Neuroprotective Properties of Citicoline: Facts, Doubts and Unresolved Issues

Citicoline displays negligible toxicity. The compound is quickly catabolized (Fig. 2), and the products arising are subsequently available for diverse biosynthetic pathways and ultimately excreted as carbon dioxide. The lack of acute and chronic toxicity of citicoline has been repeatedly confirmed in rodents and dogs (see the most recent report by Schauss et al. [15] and the references quoted therein). An impressive example is the median lethal dose (LD50) of an acute single intravenous application of citicoline, which equals 4,600 and 4,150 mg/kg in mice and rats, respectively. The LD50 for ingested citicoline is even higher at approximately 8 g/kg in both mice and rats. For comparison, in mice, the LD50 of an acute single intravenous dose of sodium chloride is 645 mg/kg, and that of vitamin C is 518 mg/kg. In a 90-day rat oral toxicity study of 100–1,000 mg/kg daily doses, increases in serum creatinine and in renal tubular mineralization, likely caused by phosphate liberation from citicoline, were found, without concomitant degenerative or inflammatory reactions.

Presumed catabolism of citicoline (Cyt-P-P-Cho) in the rodent intravascular compartment. In the first step, hydrolysis of the pyrophosphate bridge takes place. In the second step, cytidine monophosphate (Cyt-P) and phosphocholine (Cho-P) are dephosphorylated to cytidine (Cyt) and choline (Cho), respectively; supposedly, a large part of the liberated choline is taken up by the liver (which may explain the unexpectedly low cholinergic toxicity of citicoline)

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The usual daily therapeutic dosage of citicoline in humans is 500–2,000 mg—that is, 7–28 mg/kg in a person of average bodyweight (70 kg). Data from clinical trials have corroborated preclinical toxicological findings, revealing a favourable safety profile, with only a few reports of adverse events, mostly related to digestive disturbances following oral intake. In adult and elderly stroke patients, the drug has lacked significant adverse events (see, for example, the study by Cho and Kim [16] in 4,191 Korean stroke patients), and a meta-analysis of placebo-controlled trials has shown that the overall frequency of adverse effects was comparable between groups comprising 1,652 actively treated and 686 placebo-treated subjects [17]. There are no data concerning the effects of liver or kidney insufficiency on the safety profile and pharmacokinetics of citicoline although, on the basis of the aforementioned toxicology data, an enhanced threat of hyperphosphataemia may be predicted in patients with kidney failure.

Upon administration, citicoline is relatively quickly catabolized and is the source of choline that appears in the blood. Administered parenterally or orally, citicoline is relatively quickly (i.e. within minutes rather than hours) converted to its cholinergic and pyrimidinergic catabolites. In the perfused rat liver, citicoline disappeared from the perfusate within 10 min [18, 19]. Since phosphorylated substrates are considered unable to penetrate cell membranes, it is usually assumed that cytidine monophosphate (CMP) and phosphocholine (PCho) yielded from hydrolysis of citicoline are further dephosphorylated by phosphatases in blood plasma. In agreement with this assumption, citicoline given orally to rats produced pronounced increases in plasma cytidine and choline, although it should be noted that the rise of cytidine was several-fold larger than that of choline [20]. A recently published paper [21] reported on the use of a liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) method to evaluate the pharmacokinetics of choline in blood from human volunteers following ingestion of 1,000 mg citicoline tablets. Biphasic concentration–time curves of choline in plasma have been recorded, with a large peak of 2 μg/mL at approximately 2 h and a second, slightly smaller but much broader peak with a maximum at approximately 24 h following ingestion. Unfortunately, no data on the initial plasma level of choline were shown, which makes the whole picture a bit unclear. One older source reported an arithmetic mean plasma choline level oscillating around 1.36 μg/mL [22] in a healthy human subject, and this was confirmed recently [23].

Is citicoline carcinogenic? In a wide variety of cancers, choline phospholipid metabolism is altered in such a way that cancer cells display elevated levels of phosphocholine, as well as total choline-containing compounds [24]. One may therefore pose a question as to whether increasing choline exposure doesn’t induce carcinogenesis and/or accelerate cancer growth. However, choline chloride has displayed no mutagenic potential when tested in vitro (using Ames testing, yeast gene conversion, clastogenicity and sister chromatid exchange) [25], and similar negative data have been obtained for citicoline [26]. Moreover, epidemiological data have shown that the associations between choline intake and cancer—if any—are weak. For example, Johansson et al. [27] found that elevated plasma concentrations of choline may be associated with a slightly increased risk of prostate cancer, but a similar (even slightly stronger) association has been found for vitamin B2. On the other hand, Lee et al. [28] did not find any association between choline (or betaine) intake and the risk of colorectal cancer, whereas Xu et al. [29] found that dietary choline intake was inversely associated with breast cancer risk.

Citicoline is neuroprotective in various animal (preclinical) experimental paradigms. The compound has offered marked neuroprotection in several in vitro and in vivo models of acute and chronic brain ischaemic and neurodegenerative diseases, including brain hypoxia, ischaemia and intracerebral haemorrhage (reviewed by Adibhatla and Hatcher [30]), brain and spinal cord trauma [31], in vitro glutamate excitotoxicity [32, 33] and in vivo amyloid toxicity [34]. However, the mechanisms of this neuroprotection are far from being understood.

One major effect of citicoline is believed to be stimulation of the synthesis and increase in the content of brain phospholipids. Increases in brain phospholipids following oral administration of citicoline have also been observed in humans, with use of phosphorous magnetic resonance spectroscopy [35]. The relevant hypothesis assumes that the citicoline breakdown products cytidine and choline enter the brain separately and, inside brain cells, they act as substrates for resynthesis of CDP-choline. This, in turn, is believed to result in slowing down of phospholipid breakdown and acceleration of phospholipid resynthesis necessary for membrane repair [36]. However, since citicoline is devoid of cholinergic toxicity (see below), a significant rise in brain choline following therapeutic doses of citicoline in humans does not seem probable—indeed, a decrease in choline in the brains of older subjects and no change in those of younger subjects have been observed following oral citicoline, with use of proton magnetic resonance spectroscopy (MRS) [37].

The other mechanisms suggested to be involved in the neuroprotective effects of citicoline in stroke models include prevention of activation of phospholipase A2 (PLA2) [38]. The related effects comprise attenuation of the increase in hydroxyl radical generation, preventing loss of cardiolipin (an exclusive inner mitochondrial membrane phospholipid essential for mitochondrial electron transport, which is degraded in response to cellular insults and disrupts the mitochondrial respiratory chain). In aged rats, an increase in the brain level of platelet-activating factor (a bioactive phospholipid implicated in neuronal excitotoxic death) has also been noted [39]. In rats, attenuation of mitogen-activated protein kinases (MAPKs) and caspase activation have been observed following citicoline administration [40, 41]. Last, but not least, according to the most recent report [42], treatment with citicoline has been found to increase sirtuin-1 (SIRT1) protein levels in cultured neurons, in circulating blood mononuclear cells and in the brain. This effect seems to be of critical importance for neuroprotection in experimental stroke because sirtinol, a specific inhibitor of SIRT1 which, by itself, does not influence infarct volume, has been shown to abolish the neuroprotection offered by citicoline. Citicoline displayed a potent synergistic effect with resveratrol (which is known to be a SIRT1 activator), leading to a 60 % reduction in the experimental infarct volume in rats when both drugs were used in doses that were individually ineffective. Moreover, citicoline was ineffective in SIRT1 knock-out homozygotic mice subjected to focal brain ischaemia. However, detailed mechanistic explanations for all of these effects are lacking. For example, there is no explanation as to how citicoline administration leads to attenuation of MAPK activity and increases sirtuin-1 protein content in brain tissues; in particular, does the drug act extracellularly, or is resynthesis of CDP-choline inside brain cells a prerequisite?

Citicoline is not beneficial in patients with stroke and traumatic brain injury. Positive results of preclinical studies with animal models of neurodegenerative diseases have prompted clinical trials with citicoline as a treatment for human brain diseases. Whereas several previous small clinical studies had achieved promising results, two recent large randomized multicenter trials—the COBRIT (Citicoline Brain Injury Treatment) trial performed in 1,213 patients with traumatic brain injury [43], and the international, randomized, multicentre, placebo-controlled sequential ICTUS (International Citicoline Trial on Acute Stroke) trial performed in 2,298 patients with moderate-to-severe acute ischaemic stroke [44]—led to the conclusion that citicoline is not efficacious in these clinical settings. The negative outcomes of these studies were deemed surprising and prompted a few comments, which focused mostly, although not exclusively, on methodological aspects of the evaluation of the clinical effects of the drug [45–47]. What was not commented on was the lack of a mechanistic explanation for the putative neuroprotective properties of citicoline.

Citicoline treatment seems beneficial in some chronic neurodegenerative diseases. Some recent data are suggestive that prolonged intake of citicoline, given orally or by injection, may be significantly effective in certain slowly developing neurodegenerative diseases. One is glaucoma, currently considered a neurodegenerative disease, which involves the entire central visual pathway. In glaucoma patients with moderate visual defects, citicoline treatment improved retinal function and neural conduction, and continuation of treatment for 2–8 years significantly slowed, stabilized or even improved glaucomatous visual dysfunction [48, 49]. The other is mild vascular cognitive impairment. In the open IDEALE (Studio di Intervento nel Decadimento Vascolare Lieve) study [50], oral citicoline taken for up to 9 months significantly improved the Mini-Mental State Examination score and positively influenced mood; the latter effect could have been related to increases in noradrenaline and dopamine levels, which would be expected on the basis of animal experiments (see the paper by Rejdak et al. [51] and the references cited therein).

Also, it has recently been shown that in sub-acute ischaemic cerebrovascular disease, administration of citicoline in an intravenous dose of 2,000 mg for 5 or 10 days improves functional independence and reduces the burden of care [52]. The uniqueness of citicoline may lie not only in its negligible toxicity and virtual lack of side effects but also in the fact that it appears to deliver a significant subjective improvement and mood-enhancing effect.