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ISSN 0379-0355

Vol ume 28, Suppl . ?, Se pte mbe r 2006

Citicoline:

P h a rma c o lo g i c al a n d C l i n ic a l R ev i ew,

2006 Update

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transmission (49, 50) and excitotoxic aggression (51, 52)are also involved. Changes in phospholipid metabolism,

particularly phosphatidylcholine, have recently beenimplicated as mechanisms inducing apoptosis (53-58).Because of these pathophysiological conditions, there isagreement on the need for having drugs that may accel-erate and/or increase synthesis of membrane structural

phospholipids in such situations, that is, having a restora-tive or reparative activity (59-61).

Cytidine diphosphocholine (CDP-choline) is amononucleotide consisting of ribose, cytosine, pyrophos-

phate, and choline whose chemical structure (Figure 1)corresponds to 2-oxy-4-aminopyrimidine (62). CDP-choline is involved as an essential intermediate in thesynthesis of structural phospholipids of cell membranes(4, 63-75), and formation of this compound from phos-

phorylcholine is the rate-limiting step of this biosynthet-ic pathway (66, 76-85). As shown in Figure 2,CDP-choline is also related to acetylcholine metabolism.Thus, administration of CDP-choline is an exogenouscholine source for acetylcholine synthesis, as will be dis-cussed later.

PHARMACOLOGICALACTIONS

Traumatic lesions and experimental cerebral edema

Horrocks and Dorman (86) have shown thatCDP-choline and CDP-ethanolamine prevent degrada-

There are various conditions in which a phospholipidloss or decreased synthesis occurs, leading to an impair-ment in cell functions that may have a pathophysiologi-cal impact (1, 6).

At central nervous system levels, structural phospho-lipids of the neuronal membrane are essential for ade-quate brain maturation (7-9). Impaired cell membraneand phospholipid metabolism have been implicated inthe pathophysiology of cerebral edema and traumaticcerebral lesion (10-19), as well as cerebral hypoxia (20,21) and ischemia (22-32). Moreover, it has been shownthat there are certain changes in neuronal membranes andmetabolism of structural phospholipids associated to

brain aging (33-35) and certain neurodegenerative dis-eases such as senile dementia of the Alzheimer type (31,36-48), and in other conditions where changes in neuro-

2 Julio J. Secades, Jos Luis Lorenzo

N

O

OH

N

O

OH

O

NH2

P

O

OH

O

P

O

O

O

N+

CH3

CH3

CH3

Fig. 1. Chemical structure of CDP-choline or citicoline.

Fig. 2. Relation of CDP-choline with choline metabolism, cerebral phospholipids and acetylcholine.

Phosphatidyl ethanolamine

S-adenosyl methionine

Base exchangereaction with serine orethanolamine

Citrate Glucose

AcetilCoA

Acetate

ACh

Choline

Glycerol phosphate

Glycerophosphocholine

Fatty acids

CTP

CITICOLINE

Diglyceride

Phosphatidylcholine

Phosphorylcholine

Choline in circulation(lipid-bound and free)

Choline

Betaine

Serine

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ate increase occurred in cholinephosphotransferase andwas associated to a greater increase in phospholipaseA2 and several lysosomal hydrolases. They also found anincreased number and size of lysosomes during neuronalregeneration. Arrigoni et al. (94) have shown citicoline to

be able to completely inhibit activation of phospholipaseA2 without altering cholinephosphotransferase activity.On the other hand, Freysz et al. (95) showed that, in addi-tion to decreasing phospholipase A1 and A2 activity, citi-coline decreases free fatty acid release under hypoxicconditions, thus adding a protecting effect to its activat-ing capacity of phospholipid reconstruction. Massarelliet al. (96) also showed citicoline action upon phospholi-

pase A1, and agreed with all other authors in their con-clusions. Kitazaki et al. (97) also showed the inhibitoryeffect of citicoline upon membrane-associated phospho-lipase A

1

in rat brain cortex. Based on these characteris-tics, citicoline has been considered a non-specificinhibitor of phospholipase A1 at intracellular level (98).

Algate et al. (99) tested the effects of citicoline in anexperimental model of epidural compression in anes-thetized cats. They noted that animals treated with citico-line had a greater resistance to the effects of mechanic

brain compression as compared to animals in the controlgroup, electroencephalographic changes occurring athigher compression levels. They also found that respira-tory and cardiovascular changes were less intense intreated animals, and concluded that citicoline provides asignificant protection against the lethality of epiduralcompression. These results agreed to those obtained byHayaishi (100) and Kondo (101), who showed animprovement in the EEG tracing following administra-tion of citicoline to cats undergoing experimental braincompression, and also in survival quality.

Tsuchida et al. (102) administered 3H-citicoline bythe intraperitoneal route to rats subjected to cerebralcryogenic lesion by dry ice application on the scalp, andconfirmed the presence of the labeled drug in brain

parenchyma, particularly in the white matter, and aboveall in damaged areas.

Boismare (11, 103) conducted an experimental modelof craniocervical trauma without direct blow(whiplash) in order to assess the effects occurring uponcentral catecholamine levels, and found increaseddopamine levels and decreased norepinephrne levels inthe brain following trauma. This type of lesion causes

postural dysregulation of brain supply and behavioraland learning disorders, that are related to accelerateddegradation of cerebral norepinephrine. In animals treat-ed with citicoline, trauma did not change the levels ofthese amines. The author stressed the protective role ofciticoline, due to this stabilizing effect of catecholamine

brain levels.Clendenon et al. (104) showed that the decrease in

Mg++-dependent ATPase activity in the mitochondrialand synaptosomal membrane occurring in traumaticlesions is prevented by citicoline administration.

tion of choline and ethanolamine phospholipids duringdecapitation ischemia in rats, and induce a partial rever-sion of free fatty acid release during reperfusion afterexperimental global ischemia in gerbils. CDP-cholineand CDP-ethanolamine, when administered together,have a synergistic effect and stimulate resynthesis ofcholine, ethanolamine, and inositol phospholipids, mar-kedly decreasing free arachidonic acid levels.

In an experimental rat model of acute inducedischemia, LePoncin-Lafitte et al. (87) assessed integrityof the blood-brain barrier (BBB) with labeled iodinatedalbumin, and brain metabolism using histoenzymologicalstudies. In this experimental model, administration ofciticoline was able to reduce vasogenic cerebral edemaand to restore BBB integrity. Authors also found that thesize of induced infarctions was smaller with citicoline,and this compound decreased the activity of lactate dehy-

drogenase, succinyl dehydrogenase, monoamine oxidase,and acid phosphatase, emphasizing its protective rolethrough a direct action at cell membrane level.

Mykita et al. (88) found in neuronal cultures thataddition of citicoline after a hypocapnic lesion resulted inculture protection. Hypocapnia increases incorporationof labeled choline into phospholipids, while this processis slowed in the presence of citicoline. These authorsconcluded that citicoline is able to protect neurons underalkalosis conditions and may promote cell proliferation.

Yasuhara et al. (89, 90), in an electrophysiologicalstudy in rabbits, showed that citicoline decreased in par-allel the threshold for the arousal reaction and the thresh-

old for muscle discharge, and concluded that this is avaluable drug for treatment of brain lesions because of itseffects on consciousness and on the motor activity of the

pyramidal system and its afferent pathways.Mart Viao et al. (91) compared the effects of

pyriglutine, piracetam, centrophenoxine, and citicoline ina study on antagonism of barbiturate coma in mice. Nodifferences were seen in animals treated with pyriglutine,

piracetam, or centrophenoxine as compared to the con-trol group, while with citicoline both coma duration anddepth, as well as respiratory depression, were decreasedas compared to all other groups. Arousal effects of citi-coline were found to be due to increased cerebral blood

flow (CBF), improved O2 cerebral uptake and utilizationof energy metabolism, and enhanced mitochondrialbreathing.

Ogashiwa et al. (92), in an experimental model ofhead trauma in monkeys, established a significantdose-effect relationship between citicoline dose andcoma duration, that started to be significant at doses of60 mg/kg (p < 0.05).

Watanabe et al. (93), studying the effects of severalactivators of brain metabolism, found that citicolineincreased glucose incorporation and metabolism anddecreased lactate accumulation in the brain, and alsoslightly increased CBF.

Alberghina and Giuffrida (10), in a study on nerve tis-sue response to a contusion lesion, showed that a moder-

Citicoline: Pharmacological and Clinical Review, 2006 Update 3

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fluid drainage to ventricles, i.e. increasing cerebral com-pliance. Authors concluded that CDP amines are helpfulto control tissue lesions related to increased free fattyacids and to restore cell energy metabolism by restartingthe Na+/K+pump.

Makem et al. (108) assessed the electroencephalo-graphic changes occurring in rats when cryogenic edemais induced, and how such EEG changes were modified by

citicoline administration. These authors noted a signifi-cant increase in the theta frequency band during theawake state, with decreased delta and slow alpha bandsand a lesser interindividual scatter of the overall frequen-cy bands, which resulted in a greater electrogenic cere-

bral stability. They concluded that citicoline protectedfrom the effects of cryogenic cerebral edema.

Roda (109), in an experimental model of cryogeniccerebral edema, measured extravasation of Evans bluethrough the BBB and fluorescein uptake by astrocytesand neurons, and found that citicoline administration sig-nificantly reduced both processes as compared to controlanimals, thus allowing to state that citicoline has a direct

effect upon transmembrane transport of sodium, potassi-um, water, and proteins at both BBB endothelial celllevel and astrocyte and neuron level. Though the exactmechanism of this action is not completely understood,its effect appears to occur at two levels: on the interfaceseparating capillaries from the neuroglia and on cellmembranes.

Dixon et al. (110) analyzed the effects of exogenousadministration of citicoline on motor deficits, spatialmemory capacity, and acetylcholine levels in dorsal hip-

pocampus and neocortex in a model of traumatic brainlesion in rats, induced by a controlled lateral impact.Citicoline was administered by the intraperitoneal routeat a dose of 100 mg/kg for 18 days from the first day

Cohadon et al. (13, 14, 105), in a series of studies ona model of cryogenic cerebral edema in rabbits, showedthat treatment with citicoline 20 mg/kg/d:

Slowed the drop in enzymatic activity of mitochon-drial ATPase.

Restored Na+/K+ ATPase activity (Figure 3). Restored oligomycin-sensitive ATPase activity.

Accelerated cerebral edema reabsorption, with nor-mal values achieved in the fourth day, while suchlevels were not reached until the tenth day withspontaneous resorption.

These authors stated that the beneficial activity ofciticoline in cerebral edema occurred by two mecha-nisms: by restoring insertion of membrane enzymes andenhancing their activity, and by acting upon edema byreducing water imbibition of brain parenchyma.

Lafuente and Cervs-Navarro (106, 107) conducted amicrogravimetric study in experimental cerebral edemainduced by ultraviolet radiation in cats to assess the

effect of citicoline in this situation. They divided animalsinto 3 groups: the first group received citicoline at dosesof 20 mg/kg, the second group at 100 mg/kg, and thethird group served as control group. After 48 h, authorstook a coronal section of the necrotic area and divided itinto 6 depth levels, so that level I corresponded to thecortex, and level VI to the periventricular area. By mea-suring the extent of edema at each level by micro-gravimetry, the authors found edema reductions, as com-

pared to the control group, in levels I to V at the highestdose, while there was a paradoxal increase in edema atlevel VI with both treatment schedules. These resultssuggested an action of citicoline decreasing the amountof edema, enhancing fluid reabsorption and accelerating


0,2 -

0,1 -

0 -

- 5

- 4

- 3

CDP-choline

Controls

0 48

Hours

96

Edema

Control

CDP-choline

ATPase

CDP-choline

Edema

Control

ATPase

Na

/KATPase

nmo

lP

I/m

inmgpro

tein

Water weight

Dry weight

Fig. 3. Kinetics of Na+/K+ ATPase activity in relation to time and the amount of water in cerebral edema.

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In a more recent study, Dempsey and Rao (113),using an experimental model of controlled lateral impactin rats, have shown that intraperitoneal administration ofciticoline 200-400 mg/kg following induction of the trau-matic brain lesion prevents neuronal damage in hip-

pocampus associated to the traumatic lesion, decreasescortical contusion volume, and improves neurologicalrecovery.

Effect of citicoline upon traumatic medullary lesionwas also studied, and it was shown that intraperitoneal(i.p.) administration of citicoline 300 mg/kg 5 minutesafter lesion induction significantly reduced lipid peroxida-tion and improved motor function in treated animals (114).

Because of its biochemical, pharmacological, andpharmacokinetic characteristics, citicoline is a potential-ly useful drug for the treatment of traumatic cerebrallesions (115).

Cerebral hypoxia and ischemia

In vitro studies using nerve tissues have shown anox-ia to induce a decrease in the synthesis of structural phos-

pholipids that is time-dependent, i.e. the longer thehypoxia the stronger the impact upon neuronal phospho-lipid metabolism (116). Moreover, a decreased incorpo-ration of marked precursors into phospholipids of neu-ronal subcellular fractions obtained from animalssubjected to experimental hypoxia has also been shown(20). It is also known that, when cerebral ischemia isexperimentally induced, glycerophospholipids in cellmembranes are broken down by the action of different

phospholipases, producing free fatty acids and arachi-donic acid derivatives. With prolonged ischemia,induced aggression upon membranes becomes moreintense and membranes lose their functions. Na+ andCa2+ accumulate inside the cell, invariably leading to celldeath (6, 27, 31, 98, 117).Under ischemia conditions, with the attendant neuronaldistress, endogenous citicoline synthesis is compromised

because the cell, under such conditions, lacks thehigh-energy phosphate compounds necessary for this

biosynthetic route (31, 118).Because of the importance of restoring neuronal

activity following cerebral ischemia (4) and based onthe experimental data discussed, various authors haveinvestigated the effects of citicoline administration invarious experimental models of cerebral ischemia and/orhypoxia.

Boismare et al. (119) reported that treatment withciticoline 20 mg/kg by the i.p. route in rats induced, dur-ing acute hypoxia, a decrease in vegetative responses,

protection from conditioned avoidance responses, andstabilization of dopamine and norepinephrine brain lev-els. This same group (120) found in dogs subjectedto normobaric hypoxia increases in blood pressure, heartrate, cardiac output, and regional blood flows, whileno changes occurred in total peripheral resistance.Administration of citicoline abolished these hemody-

following induction of the traumatic lesion. Anothergroup of animals was treated with saline. Motor assess-ment was performed using a balance test for which ani-mals had previously been trained, and cognitive assess-ment was made with a variant of the Morris maze test,that is sensitive to cholinergic function. Microdialysismethods were also used to analyze the effects uponacetylcholine release. In the motor function study, citico-line-treated animals showed on day 1 after the lesion asignificantly longer balance period as compared toanimals receiving saline (39.66 3.2 seconds versus30.26 2.9 seconds;p < 0.01). In addition, animals treat-ed with citicoline had significantly less cognitive deficitsas compared to animals treated with saline. In microdial-ysis studies, after a single administration of citicoline bythe intraperitoneal route, a rapid increase in acetylcholine

production was seen as compared to baseline, that wasmaintained for up to 3 hours, in both dorsal hippocampus(p < 0.014) and neocortex (p < 0.036), while no changeswere noted in animals receiving saline. Authors conclud-ed that post-traumatic deficits in spatial memory functionare due, at least partly, to deficiency changes in choliner-gic transmission, that are attenuated with citicolineadministration.

Plataras et al. (111) analyzed the effects of differentciticoline concentrations (0.1-1 mM) upon the activitiesof acetylcholinesterase, Na+/K+-ATPase, and Mg++-ATPase in total brain homogenates from rats and extractsof non-membrane bound pure enzymes. Following 1-3 h

preincubation with citicoline, peak stimulations of20%-25% (p < 0.001) and 50%-55% (p < 0.001) are seenfor acetylcholinesterase and Na+/K+-ATPase respective-ly, while no significant effects are seen on Mg++-ATPase.Authors concluded that citicoline may stimulate cerebralacetylcholinesterase and Na+/K+-ATPase independentlyfrom acetylcholine and norepinephrine, which could

partly account for the clinical effects of the drug.Baskaya et al. (112) examined the effects of citicoline

upon cerebral edema and rupture of the blood-brain bar-rier in a rat model of traumatic brain lesion. Animalsreceived citicoline (50, 100, 400 mg/kg) or saline by the

intraperitoneal route twice following induction of thetraumatic brain lesion. Induction of the traumatic lesioncaused an increase in water content percentage andEvans blue extravasation (a marker of BBB rupture) atthe damaged cortex and ipsilateral hippocampus. The50 mg/kg dose of citicoline was not effective, while at100 mg/kg a reduction was seen in Evans blue extrava-sation in both regions, although this dose only decreasedcerebral edema in the damaged cortex. The 400 mg/kgdose of citicoline significantly reduced cerebral edemaand caused BBB rupture in both regions. Authors con-cluded that these results suggest citicoline to be an effec-tive neuroprotective agent upon secondary lesions occur-ring in association to traumatic cerebral lesion.


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citicoline and CDP-ethanolamine decreased free fattyacid release and increased synthesis of the correspondingglycerophospholipids, suggesting an involvement ofcholine and ethanolamine phosphotransferases.Trovarelli et al. (125, 126), using an experimental globalischemia model consisting of bilateral carotid ligation ingerbils, found that intraperitoneal citicoline administra-tion partially prevents changes in lipid metabolisminduced by cerebral ischemia, correcting the increase infree fatty acids, changes in neutral lipids such as diacyl-glycerol, and the decrease in phosphatidylcholine. Sunoand Nagaoka (128) experimentally studied in rats theeffects of citicoline administration upon free fatty acidrelease caused by total cerebral ischemia lasting 5 min-utes. It was shown that the tested drug reduced theincrease in free fatty acids, and that the intensity of thiseffect depended on the dose used. Arachidonic acid con-

tents in brains from control group animals subjected toischemia was 174 22 mmol/g, as compared to 119 8mmol/g and 61 8 mmol/g in animals receiving 200 and1000 mg/kg i.p. of citicoline respectively (Figure 4).Authors concluded that these results suggest that admin-istration of citicoline may prevent ischemic cerebraldamage. Agut and Ortiz (130) treated male rats weighing190-200 g with 4 mg/kg of 14C methyl citicoline (50mCi) by the oral route. At 24 hours, brain radioactivitylevels and the presence of labeled phospholipids wereassessed under conditions of normoxia, hypoxia, andhypoxia following additional administration of 100mg/kg of unlabeled citicoline. Investigators found a

marked incorporation of radioactivity into the brains ofnormoxic and hypoxic animals, mostly associated to

phosphatidylcholine. In addition, administration of unla-beled citicoline reduced the elevation in cerebral

namic effects induced by acute hypoxia, suggesting thatthis action was correlated to a dopaminergic agonisticeffect of the drug. In cats subjected to short periods ofcerebral ischemia, these authors (121) noted that adepression occurred in cortical evoked potentials. Suchdepression was attenuated by prior administration of citi-coline by the intracarotid route. These authors think thatthe protective effects of citicoline are metabolic ratherthan hemodynamic in origin, and do not rule out a directaction of the drug upon central dopaminergic structures.

Alberghina et al. (122) investigated the effect of citi-coline upon incorporation of labeled precursors into cere-

bral phospholipids of guinea pigs subjected to hypoxia. Agroup of animals were given 100 mg/kg of citicoline bythe i.p. route. Ten minutes later, the labeled precursors[2-3H]glycerol and [1-14C]palmitate were administered

by the intraventricular route. Another group of animalsreceived the precursors only, and acted as control group.Investigators noted that, as compared to the controlgroup, citicoline-treated animals showed an increase inspecific radioactivity of total lipids and phospholipids in

purified mitochondria obtained from brain hemispheres,cerebellum, and brain stem. In a subsequent study, thissame investigating team (123) showed citicoline to beable to revert the effects of hypoxia upon incorporationof labeled precursors into RNA and proteins, particularlyat nuclear and mitochondrial level.

Various experimental studies have shown citicoline toprevent fatty acid release during cerebral ischemia and

hypoxia, and to increase synthesis of structural phospho-lipids (124-143). Horrocks et al. (124, 127, 129), usingan experimental model of global cerebral ischemia bydecapitation, showed that administration of a mixture of


Ischemia + 1 gCDPc (**)

Ischemia + 0.2 gCDPc (*)

Ischemia

Control

0 50 100

Arachidonic acid (nmol/g tissue)

150 200

Fig. 4. Effect of CDP-choline on arachidonic acid release in the ischemic brain of the rat. CDP-choline (200 and 1000 mg i.p.) was administered

10 min before decapitation. Five min later, the free fatty acids were extracted. Arachidonic acid was determined by gas chromatography. *p

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Narumi and Nagaoka (147) investigated the effects ofciticoline administration upon metabolism of cerebralmonoamines in two rat models of global cerebral is-chemia. In the first model they performed cerebralischemia, using bilateral carotid occlusion, for 30 min-utes in spontaneously hypertensive rats and noted that asignificant decrease occurred in norepinephrine levels inthe brain cortex. In this model, administration of1000 mg/kg of citicoline decreased dopamine contents instriatum and diencephalon, normalizing the decrease inthe dopamine metabolites/dopamine ratio induced byischemia. In the second model, bilateral carotid occlusionwas also performed 24 hours after electrocauterization of

both vertebral arteries in Wistar rats. In this model, nor-epinephrine, dopamine, and serotonin levels decreased70%-80% in the brain cortex. Similar decreases were

also seen in norepinephrine and serotonin levels in hip-pocampus, in dopamine levels in the nucleus accumbens,in dopamine and serotonin levels in striatum, and in nor-epinephrine levels in diencephalon and brain stem.Administration of citicoline at a dose of 500 mg/kg sig-nificantly enhanced the ischemia-induced decrease instriatal dopamine levels. These authors therefore suggestthat citicoline appears to restore dopamine turnover inthe striatum of rats subjected to experimental cerebralischemia.

Nagai and Nagaoka (148) reported the results of aninteresting study investigating the effect of citicoline

upon glucose uptake in different brain areas from ratsinduced global cerebral ischemia by occlusion of bothcarotid arteries for 30 minutes after electrocauterizationof both vertebral arteries. Glucose uptake by the brainwas measured four days after recirculation. Without citi-coline administration, global cerebral uptake was foundto be reduced to 81% of the normal value. With adminis-tration of citicoline at a dose of 250 mg/kg i.p. twicedaily for 3 days after the start of recirculation, postis-chemic reduction of glucose uptake was significantlylower in the brain cortex (Table I). This suggests that citi-coline improves energy metabolism in the brain underischemic conditions.

lysophosphatidylcholine caused by hypoxia. Rao et al.(134) showed that citicoline significantly decreased

blood-brain barrier dysfunction after ischemia with a6-hour reperfusion in gerbils and, in the same model oftransient cerebral ischemia, considerably reduced theincrease in arachidonic acid and leukotriene C4 synthesis24 hours after ischemia induction. They also showed thatthe cerebral edema volume was substantially lower at3 days in animals treated with citicoline. Following6 days of reperfusion, ischemia was seen to cause 80 8% neuronal death at the hippocampal CA1 layer level,and citicoline provided a neuroprotection of 65 6%. Ina subsequent study, these authors (135) showed citicolineto be able to significantly restore phosphatidylcholine,sphingomyelin, and cardiolipin levels after induction oftransient cerebral ischemia in gerbils. For these authors,the main action mechanism of citicoline would be inhibi-tion of stimulation of phospholipase A2 activity inischemia conditions, though they also stress its effectsupon glutathione synthesis and glutathione reductaseactivity. Thus, the drug would prevent membranedestruction, decrease free radical generation, and pre-serve the natural defenses of the nervous system againstoxidative damage (136-140). More recently, this investi-gating team has also shown that citicoline enhances

phosphatidylcholine synthesis, that is impaired underischemia conditions, attenuating the loss of CTP-phos-

phocholine cytidyltransferase activity (141, 142). Thus,the drug has effects preventing phospholipid degradationand its implications and promoting regeneration of cere-

bral phosphatidylcholine, effects that are seen to result ina decreased volume of the cerebral ischemic lesion (143).

Tornos et al. (144) conducted a pharmacologicalstudy on the protective effect of citicoline against toxi-city in an experimental model of hypoxia induced by

potassium cyanide. They found that treatment with oralciticoline for 4 days before hypoxia induction had a pro-tective effect, demonstrated by a longer survival time intreated animals.

These benefits of citicoline may also be ascribed tothe activation of the cerebral energy metabolism (145)and the increased activity of mitochondrial cytochromeoxidase (146) induced by this drug.


TABLE I. Effect of CDP-choline on glucose uptake by different cerebral regions in rats subjected to experimental cerebral ischemia. All the

values represent the mean SE of seven rats.

Glucose uptake (mg/g/10 min)

Region Normal rats Rats subjected to cerebral ischemiaSaline solution CDP-choline

Frontal cortex 3.317 0.106 2.546 0.144** 2.931 0.090#

Parietal cortex 3.250 0.114 2.232 0.145** 2.652 0.031#

Occipital cortex 3.250 0.118 2.175 0.160** 2.563 0.105#

Temporal cortex 2.671 0.042 2.106 0.106** 2.410 0.041##

Striate nucleus 2.703 0.074 2.152 0.192* 2.249 0.053Thalamus 3.130 0.132 2.421 0.141** 2.655 0.139

*p

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minutes and reperfusion. These results suggest that citi-coline has a neuroprotective role against cerebralischemia and reperfusion.

Saligaut and Boismare (154) studied the effects ofciticoline, administered at a dose of 1000 mg/kg p.o., inWistar rats undergoing acute hypobaric hypoxia (15 min-utes at a simulated altitude of 7180 meters), assessing a

behavior-conditioning test, striatal dopamine uptake, andlevels of this neurotransmitter and its metabolites in thestriatum. In the behavior-conditioning test, citicoline wasseen to protect against hypobaric hypoxia in a differentway and to a greater extent than apomorphine.Biochemical studies showed a presynaptic effect, proba-

bly because of activation of tyrosine hydroxylase, induc-ing changes in dopamine uptake, as well as an improveddopamine release. Similar results on the effect of citicol-

ine on tyrosine hydroxylase activity have been obtainedby other teams (155).LePoncin-Lafitte et al. (75) studied the effects of citi-

coline on various histological brain changes in an exper-imental model of multifocal cerebral ischemia in cats, inwhich ischemic lesion was caused by introducing in theinternal carotid artery calibrated microspheres, that will

produce cerebral microinfarctions, characterized by hav-ing a central necrosis area surrounded by a penumbraarea, together with edema due to rupture of the

blood-brain barrier. Citicoline administration consider-ably decreased the number of lesions, and also theamount of extravasated albumin, which confirms, for

these authors, that citicoline exerts its neuroprotectiverole against ischemia by acting upon cell membranes.Araki et al. (156) also found some neuroprotective

effect of citicoline in complete cerebral ischemia inducedby decapitation and potassium cyanide poisoning inmice.

Aronowski et al. (157) evaluated the effects of chron-ic citicoline administration (500 mg/kg) upon recovery inspontaneously hypertensive rats undergoing occlusion ofthe middle cerebral artery for 30 to 120 minutes. Drug orsaline were administered by the intraperitoneal routefrom 15 minutes after ischemia induction and were con-tinued for 14 days. Morphological lesion and neurologi-

cal disorders (motor and sensorimotor capacities) wereanalyzed by measuring the maximum morphologicallesion volume, maximum neurological change, andischemia duration causing half the maximum morpho-logical lesion or maximum neurological change.Maximum morphological lesion volume was not affect-ed by citicoline (101.6 11.4 mm3 for citicoline, 103.3 13.6 mm3 for saline); however, citicoline significantlyincreased ischemia duration required to cause half themorphological lesion, that changed from 38.3 5.9 to60.5 4.3 min (p

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5 mg/kg, 5) rTPA 5 mg/kg + citicoline 250 mg/kg, and(6) rTPA 5 mg/kg + citicoline 500 mg/kg. Treatment withrTPA was given at a suboptimal dosage (5 mg/kg infusedover 45 minutes, starting treatment 45 minutes afterembolization). Citicoline was administered daily by theintraperitoneal route for 4 days. Brains from survivinganimals were fixed at four days and infarction volume,calculated as percentage of the total volume of the hemi-sphere affected, was measured using a microscope. Meaninfarction volume values suggested that high-dose citico-line and the combination of citicoline with rTPAdecreased the size of ischemic lesion. In the controlgroup, mean infarction volume was 41.2% (5.9-87.0%).In groups treated with citicoline alone, values were30.4% (1.0-70.0%, n.s.) in group 2, and 22.2%(0.7-76.6%,p < 0.05) in group 3. With rTPA alone (group4), mean volume was 24.5% (1.4-71.1%, n.s.), while

with combined treatment, mean volumes were 13.5%(0.2-47.8%, p = 0.002) in group 5 and 29.2%(0.11-72.1%, n.s.) in group 6. This study showed thathigh-dose citicoline and a combination of citicoline atlower doses with rTPA significantly reduced the size of

brain infarctions. Dez-Tejedoret al. (160) reported sim-ilar results, stating that results of this association areimproved when citicoline is administered immediatelyafter rTPA administration. Shuaib et al. (161) investigat-ed the neuroprotective effects of citicoline alone or com-

bined with urokinase in a rat model of focal cerebralischemia induced by embolization at the origin of themiddle cerebral artery. Both drugs were administered

2 hours after ischemia induction. Animals were killed at72 hours. In saline-treated animals, infarction volumewas 33.1 9.7%. Citicoline-treated animals were divid-ed into two groups, one of which was given a single doseof citicoline 300 mg/kg, while the other group received adaily dose of 300 mg/kg for 3 days, both by the intraperi-toneal route. A significant reduction in infarction volumewas seen in both groups (20.9 9.7% with single doses,p = 0.01; 18.9 11.4% with multiple doses,p = 0.008).Animals treated with urokinase alone, at doses of 5000U/kg, also had a smaller infarction volume (19.5 12.5%, p = 0.01). However, the greatest volume reduc-tion was achieved in the group of animals treated with

the combination of citicoline and urokinase (13.6 9.1%,p = 0.0002). These authors concluded that citicol-ine provides a significant neuroprotective effect that may

be enhanced by association with a thrombolytic (Figure6). nal et al. (162) investigated the potential synergisticneuroprotective effects of chronic citicoline treatmentand administration of the N-methyl-D-aspartate receptorantagonist, MK-801, or dizocilpine in a rat model of tran-sient cerebral ischemia. Ischemia was induced using amodel of intraluminal suture for occlusion of the middlecerebral artery, with a 90-minute ischemia period.Forty-eight Sprague-Dawley rats were used, distributedinto 4 groups: (1) animals treated with physiologicalsaline; (2) animals treated with 0.5 mg/kg of MK-801 asan intravenous bolus 60 minutes after ischemia induc-

half the maximum neurological change from 41.9 4.6to 72.9 24.5 min (p < 0.05). According to these authors,citicoline shows a greater efficacy in animals who expe-rience a submaximal lesion, occurring in this model with30-75 minutes of ischemia.

Schbitz et al. (158) evaluated the effects oflong-term treatment with citicoline in a model of tran-sient focal ischemia (2 hours) in rats. Ten animals wererandomly assigned to each of the groups: placebo (saline0.3 ml/d/7 d), low dose (citicoline 100 mg/kg/d/7 d i.p.)and high dose (500 mg/kg/d/7 d i.p.). Treatment was start-ed at the time of reperfusion, once the 2-hour ischemia

period had ended. Daily neurological assessments weremade (modified Zea Longa scale), and surviving animalswere killed on day 7, after which cerebral edema andinfarction volume were calculated. No differences wereseen in neurological assessment of animals at study end,

but a more favorable trend was noted in the citicolinehigh-dose group. Mean infarction volume was 243.5 88.6 mm3 in the placebo group, 200.2 19.9 mm3 in thelow-dose group, and 125.5 45.2 mm3 in the high-dosegroup. These differences were statistically significant(p < 0.01). A dose-dependent decrease in cerebral edemavolume was also seen, but did not reach statistical signif-icance (Figure 5).

In a series of recently conducted studies, citicolinewas shown to have a synergistic effect with other drugsin the treatment of cerebral ischemia, such as throm-

bolytic (159-161) and neuroprotective drugs (162-165).Andersen et al. (159) conducted an experimental study in

a rat model of carotid embolism to evaluate the effect ofdifferent doses of citicoline, administered alone or com-

bined with recombinant tissue plasminogen activator(rTPA), on infarction size. Ninety Sprague-Dawley ratssubjected to embolism in the carotid territory were ran-domized into 6 groups: (1) saline-treated animals, (2)citicoline 250 mg/kg, (3) citicoline 500 mg/kg, (4) rTPA


Control

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Fig. 5. Effect of citicoline at a low dose (100 mg/kg) and high dose (500mg/kg) on infarction volume. The values represent the mean SD.

Infarct volume was significantly smaller (p

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infarction volume was decreased in the group treated withthe drug combination as compared to the other groups(175.2 89.3 mm3 in control group; 179.1 78.5 mm3 in theMK-801 group; 163.9 73.7 mm3 in the citicoline group;84.7 56.8 mm3 in the combined treatment group;p < 0.02

for the overall comparison andp < 0.05 for the comparisonof the combined treatment to the control) (Figure 7). No

tion, followed by treatment with 1 ml/kg/d/7 d of physio-logical saline solution by the intraperitoneal route; (3) ani-mals treated with physiological saline solution by the intra-venous route 60 minutes after ischemia, followed by citico-line 250 mg/kg/d/7 d by the intraperitoneal route; and (4)

animals treated with a combination of both drugs (citicol-ine 250 mg/kg/d/7 d i.p. + MK-801 0.5 mg/kg i.v.). Mean


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Fig. 6. Change in the volume of cerebral infarct in four groups of treated animals compared with the control group. Combination: urokinase (5000U/kg) + citicoline (300 mg/kg). *p = 0.01; **p = 0.008; ***p = 0.0002.

0 50 100

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Fig. 7. Mean infarct volume in each group (n = 12 in each group). The infarct volume was significantly lower in the group of combined treatment(p

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by contrast, has an abundant cholinergic nerve supply.Changes in perfusion pressure were measured during adose-response curve to acetylcholine and following infu-sion of 1 mg/min/30 min of citicoline. Authors noted thatciticoline caused relaxation in both vascular beds, whichwould suggest the presence of muscarinic receptors. Inthe internal carotid vascular bed, citicoline infusion for30 minutes significantly shifted to the left thedose-response curve to acetylcholine, enhancing relax-ation. However, this did not occur in the external carotid

bed. The effect of citicoline was masked when it wasjointly infused with hemicolinium. According to theseauthors, results suggest that citicoline would act byincreasing choline levels at cholinergic endings, increas-ing acetylcholine synthesis and/or release.

Clark et al. (176) examined whether citicoline was

able to reduce ischemic damage and improve the func-tional neurological result in an intracerebral hemorrhagemodel in mice. They caused hemorrhage in 68 Swissalbino mice by injecting them collagenase at the caudatenucleus. Animals randomly received saline or citicoline500 mg/kg i.p. before administration of collagenase andat 24 and 48 hours. Mice were assessed using a 28-itemneurological scale and were killed at 54 weeks to assesshematoma volume, total damage, and surroundingischemic damage. As regards neurological course, citi-coline-treated animals had a better score that

placebo-treated animals (10.4 2.0 versus 12.1 2.4;p< 0.01). No differences were seen in hematoma volumes,

but a significant reduction in the volume of the sur-rounding ischemic damage was noted in animals treatedwith citicoline, with values being 13.8 5.8 mm3 (10.8 4.3% of hemisphere) and 17.0 7.1 mm3 (13.3 5.1%)for placebo, with p < 0.05. According to authors, theseresults support a potential role of citicoline for treatmentof intracerebral hemorrhage.

In recent years, apoptotic mechanisms have beenshown to play a primary role in the pathophysiology ofcerebral ischemic damage both at experimental level(177-181) and in humans (182, 183). We therefore inves-tigated (184) whether citicoline could influence apoptot-ic mechanisms following focal cerebral ischemia. A

model of permanent distal occlusion of the middle cere-bral artery was used in Sprague-Dawley rats. Animalswere randomized into 4 groups: B+A, citicoline 500mg/kg i.p. 24 and 1 hours before occlusion and 23 hoursafter occlusion; A, citicoline 500 mg/kg i.p. within 30minutes and 23 hours following occlusion; C, salinesolution i.p.; D, sham-operated. Animals were killed at12 (7 animals per group) and 24 hours (7 animals pergroup) following occlusion. Immunohistochemistry for

procaspases 1, 2, 3, 6, and 8 was performed using goatpolyclonal antibodies and, using gel electrophoresis andWestern blotting, specific substrates for caspase actionwere tested using poly-ADP-ribose polymerase (PARP)antibodies. Ischemia induced expression of all procas-

increased survival in animals treated with this citicolineformulation (166-168), and more recently, that this samedrug formulation significantly reduces the maturation

phenomenon, that is, delayed cerebral neurodegenerativelesion, occurring after an ischemic event, resulting in asignificant improvement in brain function (169). Theseresults agree with previously discussed results (143)showing that administration of liposomal citicoline ismore effective as compared to non-liposomal citicoline.

In recent years, citicoline has also been shown tohave a neuroprotective effect against neurotoxic damageinduce by kainic acid in retinal cells (170, 171).

Hamdorf and Cervs-Navarro (172) exposed 48 ratsfor 103 days to a decreasing amount of oxygen, i.e. theywere exposed to chronic hypoxia. Animal behavior in anopen field was determined for each hypoxia level (15%,

12%, 10%, 8%, and 7% inspired oxygen). Twenty-fouranimals received citicoline at doses of 100 mg/kg withfeed. During hypoxia conditions, reactions indicating animpaired vigilance were recorded. Citicoline showed a

protective effect by increasing vigilance under moderatehypoxic conditions (15% O2). In a subsequent study,these same authors (173) analyzed the effects of citicol-ine in Wistar rats subjected to hypoxia for 5 months.Eighty rats were kept in a gradually hypoxic environ-ment. Hypoxia levels in the order of 15%, 12%, 10%,and 7% O2 in inspired air were established, and rat

behavior in an open field was subsequently recorded. Agroup of 40 animals received citicoline with feed at a

dose of 100 mg/kg. Behavioral changes induced byhypoxia were attenuated in the group or animals treatedwith citicoline. Interestingly, therapeutic administrationof citicoline was found to be more effective than prophy-lactic administration. In addition, under extreme hypoxiaconditions, citicoline showed a protective effect bylengthening survival.

On the other hand, Masi et al. (174) have shown citi-coline to have a certain antiplatelet aggregant effect, thatmay provide an additional benefit for the treatment ofcerebral vascular disease. These authors analyzed theeffects of acute (250 mg/kg) and chronic (250 mg/kg/d/2w) citicoline administration on platelet aggregation,

thromboxane formation, and antiplatelet aggregant activ-ity in rat thoracic aorta. Acute administration mainlycaused a decrease in platelet reactivity to aggregantagents, while no changes were shown in thromboxane

production. After chronic treatment, the main effect seenwas an increased antiaggregant activity in the vascularwall, while platelet function was not changed. Thus, citi-coline has favorable effects, particularly in acute treat-ment, leading to a reduction in platelet reactivity.

Pinardi et al. (175) investigated in Sprague-Dawleyrats the effects of citicoline infusion on relaxationinduced by exogenous acetylcholine in the isolated exter-nal carotid vascular bed, having no cholinergic nervesupply, and the isolated internal carotid vascular bed that,


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tral nervous system. Citicoline stimulates dopamine syn-thesis in nigrostriatal areas and antagonizes changes indopamine and norepinephrine levels caused by variousnoxa at central nervous system level. Citicoline stimu-lates cholinergic neurotransmission. In various brain dis-ease models, citicoline has improved or normalized bio-chemical and functional parameters of the centralnervous system. Citicoline stimulates the synthesis andinhibits catabolism of cerebral phospholipids, and also

has a protective effect upon membrane ATPase andenzymes involved in brain energy metabolism, particu-larly succinyl dehydrogenase and citrate synthetase, aswell as protein and nucleic acid metabolism, increasingRNA biosynthesis at certain brain regions. In chroniccerebral ischemia models, citicoline improves neurologi-

pases and PARP in both the infarction and the penumbraareas 12 and 24 hours following ischemia. Citicolinereduced expression of all procaspases at 12 and 24 hoursfollowing ischemia, except for procaspase 3 at 24 hoursin group A (Figs. 9 and 10) and PARP expression (Figure11), and results were more clear in group B+A, suggest-ing a certain prophylactic role of citicoline. Citicolinehas recently been shown to be able to inhibit certainintracellular signals involved in apoptotic processes

(185) and to maintain these inhibitory effects in differentexperimental models to study apoptosis (186-188).

According to Drago et al. (189), citicoline is a drug ofchoice for treatment of cerebral vascular disease, partic-ularly in its chronic form, because its clinical use is jus-tified by the pharmacological actions it exerts on the cen-


Control

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Procaspase 1 Procaspase 2 Procaspase 3 Procaspase 6 Procaspase 8

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Fig. 9. Procaspase expression 12 h after inducing ischemia. B+A: group treated before and after ischemia; A: group treated only after ischemia.*p

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norepinephrine, and serotonin was expressed as a con-version index equal to the ratio between the amount oflabeled neurotransmitter per gram of brain (cpm/g) andthe tyrosine- or tryptophan-specific radioactivity(cpm/mmol) in brain. As shown in Figure 12, citicolinesignificantly increased dopamine levels and synthesis ratein the striate body, and the effect exerted on tyrosine lev-els was very similar. Norepinephrine levels were increasedin cortex, but showed no changes from control in the brainstem. As regards effects on serotonin, the drug was seen tocause decreases in the levels and synthesis rate of this neu-

rotransmitter in the brain stem and hypothalamus, and nochanges were seen in the cortex or striate. According tothese authors, increased dopamine synthesis could beattributed to an enhancing effect of citicoline upon tyro-sine hydroxylase activity, the rate-limiting step indopamine synthesis. This activation of tyrosine hydroxy-lase would lead to an inhibition of dopamine reuptake atthe synapse, an action that has been shown in ex vivo stud-ies (191, 192). By contrast, the increase seen in dopaminesynthesis does not appear to be related to increased levelsof tyrosine, since this completely saturates tyrosinehydroxylase under physiological conditions. The effects ofciticoline upon striatal dopamine synthesis are particularly

interesting because changes in dopamine synthesis byextrapyramidal dopaminergic neurons are in the origin ofParkinson disease.

Saligaut et al. (193) obtained results in agreementwith the previous ones when studying dopamine reuptakein synaptosomes taken from the striate body of rats pre-viously treated with citicoline. Following long-termtreatment with this drug, a decreased dopamine reuptake

by synaptosomes was seen, and authors related this factto the increase in tyrosine hydroxylase activity, thatwould involve an increased dopamine synthesis. Theyalso think that a structural change in neuronal mem-

branes, mainly of phospholipid levels, could be one ofthe factors responsible for the change in synaptosomal

cal deficits and prolongs animal survival. In addition,citicoline promotes neuronal synaptogenesis and has aninhibitory action on the activity of some phospholipases.To sum up, citicoline:

Interferes positively with brain energy metabolism. Stimulates central neurotransmission. Activates cell repair mechanisms Decreases ischemic lesion size. Inhibits apoptosis associated to ischemia. Has synergistic effects with thrombolytic and neu-

roprotective drugs.

These characteristics confer citicoline a suitablepharmacological profile for the treatment of cerebralischemia.

Synaptic transmission and neurotransmitter levels

As previously discussed, citicoline exerts some of itseffects through its action on the levels of certain neuro-transmitters. This section will discuss these specificeffects upon neurotransmission. As will be seen below,most studies have focused on analyzing the effect of citi-coline on central dopaminergic transmission.

Martinet et al. (190) conducted a study in which theeffects of citicoline administration on norepinephrine,dopamine, and serotonin levels were assessed in differentrat brain regions. For this, conversion of3H-tyrosine and3H-tryptophan, administered by the intravenous route,into 3H-norepinephrine, 3H-dopamine, and 3H-serotoninwas measured, comparing the results obtained withadministration of saline to those obtained after adminis-tration of citicoline at different doses. Metabolism ofeach neurotransmitter was studied in the brain regionswhere it has functional activity. Thus, for catecholaminesciticoline action was studied in the striate body, braincortex, and midbrain, while for serotonin the hypothala-mus was also studied. The synthesis rate of dopamine,


Fig. 11. Band densitometry analysis for PARP by Western blotting in different groups of rats in the infarct zone and penumbra zone 12 and 24 h afterischemia. ***p

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series of experiments, these authors examined the effectsof citicoline on catecholamine metabolism in the striateand hypothalamus from rats subjected to acute hypobar-ic hypoxia (195). Hypoxia induces decreases in striatallevels of homovanillic acid, dihydrophenylacetic acid,and 3-methoxytyramine, and in norepinephrine levels inhypothalamus, but increases dopamine levels in both

sites. Administration of citicoline at doses of 1000 mg/kgp.o. was able to partially revert the effects of hypoxiaupon 3-methoxytyramine, dopamine, and norepinephrinewhile enhancing the effects upon homovanillic acid anddihydrophenylacetic acid. These results show that citico-line partially counteracts the effects of hypoxia upon therelease and metabolism of certain neurotransmitters. Inanother study, Saligaut et al. analyzed the effects of citi-coline in rats with unilateral nigrostriatal lesion induced

by 6-hydroxydopamine (196). In damaged animals,amphetamine administration induced an ipsiversive cir-cling behavior, while such circling behavior was contra-versive with administration of levodopa and apomor-

phine. This appears to be mediated by the development in

reuptake of the neurotransmitter induced by citicoline.Hypobaric hypoxia was also seen to antagonize theinhibitory effect of citicoline on dopamine reuptake bysynaptosomes. This antagonism may be explained by thefact that hypoxia decreases activity of tyrosine hydroxy-lase, an enzyme that requires oxygen, thus counteractingenzyme activation exerted by citicoline. This leads to a

decreased dopamine synthesis and a subsequent increasein dopamine reuptake. These same authors studied citi-coline action in the experimental oxotremorine-inducedcholinergic syndrome in mice (194), and showed thatciticoline pretreatment does not potentiate this syndrome,

but inhibits salivation induced by oxotremorine.Levodopa antagonized brain symptoms such astremor-akinesia induced by oxotremorine. However, thisantagonism disappeared in animals under long-term oraltreatment with citicoline, thus confirming the action ofciticoline on dopaminergic pathways. Citicoline effectsappear to be mediated by hypersensitivity of somedopaminergic receptors, rather than by a direct stimulat-ing effect on striatal dopaminergic receptors. In another


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Fig. 12. Influence of citicoline (30 mg/kg i.v.) on catecholamine synthesis at different timepoints after administration. The graphs show variations incatecholamine concentrations and rates of synthesis, in percent with respect to control, at different locations. l corpus striatum; n cortex; sbrain-stem-mesencephalon; *p

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Action of citicoline upon the dopaminergic systemhas also been studied by investigating its pharmacologi-cal actions in experimental models used for that purpose,such as hypothermia induced by apomorphine, tardivedyskinesia induced by haloperidol, or acrylamide-induced lesion. Agut et al. (199) studied the effect of citi-coline administration on hypothermia induced by apo-morphine, considered to be the result of the agonistaction of apomorphine on D2 receptors. Experimentalanimals received, in addition to apomorphine, haloperi-dol at a sufficient dose to partially block apomor-

phine-induced hypothermia in order to obtain a pharma-cological system sensitive to citicoline action upon thedopaminergic system. A group of animals received a doseof citicoline 100 mg/kg p.o., and haloperidol 0.15 mg/kgwas administered at 30 minutes by the intraperitoneal

route. Thirty minutes later, rectal temperature was mea-sured and apomorphine 1 mg/kg was administered by thesubcutaneous route. Rectal temperature was again mea-sured at 30, 60, and 90 minutes. Another group of ani-mals received water instead of citicoline using the samescheme. Effects of chronic administration of citicoline ata dose of 100 mg/kg/d p.o. for 5 days were also analyzed.The same protocol as for acute administration was fol-lowed on the last day. Table III shows the mean temper-ature decrease seen in each animal batch and at the dif-ferent evaluation time points. Acute administration ofciticoline causes hypothermia, that is significant for allcontrol time points. Chronic administration only

achieves a significant result at 90 minutes. Authors con-cluded that a 100 mg/kg dose of citicoline administeredacutely by the oral route has a hypothermizing effectsimilar to the one reported for various dopaminergic ago-nists. On the other hand, they considered that the fact thatchronic citicoline administration only caused a signifi-cant hypothermia in the last time point analyzed proba-

bly reflects that, with this form of administration, thetested product predominately acts upon phospholipidrather than acetylcholine synthesis. This second action

pathway of citicoline would predominate with acuteadministration, as this would involve a relatively rapidutilization of the choline provided, that would be used for

acetylcholine synthesis, thereby increasing tyrosinehydroxylase activity through cholinergic interneurons.By contrast, chronic administration of citicoline wouldresult in a progressively greater availability of cytidine,and would therefore divert cerebral choline toward thesynthetic pathway of citicoline and phospholipids, whichwould indirectly result in a dopaminergic agonisticeffect. These authors developed an experimental modelof tardive dyskinesia induced by haloperidol (2mg/kg/d/7 d) in rats in a study including chronic admin-istration of haloperidol or water to a total of 120 animals(200). After treatment discontinuation for 7 days, animalsreceived water or apomorphine and their motor activitywas measured. Animals treated with apomorphine 0.25

the damaged side of a supersensitivity of postsynapticdopaminergic receptors. Subchronic treatment with citi-coline did not induce behavioral effects. Citicoline didnot change the stimulating effect of apomorphine, but

potentiated the effects of levodopa and amphetamine.These data show that citicoline effects are mediated by a

presynaptic mechanism. Although potentiation of lev-odopa may not be explained by an activation of tyrosinehydroxylase, this effect appears to be related to animproved release of dopamine synthetized from exoge-nous levodopa.

Agut et al. (197) indirectly studied the effect of citi-coline upon dopamine synthesis in the striate body bymeasuring local levels of dopamine metabolites in ani-mals in which blockade of dopaminergic receptors had

been induced by administration of haloperidol.Pretreatment with citicoline 100 mg/kg/d/5 d significant-

ly increased levels of homovanillic acid (HVA) and3,4-dihydroxyphenylacetic acid (DOPAC) in the striateof treated animals as compared to the control group(Table II). Increase in levels of these metabolites waseven stronger in a group of animals also receiving apo-morphine. Results obtained in this study suggest that citi-coline increases dopamine synthesis in the striate of ratsin which activation of such synthesis has been experi-mentally induced by haloperidol administration. Thissame investigating team subsequently conducted a studyto examine whether citicoline alone, without provokingan increased dopamine demand by dopaminergic recep-tors, caused an increased synthesis of this neurotransmit-

ter, resulting in increased striatal levels of its mainmetabolites, HVA and DOPAC (198). Two groups of5 animals each were administered oral citicoline at dosesof 100 mg/kg and 2 g/kg respectively for three consecu-tive times at one-hour intervals. A third group with simi-lar characteristics received 10 ml/kg of water accordingto the same protocol. Thirty minutes after the last admin-istration, animals were killed, their brains were extracted,and HVA and DOPAC levels in the striate body weremeasured. Striatal levels of DOPAC increased 60% (p

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