Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1 2 9 6 -1 0 1 A ~251782
NOVEL LIPID FR~CTION, ITS PREPARATION ~lD
PHARMACEUTICAL COMPOSITIONS CONTAINI~JG SA~SE
Field of the Invention
The present invention relates to a process for ~he
fractionation of lipids from natural sources into various
fractions, the fraction of choice being one with a
subs~an~ially increased potential for fluidization as well
as for the restoration of impaired functions of biological-
membranes, when compared with other lipid preparations.
The invention further relates to the fractions thus
separated, and to pharmaceutical compositions comprising
such fractionsO The invention further relates to the
treatment of various disorders connected with abnormalities
in the structure and dynamics of membranes such as in
aging, dysfunction of the immune system, mental and
- neurological disorders, drug addiction and alcoholism, and
hyperlipidemic disorders such as gallstones; hypertension
and atherosclerosis. The in vivo treatment comprises
administering an effective quantity of such fractionated
lipids. Treatment of other dysfunctions, such as sperm
infertility, can also be carried out in vitro.
Other and further objects of the invention will become
apparent hereinafter.
.~Z5~7
--2--
Background of the In~ention
The lipid fluidity (reciprocal of misroviscosit~-n~ of
biological membranes is determined by their s~ructure ana
chemical çomposition and, in particular, the mole ratio of
cholesterol to phospholipids (C/PL), the mole ratio of
sphingomyelin to 'ecithin (S/L) and the degree of
unsaturation of the phospholipid acyl chains ~Shini~zXy
and Henkart, Int.Rev.Cytol. 60, 121 (1979); Cooper, J.,
Supramol.Struct. 8, 413 (1978)).
The membrane lipid fluidity, in turn determines many
of the physiological properties of receptors (Muller and
Shini~zky, Brit~J.Haematol. 42, 355 (1979); Heron, et al.,
Proc~NatlOAcadOSciO USA, 77, 7463 (1980); Heron et al., in
"Recep~ors and their Neurotransmitters", eds. Littauer et
al~, John Wiley, London (1980); Heron et alO,
EurOJ~Pharmacol~ (in press), anti-gens (Shinitzky and
Sourojon, Proc.Natl.AcadOSci. USA, 76, 4438 (1979) e~zymes-
(Sandermann, Biochim.BiophysOActa, 515, 209 (1978); Rimon
et alO, Nature, 270, 267 (1977), transport carriers
(Kimelberg, Biochim.BiophysOActa, 413, (1975), ion channels
(Stephens and Shinitzky, Nature, 270, 267 (1977), and
ribosomes (Towers et al., Biochim.Biophys.Acta, 287, 301
(1972) which are bound to these membranes in the brain and
other organs. This subject was recently extensively
reviewed (Shinitzky, Physiol. Rev. in press).
The final response of target cells depends, therefore,
on the structural and dynamic properties of their
membranes, which are determined by their lipid composition.
One may, therefore, expect an optimal lipid fluidity for
the maximal response of each target cell (Heron et al.,
Proc~Natl.Acad.Sci. USA, 77, 7463 (1980); Heron et al., in
"Receptors and their Neurotransmitters", eds. Littauer et
al., John Wiley, London (1980); Yuli et al., Biochemistry,
20, 4250, (1980): Shinitzky, Physiol. Rev., in press).
In many disorders, the pathogenesis involves changes
in membrane lipid composition or lipid metabolism (Cooper,
~ 25 ~7
--3--
N.Engl.J.Med., 297, 371 (1977)). These changes have been
correlated in many cases to an increase in membrane lipid
miscroviscosity of various tissues due to an incrPase in
C/PL or S/L or a decrease in the degree of unsaturation of
the phospholipid acryl chains or any combination of the
three. Lipid peroxidation can al30 affect the dynamics of
cell membra~e proteins and consequently the overt
physiological functions ~Sagai and Ichinose, Life Sci., 27,
731 (1980)). The following is a list of such disorders
mediated by lipid imbalances, all of which are amenable to
lipid manipulations.
~1) Aging and senescence (Yamamoto, Lipids, 3, 284
U968~; RiYnay et alO, Mech. Age.DevO 10, 71 (1979); Heron
et al., to be published; see also Table 4 in this
specification; Araki and Rifkind, Life Sci. 26, 2223
(19~0); Hershkowitz et al., Progress in Brain Research,
Elsevier-North Holland, in press~, Ronser et al., Adv Lipid
Res. 10, 262 (1972). -~
(2) Withdrawal symptoms of drug and alcohol addiction
(Johnson et al., Mol~Pharmacoll, 15, 739 ~1979); Chin and
Goldstein, Science, 196, 684 (1979); Littleton and John,
JOPharm.Pharmac.,~29, 579 (1977); Heron et al., Biochem,
Phanmacol. in press (1982); (see also Table 3 in this
specification).
t3) Hyperlipidemic disorders such as hypertension,
atherosclerosis, gallstones, cirrhosis, and obesity
(Montenay et al., Biochem.Biophy.Res.Comm. 100, 660 (1981);
Cooper, N., Engl.J.Med., 297, 371 (1977); Miettinen et al.,
Lancet 2, 835 (1972). See also Table 8 in this
specification.
(4) Sperm infertility (Davis et al., Biochim.Biophys.
Acta, 558, 257 (1979): Davis, Proc.Soc.Exp.3iol.Med., 152r
257 (1~76)).
(5) Impaired immune function such as in aging,
obesity and certain cases of allergies (Rivnay et al.,
~ech.Age.Dev., 12, 119 (1980); Rivnay et al., Mech.Age.Dev.
10, 71 (1979). See also Table 9 in this specification.
~5~7~2
--4--
We have also shown that synaptic membrane
miscroviscosity increases as a result of surgical or
chemical lesions of specific pathways in the brain ~H~ron
et al., Biochem.Pharmacol., in press (1982). Thçse findings
may apply to other degenerative or orgnaic damages 5uch as
Alzheimer's disease, Parkinsonism, Tardive dyskinesia,
Huntingtcnls chorea, tremor, ataxia, and epilepsy and
certain cases of mental retardation, all of which could in
principle be treated by lipid manipulation.
It is also generally accepted that certain mental
disorders such as mania, depression and schizophrenia are
related to a chemical imbalance in the turnover rate of
neutrotransmitters in the brain. There is evidence to
suggest that the biogenic amines (dopamine, norephinephrine
and serotonin) are primarily involved5 The receptors and
membranes bound enzymes concerned with the turnover of
these transmitters can be altere~ by changes in membrane
fluidity (Hershkowitz et al., Progress in Brain Rese~rch,
Elsevier~North Holland, in press; fferon et al.,
Proc.Natl.Acad~Sci. USA 77, 7463 (1980), ~eron et al~, in
"Receptors and Their Neurotransmittersn, eds. Littauer et
al~, John Wiley, London (1980); Heron et al., Eur.
JOPharmacol., 72, 361 (1981), adn therefore also falls into
the category of disorders amenable to lipid manipulations.
Modulation of function by lipid manipulations can also
be carried out in vitro. This could be applicable to
modulation of viral infectivity for use in vaccinations
(Pal et al., Biochemistry 20, 530 (1981), and antigenicity
(Shinitzky and Souroujon, Proc. Natl.Acad.Sci., USA 76,
4438 (1979)), which could reduce tissue rejection and
facilitate transplatations.
We have found that some of the adverse effects
mediated by lipid imbalances could be rectified by a form
of "membrane engineering", through tne use of an action
fraction of lipids from natural sources. This fraction
(which contains a substantial portion of lecithin) can
operate via several possible mechanisms:
~LZ5~7~:2
--5--
(1) Extraction of excess cholesterol b~ pa3siJe
translocation (Cooper, J.Supramol.Struct., 8, 413
(1978), Miettinin et al., Lancet, 2, 835 (1~72);
Morrison, Geriatrics 13, lZ (1958); Caoper e~ al,,
J.Clin.Invest. 55, 115 (1975)).
(2) Exchange with membrane lipids of higher micro-
viscosity (Wirtz and Zilversmit, Biochim.BiophyO.
Acta 193, 105 (1969).
(3) Net incorporation into or replacement of damaged
lipids (e.g. peroxidized) (Bakardjieva et al.,
Biochemistry 18, 3016 (1979)). This could restore
the structure and function of degenerate
membranes.
(4) Precursors in various metabolic pathways (e.g.
prostaglandins, vitamin D and acetylcholine).
However, diets having a hign content of lecithin,
which are frequently recommended -for a variety of disorders
(Cobb et al~, Nutr~Metab., 24, 228 (1980); Blas~ -~
(Cornall-Burke Rehabilitation Center); Gershon (Lafayette
Clinic, Detroit); ~eyman (Duke University Med. Center);
Sul~ivan et al., (M~IoT~ and Tufts Univ.), in "Proceedings
of the International Study Group on the Pharmacology of
Memory Disorders Associated with Aging", Zurich (1981)),
are not very effective in all viating symptoms associated
with lipid imbalances and in restoring membrane lipid
fluidity to normal. The reasons for this are not yet
clearO It seems that the previously proposed rationale for
these lecithin treatements, which are based either on its
acetycholine precursor role or on the high degree of
unsaturation covers only a minor aspect of this approach
(~erring et al., Biochim.Biophys.Acta 602, 1 (1980),
Shinitzky and Henkart, Int. Rev. Cytol. 60, 121 (1979)).
It is plausible that the process of lipid manipul~tion
combines several prerequisites such as the following:
(1) Fluidization is effected by a well defined portion
of the lipids, while the rest serve as essential
~2S~78~
-6-
carriers which facilitate transport and abDorp~ion
into the membranes.
(2) The assembly of the active ~nd the carrier
components is of defined physico-chemical
characteristics, such as the surface density and
charge distribution.
(~) These characteristics could be optimal for prop~r
tranSportation~ associated with cell surfaces,
disintegration, unloading or exchange, as dictated
by the site of interaction.
(4) The various lipid components could act
synergistically to efPect the activities described
above.
(5) ~he degree of unsaturation i5 optimal, i e. it has
the necessary fluidity charac~istics (the transi-
tion from fully saturated to mono-unsa urated is
the most critical to flu-idizin~ ability, while the
transition from mono to poly-unsaturated do-es not
significantly change the fluidizing ability
(~ubbel and McConnell, J Am.Chem Soc~ 93, 314
(lg71); Stubbs et al~, Biochemistry 20, 4257
(1981)), and yet not too unsaturated, thus less
vulnerable to oxidationO
Summary_of the Invention
According to the present invention there is provided a
novel process for the fractionation of lipids and
preferably of lipid extract, from natural sources, into at
least two fractions, one of these, the one to be used for
purposes of the present invention, having a substantially
higher potential for membrane fluidization than other
preparations. This fraction is hereafter referred to as
"active lipid", (AL), and is a novel composition of matter,
which is characterized by a high potency of membrane
fluidization.
~LZ5~7~
--7--
The invention further relates to the use of AL in
the treatment of various diseases and anomalous stat~s in
mammals and also in humans and to pharmaceutical composi-
-tions for such treatments. Amongst conditions amenable
to treatment by means of the compositions according to
the invention are the followiny:
1. Various symptoms of aginy and senescence (e.y. loss
of mental functions and libido, increased vulnerab-
ility to bacterial contaminations, etc );
2. Dysfunctions of the immune system;
3. Allergies;
4. Mental disorders such as manic-depression and schizo-
phrenia and the like;
5. Mental retardation;5 6. Neurological disorders such as Alzheimer's disease,
Parkinsonism, Tardive dyskinesia, Huntington's phorea,
tremor, ataxia, epilepsy and the like;
7. Hyperlipidemic states such as hypertension, athero-
sclerosis, gallstones, cirrhosis and obesity, and the
like;
8. Symptoms of withdrawal from alcohol and other drugs;
9. Prevention of tolerance to drugs.
The invention further relates to the use of AL (in vitro
or _ vivo) in the treatment of infertiliiy, viral and
microbial contaminations, and reduction of tissue anti-
genicity which could reduce tissue rejection and facili-
tate transplantations etc.
The invention further relates to the treatment of the
above mentioned disorders, by administering (either in vivo
or ln vitro) effective quantities of AL having a substant-
ially increased capacity for membrane fluidization compared
with other lipid preparations.
Statement of the Invention
According to the invention, as described and claimed
herein, there is provided a novel composition of matter
comprising a lipid fraction derived from natural sources
(AL), said lipid fraction containing 40-80 weight percent
~25~7b~2
-7a-
glycerides, 3-5 weight percent cholesterol, 10-30 neiyh'
percent lecithin (phosphatidyl choline), 5-15 weight per-
cent phosphatidyl ethanolamine and 2-5 w2ight percent
negatively charged phospholipids, wherein the ratio of
unsaturated to saturated fatty acids is at least 1:1.
According to a further feature of the invention, as
claimed herein, a preferred novel composition compri~es
a lipid fraction (AL) derived from natural sources, said
lipid fraction containing 60-70 weight percent glycerides,
3-5 weight percent cholesterol, 15-25 weight percent lec-
ithin (phosphatidyl choline), 5-10 weight percent phos-
phatidyl ethanolamine and 2-3 weight percent neyativel~
charged phospholipids wherein the ratio of unsaturated to
saturated fatty acids is at least 1:1.
According to still a further feature of the invention,
as claimed herein, a more preferred composition comprises
a lipid fraction (AL), as defined in the immediately pre-
ceding paragraph, wherein the fatty acid composition of
the lipid fraction is such that there is present the fol-
lowing fatty acids: palmitic acid 35-45%, oleic acid 35-
45%, linoleic acid 5-10%, stearic acid 5-7%, palmitoleic
acid 2-3%, arachidonic acid 0.2-1%. In such a preferred
composition, it is further most preferred that the ratio
of unsaturated to saturated fatty acids is at least 2:1.
The fractionation is effected by dissolving a lipid
extract from a biological source (e.g. egg yolk, soybean)
in a suitable solvent, evaporating the solvent to almost
complete dryness and precipitating a fraction of the
dissolved lipids by addition of an organic solvent, and
~ ~S ~7 ~Z
--8--
recovering the desired fraction frotn the sup~rnatant ~y
evaporation of the solvent. This fraction i5 supplemented
with a 0.5% (w/w) tocopherol or any other suitable
antioxidant. Alternatively, the fractionation is effected
by treating the lipid source (e.g. egg yolk, soy~ean~ ~ith
a suitable solvent, removing the precipitate and then
dissolving it again in a suitable solvent and recovering
the supernatant. The desired fraction is then recovered
from the supernatant either by evaporation of the solvent
or by precipitation in the cold and then evaporation of the
traces of the solvent. This fraction is also supplemented
with 0.5% (w/w) tocopherol or any other suitable
antioxidant.
The three preferred embodiments of the invention are
the following
(1) The lipid extract from egg yolk (eOg. crude
lecithin) is dissolved in chlorof~rm, evaporated to almost-
dryness, acetone is added to effect a precipitation ~~f a
certain part of the lipid, and the supernatant is removed,
evaporated and the solvent is removed to complete dryness,
leaving a fraction of about 5 weight percent of the initial
quantity of the untreated egg-yolk, which is the desired
fraction A~. An antioxidant such as tocopherol is added to
a final concentration of about 0~5% (w/w). Analysis of the
lipid composition of this fraction (Preparatin 1) is given
in Table 1.
(2) A natural lipid source (e.g. egg yolk, soybean) is
first mixed with acetone to remove excess undesired lipids.
The precipitate is then treated again with acetone, and the
supernatant is collected, evaporated to complete dryness,
leaving a fraction of a~out 10-15 weight percent of the
initial quantity of the untreated egg-yolk, which is the
desired fraction AL. An antioxidant such as tocopherol is
added to a final concentration of about 0.5% (w/w).
Analysis of the lipid composition of this fraction
(Preparation 2) is also given in Table 1. Arnongst other
solvents which can be used there may be mentioned:
~zs~
g_
chloroform-methanol l:l v/v, hexane, tetrahydrof~ran,
acetonitrile, ethanol, methanol, diethyl ~fher and di~th~l
ketone.
(3) A natural lipid source ~e.g. egg-yolk, so~bean~ i~
first mixed with acetone to remove excess undesired lipid3.
The precipitate is then treated again with acetone, and fhe
supernatant is collected and cooled below 0C, uporl which
the desired fraction (AL) amounting to about 10-15 weight
percent of the initial quantity of the untreated egg-yolk,
is precipitated and collected. An antioxidant such as
tocopherol is added to a final concentration of about 0.5~
(w/w). Analysis of the lipid composition of this fraction
(preparation 3) is given in Table 1 and 2. Amongst other
solvents which can be used there may be mentioned:
lS chloroform-methanol 1:1 v/v, hexane, tetrahydrofuran,
acetonitrile, ethanol, methanol, diethyl ether and diethyl
ketoneO - -
Description of the Pre~erred Embodiment
.. . . ... _ . . ....
The lipids are extracted from a quantity of lO g dried
egg yolk by first mixing with 50 ml of acetone. The
precipitate is removed and then treated with 50 ml
chloroform and the solution, which contains the lipid
extract, is collected. The chloroform is then removed
under reduced pressure to almost dryness. A quantity of 50
ml of cold (5-10C) acetone is added, and this results in a
precipitation of the majority of the lipids within 1-3
hours. This precipitate is discarded, the supernatant is
collected and the acetone is completely evaporated. There
remains the desired fraction of active lipids (AL) weighing
0.8-1.2 grams. This fraction is supplemented with 0.5~
(w/w) tocopherol. The composition of this preparation (~l)
is given in Table l.
A modified procedure (#2) which yields similar results
is described in the following: lO ml of fresh egg yol:~ is
mixed with 30-40 ml acetone and stirred for S minutes at
~ 25~7~
--1 o--
room temperature. The precipitate is collected and
extracted with 30-40 ml of fresh acetone at 40-45C for
30-60 minutes. The supernatant is collected and evaporated
to complete dryness. There remains the desired frac~ion of
1.0-1.5 gram active lipids ~AL). This fraction is
supplemented with 0.5% tocopherol. The composition of thio
preparation (#2) is also given in Table 1.
Preparation #3 is prepared as follows:
One volume of fresh egg-yolks is mixed with 2-3 volumes of
acetone containing 1 mg/ml vitamin E (a-tocopherol asetate)
at room temperature for 5 minutes. The solid material i3
separated and treated with 2 volumes of fresh acetone at
40-45C for 1 hour. The acetone extract is separated from
the solid residue by fast filtration and is cooled to -20~C
for 16 hours, upon which the Active Lipid (AL) precipitates
out5 AL is separated by fast filtration/ washed with
ethanol and exhaustively dried (under vacuum). The product
is supplemented with 0.5% vitamin E~ The yield is 1-~-15 gr
out of 100 gr wet egg yolks.
~2~
Table 1: Composition of AL
Preparation #l Preparatlon #~ Pr~para~r,n $
weight % weight % ,~ight
Neutral lipids 50-70 70-80 65-75
(total~
(a) glycerides 40-60 60-70 60-70
(b) cholesterol 3-5 3-5 3-5
(c) others less than 5 less than 5 less than 5
Lecithin (phosp'natidyl20-50 10-20 15-25
choline)
~hosphatidyl aEhanolamine 10-lS 5~10 5 10
~ga~ively charged 2~5 2-5 2-3
Phospholipids
Unsatura~ed/saturat~dabove 2 ~ above 2 above 1
--1 2--
Table 2: Fatt~ A~id C~ition o~ PrsparatirJn ~3
(h Typ~cal E~7UarD1e)
Fatty acid AL NL FC P2
diglycerides triglycerid;os
1600 39.8 42.5 27.6 ~2.~ 33.3
16:1 2.5 S.O 14.5 -- --
18:0 6.6 5.8 12.3 13.5 15.1
18:1 42.5 40.0 26.3 34.8 42.7
18:2 8.2 6.6 19.3 8.9 8.9
2~ 4 004 - - - _
~5~7~Z
-13
The activity of AL as a lipid fluidizPr, is demon3trafed
in the following experimer.ts. .~ouse brain mPm~ranes ~Crud?
mitochondrial fraction - P2m, prepared from the r,ouse
forebrain) was incubated with lipid dispersion in 50 mE~
Tris- HCl buffer pH 7.4 containing 3.5~ polyvin~l
pyrrolidone (PVP) (0.2 mg/ml) for 30 minutes at room
temperature, with constant shaking (Heron, et ai., Proc.
Natl. Acad. Sci. U~A 77, 7463 (1980). The final
concentration of lipids was 0.04 mg lipids per 1 mg P2m
membranes. The membranes were then extensively washed and
the lipid microviscosity (n) was determined according to
Shinitzky and Barenholz, Biochim. Biophys. Acta 515, 367
(1978). The cholesterol and phospholipid content were
determined according to Bartlett, J~ Biol. ChemO 234, 466
(1959), and Brown et alO, AnalO ChemO 26, 367 (lg54),
respectively~ It can be clearly seen in Table 3 that AL is
superior to all other lipids tested in its fluidizing
potency. It can be also seen in Table 3 that the --~
fluidization is effected ~oth by cholesterol extraction and
by net incorporation of the phospholipidsO Similar
experiments with mouse spleen cells were carried out. The
cells (10~/ml) were incubated with lipid dispersion (0.3
mg/ml) in phosphate-buffered saline (PBS) containing 3.5%
PVP for 2 hours at 37C, and washed extensively
(Shinitzky et al. Proc. Natl. Acad. Sci. USA 76, 5313
(1979)o The results are summarized in Table 4. Again, it
can be clearly seen that AL is much more potent than PC in
its fluidizing capacity.
~L~25~7~
-14-
Table 3
The effects of various lipids Gn the microvis~sit~ ( O of m~u3e
membranes(P~m) and their cholesterol (C) and phosp'rlolipid (PL) o~ntent
Treatment n C/pr~tein P~prokein C~EL
_ (25C, poise) _ _ _ (w/w) (w/w)~ /M)
Control 4.7+0.2 4.1~rJ.31.0~0.2 0.3310.4
(Vehicle treated)
AL 2.8+0.3 2.3+0.31.~+0.3 0.11~0.2
(Preparation #3)
Crude egg-lechithin 4.0+0.1 3.6+0.21.1~0.2 0.27+0.4
("Sigma", Grade II)
Pure egg~lecithin 4O1+0.2 3.3+0.41.5+0.3 0.18~0~3
(PC) ("Lipid-Products"~
Nutfi~ld~ England)
Dipalmit~yl~leci~hin 6~4~0O2
(DPL)
(Koch=Light Labs,
Golnbrook, England _O
The results represen~ the mean~OD~ of ~t least lO experiments, each wi~h a
different batch of AL.
~;25~7~
- 1 s
Table 4
The effect of various lipids on the micro~iscosit~
(n) of mouse spleen cells.
~ . . - ~
Treatment rl
_ (259C, poise)
Control 3~5+0.2
Vehicle treated
hL 2.3~0.3
(Preparation #3)
PC 3.3~0.2
( n Lipid p~oducts n
Nutfield~ Enyland)
The results r~present the mean+SOD. of at least 5
experiments r each with a diff~rent batch of ~L.
25~7~
- In addition to being use~ul or ln ~i ro
manipulations, as described above, the above ~ fract~ on3
can be used as an active ingredient in drugs ~dminiatGred
to warm blooded mammals for the trea~ment of condition,
where the structure and dynamics of the membrane lipids i3
impaired.
The effective quantities of the fraction vary with th~
condition treated and the needs of t~e patient, but the
effecti-~e quantities for waxm blooded mammal~ are in th~
order of from 1 g to 20 g per patient per day. The novel
fraction is advantageously administered inkraveneously in
the form of lipid suspension in saline (10-100 mg/ml).
For the in vitro manipulatio~s the effective
quantities are in the order of 50-200 mg/106 cells in 1
ml medium. Such in itro manipulations may be used to
treat sperm infertility, facilitate tissue transplantations
or to modulate v-ral infectivity for use in vaccinations.
Suppo~tive Res~lts
~1) Reduction of the Withdrawal Symptoms in Morphine
Addict2d Mice
Four groups of male Balb/C mice were injected
subcutaneously with morphine (between 40 and 200 mg/kg
twice daily for eight days). On the 9th day each group was
injected intraperitoneally with (a) saline (0.3 ml), (b)
dipalmitoyl lecithin (a synthetic fully saturated membrane
rigidifying agent), (c) AL by i.p. injection, or (d) AL
given in the diet. All four groups were then injected with
2.5 mg/kg naloxone, a morphine antagonist known to
precipitate withdrawal symptoms. These symptoms were then
scored in an observation chamber and the results are shown
in Table 5.
The microviscosity of the synaptic membranes from the
different regions of the brain, was measured by
fluorescence polarization using diphenylhexatriene (DPH) as
a probe, by the method of Shinitzky and Barenholz, Biochim.
~5'~7~
-11-
3iophs.Acta 515, 367 (1978). The results are also gi~en ir
Table 5, and are compatible with the suggestion that
membrane microviscosity ( ) is increased during chronic
morphine intake. This is probably due to an increase in
5 C/PL so as to compensate for the fluidizing effects of the
drug. Similar results have been reported b~f others
~Johnson et al., Mol.Pharmaco . 15, 739 (1979); Chin and
Goldstein, Science 196, 684 (1977)) for alcohol addiction.
It can be seen in Table 5 that the withdrawal symptom3
were aggravated by dipalmitoyl lecithin (which induces an
increase in membrane microviscos:ity), and reduced, or almost
entirely eliminated, by AL - both when injected or given in
the diet, with concomitant dec~eases in membrane
microviscosity.
As was mentioned above, chronie alcoholism also
involves increased cholesterol in synaptic membranes, in
order to compensate for the fluidizing effects of alcohol,
and therefore alcohol withdrawal is also amenable to -
treatment by AL~
Finally, since the process of adaptation (i.e.
tolerance) to morphine and other drugs involves increase in
C~PL mole ratio in the membranes, ~L given in conjunction
with drugs such as morphine etc. could prevent t~e
development of tolerance and therefore the decreased
potency of such drugs~ This approach could be of paramount
importance, for example in cases of terminal cancers
receiving morphine to ease the pain.
~L2~i~7~Z
--1 8--
Table 5
The effect of lipids on naloxone-precipi'cated ~"ithdrawal
symptoms and on brain membrane lipid 1uidit~y in m~rphine
5 dependent mice.
AL Saline Dipal}nitoyAl Diet
(Control3 Lecithin
n=32 n-28 n-16 n=10
J~ps 3+.07(*) 21~4 35+8(t) 11+4,4(~)
Body shakes 4~.9(*) 17~2.5 19~2.3(n.0)4+1.6~*~
Forelinb tremorl<40% >80~ >90% C60%
Diarrhea2 ~50% ~go~ ~90~6 ~75~6
Writhing3 <50% >75% >90% <50%
Penile ejaculation4 >75% >50% >5096 >75%
n, 25~C (poise)
E11ppocampus5.95+0.03(*) 6041~0.04 6O58+0~07(Y) 6010*0.7(*)
Caudate 6041+0.06(*) 6.75 .05 6.75+0008(nOs.) 6.58+0.09(-t)
Values represent the mean S.E.M. from four separate
experiments. "n" is the total number of animals tested.
The microviscosity ( ~i) values of hippocampus and caudate
from naive mice (n=20) were 5.80+0.03 and 6.10+0.05,
35 respectively.
1. Percent of animals showing continuous and strong tremor
of forelimbs (more than 70 episodes).
2. Percent of animals showing severe diarrhea with soft
liquid feces.
40 3. Percent of animals showing more than 10 episodes.
4. Percent of animals showing more than 5 episodes.
Other symptoms such as rearing, grooming, sniffing,
biting, etc. were also less prominant in the AL groups
compared to saline or dipalmitoyl lecithin. In general,
45 the AL groups were very calm most of the time and with
weaker symptoms, while the diapalmitoyl lecithin groups
~5~7~
- 1 9 -
were strange and aggres~ e even before the naloxone
injections, and afterwards showed the mos~ ~e~ere s~mptoms
of all groups.
Student t-test significant levels:
(*) - p<0.001, ( ) - p~0.025,
(~) - p<0~05, (#) ~ p<0.1,
(n.s.) - not significant
2. Reversal of MicroviscositY of Brain ~mbranes
of Old Animals by AL Diets
Four groups of mice were used in this experiment, in a
classic T-square design. Two groups consisted of young
(2-3 months) and two of old mice (24-27 months), of which
group was treated with AL given in the diet (mixed with the
Purina Chow) for 1~-20 daysO The results are shown in
Table 60
7~,~
-20-
Table 6
Membrane lipid microvisocisty (n) of various brain
preparation from various brain regions of young (2-.
months) and old (24-28 months) Eb/Bl mice before and after
treatment with AL
_ _ .
Prep~ration Brain ~, 25~C ~poise)
Region
Old (AL) Old (control) Young Young(AL)sf
(control )
SPM ~orebrain 5.4+û.4(20)t 6.4+U.8(20)~ 5.0+0.2(20) 4.9+0.2(6)n.3.
Mitochondria " 3.4+0.3(14)t 4.0+0.2(14)~ 3.4+0.2(14) --
Mic~osome~ " 4r9~0.3(12)n~sO 5.1~0.2(12)n.s. 5.0+0.1(12) --
C~uds nuclei " 7O5~0~4(12)1 7O9 0.4(12)~ 7.5+0.2(12) -- ~act ion
My~lin " 9.3~0.6(1~)nOc. ~.3+LA(12)n.~. 9.1+û.4(12) ~
Crud~ " 508~001(14)t 6.1+002(14)* 5.5+0.2(2û) 5.4+0.2(12)n.s.
homog0nat2
Dissociated Hippocampus 6.0+0.2(20)t 6~4~0O2(20)~ 504+0o2(24) 5.4~0.2(12)n. i.
cell~
1~ Caudate 6.2~0.2(20)n.~. 6.4+0.2(20)* 5.5+0.2(24) 5.5~0.3(12)n.s.
The data represent the mean +S~D. of 4-5 separate
experiments, each group included 4-6 animals. Numbers in
parenthesis represent the number of determinations, carried
out in duplicate on samples prepared from 2 pooled animal
brains.
* ~ p<0.01 old (control) as compared to young (control)
t - p<0.01 old (AL) as compared to old (control)
n.s. - not significant
** - young (AL) compared to young (control).
It can be clearly seen that AL reversed the
hyperviscosity of various brain preparations from old
animals, especially of SPM (synaptic plasma membranes) and
~5~7
--2 1 --
mitochondria, while those taken from young animal3 "ere no~
affected at all by AL treatment. Similar "rejutenating"
effects were found also in the binding charact~ristic3 of
receptors such as serotinin receptors and in protein
phosphorylation ~Hershkowitz et al., Progre~s in Brain
Research, Elsevier-llorth Holland, in press) in the brains
of old animals, while no effects o AL treatment of young
animals were observed. This fact implies that in ~oung
animals with normal membrane microviscosity, loading "ith
1~ lipids (e.g., AL treatment) has no effect due to efficient
regulatory processes. In aged animals, however,
"homeoviscous adaptation" (Sinensky, J.CellOBiol., 85, 166
(19&0)), is impairedO This implies that
there is no danger of AL overdose, since excess AL is
either removed or compensated for by changes in other
lipidso The clinical implications of these results are
obvious~ -
It should be mentioned that in all cases of ani^mal
treatments ~by diet), no discernible toxic or side efîects
were observed~
Finally, it should be mentioned that protein synthesis
by membrane bound ribosomes was found to be substantially
decreased in the cerebeilum of old animals. These changes
are probably due to the changes in membrane composition and
structure.
Preliminary results show that treatments of old
animals with ~L (in diet) caused a non-specific general
increase in protein synthesis in the cerebellum of these
animals~
3. The_Effect of AL on the Immune Function
The main immune mechanism operating against bacterial
infections, is the ingestion of bacteria by macrophages.
We have followed this process after treatment with AL in
vitroO The results of a representative experiment are
shown in Table 7.
-22-
Table 7: Number of Staehilococcus Auereu3 Colonies
0 hour 1 hour 2 hour
Young donor ~1 380 50 20
Young donor #1 ~ AL 375 55 18
Young donor #2 385 62 17
Young donor ~2 + AL 380 61 31
Old donor #1 390 372 352
Old donor ~1 + AL 375 150 92
Old donor ~2 383 3~1 348
Old donor X2 + AL 381 180 70
-~
AL was added to whole blood (heparinized) from old or young
donors to a final concentration of 400 ~g/ml. The blood
~as then incubated at 37C for up to 2 hours. ~g ml of
whole blood either treated or untreated from the donors was
lS then added to 71 ml of 1.1000 dilution of a 0~6 O.D. (620
nm3 suspension of Staph. aureus in PBS. At indicated times
10 Ul of above b~ood mixture was added to 5.5.3 agar
maintained at 60C. The agar-blood mixture was vortexed at
high speed and poured into a petri dish and allowed ~o
cool. The plates were incubated overnight at 35C and
colonies counted the following morning (Kensel, et al.,
J.Infect.Dis. 131, 584 (1975)).
The number of colonies indicate the number of
surviving bacteria. It can be clearly seen that the number
of surviving colonies was very much reduced in the cases of
blood from young donors, indicating an efficient immune
response. AL had no effect in these cases. In the cases
of blood from old donors the number of surviving colonies
only slightly decreased indicating an impaired iminune
'~ ~5
-23-
response. AL had ~rejuvenating'7 effects on the immune
system which showed restoration of function. The clinical
implications are obvious.
~. Effect of AL on human lymphocytes activity in ~itro
~ . . .
Peripheral blood lymphocytes from old males (70-75
years) were mixed with irradiated lymphocytes froM ysung
(30-40 years) in a classical mixed lymphocytes assay (MLC).
Sensitization of the lymphocytes was assessed by
incorporation of 3H-thymidine. In the presence of 0.2
mg/ml AL the thymidine incorporation by the lymphocytes
from old men increased by 70-300~ indicating a marked
increase in immunological responsivenessO
A_ Effects on H~pertensive Rats
.
Spontaneously hypertensive ,emale rats (SHR) were
purchased from Charles Rivers (N~Y.) and raised locally
until reaching 5 months of aye. -One group of 10 ra~s
received a diet supplemented with 5~ (w/w) AL for 3 weeks.
The control group (9 rats) were fed in a similar manner but
without AL supplementation. The mean art2rial blood
pressure (~.A.B.P~ was then measured. In the control group
the M~A~B~Po was 125 +16 (mm~g) while that of ~he AL
treated animals was 110+12. AL signiEicantly (p<0.05)
reduced the MABP of the hypertensive rats, Concomitantly
it also reduced the microvoscosity of P2m membranes taken
from the striatum from 6.5+0.2 poise (25C) to 5.5+0.2
poise. There was no significant difference between the two
groups in heart rate or weight.
6~ Other Effects of AL:
Preliminary results indicate that the various symptoms
of amphetamine-induced psychosis in rats, were eased or
almost entirely eliminated by AL (done in collaboration
with G. Ellison, Brain Research Ins., UCLA).
~2~7~2
--2~--
7. Procedure for Preparation of Active Lipid
A. Materials: Fresh big hen eggs, distilled a~etor.e;
0.1 gr/ml tocopherol acetate ~vitamine E~ in
ethanol, 0.1 m M~C12, 0.1 m CaC12 in ~,Jater ~salt
solution).
B. Procedure:
~1~ Mix 1 liter of egg yolks (approx. 60 eggs)
with 2 liters of distilled acetone, 5 ml
vitamine E at room temperature for 5 minutQs.
Collect the precipikate on a synter glass
funnel.
~2~ Transfer the precipitate to 2 lit~rs of
acetone preheated to 45 degree in a thermo-
stated bath~ ~dd 60 ml sal~ solution and
mix well while at 45 degree ~or 1 hr. Eilter
fast hrough synter glass funnel and collect
~he liquidO
~3~ Transfer the liquid to an acetone dry ice
cooling bath (between 60 degree to -~7~
2~ degree C) whereupon a precipitate is formed.
After 3 hrs. in the cold, collect the
precipitate by fast filtration. The weight
ratio of phospho-lipids to glycerides should
be around 1-3. This is determined by
phosphate analysis and should be checked
for each production scale (see comments).
~4) Thaw the precipitate by warming and transfer
to an evaporator vessel and add 5 ml of
vitamine E. Evaporate the sludge to complete
dryness under high vacuum until no trace of
acetone is left. Transfer the product to
brown containers. The yield is about 1 gr
per egg.
(5) Analyze for phosphate (see below), multiplv
by 26 which gives the approximate weight of
~ 2~
the phospnolipids. This shou'd amo-ln~ o
25 - 50% of the total ~,/eight.
C. Co~ments: The ~procedure described i3 ~or a
laboratory scale and some varlation should bQ
considered for longer scales. The main v~riables
are the volume of acetone (2) the volume of added
salt solution (2) and the cooling ~etllp and
length ~3). We still find tha~ 20 - 30~ pho3pho-
lipids is optimal, ~hough it could be that special
purposes will require a different le~tel of
phospholipids. Currently, we have the technical
information on how to divert the final phospho-
lipid content between 5% to 75%. In principle
it is possible to prepare any intermediate con-
centration by mixing low and high phospholipid
batchesO
Do Analyseso
~1) Phospholipids~ This is determined by phos-
phoxous assay accordlng to Bartlett J~ Biol.
Chem. 234 466 (l9S9)- with the modification
of Bottcher et alO Anal Chim. Acta 24 203
~1961)o Mole of phosphorous corresponds
to mole o~ phospholipid. The average
molecular weight of phospholiplds is taken
as 800 therefore the weight of phospho-
li~ids is 26 times the weight of phosphorous
obtained in the analysis.
(2) Glycerides. This analysis is not easy and is
actually not essential since over 90~ o AL
is composed of phospholipid glycerides and
the latter can be estimated through the
phospholipid content. The method is given
in Kates, Technlques of Lipidology P 373-374.
~3) Dispersibility. The main physical character-
istics of AL is its abllity to form a homogenous
~ 7 ~Z
-2~-
dispersisn in water. Mix ~0~ m5 ~L in
5 ml water and place in a soni ying batn
and sonicate for 3 min, A mil~y sus2ension,
which is stable for at leas~ a ~e-,J days,
is formed. Such a dispersion sho~lld pro-
vide the basis to material for intravenous
injection.
(4~ Biological Potency. AL increases the
responsiveness of leukosytes to mitogens
(e.g. Con A). This can be assayed either
in vivo or ln vitro. Currently the
following ln vivo test is the most satis-
factory. Blood is drawn form a 5 - 8
month old female rabbit and immediately
~sed for mitogenic stimulation assay.
Immediately after blood drawing, sonicated
200 mg AL in 2 ml saline is inje~ted in-
travenously into the same rabbit. Twenty-
four hours later, blood is drawn and the
mitogenic response is assayed again. ~er
a 2 fold increase in mitogenic response
indicates a potent AL batcnO
