An oral solution based on citicoline and nicotinamide for use in the treatment of glaucoma.

Abstract

The present invention relates to a composition for oral use in the form of an aqueous solution comprising citicoline and nicotinamide, and said composition for use in the treatment of glaucoma.The present invention further relates to a pharmaceutical form in aqueous solution characterized by high bioavailability.

Description

[Technical Field]

[0001] The present invention generally relates to a composition for oral use in the form of an aqueous solution containing citicoline and nicotinamide. The composition is used for the treatment of neurodegenerative conditions, and in particular for the treatment of glaucoma. [Background technology]

[0002] Glaucoma is a chronic neurodegenerative ophthalmic condition characterized by damage to retinal ganglion cells (RGCs), optic nerve fibers, and progressive visual field loss. It is the second leading cause of irreversible blindness, and the most frequent and insidious form is open-angle glaucoma (OAG). Age, proficiency, severe myopia, and high intraocular pressure (IOP) are just some of the risk factors that can increase the likelihood of developing the disease. High IOP represents the only modifiable risk factor, and therefore, the primary treatment approach is based on eye drops, or, if insufficient, laser or surgery.

[0003] Histological and in vivo imaging studies in humans have demonstrated that, in the presence of glaucoma, the entire optical pathway is involved in synaptic degenerative processes that damage the optic nerve and brain structures, such as the geniculate nucleus and visual cortex, which are the seat of the primary visual region. Such processes, activated by high-voltage injury, result in impaired visual function characterized by progressive thinning of the retinal nerve fiber layer, progressive erosion of the optic disc, and subsequent onset and progressive enlargement of scotomas (non-visual areas) in the visual field, both anatomically and functionally.

[0004] Recent research has demonstrated that glaucoma is a neurodegenerative condition similar to Alzheimer's and Parkinson's disease. These are diseases in which neurons located at different loci are involved in degeneration and death through apoptosis. In Alzheimer's disease, this process begins in the hippocampus; in Parkinson's disease, it begins in the substantia nigra; and in glaucoma, it begins at the level of retinal ganglion cells.

[0005] Following primary injury with a high-pressure nature, apoptosis of neurons is triggered at the capillary level of this structure, disrupting normal blood supply and impairing the regular axonal transport of metabolites and neurotrophins essential for ganglion cell survival, both anterograde and retrograde. Apoptosis is the cause of secondary injury related to a mechanism of local excitotoxicity resulting from excessive stimulation of NMDA receptors by glutamate released by the cell during apoptosis. In fact, glutamate, when present in excess concentration in the extracellular space, can cause Ca 2+ Overstimulation of NMDA receptors on the surface of surrounding neurons, which determines the opening of the channel, triggers a biochemical cascade leading to apoptosis of the neuron itself, and subsequently establishes a mechanism for self-supply even without primary injury.

[0006] Glaucoma has been shown to damage neurons through different mechanisms among oxidative stress, neuroinflammation, and mitochondrial dysfunction. Oxidative stress, associated with the accumulation of free radicals in the presence of high IOPs, affects particularly sensitive cells, such as CGR, by jeopardizing the functionality of mitochondria, intracellular organelles that provide energy and ensure different functions (e.g., regulation of cellular homeostasis). Retinal ganglion cells have more mitochondria than any neurons in the central nervous system because they require very high and continuous energy intake for their precise operation. When oxidative stress damages mitochondria, they fail to supply the amount of energy necessary for retinal ganglion cells, which become dysfunctional and, in the most severe cases, undergo apoptosis.

[0007] The functional consequence of this degeneration, which involves the optical path, is a gradual narrowing of the visual field, with non-visual areas (scotomas) generally appearing first in the periphery and then extending to the central region until complete vision loss occurs.

[0008] Despite effective pressure control, several clinical studies have confirmed that glaucoma patients experience progressive damage that can persist for several years, resulting in visual field loss. The Early Manifest Glaucoma Trial (EMGT) study conducted by the National Institutes of Health highlighted that 45% of glaucoma patients had progressive visual field loss, even in low blood pressure controls.

[0009] Several studies have evaluated the possibility of reducing or at least slowing down such degenerative processes by taking molecules called neuroprotective molecules, which are directly aimed at maintaining CGR functionality and reducing vulnerability, in addition to antihypertensive drugs.

[0010] Particular interest in the field of neuroprotection has been directed towards citicoline (cytidine-5'-diphosphocholine) and nicotinamide (i.e., vitamin B3), focusing on their mechanisms of action and experimental and clinical results obtained in glaucoma patients.

[0011] The neuroprotective properties of citicoline in glaucoma were evaluated for the first time using an injectable formulation that has been used for some time in neurodegenerative conditions of the central nervous system. Virno et al. demonstrated, in a 10-year follow-up study, a reduction in peripheral defects, assessed as unperceptible areas, obtained by treatment with intramuscular citicoline in glaucoma patients. These results were confirmed by a further long-term study (8-year follow-up), which confirmed the efficacy of citicoline through significant improvements in retinal and cortical bioelectrical responses in OAG patients.

[0012] Intramuscular administration of citicoline to treat glaucoma has proven effective in glaucoma patients, even though it is clearly very uncomfortable for chronic patients and for those who need to be able to administer injections.

[0013] The results obtained in systemic studies facilitated the development of citicoline-based oral formulations, resulting in increased compliance with treatment in glaucoma patients. In patients with controlled IOP (<18 mmHg) treated with b-blockers, a study showed that treatment with oral citicoline induced improvement in PERG signal width (p<0.01) and reduction in VEP latency time (p<0.01) compared to checks against baseline. Based on these results, an oral solution formulation was developed, and the following clinical trial (1), conducted at the University of Palermo, demonstrated that citicoline in oral solution (500 mg / die) delayed nerve fiber loss in the retinal layer via GDX in patients treated with antihypertensive therapy alone, in a double-blind, placebo-free environment. It was further demonstrated that cycling of citicoline in oral solution enables delaying electrophysiologically demonstrated loss of visual function (latency time in pEV and width in pERG). Citicoline in oral solution actually improved electrical function parameters, particularly by increasing the width of the P50-N95 region of the pERG by 100%.

[0014] As far as nicotinamide is concerned, a very recent experimental study conducted at the Jackson Laboratory / Howard Hughes Medical Institute and Bascom Palmer Eye Institute in Miami, whose results were published in the journal Science, demonstrated that vitamin B3 reduces the incidence of glaucoma in the treated group (2). The researchers performed a series of genetic, metabolic, and neurobiological tests on a group of mice genetically modified to be susceptible to glaucoma, as well as a group of healthy controls. The results showed that nicotinamide levels decreased with age, and that administration of this molecule in addition to drinking water protected the mice from developing glaucoma.

[0015] In light of the above, the problem of providing a novel composition for treating glaucoma remains very real. [Overview of the project]

[0016] The technical problem addressed and solved by the present invention is to provide a composition for oral use for the treatment of glaucoma, characterized in particular by high bioavailability comparable to that of intravenously administered solutions.

[0017] These problems are solved by compositions for oral use in the form of aqueous solutions based on citicoline and nicotinamide. Glaucoma is actually multifactorially pathogenic and is characterized by intersynaptic degeneration that runs along the optical pathway from retinal ganglion cells to the visual cortex, leading to gradual loss of visual field.

[0018] Advantageously, the authors of this invention have found that the formulations described herein in aqueous solution determine the very high bioavailability of citicoline and nicotinamide, compared to those obtained when these active ingredients are administered intravenously. When the active ingredients are dissolved, in practice, they are in a form readily available for the absorption step, which ensures higher bioavailability compared to when they are administered in a solid state, for example, as tablets or suspensions.

[0019] Therefore, compared to prior art formulations, the compositions of the present invention have enormous advantages in that they combine the high bioavailability of these two active ingredients, comparable to that of intravenous administration, with the classic advantages of oral routes, such as high compliance, low cost, and safety. As highlighted by the results of clinical studies presented in the experimental section of this application, administration of the compositions for oral use in the form of aqueous solutions as described herein to patients with glaucoma has been found to be remarkably more effective, particularly in terms of improving visual function and structural protection of the retinal layer, compared to what could be predicted based on the combined effects of citicoline and nicotinamide administered individually, by confirming the synergistic action between the two active ingredients in the compositions of the present invention.

[0020] Therefore, the present invention relates to a composition for oral use in the form of an aqueous solution containing choline and nicotinamide, and to its use in the treatment of glaucoma.

[0021] The present invention also relates to a method for preparing the composition.

[0022] Preferred features of the present invention are described in the dependent claims.

[0023] Other advantages, features and forms of use of the present invention will become apparent from the following detailed description of some embodiments shown by way of example and not for limitation.

[0024] The following detailed description of the present invention and preferred embodiments can be better configured with reference to the following figures.

Brief Description of the Drawings

[0025] [Figure 1] Bioavailability curves of choline (1a) and nicotinamide (1b) administered intravenously, orally as a solution and in tablets. The bioavailability of choline and nicotinamide in the oral solution is close to 100%. [Figure 2] Flow diagram of the method for manufacturing an aqueous solution according to a preferred embodiment. [Figure 3] Quantitative analysis of the effect of several treatments described in Example 2 on electroretinogram abnormalities evaluated by ERG. [Figure 4] Effect of several treatments described in Example 2 on cell apoptosis (TUNEL assay). [Figure 5] Effect of several treatments described in Example 2 on the level of caspase-3. [Figure 6] Comparison of immunofluorescence of synaptophysin (red) between healthy control group, placebo-treated group, and choline + nicotinamide-treated group.

Modes for Carrying Out the Invention

[0026] The present invention relates to a composition for oral use in the form of an aqueous solution containing citicoline and nicotinamide.

[0027] Citicoline has the following structure: [ka] It is a nootropic drug with widely recognized efficacy in central nervous system conditions such as Parkinson's disease, multiple sclerosis, trauma and brain injury, cerebrovascular events, and cognitive impairment.

[0028] Citicoline acts at various levels through a multifactorial mechanism: it preserves levels of cardiolipin and sphingomyelin (phospholipid components of the mitochondrial inner membrane and retinal ganglion cell axon membranes, respectively), restores phosphatidylcholine (one of the most abundant phospholipids in nerve cell membranes), preserves mitochondrial function by preventing oxidative damage and the initiation of neuronal apoptosis, prevents glutamate excitotoxicity by stimulating glutathione synthesis and reducing its concentration, stimulates myelin synthesis, improves neuronal membrane integrity, increases the synthesis of neurotransmitters such as acetylcholine and dopamine, and prevents endothelial dysfunction. In particular, its effects on phospholipids are the basis for its structural effects, which are accompanied by further functional effects on neurotransmitters.

[0029] Recent evidence demonstrates that citicoline can interact with the proteasome (a molecular complex involved in protein degradation at the cellular level). Dysfunction affecting the proteasome leads to protein accumulation, as is typical in neurodegenerative conditions. Citicoline significantly stimulates the enzymatic properties of the complex with respect to proteins, including amyloid-forming proteins (α-synuclein), and thus exerts neuroprotective effects.

[0030] Citicoline, when ingested orally or parenterally, is immediately broken down into P-choline and cytidine-5'-monophosphate (CMP). Since phosphorylated compounds do not cross the blood-brain barrier, P-choline and CMP are further dephosphorylated to choline and cytidine. Choline crosses the blood-brain barrier and is phosphorylated to P-choline at the SNC, while cytidine is converted to uridine in humans, then crosses the blood-brain barrier again, is phosphorylated to uridine triphosphate (UTP) at the SNC, and then converted to CTP. P-choline and CTP are reconstituted as citicoline thanks to the action of the cytidine-5'-triphosphate phosphocholinecytidyl-transferase enzyme, a reversible reaction whose direction and rate depend on the substrate concentration. From this, a higher fundamental importance of the bioavailability of the collected preparations compared to other compounds is derived.

[0031] Regarding excretion, excretion from feces was minimal (an indicator of optimal absorption), while excretion via the respiratory (CO2) and urinary pathways was observed. Demonstrating that the catabola were incorporated into biological structures, only 16% of the marked product was excreted over 5 days. Excretion was biphasic, with the first phase occurring within a few hours and the second phase being very slow. This process further demonstrates that absorbed citicoline is incorporated into the cell membrane and then excreted according to the physiological metabolic fate of the biological structure.

[0032] Nicotinamide, or vitamin B3, has a structure shown in the image below. [ka] Nicotinamide belongs to the group of water-soluble vitamins that must be regularly obtained through food. It plays an important role in oxidation / reduction reactions and is a precursor of NAD and NADP oxidative reductase coenzymes involved in catabolic and anabolic processes. Thanks to the mechanism of action described above, nicotinamide explains various functions, and in particular, it plays a fundamental role in the functioning of the nervous system by promoting synaptic plasticity and axonal growth.

[0033] Similar to aging, nicotinamide deficiency and the resulting decrease in NAD and NADP make the optic nerve and nerve cells more susceptible to increased intraocular pressure, thus predisposing to the development of glaucoma.

[0034] Intestinal absorption of orally ingested nicotinamide is high, reaching up to 70%. This occurs mostly by diffusion mediated by sodium-dependent carriers. Nicotinamide is the primary form of vitamin B3 present in the bloodstream. From the blood, it moves across cell membranes by simple diffusion, but transport in the renal tubules and red blood cells requires carriers. Within cells, nicotinamide can be used to synthesize NAD, which can then be phosphorylated to NADP. Both can accept two electrons and one proton, forming NADH and NADPH. Nicotinamide is converted to NAD by reaction with 5-phosphoribosyl-1-pyrophosphate, and ATP and NAD are converted to NADP by reaction with ATP. The main pathway of nicotinamide catabolism is methylation and subsequent oxidation in the liver. Nicotinamide is metabolized to N-methylnicotinamide (NMN), which is then converted to ATP and Mg 2+ Furthermore, S-adenosylmethionine is involved as a methyl donor. NMN can be oxidized to N-methyl-2-pyridone-carboxamide (2-Pyr) and N-methyl-4-pyridone-carboxamide (4-Pyr), both of which are present in plasma and urine.

[0035] According to one embodiment of the present invention, citicoline is present in the composition at a concentration in the range of 25 mg / ml to 100 mg / ml, preferably 49.75 to 50.25 mg / ml, or at about 50 mg / mL, and nicotinamide is present at a concentration in the range of 5 mg / ml to 20 mg / ml, preferably 9.95 to 10.05 mg / ml, or at about 10 mg / mL.

[0036] In further embodiments, the composition has a ratio of citicoline free acid to nicotinamide, preferably contained in a ratio of 2.5:0.5 to 10:2.

[0037] According to a preferred embodiment, the composition has a ratio of citicoline free acid to nicotinamide equal to 5:1.

[0038] Another embodiment of the present invention, according to other embodiments described herein, may contain potassium sorbate in concentrations ranging from 1.34 mg / ml to 3 mg / ml, preferably from 2.67 mg / ml to 2.69 mg / ml, or about 2.68 mg / mL.

[0039] Potassium sorbate is represented by the following formula: [ka] It is used as a preservative, particularly in the cosmetics and food industries, for its antifungal and antibacterial properties. It is active only at acidic pH and is highly soluble in water. It is synthesized by reacting naturally occurring ascorbic acid with potassium hydroxide (KOH).

[0040] According to one embodiment of the present invention, the composition may further contain 60% sodium lactate at a concentration in the range of 2.5 mg / ml to 7 mg / ml, preferably 4.975 mg / ml to 5.025 mg / ml, or at about 5 mg / mL.

[0041] The formula for sodium lactate is shown in the image below. [ka] It is a sodium salt of lactic acid, which has naturally occurring antioxidant properties. It also acts as an acidity modifier, wetting agent, antistatic agent, emulsifier, and / or thickener. It is a water-soluble compound and is not particularly sensitive to high temperatures.

[0042] According to another embodiment of the present invention, the composition may further contain fructose in a concentration ranging from 150 mg / ml to 400 mg / ml, preferably from 298.5 mg / ml to 301.5 mg / ml, or about 300 mg / mL.

[0043] Fructose has the following structural formula: [ka] It is a topological isomer of the glucose monosaccharide and is distinguished from aldoses by being a ketose. It is important in human and animal nutrition and is often used as a sweetener because, compared to sucrose, it has a greater sweetness, slightly lower calorie intake, and a lower blood glucose index.

[0044] Further embodiments of the present invention may include concentrations ranging from 0.92 mg / ml to 2.00 mg / ml, preferably from 1.82 mg / ml to 1.84 mg / ml, or even about 1.83 mg / mL of acid anhydride.

[0045] The citric acid shown in the image below is [ka] It is used as a flavoring and preservative in food and beverages. Furthermore, it is used as an acidulant, emulsifier, lemon juice substitute, and to correct the pH of dyes. It is also used in combination with sodium bicarbonate in effervescent preparations.

[0046] A preferred embodiment provides that a composition according to any one of the described embodiments comprises, or is substituted with, citicoline at a concentration equal to 50 mg / mL, nicotinamide at a concentration equal to 10 mg / mL, potassium sorbate at a concentration equal to 2.68 mg / mL, 60% sodium lactate at a concentration equal to 5 mg / mL, fructose at a concentration equal to 300 mg / mL, and citric acid at a concentration equal to 1.83 mg / mL.

[0047] According to one embodiment of the present invention, the composition may further comprise one or more excipients and / or additives, according to other embodiments described herein. Characteristic examples of excipients for compositions for oral use in liquid form include sorbitol, glycerol, sucrose, glycerin, sodium phosphate, methyl p-oxybenzoate, propyl p-oxybenzoate, and water-soluble fragrances.

[0048] One embodiment of the present invention provides that the composition, according to any one embodiment described herein, has high bioavailability of citicoline, in particular, that its bioavailability is 90% to 100%, preferably at least 95%, and preferably at least 98%.

[0049] Under the term "bioavailability," this refers to the amount of the active ingredient in the bloodstream, in this case citicoline, which defines its biological availability. It is assumed that there is a correspondence between tissue concentration and plasma concentration, since equilibrium is established between the central compartment and peripheral tissues. The bioavailability profile is described by a plasma concentration-versus-time curve, and the pharmacokinetic parameters used to describe it are the maximum plasma concentration (C), which is directly proportional to the absorption rate. max ), the time (t) to reach the maximum plasma concentration which is inversely proportional to the absorption rate max Bioavailability is the area under the curve (AUC), which is directly proportional to the amount of active ingredient absorbed. Bioavailability can mean both absolute and relative.

[0050] In particular, the term "absolute bioavailability" refers to the ratio of the area under the curve for the extravascular route (in this case, the oral route) to the area under the curve for the intravenous route, each corrected according to the administered dose. This parameter allows us to understand how much less citicoline and nicotinamide are absorbed when administered orally, considering that 100% bioavailability is achieved only intravenously and the absorption step is missing.

[0051] In contrast, the term "relative bioavailability" refers to the ratio of the areas under the curve produced by the same active ingredient administered via two different routes other than intravenous administration, or by the same active ingredient administered via the same route but in different formulations (e.g., an active ingredient formulated as a tablet and an oral solution).

[0052] Unless otherwise specified herein, percentages of bioavailability refer to absolute bioavailability.

[0053] According to a preferred embodiment, the composition is formulated as an aqueous solution.

[0054] Compositions for oral use can vary considerably among them in terms of bioavailability, rate, and absorption profile, and the formulation is the determining factor, given that the active ingredient is the same. Formulations for oral administration can be solid (tablets, capsules, late formulations, gastric protective formulations, etc.) or liquid (suspensions and solutions). Regardless of the formulation used, drugs administered orally must be dissolved in gastrointestinal fluid for absorption. The oral solid formulation then must release the active ingredient. This is a two-step process involving the breakdown of the solid formulation and the solubilization of the drug itself. Different solid forms of the same active ingredient can have different absorption profiles depending on the breakdown and solubilization time, which can vary significantly depending on the excipients used, external factors, or chemical-physical properties.

[0055] Liquid formulations (such as syrups, phases, and sachets to be dissolved) can be suspensions or solutions, and in fact, the latter can guarantee greater bioavailability. Formulations in suspension actually have the active ingredient in the form of fine solid particles suspended in a liquid phase, in which case the particles should enter the solution to be absorbed without the need for a disruption step. Instead, solutions advantageously have the active ingredient already dissolved and then readily available for the absorption step, which guarantees greater bioavailability.

[0056] According to further embodiments of the present invention, based on any one of the embodiments described herein, the aqueous solution has a pH in the range of 3.9 to 4.1, preferably 4.00.

[0057] According to further embodiments of the present invention, based on any one of the embodiments described herein, the aqueous solution has a density of 1.116 to 1.156 g / ml, preferably 1.136 g / ml.

[0058] According to one embodiment of the present invention, the composition is for use in treating neurodegenerative conditions.

[0059] Under the term "neurodegenerative diseases," a range of conditions affect all neurons, particularly those in the central nervous system. These are debilitating and incurable conditions that cause progressive degeneration and / or death of nerve cells.

[0060] According to a preferred embodiment of the present invention, the composition is intended for use in the treatment of glaucoma. Scientific literature has demonstrated that citicoline has a neuroprotective effect by slowing the progression of visual field damage in glaucoma patients, and that nicotinamide improves visual field by increasing the functionality of the inner retinal layers. Since the action of nicotinamide is combined with the neuroprotective effect of citicoline in certain formulations with high bioavailability, the composition of the present invention offers the potential to provide neuroprotection in two ways.

[0061] According to one embodiment of the present invention, the composition is administered once a day, preferably in the morning, in a volume of 5 ml to 20 ml, or about 10 ml, at the above-mentioned dosage.

[0062] The present invention further relates to a method for preparing a composition described in any one of its embodiments. In particular, such a method provides dissolution in water and mixing of the elements constituting the composition.

[0063] example Example 1. Preparation of a composition according to the present invention and bioavailability testing. An aqueous solution containing potassium sorbate (2.6 mg / mL), 60% lactic acid solution (5 mg / mL), fructose (300 mg / mL), citicoline free acid (50 mg / mL), nicotinamide (10 mg / mL), and anhydrous citric acid (1.83 mg / mL). Appearance: Transparent solution Color: Colorless pH: 4.00 Density: 1.136g / ml

[0064] Preparation method: An aqueous solution was prepared using citicoline free acid, nicotinamide, and excipients commonly used in this type of formulation, such as potassium sorbate as a preservative, sodium lactate and citric acid as acidity adjusters, and fructose as a sweetener. For relative solubility in water, the components were weighed and mixed according to the above amounts. The pH and density of the solution were adjusted to the above values. The composition was prepared according to the flow chart shown in Figure 2.

[0065] method Bioavailability research The bioavailability of citicoline present in the composition according to the present invention was investigated by bioavailability testing in Sprague-Dawley rats. In particular, 30 male Sprague-Dawley rats weighing 175g and 225g were fasted 16 hours prior to administration. The animals were divided into three groups: Group 1: 10 rats treated intravenously with citicoline + nicotinamide. Group 2: 10 rats treated with citicoline + nicotinamide in oral solution. Group 3: 10 rats treated with citicoline + nicotinamide in tablet form.

[0066] 14C-methylciticoline and 714C-nicotinamide were used to prepare the radiolabeled formulation.

[0067] The preparations were purified by ion exchange chromatography using isobutyric acid:water:ammonia:EDTA (500:280:21:8), n-butanol:acetic acid:water (12:3:5), ethanol:1 mol / l ammonium acetate pH 7 (5:2), and ethanol:saturated borax (8%):0.5 mol / l EDTA:5 mol / l ammonium acetate (220:80:0.5:20).

[0068] 14 The sample was analyzed using a liquid scintillator counter (LSC) according to program C.

[0069] For quantitative analysis, Packard soluene (solubilizer), H2O2 (bleaching agent), and Packard Instagel (scintillator) were used as reagents.

[0070] Each formulation was administered at a dose of 5 mg / kg: Group 1, intravenously administered into the jugular vein; Group 2, administered orally via a nasogastric tube; Group 3, administered as a tablet dissolved in physiological saline via a nasogastric tube.

[0071] Approximately 100 ml of blood samples were collected at programmed times (after administration at 10', 20', 30', 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, and 24 hours).

[0072] Absolute bioavailability was calculated by the ratio of the area under the curve (AUC) of the mean values ​​obtained for intravenous administration / aqueous solution and intravenous administration / tablet administration, respectively (Figure 1).

[0073] Among the pharmaceutical forms for oral administration, the formulation in an aqueous solution based on citicoline + nicotinamide showed a greater bioavailability of its active ingredients compared to tablets. The bioavailability of citicoline + nicotinamide administered in an aqueous solution resulted in a bioavailability comparable to that of intravenous administration. After administration of an aqueous solution based on citicoline + nicotinamide 14 Bioavailability of citicoline:

Number

Number

[0074] The results obtained demonstrated the bioavailability of citicoline and nicotinamide formulated as a composition for oral use in an aqueous solution, which is close to 100%. Such results are due to the pharmaceutical form, as the oral solution makes the active ingredients immediately available for absorption and guarantees a bioavailability comparable to the injection route (Figure 1).

[0075] Example 2. In vivo study on an experimental model of neurodegeneration in animals Method The neuroprotective effect of the citicoline + nicotinamide association in an oral solution was evaluated in an experimental study conducted on db / db mice. Db / db mice have a mutation in the leptin receptor gene and are an animal model of retinal neurodegeneration. Such a model is useful for mimicking the pathophysiological events seen in neurodegenerative pathologies such as glaucoma.

[0076] Twenty db / db rats (BKS.Cg-+Leprdb / +Leprdb / OlaHsd, male, 10 weeks old) were randomized into five groups and treated orally for 15 days with an oral solution based on the following criteria. 1. Administer 0.5% citicoline (Cit.) in a 10% lactose solution via nasogastric tube (SNG). 2. Nicotinamide 0.1% (Nic.) in 10% lactose solution is administered via SNG. 3. Administer citicoline 0.5% + nicotinamide 0.1% (Cit+Nic) in a 10% lactose solution via SNG. 4. 10% lactose solution (placebo) was administered via SNG. 5. Healthy, non-diabetic control group (control group).

[0077] On day 15, one hour after administration of several treatments, mice were subjected to (i) very high-resolution spectral domain optical coherence tomography (SD-OCT) to obtain detailed images of the morphology of different retinal layers in vivo. Mice were anesthetized by a single intraperitoneal injection of 80 mg / ml ketamine and 12 mg / ml xylazine (Sigma-Adrich). Subsequently, one drop of 0.5% tropicamide was added to induce pupillary dilation, and then each mouse was placed in front of the OCT objective lens.

[0078] For all animals, only the right eye was examined, and the average of three consecutive retinal scans for each eye was considered for each layer. OCT images were acquired using ART (Automated Real-Time) with a 30-degree field of view focused on the head of the optic nerve, combined with noise reduction software and two-dimensional scanning (B-scan). After the OCT examination, the animals were euthanized by cervical dislocation, and the eyes were immediately removed.

[0079] (ii) Retinal cell electrical activity and then functionality were evaluated via full-field electroretinography (ffERG). Recordings were made using the Ganzfeld ERG platform (Phoenix Research Laboratories, Pleasanton, CA, USA) and measured according to the recommendations of ISCEV (International Society for Clinical Electrophysiology of Vision). We studied neurodegenerative histological markers (glial activation and apoptosis) through immunohistochemical assays:

[0080] (iii) Glial activation was evaluated by confocal laser scanning microscopy using an antibody against glial fibrillary acid protein GFAP (rabbit anti-GFAP). A scoring system (1-5) was used based on the elongation of GFAP (glial fibrillary acid protein) staining to assess the level of glial activation.

[0081] Sections were fixed in methanol (-20°C) for 2 minutes, followed by three washes with physiological saline phosphate buffer (PBS) (5 minutes each). Sections were treated with Tris Saline Buffer TBS-Triton X-100 (0.025%) and incubated at room temperature for 2 hours in a 1% solution of bovine albumin serum BSA and 10% goat serum in PBS. Sections were then incubated with rabbit anti-GFAP overnight at 4°C in a moist atmosphere. After three washes with PBS (5 minutes each), sections were incubated with the secondary antibody Alexa AF488 goat anti-rabbit (dilution 1:200 prepared in blocking solution). Sections were washed with PBS, stained with Hoestch, and assembled on Fluorescence Mounting Medium with coverslips. Once the mounting support was dry, comparative digital images of the samples were recorded using a confocal laser scanning microscope, Fluoview FV 1000 Olympus (Olympus, Shinjuku-ku, Tokyo, Japan), with the same brightness and contrast settings.

[0082] (iv) Cellular apoptosis events were investigated using a TUNEL assay with the Kit DeadEnd® Fluorometric TUNEL System (PROMEGA, Madison, WI, USA). TUNEL (Terminal Transferase dUTP Nick-End Labeling) staining was performed using the Kit System TUNEL Fluorometric DeadEnd (PROMEGA, Madison, WI, USA). Frozen sections of the retina were permeabilized by incubation with 0.1% Triton X-100 in 0.1% sodium citrate on ice for 2 minutes. The secondary antibody was Alexa 488 goat anti-rabbit. Evaluation by confocal laser scanning microscopy showed an excitation wavelength of 488 nm (detected in the range of 515-565 nm (green)).

[0083] (v) Caspase-3 and synaptophysin were quantified by immunofluorescence. Sections were fixed in methanol (20°C) for 1 minute and washed with 0.01 M buffered saline (PBS) at pH 7.4. The sections were then incubated in 10% solution NGS, 0.1% Triton X-100, and PBS at room temperature for 1 hour, followed by incubation with specific primary antibodies (rabbit anti-caspase-3 and anti-synaptophysin monoclonal antibodies, respectively) overnight at 4°C. The following day, after washing, the sections were incubated with the fluorescent secondary antibody Alexa 594 (anti-rabbit - Life Technologies SA, Madrid, Spain) for 1 hour, followed by washing. Finally, the nuclei were stained with Hoechst and assembled on Fluorescence Mounting Medium (Prolunga, Invitrogen) with coverslips. Images were acquired using a confocal laser scanning microscope (FV1000; Olympus, Hamburg, Germany). Five visual fields (three corresponding to the central retina and two corresponding to the peripheral retina) were analyzed. Fluorescence intensity of the images was quantified using ImageJ software.

[0084] Statistical analysis was performed using Student's t-test and ANOVA tests on independent data. A value of p < 0.05 was considered statistically significant.

[0085] result (i) Evaluation of retinal thickness OCT: The results obtained from measurements using SD-OCT (shown in Table 1) highlighted significant changes in the thickness of the RNFL (retinal nerve fiber layer), GC / IPL (ganglion cell / internal plexus layer), and INL (internal core layer) in the placebo group (untreated db / db mice) compared to healthy controls. Diabetic mice treated with citicoline alone and those treated with nicotinamide alone showed less reduction in layer thickness, and significant differences were observed in the RNFL and GC / IPL layers compared to the placebo group (p<0.05). Surprisingly, such changes were reversed in the group treated with citicoline + nicotinamide, highlighting the absence of the aforementioned difference in layer thickness compared to healthy controls, thus suggesting a synergistic protective effect of the combination on retinal ganglion cells (axons and somatic cells) against the toxic effects of long-term diabetes (p<0.01). [Table 1]

[0086] (ii) Evaluation electroretinogram: Figure 3 shows a quantitative analysis of wave b width in response to flash intensity in various groups. In db / db mice, a decrease in wave b width in the ERG was observed in response to both low intensity (10 cd s / m2) and average intensity (40 cd s / m2) stimuli. Such a decrease was attenuated compared to placebo in both the Cit. and Nic groups. This effect was synergistically enhanced in the Cit+Nic group and was statistically significant compared to the placebo group (p<0.05).

[0087] (iii-v) Immunohistochemical assay: (iii) In the healthy control group, GFAP protein expression was observed mainly at the GCL layer level. In contrast, placebo-treated diabetic mice highlighted a significant increase in all retinal layers, indicating glial hyperactivation due to neurodegenerative inflammatory processes. Indeed, 100% of diabetic mice in the placebo group showed a GFAP score of ≥3 (Table 2). Administration of citicoline and nicotinamide resulted in a significant reduction in reactive gliosis, with GFAP scores of ≤3 in both cases. Surprisingly, such scores showed considerable improvement in the group treated with Cit.+Nic combination, reaching 1 in 94% of diabetic mice (≤2 in 100%). [Table 2]

[0088] (iv) Through the TUNEL assay, the percentage of apoptotic cells in the GCL retinal layer was significantly higher in diabetic mice than in non-diabetic control mice, thus demonstrating a significant increase in the apoptotic phenomenon affecting retinal ganglion cells after the neurodegenerative process induced by diabetes (Figure 4). Diabetic mice treated with citicoline only had a lower apoptosis rate compared to diabetic mice treated with vehicle (p<0.05). Even mice in the Nic. group showed lower apoptosis levels than diabetic mice treated with vehicle, but not at a statistically significant level. Surprisingly, the results for the Cit+Nic group highlighted a synergistic effect in reducing and preventing apoptosis, with statistically significant differences compared to single treatment and the placebo group, but not statistically significant compared to the control group. Such results can be compared to the synergistic effect of the multifactorial action mechanism of the two active ingredients: citicoline and the antioxidant cytotoxic effect of nicotinamide. Such a synergistic effect is involved in a potent protective effect on cellular structure and prevention of apoptosis, thus guaranteeing an important neuroprotective effect.

[0089] (5) The anti-apoptotic and protective role of synapses was confirmed by results related to the levels of caspase-3 and synaptophysin observed in various groups. Caspase-3 is an enzyme involved in programmed cell death that actively participates in the apoptotic process, and its levels reliably increase during the neurodegenerative process. In db / db mice, a statistically significant increase in caspase-3 levels was highlighted compared to the control group. Such levels were considerably reduced in the Cit+Nic group, showing a statistically significant difference not only compared to the placebo group but also compared to single treatment (Cit.;Nic) administered alone (Figure 5). Synaptophysin is a protein present in presynaptic vesicles and is the primary indicator of synaptic loss and the resulting neuronal death. In placebo-treated db / db mice, a statistically significant downregulation of synaptophysin was observed compared to the control group (p<0.05). In both the Cit. and Nic. groups, a statistically significant increase in synaptophysin levels was observed, reaching levels comparable to those of healthy controls in the Cit+Nic group, demonstrating higher efficacy against the same single-dose molecule (Figure 6). These results reaffirm the potent synergistic effect of citicoline + nicotinamide association, particularly its anti-apoptotic mechanism, which would represent an important neuroprotective therapy for neurodegenerative conditions where apoptosis is the primary cause of the neurodegenerative process.

[0090] Example 3. Clinical observation of a patient with open-angle glaucoma. The neuroprotective effects of the citicoline + nicotinamide combination in an oral solution according to the present invention were evaluated in clinical observations conducted on patients with open-angle glaucoma. Selected patients had to have been diagnosed with open-angle glaucoma for at least two years, be controlled in terms of intraocular pressure (IOP ≤ 18 mmHg) with any hypotensive drug therapy, and have a rate of progression of visual field damage (rate of progression - RoP) of at least 1 dB of mean visual field loss (MD) per year, demonstrated across at least four visual fields in the past two years.

[0091] The 12 randomized patients were divided into four groups: • CIT group - Three patients treated with citicoline in an oral solution at a dose of 10 ml / die, equivalent to 500 mg / die of citicoline, in addition to antihypertensive therapy; • NIC group - Three patients treated with nicotinamide in 10 ml / die oral solution equivalent to 100 mg / die of dinicotinamide, in addition to antihypertensive therapy; • CIT.+NIC. group - Three patients treated with citicoline + nicotinamide in an oral solution equivalent to 500 mg / die of disiticoline and 100 mg / die of nicotinamide, in addition to antihypertensive therapy. • NT group - 3 patients treated with antihypertensive therapy alone.

[0092] Patients were treated and followed for 6 months. At baseline and 6 months after treatment: a complete ophthalmological examination including visual acuity, control of intraocular pressure (IOP) by Goldmann tonometry (GAT), visual field testing, and electrofunctional and morphological examinations were performed.

[0093] (1) Visual field testing was performed using automated perimetry (HFA, Sita Standard 24-2 protocol; Zeiss, San Leandro, CA, USA). Such analysis represents the gold standard for the diagnosis and follow-up of glaucoma patients and allows for a schematic representation of the spatial expansion of visual ability, even by measuring their peripheral visual field. Two analyses were performed for each test, with a second test considered for analysis. To conduct the tests, patients were positioned in front of the apparatus. After covering the eye not being tested and placing their forehead and chin inside the dome, they began pressing the button, fixing their focus on the central target each time they perceived a light stimulus toward themselves, even if it was of insufficient intensity. MD was considered the peripheral target.

[0094] Next, (2) electrical function tests: electroretinography was performed from PERG patterns and PEV visual evoked potentials to evaluate the bioelectrical responses of retinal ganglion cells and the visual cortex, respectively, evoked by visual stimuli. Patients were placed in an acoustically insulated semi-dark room in front of a display surrounded by a uniform field of 5 cd / m2 brightness. Recordings for PERG and PEV were performed by employing the same visual stimulus: on an 18×18 degree TV monitor, a chessboard-like contrast modulation was shown, with white and black elements alternating periodically over time (18 degree visual arc, 80% contrast, and a reversal rate of 2 times per second). Such a method allows for the detection of the maximum sensitivity and specificity of PERG and PEV in detecting retinal and postretinal dysfunction in glaucomatous eyes. Signals for PERG and PEV were recorded using small Ag / AgCl electrodes placed on the lower eyelid and scalp, respectively. Transient responses to PERG are characterized by several waves with three subsequent peaks of negative, positive, and negative polarity, respectively. In visually normal subjects, these peaks have the following latency times: 35.50 and 95 ms (N35, P50, N95 - letters specify polarity and latency time). Peak width was measured for each wave using PERG P50-N95. Transient responses to PEV are characterized by several waves with three subsequent peaks of negative, positive, and negative polarity, respectively. In visually normal subjects, these peaks have latency times of 75, 100, and 145 ms (N75, P100, N145). The PEV P100 latency time was measured for each of the average waves displayed in the recording. During the recording session, simultaneous PERG and PEV were recorded at least twice (2-6 times), and the resulting waveforms were superimposed to verify the reproducibility of the results.During the recording session, two subsequent waves could be superimposed where the difference in ms (for latency time at PEV P100) and μV (for width at PERG P50-N95 and PEV N75-P100) was lower than the intra-individual variability (2 ms per pev P100 implicit time, and approximately ±0.18 μV for width at PERG P50-N95 and PEV N75-P100), and were therefore considered reproducible.

[0095] (3) The thickness of the RNFL nerve fiber layer was evaluated using spectral domain optical coherence tomography (SD-OCT) (RTVue Model-RT100 version 3.5; Optovue Inc, Fremont, CA, USA). Such analysis allows for tomographic surveys of the retina that can highlight the structural details of the cell layer and their nerve plexus through high-resolution sections, enabling qualitative and quantitative analysis of retinal changes. The patient was positioned in front of the instrument and instructed to observe the luminescent target: the instrument performed the scan once the ocular structure to be analyzed was focused. In the OCT results, the mean of RNFL-T of four measurements per quadrant was considered: high (RNFL-TS), low (RNFL-TI), nasal (RNFL-TN), and temporal (RNFL-TT). All data obtained in all quadrants (mean of 16 values) were identified as general RNFL (RNFL-TO).

[0096] Finally, patients were given questionnaires to assess treatment compliance and potential adverse effects.

[0097] Only data related to the worst-performing eye were considered. The significance level was fixed at p<0.05 for all studies.

[0098] result Table 3 shows the baseline characteristics of 12 patients. [Table 3]

[0099] Table 4 shows the 6-month results related to IOP, MD, PERG, and PEV, as well as values ​​related to RNFL thickness. [Table 4]

[0100] In the group treated with citicoline alone (Cit.), a significant improvement in MD compared to baseline was highlighted. Such improvements were statistically significant even compared to the untreated group. In the group treated with nicotinamide alone (Nic.), the inventors observed a lower reduction in MD values ​​compared to the untreated group (NT) at the end of the study. In the group treated with the Cit.+Nic. combination in oral solution, the inventors surprisingly observed a remarkable improvement in MD. Comparison with the administration of various molecules tested individually revealed the synergistic effect of the combination by demonstrating the higher efficacy of the combination on visual function.

[0101] Electrical function assessment highlighted improvements in parameters related to the range to PERG and latency time to PEV in all treated patients. In the untreated group, the inventors observed a significant deterioration in these parameters. The greater efficacy of Cit+Nic combination in the oral solution was highlighted, with a statistically significant difference observed not only compared to the untreated group but also to the single treatment (p<0.05).

[0102] Finally, morphological examinations confirmed the neuroprotective efficacy of various treatments on the retinal layer. In particular, the results of the Cit.+Nic. combination were surprisingly the most effective, halting nerve fiber thickening in a statistically significant manner across all groups. Even in this case, the improvement achieved by the combination was greater than the sum of the effects of the individually administered single molecules, confirming the synergistic action of the two active ingredients. Such thickening was considerably more pronounced in the untreated group. No adverse reactions were recorded for any of the treatments.

[0103] References 1.Morreale Bubella R et al.Neuroprotection of patient with open-angle chronic Glaucoma:role of citicoline in oral solution”.Ottica Fisiopatologica 2011. Neuroprotection in patients with open-angle glaucoma: The role of citicoline in oral solutions 2.Williams PA,et al.Vitamin B3 modulates mitochondrial vulnerability and prevents glaucoma in aged mice.Science 2017;355,756-760. Vitamin B3 regulates mitochondrial vulnerability and prevents glaucoma in aging mice.

Claims

1. A composition for oral use in the form of an aqueous solution, comprising citicoline and nicotinamide, for use in the treatment of glaucoma.

2. The composition according to claim 1, wherein the citicoline is present in a concentration of 25 mg / ml to 100 mg / ml.

3. The composition according to claim 1, wherein the nicotinamide is present in a concentration of 5 mg / ml to 20 mg / ml.

4. The composition according to claim 1, wherein the ratio of citicoline free acid to nicotinamide is within the range of 2.5:0.5 to 10:

2.

5. The composition according to claim 1, further comprising one or more of the following compounds: potassium sorbate, sodium lactate, fructose, and citric acid.

6. The composition according to claim 5, wherein the potassium sorbate is present at a concentration of 1.34 mg / ml to 3.00 mg / ml, and / or the 60% sodium lactate is present at a concentration of 2.5 mg / ml to 7 mg / ml, and / or the fructose is present at a concentration of 150 mg / ml to 400 mg / ml, and / or the anhydrous citric acid is present at a concentration of 0.92 mg / ml to 2.00 mg / ml.

7. The composition according to any one of claims 1 to 6, further comprising one or more excipients and / or additives.

8. The composition according to any one of claims 1 to 6, wherein it has high bioavailability, where the high bioavailability is the bioavailability of citicoline and nicotinamide, which is comparable to the bioavailability for intravenous administration and is contained in 90% to 100% of the composition.

9. The composition according to any one of claims 1 to 6, wherein the pH is within 3 to 5.

10. The composition according to any one of claims 1 to 6, wherein the density is contained in 1.116 to 1.156 g / ml.

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