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Pharmacological properties of phospholipid liposomes
Pharmacological Research Communications, Vol. 12, No. 9, 1980
829
PHARMACOLOGICAL PROPERTIES OF PHOSPHOLIPID LIPOSOMES.
G. Toffano and A. Bruni ~ Fidia Research Laboratories~ Abano Terme~ Italy ~Institute of Pharmacology~ University of Padova9 Italy Receivedin final form 4 June 1980
INTRODUCTION The use of natural phospholipids as therapeutic agents, has been introduced from many years and considerable effort has been made to detect and eventual ly define pharmacological effects "in vivo". These studies have been based on two general hypothesis= (a) alteration of the composition of phospholipid containing structures (e. 8. cellular membranes9 lipoprotein) may result in controlled variations of biological activity and (b) supply of exogenous phospholipids to the tissues may improve cellular activities when pathological processes or natural aging reduce the renewal of the phospho]ipid membrane component. Support to these hypothesis is provided by the development of knowledges on membrane structure9 composition and function. It has been established that the composition of the bilamellar leaflet forming the network of cellular membranes is asymmetric and that a different function is likely assigned to the phospholipids present in the inner and outer layer (Verkleij et al., 1973). Depending on the phospholipid composition it is possible to induce the transient formation of non-bilayer lipid configurations with potential ion-transporting activity (Cullis and De Kruyff~ 1978). Also pertinent to the purpose of this review is the observation that the phospholipid fluidity influences the mobility of membrane proteins and therefore their catalytic activity and the possibility to interact with specific ligands (Hirata and Axelrod,
1973; Hirata et a l .
1979; Hirata et al.,
1979). Moreover,
administration of large amount of phosphatidylcholine enhances the synthesis of acetylcholine in the nervous tissue (Cohen and Wurtman~ 1976; Hirsch and 0031--6989/80/090829-17~02.00/0
© 1980 The Italian Pharmacological Society
Pharmacological Research Communications, VoL 12, No. 9, 1980
830
Wurtman, 1978; Wurtman et al., 1977). This obsm'va'tion which awaits confir'mation, implies the possibility to influence the synthesis of neurotransmitters Dy exogenous phospholipids. Furthermore,
the clarification
of phenomena
underlying membrane fusion (Papahadjopoulos, 1978) may provide the experimental support to the possibility that the injected phospholipids may reach and fuse with cellular membranes altering their composition and function. A parallel line of investigation on liposome pharmacology has been developed and is based on the property of phospholipid..liposomes to serve as drug carriers (Gregoriadis,
1976). Attention has been focused also on the influence of
liposomes on immune reactions (Allison and Gregoriadis, 197/4) and on the possibility to use these structures as chemotherapeutic agents (Alving, 1979). For a detailed survey of these recent developments, the reader is referred to the review of Tyrrel et al. (]976) and to the book "Liposomes in Biological Systems" (Gregoriadis and Allison, 1980) Since the biological activity of phosphol~pids is dependent on the structure formed in aqueous solution, a brief survey of this field will be presented first. Subsequently, data on the distribution of these compounds will be reported. Finally,
the pharmacological
properties
of phospholipids,
with
particular
emphasis to phosphatJdylserine, will be reviewed. Phosphatidylserine has been the most extensively
investigated
in our laboratories
and the activities
detected so far are not shared by other natural occurring phospholJpids. Self-assembly of p hospholipids Phospholipids are amphiphilic
molecules
provided
with
a hydrophobic
carbon tail and a polar head-group. Because of the thermodynamic unfavourab'e interaction of water with the hydrocarbon chains, phospholipids aggregate in defined structures above a concentration denoted as "the critical micellar concentration"
(reviewed
by Israelachvili
et al.,
1977). The hydrophobic
bonding among phospholipid aliphatic chains are balanced by the electrostatic bonding between the polar head groups and the water dipoles. [3epending on the number and lenght of hydrocarbon chains, phospholipids yield different structures in aqueous solution. For the purpose of this review three of them are relevant: micelle$, multilamellar bilayers (liposomes), vesicles (Fig. I). Micelles can be spherical or elongated. They have a polar external surface
Pharmacological Research Communications, VoL 12, No. 9, 1980 LIPOSOME (multilamellar)
VESICLE
1L monomer cone. 10"3M
monomer conc. 10"10M
Fig. l : Diagramatic representation of micelle, liposorne and vesicle forrnation in water. (modified from Hokin, 1969). and an apolar core in a liquid-like state. In these structures the inner aqueous compartment is not delimited. Liposomes and vesicles have in common a phospholipid bilayer organization which delimits separated aqueous compartments. They differ in the numl~er o[ phospholipid layers and in the radius o5 bilayer curvature. Liposomes are spontaneously formed when water is added to a dry film of phospholipids. They are generally multilayers with a large radius of curvature (planar bilayer). On sonication~ liposomes reorganize in structures with reduced number of biJayers. Small (200 ~) unilamellar vesicles can be obtained. The unJlamellar vesicles mantain an aqueous compartment inside while the small radius of curvature will favour the asymmetric distribution of phospholipids in the bilayer. Of relevance is the analysis oJ the factors determining the phospholipjds self assembly whether in micelles or bilayers. In both cases the driving force is the hydrophobic bonding among the hydrocarbon chains which depends on the lensht of the acyl-chains. Short acyl-chains yield small and unstable aggregates (micelles)in rapid equilibrium with a relatively high monomer concentration. Long acyl-chains yield a bilayer organization slowly equilibrating with a low monomer concentration (approx. 10-10M, Smith and Tanford~ 1972). The significant differences between the micellar and the bilayerorganizations are (i) greater dimension of bilayer aggregates,(ii) lower stability of the micelles and (iii) higher monomer concentration in the micellar dispersions.
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Pharmacological Research Communications, VoL 12, No. 9, 1980
832
Table I - Effect of BC-PL dispersion and specificity of phosphatidylserine and lyso-phosphatidylserine on brain glucose accumulation. (from: Bruni et al., 1976, a; Bigon et a l . 19799 a).
Phospholipid
Dose mol/kg i.v.
Brain glucose
tool~ g wet wt
--
1.1
BC-PL (homogenization)
150
1.2
B C - P L (sonication)
150
2.7 ~
None
Brain Phosphatidylserine
50
3.69 ~
Brain Phosphatidylethanolamine
50
1.0#
Brain Phosphatidylcholine
50
0.96
Brain Phosphatidylinositol
50
1.17
Heart diphosphatidylglycerol
50
0.89
Lyso-phosphatidylserine
10
4.50 ~
Lyso-phospha tidylethanolamine
20
1.50
Lyso-phosphatidylcholine
20
1.63
* P < O.Ol
Pharmacokinetics Because of the differences outlined above, distinct pharmacokinetics are expected after administration in vivo of a micellar or a liposomal dispersion. The high monomer concentration in the micellar dispersion is a favourable condition for extensive binding to plasma proteins. The decrease of monomer concentration below the critical micellar concentration as a consequence of protein binding, may lead to dissolution of phospholipid aggregates. For the same reason micellar dispersions have greater potential activity in comparison to liposomes to cross membrane barriers. Liposomes and vesicles are more stable in the blood stream° Their stability cholesterol
is further
increased provided
is included in their composition (Bireisblatt and Ohki,
[976).
Pharmacological Research Communications, Vol. 12, No. 9, 1980 However, the plasma levels of liposomes decrease rapidly after their administration mainly as consequence of accumulation in the liver and in the spleen. Large part of lJposomes are incorporated in phagocytic cells. A comprehensive review dealing with the liposome distribution in vivo is available (Tyrell et al., 1976). The large size of liposomes and unilamellar vesicles prevents these structures to reach a large volume of distribution. Within this limitation, remarkable differences in pharmacological activity are observed between unsonicated and sonicated liposomes (15runi et al., 1976). As shown in Table 1, the capacity of a phospholipid suspension to increase the brain glucose content becomes apparent only upon sonication of phospholipids. Clearly, the size of liposomal particles affects both the distribution and the activity o5 phospholipids. Pharmacological eff.ect_s of phospholipid.liposome~
role of phosphatidylserine.
In 1975-76 it has been observed that the intravenous injection of a sonicated phospholipid mixture (BC-PL) extracted from bovine brain influenced the cerebral metabolism. The mixture of bovine brain phospholipids was composed of 6.6 96 phosphatidic acid, 20.2 96 phosphatidylethanolamine, 15.7 96 phosphatidylserine, 15 96 lysophosphatidylethanolamin e (serine), 29.9 96 phosphatidylcholine, 9.4 96 sphyngomyelin and trace amount of cerebroside (gruni et al., 1976). increased catecholamine turnover (Leon and Toffano, 1976), glucose accumulation (Bruni et al., 1976) and enhanced acetylcholine output from cerebral cortex (Mantovani et al., 1970) have'been described. All these studies demonstrated that phosphatidylserine was the active ingredient. On these basis the suggestion has been made (Bruni et al., 1980) that deacylaton and reacylation of phosphatidylserine may have a regulatory influence in vivo on specialized cells. Detection of high biological activity in a phosphatidylserine dispersion is not surprising considering that this phospholipid has a crucial role in several membrane linked activities such as enzyme activation, (Wheeler and Whittman, 1970; Lloyd and Kaufman, 197t~; Raese et'al., 1976) liposome fusion (Papahadjopoulos et al., 1973), ion permeability (Hauser and Dawson, 1967; Newton et al, 1978), histamine release (Goth et al.~ 1971) and recognition phenomena (Abood and Takeda, 1976; Abood et al., 1975).
833
Pharmacological Research Communications, Vol. 12, No. 9, 1980
834
Parallel studies have shown that the administration 6f BC-PL dispersions to
mice produced a marked inhancement of 32p incorporation into brain phospholipids after few hours (Orlando et al., 1975).The increased incorporation ranged from ~5 to 75 96. Among individual phospholipid classes, increase of specific activities has been found in phosphatidylcholJne, pliosphatidylethanolamine and phosphatidylserine. By contrastt phosphatidic acid and phosphatidylinositol showecJ a decrease. Since in the meantime the 32p incorporation in the phosphorylated precursors was not changed, it has been assumed that the increased 32p incorporation in the lipidic fraction was due to a stimulation of phospholipid synthesis. The ef:fect of BC-PL on phospholipJd synthesis has been confirmed in mice receiving a cholesterol supplemented diet (Toffano e t a ] . , treatment
reduced the 32p and 3H-choline
J976, 1977). This
incorporation
into the brain
phospholipids. It has been found that BC-PL t r e a t m e n t almost completely restored
the incorporation
rate of both 3H-choline
and 32p into brain
phospholipid. Simultaneously it was observed a partial restoration of catecholamine, cAMP content and behavioral performances. Effect on .aminer~ic .system. Upon administration of BC-PL to mice the content of brain noradrenaline and dopamine rapidly decrease by about 20 and /40 96 respectively and returnes to normal values within 30 minutes. The parallel increase of homovanillic acid,
a catabolite of dopamine, produces a shift in tl-..? dopamine/homovanillic acid ratio
indicative
of an increased catecholamine
turnover rate (Leon and
Toffano, 1976; Leon eta]., "1978; To~'fano et al., 1976). Associated with the increased catecholamine turnover, a significant stimulation of both the NaF-dependent and the dopamine dependent-adenylate cyclase is also detected. The unstimulated activity is not affected. Such a stimulation is accompanied by a dose-dependenl higher level of cAMP, which is abolished by haloperidol treatment. The phospholipid effect op the NaF dependent adenylate cyclase is maintained at high dosage of BC~-PL, whereas the effect on dopamine-dependent adenylate cyclase disappears Leon et al., 1978). This is reminiscent of the influence produced by increased doses of apomorphine (Kebabian et al., 197.~).These experiments support the conclusion
Pharmacological Research Communications, Vol. 12, No. 9, 1980
835
Table 2: Effect of PS on Ach output from rat cerebral cortex in vivo.
Ach output Spontaneous After drug
Treatment
Pretreatrnent
96 change
none
Tris-buffer
1.15 _+ 0.20 ~"
0.92 _+ 0.07
- 20 n.s.
none
PS (150 mg)
1 . 0 7 + 0.16
1.87 + 0 . I 6
+ 75 P<0.01
P i m o z i d e ( l mg)
PS(150mg)
1,02+0.22
0,85+0.20
- 16 n,s,
Septallesion
PS(150mg)
1.20+0.19
1,03+0,16
- 16 n,s,
(12 days before) values are expressed as ng x rain -I x c m
2
+ S.E.M.
m
(taken from Casamenti et al., 1979). that catecholamines
may be involved
in the pharmacological
effect
of
exogenous phospholipids. Since the active component of phospholipid mixture is phosphatidylserine~ the influence of this phospholipid on brain catecholamine turnover has been also investigated. The employment of rat has permitted to analyze separately the hypothalamus, the cerebral cortex and the striatum. The hypothalamus and the cerebral cortex have been selected because they are the major areas of innervation of the ventral and dorsal noradrenaline bundle respectively, while striatum is largely innervated by the nigro striatal dopamine system. These pathways represent large part of cerebral noradrenaline and dopamine neurons
(Ungerstedt, 1971; Bjorklund et al., 197.5). Phosphatidylserine injection decreases the hypothalamic noradrenaline content and doubled the content of its major metabolite Phenyl-Glycol. Sulphate at doses ineffective
Methoxy-Hydroxy-
on cortical noradrenaline and
striatal dopamine. Only at much higher doses phosphatidylserine is capable to rise striatal dopaminergic and cortical noradrenergic catabolite levels, suggesting a specific
effect
of phosphatidylser~e
at hypothalamic level.
The
ineffectiveness of low phosphatidylserine doses on cortical noradrenaline and striatal dopamine may reflect its difficulty to enter the brain. The hypothala-
Pharmacological Research Communications, Vol. 12, No. 9, 1980
836 DOSE-RESPONSE 20 t
TIM~.COURSE
MHPG,S04 *
',01<
.6OO tO O.
300 (/) C~
,300 Z
-r
O.0
=E ,
o
,
,
do
(mglkg)
G O - ~
,oo
,
o
-- •-~
,
2
time
4 (hrs)
Fig. 2: Effect of PS on hypothalamic NE and MHPG*SO4 concentration. (modified from Toffano et al., 1978). mic region is known to be less efficiently protected by the blood brain harrier. The effect of phosphatidyJserine on hypothalamic noradrenaline has been further explored in animals pretreated with a MAO inhibitor agent (phenylethylhydrazine) or with an inhibitor of catecholamine synthesis ((7.-methyl-ptyrosine). Phosphatidylserine further increases the accumulation or the disappearance of noradrenaline following the administration of MAO inhibitor agent or the inhibitor of catecholamine synthesis, respectively. That brain catecholamine are involved in the phosphatidylserine effect
is confirmed by the
stimulation of cAMP synthesis in the hypothalamus (Nathanson, 1977). Reserpine treatment prevents the
phosphatidy]serine-induced hypothalamic cAMP
accumulation. Similar effect is produced by propranolol but not by phentolamine. This suggested that cAMP accumulation occurred through the involvement of a ~-adrenergic receptor. A noradrenaline sensitive adenyiate cyclase has been identified in the hypothalamus (Nathanson, 1977). Parallel investigations have shown that phosphatidylserine administration increases the acetylcholine output from cerebral cortex (Casamenti et al., 1979). Also in this case phosphatidylserine has been the most active phospholipid tested. Phosphatidylcholine and phosphatidylethanolamine have I~een ineffective. The enhanced acetylcholine output from the cerebral cortex is # completely prevented by a septal lesion capable of inhibiting the stimulatory effect of amphetamine or levo dopa. Consistently, pimozide, a dopamine
Pharmacological Research Communications, VoL 12, No. 9, 1980
837
receptor blocking agent, abolishes the effect of phosphatidylserine on the acetylcholine output (Tab. 2). The prevention of phosphatidylserine effect by a septal lesion or by pimozide suggests that phosphatidylserJne might stimulate the acetylcholine eutput from the cerebral cortex through an indirect mechanism possibly involving a monoaminergic link. In accord, the effect of phosphatidylserine on acetylcholine release is absent when the effect is studied in vitro on brain slices.
Effect on carbohydrate rnetab_oli_sm When BC-PL or phosphatidylserine
sonicated dispersions are injected
intravenously to mice, extensive m~dification of the carbohydrate metabolism is produced, The blood' glucose and the brai~, free-glucose levels increase whereas the liver glycogen decreases (Bruni et al., 1976). Other natural phospholipids including phosphatidylcholine, phosphatidylethanolamine~ phosphatidylinrsitol and diphosphatidylglycerol are without effect (Tab. l). Deterruination of cerebral content of glycolytJc intermediates and high-energy compounds have disclosed that the glucose .accumulation in the brain is the consequence of reduced energy expenditure by the nervous tissue (Bigon et ah,
1979a). Further investigations have led to the identification of two fundamental steps in the effect of phosphatidylserJne on brain energy metabolism= a) the phospholipid is activated by a metabolic conversion "in vivo" and (b) the metabolic product of phosphatidylserine becomes in t~'n a potent activator of processes taking place in specialized cells (BJgon et al.~ 1979 b; Bigon et al, 1980; Bruni et al., 1980). Phosphatidylserine activation is due to deacylation catalysed by a phospholipase A 2 enzyme. The resulting product is lysophosphatidylserine.
The
splitting of the acyl-chain in position 2 of phosphatidylserine greatly enhances the activity on carbohydrate mobilization in mice. High activity has been detected at remarkable low doses (l-2/Jmoles/kg, corresponding to 0.5-1.0 w
mg/kg). The possibi.lity that lyso-phosphatidylserine acts by an aspecific detergent effect common to all lysophospholipids has been ruled out by the ineffectiveness of lysophosphatidylcholine and lysosphosphatidylethanolamine
Pharmacological Research Communications, VoL 12, No. 9, 1980
838 (Tab.
I). Moreover,
the low amount of this compound required for full
effectiveness excludes that the critical micellar concentration of lyso-phosl
phatidy]serine (approx. 0.01mM, Martin and Lagunoff, 1979) is reached in the body fluids. A transfer of lysophosphatidylserine across the blood-brain barrier can be accomplished by the phospholipid in form of monomer. Experiments in v!tro on isolated mast cells have been performed to explain the mechanism of the lysophosphatidylserine effect. On this preparation the phospholipid induces extensive histamine release, when calcium and mouse plasma are added (Bigon et al., 1980). This demonstrates that this phospholipid does not act directly but requires the presence of plasma factor(s) to promote the release of amines from specialized cells. Neuroendocrine effects. Since the hypothalamic neurotransmitters influence the pituitary hormon secretion, the' effect of phosphatidylserine on plasma prolactin level has been studied in vivo and in vitro. In rats phosphatidylserine9 upon acute and chronic treatment~ reduces plasma prolactin levels during different phases of circadian rhythm (Canonico et al., 1980). The phospholipid is able to inhibit the increase of plasma level occurring in cycling female rats on the afternoom of the proestrus. The time course of phosphatidylserine effect on prolactin secretion is characterized by a short latency (15 rnin) and a long duration of action (6 hours). In rats with hypothalamic deafferentation, phosphatidylserine partially reduces the increase of circulating prolactm induced by a pretreatment with
(~-methyl-p-
tyrosine. Behavioral effect. It is of interest to know whether phosphatidylserine modifies
the animal
behavior and to investigate the correlation between the behavioral patterns and the changes in brain neurotransmitter concentration. Since phosphatidylserine modifies catecholamine and acetylcholine turnover it might be expected that the specific behavior depending on catecholamine and acetylcholine activity can be influenced. Preliminary results indicate that phosphatidylserine administration in rats
Pharmacological Research Communications, Vol. 12, No, 9, 1980
839
30.
o
Bc-P
(2.2 rng~kg)
25-
/! \ Ii
20-
CPZ
.~E 15.
j.
•
T
, ~'---....,, .~ lO.
E
5-
O-10 0
*30
*60 *90 .130 Time (rain)
.150
-180
.210
*P
Fig. 3: Effect of BC-PL on Prl serum level in.chlorpromazine (CPZ) treated subjects.
is followed by a dose dependent delay of morphine catalepsy onset (Tof fano, 1979) and a reduction of the scopolamine effect on spontaneous alternation in a T maze (Pepeu and Casamenti, 1979). Morphine catalepsy is believed to be the consequence of a stimulation of striatum
opiate receptors. On this basis it can be suggested that
the
antagonism by phosphatidylserine occurs in the same brain area. Stimulation of dopamine receptors in this area or decreased morphine binding to its specific sites may explain this effect. The counteracting effect of phosphatidylserine on the action of scopolamine on the alternation behaviour suggests an effect of
phosphatidylserine on subcortical
cholinergic
pathways, possibly the
hippocampus. Clinical pharmacology Data on the influence of BC-PL on homovanillic acid and ~-hydroxyindoleacetic acid
concentrations in the human cerebrospinal fluid and on prolactin
840
Pharmacological Research Communications, Vol. 12, No. 9, 1980
and growth-hormon levels in human plasma are availal~le (Toffano et al., 1979; Nizzo et a l , 1978). In healthy human subjects it has been shown that BC-PL significantly increase the plasma level o£ growth-hormon whereas the prolactin level is decreased. The hypoprolactinem!zJng effect by 15C-PL may be compared with a similar influence of levo dopa~ bromocriptine and piribedil (Masturzo et al,
1977; 13arreca et al.9 1977). Of
more interest
is the
observation that BC-PL antagonize the prolactin-enhancing effec~ of chlorpromazine (Masturzo et al., 1977) (Fig. 3) and haloperidol (Polleri et al., 1979) at doses insufficient to modify the physiological prolactin levels (Tab. 3). This is in accord with a dopaminergJc effect of BC-PL. The same effect is indicated also by the increase of homovanillic acid in the cerebrospinal fluids since it is known that the concentration of this dopamine catabolite in the lumbar fluid correlates with that in,the brain (Garelis et al.~ 1974; Moir et a l , 1970). From these results no conclusion as to the site of the phospholipid e f f e c t can be %f drawn. Homovanillic acid concentrations in the cerebrospinal fluids may not simply reflect the dopamine turnover in the hypothalamus. Furthermore dopaminergic pathways involved in the regulation of endocrine events may originate in other brain areas. The possibility that 13C-PL act directly on anterior pituitary cannot equally be excluded. The finding that 5-hydroxyindoleacetic acid concentration raises in lumbar fluid after BC-PL administration indicates that also serotonin metabolism nlay be influenced (Nizzo et al.~ 1978). Whether the increase of serotonin level is the consequence of an effect on the spinal cord or brain areas can not be decided at present (Bulat and Zivcovic9 1971). Finally it has also been shown that BC-PL failed to counteract the benserazide-induced hyperprolactinemia (Murialdo et al., 1979). Discussion and future perspect!ves Several lines of investigation have shown that cerebral metabolism in animal and in man is inf!uenced by the intravenous injection of exogenous phospholipids. These effects corroborate the original hypothesis (see introduction) that modifications of cellular activity may be obtained by the administration of exogenous phospholipids. Among the brain functions~ the activity of the adrenergic systemp particularly the turnover of catecholamines, is greatly affected. Several evidence supporting this conclusion have been collected in
Pharmacological Research Communications, VoL 12, No, 9, 1980
841
direct and indirect studies, Both in rats and in mice~ an increased turnover of noradrenaline and dopamine has been detected (Leon and Toffan% 1976; Leon et al., 1978; Toffano et a l ,
1978). The effect
is more manifest at the
hypothalamic level, presumably as a consequence of greater accessibility of this area to compounds circulating in the blood, The increased catecholamJne turnover
is accompanied by adenylate cyclase activation
in accord with
current hypothesis on the ~,ffects of these neurotransmitters. Relevant consequences of adrenergic system activation are the enhancement of acetylcholine output from the cerebral cortex (Mantovani et al., 1976; Casamenti et al., 1979) and the decreased prolactin secretion (Canonico et a l , 1980). All these effects are worth of attention for potential therapeutic significance. Additional interest is provided by the finding that phosphatidylserine is the active component of the BC-PL mixture. This phospholipid has a relevant physiological role since it is able to interact with several membrane bound enzymes (Wheeler and Whittam~ 1970; Lloyd and Kaufman~ 197k~ Raese et a l , 1976) or to promote fusion among membranes (Papahadjopoulos et al.,
1973). The
pharmacological effects detected in our studies may be a reflection of these properties. The observation that metabolic conversion of phosphatidyJserine to lyso-phosphatJdylserine greatly enhances the pharmacological activity indicate that this lysocornpound may play a preminent role in the effects of the brain phospholipid mixture, The generation of active lyso-phosphatidylserine from the phospholipid mixture is possible in the blood stream of animal possessing plasma phospholipase A 2 activity. Additional sites for lyso-phosphatidylserine generation can be found in several organs considering the ubiquitous distribution of phospholipid-deacylating systems. Under optimal conditions the influence on carbohydrate metabolism can be detected at doses as low as 0.5 mg/kg. These observations together with the possibility o f lyso-phosphatidylserine generation from the endogenous phosphatidylserine indicate tha~ this lyso derivative deserves consideration as endogenous messenger to specialized cells under particular circumstances. The membrane bound phosphatidylserlne may be the natural source of lyso-phosphatidylserine and the effect of the lyso-
compound can be terminated
by acylation.
A particular aspect of lyso-
phosphatidylserine pharmacology is the histamine release in mice (Bigon et a l .
842
Pharmacological Research Communications, Vol. 12, No. g, 1980
1980). This effect is apparently linked to the exhisteni:e in this animal of a plasma factor capable to enhance the sensitivity o£ mast cells to lysophosphatidylserine. In conclusion, the field of pharmacological action of phospholipids is open to fruitful investigations. These compounds appear as drubs capable of activating physiological mechanisms requiring the release of intracellular amine stores. Among these, catecholamine stores are influenced.
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Pharmacological Research Communications, Vol. 12, No. 9, 1980
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Pharmacological Research Communications. VoL 12, No. 9. 1980
Mantovani, P., Pepeu, G. and Amaducci, L. (J976) A'dvances Jn Experimental Medicine and Biology 72, 2g`5..292. Martin, T.V/. and Lagunoff, D. ([979) Nature (London) 29_7, 2`50-2`52. Masturzo, P., GaltamJnJ, A., Murialdo, G., Nizzo, M.C. and Tc~ifano, G. (1977) N. Engl..1. Med, 297, 338-339. Moir, A.T,B., Ashcroft, G.W., Grawford, T.B.B., Ecc|eston, D. and GuIdsberg, H. ([970) Brain 93, 357-368. Muriaido, G., Masturzo, P., Polleri, A., Carolei, A. and Toffano, G. (1979) Neuropsychobiology 5, 3[ 7-32 [. Nathanson, J.A. (1977) Physiol. Rev. 577, 157-256. Newton, C., Pangborn, W., Nir, S. and Papahadjopoulos, I"3. (1978) Biochim. Biophys. Acta .506, 281-287.
Nizzo, M.C., Tegos, S., Gallamini, A., Toffano~ G., PoJieri, A. and Massarotti, M. (|978) 3. Neural Transm. #_3,93-102. Orlando, P., Cerrito, F. and Massari, P. (197`5) Acta Med. Rom. ]3, 66-101. Papahadjopoulos, D. (197~) [n "Membrane Fusion", Poste, G. and Nicolson, G.L. eds., North-Holland Publishing, Amsterdam, pp. 76`5-790. Papahadjopoulos, D., Poste, G. and Schaeffer, B.E. (1973) l~iochim. ~iophys. Acta 323, 23-t~2. Pepeu, G. and Casamenti, F. (1979) Seventh Meeting of the International Society for Neurochemistry, 3erusalem, Israel, September 2-6. Abstract 159. Polleri, A., Masturzo, P., Murialdo, G., Baldassarre, M., Martucci, N., Nappi, G., Savoldi~ F., Muratorio, A., Murri, L. and Gasparetto, i5. (1979) In "Central and Peripheral Regulation o! Prolactin Function" Taormina, october 12-1#. l~.aese, 3., Patrick, R.L. a~cl Barchas, 3.D. (1976) Biochem. Pharmacol. 25, 2245-2250. Smith, R. and Tanford, C. (1972) ], Mo]. Biol. 6~7, 75-g3. Toffano, G. (1979) Seventh Meeting of the International Society for Neurochemistry. 3erusalem, Israel, September 2-6. Abstract 155. Tof~ano, G., GaIlamini, A., Massarotti, M. and Savoldi, F. (]979) In "Neuroendocrinology: Biological and Clinical Aspects" Polleri, A. and MacLeod, R.M. eds., Academic Press, New York and London, pp. 3qS-3t~9.
Pharmacological Research Communications, VoL 12, No, 9, 1980
ToHan% G., Leon, A., Benvegn~J, D., Boarato, E. and Azzone, G.F. (1976) Pharma~ol. Res. Commun. 8, 581-590. Toffano, G., Leon, A., Benvegn~, D. and Cerrito, F. (1977) Atherosclerosis, 266, 59-66.
ToHano, G., Leon, A., MazzarJ, S., Savoini, G., Teolato, S. and Orlando, P. (1978) Life Sciences 23, 1093-1102. Tyrell, D.A., Heath, T.D., Colley, C.H. and Ryman, B.E. (1976) Biochim. Biophys. Acta, t~.57, 259-302. Ungerstedt, S. (1971) Acta Physiol. Stand., Suppl. 367, 1-45. Verkleij, A.3., Zwaal, R.F.A., Roelofsen, B., Comfurius, P., Kastelijn, D. and Van Deenen, LL.M. (1973) Biochim. Biophys. Acta,<323, 178-193. Wheeler, K.P. and Whittam, R. (1970) Nature, 225, 0#9-050. Wurtman, R.3., Hirsch, M.3. and Gro~vdon, 3.H. (1977) Lancet, II, 68-69.
845
829
PHARMACOLOGICAL PROPERTIES OF PHOSPHOLIPID LIPOSOMES.
G. Toffano and A. Bruni ~ Fidia Research Laboratories~ Abano Terme~ Italy ~Institute of Pharmacology~ University of Padova9 Italy Receivedin final form 4 June 1980
INTRODUCTION The use of natural phospholipids as therapeutic agents, has been introduced from many years and considerable effort has been made to detect and eventual ly define pharmacological effects "in vivo". These studies have been based on two general hypothesis= (a) alteration of the composition of phospholipid containing structures (e. 8. cellular membranes9 lipoprotein) may result in controlled variations of biological activity and (b) supply of exogenous phospholipids to the tissues may improve cellular activities when pathological processes or natural aging reduce the renewal of the phospho]ipid membrane component. Support to these hypothesis is provided by the development of knowledges on membrane structure9 composition and function. It has been established that the composition of the bilamellar leaflet forming the network of cellular membranes is asymmetric and that a different function is likely assigned to the phospholipids present in the inner and outer layer (Verkleij et al., 1973). Depending on the phospholipid composition it is possible to induce the transient formation of non-bilayer lipid configurations with potential ion-transporting activity (Cullis and De Kruyff~ 1978). Also pertinent to the purpose of this review is the observation that the phospholipid fluidity influences the mobility of membrane proteins and therefore their catalytic activity and the possibility to interact with specific ligands (Hirata and Axelrod,
1973; Hirata et a l .
1979; Hirata et al.,
1979). Moreover,
administration of large amount of phosphatidylcholine enhances the synthesis of acetylcholine in the nervous tissue (Cohen and Wurtman~ 1976; Hirsch and 0031--6989/80/090829-17~02.00/0
© 1980 The Italian Pharmacological Society
Pharmacological Research Communications, VoL 12, No. 9, 1980
830
Wurtman, 1978; Wurtman et al., 1977). This obsm'va'tion which awaits confir'mation, implies the possibility to influence the synthesis of neurotransmitters Dy exogenous phospholipids. Furthermore,
the clarification
of phenomena
underlying membrane fusion (Papahadjopoulos, 1978) may provide the experimental support to the possibility that the injected phospholipids may reach and fuse with cellular membranes altering their composition and function. A parallel line of investigation on liposome pharmacology has been developed and is based on the property of phospholipid..liposomes to serve as drug carriers (Gregoriadis,
1976). Attention has been focused also on the influence of
liposomes on immune reactions (Allison and Gregoriadis, 197/4) and on the possibility to use these structures as chemotherapeutic agents (Alving, 1979). For a detailed survey of these recent developments, the reader is referred to the review of Tyrrel et al. (]976) and to the book "Liposomes in Biological Systems" (Gregoriadis and Allison, 1980) Since the biological activity of phosphol~pids is dependent on the structure formed in aqueous solution, a brief survey of this field will be presented first. Subsequently, data on the distribution of these compounds will be reported. Finally,
the pharmacological
properties
of phospholipids,
with
particular
emphasis to phosphatJdylserine, will be reviewed. Phosphatidylserine has been the most extensively
investigated
in our laboratories
and the activities
detected so far are not shared by other natural occurring phospholJpids. Self-assembly of p hospholipids Phospholipids are amphiphilic
molecules
provided
with
a hydrophobic
carbon tail and a polar head-group. Because of the thermodynamic unfavourab'e interaction of water with the hydrocarbon chains, phospholipids aggregate in defined structures above a concentration denoted as "the critical micellar concentration"
(reviewed
by Israelachvili
et al.,
1977). The hydrophobic
bonding among phospholipid aliphatic chains are balanced by the electrostatic bonding between the polar head groups and the water dipoles. [3epending on the number and lenght of hydrocarbon chains, phospholipids yield different structures in aqueous solution. For the purpose of this review three of them are relevant: micelle$, multilamellar bilayers (liposomes), vesicles (Fig. I). Micelles can be spherical or elongated. They have a polar external surface
Pharmacological Research Communications, VoL 12, No. 9, 1980 LIPOSOME (multilamellar)
VESICLE
1L monomer cone. 10"3M
monomer conc. 10"10M
Fig. l : Diagramatic representation of micelle, liposorne and vesicle forrnation in water. (modified from Hokin, 1969). and an apolar core in a liquid-like state. In these structures the inner aqueous compartment is not delimited. Liposomes and vesicles have in common a phospholipid bilayer organization which delimits separated aqueous compartments. They differ in the numl~er o[ phospholipid layers and in the radius o5 bilayer curvature. Liposomes are spontaneously formed when water is added to a dry film of phospholipids. They are generally multilayers with a large radius of curvature (planar bilayer). On sonication~ liposomes reorganize in structures with reduced number of biJayers. Small (200 ~) unilamellar vesicles can be obtained. The unJlamellar vesicles mantain an aqueous compartment inside while the small radius of curvature will favour the asymmetric distribution of phospholipids in the bilayer. Of relevance is the analysis oJ the factors determining the phospholipjds self assembly whether in micelles or bilayers. In both cases the driving force is the hydrophobic bonding among the hydrocarbon chains which depends on the lensht of the acyl-chains. Short acyl-chains yield small and unstable aggregates (micelles)in rapid equilibrium with a relatively high monomer concentration. Long acyl-chains yield a bilayer organization slowly equilibrating with a low monomer concentration (approx. 10-10M, Smith and Tanford~ 1972). The significant differences between the micellar and the bilayerorganizations are (i) greater dimension of bilayer aggregates,(ii) lower stability of the micelles and (iii) higher monomer concentration in the micellar dispersions.
831
Pharmacological Research Communications, VoL 12, No. 9, 1980
832
Table I - Effect of BC-PL dispersion and specificity of phosphatidylserine and lyso-phosphatidylserine on brain glucose accumulation. (from: Bruni et al., 1976, a; Bigon et a l . 19799 a).
Phospholipid
Dose mol/kg i.v.
Brain glucose
tool~ g wet wt
--
1.1
BC-PL (homogenization)
150
1.2
B C - P L (sonication)
150
2.7 ~
None
Brain Phosphatidylserine
50
3.69 ~
Brain Phosphatidylethanolamine
50
1.0#
Brain Phosphatidylcholine
50
0.96
Brain Phosphatidylinositol
50
1.17
Heart diphosphatidylglycerol
50
0.89
Lyso-phosphatidylserine
10
4.50 ~
Lyso-phospha tidylethanolamine
20
1.50
Lyso-phosphatidylcholine
20
1.63
* P < O.Ol
Pharmacokinetics Because of the differences outlined above, distinct pharmacokinetics are expected after administration in vivo of a micellar or a liposomal dispersion. The high monomer concentration in the micellar dispersion is a favourable condition for extensive binding to plasma proteins. The decrease of monomer concentration below the critical micellar concentration as a consequence of protein binding, may lead to dissolution of phospholipid aggregates. For the same reason micellar dispersions have greater potential activity in comparison to liposomes to cross membrane barriers. Liposomes and vesicles are more stable in the blood stream° Their stability cholesterol
is further
increased provided
is included in their composition (Bireisblatt and Ohki,
[976).
Pharmacological Research Communications, Vol. 12, No. 9, 1980 However, the plasma levels of liposomes decrease rapidly after their administration mainly as consequence of accumulation in the liver and in the spleen. Large part of lJposomes are incorporated in phagocytic cells. A comprehensive review dealing with the liposome distribution in vivo is available (Tyrell et al., 1976). The large size of liposomes and unilamellar vesicles prevents these structures to reach a large volume of distribution. Within this limitation, remarkable differences in pharmacological activity are observed between unsonicated and sonicated liposomes (15runi et al., 1976). As shown in Table 1, the capacity of a phospholipid suspension to increase the brain glucose content becomes apparent only upon sonication of phospholipids. Clearly, the size of liposomal particles affects both the distribution and the activity o5 phospholipids. Pharmacological eff.ect_s of phospholipid.liposome~
role of phosphatidylserine.
In 1975-76 it has been observed that the intravenous injection of a sonicated phospholipid mixture (BC-PL) extracted from bovine brain influenced the cerebral metabolism. The mixture of bovine brain phospholipids was composed of 6.6 96 phosphatidic acid, 20.2 96 phosphatidylethanolamine, 15.7 96 phosphatidylserine, 15 96 lysophosphatidylethanolamin e (serine), 29.9 96 phosphatidylcholine, 9.4 96 sphyngomyelin and trace amount of cerebroside (gruni et al., 1976). increased catecholamine turnover (Leon and Toffano, 1976), glucose accumulation (Bruni et al., 1976) and enhanced acetylcholine output from cerebral cortex (Mantovani et al., 1970) have'been described. All these studies demonstrated that phosphatidylserine was the active ingredient. On these basis the suggestion has been made (Bruni et al., 1980) that deacylaton and reacylation of phosphatidylserine may have a regulatory influence in vivo on specialized cells. Detection of high biological activity in a phosphatidylserine dispersion is not surprising considering that this phospholipid has a crucial role in several membrane linked activities such as enzyme activation, (Wheeler and Whittman, 1970; Lloyd and Kaufman, 197t~; Raese et'al., 1976) liposome fusion (Papahadjopoulos et al., 1973), ion permeability (Hauser and Dawson, 1967; Newton et al, 1978), histamine release (Goth et al.~ 1971) and recognition phenomena (Abood and Takeda, 1976; Abood et al., 1975).
833
Pharmacological Research Communications, Vol. 12, No. 9, 1980
834
Parallel studies have shown that the administration 6f BC-PL dispersions to
mice produced a marked inhancement of 32p incorporation into brain phospholipids after few hours (Orlando et al., 1975).The increased incorporation ranged from ~5 to 75 96. Among individual phospholipid classes, increase of specific activities has been found in phosphatidylcholJne, pliosphatidylethanolamine and phosphatidylserine. By contrastt phosphatidic acid and phosphatidylinositol showecJ a decrease. Since in the meantime the 32p incorporation in the phosphorylated precursors was not changed, it has been assumed that the increased 32p incorporation in the lipidic fraction was due to a stimulation of phospholipid synthesis. The ef:fect of BC-PL on phospholipJd synthesis has been confirmed in mice receiving a cholesterol supplemented diet (Toffano e t a ] . , treatment
reduced the 32p and 3H-choline
J976, 1977). This
incorporation
into the brain
phospholipids. It has been found that BC-PL t r e a t m e n t almost completely restored
the incorporation
rate of both 3H-choline
and 32p into brain
phospholipid. Simultaneously it was observed a partial restoration of catecholamine, cAMP content and behavioral performances. Effect on .aminer~ic .system. Upon administration of BC-PL to mice the content of brain noradrenaline and dopamine rapidly decrease by about 20 and /40 96 respectively and returnes to normal values within 30 minutes. The parallel increase of homovanillic acid,
a catabolite of dopamine, produces a shift in tl-..? dopamine/homovanillic acid ratio
indicative
of an increased catecholamine
turnover rate (Leon and
Toffano, 1976; Leon eta]., "1978; To~'fano et al., 1976). Associated with the increased catecholamine turnover, a significant stimulation of both the NaF-dependent and the dopamine dependent-adenylate cyclase is also detected. The unstimulated activity is not affected. Such a stimulation is accompanied by a dose-dependenl higher level of cAMP, which is abolished by haloperidol treatment. The phospholipid effect op the NaF dependent adenylate cyclase is maintained at high dosage of BC~-PL, whereas the effect on dopamine-dependent adenylate cyclase disappears Leon et al., 1978). This is reminiscent of the influence produced by increased doses of apomorphine (Kebabian et al., 197.~).These experiments support the conclusion
Pharmacological Research Communications, Vol. 12, No. 9, 1980
835
Table 2: Effect of PS on Ach output from rat cerebral cortex in vivo.
Ach output Spontaneous After drug
Treatment
Pretreatrnent
96 change
none
Tris-buffer
1.15 _+ 0.20 ~"
0.92 _+ 0.07
- 20 n.s.
none
PS (150 mg)
1 . 0 7 + 0.16
1.87 + 0 . I 6
+ 75 P<0.01
P i m o z i d e ( l mg)
PS(150mg)
1,02+0.22
0,85+0.20
- 16 n,s,
Septallesion
PS(150mg)
1.20+0.19
1,03+0,16
- 16 n,s,
(12 days before) values are expressed as ng x rain -I x c m
2
+ S.E.M.
m
(taken from Casamenti et al., 1979). that catecholamines
may be involved
in the pharmacological
effect
of
exogenous phospholipids. Since the active component of phospholipid mixture is phosphatidylserine~ the influence of this phospholipid on brain catecholamine turnover has been also investigated. The employment of rat has permitted to analyze separately the hypothalamus, the cerebral cortex and the striatum. The hypothalamus and the cerebral cortex have been selected because they are the major areas of innervation of the ventral and dorsal noradrenaline bundle respectively, while striatum is largely innervated by the nigro striatal dopamine system. These pathways represent large part of cerebral noradrenaline and dopamine neurons
(Ungerstedt, 1971; Bjorklund et al., 197.5). Phosphatidylserine injection decreases the hypothalamic noradrenaline content and doubled the content of its major metabolite Phenyl-Glycol. Sulphate at doses ineffective
Methoxy-Hydroxy-
on cortical noradrenaline and
striatal dopamine. Only at much higher doses phosphatidylserine is capable to rise striatal dopaminergic and cortical noradrenergic catabolite levels, suggesting a specific
effect
of phosphatidylser~e
at hypothalamic level.
The
ineffectiveness of low phosphatidylserine doses on cortical noradrenaline and striatal dopamine may reflect its difficulty to enter the brain. The hypothala-
Pharmacological Research Communications, Vol. 12, No. 9, 1980
836 DOSE-RESPONSE 20 t
TIM~.COURSE
MHPG,S04 *
',01<
.6OO tO O.
300 (/) C~
,300 Z
-r
O.0
=E ,
o
,
,
do
(mglkg)
G O - ~
,oo
,
o
-- •-~
,
2
time
4 (hrs)
Fig. 2: Effect of PS on hypothalamic NE and MHPG*SO4 concentration. (modified from Toffano et al., 1978). mic region is known to be less efficiently protected by the blood brain harrier. The effect of phosphatidyJserine on hypothalamic noradrenaline has been further explored in animals pretreated with a MAO inhibitor agent (phenylethylhydrazine) or with an inhibitor of catecholamine synthesis ((7.-methyl-ptyrosine). Phosphatidylserine further increases the accumulation or the disappearance of noradrenaline following the administration of MAO inhibitor agent or the inhibitor of catecholamine synthesis, respectively. That brain catecholamine are involved in the phosphatidylserine effect
is confirmed by the
stimulation of cAMP synthesis in the hypothalamus (Nathanson, 1977). Reserpine treatment prevents the
phosphatidy]serine-induced hypothalamic cAMP
accumulation. Similar effect is produced by propranolol but not by phentolamine. This suggested that cAMP accumulation occurred through the involvement of a ~-adrenergic receptor. A noradrenaline sensitive adenyiate cyclase has been identified in the hypothalamus (Nathanson, 1977). Parallel investigations have shown that phosphatidylserine administration increases the acetylcholine output from cerebral cortex (Casamenti et al., 1979). Also in this case phosphatidylserine has been the most active phospholipid tested. Phosphatidylcholine and phosphatidylethanolamine have I~een ineffective. The enhanced acetylcholine output from the cerebral cortex is # completely prevented by a septal lesion capable of inhibiting the stimulatory effect of amphetamine or levo dopa. Consistently, pimozide, a dopamine
Pharmacological Research Communications, VoL 12, No. 9, 1980
837
receptor blocking agent, abolishes the effect of phosphatidylserine on the acetylcholine output (Tab. 2). The prevention of phosphatidylserine effect by a septal lesion or by pimozide suggests that phosphatidylserJne might stimulate the acetylcholine eutput from the cerebral cortex through an indirect mechanism possibly involving a monoaminergic link. In accord, the effect of phosphatidylserine on acetylcholine release is absent when the effect is studied in vitro on brain slices.
Effect on carbohydrate rnetab_oli_sm When BC-PL or phosphatidylserine
sonicated dispersions are injected
intravenously to mice, extensive m~dification of the carbohydrate metabolism is produced, The blood' glucose and the brai~, free-glucose levels increase whereas the liver glycogen decreases (Bruni et al., 1976). Other natural phospholipids including phosphatidylcholine, phosphatidylethanolamine~ phosphatidylinrsitol and diphosphatidylglycerol are without effect (Tab. l). Deterruination of cerebral content of glycolytJc intermediates and high-energy compounds have disclosed that the glucose .accumulation in the brain is the consequence of reduced energy expenditure by the nervous tissue (Bigon et ah,
1979a). Further investigations have led to the identification of two fundamental steps in the effect of phosphatidylserJne on brain energy metabolism= a) the phospholipid is activated by a metabolic conversion "in vivo" and (b) the metabolic product of phosphatidylserine becomes in t~'n a potent activator of processes taking place in specialized cells (BJgon et al.~ 1979 b; Bigon et al, 1980; Bruni et al., 1980). Phosphatidylserine activation is due to deacylation catalysed by a phospholipase A 2 enzyme. The resulting product is lysophosphatidylserine.
The
splitting of the acyl-chain in position 2 of phosphatidylserine greatly enhances the activity on carbohydrate mobilization in mice. High activity has been detected at remarkable low doses (l-2/Jmoles/kg, corresponding to 0.5-1.0 w
mg/kg). The possibi.lity that lyso-phosphatidylserine acts by an aspecific detergent effect common to all lysophospholipids has been ruled out by the ineffectiveness of lysophosphatidylcholine and lysosphosphatidylethanolamine
Pharmacological Research Communications, VoL 12, No. 9, 1980
838 (Tab.
I). Moreover,
the low amount of this compound required for full
effectiveness excludes that the critical micellar concentration of lyso-phosl
phatidy]serine (approx. 0.01mM, Martin and Lagunoff, 1979) is reached in the body fluids. A transfer of lysophosphatidylserine across the blood-brain barrier can be accomplished by the phospholipid in form of monomer. Experiments in v!tro on isolated mast cells have been performed to explain the mechanism of the lysophosphatidylserine effect. On this preparation the phospholipid induces extensive histamine release, when calcium and mouse plasma are added (Bigon et al., 1980). This demonstrates that this phospholipid does not act directly but requires the presence of plasma factor(s) to promote the release of amines from specialized cells. Neuroendocrine effects. Since the hypothalamic neurotransmitters influence the pituitary hormon secretion, the' effect of phosphatidylserine on plasma prolactin level has been studied in vivo and in vitro. In rats phosphatidylserine9 upon acute and chronic treatment~ reduces plasma prolactin levels during different phases of circadian rhythm (Canonico et al., 1980). The phospholipid is able to inhibit the increase of plasma level occurring in cycling female rats on the afternoom of the proestrus. The time course of phosphatidylserine effect on prolactin secretion is characterized by a short latency (15 rnin) and a long duration of action (6 hours). In rats with hypothalamic deafferentation, phosphatidylserine partially reduces the increase of circulating prolactm induced by a pretreatment with
(~-methyl-p-
tyrosine. Behavioral effect. It is of interest to know whether phosphatidylserine modifies
the animal
behavior and to investigate the correlation between the behavioral patterns and the changes in brain neurotransmitter concentration. Since phosphatidylserine modifies catecholamine and acetylcholine turnover it might be expected that the specific behavior depending on catecholamine and acetylcholine activity can be influenced. Preliminary results indicate that phosphatidylserine administration in rats
Pharmacological Research Communications, Vol. 12, No, 9, 1980
839
30.
o
Bc-P
(2.2 rng~kg)
25-
/! \ Ii
20-
CPZ
.~E 15.
j.
•
T
, ~'---....,, .~ lO.
E
5-
O-10 0
*30
*60 *90 .130 Time (rain)
.150
-180
.210
*P
Fig. 3: Effect of BC-PL on Prl serum level in.chlorpromazine (CPZ) treated subjects.
is followed by a dose dependent delay of morphine catalepsy onset (Tof fano, 1979) and a reduction of the scopolamine effect on spontaneous alternation in a T maze (Pepeu and Casamenti, 1979). Morphine catalepsy is believed to be the consequence of a stimulation of striatum
opiate receptors. On this basis it can be suggested that
the
antagonism by phosphatidylserine occurs in the same brain area. Stimulation of dopamine receptors in this area or decreased morphine binding to its specific sites may explain this effect. The counteracting effect of phosphatidylserine on the action of scopolamine on the alternation behaviour suggests an effect of
phosphatidylserine on subcortical
cholinergic
pathways, possibly the
hippocampus. Clinical pharmacology Data on the influence of BC-PL on homovanillic acid and ~-hydroxyindoleacetic acid
concentrations in the human cerebrospinal fluid and on prolactin
840
Pharmacological Research Communications, Vol. 12, No. 9, 1980
and growth-hormon levels in human plasma are availal~le (Toffano et al., 1979; Nizzo et a l , 1978). In healthy human subjects it has been shown that BC-PL significantly increase the plasma level o£ growth-hormon whereas the prolactin level is decreased. The hypoprolactinem!zJng effect by 15C-PL may be compared with a similar influence of levo dopa~ bromocriptine and piribedil (Masturzo et al,
1977; 13arreca et al.9 1977). Of
more interest
is the
observation that BC-PL antagonize the prolactin-enhancing effec~ of chlorpromazine (Masturzo et al., 1977) (Fig. 3) and haloperidol (Polleri et al., 1979) at doses insufficient to modify the physiological prolactin levels (Tab. 3). This is in accord with a dopaminergJc effect of BC-PL. The same effect is indicated also by the increase of homovanillic acid in the cerebrospinal fluids since it is known that the concentration of this dopamine catabolite in the lumbar fluid correlates with that in,the brain (Garelis et al.~ 1974; Moir et a l , 1970). From these results no conclusion as to the site of the phospholipid e f f e c t can be %f drawn. Homovanillic acid concentrations in the cerebrospinal fluids may not simply reflect the dopamine turnover in the hypothalamus. Furthermore dopaminergic pathways involved in the regulation of endocrine events may originate in other brain areas. The possibility that 13C-PL act directly on anterior pituitary cannot equally be excluded. The finding that 5-hydroxyindoleacetic acid concentration raises in lumbar fluid after BC-PL administration indicates that also serotonin metabolism nlay be influenced (Nizzo et al.~ 1978). Whether the increase of serotonin level is the consequence of an effect on the spinal cord or brain areas can not be decided at present (Bulat and Zivcovic9 1971). Finally it has also been shown that BC-PL failed to counteract the benserazide-induced hyperprolactinemia (Murialdo et al., 1979). Discussion and future perspect!ves Several lines of investigation have shown that cerebral metabolism in animal and in man is inf!uenced by the intravenous injection of exogenous phospholipids. These effects corroborate the original hypothesis (see introduction) that modifications of cellular activity may be obtained by the administration of exogenous phospholipids. Among the brain functions~ the activity of the adrenergic systemp particularly the turnover of catecholamines, is greatly affected. Several evidence supporting this conclusion have been collected in
Pharmacological Research Communications, VoL 12, No, 9, 1980
841
direct and indirect studies, Both in rats and in mice~ an increased turnover of noradrenaline and dopamine has been detected (Leon and Toffan% 1976; Leon et al., 1978; Toffano et a l ,
1978). The effect
is more manifest at the
hypothalamic level, presumably as a consequence of greater accessibility of this area to compounds circulating in the blood, The increased catecholamJne turnover
is accompanied by adenylate cyclase activation
in accord with
current hypothesis on the ~,ffects of these neurotransmitters. Relevant consequences of adrenergic system activation are the enhancement of acetylcholine output from the cerebral cortex (Mantovani et al., 1976; Casamenti et al., 1979) and the decreased prolactin secretion (Canonico et a l , 1980). All these effects are worth of attention for potential therapeutic significance. Additional interest is provided by the finding that phosphatidylserine is the active component of the BC-PL mixture. This phospholipid has a relevant physiological role since it is able to interact with several membrane bound enzymes (Wheeler and Whittam~ 1970; Lloyd and Kaufman~ 197k~ Raese et a l , 1976) or to promote fusion among membranes (Papahadjopoulos et al.,
1973). The
pharmacological effects detected in our studies may be a reflection of these properties. The observation that metabolic conversion of phosphatidyJserine to lyso-phosphatJdylserine greatly enhances the pharmacological activity indicate that this lysocornpound may play a preminent role in the effects of the brain phospholipid mixture, The generation of active lyso-phosphatidylserine from the phospholipid mixture is possible in the blood stream of animal possessing plasma phospholipase A 2 activity. Additional sites for lyso-phosphatidylserine generation can be found in several organs considering the ubiquitous distribution of phospholipid-deacylating systems. Under optimal conditions the influence on carbohydrate metabolism can be detected at doses as low as 0.5 mg/kg. These observations together with the possibility o f lyso-phosphatidylserine generation from the endogenous phosphatidylserine indicate tha~ this lyso derivative deserves consideration as endogenous messenger to specialized cells under particular circumstances. The membrane bound phosphatidylserlne may be the natural source of lyso-phosphatidylserine and the effect of the lyso-
compound can be terminated
by acylation.
A particular aspect of lyso-
phosphatidylserine pharmacology is the histamine release in mice (Bigon et a l .
842
Pharmacological Research Communications, Vol. 12, No. g, 1980
1980). This effect is apparently linked to the exhisteni:e in this animal of a plasma factor capable to enhance the sensitivity o£ mast cells to lysophosphatidylserine. In conclusion, the field of pharmacological action of phospholipids is open to fruitful investigations. These compounds appear as drubs capable of activating physiological mechanisms requiring the release of intracellular amine stores. Among these, catecholamine stores are influenced.
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