The CITIDEMAGE Study: Combined Treatment with a Cholinergic Precursor in Dementia Patients

Certain previous studies have demonstrated the benefits of the addition of citicoline, a cholinergic precursor, to acetylcholinesterase inhibitors (AchEIs) or memantine in patients with Alzheimer’s disease (AD). groups, memantine and AchEIs were used at the highest dosage tolerated. Tests were administered at baseline (T0), after 6 (T1), and 12 months (T2) and included MMSE, ADL, IADL, NPI, CIRS, GDS-short form, the EuroQoL, and the Sleep questionnaire. The primary outcomes were the effects of combined treatment versus AchEIs plus memantine on cognitive functions assessed by MMSE. The secondary outcomes were possible side effects of treatment in both groups, the influence on daily life functions, behavioral symptoms, quality of life, and sleep. Patients in group A showed a mild increase in MMSE at 6 (15.85 ±2.86 vs. 16.39 ±2.93) and 12 months (16.39 ±2.93 vs. 16.43 ±3.08). On comparing the two groups, the difference in MMSE score was significant, both at T1 ( p = 0.003) and T2 ( p = 0.011). Moreover, a significant improvement in GDS and EuroQoL scores was observed. No differences in secondary outcomes, including side effects, were observed between the two groups. This study strengthens the role of citicoline plus AchEIs plus memantine in patients with AD.


Introduction
In the majority of countries, the population is rapidly becoming older; as a consequence, the prevalence of age-related disorders has been increasing. Alzheimer's disease (AD) is the most common form of dementia; its prevalence rate increases exponentially with age, particularly increasing markedly after 65 years and accounting for 60 to 80% of cases. Between the age of 60 and 85 years, a 15-fold increase in the prevalence of dementia, mainly AD, was assessed [1].
The prevalence of AD appears to be considerably higher in the US when compared with Africa, Asia, and Europe, even if it may relate to the methods of diagnosis. About 5.8 million Americans aged 65 years and older were found to be affected with AD in 2020, and 80% were 75 years old or older [2]. According to future projections, the number of people with a diagnosis of AD will increase up to 13.8 million individuals in 2050 [3].
None of the pharmacologic medications available today for AD can slow or prevent the destruction of neurons that cause Alzheimer's symptoms. Drugs approved for the treatment of AD include acetylcholinesterase inhibitors (AchEIs) rivastigmine, galantamine, donepezil, and the Nmethyl-D-aspartate (NMDA) receptor antagonist memantine. The Food and Drug Administration (FDA) also approved a combination of memantine and donepezil. AchEIs are widely used in patients with AD and have shown symptomatic efficacy [4][5][6]. They temporarily improve the cognitive symptoms by increasing the amount of acetylcholine in the brain. Memantine blocks NMDA receptors in the brain from excess stimulation that can damage the nerve cells. The effectiveness of these drugs varies from individual to individual and is limited in duration.
Therefore, AChEIs and memantine may have a complementary and synergistic action in the treatment of AD [7]. As described above, memantine and AChEIs target two different pathological pathways, i.e., the dysfunctional glutamatergic and cholinergic transmitter systems, respectively. In addition, preclinical studies have demonstrated that memantine has neuroprotective effects because it can prevent glutamatergic overstimulation and the consequent neurotoxicity, and prevent the cholinergic fiber loss in the parietal cortex [8,9]. In particular, it inhibits calcium influx into cells that are normally caused by chronic NMDA receptor activation by glutamate [9].
Neuroinflammation is the first step in neurodegeneration as well as in cognitive impairment; it is characterized by non-neuronal cells activation (microglia and astrocytes), leading to toxic mediator release (i.e., reactive oxygen species [ROS], inflammatory cytokines) [10]. However, the final target is represented by the cholinergic neuron; indeed, the cholinergic system is one of the primary system's failures that contribute to the pathogenesis of AD [11]. Furthermore, the role of cholinergic precursors in combination therapy with cholinesterase inhibitors has opened a new possible therapeutic option for the treatment of AD. In contrast, up to now, we do not have any drugs against this disease other than cholinesterase inhibitors and memantine. Aducanumab was recently approved by the Food and Drug Administration (FDA), even if its true efficacy is somewhat controversial [12].
The ASCOMALVA study was a trailblazer, showing the efficacy of donepezil plus choline alphoscerate [13]. However, we have shown the effects of another cholinergic precursor, citicoline. Some years ago, we had reported the benefits on cognition of citicoline 1 g plus AchEIs (The CITIRIVAD and the CITICHOLINAGE studies) [14,15] and recently of citicoline 1 g plus memantine (CITIMEM study) [16]. Recent retrospective studies have demonstrated the effectiveness of citicoline in a combined treatment with AchEIs and memantine [17,18]. Citicoline exerts beneficial effects both in degenerative and vascular cognitive decline; it works through an increase in acetylcholine intra-synaptic levels and by promoting phospholipid synthesis (chiefly phosphatidylcholine), cellular function, and neuronal repair [15,16].
Therefore, on the route traced by our previous studies, we have postulated that coadministration of AchEIs, memantine, and citicoline exert a synergistic action on patients with AD.
The present study showed the effectiveness of oral citicoline plus AchEIs plus memantine in outpatients with AD. This study is called the CITIDEMAGE study (CITIcoline in combination treatment in DEMentia AGEd patients).

Sample Selection
This was a retrospective case-control multicentric study performed between 2016 and 2018 on 169 outpatients with AD aged 65 years old or older (mean age: 78.3 ±5.68 years old). The study involved four different centers for dementia all over Italy.
Patients affected with AD and treated with combination therapy (citicoline + memantine + an AchEI) or memantine + AchEI were randomly selected in a 3-year time range from an original sample of 320 patients.
The inclusion criteria are reported below: -Age of 65 years or older; -People affected with AD; -People had to be on treatment with memantine + AchEI or the combination therapy with citicoline sodium salt 1000 mg/day for at least 3 months, from the scheduled start.
The exclusion criteria were: -Patients with other neurological disorders or taking medications potentially able to interfere with the results of the study (for example, drugs used for cognitive impairment, i.e., cognitive enhancers such as choline alphoscerate, choline bitartrate, L-α-glycerophosphorylethanolamine, homotaurine).
In both groups, memantine and AchEIs were used at the highest dosage tolerated. Memantine tablets 10 and 20 mg were used; among the AchEIs, we included only patients with donepezil (oral tablets 5 and 10 mg) and rivastigmine (transdermal patches: 4.6, 9.5, and 13.3 mg) because of the reduced availability of galantamine.
Diagnosis of AD has been made according to clinical symptoms, neuropsychological tests assessing different cognitive areas, such as memory, language, praxis (i.e., Mini-Mental State Examination, Montreal Canadian Assessment, IWG-2 (International Working Group-2) criteria [19], the presence of atrophy of the medial temporal lobe on brain imaging (CT scan or MRI scan) and/or, if needed, hypometabolism of the temporoparietal cortex, and posterior cingulate/precuneus cortex on 18-fluorodeoxyglucose-PET (FDG PET) [19].
The research was in accordance with national requirements and conform to the principles embodied in the 1964 Declaration of Helsinki (http:/www.wma.net) as well as to the International Ethical Guidelines for Biomedical Research Involving Human Subjects and the International Guidelines for Ethical Review for Epidemiological Studies (http:/www.cioms.ch). Written informed consent was obtained from all patients, or their legal representatives.

Tests
Cognitive functions were assessed by MMSE [20], daily life functions by Activities of Daily Living (ADL) and Instrumental ADL (IADL) [21,22], behavioral symptoms by Neuropsychiatric Inventory Scale (NPI) [23], comorbidities by CIRS (comorbidity index) [24], and mood by Geriatric Depression Scale (GDS)-short form [25]. In addition, the Euro quality of life (EuroQoL-5D) [26] and the Sleep questionnaire were used [27]. EuroQoL-5D describes the health-related quality of life by reporting five dimensions, including mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. It also includes a Visual Analog Scale (VAS), reporting the perceived health status ranging from 0 to 100 (respectively the worst and the best possible health status) [26]. The Sleep questionnaire is an 8-item screening tool reporting concerns about getting to sleep, remaining asleep, sleep quality, daytime personal functioning, daytime performance, duration of sleep problem, nights per week having a sleep problem, and extent troubled by poor sleep [27].

Outcomes
The primary outcomes were: -Effects of combined treatment versus AchEIs plus memantine alone on cognitive functions assessed by MMSE.
The secondary outcomes were: -Possible side effects of combination therapy versus AchEIs plus memantine alone; -Influence on daily life functions, mood, and behavioral symptoms, sleep, and quality of life. Informed consent was signed by all participants or their legal representatives for using their data and medical records.
The study protocol was approved by the Ethics Committee (Italy, Calabria Region, registered protocol on June 21, 2018).

Statistical Analysis
Data are expressed as mean ±standard deviation (SD). Student's t-test or Chi-square test, as appropriate, was used for performing comparisons among groups. Repeated measure analysis of variance (ANOVA) was applied to assess the differences in changes between data values at baseline. ANOVA was also used for assessing the difference between the two groups at 6 (T1) and 12 (T2) months. Significant differences were assumed to be present at p < 0.05.
Statistical Package for the Social Sciences (SPSS) software, program version 23.0 for Windows, was used for performing all analyses.

Results
Age, gender, education, as well as different tests used, MMSE, CIRS, ADL, IADL, NPI, GDS-short form, and EuroQoL at baseline were not significantly different at baseline (Table 1).   The difference in MMSE score was significant when Group A was compared with Group B over time, both at T1 (p = 0.003) and T2 (p = 0.011) (Figure 1).

Figure 1
Inter-group changes in MMSE score over time are shown.
There was also a significant improvement in GDS in patients taking the combined treatment with citicoline (p = 0.000 at T1, 0.008 at T2), but not between T1 and T2 (p = 0.37) (Figure 2) and in EuroQoL scores both at T1 (p = 0.05) and T2 (p = 0.000) (Figure 3) in patients taking the combined treatment.  No significant intra-and inter-group changes were found on ADL, IADL, NPI, and sleep questionnaire over time, as shown in Table 2, or GDS and EuroQoL (Table 3).  Significant differences were found in MMSE, GDS-5 items, EuroQoL between Group A and Group B. Interestingly, regarding MMSE, a non-significant increase was found at T1 and T2 in Group A. A significant difference was found when comparing Group A and Group B.
Regarding GDS and EuroQoL, a significant decrease was recorded in Group A (patients taking the triple therapy) at T1 and T2 (this was indicated by the single asterisk *). The difference was highly evident when comparing Group A and Group B (that is patients taking the triple therapy vs. those taking memantine plus an AchEI) at T1 and T2 (this was indicated by the double-asterisk **).
Side effects were few and superimposable in the two groups, as reported in Table 3. No differences in the side effects rate were recorded between Group A and Group B (14.2% vs. 14.1%, respectively). The main side effects reported were irritability, restlessness, epigastralgia, diarrhea, constipation, dizziness, and itch (Table 3).

Discussion
As previously stated, the potential role of citicoline in a combination treatment with AchEIs and memantine is worth investigating [16]. We conducted this study and preliminary results were presented a few months ago at the 2020 AAIC (Alzheimer's Association International Conference) [28].
The CITIDEMAGE study showed that a combined treatment of citicoline 1 g plus AchEIs plus memantine led to a mild but non-significant increase in MMSE at T1 and T2. Interestingly, the difference in the MMSE score was significant when cases and controls were compared over time, both at T1 and T2.
Furthermore, the benefits of combined treatment of AchEIs plus memantine with citicoline vs. AchEIs plus memantine alone were found on GDS and EuroQoL scores. Indeed, there was a significant improvement in the mood and quality of life perceived and a significant difference in the same scores in the control group at 6 and 12 months. The effects of the triple therapy on cognition, mood, and EuroQoL could be due to the synergistic action of citicoline, AchEIs, and memantine; if on the one hand AchEIs plus memantine exert an effective action on cognition through different mechanisms, the add-on therapy with citicoline, on the other hand, has a remarkable effect on cholinergic, noradrenergic, and dopaminergic neurotransmission, and the synthesis of serotonin via S-adenosyl-methionine (see the following text for details). We believe that the first-ever reported effects on the quality of life could be the consequence of an improved mood.
Some years ago, the Memage Study showed that memantine plus AchEIs slowed down cognitive impairment and decreased agitation and aggression [9]. This is in line with other studies showing the effects of these drugs acting on glutamatergic and cholinergic pathways, respectively [4][5][6][7][8].
In contrast, we have already demonstrated the role of combined treatment of citicoline 1 g with memantine [16] or AchEIs [14,15], respectively. In particular, the CITIMEM Study was the first study assessing the efficacy of citicoline and the N-methyl-D-aspartate (NMDA) receptor antagonist memantine in significantly slowing down the progression of disease in AD and MD patients vs. memantine alone [16]. The effects were found after 6 months and lasted even after 1 year, showing that chronic administration of oral 1 g citicoline is remarkable for obtaining the outcome. This is in line with several different previous studies that have demonstrated the need for chronic administration of this cholinergic precursor [14-16, 33, 34]. The CITIRIVAD and the CITICHOLINAGE Studies showed the efficacy of combination treatment with citicoline 1 g and AchEIs (rivastigmine and rivastigmine or donepezil, respectively) in slowing down cognitive impairment after 3 and 9 months [14,15]. Our remarkable starting points were the VITA and IDEALE studies [46,47], where we demonstrated the effects of parenteral (2 g intravenously) and oral (1 g) administration of citicoline, respectively in vascular cognitive impairment.
Overall, the CITIDEMAGE study has a characteristic that makes it different from all previous and recent studies on combined treatment with citicoline in dementia patients: this is the first study showing the improvement of mood and quality of life perceived with combination therapy in people with AD. Alvarez-Sabin et al. first showed that long-term treatment (2 years) with 1 g oral citicoline significantly improved the cognitive status (p = 0.005) and even the quality of life assessed through the EuroQoL-5D after a first ischemic stroke [48,49].
The putative reasons for explaining why citicoline acts on cognition, mood, and quality of life in AD patients may be substantially assumed to be the following three factors: 1. It is neuroprotective due to the activation of sirtuin, as well as other important factors such as mitochondria membrane stabilization, anti-apoptotic role, and synthesis of membrane phospholipids. Sirtuin-1 is a NAD-dependent protein deacetylase that regulates adult hippocampal neural stem cells, reduces Aβ plaques and Aβ amyloid, activates the transcription of the good secretase, ADAM-10 (a protein with α-secretase activity for proteolytic processing of amyloids), and inhibits NF-кβ, a protein complex that controls DNA transcription and is implicated in synaptic plasticity and memory [36,38,39]; 2. Its metabolic pathway leads to S-adenosyl-methionine, which is a precursor of serotonin and increases the intra-synaptic levels of acetylcholine, noradrenaline, and dopamine [34]; 3. The improved mood might be considered a key factor for the perceived quality of life, henceforth this was the first-ever study showing improved EuroQoL scores after combination treatment with citicoline plus memantine plus an AchEI. Citicoline combined with an AchEI and memantine together could not only potentiate cholinergic neurotransmission but work as possible excitatory amino acid transporter activator (EAAT2), thus reducing the glutamate efflux [50]. Its effects on mood have already been postulated through the above-mentioned mechanism on the increase in S-adenosyl-methionine and have been confirmed by a recent study that focused on post-stroke patients [51]. This article showed the effects of cholinergic precursors, such as citicoline, on post-stroke depression through its pleiotropic mechanisms [51].
However, the CITIDEMAGE study presents strong limitations: 1) sample size limited to a few patients; 2) kind of the study, retrospective, unblinded, and observational; 3) the need for further confirmation for our hypotheses, which could derive from an openlabel, blinded, and wider study. We strongly argue that this old drug has been misused so far, especially because of unproportionate expectations from short-time administration. 4) the presence of a possible recall bias due to the observational and retrospective nature of the study.

Conclusions
In light of the importance of maximizing the pharmacological means in AD, the present study encourages the role of combined administration of AchEIs plus memantine plus citicoline in disease management. This is one of the first studies assessing the effectiveness of combination treatment with citicoline after 12 months. Furthermore, unlike certain previous studies or even recent studies that have tried to focus on the effectiveness of citicoline, this is one of the first studies assessing the benefits on the mood and quality of life perceived. Our study showed an improvement in mood and the quality of life, both at 6 months and 1 year.
The identification of biomarkers for AD enables early detection of the disease and could accelerate the development of new treatments. In the future, more will be understood about the most effective therapies against AD. In the meanwhile, our duty as medical doctors taking care of patients affected with dementia is to try to optimize the available treatments. To this purpose, using all the drugs available, including combination treatment with citicoline, appears a reasonable tool to slow down the disease progression.