CA1235659A - Pharmaceutical compositions and method for preparing phosphatidylserine compositions useful in treating central nervous system disorders without effects on blood coagulation - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Processes are provided herein for the preparation of phospholipid compositions comprised of a mixture of phosphatidylserine and phosphatidyl-ethanolamine in a specified ratio. Such compositions have activity against disorders of the central nervous system but without secondary haematic coagulation effects. Particularly useful synergistic compositions comprise from 60 to 75% by weight of phosphatidylserine and from 40 to 25% by weight of phosphatidylethanolamine.

Description

lZ~56S9 The present invention relates to a phospholipid composition which is administered by various routes for treating disorders of the central nervous system, particularly those connected with aging of the brain and to processes for preparing such phospholipid compositionS.
The invention also relates to novel synergistic phospholipid pharmaceutical compositions.
Phospholipids are a large class of lipids characterized by a glycerol portion which binds phosphate and fatty acids. The general formula in phospholipids is as follows:

10 ~
~ ~1 Head \(~/ \(~) I
Group 0 ~ ~ ~ POLAR RÉGION

~0 ~ o~ I
C-O

HYDROCARBON
REG I ON

3~

i~35~
.

or, -O Polar Head O---P--O

Clt~_2CI~--C~2 ~-O 1 0

2 t$2 l Ta i 1 ~L~35~5~

wherein X is a particular polar head group charact~rizing the different phospholipid cl~sses. (See Lehningert Biochem~stry, 2 Ed.o p. 228).
It is understood that the role of phospholipids in haematic coagulation is crucial but not yet altogether clear. Indeed, on a molecular level, the interaction of phosphol}pids during this process with coagulation factors is still to be explained.
By outlining the intrinsic and extrinsic coagulation pathways it is possible to observe that phospholipids play a key role in the coagulation cascade. In fact, phospholipids constitute 30-s0% of crude thromboplastin preparations, and their composition varies according to the origin of the preparation. The ma~or part, however, is constituted by phosphatidylcholine (PC) and phosphatidylethanolamine (PE), while ne~atively charged phospholipids such as phosphati-dylserine (PS) and phosphatidylinositol (PI) account for a small part (Liu D. T. H. et al. Thromb. Res. 7 (1975) 213-221).
The polar group of the phospholipid plays an essential role in the formation of the complexes in the coagulation pathway (Otnaes A. B. et al. Eur. J. Biochem. 27, (1972), 238-243;
Nemerson Y. Adv. Exp. Med. Biol. 63, (1975) 245-253).
Regarding the specificity of the polar head, it has been observed that by recombining the tissue factor apoprntein with PE, thromboplastic activity is completely restored (Nemerson Y., supxa; Liu D. T. H. et al., ~y~). PC proves to be less active, while other phospholipid fractions and PE
have no activity at all, despite the fact that they bind to the apoprotein frac~ion. According to Wijngaards et al.
(biochem. Biophys. Acta 488, (1977), 161-171) lipid mixtures with moderate negative charges are necessary for a complete restora~ion of thromboplastin activity.

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Also, the role of externally added phospholipid~ is not altogether clear. Indeed, there is evidence in the literature that phospholipid preparations from cerebral tissue, prepared by extraction in chloroform and dried in acetone, act in vitro as strong coagulants ~Bell, W. M. et al., Nature 174 (1954), 880-881), while phospholipid mixtures extracted from cerebral tissue with chloroform:methanol followed by removal of the nonlipidic parts with saline have proved to he virtually inactive (Folch, H. et al., J.
Biol. Chem. 226, (1957), 497-509). It should be noted that in the first case cerebral tissue proteins were present, while in the second case they had been eliminated by the parti-tioning process.
The composition and purity of the phospholipids used is still the fundamen~al factor in understanding their exact `role, considering also the fact that different activities have been r~ported for the individual particular classes of phospholipid. It ist however, the correlation between negative charge and coagulant activity which makes phospha-tidylserine particularly interesting for these phenomena.
The activity of PS is debated with some authors in fact claiming that PS has a catalytic action (Marcus A. J. Adv.
Lip. Res. 4 (1966) 1-37), while others report an inhibitory activity of P5 (Turner D. L. et al. J. Lip. Res. 5 (1964) 616-623) due to the formation of inactive complexes. Mustard et al. al~o report (Nature, 196 (1962~ 1963-1065) an anti-coagulant activity of PS when injected in vivo in dogs in a range of dosage~ between 30 and 50 mg/kg. In addition, it has been reported that PE ha~ a marked coagulant activity (Rouser et al. Biochem. Biophys. Acta 28 (1958) 71-80; Turner D. L~ et al. J. Lip. Res. 4 (1963) 52-56). The activity of a mixture of at lea~t two clas~e~ of phospholipid is, however, probably more interesting than that of the single classes.
i ~35~

Some preparations with considerable activity are binary compositions with PE/PS ( 65/35 w/w), PC/PS ~ 57/43 w/w/), PC/PG 148/52 w/w) and PG/stearoylamine (85/15 w/w) (Zwaal R.
F. A. Biochem. Biophys. Acta 515, ~1978), 163 205).
The binary mixture of PS/PC in the presence of calcium seems important in the formation of the coagulation pathway complexes (Subbaiah E. V. et al . Biochem. Biophys. Acta 444, (1976), 131-146~. When PS constitutes 25P6 of the mixture with other phospholipids it proves to ~e critical in deter-lo mining, in this range, the maximum biological activity in theconversion from prothrombin and thrombin (M~ F. Lecompte et al. J. Electro. Anal. Chem. lO4 (1979) 537-541).
It should be observed that this phospholipidic fraction, namely phosphatidylserine, is also interesting for other biological and pharmacological activities.
In fact, a wide range of implications of phosphatidyl-serine in various biological processes has been reported in the literature. In summary, the biological implications of PS in physiological phenomena are the following:
a) PS restores ATPase activity associated with isolated neuronal membranes (Wheeler and Whittam ~1970), Nature 225, 449-450; Palatini et al. (1977) Biochem. Biophys.
Acta 466, 1-9).
b) PS stimulates activity of isolated tyrosine hydroxylase enzyme (Lloyd and Kaufmann (1974) ~iochem. Biophys.
Res.. Comm. 59, 1262-1269; Raese et al. (1976) Biochem.
Pharmacol. 25, 2245-22501.
c) PS liposomes induc~ cell fu~ion (Papahadjopoulos et al.
(1973) Biochem. Biophys. Acta 323, 23-42).
In addition to the biochemical effects observed, several lines of investigation have shown that cerebral metabolism is influenced by the in vivo aamin~stration of PS; the activity of dopaminergic and cholinergic ~y~tems is greatly affected, i ~35~S~

probably by an intervention on presynaptic mechanisms regulating neurotransmitter release. This activity is reflec~ed in measurable effects on several neuroendocrine, electrophysiological and behavioral correlates.
In particular, Bruni et al. (Nature 260 (1976) (5549), 331-333) reported a significant biological effect of PS in vivo on cerebral carbohydrate metabolism by measuring an increase of the cerebral glucose haematic ratio. Other phospholipids proved unable to induce this effect.
This cerebral activity after injection in vivo of PS
was confirmed by Toffano et al. (Life Sciences 23, (1978) 1093-1102), who ~howed that in cerebral neurotransmitter systems the intravenous (i.v.) injection of PS increased the turnover ra~e of norepinephrine in the hypothalamus. This hypothalamic effect was accompanied by an increase in the affinity of tyrosine hydroxylase for its synthetic pteridine cofactor (Toffano, Battistin, "Neurochemistry and Clinical Neurology", (1980) 205-214) and by an increase of cAMP.
These effects are correlated with an increase of ace-tylcholine (ACn) output from the cerebral cortex in urethane-anaesthetised rats (Casamenti et al. J. Neurochem. (32 (1979) 529-533~. When ACh levels in rat cerebral cortex were measured, it was found that PS (30 mg/kg l/p. for 10 days) potentiated the effect of the antimuscarinic drug scopolamine (Mantovani et al., Phospholip_ds in the Nervous System -Vol. 1, (1981) 165-172).
The effect of PS on cerebral neurotransmitter systems is supported by neuroendocrine, electrophysiological and beh~viorial correlates. Canonico et al. (Neuroendocrinology

3 33, (1981) 358-362) described the effects of PS on prolactin secretion in rats. The drug, in acute or repeated adminis-tration, reduced plasma prolactin levels during different phases of the circadian rhythm and inhibited the plasma pro-lactin surge typical of the proestrus afternoon ~n female rats.

~23s6sg On ~he other hand, a series of experiments (Mantovani et al., supra; Toffano et al., New Trends in Nutrition LiRid Researchl and Cardiovascular Di~eases, (1981) 91-99) showed that the typical disruption of EEG patter~ induced by the anticholinergic drug scopolamine was efficiently antagonized ~y previous or simultaneous administration of PS.
Subsequently, Aporti et al. (Res. Comm. Psychol. Psych.
Behav. 7 (1982) (1), 131-143) extended the EEG investigations by using computerized analysis of EEG recorded by means of implanted cortical and subcortical electrodes in freely moving rats. Acute administration of PS produced, at a cortical level, a reversal of the pre-drug ratios of the amplitudes in the right and left hemispheres. The effect became most pronounced at the end of 10 days of chronic administration.
This effect is correlated to a typical instinctive exploratory behavior, defined as alternation, and exhibited by rats placed in a T-maze set-up. Also this typical behavior is disrupted by the anticholinergic drug scopolamine~
Pepeu et al. (aging Br. Dementia 13, (1980) 271-274) reported that intraperental (i.p.) administratiion of PS completely antagonized the disrupting effect of scopolamine on spontan-eous alternation in a T-maze.
More recently, Toffano et al. (International Multidisci-plinar Seminar: "Cerebral Pathology in Old Age", Second Edition (In pxess 1983)1 reviewed pharmacological data ob-tained in aged animals, in order to evaluate the protective effects of PS on parameter~ which undergo changes with age.
In~ection of PS favorably influenced s~veral biochemical membrane parameter~, including stimulation of (Na+K~)-ATPase, and antagonism of age-related alterations of the cholesterol to phospholipid molar ratio. Concomitantly, PS prevented the ~ge-dependent decrea~e of dopamine and it~ catabolites in rat ~tr~a~um and limbic ~reas.

1~35~i5gl In addition, a series of studies confirmed a favorable effect of PS on learning and memory processes in aged rats.
Positive effects of PS on cognitive functions in aged rats were reported by Drago and Scapagnini, NeurobiologY of Aginq 2, (1981) 209-213.
In summary, pharmacological data reported in the liter-ature make it evident that administration to man of pharma-ceutical compositions containing PS of cerebral origin and with a high degree of purity may represent a valid therapeutic lo means for treating aging of the brain where a diminishing of dopaminergic control is well known. On the other hand, data reporting the effects of PS on blood coagulation suggest a grave risk of haemorrhage upon administration of PS. This is a particular disadvantageous risk in elderly patients who often already suffer from vascular complications.
~ he present invention basically resides in identifying new phospholipid compositions by means of which highly pure phosphatidylserine can be administered to humans by various routes with pharmacological activity on the nervous system, but without undesirably altering coagulation of the blood.

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By one broad aspect of this invention, a process is provided for preparing a phospholipid composition comprising: extracting phosphatidyl-serine from animal brain tissue to provide a phosphatidylserine extract;
extracting phosphatidylethanolamine from; animal brain tissue to provide a phosphatidylethanolamine extract; combining a specified amount of the phosphatidylserine extract with a specified amount of the phosphatidylethano-lamine extract in an appropriate solvent to provide a resultant solution;
agitating the resultant solution to provide an agitated solution; filtering the agitated solution to provide a filtered solution; and precipitating the filtered solution, thereby to obtain a phospholipid composition compris-ing phosphatidylserine and phosphatidylethanolamine.
The appropriate solvent may be a 2:1 mixture of methanol:chloro-form. The precipitation is preferably conducted in acetone. In one embodiment, 3 parts of phosphatidylserine are combined with 1 part of phosphatidylethanolamine, thereby to obtain a phospholipid composition comprising 75 weight percent phosphatidylserine and 25 weight percent phosphatidylethanolmine. In a second embodiment, 65 weight percent of phosphatidylserine is combined with 35 weight percent of phosphatidylethano-lamine, thereby to obtain a phospholipid composition comprising 65 weight percent phosphatidylserine and 35 weight percent phosphatidylserine and weight percent phosphatidylethanolmine. In a third embodiment, 60 weight percent phosphatidylserine is combined with 40 percent of phospha-tidylethanolamine, thereby to obtain a phospholipid composition comprising weight percent phosphatidylserine and ~iO weight percent phosphatidyl-ethanolamine.
By another aspect of this invention, a process is provided for preparing a phospholipid composition comprising: extracting phosphatidyl-serine from animal brain tissue, thereby to provide a phosphatidylserine extract; extracting phosphatidylethanolmine from animal brain tissue, _ 9 _ :~3S6~59 thereby to provide a phosphatidylethanolamine extract; combining 3 parts of the phosphatidylserine extract with I part of the phosphatidylethanolmine extract in a mixture of methanol:chloroform (2:1) thereby to provide a resultant solution; agitating the resultant solution, thereby to provide an agitated solution; filtering the agitated solution, thereby to provide a filtered solution; and precipitating the filtered solution in acetone, thereby to obtain a phospholipid composition comprising 75 weight percent phosphatidylserine and 25 percent phosphatidylethanolamine.
By still another aspect of this invention a process is provided for preparing a phospholipid composition comprising: extracting phosphatidyl-serine from animal brain tissue, thereby to provide a phosphatidylserine extract; extracting phosphatidylethanolamine from animal brain tissue, thereby to provide a phophatidylethanolamine extract; combining 65 weight percent of the phosphatidylserine extract with 35 weight percent of the phosphatidylethanolamine extract in a mixture of methanol: chloroform (2:1), thereby to provide a resultant solution; agitating the resultant solution, thereby to provide an agitated solution; filtering the agitated o solution, thereby to provide a filtered solution; and ?-resipjtating the filtered solution in acetone, thereby to obtain a phospholipid composition comprising 65 weight percent phosphatidylserine and 35 weight percent phosphatidylethanolamine.
By yet another aspect of this invention a process is provided for preparing a phospholipid composition comprising: extracting phosphatidyl-serine from animal brain tissue, thereby to provide a phosphatidylserine extract; extracting phosphatidylethanolmine from animal brain tissue, thereby to provide a phosphatidylethanolamine extract; combining 3 parts of the phosphatidylserine extract with 2 parts of the phosphatidylethanola-mine extract in an appropriate solvent, thereby to proviùe a resultant _ 9 .l -~3S6S9 solution; agitating the resultant solution, thereby to provide an agitated solution; filtering the agitated solucion, thereby to provide a filtered solution; and precipitating the filtered solution, thereby to obtain a phospholipid composition comprising 60 weight percent phosphatidylserine and 40 weight percent phosphatidylethanolamine.
By still a further aspect of this invention, a synergistic pharmaceutical composition is provided comprising an effective dopaminergic control modulation amoung of phosphatidylserine and phosphatidylethanolamine, together with a pharmaceutically acceptable carrier, diluent or excipient wherein the phosphatidylserine is present in an amount of from 60 to 75 percent, and the phosphatidylethanolamine is present in an amount of from 40-25 weight percent, the composition being substantially free of secondary hematic coagulation effects.
Three embodiments of such synergistic pharmaceutical composition are: wherein the phosphatidylserine is present in an amount of 75 weight percent nd the phosphatidylethanolamine is present in an amount of 25 weight percent; wherein the phosphatidylserine is present in an amount of 65 weight percent and the phosphatidylethanolamine is present in an amount of 35 weight percent; and wherein the phosphatidylserine is present in an amount of 60 weight percent and the phosphatidylethanolamine is present in an amount of 40 weight percent.
The specific pharmacological proEile of the compositions consists of dopamineric control modulation. The compositions are, therefore, particularly useful for treatment of pathologies which are characterized by decreased dopaminergic control; namely involution ot the senile psycho-motor syndromes vascular cerebropathy, chronic and prevalent psychic component in old age, senile dementia and pre-senile syndromes subjective to post-trauma, post-anoxic cerebropclthy, and extrapyramidal syndromes.

- 9 b -.1 -- 1() --~23~65i9 In treating these various pathologies, it is necessary to eliminate wha~ever secondary haematic coagulation effects may arise upon administration of the compositions. In fact it is well known tha~ vascular complications often occur in old age, so that, haematic coagulation effects must be considered in the treatment of these pathologies. The phos-pholipid compositions of the invention are comprised of phosphatidylserine which is extracted from the brain tissue of animals. These particular compositions do not have any lo undesirable effects on blood coagulation.
Detailed studies have been conducted on the effect on coagulation of a mixture of phospholipids extracted and puri-fied from bovine brain cortex. These studies have also determined the effects of purified phosphatidylserine and binary mixtures of phosphatidylserine with other single classes of phospholipids (PE, PC and sphingomyelin). This research was conducted with an aim toward obtaining a pharma-cologically active phosphatidylserine preparation which has an undesirable side effects on haematic coagulation.
Specific evaluation was made of the eentral nervous system activity effects of in vivo administration of the pho~pho-lipid compositions together with any side effects on haematic coagulation which may occur.
Methods:
The activity of the various products has been evaluated in vivo by intravenously injecting various phospholipid preparationæ in New Zealand rabbits weighing 1.5 - 2 kg, at doages ranging from 9.5 mg/kg to 50 mg/kg. The phospholi-pid~s were placed in a vehicle of Tris HCl (50 mM, pH 7.4) and sonicated until the ~uspension became clear (all possible traces of titanium from the sonicator were eliminated by means of centrifuga~ion). After treatment, blood was drawn from the animals and tested for coagulation. Determinations ~3565~

were carried out using hemocoagulation kits supplied by Biochemia ~Boheringer, Mannheim) with a Digiclot Elvi 818 apparatuS, on plasma obtained by centrifugation at a speed of 4,200 r.p.m. for 10 minutes of blood in 3~8% sodium citrate at a ra~io of 1:9. The parameter considered was prothrombin time according to Quick.
~he pharmacological activity of the individual product compositions tested was evaluated by measuring mouse cerebral glucose in the same dosage range, considering this parameter lo as an indication of cerebral activation~ For this experiment Swiss mice weighing between 20 - 25 gr were treated and sacrificed 30 minutes after injection. Glucose levels were measured as described by Bruni et al. in Nature 260, (1976) The individual compositions tested were as follows:
(1) phosphatidylserine (PS) from the bovine brain cortex with a titer of over 90%;
~2) phosphatidylserine + phosphatidylethanolamine (BC-PS) with 75~ PS and 25% PE w/w;
(3) phosphatidylserine + phosphatidylethanolamine (BC-PSl) with a 65% PS and 35~ PE w/w;

(4) phosphatidylserine + phosphatidylenthanolamine (BC-PS2) with a 60% PS and 4~ PE w/w;

(5) phosphatidylserine + phosphatidylethanolamine (BC-PS3) with a 50% PS and 50~ PE w/w;

(6) phosphatidylserine ~ phosphatidylethanolamine (BC-PS4) with a 40~ PS and 60% PE w/w;

(7) phosphatidylserine + phosphatidylethanolamine ~BC-PSs) with a 25% PS and 75% PE w/w;

(8) phosphatidylserine + phosphatidylethanolamine (BC-PS6) with a B5~ PS and 15% PE w/w;
~9) phosphatidylserine + phosphatidylcholine (BC-PS7) with a 75% PS and 25~ PC w/w;
(10) pho~phatidylserine ~ spingomyelin ~BC PSg) with a 75 PS ~nd 25~ SF w/w.
Results:
.. . ...
The results from the above-~escribed tests are summarized below in Table 1 and 2.

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T~ble 1 Activity of various phospholipid compositions on haematic coagulation and on cerebral glucose recorded 30 minutes after in vivo treatment at a dosage of 20 mg/kg i,v. of different mixtures.

Percentage of Phospho- Dosaqe (mq/kq)a lipids in Total Dose Compound Prothrombin Cerebral the composi- of Composi- Dose of time according glucose tion mixture tion Mixture PS to Quick (sec.) (umole/g) . _ . , .
Controls - - 7.~3 + 0.07 1.3 PS 20 20 12.34 + 0.8 4.1 BC-PS (PS 7S
PE 25) 20 15 7.36 + 0.14 3.7 BC-PSl (PS 65 PE 35) 20 1~ 7.5 + 0.3 3.5 BC-PS2 (PS 60 PE 40) 20 12 7.8 ~ 0.5 3.2 BC-PS3 (PS 50 PS 503 ~0 10 8.5 + 0.2 3.0 BC-PS4~(PS 40 PE 60) 20 8 6.7 + 0.9 2.5 BC-PSs (PS 25 PE 75) 20 5 6.5 + 0.2 1.9 BC-PS6 (PS 85 PE 15) 20 17 11.3 + 1.2 2.9 BC-PS7 (PS 75 PC 25) 20 15 9.3 + 0.7 3.0 BC-PSg (PS 75 SF 25) 20 15 14.7 + 0.9 3.1 .
.. ___ . ... _ _. _ _ ~ , . ___ _ a The ~osage o~ the compo~ition mixture was m~intaine~ at a constant rate of 20 mg/kg. Therefore the do~age of PS varied according to the corresponding percentage of PS in each mixture.

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Table 2 Activity of the various phospholipid compositions on hematic coagulation and on cerebral glucose recorded 30 minutes after in vivo treatment of a do~age of 20 mg/kg i~v~ of PS.

__ Dosage (mg/kg)a Total Dose Cerebral Compound of Composi- Pxothrombin glucose Dose of PS tion Mixture time Isec.) (umole/kg) Control - - 7.43 + 0.07 1.3 PS 20 20 12:34 + 0.8 4.1 BC-PS 20 27 7.5 + 0.20 4.3 BC-PSl 20 30.7 8.0 + 0.2 4.1 BC-PS~ 20 33.3 8.2 + 0.7 3.B
BC-PS3 20 40 9.5 + 0.3 3.8 BC-PS4 20 50 4.2 + 9.3 4.0 BC-PSs 20 80 3.5 + 0~4 3.3 BC-PS6 20 23.5 13.3 + 0.2 3.6 BC-PS7 20 27 11.5 + 0.3 3.8 BC-PSg 20 27 12.3 + 0.5 3.9 .
From the results measuring the coagulation parameters, it is possible ~o observe a clear anticoagulant activity after administration of pure phosphatidyl~erine in vivo, which confirms previous observations of Mustard et al. (Nature 196 pages 1063-1065 (1965)).

aThe dosage of PS was maintained at a constant rate of 20 mg/kg.
In order to achiev~ this constant do~age rate of PS, the dosage rate of the total composition mixture varied according to the corres~onding percentage of PS ~n each mixture.

~23S~5~) In the case of the binary mixtures of PS plus other phospholipids, the anticoagulant activity varies with diffexent rates according to the particular type of phos-pholipid associated with PS. But in the case of BC-PS, the measured prothrombin time is essentially ~he same as that for the controls, indicating that this composition has no un-deslrable effect on blood coagulation.
Further analysis of the results reported in Tables l and 2 shows that phosphatidylserine is the most active fractlon for increasing brain gluco~e levels. This eff2ct can also be observed in the case of the binary mixtures of PS with other phospholipids, in a manner similar to that for pure PS. By considering the data reported in Table 2 with constant doses of 20 mg/kg i.v. of pure PS, it can be seen that cerebral activity is enhanced for all of the binary mixtures of phospholipids, as compared to the controls. However, the activity on blood coagulation is extremely varied depending upon the particular binary composition with some compositions shortening and others lengthening the measured times as compared to controls.
It is, therefore, of particular importance to note that the use of a binary mixture of PS and PE (particularly 75~ PS
plus 25~ PE) exhibits no undesirable activity on coagulation, while it does have good activity on the central nervous sys-tem (CNS). That is, the PS composition comprised of a mix-ture of phospholipids exhibits good CNS activity as measured from the cerebral glucose level, but this composition also has unde~irable hematic coagulation side effect~ as shown by the extended prothrombin time. On the other hand, the BC-PS4 and BC-PSs compositions admini~tered at constant dosage levels o~ the binary mixture exhibited decreased prothxombin times in a more acceptable range as reported in Table 1.
However, these compositions (particularly BC-PSs~ did not ~35~

exhibit sufficiently desirable CNS activ~ty as evidenced by the low cerebral glucose levels. Moreover, in administering constant dosages of PS in a binary mixture, compositions of BC-~S4 and BC-PSs al~o exhibited marked decreased prothrombin tim~s ~uch that the low prothrombin tlmes for the BC-PS5 composition actually represent an undesirable side effect in causing coagulation of the blood at a rate that is too fast. This effect would lead to a risk of thrombosis in patients treated with these mixtures.
The fractions of PS with PC, and PS with SF were also found to be undesirable in tha~ they exhibited extended prothrombin times.
It has, however, been determined by the present inventors that the BC-PS, BC-PSl and BC-PS2 compositions do represent useful pharmaceutical compositions. All three of these compositions exhibit an acceptable prothrombin time substantially reduced from the prothrombin time of PS and comparable to that of the controls, while at the same time exhibiting good CNS activity as shown by the cerebral glucose levels.
It has, therefore, been determined by the present inventors ~that a preferred phospholipid compo-sition having good CNS activity and without undesirable hematic coagulation effects is obtained by preparing a mixture of from 60 to 75% PS with from 40 to 25% PE. More preferably, the composition should contain 75% PS and 25% PE. The following examples describe specific pharmaceut~cal compo-sitions useful for treating various disorders of the central nervous sy~tem by dopaminergic control modulation.
Example 1 - P_eparation of the phos~holipid composition BC-PS
1000 g of phosphatidylserine ~PS) extracted from bovine nervou~ tissue with ~ titer of more than 95~ are di~solved in 5 l~ters of a mixture o~ methanol:chloroform (2:1). 333 g of phosphatidylethhnolamine (PE) with a titer of more than 80% are added to the 801ution.

~35~iS~

The solution is kept in agitation for 30 minutes, fil-tered ~hrough a sterilizing membrane filter and precipitated in 5 volumes of acetone under suitable agitation.
The precipitate is separated and dried in vacuum at low temperature.
About 1250 g of a BC-PS compositlon are obtained in this way.
Example 2 - Preparation of the phospholipid composition BC-PS
In the same manner as in Example 1, 1000 g of PS with a titer of more than 95% are dissolved in 5 liters of a mixture of methanol: chloroform (2:1) and 538 g of PE with a titer of more than 80% are added to the solution.
After agitation, filtering, precipi~ation, and drying, about 1442 g of a BC-PSl composition are obtained.
Exam~le 3 - Preparation_of ~he-phospholipid composition BC-PS2 In the same mannex as in Example 1, 1000 g of PS with a titer of more than 95% are dissolved in 5 liters of a mixture of methanol:chloroform (2:1) and 666 g of PE with a titer of more than 80% are added to the solution.
After agitation, filtering, precipitation, and drying, about 1562 g of a BC-PS2 composition are obtained.
Example 4 - ExamPles of iniectable pharmace~tical compositions a) One 2 ml vial contains:
- Liposomes of phospholipid composition BC-PS: 66.5 mg - monobasic sodium phosphate: 2.4 mg - dibasic sodium phosphate: 2.26 mg - pyrogen-free twice-distilled water: q.s. 2 ml b) One 5 ml vial contains:
- Liposomes of phospholipids composition BC-PS: 332.5 mg - monobasic sodium phosphate: 5.35 mg - dibasic odium phosphate: 6.65 mg - pyrogen-free twice~distilled water:q.s. 5 ml ~3S65~

c) One 3 ml vial contains:
Liposomes of phospholipid composition BC-PS: 199.5 mg - monobasic sodium phosphate: 3.21 mg - dibasic sodium phosphate: 3.39 mg - mannitol: 30 mg The products described above in Example 4 can be utilized in emergency therapy against pathologies of the central nervous system. More specifically, these formulations can be used acutely in anoxic cerebropathy and extrapyramidal traumatic syndromes~ pre-senile and senile dementia, as well as metabolic encephalopathy.
Example 5 - Examples of oral Pharmaceutical compositions a) Each gelatinous capsule contains:
- phospholipid composition BC-PS: 133 mg - vegetable oil~ 270 mg - beeswax: 1 mg b) Each gelatinous capsule contains:
- phospholipid composition BC-PS332.5 mg - vegetable oil~ mg - beeswax: mq c) Each pill contains:
- phospholipid composition BC-PS:66.5 mg - mannitol: 100 mg - microcrystalline cellulose: 25 mg - starch: 5 mg - saccharose: 30 mg - lacquer: 5 mg d) Each operculum contains:
- phospholipid composition BC-PS:199.5 mg _ mannitol 100 mg - lactose 100 mg ~23S65g The products described above in Example 5 can be used in chronic therapies. Specifically these formulations can be used to treat involu~ion of senile psycho-motor syndromes, chronic vascular cerebropathy, metabolic encephalopathy in old age, senile dementia and pre-senile syndromes subjective to post-trauma, post-anoxic cerebropathy and extrapyramidal syndromes.
Although the above pharmaceutical compositions have been lo described in particular physical forms with particular carriers, it will be recognized that ~he pharmaceutical compositions of the invention can be formulated in other standard physical forms combined with other known pharmaceutically acceptable carriers, excipients and diluents.
From the above, it can be seen that the present invention pro-vides processes for preparing pharmaceutical compositions useful for treating disorders of the central nervous system which are specifirelated to aging of the brain. Such compositions have good central nervous sys-tem activity while, at the same time, they do not exhibit undesirable side effects with respect to haematic coagulation. This discovery is particu-larly important in providing a means for treating central nervous system disorders with phospholipid compositions, since compositions containing only phosphatidylserine have such an undesirable effect on blood coagu-lation as to be non-usable as a pharmaceutical composition. The present inventors, however, have overcome this problem by discovering that a par-ticular binary composition of phosphatidylserine with phosphatidylethola-mine can provide a useful active pharmaceutical composition without the undesirable side effects encountered with administration of phosphatidyl-serine alone.

Claims (14)

1. THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
   PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
   l. A process for preparing a phospholipid composition comprising:
   extracting phosphatidylserine from animal brain tissue to provide a phosphatidylserine extract;
   extracting phosphatidylethanolamine from animal brain tissue to provide a phosphatidylethanolamine extract;
   combining a specified amount of said phosphatidylserine extract with a specified amount of said phosphatidylethanolamine extract in an appropriate solvent to provide a resultant solution;
   agitating said resultant solution to provide an agitated solution;
   filtering said agitated solution to provide a filtered solution;
   and precipitating said filtered solution, thereby to obtain a phospho-lipid composition comprising phosphatidylserine and phosphatidylethanolamine.
2. 2. A process as claimed in claim 1, wherein said appropriate solvent is 2:1 mixture of methanol:chloroform.
3. 3. A process as claimed in claim 1, wherein said precipitation is conducted in acetone.
4. 4. A process as claimed in claim 1 wherein 60-75 weight percent of said phosphatidylserine extract is combined with 40-25 weight percent of said phosphatidylethanolamine extract, thereby to obtain a phospholipid composition comprising 60-75 weight percent phosphatidylserine and corres-pondingly 40-25 weight percent phosphatidylethanolamine.
5. 5. A process as claimed in claim 1, wherein 3 parts by weight of said phosphatidylserine extract are combined with 1 part by weight of said phosphatidylethanolamine extract thereby to obtain a phospholipid composition comprising 75 weight percent phosphatidylserine and 25 weight percent phosphatidylethanolamine.
6. 6. A process as claimed in claim 1, wherein 65 weight percent of said phosphatidylserine extract is combined with 35 weight percent of said phosphatidylethanolamine extract, thereby to obtain a phospholipid composition comprising 65 weight percent phosphatidylserine and 35 weight percent phosphatidylethanolamine.
7. 7. A process as claimed in claim 1, wherein 60 weight percent of said phosphatidylserine extract is combined with 40 percent of said phosphatidylethanolamine extract thereby to obtain a phospholipid composition comprising 60 weight percent phosphatidylserine and 40 weight percent phosphatidylethanolamine.
8. 8. A process for preparing a phospholipid composition comprising:
   extracting phosphatidylserine from animal brain tissue, thereby to provide a phosphatidylserine extract;
   extracting phosphatidylethanolamine from animal brain tissue, thereby to provide a phosphatidylethanolamine extract;
   combining 3 parts of said phosphatidylserine extract with part of said phosphatidylethanolamine extract in a mixture of methanol:chloro-form (2:1) thereby to provide a resultant solution;
   agitating said resultant solution, thereby to provide an agitated solution;
   filtering said agitated solution, thereby to provide a filtered solution; and precipitating said filtered solution in acetone, thereby to obtain a phospholipid composition comprising 75 weight percent phosphatidyl-serine and 25 percent phosphatidylethanolamine.
9. 9. A process for preparing a phospholipid composition comprising:
   extracting phosphatidylserine from animal brain tissue, thereby to provide a phosphatidylserine extract;
   extracting phosphatidylethanolamine from animal brain tissue, thereby to providde a phosphatidylethanolamine extract;
   combining 65 weight percent of said phosphatidylserine extract with 35 weight percent of said posphatidylethanolamine extract in a mixture of methanol: chloroform (2:1), thereby to provide a resultant solution;
   agitating said resultant solution, thereby to provide an agitated solution;
   filtering said agitated solution, thereby to provide a filtered solution; and precipitating said filtered solution in acetone, thereby to obtain a phospholipid composition comprising 65 weight percent phosphatidyl-serine and 35 weight percent phosphatidylethanolamine.
10. 10. A process for preparing a phospholipid composition comprising:
    extracting phosphatidylserine from animal brain tissue, thereby to provide a phosphatidylserine extract;
    extracting phosphatidylethanolamine from animal brain tissue, thereby to provide a phosphatidylethanolamine extract;
    combining 3 parts of said phosphatidylserine extract with 2 parts of said phosphatidylethanolamine extract in an appropriate solvent, thereby to provide a resultant solution;
    agitating said resultant solution, thereby to provide an agitated solution;
    
    filtering said agitated solution, thereby to provide a filtered solution; and precipitating said filtered solution, thereby to obtain a phospho-lipid composition comprising 60 weight percent phosphatidylserine and 40 weight percent phosphatidylethanolamine.
11. 11. A synergistic pharmaceutical composition comprising an effective dopaminergic control modulation amount of phosphatidylserine and phospha-tidylethanolamine, together with a pharmaceutically acceptable carrier, diluent or excipient wherein said phosphatidylserine is present in an amount of from 60 to 75 percent, and said phosphatidylethanolamine is present in an amount of from 40 to 25 weight percent, said composition being substantially free of secondary hematic coagulation effects.
12. 12. The synergistic pharmaceutical composition as claimed in claim 11, wherein said phosphatidylserine is present in an amount of 75 weight percent and said phosphatidylethanolamine is present in an amount of 25 weight percent.
13. 13. The synergistic pharmaceutical composition as claimed in claim 11, wherein said phosphatidylserine is present in an amount of 65 weight percent and said phosphatidylethanolamine is present in an amount of 35 weight percent.
14. 14. The synergistic pharmaceutical composition as claimed in claim 11, wherein said phosphatidylserine is present in an amount of 60 weight percent and said phosphatidylethanolamine is present in an amount of 40 weight percent.