Title of Invention

A SERINE GLYCEROPHOSPHOLIPID CONJUGATE WITH EPA AND DHA OR A MIXTURE THEREOF AND A PHARMACEUTICAL COMPOSITION, COMRISING IT

Abstract The invention discloses a serine glycerophospholipid conjugate with EPA and DHA. of formula (I) or a mixture of such conjugates, useful for enhancing omega-3 fatty acids bioavailability to the brain, improving memory, improving learning abilities and treating Attention Deficit Hyperactivity Disorder (ADHD): wherein R" is serine; wherein each of R and R' may be identical or different and may be hydrogen, acyl EPA or acyl DHA with the proviso that R and R' cannot simultaneously represent hydrogen: wherein the amount of EPA and DHA present in the conjugate constitutes 10-50% by weight of the total fatty acids content of said conjugate; and wherein said conjugate is not identical to naturally occurring human or mammalian brain PS; The invention is also for a pharmaceutical composition, nutraceutical composition and functional food comprising said conjugate.
Full Text A SERINE GLYCEROPHOSPHOLIPID CONJUGATE WITH EPA
AND DHA OR A MIXTURE THEREOF AND A PHARMACEUITCAL
COMPOSITION, COMPRISING IT
Field of the Invention
The present invention, relates to. phospholipids and polar hpids
preparations which are enriched with omega-3 and/or omega-6 fatty acids
covalently attached to the lipid backbone.' The phospholipid preparations
of the invention are particularly useful as nutraceuticals, food additives
and/or pharmaceutical agents for the treatment of varioua oonditions, in
particular related to cognitive functions.
Background of the Invention
Lipids, and especially polar LIpids, nitrogen containing lipids, and
carbohydrate containing hpids (phospholipids, aphingosines, glycohpids,
ceramides, sphingomyelins) are the major building blocks of cell
membranes, tissues, etc. Additionally they play important roles in signal
transduction processes and in a variety of biochemical and biosynthetic
pathways.
Glycerophospholipids, lipids based on a glycerol backbone and containing
a phosphate head group, are the main building blocks of cell membranes.
Since most, if not all, biochemical processes involve ceU membranes, the
structiural and physical properties of membranes in different tissues is
crucial to the normal and efi&cient ftmctioning of membranes in all
biochemical processes.
In light of the emerging functional foods category in the area of dietary
lipids many health benefits have been attributed to the consumption of
certain fatty acids. For example, it has been reported in many research
studies that polyunsaturated fatty acids (PUFA) of the type omega-3 and
omega-6, have several health benefits on cardiovascular disease, immune
disorders and inflammation, renal disorders, allergies, diabetes, and

cancer. These typea of fetty adds are naturally occtirring mainly in fish
and algae, where they are randomly distributed on the sn-1, sn-2, and sn-3
positions of the glycerol backbone of triglycerides.
The professional literature emphasizes the importance of an adequate diet
containing omega-3 fatty acids. Extensive clinical studies investigating the
importance of Docosahexaenoic add (DHA), one of the most important
omega-3 fatty acids, in the brain, found that low levels of DHA are
assodated with depression, memory loss, dementiEi, and visual problems.
All studies showed a dramatic improvement in the elderly brain function
as blood levels of DHA increased.
Other known benefits of DHA indude: lower risk of arrhj^hmias,
reduction in the risk of sudden cardiac death, lower plasma triglyceride
levels and reduced blood clotting tendency. Furthermore, DHA may have
importance in the field of brain functioning enhancement, baby formula
fortification, diabetics and cancer. Nutritional studies, investigating the
importance of DHA in the brain, found that low levels of DHA Eire
assodated with depression, memory loss, cognitive impairment, dementia
and visual problems.
The human body does not adequately synthesize DHA. Therefore it is
necessary to obtain it firom the diet Humans obtain DHA from their diets,
initially through the placenta, then firom breast milk, and later through
dietazy soiut:es, such as fish, red meats, animal organ meats and eggs.
Popular fish like tuna, salmon and sardines are rich soiirces. Until
recentiy, the primary source of DHA dietary supplements has been fiah
oils. The ability of enzymes to produce the omega-6 and omega-3 family of
products of linoleic and alpha-Unolenic acid declines with age. Because
DHA synthesis declines with age, as we get older our need to acquire DHA
directly firom diet or supplements increases. In fact, several recent
publications suggested DHA to be considered as essential fatty acid [for
example, Muskiet, P. et al (2004) JNiUr, 134(1): 183-6].
Because DHA is important for signal transmission in the brain, eye and
nervous system, many consumers concerned with maintaining mental
acuity are searching for a pure, safe way td supplement their DHA levels.
Polyunsaturated acids, in particular long chain, such as omega-3 and 6,
have been shown to confer many valuable health benefits on the
population. The global market for long-chain PUFAs, including the food
segment, is rapidly growing.
The majority of efforts in the industry are however invested in the
improvement of PUFA processing techniques and in the creation of higher
concenfarated grades of PUFA derivatives to accommodate dietary
supplements and fiinctional foods needs.
The academic and industrial commimities are less concerned regarding
the evaluation of different delivery approaches of PUFA in order to
enhance their bio-avaUability and their efficacy in term of their known
variety of health benefits. These benefits range from prevention and
treatment of CVD, diabetes, cognitive disorders and/or decline, visual
disorders, skin conditions, learning disorders, etc. Additionally, PUFAs
have been shown to assist in the cognitive and visual development of
in&nts.
PUFA-toids
PS-PUFA
Phosphatidylserine, also known as PS, is a natural phospholipid with bio-
functionality that has made it one of the most promising dietary
supplements in the field of brain nutrition. PS and its health benefits have
beea known to tho sdentifu: and. nutrition communities since the 1970's.
Numerous studies have been conducted in order to establish this efficacy
in a variety of cognitive and mental functions. Those studies have shown
that PS can improve memoiy, fight dementia, fight early stages of
Alzheimer's disease, reduce stress and tension, improve attention span,
enhance mood and fight depression, to name but few.
PS is one of the most important building blocks of cell membranes in the
brain. Hence, the level of PS in brain cell membranes ensures the fluidity
and structure of these membranes. The normal level ensures normal and
efficient signal transduction processes, efficient glucose consumption, and
other biological pathways that result in normal cognitive and mental
fiinctions.
Since PS is not abundant in human nutrition and since in many people,
especially the elderly, the biosynthetic pathways responsible for the
production of PS are malfunctioning, the levels of PS in the body and brain
are low. This results in a variety of cognitive and mental disorders, such
as depression, memory loss, short attention span, learning difficulties, etc.
The supplementation of PS in the diets of elderly people with such
disorders has resulted, in many cases, in dramatic improvements of these
disorders. Over the recent years, studies have shown that even younger
people can benefit firom dietary supplementation of PS. PS has been shown
to improve the learning capabilities of students, improve memory and
attention span, etc.
It is there&re an object of the present invention to provide special
preparations of PS, £3r use mainly as nutraceuticals and as functional food
additives.
PC-PUPA
As mentioned betore. phoepholipide are eeaential components of all
cellular and sub-cellular membranes. Pboephatidylcholine and
pbosphatidylethanolamine predominate quantitatively, substantially
constituting the typical bilayer configuration. Phoepbolipids belong to the
amphipathic moleailes with a water-soluble and a fat-soluble component.
In the bilayer configuration the hydrophilic groups are arranged at the
outer and inner side of the membrane toward the surrounding medium;
the lipophilic groups, in contrast, £ace each other at the inner side of the
bilayer configuration.
Other important constituents of biological membranes are cholesterol,
glyoolipids. and peripheral and integral proteins. The basic structure of
biological membranes is thus a series of recurrent unitiee of lipid-protein
complexes. The membrane is asymmetric. The function of the external
(cellular) and internal (sub cellular) membrane systems depends on their
composition and on the integrity of their phospholipid structure. In
addition to their presence in cell membranes, phospholipids constitute
stnictxiral and functional elements of the surface mono-layers of
lipoproteins and of sur&ctants.
Of utmost importance for the function of biological membranes is their
fluidity, which is decisively influenced by phospholipids. Besides the
content in cholesterol and proteins and the nature and charge of the polar
head groups of phospholipids in the system, membrane fluidity depends on
the length of the chains of Catty add residues in the phospholipid
molecxile, as well as on the number and type of pairing of their double
bonds.


Phospholipids containing poly-unsaturated fatty acids supply the
organism with important bmlding blocks which improves membrane
fluidity.
Studies conducted with PUPA-containing phospholipids have shown the
following:
1. They axe high-energy, basic, structural, and funcfcLonal elements of all
biological membranes, such as cells, blood corpuscles, lipoproteins, and the
surfactant.
2. They are indispensable for cellular differentiation, proliferation, and
regeneration.
3. They maintain and promote the biological activity of many membrane-
bound proteins and receptors.
4. They play a decisive role £}r the activity and activation of numerous
membrane-located enzymes, such as sodium-potassium-ATPase, adenylate
cyclase and lipoprotein lipase.
6. They are important for the transport of molecules through membranes.
6. They control membrane-dependent metabolic processes between the
intracellular and intercellular space.
7. The polyunsaturated fatty acids contained in them, such as linoleic acid,
are precvuraors of the c3^protective prostaglandins and other eicosanoids.
8. As choline and fatty acid donors they have an influence in certain
neurological processes.
9. They emulsify fat in the gastrointestinal, tract.
10. They are important emulsifiers in the bile.
11. They codetermine erythrocyte and platelet aggregation.
12. They influence immunological reactions on the cellular level.
Phospholipids containing PUFA are theoretically of importance in all
those diseases in which damaged membrane structures, reduced
phospholipid levels, and/or decreased membrane fluidity are present. This
hypothesis is supported by experimental and clinical investigations of
various membrane-associated disorders and illnesses.
Studies on the active principle as well as pharmacological and clinical
trials are available on a variety of disturbances and diseases related to
membrane damages. For example, in liver diseases the hepatocyte
structures are damaged by, for example, viruses, organic solvents, alcohol,
medicaments, drugs, or fatty food. As a consequence, membrane fluidity
and permeability may be disturbed, and membrane-dependent metabolic
processes as well as membrane-associated enzjnne activities may be
impaired. This considerably inhibits the metabolism of the hver.
Other examples include hyperlipoproteinemia with or without
atherosclerosis, hemorrheobgical disturbances with an elevated
cholesterol/phospholipid ratio in the membranes of platelets smd red blood
cells, neurologicfd diseases, gastro intestinal inflammations, kidney
diseases, and in a variety of aging symptoms.
All these very dii&rent diseases have in common comparable membrane
disorders. With polyunsatmrated phosphatidylcholine molecules such
disorders may be positively influenced, eliminated, or even improved
beyond normal due to the high content in polyunsatmrated fatty acids.
Following are some examples of the mechanisn:is that mediate this
phenomenon:
1. EDDL particles enriched with PUFA-containing-phosphatidylchohne are
able to take up more cholesterol fifom low-density lipoprotein (LDL) and
tissues. More cholesterol can be transported back to the liver. This action
on the cholesterol reverse transport is unique. All other lipid-lowering
agents reduce either the cholesterol absorption in the body or the
cholesterol synthesis in the liver and its distribution to the periphery.
These substances, however, do not physiologically mobilize the cholesterol
already present in the periphery.
2. The cholesterol/phospholipid ratio in membranes, platelets, and red
blood cells decreases and membrane function is improved up to
normalization.
3. Peroxidative reactions are reduced, damaged hepatocyte membrane
structures restored, membreme fluidity and function stabilized, immuno-
modulation and cell protection improved, and membrane-associated liver
fiinctions enhanced.
4. With the normalization of the cholesterol/phospholipid ratio, the bile is
also stabilized.
5. Due to its specific property as a surface-active emulsifier, PUFA-
containing-phosphatidylcholine solubOize fat and is used in reducing the
risk and treatment of fat embolism.
6. The substitution with poly-unsaturated-fatty-acids and choline may
have a cytoprotective effect in the brain and activate neuronal processes.
7. Liposomes with polyunsaturated phosphatidylcholine molecules may act
as drug carriers, such as of vitamin £.
Liver Disease
Experimental and clinical results support the assiunption that the
therapeutic application of PUFA-containing-phosphatidylcholine has
protective and even curative and regenerative effects on biological
membranes of sinus endotheUal cells and hepatocytes. The cytoprotective
effect of PUFA-containing-phosphatidylcholine has been corroborated in 7
in vitro and in 56 in vivo esiperiments, in which 20 different models with
five different animal species were used. Types of intoxication that are
known to play a role in the etiology of liver disease have mostly been
applied: chemical substances, medicaments, alcohol, cholestasis,
immunological phenomena, esposxure to radiation, and so on.

The liepato-protective effects of PUFA-contaimng'-phosphatidylchoIine
have been confirmed and were the more pronounced the earlier PUFA-
containing-phosphatidylcholine was administered:
1. Structures of membranes were normal or largely normalized.
2. Fatty infiltrations and hepatocyte necrosis could be diminished or even
eliminated
3. Corresponding data were found for lipid peroxidation, transaminase
and cholinesterase activity, and for serum lipids,* liver cell metabolism
increased.
4. The increase of BNA and pretein synthesis and of the liver cell glycogen
content indicated a stimulation of the liver cells.
5. Reduced collagen production, collagen/DNA ratio, and liver
hydro^grproline content indicated a reduced formation of connective tissue.
The dosage of PUFA-containing-phosphatidylcholine ranged from 525 to
2,700 mg/day when administered orally, and from 500 to 3,000 mg/day in
intravenous application. The dvuration of treatment lasted £rom a few
weeks to up to 30 months. The main liver indications were acute hepatitis,
chronic hepatitis, fatty liver, toxic liver damage, cirrhosis of the liver, and
hepatic coma.
The clinical findings, showing the effectiveness of PUFA-containing-
phosphatidylcholine, can be siunmarized generally as follows:
1. Accelerated improvement or normalization of subjective complaints, of
clinical findings, and of several biochemical values
2. Better histological results as compared with the control groups
3. A shortened duration of hospitalization
Premising results were obtained also in renal disorders, chronic
ambulatory peritoneal dialysis, hyperlipoproteinemia/atheroscleresis,
gastreintestinal inflammation, psoriasis, and more.
Recent research studies have shown that PUFA-enriched phospholipids,
isolated from rainbow trout embryos, have novel health benefits. Some of
these benefits include the treatment of tumor ceUs, inhibition of 5-
lipo3Q^genase activity, reduction of neutral £at levels (such as cholesterol).
There is proof that a person who receives enriched phospholipids
nutritionally, these phospholipids cross the intestinal barrier and the
blood-brain barrier, thus reaching the brain. Recently, investigators from
Ponroy Laboratories had described an experiment in which mice lacking
essential fatty acids, i.e. linoleic acid (18:2 n-6) and ot-linolenic acid (18:3
n-3), which serve as the sole sources for LC-PUFA, were fed cerebral
phospholipids and the quantity of phosphoHpids in each part of the brain
measured. These phosphoUpids were found in the cytoplasm, in the
synapses, and in other parts of the brain [Carrie et al., (2000) J. Lipid. Res.
41, 465-472].
The utilization of phospholipids enriched with PUPA holds many potential
advantages from a clinical point of view. The phosphohpid may deHver the
essential fatty acid to specific organs or body parts, such as the brain, and
assist in the incoirporation of these &Ltty acids in membranes. Other
advantages may arise from the fact that phospholipids enriched with
PUFA will not have odor problems such as foimd in the major current
nutraceutical source, the fish oils. Furthermore, some preliminary clinical
studies have shown that PUFA incorporated in phospholipids possess
superior efficacy than PUFA carried by tr^flycerides. [Song et al. (2001)
Atherosclerosis, 155, 9-18].
Further studies have shown that the activity of DHA-rich phosphohpid
was different from that of DHA-zich triacylglycerol in spontaneously
hypertensive rats pirukayama-Tomobe et al. (2001) Journal of Oleo
Science, 50(12), 945-950]. Spontaneously hypersensitive rats (SHR) were
fed test lipid diets for six weeks, which contained 30%-docosahexaenoic
add (DHA) phospholipid (DHA-PL) extracted from fish roe or 30%-DHA
fish oil CDHA-TG). The control diet contained com oil in the presence of
test lipids. After feeding, blood pressure in the DHA-TG and DHA-PL diet
groups was found significantly lower compared to the control. Serum fatty
add content of dihomo-linoleic add (DHLnA) and Arachidonic acid (AA) of
the DHA-PL diet group was significantly less than the control or DHA-TG
diet group. Serum triacylglycerol, phospholipid and total cholesterol in
the DHA-TG and DHA-PL diet groups were significantly less than in the
control. Liver total cholesterol in DHA-PL was twice that in the DHA-TG
diet group and control. The mechanism for cholesterol removal from blood
by DH-PL would thus appear to differ firam that by DHA-TG. Serum lipid
peroxide (LPO) in the DHA-TG and DHA-PL diet groups was essentially
the same as in the control.
Many PUFA-containing agents sufGsr from stability and quality problems
due to the high degree of oxidation of the polyunsaturated fatty acids.
These problems require the incorporation of antioxidants as well as the
utilization of special measures which attempts to reduce this oxidation.
The utilization of phospholipids as carriers of PUFA may result in
enhanced stability of such products due to the anti-oxidative properties of
phospholipids.
It seems that one of the most effective transport mechanism for such
essential fatty adds is the attachment of these groups to phospholipid
molecules. The phospholipids have been shown to pass through the blood-
brain barrier and transport the DHA where it is needed.
Organoletitic concerna
PJJFAa are traditionally extracted from coldwater fish. Despite the
healthy image, one of the problems of consumer acceptance has been the
resulting strong, fishy taste. To address this, microencapsulated forms of
omega-3 have been pioneered in the last 16 years. A further step was the
development of egg-containing products such as DHA-enriched
mayonnaise and pasta. DHA-enriched yogurts, baked goods and broilers
were also envisaged.
There is no other nutritional product or ingredient that is considered to be
an agent of FUFA delivery. All current commercial products are based on
the fatty acids themselves in an encapsulated form or on foods enriched
with PUFA through special animal/crop feed.
It is therefore an object of the present invention to provide lipid
preparations enriched with omega-3 or omega-6 fatty acids, for use mainly
as nutraceuticals and as fiinctional food additives. The composition of said
preparation is such that it provides the preparation with the property of
enhancing the bioavailability of PUFAs. Thus upon its consumption,
prefsrably in the form of nutraceuticals, food additives or pharmaceutical
compositions, the organism may, in the most e£Eicient way, enjoy the
benefits provided by said preparation, as will be described in detail below.
This and other objects of the invention will become apparent as the
description proceeds.
Summary of the Invention
In a first aspect the present invention provides a lipid preparation,
wherein said lipid is selected from glycerophospholipids and their salts,
conjugates, and derivatives and any mixture thereoi^ and poly-
unsaturated fatty add (PUFA) acyl groups, particularly long-chain poly-
unsaturated fatty acid (LC-PUFA) acyl groups, preferably omega-3 and/or
omega-6 acyl groups, at a concentration of least 5% (w/w) of total fatty
acids content of said preparatioa, preferably more than 10% (w/w), more
preferably 20-50% (w/w), wherein said PUFA is covalently bound to said
lipid.
Said lipid may be a naturally occvirring lipid, or a synthetic hpid.
Preferably, said lipid is a glycerophosphoUpid in which at least some of the
sn-1 or sn-2 groups of the g^cerol backbone are substituted with s&dd poly-
unsaturated fetly acid (PUFA) acyl groups.
In one particular embodiment, said Hpid is a glycerophosphlipid of formula
I:
wherein R" represents a moiety selected from serine (PS), choline (PC),
ethanolamine (PE), inositol (PI), g^rcerol (PG) and hydrogen O?hosphatidic
acid - PA), and R and R', which may be identical or different,
independently represent hydrogen or an acyl group, wherein said acyl
group is selected from saturated, mono-unsaturated or poly-unsaturated
acyl groups (PUFA), particularly long-chain poly-unsaturated fatty acids
(LC-PUFA), more preferably omega-3 and/or omega-6 acyl groups, and
salts thereof with the proviso that R and R' cannot simultaneously
represent hydrogen, and wherein said polyunsaturated acyl groups
comprise at leaat 6H (w/w) of total lipid £atty adds, preferably more than
lOH (w/w), and partioilarly 20-60H (w/w).
In one more particular embodiment of said preparation, R represents
hydrogen and R' represents an acyl group. Alternatively, R' represents
hydrogen and R represents sn acyl group.
Considering these latter embodiments, when said acyl group is preferably
an omega-3 acyl group, it may be an eicoeapentaenoyl (EPA), a
docosahezaenoyl (DHA) group, or linolenic omega-3 group. And, when said
acyl group is preferably an omega-6 acyl group, it may be an arschidonoyl
(AHA) group, or a linoleic omega-6 group. A further possibility is that said
acyl group may be a linolenoyl (18:3) group.
In a yet further embodiment of the preparation of the invention, R" may
be any one of serine, choline, ethanolamine, inositol or glycerol.
In a further particular embodiment, the identity and content of R and R'
are predetermined.
The preparation of the invention which comprises the compound of
formula I in which R" is serine, mimift* the composition of human brain
PS.
Nonetheleee, the invention also refers to preparations comprising the
compound of fermula I in which R" is serine, which are difiEsrent from
human brain PS, but still have an improved bioactivity, particularly as
compared to soybean-PS. This improved bioactivity results in beneficial
efibcts on both the learning and working memory in elderly population, in
particularly in cholinergic impaired conditions like Alzheimer's disease.
The invenidon also relates to preparation PS preparation which mimics
the human hrain PS, is effective at lower dosage (2-3 fold) compared to
soybean-PS, while having similar or improved bioactiviiy compared to
soybean-PS.
The PS may be of plant, animal or microoi^amsm source, and is enriched
with PS of formula I, wherein R" represents a serine moiety.
The preparation of the invention may be further enriched with PS of
formula I, characterized in havii^ reduced or absent of fish-related
organoleptic effects. Such preparation may be particularly sviitable for
incorporation into chocolate-containing or dedry-based food articles
(including concentrated milk).
The preparation of the invention may be used in the improvement and
treatment of cognitive and mental conditions and disorders as well as the
maintenance of normal functions of brain-related systems and processes,
preferably ADHD, aging, Alzheimer's disease, Parkinson's disease,
multiple sclerosis (MS), dyslexia, depression, learning capabilities,
intensity of brain waves, stress, anxiety, mental and psychiatric disorders,
concentration and attention, mood, brain glucose utilization, general
cognitive and mental well being, neurological disorders and hormonal
disorders.
The preparation of the invention ia particularly useful in enhancing the
bioavailability of omega-3 and omega-6 fatty acids.
The preparation of the invention may be used in combined improvement of
cognitive and mental functions together with improvement of additional
health disorders or conditions. Such additional health disorders or
conditions may be at least high blood cholesterol levels, high triglycerides
levels, h^h blood fibrinc^en levels, HDL/LDL ratio, diabetes, metabolic
syndrome, menopaiisal or post-menopausal conditions, hormone related
disorders, vision disorders, inflammatory disorders, immune disorders,
liver diseases, chronic hepatitis, steatosis, phospholipid deficiency, Upid
peroxidation, dysrhjrthmia of cell regeneration, destabilization of cell
membranes, coronary artery disease, high blood pressure, cancer,
hypertension, aging, kidney disease, skin diseases, edema,
gastrointestinal diseases, peripheral vascular system diseases, allergies,
neurodegenerative and psychiatric diseases.
The preparation of the invention may also be used in the reduction and/or
prevention of serum oxidative stress leading to atherosclerosis,
cardiovascular disorders and/or coronary heart disease.
The invention further relates to nutraceutical compositions comprising a
Upid preparation in accordance with the invention. The nutraceutical
composition may be in the form of softgel capsules, tablets, syrups, or any
other common dietary supplement deUvery system.
Still further, the invention relates to &inctional fisod article comprising the
lipid preparation of the invention. Such functional food article may be
selected &om dairy products, dairy drinks, ice-creams, bakery products,
confectionary products, biscuits, soy products, pastry and bread, sauces,
condiments, oils and fats, margarines, spreads, cereals, drinks and shakes,
oils and fats, in&nt formulas, infant foods (biscuits, mashed vegetables
and fruits, cereals), bars, snacks, candies and chocolate products.
In yet a further aspect, the invention relates to pharmaceutical
compositions comprising the lipid preparation of the invention, and
optionally further comprising at least one pharmaceutically acceptable
additive, diluent or excipient. The pharmaceutical composition of the
invention may Airther optionally comprise at least one pharmaceutically
active agent
^ACCOMPANYING -/^' "" ' ^
Brief Description of th^Figures
FigurelA-D: Performance of rats in acquisition of the spatial
Morris maze task.
Latency time to platform in the three days of acquisition (2 sessions per
day) of aged rats supplemented for three months with various
supplements as detailed below was analsrzed using video camera, with
(open squares) or without (closed circuits) pretreatment of 1 mg/kg of
scopolamine.
Fig. lA: Rats supplemented with MCTT, P Fig. IB: Rata supplemented with PS- wS, P Fig. IC: Rats supplemented with SB-PS, P Fig. ID: Rats supplemented with LC-PXJFA, P Values represent mean ± S.E.M of four to five rats per supplement.
Abbreviations: Lat. T., latency time; sec., seconds.
Figure 2. Performance of scopolamine-treated rats in the Morris
water maze task in the spatial probe test.
This graph represents percentage of time (T.) that aged rats,
supplemented for three months with MCT (open bars), PS- w3 (solid bars),
SB-PS (dotted bars) or LC-PUFA (striped bars), spent in different areas
after the platform being removed, was analyzed using video camera,
followiz]^ pre-treatment of 1 mg/kg of scopolamine. Values represent mean
± S.E.M of four to five rats per supplement. Significance compared to
control group (MCT) * P Figure 3A-D: Performance of scopolanaine-induced rats in locating
the platform after its reposition.
Latency time to platfarm on the fifbh day of the water maze test, in which
the platform was repositioned between the sessions, in aged rats
supplemented for three months with difforent supplements as specified
below, wsis analyzed using video camera,'with (open squares) or without
(closed circuits) pretreatment of 1 mg/kg of scopolamine.
Fig. 3A: Rats supplemented with MOT.
Fig. 3B: Rats supplemented with PS- to3.
Fig. 3C: Rats supplemented with SB-PS.
Fig. 3D: Rats supplemented with LC-PUFA.
Values represent mean ± S.E.M of four to five rats per supplement.
Abbreviations: Lat. T., latency time; sec, seconds; tr., trials.
Figure 4A-B: Phospholipid levels in rat tissues as measured using
81P-NMR.
Lipids were extracted firom tissues of aged rats that were supplemented
for three months with MCT (open bars), PS- m3 (solid bars), SB-PS (dotted
bars) or LC-PUFA (striped bars). Phospholipids levels were analyzed
using a ^ip-NMR machine and the relative levels of phosphatidylchohne of
the different treatments are presented.
Fig. 4A: Analysis of lipids extracted from the liver.
Fig. 4B: Analysis of lipids extracted fiixim the brain (cortex region).
Values represent mean ± S.D. of four to five rat tissues per supplement.
Significance compared to control group (MCT) *P Abbreviations: Tot. PL, total phospholipids.
Figure 5: Parental scores of ADHD children according to
behavioral rating scales.
The graph represents percentage of ADHD children that demonstrated
improvement or lack of improvement in a parental view following two
months of supplementation with canola oil (open bars), DHA (solid bars)
or PS- id3 (hatched bars). Rating includes remarks regarding behavioral
tendencies at home, at school, with sibling^ or peeirs and teachers
feedback. Values represent percentage of twenty to twenty-five ADHD
children scores per supplement. Note that twelve parents decline to
respond to the questioner and six children did not complete the
supplementation period due to poor taste or severe discipline problems
(mostly the control group).
Abbreviations: Improv., improvement; Marg. Improve., marginal
improvement; n.c., no change; Deter., deterioration.
Figure 6: Effect of PC-DHA on the serum oxidative stress.
Apo E* mice were fed for 10 weeks with placebo (open bars) or PC-DHA
(solid bars). Serum lipid peroxide (Ser. per.) levels were measured using a
spectrophotometric assay. Values represent mean ± S.D. of 5 mice per
treatment.
Detailed Description of the Invention
In a first aspect the present invention provides a lipid preparation,
wherein said lipid is a glycerophospholipid, a salt, conjugate, and
derivative thereof and any mixture thereof and poly-unsattirated fatty
add (PUFA) acyl groups, particularly long-chain poly-vmsaturated fatty
acid (LC-PUFA) acyl groups, preferably omega-3 and/or omega-6 acyl
groups, at a concentration of least 5% (w/w) of total fatty acids content of
said preparation, preferably more than 10% (w/w), more preferably 20-
50% (w/w), wherein said PUFA is covalently bound to said
glycerophospholipid.
Said lipid may be a naturally occurring lipid, or a synthetic Hpid.
Preferably, said lipid is a giycerophospholipid in which at least some of the
sn-1 or sn-2 groups of the glycerol backbone are substituted with said poly-
unsaturated fetty acid (PUFA) acyl groups.
In one particular embodiment, said lipid is a glycerophosphlipid of formula
I:
wherein R" represents a moiety selected from serine (PS), choline (PC),
ethanolamine (PE), inositol (PI), glycerol (PG) and hydrogen (phosphatidic
acid - PA), and K and R', which may be identical or different,
independently represent hydrogen or an acyl group, wherein said acyl
group is selected from saturated, mono-unsaturated or poly-unsaturated
acyl groups (PUFA), particularly long-chain poly-unsaturated fatty acids
(LC-PUFA), more preferably omega-3 and/or omega-6 acyl groups, and
salts thereof, with the proviso that R and R' cannot simultaneously
represent hydrogen, and wherein said polyunsaturated acyl groups
comprise at least 5% (w/w) of total lipid fatty acids, preferably more than
10% (w/w), andpartiodarly 20-50% (w/w).
In one more particular embodiment of said preparation, R represents
hydrogen and R' represents an acyl group. Alternatively, R' represents
hydrogen and R represents an acyl group.
Considering these latter embodiments, when said acyl group is preferably
an omega-3 acyl group, it may be an eicosapentaenoyl (EPA), a
docosahexaenoyl (DHA) group, or linolenic omega-3 group. And, when said
acyl group is preferably an omega-6 acyl group, it may be an arachidonoyl
(ASA) group, or a linoleic omega-6 group. A further possibility is that said
acyl group may be a linolenoyl (18:3) group.
In a yet further embodiment of the preparation of the invention, R" may
be any one of serine, choline, ethanolamine, inositol or glycerol.
In a further particular embodiment, the identity and content of R and R'
are predetermined.
The preparation of the invention which comprises the compound of
fbrmiila I in which R" is serine, mimics the composition of human brain
PS.
Nonetheless, the invention also refers to preparations comprising the
compound of formula I in which R" is serine, which are different &om
human brain PS, but still have £ui improved bioactivity, partictdarly as
compared to soybean-PS.
Traditionally, PS active ingredients used as dietary supplements were
produced by the extraction of animal brains, particularly bovine brains.
The PS extracted from animal brain tissues, similarly to human brain PS,
has a fatiy add composition which is characterized by relatively higher
levels of omega-3 moieties, compared to the levels of omega-3 found in
plant phospholipids.
PS has the £bUowmg structure:
Human brain PS is characterized by over 20-30% PS containing omega-3
£atty acyls, preferably at the sn-2 position of the glycerol moiety, and
mainly DHA or EPA. As mentioned above, phosphoHpids, and PS in
particular, are responsible for membrane structure and physical
properties. One of the major physical properties governed by phospholipids
is the fluidity of these membranes. Omega-3 fatty acids, DHA and EPA in
particular, also have a crucial role in membrane fluidity io. Hght of their
unique 3D structure. Therefore, PS with omega-3 fatty acyl moieties, DHA
and EPA in particular, has unique bio-fiinctionality which cannot stem
from just the basic phospholipid skeleton of this phospholipid.
Considering the risks involved with prion diseases, particularly bovine
spongiform encephalopathy (BSE), as well as other disadvantages
associated with ingredients obtained from animal sources, PS
supplements are usually prepared using PS originating from soybean
lecithin. This lecithin is enriched, usualti^ enzymatically, with PS. This
method of production results in PS with a fatty acid profile of soybean
phospholipids, which is characterized by low level of omega-3 fatty acids,
and almost no DHA and EPA. This PS active ingredient is also known as
soybean-PS.
Although the bio-functionalily of soybean-PS in the improvement of
cognitive function has been shown to be similar to that of bovine-PS, it is
still different £rom human brain PS. It is a purpose of the present
invention to provide a PS ingredient with a predetermined fatty acid
composition that mimics the fatty add composition of the human brain PS.
It is a further object of the present invention to provide a PS ingredient
which, while not identical to naturally occurring brain PS, is characterized
by improved functionality, particularly in comparison with soybean-PS.
This improved PS ingredient has a predetermined fatty acid composition.
The PS ingredient of the present invention is enriched with omega-3 fatty
acyls, preferably DHA, EPA or linolenic omega-3. Furthermore, the PS of
this invention is enriched with omega-3 fatty acyls covalently bonded to
either or both of the sn-1 or sn-2 positions of the glycerol moiety in the PS
backbone.
The present invention is also related and describes other phospholipids,
such as phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidyl-inositol (PI), phosphatidylglycerol (PG) and phosphatidic
acid (PA), enriched with omega-3 i&tty acids, preferably DHA, EPA, or
Unolemc add which are covalently bonded at either or both of the an-1 or
sn-2 positions of the glycerol moiety of the phosphohpid. Alternatively, the
phospholipids of the invention are enriched with omega-6 fatty adds.
When referring to PS in the present description, it should be taken to
mean also any other lipid, such as, but not limited to, the polar lipids
listed above.
In a preferred embodiment, the amount of omega-3 (particularly EPA,
DHA or linolenic acid) or omega-6 particularly ABA and linoleic acid)
fatty acids in the PS ingredient of the invention is greater than 10% at
either or both of the an-l or sn-2 positions, preferably at the sn-2 position,
preferably over 20% and most preferably above 40%.
As mentioned, the desired omega-3/omega-6 fatty acyls can be bonded at
both or only one of the sn-l and sn-2 positions.
The £atty add composition of the PS preparation of this invention can
have a predetermined fatty add composition similar to or difiEerent &om
the fatty acid composition found in normal healthy human brain, provided
it has enhanced activity, particularly compared to the activity of plant PS,
for example soybean-PS.
The preparation of the omega-3/omega-6-enriched PS preparation of this
invention can be enzymatic, chemical or by molecular biology methods.
Briefly, the PS can be enriched with omega-3 or omega-6 moieties by
enzymatic processes, e.g. enrichment of a natural phospholipidyiecithin
with omega-3 fatty acids by enzymatic transesterification/esterification
followed by transformation of the head group to serine (using PLD
enzymes) to obtain a PS-omega-3/omega-6 conjugate. Another enzymatic
pathway is to obtain a ledthin or phospholipid source which is naturally
rich in omega-3 acids, such as krill phospholipids, and transform their
head groups to serine. It is to be noted that the fatty acid composition of
the PS obtained by this method has an omega-3 composition which is
predetermined by the source of choice (fish, knll, algae, etc.). Such
methods have been thoroughly described in Applicant's co-pending POT
Application claiming priority from IL168553.
The PS-omega-3/omega-6 ingredient of the present invention can also be
prepared by chemical transesterification/esterification methods that will
enrich the sn-l and 2 positions with omega-3 or omega-6 acyl residues.
Such methods of preparation of PS-omega-3 and PS-omega-6 have been
described in Applicant's co-pending PCT Application claiming priority
from IL15d553.
Alternatively, the PS ingredient of the present invention can be prepared
by GMO (geneticaify modified organism8)/biotechnology methods, for
example, providing phospholq>id8-producii^ oi^anisms with omega-3 or
omega-6 fatty adds to obtain phospholipids enriched with omega-3 or
omega-6 PS. It may be preferred to use genetically engineered plants or
micax>orgaiusm8, to avoid use of animal sources.
The PS of this invention can have the omega-3 or omega-6 fiatty acid
composition of a spedfic ledthin raw material, relatively rich with omega-
3 or omega-6 fatty adds, enriched with PS to yield a PS ingredient with
elevated omega-3 or omega-6 fatty acids levels, compared to soybean-PS.
Such is the case, for example, when phospholipids from krOl are used as
the starting material, as described above.
In a preferred embodiment the PS enriched with omega-3 or omega-6 can
be soybean-PS or any other PS, from plant, animal, for example krill, or
microorganism source. In a further preferred embodiment the omega-3 or
omega-6 enrichment can be performed on a lecithin, which in turn is
enriched with PS by transphosphatidylation.
It is the purpose of this invention to provide a novel PS ingredient,
enriched with omega-3 fisitty adds, reaidting in an ingredient with
improved efBcacy compared to ingredients containing natural or simply
enriched PS.
The improved PS preparation of this invention exhibits enhanced activity
in the improvement and treatment of cognitive and mental conditions and
disorders as well as the maintenance of normal functions of brain related
systems and processes. These include, but are not limited to ADHD,
multiple sclerosis (MS), dyslexia, depression, learning capabilities,
intensity of brain waves, stress, mental and psychiatric disorders,
neurological disorders, hormonal disorder, concentration and attention,
mood, brain glucose utilization, and general cognitive and mental well
being.
The novel lipid preparation of this invention exhibits enhanced activity in
the improvement of cognitive functions, as detailed hereiuider, over
omega-3 or omega-6 lipids per se or soybean-PS. Fiurthermore, under
certain conditions or for all or specific disorders, the lipid preparation of
the invention is efifective at a dosage of less than 100 mg/day. This is lower
that the current recommended daily dosage of soybean-PS (100-
300mg/day) or omega-3 lipids (approx. l-2g/day or more) currently
available in the market. Nonetheless, dosages of 100-600mg/day are
pre&rred for enhanced efEicacy of the Hpid preparation of the invention.
An important advantage of the PS preparation of the invention is that it
exhibits multiftinctional activity. This multi-:ftinctionality is exhibited by
improvement in cognitive and mental functions, together with
improvement of other health disorders or conditions.
The enhanced activity of this PS ingredient, as well as its multi-
functionality, may arise from the unique structure of this ingredient and
its influence on the physical and chemical properties of cell membranes in
brain tissues as weU as other organs and tissues.
The enhanced activity of this PS ingredient, as well as its multi-
functionality, may also be attributed to the enhanced bioavailability of the
omega-3 fatty acids, due to their incorporation in the PS skeleton. Thus,
the omega-3 fatty adds can be delivered to the brain across the blood-
brain barrier, being a part of the PS molecule, which readily passes this
barrier. The PS functions as a delivery platform for the fatty acids bound
thereto, to various oi^ans and tissues, thereby enhancing their
bioavailabiliiy.
The additional health disorders or. conditions which are affected by the
multifunctional PS preparation of the invention include, but are not
limited to high blood cholesterol levels, high triglycerides levels, high
blood fibrinogen levels, HDI/LiDL ratio, diabetes, metabolic syndrome,
menopausal or post-menopausal conditions, hormone related disorders,
vision disorders, inflammatory disorders, immune disorders, liver
diseases, chronic hepatitis, steatosis, phospholipid deficiency, lipid
peroxidation, dysrhythmia of cell regeneration, destabilization of cell
membranes, coronary artery disease, high blood pressure, cancer,
hypertension, aging, kidney disease, skin diseases, edema,
gastrointestinal diseases, peripheral vascular system diseases, allergies,
airways diseases, neurodegenerative and psychiatric diseases.
The new ingredients of the invention can be delivered and utilized in a
variety of products. Such products include dietary supplements, &inctional
fobds, pharmaceutical delivery systems, etc.
The preparation of pharmaceutical compositions is well known in the art
and has been described in many articles and textbooks, see e.g., Gennaro
A. R. ed. (1990) Remington's Phca-maceutical Sciences, Mack Pubhshing
Company, Easton, Pennsylvania, and especially pages 1521-1712 therein.
As dietary supplements, the preparations of the invention may be used in
the form of soft gel capsules, tablets, syrups, and other common dietary
supplements delivery systems.


As functional faods, the preparations of the invention can be iacorporated
and used in a variety of foods, such as dairy products, ice-creams, biscuits,
soy products, pastry and bread, sauces, condiments, oils and fats,
margarines, spreads, cereals, drinks and shakes, infant formulas, infant
fix)ds (biscuits, mashed vegetables and fruits, cereals), bars, snacks,
candies, chocolate products.
As pharmaceutical products, the preparations of the invention can be
delivered orally, intravenously, or by any other conventional or special
route of administration.
The new preparations of the invention may be in the form of fluid oU,
powder, granules, wax, paste, oil or aqueous emulsion, and any other form
that will enable its use in the target applications.
Pharmaceutical or nutraceutical formulations comprising the PS
preparation of the invention may include physiologically acceptable &ee
flowing agents, other additives, excipients, dessicants and diluents,
colorants, aroma and taste ingredients, and any ingredients that control
physical, organoleptic, and other properties, as well as additional active
ingredients, for example minerals, vitamins, other nutritioned additives.
The utilization of omega-3 Upids in a variety of applications, and
especially £is ingredient of fimctional foods, is hindered due to their
distinct fish odor. Thus, another advantage of the omega-3 enriched
phospholipids ingredients of the invention is that they have reduced odor
or taste of omega-3 acyl moieties, due to the covalent binding of these
groups to the PS backbone. This increases the vapor pressure of these
materials, hence reducing their distinct aroma. Thus, the covalent binding
of the omega-3 fatty acids to the phospholipid backbone, especially PS,
alters and improves their taste properties. Moreover, the PS ingredient of
the invention also oiBfers enhanced stability to the oxidation sensitive
omega-3 fatly acids. Phospholipids in general, and PS in particular, are
known to act as anti-oxidants and stabilizers.
These benefits make the lipid preparation of the invention highly
beneficial and important in a variety of applications and especially in
functional foods, where stability, aroma and taste are fundamental
requirements.
Furthermore, these novel ingredients can be formulated with additional
lipids for an even enhanced bio-functionality and ef&cacy.
The polar hpids derivatives of PUFA, such as the PS-PUFA derivatives
have exhibited high stabiUly as a preparation and additionally in several
food applications, used in the clinical trials of the present invention. The
stability of these sensitive compounds is emerging firom the covalent
combination of phospholipids, known in the past to be used as
preservatives and of the un-stable PUFA moieties.
The new ingredients of the invention can be delivered and utilized in a
variety of products. Such products include dietary supplements, functional
&)ods, pharmaceutical dehveiy systems, etc.
Disclosed and described, it is to be understood that this invention is not
limited to the particular examples, process steps, and materials disclosed
herein as such process steps and materials may vary somewhat. It is also
to be understood that the terminology used herein is used for the purpose
of describii^ particular embodiments only and not intended to be hmiting
since the scope of the present invention will be limited only by the
appended claims and equivalents thereof
It must be noted that, as used in this specification and the appended
claims, the singular forms "a", "an" and "the" include plural referents
unless the content clearly dictates otherwise.
Throughout this specification and the claims which follow, unless the
context requires otherwise, the word "comprise", and variations such as
"comprises" and "comprising", will be understood to imply the inclusion of
a stated integer or step or group of integers or steps but not the exclusion
of any other integer or step or group of integers or steps.
The following Examples are representative of techniques employed by the
inventors in carrying out aspects of the present invention. It shovild be
appreciated that while these techniques are exemplary of preferred
embodiments for the practice of the invention, those of skill in the art, in
Hght of the present disclosure, will recognize that numerous modifications
can be made without departing £rom the spirit and intended scope of the
invention.
Examples
Example 1
Methods:
Animals and diet
Male Wistar rats originated from the same colonies were obtained from
Harlen. Fifty rats were randomly divided into five dietary supplemented
groups, in addition to their normal diet: (i) a group fed O.lg medium-chain
triglycerides (MCT)/lml supplement matrix (MCT group); (ii) a group fed
O.lg DHA/EPA (20/30% of total fatty adds composition, diluted with MCT
to generate 30% (w/w) LC-PUFA compound) triglycerides/lml supplement
matrix (LC-PUFA group); (iii) a group fed O.lg soybean lecithin-derived
PS (20% SB-PS w/w)/l ml supplement matrix (SB-PS group); and (iv) a
group fed O.lg PS- mS (20% PS w/w, and total LC-PUFA composition of
30%)/l ml supplement matrix (PS group). The supplement matrices were
stored at -20'C, and £resh portions were fed to the rats every day. All
supplements were handled so as to minimize oxidation of the fatty acids.
Rats consumed the diet and water ad libitum. All rats were housed in a
standard environment, in which temperature was maintained at 24 ±
0.5°C, and the relative humidiiy was kept at 65 ± 5% with 12-h periods of
hght and dark. Body weight was measured at the beginning and the end of
the treatment period.
The PS-told compound used in this study mimics the fatty acids
composition of the mammalian brain PS, with respect to its DHA content
(20%). Generally, in animal cells, the fatty acid composition of PS varies
from tissue to tissue, but does not appear to resemble the precursor
phospholipids, either because of selective utilization of specific molecular
species for biosynthesis or because or re-modeling of the lipid via
deacylation-reacylation reactions. In human plasma, l-3te£iroyl-2-oleoyl
and l-stearoyl-2-arachidonoyl species predominate, but in brain and many
others related tissues l-stearoyl-2-doco8ahexaenoyl species are very
abundant [O'Brien et al (1964) J Lipid Res. 5(3):329-38]. An early work by
Yabuuchi et al. [Yabuuchi et al. (1968) J Lipid Res. 9(l):65-7] established
that the DHA content in bovine gray matter is up to 30% of the total fatty
acids composition; most of the total amoimt of DHA was located at the sn-
2 position (60%). It was the bovine brain PS that Tofiano and Bruni
reported in the early 1980's to be a pharmacologically active compound,
which counteracts age-related changes in the central nervous system
[TofEano et al. (1980) PkarmacolRes. Commun. 12:829-845].
Behavioral testing
Water maze test, which was developed by Morris [Stewart, CA. and
Morris, EG. (1993) The water maze. In: Behavioural Neuroscience: A
Practical Approach. Vol. 1 (Saghal, A., ed.), pp. 107-122. Oxford
University Press, New York, NY.], uses a circular tank (137 cm diameter,
36 cm deep) constructed of opaque white plastic. It is filled with water
(21-22*'C) to a depth of 28 cm, and the water is rendered opaque by the
addition of soluble, nontoxic white latex paint In the place version of the
maze, the rat develops a spatial map of the extra-maze cues, which it then
uses to locate the platform. Thus the distance swum to the platform and
the time taken ia doing so should decrease over testing sessions (days) as
the rat learns the location of the platform. Moreover, it is expected that if
the rat has learned the location of the platform in relation to the extra-
maze cues, its initial response on the probe trial wiU be to swim directly to
the quadrant in which it e3q>ects to find the platform. Thus the distance
swum (and time spent) in the target quadrant should be greater than that
in the other two quadrants (excluding the start quadrant). The distance
swum to the platform as well as the latency to reach the platform were
monitored using the video-based tracking system. The behavioral testing
was conducted during the dark cycle, when rats are normally most active.
The pool was located in a test room in which there were many extra-maze
spatial cues. On the first three days, the rats were required to locate the
hidden platform (15.5 cm x 15.6 cm) situated 1 cm below the surface of the
water. There were two acquisition testing sessions per day, with four trials
per session. On each trial, the rat was placed, facing the w£dl, in one of the
four quadrants in the tank, and allowed to swim for a maximum of 60
seconds. Once the rat found the platform, it remained there for 6 seconds
before being retiurned to the holding cage, which was kept warm on a
heatiog pad. If the rat failed to find the platform in that time, it was
placed on it for 5 seconds before being returned to the holding cage. Each
of the eight trials conducted each day was started from a different
quadrant, with the order determined pseudorandomly (not twice £rom the
same quadrant) and varying firom day to day. The intertrial interval (TTI)
was 120 seconds, counted £com the end of one trial to the beginning of the
next. On fourth day, followed by a session as abovementioned, the
platform was removed from the tank, and a probe trial was conducted by
placing the rat in the quadrant opposite to that of the platform and then
allowing it to swim for 60 seconds. The day following the probe trial, the
rats were tested with a session in which the maze was set up as previously
described, followed by a session in which the platform was repositioned to
the center of the opposite quadrant. The latency to find the platform on
each trial was recorded. Scopolamine (Img/Kg) was intraperitoneally (i.p.)
administered 30 minutes before the indicated trials.
Lipid extraction and NMR analyses
At the end of the behaviorsd testing, the rats were anesthetized with
Halothane and then decapitated. Liver and brain tissues were quickly
removed and stored (at -80°C). The hpid fraction of the rat tissues were
extracted using a modified version of the technique described by Bhgh and
Dyer 1959 [BUgh and Dyer, (1959) Can. J. Biochem. PhysioL 37, 911-917].
Briefly, 500-700 mg and 300-1200 mg of liver and brain tissues,
respectively, were homogenized in a solution of CDC13, methanol and CS-
EDTA (1:2:2 v:v:v). The homogenates were further agitated using
ultrasonic bath (10 min, 80°C!), followed by additional vigorous shaking (20
min). The relative ratio of the phospholipids in the homogenates was
measiured using h^h-resolution ^ip-NMR at 121.MHZ usii^ a 7.06 Tesla
General Electric spectrometer.
These homogenates were further analyzed for their fatty acids
distribution. First, the lipids extracts were desalted by reverse-phase
chromatography using an HP-18 colxunn [Williams et al. (1980) J.
Neurochem.; 36, 266-269]; diheptadecanoyl phosphatidylcholine was
added as internial standard before the loading on the column.
Phospholipids were separated &om neutral Upids, such as cholesterol, on
silica gel plates (Merck 60) developed in isohexane: ether: formic acid
80:20:2 (v:v:v). The phospholipids spot was visualized by spraying
primuJin solution and compared with authentic phosphoHpids standards.
Henicasonoic methyl ester (C21:0) was added as a 2nd internal standard
and the phosphoHpids were converted to methyl esters by mild acid
hydrolysis with 1% methanolic H2S04 overnight at 50°C. The fatty acids
projGle of the different samples was determined by gas-liquid
chromatography.
Results
Anti-dementia effects of bovine brain cortex-derived PS (BC-PS) has been
demonstrated by several double-blind, placebo-controlled studies, see
review by [Eidd P. (1996) Alt Med Rev. l(2):70-84]. In the past decade both
BC-PS and soybean lecithin transphosphatidylated PS (SB-PS) were
shown to recover the scopolamine-induced amnesia in rodent, although the
fatty acids composition is considerably different between these compounds
[Zanotti A et al. (1986) Psychopharmacology (BerV). 90(2):274-5.; Claro F.
et al. (1999) Physiol Behav. 67(4):551-4; Sakai M. (1996) Nutr Sci
Vitaininol (Tokyo) 42(l):47-54; Furushiro M et al. (1997) Jpn J
Pharmacol. 76(4):447-50]. The means of PS administration in these
studies was predominantly intravenous or intraperitoneal; although
Furushiro et al. described also oral administration of SB-PS that
antagonized amnesic effects of scopolamine. However, in the latter study
the investigator used a considerable high dose of SB-PS, ranging between
60 to 240 mg/Kg.
In the presented study, rat diet was supplemented with the above-
mentioned treatments (diets i, ii, iii, iv and v) for three months be&re the
maze test was performed. In the acquisition stage (Figure lA-lD) there is
an expected and marked increase in the latency time to find the platform
after the administration of scopolamine (Img/Kg) of all groups. Although
the latency curves of MCT and PS-id3 groups are similar, there is a
statistically smaller difference in the latency change, induced by
scopolamine, in the PS- tnS group with resjpect to the latency presented by
the MCT group (P-value Similarly, the groups treated with SB-PS or LC-PUFA, demonstrated a
reduced e£fect of scopolamine on their learning curves, with respect to the
MCT group (see Figure lA-lD). Having all groups learn the task at a
similar rate, resembles data presented by Blokland et al. [Blokland et al.
(1999) Nutrition 15(10): 778-83], which showed no difference between PS
obtained firom dififerent sources and the empty vehicle, in a water maze
test.
What is particular to the present trial is the accelerated rate in learning
the task under the scopoleunine sedation. This was not demonstrated
previovisly [Furushiro et al. (1997) id ibid.; Suzuki et al., (2000) Jpn. J.
Pharmacol. 84, 86-8]. Note that in these studies the rodent faced a
difEerent task (passive avoidance). In Suzuki et aL 2001 (J. Nutr. 131:
2961-6) the investigators utilized considerably older rats (24-25 months
old) than the ones tested in the present trial. The latency time in the
acquisition step was considerably longer for the aged rats compared to the
young ones that were tested (eight weeks). Interestingly, although the
latency time in the present trial of non-sedated rats is somewhat
comparable to the younger rats tested by Sxiziiki et al. [Suzuki et al. (2001)
id ibid.], the scopolamine-induced amnesia latency time in the MCT group
resembles the one obtained at the described study for elderly rats. In
conclusion, scopolamine induced a comparable long latency time in the
control group (MCT). This e£Eect was augmented to a different extent by
long-term treatment of rats with either PS or LC-PUFA.
In the probe trial, the rats treated with PS- tn3 showed a distinctively
higher tendency than MCT-treated ones (P zone in which the platform was located during the acquisition of the task
(Figure 2), indicating that the rats had learned the spatial location of the
platform. Moreover, PS-ot3 treated rats presented a reduced tendency
(P zone. These latter indications, presented by the PS- td3 group are related
to a higher adventurous characteristic and could be somewhat correlated
with the open field behavior trial. Interestingly, in BloMand et al.
[Blokland et al. (1999) id ibid.] BC-PS treated mice demonstrated a non-
significant but clear tendency to be less adventurovis in the open field
behavior trial, by spending less time in the center area. With respect to
the remarkable learning abilities demonstrated by the rats that were
treated with PS- tuS, it is interesting to compare their performance in the
Morris water maze task in the spatial probe test to the one obtained by the
SB-PS treated animals by Suzuki et al [Suzuki et al (2001) id ibid.].
Though the percent of time spent in the quadrant where the platform was
located is similar (~45%), it is remarkable that the dosage in the current
study was merely one third of the administration levels in Suzuki et al
2001 (20mg/kg vs. 60mg/kg, respectively). Indeed, in the present study
there was no significant change in the time that the SB-PS (20mg/kg)
treated rats spent in this quadrant when compared with the values
obtained by the MCT-treated group [Fig.lC and Fig. lA, respectively]. In
summary, the PS-tuS treated group learning abilities were markedly
higher than the control, in a considerably low level of PS administration.
In addition, the rats treated with PS- tdS were less conservative and more
adventurous in studying the maze in the absence of the platform.
Finally, the most prominent and outstanding data obtained in the present
study was the response to the repositioning of the platform. All groups
presented a shorter latency in finding the platform at the first session,
when compared to the one obtained by the MCT-treated group, under
scopolamine sedation (Figure 3A-3D). These data suggest that LC-FUFA,
and more potently PS, can attenuate scopolamine-induced amnesia, as
previously presented by other studies (see selected references above).
Siuprisingly, in the second session, there were no differences between the
latency in finding the plat&rm after its repositioning in all groups but the
PS-htS treated group. In fact, it seemed that in all treatments but the PS-
w3 there was no learning process of the position of the platform. The FS-
11x3 group presented a remarkably different behavior; it seemed that there
was no lag in the learning of the repositioned platform in the rat treated
with this anti-muscarinic drug. The ability of the PS-iuS treated group to
locate the platform after it had been repositioned seemed to be
contradictory with the result obtained earher in ihe spatial probe test
(Fig. 2), where these rats showed preference for the third quadrant. Pearce
and colleagues [Pearce et al. (1998) Nature 396: 75-77] attempted to
resolve this discrepancy, by describing two means for memorizdng a
specific spatial location. One is to use a cognitive map that encodes
information about the geometric relationship between the object ahd
several land marks (the cognitive map method) and the other is the use of
heading vectors that specii^ the direction and distance from a single
landmark to the object (the heading vector method). In the present test,
the rats coiild locate the platform firom the above-mentioned cues and/or
from the distance and direction with respect to the walls. In the
acquisition and the spatial probe test, both methods contributed to the
score of finding the platform. However, in the repositioning test, the
cognitive abilities which are related to the heading vector method and the
short-term memory (workiag memory), made the difference. The heading
vector method, because the distance from the wall was not effected by the
repositioning (jxist the quadrant), and the working memory due to the
benefits in memorizing the areas already explored that enable an e£fective
search in the pool.
It has been previously reported that the mechanism by which PS
attenuates the scopolamine effect could be attributed not only to a
beneficial effect on the cholinergic circuitry, but PS could also have an
effect on the serotonergic neuronal system [Furushiro et al. (1997) id
ibid.]. It appears that the presented data could be the result of more than
one neiuronal system alteration, possibly the dopaminergic. In an earlier
study Prago et al. (1981) Neurobiol A&ng, 2(3):209-13], it was suggested
that the alteration in the obtained behavioral changes between BC.-PS
treated aged rats to their control coiild be attributed not only to the
modifications in cholinergic and serotonergic transmission, as described
above, but also through affecting the catecholaminergic (like dopamine)
system. In this study the facilitated acquisition of active avoidance
behavior as studied in shuttle-box and pole jumping test situations, and
the retention of active and passive avoidemce responses were improved in
the PS-treated rats. Tsakiris [Tsakiris, S. (1984) ZNaturforsch [C\, 39(11-
12): 1196-8] reported on an indirect effect of PS on the dopamine related
adenylyl cyclase, through membrane fluidity mechanism. Interestingly, it
has also been reported [Chalon, et al. (1998) JNuir.; 128(12):2612-9] that
enriched diet with high level of (n-3) PUFA could result in an effect on the
cortical dopaminergic function. It is conceivable that the existence of LC-
PUFA on the backbone of the phospholipids was highly beneficial in terms
of such a multi-neurotransmitter mechanism.
The biochemical analyses of the present results in Uver tissues (Fig. 4A.)
shows that in rats supplemented with.PS for three months (SB-PS and
PS-to3) there was a notable increase in the levels of the primer
phospholipids, i.e. phosphatidylcholine (PC). These data is consistent with
early observations regarding the liver and its major role in the
phospholipids uptake and the primazy metabolism of most fatty acids.
Wijendran and colleagues [Wijendran et cd. (2002) Pediatr. Res. 51:266-
272] described a study in which baboons were fed labeled LC-PUFA on the
backbone of PC and triglycerides, and demonstrated that the levels of
incorporation of LC-PUFA on a phospholipid backbone to the liver was
higher than the extent of incorporation of LC-PUFA on the triglycerides
backbone. In addition, PS levels of rats fed with PS- vi 3 were elevated in
cortex tissues analyses of phospholipids distribution (Figiire 4B),
comparir^ with MCT. Interestingly, the phospholipids fetty adds profile
of these cortices (Table 1) demonstrate a marked elevation in the DHA
content of the rats fed with PS-ro3 (E*=0.007). Similar elevation was noted
for LC-PUFA fed rats, however to a reduced extent compared with PS- tnS
treatment (14.6 versus 17.5, respectively P=:0.03) and MCT (14.6 versus
12.3, respectively P=0.02). This difference in the DHA levels between the
two omega-S groups might suggest enhanced bioavailability of DHA when
it is esterified to the backbone of phospholipids rather than to
triglycerides. Similar conclusions were drawn by Lemaitre-Delaunay and
colleagues [Lemaitre-Delaimay et al. (1999) J. Lipid Res.; 40:1867-1874],
when they had study the kinetics and metabolic fate of labeled DHA on
triglycerides versus its enrichment in lysophsphasrtidylchoHne, and by
Wijendran et al. [Wijendran et al. (2002) id ibid.] in the above-mentioned
baboons study.
Interestingly, this increase la DHA content in the cortices of both PS- vs3
and LC-PUFA fed rats is accompanied with a statistically significant
decrease in the levels of oleic adds and to somewhat lower extent of
hnoleic add (Table 1) in the phospholipids fraction. Similar changes in the
ratios of the fetty acids profile was demonstrated by others, by feeding
rodents with dietary fets enriched with LC-PUFA [for example: Yamamoto
et al. (1987) J. Lipid Res. 28: 144-151]. The SB-PS group showed a very
similar profile to the MCT group.
In sum, the improved perfbrmazjce in the Monis water maze test of the
PS-133 treated rats under scopolamine sedation strongly supports the
potency of PS-tsS as an anti-dementia and age-associated memory
impairment effects. This cognitive enhancement is further supported by
the biochemical evidence of the elevated phospholipids levels in the Hver
and brain tissues (Fig. 4A-4B), and with elevated levels of DHA attached
to the phospholipids from the cortex of the PS- tuS &d rats.
Table 1 summarizes the effect of dietary LC-PUFA from different sources
on the fritty acids profile in cerebral phospholipids from elderly Wistar
rats. Fatty acids from the ptirified phosphohpids fraction were analyzed by
gas-liquid chromatography. The major fatty acids are expressed as % of
total fatty acids in the phospholipids. Values represent mean±S.D. of fovu:
different rats per treatment. Statistical significant between different
supplements and MOT group is presented as followed: * P P Table 1
i
Example 2 - PS-omega-3 in the treatment of ADHD children
Attention-deficit/hyperactivity disofder (ADHD) encompasses a broad
constellation of behavioural and learning problems and its definition and
diagnosis remain controversial [Kamper (2001) J. Pediatr. 139:173-4;
Richardson et al. (2000) Prostaglandins Leukot. Bsaent. Fatty Acids, 63(1-
2):79-87]. The etiology of ADHD is acknowledged to be both complex and
miilti-factoriaL Traditionally, ADHD is the diagnosis used to describe
children who are inattentive, impulsive, and/or hyperactive. Roughly 20-
25% of children with ADHD show one or more specific learning disabilities
in math, reading, or spelling [Barkley, RA. (1990) Attention-deficit
hyperactivity disorder: a handbook for diagnosis and treatment. New York:
Guilford Press]. Children with ADHD often have trouble performing
academically Eutid paying attention, and may be disorganized, have poor
self-discipline, and have low self-esteem. A conservative estimate is that
3-5% of the school-age population has ADHD [American Psychiatric
Association. Diagnostic and statistical mtuiual of mental disorders. 4th ed.
(DSM-IV) Washington, DC: American Psychiatric Association, 1994].
Treatments for ADHD include behavior therapy £md medications, mainly
methylphenidate (Ritalin*^. Psychostimidant drugs and antidepressants
are often used to calm children with ADHD, with an efEectiveness rate of
~75% (Swanson et al. Except Child 1993; 60:154-61). The advantages of
using these medications include rapid response, ease of use, efEectiveness,
and relative safely. Disadvantages include possible side effects, including
decreased appetite and growth, insomnia, increased irritability, and
rebound hypei^^ictivity when the drug wears off [Ahmann et al. (1993)
Pediatrics; 91:1101-6]. Moreover, these medications do not address the
underlying causes of ADHD. Thus, studies to elucidate the potential
contributors to the behavior problems in ADHD may lead to more effective
treatment strategies for some children.
Omega-3 fatty acids are specifically implicated in maintaining central
nervous system function. Deficiency of n-3 fatty acids in rats and monkeys
has been associated with behavioral, sensory, and neurological
dysfunction [Yehuda et al. (1993) Proc. Natl. Acad. Sci. USA; 90:10345-9;
Reisbick et al. (1994) Physiol. Behav. 65:231-9; Enslen et al. (1991) Lipids;
26:203-8]. Several studies have fixsused on essential fatty acid metabolism
in children with ADHD [Colquhoun et al (1981) Med Hypotheses; 7:673-
679]. Children with hyperactivity have been reported to be more thirsty
than normal children and have symptoms of eczema, asthma, and other
allei^es [Mitchell et al (1987) Clin. Pediatr.; 26:406-11]. For example, in
a. cross-sectional study in 6-12-y-old boys recruited from central Indiana,
it was showed that 53 subjects with ADHD had significantly lower
proportions of key fatty acids in the plasma polar lipids [arachidonic acid
(AA; 20:4n-6), eicosapentaenoic acid (EPA; 20:5n-3), and docosahexaenoic
acid (DHA; 22:6n-3)J and in red blood cell total lipids (20:4n-6 and 22:4n-6)
than did 43 control subjects [Stevens et al (1995) Am. J. Clin. Nutr.;
62:761-8]. However, recent publications [Elirayama et al (2004) Eur. J.
Clin. Nutr.; 58(3):467-73; Voigt et al (2001) J Pediatr.; 189(2): 189-96] that
investigated whether DHA supplementation would result with ameliorate
the sjnnptoms in ADHD children, suggested that careful attention should
be paid as to which fatly acid(s) is used. In these studies DHA
supplementation had demonstrated only marginal if any beneficial effects.
Recently, it has been suggested that one of the possible solutions to the
nutrient deficiencies which are common in ADHD, could be FS
supplementation (Kidd (2000) Altern Med Rev.; 5(5):402-28].
Method
Subjects and diet
Nineiy 8-to-13-year old children diagnosed according to the DSM-IV as
ADHD, were assigned randomly, in a double-bUnd fashion to receive PS-
tnS (300 mg/d; containing total 450 mg/d DHA/EPA), 450 mg/d DHA/EPA
or canola oil (30 per group) fi)r two months, while not taking stimulant
medication or other supplements. Characterizing the subject as ADHD
included a score lower than -1.8 in the Test of Variables of Attention.
Data Analysis
At the conclusion of the trial, ADHD children were scored according to
parental behavioural rating scales (Connors' Rating scale).
Results and discussion
Use of complementary therapies is particularly common among patients
with chronic, incurable, or frequently relapsing conditions. For example,
use of complementary and alternative medical therapies (CAM) is common
in children with cancer, asthma, and cystic fibrosis. Parents or subjects
who seek CAM typically do so because such therapies are more consistent
with their values, are more empowering, and are perceived as more
natural and less risky than conventional treatments. The majority of these
patients do not abandon mainstream therapies but use herbs and other
forms of CAM as adjunctive treatments. Only a minority ( their pediatricians about their use of CAM. Because of the stigma and side
effects that accompany use of stimulant medications, many families turn
to CAM to treat ADHD. Typically, only 70% of children respond to
stimulants such as Bitalin'™, and of those who do, approximately half
report side effects from their medications. In an Austrahan survey of 290
families seen at a multidisciplinary referral center for ADHD, 64% had
tried at least one "other therapy," most conunonly dietary restriction,
multivitamin supplementation, and occupational therapy [Stubberfield et
al. (1999) JPaediatr Child Health;36:460-3].
In the presented study the different supplementation was formulated into
a popular chocolate paste (see below). Using this matrix enable the
parents to administer the treatments in a non-conventional form to their
children and provided a reduced organoleptic effect characteristic of the
marine-derived compounds (see below).
The parental rating survey, at the end of the treatment period, measured
the attention deficit, hyperactivity and impulsivity of the children, as w^ell
as the aggression as assessed by parents, teachers, siblings and peers. The
results indicate a distinctively large placebo effect. This efEect is somewhat
reduced if the placebo-treated ADHD children that fiEdled to complete the
study due to severe behavioral deterioration are taken into consideration.
It seemed that most of these children insisted on reassigning for Ritalin^^
administration. However, the present data also clearly demonstrate PS-
0x3 as a potent agent. AU in all, ~70% of the parents of the PS- rnS treated
ADHD children indicated some improvement in the behavioural score of
their children, whereas 50% of these parents provided clear indications for
multiple beneficial effect of the supplement on their children behavior.
This prominent e£fect is 2.2-fold higher than the improvement obtained by
placebo (~30%). Comparison of the parental scoring of LC-PUFA on ADHD
children behavior with the parallel rating that followed three months of
PS- tD3 administration, point at the latter to have a higher score. While
both compoimds demonstrated similar extent of marginal improvement,
PS- m3 had a marked higher rate of substantial improvement (47% versus
35%, respectively) with the lowest rats of lack or deteriorating effects (21%
& 11% versus 26% and 17%, respectively). These effects of PS-bj3
supplementation could be attributed to both enhanced bioavailability of
omega-3 fatty adds and through PS well docxuaented effects on mood,
stress and anxieiy.
Example 3 - Effect of PC-DHA consiunption in Apo£° mice
Methods
Animal diet
Apolipoprotein E deficient (ApoE°) mice [Hayek T. et al. (1994) Biochem.
Biophys. Res. Commun. 201:1567-1574] at 8 weeks of age, were assigned
randomly (5 mice each) to LC-PUFA enriched lecithin (30% omega-3 of
total fetty acids composition; PC-DHA group) or placebo. The mice were
&d, besides the regular chow diet, once every three days with either 25 \il
PC-DHA or PBS, via oral gavage, during 10 weeks.
Each mouse consumed approximately 5 mL of water/day, and 5 g of
chow/day.
Serum lipids perosddation
Serum was diluted 1:4 in PBS. Serum susceptibility to oxidation was
determined by incubating serum sample with lOOmM of the free radical
generating compound, 2'-2'-azobis 2'-amidinopropane hydrochloride
(AAPH), which is an aqueous soluble azo compound that thermally
decomposes to produce peroxyl radicals at a constant rate. The formation
of thiobarbituric reactive substances (TBABS) and of lipid peroxides was
measured and compared to serum that was incubated under similar
conditions, but without AAPH.
Results and Discussion:
ApoE° mice are wide^ used as an animal model for atherosclerosis as they
develop severe hypercholesterolemia and atherosclerotic lesions on a chow
diet. Moreover, accelerated atherosclerosis is associated with increased
lipid peroxidation of plasma lipoproteins and arterial cells in these mice
[Hayek T. et aL (1994) id ibid.; Keidar S. (1998) Life Sci. 63:1-11].
Figure 6 shows how prolonged PC-DHA consumption by ApoEo mice
resulted in a clear tendency (P AAPH-induced oxidation by 16% (in comparison to placebo).
Organoleptic issues
The utilization of omega-3 lipids in a variety of applications, and
especially as ingredient of functional foods, is hindered due to their
distinct fish odor. Thus, another advantage of the omega-3 enriched
phospholipids ingredients of the invention is that they have reduced odor
or taste of omega-3 Acyl moieties, due to the covalent binding of these
groups to the PS backbone. This increases the vapor pressure of these
materials, hence reducing their distinct aroma. Thus, the covalent binding
of the omega-3 fatty acids to the phospholipid backbone, especially PS,
alters and improves their taste properties. Moreover, the PS ingredient of
the invention also offers enhanced stability to the oxidation sensitive
omega-3 fatty acids. Phospholipids in general, and PS in particvdar, are
known to act as anti-oxidants and stabilizers.
These benefits make this novel phospholipids' preparation of the invention
highly beneficial and important in a variety of applications and especially
in functional foods, where stability, aroma and taste are fiindamental
requirements.
Furthermore, these novel ingredients can be formulated with additional
lipids for an even enhanced bio-fimctionality and efficacy.
The starting compound used for the above-mentioned clinical trial in
ADHD patients, was LC-PUFA enriched PS mixed with fish oH.
Originally, this product and the control fish oil were formulated in food
products like energy bars; however the responses horn, expert panels were
categorically devastating, pointing at severe organoleptic problems. In
order to overcome this taste barrier the PS- in3 product of the invention
was de-oiled. The end-product of this process was a paste that when
reformulated with either inert or dominant - oiganoleptic satvirated fats
could be easUy formulated in chocolate bars, chocolate spread, chocolate
coated cornflakes, low-fat dairy products or concentrated milk. Each one of
these formulations had an evidently reduced organoleptic objection firom
both the expert panels and the trial volunteers.
The polar lipids derivatives of PUFA, such as the PS-PUFA derivatives
have exhibited high stability as a preparation and additionally in several
food applications, used in the clinical trials of this invention. This
stabiliiy, of these sensitive compoiinds is emerging &om the covalent
combination of phosphol^)ids, known in the past to be used as
preservatives and of the tin-stable PUFA moieties.
The stability of a commercially prepared fish oil (omega-3 fatty acid) for
laboratory rodent diet jLytle et al. (1992) Nutr Cancer; 17(2):187-94] or as
an enrichment in spreadable fats [Kolanowski et al. (2001) Int J Food Sci
Nutr.; 62(6):469-76] was addressed by several studies as the public
awareness towards the beneficial efEects of LC-PUFA increased. A major
effort was directed at maintaining the oxidative stability of the fish oU, as
these fatly acids are subject to rapid and/or extensive oxidation and other
chemical changes by eiqposure to air, light, or heat dviring processing or
when stored for various lengths of time. The common solution presented in
these studies was supplementation the fish oil matrix with antioxidants
like butylated hydro:Qdx)luene, butylated hydrosyquinone and alpha-
tocopherol, or alternatively, dilution of concentrated fi^h oil to a limit of
1% in a saturated fats matrix. However, Song and colleagues [Song et al.
(1997) Biosci Biotechnol Biochem.; 61(12):2085-8] had already evaluated
the peroxidative stability of DHA-containing oils the form of
phosphoUpids, triglycerides, and ethyl esters in the dark at 25°C in a bvdk
phase durii^ 10 weeks storage. They had shown that DHA-containing oil
in the form of phospholipids was more resistant to the oxidative
degradation of DHA than that in the &rm of triglycerides and ethyl esters
in a bulk phase.
The abovementioned PS-ta3 containing products utilized for the clinical
studies were tested for their shelf-life and stability in room temperature.
The enriched PS-ia3 formulated in condensed mWV (i g product per 10 ml
milk) was anal^rzed by ^iP-NMB for stability in cycles of freeze-thawing for
a week, and was found to be stable. In the second phase, PS-m3 in a
chocolate paste matrix (0.75 g product per 20 g chocolate spread) was
tested for stability after two weeks storage in room temperature. This
formulation also presented a stable percentage of PS, in 3ip.i analysis. In conclusion, we had been able to establish that Tn-3 containing
phospholipids are highly stable in room temperatiure, as well as in
freezing-thawing cycles, as oppose to tiT-3 containing triglycerides known to
rapidly decay after antioxidant consumption.
WE CLATM:
1. A serine glycerophospholipid conjugate with EPA and DHA, of formula (I) or a
mixture oi' sucii conjugates, useful for enhancing omega-3 fatty acids
bioavailability to the brain, improving memory, improving learning abilities and
treating Attention Deficit Hyperactivity Disorder (ADHD):
wherein R" is serine;
wherein each of R and R' may be identical or different and may be hydrogen, acyl EPA or
acyl DHA with the proviso that R and R' cannot simultaneously represent hydrogen;
wherein the amount of EPA and DHA present in the conjugate constitutes 10-50% by
weight of the total fatty acids content of said conjugate; and
wherein said conjugate is not identical to naturally occurring human or mammalian brain
PS;
2. The conjugate as claimed in claim 1 wherein the amount of EPA and DHA
present in the conjugate constitutes 20-50% by weight of the total fatty acids
content of said conjugate.
3. The conjugate as claimed in claim 1, wherein said glycerophospholipid conjugate
is prepared by enzymatic transphosphatidylation of a lipid source, preferably any
one of plant, animal or microorganism source.
4. A pharmaceutical composition comprising the conjugate as claimed in claim 1 or
a mixture thereof.
5. The pharmaceutical composition as claimed in claim 4, wherein the composition
optionally comprises at least one pharmaceutically acceptable additive, diluent or
excipient.
6. The pharmaceutical composition as claimed in claim 4, wherein the composition
optionally comprises at least one additional pharmaceutically active agent.

7. The pharmaceutical composition as claimed in claim 4, wherein said composition
is administrable orally or intravenously.
8. A nutraceutical composition comprising the conjugate as claimed in claim 1 or
mixture thereof.
9. The nutraceutical composition as claimed in claim 8, wherein said composition is
in the form of a softgel capsule, tablet, syrup, or other dietary supplement delivery
system.
10. A functional food comprising the conjugate as claimed in claim 1 or a mixture
thereof.
11. The composition as claimed in claim 10, wherein the functional food is a dairy
product, ice-cream, biscuit, soy product, bakery, pastry, bread, sauce, soup,
prepared food, frozen food, condiment, confectionary, oil, fat, margarine, spread,
filling, cereal, instant product, drink, shake, infant formula, infant food, bar,
snack, candy or chocolate product.

The invention discloses a serine glycerophospholipid conjugate with EPA and DHA. of
formula (I) or a mixture of such conjugates, useful for enhancing omega-3 fatty acids
bioavailability to the brain, improving memory, improving learning abilities and treating
Attention Deficit Hyperactivity Disorder (ADHD):

wherein R" is serine;
wherein each of R and R' may be identical or different and may be hydrogen, acyl EPA or
acyl DHA with the proviso that R and R' cannot simultaneously represent hydrogen:
wherein the amount of EPA and DHA present in the conjugate constitutes 10-50% by
weight of the total fatty acids content of said conjugate; and
wherein said conjugate is not identical to naturally occurring human or mammalian brain
PS;
The invention is also for a pharmaceutical composition, nutraceutical composition and
functional food comprising said conjugate.

Documents:

01297-kolnp-2006 assignment.pdf

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01297-kolnp-2006-abstarct.pdf

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1297-kolnp-2006-claims.pdf

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1297-KOLNP-2006-PETITION UNDER RULE 137.pdf

1297-KOLNP-2006-REPLY TO EXAMINATION REPORT.pdf

1297-kolnp-2006-specification.pdf


Patent Number 236010
Indian Patent Application Number 1297/KOLNP/2006
PG Journal Number 38/2009
Publication Date 18-Sep-2009
Grant Date 15-Sep-2009
Date of Filing 16-May-2006
Name of Patentee ENZYMOTEC LTD.
Applicant Address RAMAT GAVRIEL INDUSTRIAL PARK, P.O. BOX 6, 23106 MIGDAL HAEMEK
Inventors:
# Inventor's Name Inventor's Address
1 BEN DROR, GAI P.O. BOX 126, 30835 MOSHAV OFER
2 PLATT, DORIT P.O. BOX 249, 17906 SHIMSHIT
3 FARKASH, ORLY P.O. BOX 217, 17906 SHIMSHIT
4 ZUBAI, RASSAN VILLAGE 19320 KFAR NEEN
5 BAR-ON, ZOHAR 32 YODFAT STREET, 21950 KARMIEL
6 SHULMAN, AVIDOR 29 HAGOMEH STREET, 36090 KIRYAT TIVON
7 PELLED, DORI 29 HASHSHAR STREET, 45325 HOD HASHARON
PCT International Classification Number A61K 31/683
PCT International Application Number PCT/IL2004/000957
PCT International Filing date 2004-10-21
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 158552 2003-10-22 Israel