A REVIEW OF PHOSPHATIDYLSERINE PHARMACOLOGICAL AND CLINICAL EFFECTS. IS PHOSPHATIDYLSERINE A DRUG FOR THE AGEING BRAIN?

Pharmacological Research, Vol. 33, No. 2, 1996

A REVIEW OF PHOSPHATIDYLSERINE PHARMACOLOGICAL AND CLINICAL EFFECTS. IS PHOSPHATIDYLSERINE A DRUG FOR THE AGEING BRAIN? GIANCARLO PEPEU, ILEANA MARCONCINI PEPEU and LUIGI AMADUCCI* Department of Preclinical and Clinical Pharmacology and *Department of Neurological Sciences, University of Florence, Viale G.B. Morgagni 65-80, 50134 Florence, Italy Accepted 15 December 1995 1996 The Italian Pharmacological Society

INTRODUCTION

the first compound have been demonstrated on the CNS [7], and of the second on immune responses [8].

The history of phosphatidylserine (PtdSer) began more than 50 years ago with its identification by Folch [1], as a constituent of the cephaline fraction, up to its clinical use for the treatment of the ‘ageing brain’ [2]. This use remains a matter of debate [3]. During these 50 years PtdSer has been the object of a large number of investigations aimed at defining the physiological role of endogenous PtdSer and the pharmacological effects following its administration in animals and man. The purpose of this review is to analyse critically our present knowledge on PtdSer, and in particular its actions on the central nervous system. It is hoped that the identification of unanswered questions might prompt new research on this pharmacologically active membrane constituent.

BIOCHEMISTRY PtdSer is an acidic phospholipid widely distributed, in small amounts, in animals, higher plants and microorganisms. It is located mainly on the internal leaflet of cell membranes and comprises 10–20% of the total phospholipids in the cell membrane bilayer [4] where it exerts important functions. Different fatty acids can constitute the acyl chains of the phospholipid, depending on its source. The main source of PtdSer for experimental and clinical trials has been bovine brain. This PtdSer has been frequently indicated as BC-PS (Bovine Cortex Phosphatidyl Serine). Like other phospholipids, PtdSer is deacylated by phospholipase A 2 [5]; and lysophosphatidylserine (lysoPtdSer) is formed. The latter also exerts biochemical and pharmacological actions. Finally, synthetic phosphatidylserines have been prepared [6], such as the 1,3dipalmitoyl-sn-glycerol-2-phosphorylserine, and cholesterylphosphorylserine. Pharmacological actions of

Correspondence to: Prof. Giancarlo Pepeu, Department Pharmacology, Viale Morgagni 65, 50134 Florence, Italy. 1043–6618/96/020073–08/$18.00/0

of

BIOCHEMICAL ACTIONS PtdSer is the most effective acidic phospholipid in activating the different protein kinase C isoforms [9]. A stimulation of the (Na+−K +)-dependent ATPase by mM concentrations of PtdSer has been observed in rabbit kidney preparation by Specht and Robinson [10]. A marked increase in membrane-bound (Na++K +)-stimulated ATPase and acetylcholinesterase activity has been reported after incubation of brain synaptosomes plasma membranes with PtdSer liposomes [1–5 µmol (mg protein)−1] [11]. It has been also shown that the addition of PtdSer to the incubation mixture strongly activates tyrosine hydroxylase from rat brain striatal synaptosome by decreasing the Km for the reduced pteridine cofactor [12]. A role in regulating Ca2+ uptake through passive influx, depolarization, and Na+/Ca2+ exchange [13] has been demonstrated by adding 0.1–0.3 µmol to rat brain synaptosomes in vitro. The addition of PtdSer to synaptic membrane has been shown to modulate flunitrazepam binding (PtdSer concentrations from 0.5 to 130 µM) [14], enhance opiate binding (100 µg ml−1) [15] and inhibit glutamate binding (100 µg ml−1 ) [16]. These findings indicate that modification of the phospholipid environment may affect ligand binding to their receptors.

PHARMACOLOGICAL ACTIONS ON PERIPHERAL TISSUES The identification of PtdSer in cell membranes has led to investigation as to whether an increase in its concentration could modify cell functions. The first action reported was the potentiation by PtdSer of histamine release from rat peritoneal mast cells induced by dextran and protein antigen [17], and by antigen from rat mesentery and lung, and guinea pig lung [18]. 1996 The Italian Pharmacological Society

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According to Foreman and Mongar [19] the addition of an emulsion containing from 1 to 100 µg ml−1 of PtdSer is able to increase the efficacy of calcium in triggering histamine release from mast cells, since PtdSer presumably forms at least part of the membrane carrier for calcium in the mast cell. The finding that PtdSer is active on mast cells has encouraged research into the possible effects on inflammatory and immune responses. A detailed description of these effects is outside the scope of this review. However, we would like to mention that PtdSer liposomes injected intravenously have been shown to decrease the primary humoral immune response in mice [20], possibly by inhibiting the steps of T-cell activation [21]. Furthermore it has been demonstrated that PtdSer liposomes (30 mg kg −1 for 3–5 days i.p. or i.v.) reduce lipopolysaccharideinduced production and release of tumor necrosis factor (TNT) in mice [22], and prevent autoimmune demyelination, presumably through a reduction of TNT production [23]. Similarly, PtdSer (30 mg kg−1 day−1 i.p.) administered for 14 days, beginning at the onset of the disease, markedly reduced the clinical severity and the mortality caused by myelin-induced experimental allergic neuritis in Lewis rats [24].

PHARMACOLOGICAL ACTIONS ON CNS

Young and adult animals The first CNS effect observed following administration of PtdSer liposomes (25–75 mg kg−1 i.v.) to mice was an increase in brain glucose, associated with, but independent from, an increase in blood glucose, and a slight decrease in adrenal catecholamines [25]. Evidence has been presented that the injected PtdSer is transformed in lysoPtdSer which in turn increases brain glucose through the release of catecholamines and histamine from peripheral stores [26]. Investigations of phospholipid effects on cortical acetylcholine (ACh) in anaesthetized rats using the cortical cup technique, have demonstrated [27, 28] that the administration of PtdSer liposomes (75–150 mg kg−1 i.p.) is followed by a large increase in ACh release which was calcium-dependent and could be blocked by the dopamine receptor antagonist pimozide. Phosphatidylethanolamine (PtdEth) shows a slight effect while phosphatidylcholine is inactive. The demonstration that PtdSer administration enhances brain ACh release prompted to study whether it can antagonize the behavioural effects of anticholinergic agents. It was shown that single administrations of PtdSer at doses of 75 and 30–60 mg kg−1 i.p., respectively, are able to restore in the rat the spontaneous alternation [29], and passive avoidance conditioned response [30] disrupted by scopolamine. Attenuation of the electroencephalographic changes induced by scopolamine were also

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observed in rats after a single dose (30 mg kg −1 i.p.) of PtdSer, and in rabbits after repeated doses (30 mg kg−1 i.p. for 10 days) [31]. A facilitation of avoidance response acquisition was observed in young rats born of mothers treated with PtdSer liposomes during pregnancy and treated for the first 5 days after birth [32], and in adult low responders rats treated with 15 mg kg −1 i.p. for 30 days [33]. These findings demonstrated that PtdSer can directly affect cognitive mechanisms. Finally, perfusion of turtle olfactory epithelium with PtdSer in vivo greatly lowered the threshold for olfactory bulbar responses to odorants and enhanced their size [34]. The acute administration of PtdSer not only stimulates the cholinergic system, but a decrease in brain content of noradrenaline and dopamine and an increase in homovanillic acid has been observed in mice after i.v. injection of 75 mg kg−1 of PtdSer liposomes [35]. Moreover, the i.v. injection of 50 mg kg−1 of PtdSer liposomes has been reported to enhance the activity of DA-stimulate adenylate cyclase in the mouse brain [36].

Aged animals The late 1970s and early 1980s witnessed a burgeoning in the search for drugs potentially useful as treatment of age-associated cognitive deficits, mostly using the ’cholinergic hypothesis of geriatric memory dysfunction’ [37] as a starting point. The neurochemical and pharmacological properties of PtdSer made it potentially useful in these conditions. Animal experiments confirmed that repeated administrations of PtdSer improved acquisition and retention of passive and active avoidance tasks in aged Wistar rats [38]. A significant dose-related (range 12.5–50 mg kg−1 i.p.) effect on retention was also observed [39] when PtdSer was given to aged Fischer 344 rats both prior to training and retention or only 30 min prior to training, as well only 5 min following training. No improvement was observed in the psychomotor deficits. PtdSer (15 mg kg−1 day−1 i.p.) administered for 55 days to ageing Sprague–Dawley rats reduced the number of age-associated bursts of seizure-like EEG patterns, improved passive avoidance conditioned responses and restored spontaneous alternation [40]. Vannucchi et al. [41] demonstrated that recovery of passive avoidance impairment occurring in 18-monthold rats could be obtained with PtdSer administration (15 mg kg−1 day−1 i.p.) per 7 days only. In all the experiments reported in the above paragraph PtdSer was administered by i.p. injections. However, it has been shown [42] that in 21–24month-old rats chronic oral administration (50 mg kg−1 day −1 for 12 weeks) is also followed by an improvement of the deficit in spatial memory, evaluated by the Morris maze test, and passive avoidance tests. The hypothesis that the behavioural improvement observed in aged rats might be correlated to that of

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age-associated cholinergic hypofunction was tested by Pedata et al. [43]. It was shown that ACh release evoked by electrical stimulation from cortical slices prepared from 24-month-old rats is approximately 50% lower than that in 3-month-old rats. PtdSer (15 mg kg−1 day−1 i.p. for 30 days) completely restored the release. Phosphatidylcholine was inactive and the same PtdSer treatment did not increase ACh release in young rats. In further investigations on the effects of PtdSer on the cholinergic deficit in ageing rats, it was shown [44] that in 16-month-old rats 7 days of treatment (15 mg kg−1 i.p.) with PtdSer liposomes are sufficient to restore ACh release evoked by electrical stimulation from cortical slices to the levels of 3month-old rats. The marked decrease in ACh content found after the stimulation period in cortical slices from the ageing rats was also prevented by PtdSer treatment, a finding suggesting that PtdSer restores ACh release in ageing rats by maintaining an adequate ACh supply. In order to understand through which mechanism PtdSer may stimulate ACh formation in ageing rats, ACh synthesis was investigated by incubating electrically stimulated cortical slices with [3 H] choline and measuring total and [3H]ACh release. PtdSer administration (15 mg kg −1 day−1 i.p.) for 1 week to 18-month-old rats prevented the reduction in total ACh release but not the reduction in evoked [3 H]ACh release. This finding indicates that PtdSer is able to increase the availability of endogenous choline for de novo ACh synthesis and release [41]. Finally, confirmation that PtdSer corrects the age-associated cholinergic impairment was sought in vivo by microdialysis measurement of ACh release. It was demonstrated that PtdSer administration (15 mg kg−1 day−1 i.p.) for 8 days to 19-month-old rats significantly attenuates the decrease in ACh release occurring in these rats in comparison with 4-month-old rats. Similar treatments with phosphatidylcholine or o-phosphodl-serine had no effect [45]. Other actions of PtdSer in ageing animals have been reported. K+-depolarized cortical synaptosomes prepared from 22-month-old rats show a marked decrease in 45Ca2+ uptake. The treatment with PtdSer liposomes (15 mg kg−1 i.p.) for 30 days restored the uptake [44]. In aged male rabbits, a 30-day treatment with PtdSer (15 mg kg −1 i.p.) restored the number of prolactin receptors in the hypothalamus and substantia nigra, which is reduced in the old animals, and decreased prolactin plasma levels in both ageing and young animals [46]. Oral administration of PtdSer (50 mg kg−1 day −1 for 30 days) restored the hypotensive effect and bradycardic effect of clonidine infused in the nucleus tractus solitarii which is reduced in 16month-old rats. PtdSer neither affected clonidine actions in young rats, nor restored it in 24-month-old rats [47]. According to this paper, during ageing there is a decreased sensitivity of α2 receptors. Such an alteration can be reversed by chronic treatment with PtdSer during the initial but not the later stages of

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ageing [48]. It was also observed that novelty-induced grooming increases in ageing-rats. PtdSer (20 mg kg−1 day −1 i.p. for 20 days) administration to ageing rats not only normalizes grooming behaviour but also reduces hyperthermia and the number of gastric ulcers induced by restraint stress. Finally, PtdSer (50 mg kg−1 day−1 o.s.), suspended in tap-water, administered to rats between the ages of 3 and 27 months prevented the age-associated loss of dendritic spines of CA1 hippocampal pyramidal cells [49]. In conclusion, the experiments carried out in ageing rats demonstrates that PtdSer either administered i.p. or orally may reduce the severity of behavioural, physiological, neurochemical and morphological changes associated with ageing. Although direct evidence of a relationship between the improvement in age-related cholinergic deficit and cognitive impairment has not been presented, it has been shown that the improvement in cholinergic hypofunction and behavioural deficits show the same time course and occur with the same PtdSer doses [41]. The effective doses of PtdSer range from 15 mg kg −1 i.p. to 50 mg kg−1 o.s. daily and the duration of the treatment from 7 days i.p. to 24 months p.o. Unfortunately, the dose–effect relationship has only been investigated in the study of Corwin et al. [39] In this study PtdSer behavioural effects in ageing rats were seen even after one or two administrations. Similarly, in one study only [50], the shortest duration of the effective i.p. treatment (15 mg kg−1 day−1 ) was established and was found to be 7 days, a demonstration that PtdSer can be remarkably active in correcting some of the agedependent deficits. Finally, it is important to stress that none of the effects of PtdSer observed in ageing animals has been obtained with phosphatidylcoline.

CLINICAL ACTIONS OF PtdSer Since PtdSer administration is able to improve several age-associated behavioural and neurochemical alterations in rats and rabbits, the question arises as to whether PtdSer can also improve the cognitive decline in the elderly including that accompanying Alzheimer’s disease (AD). Several clinical trials have attempted to answer this question. In this review only the double blind, controlled trials will be mentioned and discussed. Few trials demonstrated therapeutic efficacy in subjects meeting the clinical criteria for probable Alzheimer’s disease. The largest [51] involved 142 subjects beetween 40 and 80 years of age, with a gradual, progressive decline in mental capacity, meeting the NINCDS–ADRDA diagnostic criteria. The subjects treated with PtdSer (200 mg day−1 p.o.) for 3 months showed a statistically significant improvement in several items of the Blessed Dementia Scale, block tapping test and Clifton Assessment Scale. The differences between the two groups were not striking but

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related to the subgroup of patients with higher impairment. The effect was long lasting, and in some cases even more evident three months after cessation of the therapy. In a group of 51 patients, which also met the NINCDS–ADRDA criteria for probable AD and DMS-III for primary degenerative dementia, the treatment with PtdSer (100 mg t.i.d. p.o.) for 3 months brought about a statistically significant improvement over placebo on several cognitive measures [52]. Contrary to the previous trial, differences between treatment groups were most apparent among patients with less severe cognitive impairment and were defined by the authors as a ’mild’ therapeutic effect. Similar results were obtained by Engel et al. [53] on 42 patients between 55 and 75 years of age with mild primary degenerative dementia according to DMS-III, and a Mini Mental State score between 15 and 27. Clinical global improvement ratings showed significantly more patients improving under PtdSer (300 mg day−1 for 8 weeks) than under placebo. The improvement carried over to the following wash-out and treatment phases, similarly to what had been observed in the trial by Amaducci et al. [51] There was no improvement in the dementia GBS scale, psychometric tests or P300-latency. However PtdSer shifted the EEG power, higher than normal in the patients, more versus normal levels. A significant reduction of relative delta power, associated with an increase in global power was observed in a group of 18 patients with probable AD according to the NINCDS–ADRDA criteria who were treated for 6 months with PtdSer (200 mg twice per day p.o.) and cognitive training, in comparison with AD patients receiving only cognitive training. The EEG changes were associated with an increase in glucose metabolism, evaluated by positron emission tomography with 18F-2-fluoro-2-deoxy-dglucose, in the visual association areas, during functional activation and in the left superior temporal girus. The group treated with PtdSer showed that 50% of responders had improvement of the neuropsychological tests which was most evident after 8 and 16 weeks of treatment [54]. The therapeutic effect of PtdSer was also observed [2] in 149 subjects (mean age 63 years) meeting ageassociated memory impairment (AAMI) inclusion and exclusion criteria. Patients treated with the drug (100 mg t.i.d. p.o. for 12 weeks) improved relative to those treated with placebo on performance tests related to learning and memory tasks of daily life. It is interesting that individuals within the sample who performed at a relatively low level prior to treatment were mostly likely to respond to PtdSer. Cenacchi et al. [55] evaluated the therapeutic efficacy of PtdSer by treating for 6 months (300 mg day −1 ) 247 patients who were more than 65 years old with cognitive decline from moderate to severe as shown by a score of 10–23 in the Mini Mental State examination, and stages 4–6 of the Reisberg Global Deterioration Scale. Severe Alzheimer’s disease was included in the

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exclusion criteria. The efficacy of the treatment was measured using the Plutchik Gariatric Rating Scale and the Buschke Selective Reminding Test. Statistically significant improvements in the PtdSer-treated group compared to placebo were observed both in terms of behavioural and cognitive parameters. No side effects were observed. Neuropsychological improvement in elderly patients, affected by what is generically defined mental or intellectual deterioration, has been also reported by [56, 57] after long-term PtdSer treatment. In conclusion, this group of controlled clinical trials carried out on patients with probable AD, AAMI and ’mental deterioration’ indicates that a prolonged treatment with PtdSer is followed by a consistent, albeit slight, improvement detected with neuropsychological and instrumental tests in subgroups of patients. To what extent the improvement translates into significant advantages in the daily life of the patients and the care-givers has not yet been clearly established. However, if one considers that no side effects have been reported in over hundreds of patients treated with PtdSer, and the limited therapeutic results so far obtained with the cholinesterase inhibitor tacrine, the only therapeutic agent for AD, according to the Food and Drug Administration [58], the therapeutic possibilities offered by PtdSer should not be dismissed. The observations that PtdSer administration affects neuroendocrine functions in animals [46, 48, 59] prompted investigations of the effects of PtdSer on neuroendocrine responses in man. Intravenous injection of 50 and 75 mg significantly blunted the ACTH and cortisol responses to physical stress elicited in healthy men by physical exercise with a bicycle ergometer [60]. Similar effects were obtained after administration of 800 mg day−1 for 10 days [61]. These results suggest that PtdSer may inhibit the stress-induced activation of the hypothalamo-pituitary-adrenal axis in man.

ABSORPTION, FATE AND MECHANISM OF ACTION OF PtdSer The attempts to propose one or more mechanisms of action for the pharmacological and clinical effects following PtdSer administration raise the question whether exogenous PtdSer can reach the inner leaflet of cell membrane and exert the biochemical actions attributed to endogenous PtdSer, namely, to activate protein kinase C, (Na+−K +)-dependent ATPase, tyrosine hydroxylase, and facilitate calcium uptake. The addition of PtdSer to cell cultures results in an efficient incorporation of the exogenous phospholipid [62]. Incorporation of PtdSer into olfactory epithelial cells [34], and brain synaptosomes [13] has been also demonstrated. According to Foreman and Mongar [19] PtdSer is incorporated into the mast cell mem-

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branes and may form at least a part of the membrane binding site or carrier of calcium. However, most of the effects described in this review were observed after parenteral or oral administration. According to Bruni et al. [63], i.v. injected phosphatidy[14C]serine vesicles are rapidly removed from plasma since they are taken up by macrophages and internalized by lymphocyte and presumably endothelial cells. Therefore the actions on the immune system, and by extension on the pituitary gland, can be explained by direct effects of exogenous PtdSer on cell membranes. According to Palatini et al. [64], the irreversible uptake of PtdSer liposomes by the monuclear phagocyte system, explains the rapid initial decline in PtdSer plasma concentration observed following bolus i.v. injection of radiolabelled PtdSer in rats. The same authors demonstrated that a large fraction of PtdSer is hydrolized at the injection site probably by phospholipase A 2 and other hydrolytic enzymes released by platelets. As shown by the comparative analysis of the biotransformation products found in tissues after administration of either [3H]-glycerol-PtdSer or [14 C]serine-PtdSer, parenterally administered PtdSer follows two metabolic pathways, decarboxylation to phosphoethanolamine, and extensive hydrolytic degradation. These pathways probably reflect the two main mechanisms of PtdSer uptake, incorporation into plasma membrane and internalization by endocytosis, respectively. Four hours after i.p. injection of phosphatidyl[14C]serine vesicles, 10% of the dose has not been absorbed and is present mostly as PtdSer; 7.9% of the dose injected is found in the tissues in a liposoluble form, approximately 60% of which is PtdSer. However, the largest part is in the liver and 0.01% only has been detected in the brain [65]. It should be also mentioned that a specific phospholipase A1 in bovine plasma which converts PdtSer in lysoPtdSer, and after a longer incubation in 2-acyl lysophosphatidylserine has been detected [66]. The latter phospholipid might be in turn considered a precursor of PtdSer. The absorption after i.p. route seems therefore large enough to explain PtdSer actions on peripheral tissues but the small amount detected in brain tissue raises the question of how it acts on the CNS. The fate of orally administered PtdSer oral administration was investigated in the rats after duodenal infusion of 50 mg kg −1 of [3H]-glycerol-PtdSer [67]. The liposoluble radioactivity recovered in the lymph over 5 h accounted for 19% of the given dose. Triglycerides formed 80% of this fraction, suggesting that part of PtdSer was completely degraded and the labelled glycerol used for the de novo synthesis of phospholipids and acylglycerols. Phospholipids constituted 11% of liposoluble radioactivity and PtdEth (66%) was the major component followed by phosphatidylcholine (8%) and PtdSer (6%). Although most of the labelled phospholipid was hydrolysed or con-

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verted into PtdEth in the intestinal tract, a small fraction reached the systemic circulation as a component of the phospholipid pool. No information is available on plasma and tissue concentrations, except for the intestinal wall, after oral administration of PtdSer. PtdSer has been found in the intestinal cells and Payer’s patches 1 and 4 h after administration. Presumably the molecules escaping complete degradation in the intestinal lumen are absorbed in the form of lyso derivatives, as in the case of other phospholipids, and are then promptly reacylated when they reach the inner membrane [63]. From the data available it appears therefore that the amount of PtdSer which reaches the CNS after intraperitoneal injection or oral administration is very small. However, most of the behavioural and neurochemical effects were observed, particularly in ageing animals, after repeated or chronic i.p. and oral PtdSer administrations. It may therefore be possible that an accumulation of the small amounts of PtdSer penetrating into the brain is needed in order to induce the effect. The hypothesis of an accumulation could also explain the persistence of the effect after treatment interruption, as shown in the rat [50], and man [51, 53]. Still if we admit that 0.01% only of the injected dose is found in the brain, less than 1 g per day of PtdSer would accumulate in the brain of ageing rats. Maximal potentiation of anaphylactic histamine release from isolated mast cells was obtained with 10 µ g l−1 of PtdSer [19], stimulation of calcium uptake by brain synaptosomes [13] and activation of protein kinase C 39 with µM concentrations, and (Na++K +)dependent ATP-ase [13] with mM concentrations. On the basis of these considerations, the most likely mechanisms of action of PtdSer administered parenterally, and orally, could be an interaction with calcium uptake and/or an activation of protein kinase C. In this regards it may be mentioned that PtdSer administration (15 mg kg−1 day −1 i.p.) to ageing rats restores calcium uptake by brain synaptosomes [44]. In favour of a possible effect of exogenous PtdSer on protein kinase C, stands the observation that PtdSer (15 mg kg−1 day−1 i.p.) administration for 30 days restores protein kinase C activity which is reduced in ageing rats [68]. It remains to be explained through what chain of events the changes in calcium uptake and/or the activation of protein kinase C lead to the large number of PtdSer actions described in this review. However, a protein kinase C facilitation of ACh release has been demonstrated [69].

CONCLUSIONS From the scientific literature examined in this review, PtdSer emerges as a substance endowed with remarkable pharmacological activities, particularly on the immune system and CNS. In the rat the improvement of age-dependent behavioural impairment and chol-

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inergic hypofunction brought about by repeated PtdSer administrations is particularly well documented. This improvement is reminiscent of that reported in old rats after nerve growth factor (NGF) intracerebroventricular injections [70, 71]. However, even if PtdSer actions on the CNS have been clearly and consistently demonstrated, our knowledge remains mostly at a descriptive level. More dose–effect, time-course and pharmacokinetic studies are needed for their full understanding, and for proposing convincing mechanisms of actions. The few controlled studies carried out in elderly subjects with cognitive deficits, in some cases caused by AD, indicate a possible interesting therapeutic effect of PtdSer, in line with the animal experiments, apparently associated with few side effects. However, a strict cost versus benefit policy in health systems makes it mandatory to remove any reasonable doubt about PtdSer cost/benefit ratio in the treatment of the ageing brain. This can be achieved only with further clinical trials, provided that the bovine PtdSer, on which the cloud of the spongiform encephalopathy looms, can be substituted with PtdSer from other sources. In the meantime, basic research on the physiological and pharmacological actions of PtdSer and its analogues should be continued in order to understand their mechanisms of action. REFERENCES 1. Folch J, Schneider HA. An amino acid constituent of ox brain cephalin. J Biol Chem 1941; 137: 51–60. 2. Crook TH, Tinkleberg J, Yesavage J, Petrie W, Nunzi MG, Massari DC. Effects of phosphatidylserine in ageassociated memory impairment. Neurology 1991; 41: 644–9. 3. Soares JC, Gershon S. Advances in the pharmacotherapy of Alzheimer’s disease. Eur Arch Psych Clin Neurosci 1994; 244: 261–71. 4. Freysz L, Dreyfus C, Vincendon C. Asymmetry of brain microsomal membranes. In: Horrocks L, ed. Phospholipids in the nervous system. v. 1. New York: Raven Press, 1982: 37–47. 5. Gurr MI, James AT. Lipid biochemistry. London: Chapman and Hall, 1971: 125–48. 6. Hermetter A, Paltauf F, Hauser H. Synthesis of diacyl and alkylacyl glycerophosphoserines. Chem Phys Lipids 1982; 50: 35–45. 7. Pepeu G, Casamenti F, Scali C, Jeglinski W. Effect of serine phospholipids on memory and brain cholinergic mechanisms in ageing rats. Neurosci Res Comm. 1993; 13(Suppl 1): S63–6. 8. Bruni A, Mietto L, Secchi FE, et al. Inhibition by cholesterylphosphorylserine of T-cell-mediated immune responses in mice. Int J Immunopharmacol 1995; 17: 517–21. 9. Kaibuchi K, Takay Y, Nishizuka Y. Cooperative roles of various membrane phospholipids in the activation of calcium-activated, phospholipid-dependent protein kinase. J Biol Chem 1981; 256: 7146–9. 10. Specht SC, Robinson JD. Stimulation of the (Na ++K+)dependent adenosine triphosphatase by amino acids and phosphatidylserine: chelation of trace metal inhibitors. Arch Biochem Biophys 1973; 154: 314–23.

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