the 18:4 n-3 acid and the names eicos~rPnt~PnCIic acid and ~oco~hPY~nPn~ic acid as such are also used.
It is becoming a~a-cnt that in many different disease states there are abnorm~litiçs of EFA biochPmictry leading to abnormal EFA levels in various lipid fr~-~tion~ and in various tissues. These ~ e~Ps include ~ P~Ps of the heart and circulation such as hy~c~Lellsion and co~ aly and peripheral vascular ~ P~P, e~PS of infl~mm~tinn and immnnity such as atopic disorders, osteoa Lll-ilis, rh~llm~toid arthritis, ulcerative colitis, Crohn's disease and various disordersgoing under the general cl~c~ific~tion~ of infl~mm~tory or auto-immllnç, neurological disorders such as ~l7hPimp-rls disease, Parkinson's disease and multiple sclerosis, disorders of the kidney, disorders of the skin, disorders ofthe gastrointestin~l tract, disorders of metabolism of calcium and other minPr~lc, disorders of bone and colme~;Li~re tissue, disorders of the reproductive and endocrine systems, psychiatric disorders in~hl~ing scl,i~ -cnia, and disorders of aging.
Different ~ P~ees have different problems in EFA metabolism and exhibit di,rr~lcl~t abnormal p~tt~-rn~ of EFAs and it is therefore desirable in some situations to give two or more of the EFAs simultaneously.
FurthPrmore, the EFAs are exceptionally susceptible to oxidation and so it may be appr~-iale to co-~-lmini~tP-r the EFAs with oleic acid (OA) which has potent p.~c.Lies as an antioxidant.

CA 022172~8 1997-10-02 W 096/31457 PCT/GB9G/'~D~-VVhile the EFAs can be supplied in various forms and in various ll.iXLul~;s, it is in principle convenient in both nutrition and in mPlic~1 tre~tment to be able to supply the fatty acids as predett:illlined, particular molP~ulP~ This is particularly true with respect to pharm~e~tic~ where reg--l~tion~ and directives covering comhin~tinn products are becoming steadily more restrictive. For çy~mrlP, in order to win governmPnt approval for a combination drug product co.~l; ining compounds A, B and C, it is now no longer adequate to mix the three compounds together in fnrm~ ti-m X, and then to colllpart; X with placebo, P.
Many gove~nmPnt~ now require proof of the value of each individual chPmic~
entity, whether or not the whole point of a proposal is a synergistic action of dirr~ nt entities or a newly discovered ~imlllt~npQus lack of more than one entity.
Therefore at the very least clinical studies have to be set up co- ~ ;llg P with X, with A alone, with B alone and with C alone. Some governmPntc might also require co-- p ~- ;.con~ with A + B, A + C and B + C. Thus at least five and possibly eight groups would be required for testing with an enormous esc~l~tion of cost. In order to avoid this ~itll~tinn, it would be a~r~liate instead of having a IllixLule of A, B and C, to have a single molecule in which A, B and C
are found together in the same c-hP-mic~l compound, Y, allowing direct and simple testing of Y against P with only two groups required. For this purpose triglycçri~les, which can contain three fatty acids, are proposed.

Triglyceride Structures In triglycerides the different EFAs and oleic acid may be present in the same molPcule, either randomly distributed among the 1, 2 and 3 positions or with a particular EFA being found spP~-ific~lly in one of the positions on the molecule. With each triglyceride one or two positions will be occupied by one fatty acid while the other one or two positions will be occupied by one or two other fatty acids.

W 096/31457 PcT/GB96lc~?x In Preparing Triglycerides The course of esterifi~tion may in principle be directed to favour a desired isomer, but if it is not, then the position of individual acid residues in the triglycerides produced from starting ~ lules of two acids is one of the several pos~ihiliti~s: -TABLE 2a AAA AAB*ABA ABB*
BAB
BBB
and for three different acids:-TABLE 2b AAA AAB*AAC*
ABA ABB* ABC*
ACA ACB* ACC*

BAB BAC*
BBB BBC*
BCB BCC*

CAC
CBC
CCC

W 096/31457 PcTlGB9G~ 2x with either isomer equally likely to be formed where C2 is chiral, at least in c~ r~l as opposed to enzymatic synthesis. What the two or three acids are, of course, dep~nfls on the choices made from the possible acids. What the ;one of the isomers are is calculable, for undir~;led synthesis.
A previous filing has described in detail a range of triglycerides for use in ph~rTn~ce~ltic~lc, mltriti~ n~l supplçmçnte~ foods and skin care ~lc~ ;one In that filing it was suggested that the parent eee~--nti~l fatty acids, linoleic acid and alpha-linolt-nic acid had few biological actions themeelves and so those fatty acids were eYrl-lded from the triglycerides described. The described triglycerides cont~ined fatty acids which were divided into one of the following four cal~golies:
a) GLA and DGLA.
b) AA, adrenic acid and the 22:5n-6 acid.
c) Stearidonic acid and the 20:4n-3 acid.
d) EPA, the 22:5n-3 acid and DHA.
Triglycerides were dçs~ rihed which cont~ined either two residues of a fatty acid from one group, and one residue sPlçcted from a different group, or which cont~ined three dirr~lc, t fatty acids st~-lect~ from three dirrel~nt groups. These triglycerides optionally contained oleic acid as one of the fatty acid residues due to its potent antioxidant pl~elLies.
However, the concept that LA and ALA thPmep~lves have little value may have been uvel~L~ted and it is probable that LA and ALA do have i,llpolL~lt actions in their own right. For t-Y~mpl~, linoleic acid appears to have specific actions on the skin (Prottey et al, Br J Dermatol 1977; 97:29-38) and recent unpl-bliehçd studies (AC Buck) intlic~te that it may stimulate c~lcillm absorption from the gut. ALA
itself may have actions which result in a coneidç~hle reduction in the risk of myocardial infarction (M de Lorgeril et al, Lancet 1994; 343: 1454-9). It therefore seems that LA and ALA may have useful actions in their own right and that at thevery least they do not have adverse effects and can act as useful carriers for the other fatty acids.

CA 022172~8 1997-10-02 W 096/31457 PcTl~9GlQo82x Furthermore, it may also be a~pl~-iate to co-~dmini.ctPr the EFAs with a fatty acid other than oleic acid or the twelve çccPnti~l fatty acids of the n-6 and n-3 series. Such fatty acids might include stearic acid, p~lmitic acid, lauric acid,myristic acid or any other fatty acid conf~ g 8 to 26 carbon atoms, whether ~ tr~ mono-lln~tllratpl1 or polyun.~~ tP~, and whether in the case of un.saturated fatty acids having its double bonds in either cis or trans config--rationc.

The Invention In the light of the above the invention provides, as groups of isomers or singly, triglycerides Containinf~:-a) one residue of an acid st-lPct~ from oleic acid and the following groups of acids:-Group i) gamma-linol~-nic acid (GLA) and dihomo-gamma linQlçnic acid (DGLA) Group ii) arachidonic acid (AA), adrenic acid, and the 22:5 n-6 acid Group iii)stp~ritlonic acid and the 20:4 n-3 acid Group iv) eico~penta~noic acid (EPA) the 22:5 n-3 acid, and dQco.cahPY~Prloic acid (DHA), one residue of an acid sçlçctPd differently therefrom and one residue of an acid sçlPctçd from linoleic acid, alpha-linolenic acid or any other fatty acid, other than oleic acid or the twelve n-6 and n-3 series P-ssential fatty acids, Cont~ining 8 to 26 carbon atoms whether 5~hl~t~i, mono--lncatllratPd or polyunsaturated and whether in the case of lm.c~hlratP~ fatty acids having its double bonds in either cis or trans configurations; or b) two residues of the same acid sP-lPctçd from oleic acid and the acids of groups i) to iv) above and one residue of an acid SPl~-ctP~ from alpha-linolP.nic acid or any other fatty acid, other than oleic acid or the twelve n-6 and n-3 series çc.cP.ntial fatty acids, containing 8 to 26 carbon atoms whether satllr~tP~l, mono-CA 022172~8 1997-10-02 WO96/31457 PCT/GB9~ B?~

l~n~hlr~tyl or polyl~n~t~lr~t~A and whether in the case of un~t~r~t~d fatty acids having its double bonds in either cis or trans configurations; or c) one residue of linoleic acid, one residue of alpha-linolenic acid and one residue of an acid s~lPct~ from oleic acid and the acids of groups i) to iv)above; or d) one residue of an acid s~ t~d from oleic acid and the acids of groups i) to iv) above, one residue of an acid sP~ ted from linoleic acid and alpha-linol~.nic acid and one residue of any other fatty acid, other than oleic acid or the twelve n-6 and n-3 series ç~enti~l fatty acids, CQl.~ g 8 to 26 carbon atoms whether saturated, mono-unsaturated or polyunsaturated and whether in the case of n~ r~ fatty acids having its double bonds in either cis or trans configurations.The groups of isomers so defined comprise ~ Lulc;s of position~l and/or optical isomers, which may be in the plupolLions arising from directed or undi~cled synthçsi.~, or in ~l~,polLions arising from tre~tm~-nt of as-synth~ çd llli~lulcs to ~-.nh~nce the p~olLion of particular isomers or groups of isomers. Further, according to the method of synthesis and degree of enh~nsçm~nt if any, varying amounts of triglycerides other than those defined may also be present.
The selection of desired groups of isomers may also be tabulated as below, with albitld-y reference numbers for the triglycerides Cl Gs), or rather possible groups of t~glyceride isomers repres~ t~i. For example TG45 is the possible mono-linoleoyl - mono-alpha linolenoyl - mono- Group (iv) glycerides eg. the mono-linoleoyl) - mono - (alpha linolenoyl) - mono- (eicosapentanoyl) glyceride.The table is:-CA 022172~8 1997-10-02 w 096131457 PCT/Gs96l00828 1~. LA ALA Oleic Grp.l Grp.2 Grp.3 Grp.4 Other FA

1~ - 1 - - 1 1 - -CA 022172~8 1997-10-02 W 096/31457 PCT/GB96/00~2 able 3 cont'd ) ALA Qleic G~. l Grp.2 Grp.3 Grp.4 Other FA

CA 022172~8 1997-10-02 W 096/31457 PCT/~b9C~8?8 (Table 3 cont'd ) TG LA ALA Oleic Grp. 1 Grp.2 Grp.3 Grp.4 Other FA

As well as in structural terms as above, the invention may be considered in terms of starting mixtures of acids, sPlPcted from oleic acid and the acids of Groups i) - iv) above, linoleic acid, alpha-linolenic acid and other Cg 26 fatty acids, namely in molar terms (33% stands for one third, 66% stands for two thirds):-i) 33% of an acid SPlPctPll from oleic acid and the acids of Groups i), ii), iii) and iv), 33% of a different acid sPlPcted therefrom and 33% of linoleic acid, alpha lino1enic acid, or any other Cg 26 fatty acid other than oleic acid and the twelve n-6 and n-3 e~onti~l fatty acids; or ii) 66% of an acid selected from oleic acid and the acids of Groups i), ii), iii) and iv) and 33% of an acid sP-lect~Pd from alpha-linolenic acid or other Cg 26 fatty acid other than oleic acid and the twelve n-6 and n-3 e~Pnti~l fatty acids; or W 096/31457 PCT/GB~G~ 2 iii) 33 % of linoleic acid; 33 % of alpha-linol~ni~- acid; and 33 % of an acid s-PlP~ted from oleic acid and the acids of groups i), ii), iii) and iv); or iv) 33% of an acid sPlP~t-Pri from oleic acid and the acids of Groups i), ii), iii) and iv), 33% of linoleic acid or alpha-linolenic acid and 33% of a Cg 26 fatty acid other than oleic acid and the twelve n-6 and n-3 e~ n~;~l fatty acids.
Preferred starting ll~Lùl~S, with ~I,iLldl~/ reference nllmbPrs for the triglycPri~es, or rather possible groups of triglyceride isomers (TGs), that they formally ~ esent, are derived from Table 3, specifying numbers of re~i~luPc, by reading 33 mole % (one third) for '1' and 66 mole % (two thirds) for '2'.
As the desire is to give mixed triglycerides of two or three acids, species AAA and BBB of Tables 2a and 2b are for example unwanted colllpollents of the synthP-ci7P~ Illi~Lul~ but the mixed species predolllh~ate and the as-synthPci7~d llli~Lulcs are therefore valuable. Where the desire spe~ific~lly is to give mixed triglycerides of three acids, such species do not predominate but are still present in a valuable ~lu~?ollion. In either case, desired species can be sep~r~tPd or part s~ ed from others by chromatographic or other methods known in thP.mcP,lveS.
Individual triglycerides either cont~ining three different fatty acids or two fatty acids in a 2:1 ratio, may thus be manufactured by ~hemic~l or enzymatic means bymethods known in thPmct-lves to those skilled in the art. If the method of synthesis or m~nllf~rtllre does not provide an adequate concPntr~tic n of the desired triglyceride, then that triglyceride may be concentrated and purified by a~lu~liat~
techniques as outlined later.
As far as we are aware, all the groups of triglyceride isomers de-fine~ as aboveconsist of new triglycerides which do not appear in nature and have not previously been described. They may broadly be ~l~al~d as follows:
a) The individual fatty acids are purified from natural animal, vegetable or microbial sources or are çhPmic~lly synthPci7Pd, there being methods known in themCp-lves to those skilled in the art.

W 096t31457 PCT/GB~6/00828 b) The individual fatty acids are then est~rifi~d with glycerol by çh~-.mic~l or enzylllatic methods, therebeing again methods known in th~m.cPlves to those skilled in the art. For eY~mrle, the fatty acids and glycerol may be allowed to react together in thepresence of one of a number of ayployliale enzymes, or of p-toluene s~lphonic acid hydrate.
c) If required, the specific triglycerides are further purified by ayyn~yliate me.tho-lc, again known to those skilled in the art, in particular high yl~;s~ulc liquid chromatography or other a~yn~pliate forms of chromatography; low ~ulpe-dlul~ cryst~llic~tit n; or the use of solvents which dirrelentially select triglycerides of particular composition.

In the product, desirably a sper-ifie~ particular triglyceride or group of triglycerides forms more than 10%, preferably more than 30% very preferably more than 70% and ideally more than 90% of the triglycerides present in any triglyceride I~ lul~; used for thepr~ n of pharm~reutic~l compo.citis)nc, foods, or skin careproducts. The triglycerides may be made up into a~ropliate pharm~.eutit~l.c or foods so as to provide a daily dose of lmg to lOOg per day, preferably lOmg to lOg and very preferably 500mg to 4g. ~lt~rn~tively in foods or skin care products the triglycerides may be inco-~u-d~ed in conrçntr~tions of 0.001 to 50%, preferably 0.05 to 20% and very prerc;l~bly 0.1 to 5 % .
The spel--ifi~d triglycerides have a wide variety of possible uses. They ma.y be used as pharm~-euti~lc for the tre~tmçnt or prevention of disease in which abnorm~litiPs of EFAs have been ide-ntifiP l They may be added to foods or be added to or used as nutritional supplçmPnt.c for those who require theparticularEFAs for the tre~tmPnt or prevention of .li.cP~c~- They may also be used in foods or ph~rm~eutic~l.c for vet~-rin~ry use. They may be used for skin care.
The triglycerides may be formulated in any way ~pro~liate, as well known to those skilled in the art of p-~a ing pharm~eutic~l.c, skin care products W 096/31457 PCTIGB~G~ 2 or foods. They may be ~tlminictpred orally, enterally, topically, parenterAlly, (subcllt~neQusly, intr~mtlscu1~rly, intravenously or by any other route), rectally, vaginally or by any other a~plopliate route.

Synthetic FY~m The following are eY~mrles of synthesis of the triglyceri~
Several triglycerides have been plc~al~d as examples of the range of tnglycerides outlined in Table 3, as summ~riced in Table 4:-Code Triglyceride TG number (as in Table 3) GocLE GLA/ALA/EPA 17 aLLG ALA/LA/GLA 42 There are a large number of synthetic routes to triglycerides r~olLed in thelitP,AI,,-e but two of these methods are of particular use. The first uses glycerol mon~lole~;led with a 4-methoxybenzyl group as the starting point. At a later stage in the synthesis this group is removed using a boron reagent, but while this very efficiPntly removes the ~ te~;Ling group and minimiCPs acyl group scrambling it also causes cis-trans iSompric~tion of the fatty acid double bonds. This fact coupled with the expense of the reagent severely limits the applicability of this route. The second main route starts with a base-catalysed epoxide ring opening of a glycidol by a fatty acid to yield a monoacyglycerol. This route does lead to a Illib~Lule ofpositional isomers but nevertheless has good potential for larger scale synth~ses Direct reaction between glycerol and a mixture of fatty acids m~Ai~t~A either byDCC/DMAP or p-tol-lenPculfonic acid, using a mixture of two dirr~,c;l~t fatty acids W 096t31457 PCT/GB9~C~828 is a further useful synthetic pathway. ,A~ ming that both fatty acids react at equal rates simple probability theory can be applied to predict the distribution of triglyceride products. For example when equal parts of 2 dirr~ent fatty acids (A,B) are reacted with glycerol using p-toll-enes--lfonic acid catalysis. Four classes of triglyceride will be formed:

AAA 12.5 %
AAB,ABA,BAA 37.5 %
ABB,BAB,BBA 37.5 %
BBB 12.5 %
If the fatty acids are in a ratio of 2 parts A:l part B the theoretical prep~r~tion.~ are:-AAA 29.6%
AAB,ABA,BAA 44.4%
ABB,BAB,BBA 22.2%
BBB 3.7 %
In actual measurements it should be noted that due to dirre,~ t eYtinction coefflciPnt.~ at 210nm (the monitoring wavelength used for the hplc analysis) the pe.cell~ges measured by hplc are dirr~l~nt to the theoretical values.
The same approach can be used to PY~minP the distribution when 3 different fatt.y acids are used, when in reaction as above ten classes of triglyceride will be formed:
AAA 3.7 %
BBB 3.7 %
C C C 3.7 %
AAB,ABA,BAA 11.1%
AAC,ACA,CAA 11.1%
BBA,BAB,ABB 11.1%
BBC,BCB,CBC 11.1%

W 096/31457 PCT/GB~G/OQ8~X

CCA, CAC, ACC 11.1 %
CCB, CBC, BCC 11.1 %
ABC, ACB, BAC, BCA, CAB, CBA 22.2%

The invention is illll$t~t~ by the following pr~a.dLi~Je s~-h~m~, in which the following abbreviations occur:-DCC = dicyclohexylcarbo~liimi.lç
DMAP = 4-N,N-dimethylaminopyridine LA = linoleic acid (cis, cis - 9, 12 - oct~-lec~-liPnoic acid) ALA = alpha-linolenic acid (cis, cis, cis - 9, 12, lS -oct~ tri~n- ic acid) GLA = y-linnl~nic acid (cis,cis,cis - 6~9~l2-octzltlec~tri~noic acid) EPA = cis,cis,cis,cis,cis-5,8,11,14,17-eicosd~ t~Pnoi~ acid DHA = cis,cis,cis,cis,cis,cis, - 4,7,10,13,16,19-docos~heY~Pnni( acid The synthetic route for the p~ey~l;tm of the triglycerides LGE, GaLE and aLLG is as follows:-Step (i) A mixture of GLA (95%, 2.0g, 7.2mmol), glycidol (560mg,7.5mmol) and tri n-butylamine (40,u1, 0.16mmol) was heated at 85~C under nillogell for 5h. The reaction was then cooled and purified by flash ch.ulll-dlography (5% meth~nol / methylene chloride) to yield the product monoacylglycerol as a colourless oil. (Yield = lg, 38 %) .
Step (ii) A solution of DCC (340mg, 1.63mmol) and DMAP (200mg, 1.63mmol) in methylene chloride (5 ml) was added to a solution of the monoacylglycerol from (i) (SOOmg, 1.42mmol) and LA (380mg, 1.35mmol) in methylene ~hl~ le (15 ml) at room ~elllpeldture under niL,ogen. After 3h the reaction ~ lule was diluted with hexane (25 ml), filtered, con~entr~t~d and CA 022172~8 1997-10-02 W 096131457 PcTlGB~lcc82x purified by flash chromatography (2% methanol / methylene r-hloricle) to yield an isomeric ~ Lulc of diacylglycerols as a colourless oil. (Yield = SOOmg, 58%.) Step (iii) A solution of DCC (340mg, 1.63mmol) and DMAP (200mg, 1.63mmol) in methylene rhlon(le (Sml) was added to a soll~ti~m of the ~ mono~ylglycerol from (i) (SOOmg, 1.42mmol) and ALA (380mg, 1.35mmol) in methylene chlori~ie (lS ml) at room lclll~cldlulc under nitrogen. After 3h the reaction mixture was diluted with hexane (25 ml), filtered, concen~dted and purified by flash chlu- ~logr~rhy (2% mPth~nol / methylene ~hlon~e) to yield an isomeric llli~lure of diacylglycerols as a colourless oil. (Yield = SOOmg, 58%.)Step (iv) A solution of DCC (llOmg, 0.53mmol) and DMAP (65mg, 0.53mmol) in methylene çhloricle (S ml) was added to a solution of the diacylglycerol (prepared as in (ii)) (250mg, 0.41mmol) and EPA (95%, 145mg, 0.47mmol) in methylene chloride (lOml) and the mixture was stirred at room telll~cldlul~c under nill~,cll. After 2h the reaction mi~lulc was diluted with hexane (SO ml), filtered, co~ce~ t~l and purified by flash chromatography (5% ethyl acetate / hexane) to yield the pure triglyceride LGE as a colourless oil. (Yield =
300mg, 82%) Step (v) A sol~ltion of DCC (llOmg, 0.53mmol) and DMAP (65mg, 0.53mmol) in methylene chloride (S ml) was added to a solution of the diacylglycerol (prepared as in (ii)) (250mg, 0.41mmol) and ALA (130mg, 0.47mmol) in methylene chloride (10 ml) and the Illi~lul~ was stirred at room lt;l,l~cldture under nil-ugen. After 2h the reaction Illi~lulc was diluted with hexane (50 ml), filtered, con~P-ntr~ttod and purified by flash chromatography (5% ethylacetate / hexane) to yield the pure triglyceride aLLG as a colourless oil. (Yield 293mg, 79%).
Step (vi) A solution of DCC (220mg, 1.07mmol) and DMAP (130mg, 1.07mmol) in methylene chloride (10 ml) was added to a solution of the diacylglycerol (~l~alc~d as in (iii)) (SOOmg, 0.82mmol) and EPA (95%, 285mg, 0.94mmol) in methylene chloride (20 ml) and the mixture was stirred at room W 096/31457 PCT/GB~6/00~2 ,c.,,t-lre under nitrogen. After 2h the reaction ~ Luie was diluted with hexane (50 ml), filtered, concentrated and purified by flash chromatography (5% ethyl acetate / hexane) to yield the pure triglyceride GaLE as a colourless oil. (Yield =
640mg, 87%).
The products of the above examples have been subject to hplc triglyceride analysis and gc fatty acid analysis using the following meth~1c.

HPLC method for triglycerides Column: Techsphere 50DS
25cm x 4.6mm i.d., PP 55733 Solvent: acetonitrile (65 %): isopiuyallol (35 %) Flow: 1.5 ml/min Det~tion: 210 nm Gc method for fatty acid methyl esters Column: SupelcowaxTM 10 fused silica capillary column 30m x 0.53mm i.d.
l.OO~m film thicknpc~
Conditions: Initial le-llpt;ldture 165~C (5 min) Level 1 gr~ nt 2~C /min to 210~C
Final temperature 210~C (15 min) Summary of HPLC analyses Trzglyceride Retention time % purity ALA / LA / GLA 9.33 min. 94.9 GLA / ALA / EPA 6.42 min. 85.3 LA / GLA / EPA 8.05 min. 88.9 ~c~lmm~ry of gc analyses ALA / LA / GLA
LA. methyl ester 35.45%
GLA methyl ester 32.4%
ALA methyl ester 32.15 %
GLA / ALA / EPA
GLA methyl ester 32.13 %
ALA methyl ester 33.12%
EPA methyl ester 33.04%
LA / GLA / EPA
LA methyl ester 33.95%
GLA methyl ester 31.71%
EPA methyl ester 33.16%

CA 022l72',8 l997-l0-02 Use Fy~mrl~c The following are examples of modes of use of the triglycPrid~s.
1. Any one or any ~ Lulc; of the triglycerides specified in Table 3 made up in soft or hard gelatin c~psllleS of any size belw~n lOOmg and lg and zl~lminictered to provide a daily dose of between lOOmg and lOg.

2. Any one or any mixture of the specified triglycerides micrornr~rs--l~tff~ in gelatin or agar or any other a~plopiiate m~tPri~l, or inco~o,~L~d into any a~pr~liate m~t~ri~l to form a powder which can be taken orally, added to foods, t~hlett~d, encapslll~t~d, packed in sachets or any other a~,~-iate form.

3. Any one or more of the specified triglycerides made up in a whip, liquid, cream or other ~pro~,iate form for oral ~-lminictr~tinn 4. Any one or more of the specified triglycerides made into a cream, ointm~nt or other topical ~ tion at a conr~-ntr~fion ranging from 0.1 to 30%.

5. Any one or more of the specified triglycerides made up into an emlllcion suitable for parenteral ~lminictration.

6. Any one or more of the specified triglycerides added to any a~plo~liate food m~teri~l such as a spread, drink, candy, cereal, infant food or bakery product.

Claims (9)

1. As groups of isomers or singly, triglycerides containing:-a) one residue of an acid selected from oleic acid and the following groups of acids:-Group i) gamma-linolenic acid (GLA) and dihomo-gamma linolenic acid (DGLA) Group ii) arachidonic acid (AA), adrenic acid, and the 22:5 n-6 acid Group iii)stearidonic acid and the 20:4 n-3 acid Group iv) eicosapentaenoic acid (EPA) the 22:5 n-3 acid, and docosahexaenoic acid (DHA), one residue of an acid selected differently therefrom and one residue of an acid selected from linoleic acid, alpha-linolenic acid or any other fatty acid, other than oleic acid or the twelve n-6 and n-3 series essential fatty acids, containing 14 to 26 carbon atoms whether saturated, mono-unsaturated or polyunsaturated and whether in the case of unsaturated fatty acids having its double bonds in either cis or trans configurations; or b) two residues of the same acid selected from oleic acid and the acids of groups i) to iv) above and one residue of an acid selected from alpha-linolenic acid or any other fatty acid, other than oleic acid or the twelve n-6 and n-3 series essential fatty acids, containing 14 to 26 carbon atoms whether saturated, mono-unsaturated or polyunsaturated and whether in the case of unsaturated fatty acids having its double bonds in either cis or trans configurations; or c) one residue of linoleic acid, one residue of alpha-linolenic acid and one residue of an acid selected from oleic acid and the acids of groups i) to iv) above; or d) one residue of an acid selected from oleic acid and the acids of groups i) to iv) above, one residue of an acid selected from linoleic acid and alpha-linolenic acid and one residue of any other fatty acid, other than oleic acid or the twelve n-6 and n-3 series essential fatty acids, containing 14 to 26 carbon atoms whether saturated, mono-unsaturated or polyunsaturated and whether in the case of unsaturated fatty acids having its double bonds in either cis or trans configurations.

2. Triglycerides according to claim 1, containing acid residues selected as set out in Table 3 herein under any one of the references TG1 to TG50.

3. Triglycerides according to claim 1 or 2, in the form of mixtures with other triglycerides, made by reaction of acid and glycerol starting materials in kinds and proportions corresponding to the selected residues.

4. Triglycerides according to claim 3, wherein the triglycerides according to claim 1 or 2 constitute more than 10 preferably more than 30 very preferably more than 70 and ideally more than 90 molar percent of the mixtures.

5. Triglycerides according to any preceding claim in the form of a pharmaceutical composition generally.

6. Triglycerides according to any of claims 1 to 4 in the form of a nutritional supplement or food composition.

7. Triglycerides according to any of claims 1 to 4 in the form of a topical pharmaceutical, skin care or cosmetic composition.

8. Triglycerides according to claims 5, 6 or 7 wherein the composition is in a form to provide a daily dose of 1mg to 100g preferably 10mg to 10g and very preferably 500mg to 4g of triglyceride according to claim 1 or 2.

9. Triglycerides according to claims 5, 6 or 7 wherein the composition comprises0.001 to 50% preferably 0.05 to 20% and very preferably 0.1 to 5% by weight of triglyceride according to claim 1 or 2.