Citicoline: pharmacological and clinical review, 2022 update

This review is based on the previous one published in (Secades JJ. Citicoline: pharmacological and clinical review, update. Rev Neurol ; 63 (Supl 3): S1-S73), incorporating 176 new references, having all the information available in the same document to facilitate the access to the information in one document. This review is focused on the main indications of the drug, as acute stroke and its sequelae, including the cognitive impairment, and traumatic brain injury and its sequelae. There are retrieved the most important experimental and clinical data in both indications.

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Citicoline is the generic name of the pharmaceutical substance that chemically is cytidine-5&#;-diphosphocholine (CDP-choline), which is identical to the natural intracellular precursor of phospholipid phosphatidylcholine [ 78 ]. CDP-choline is a mononucleotide consisting of ribose, cytosine, pyrophosphate, and choline whose chemical structure ( ) corresponds to 2-oxy-4-aminopyrimidine [ 79 ]. CDP-choline is involved as an essential intermediate in the synthesis of structural phospholipids of cell membranes [ 4 , 78 - 92 ], and formation of this compound from phosphorylcholine is the rate-limiting step of this biosynthetic pathway [ 82 , 93 - 104 ]. The CDP-choline cycle is integrated into a larger metabolic network and its interruption can affect the distribution of lipid-related metabolites in several other pathways [ 105 ]. As shown in , CDP-choline is also related to acetylcholine metabolism. Thus, administration of CDP-choline is an exogenous source of choline and cytidine. Choline participates in several relevant neurochemical processes. It is the precursor and metabolite of acetylcholine, plays a role in single-carbon metabolism and is an essential component of different membrane phospholipids [ 106 ]. The cytidine fraction, once transformed in uridine, is used for DNA and RNA synthesis as well as for the synthesis of membrane constituents and glycosylation, also having an important effect on purinergic receptors [ 107 ].

There are various conditions in which a phospholipids loss or decreased synthesis occurs, leading to an impairment in cell functions that may have a pathophysiological impact [ 1 , 10 ]. At central nervous system levels, structural phospholipids of the neuronal membrane are essential for adequate brain maturation [ 11 - 14 ], including astroglial cells [ 15 ]. Phosphatidylcholine has been proposed as an important molecule for neurite growth and neuronal regeneration [ 16 ]. Impaired cell membrane and phospholipid metabolism have been implicated in the pathophysiology of cerebral edema and traumatic brain injury [ 17 - 26 ], as well as cerebral hypoxia [ 27 , 28 ] and cerebral ischemia [ 29 - 42 ]. Moreover, it has been shown that there are certain changes in neuronal membranes and metabolism of structural phospholipids associated to brain aging [ 43 - 45 ] and certain neurodegenerative diseases such as cognitive impairment, vascular dementia and senile dementia of the Alzheimer type [ 39 , 46 - 58 ], contributing to the neuroplasticity mechanisms [ 59 ], and in other conditions where changes in neurotransmission [ 60 - 63 ] and excitotoxic aggression [ 64 , 65 ] are also involved. Changes in phospholipid metabolism, particularly phosphatidylcholine, have been implicated as mechanisms triggering the apoptotic cascade in several conditions [ 62 - 71 ]. Because of these pathophysiological conditions, there is an agreement on the need for having drugs that may accelerate and/or increase synthesis of membrane structural phospholipids in such situations, that is, having a protective and a restorative or reparative activity on the nervous system [ 72 - 77 ].

The chemical structure of a phospholipid shows esterification of a polyalcohol (glycerol or sphingosine) with two long-chain fatty acids and a molecule of phosphoric acid that is in turn esterified with nitrogenated bases (choline, ethanolamine), amino acids (serine), or inositol [ 3 , 4 ]. The main phospholipids in humans are phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and sphingomyelin [ 4 ]. The major phospholipid present in most eukaryotic membranes is phosphatidylcholine (PC), comprising ~50% of phospholipid content [ 5 ]. The main function of the phospholipids is to be part of cell membrane structures, and these compounds are indispensable to fulfil membrane functions, particularly maintenance of homeostasis and cell compartmentalization, as well as enzymatic activities associated to membrane systems, and coupling between receptor and intracellular signal [ 1 ]. Thus, phospholipid plays a pivotal role in regulating physiological functions and maintaining cellular membrane structures [ 6 ], also serves as a source of several lipid mediators [ 7 ] and orchestrates humoral immunity, highlighting the metabolic control of context-dependent immune signaling and effector programs [ 8 ]. Additional specific functions of the neuronal membrane include nerve impulse conduction and neurotransmission [ 1 , 9 ].

Zhong et al [ 430 ] concluded that citicoline can protect against neomycin-induced hair cell loss by inhibiting reactive oxygen species aggregation and thus preventing apoptosis in hair cells, and this suggests that citicoline might serve as a potential therapeutic drug in the clinic to protect hair cells and thus preventing hearing loss associated to aminoglycoside use.

Aminzadeh and Salarinejad [ 426 ] analyzed the effect of citicoline on lead-induced apoptosis in PC12 cells and their findings revealed that citicoline exerts a neuroprotective effect against lead-induced injury in PC12 cells through mitigation of oxidative stress and at least in part, through suppression of mitochondrial-mediated apoptotic pathway. Gudi et al [ 427 ] tried to confirm previous results showing that citicoline improves remyelination and to determine the potential regenerative effects of lower doses of citicoline (100 and 50 mg/kg). The effects of citicoline were investigated in the toxic cuprizone-induced mouse model of de- and remyelination. The authors found that even low doses of citicoline effectively enhanced early remyelination. The beneficial effects on myelin regeneration were accompanied by higher numbers of oligodendrocytes. They concluded that citicoline could become a promising regenerative substance for patients with multiple sclerosis and should be tested in a clinical trial. Shaffie and Shabana [ 428 ] indicated that citicoline treatment can protect against toluene-induced toxicity in rats.

Characteristic findings of fetal alcohol syndrome include delayed maturation and late development of dendrites in neocortex, hippocampus, and cerebellum. Based on these data, Patt et al [ 417 ] conducted a study to investigate the effects of citicoline on Purkinje cells from rats newborn from alcoholic dams, showing that this stabilizing agent of neuronal membranes decreases the harmful effect of alcohol on the central nervous system. Wang and Bieberich [ 418 ] demonstrated that prenatal alcohol exposure triggers ceramide-induced apoptosis in neural crest-derived tissues concurrent with defective cranial development and that treatment with CDP-choline may alleviate the tissue damage caused by alcohol. Petkov et al [ 419 ] have also shown that citicoline decreases mnesic deficits in rats pre- and post-natally exposed to alcohol, which may be related with the beneficial effects upon acetylcholine synthesis and release shown using cerebral microdialysis in rats chronically exposed to alcohol [ 420 , 421 ]. Citicoline has also shown a protective effect in nicotine intoxication [ 422 ] and in mercury intoxication [ 423 ]. Buelna-Chontal et al [ 424 ] demonstrated that citicoline circumvents mercury-induced mitochondrial damage and renal dysfunction in a model of renal failure in rats. Laksmita et al [ 425 ] demonstrated the potential benefits of citicoline for management of ocular methanol intoxication in an experimental rat model.

If citicoline 300 mg is injected by the intracarotid route to cats, effects similar to those seen with administration of 2 mg of morphine by the same route are obtained. The animal shows symptoms of anger and alertness, and the tail is placed in a rigid and upright position. This finding led to think that both substances could have some parallel effects at neuroreceptors of endogenous opiates, and that administration of citicoline could be of value in the opiate withdrawal syndrome by slowing the effects of sudden drug discontinuation [ 414 ]. Tornos et al [ 415 ] studied the effects of citicoline administration upon experimental withdrawal syndrome by analyzing various methods, such as the jumping test in mice and studies of the behavior and body temperature changes in rats. The withdrawal syndrome caused by administration of naloxone to morphine-dependent mice was assessed based on the number of jumps by the animals. A decrease in severity was seen in the group of animals treated with citicoline 2 g/kg p.o. as compared to the untreated animal group. This decreased severity of the withdrawal syndrome was demonstrated by a 39% decrease in the mean number of jumps by the animals within 10 minutes of administration of the opiate antagonist. Similarly, the behavioral study in morphine-dependent rats showed that administration of an oral dose of citicoline 2 g/kg at the same time as naloxone was able to significantly decrease the severity of manifestations that characterize the withdrawal picture provoked. As regards hypothermia caused by naloxone administration in morphine-dependent rats, administration of a single oral dose of citicoline neutralizes such effect almost completely. Nejati et al [ 416 ] studied the effects of intraperitoneal injections of citalopram and citicoline on morphine-induced anxiolytic effects in non-sensitized and morphine-sensitized mice and demonstrated a synergistic effect of citalopram and citicoline upon induction of anti-anxiety behavior in non-sensitized and morphine-sensitized mice.

Because of such actions, various studies have shown the positive effects of citicoline on learning and memory in aged animals [ 373 , 407 , 408 ]. Based on these effects and the effects on neuroplasticity [ 409 ] and on proliferation and differentiation of astroglial cells [ 15 , 410 ] it has been postulated the use of citicoline in neurodegenerative diseases, but there are some exceptions, such the no protective effect of the drug in a model of Huntington&#;s disease [ 411 ] and in a model of amyotrophic lateral sclerosis [ 412 ]. Gromova et al [ 413 ] considered that the pharmacological effects of CDP-choline are realized via multiple molecular mechanisms contributing to the nootropic actions of this molecule in different experimental models.

There are multiple morphological, neurochemical, and physiological changes characterizing brain aging in mammals. General agreement exists between investigators on the existence of aged-related changes in certain neurochemical parameters, such as enzyme activity, receptor binding, and neurotransmission. Biochemical evidence is available of the existence of a component of cholinergic dysfunction and impaired cerebral phospholipids metabolism in the pathophysiology of brain aging [ 1 , 4 , 5 ]. De Medio et al [ 382 ] investigated the effects of citicoline upon changes in lipid metabolism in the brain during aging. Thus, they measured in vivo lipid synthesis in different brain areas from 12-month-old male rats. For this, they administered, by injection in the lateral cerebral ventricle, a mixture of (2- 3 H)glycerol and (Me- 14 C)choline, as lipid precursors, and measured, one hour after isotope administration, incorporation of these precursors into the fractions of total lipids, water-soluble intermediates, and choline phospholipids. In another series of experiments, citicoline was injected intraventricularly to aged rats 10 minutes before killing, and the same radioactivity tests as described above were performed. Distribution of the radioactivity contained in citicoline in the brain 10 minutes following administration showed enrichment, in the studied areas, of nucleotides and related water-soluble compounds. Incorporation of labelled glycerol, that is greatly decreased in aged rats, increased in all areas. Incorporation of labelled choline also decreases with aging, and citicoline was able to increase such incorporation in the cortex. As a result, the 3 H/ 14 C ratio was increased in total lipids and in phosphatidylcholine and choline plasmalogens following citicoline treatment. Following this line of research, López-Coviella et al [ 383 ] studied the effects of oral citicoline on phospholipids content in mouse brain. These authors supplemented animal diet with citicoline 500 mg/kg/day for 27 months in 3-month-old mice, and for 90, 42, and 3 days in 12-month-old mice, after which phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine levels, and contents of phosphatidylinositol plus phosphatidic acid were measured in brain cortex. After 27 months of treatment, phosphatidylcholine and phosphatidylethanolamine levels significantly increased by 19 and 20% respectively, while phosphatidylserine levels increased by 18%, but statistical significance was not reached ( ). Similar increases were noted when 12-month-old animals were treated for three months, but not with shorter treatment periods. These results suggest that chronic administration of citicoline may have significant effects on phospholipid composition of the brain that may partly be responsible for the reported therapeutic efficacy of this drug. Wang and Lee [ 384 ] obtained similar results in their study. Plataras et al [ 385 ] showed citicoline to be able to restore activity of hippocampal acetylcholinesterase and Na + /K + pumps, involving these mechanisms in the improvement of memory performance exerted by citicoline. Zhang et al [ 386 ] suggest the citicoline could play a role in improving memory performance and exert protective effects against Alzheimer&#;s disease by increasing expression or activity of Na + /K + -ATPase. Giménez et al [ 387 ] showed that citicoline, administered for two months to aged rats, caused a significant activation of cytidine triphosphate:phosphocholine cytidyltransferase, which according to authors would explain the reparative effects of the drug on damaged membranes of aged animals. This same investigating team made a more extensive study of the effects of citicoline on the activity of this enzymatic system and showed that, in addition to its effect on phospholipid metabolism, citicoline also has a regulatory effect upon platelet activating factor levels in the brain [ 357 , 358 ]. All such effects occur with no changes in plasma levels of homocysteine, a known risk factor [ 388 ]. However, citicoline also offers beneficial actions on brain metabolism of nucleic acids and proteins [ 385 , 389 - 391 ], on dopaminergic, nicotinic and muscarinic receptors [ 324 ], and on neuroendocrine and neurosecretory changes [ 392 - 394 ] in experimental aging models, as well as a neuroprotective effect against neurotoxic aggressions [ 394 - 402 ], an immunomodulatory effect [ 403 ], and an antiapoptotic effect [ 404 , 405 ] in various models of neurodegeneration and cerebrovascular dementia. Sahraiian and Khazali [ 406 ] showed that citicoline, as ghrelin, improves passive avoidance learning by altering the N-methyl-D-aspartate receptor (NMDAR1) and the serotonin receptor 1A (HTR1a) expression in the hippocampus in adult male rats.

Safavi et al [ 381 ] compare the individual effects of benfotiamine and citicoline and their co-administration on memory impairments in diabetic mice. Diabetes was induced by a single dose of streptozotocin (140 mg/kg, intraperitoneal) and benfotiamine and/or citicoline were administered for three weeks. Memory was evaluated using the object recognition task and passive avoidance test. In passive avoidance test, co-administration of benfotiamine and citicoline was more effective than either alone in improving memory. Regarding object recognition task, although benfotiamine added to citicoline improved memory notably, the difference between combination therapy and single-drug therapy was not considerable.

Cakir et al [ 380 ] investigated the effects of citicoline on the well-known negative effects of rapid eye movements sleep deprivation on learning and memory in adult male Wistar albino rats, and the results obtained suggest that citicoline reduces rapid eye movements sleep deprivation-induced impairment in memory, at least in part, by counteracting the disturbances in biochemical and molecular biological parameters.

In a model of scopolamine-induced memory impairment, Petkov et al [ 368 ] showed citicoline to be able to prevent amnesia induced by scopolamine. Subsequently, Mosharrof and Petkov [ 369 ] showed that citicoline 100 mg/kg completely prevented amnesia induced by scopolamine, as did the association of citicoline 50 mg/kg and piracetam 500 mg/kg, also causing a significant increase in retention. Authors suggested that this effect is mediated by drug actions on neurotransmission. Takasaki et al [ 370 ] suggest that citicoline has ameliorative effect on the impairment of spatial memory induced by scopolamine, reducing the neuronal death and improving the impaired cholinergic signal. Citicoline acts as a memory-enhancing drug, and this effect is particularly marked in animals with memory deficits [ 371 ]. On the other hand, Álvarez et al [ 372 ] showed that citicoline antagonized amnesia induced by bromazepam in rats. Bruhwyler et al [ 373 ] found that chronic administration of citicoline has facilitating effects on learning and memory processes in dogs, but does not affect the established capacities and does not show, in this model, any effect on the motor, neurovegetative, or motivational systems. According to these authors, this represents an argument in favor of the selectivity of drug action in memory processes. Citicoline has even been shown to have a protective effect against mnesic disorders in aged animals [ 374 ] and in animals in isolation conditions [ 375 ] when administered as a dietary supplement, as well as in spontaneously hypertensive rats [ 376 ]. Ahmad et al [ 377 ] compare the relative efficacy of nootropics like piracetam, modafinil and citicoline on learning and memory in rats using the Morris water maze test. A total of 30 Wistar rats were used for the study. The animals were divided into five groups (n = 6). The groups I to V received gum acacia orally, scopolamine 2 mg/kg intraperitoneally, piracetam (52.5 mg/kg), modafinil (2.5 mg/kg), citicoline (25 mg/kg) respectively orally for 20 days. Learning and memory was evaluated using the Morris water maze test. The animals were trained in the Morris water maze on the last five days of dosing. Scopolamine 2 mg/kg was administered intraperitoneally to the above groups of animals (except groups I and II) for induction of amnesia, 45 minutes before the behavioural test. Scopolamine induced marked impairment of memory evidenced by significant reduction (p < 0.01) in the number of entries and time spent in the target quadrant when compared to the control group. There was significant (p < 0.05) increase in the number of entries and time spent in target quadrant of the Morris water maze in the animals who were pre-treated with piracetam, modafinil and citicoline, in comparison to the scopolamine treated group. Amongst the three nootropics, modafinil and citicoline showed significant (p < 0.05) memory enhancement in comparison to piracetam. Abdel-Zaher et al [ 378 ] investigated the potential protective effect of citicoline on aluminum chloride-induced cognitive deficits in rats, and demonstrated, for the first time, that citicoline can protect against the development of these cognitive deficits through inhibition of aluminum-induced elevation of glutamate level, oxidative stress, and nitric oxide overproduction in the hippocampus. Hosseini-Sharifabad et al [ 379 ] showed that magnesium increases the protective effect of citicoline on aluminum chloride-induced cognitive impairment in mice.

Drago et al [ 367 ] administered citicoline 10-20 mg/kg/day intraperitoneal for 20 days to 24-month-old Sprague-Dawley male rats from a strain showing cognitive and motor deficits. The drug was also given to rats with behavioral changes induced by a single injection of scopolamine, a cholinergic antagonist, by prenatal exposure to methylazoxymethanol, or by bilateral injections of kainic acid into the magnocellular basal nuclei. In all cases, citicoline improved learning and memory performance, evaluated using active and passive avoidance tests. In the old rat group, improved motor capacity and coordination was also seen. For these authors, these results suggest that citicoline affects the central mechanisms involved in cognitive behavior, probably through a cholinergic action.

It has been shown that hypobaric hypoxia decreases learning performance in rats undergoing sound avoidance conditioning, and that this effect may be antagonized by pretreatment with apomorphine or other dopaminergic agonists. These effects of hypoxia appear in relation to an inhibition of metabolism of cerebral catecholamines that would be ultimately responsible for an understimulation of central postsynaptic dopaminergic receptors. Based on these assumptions, Saligaut and Boismare [ 206 ] conducted a study on the effects of citicoline administration upon learning performance in rats subjected to hypobaric hypoxia. Under hypoxic conditions, citicoline was administered at 300 mg/kg/day for 12 days to a group of rats that underwent learning tests of a sound avoidance conditioning in the last five days of treatment. Effects seen in this group were compared to those seen in another group receiving apomorphine 0.5 mg/kg 30 minutes before each daily conditioning session and to those recorded in animals receiving both treatments. A group of animals acted as control and received an ascorbic acid solution under the same experimental conditions. Citicoline partially restored learning performance. The same effect, but to a lesser extent, was seen with administration of apomorphine and with combined administration of both drugs. These results suggest that administration of citicoline counteracts, as with dopaminergic agonists, the effects of hypoxia. Previously we commented the protective effect of citicoline against the cognitive impairment induced by chronic cerebral hypoperfusion [ 253 ].

To sum up, the effects of citicoline in the experimental models used to reveal pharmacological actions upon the dopaminergic system have been studied. Citicoline has been shown to act as a dopaminergic agonist and has a particularly significant effect upon levels of dopamine and its metabolites in the corpus striatum. The results obtained suggest that, with citicoline administration, striatal dopamine synthesis is increased, probably through tyrosine hydroxylase activation. Increase in dopamine levels would partly result from inhibition of dopamine reuptake, possibly related to citicoline action upon phospholipids synthesis. In addition, citicoline also has some effects upon the other monoamines, serotonin and norepinephrine, muscarinic and nicotinic receptors, and glutamate, opioids and gamma-aminobutyric acid, together to important modulating effects on several intracellular signaling processes.

Citicoline, administered prior to thiopental sodium anesthesia, can improve brain function by decreasing the duration of lack of response to corneal reflex and also increasing the effect on analgesia duration [ 349 ], and a significant increase in heart and respiration rate, an insignificant increase in oxygen saturation and an insignificant decrease of rectal temperature in animals [ 350 ]. Citicoline also has a protective effect in models of epilepsy induced by xylocaine [ 351 ] and pentylenetetrazol [ 352 , 353 ], but not when the epilepsy is induced by pilocarpine [ 354 ]. Bekhet et al [ 355 ] aimed to formulate citicoline-loaded niosomes for efficient brain delivery via the intranasal route to improve management of epilepsy. The protection against pentylenetetrazol-induced generalized seizures and mortality were determined in rats and compared with the oral drug solution at the exact dosage. The in vivo results revealed that a low dose of citicoline-loaded niosomes in situ gel had a powerful protective effect with delayed the latency for the start of convulsions and this can be considered as a method to boost the efficacy of citicoline in epilepsy management.

Roohi-Azizi et al [ 340 ] concluded that administration of citicoline, as an adjuvant drug, in combination with citalopram, enhanced the effectiveness of selective serotonin reuptake inhibitors drugs for depression treatment in a mice model of depression. Khakpai et al [ 341 ] concluded that the administration of citicoline, as an adjuvant drug, in combination with imipramine increased the efficacy of tricyclic antidepressant drugs for modulation of pain and depression behaviors in mice.

Shibuya et al [ 303 ] measured, using fluorometry, striatal dopamine levels after administration of citicoline in a single dose of 500 mg/kg intraperitoneal, and found that a significant (p < 0.05) increase occurred in striatal dopamine levels one hour after injection. On the other hand, Stanzani [ 304 ] showed citicoline to have a neuroprotective effect in substantia nigra, noting how citicoline protects this area against lesion induced by peroxydases (horse radish), achieving an increased number of surviving cells. Porceddu and Concas [ 305 ] also reported a trophic and/or stimulating effect of citicoline upon nigrostriatal dopaminergic neurons in a model of lesion induced by kainic acid. Also, there are experimental studies showing the protective effect of citicoline in cultures of dopaminergic neurons exposed to 6-hydroxydopamine [ 306 ], 1-methyl-4-phenylpyridinium [ 307 , 308 ], and glutamate [ 307 ]. Miwa et al [ 309 ] suggested that citicoline may act as a dopamine reuptake inhibitor after administration of a single dose, and that this drug may change the activity of dopaminergic neurons through changes in compositions of the neuronal membrane following repeated administration. In addition, these authors found citicoline to have certain muscarinic effects. In this regard, Giménez et al [ 310 ] show that chronic administration of citicoline to aged mice promotes partial recovery of the function of dopaminergic and muscarinic receptors, that normally decreases with aging, and think that this action may be explained based on mechanisms involving fluidity of neuronal membrane, in agreement with the results obtained by Petkov et al [ 311 ]. This latter investigating team, when comparing the effects of citicoline to those of the nootropic drugs adafenoxate and meclofenoxate upon the levels of the cerebral biogenic monoamines norepinephrine, dopamine, and serotonin in the frontal cortex, striate, hippocampus, and hypothalamus of rats [ 312 ], found that adafenoxate increased norepinephrine levels in striate and decreased norepinephrine levels in hypothalamus, increased dopamine levels in the cortex and hypothalamus and decreased them in the striate, and increased serotonin levels in the cortex but decreased them in the hippocampus. Meclofenoxate induced decreases in norepinephrine levels in the cortex and hypothalamus, while it increased dopamine levels in hippocampus and hypothalamus, and serotonin levels in the cortex, striate, hippocampus, and hypothalamus. Administration of citicoline has also recently been shown to increase dopamine levels in the retina [ 313 ]. Mao et al [ 314 ] showed that an intraperitoneal injection of citicoline could retard the myopic shift induced by form deprivation in guinea pigs, which was mediated by an increase in the retinal dopamine level. Citicoline increases norepinephrine levels in cortex and hypothalamus, dopamine levels in striate, and serotonin levels in cortex, striate, and hippocampus, having a slightly different profile as compared to nootropic drugs. As regards action of citicoline upon norepinephrine, a study by López-Coviella et al [ 315 ] shows that administration of citicoline increased total urinary excretion of 3-methoxy-4-hydroxyphenylglycol, reflecting noradrenergic activity, in rats and humans, suggesting that citicoline increases norepinephrine release. Recently, citicoline has also been experimentally shown to be able to influence the relationship between excitatory (glutamate) and inhibitory (gamma-aminobutyric acid) amino acids at the brain cortex of rats [ 316 ]. A series of experiments assessed the potential of citicoline to produce a central cholinergic activation. Intracerebroventricular administration of citicoline was shown to cause an increase in levels of vasopressin [ 317 ] and other pituitary hormones [ 318 ], due mainly to central cholinergic activation. Same effect has been demonstrated after intravenous administration [ 319 ]. Citicoline has also been shown to have a pressor effect in cases of hypotensive animals [ 320 ], or even in cases of hypotension due to hemorrhagic shock [ 321 , 322 ]. Amín et al [ 323 ] study the effects of citicoline on cardiovascular function in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) -treated albino rats. MPTP is a chemical that changes into the neurotoxin 1-methyl-4-phenylpyridinium, which causes catecholamine depletion. In this model, citicoline increased cardiac norepinephrine and tyrosine hydroxylase and improved markers related to Reactive oxygen species scavenger, mitochondrial permeability, calcium homeostasis on the cellular level, metabolic homeostasis, and mitochondrial biogenesis. Authors conclude that citicoline improved cardiovascular dysautonomia and that was reflected on cardiac contractility, electrical activity, blood pressure, and vascular reactivity. Also, a contribution of the central histaminergic system is involved in this effect of citicoline [ 324 ]. The central cholinergic activating effect exerted by citicoline was again emphasized, involving this effect to explain the cardiovascular [ 325 - 328 ] and metabolic effects [ 329 - 332 ] of the drug. Citicoline also modulates cerebral metabolism through glutamate-linked enzyme activities [ 333 ]. Sbardella et al [ 334 ] shown that citicoline greatly affects the proteolytic activity of the 20S proteasome, functioning as a bimodal allosteric modulator, likely binding at multiple sites, suggesting its potential role as a regulator of proteostasis in nervous cells. Ilcol et al [ 335 ] observed that citicoline treatment alters serum lipid responses to endotoxin and prevents hepatorenal injury during endotoxemia through a nicotinic acetylcholine receptor mediated mechanism. CDP-choline attenuates scopolamine induced disruption of prepulse inhibition in rats thanks to the involvement of central nicotinic mechanisms [ 336 ]. Yilmaz et al [ 337 ] show that citicoline administration restores the abnormalities in the primary, secondary, and tertiary hemostasis and prevents the development of disseminated intravascular coagulation during experimental endotoxemia in dogs probably by increasing both neuronal and nonneuronal cholinergic activity. Kocaturk et al [ 338 ] show that treatment with citicoline improves functions of cardiovascular and respiratory systems in experimental endotoxemia in dogs and suggest that they may be useful in treatment of endotoxin shock in clinical setting. Doolittle et al [ 339 ] demonstrated that citicoline corrects alveolar type II cell mitochondrial dysfunction in influenza-infected mice through preventing the declines in oxidative phosphorylation, mitochondrial membrane potential, and cardiolipin synthesis.

Action of citicoline upon the dopaminergic system has also been studied by investigating its pharmacological actions in experimental models used for that purpose, such as hypothermia induced by apomorphine, tardive dyskinesia induced by haloperidol, or acrylamide-induced lesion. Agut et al [ 300 ] studied the effect of citicoline administration on hypothermia induced by apomorphine, considered to be the result of the agonist action of apomorphine on D 2 receptors. Experimental animals received, in addition to apomorphine, haloperidol at a sufficient dose to partially block apomorphine-induced hypothermia in order to obtain a pharmacological system sensitive to citicoline action upon the dopaminergic system. A group of animals received a dose of citicoline 100 mg/kg p.o., and haloperidol 0.15 mg/kg was administered at 30 minutes by the intraperitoneal route. Thirty minutes later, rectal temperature was measured and apomorphine 1 mg/kg was administered by the subcutaneous route. Rectal temperature was again measured at 30, 60, and 90 minutes. Another group of animals received water instead of citicoline using the same scheme. Effects of chronic administration of citicoline at a dose of 100 mg/kg/day p.o. for five days were also analyzed. The same protocol as for acute administration was followed on the last day. shows the mean temperature decrease seen in each animal batch and at the different evaluation time points. Acute administration of citicoline causes hypothermia, that is significant for all control time points. Chronic administration only achieves a significant result at 90 minutes. Authors concluded that a 100 mg/kg dose of citicoline administered acutely by the oral route has a hypothermizing effect similar to the one reported for various dopaminergic agonists. On the other hand, they considered that the fact that chronic citicoline administration only caused a significant hypothermia in the last time point analyzed probably reflects that, with this form of administration, the tested product predominately acts upon phospholipids rather than acetylcholine synthesis. This second action pathway of citicoline would predominate with acute administration, as this would involve a relatively rapid utilization of the choline provided, that would be used for acetylcholine synthesis, thereby increasing tyrosine hydroxylase activity through cholinergic interneurons. By contrast, chronic administration of citicoline would result in a progressively greater availability of cytidine, and would therefore divert cerebral choline toward the synthetic pathway of CDP-choline and phospholipids, which would indirectly result in a dopaminergic agonistic effect. These authors developed an experimental model of tardive dyskinesia induced by haloperidol (2 mg/kg/day/7 days) in rats in a study including chronic administration of haloperidol or water to a total of 120 animals [ 301 ], finding that the administration of citicoline plus apomorphine in rats treated with haloperidol induced a motor activity similar to the activity seen in the group receiving citicoline only. Data provided in this study show that, in a model of haloperidol-induced dopaminergic hypersensitivity, administration of oral citicoline induces hypermotility; this may induce a phenomenon of competition against other agonists, leading to a partial reduction of the effect of apomorphine in animals pretreated with citicoline. In the model of acrylamide-induced lesion, these same authors [ 302 ] show that administration of low oral doses of citicoline, 50 mg/kg, is effective for correcting the neurological syndrome induced by acrylamide. Simultaneous administration of both substances, inducing an obvious weight loss in mice, has also been shown to cause an activation of the dopaminergic system, as seen in the results obtained with the apomorphine stereotype test.

Agut et al [ 298 ] indirectly studied the effect of citicoline upon dopamine synthesis in the striate body by measuring local levels of dopamine metabolites in animals in which blockade of dopaminergic receptors had been induced by administration of haloperidol. Pretreatment with citicoline 100 mg/kg/day/5 days significantly increased levels of homovanillic acid and 3,4-dihydroxyphenylacetic acid in the striate of treated animals as compared to the control group. Increase in levels of these metabolites was even stronger in a group of animals also receiving apomorphine. Results obtained in this study suggest that citicoline increases dopamine synthesis in the striate of rats in which activation of such synthesis has been experimentally induced by haloperidol administration. This same investigating team subsequently conducted a study to examine whether citicoline alone, without provoking an increased dopamine demand by dopaminergic receptors, caused an increased synthesis of this neurotransmitter, resulting in increased striatal levels of its main metabolites, homovanillic acid and 3,4-dihydroxyphenylacetic acid [ 299 ].

Saligaut et al [ 290 ] obtained results in agreement with the previous ones when studying dopamine reuptake in synaptosomes taken from the striate body of rats previously treated with citicoline. Following long-term treatment with this drug, a decreased dopamine reuptake by synaptosomes was seen, and authors related this fact to the increase in tyrosine hydroxylase activity, that would involve an increased dopamine synthesis. They think that a structural change in neuronal membranes, mainly of phospholipid levels, could be one of the factors responsible for the change in synaptosomal reuptake of the neurotransmitter induced by citicoline. Hypobaric hypoxia was also seen to antagonize the inhibitory effect of citicoline on dopamine reuptake by synaptosomes. This antagonism may be explained by the fact that hypoxia decreases activity of tyrosine hydroxylase, an enzyme that requires oxygen, thus counteracting enzyme activation exerted by citicoline. This leads to a decreased dopamine synthesis and a subsequent increase in dopamine reuptake. These same authors studied citicoline action in the experimental oxotremorine-induced cholinergic syndrome in mice [ 291 ] and showed that citicoline pretreatment does not potentiate this syndrome, but inhibits salivation induced by oxotremorine. Levodopa antagonized brain symptoms such as tremor-akinesia induced by oxotremorine. However, this antagonism disappeared in animals under long-term oral treatment with citicoline, thus confirming the action of citicoline on dopaminergic pathways. Citicoline effects appear to be mediated by hypersensitivity of some dopaminergic receptors, rather than by a direct stimulating effect on striatal dopaminergic receptors. In another series of experiments, these authors examined the effects of citicoline on catecholamine metabolism in the striate and hypothalamus from rats subjected to acute hypobaric hypoxia [ 292 ]. The results show that citicoline partially counteracts the effects of hypoxia upon the release and metabolism of certain neurotransmitters. In another study, Saligaut et al analyzed the effects of citicoline in rats with unilateral nigrostriatal lesion induced by 6-hydroxydopamine [ 293 ]. In damaged animals, amphetamine administration induced an ipsiversive circling behavior, while such circling behavior was contraversive with administration of levodopa and apomorphine. This appears to be mediated by the development in the damaged side of a supersensitivity of postsynaptic dopaminergic receptors. Subchronic treatment with citicoline did not induce behavioral effects. Citicoline did not 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 levodopa may not be explained by an activation of tyrosine hydroxylase, this effect appears to be related to an improved release of dopamine synthetized from exogenous levodopa. Kashkin et al [ 294 ] evaluate the effect on the combination of citicoline with levodopa/carbidopa in the rotenone model of Parkinson&#;s disease in rats, confirming that the combination therapy had more pronounced therapeutic effect on extrapyramidal disorders than monotherapy.

Martinet et al [ 287 ] conducted a study in which the effects of citicoline administration on norepinephrine, dopamine, and serotonin levels were assessed in different rat brain regions. For this, conversion of 3 H-tyrosine and 3 H-tryptophan, administered by the intravenous route, into 3 H-norepinephrine, 3 H-dopamine, and 3 H-serotonin was measured, comparing the results obtained with administration of saline to those obtained after administration of citicoline at different doses. Metabolism of each neurotransmitter was studied in the brain regions where it has functional activity. Thus, for catecholamines citicoline action was studied in the striate body, brain cortex, and midbrain, while for serotonin the hypothalamus was also studied. The synthesis rate of dopamine, norepinephrine, and serotonin was expressed as a conversion index equal to the ratio between the amount of labelled neurotransmitter per gram of brain (cpm/g) and the tyrosine or tryptophan-specific radioactivity (cpm/mmol) in brain. As shown in , citicoline significantly increased dopamine levels and synthesis rate in the striate body, and the effect exerted on tyrosine levels was very similar. Norepinephrine levels were increased in cortex, but showed no changes from control in the brain stem. As regards effects on serotonin, the drug was seen to cause decreases in the levels and synthesis rate of this neurotransmitter in the brain stem and hypothalamus, and no changes were seen in the cortex or striate. According to these authors, increased dopamine synthesis could be attributed to an enhancing effect of citicoline upon tyrosine hydroxylase activity, the rate-limiting step in dopamine synthesis. This activation of tyrosine hydroxylase would lead to an inhibition of dopamine reuptake at the synapse, an action that has been shown in ex vivo studies [ 288 , 289 ]. By contrast, the increase seen in dopamine synthesis does not appear to be related to increased levels of tyrosine, since this completely saturates tyrosine hydroxylase under physiological conditions. The effects of citicoline upon striatal dopamine synthesis are particularly interesting because changes in dopamine synthesis by extrapyramidal dopaminergic neurons are in the origin of Parkinson&#;s disease.

As previously discussed, citicoline exerts some of its effects through its action on the levels of certain neurotransmitters and on some intracellular signaling processes. This section will discuss these specific effects upon neurotransmission and on intracellular signaling processes. As will be seen below, most studies have focused on analyzing the effect of citicoline on central dopaminergic transmission and on nicotinic cholinergic neurotransmission.

These characteristics confer citicoline a suitable pharmacological profile for the treatment of cerebral ischemia [ 39 , 40 , 281 - 283 ]. Also, it has been postulated a role of citicoline in the treatment of complications of infectious diseases, such as cerebral malaria [ 284 , 285 ]. Abdel-Aziz et al [ 286 ] conclude that citicoline avoid both short and long-term damages to the neuroendocrine disturbances, oxidative stress, inflammation, and apoptosis induced by head irradiation in a rat model.

According to Drago et al [ 280 ], citicoline is a drug of choice for treatment of cerebrovascular diseases, particularly in its chronic form, because its clinical use is justified by the pharmacological actions it exerts on the central nervous system. To sum up, citicoline ( ):

Fiedorowicz et al [ 277 ] found that citicoline can attenuate brain damage in a rat model of birth asphyxia. It has been demonstrated that meta-analysis provides an effective technique for the aggregation of data from experimental stroke studies. With this technique, Bustamante et al confirm that citicoline reduces the infarct volume and improves outcome [ 278 ], pointing doses of 300-500 mg/kg as the optimal dose to be translated into a candidate neuroprotective drug for human stroke [ 279 ].

Apoptotic mechanisms have been shown to play a primary role in the pathophysiology of cerebral ischemic damage both at experimental level [ 262 - 266 ] and in humans [ 267 , 268 ]. We therefore investigated [ 269 ] whether citicoline could influence apoptotic mechanisms following focal cerebral ischemia. A model of permanent distal occlusion of the middle cerebral artery was used in Sprague-Dawley rats. Animals were randomized into four groups: B + A, citicoline 500 mg/kg intraperitoneal 24 hours and one hour before occlusion and 23 hours after occlusion; A, citicoline 500 mg/kg intraperitoneal within 30 minutes and 23 hours following occlusion; C, saline solution intraperitoneal; D, Sham-operated. Animals were killed at 12 (seven animals per group) and 24 hours (seven animals per group) following occlusion. Immunohistochemistry for procaspases 1, 2, 3, 6, and 8 was performed using goat polyclonal antibodies and, using gel electrophoresis and Western blotting, specific substrates for caspase action were tested using poly-ADP-ribose polymerase (PARP) antibodies. Ischemia induced expression of all procaspases and PARP in both the infarction and the penumbra areas 12 and 24-hours following ischemia. Citicoline reduced expression of all procaspases at 12 and 24-hours following ischemia, except for procaspase 3 at 24 hours in group A and PARP expression ( ), and results were clearer in group B + A, suggesting a certain prophylactic role of citicoline, results that have been confirmed recently [ 270 ]. Citicoline has been shown to be able to inhibit certain intracellular signals involved in apoptotic processes [ 271 ] and to maintain these inhibitory effects in different experimental models to study apoptotic mechanisms [ 142 , 226 , 272 - 276 ].

Clark et al [ 261 ] examined whether citicoline was able to reduce ischemic damage and improve the functional neurological result in an intracerebral hemorrhage model in mice. They caused hemorrhage in 68 Swiss albino mice by injecting them collagenase at the caudate nucleus. Animals randomly received saline or citicoline 500 mg/kg intraperitoneal before administration of collagenase and at 24 and 48 hours. Mice were assessed using a 28-item neurological scale and were killed at 54 weeks to assess hematoma volume, total damage, and surrounding ischemic damage. As regards neurological course, citicoline-treated animals had a better score than placebo-treated animals (10.4 ± 2 versus 12.1 ± 2.4; p < 0.01). No differences were seen in hematoma volumes, but a significant reduction in the volume of the surrounding ischemic damage was noted in animals treated with citicoline, with values being 13.8 ± 5.8 mm 3 (10.8 ± 4.3% of hemisphere) and 17 ± 7.1 mm 3 (13.3 ± 5.1%) for placebo, with p < 0.05. According to authors, these results support a potential role of citicoline for treatment of intracerebral hemorrhage.

On the other hand, Masi et al [ 259 ] have shown citicoline to have a certain antiplatelet aggregant effect, that may provide an additional benefit for the treatment of cerebral vascular disease. Pinardi et al [ 260 ] investigated in Sprague-Dawley rats the effects of citicoline infusion on relaxation induced by exogenous acetylcholine in the isolated external carotid vascular bed, having no cholinergic nerve supply, and the isolated internal carotid vascular bed that, by contrast, has an abundant cholinergic nerve supply. Changes in perfusion pressure were measured during a dose-response curve to acetylcholine and following infusion of 1 mg/minute/30 minutes of citicoline. Authors noted that citicoline caused relaxation in both vascular beds, which would suggest the presence of muscarinic receptors. In the internal carotid vascular bed, citicoline infusion for 30 minutes significantly shifted to the left the dose-response curve to acetylcholine, enhancing relaxation. However, this did not occur in the external carotid bed. The effect of citicoline was masked when it was jointly infused with hemicholinium. According to these authors, results suggest that citicoline would act by increasing choline levels at cholinergic endings, increasing acetylcholine synthesis and/or release.

Other mechanisms proposed to explain the neuroprotective effects of citicoline are the restoration of the barrier function of endothelial cells [ 254 ], the inhibition of mitochondrial permeability transition [ 255 , 256 ], and providing neuronal membrane integrity and protection of membrane stability in cortical spreading depression [ 257 ]. Another mechanism investigated has been the participation of sirtuin 1 in the neuroprotective actions of CDP-choline [ 258 ]. Treatment with CDP-choline increased sirtuin 1 protein levels in brain concomitantly to neuroprotection. Treatment with sirtinol blocked the reduction in infarct volume caused by CDP-choline, whereas resveratrol elicited a strong synergistic neuroprotective effect with CDP-choline. These results demonstrate a robust effect of CDP-choline like sirtuin 1 activator by up-regulating its expression.

Hamdorf and Cervós-Navarro [ 251 ] exposed 48 rats for 103 days to a decreasing amount of oxygen, i.e., they were exposed to chronic hypoxia. Citicoline showed a protective effect by increasing vigilance under moderate hypoxic conditions (15% O 2 ). In a subsequent study, these same authors [ 252 ] analyzed the effects of citicoline in Wistar rats subjected to hypoxia for five months. Behavioral changes induced by hypoxia were attenuated in the group or animals treated with citicoline. Interestingly, therapeutic administration of citicoline was found to be more effective than prophylactic administration. In addition, under extreme hypoxia conditions, citicoline showed a protective effect by lengthening survival. Lee et al [ 253 ] demonstrated that citicoline protects against cognitive impairment in a rat model of chronic cerebral hypoperfusion.

Citicoline has also been shown to have a neuroprotective effect against neurotoxic damage induce by kainic acid in retinal cells [ 244 - 247 ] and in in vitro models of retinal neurodegeneration [ 248 ]. Komnatska et al [ 249 ] demonstrate that citicoline restores the microcirculation in the vessels of the ciliary body in rabbits, measured with laser Doppler flowmetry. Bogdanov et al [ 250 ] suggest that topical administration of citicoline in liposomal formulation could be considered as a new strategy for treating the early stages of diabetic retinopathy.

Fresta et al conducted a series of experiments in models of transient cerebral ischemia in rats using liposomal citicoline, in which they showed a significantly increased survival in animals treated with this citicoline formulation [ 234 - 236 ], and more recently, that this same drug formulation significantly reduces the maturation phenomenon, that is, delayed cerebral neurodegenerative lesion, that occurs after an ischemic event, resulting in a significant improvement in brain functions [ 237 ]. These results agree with previously discussed results [ 192 ] showing that administration of liposomal citicoline is more effective as compared to non-liposomal citicoline [ 238 - 240 ]. Other ways to improve neuroprotective efficiency of citicoline are the stereotactic delivery [ 241 ], nanocarriers [ 242 ] or simple diffusion delivery via brain interstitial route [ 243 ].

In a series of conducted studies, citicoline was shown to have a synergistic effect with other drugs in the treatment of cerebral ischemia, such as thrombolytic [ 211 - 215 ] and neuroprotective drugs [ 216 - 224 ]. Andersen et al [ 211 ] conducted an experimental study in a rat model of carotid embolism to evaluate the effect of different doses of citicoline, administered alone or combined with recombinant tissue plasminogen activator (rTPA), on infarction size. Ninety Sprague-Dawley rats subjected to embolism in the carotid territory were randomized into six groups: a) saline-treated animals; b) citicoline 250 mg/kg; c) citicoline 500 mg/kg; d) rTPA 5 mg/kg; e) rTPA 5 mg/kg + citicoline 250 mg/kg; and f) rTPA5 mg/kg + citicoline 500 mg/kg. Treatment with rTPA was given at a suboptimal dosage (5 mg/kg infused over 45 minutes, starting treatment 45 minutes after embolization). Citicoline was administered daily by the intraperitoneal route for four days. Brains from surviving animals were fixed at four days and infarction volume, calculated as percentage of the total volume of the hemisphere affected, was measured using a microscope. Mean infarction volume values suggested that high-dose citicoline and the combination of citicoline with rTPA decreased the size of ischemic lesion ( ). In the control group, mean infarction volume was 41.2% (5.9-87%). In groups treated with citicoline alone, values were 30.4% (1-70%, n.s.) in group 2, and 22.2% (0.7-76.6%; p < 0.05) in group 3. With rTPA alone (group 4), 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 that high-dose citicoline and a combination of citicoline at lower doses with rTPA significantly reduced the size of brain infarctions. Díez-Tejedor et al [ 212 , 213 ] reported similar results, stating that results of this association are improved when citicoline is administered immediately after rTPA administration. The same team [ 214 ] compared the effects of high doses of CDP-choline (1,000 mg/kg) with rTPA (5 mg/kg) in an experimental animal model of embolic stroke. CDP-choline and rTPA produced a significant reduction in brain damage considering infarct volume, cell death, and inflammatory cytokines (tumour necrosis factor-alpha and interleukin 6) compared with the infarct group. Additionally, CDP-choline significantly decreased infarct volume, cell death, and interleukin 6 levels with respect to the rTPA group. From these results, they concluded that high-dose CDP-choline may be an effective treatment for acute ischaemic stroke even in absence of thrombolysis. Shuaib et al [ 215 ] investigated the neuroprotective effects of citicoline alone or combined with urokinase in a rat model of focal cerebral ischemia induced by embolization at the origin of the middle cerebral artery. Both drugs were administered two hours after ischemia induction. Animals were killed at 72 hours. In saline-treated animals, infarction volume was 33.1 ± 9.7%. Citicoline-treated animals were divided into two groups, one of which was given a single dose of citicoline 300 mg/kg, while the other group received a daily dose of 300 mg/kg for three days, both by the intraperitoneal route. A significant reduction in infarction volume was 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 5,000 u/kg, also had a smaller infarction volume (19.5 ± 12.5%; p = 0.01). However, the greatest volume reduction was achieved in the group of animals treated with the combination of citicoline and urokinase (13.6 ± 9.1%; p = 0.). These authors concluded that citicoline provides a significant neuroprotective effect that may be enhanced by association with a thrombolytic. Synergistic effects have also been shown with the association of citicoline with MK-801 or dizocilpine [ 216 ], basic fibroblast growth factor [ 217 ], lamotrigine [ 218 ], nimodipine [ 219 , 220 ], N-nitro L-arginine methyl ester [ 221 ], homotaurine [ 222 ], docosahexaenoic acid [ 223 ], and azelnidipine [ 224 ], but no with piracetam [ 225 ] in models of cerebral ischemia. It has been demonstrated that citicoline with hypothermia is more effective than used alone in ameliorating cerebral damage after transient focal ischemia [ 226 ]. Also, it has been demonstrated that pre-conditioning with CDP-choline attenuates oxidative stress-induced cardiac myocyte death in a hypoxia/reperfusion model [ 227 ]. Zazueta et al [ 228 ] demonstrate that citicoline protects liver from ischemia/reperfusion injury preserving mitochondrial function and reducing oxidative stress. Also, it is known that citicoline and mesenchymal stem cells administration show equal efficacy in the neurological recovery, the decrease of neuronal death and the increase of neuronal repair in a model of cerebral infarction in rats, but the combination does not increase the benefit [ 229 ], despite that citicoline treatment induces brain plasticity markers expression in experimental animal stroke [ 230 ]. Diederich et al [ 231 ] designed a study to check whether citicoline also enhances neuroregeneration after experimental stroke. Animals were subjected to photothrombotic stroke and treated either with daily injections of CDP-choline (100 mg/kg) or vehicle for 10 consecutive days starting 24 hours after ischemia induction. Sensorimotor tests were performed after an adequate training period at days 1, 10, 21, and 28 after stroke. Then brains were removed and analyzed for infarct size, glial scar formation, neurogenesis, and ligand binding densities of excitatory and inhibitory neurotransmitter receptors. Animals treated with citicoline showed a significantly better neurological outcome at days 10, 21, and 28 after ischemia, which could not be attributed to differences in infarct volumes or glial scar formation. However, neurogenesis in the dentate gyrus, subventricular zone, and peri-infarct area was significantly increased by CDP-choline. Furthermore, enhanced neurological outcome after citicoline treatment was associated with a shift toward excitation in the perilesional cortex. The present data demonstrate that, apart from the well-known neuroprotective effects in acute ischemic stroke, CDP-choline also possesses a substantial neuroregenerative potential. Also, citicoline potentiates angiogenesis [ 232 ] and astroglial cell proliferation and differentiation [ 233 ], both mechanisms involved in neuroplasticity.

Schäbitz et al [ 210 ] evaluated the effects of long-term treatment with citicoline in a model of transient focal ischemia (two hours) in rats. Ten animals were randomly assigned to each of the groups: placebo (saline 0.3 mL/day/7 days), low dose (citicoline 100 mg/kg/day/7 days intraperitoneal) and high dose (500 mg/kg/day/7 days intraperitoneal). Treatment was started at the time of reperfusion, once the two-hour ischemia period had ended. Daily neurological assessments were made (modified Zea Longa scale), and surviving animals were killed on day 7, after which cerebral edema and infarction volume were calculated. No differences were seen in neurological assessment of animals at study end, but a more favorable trend was noted in the citicoline high-dose group. Mean infarction volume ( ) was 243.5 ± 88.6 mm 3 in the placebo group, 200.2 ± 19.9 mm 3 in the low-dose group, and 125.5 ± 45.2 mm 3 in the high-dose group. These differences were statistically significant (p < 0.01). A dose-dependent decrease in cerebral edema volume was also seen, but did not reach statistical significance.

Aronowski et al [ 209 ] evaluated the effects of chronic citicoline administration (500 mg/kg) upon recovery in spontaneously hypertensive rats undergoing occlusion of the middle cerebral artery for 30 to 120 minutes. Drug or saline were administered by the intraperitoneal route from 15 minutes after ischemia induction and were continued for 14 days. Morphological lesion and neurological disorders (motor and sensorimotor capacities) were analyzed by measuring the maximum morphological lesion volume, maximum neurological change, and ischemia duration causing half the maximum morphological lesion or maximum neurological change. Maximum morphological lesion volume was not affected by citicoline (101.6 ± 11.4 mm 3 for citicoline, 103.3 ± 13.6 mm 3 for saline); however, citicoline significantly increased ischemia duration required to cause half the morphological lesion, that changed from 38.3 ± 5.9 to 60.5 ± 4.3 minutes (p < 0.05). Similarly, citicoline did not change the value of maximum neurological change (8.5 ± 0.7 for citicoline, 10.1 ± 4.0 for control), but did significantly increased ischemia duration required to cause half the maximum neurological change from 41.9 ± 4.6 to 72.9 ± 24.5 minutes (p < 0.05). According to these authors, citicoline shows a greater efficacy in animals that experience a submaximal lesion, occurring in this model with 30-75 minutes of ischemia.

LePoncin-Lafitte et al [ 110 ] studied the effects of citicoline on various histological brain changes in an experimental model of multifocal cerebral ischemia in cats, in which ischemic lesion was caused by introducing in the internal carotid artery calibrated microspheres, that will produce cerebral microinfarctions, characterized by having a central necrosis area surrounded by a penumbra area, together with edema due to rupture of the blood-brain barrier. Citicoline administration considerably decreased the number of lesions, and also the amount of extravasated albumin, which confirms, for these authors, that citicoline exerts its neuroprotective role against ischemia by acting upon cell membranes. Araki et al [ 208 ] also found some neuroprotective effect of citicoline in complete cerebral ischemia induced by decapitation and potassium cyanide poisoning in mice.

Saligaut and Boismare [ 206 ] studied the effects of citicoline, administered at a dose of 1,000 mg/kg p.o., in Wistar rats undergoing acute hypobaric hypoxia (15 minutes at a simulated altitude of 7,180 meters), assessing a behavior-conditioning test, striatal dopamine uptake, and levels of this neurotransmitter and its metabolites in the striatum. In the behavior-conditioning test, citicoline was seen to protect against hypobaric hypoxia in a different way and to a greater extent than apomorphine. Biochemical studies showed a presynaptic effect, probably because of activation of tyrosine hydroxylase, inducing changes in dopamine uptake, as well as an improved dopamine release. Similar results on the effect of citicoline on tyrosine hydroxylase activity have been obtained by other teams [ 207 ].

Nagaoka [ 205 ] studied the effects of citicoline on stroke onset and mortality in spontaneously hypertensive rats subjected to cerebral ischemia. Ischemia was induced by occluding both common carotid arteries. Citicoline (200-1,000 mg/kg intraperitoneal), administered before ischemia induction, caused a dose-dependent delay in the onset of stroke and respiratory arrest. These effects were also seen in animals treated after ischemia induction. In addition, citicoline 500 mg/kg intraperitoneal improved the neurological status in rats undergoing brain ischemia for 40 minutes and reperfusion. These results suggest that citicoline has a neuroprotective role against cerebral ischemia and reperfusion.

Kakihana et al [ 202 ] investigated distribution of labelled citicoline and its effects on acetylcholine synthesis from glucose in the brain cortex of rats subjected to 30 minutes of ischemia followed by reperfusion. Treatment with citicoline improved glucose metabolism and significantly restored acetylcholine synthesis from glucose. For these authors, the results obtained suggest that citicoline improves brain energy metabolism in ischemia conditions. These authors [ 203 ] subsequently evaluated the effects of citicoline on neurological sequelae and glucose metabolism in the brain in an experimental rat model of transient cerebral ischemia, showing that high-dose citicoline improved the neurological state of animals subjected to ischemia, which was correlated to an improved brain energy metabolism and to drug incorporation in the fraction of membrane phospholipids. These results agree with those obtained by Fukuda et al [ 204 ] in a preliminary study.

Hurtado et al [ 198 ] have shown that administration of citicoline significantly increased adenosine triphosphate (ATP) brain levels in both healthy and ischemic animals, and that this increase in ATP was correlated to a positive effect on glutamate transporters, restoring their normal activity and therefore decreasing both brain parenchymal and circulating glutamate levels. This was correlated to a decreased cerebral infarction volume. The same authors demonstrated that citicoline redistributes the glutamate transporter EAAT2 to lipid raft microdomains and improves glutamate uptake and this effect is also found after experimental stroke, when citicoline is administered four hours after the ischemic occlusion [ 199 ]. In another study [ 200 ], they found that a chronic treatment with citicoline, initiated 24 hours after the insult, is able to increase the neuronal plasticity as well as to promote functional recovery ( ). Zhao et al [ 201 ] also showed a positive effect of citicoline on spatial learning and memory of rats after focal cerebral ischemia.

Kim et al [ 197 ] investigate the effect of citicoline in the context of hypoglycemia-induced neuronal death in a rat model with insulin-induced hypoglycemia. Acute hypoglycemia was induced by intraperitoneal injection of regular insulin (10 U/kg) after overnight fasting, after which isoelectricity was maintained for 30 minutes. Citicoline injections (500 mg/kg, intraperitoneal) were started immediately after glucose reperfusion. Treatment with citicoline resulted in significantly reduced neuronal death, oxidative injury, and microglial activation in the hippocampus when compared to vehicle-treated control groups at seven days after induced hypoglycemia. Citicoline administration after hypoglycemia decreased immunoglobulin leakage through blood brain barrier disruption in the hippocampus when compared to the vehicle group. Citicoline increased choline acetyltransferase expression for phosphatidylcholine synthesis after hypoglycemia. These findings suggest that neuronal membrane stabilization by citicoline administration can save neurons from the degeneration process after hypoglycemia, as seen in several ischemia studies. Therefore, these results suggest that citicoline may have therapeutic potential to reduce hypoglycemia-induced neuronal death.

Nagai and Nagaoka [ 196 ] reported the results of a study investigating the effect of citicoline upon glucose uptake in different brain areas from rats with global cerebral ischemia induced by the occlusion of both carotid arteries for 30 minutes after electrocauterization of both vertebral arteries. Glucose uptake by the brain was measured four days after recirculation. Without citicoline administration, global cerebral uptake was found to be reduced to 81% of the normal value. With administration of citicoline at a dose of 250 mg/kg intraperitoneal twice daily for three days after the start of recirculation, postischemic reduction of glucose uptake was significantly lower in the brain cortex. This suggests that citicoline improves energy metabolism in the brain under ischemic conditions.

Narumi and Nagaoka [ 195 ] investigated the effects of citicoline administration upon metabolism of cerebral monoamines in two rat models of global cerebral ischemia. In the first model they performed cerebral ischemia, using bilateral carotid occlusion, for 30 minutes in spontaneously hypertensive rats and noted that a significant decrease occurred in norepinephrine levels in the brain cortex. In this model, administration of 1,000 mg/kg of citicoline decreased dopamine contents in striatum and diencephalon, normalizing the decrease in the dopamine metabolites/dopamine ratio induced by ischemia. In the second model, bilateral carotid occlusion was also performed 24 hours after electrocauterization of both vertebral arteries in Wistar rats. In this model, norepinephrine, dopamine, and serotonin levels decreased 70%-80% in the brain cortex. Similar decreases were also seen in norepinephrine and serotonin levels in hippocampus, in dopamine levels in the nucleus accumbens, in dopamine and serotonin levels in striatum, and in norepinephrine levels in diencephalon and brain stem. Administration of citicoline at a dose of 500 mg/kg significantly enhanced the ischemia-induced decrease in striatal dopamine levels. These authors therefore suggest that citicoline appears to restore dopamine turnover in the striatum of rats subjected to experimental cerebral ischemia.

Tornos et al [ 192 ] conducted a pharmacological study on the protective effect of citicoline against toxicity in an experimental model of hypoxia induced by potassium cyanide. They found that treatment with oral citicoline for four days before hypoxia induction had a protective effect, demonstrated by a longer survival time in treated animals. These benefits of citicoline may also be ascribed to the activation of the cerebral energy metabolism [ 193 ] and the increased activity of mitochondrial cytochrome oxidase [ 194 ] induced by this drug.

Trovarelli et al [ 173 , 174 ], using an experimental global ischemia model consisting of bilateral carotid ligation in gerbils, found that intraperitoneal citicoline administration partially prevents changes in lipid metabolism induced by cerebral ischemia, correcting the increase in free fatty acids, changes in neutral lipids such as diacylglycerol, and the decrease in phosphatidylcholine. Suno and Nagaoka [ 176 ] experimentally studied in rats the effects of citicoline administration upon free fatty acid release caused by total cerebral ischemia lasting five minutes. It was shown that the tested drug reduced the increase in free fatty acids, and that the intensity of this effect depended on the dose used. Arachidonic acid contents in brains from control group animals subjected to ischemia was 174 ± 22 mmol/g, as compared to 119 ± 8 mmol/g and 61 ± 8 mmol/g in animals receiving 200 and 1,000 mg/kg intraperitoneal of citicoline respectively ( ). Authors concluded that these results suggest that administration of citicoline may prevent ischemic cerebral damage [ 179 - 181 ]. Agut and Ortiz [ 178 ] treated male rats weighing 190-200 g with 4 mg/kg of 14 C-methyl-citicoline (50 μmCi) by the oral route. At 24 hours, brain radioactivity levels and the presence of labelled phospholipids were assessed under conditions of normoxia, hypoxia, and hypoxia following additional administration of 100 mg/kg of unlabelled citicoline. Investigators found a marked incorporation of radioactivity into the brains of normoxic and hypoxic animals, mostly associated to phosphatidylcholine. In addition, administration of unlabelled citicoline reduced the elevation in cerebral lysophosphatidylcholine caused by hypoxia. Rao et al [ 182 ] showed that citicoline significantly decreased blood-brain barrier dysfunction after ischemia with a six-hour reperfusion in gerbils and, in the same model of transient cerebral ischemia, considerably reduced the increase in arachidonic acid and leukotriene C 4 synthesis 24 hours after ischemia induction. They also showed that the cerebral edema volume was substantially lower at three days in animals treated with citicoline. Following six days of reperfusion, ischemia was seen to cause 80 ± 8% neuronal death at the hippocampal CA 1 layer level, and citicoline provided a neuroprotection of 65 ± 6%. In a subsequent study, these authors [ 183 ] showed citicoline to be able to significantly restore phosphatidylcholine, sphingomyelin, and cardiolipin levels after induction of transient cerebral ischemia in gerbils. For these authors, the main action mechanism of citicoline would be inhibition of stimulation of phospholipase A 2 activity in ischemia conditions, though they also stress its effects upon glutathione synthesis and glutathione reductase activity. Thus, the drug would prevent membrane destruction, decrease free radical generation, and preserve the natural defenses of the nervous system against oxidative damage [ 184 - 188 ]. More recently, this investigating team has also shown that citicoline enhances phosphatidylcholine synthesis, which is impaired under ischemia conditions, attenuating the loss of CTP-phosphocholine cytidyltransferase activity [ 189 , 190 ]. Thus, the drug has effects preventing phospholipid degradation and its implications and promoting regeneration of cerebral phosphatidylcholine, effects that are seen to result in a decreased volume of the cerebral ischemic lesion [ 191 ].

Various experimental studies have shown citicoline to prevent fatty acid release during cerebral ischemia and hypoxia, and to increase synthesis of structural phospholipids [ 172 - 191 ]. Horrocks et al [ 172 , 175 , 177 ], using an experimental model of global cerebral ischemia by decapitation, showed that administration of a mixture of citicoline and CDP-ethanolamine decreased free fatty acid release and increased synthesis of the corresponding glycerophospholipids, suggesting an involvement of choline and ethanolamine phosphotransferases.

Alberghina et al [ 170 ] investigated the effect of citicoline upon incorporation of labelled precursors into cerebral phospholipids of guinea pigs subjected to hypoxia. A group of animals were given 100 mg/kg of citicoline by the intraperitoneal route. Ten minutes later, the labelled precursors [2- 3 H]glycerol and [1- 14 C]palmitate were administered by the intraventricular route. Another group of animals only received the precursors, and acted as control group. Investigators noted that, as compared to the control group, citicoline-treated animals showed an increase in specific radioactivity of total lipids and phospholipids in purified mitochondria obtained from brain hemispheres, cerebellum, and brain stem. In a subsequent study, this same investigating team [ 171 ] showed citicoline to be able to counteract the effects of hypoxia upon incorporation of labelled precursors into RNA and proteins, particularly at nuclear and mitochondrial level.

Boismare et al [ 167 ] reported that treatment with citicoline 20 mg/kg by the intraperitoneal route in rats induced, during acute hypoxia, a decrease in vegetative responses, protection from conditioned avoidance responses, and stabilization of dopamine and norepinephrine brain levels. This same group [ 168 ] found in dogs subjected to normobaric hypoxia increases in blood pressure, heart rate, cardiac output, and regional blood flows, while no changes occurred in total peripheral resistance. Administration of citicoline abolished these hemodynamic effects induced by acute hypoxia, suggesting that this action was correlated to a dopaminergic agonistic effect of the drug. In cats subjected to short periods of cerebral ischemia, these authors [ 169 ] noted that a depression occurred in cortical evoked potentials. Such depression was attenuated by prior administration of citicoline by the intracarotid route. These authors think that the protective effects of citicoline are metabolic/biochemical rather than hemodynamic in origin, and do not rule out a direct action of the drug upon central dopaminergic structures.

In vitro studies using nerve tissues have shown anoxia to induce a decrease in the synthesis of structural phospholipids that is time-dependent, i.e., the longer the hypoxia the stronger the impact upon neuronal phospholipids metabolism [ 164 ]. Moreover, a decreased incorporation of marked precursors into phospholipids of neuronal subcellular fractions obtained from animals subjected to experimental hypoxia has also been shown [ 26 ]. It is also known that, when cerebral ischemia is experimentally induced, glycerophospholipids in cell membranes are broken down by the action of different phospholipases, producing free fatty acids and arachidonic acid derivatives. With prolonged ischemia, induced aggression upon membranes becomes more intense and membranes lose their functions. Na + and Ca 2+ accumulate inside the cell, triggering the ischemic cascade and invariably leading to cell death [ 7 , 33 , 37 , 41 , 121 , 165 ].

Also, it has been observed some beneficial effects of citicoline in experimental models of partial optic nerve crush in the rat [ 151 ]. Kitamura et al [ 152 ] investigated the effectiveness of a single or a combination of topical neurotrophic factors, including citicoline, in protecting retinal ganglion cells in the rat optic nerve crush model, and conclude that the combination of the three neurotrophic factors, including citicoline, was the most effective way to protect retinal ganglion cells after the optic nerve crush. Also, there are some data suggesting that citicoline promotes nerve regeneration and reduces postoperative scarring after peripheral nerve surgery [ 153 ]. Aslan et al [ 154 ] demonstrated that CDP-choline improves the functional recovery and promotes the regeneration of injured sciatic nerves treated with immediate or delayed surgical repair in rats. The same team [ 155 ] demonstrated that intraperitoneal administration of CDP-choline improves nerve regeneration and functional recovery in a rat model of sciatic nerve injury, also improving nerve adherence and separability. Kaplan et al [ 156 ] concluded that citicoline exhibits dose-dependent effects on axonal regeneration and recovery without scar formation in a rat model of peripheral nerve incision and primary anastomosis. In this context, CDP-choline modulates matrix metalloproteinase activity and promotes the expression of tissue inhfibitor of metalloproteinases to stimulate axonal regeneration [ 157 ]. These data help to explain one mechanism by which CDP-choline provides neuroprotection in peripheral nerve injury. Samadian et al [ 158 ] described a role of citicoline for peripheral nerve regeneration. Emril et al [ 159 ] demonstrated that in situ administration of 0.4 mL of 100 μmol/L CDP-choline prevents the occurrence of neuropathic pain and induces motoric recovery four weeks after sciatic nerve injury. Ahlawat and Sharma [ 160 ] concluded that simultaneous administration of S-methylisothiourea sulfate (a selective iNOS inhibitor) and citicoline may provide potential therapeutics for diabetic neuropathic pain. Savran et al [ 161 ] demonstrated that CDP-choline may be effective for preventing postoperative epidural fibrosis in an experimental model. After a systematic review of the literature on rodent models, Wang et al [ 162 ] consider that CDP-choline is one of the most effective adjuvant treatments after surgery in peripheral nerve laceration.

Effect of citicoline upon traumatic spinal cord lesion was also studied, and it was shown that intraperitoneal administration of citicoline 300 mg/kg five minutes after lesion induction significantly reduced lipid peroxidation and improved motor function in treated animals [ 146 ], having the same efficacy than methylprednisolone in the behavioral and neuroanatomical recovery [ 147 ]. It has been demonstrated that the administration of repeated doses of citicoline prevents the tissue damage associated with the spinal cord shock in acute phase [ 148 ], and that the combination of ischemic postconditioning with citicoline confers protection in a model of ischemic spinal cord lesion [ 149 ], through the inhibition of the caspases pathway and the increase of antiapoptotic proteins. In a model of spinal cord injury, Paulose et al [ 150 ] suggest that the neurotransmitters combination along with bone marrow or citicoline with bone marrow can reverse the muscarinic receptor alterations in the spinal cord of spinal cord injured rats, which is a promising step towards a better therapeutic intervention for spinal cord injury because of the positive role of cholinergic system in regulation of both locomotor activity and synaptic plasticity.

Abdolmaleki et al [ 144 ] evaluated the anticonvulsant effect of citicoline in the pentylenetetrazole seizure model. In this study it was showed that the acute administration of citicoline has anticonvulsant activity and sedative effect, suggesting a positive effect of citicoline on post-traumatic epileptogenesis. Rasooli et al [ 145 ] indicated that citicoline has anticonvulsant effects probably through the inhibition of nitric oxide.

Qian et al [ 142 ] demonstrate the protection of citicoline against white matter and grey matter damage due to closed head injury through suppressing oxidative stress and calpain overactivation, providing additional support to the application of citicoline for the treatment of traumatic brain injury. Gan et al [ 143 ] in a zebrafish traumatic brain injury model assessed the anti-inflammatory actions of citicoline. In this model the authors demonstrate that citicoline could activate microglia, reduces neuronal apoptosis and promotes neuronal proliferation around the lesioned site.

Jacotte-Simancas et al [ 141 ] examined the effects of citicoline and of voluntary physical exercise in a running wheel (three weeks), alone or in combination, on traumatic brain injury-related short-term (three hours) and long-term (24 hours) object recognition memory deficits, and on neurogenesis and neuroprotection, using a rodent model of traumatic brain injury (controlled cortical impact injury). Citicoline improved memory deficits at the two times tested, while physical exercise only in the long-term test. Some degree of neuroprotection of citicoline was suggested by reduced interhemispheric differences in the volume of the hippocampal formation. But, contrary to what was expected, the effects of citicoline and physical exercise did not sum up.

Baskaya et al [ 138 ] examined the effects of citicoline upon cerebral edema and rupture of the blood-brain barrier in a rat model of traumatic brain injury. Animals received citicoline (50, 100, 400 mg/kg) or saline by the intraperitoneal route twice following induction of the traumatic brain lesion. Induction of the traumatic lesion caused an increase in water content percentage and Evans blue extravasation (a marker of blood-brain barrier rupture) at the damaged cortex and ipsilateral hippocampus. The 50 mg/kg dose of citicoline was not effective, while at 100 mg/kg a reduction was seen in Evans blue extravasation in both regions, although this dose only decreased cerebral edema in the damaged cortex. The 400 mg/kg dose of citicoline significantly reduced cerebral edema and the blood-brain barrier rupture in both regions. Authors concluded that these results suggest citicoline to be an effective neuroprotective agent upon secondary lesions occurring in association to traumatic cerebral injury.

Plataras et al [ 137 ] analyzed the effects of different citicoline concentrations (0.1-1 mM) upon the activities of acetylcholinesterase, Na + /K + -ATPase, and Mg ++ -ATPase in total brain homogenates from rats and extracts of non-membrane bound pure enzymes. Following 1-3 hours preincubation with citicoline, peak stimulations of 20-25% (p < 0.001) and 50-55% (p < 0.001) are seen for acetylcholinesterase and Na + /K + -ATPase respectively, while no significant effects are seen on Mg ++ -ATPase. Authors concluded that citicoline may stimulate cerebral acetylcholinesterase and Na + /K + -ATPase independently from acetylcholine and norepinephrine, which could partly account for the clinical effects of the drug.

Dixon et al [ 136 ] analyzed the effects of exogenous administration of citicoline on motor deficits, spatial memory capacity, and acetylcholine levels in dorsal hippocampus and neocortex in a model of traumatic brain lesion in rats, induced by a controlled lateral impact. Citicoline was administered by the intraperitoneal route at a dose of 100 mg/kg for 18 days from the first day following induction of the traumatic lesion. Another group of animals was treated with saline solution. Motor assessment was performed using a balance test for which animals had previously been trained, and cognitive assessment was made with a variant of the Morris maze test, that is sensitive to cholinergic function. Microdialysis methods were also used to analyze the effects upon acetylcholine release. In the motor function study, citicoline-treated animals showed on day 1 after the lesion a significantly longer balance period as compared to animals receiving saline (39.66 ± 3.2 seconds versus 30.26 ± 2.9 seconds; p < 0.01). In addition, animals treated with citicoline had significantly less cognitive deficits. In microdialysis studies, after a single administration of citicoline by the intraperitoneal route, a rapid increase in acetylcholine production was seen as compared to baseline, that was maintained for up to three hours, in both dorsal hippocampus (p < 0.014) and neocortex (p < 0.036), while no changes were noted in animals receiving saline. Authors concluded that post-traumatic deficits in spatial memory function are due, at least partly, to deficiency changes in cholinergic transmission, that are attenuated with citicoline administration.

Roda [ 132 ], in an experimental model of cryogenic cerebral edema, measured extravasation of Evans blue through the blood-brain barrier and fluorescein uptake by astrocytes and neurons, and found that citicoline administration significantly reduced both processes as compared to control animals, thus allowing to state that citicoline has a direct effect upon transmembrane transport of sodium, potassium, water, and proteins at both blood-brain barrier endothelial cell level and astrocyte and neuron level. Though the exact mechanism of this action is not completely understood, its effect appears to occur at two levels: on the interface separating capillaries from the neuroglia and on cell membranes. Citicoline reduces microvascular permeability during experimental endotoxemia [ 133 ] and in early burn edema in rats [ 134 ]. Farshad et al [ 135 ] propose citicoline as a potential protective agent in a model of hepatic encephalopathy, a known cause of cerebral edema. They found that citicoline supplementation enhanced the animal&#;s locomotor activity and improved brain tissue markers of oxidative stress, concluding that the effects of citicoline on oxidative stress markers could play a fundamental role in its neuroprotective properties.

Majem et al [ 131 ] assessed the electroencephalogram changes occurring in rats when cryogenic edema is induced, and how such electroencephalogram changes were modified by citicoline administration. These authors noted a significant increase in the theta frequency band during the awakening state, with decreased delta and slow alpha bands and a lesser interindividual scatter of the overall frequency bands, which resulted in a greater electrogenic cerebral stability. They concluded that citicoline protected from the effects of cryogenic cerebral edema.

Lafuente and Cervós-Navarro [ 129 , 130 ] conducted a microgravimetric study in experimental cerebral edema induced by ultraviolet radiation in cats to assess the effect of citicoline in this situation. The results suggested an action of citicoline decreasing the amount of edema, enhancing fluid reabsorption and accelerating fluid drainage to ventricles, i.e., increasing cerebral compliance. Authors concluded that CDPamines are helpful to control tissue lesions related to increased free fatty acids and to restore cell energy metabolism by restarting the Na + /K + pump.

Boismare [ 18 , 126 ] conducted an experimental model of craniocervical trauma without direct blow (&#;whiplash&#;) in order to assess the effects occurring upon central catecholamine levels and found increased dopamine levels and decreased norepinephrine levels in the brain following trauma. This type of lesion causes postural dysregulation of brain supply and behavioural and learning disorders, that are related to accelerated degradation of cerebral norepinephrine. In animals treated with citicoline, trauma did not change the levels of these amines. The author stressed the protective role of citicoline, due to this stabilizing effect of catecholamine brain levels.

Tsuchida et al [ 125 ] administered 3 H-citicoline by the intraperitoneal route to rats subjected to cerebral cryogenic lesion by dry ice application on the scalp and confirmed the presence of the labelled drug in brain parenchyma, particularly in the white matter, and above all in damaged areas.

Algate et al [ 122 ] tested the effects of citicoline in an experimental model of epidural compression in anesthetized cats. They noted that animals treated with citicoline had a greater resistance to the effects of mechanic brain compression as compared to animals in the control group. They also found that respiratory and cardiovascular changes were less intense in treated animals and concluded that citicoline provides a significant protection against the lethality of epidural compression. These results agreed to those obtained by Hayaishi [ 123 ] and Kondo [ 124 ] who showed an improvement in the electroencephalogram tracing following administration of citicoline to cats undergoing experimental brain compression, and also in survival quality.

Alberghina and Giuffrida [ 17 ], in a study on nerve tissue response to a contusion lesion, showed that a moderate increase occurred in the activity of cholinephosphotransferase and was associated to a greater increase in the activity of phospholipase A 2 and several lysosomal hydrolases. They also found an increased number and size of lysosomes during neuronal regeneration. Arrigoni et al [ 117 ] have shown citicoline to be able to completely inhibit activation of phospholipase A 2 without altering cholinephosphotransferase activity. On the other hand, Freysz et al [ 118 ] showed that, in addition to decreasing phospholipase A 1 and A 2 activity, citicoline decreases free fatty acid release under hypoxic conditions, thus adding a protecting effect to its activating capacity of phospholipid reconstruction. Massarelli et al [ 119 ] also showed citicoline action upon phospholipase A 1 and agreed with all other authors in their conclusions. Kitazaki et al [ 120 ] also showed the inhibitory effect of citicoline upon membrane-associated phospholipase A 2 in rat brain cortex. Based on these characteristics, citicoline has been considered a non-specific inhibitor of phospholipase A 2 at intracellular level [ 121 ].

Martí Viaño et al [ 114 ] compared the effects of pyriglutine, piracetam, centrophenoxine, and citicoline in a study on antagonism of barbiturate coma in mice. No differences were seen in animals treated with pyriglutine, piracetam, or centrophenoxine as compared to the control group, while with citicoline both coma duration and depth, as well as respiratory depression, were decreased as compared to all other groups. Arousal effects of citicoline were found to be due to increased cerebral blood flow, improved O 2 cerebral uptake and utilization of energy metabolism, and enhanced mitochondrial breathing.

Yasuhara et al [ 112 , 113 ], in an electrophysiological study in rabbits, showed that citicoline decreased in the threshold for the arousal reaction and the threshold for muscle discharge, and concluded that this is a valuable drug for treatment of brain lesions because of its effects on consciousness and on the motor activity of the pyramidal system and its afferent pathways.

Mykita et al [ 111 ] found in neuronal cultures that addition of citicoline after a hypocapnic lesion resulted in culture protection. Hypocapnia increases incorporation of labelled choline into phospholipids, while this process is slowed in the presence of citicoline. These authors concluded that citicoline is able to protect neurons under alkalosis conditions and may promote cell proliferation.

In an experimental rat model of acute induced ischemia, LePoncin-Lafitte et al [ 110 ] assessed integrity of the blood-brain barrier with labelled iodinated albumin, and brain metabolism using histoenzymological studies. In this experimental model, administration of citicoline was able to reduce vasogenic cerebral edema and to restore blood-brain barrier integrity. Authors also found that the size of induced infarctions was smaller with citicoline, and this compound decreased the activity of lactate dehydrogenase, succinyl dehydrogenase, monoamine oxidase, and acid phosphatase, emphasizing its protective role through a direct action at cell membrane level.

Horrocks and Dorman [ 109 ] have shown that citicoline and CDP-ethanolamine prevent degradation of choline and ethanolamine phospholipids during decapitation ischemia in rats and induce a partial reversion of free fatty acid release during reperfusion after experimental global ischemia in gerbils. Citicoline and CDP-ethanolamine, when administered together, have a synergistic effect and stimulate resynthesis of choline, ethanolamine, and inositol phospholipids, markedly decreasing free arachidonic acid levels.

Javaid et al [ 108 ] described the pathophysiological changes in brain phospholipids induced by traumatic brain injury, specially of choline-containing phospholipids such as phosphatidylcholine, and they highlight the role of choline-specific therapeutic strategies, such as the administration of citicoline, for the amelioration of traumatic brain injury.

Citicoline was administered to albino rabbits at a dose of 800 mg/kg during the organogenesis phase, i.e., from days 7 th to 18 th of pregnancy. Animals were killed on day 29 th , and a detailed examination was made of fetuses and their mothers. No signs of maternal or embryofetal toxicity were seen. Effects on organogenesis were imperceptible, and only a slight delay in cranial osteogenesis was seen in 10% of treated fetuses (unpublished data).

Chronic oral (1.5 g/kg/day for 6 months in dogs) and intraperitoneal (1 g/kg/day for 12 weeks in rats) toxicity studies did not reveal either significant abnormalities related to drug administration [ 432 , 438 ]. Intravenous administration of citicoline 300-500 mg/kg/day for three months in dogs only caused toxic signs immediately after injection, including vomiting and occasional diarrhea and sialorrhea [ 435 ]. In a 90-day study in rats, 100, 350, and 1,000 mg/kg/day oral doses resulted in no mortality. In males, slight significant increases in serum creatinine (350 and 1,000 mg/kg/day) and decreases in urine volume (all treated groups) were observed. In females, slight significant increases in total white blood cell and absolute lymphocyte counts (1,000 mg/kg/day), and blood urea nitrogen (100 and 350 mg/kg/day) were noted. A dose-related increase in renal tubular mineralization, without degenerative or inflammatory reaction, was found in females (all treated groups) and two males (1,000 mg/kg/day). Renal mineralization in rats (especially females) is influenced by calcium:phosphorus ratios in the diet. A high level of citicoline consumption resulted in increased phosphorus intake in the rats, and likely explains this result [ 436 ].

Intraperitoneal administration to rats of doses up to 2 g/kg/day of citicoline for 4.5 weeks did not results in clinical toxicity signs or significant changes in the hematological, biochemical, or histological parameters analyzed. A slight decrease in intake and weight gain was only seen from two weeks of the study [ 433 ]. Similar results were seen following subcutaneous administration to male rats of up 1 g/kg for four weeks [ 432 ]. Oral administration of 1.5 g/kg/day to rats for 30 days did not cause weight, hematological, biochemical, or histological changes [ 437 ].

Acute toxicity from single citicoline administration has been studied in various animal species and using different administration routes. The intravenous LD 50 in mice, rats, and rabbits is 4.6, 4.15, and 1.95 g/kg, respectively [ 431 , 432 ]. Oral LD 50 is 27.14 g/kg in mice and 18.5 g/kg in rats [ 433 ]. The intravenous LD 50 of citicoline is approximately 44 times higher than the LD 50 of choline hydrochloride at equivalent doses, and it has been shown that choline doses inducing cholinergic crises do not cause any toxicity sign when equivalent doses of citicoline are administered [ 434 , 435 ]. This suggests that administration of choline has metabolic implications clearly different from those of exogenous choline administration. The administration of 2,000 mg/kg of citicoline p.o. during 14 days was well tolerated [ 436 ].

Two phases are differentiated in urinary elimination of the drug: a first phase, lasting approximately 36 hours, in which excretion rate decreases rapidly, and a second phase in which excretion rate decreases much more slowly. The same occurs with expired CO 2 , whose elimination rate decreases rapidly for the first 15 hours, approximately, after which a slower decrease is seen.

In conclusion, these studies show that the citicoline administered is widely distributed in brain structures, with a rapid incorporation of the choline fraction into structural phospholipids, and of the cytosine fraction into cytidine nucleotides and nucleic acids. Citicoline reaches the brain and incorporates actively into the cytoplasmic and mitochondrial cell membranes, being part of the structural phospholipid fraction [ 441 , 450 , 451 ].

In another test battery, the presence of the drug in various brain areas and its distribution in cerebral ultrastructures was measured following administration of (methyl 14 C) citicoline [ 445 - 449 ]. In a study performed with high-performance autoradiography in mouse brain 24 hours following administration of labelled citicoline [ 445 ], the radioactive marker was seen to be widely incorporated into the different cerebral areas studied, brain cortex, white matter, and central grey nuclei. It was found in both intra and extracellular spaces, with a particular presence in cell membranes. In the same experimental model, but 10 days following administration of the labelled drug [ 446 ], concentration of radioactivity in the more myelinated areas was seen, as well as a marked uptake by the cerebellar Purkinje cells. Using low-performance autoradiography, distribution of radioactivity of labelled citicoline in rat brain was analyzed five and 24 hours after drug administration [ 447 ]. At 24 hours, most radioactivity was detected at intracellular level. In another study, incorporation of radioactivity from (methyl 14 C) citicoline after oral administration to male Sprague-Dawley rats was analyzed in the different cerebral phospholipid fractions [ 448 ]. Of total radioactivity measured in brain, 62.8% was found to be part of brain phospholipids, particularly phosphatidylcholine and sphingomyelin, showing that citicoline administered by the oral route influences the synthesis of structural phospholipids of cell membranes. These results agree with those obtained by Aguilar et al [ 449 ], who showed radioactivity from labelled citicoline to be associated to cytoplasmic and mitochondrial membranes in brain homogenate.

In a group of animals, radioactivity levels of the labelled compounds were measured in the brain at 0.5, 1, 4, and 48 hours of administration of dually labelled citicoline. Radioactivity corresponding to 3 H in the brain was mainly concentrated in cytidine nucleotides at the beginning, but subsequently concentrated in nucleic acids. As regards compounds labelled with 14 C, the highest levels initially corresponded to betaine, choline, and phosphorylcholine, while at four hours 14 C-methionine and 14 C -phospholipids accounted for 26.4 and 24.2% respectively of total cerebral radioactivity corresponding to 14 C. At 48 hours, this radioactivity mainly concentrated in phospholipids and proteins. Thus, labelled phospholipids were seen to continuously increase in the 48 hours following administration of dually labelled citicoline. As shown in , such increase is fast in the first five hours, but then becomes slower.

Tissue diffusion of citicoline and its components has been studied in rats intravenously administered (methyl 14 C, 5- 3 H) citicoline, labelled in the choline and the cytidine fraction [ 443 , 444 ]. In the same battery test, plasma radioactivity levels were measured for 30 minutes following administration. Renal and fecal excretion of labelled metabolites was also measured for 48 hours. As early as two minutes following injection, less than 10% of administered radioactivity was found in plasma. In addition, radioactivity excreted by the kidney during the first 48 hours only accounted for 2.5% of 14 C and 6.5 % of 3 H administered. In the same time interval, fecal excretion did not exceed 2% of the administered dose. These results suggest that citicoline rapidly diffuses to tissue following administration and is actively used by tissues. shows the radioactivity levels found in liver, brain, and kidney at different time points following intravenous administration of dually labelled citicoline. There is a special interest in changes in brain levels. Radioactivity uptake by the brain gradually increases for the first 10 hours after drug administration, and the levels achieved remain unchanged at 48 hours.

López-Coviella et al [ 440 ] studied the effects of citicoline on plasma levels of cytidine, choline, and CDP-choline in healthy volunteers receiving the substance by the oral or intravenous route and in rats treated by the intravenous route. Two hours following administration of a single oral dose of citicoline 2 g, choline plasma levels increased 48%, and cytidine plasma levels 136% ( ). In individuals receiving three 2 g doses at two-hour intervals, choline plasma levels reached a peak, representing approximately 30% of baseline value, four hours after administration of the initial citicoline dose, while cytidine plasma levels increased up to six hours ( ) and were five-fold higher than the baseline value (p < 0.001). Citicoline administered by the intravenous route was rapidly hydrolyzed in both humans and rats [ 441 ]. In healthy individuals receiving a citicoline infusion of 3 g in 500 mL of physiological saline over 30 minutes, CDP-choline levels were virtually undetectable just after the end of the infusion period, when plasma levels of cytidine and choline reached a peak, though their concentrations remained significantly increased up to six hours after the start of infusion ( ). These observations show that citicoline, administered by both the oral and intravenous routes, is converted into two major circulating metabolites, cytidine and choline. However, plasma cytidine is converted in humans to uridine, its circulating form, that is transformed in the brain to uridine phosphate, that will in turn be converted to cytidine triphosphate at neuronal level [ 442 ].

Labelled citicoline (methyl 14 C) was administered to rats at a dose of 4 mg/kg by jugular vein injection and by the oral route using a nasogastric tube [ 439 ]. The results obtained, expressed as percent radioactivity in 10 mL of blood for each administration route, are shown in . From these data, the ratio between bioavailability of the oral and the intravenous administration route was estimated and found to be virtually one, which agrees with the fact, demonstrated in the same study, that no residual radioactivity is found in feces excreted in the 72 hours following oral administration.

Clinical experience

Acute cerebrovascular disease and sequelae

The neurobiological processes involved in the pathophysiology of the cerebral ischemia are extremely complex [498]. For this reason, some authors postulate the need to use multifunctional treatments for this disease [499-504], for intracerebral hemorrhages [505,506], and for the recovery phase [507,508]. As experimentally shown, citicoline is a drug having pleiotropic actions including activation of neuronal metabolism, stabilization of neuronal membranes and their function, and normalization of neurotransmission [20,39-41,180,281, 282]. Various studies with citicoline conducted in the sixties suggested its efficacy to reduce neurological symptoms in patients with cerebral ischemia [509,510].

Hazama et al [511] conducted a double-blind study to assess the effect of citicoline on functional recovery from hemiplegia in 165 patients with cerebrovascular disease. These authors showed that citicoline, at a dose of 1,000 mg/day for eight weeks, was superior to placebo, particularly for motor recovery in the upper limbs, and concluded that citicoline promotes natural recovery from hemiplegia.

Goas et al [512] conducted a double-blind study comparing citicoline (750 mg/day/10 days intravenous) versus placebo in 64 patients with cerebral infarction starting less than 48 hours before. Assessment at three months showed citicoline to be superior to placebo for improving motor deficit (p < 0.05), hypertonia (p < 0.03), gait recovery (p < 0.02), changes over time in electroencephalographic tracing (p < 0.01) and psychometric tests (p < 0.05), achieving a higher number of independent states (51.6% with citicoline; 24.24% with placebo) ( ). In a study with the same characteristics, Boudouresques et al [513] achieved similar results. This study included 52 patients, of whom 27 patients received citicoline (750 mg/day/10 days intravenous) and 25, placebo. An assessment was made at 10 days and showed that citicoline-treated patients had a better course as regarded consciousness disorders, with recovery of consciousness in 66.7% of cases as compared to 32.0% in the placebo group (p < 0.01), and deficit syndromes (82.6 and 54.5% of patients recovered with citicoline and placebo respectively; p < 0.04) and electroencephalographic tracings (83.3% with citicoline versus 35.3% with placebo; p < 0.01). In both studies, citicoline tolerability was rated as excellent by investigators.

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Corso et al [514], in a double-blind study of citicoline (1 g/day/30 days intravenous) versus placebo in a sample of 33 patients, noted that at the end of the study the deficit syndrome had improved in 76.5% of patients treated with citicoline (p < 0.01 versus placebo), while an improved electroencephalographic tracing was seen in 70.6% of patients (p < 0.01 versus placebo).

Tazaki et al [515] performed a double-blind, prospective, multicentre, placebo-controlled study on the value of citicoline for the treatment of acute cerebral infarction. Sixty-three Japanese academic centers participated in this study, in which a total of 272 patients were enrolled following strict inclusion criteria. Patients were randomized to receive 1 g/day intravenous of citicoline or saline (placebo) for 14 days. At the end of treatment, citicoline was shown to significantly improve consciousness (51% versus 33% for placebo; p < 0.05) and overall improvement (52% versus 26%; p < 0.01) and overall usefulness rates (47% versus 24%; p < 0.001). In addition, fewer complications occurred in the citicoline-treated patient group (1%) as compared to the placebo group (8.1%). These authors concluded that citicoline is an effective and safe drug for the treatment of acute cerebral infarction. These results agree to those reported by other authors [516-519].

Guillén et al [520] reported a comparative, randomized study on the efficacy of citicoline for treating acute ischemic stroke as compared to conventional therapy, showing a significantly higher improvement in the citicoline group as compared to the control group. In the open label studies by Bruhwyler et al [521] and Fridman et al [522], results favoring citicoline were also achieved, with a significant clinical improvement of patients and an excellent safety profile of the drug. Alviarez and González [523] reported the beneficial effects with citicoline in a double-blind study conducted in Venezuela. León-Jiménez et al [524] evaluated the correlation between citicoline exposure and functional outcome at discharge and at 30 and 90 days post-stroke, in a retrospective case-control design on systematic descriptive databases from three referral hospitals in Mexico.

In the second half of the 90s, study of oral citicoline for the treatment of acute ischemic stroke was started in the United States. The first clinical trial was a randomized, dose-response study [525]. This double-blind, randomized, multicentre study compared three citicoline doses (500 mg, 1,000 mg, and 2,000 mg by the oral route) to placebo to document drug safety, find the optimum dose, and collect data on the efficacy of citicoline for the treatment of acute ischemic stroke. A total of 259 patients with ischemic stroke in the territory of the middle cerebral artery were recruited within 24 hours of the start of symptoms. Patients were randomized into four groups: administration of placebo or 500, 1,000, or 2,000 mg/day of oral citicoline for six weeks. Patient recovery at the end of the six-week treatment period and after a subsequent follow-up period of six additional weeks was assessed. The main efficacy endpoint was Barthel Index (BI) at 12 weeks. Secondary endpoints included the modified Rankin Scale (mRS), the National Institutes of Health Stroke Scale (NIHSS), the Mini-Mental State Examination (MMSE), hospital stay duration, and mortality. A significant difference favoring citicoline was found between the groups in functional status (BI, mRS), neurological assessment (NIHSS), and cognitive function (MMSE). In a regression analysis of BI including as covariate baseline NIHSS score, a significant effect of citicoline treatment was found at 12 weeks (p < 0.05). The proportions of patients who achieved a BI score ranging from 85 and 100 were 39.1% for placebo, 61.3% for the 500 mg dose, 39.4% for the 1,000 mg dose, and 52.3% for the 2,000 mg dose. Odds ratios for an improved outcome were 2.0 for the 500 mg dose and 2.1 for the 2,000 mg dose. The lack of efficacy seen in the 1,000 mg group could be due to the greater overweight of patients included in this group and their poorer neurological status at baseline. Mean score in the mRS was 3.1 with placebo, 2.5 with citicoline 500 mg, 3.1 with 1,000 mg, and 2.6 with 2,000 mg, with a significant difference being found between the 500 mg and placebo groups (p < 0.03). No citicoline-related serious adverse events or deaths were seen. According to these results, oral citicoline treatment achieves a better functional outcome, and 500 mg is the most effective dose of citicoline.

A second multicentre, double-blind, placebo-controlled, randomized study [526] recruited 394 patients with acute ischemic stroke arising in the middle cerebral artery less than 24 hours before and with a NIHSS score of 5 or higher. Patients were assigned oral administration of placebo (n = 127) or citicoline 500 mg/day (n = 267). Treatment was continued for six weeks, and follow-up was subsequently conducted for six additional weeks. Mean entry time was 12 hours after the stroke, and mean patient age was 71 years in the placebo group and 71 years in the citicoline group. While the mean baseline NIHSS score was similar in both groups, a greater proportion of patients had a baseline NIHSS <8 (34% versus 22%; p < 0.01). The planned primary endpoint (logistic regression for five BI categories) did not meet the proportional odd assumption and was therefore not reliable. No significant between-group differences were seen in any of the planned secondary variables, including a BI of 95 or higher at 12 weeks (placebo 40%, citicoline 40%) or mortality rate (placebo 18%, citicoline 17%). However, a post hoc subgroup analysis showed that in patients with moderate to severe stroke, defined by a baseline NIHSS score of 8 or higher, treatment with citicoline conferred a greater chance of achieving a complete recovery, defined as a BI &#;95 at 12 weeks (21% placebo, 33% citicoline; p = 0.05), while no differences were found in patients with mild stroke, i.e. with a baseline NIHSS score <8. No serious adverse events attributable to the drug were detected, which attests to its safety. Based on these data, citicoline may be considered a safe drug that may induce favorable effects in patients with moderate to severe acute ischemic stroke.

The last clinical study conducted in the US was the ECCO study [527]. This study, having similar characteristics to the previous ones, enrolled 899 patients with moderate to severe acute ischemic stroke (baseline NIHSS score &#;8) arising in the middle cerebral artery within the past 24 hours. Patients were randomized to receive citicoline 2,000 mg/day (n = 453) or placebo (n = 446) by the oral route for six weeks, with a subsequent follow-up for six additional weeks. The primary study endpoint was the proportion of patients having a reduction by 7 or more points in the NIHSS scale at 12 weeks. At the end of the study, 51% of patients in the placebo group and 52% of those in the citicoline group had achieved the reduction by 7 or more points in the NIHSS scale, with no significant between-group differences. By contrast, there was a trend favoring citicoline in achievement of a complete neurological recovery, defined by a score in the NIHSS scale of 1 or less (40% with citicoline versus 35% with placebo; p = 0.056), and in complete functional recovery, defined by a BI score of 95 or higher (40% with citicoline versus 35% with placebo; p = 0.108). With regard to mRS, 20% of patients in the placebo group achieved a complete recovery (mRS &#;1), as compared to 26% of patients in the citicoline group, the difference being statistically significant (p = 0.025). There were no differences between treatments in mortality or incidence of serious adverse events, but a significant decrease was seen in stroke worsening (3% with citicoline versus 6% with placebo; p = 0.02). On the other hand, occurrence of new stroke was decreased in patients treated with citicoline (2.9% with placebo versus 1.8% with citicoline), i.e., a 62.1% risk reduction. A post hoc analysis assessed the effect of citicoline in a multiple outcome global assessment, using the method of Generalized Estimating Equations defined by Tilley et al [528], considering the proportion of patients who had a complete recovery in all 3 scales used, i.e., achieved scores of 0-1 in the NIHSS scale, 0-1 in mRS, and &#;95 in BI at 12 weeks. Citicoline was shown to be significantly superior to placebo, achieving this complete recovery in 19% of the cases, as compared to 14% in the placebo group (OR 1.32; 1.03-1.69; p = 0.03).

Citicoline effects on reduction of cerebral infarction volume were investigated in parallel. The first analysis conducted was a pilot study to assess citicoline effects on lesion volume measured by diffusion-weighted magnetic resonance imaging in patients with acute cerebral infarction [529]. This study recruited 12 patients from the first clinical study on citicoline in the United States [525]. Lesion growth was seen in three of the four patients treated with placebo, while a decrease in lesion volume was noted in seven of the eight patients treated with citicoline (p < 0.01, with baseline NIHSS score as covariate). A second, double-blind study designed for this purpose, i.e., to measure changes in lesion volume using diffusion-weighted techniques, recruited 100 patients who were randomized to receive citicoline 500 mg/day or placebo by the oral route for six weeks [530]. These patients should be enrolled within 24 hours of symptom onset and have a baseline NIHSS score of 5 points or more and a lesion volume in cerebral grey matter of 1-120 cm3 in diffusion-weighted magnetic resonance imaging. Neuroimaging techniques (diffusion-weighted magnetic resonance imaging, T2-weighted magnetic resonance imaging, perfusion-weighted magnetic resonance imaging, and magnetic resonance imaging angiography) were performed at baseline and on weeks 1 and 12. Main endpoint was progression of ischemic lesion from baseline to final assessment at 12 weeks as measured by magnetic resonance imaging. The primary analysis planned could be performed in 41 patients treated with citicoline and 40 patients treated with placebo, and no significant differences were found. From baseline to 12 weeks, ischemic lesion volume expanded by 180 ± 107% in the placebo group and 34 ± 19% in the citicoline group. A secondary analysis showed that, from week 1 to week 12, lesion volume decreased by 6.9 ± 2.8 cm3 in the placebo group and by 17.2 ± 2.6 cm3 with citicoline (p < 0.01). A significant finding in this study was the great correlation existing, regardless of treatment, between lesion volume reduction and clinical improvement, supporting the idea of using this methodology for assessing stroke treatments. In the ECCO study [527], a substudy was conducted to assess the effects of citicoline on lesion volume [531]. This substudy had three objectives. The first objective was to assess the effects of the drug on chronic lesion volume, as measured using magnetic resonance imaging T2 sequences in the whole patient sample, although this assessment could only be made in 676 patients. The second objective was to analyze citicoline effects on change in lesion volume, using diffusion-weighted magnetic resonance imaging performed at baseline and week 12. One hundred and eighty-one patients were recruited for this second objective, of whom only 134 patients were evaluable. The third objective was methodological in nature, that is, an attempt was made to correlate clinical changes to volume changes and to check if lesion volume reduction was associated to clinical improvement. No significant differences were found in assessment of chronic lesion volume (median of 25 cm3 for citicoline; median of 31.3 cm3 for placebo). The diffusion-weighted study showed that in the placebo group (n = 71) lesion the increased 30.1 ± 20.5%, with a median of &#;8.7%, while the change occurring in the citicoline group (n = 63) was 1.3 ± 14.3%, with a median of &#;22.9%, a non-significant difference (p = 0.077). However, when the logarithm of change was analyzed and the baseline NIHSS score was introduced as covariate, the difference was significant (p = 0.02). In this diffusion-weighted substudy, 54% of patients in the placebo group and 675 of citicoline-treated patients were shown to have a decreased lesion volume compared to baseline, though the difference was not significant (p = 0.122). In patients having at baseline a cortical lesion with a volume ranging from 1-120 cm3 were analyzed, a lesion increase by 40.5 ± 28.7% was seen in patients treated with placebo (n = 47), with a median of 4.5%, while in patients receiving treatment with citicoline (n = 43) the lesion increased by 7.3 ± 19.9%, with a median of &#;23.9%. The difference between the groups was statistically significant (p = 0.006, median comparison). In this patient subgroup with initial cortical lesions with a volume of 1-120 cm3, a decrease in lesion volume occurred in 47% of patients in the placebo group and in 70% of patients in the citicoline group. The difference was significant, with a value of p = 0.028. The decrease in volume was also seen to be significantly correlated to the clinical improvement of patients.

Although the results obtained in studies conducted in the United States with oral citicoline for treatment of acute ischemic stroke were not conclusive for citicoline efficacy, it may be seen that, in addition to drug safety, there is a certain trend to an improved prognosis of treated patients. Since there was currently no neuroprotective drug that has been shown to be effective for the treatment of this severe condition [532], it was decided to conduct a meta-analysis of the results obtained with oral citicoline in the treatment of acute ischemic stroke to examine the effects of the drug on neurological and functional recovery of patients [533]. For this, following the methods of the Cochrane Library [534] and the guidelines of the International Conference on Harmonization [535], a comprehensive literature search was made in both Medline and our own literature database. This search found that only four double-blind, randomized clinical studies had been conducted with oral citicoline for the treatment of acute ischemic stroke, namely the four trials performed in the United States [525-527,529]. The total sample comprised 1,652 patients, 686 patients in the placebo group and 966 patients in the citicoline group (381 with 500 mg/day, 66 with 1,000 mg/day, and 519 with 2,000 mg/day). The first analysis was performed irrespective of the dose and in the total patient sample. As regards complete neurological recovery (NIHSS &#;1) at three months, the odds ratio was 1.22 (0.98; 1.52), not reaching statistical significance (p = 0.07); by contrast, significant differences favoring citicoline were obtained in an analysis of patients who achieved a virtually complete recovery in activities of daily living (BI &#;95) at three months &#;OR, 1.26 (95% CI, 1.02-1.55); p = 0.01&#; and functional recovery at three months, defined as a score of 1 or less in the mRS &#;OR 1.36 (95% CI, 1.06; 1.74), p = 0.01&#;. Since the experience gathered in the above clinical studies suggests that the drug is more effective in patients with moderate to severe acute ischemic stroke (baseline NIHSS &#;8), databases from the original studies were obtained, and patients who met this criterion and had an optimum functional status before the stroke (mRS &#;1) were selected. Of the whole sample, 1,372 patients met these criteria and therefore underwent the same assessment. In this case, the meta-analysis found statistically significant differences for all variables analyzed ( ).

Table VIII

Studies (n)Patients (n)Peto odds ratio (95% CI) p NIHSS &#; 141,.34 (1.05-1.71)0.020 mRS &#; 141,.45 (1.11-1.90)0.007 BI &#; ,.28 (1.03-1.59)0.003Open in a separate window

To continue with analysis of these data, it was decided to perform a pooling data analysis [536], using individual data from each patient. This additional analysis included the sample of 1,372 patients who met the established criteria of severity (baseline NIHSS &#;8), prior functional status (mRS &#;1), therapeutic window not longer than 24 hours, and consistent neuroimage. The efficacy endpoint selected was total recovery at 3 months in the three scales analyzed (mRS &#;1 + NIHSS &#;1 + BI &#;95), using the previously described Generalized Estimating Equations analysis [527]. Among the 1,372 patients, 583 received placebo and 789 citicoline (264 patients 500 mg, 40 patients 1,000 mg, and 485 patients 2,000 mg). Total recovery at three months was achieved in 25.2% of patients treated with citicoline and 20.2% of patients in the placebo group &#;OR, 1.33 (95% CI, 1.10-1.62); p = 0.003&#;, and the dose shown to be most effective was 2,000 mg. This dose resulted in complete recovery at three months in 27.9% of patients who received it &#;OR, 1.38 (95% CI, 1.10-1.72); p = 0.004&#; ( ). In addition, citicoline safety was similar to placebo.

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The preliminary results of a Cochrane review on the effects of choline precursors, including citicoline, in the treatment of acute and subacute stroke were reported in [537]. This meta-analysis collected data from eight double-blind studies conducted with citicoline at doses ranging from 500 and 2,000 mg daily, administered by both the oral and intravenous routes. Despite study heterogeneity, citicoline treatment was associated to decreases in late mortality and disability rates: citicoline 611/1,119 (64.6%) versus placebo 561/844 (54.4%) &#;OR, 0.64 (95% CI, 0.53-0.77); p < 0.&#;. In order to decrease heterogeneity, analysis was restricted to the four studies with a greater sample size (n > 100), and the positive effect seen persisted: citicoline 574/1,048 (54.58%) versus placebo 500/773 (64.7%) &#;OR, 0.70 (95% CI, 0.58-0.85); p < 0.&#;. In the safety analysis, no differences were found between citicoline and placebo in the mortality rate. Authors concluded that the formal meta-analysis of citicoline studies in acute and subacute stroke suggests a beneficial and substantial effect of the drug, with absolute reductions by 10%-12% in the long-term disability and mortality rate, i.e., the number of patients with a score of 3 or higher in the mRS is significantly decreased. These results agree with those previously reported for the pooled data analysis [536].

A pooled data analysis evaluating the effect of citicoline on increase of cerebral infarction size is also available [538]. Data used in this analysis come from two studies in which neuroimaging data had been obtained using magnetic resonance imaging techniques [527,530]. The primary endpoint in this analysis was percent change in infarction size from the start to the end of the study at three months. Data were available for 111 patients receiving placebo, 41 patients treated with citicoline 500 mg/day/6 weeks, and 62 patients treated with citicoline 2,000 mg/day/6 weeks. Patients receiving placebo experienced a mean increase by 84.7 ± 41.2%, while a dose-dependent effect was seen associated to citicoline: mean increase by 34.0 ± 18.5% with citicoline 500 mg and by 1.8 ± 14.5% with citicoline 2,000 mg.

These benefits shown in these systematic reviews were also associated to a reduction in the costs of integral treatment of patients with acute ischemic stroke [539]. Same results on cost-efficacy of citicoline have been obtained in Russia [540, 541].

Sobrino et al [542] investigate if an administration of citicoline, started in the acute phase of stroke, could increase the endothelial progenitor cell concentration in patients with ischemic stroke. Forty eight patients with a first-ever non-lacunar ischemic stroke were prospectively included in the study within 12 hours of symptoms onset. Patients received treatment (n = 26) or non-treatment (n = 22) with oral citicoline (2,000 mg/day/six weeks. Endothelial progenitor cell colonies were quantified as early outgrowth colony forming unit-endothelial cell (CFU-EC) at admission (previous to citicoline treatment) and day seven. The endothelial progenitor cell increment during the first week was defined as the difference in the number of CFU-EC between day seven and admission. CFU-EC were similar at baseline between patients treated and non-treated with citicoline (7.7 ± 6.1 versus 9.1 ± 7.3 CFU-EC; p = 0.819). However, patients treated with citicoline and recombinant tissue-plasminogen activator (rt-PA) had a higher endothelial progenitor cell increment compared to patients treated only with citicoline or non-treated (35.4 ± 15.9 versus 8.4 ± 8.1 versus 0.9 ± 10.2 CFUEC; p < 0.). In a logistic model, citicoline treatment (OR, 17.6; 95% CI, 2.3-137.5; p = 0.006) and co-treatment with citicoline and rt-PA (OR, 108.5; 95% CI, 2.9-.2; p = 0.001) were independently associated with an endothelial progenitor cell increment &#;4 CFU-EC. The authors concluded that the administration of citicoline and the co-administration of citicoline and rt-PA increase endothelial progenitor cell concentration in acute ischemic stroke. However, the molecular mechanism by which citicoline increases the concentration of endothelial progenitor cells remains to be clarified.

Regarding safety, a drug surveillance study involving 4,191 acute stroke patients treated with citicoline has been finished in South Korea [543]. The aim of this study was to determine the efficacy and safety of oral citicoline in Korean patients with acute ischemic stroke. Oral citicoline (500-4,000 mg/day) was administered within less than 24 hours after acute ischemic stroke in 3,736 patients (early group) and later than 24 hours after acute ischemic stroke in 455 patients (late group) for at least six weeks. For efficacy assessment, primary outcomes were patients&#; scores obtained with a short form of the National Institutes of Health Stroke Scale (s-NIHSS), a short form of the Barthel Index of activities of daily living (s-BI) and a modified Rankin Scale (mRS) at enrolment, after six weeks and at the end of therapy for those patients with extended treatment. All adverse reactions were monitored during the study period for safety assessment. All measured outcomes, including s-NIHSS, s-BI and mRS, were improved after six weeks of therapy (p < 0.05). Further improvement was observed in 125 patients who continued citicoline therapy for more than 12 weeks when compared with those who ended therapy at week six. Improvements were more significant in the higher dose group (&#;2,000 mg/day) (p < 0.001). s-BI scores showed no differences between the early and late groups at the end of therapy. Citicoline safety was excellent; 37 side effects were observed in 31 patients (0.73%). The most frequent findings were nervous system-related symptoms (8 of 37, 21.62%), followed by gastrointestinal symptoms (5 of 37, 13.5%). Oral citicoline improved neurological, functional and global outcomes in patients with acute ischemic stroke without significant safety concerns.

A pilot study has been published on the safety and efficacy of citicoline for the treatment of primary intracerebral hemorrhage [544]. This study recruited 38 patients aged 40 to 85 years, who should be previously independent and be enrolled within six hours of the onset of symptoms caused by primary intracerebral hemorrhage, as diagnosed by neuroimaging tests (computed tomography or magnetic resonance imaging). Patients should have a baseline severity as determined by a score higher than 8 in the Glasgow Coma Scale and higher than 7 in the NIHSS. Patients were randomized to 1 g/12 hours of citicoline or placebo by the intravenous or oral route for two weeks. The primary study objective was to assess treatment safety based on the occurrence of adverse events. The efficacy endpoint selected was the proportion of patients who had a score of 0-2 in the mRS at three months. Nineteen patients were included in each of the groups, that were perfectly matched as regarded baseline characteristics. Adverse event rate did not differ between the groups (four cases each). About efficacy, a patient from the placebo group was rated as independent (mRS <3), as compared to five patients from the citicoline group (OR, 5.38; 95% CI, 0.55-52, n.s.). As a conclusion, it may be stated that citicoline appears to be a safe drug in patients with primary intracerebral hemorrhage, which may allow citicoline to be given to patients with clinical signs suggesting stroke before neuroimaging tests are performed, at an earlier time than usual. As regards efficacy, highly promising data have been obtained, but should be confirmed in a larger study. Also, recently Eribal and Chua [545] communicated the results of the RICH trial performed in the Philippines. This study was conceived to investigate the role of neuroprotectants, particularly citicoline, in intracerebral supratentorial hemorrhage which to date, still has paucity of data on proven effective therapy. This was a randomized double-blind, placebo-controlled, multicentre, parallel group study on patients with first ever supratentorial intracerebral hemorrhage given either 4 g citicoline or placebo for 14 days from index stroke. A total of 182 patients were enrolled into the study. The mean age of both groups was similar 56.90 ± 11.45 citicoline and 57.61 ± 11.83 for placebo. Comorbidities were similar except for the significantly higher number of diabetes patients in citicoline group. Results showed there were more patients with favorable BI scores (2.2 versus 0, 9.2 versus 8.5, and 50.8 versus 31.9) in the citicoline group than in the placebo group respectively. However, the difference was only clinically significant after day 90. Patients had favorable mRS score (7.9 versus 13.4, 18.2 versus 20.3, and 46.1 versus 33.8) in the citicoline that in the placebo group only on the day 90th. This was however not statistically significant. The NIHSS did not differ in both groups with scores of 76.3 versus 75.6, 93.9 versus 91.9, and 96.8 versus 94.3 respectively. Mortality was slightly higher in the citicoline group (11 patients) than in the placebo group (10 patients) but this was not statistically significant. The incidence of adverse in both groups was not different statistically. For the authors, citicoline is effective in improving the BI, and mRS scores on the attainment of functional independence beginning on the 90th day post stroke compared to placebo. Iranmanesh and Vakilian demonstrated the efficiency of citicoline in increasing muscular strength of patients with nontraumatic cerebral hemorrhage in a double-blind randomized clinical trial [546]. Thus, citicoline could play a role in the pharmacological treatment of patients with intracerebral hemorrhages [547, 548], and also in subarachnoid hemorrhage [549]. Zhu [550] investigates the clinical efficacy of citicoline and oxiracetam in patients with cerebral hemorrhage and concludes that this therapeutic combination can affectively promote the absorption of the hematoma, improve the outcome and the quality of life of this kind of patients.

In a study-based meta-analysis, including all the double-blind studies performed with citicoline in acute stroke patients, Saver [551,552] suggests again the beneficial effect of citicoline on the long-term death and disability in this kind of patients ( ).

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Several publications from different countries about the use of citicoline in the treatment of acute stroke have been published in the last years [553-563], and, in some cases, assessing the major efficacy when associated with other neuroprotective drugs [564].

From to , a large trial was conducted in Europe with the objective to corroborate the data obtained with citicoline, but under the current circumstances. This was the ICTUS trial [565-568]. It was a randomised, placebo-controlled, sequential trial in patients with moderate-to-severe acute ischemic stroke admitted at university hospitals in Germany, Portugal, and Spain. Using a centralised minimisation process, patients were randomly assigned in a 1:1 ratio to receive citicoline or placebo within 24 hours after the onset of symptoms &#;1,000 mg/12 hours intravenously during the first three days and orally thereafter for a total of six weeks [2 × 500 mg/12 hours&#;. All study participants were masked. The primary outcome was recovery at 90 days measured by a global test combining three measures of success: NIHSS &#;1, mRS &#;1, and BI &#;95 [567]. Safety endpoints included symptomatic intracranial hemorrhage in patients treated with rTPA, neurological deterioration, and mortality. This trial was registered, NCT. 2,298 patients were enrolled into the study. 37 centers in Spain, 11 in Portugal, and 11 in Germany recruited patients. Of the 2,298 patients who gave informed consent and underwent randomisation, 1,148 were assigned to citicoline and 1,150 to placebo. The trial was stopped for futility at the third interim analysis on the basis of complete data from 2,078 patients. The final randomised analysis was based on data for 2,298 patients: 1,148 in citicoline group and 1,150 in placebo group. Global recovery was similar in both groups (OR, 1.03; 95% CI, 0.86-1.25; p = 0.364). No significant differences were reported in the safety variables nor in the rate of adverse events. Thus, under the circumstances of the ICTUS trial, citicoline is not efficacious in the treatment of moderate-to-severe acute ischaemic stroke. But when the results of the ICTUS trial were placed in the context with the previous data, the interpretation of the study was that on top of the best treatment possible, citicoline does not show any clinical improvement but, as shown an the updated fixed-effects meta-analysis included in the original paper, the effect of the drug remains significant (OR, 1.14; 95% CI, 1-1.3). Heterogeneity coming from the older studies suggests that the beneficial effect of citicoline over time was diluted in parallel with the improvement of the standard of care of acute ischaemic stroke. One of the points to consider interpreting the results of this study is that more than 46% of patients were treated with rTPA. Clinical guidelines for the treatment of ischaemic stroke should be updated in light of the salutary results of ICTUS [569].

A new updated meta-analysis [570] was done to assess whether starting citicoline treatment within 14 days after stroke onset improves the outcome, measured as a mRS score of 0-2 or equivalent, in patients with acute ischemic stroke, as compared with placebo. Additionally, to explore if the effect of citicoline has decreased along with improvements in the standard of care. A systematic search of the adequate terms was performed on Medline, PubMed, Embase, Cochrane Specialised Register of Clinical Trials, Clinicaltrials.gov, Internet Stroke Center and Grupo Ferrer database to identify all published, unconfounded, randomized, double-blind and placebo-controlled clinical trials of citicoline initiated within the first 24 h and up to 14 days of onset in acute ischemic stroke patients. Ten randomized clinical trials (n = 4,436, but only 4,420 were valid for analysis) met the inclusion and quality criteria. The studies used citicoline with doses ranging from 500 to 2,000 mg daily administered by oral and/or intravenous route. Heterogeneity among studies was observed, reflecting the time gap of 32 years between the studies included in the meta-analysis. The administration of citicoline was associated with a higher rate of independence ( ), independently of the method of evaluation used (OR, 1.56; 95% CI, 1.12-2.16 under random effects; OR, 1.20; 95% CI, 1.06-1.36 under fixed effects). The results obtained with the subgroup of patients not treated with rtPA (OR, 1.63; 95% CI, 1.18-2.24 under random effects; OR, 1.42; 95% CI, 1.22-1.66 under fixed effects), and the results of patients not treated with rtPA and receiving the highest dose of citicoline (2g/day/6 weeks) started in the first 24 hours after onset (OR, 1.27; 95% CI, 1.05-1.53) demonstrated that the effect of citicoline is diluted when paralleled with improved standards of care. In conclusion, this systematic review shows the benefits of citicoline in the treatment of acute ischemic stroke, increasing the rate of independence. This effect is stronger in the case of patients not treated with rtPA. Yu and Zelterman [571], using a new method of parametric meta-analysis, confirmed the results reported in this meta-analysis. Sanossian and Saver [572] replicated our previous meta-analysis and concluded that treatment with citicoline was associated with an increased frequency of functional independence at long-term follow-up, 36.4% versus 31.6%, with an OR of 1.20 (95% CI, 1.05-1.55) and p =0.02. Agarwal and Patel [493] in their systematic review on the role of citicoline in the management of patients with acute ischemic stroke concluded that functional outcomes were significantly improved by citicoline in these patients. However, another meta-analysis based only on studies published in Chinese and English, that is with an important bias, citicoline cannot reduce long-term mortality and dependence rate in the treatment of acute stroke [573], results similar to those obtained by Pinzon and Sanyasi [574]. Martí-Carvajal et al [575] published a controversial Cochrane review about the use of citicoline in the management of acute ischemic stroke. They concluded that the findings of the review suggest there may be little to no difference between citicoline and its controls regarding all-cause mortality, disability or dependence in daily activities, severe adverse events, functional recovery and the assessment of the neurological function, based on low-certainty evidence. The most relevant &#;problems&#; with this review were: the authors were also the reviewers, the selection of trials was biased and incomplete, and some of the comments included in the review were not true, such as when the authors mention that &#;citicoline has been banned in the United States and Canada&#;, when citicoline is marketed in the United States as a medical food.

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Touré et al [576] in a study performed in Senegal, confirmed the efficacy of citicoline in the management of acute stroke patients, reflected by an improvement on the functional outcome. Charan et al [577] did a comparative study of citicoline versus cerebroprotein hydrolysate in ischemic and hemorrhagic stroke patients and concluded that both treatments have a similar efficacy. Kobets [578] confirms the efficacy of citicoline in the management of acute ischaemic stroke. Sergeev et al [579] concluded that maximal effect of citicoline is seen when it is administered as soon as possible after stroke onset in patients who are not eligible for reperfusion therapy. Seifaddini et al [580] concluded that prescription of citicoline for treatment of acute ischemic stroke is associated with hemodynamic changes in cerebral arteries and that this finding can be one of the citicoline&#;s mechanisms of action in ischemic stroke process. Mehta et al [581] did a prospective, single center, single-blinded, and hospital-based study with the purpose to evaluate the efficacy of citicoline, edaravone, minocycline, and cerebrolysin compared with placebo in patients with a cerebral infract at the middle cerebral artery territory with 20 patients in each group. There was significant improvement in the functional outcome of patients with acute ischaemic stroke involving middle cerebral artery territory at 90 days receiving citicoline, edaravone, and cerebrolysin. However, minocycline did not offer the same efficacy as compared with other neuroprotective agents. Diana et al [582] investigate motoric improvement in acute ischemic stroke patients in Siti Khodijah Sepanjang Hospital in an observational retrospective case-control study and concluded that citicoline 500 mg/day/5 days significantly improved motor function in acute ischaemic stroke patients. Kuryata et al [583] performed a study was to estimate the effects of citicoline therapy on the levels of circulating neurospecific protein markers in serum of the patients with ischemic stroke and atrial fibrillation. The results obtained allow the authors to hypothesize that therapeutic benefit of citicoline in patients with ischemic stroke and atrial fibrillation can be mediated through increasing neuronal viability, protecting against axonal injury, decreasing the level of reactive astrogliosis, preventing deficiencies in the blood-brain integrity, and reducing the intensity of demyelination. Mazaheri et al [584] investigate the efficacy of citicoline in acute stroke patients in a randomized clinical trial on 160 patients with hemorrhagic and ischemic stroke. The participants were randomly assigned into two groups of intervention and control. The intervention group daily received 1 g citicoline injections for 10 days, in addition to the standard therapy. Regardless of the type of stroke, the severity of the disease decreased over time in both groups. However, at the end of the study (the 90th day), the intervention group had lower disease severity, compared to the control group (p < 0.05). In terms of the ischemic stroke patients, the severity of the disease was significantly lower in the intervention group on the 90th day, compared to that in the control group. According to the authors, the long-term administration of citicoline could result in significant impacts on the treatment of the patients, especially those with ischemic stroke, and improvement of their efficacy.

Agarwal et al [585] published a pilot study to determine whether administration of citicoline immediately after recanalization therapy for acute ischaemic stroke would improve clinical and radiological outcome at three months compared to standard treatment alone. They recruited participants with acute ischaemic stroke undergoing recanalization therapy and randomly assigned them to receive either citicoline or placebo in 1:1 ratio. Citicoline arm patients received citicoline 1 g intravenous twice a day for three days, followed by oral citicoline 1 g twice a day for 39 days. Placebo arm patients received 100 mL intravenous normal saline for three days, followed by multivitamin tablet twice a day for 39 days. Authors did not find any significant difference between the citicoline or placebo arms with respect to either our primary or secondary outcomes. Reasons for the failure included:

• &#; The ceiling effect of maximum benefit achieved by thrombolysis and mechanical thrombectomy.

• &#; The low power of the study (small sample size) to detect a meaningful difference in functional outcomes

• &#; The study couldn&#;t achieve the planned sample size causing a reduction in power with a possibility of type 2 error.

• &#; Even for the surrogate outcome of the stroke volume it was not possible achieve the sample size due to poor recruitment during COVID-19 pandemic.

• &#; Other reason could be that lacunar stroke was the common stroke subtype in the study.

Premi et al [586], in a pilot randomized, single-blind experimental study, evaluated if the treatment with citicoline was able to restore intracortical excitability measures, evaluated through transcranial magnetic stimulation protocols, in patients with acute ischemic stroke. The authors conclude that the eight-week treatment with citicoline after acute ischemic stroke may restore intracortical excitability measures, which partially depends on cholinergic transmission.

Abou Zaki and Lokin [587] communicated a meta-analysis aims to evaluate the degree of effect and safety of the neuroprotectants citicoline, cerebrolysin, edaravone and MLC601 (NeuroAid) in the recovery of patients with cerebral infarcts. The analysis showed that the outcome of patients with acute ischemic strokes improved significantly when receiving neuroprotectants versus placebo (OR, 0.29; CI 95%, 0.09-0.5). According to the authors, this study supports the use of neuroprotectants in patients with acute ischemic strokes unable to receive thrombolysis or thrombectomy to improve long term functional outcomes and ultimately quality of life.

In the sequelar phase, some studies have been shown that citicoline potentiates the effects of motor rehabilitation [511,546,588,589]. In a published meta-analysis [590], it was shown how citicoline is able to increase the efficacy of motor rehabilitation in upper limbs in hemiplegic patients after ischemic stroke. Citicoline could play a relevant role in neurorehabilitation [591]. Mushba et al [592] evaluated the effect of citicoline on the efficacy of rehabilitation measures in ischemic stroke patients and concluded that citicoline significantly improves cognitive function, which in turn has a positive effect on the efficacy of remediation and indirectly improves cerebral perfusion measured with single photon emission computed tomography in patients with hemispheric ischemic stroke. Kostenko and Petrova [593] presented the results of their own observation of the use of citicoline in the complex program of medical rehabilitation of patients after ischemic stroke describing that the high efficiency of citicoline application in complex rehabilitation of patients in the early recovery period of ischemic stroke is shown in the form of improving walking function, increasing functional independence, daily activity and quality of life. Szelenberger et al [594] postulate citicoline among the pharmacological interventions for enhancing brain self-repair and stroke recovery. Singh et al [595] did a prospective study to test the role of citicoline in stroke patients in terms of cognition, memory and post stroke disability. Patients received either a placebo (n = 40) or 500 mg/12 hours citicoline (n = 35) for 12 weeks (orally or intravenously). Citicoline shows beneficial effects in stroke in terms of cognition, memory and post stroke disability. Alizadeh et al [596] showed that citicoline is more useful drug for improving speech and language skills than piracetam in post-stroke aphasia. Shulginova et al [597] described a role of citicoline alone or in combination with other drugs in patients with chronic cerebral ischemia with disorders of the immune status.

Corallo et al [598] conducted a narrative review to investigate whether antidepressant therapy, including the use of selective serotonin reuptake inhibitors or serotonin-norepinephrine reuptake inhibitors or the use of supportive drugs (i.e., citicoline or choline alfoscerate) as a substitute for antidepressant therapy, reduces depression in patients with cerebrovascular diseases. The authors concluded that the findings support the efficacy of citicoline as a treatment for depression. Arcadi et al [599] in a retrospective cohort study concluded that the administration of nootropic drugs, such as citicoline, could be a valid therapeutic strategy to manage post-stroke patients suffering from mild-moderate anxiety or anxious-depressive syndrome. Tykhomyrov et al [600] indicated for the first time that citicoline protects both astrocytes and neurons and improves angiogenic capacity through down-regulation of angiostatin in post-ischemic patients with atrial fibrillation after acute ischemic stroke.

In conclusion, it may be stated that it has been adequately shown that patients with acute stroke, as well as with sequelae, may benefit from citicoline treatment by achieving a better functional and neurological recovery, and that this is a safe and well tolerated treatment, as recognized by various authors [601-615] and some agencies [616,617].

Other clinical experiences

Parkinson&#;s disease

While levodopa continues to be the central therapeutic agent in Parkinson&#;s disease, it has well-known limitations, the main of which is a progressive loss of efficacy, that is often already evident at 3-5 years of treatment. It seems therefore warranted to use other drugs that, associated to levodopa, allow for decreasing its dosage or may even be administered as the only medication in the early stages of the disease. In this regard, use of citicoline has been tested because of its previously analyzed capacity to increase dopamine availability in the striatum and to act as a dopaminergic agonist. Citicoline has been shown to be effective in various experimental models, and its use in Parkinson&#;s disease is therefore accepted [735].

Ruggieri et al [736], in a double-blind, crossover study conducted on 28 parkinsonian patients comparing citicoline 600 mg/day/10 days intravenous to placebo, showed citicoline to be an effective treatment for these patients, achieving improvements in the assessment of bradykinesia, rigidity, and tremor, and also in the scores of the Webster scale and the Northwestern University disability scale. These same investigators later obtained very similar results in an extension of the above [737]. They subsequently tested the effects of citicoline in two groups of patients with Parkinson&#;s disease [738]. The first group included 28 patients who had not previously received treatment, while the second group included 30 patients who were already receiving treatment with levodopa and carbidopa since at least two months before, and in whom dosage had been stabilized at the minimum effective level. The same methods as in previous studies by these investigators were used, that is, a double-blind, crossover study comparative to placebo. Treatment was administered for 20 days at a dose of 500 mg/day by the parenteral route. Clinical assessments were performed on days 10 and 20, coinciding with change in treatment, according to the study design. Treatment with citicoline provided statistically significant improvements in Webster scale, Northwestern University disability scale, and assessment of bradykinesia in both patient groups. Rigidity also improved in both groups, although this improvement only reached statistical significance in the previously treated group of patients. Tremor also improved in both groups, but the desired statistical significance was not reached.

Eberhardt et al [739-741] have shown that association of citicoline to levodopa treatment allows for reducing levodopa dose by 50%, thus minimizing the side effects associated to levodopa therapy. Thus, for this group of investigators, citicoline represents a useful alternative in patients requiring a reduction in levodopa doses and, moreover, addition of citicoline to a treatment with levodopa may relieve decompensation states in the course of parkinsonism [742].

Loeb et al [743] conducted a multicentre, double-blind study with citicoline for the treatment of parkinsonian patients. In this study, 65 patients were randomized to a group to which citicoline 1 g/day intravenous was added or to a placebo group. Treatment lasted 21 days. All patients continued their underlying treatment with levodopa plus mianserin or benserazide for at least eight weeks. Authors found significant differences between citicoline and placebo at the controls performed after 14 and 21 days of treatment in all parameters assessed by the Webster and Northwestern University disability scales. They also noted that patients treated with citicoline experienced a significant worsening 45 days after the medication was discontinued, thus showing the efficacy of citicoline as adjuvant treatment to levodopa in patients with Parkinson&#;s disease.

Acosta et al [744] treated with citicoline 61 parkinsonian patients, of whom 48 patients were already receiving treatment with levodopa. Each patient received two treatment courses. In the first 10-day phase, citicoline 500 mg daily were administered by the intramuscular route. This was followed in a second phase by oral treatment at the same dose for 14 weeks. Patients treated with levodopa continued taking this medication at the same dose in a first period, after which an attempt was made to decrease it. Parkinsonian symptoms were assessed using the Webster scale. Among patients receiving levodopa, 36% improved when citicoline was added, with the greater percent improvements being obtained in bradykinesia, rigidity, posture, gait, and limb sway. In patients who had been treated with levodopa for less than two years, percent improvements amounted to 42.12%, as compared to 19.08% of improvements in patients with more than two years of levodopa therapy. Levodopa doses could be decreased by 20 to 100% in 35.3% of patients with less than two years of treatment. In patients with more than two years of levodopa treatment, levodopa dose could be reduced by 25-33% in 10% of the cases. Authors concluded that citicoline treatment allows for delaying the start of levodopa therapy in the early disease stages, and for decreasing or maintaining levodopa dosage in already treated subjects.

Cubells and Hernando [745] tested citicoline in 30 parkinsonian patients who were already being treated with levodopa. The dose administered was 500 mg/day by the intramuscular route for two months and was reduced to a third at the end of the first month of treatment. Changes in parkinsonian symptoms, according to the Yahr scale, showed after the first month of treatment a moderate improvement in facial expression and digital skills, and an obvious improvement in postural stability, motor changes and bradykinesia. A greater stabilization of therapeutic response was also seen, with a decreased incidence of &#;wearing-off&#; and &#;on-off&#; phenomena, although dyskinesia increased. When levodopa dose was decreased during the second study month, clinical improvement was maintained, and incidence of dyskinesia was decreased. Measurements of various electrophysiological parameters using an original technique by the authors revealed recovery from hyporeflexia and hypotonia after one month of treatment with citicoline, and a major improvement in active muscle contraction, decreased muscle fatigue, and an obvious recovery of contractile speed, a parameter that was greatly decreased before treatment with citicoline was started. Authors stated that the increase in levodopa plasma levels was so significant that it could not be interpreted as due only to an increased release of dopamine stored in presynaptic vesicles. They therefore assumed that citicoline exerts an action upon the synthetic mechanism of dopamine, acting through the tyrosine hydroxylase enzymatic system. In addition, the increase in dopamine receptors quantified in lymphocytes suggests, according to authors, a promoting role of citicoline upon the availability of postsynaptic dopamine receptors.

Martí-Massó and Urtasun [746] examined the effects of citicoline in 20 parkinsonian patients treated with levodopa for more than two years. These patients were administered citicoline 1 g/day/15 days intramuscular, and then continued with half the dose for 15 additional days. A progressive symptom improvement was achieved. Thus, 4.16% and 7.26% overall improvements were achieved in the Columbia University scale at 15 days and at the end of treatment respectively. Partial improvements achieved in ambulation, turning time in bed, and writing time should be particularly noted. In assessment conducted by relatives, improvements achieved in agility, ambulation, and general patient status deserved special mention.

García-Mas et al [747] conducted a study with quantified electroencephalography using fast Fourier transforms in two groups of patients with idiopathic Parkinson&#;s disease, one of which showed cortical cognitive impairment. Study of specific quantified electroencephalography indices allowed for establishing some parameters differentiating patients with and without cortical impairment. Specifically, differences were found in global potencies of delta and alpha rhythms, the alpha/theta index, posterior activities, anteriorization index of delta and alpha rhythms and finally, spatialization index of alpha rhythm. Administration of citicoline 2 g intravenous in these patients achieves a global increase in potencies corresponding to posterior rhythms, particularly alpha rhythm, that is a marker of cognitive activity in dementia processes. As shown previously, citicoline is an adjuvant therapy on mild cognitive impairment in Parkinson&#;s disease [699]. On the other hand, citicoline significantly improved essential tremor [748].

Based on the reported and discussed studies, it may be stated that citicoline represents an effective treatment for Parkinson&#;s disease in both untreated patients and patients already treated with levodopa, in whom it also allows for reducing levodopa dose. In patients with Parkinson&#;s disease and cognitive impairment, administration of citicoline induces a trend to normalization of deficits and the main electrophysiological parameters altered. Que and Jamora [749] did a systematic review with the aim to synthesize current existing evidence on the efficacy of citicoline adjunctive therapy in improving Parkinson&#;s disease symptoms and concluded that citicoline adjuvant therapy has beneficial effects as an adjuvant therapy in patients with Parkinson&#;s disease. However, due to the heterogeneity of the studies, there is a need for more high-quality studies.

Alcoholism and drug addiction

Clinical experience with citicoline in alcoholism and drug addictions is not very extensive, but there is some evidence of its efficacy in these applications.

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Chinchilla et al [750] conducted a randomized, double-blind study on the effects of citicoline in 20 patients with alcohol withdrawal syndrome. At the end of the study, i.e., at two months, a significant improvement in attention-concentration and time and space orientation in the group of patients receiving citicoline suggesting, according to the authors, that the drug may be useful for the treatment of chronic alcoholism. Brown et al [751] reported a neutral study in patients with alcohol use disorder. Kang and Choi [752], despite the neutral results of the study by Brown et al [751], consider that the use of citicoline in the management of alcohol use disorder remains at investigational stages and needs further studies, and Shen [753] considers citicoline among the anticraving therapies for alcohol use disorder.

Renshaw et al [754-756] published a double-blind pilot study of patients addicted to cocaine, showing that after 14 days of treatment with 500 mg/12 hours of citicoline or placebo, the patients in the citicoline group experienced a reduction in craving for cocaine. Consequently, citicoline appears to be a promising therapy for this type of affliction, that do not perturb sleep/wake cycles [757] and may decrease cocaine use and enhance cognition [758]. But Licata et al [759] reported that citicoline is not an effective treatment reducing craving for heavy cocaine users. Also have been reported positive effects in patients with mood disorders related with the use of cocaine [760,761], antidepressant properties in methamphetamine dependence [762], and a role facilitating the treatment of marijuana use disorders by improving the cognitive skills necessary to fully engage in comprehensive treatment programs [763]. There is a clear implication of the cerebral metabolism in the drug addiction pathophysiology [764,765].

The application of brain imaging to study drug addictions has offered new insights into the fundamental factors that contribute to their use and abuse [766]. That is the case of Jeong et al [767], who performed a randomized, double-blind, placebo-controlled study to assess the effects of citicoline on the brain structures and their associations with craving and methamphetamine use. Methamphetamine users (n = 44) were randomized to receive 2 g/day of citicoline (n = 22) or placebo (n = 22) for eight weeks. Patients underwent brain magnetic resonance imaging at baseline and eight-week follow-up. Healthy individuals (n = 27) were also examined using brain magnetic resonance imaging at the same interval. Voxel-based morphometry analysis was conducted to examine changes in gray matter volumes and their associations with craving and methamphetamine use. Craving for methamphetamine was significantly reduced after the eight-week treatment with citicoline (p = 0.01), but not with the placebo treatment (p = 0.10). There was no significant difference in the total number of methamphetamine negative urine samples between the two groups (p = 0.19). With citicoline treatment, gray matter volumes in the left middle frontal gyrus (p = 0.001), right hippocampus (p = 0.009), and left precuneus (p = 0.001) were significantly increased compared to the placebo and control groups. Increased gray matter volumes in the left middle frontal gyrus with citicoline treatment were associated with reduced craving for methamphetamine (Spearman&#;s ρ = 0.56; p = 0.03). In addition, the right hippocampal volume increases were positively associated with the total number of methamphetamine -negative urine results in the citicoline group (Spearman&#;s ρ = 0.67; p = 0.006). Then, these results suggest that citicoline-induced gray matter volume increases may contribute to decreases in methamphetamine use and craving.

And there is some data suggesting a potential usefulness of citicoline in modulating appetite [768]. Preuss et al [769] consider citicoline among the therapies for bipolar disorder and comorbid use of illicit substances. Despite the limited research on the efficacy of citicoline for addictive disorders, the available literature suggests promising results [770].

Amblyopia and glaucoma

There is clinical evidence that citicoline improves visual acuity in patients with amblyopia [771-786], visual function in patients with glaucoma [787-814], in patients with non-arteritic ischemic optic neuropathy [815,816], and in early diabetic retinopathy [817].

Now there are citicoline in eye drops formulations for the management of glaucoma [818-821]. Tokuc et al [822] demonstrate the protective effects of citicoline eye drops on ultraviolet B radiation-induced corneal oxidative damage in a rat model. Parisi et al [823] reported the results of a pilot study evaluating the long-term efficacy of citicoline and vitamin B12 eye drops on macular function in patients with type 1 diabetes with mild signs of non-proliferative diabetic retinopathy. Treatment with citicoline and vitamin B12 eye drops for a 36-month period achieved an improvement of the macular bioelectrical responses, whereas, during the same period of follow-up, patients treated with placebo showed a worsening of the macular function. Topical citicoline and vitamin B12 ameliorated both morphology and function of corneal nerves in patients with diabetic neuropathy [824]. Topically administered citicoline eye drops had beneficial effects in the early recovery of corneal sensitivity during the first six weeks after LASIK, suggesting that citicoline may play a significant role in accelerating corneal reinnervation [825]. Despite their neuroprotective effect, topical citicoline drops had no significant effect on the superficial and deep microvascular structures of the retina or choriocapillaris [826].

Oddone et al [827] recently published an extensive review on the role of citicoline in ophthalmological neurodegenerative disease.

Other uses

There are positive results reported for citicoline in the treatment of facial neuritis [828], X-chromosome-linked ichthyosis [829], delayed-onset encephalopathy caused by carbon monoxide poisoning [830], epilepsy [831], vertigo [832], major depressive disorder [833,834], schizophrenia [835-837], fragile X-associated tremor/ataxia syndrome [838], an COVID-19 [839]. Recently, a new mechanism to enhance central nervous system remyelination via the choline pathway has been described. Due to its regenerative action combined with an excellent safety profile, CDP-choline could become a promising substance for patients with multiple sclerosis as an add-on therapy [840-844].

Pediatric use. The experience in children is limited; therefore, it may only be administered when the expected therapeutically benefit is higher than any possible risk.

There are some studies published in pediatric populations with citicoline in traumatic brain injuries [456], organic brain syndromes [845-847], neonatal hypoxic-ischaemic encephalopathy [848-852], visual impairment [853], refractive amblyopia [854], neurophysiologic abnormalities in developmental dysphasias [855], choline kinase beta deficiency [856], children with post cardiac arrest [857], learning disturbances [858,859], and autism and Asperger&#;s syndrome [860]. There is a review on the use of citicoline in pediatric neurology and pediatric psychiatry [861]. No safety concerns related with the use of citicoline were reported in these studies.

Safety

Dinsdale et al [862] administered citicoline or placebo to 12 healthy volunteers in two oral regimens repeated at short-term intervals (600 mg/day and 1 g/day), every day for five days. The only adverse effects that appeared were self-limiting headaches in four and five subjects with high and low doses, respectively and in one subject who was given placebo. The results of hematological and clinical analyses did not show any abnormality associated to citicoline administration. No clinically significant electrocardiogram and electroencephalogram abnormalities were registered. Empirical neurological tests, tendon reflexes, blood pressure and heart rate were not affected by any dose of the drug or placebo.

In addition to an excellent tolerability in healthy individuals, as demonstrated in the above study, all of the authors of clinical trials using citicoline that have been reviewed in this present article, agree in rating the safety of this drug as excellent without serious side effects being reported. In some cases, the appearance of digestive intolerance has been reported and occasional excitability or restlessness in the first days of treatment. For instance, Lozano [863] monitored a study of the efficacy and safety of citicoline in 2,817 patients of all ages, with a predominance of patients between 60 and 80 years, who had different neurological processes, mostly cognitive disorders of diverse origin. The duration of citicoline Treatment ranged from 15 to 60 days and the mean dose administered was 600 mg/day orally. Only 5.01% of the patients had collateral effects associated with citicoline treatment, most often digestive intolerance (3.6%). In no case was it necessary to interrupt treatment for side effects attributable to citicoline use.

In the pooled analysis of citicoline in the treatment of acute ischemic stroke in the United States [536], there were few adverse events that were reported in more than the 5 %. These adverse events are listed in .

Table XII

PlaceboCiticoline n % n % p Adverse events with incidence > 5% in the citicoline group &#;Anxiety589..690.036 &#;Leg oedema386..760.032 Adverse events with incidence > 5% &#;Accidental injury..11n.s. &#;Agitation..32n.s. &#;Constipation..25n.s. &#;Coughing..31n.s. &#;Diarrhoea..83n.s. &#;Dizziness467..13n.s. &#;ECG abnormality579..38n.s. &#;Fever..54n.s. &#;Auricular fibrillation..66n.s. &#;Headache..08n.s. &#;Haematuria539..53n.s. &#;Hypertension..60n.s. &#;Hypokalemia..08n.s. &#;Hypotension559..41n.s. &#;Urinary tract infection..77n.s. &#;Insomnia..38n.s. &#;Joint pain488..89n.s. &#;Nausea..90n.s. &#;Pain..77n.s. &#;Back pain457..38n.s. &#;Chest pain559..39n.s. &#;Rash..20n.s. &#;Restlessness498..38n.s. &#;Shoulder pain..31n.s. &#;Vomiting..07n.s. Adverse events with incidence > 5% in the placebo group &#;Depression..560.038 &#;Falling down..550.002 &#;Urinary incontinence..520.047Open in a separate window

In the South Korean drug surveillance study [543], the safety of the product was considered as excellent, with only 37 side effects in 31 cases among the 4,191 patients treated, that is a rate of side effects of 0.73 %.

Also, in the Cochrane review [692], it was demonstrated a lower rate on the incidence of adverse events related with citicoline in comparison with placebo.

In front of the question: Can citicoline cause depression?, Tardner [864] did a literature review and conclude that not only are there no recorded cases of citicoline causing depression in anybody, regardless of medical or psychiatric history. If anything, the clinical evidence suggests that citicoline may have antidepressant properties.

Synoradzki and Grieb [865] explained why, on a molar mass basis, citicoline is significantly less toxic than choline that has been associated with concerning its contribution to normal lipid metabolism, maintenance of normal liver function, and normal homocysteine metabolism. Choline in citicoline is less prone to conversion to trimethylamine and its putative atherogenic N-oxide, then the choline supplementation with citicoline may be safer and more efficacious.

In conclusion, the tolerability of citicoline is excellent and the side effects attributable to this drug are infrequent. In any case, side effects are never severe and consist, mainly, in gastrointestinal discomfort and restlessness.

Conclusions: The eight-week treatment with citicoline after acute ischemic stroke may restore intracortical excitability measures, which partially depends on cholinergic transmission. This study extends current knowledge of the application of citicoline in acute ischemic stroke.

Results: A total of thirty participants (mean [SD] age, 68.1 [9.6] years; 11 women [37%]) completed the study. We did not observe significant changes in clinical scores after CDP-choline treatment (all p > 0.05), but we observed a significant improvement in short-interval intracortical inhibition (SAI) ( p = 0.003) in the TG group compared to the CG group.

Methods: Patients with acute ischemic stroke were recruited and assigned to an eight-week therapy of standard treatment (control group - CG) or CDP-choline (Rischiaril ® Forte, containing 1,000 mg of citicoline sodium salt) added to conventional treatment (treatment group - TG). Each subject underwent a clinical evaluation and neurophysiological assessment using TMS, pretretament and posttreatment.

Objectives: The objective of this randomized, single-blind experimental study was to evaluate whether the treatment with Rischiaril ® Forte was able to restore intracortical excitability measures, evaluated through transcranial magnetic stimulation (TMS) protocols, in patients with acute ischemic stroke.

Background: Recent research on animal models of ischemic stroke supports the idea that pharmacological treatment potentially enhancing intrinsic brain plasticity could modulate acute brain damage, with improved functional recovery. One of these new drugs is citicoline, which could provide neurovascular protection and repair effects.

Introduction

Ischemic stroke is one of the most devastating diseases (often involving severe physical damage) with more than 50% of stroke survivors presenting persistent disability, and about 30% still living with partial dependency on daily living activities 6 months after stroke (1, 2). Another post-stroke complication consisted of a series of syndromes from mild cognitive impairment to dementia, with an increased risk by at least five to eight times (3). For these reasons, stroke has been classed as a medical emergency and it is important to find new protective therapies beyond the acute phase (1, 4). Within the last few years, recent research on animal models of ischemic stroke supports the idea that pharmacological treatments potentially enhancing intrinsic brain plasticity could modulate acute brain damage, improving functional recovery, even when they are administered several h after the onset (5&#;7). In this scenario, it has been demonstrated that citicoline could provide neurovascular protection and repair effects in patients suffering from stroke (8).

Citicoline (or CDP-choline) is physiologically present in all human cells, and it acts as a neuroprotective compound as well as an intermediate in membrane phosphatide biosynthesis (9). In human, citicoline is degraded to cytidine and choline through hydrolysis and dephosphorylation. Thus, cytidine and choline represent substrates for the synthesis of phosphatidylcholine and CDP-choline in neurons (10, 11).

Up to now, citicoline has been widely studied in patients with various neurological conditions (12, 13). Considering patients suffering from stroke, contrasting findings were reported, with different studies that supported a beneficial effect of citicoline on clinical measures (13) but at least one sizeable multicentre study did not (4). However, no study has investigated potential beneficial effects on brain neurotransmitters circuits to further corroborate citicoline efficacy.

One of the latest approaches which may help to understand the neurophysiology of acute ischemic stroke is transcranial magnetic stimulation (TMS), which allows to indirectly assess neuronal circuits by applying paired-pulse TMS protocols (14).

In particular, short-afferent latency inhibition (SAI) allows to indirectly assess cholinergic circuits, while short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF) protocols assess GABAergic and glutamatergic neurotransmission, respectively.

Overall, TMS is safe and well-tolerated and can be exploited as a non-invasive tool that can evaluate in vivo the cortical excitability, the propension to undergo neural plastic phenomena, and the underlying transmission pathways. In particular, patients suffering from stroke are characterized by lower motor excitability in the affected hemisphere (15) with also an interhemispheric imbalance in motor primary areas of both hemispheres, resulting in an asymmetric inhibition from the unaffected hemisphere (16). The objective of the present study was to evaluate the effects of citicoline on neuronal circuits, evaluated by TMS. To this, we carried out a pilot, randomized, single-blind clinical trial in a cohort of patients with acute ischemic stroke.

Methods

Participants

A total of thirty patients with acute ischemic stroke were recruited from the Stroke Unit, ASST Spedali Civili Hospital, Brescia, Italy within 36 h after the onset of symptoms and entered the study.

For each patient, past medical history was carefully recorded, and each patient underwent clinical and neurological examination, as well as brain structural imaging.

The inclusion criteria consisted of patients older than 60 years old and with a National Institutes of Health Stroke Scale (NIHSS) <14 and not treated with reperfusion treatments (thrombolysis with intravenous recombinant tissue plasminogen activator (rTPA) and/or mechanical thrombectomy) for known contraindication (time window and/or clinical/anamnestic factors that increased the hemorrhagic risk) (4, 17). We excluded cases with severe head trauma in the past, history of seizures, ischemic or hemorrhagic stroke, intracranial expansive process, pacemaker, metal implants in the head/neck region, and severe comorbidity (i.e., cancer in the past 5 years, non-controlled hypertension).

Full written informed consent was obtained from all participants according to the Declaration of Helsinki. The study protocol was approved by the local ethics committee (Brescia Hospital, #NP).

Study Design

Patients were randomly assigned to two groups with a 1:1 ratio; the control group (CG) received conventional treatment (antiplatelet or anticoagulant drugs, statin, antihypertensive therapy according to current guidelines), and the treatment group (TG) received CDP-choline (Rischiaril® Forte, containing 1,000 mg of citicoline sodium salt) in addition to conventional treatment for 8 weeks.

At baseline (T0) and at 8-weeks follow-up (T1), each participant underwent a standardized assessment of neurological deficits and cognitive functions and a standardized TMS protocol.

Neurological deficits were evaluated using the NIHSS (17), a 15-item scale that measures the level of neurological impairment, and the modified Rankin score (mRs) (18), a measure of functional disability. A brief cognitive evaluation was performed with the Mini-Mental State Examination (MMSE) (19). TMS protocols were carried out as described below.

The primary endpoint was defined as a significant change from baseline in neurophysiological measures, evaluated indirectly with TMS. The secondary endpoint was defined as changes from baseline in clinical assessment.

The examiners were blinded regarding the type of treatment when performing clinical ratings (EP, NG, AC, ID, RS, and MG) and TMS protocols (VC).

MM was responsible for random allocation sequences, participants' enrolment, and participants' assignation to specific interventions. Computer-assisted randomization was applied to allocate subjects into groups.

Transcranial Magnetic Stimulation Assessment

A TMS figure-of-eight coil (each loop diameter 70 mm &#; D702 coil) connected to a monophasic Magstim Bistim2 system (Magstim Company, Oxford, UK) was employed for all TMS paradigms, as previously reported (20). Patients were stimulated on the ischemic lesion side. Electromyographic (EMG) recordings were performed from the contralateral first dorsal interosseous muscle using 9 mm diameter Ag-AgCl surface-cup electrodes. The active electrode was placed over the muscle belly and the reference electrode over the metacarpophalangeal joint of the index finger. Responses were amplified and filtered at 20 and 2 kHz with a sampling rate of 5 kHz. The TMS coil was held tangentially over the scalp region corresponding to the primary hand motor area contralateral to the target muscle, with the coil handle pointed 45° posteriorly and laterally to the sagittal plane.

Resting motor threshold (RMT) was determined as the minimum intensity of the stimulator required to elicit motor evoked potentials (MEPs) with a 50 μV amplitude in 50% of 10 consecutive trials, recorded during full muscle relaxation (21).

SICI-ICF and SAI were studied using a paired-pulse technique, employing a conditioning-test design. For all paradigms, the test stimulus (TS) was adjusted to evoke an MEP of approximately 1 mV amplitude.

For SICI and ICF, the conditioning stimulus (CS) was adjusted at 70% of the RMT, employing multiple interstimulus intervals (ISIs), including 1, 2, and 3 ms for SICI and 7, 10, and 15 ms for ICF (22, 23). SAI was evaluated employing a CS of single pulses (200 μs) of electrical stimulation delivered to the right median nerve at the wrist, using a bipolar electrode with the cathode positioned proximally, at an intensity sufficient to evoke a visible twitch of the thenar muscles (24). Different ISIs were implemented (0 and +4), which were fixed relative to the N20 component latency of the somatosensory evoked potential of the median nerve (24).

For each ISI and for each protocol, ten different paired CS-TS stimuli and fourteen control TS stimuli were delivered to all participants in a pseudo-randomized sequence, with an inter-trial interval of 5 secs (±10%).

The conditioned MEP amplitude, evoked after delivering a paired CS-TS stimulus, was expressed as a percentage of the average control MEP amplitude. Average values for SICI (1, 2, and 3 ms ISI), ICF (7, 10, and 15 ms ISI), and SAI (0 and +4 ms ISI) were used for analysis.

Stimulation protocols were conducted in a randomized order. Audio-visual feedback was provided to ensure muscle relaxation during the entire experiment and trials were discarded if EMG activity exceeded 100 μV in the 250 ms prior to TMS stimulus delivery. Less than 5% of trials were discarded for each protocol. All of the participants were capable of following instructions and reaching complete muscle relaxation; if, however, the data were corrupted by patient movement, the protocol was restarted and the initial recording was rejected.

Statistical Analyses

Continuous and categorical variables are reported as mean (± standard deviation) and percentage (number), respectively. Demographic and clinical data were assessed using the Mann-Whitney U test for continuous variables and Chi-square test for categorical variables, as appropriate. To assess the effect of CDP-choline treatment on neurophysiological or clinical measures over time, we used a two-way repeated-measures ANOVA with TIME (T0 and T1) and TREATMENT (CG and TG groups) as within-subjects factors. Statistical analyses were performed using SPSS version 21 (SPSS, Inc., Chicago, IL, USA).

Data Availability

All study data, including study design, protocol, statistical analysis plan, and results, are available from the corresponding author upon reasonable request.

Results

Participants

A total of thirty-three participants with acute ischemic stroke entered the study; three patients were excluded from analyses due to meeting the exclusion criteria (n = 2 with unexcitable motor cortex and n = 1 carrying a pacemaker). A final count of 30 patients (16 with the right-sided lesions, 14 with left-sided lesions) was considered in the present study and was randomized. The two groups did not differ in demographic and clinical characteristics at baseline as well as at follow-up. The location of the stroke and the subtype classification [according to TOAST criteria (25)] did not differ among groups (see Table 1).

TABLE 1

Table 1. Demographic and clinical characteristics of included patients.

Effect of CDP-Choline Treatment on Neurophysiological and Clinical Assessment

Baseline and follow-up clinical and neurophysiological scores are reported in Table 2. No statistically significant differences in clinical measures (at baseline, follow-up, or at the TIME × TREATMENT interaction) were evident.

TABLE 2

Table 2. Clinical and neurophysiological parameters of included patients before and after CDP-choline or standard treatment.

For SAI, there was a statistically significant TIME × TREATMENT interaction at the repeated measures ANOVA (F = 9.94, p = 0.004, partial η2 = 0.29), with a significantly restored cholinergic transmission at T1 (average.51 ±0.18) compared to T0 (average.81 ±0.21) in the CDP-choline treatment group (Figure 1 and Table 2). No statistically significant TIME × TREATMENT differences were observed for SICI (F= 1.56, p = 0.223, partial η2 = 0.03) and ICF (F = 3.75, p = 0.063, partial η2 = 0.04).

FIGURE 1

Figure 1. SAI measures before and after exposure to CDP-choline treatment. SAI, short-latency afferent inhibition. Error bars represent standard errors. *Significant difference.

Discussion

The treatment of acute ischemic stroke remains a page still largely to be written, given that, unfortunately, it continues to be a fearful disease, especially for its disabling results.

Stroke has been classed as a medical emergency and it is important to find new effective protective therapies.

In this randomized, single-blind pilot study, we demonstrated the beneficial effect of citicoline in restoring SAI in patients with acute ischemic stroke. SAI is a marker of sensorimotor integration which partially and indirectly reflects cholinergic inhibition mediated by GABAA receptors (26, 27). Literature data on the relationship between stroke and the cholinergic system reported an impaired cholinergic activity (choline acetyltransferase and acetylcholinesterase) in patients suffering from stroke (28). Moreover, SAI has been shown to correlate with the degree of motor impairment after stroke (29). Interestingly, in our study, the impairment of SAI persisted after 8 weeks in patients in the control group. Citicoline has been proved to modulate different neurotransmitter pathways in clinical studies as well as in animal models of disease (13, 30, 31). Recently, the cholinergic system and the extended hippocampal network (primarily involving the nucleus basalis of Meynert) have been identified as the main players in cognitive recovery after stroke, supporting the idea that targeted therapeutic strategies could enhance spontaneous mechanisms of recovery (32, 33). Previous randomized clinical trials on citicoline in stroke have reported a mixed effect (34), with specific clinical factors (patients >70 years of age, moderate stroke severity, utilization of recanalization treatments (i.e., rt-PA thrombolysis and/or mechanical thrombectomy) potentially affecting clinical efficacy of citicoline in the acute phase of ischemic stroke (4). Moreover, clinical trials on the cholinergic modulation in stroke (using Donepezil) have reported inconclusive results (35&#;38). Interestingly, TMS in vascular cognitive impairment demonstrated increased cortical excitability and synaptic plasticity as adaptative responses potentially related to disease progression (39). Thus, TMS could be used to forecast cognitive deterioration in subjects &#;at-risk&#; for dementia (chronic vascular encephalopathy, leukoaraiosis, etc.) (39), in light of disease-modifying/neuromodulatory treatments. From this perspective, TMS assessment (considering the SAI protocol) may represent an effective and feasible tool to detect those patients with an established cholinergic deficit that could benefit more from a targeted treatment for cholinergic restoration, as already studied in vascular cognitive impairment (39&#;42). Thus, for the first time, the present study demonstrated in vivo modulation of the cholinergic system by the utilization of citicoline in patients with ischemic stroke, paving the way for a personalized medicine approach to potentiate the clinical recovery after ischemic stroke.

Therefore, TMS can be exploited, as in this case, to evaluate the response to specific pharmacological treatments in the attempt to not only identify new therapeutic targets but also to predict cognitive deterioration caused by stroke. The role of TMS in cerebrovascular diseases is catching on cortical excitability, plasticity, and connectivity, also providing new clues on the pathophysiology of the impairment with a translational perspective toward novel treatments for these patients (27).

We acknowledge that the present pilot study entails some limitations. First, the group sample is limited, even though well characterized. Moreover, correlations between neurophysiological and clinical variables should be considered in larger samples (also considering the potential modulating effect of recanalization treatments) to corroborate the present findings. Taking into account these caveats, the present approach for the evaluation and modulation of the cholinergic system in ischemic stroke should warrant further studies.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics Statement

The studies involving human participants were reviewed and approved by Comitato Etico, ASST Spedali Civili, Brescia. The patients/participants provided their written informed consent to participate in this study.

Author Contributions

EP: conception and design of the work, acquisition of the data, statistical analysis, and draft of the manuscript. VC: acquisition of the data, statistical analysis, and draft of the manuscript. AB: conception and design of the work, acquisition of the data, statistical analysis, and revision of the manuscript. NG, VV, ID, MG, RS, and AC: acquisition of the data and revision of the manuscript. AP: critical revision of the manuscript for intellectual content. BB: conception and design of the work and draft of the manuscript. MM: conception and design of the work and revision of the manuscript. All authors contributed to the article and approved the submitted version.

Funding

AB was partially supported by the Airalzh-AGYR and by Fondazione Cariplo, grant n&#; -.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

The authors wish to thank patients for participating in this study.

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