A citicoline pharmaceutical composition and its uses

Abstract

The present invention discloses a citicoline pharmaceutical composition and its use. The pharmaceutical composition is composed of citicoline or its pharmaceutically acceptable salt and nicotinamide mononucleotide NMN / nicotinamide adenine dinucleotide NAD+ or its pharmaceutically acceptable salt; and its application in the preparation of a drug for preventing and / or treating vascular dementia. The present invention discovers that the combined administration of citicoline and NMN / NAD+ has better curative effects than the single administration of citicoline or NMN / NAD+, promotes the significant growth of the axon length of mouse neuroblastoma cells Neuro-2a, significantly increases the number of axon cells, and promotes the extension and regeneration of the processes of primary mouse cortical neurons; it can significantly shorten the escape latency of rats in the Morris water maze, increase the number of times of crossing the platform position, the swimming distance and time in the platform quadrant, and increase the number of times and time of exploring new objects; in addition, it can significantly improve the immune-inflammatory microenvironment in the brains of rats.

Description

Technical Field

[0001] The present invention relates to a new application of the combined administration of citicoline and nicotinamide mononucleotide NMN / nicotinamide adenine dinucleotide NAD + in the preparation of drugs, mainly involving the combined administration of citicoline and NMN / NAD + in the preparation of drugs for the treatment of vascular dementia, belonging to the field of medical technology. Background Art

[0002] Vascular Dementia (VaD) refers to a severe cognitive impairment syndrome caused by ischemic stroke, hemorrhagic stroke, and cerebrovascular diseases that result in low perfusion in brain regions such as memory, cognition, and behavior. According to epidemiological surveys, vascular dementia is the second most common cause of dementia after Alzheimer's disease, accounting for approximately 15% of cases. The incidence increases with age, and the risk of vascular dementia approximately doubles every 5.3 years. In addition, approximately 15 - 30% of subjects develop dementia within 3 months after a stroke. VaD not only reduces the quality of life of the patients themselves but also brings a heavy burden to their families and society. However, different from Alzheimer's disease, there is no licensed treatment for vascular dementia. Therefore, there is an urgent clinical need to find a low-risk and effective drug for the treatment of vascular dementia. Summary of the Invention

[0003] The pathogenesis of vascular dementia is mainly related to the continuous decline of cerebral blood flow. Chronic hypoperfusion and thromboembolism lead to a continuous decline in cerebral blood flow, hypoxia, oxidative stress, and trigger an inflammatory response. The periventricular white matter region, basal ganglia, and hippocampus are extremely vulnerable to lesions caused by insufficient perfusion. The interruption of the prefrontal-basal ganglia circuit leads to cognitive defects. The white matter of the brain is extremely vulnerable to hypoxia-induced damage, causing demyelination. Demyelination delays nerve signal transmission and results in cognitive loss. Inflammatory factors further exacerbate white matter damage (demyelination, axonal loss, oligodendrocyte degeneration), impair neurogenesis, neuronal progenitor cell proliferation, synaptic plasticity, and dendritic spine density, leading to neurodegeneration and cell death.

[0004] Citicoline is a natural endogenous compound and a precursor for the synthesis of phosphatidylcholine, one of the components of cell membranes. Citicoline administration can protect cell membranes by reducing the breakdown of phosphatidylcholine. Once absorbed, citicoline is converted into choline and cytidine, which circulate in the body, enter the systemic circulation and cross the blood-brain barrier, and are re-synthesized into citicoline in the brain. Multiple studies have shown that citicoline can effectively treat central nervous system diseases, including acute and chronic cerebral ischemia, cerebral hemorrhage, global cerebral hypoxia, and neurodegenerative diseases. Citicoline treatment can reduce the infarct area (the area of infarcted tissue), lower the free fatty acid concentration, reduce neurological deficits, restore the learning ability of animals, reduce glutamate-mediated damage, maintain phosphatidylcholine levels, and increase neuron survival rate.

[0005] β-cotinamide mononucleotide is a naturally occurring bioactive nucleotide and a precursor of coenzyme 1 NAD + (nicotinamide adenine dinucleotide). Under normal and pathophysiological conditions, intraperitoneal administration of NMN rapidly (within 15 minutes) increases the NAD + levels in brain regions such as the hippocampus and hypothalamus, indicating that NMN can cross the BBB and contribute to NAD + biosynthesis in the brain. Recent studies have shown that NMN can improve many neuronal functions in the brain. NMN administration improves cognition and memory in mouse and rat models of Alzheimer's disease. NMN protects neurons from cell death after ischemia or cerebral hemorrhage and improves the loss of BBB integrity and tissue plasminogen activator-induced hemorrhagic transformation in cerebral ischemia.

[0006] In recent years, studies have found that combined use of brain protectants such as citicoline is effective in restoring brain function and has a certain effect on improving the cognitive ability and intellectual level of patients. As a brain cell metabolism agent, sodium citicoline promotes brain substance metabolism and improves cerebral blood circulation by reducing the resistance of cerebral blood vessels and increasing cerebral blood flow. It can also enhance the function of the pyramidal system, improve motor paralysis, promote the recovery of brain function and promote arousal, and gradually restore the function of the limbs in hemiplegia caused by stroke. It is used for the treatment of ischemic cerebrovascular diseases and vascular dementia. In addition, sodium citicoline can promote the synthesis of phosphatidylcholine and improve brain cell nutrition. Cerebral ischemia consumes NAD + in brain tissue, + leading to bioenergetic failure and cell death. The NAD +Horizontally promotes the activation of SIRT1 in the neurovascular unit. In addition, nicotinamide mononucleotide also has the effect of protecting mitochondria in brain cells, generating sufficient energy to restore nerve conduction function. The purpose of the combination of the two drug components is to synergistically cooperate in terms of energy guarantee and brain cell nutrient metabolism, enabling cerebrovascular vessels to promote brain cell metabolism through citicoline sodium and providing NAD by nicotinamide mononucleotide + , enabling the brain cells to restore normal functions and wake up, thereby producing a long-term and effective therapeutic effect on cerebrovascular diseases and avoiding the poor therapeutic effect and easy recurrence of single drug treatment. Therefore, the present invention combines citicoline with NMN / NAD + to prepare a drug for preventing vascular dementia, which may produce good clinical effects.

[0007] So far, there have been no relevant papers elaborating on the application of citicoline and NMN / NAD + in vascular dementia. The core innovative content of the present invention lies in that the combination of citicoline and NMN / NAD + can be used to treat vascular dementia, can improve the behavioral cognitive ability of rats with chronic cerebral hypoperfusion, and can improve the neuroimmune inflammatory microenvironment in the brains of rats, which is a new use that has not been proven so far.

[0008]

[0009]

[0010] Based on the above situation, the present invention provides a citicoline pharmaceutical composition and its use, thereby providing a new therapeutic drug for the treatment of vascular dementia.

[0011] For this reason, the present invention provides the following technical solutions:

[0012] A pharmaceutical composition, which is composed of citicoline or a pharmaceutically acceptable salt thereof and nicotinamide mononucleotide NMN / nicotinamide adenine dinucleotide NAD + or a pharmaceutically acceptable salt thereof; the molar ratio of citicoline or a pharmaceutically acceptable salt thereof to NMN / NAD + or a pharmaceutically acceptable salt thereof is 1:1 to 10:1, wherein citicoline or a pharmaceutically acceptable salt thereof is calculated as citicoline, and NMN / NAD + or a pharmaceutically acceptable salt thereof is calculated as NMN / NAD + calculated.

[0013] Among them, the citicoline or a pharmaceutically acceptable salt thereof and NMN / NAD +The molar ratio of the citicoline or its pharmaceutically acceptable salt can be any ratio from 1:1 to 10:1, including but not limited to 1:1, 2:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.33:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc.

[0014] In some embodiments, the molar ratio of the citicoline or its pharmaceutically acceptable salt to NMN / NAD + or its pharmaceutically acceptable salt is 1:1 to 8:1, preferably 2:1 to 6:1, more preferably 2:1 to 4:1, and most preferably 3.3:1.

[0015] In some embodiments, the pharmaceutically acceptable salt of citicoline is selected from sodium citicoline.

[0016] In a second aspect, there is provided the use of the described pharmaceutical composition in the preparation of a medicament for preventing and / or treating vascular dementia.

[0017] The pharmaceutical composition can promote axon regeneration of nerve cell lines, significantly increase the axon length of mouse neuroblastoma cells Neuro-2a, increase the number of axon-bearing cells, and promote the extension and regeneration of processes of primary mouse cortical neurons.

[0018] The pharmaceutical composition can significantly shorten the escape latency of rats in the Morris water maze, increase the number of times of crossing the platform position, the swimming distance and time in the platform quadrant, and increase the number and time of exploration of new objects.

[0019] The pharmaceutical composition can significantly improve the neuroimmune inflammatory microenvironment in the rat brain and play a role in treating vascular dementia. The pharmaceutical composition is safe and non-toxic and has no effect on body weight and organ coefficients.

[0020] There is provided the use of different doses of citicoline and NMN and the combination of citicoline and NMN / NAD + in the preparation of a medicament for a rat model of vascular dementia, wherein the treatment of rat vascular dementia means that the pharmaceutical composition can shorten the escape latency of rats in the Morris water maze, increase the number of times of crossing the platform position, the swimming distance and time in the platform quadrant.

[0021] Preferably, the daily dosage of citicoline is 0.1 to 2 mmol / kg, and the daily dosage of NMN is 0.1 to 2 mmol / kg.

[0022] On the basis of the above scheme, most preferably, the daily intraperitoneal injection dose of citicoline is 0.2 - 0.4 mmol / kg, and the daily intraperitoneal injection dose of NMN is 0.1 - 0.4 mmol / kg.

[0023] The combined use of citicoline and NMN of the present invention has the following effects on the rat vascular dementia model:

[0024] Experiments show that the combined use of citicoline and NMN can promote axon regeneration of neural cell lines, significantly increase the axon length of mouse neuroblastoma cells Neuro-2a, increase the number of axon-bearing cells, and promote the extension and regeneration of mouse primary cortical neuron processes.

[0025] The combined use of citicoline and NMN can significantly shorten the escape latency of rats in the Morris water maze, increase the number of times of crossing the platform position, the swimming distance and time in the platform quadrant, and increase the number and time of exploring new objects.

[0026] The combined use of citicoline and NMN can significantly improve the immune-inflammatory microenvironment in the rat brain and play a role in treating vascular dementia.

[0027] The third aspect of the technical solution of the present invention is to provide a pharmaceutical preparation, the active ingredients of which are citicoline and NMN / NAD + drug combination, adding conventional excipients, and preparing into clinically acceptable gastrointestinal dosage forms, injection dosage forms, etc.

[0028] The present invention discloses the mechanism of action of the combined administration of citicoline and NMN / NAD + in the treatment of the rat vascular dementia model, and it is found that the combined administration of citicoline and NMN / NAD + has better curative effects than the administration of citicoline or NMN / NAD alone + in promoting the significant increase in the axon length of mouse neuroblastoma cells Neuro-2a, the significant increase in the number of axon-bearing cells, and the extension and regeneration of mouse primary cortical neuron processes; it can significantly shorten the escape latency of rats in the Morris water maze, increase the number of times of crossing the platform position, the swimming distance and time in the platform quadrant, and increase the number and time of exploring new objects; in addition, the combined administration of citicoline and NMN / NAD + has better curative effects than the administration of citicoline or NMN / NAD alone + and can also significantly improve the immune-inflammatory microenvironment in the rat brain. Therefore, the combined administration of citicoline and NMN / NAD + can be used to treat vascular dementia, reduce adverse reactions, provide a safe and effective method for the treatment of vascular dementia, and has an unexpected effect of 1 + 1 > 2. Description of the Drawings

[0029] Figure 1 . Effects of single administration of citicoline and NMN on the axonal regeneration model of Neuro-2a cells induced by low serum. A. Axonal length of Neuro-2a cells measured by Image J software after single administration of citicoline; B. Percentage of cell axons calculated by Image J software after single administration of citicoline; C. Axonal length of Neuro-2a cells measured by Image J software after single administration of NMN; D. Percentage of cell axons calculated by Image J software after single administration of NMN; * P < 0.05, ** P < 0.01 and *** P < 0.001 compared with the blank group; # P < 0.05, ## P < 0.01 and ### P < 0.001 compared with the model group, N = 10.

[0030] Figure 2 . Effects of combined administration of citicoline and NMN on the axonal regeneration model of Neuro-2a nerve cells induced by low serum. A. Axonal length of Neuro-2a cells measured by Image J software after combined administration of citicoline and NMN; B. Percentage of cell axons calculated by Image J software after combined administration of citicoline and NMN; * P < 0.05, ** P < 0.01 and *** P < 0.001 compared with the blank group; # P < 0.05, ## P < 0.01 and ### P < 0.001 compared with the model group, N = 10.

[0031] Figure 3 . Effects of combined administration of citicoline and NMN on the axonal regeneration model of primary mouse neurons induced by low serum. A. Axonal length of primary mouse neurons measured by Image J software after single and combined administration of citicoline and NMN; B. Percentage of nerve cell axons calculated by Image J software after combined administration of citicoline and NMN; # P < 0.05, ## P < 0.01 and ### P < 0.001 compared with the blank group; & P < 0.05, && P < 0.01 and &&& P < 0.001 compared with the Citicoline (2 mM) group, N = 10.

[0032] Figure 4. Effects of single and combined administration of citicoline and NMN on learning and memory abilities in a rat model of vascular dementia. A. Schematic diagram of the behavior of rats in each group during the water maze place navigation experiment; B. Escape latency of rats in each group during the water maze place navigation experiment; C. Exploration time of rats in each group for new objects in the novel object recognition experiment; D. Schematic diagram of the behavior of rats in each group during the water maze spatial exploration experiment; E. Number of platform position crossings, swimming distance, and time in the platform quadrant of rats in each group during the water maze spatial exploration experiment; * P < 0.05, ** P < 0.01 and *** P < 0.001 compared with the blank group; # P < 0.05, ## P < 0.01 and ### P < 0.001 compared with the model group; & P < 0.05, && P < 0.01 and &&& P < 0.001 compared with the Citicoline (200 mg / kg) group, N = 6 - 15.

[0033] Figure 5 . Effects of single and combined administration of citicoline and NMN on the gene expression of TNF-α, IL-1β, IL-6, CD206, and IL-10 in the brain tissue. A. Effects on TNF-α gene expression after administration; B. Effects on IL-1β gene expression after administration; C. Effects on IL-6 gene expression after administration; D. Effects on CD206 gene expression after administration; E. Effects on IL-10 gene expression after administration; * P < 0.05, ** P < 0.01 and *** P < 0.001 compared with the blank group; # P < 0.05, ## P < 0.01 and ### P < 0.001 compared with the model group; & P < 0.05, && P < 0.01 and &&& P < 0.001 compared with the Citicoline (200 mg / kg) group, N = 4.

[0034] Figure 6 . Safety evaluation of single and combined administration of citicoline and NMN in rats. A. Body weight statistics of rats in each group; B. Organ coefficient of the liver in rats in each group; C. Organ coefficient of the spleen in rats in each group; * P < 0.05, ** P < 0.01 and *** P < 0.001 compared with the blank group; # P < 0.05,## P < 0.01 and ### P < 0.001 compared with the model group, N = 6 - 15. Detailed implementation manners

[0035] To further clarify the present invention, a series of embodiments are given below. These embodiments are completely illustrative and are only used to specifically describe the present invention, and should not be construed as a limitation to the present invention.

[0036] The following makes a detailed description of the present application in conjunction with specific embodiments. In the embodiments, citicoline sodium salt (molecular weight 510.31) is selected as an example of citicoline or its pharmaceutically acceptable salt, abbreviated as Cit, NMN / NAD + Or its pharmaceutically acceptable salt selects NMN (molecular weight 334.22) as an example in the embodiments.

[0037] According to the experimental results of the combined administration of citicoline and NMN on the axonal regeneration of Neuro-2a neurons, the optimal molar ratio of citicoline and NMN is 3.33:1. The Q value formula is used to calculate the synergy index of the drug: Q = E(a + b) / (Ea + Eb - Ea * Eb), where E(a + b) represents the inhibition rate of the combination of drug a (citicoline) and drug b (NMN) (here refers to the axonal growth rate of neurons), and Ea and Eb respectively represent the inhibition rates of drug a (citicoline) and drug b (NMN) (here refers to the axonal growth rate of neurons). Judgment of the drug synergy index result: Q < 0.85 is defined as antagonism, 0.85 < Q < 1.15 is defined as additive effect, and Q > 1.15 is defined as synergistic effect. According to the in vitro neuron axonal growth experimental data, E(citicoline) = 449.76%, E(NMN) = 20.30%, E(citicoline + NMN) = 671.04%, and finally Q = 1.772 > 1.15 is calculated, indicating that citicoline and NMN have good pharmacodynamic synergy.

[0038] According to the in vivo experimental results, preferably, the daily dosage of citicoline is 0.1 - 2 mmol / kg, and the daily dosage of NMN is 0.1 - 2 mmol / kg.

[0039] On the basis of the above scheme, most preferably, the daily intraperitoneal injection dose of citicoline is 0.2 - 0.4 mmol / kg, and the daily intraperitoneal injection dose of NMN is 0.1 - 0.4 mmol / kg.

[0040] Example 1

[0041] A pharmaceutical composition is composed of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of the citicoline sodium salt to the nicotinamide mononucleotide NMN is 1:1.

[0042] Example 2

[0043] A pharmaceutical composition is composed of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 2:1.

[0044] Example 3

[0045] A pharmaceutical composition is composed of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 3.33:1.

[0046] Example 4

[0047] A pharmaceutical composition is composed of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 4:1.

[0048] Example 5

[0049] A pharmaceutical composition is composed of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 6:1.

[0050] Example 6

[0051] A pharmaceutical composition is composed of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 8:1.

[0052] Example 7

[0053] A pharmaceutical composition is composed of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 10:1.

[0054] Example 8. Effects of single administration of citicoline and NMN on the axonal regeneration model of Neuro-2a neurons induced by low serum.

[0055] Experimental method: Neuro-2a cells were seeded in culture dishes and induced to regenerate axons with MEM medium containing 0.1% FBS. Then, different concentrations (0.2 mM, 0.6 mM, 2 mM) of citicoline and NMN were applied to Neuro-2a cells for 12, 24, and 48 h respectively, and bright-field images were taken to detect whether the axon length and the number of axon-bearing cells increased significantly.

[0056] The results are shown in Table 1, Table 2 and Figure 1As shown, when the action time was 24 h, citicoline (2 mM) could significantly promote the obvious growth of the axon length of mouse neuroblastoma cells Neuro-2a, and the number of axon-bearing cells increased significantly. * P < 0.05, ** P < 0.01 and *** P < 0.001 compared with the blank group; # P < 0.05, ## P < 0.01 and ### P < 0.001 compared with the model group, N = 10.

[0057] Group Axon length Percentage of the axon cells Control 6.26±2.89 6.98±1.48 Model <![CDATA[27.97±4.71 ** > <![CDATA[15.58±3.94 ** > Citicoline(0.2mM) <![CDATA[38.87±7.78 # > <![CDATA[23.48±3.15 ## > Citicoline(0.6mM) <![CDATA[96.56±7.99 ### > <![CDATA[41.86±5.55 ### > Citicoline(2mM) <![CDATA[145.83±14.47 ### > <![CDATA[50.63±8.29 ### >

[0058] Table 1

[0059] Group Axon length Percentage of the axon cells Control 7.59±1.28 7.21±2.48 Model <![CDATA[25.54±6.19 ** > <![CDATA[14.96±3.91 ** > NMN(0.2mM) 26.74±3.86 <![CDATA[18.28±2.47 # > NMN(0.6mM) <![CDATA[32.91±3.32 # > <![CDATA[21.87±2.39 ### > NMN(2mM) <![CDATA[42.06±3.39 ### > <![CDATA[25.43±3.35 ### >

[0060] Table 2

[0061] Example 9. Effect of combined administration of citicoline and NMN on the axon regeneration model of Neuro-2a nerve cells induced by low serum.

[0062] Experimental method: Neuro-2a nerve cells were inoculated in a culture dish, and their axon regeneration was induced with MEM medium containing 0.1% FBS. Then, different ratios (C1 or N1: 2 mM; C2 or N2: 0.6 mM; C3 or N3: 0.2 mM; C4 or N4: 0.06 mM; C5 or N5: 0.02 mM) of citicoline and NMN were respectively applied to Neuro-2a cells for 12, 24, and 48 h, and bright-field photographs were taken to detect whether the axon length and the number of axon-bearing cells increased significantly.

[0063] The results are shown in Table 3 and Figure 2 as shown, when the action time was 24 h, citicoline (2 mM) + NMN (0.6 mM) could significantly promote the obvious growth of the axon length of mouse neuroblastoma cells Neuro-2a, and the number of axon-bearing cells increased significantly. * P < 0.05, ** P < 0.01 and *** P < 0.001 compared with the blank group; # P < 0.05, ## P < 0.01 and ### P < 0.001 compared with the model group, N = 10.

[0064] Group Axon length Percentage of the axon cells Control 6.14±2.46 5.86±2.6 Model <![CDATA[26.53±5.79 ** > <![CDATA[14.11±1.99 ** > C1N1 <![CDATA[194.49±12.36 ### > <![CDATA[55.85±5.53 ### > C1N2 <![CDATA[224.53±16.51 ### > <![CDATA[68.47±5.32 ### > C1N3 <![CDATA[160.42±12.3 ### > <![CDATA[54.55±7.31 ### > C1N4 <![CDATA[153.96±9.92 ### > <![CDATA[53.33±6.24 ### > C1N5 <![CDATA[142.06±10.08 ### > <![CDATA[48.56±2.99 ### > C2N1 <![CDATA[102.72±8.65 ### > <![CDATA[35.18±2.99 ### > C2N2 <![CDATA[94.76±8.89 ### > <![CDATA[32.63±4.05 ### > C2N3 <![CDATA[62.66±10.17 ### > <![CDATA[31.41±4.77 ### <!-- 6 -->]]> C2N4 <![CDATA[54.05±8.46 ### > <![CDATA[28.88±4.41 ### > C2N5 <![CDATA[58.29±9.91 ### > <![CDATA[28.4±2.61 ### >

[0065] Table 3

[0066] Example 10. Effect of combined administration of citicoline and NMN on the axonal regeneration model of primary cortical neurons in mice induced by low serum.

[0067] Experimental method: Pregnant rats on the 15th day of pregnancy were decapitated to remove the brain, and the meninges were dissected. The white matter other than the cortex was removed, digested with 0.25% trypsin, filtered through a sieve, centrifuged and resuspended, and inoculated on a 24-well plate coated with L-polylysine at a density of 1×10 6 / ml. Then, citicoline and NMN at different ratios (1:1, 2:1, 4:1, total dose 2 mM) were applied to the cells for 12, 24, and 48 h respectively, and bright-field images were taken to detect whether the axonal length of nerve cells and the number of cells with axons increased significantly.

[0068] Results are shown in Table 4 and Figure 3 as follows. When the action time was 24 h, Citicoline (1.6 mM) + NMN (0.4 mM) could significantly promote the significant growth of the axonal length of primary cortical neurons in mice and the significant increase in the number of cells with axons. # P<0.05, ## P<0.01 and ### P<0.001 compared with the blank group; & P<0.05, && P<0.01 and &&& P<0.001 compared with the Citicoline (2 mM) group, N = 10.

[0069] Group Axon length Percentage of the axon cells Control 30.41±5.57 6.96±3.48 Citicoline(2mM) <![CDATA[176.67±16.13 ### > <![CDATA[26.37±5.49 ### > NMN(2mM) 54.21±13.76 <![CDATA[18.83±2.65 ### > Cit(1mM)+NMN(1mM) <![CDATA[250.41±32.56 ### > <![CDATA[36.67±3.74 ### > Cit(1.33mM)+NMN(0.67mM) <![CDATA[318.44±11.67 ### > <![CDATA[44.59±7.62 ### > Cit(1.6mM)+NMN(0.4mM) <![CDATA[372.31±25.66 ###&&& > <![CDATA[54.62±7.45 ###&&& >

[0070] Table 4

[0071] Example 11. Effects of single and combined administration of citicoline and NMN on the learning and memory ability of a rat model of vascular dementia.

[0072] Experimental materials: Male SD rats, weighing 220 - 250 g, were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd.

[0073] Animal grouping: 90 male SD rats were randomly divided into 6 groups of 15 rats each, namely the blank group, the model group, the Citicoline (200 mg / kg) group, the NMN (500 mg / kg) group, the Citicoline (133.33 mg / kg) + NMN (66.67 mg / kg) group, and the Citicoline (160 mg / kg) + NMN (40 mg / kg) group. Except for the blank group, in the other 5 groups, the skin and subcutaneous tissue were incised along the midline of the neck with sterile instruments, and the common carotid artery on one side was bluntly dissected and ligated in the space between the anterior cervical muscle and the lateral muscle. The same operation was performed on the other side, and the skin was sutured. In the blank group, only the muscle was bluntly dissected and then the skin was sutured. Seven days after the surgery, the cerebral blood flow of the rats became stable and decreased to 65% of that before the surgery, and then the administration started. The blank group and the model group were intraperitoneally injected with an equal amount of normal saline every day, and the other 4 groups were injected with the corresponding doses of drugs for 28 consecutive days. After 28 days of the surgery, the water maze test and the new and old object recognition experiment were started, and the rats were sacrificed to obtain the brain at 35 days after the surgery.

[0074] The results are shown in Tables 5, 6, 7 and Figure 4 as follows. After the surgical model was established, the cognitive ability of male rats decreased. However, Citicoline (160 mg / kg) + NMN (40 mg / kg) could significantly shorten the escape latency of rats in the Morris water maze, increase the number of times of crossing the platform position, the swimming distance and time in the platform quadrant, and increase the number and time of exploring new objects. Moreover, it had a significant advantage compared with the administration of Citicoline (200 mg / kg) or NMN (500 mg / kg) alone, and the therapeutic effect on vascular dementia was more significant. * P < 0.05, ** P < 0.01 and *** P < 0.001 compared with the blank group; # P < 0.05, ## P < 0.01 and ### P < 0.001 compared with the model group; & P < 0.05, && P < 0.01 and &&& P < 0.001 compared with the Citicoline (200 mg / kg) group, N = 6 - 15.

[0075]

[0076] Table 5

[0077]

[0078] Table 6

[0079]

[0080] Table 7

[0081] Example 12. Effects of single and combined administration of citicoline and NMN on the gene expression of TNF-α, IL-1β, IL-6, CD206 and IL-10 in brain tissue.

[0082] The animal model establishment and administration method were the same as those in Example 7. Take brain tissue, weigh about 20 mg on an electronic balance, add Trizol for lysis, extract the total RNA of brain tissue, reverse transcribe it into cDNA, prepare the reaction system, and perform amplification to detect the changes in the gene expression of TNF-α, IL-1β, IL-6, CD206 and IL-10 in brain tissue.

[0083] The results are shown in Table 8, Table 9 and Figure 5 As shown, after the surgical model was established, the mRNA expression levels of TNF-α, IL-1β, and IL-6 in the rat brain tissue increased. The combined administration of citicoline (160 mg / kg) and NMN (40 mg / kg) could significantly reduce the mRNA expression levels of TNF-α, IL-1β, and IL-6 in brain tissue, and had a significant advantage compared with the single administration of citicoline (200 mg / kg) or NMN (500 mg / kg); after administration after the surgical model was established, the combined administration of citicoline (160 mg / kg) and NMN (40 mg / kg) could significantly increase the mRNA expression levels of CD206 and IL-10 in brain tissue, and had a significant advantage compared with the single administration of citicoline (200 mg / kg) or NMN (500 mg / kg). These results suggest that compared with the single administration of citicoline or NMN, the combined administration of citicoline and NMN can more significantly improve the immune microenvironment in the rat brain and has a more significant therapeutic effect on vascular dementia. * P < 0.05, ** P < 0.01 and *** P < 0.001 compared with the blank group; # P < 0.05, ## P < 0.01 and ### P < 0.001 compared with the model group; & P < 0.05, && P < 0.01 and &&& P < 0.001 compared with the citicoline (200 mg / kg) group; $ P < 0.05, $$ P < 0.01 and $$$ P < 0.001 compared with the NMN (500 mg / kg) group, N = 4.

[0084] Group TNF-α IL-1β IL-6 Sham 1±0.12 1±0.19 1±0.44 Vehicle <![CDATA[2.97±0.75 *** > <![CDATA[5.07±0.37 *** > <![CDATA[4.07±0.69 *** > Citicoline(200mg / kg) 2.76±0.43 <![CDATA[3.51±0.64 # > 3.94±0.45 NMN(500mg / kg) 2.57±0.41 <![CDATA[3.32±0.31 ## > 3.64±0.48 Cit(160mg / kg)+NMN(40mg / kg) <![CDATA[1.4±0.31 ##&$ > <![CDATA[2±0.49 ###&&$ > <![CDATA[2.15±0.41 #&$ >

[0085] Table 8

[0086] Group CD206 IL-10 Sham 1±0.17 1±0.17 Vehicle 1.14±0.18 1.15±0.19 Citicoline(200mg / kg) 1.24±0.25 1.31±0.3 NMN(500mg / kg) 1.69±0.31 1.44±0.25 Cit(160mg / kg)+NMN(40mg / kg) <![CDATA[2.62±0.4 ###&&&$$ > <![CDATA[2.24±0.52 ##&$ >

[0087] Table 9

[0088] Example 13. Investigation of the toxicity of citicoline and NMN administered alone and in combination to rats.

[0089] The animal model establishment and drug administration method were the same as those in Example 7. The body weights of the rats in each group were statistically counted daily. After the animals were sacrificed, the spleen weights and liver weights of each rat were weighed, and the organ coefficients of each organ were calculated and statistically analyzed.

[0090] The results are shown in Table 10 and Figure 6 As shown, the body weights of each group increased slowly according to the number of days, and there were no significant differences in the statistical analysis of the organ coefficients, indicating that each dose of citicoline and NMN was non-toxic and met the clinical safety drug use standards.

[0091]

[0092]

[0093] Table 10

[0094] To sum up, in the present invention, a vascular dementia model was established by ligating the bilateral common carotid arteries in the neck of rats to cause persistent cerebral hypoperfusion and hypoxic-ischemic brain injury, and the therapeutic effect of the combined administration of citicoline and NMN on the vascular dementia model was investigated. The results showed that in vitro, the combined administration of citicoline and NMN could significantly promote axon regeneration of the nerve cell line, significantly increase the axon length of mouse neuroblastoma cells Neuro-2a, significantly increase the number of cells with axons, and promote the extension and regeneration of the processes of primary mouse cortical neurons, showing significant advantages compared with the administration of citicoline or NMN alone. In vivo, the combined administration of citicoline (160 mg / kg) and NMN (40 mg / kg) could significantly shorten the escape latency of rats in the Morris water maze, increase the number of times of crossing the platform position, the swimming distance and time in the platform quadrant, and increase the number and time of exploring new objects; in addition, citicoline (160 mg / kg) and NMN (40 mg / kg) could significantly improve the neuroimmune inflammatory microenvironment in the brains of rats, which has certain reference significance for the treatment of vascular dementia. These results all indicate that the combined use of citicoline and NMN has certain pharmacological and pharmacodynamic synergistic effects in the treatment of vascular dementia. The combined use of citicoline and NMN can further increase the drug effect and reduce adverse reactions, providing an economical, stable, safe and reliable solution for the treatment of vascular dementia diseases, with an unexpected effect of 1 + 1 > 2.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to be limiting. Although the present invention has been described by referring to the preferred embodiments of the present invention, those of ordinary skill in the art should understand that various changes can be made in form and details without departing from the spirit and scope of the present invention defined by the appended claims.

Claims

1. A pharmaceutical composition for preventing and / or treating vascular dementia, characterized in that, The pharmaceutical composition is composed of citicoline or a pharmaceutically acceptable salt thereof and nicotinamide mononucleotide (NMN) or a pharmaceutically acceptable salt thereof; the molar ratio of citicoline or a pharmaceutically acceptable salt thereof to NMN or a pharmaceutically acceptable salt thereof is 2:1 to 6:1, wherein citicoline or a pharmaceutically acceptable salt thereof is calculated as citicoline, and NMN or a pharmaceutically acceptable salt thereof is calculated as NMN.

2. The pharmaceutical composition according to claim 1, wherein The molar ratio of citicoline or a pharmaceutically acceptable salt thereof to NMN or a pharmaceutically acceptable salt thereof is 2:1 to 4:

1.

3. The pharmaceutical composition according to claim 1, wherein The molar ratio of citicoline or a pharmaceutically acceptable salt thereof to NMN or a pharmaceutically acceptable salt thereof is 3.3:

1.

4. The pharmaceutical composition according to claim 1, wherein The pharmaceutically acceptable salt of citicoline is selected from sodium citicoline.

5. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a medicament for preventing and / or treating vascular dementia.

6. The application according to claim 5, characterized in that, The pharmaceutical composition can significantly increase the axon length of mouse neuroblastoma cells Neuro-2a and significantly increase the number of axon-containing cells. and / or, the pharmaceutical composition can promote axon regeneration of nerve cells and promote the extension and regeneration of processes of primary mouse cortical neurons. and / or, the pharmaceutical composition can significantly shorten the escape latency of rats in the Morris water maze, increase the number of times of crossing the platform position, the swimming distance and time in the platform quadrant, and increase the number and time of exploration of new objects. and / or, the pharmaceutical composition can significantly improve the immune-inflammatory microenvironment in the rat brain and play a role in treating vascular dementia.

7. A pharmaceutical preparation, which contains a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1-4 and a pharmaceutically acceptable carrier, adjuvant or vehicle.

8. The pharmaceutical preparation according to claim 7, wherein The dosage form of the pharmaceutical preparation is a gastrointestinal administration dosage form, an injection dosage form or a topical dosage form, including capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, creams, ointments, suppositories or patches.

9. Use of the pharmaceutical preparation according to claim 7 or 8 in the preparation of a medicament for preventing and / or treating vascular dementia.

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