CA2109611A1 - Phospholipids - Google Patents
PhospholipidsInfo
- Publication number
- CA2109611A1 CA2109611A1 CA002109611A CA2109611A CA2109611A1 CA 2109611 A1 CA2109611 A1 CA 2109611A1 CA 002109611 A CA002109611 A CA 002109611A CA 2109611 A CA2109611 A CA 2109611A CA 2109611 A1 CA2109611 A1 CA 2109611A1
- Authority
- CA
- Canada
- Prior art keywords
- phospholipids
- fatty acids
- omega
- emulsion
- amount
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
- A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
- A23D7/00—Edible oil or fat compositions containing an aqueous phase, e.g. margarines
- A23D7/01—Other fatty acid esters, e.g. phosphatides
- A23D7/011—Compositions other than spreads
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J7/00—Phosphatide compositions for foodstuffs, e.g. lecithin
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/115—Fatty acids or derivatives thereof; Fats or oils
- A23L33/12—Fatty acids or derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
Abstract
The invention relates to an emulsion which comprises phospholipids containing omega-3-fatty acids such as DHA and EPA in a high amount and a vegetable oil and/or marine oil. It also relates to the use of phospholipids containing the omega-3-fatty acids such as DHA and EPA in high amount for the manufacturing of a nutritive emulsion giving low serum triglyceride and cholesterol levels and for the manufacturing of a medicament with anti-inflammatory and immunosuppressive effects. The invention also discloses phospholipids containing omega-3-fatty acids such as DHA and EPA with therapeutic effects such as effects on inflammatory and immunologically active cells, e.g. rheumatoid arthritis and sepsis and an effect on normal brain and retina development and function and cardiovascular diseases. Also disclosed are pharmaceutical and nutritive compositions as well as lipid particles comprising the phospholipids.
Description
WO 92/21335 1 210 9 X 1 ~Cr/5E9~/00333 E~O~O~PIDS
~V31 The pre~ent inYention relates to emulsions containing phospholipids of ma~ine and/or synthetic orig~n with a high amolLnt o~ (at leas~ 30~o (w/w) of its total fatty acid conteIlt) omega-3 (hereinafter written as) w3-fatty acids, with docosahea~aenoic acid, 22:6w3 (hereinai~ter wIitten as DHA) and eicosapentaenoic acid, 20:5w3 (hereinafter written as EPA) in combination, as well as the use of these phospholipids. The phospholipids are used as emulsifiers, nutritive ~ubstrates, as a pharmacologic~lly active agent in an emul~ion, aæ a component in pharmaceutical compositions or a~ a component in lipid particles.
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It is known that lipid e~sion~ ca~ be used irltravenou~ly to const~tute an energy de~se calo~c source and a source of e~sential fat~y acids for those patie~ts who have difficulties in using orally admi~istered nutrition.: : :~
Since ~ beginning of t~e 1960s, fat emulsions which are iIltended for :intr~lvsDou6 nutriellt supply:~ d:~ h exhibit i~significant secondary effe~ta have bes~ available:~Wretli~d, A. Develo~m~nt ~fiat emulsions, dPEN 5:~No 3, 23~3~, 1981).
Tbis developmental :work has investi~ated the effect of e~ulsi~ns whic~
contain a numlber of:diflferent fats, such as soy bean oil7 mai~e oil~
ssf~lswer oil, cottoIIseed oil etc. and di~ereIlt e~ul~ifiers, such as soy bean phospholipids, egg yolk;phospholipids etc.
Emulsion~ for nut~tive or therapeutic use are, for exampIe, described in US patent 4 168 308.
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The nutri~ re emulsions DOW most commonly used contain a vegetable oil such~ as ~oybe~n oil and/or s~lower oil, an emulsifyi~g agent such as an :~ : egg yolk phospholipid aIld water together with gIycerQI. An e:gample of ~ ~ ~: such an emulsion is Intralipid~), manufactured since 1962 and sold by WO g2/21335 2 pcr/sE92/oo333 Kabi Pha~macia AB. Intralipid~ 10 % contains 10 % oil as soy bean oil and 1,2 % egg yolk phospholipids.
Different fatty acids in the lipids have different physiological, biochemical and pharmacological properties and during the last years great interest has been concentrated on the importance of the w3-fatty acids, containing 18-22 carbon atoms.
The w3-&tty acids eicosapentaenoic acid (20:5w3, EPA) and docosahexaenoic acid (22:6w3, DHA) are essential fatty acids in man.
Beside~ their nutritional value, they are also known to possess pharmacological effects. The most known and important are the cardiovascular effects, the benefici~al effects on inflammatory and autoimmune diseases and the necessity of these fatty acids for the normal development of brain and retina functions.
These effects have such an importance that a lot of work has been done to find good nutritional compositions containing a high amount of w3-fatty acids. &e e g~W0 87/02247~(Baxter) and US 4 820 731 (New England Deaconess Hospital) in which marine oils are used which contain a high amount of the ~w3-fatt~r acid~EPA and DHA.
The patient also needs ~omeg~-6 fatty acids (hereafter written as w6-fatty acids) which are found,~for example, in vegetable oils. Nu~ients given to patients should therefore ~also contain ~an appropriate vegetable oil.
Infusion of lipid emuIsion6~containiDg w6-fatty acids results, however, in a rai~ed level~ of eholesterol and triglycendes in some patients, which should b~avoided. Until now-;some patients depending on parenteral -~n rition have not been able to avoid~a certain increase of total chclesterol and~ t~glycendes when ~an ~emulsion containing mai~y w6-fatty a~ds is given. ;w6 Fa~ acids~ also ~increase the level of eicosanoides and leucotnenes, which when overproduced in some patients, eg with overactivè inflammato~y andlimmunological reàctions, may have deleterious e~ects.
Phospholipids contaiI~ing ~EPA or DHA are known as being useful in various fields, such as foods, cosmetics, medicines, agriculture etc. and dif~erent~methods for~their manufacture have been disclosed. See eg. JP
2097393 and JP 1050890.
WO 92/21335 3 Pcr/sEs2/oo333 ~lD!g~l~
The use of phospholipids, contsining EPA or DHA, a~ emulsifier for an EPA-triglyceride-emulsion and a DHA-triglyceride-emulsion, respectively, has been i~vestigated by EIamazaki T et al in Biochem and Biophys Res Comm. Vol 151, No 3, 1386-1394, 1988; in LIPIDS Vol 22 No 12, ~031-1034, 1987 and in Thrombosis Research Vol 44, 1986, (673-682).
Hsmazalci found, for example, that both the EPA and DHA levels in platelets and RBC (red blood cell) membranes increased significantly when either the EPA or the DHA emulsion, respectively, were infused intravenously for a short time. Blood lipids remained unchanged, except for free fatty acids which decreased. Platelet aggregation and leucocyte adhesion were depressed mai ly af~er administration of the EPA
containing emulsion.
The Swedish patent application SE 8705122-3 is related to a method for manufacturing fatty emul~ions with phospholipids from eggs as an emulsifier consisting of at least 10 %~ (w/w) w3-fatty acids, wherein the phospholipids are derived from eggs of animals fed with a diet rich in marine oils.
ThiB method of incorpora~g w3-fatty acids in pho~pholipids i~ however provided with natural limitations. It has not been possible to exceed about 15 % (w/w) of w3-fatty acids in phospholipids derived by thi~ method, with the level of EP~ about 2%~(w/w), which in the conventional lipid emulsions described is to~be ~considered t~ be below the leve! for therapeutic efflcts.
The egg phospb~lipid8 de6cribed in the above mentioned ~wedish patent application does ~ot nece6sarily have the therapeutic effects pres~med.
Since there i~ evidence ~which 6uggests that dietary EPA can provide some cIinical benefit in treatment of inflammatory diseases (Salmon, n-3 News, Vol II (3) 1987), it is;importane to have a high amount of DHA as well as EPA in the phosphol~ipids.
DE 334~269 describes a method of how to synthetically manu~acture lecithiile and lyso-lecithine containing EPA and/or DHA, but does not reveal anytbing about the total amount inco~porated of w3-fatty acids or the therpeutical use of the pro~duct.
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Wo 92/21335 4 PCr/SEg2/00333 ~, ... ..
No one has, however, investigated the biological effects and potency of phospholipids, and compred them with those of triglycerides, contaiNng the w3-fatty acids such as EPA and DHA in combination in a high amount in an emulsion with an w6-fatty acid containing vegetable oil or the therapeutic use of phospholipids containing the ~,v3-fatty acids such as EPA and DHA in combination.in high amount Descr~1ion of t~e invention We have now found that when using the w3-fatty acids DHA and EPA in high amount in combination in phospholipids from marine or synthetic origin, instead of using them as triglycerides, but together with a vegetable oil contaiMng~ w6-fatty acids in a nutritive lipid emulsion, the amount of serum cholesterol and triglycerides are surprisingly lower than with the eame amount~ of w3-fatty acids given as fish oil. By using this origi~ of w3-fatty~ acids ;in~ phospholipids the amount of all the impDrtant w3-fatty ac~ together with all the important and essential w6-fatty;~acids was increased in~biological membranes. Furthermore, the incorporation of w3-fatty acids into biological membranes is une~pectedly increased.
I t is also tot~ une~pected;that~ the incorporation in biological membranes of w3-fatty ;acids~as well as w6-fatty acids is more efficient, and~e ~pDten~;y is higher, by llsing the ~Iv3-fatty acit- rich phospholipid (accordi~g to the inrention) han with the same amount of~w3- and w6-fatty acids given as marine oil in emulsion or as ~egetable oils in emulsion. ~
Most surprisingly and important is the finding that DHA is specifically increased- in membrane phos~ olipids.` This is of utmost importance siIlce DEA, being the most important w3-fatty acid in phospholipids in biologi~al m~mbranes, does~not compete with and decrease the level of arachidonic acid, the st~important w6-fatty acid in phospholipids in biological membranes, as much as EPA does. In cholesterol esters also the level of EPA and alpha~ olenic acid are increased.
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W O 92/21335 5 PC~r/SE92/00333 2iO9611 , ~ .
In all lipid fractions the level of arachidonic acid, which is the most important w6-fatty acid in biological membranes, is maintained constant.
This is in contrary to the reduction of arachidonic acid in biological membranes, which is observed when using therapeutic doses of marine oils, cont~ining a high amount of EPA. After the admini~tration of fish phospholipids (according to the invention3, however, even the levels in triglycerides of the w6-fatty acids are increased.
This indicates that the metabolism of arachidonic acid to eicosanoides is reduced and thus the w6-fatty acids are in good balance with the w3-fatty acids and are spared as important components in biological membranes.
The findings are of utmost interest for nutritive emulsions as the amount of total serum cholesterol and triglycerides should be kept as lo~r as possible and the levels of w6- and w3-fatty acids in biological membranes should be kept in balance.
The findings are also of u tmost interest to obtain normal and well balanced levels of essential fatty acids, w3-fatty acids as well as w6-fatty a~ids, for~nutritive emuls-ons to prematureinewbo~ babies and in long-te~n TPN (total parenteral n:trition). The specific increase in DHA in phospllolipids together with the increase of all important w3- and w6-fatty acids in cholesterol esters ;and tliglycerides fuifill the nutritive requirements of a well-balari~d, increased level of w3-fatty acids as well as w~fatty acids. &ch emulsions can thus be useful nutritionally for example,~ in long ;term l~tal p ren~ral nutrition (TPN) and in p~ernature~/newbonl patients, who need w3-fatty acids as well as w6-fatty acids for nonnal brain~ ~d retinal ~development.
Love et al, Annals ofthe Rheurnatic Diseases, 1990, 49, pp 611-614 has shown that egg phospholipid~ are accumulated in immlmologically active cells. Billiar T K et al ("Fatty!acid intake and Kupffer cell function; Fish oil alters eicosanoid and monokine production to endotoxin stimulation"
Surgery, 104, 343-349 1988)~has shown that w3-fatty acids are incoIporated into and have anti-inflammatory effects on Kup~er cells, when fish oil with w3-fatty acids was given orally. We have now surpIisingly found that w3-fatty acid containing~ma~ne phospholipids accumulate in Kupffer cells and that DHA from the marine phospholipids is incorporated in wo 92/21335 6 PCr/SE92/00333 ,; ' '; ~$;.~
~ 2109611 membrane phospholipids with an unexpectedly high specificity and potency. ~so the other essential w3-fatty acids, EPA and alpha-linolenic acid, which are increa~ed in cholesterol esters, as well as the essential w6-fatty acids are increased in neutral lipids. The increase in membrane lipids of w3-fatty acids as well as of w6-fatty acids show that the potency of the w3-fatty acids iB significantly higher after administration of w3-fatty acid containing maIine phospholipids together with vegetable oil in emulsion (the invention) than after administration of comparable amounts of w3- and w6-fatty acids in fish oil and vegetable oil, respectively, in an emulsion.
The phospholipids according to the invention accumulate in Kupf~er cells and can thus be used to reduce the w6-/w3-fatty acid ratio in stimulated immunologically active cells for the treatment of diseases with increased inflammatory and immunological reactions, e.g. sepsis, rheumatoid arthritis or other autoimmune and inflammatory diseases.
The invention thus relates~to~ an emulsion comprising vegetable oil and/or fish oil which contains phospholipids with the omega-3-fatty acids ~
and EPA in high aInount~in combination which can be used as a nutritive emuIsion to meet the requirement of essential fatty a~:ids, e g in long-term TPN and-for premature/newbo~n ba~bies. This emulsion can also be used for~ therapeutic purposes~; for a better~ w3-lw6-fatty acid balance, with 8ert~m~1ipid lowering and;anti-inflammatory effects, effects on hemo~s,~ and in higher dosages immunofiupprefisive effects.
It al80 relates to the use of phospholipids containing w-3-fatty acids for the manufacturing of a~medicament with anti-infhmmatory and immuno~uppressive effects and the use of phospholipids with the omega-~; 3-fatty acids DHA and EPA in combination for the manufacture of a ; onal emulsion ~ng low serum triglyceride and cholesterol levels and a more balanced w6-lw3-fatb acid ratio and with anti-inflammatory and i= suppressive effects and e~ects on hemostatis .
The iD~rention also relates to phospholipids containing the omega-3-fatty acids DHA and EPA with therapeutic effects on diseases with WO 92/21335 7 pcrlsEs2/oo333 overproduction of eicosanoids in inflammatory and immunologically active cells, on rheumatoid arthritis, inflammatory situations and on the development and function of normal brain and retina.
Another aspect of the invention is to use phospholipids with EPA and DHA in a high amount in combination with drugs with simil~r effects or used for diagnostic purpo~es.
The emulsion could comprise 0,5-40 % (w/v of total emulsion) oil, preferably 5-30 % (w/v), such as soybean oil, coconut oil, cottonseed oil, safflower oil, sunflower seed oil, linseed oil, borage oil, blackcurrent seed oil, canola oil, marine oil or a mixture of these. The amount of the phospholipids according to the invention could be 0,1-30 % (w/v of total emulsion), preferably 0,1-10 % (w/v). The phospholipids containing w3-fatty acids could be of marine or synthetic origin.
Other phospholipids ~ such as e~g yolk or soybean phospholipids and/or synthetic emulsifiers can ~also be i~cluded as complements in the em~sion. The total amount of emulsifier is preferably 0,1-30 % (w/v of total emulsIon).
The emulsion can also ~contain other components which normally are incorporated in emulsions e.g.: monoglycerides of fatty acids, components for adjusting of isotonic properties such as glycerol, anti-oxidants such as ~pha-tocoph_, components for adjusting stability such as amino acids and carbohy rate~ such~;~as fi!UCto8e and glucose etc. It can alfio contain one or more bioactive compounds to be administered.
The preparation of the~ emulsion is carried out in a conventional manner.
Thus the lipids are mixed with the aqueous phase, phospholipids according to the invention an;d optionally other emulsifiers and auxiliary agents in a suitable mixing device . The blend is thereafter homogenized to~ a desired particle~size. The ways to adjust the emulsion to a suitible particle size is well known to a person skilled in the art.
Our findings are of utmost interest for nutritive emulsions to keep the amount of total cholesterol and triglycerides as low as possible for the `:
WO 92/21335 8 pcr/sE92/oo333 patient and the balance of the ratio w6/w3-fatty acids in biological membranes optimal e g for newbo~lpremature infants, in long-term TPN and in situations with stimulated inflammatory and immunological reactions.
The phospholipids according to the invention are also conciëva~le as components in lipid particles ~uch as liposomes or any other mono-, bi- or multilayered vesicle.
The means and methods of how to use phospholipids to prepare such vesicles are ~vell-known to anyone skilled in the art since numerous papera and patents have been published in this technical field ~ an overview of liposome preparation can be found in Drug Dev Ind Pharm 15 (19), 152~54, 1989).
An aspect of the invention is to use ~he phospholipids ~1vith a high content of w3-fatty acids in the preparation of various lipid vesicles, either to deliver one or more bioactive components, or to be an administration form in itself for the bigbly tb~r~ potent phospholipids.
Additional bioactive com~ pone~ can be enclosed in the vesicles or be parts of their rmembra~es or can ~in certain cases be conjugated to the membrane molecules. ~ ~
These systems can be~ taylored~ indlividually for each bioactive molecule and depend on the nèt~charge,~ molecular weight and the number of hydrophilic or hydrophobic~ groups on the molecules.
The~bioactive~ compuhds may be~ such~that potentiates the t herapeutical eff~ ~the admini8tered~v3-fatty acids or any other drug, which is - -appropriate to d~eliver.~
A bioactive compound uséd ~in combination ~nth the vesicles can also be a ligand with affinity to a~ :biological receptor to create a more specific drug targeting 8yBtem. ,:: ~ " ' ! ~ , ::
The veaicles may also be ;used~for diagnostic purposestand the bioact*e compound can in such~ cases ~be a labeled or signal-carrying molecule.
The vesicIes prepared from the phospholipids according to the invention can be administered in conventional manners in pharmaceutical or .: ~
WO 92/2133~ 9 pcr/sEs2/oo333 ~21096il diagnostical preparations. The additional ingredients for adapting the preparations for oral, buccal, parenteral, intraocular, nasal, pulmonary, rectal, or transdermal use are well-known for anyone skilled in the art.
The phospholipids according to the invention can also be administered in any oral, parenteral, intraocular, nasal, pulmonary, rectal or transdermal preparation in combination with conventional carriers and/or diluents. The administration forms can also, when appropriate comprise other adjuvants and enhancers for increasing or controlling membrane penetration such as monoglycerides and compounds with surface active properties.
Following figures are included:
Figur~ 1 which shows the level of serum cholesterol (S-CHOL) after administration of di~erent~emulsions and solution6. See example 4.
Eie~2which shows~the~level~of serumtrig1ycerides (S-TG) after a(hninistra'don of di~erent emulsions and solutions. See example 4.
show 1 he 1evel in liver phospho1ipids (PL;Fig 3), in liver cholesterol~ esters (OE; Fig~4~and in liver~triglycendes ~TG; Fig 5) of the most impo nt w3-fatty acids~DHA (22:6w3), EPA (20:5 w3), alpha-linolenic acid (18:3 w3) and the w6-fatty acids dihomo-gamma-linolenic acl~ ~u:3 w6? ana gamma-l~olemc acl~
(18:3 w6) afcer~administrntion of ~difli~rent~ em~lsions and solutions. See example~4.~Statistidb~sigDifi~tincrease~p<0.05,ANOVA)af~er administration of fish~phospholipid containing emulsion (*, F-PLe~n) compared ~ fi~h oil emulsion ~FOem) and Intralipid (IL) and after FOem (+) compared to the ot;her~groups.
Fi~ures 6 shows the level of linoleic acid (18:2 w6) and Fieurè 7 shows the iev~l ~of~arachidonic acid (20:4 w6) in liver lipids after !
admilust~ation of different~emulsions and solutions. See example 4. For explanation of abbreviations, see Ngures 3-5.
Various ~modîfications and~ equivalents of the emulsion will be apparent to o ne; ~ed in the art without departing from the spi~it or scope of the , ::::
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WO 92/21335 1 0 Pcr/sE92/oo333 ' 2lin~6 i ~
invention. It is therefore to be understood that the invention i~ not to be limited to the specific examples and embodiments disclosed herein.
Fish phospholipids can be manufactured in different ways and in the following only one example of such a manufacturing is given.
E~AMP~
~_~/~ds 1,5 kg fishmeal was extracted two consecutive times with 61 and 31 95 %
ethanol. Aft~r filtration and~pooling, ethanol was evaporated from the extract (vacuum, 40C). 134 g was left (22 % of thifi was insoluble matter).
This residue was dis~o1ved in 1 volume petroleum ether, filtered, precipitated in four volumes 20C acetone, filtered and dissolved in petroleum ether. Thi8 last precipation and dissolution was made twice.
After precipitation again in four volumes of acetone, the ~olution was filtered and freeze-dried~ in a ~Ditrogen atmosphere. The yield was 27 g.
The ~prepared~ fish phospholipid had the following fatty acid content in %:
14:0~ Myristic acid~ 2 16:0 P~almitic acid 16:1 Palmitoleic acid ~ ~ 2,1 18:0 Stearic acid ~ 3,0 18:1 Oleic acid ~ ~ ~ 11,3 ;;182~ Lino1eic acid E:icoscnoic acid~ 1,6;
4 Arachidonic acid~ ; . 71~.
5~ EPA ~ 10,6 6~ ~DHA ~ 32,4 Total amount of fatty acids:; 100 % (w/w) ~ ,:
, :
WO 92/2133~ 1 1 '2 1 0 9 1; 1 ID
Exan~le X. Pre~aration of a fish ~hospholi~d emu,lsion accordin~ to the ye, ntion.
The Ssh phospholipid from example 1 was used for the manufacturing of an emulsion containing:
100 g soybean oil 12,0 g fish phospholipid 22,2 g glycerol 860 g Aq. ad inject.
3,0 ml NaOH lM~
The ingredients were mi~Eed in a "Turrax-mixer" and thereafter homogenized in a "Moillin-Gaulin Homogenizer".
The soybean oil used had the following fatty acid content in %:
16:0 Palmitic acid~ 11 18:0 Steanc acid; 4 18:1 Oleic acid~ 23 18:2 Linoleic~acid ~
:3 Alfa-linolenic~ acid ~ 7 Total alnount of f~tty acids:~ 100 % (w/w) The total umount of w3-fatty~ acids~is 12,2~ gll emulsion and the ratio w6-/w3-fi~ acids is 4,5~
EXamVIe 3. PreDarah~ of an emulsion cont~inin~ fish oil and e~F volk h~s~holi~ids. ~
This~emulsion was~prepared~according to the method described in E~ca~nple ~2, and contains a ~s uular amount of w3-fatty acids and a similar w6/w3-fàtty acid ratio as in~Exampl`e 2.~
~, , WO92/21335 ~ 1 2 pcr/sEs2/oo333 The emulsion contained:
Fish oil 10,o g Soy bean oil 90,0 g Egg yolk phospholipid 12,0 g Glycerol 22,2 g Aq. ad in~ect. 860 g NaOH, 1 M 3,0ml As antioxidant vitamin E ( :alpha-tocopherol) was added to the emulsion The fish oil used had the following fatty acid content in %:
14:0 Myristic acid ~6,3 16:0 Palmitic acid ~: 14,7 16:1 Palmitoleic acid 7,3 18:0 Stea~c acid 2,6 I8:1 Oleic: acid ~ :8, 9 18:1 ~accenic acid~ 3,1: ~ :
18:2: I,inoleic acid 18:3~ Linolen~c - acid ~ 0,7 4 Stearidonic acid~ 2,6 Eicosenoic ~acid~ 1,5 2~:4 -~Arachidonic~ acîd~ 1,4~
5:~ :EPA : ~ 17,8:;
:22:1 ~::Docosaenoic acid~ 2 ,2 :: :~
~:5~ Docosapentaenoic:acid~ 2,9 ~.6~ ~DHA~ 13,5~
T otaI~amount:of fiatty acids: ~100 % (w/w).
WO92/21335 1 3 ~ ~ O~9~ Cr/SEg2/00333 The egg yolk phospholipids used had the following fatty acid content in %:
14:0 Myristic acid 0,2 16:0 Palmitic acid 31~5 16:1 Palmitoleic acid 1,2 18:0 Stearic acid 14,1 18:1 Oleic acid 28,0 18:2 Linoleic acid 12,4 20:1 Eicosenoic acid 0,2 20:4 Arachidonic acid 4,2 22:6 DHA 5,8 The total amount of w3-fatty acids is 10,8 g/l emulsion and the ratio of w6-/w3-fatty acids is 4,8:1.
Examvle 4. Comvarativé~examDle (Fish phos~holioi~- fish oil) he purpose of thi6 exsmple was to investigate the effects of the fish pho8pholipid preparation~in~an emulsion according to~the i vention and to compare it w:th different emulsions such as a fish oil emulsion contair~ the ~same amount of w3-fatty acids, Intralipid~) and also to compare it with fish phospholipids in water solution and physiological sali~e~ solution.
Male ~8plague Dawley rà~, with weight on arrival~ of 170-190 g were used.
The~rat~ were placed indi~ridually in cages ~nd provided with a p~r~eweigh~d sm~ll 1eatherharness.;The i.v. catheterwas inserted 7-8 days latsr under anaesthesia.~After the opèration the animals were placed in the mfilslon room. Another 4 days were allowed for recovery.
r ~urgery the rats were provided with grounded laboratory stock diet R3 (Ewos~ AB; Sodert~lje, Sweden) and tap water ad libitum.
During the entire test period the rats were~ provided wi~h grounded laboral~ry stock: diet R3 and tap water ad libitum.
The~ rat~ were randomized using a :andom unit into expe2imental groups A-E.~
Six rats were used in each experimental group W092/2l335 1 4 PCr/SE92/00333;
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~ 10~6i1 All groups rece*ed 50 ml/kg body weight (b.w.)/day.
Infu6ions were administrated intravenously via a permanent central vein catheter during 20 h/day, normally from 1 p.m. to 9 a.m., v~a IMED
volumetric pumps.
Food consumption was recorded on a 24 h basis. The general appearance of the rats was recorded. The infusions were given during 9 consecutive days. On day 10 the infusions were stopped at 7.00 a.m. and the oral food withdrawn.
The groups A-E were given the following infusions:
A: 10 % Fat emulsion according to Example 2 (F-PLem, the Invention) B: ~ -~ according to Example 3 (FOem) C: 10 % Intralipid ~, containg 10 % ~w/v) oil a~ soy bean oil and 1,2 % egg yolk phospholipid D: 1.2% Phospholipid~`~olution containing: -Fifih phos~olipits~ ~ ~ 12,0g Glycerol; ~ 22,2g ~ --Aq. ad inject.~ 967 g NaOH l M~ 3 ml E: 0.99'o~NaCl-601uOon ~
The ~amount of w3- and w~fatty ~acids were as follows:
to~t w3 - FA~ tot w6 - FA w6 / w3 A =~ 2 (Invention) ~ 55,2 4,5 B = Ex 3 (Eish oil) IO,8 51,6 4,8 C - Intrslipid' ~ 7, 7 56,8 7,4 D = Fish phospholipid~ 4,8~ ~ ~ 0,3 0,1 solution Approx. 2 hours af~;erl ~topping the infusions, the rats were anaesthetized wi~h ~Mebumal~ [60 mg/kg). The blood samples were collected for analysis ;of se~ lipids.
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Wo92/21335 1 5 PCr/SE92/00333 . .
Serum lipids: From 1 ml blood, serum was taken for analysis of serum l;riglycerides and serum cholesterol and frozen at -70 C until analysis.
Serum cholesterol was measured enzymatically. Serum triglycerides were measured enzymatically after eliminating free glycerol and enzymatic hydrolysis.
Hifitopatholoev: liver, kidneys, heart, lungs, ~pleen and thymus were excised, weighed and prepared for histopathological examination by embedding in paraffin and sectioning at 4-5 micrometer, then staining with haemato~ylin-eosin. Frozen sections from all the tissues stained with Oil Red O for fat were also examined.
Fattv aci~Dfile in liver lipids. The remaining liver tissue from rats in Groups A-C was used for measurement of fatty acid profile in neutral lipid6 and phospholipids(PL). The neutral lipid fractions examined were cholesterol esters (CE)~ and triglycerides (TG).The lipid material was estracted ~and the fatty~acids d :atized and analyzed (GLC) using conventional methods. ~ ~
The resulting data on serum 1ipids~ are shown in Figure 1 (level of serum chole6terol,~ SCHOL) and;~Figure 2 (level of serum t:riglycerides, S-TG).
The 1evels of tlie most unportant ~fatty acids in liver lipidE are shown in Figures 3-7.~ Tbe results are espressed as mean values + SEM for the different emulsions ~anLd 601utions.
Figure 1 clearly shows that the serum cholesterol level is very low when the~ emuls on according to thé invention is used ~ AcB, C; p<O 05, ANOVA).The most surprising effect, however, is the result when companng the serum ~lyceride levels. See Figure 2. That level is surprisingly low (0,30 r ol/l) and only half of the value when Intralipid~, fish phospholipids in a solution or NaCl solution (0,60 ~,, mmol/l) were used (~ A<B, C, D, E; p<O.05, ANOVA).
WO92/21335 1 6 PCr/SE92/00333 ~1096i1 It is absolutely unexpected and unknown, that the small amount of w3-fatty acids which can be derived from phospholipids in a lipid emulsion, can exert physiological effects. The same amount of w3-fatty acids in triglycerides (Group B) does not exert the same biological effects or effects on fatty acid incorporation. Since the phospholipid/triglyceride ratio is about 1:10 in a 10% lipid emul6ion and 1:20 in a 20% lipid emulsion, and the threshold level for biological effects for fish oil, containing about 30 %
w3-fatty acids, is at least 10 % of the oil phase, the amount of w3-fatty acids in the phospholipid part of a lipid emulsion should be at least 30 % (w/w of total fatty acids) to be expected to exert biological effects.
Thus giving an emulsion, according to the invention allows the patient to receive all essential fatty acids such as w6- and w3-fatty acids and still remain at a very low level of serum cholesterol and serum triglycerides.
The hi6topathological data ~showed~ that the emulsions with fish oil or fish phospholipids were accumulated mainly in the Kupffer cells in the liver, whereas Intralip;dg)~ was accumul~ated~ as we}l in hepatocytes. ~ish phospholipids in water solution were however accumulated only in Kupffer cells in the liver and in ;similar cells in the spleen. These cells are immunologically active. This spec fic accumulation to immunologically active Gells fac l;tates an effect of w3-fatty acids on inflammatory and immunological reactions. ~Th~s i8 of im~ortance when using fish pho~pholip~ds for i~mmatu~y or immunological diseases such as rheumatoid a~s a~d ~sepsis ;(Love et al., 1990 and Billiar et al.,l988) or to reduce the incidence ~of arterosclerosis.
The fatty acid pattern ~in liver phospholipids and liver cholesterol esters show that EPA and DHA are incorporated better after treatment with the emulsion according to the invention when compared to the fish oil emulsion containing the~ same amount of w3-fatty acids.
The changes induced by fish phospholipid containing emulsion (F-PLem), fish oil containg emulsion (FOem) or Intralipid ~IL) in fatty acid pattern in liver phospholipids (PL), cholesterol esters ~CE) and triglycerides (TG) are W 0 92~21335 1 7 PC~r/SE92/00333 - ~109~11 shown in Figures 3-7. In the phospholipid fraction (Figs 3 and 6), the only changes seen were increases in DHA, induced by F-PLem (invention, *) p~0.05, ANOVA) and in EPA, induced by FOem (~ p~0.05, ANOVA).
The specific increase in DHA in phospholipids, the main pool for DHA, is of importance for brain and retina deve~opment.
The uptake of and the level in biological membranes of polyunsaturated fatty acids, especially DHA in brain, is well correlated with that in liver (Anderson and Connor,Lipids 1988, 23(4), 286-290) and in heart (Swanson et al.,British Journal of Nutrition, 1988, 59, 535-545). Thus a similar enhancement of uptake in brain and heart is e~pected following administration of the invented emulsion compared to the fish oil emulsion. Therefore the invented emulsion can be used also for normal brain and retina development and ~or cardiovascular di~eases.
In the liver cholssterol ester ~fraction (E igs 4 and 6) F-PLem increased DHA, EPA and li~oleic acit (18:2w6), compared to the FOem- and the I~
groups, and a-linole:nic~acid (~18:3w3), compared to the IL group. Thus, w3-fatty acids adlml~ist:~d~in the phospholipid form, are more effectively inc~rporated in membrane; lipids~ ~phospholipids and cholesterol ester~) than w3-fatty acids iD the trigIyceride form (fish oil) are. The m~n functions ofthe esse~tial~fat~y~acids~and thus also the w3-fatty acids are exerted~ in the membrane lipids.; ~ ~
No decrea~e in any of ~the~w~f~tty acids could be seen in the F-PLem - -group. On t~e contrary, the w6-fatty acids ~linoleic acid (18:2 w6, Fig 6), g_-linolenic acid (l8:3w~6)~and~dihomo-gamma-linolenic acid (20:3w6), as~ well as alpha-linolenic acid (18:3w3) (Fig 5) were increased in the t~glyceride fraction, compared to the FOem- and I~groups. The level of arachidonic acid (20:4w6) remained unchanged in all lipid fractions (Fig7i.This finding i6 of significance be~use of the importance of arachidonic acid in ~biological ~membranes.
Since the w3-fatty acid containing phospholipids are taken up more in Kupffer cells than in hepatocytes, as shown in the invention, the relative increase in incorporated w3-fatty acids is expected to be even higher in W092/21335 1 8 Pcr/sEg2/oo333 ' ~,'l'd'g''~'I'I . .
immunologically active cells (Kupffer cells and other macrophages) than in the whole liver. This is of importance for the anti-inflammatory and immunosuppress*e effect~ exerted by the w3-fatty acids.
The biological effects obtained by the w3-fatty acids in phospholipids can be used for therapeutic purposes as such or in combination with drugs with similar effects and included in the phospholipid vesicles (liposomes) Co~usion~
We have shown that marine phospholipids in an emulsion according to the invention result in surprisingly lower serum triglyceride and serum cholesterol levels when compared to fish oil emulsion, containing a similar amount of w3-fatty acids and a similar w6/w3-fatty acids ratio.
Marine phospholipids induce more effective incorporation of w3-fatty acids in biological membranes than fish oil, containing a comparable amount of w3-fatty acids, in ~n em~sion.
These results show that a ve~y favorable fatty acid pattem in membrane ;~; lipids is ob ained, with an~ increase in all important w3-fatty acids as well as in the w6-fatty acids linoleic, gamma-linolen~c and dihomo-gamma-linolenic acids. The invented w3-fa~tty acid containing phospholipid is thérefore important in all;~situations with increased need of all essential fatty acids, since it makes it possible~ to increase the w3-fatty acids (from the invented~phosph~olipid)~as~well as the w6-fatty acids fin vegetable oils) in a vvell-balanced pattern.~
Tliese~ results have implicatioDs for the use of w3-fatty acid containing phospholipids in vegetable oil emulsions for a more effective utilization and incorporation of;w3- and~w6-fatt~ acids. The use of w3-fatty acid containing phospholipids ;also allows a more specific incorporation of w3-fatty acids in immunologically active cells.
The invention may have implications specifically for situations with increased~level of serum lipids, increased inflammatory response and increased imm: ological activity a~d also for the normal development of the brain and retina.
WO 92~21335 1 9 P~/SE92/00333 9~1 The biological ef~ect~ obtained by the w3-fatty acids in phospholipids can be used for therapeutic purposes as such or in combination with drugs with similar ef~ects and included in the phospholipid vesicles (li;~osomes).
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~V31 The pre~ent inYention relates to emulsions containing phospholipids of ma~ine and/or synthetic orig~n with a high amolLnt o~ (at leas~ 30~o (w/w) of its total fatty acid conteIlt) omega-3 (hereinafter written as) w3-fatty acids, with docosahea~aenoic acid, 22:6w3 (hereinai~ter wIitten as DHA) and eicosapentaenoic acid, 20:5w3 (hereinafter written as EPA) in combination, as well as the use of these phospholipids. The phospholipids are used as emulsifiers, nutritive ~ubstrates, as a pharmacologic~lly active agent in an emul~ion, aæ a component in pharmaceutical compositions or a~ a component in lipid particles.
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It is known that lipid e~sion~ ca~ be used irltravenou~ly to const~tute an energy de~se calo~c source and a source of e~sential fat~y acids for those patie~ts who have difficulties in using orally admi~istered nutrition.: : :~
Since ~ beginning of t~e 1960s, fat emulsions which are iIltended for :intr~lvsDou6 nutriellt supply:~ d:~ h exhibit i~significant secondary effe~ta have bes~ available:~Wretli~d, A. Develo~m~nt ~fiat emulsions, dPEN 5:~No 3, 23~3~, 1981).
Tbis developmental :work has investi~ated the effect of e~ulsi~ns whic~
contain a numlber of:diflferent fats, such as soy bean oil7 mai~e oil~
ssf~lswer oil, cottoIIseed oil etc. and di~ereIlt e~ul~ifiers, such as soy bean phospholipids, egg yolk;phospholipids etc.
Emulsion~ for nut~tive or therapeutic use are, for exampIe, described in US patent 4 168 308.
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The nutri~ re emulsions DOW most commonly used contain a vegetable oil such~ as ~oybe~n oil and/or s~lower oil, an emulsifyi~g agent such as an :~ : egg yolk phospholipid aIld water together with gIycerQI. An e:gample of ~ ~ ~: such an emulsion is Intralipid~), manufactured since 1962 and sold by WO g2/21335 2 pcr/sE92/oo333 Kabi Pha~macia AB. Intralipid~ 10 % contains 10 % oil as soy bean oil and 1,2 % egg yolk phospholipids.
Different fatty acids in the lipids have different physiological, biochemical and pharmacological properties and during the last years great interest has been concentrated on the importance of the w3-fatty acids, containing 18-22 carbon atoms.
The w3-&tty acids eicosapentaenoic acid (20:5w3, EPA) and docosahexaenoic acid (22:6w3, DHA) are essential fatty acids in man.
Beside~ their nutritional value, they are also known to possess pharmacological effects. The most known and important are the cardiovascular effects, the benefici~al effects on inflammatory and autoimmune diseases and the necessity of these fatty acids for the normal development of brain and retina functions.
These effects have such an importance that a lot of work has been done to find good nutritional compositions containing a high amount of w3-fatty acids. &e e g~W0 87/02247~(Baxter) and US 4 820 731 (New England Deaconess Hospital) in which marine oils are used which contain a high amount of the ~w3-fatt~r acid~EPA and DHA.
The patient also needs ~omeg~-6 fatty acids (hereafter written as w6-fatty acids) which are found,~for example, in vegetable oils. Nu~ients given to patients should therefore ~also contain ~an appropriate vegetable oil.
Infusion of lipid emuIsion6~containiDg w6-fatty acids results, however, in a rai~ed level~ of eholesterol and triglycendes in some patients, which should b~avoided. Until now-;some patients depending on parenteral -~n rition have not been able to avoid~a certain increase of total chclesterol and~ t~glycendes when ~an ~emulsion containing mai~y w6-fatty a~ds is given. ;w6 Fa~ acids~ also ~increase the level of eicosanoides and leucotnenes, which when overproduced in some patients, eg with overactivè inflammato~y andlimmunological reàctions, may have deleterious e~ects.
Phospholipids contaiI~ing ~EPA or DHA are known as being useful in various fields, such as foods, cosmetics, medicines, agriculture etc. and dif~erent~methods for~their manufacture have been disclosed. See eg. JP
2097393 and JP 1050890.
WO 92/21335 3 Pcr/sEs2/oo333 ~lD!g~l~
The use of phospholipids, contsining EPA or DHA, a~ emulsifier for an EPA-triglyceride-emulsion and a DHA-triglyceride-emulsion, respectively, has been i~vestigated by EIamazaki T et al in Biochem and Biophys Res Comm. Vol 151, No 3, 1386-1394, 1988; in LIPIDS Vol 22 No 12, ~031-1034, 1987 and in Thrombosis Research Vol 44, 1986, (673-682).
Hsmazalci found, for example, that both the EPA and DHA levels in platelets and RBC (red blood cell) membranes increased significantly when either the EPA or the DHA emulsion, respectively, were infused intravenously for a short time. Blood lipids remained unchanged, except for free fatty acids which decreased. Platelet aggregation and leucocyte adhesion were depressed mai ly af~er administration of the EPA
containing emulsion.
The Swedish patent application SE 8705122-3 is related to a method for manufacturing fatty emul~ions with phospholipids from eggs as an emulsifier consisting of at least 10 %~ (w/w) w3-fatty acids, wherein the phospholipids are derived from eggs of animals fed with a diet rich in marine oils.
ThiB method of incorpora~g w3-fatty acids in pho~pholipids i~ however provided with natural limitations. It has not been possible to exceed about 15 % (w/w) of w3-fatty acids in phospholipids derived by thi~ method, with the level of EP~ about 2%~(w/w), which in the conventional lipid emulsions described is to~be ~considered t~ be below the leve! for therapeutic efflcts.
The egg phospb~lipid8 de6cribed in the above mentioned ~wedish patent application does ~ot nece6sarily have the therapeutic effects pres~med.
Since there i~ evidence ~which 6uggests that dietary EPA can provide some cIinical benefit in treatment of inflammatory diseases (Salmon, n-3 News, Vol II (3) 1987), it is;importane to have a high amount of DHA as well as EPA in the phosphol~ipids.
DE 334~269 describes a method of how to synthetically manu~acture lecithiile and lyso-lecithine containing EPA and/or DHA, but does not reveal anytbing about the total amount inco~porated of w3-fatty acids or the therpeutical use of the pro~duct.
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Wo 92/21335 4 PCr/SEg2/00333 ~, ... ..
No one has, however, investigated the biological effects and potency of phospholipids, and compred them with those of triglycerides, contaiNng the w3-fatty acids such as EPA and DHA in combination in a high amount in an emulsion with an w6-fatty acid containing vegetable oil or the therapeutic use of phospholipids containing the ~,v3-fatty acids such as EPA and DHA in combination.in high amount Descr~1ion of t~e invention We have now found that when using the w3-fatty acids DHA and EPA in high amount in combination in phospholipids from marine or synthetic origin, instead of using them as triglycerides, but together with a vegetable oil contaiMng~ w6-fatty acids in a nutritive lipid emulsion, the amount of serum cholesterol and triglycerides are surprisingly lower than with the eame amount~ of w3-fatty acids given as fish oil. By using this origi~ of w3-fatty~ acids ;in~ phospholipids the amount of all the impDrtant w3-fatty ac~ together with all the important and essential w6-fatty;~acids was increased in~biological membranes. Furthermore, the incorporation of w3-fatty acids into biological membranes is une~pectedly increased.
I t is also tot~ une~pected;that~ the incorporation in biological membranes of w3-fatty ;acids~as well as w6-fatty acids is more efficient, and~e ~pDten~;y is higher, by llsing the ~Iv3-fatty acit- rich phospholipid (accordi~g to the inrention) han with the same amount of~w3- and w6-fatty acids given as marine oil in emulsion or as ~egetable oils in emulsion. ~
Most surprisingly and important is the finding that DHA is specifically increased- in membrane phos~ olipids.` This is of utmost importance siIlce DEA, being the most important w3-fatty acid in phospholipids in biologi~al m~mbranes, does~not compete with and decrease the level of arachidonic acid, the st~important w6-fatty acid in phospholipids in biological membranes, as much as EPA does. In cholesterol esters also the level of EPA and alpha~ olenic acid are increased.
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W O 92/21335 5 PC~r/SE92/00333 2iO9611 , ~ .
In all lipid fractions the level of arachidonic acid, which is the most important w6-fatty acid in biological membranes, is maintained constant.
This is in contrary to the reduction of arachidonic acid in biological membranes, which is observed when using therapeutic doses of marine oils, cont~ining a high amount of EPA. After the admini~tration of fish phospholipids (according to the invention3, however, even the levels in triglycerides of the w6-fatty acids are increased.
This indicates that the metabolism of arachidonic acid to eicosanoides is reduced and thus the w6-fatty acids are in good balance with the w3-fatty acids and are spared as important components in biological membranes.
The findings are of utmost interest for nutritive emulsions as the amount of total serum cholesterol and triglycerides should be kept as lo~r as possible and the levels of w6- and w3-fatty acids in biological membranes should be kept in balance.
The findings are also of u tmost interest to obtain normal and well balanced levels of essential fatty acids, w3-fatty acids as well as w6-fatty a~ids, for~nutritive emuls-ons to prematureinewbo~ babies and in long-te~n TPN (total parenteral n:trition). The specific increase in DHA in phospllolipids together with the increase of all important w3- and w6-fatty acids in cholesterol esters ;and tliglycerides fuifill the nutritive requirements of a well-balari~d, increased level of w3-fatty acids as well as w~fatty acids. &ch emulsions can thus be useful nutritionally for example,~ in long ;term l~tal p ren~ral nutrition (TPN) and in p~ernature~/newbonl patients, who need w3-fatty acids as well as w6-fatty acids for nonnal brain~ ~d retinal ~development.
Love et al, Annals ofthe Rheurnatic Diseases, 1990, 49, pp 611-614 has shown that egg phospholipid~ are accumulated in immlmologically active cells. Billiar T K et al ("Fatty!acid intake and Kupffer cell function; Fish oil alters eicosanoid and monokine production to endotoxin stimulation"
Surgery, 104, 343-349 1988)~has shown that w3-fatty acids are incoIporated into and have anti-inflammatory effects on Kup~er cells, when fish oil with w3-fatty acids was given orally. We have now surpIisingly found that w3-fatty acid containing~ma~ne phospholipids accumulate in Kupffer cells and that DHA from the marine phospholipids is incorporated in wo 92/21335 6 PCr/SE92/00333 ,; ' '; ~$;.~
~ 2109611 membrane phospholipids with an unexpectedly high specificity and potency. ~so the other essential w3-fatty acids, EPA and alpha-linolenic acid, which are increa~ed in cholesterol esters, as well as the essential w6-fatty acids are increased in neutral lipids. The increase in membrane lipids of w3-fatty acids as well as of w6-fatty acids show that the potency of the w3-fatty acids iB significantly higher after administration of w3-fatty acid containing maIine phospholipids together with vegetable oil in emulsion (the invention) than after administration of comparable amounts of w3- and w6-fatty acids in fish oil and vegetable oil, respectively, in an emulsion.
The phospholipids according to the invention accumulate in Kupf~er cells and can thus be used to reduce the w6-/w3-fatty acid ratio in stimulated immunologically active cells for the treatment of diseases with increased inflammatory and immunological reactions, e.g. sepsis, rheumatoid arthritis or other autoimmune and inflammatory diseases.
The invention thus relates~to~ an emulsion comprising vegetable oil and/or fish oil which contains phospholipids with the omega-3-fatty acids ~
and EPA in high aInount~in combination which can be used as a nutritive emuIsion to meet the requirement of essential fatty a~:ids, e g in long-term TPN and-for premature/newbo~n ba~bies. This emulsion can also be used for~ therapeutic purposes~; for a better~ w3-lw6-fatty acid balance, with 8ert~m~1ipid lowering and;anti-inflammatory effects, effects on hemo~s,~ and in higher dosages immunofiupprefisive effects.
It al80 relates to the use of phospholipids containing w-3-fatty acids for the manufacturing of a~medicament with anti-infhmmatory and immuno~uppressive effects and the use of phospholipids with the omega-~; 3-fatty acids DHA and EPA in combination for the manufacture of a ; onal emulsion ~ng low serum triglyceride and cholesterol levels and a more balanced w6-lw3-fatb acid ratio and with anti-inflammatory and i= suppressive effects and e~ects on hemostatis .
The iD~rention also relates to phospholipids containing the omega-3-fatty acids DHA and EPA with therapeutic effects on diseases with WO 92/21335 7 pcrlsEs2/oo333 overproduction of eicosanoids in inflammatory and immunologically active cells, on rheumatoid arthritis, inflammatory situations and on the development and function of normal brain and retina.
Another aspect of the invention is to use phospholipids with EPA and DHA in a high amount in combination with drugs with simil~r effects or used for diagnostic purpo~es.
The emulsion could comprise 0,5-40 % (w/v of total emulsion) oil, preferably 5-30 % (w/v), such as soybean oil, coconut oil, cottonseed oil, safflower oil, sunflower seed oil, linseed oil, borage oil, blackcurrent seed oil, canola oil, marine oil or a mixture of these. The amount of the phospholipids according to the invention could be 0,1-30 % (w/v of total emulsion), preferably 0,1-10 % (w/v). The phospholipids containing w3-fatty acids could be of marine or synthetic origin.
Other phospholipids ~ such as e~g yolk or soybean phospholipids and/or synthetic emulsifiers can ~also be i~cluded as complements in the em~sion. The total amount of emulsifier is preferably 0,1-30 % (w/v of total emulsIon).
The emulsion can also ~contain other components which normally are incorporated in emulsions e.g.: monoglycerides of fatty acids, components for adjusting of isotonic properties such as glycerol, anti-oxidants such as ~pha-tocoph_, components for adjusting stability such as amino acids and carbohy rate~ such~;~as fi!UCto8e and glucose etc. It can alfio contain one or more bioactive compounds to be administered.
The preparation of the~ emulsion is carried out in a conventional manner.
Thus the lipids are mixed with the aqueous phase, phospholipids according to the invention an;d optionally other emulsifiers and auxiliary agents in a suitable mixing device . The blend is thereafter homogenized to~ a desired particle~size. The ways to adjust the emulsion to a suitible particle size is well known to a person skilled in the art.
Our findings are of utmost interest for nutritive emulsions to keep the amount of total cholesterol and triglycerides as low as possible for the `:
WO 92/21335 8 pcr/sE92/oo333 patient and the balance of the ratio w6/w3-fatty acids in biological membranes optimal e g for newbo~lpremature infants, in long-term TPN and in situations with stimulated inflammatory and immunological reactions.
The phospholipids according to the invention are also conciëva~le as components in lipid particles ~uch as liposomes or any other mono-, bi- or multilayered vesicle.
The means and methods of how to use phospholipids to prepare such vesicles are ~vell-known to anyone skilled in the art since numerous papera and patents have been published in this technical field ~ an overview of liposome preparation can be found in Drug Dev Ind Pharm 15 (19), 152~54, 1989).
An aspect of the invention is to use ~he phospholipids ~1vith a high content of w3-fatty acids in the preparation of various lipid vesicles, either to deliver one or more bioactive components, or to be an administration form in itself for the bigbly tb~r~ potent phospholipids.
Additional bioactive com~ pone~ can be enclosed in the vesicles or be parts of their rmembra~es or can ~in certain cases be conjugated to the membrane molecules. ~ ~
These systems can be~ taylored~ indlividually for each bioactive molecule and depend on the nèt~charge,~ molecular weight and the number of hydrophilic or hydrophobic~ groups on the molecules.
The~bioactive~ compuhds may be~ such~that potentiates the t herapeutical eff~ ~the admini8tered~v3-fatty acids or any other drug, which is - -appropriate to d~eliver.~
A bioactive compound uséd ~in combination ~nth the vesicles can also be a ligand with affinity to a~ :biological receptor to create a more specific drug targeting 8yBtem. ,:: ~ " ' ! ~ , ::
The veaicles may also be ;used~for diagnostic purposestand the bioact*e compound can in such~ cases ~be a labeled or signal-carrying molecule.
The vesicIes prepared from the phospholipids according to the invention can be administered in conventional manners in pharmaceutical or .: ~
WO 92/2133~ 9 pcr/sEs2/oo333 ~21096il diagnostical preparations. The additional ingredients for adapting the preparations for oral, buccal, parenteral, intraocular, nasal, pulmonary, rectal, or transdermal use are well-known for anyone skilled in the art.
The phospholipids according to the invention can also be administered in any oral, parenteral, intraocular, nasal, pulmonary, rectal or transdermal preparation in combination with conventional carriers and/or diluents. The administration forms can also, when appropriate comprise other adjuvants and enhancers for increasing or controlling membrane penetration such as monoglycerides and compounds with surface active properties.
Following figures are included:
Figur~ 1 which shows the level of serum cholesterol (S-CHOL) after administration of di~erent~emulsions and solution6. See example 4.
Eie~2which shows~the~level~of serumtrig1ycerides (S-TG) after a(hninistra'don of di~erent emulsions and solutions. See example 4.
show 1 he 1evel in liver phospho1ipids (PL;Fig 3), in liver cholesterol~ esters (OE; Fig~4~and in liver~triglycendes ~TG; Fig 5) of the most impo nt w3-fatty acids~DHA (22:6w3), EPA (20:5 w3), alpha-linolenic acid (18:3 w3) and the w6-fatty acids dihomo-gamma-linolenic acl~ ~u:3 w6? ana gamma-l~olemc acl~
(18:3 w6) afcer~administrntion of ~difli~rent~ em~lsions and solutions. See example~4.~Statistidb~sigDifi~tincrease~p<0.05,ANOVA)af~er administration of fish~phospholipid containing emulsion (*, F-PLe~n) compared ~ fi~h oil emulsion ~FOem) and Intralipid (IL) and after FOem (+) compared to the ot;her~groups.
Fi~ures 6 shows the level of linoleic acid (18:2 w6) and Fieurè 7 shows the iev~l ~of~arachidonic acid (20:4 w6) in liver lipids after !
admilust~ation of different~emulsions and solutions. See example 4. For explanation of abbreviations, see Ngures 3-5.
Various ~modîfications and~ equivalents of the emulsion will be apparent to o ne; ~ed in the art without departing from the spi~it or scope of the , ::::
` ~
WO 92/21335 1 0 Pcr/sE92/oo333 ' 2lin~6 i ~
invention. It is therefore to be understood that the invention i~ not to be limited to the specific examples and embodiments disclosed herein.
Fish phospholipids can be manufactured in different ways and in the following only one example of such a manufacturing is given.
E~AMP~
~_~/~ds 1,5 kg fishmeal was extracted two consecutive times with 61 and 31 95 %
ethanol. Aft~r filtration and~pooling, ethanol was evaporated from the extract (vacuum, 40C). 134 g was left (22 % of thifi was insoluble matter).
This residue was dis~o1ved in 1 volume petroleum ether, filtered, precipitated in four volumes 20C acetone, filtered and dissolved in petroleum ether. Thi8 last precipation and dissolution was made twice.
After precipitation again in four volumes of acetone, the ~olution was filtered and freeze-dried~ in a ~Ditrogen atmosphere. The yield was 27 g.
The ~prepared~ fish phospholipid had the following fatty acid content in %:
14:0~ Myristic acid~ 2 16:0 P~almitic acid 16:1 Palmitoleic acid ~ ~ 2,1 18:0 Stearic acid ~ 3,0 18:1 Oleic acid ~ ~ ~ 11,3 ;;182~ Lino1eic acid E:icoscnoic acid~ 1,6;
4 Arachidonic acid~ ; . 71~.
5~ EPA ~ 10,6 6~ ~DHA ~ 32,4 Total amount of fatty acids:; 100 % (w/w) ~ ,:
, :
WO 92/2133~ 1 1 '2 1 0 9 1; 1 ID
Exan~le X. Pre~aration of a fish ~hospholi~d emu,lsion accordin~ to the ye, ntion.
The Ssh phospholipid from example 1 was used for the manufacturing of an emulsion containing:
100 g soybean oil 12,0 g fish phospholipid 22,2 g glycerol 860 g Aq. ad inject.
3,0 ml NaOH lM~
The ingredients were mi~Eed in a "Turrax-mixer" and thereafter homogenized in a "Moillin-Gaulin Homogenizer".
The soybean oil used had the following fatty acid content in %:
16:0 Palmitic acid~ 11 18:0 Steanc acid; 4 18:1 Oleic acid~ 23 18:2 Linoleic~acid ~
:3 Alfa-linolenic~ acid ~ 7 Total alnount of f~tty acids:~ 100 % (w/w) The total umount of w3-fatty~ acids~is 12,2~ gll emulsion and the ratio w6-/w3-fi~ acids is 4,5~
EXamVIe 3. PreDarah~ of an emulsion cont~inin~ fish oil and e~F volk h~s~holi~ids. ~
This~emulsion was~prepared~according to the method described in E~ca~nple ~2, and contains a ~s uular amount of w3-fatty acids and a similar w6/w3-fàtty acid ratio as in~Exampl`e 2.~
~, , WO92/21335 ~ 1 2 pcr/sEs2/oo333 The emulsion contained:
Fish oil 10,o g Soy bean oil 90,0 g Egg yolk phospholipid 12,0 g Glycerol 22,2 g Aq. ad in~ect. 860 g NaOH, 1 M 3,0ml As antioxidant vitamin E ( :alpha-tocopherol) was added to the emulsion The fish oil used had the following fatty acid content in %:
14:0 Myristic acid ~6,3 16:0 Palmitic acid ~: 14,7 16:1 Palmitoleic acid 7,3 18:0 Stea~c acid 2,6 I8:1 Oleic: acid ~ :8, 9 18:1 ~accenic acid~ 3,1: ~ :
18:2: I,inoleic acid 18:3~ Linolen~c - acid ~ 0,7 4 Stearidonic acid~ 2,6 Eicosenoic ~acid~ 1,5 2~:4 -~Arachidonic~ acîd~ 1,4~
5:~ :EPA : ~ 17,8:;
:22:1 ~::Docosaenoic acid~ 2 ,2 :: :~
~:5~ Docosapentaenoic:acid~ 2,9 ~.6~ ~DHA~ 13,5~
T otaI~amount:of fiatty acids: ~100 % (w/w).
WO92/21335 1 3 ~ ~ O~9~ Cr/SEg2/00333 The egg yolk phospholipids used had the following fatty acid content in %:
14:0 Myristic acid 0,2 16:0 Palmitic acid 31~5 16:1 Palmitoleic acid 1,2 18:0 Stearic acid 14,1 18:1 Oleic acid 28,0 18:2 Linoleic acid 12,4 20:1 Eicosenoic acid 0,2 20:4 Arachidonic acid 4,2 22:6 DHA 5,8 The total amount of w3-fatty acids is 10,8 g/l emulsion and the ratio of w6-/w3-fatty acids is 4,8:1.
Examvle 4. Comvarativé~examDle (Fish phos~holioi~- fish oil) he purpose of thi6 exsmple was to investigate the effects of the fish pho8pholipid preparation~in~an emulsion according to~the i vention and to compare it w:th different emulsions such as a fish oil emulsion contair~ the ~same amount of w3-fatty acids, Intralipid~) and also to compare it with fish phospholipids in water solution and physiological sali~e~ solution.
Male ~8plague Dawley rà~, with weight on arrival~ of 170-190 g were used.
The~rat~ were placed indi~ridually in cages ~nd provided with a p~r~eweigh~d sm~ll 1eatherharness.;The i.v. catheterwas inserted 7-8 days latsr under anaesthesia.~After the opèration the animals were placed in the mfilslon room. Another 4 days were allowed for recovery.
r ~urgery the rats were provided with grounded laboratory stock diet R3 (Ewos~ AB; Sodert~lje, Sweden) and tap water ad libitum.
During the entire test period the rats were~ provided wi~h grounded laboral~ry stock: diet R3 and tap water ad libitum.
The~ rat~ were randomized using a :andom unit into expe2imental groups A-E.~
Six rats were used in each experimental group W092/2l335 1 4 PCr/SE92/00333;
.
~ 10~6i1 All groups rece*ed 50 ml/kg body weight (b.w.)/day.
Infu6ions were administrated intravenously via a permanent central vein catheter during 20 h/day, normally from 1 p.m. to 9 a.m., v~a IMED
volumetric pumps.
Food consumption was recorded on a 24 h basis. The general appearance of the rats was recorded. The infusions were given during 9 consecutive days. On day 10 the infusions were stopped at 7.00 a.m. and the oral food withdrawn.
The groups A-E were given the following infusions:
A: 10 % Fat emulsion according to Example 2 (F-PLem, the Invention) B: ~ -~ according to Example 3 (FOem) C: 10 % Intralipid ~, containg 10 % ~w/v) oil a~ soy bean oil and 1,2 % egg yolk phospholipid D: 1.2% Phospholipid~`~olution containing: -Fifih phos~olipits~ ~ ~ 12,0g Glycerol; ~ 22,2g ~ --Aq. ad inject.~ 967 g NaOH l M~ 3 ml E: 0.99'o~NaCl-601uOon ~
The ~amount of w3- and w~fatty ~acids were as follows:
to~t w3 - FA~ tot w6 - FA w6 / w3 A =~ 2 (Invention) ~ 55,2 4,5 B = Ex 3 (Eish oil) IO,8 51,6 4,8 C - Intrslipid' ~ 7, 7 56,8 7,4 D = Fish phospholipid~ 4,8~ ~ ~ 0,3 0,1 solution Approx. 2 hours af~;erl ~topping the infusions, the rats were anaesthetized wi~h ~Mebumal~ [60 mg/kg). The blood samples were collected for analysis ;of se~ lipids.
~: j -::: :
Wo92/21335 1 5 PCr/SE92/00333 . .
Serum lipids: From 1 ml blood, serum was taken for analysis of serum l;riglycerides and serum cholesterol and frozen at -70 C until analysis.
Serum cholesterol was measured enzymatically. Serum triglycerides were measured enzymatically after eliminating free glycerol and enzymatic hydrolysis.
Hifitopatholoev: liver, kidneys, heart, lungs, ~pleen and thymus were excised, weighed and prepared for histopathological examination by embedding in paraffin and sectioning at 4-5 micrometer, then staining with haemato~ylin-eosin. Frozen sections from all the tissues stained with Oil Red O for fat were also examined.
Fattv aci~Dfile in liver lipids. The remaining liver tissue from rats in Groups A-C was used for measurement of fatty acid profile in neutral lipid6 and phospholipids(PL). The neutral lipid fractions examined were cholesterol esters (CE)~ and triglycerides (TG).The lipid material was estracted ~and the fatty~acids d :atized and analyzed (GLC) using conventional methods. ~ ~
The resulting data on serum 1ipids~ are shown in Figure 1 (level of serum chole6terol,~ SCHOL) and;~Figure 2 (level of serum t:riglycerides, S-TG).
The 1evels of tlie most unportant ~fatty acids in liver lipidE are shown in Figures 3-7.~ Tbe results are espressed as mean values + SEM for the different emulsions ~anLd 601utions.
Figure 1 clearly shows that the serum cholesterol level is very low when the~ emuls on according to thé invention is used ~ AcB, C; p<O 05, ANOVA).The most surprising effect, however, is the result when companng the serum ~lyceride levels. See Figure 2. That level is surprisingly low (0,30 r ol/l) and only half of the value when Intralipid~, fish phospholipids in a solution or NaCl solution (0,60 ~,, mmol/l) were used (~ A<B, C, D, E; p<O.05, ANOVA).
WO92/21335 1 6 PCr/SE92/00333 ~1096i1 It is absolutely unexpected and unknown, that the small amount of w3-fatty acids which can be derived from phospholipids in a lipid emulsion, can exert physiological effects. The same amount of w3-fatty acids in triglycerides (Group B) does not exert the same biological effects or effects on fatty acid incorporation. Since the phospholipid/triglyceride ratio is about 1:10 in a 10% lipid emul6ion and 1:20 in a 20% lipid emulsion, and the threshold level for biological effects for fish oil, containing about 30 %
w3-fatty acids, is at least 10 % of the oil phase, the amount of w3-fatty acids in the phospholipid part of a lipid emulsion should be at least 30 % (w/w of total fatty acids) to be expected to exert biological effects.
Thus giving an emulsion, according to the invention allows the patient to receive all essential fatty acids such as w6- and w3-fatty acids and still remain at a very low level of serum cholesterol and serum triglycerides.
The hi6topathological data ~showed~ that the emulsions with fish oil or fish phospholipids were accumulated mainly in the Kupffer cells in the liver, whereas Intralip;dg)~ was accumul~ated~ as we}l in hepatocytes. ~ish phospholipids in water solution were however accumulated only in Kupffer cells in the liver and in ;similar cells in the spleen. These cells are immunologically active. This spec fic accumulation to immunologically active Gells fac l;tates an effect of w3-fatty acids on inflammatory and immunological reactions. ~Th~s i8 of im~ortance when using fish pho~pholip~ds for i~mmatu~y or immunological diseases such as rheumatoid a~s a~d ~sepsis ;(Love et al., 1990 and Billiar et al.,l988) or to reduce the incidence ~of arterosclerosis.
The fatty acid pattern ~in liver phospholipids and liver cholesterol esters show that EPA and DHA are incorporated better after treatment with the emulsion according to the invention when compared to the fish oil emulsion containing the~ same amount of w3-fatty acids.
The changes induced by fish phospholipid containing emulsion (F-PLem), fish oil containg emulsion (FOem) or Intralipid ~IL) in fatty acid pattern in liver phospholipids (PL), cholesterol esters ~CE) and triglycerides (TG) are W 0 92~21335 1 7 PC~r/SE92/00333 - ~109~11 shown in Figures 3-7. In the phospholipid fraction (Figs 3 and 6), the only changes seen were increases in DHA, induced by F-PLem (invention, *) p~0.05, ANOVA) and in EPA, induced by FOem (~ p~0.05, ANOVA).
The specific increase in DHA in phospholipids, the main pool for DHA, is of importance for brain and retina deve~opment.
The uptake of and the level in biological membranes of polyunsaturated fatty acids, especially DHA in brain, is well correlated with that in liver (Anderson and Connor,Lipids 1988, 23(4), 286-290) and in heart (Swanson et al.,British Journal of Nutrition, 1988, 59, 535-545). Thus a similar enhancement of uptake in brain and heart is e~pected following administration of the invented emulsion compared to the fish oil emulsion. Therefore the invented emulsion can be used also for normal brain and retina development and ~or cardiovascular di~eases.
In the liver cholssterol ester ~fraction (E igs 4 and 6) F-PLem increased DHA, EPA and li~oleic acit (18:2w6), compared to the FOem- and the I~
groups, and a-linole:nic~acid (~18:3w3), compared to the IL group. Thus, w3-fatty acids adlml~ist:~d~in the phospholipid form, are more effectively inc~rporated in membrane; lipids~ ~phospholipids and cholesterol ester~) than w3-fatty acids iD the trigIyceride form (fish oil) are. The m~n functions ofthe esse~tial~fat~y~acids~and thus also the w3-fatty acids are exerted~ in the membrane lipids.; ~ ~
No decrea~e in any of ~the~w~f~tty acids could be seen in the F-PLem - -group. On t~e contrary, the w6-fatty acids ~linoleic acid (18:2 w6, Fig 6), g_-linolenic acid (l8:3w~6)~and~dihomo-gamma-linolenic acid (20:3w6), as~ well as alpha-linolenic acid (18:3w3) (Fig 5) were increased in the t~glyceride fraction, compared to the FOem- and I~groups. The level of arachidonic acid (20:4w6) remained unchanged in all lipid fractions (Fig7i.This finding i6 of significance be~use of the importance of arachidonic acid in ~biological ~membranes.
Since the w3-fatty acid containing phospholipids are taken up more in Kupffer cells than in hepatocytes, as shown in the invention, the relative increase in incorporated w3-fatty acids is expected to be even higher in W092/21335 1 8 Pcr/sEg2/oo333 ' ~,'l'd'g''~'I'I . .
immunologically active cells (Kupffer cells and other macrophages) than in the whole liver. This is of importance for the anti-inflammatory and immunosuppress*e effect~ exerted by the w3-fatty acids.
The biological effects obtained by the w3-fatty acids in phospholipids can be used for therapeutic purposes as such or in combination with drugs with similar effects and included in the phospholipid vesicles (liposomes) Co~usion~
We have shown that marine phospholipids in an emulsion according to the invention result in surprisingly lower serum triglyceride and serum cholesterol levels when compared to fish oil emulsion, containing a similar amount of w3-fatty acids and a similar w6/w3-fatty acids ratio.
Marine phospholipids induce more effective incorporation of w3-fatty acids in biological membranes than fish oil, containing a comparable amount of w3-fatty acids, in ~n em~sion.
These results show that a ve~y favorable fatty acid pattem in membrane ;~; lipids is ob ained, with an~ increase in all important w3-fatty acids as well as in the w6-fatty acids linoleic, gamma-linolen~c and dihomo-gamma-linolenic acids. The invented w3-fa~tty acid containing phospholipid is thérefore important in all;~situations with increased need of all essential fatty acids, since it makes it possible~ to increase the w3-fatty acids (from the invented~phosph~olipid)~as~well as the w6-fatty acids fin vegetable oils) in a vvell-balanced pattern.~
Tliese~ results have implicatioDs for the use of w3-fatty acid containing phospholipids in vegetable oil emulsions for a more effective utilization and incorporation of;w3- and~w6-fatt~ acids. The use of w3-fatty acid containing phospholipids ;also allows a more specific incorporation of w3-fatty acids in immunologically active cells.
The invention may have implications specifically for situations with increased~level of serum lipids, increased inflammatory response and increased imm: ological activity a~d also for the normal development of the brain and retina.
WO 92~21335 1 9 P~/SE92/00333 9~1 The biological ef~ect~ obtained by the w3-fatty acids in phospholipids can be used for therapeutic purposes as such or in combination with drugs with similar ef~ects and included in the phospholipid vesicles (li;~osomes).
: ~ .
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Claims (17)
1. An emulsion comprising vegetable oil and/or marine oil, an aqueous phase and phospholipids as emulsifier characterized in that the phospholipids are of marine and/or synthetic origin and contain omega-3-fatty acids in an amount of at least 30 % (w/w).
2. An emulsion according to claim 1 characterized in that the omega-3-fatty acids, present in an amount of at least 30 % (w/w), are DHA and EPA.
3. An emulsion according to claim 1 or 2 characterized in that the amount of oil is 0,5-40 % (w/v) and the amount of phospholipids containing the omega-3-fatty acids is 0,1-30 % (w/v).
4. An emulsion according to claim 3 characterized in that it contains one or more bioactive compounds.
5. Use of phospholipids of marine and/or synthetic origin containing the omega-3-fatty acids in an amount of at least 30 % (w/w) for the manufacturing of a nutrition emulsion giving low blood triglyceride and cholesterol levels.
6. Use of phospholipids of marine and/or synthetic origin containing omega-3-fatty acids in an amount of at least 30 % (w/w) for the manufacturing of a medicament with anti-inflammatory and/or immunosuppressive effects.
7. Use according to claim 5 or 6 in which the omega-3-fatty acids are DHA and EPA.
8. Phospholipids of marine and/or synthetic origin containing omega-3-fatty acids in an amount of at least 30 % (w/w) with therapeutic effect.
9. Phospholipids according to claim 8 characterized in that omega-3-fatty acids are DHA and EPA in an amount of at least 30 % (w/w).
10. Phospholipids according to claim 8 or 9 with effects on inflammatory and immunological functions.
11. Phospholipids according to claim 10 in which the disease is rheumatoid arthritis or sepsis.
12. Phospholipids according to claim 8 or 9 with effect on normal brain and retina development and function.
13. A mono-, bi and/or multilayered vesicle or any mixture thereof characterized by its content of phospholipids containing the omega 3-fatty acids in an amount of more than 30 % (w/w).
14. A mono-, bi and/or multilayered vesicle or any mixture thereof according to claim 13 characterized in that the omega 3-fatty acids are EPA and DHA.
15. A compostion comprising the vesicles according to claims 13 or 14, carriers and/or diluents and optionally one or more bioactive compound(s) combined with the vesicles.
16. A pharmaceutical composition comprising the phospholipids according to claims 8 or 9 and carriers and/or diluents for adapting it to oral, nasal, pulmonary, rectal, ocular, transdermal or parenteral administration optionally in combination with one or more bioactive compound(s).
17. A nutritive composition comprising the phospholipids according to claims 8 or 9.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE9101642A SE9101642D0 (en) | 1991-05-30 | 1991-05-30 | phospholipids |
| SE9101642-8 | 1991-05-30 |
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|---|---|
| CA2109611A1 true CA2109611A1 (en) | 1992-12-10 |
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ID=20382886
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002109611A Abandoned CA2109611A1 (en) | 1991-05-30 | 1992-05-19 | Phospholipids |
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| US (1) | US5434183A (en) |
| EP (1) | EP0660708B1 (en) |
| JP (1) | JP3387498B2 (en) |
| AT (1) | ATE216227T1 (en) |
| AU (1) | AU657969B2 (en) |
| CA (1) | CA2109611A1 (en) |
| DE (1) | DE69232564T2 (en) |
| ES (1) | ES2174833T3 (en) |
| IE (1) | IE921477A1 (en) |
| NZ (1) | NZ242697A (en) |
| PT (1) | PT100524B (en) |
| SE (1) | SE9101642D0 (en) |
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| WO (1) | WO1992021335A1 (en) |
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| DE3721137A1 (en) * | 1987-06-26 | 1989-01-05 | Dietl Hans | Fat emulsion for intravenous use |
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| GB8819110D0 (en) * | 1988-08-11 | 1988-09-14 | Norsk Hydro As | Antihypertensive drug & method for production |
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1991
- 1991-05-30 SE SE9101642A patent/SE9101642D0/en unknown
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1992
- 1992-05-12 NZ NZ242697A patent/NZ242697A/en unknown
- 1992-05-19 ES ES92911596T patent/ES2174833T3/en not_active Expired - Lifetime
- 1992-05-19 JP JP51133892A patent/JP3387498B2/en not_active Expired - Fee Related
- 1992-05-19 WO PCT/SE1992/000333 patent/WO1992021335A1/en active IP Right Grant
- 1992-05-19 DE DE69232564T patent/DE69232564T2/en not_active Expired - Fee Related
- 1992-05-19 AT AT92911596T patent/ATE216227T1/en not_active IP Right Cessation
- 1992-05-19 US US08/157,024 patent/US5434183A/en not_active Expired - Lifetime
- 1992-05-19 EP EP92911596A patent/EP0660708B1/en not_active Expired - Lifetime
- 1992-05-19 SG SG1996001678A patent/SG64864A1/en unknown
- 1992-05-19 AU AU19101/92A patent/AU657969B2/en not_active Ceased
- 1992-05-19 CA CA002109611A patent/CA2109611A1/en not_active Abandoned
- 1992-05-27 PT PT100524A patent/PT100524B/en not_active IP Right Cessation
- 1992-07-01 IE IE147792A patent/IE921477A1/en not_active IP Right Cessation
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| US5434183A (en) | 1995-07-18 |
| ES2174833T3 (en) | 2002-11-16 |
| PT100524A (en) | 1993-08-31 |
| DE69232564D1 (en) | 2002-05-23 |
| SG64864A1 (en) | 2001-01-16 |
| EP0660708A1 (en) | 1995-07-05 |
| JP3387498B2 (en) | 2003-03-17 |
| AU657969B2 (en) | 1995-03-30 |
| ATE216227T1 (en) | 2002-05-15 |
| JPH06508123A (en) | 1994-09-14 |
| WO1992021335A1 (en) | 1992-12-10 |
| EP0660708B1 (en) | 2002-04-17 |
| PT100524B (en) | 1999-08-31 |
| IE921477A1 (en) | 1992-12-02 |
| DE69232564T2 (en) | 2002-09-26 |
| AU1910192A (en) | 1993-01-08 |
| SE9101642D0 (en) | 1991-05-30 |
| NZ242697A (en) | 1994-02-25 |
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