CA1251782A - Lipid fraction, its preparation and pharmaceutical compositions containing same - Google Patents
Lipid fraction, its preparation and pharmaceutical compositions containing sameInfo
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- CA1251782A CA1251782A CA000456373A CA456373A CA1251782A CA 1251782 A CA1251782 A CA 1251782A CA 000456373 A CA000456373 A CA 000456373A CA 456373 A CA456373 A CA 456373A CA 1251782 A CA1251782 A CA 1251782A
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Abstract
ABSTRACT OF THE INVENTION
A composition comprising a lipid fraction derived from natural sources (AL), the fraction containing 40-80 weight percent glycerides, 3-5 weight percent cholesterol, 10-30 weight percent lecithin (phosphatidyl choline), 5-15 weight percent phosphatidyl ethanolamine and 2-5 weight percent negatively charged phospholipids, wherein the ratio of unsaturated to saturated fatty acids is at least 1:1. The composition is useful in the treatment of various symtoms of aging and senescence, dysfunctions of the immune system, allergies, mental disorders, mental retardation, neurological disorders, hyperlipidemic states, symtoms of withdrawal from alcohol and other drugs, and the prevention of tolerance to drugs.
A composition comprising a lipid fraction derived from natural sources (AL), the fraction containing 40-80 weight percent glycerides, 3-5 weight percent cholesterol, 10-30 weight percent lecithin (phosphatidyl choline), 5-15 weight percent phosphatidyl ethanolamine and 2-5 weight percent negatively charged phospholipids, wherein the ratio of unsaturated to saturated fatty acids is at least 1:1. The composition is useful in the treatment of various symtoms of aging and senescence, dysfunctions of the immune system, allergies, mental disorders, mental retardation, neurological disorders, hyperlipidemic states, symtoms of withdrawal from alcohol and other drugs, and the prevention of tolerance to drugs.
Description
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 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 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). -~
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 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 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 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 (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).
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 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 (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~
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.
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~
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~
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
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 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
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~
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~
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
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~
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~,~
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 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.
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 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.
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~
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
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 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
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~
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~
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
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~
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~,~
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 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.
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 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~--
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
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
Claims (39)
1. A composition comprising a lipid fraction derived from natural sources (AL), said lipid fraction containing 40-80 weight percent glycerides, 3-5 weight percent cholesterol, 10-30 weight percent lecithin (phosphatidyl choline), 5-15 weight percent phosphatidyl ethanolamine and 2-5 weight percent negatively charged phospholipids, wherein the ratio of unsaturated to saturated fatty acids is at least 1:1.
2. A composition comprising 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 lecithin (phosphatidyl choline), 5-10 weight percent phosphatidyl ehtanolamine and 2-3 weight percent negatively charged phospholipids wherein the ratio of unsaturated to saturated fatty acids is at least 1:1.
3. A composition according to claim 2 where the fatty acid composition of the lipids is the following: Palmitic acid 35-45%, oleic acid 35-45%, linoleic acid 5-10%, stearic acid 5-7%, palmitileic acid 2-3%, arachidonic acid 0.2-1%.
4. A composition according to claim 3, wherein the ration of unsaturated to saturated fatty acids is at least 2:1.
5. A composition according to claim 1, containing as additive an effective quantity of a physiologically acceptable antioxidant.
6. A composition according to claim 2, containing as additive an effective quantity of a physiologically acceptable antioxidant.
7. A composition according to claim 3, containing as additive an effective quantity of a physiologically accept-able antioxidant.
8. A composition according to claim 4, containing as additive an effective quantity of a physiologically accept-able antioxidant.
9. A composition according -to claim 5, wherein said composition contains from about 0.2 to about 2 weight per-cent tocopherol as an antioxidant.
10. A composition according to claim 6, wherein said composition contains from about 0.2 to about 2 weight per-cent tocopherol as an antioxidant.
11. A composition according to claim 7, wherein said composition contains from about 0.2 to about 2 weight per-cent tocopherol as an antioxidant.
12. A composition according to claim 8, wherein said composition contains from about 0.2 to about 2 weight per-cent tocopherol as an antioxidant.
13. A process for the production of an active lipid fraction (AL) which comprises treating a natural lipid source with an organic liquid, in order to remove undesired lipids, collecting the precipitate and re-extracting the precipitate with acetone, removing the supernatant and re-covering from said supernatant the desired fraction (AL), by precipitation below 0°C.
14. A process for the production of an active lipid fraction (AL) which comprises treating a lipid source with an organic liquid, collecting the precipitate and re-ex-tracting the precipitate with acetone, removing the super-natant and recovering from the said supernatant the de-sired fraction (AL) by evaporation of the acetone.
15. A process according to claim 13, wherein the or-ganic liquid is acetone.
16. A process according to claim 14, wherein the or-ganic liquid is acetone.
17. A process according to claim 13, wherein the start-ing material is egg yolk or soybean or their crude lipid ex-tracts.
18. A process according to claim 14, wherein the start-ing material is egg yolk or soybean or their crude lipid ex-tracts.
19. A process according to claim 15 or 16, wherein the starting material is egg yolk or soybean or their crude liquid extracts.
20. A process according to claim 13, wherein an antiox-idant is added to the fraction (AL).
21. A process according to claim 14, wherein an antiox-idant is added to the fraction (AL).
22. A process according to claim 17 or 18, wherein an antioxidant is added to the fraction (AL).
23. A pharmaceutical composition for oral administration comprising as active ingredient a pharmaceutically effective amount of a composition according to claim 1, 2 or 3.
24. A pharmaceutical composition for oral administration comprising as active ingredient a pharmaceutically effective amount of a composition according to claim 7.
25. A pharmaceutical composition for oral administration comprising as active ingredient a pharmaceutically effective amount of a composition according to claim 9, 10 or 11.
26. A pharmaceutical composition for oral administration comprising as active ingredient a pharmaceutically effective amount of a composition according to claim 12.
27. A composition according to claim 1, 2 or 3, in the form of a lipid suspension in saline (10-100 mg/ml), for in-travenous injections.
28. A composition according to claim 7, in the form of a lipid suspension in saline (10-100 mg/ml), for intravenous injections.
29. A composition according to claim 9, 10 or 11, in the form of a lipid suspension in saline (10-100 mg/ml), for in-travenous injections.
30. A composition according to claim 12, in the form of a lipid suspension in saline (10-100 mg/ml), for intravenous injections.
31. A composition according to claim 1, 2 or 3, for oral administration wherein the lipid fraction (AL) is in a quantity of from 1 to 20 grams per unit dosage form.
32. A composition according to claim 7, for oral admin-istration wherein the lipid fraction (AL) is in a quantity of from 1 to 20 grams per unit dosage form.
33. A composition according to claim 9, 10 or 11, for oral administration wherein the lipid fraction (AL) is in a quantity of from 1 to 20 grams per unit dosage form.
34. A composition according to claim 12, for oral ad-ministration wherein the lipid fraction (AL) is in a quan-tity of from 1 to 20 grams per unit dosage form.
35. A composition according to claim 1, 3 or 5, where-in the lipid fraction (AL) is in a quantity of from 0.05-2 mg/ml for in vitro treatments.
36. A composition according to claim 8, wherein the lipid fraction (AL) is in a quantity of from 0.05-2 mg/ml for in vitro treatments.
37. A method of in vitro application of (AL) compris-ing administering a pharmaceutically effective quantity of a fraction of lipids from natural sources (AL), according to claim 1, 2 or 3, so as to:
a. Treat sperm infertility;
b. Facilitate tissue transplantations; and c. Modulate viral infectivity for use in vaccinations.
a. Treat sperm infertility;
b. Facilitate tissue transplantations; and c. Modulate viral infectivity for use in vaccinations.
38. A method of in vitro application of (AL) compris-ing administering a pharmaceutically effective quantity of a fraction of lipids from natural sources (AL), according to claim 4, 7 or 8, so as to:
a. Treat sperm infertility;
b. Facilitate tissue transplantations; and c. Modulate viral infectivity for use in vaccinations.
a. Treat sperm infertility;
b. Facilitate tissue transplantations; and c. Modulate viral infectivity for use in vaccinations.
39. A method of in vitro application of (AL) compris-ing administering a pharmaceutically effective quantity of a fraction of active lipids from natural sources (AL), ac-cording to claim 1, 2 or 3, said fraction of lipids further containing from about 0.2 to about 2 weight percent toco-pherol as an antioxidant, so as to:
a. Treat sperm infertility;
b. Facilitate tissue transplantations; and c. Modulate viral infectivity for use in vaccinations.
a. Treat sperm infertility;
b. Facilitate tissue transplantations; and c. Modulate viral infectivity for use in vaccinations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000456373A CA1251782A (en) | 1984-06-12 | 1984-06-12 | Lipid fraction, its preparation and pharmaceutical compositions containing same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000456373A CA1251782A (en) | 1984-06-12 | 1984-06-12 | Lipid fraction, its preparation and pharmaceutical compositions containing same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1251782A true CA1251782A (en) | 1989-03-28 |
Family
ID=4128081
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000456373A Expired CA1251782A (en) | 1984-06-12 | 1984-06-12 | Lipid fraction, its preparation and pharmaceutical compositions containing same |
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| Country | Link |
|---|---|
| CA (1) | CA1251782A (en) |
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1984
- 1984-06-12 CA CA000456373A patent/CA1251782A/en not_active Expired
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