WO2024098854A1 - Citicoline pharmaceutical composition and use thereof - Google Patents

Citicoline pharmaceutical composition and use thereof Download PDF

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WO2024098854A1
WO2024098854A1 PCT/CN2023/111202 CN2023111202W WO2024098854A1 WO 2024098854 A1 WO2024098854 A1 WO 2024098854A1 CN 2023111202 W CN2023111202 W CN 2023111202W WO 2024098854 A1 WO2024098854 A1 WO 2024098854A1
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citicoline
nmn
pharmaceutically acceptable
pharmaceutical composition
acceptable salt
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PCT/CN2023/111202
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French (fr)
Chinese (zh)
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庞涛
赵宁
谢璐阳
朱小峰
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中国药科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates to a new application of combined administration of citicoline and nicotinamide mononucleotide NMN/nicotinamide adenine dinucleotide NAD + in the preparation of medicines, and mainly relates to the application of combined administration of citicoline and NMN/NAD + in the preparation of medicines for treating vascular dementia, and belongs to the field of medical technology.
  • Vascular dementia refers to a syndrome of severe cognitive dysfunction caused by ischemic stroke, hemorrhagic stroke, and cerebrovascular disease that causes low perfusion of brain areas such as memory, cognition, and behavior.
  • vascular dementia is the second most common cause of dementia after Alzheimer's disease, accounting for about 15% of cases. The incidence rate increases with age, and the risk of vascular dementia doubles approximately every 5.3 years. In addition, approximately 15-30% of subjects develop dementia 3 months after a stroke. VaD not only reduces the patient's own quality of life, but also brings a heavy burden to the patient's family and society. However, unlike Alzheimer's disease, there is no licensed treatment for vascular dementia. Therefore, there is an urgent need to find a low-risk, effective drug to treat vascular dementia.
  • the pathogenesis of vascular dementia is mainly related to the continuous decrease in cerebral blood flow.
  • Chronic hypoperfusion and thromboembolism lead to a continuous decrease in cerebral blood flow, hypoxia, oxidative stress and inflammatory response.
  • the periventricular white matter area, basal ganglia and hippocampus are extremely susceptible to lesions caused by insufficient perfusion, and the interruption of the prefrontal lobe-basal ganglia circuit leads to cognitive deficits.
  • the white matter of the brain is extremely susceptible to hypoxia-induced damage, causing demyelination, which delays neural signal transmission and leads to cognitive loss.
  • Inflammatory factors further aggravate white matter damage (demyelination, axonal loss, oligodendrocyte degeneration), impair neurogenesis, neuronal progenitor cell proliferation, synaptic plasticity and dendritic spine density, causing neurodegeneration and cell death.
  • Citicoline is a natural endogenous compound and a precursor for the synthesis of phosphatidylcholine, a component of cell membranes.
  • Citicoline administration can protect cell membranes by reducing the breakdown of phosphatidylcholine.
  • citicoline Once absorbed, citicoline is converted into choline and cytidine, which circulate in the body, enter the systemic circulation and cross the blood-brain barrier to be resynthesized into citicoline in the brain.
  • Several studies have shown that citicoline is effective in treating central nervous system diseases, including acute and chronic cerebral ischemia, cerebral hemorrhage, global brain hypoxia, and neurodegenerative diseases.
  • Cinct size area of infarcted tissue
  • reduce free fatty acid concentrations reduce neurological deficits, restore animals' learning ability
  • reduce glutamate-mediated damage reduce glutamate-mediated damage
  • maintain phosphatidylcholine levels and improve neuronal survival.
  • Nicotinamide mononucleotide ( ⁇ -cotinamide mononucleotide) is a naturally occurring biologically active nucleotide and a precursor of coenzyme 1 NAD + (nicotinamide adenine dinucleotide).
  • NAD + nicotinamide adenine dinucleotide
  • NMN can improve many neuronal functions in the brain.
  • NMN administration improves cognition and memory in Alzheimer's disease mouse and rat models.
  • NMN protects neurons from cell death after ischemia or cerebral hemorrhage and improves loss of BBB integrity in cerebral ischemia and hemorrhagic transformation induced by tissue plasminogen activator.
  • citicoline sodium promotes brain substance metabolism and improves brain blood circulation by reducing cerebral vascular resistance and increasing cerebral blood flow. It can also enhance the function of the vertebral system, improve motor paralysis, promote brain function recovery and promote awakening. It can gradually restore the function of the limbs in hemiplegia caused by cerebral stroke, and is used for the treatment of ischemic cerebrovascular disease and vascular dementia.
  • citicoline sodium can promote the synthesis of phosphatidylcholine and improve brain cell nutrition.
  • Cerebral ischemia consumes brain tissue NAD + , leading to bioenergetics failure and cell death.
  • NAD + levels after ischemia can be supplemented by nicotinamide mononucleotide, and the increased NAD + levels promote the activation of SIRT1 in the neurovascular unit.
  • nicotinamide mononucleotide also has the function of protecting mitochondria in brain cells, generating enough energy to restore nerve conduction function.
  • the purpose of the combination of the two drug components is to ensure the energy and brain cell nutrition metabolism, so that the cerebral blood vessels promote brain cell metabolism through citicoline sodium, and provide NAD + through nicotinamide mononucleotide, so that the brain cells of the brain can restore normal function and wake up, thereby producing a long-term and effective therapeutic effect on cerebrovascular diseases, avoiding the poor effect of single drug treatment and easy recurrence. Therefore, the present invention combines citicoline with NMN/NAD + to prepare a drug for preventing vascular dementia, which may produce good clinical effects.
  • the core innovation of the present invention is that the combination of citicoline and NMN/NAD + can be used to treat vascular dementia, improve the behavioral cognitive ability of rats with chronic cerebral hypoperfusion, and improve the neuroimmune inflammatory microenvironment in the rat brain. This is a new use that has not been proven so far.
  • the present invention provides a citicoline pharmaceutical composition and its use, thereby providing a new therapeutic drug for the treatment of vascular dementia.
  • a pharmaceutical composition comprising 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 + .
  • the molar ratio of citicoline or a pharmaceutically acceptable salt thereof to NMN/NAD + or a pharmaceutically acceptable salt thereof 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 and the like.
  • the molar ratio of citicoline or a pharmaceutically acceptable salt thereof to NMN/NAD + or a pharmaceutically acceptable salt thereof 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.
  • the pharmaceutically acceptable salt of citicoline is selected from citicoline sodium salt.
  • a use of the pharmaceutical composition in the preparation of a drug for preventing and/or treating vascular dementia is provided.
  • the pharmaceutical composition can promote the regeneration of axons in nerve cell systems, promote the significant growth of axon length in mouse brain neuroma cells Neuro-2a, increase the number of axon cells, and promote the extension and regeneration of primary cortical neuron processes in mice.
  • the pharmaceutical composition can significantly shorten the escape latency of Morris water maze rats, increase the number of platform position crossings, platform quadrant swimming distance and time, and increase the number and time of exploring new objects.
  • 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.
  • rat vascular dementia model drug Provided are uses of different doses of citicoline and NMN and a combination of citicoline and NMN/NAD + in preparing a rat vascular dementia model drug, wherein the drug composition for treating rat vascular dementia can shorten Morris water
  • the escape latency of rats in the maze increased the number of platform position crossings, platform quadrant swimming distance and time.
  • the daily dosage of citicoline is 0.1 to 2 mmol/kg
  • the daily dosage of NMN is 0.1 to 2 mmol/kg.
  • the daily intraperitoneal injection dose of citicoline is 0.2-0.4 mmol/kg
  • the daily intraperitoneal injection dose of NMN is 0.1-0.4 mmol/kg.
  • citicoline and NMN can significantly shorten the escape latency of rats in the Morris water maze, increase the number of platform position crossings, the swimming distance and time in the platform quadrant, and increase the number and time of exploration of new objects.
  • the combination of citicoline and NMN can significantly improve the immune inflammatory microenvironment in the rat brain and play a role in treating vascular dementia.
  • 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, which are added with conventional excipients to prepare clinically acceptable gastrointestinal dosage forms, injection dosage forms, etc.
  • the present invention discloses the mechanism of action of the combined administration of citicoline and NMN/NAD + in treating a rat vascular dementia model, and finds that the combined administration of citicoline and NMN/NAD + has better therapeutic effect than the single administration of citicoline or NMN/NAD + , promotes the significant growth of the axon length of the mouse brain neuroma cell Neuro-2a, significantly increases the number of axonal cells, and promotes the extension and regeneration of the processes of primary cortical neurons in mice; can significantly shorten the escape latency of Morris water maze rats, increase the number of platform position crossings, the platform quadrant swimming distance and time, and increase the number and time of exploring new objects; in addition, the combined administration of citicoline and NMN/NAD + can also significantly improve the immune inflammatory microenvironment in the rat brain compared with the single administration of citicoline or NMN/NAD + . Therefore, the combined administration of citicoline and NMN/NAD + can be used to treat vascular dementia,
  • Figure 1 Effects of single administration of citicoline and NMN on the low serum-induced Neuro-2a cell axon regeneration model.
  • A. The length of Neuro-2a cell axons was measured using Image J software after single administration of citicoline;
  • Figure 2 Effect of combined administration of citicoline and NMN on the low serum-induced Neuro-2a neuronal axon regeneration model.
  • A. The length of Neuro-2a cell axons was measured using Image J software after combined administration of citicoline and NMN;
  • FIG. 3 Effect of combined administration of citicoline and NMN on axon regeneration model of primary neurons induced by low serum in mice.
  • A. The axon length of primary neurons in mice was measured using Image J software after single administration of citicoline and NMN and combined administration;
  • Figure 4 Effects of single and combined administration of citicoline and NMN on learning and memory ability of rat vascular dementia model.
  • A Schematic diagram of the behavior of rats in each group in the water maze navigation test;
  • B Escape latency of rats in each group in the water maze navigation test;
  • C Time of exploration of new objects by rats in each group in the new and old object recognition test;
  • D Schematic diagram of the behavior of rats in each group in the water maze space exploration test;
  • E Schematic diagram of the behavior of rats in each group in the water maze space exploration test.
  • FIG. 1 Effects of single and combined administration of Citicoline and NMN on the gene expression of TNF- ⁇ , IL-1 ⁇ , IL-6, CD206 and IL-10 in brain tissue.
  • 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 liver of rats in each group;
  • the optimal molar ratio of citicoline and NMN is 3.33:1.
  • the daily dosage of citicoline is 0.1-2 mmol/kg
  • the daily dosage of NMN is 0.1-2 mmol/kg.
  • the daily intraperitoneal injection dose of citicoline is 0.2-0.4 mmol/kg
  • the daily intraperitoneal injection dose of NMN is 0.1-0.4 mmol/kg.
  • a pharmaceutical composition consisting of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 1:1.
  • a pharmaceutical composition consisting of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 2:1.
  • a pharmaceutical composition consisting of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 3.33:1.
  • a pharmaceutical composition consisting of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 4:1.
  • a pharmaceutical composition consisting of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 6:1.
  • a pharmaceutical composition consisting of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 8:1.
  • a pharmaceutical composition consisting of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 10:1.
  • Example 8 Effects of single administration of citicoline and NMN on the low serum-induced Neuro-2a neuronal axon regeneration model.
  • Neuro-2a cells were inoculated in a culture dish and induced to regenerate axons using MEM medium containing 0.1% FBS. Then, different concentrations (0.2mM, 0.6mM, 2mM) of citicoline and NMN were applied to Neuro-2a cells for 12, 24 and 48h, respectively, and bright field images were taken to detect whether the axon length and the number of cells with axons increased significantly.
  • Example 9 Effect of combined administration of citicoline and NMN on the low serum-induced axon regeneration model of Neuro-2a neurons.
  • mice were seeded in culture dishes and MEM medium containing 0.1% FBS was used to induce axon regeneration. Then, different ratios of citicoline and NMN (C1 or N1: 2mM; C2 or N2: 0.6mM; C3 or N3: 0.2mM; C4 or N4: 0.06mM; C5 or N5: 0.02mM) were applied to Neuro-2a cells for 12, 24 and 48h, and bright field images were taken to detect whether the axon length and the number of cells with axons increased significantly.
  • citicoline and NMN C1 or N1: 2mM; C2 or N2: 0.6mM; C3 or N3: 0.2mM; C4 or N4: 0.06mM; C5 or N5: 0.02mM
  • Example 10 Effect of combined administration of citicoline and NMN on the low serum-induced axon regeneration model of primary cortical neurons in mice.
  • mice Take fetal mice at 15 days of gestation, decapitate them, remove the brain, and peel off the meninges. Remove the white matter except the cortex, and use The cells were digested with 0.25% trypsin, sieved through a filter, resuspended by centrifugation, and seeded on a 24-well plate coated with L-polylysine at a density of 1 ⁇ 10 6 /ml. Then, different ratios of citicoline and NMN (1:1, 2:1, 4:1, with a total dose of 2 mM) were applied to the cells for 12, 24, and 48 h, respectively. Bright field images were taken to detect whether the length of the axons of the nerve cells and the number of cells with axons increased significantly.
  • Example 11 Effects of single and combined administration of citicoline and NMN on the learning and memory abilities of a rat vascular dementia model.
  • Animal grouping 90 male SD rats were randomly divided into 6 groups, 15 rats in each group, including blank group, model group, Citicoline (200 mg/kg) group, NMN (500 mg/kg) group, Citicoline (133.33 mg/kg) + NMN (66.67 mg/kg) group, and Citicoline (160 mg/kg) + NMN (40 mg/kg) group. Except for the blank group, the skin and subcutaneous tissue were cut along the midline of the neck with sterile instruments in the other 5 groups, and the common carotid artery on one side was bluntly separated and ligated between the anterior neck muscle and the lateral muscle on one side. The operation on the other side was the same as above, and the skin was sutured.
  • the blank group only bluntly separated the muscles and then sutured the skin. Seven days after the operation, the cerebral blood flow of the rats tended to be stable and decreased to 65% of the preoperative level, and the drug was 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 corresponding doses of drugs for 28 consecutive days. Water maze test and new and old object recognition experiment were performed 28 days after the operation, and the rats were killed and their brains were removed 35 days after the operation.
  • Example 12 Effects of single and combined administration of citicoline and NMN on the gene expressions of TNF- ⁇ , IL-1 ⁇ , IL-6, CD206 and IL-10 in brain tissue.
  • Example 7 Animal modeling and administration methods were the same as in Example 7. Brain tissue was taken and weighed on an electronic balance to about 20 mg, and Trizol was added for lysis, and total RNA of brain tissue was extracted, reverse transcribed into cDNA, and a reaction system was prepared for amplification to detect changes in the gene expression of TNF- ⁇ , IL-1 ⁇ , IL-6, CD206 and IL-10 in brain tissue.
  • the mRNA expression levels of TNF- ⁇ , IL-1 ⁇ and IL-6 in rat brain tissue increased after surgical modeling.
  • the combined administration of citicoline (160 mg/kg) and NMN (40 mg/kg) can significantly reduce the mRNA expression levels of TNF- ⁇ , IL-1 ⁇ and IL-6 in brain tissue, and has significant advantages over the single administration of citicoline (200 mg/kg) or NMN (500 mg/kg); after surgical modeling, the combined administration of citicoline (160 mg/kg) and NMN (40 mg/kg) can significantly increase the mRNA expression levels of CD206 and IL-10 in brain tissue, and has significant advantages over the single administration of citicoline (200 mg/kg) or NMN (500 mg/kg).
  • Example 13 Study on the toxicity of single and combined administration of citicoline and NMN to rats.
  • the animal modeling and drug administration methods were the same as those in Example 7.
  • the body weight of rats in each group was counted daily. After the animals were sacrificed, the spleen and liver weights of each rat were weighed, and the coefficients of each organ were calculated and counted.
  • the present invention uses bilateral common carotid artery ligation in the rat neck to cause persistent cerebral hypoperfusion and hypoxic-ischemic brain damage, to prepare a vascular dementia model, and to investigate the therapeutic effect of combined administration of citicoline and NMN on the vascular dementia model.
  • the results showed that combined administration of citicoline and NMN in vitro can significantly promote axon regeneration in the neural cell system, promote a significant increase in the axon length of mouse brain neuroma cells Neuro-2a, significantly increase the number of axon cells, and promote the extension and regeneration of primary cortical neuron processes in mice, which has significant advantages compared with the administration of citicoline or NMN alone.
  • citicoline 160 mg/kg
  • NMN 40 mg/kg
  • the combined administration of citicoline (160 mg/kg) and NMN (40 mg/kg) can significantly shorten the escape latency of Morris water maze rats, increase the number of platform position crossings, platform quadrant swimming distance and time, and increase the number and time of exploring new objects; in addition, citicoline (160 mg/kg) and NMN (40 mg/kg) can significantly improve the neuroimmune inflammatory microenvironment in the rat brain, which has a certain reference significance for the treatment of vascular dementia.
  • the combined use of citicoline and NMN can further increase the efficacy and reduce adverse reactions, providing an economical, stable, safe and reliable solution for the treatment of vascular dementia, with an unexpected effect of 1+1>2.

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Abstract

Disclosed are a citicoline pharmaceutical composition, use thereof, and use thereof in the preparation of a medicament for preventing and/or treating vascular dementia. The pharmaceutical composition consists of citicoline or a pharmaceutically acceptable salt thereof, and nicotinamide mononucleotide (NMN)/nicotinamide adenine dinucleotide NAD+ or a pharmaceutically acceptable salt thereof. It is found that the combined administration of citicoline and NMN/NAD+ has a better therapeutic effect than the administration of citicoline or NMN/NAD+ alone, promoting a significant increase in the axon length of mouse brain neuroma cells Neuro-2a, remarkably increasing the number of axon cells, and thus promoting the extension and regeneration of mouse primary cortical neurites. The escape latency of rats in the Morris water maze can be remarkably shortened, thereby increasing the number of platform position crossings and the swimming distance and time in the platform quadrant, and increasing the number and duration of explorations on a new object. In addition, the immune inflammatory microenvironment in the rat brain can be remarkably improved.

Description

一种胞磷胆碱药物组合物及其用途A citicoline pharmaceutical composition and its use 技术领域Technical Field
本发明涉及胞磷胆碱(Citicoline)和烟酰胺单核苷酸NMN/烟酰胺腺嘌呤二核苷酸NAD+联合给药在制备药物中的新应用，主要涉及胞磷胆碱和NMN/NAD+联合给药在制备治疗血管性痴呆的药物中的应用，属于医药技术领域。The present invention relates to a new application of combined administration of citicoline and nicotinamide mononucleotide NMN/nicotinamide adenine dinucleotide NAD + in the preparation of medicines, and mainly relates to the application of combined administration of citicoline and NMN/NAD + in the preparation of medicines for treating vascular dementia, and belongs to the field of medical technology.
背景技术Background technique
血管性痴呆(Vascular Dementia,VaD)是指由缺血性卒中、出血性卒中和造成记忆、认知和行为等脑区低灌注的脑血管疾病所致的严重认知功能障碍综合征。根据流行病学调查显示，血管性痴呆是继阿尔茨海默病之后导致痴呆的第二大常见原因，约占15％的病例。发病率随着年龄的增长而上升，血管性痴呆的风险大约每5.3年翻一倍。此外，大约15-30％的受试者在中风后3个月出现痴呆。VaD不仅降低了患者自身的生存质量，而且也给患者家庭和社会带去沉重的负担。然而，与阿尔茨海默病不同，血管性痴呆没有获得许可的治疗方法。因此，临床迫切需要找到一种低风险、有效的药物来治疗血管性痴呆。Vascular dementia (VaD) refers to a syndrome of severe cognitive dysfunction caused by ischemic stroke, hemorrhagic stroke, and cerebrovascular disease that causes low perfusion of brain areas 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 about 15% of cases. The incidence rate increases with age, and the risk of vascular dementia doubles approximately every 5.3 years. In addition, approximately 15-30% of subjects develop dementia 3 months after a stroke. VaD not only reduces the patient's own quality of life, but also brings a heavy burden to the patient's family and society. However, unlike Alzheimer's disease, there is no licensed treatment for vascular dementia. Therefore, there is an urgent need to find a low-risk, effective drug to treat vascular dementia.
发明内容Summary of the invention
血管性痴呆的发病机制主要与脑血流持续下降有关，慢性低灌注和血栓栓塞导致脑血流持续下降，缺氧，氧化应激并引发炎症反应。脑室周围白质区域、基底神经节和海马极易受到灌注不足引起的病变影响，前额叶-基底神经节回路的中断导致认知缺陷。脑白质极易受到缺氧诱发的损伤，引起脱髓鞘，脱髓鞘会延迟神经信号传递并导致认知丧失。炎症因子会进一步加重白质损伤(脱髓鞘、轴突缺失、少突胶质细胞变性)，神经发生、神经元祖细胞增殖、突触可塑性和树突状脊柱密度受损，引起神经变性和细胞死亡。The pathogenesis of vascular dementia is mainly related to the continuous decrease in cerebral blood flow. Chronic hypoperfusion and thromboembolism lead to a continuous decrease in cerebral blood flow, hypoxia, oxidative stress and inflammatory response. The periventricular white matter area, basal ganglia and hippocampus are extremely susceptible to lesions caused by insufficient perfusion, and the interruption of the prefrontal lobe-basal ganglia circuit leads to cognitive deficits. The white matter of the brain is extremely susceptible to hypoxia-induced damage, causing demyelination, which delays neural signal transmission and leads to cognitive loss. Inflammatory factors further aggravate white matter damage (demyelination, axonal loss, oligodendrocyte degeneration), impair neurogenesis, neuronal progenitor cell proliferation, synaptic plasticity and dendritic spine density, causing neurodegeneration and cell death.
胞磷胆碱(Citicoline)是一种天然的内源性化合物，也是合成磷脂酰胆碱(细胞膜的成分之一)的前体。胞磷胆碱给药可以通过减少磷脂酰胆碱的分解来保护细胞膜。一旦被吸收，胞磷胆碱就会转化为胆碱和胞苷，它们在体内循环，进入全身循环并穿过血脑屏障，在大脑中重新合成为胞磷胆碱。多项研究表明，胞磷胆碱可有效治疗中枢神经系统疾病，包括急性和慢性脑缺血、脑出血、全脑缺氧和神经退行性疾病。胞磷胆碱治疗可减少梗塞面积(梗死组织区域)、降低游离脂肪酸浓度、减少神经功能缺损、恢复动物学习能力、减少谷氨酸介导的损伤、保持磷脂酰胆碱水平并提高神经元存活率。Citicoline is a natural endogenous compound and a precursor for the synthesis of phosphatidylcholine, a component 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 to be resynthesized into citicoline in the brain. Several studies have shown that citicoline is effective in treating central nervous system diseases, including acute and chronic cerebral ischemia, cerebral hemorrhage, global brain hypoxia, and neurodegenerative diseases. Citicoline treatment can reduce infarct size (area of infarcted tissue), reduce free fatty acid concentrations, reduce neurological deficits, restore animals' learning ability, reduce glutamate-mediated damage, maintain phosphatidylcholine levels, and improve neuronal survival.
烟酰胺单核苷酸(β-cotinamide mononucleotide)是一种自然存在的生物活性核苷酸，是辅酶1NAD+(烟酰胺腺嘌呤二核苷酸)的前体。在正常和病理生理条件下，腹腔给药 NMN会迅速(在15分钟内)增加海马和下丘脑等大脑区域的NAD+水平，表明NMN可以通过BBB并有助于NAD+大脑中的生物合成。最近的研究表明，NMN可以改善大脑中的许多神经元功能。NMN给药改善了阿尔茨海默病小鼠和大鼠模型的认知和记忆。NMN保护神经元免受缺血或脑出血后的细胞死亡，并改善脑缺血中BBB完整性的丧失和组织纤溶酶原激活剂诱导的出血性转化。Nicotinamide mononucleotide (β-cotinamide mononucleotide) is a naturally occurring biologically active nucleotide and a precursor of coenzyme 1 NAD + (nicotinamide adenine dinucleotide). Under normal and pathological physiological conditions, intraperitoneal administration NMN rapidly (within 15 minutes) increases NAD + levels in brain regions such as the hippocampus and hypothalamus, indicating that NMN can pass 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 Alzheimer's disease mouse and rat models. NMN protects neurons from cell death after ischemia or cerebral hemorrhage and improves loss of BBB integrity in cerebral ischemia and hemorrhagic transformation induced by tissue plasminogen activator.
近年研究发现，胞磷胆碱等脑保护剂联合用药对恢复脑功能有效，对提高患者的认知能力和智力水平有一定的作用。胞磷胆碱钠作为脑细胞代谢剂，通过降低脑血管的阻力，增加脑部血流来促进脑物质代谢，改善脑部血液循环，也可增强椎体系统的功能，改善运动麻痹，有促进大脑功能恢复和促进苏醒的作用，对脑中风所致的偏瘫可逐渐恢复四肢的功能，用于缺血性脑血管病和血管性痴呆的治疗。此外，胞磷胆碱钠可促进磷脂酰胆碱的合成，改善脑细胞营养。脑缺血会消耗脑组织NAD+导致生物能量学衰竭和细胞死亡。缺血后NAD+水平可以通过烟酰胺单核苷酸来补充，增加的NAD+水平促进神经血管单元中SIRT1的激活。此外，烟酰胺单核苷酸还具有保护脑细胞内线粒体的作用，产生足够能量以恢复神经传导功能。两种药物组分配伍的目的在于能量保证及脑细胞营养代谢两方面的互相协同，使得脑血管通过胞磷胆碱钠促进脑细胞代谢，并通过烟酰胺单核苷酸提供NAD+，使大脑的脑细胞恢复正常功能和苏醒，从而对脑血管疾病产生长久有效的治疗效果，避免单种药物治疗效果差、易复发。因此，本发明将胞磷胆碱与NMN/NAD+联用来制备预防血管性痴呆的药物，可能会产生良好的临床效果。Recent studies have found that the combined use of brain protective agents such as citicoline is effective in restoring brain function and has a certain effect on improving patients' cognitive ability and intelligence level. As a brain cell metabolic agent, citicoline sodium promotes brain substance metabolism and improves brain blood circulation by reducing cerebral vascular resistance and increasing cerebral blood flow. It can also enhance the function of the vertebral system, improve motor paralysis, promote brain function recovery and promote awakening. It can gradually restore the function of the limbs in hemiplegia caused by cerebral stroke, and is used for the treatment of ischemic cerebrovascular disease and vascular dementia. In addition, citicoline sodium can promote the synthesis of phosphatidylcholine and improve brain cell nutrition. Cerebral ischemia consumes brain tissue NAD + , leading to bioenergetics failure and cell death. NAD + levels after ischemia can be supplemented by nicotinamide mononucleotide, and the increased NAD + levels promote the activation of SIRT1 in the neurovascular unit. In addition, nicotinamide mononucleotide also has the function of protecting mitochondria in brain cells, generating enough energy to restore nerve conduction function. The purpose of the combination of the two drug components is to ensure the energy and brain cell nutrition metabolism, so that the cerebral blood vessels promote brain cell metabolism through citicoline sodium, and provide NAD + through nicotinamide mononucleotide, so that the brain cells of the brain can restore normal function and wake up, thereby producing a long-term and effective therapeutic effect on cerebrovascular diseases, avoiding the poor effect of single drug treatment and easy recurrence. Therefore, the present invention combines citicoline with NMN/NAD + to prepare a drug for preventing vascular dementia, which may produce good clinical effects.
迄今为止，并没有相关论文阐述胞磷胆碱和NMN/NAD+在血管性痴呆上的应用，本发明的核心创新内容就在于胞磷胆碱和NMN/NAD+联用可以用于治疗血管性痴呆，可以改善慢性脑低灌注大鼠的行为认知能力，并改善大鼠脑内的神经免疫炎性微环境，这是目前为止尚未证明的新用途。

So far, there is no relevant paper describing the application of citicoline and NMN/NAD + in vascular dementia. The core innovation of the present invention is that the combination of citicoline and NMN/NAD + can be used to treat vascular dementia, improve the behavioral cognitive ability of rats with chronic cerebral hypoperfusion, and improve the neuroimmune inflammatory microenvironment in the rat brain. This is a new use that has not been proven so far.

基于以上情况，本发明提供了一种胞磷胆碱药物组合物及其用途，从而为血管性痴呆的治疗提供新的治疗药物。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.
为此，本发明提供了如下技术方案：To this end, the present invention provides the following technical solutions:
一种药物组合物，所述药物组合物由胞磷胆碱或其药学上可接受的盐与烟酰胺单核苷酸NMN/烟酰胺腺嘌呤二核苷酸NAD+或其药学上可接受的盐组成；所述胞磷胆碱或其药学上可接受的盐与NMN/NAD+或其药学上可接受的盐的摩尔比为1:1至10:1，其中胞磷胆碱或其药学上可接受的盐以胞磷胆碱计，NMN/NAD+或其药学上可接受的盐以NMN/NAD+计。A pharmaceutical composition, comprising 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 + .
其中，所述胞磷胆碱或其药学上可接受的盐与NMN/NAD+或其药学上可接受的盐的摩尔比可以是1:1至10:1中的任何一个比例，包括但并不仅限于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等。Among them, the molar ratio of citicoline or a pharmaceutically acceptable salt thereof to NMN/NAD + or a pharmaceutically acceptable salt thereof 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 and the like.
在一些实施例中，所述胞磷胆碱或其药学上可接受的盐与NMN/NAD+或其药学上可接受的盐的摩尔比为1:1～8:1，优选为2:1～6:1，更优选2:1～4:1，最优选为3.3：1。In some embodiments, the molar ratio of citicoline or a pharmaceutically acceptable salt thereof to NMN/NAD + or a pharmaceutically acceptable salt thereof 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.
在一些实施例中，胞磷胆碱药学上可接受的盐选自胞磷胆碱钠盐。In some embodiments, the pharmaceutically acceptable salt of citicoline is selected from citicoline sodium salt.
第二方面，提供所述的药物组合物在制备预防和/或治疗血管性痴呆的药物中的应用。In a second aspect, a use of the pharmaceutical composition in the preparation of a drug for preventing and/or treating vascular dementia is provided.
所述药物组合物能够促进神经细胞系轴突再生，促进小鼠脑神经瘤细胞Neuro-2a轴突长度明显增长，有轴突细胞的数目增加，促进小鼠原代皮层神经元突起的延长及再生。The pharmaceutical composition can promote the regeneration of axons in nerve cell systems, promote the significant growth of axon length in mouse brain neuroma cells Neuro-2a, increase the number of axon cells, and promote the extension and regeneration of primary cortical neuron processes in mice.
所述药物组合物能够显著缩短Morris水迷宫大鼠的逃避潜伏期，增加平台位置穿越次数、平台象限游泳距离和时间，增加对新物体的探索的次数和时间。The pharmaceutical composition can significantly shorten the escape latency of Morris water maze rats, increase the number of platform position crossings, platform quadrant swimming distance and time, and increase the number and time of exploring new objects.
所述药物组合物能够显著改善大鼠脑内的神经免疫炎性微环境，发挥治疗血管性痴呆的作用。所述药物组合物安全无毒，对体重及脏器系数没有影响。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.
提供了不同剂量的胞磷胆碱和NMN以及胞磷胆碱和NMN/NAD+联用在制备大鼠血管性痴呆模型药物上的应用，所述的治疗大鼠血管性痴呆是指药物组合物可以缩短Morris水 迷宫大鼠的逃避潜伏期，增加平台位置穿越次数、平台象限游泳距离和时间。Provided are uses of different doses of citicoline and NMN and a combination of citicoline and NMN/NAD + in preparing a rat vascular dementia model drug, wherein the drug composition for treating rat vascular dementia can shorten Morris water The escape latency of rats in the maze increased the number of platform position crossings, platform quadrant swimming distance and time.
优选的，胞磷胆碱每日用药量是0.1～2mmol/kg，NMN每日用药量是0.1～2mmol/kg。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.
在以上方案的基础上，最优选的是，胞磷胆碱每日腹腔注射剂量是0.2～0.4mmol/kg，NMN每日腹腔注射剂量是0.1～0.4mmol/kg。Based on the above scheme, it is most preferred that 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.
本发明的胞磷胆碱和NMN联用对大鼠血管性痴呆模型具有以下效果：The combined use of citicoline and NMN of the present invention has the following effects on the rat vascular dementia model:
通过实验表明：胞磷胆碱和NMN联用能够促进神经细胞系轴突再生，促进小鼠脑神经瘤细胞Neuro-2a轴突长度明显增长，有轴突细胞的数目增加，促进小鼠原代皮层神经元突起的延长及再生。Experiments have shown that the combination of citicoline and NMN can promote axon regeneration in neural cell lines, significantly increase the length of axons in mouse brain neuroma cells Neuro-2a, increase the number of axonal cells, and promote the extension and regeneration of processes in primary cortical neurons of mice.
胞磷胆碱和NMN联用能够显著缩短Morris水迷宫大鼠的逃避潜伏期，增加平台位置穿越次数、平台象限游泳距离和时间，增加对新物体的探索的次数和时间。The combined use of citicoline and NMN can significantly shorten the escape latency of rats in the Morris water maze, increase the number of platform position crossings, the swimming distance and time in the platform quadrant, and increase the number and time of exploration of new objects.
胞磷胆碱和NMN联用能够显著改善大鼠脑内的免疫炎性微环境，发挥治疗血管性痴呆的作用。The combination of citicoline and NMN can significantly improve the immune inflammatory microenvironment in the rat brain and play a role in treating vascular dementia.
本发明技术方案的第三方面是提供了一种药物制剂，活性成分为胞磷胆碱和NMN/NAD+药物组合，加入常规辅料，制备成临床可接受的胃肠道给药剂型、注射给药剂型等。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, which are added with conventional excipients to prepare clinically acceptable gastrointestinal dosage forms, injection dosage forms, etc.
本发明公开了胞磷胆碱和NMN/NAD+联合给药在治疗大鼠血管性痴呆模型上的作用机制，发现胞磷胆碱和NMN/NAD+联合给药比胞磷胆碱或NMN/NAD+单独给药具有更好的疗效，促进小鼠脑神经瘤细胞Neuro-2a轴突长度明显增长，有轴突细胞数目明显增加，促进小鼠原代皮层神经元突起的延长及再生；可以显著缩短Morris水迷宫大鼠的逃避潜伏期，增加平台位置穿越次数、平台象限游泳距离和时间，增加对新物体的探索的次数和时间；另外，胞磷胆碱和NMN/NAD+联合给药比胞磷胆碱或NMN/NAD+单独给药还可以显著改善大鼠脑内的免疫炎性微环境。因此，胞磷胆碱和NMN/NAD+联合给药可以用于治疗血管性痴呆，减少不良反应，为血管性痴呆治疗提供安全、有效的方法，具有1+1>2的意想不到的效果。The present invention discloses the mechanism of action of the combined administration of citicoline and NMN/NAD + in treating a rat vascular dementia model, and finds that the combined administration of citicoline and NMN/NAD + has better therapeutic effect than the single administration of citicoline or NMN/NAD + , promotes the significant growth of the axon length of the mouse brain neuroma cell Neuro-2a, significantly increases the number of axonal cells, and promotes the extension and regeneration of the processes of primary cortical neurons in mice; can significantly shorten the escape latency of Morris water maze rats, increase the number of platform position crossings, the platform quadrant swimming distance and time, and increase the number and time of exploring new objects; in addition, the combined administration of citicoline and NMN/NAD + can also significantly improve the immune inflammatory microenvironment in the rat brain compared with the single administration of citicoline or NMN/NAD + . Therefore, the combined administration of citicoline and NMN/NAD + can be used to treat vascular dementia, reduce adverse reactions, and provide a safe and effective method for the treatment of vascular dementia, with an unexpected effect of 1+1>2.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1.胞磷胆碱和NMN单给药对低血清诱导Neuro-2a细胞轴突再生模型的作用。A.胞磷胆碱单给药后用Image J软件测量Neuro-2a细胞轴突长度；B.胞磷胆碱单给药后用Image J软件计算细胞轴突的百分比；C.NMN单给药后用Image J软件测量Neuro-2a细胞 轴突长度；D.NMN单给药后用Image J软件计算细胞轴突的百分比；*P<0.05，**P<0.01和***P<0.001与空白组相比；#P<0.05，##P<0.01和###P<0.001与模型组相比，N＝10。Figure 1. Effects of single administration of citicoline and NMN on the low serum-induced Neuro-2a cell axon regeneration model. A. The length of Neuro-2a cell axons was measured using Image J software after single administration of citicoline; B. The percentage of cell axons was calculated using Image J software after single administration of citicoline; C. The percentage of Neuro-2a cell axons was measured using Image J software after single administration of NMN Axon length; D. The percentage of cell axons was calculated using 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.
图2.胞磷胆碱和NMN联合给药对低血清诱导Neuro-2a神经细胞轴突再生模型的作用。A.胞磷胆碱和NMN联合给药后用Image J软件测量Neuro-2a细胞轴突长度；B.胞磷胆碱和NMN联合给药后用Image J软件计算细胞轴突的百分比；*P<0.05，**P<0.01和***P<0.001与空白组相比；#P<0.05，##P<0.01和###P<0.001与模型组相比，N＝10。Figure 2. Effect of combined administration of citicoline and NMN on the low serum-induced Neuro-2a neuronal axon regeneration model. A. The length of Neuro-2a cell axons was measured using Image J software after combined administration of citicoline and NMN; B. The percentage of cell axons was calculated using 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.
图3.胞磷胆碱和NMN联合给药对低血清诱导小鼠原代神经元轴突再生模型的作用。A.胞磷胆碱和NMN单给药以及联合给药后用Image J软件测量小鼠原代神经元轴突长度；B.胞磷胆碱和NMN联合给药后用Image J软件计算神经细胞轴突的百分比；#P<0.05，##P<0.01和###P<0.001与空白组相比；&P<0.05，&&P<0.01和&&&P<0.001与Citicoline(2mM)组相比，N＝10。Figure 3. Effect of combined administration of citicoline and NMN on axon regeneration model of primary neurons induced by low serum in mice. A. The axon length of primary neurons in mice was measured using Image J software after single administration of citicoline and NMN and combined administration; B. The percentage of axons of nerve cells was calculated using 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 (2mM) group, N=10.
图4.胞磷胆碱和NMN单给药与联合给药对大鼠血管性痴呆模型的学习记忆能力的影响。A.各组大鼠在水迷宫定位航行实验中的行为示意图；B.各组大鼠在水迷宫定位航行实验中的逃逸潜伏期；C.各组大鼠在新旧物体识别实验对新物体的探索的时间；D.各组大鼠在水迷宫空间探索实验中的行为示意图；E.各组大鼠在水迷宫空间探索实验中的平台位置穿越次数、平台象限游泳距离和时间；*P<0.05，**P<0.01和***P<0.001与空白组相比；#P<0.05，##P<0.01和###P<0.001与模型组相比；&P<0.05，&&P<0.01和&&&P<0.001与Citicoline(200mg/kg)组相比，N＝6-15。Figure 4. Effects of single and combined administration of citicoline and NMN on learning and memory ability of rat vascular dementia model. A. Schematic diagram of the behavior of rats in each group in the water maze navigation test; B. Escape latency of rats in each group in the water maze navigation test; C. Time of exploration of new objects by rats in each group in the new and old object recognition test; D. Schematic diagram of the behavior of rats in each group in the water maze space exploration test; E. Number of platform position crossings, platform quadrant swimming distance and time of rats in each group in the water maze space exploration test; * 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.
图5.胞磷胆碱和NMN单给药与联合给药对脑组织TNF-α、IL-1β、IL-6、CD206和IL-10基因表达的影响。A.给药后对TNF-α基因表达的影响；B.给药后对IL-1β基因表达的影响；C.给药后对IL-6基因表达的影响；D.给药后对CD206基因表达的影响；E.给药后对IL-10基因表达的影响；*P<0.05，**P<0.01和***P<0.001与空白组相比；#P<0.05，##P<0.01和###P<0.001与模型组相比；&P<0.05，&&P<0.01和&&&P<0.001与Citicoline(200mg/kg)组相比，N＝4。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 brain tissue. A. Effect on TNF-α gene expression after administration; B. Effect on IL-1β gene expression after administration; C. Effect on IL-6 gene expression after administration; D. Effect on CD206 gene expression after administration; E. Effect 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.
图6.胞磷胆碱和NMN单给药与联合给药对大鼠安全性评价。A.各组大鼠体重统计；B.各组大鼠肝脏器系数；C.各组大鼠脾脏器系数；*P<0.05，**P<0.01和***P<0.001与空白组相比；#P<0.05，##P<0.01和###P<0.001与模型组相比，N＝6-15。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 liver of rats in each group; C. Organ coefficient of spleen of 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.
具体实施方式Detailed ways
为了进一步阐明本发明，下面给出一系列实施例，这些实施例完全是例证性的，它们 仅用来对本发明具体描述，不应当理解为对本发明的限制。In order to further illustrate the present invention, a series of examples are given below, which are purely illustrative. It is only used to specifically describe the present invention and should not be understood as limiting the present invention.
下面结合具体实施例对本申请做出详细说明。胞磷胆碱或其药学上可接受的盐在实施例中选取胞磷胆碱钠盐(分子量510.31)为例，简写为Cit，NMN/NAD+或其药学上可接受的盐在实施例中选取NMN(分子量334.22)为例。The present application is described in detail below in conjunction with specific examples. Citicoline or its pharmaceutically acceptable salt in the examples is selected as citicoline sodium salt (molecular weight 510.31), abbreviated as Cit, and NMN/NAD + or its pharmaceutically acceptable salt in the examples is selected as NMN (molecular weight 334.22).
根据胞磷胆碱和NMN联合给药对Neuro-2a神经细胞轴突再生的实验结果显示，胞磷胆碱和NMN的最优摩尔比为3.33:1。使用Q值公式计算药物的协同指数：Q＝E(a+b)/(Ea+Eb–Ea*Eb)，其中E(a+b)表示药物a(胞磷胆碱)与药物b(NMN)结合的抑制率(此处指神经细胞轴突增长率)，Ea和Eb分别表示药物a(胞磷胆碱)和药物b(NMN)的抑制率(此处指神经细胞轴突增长率)。药物协同指数结果判断：Q<0.85定义为拮抗作用，0.85<Q<1.15定义为相加作用，Q>1.15定义为协同作用。根据体外神经细胞轴突生长实验数据得出E(胞磷胆碱)＝449.76％，E(NMN)＝20.30％，E(胞磷胆碱+NMN)＝671.04％，最终计算出Q＝1.772>1.15，说明胞磷胆碱和NMN具有良好的药效协同作用。According to the experimental results of the combined administration of citicoline and NMN on the axon regeneration of Neuro-2a nerve cells, the optimal molar ratio of citicoline and NMN is 3.33:1. The synergistic index of the drug is calculated using the Q value formula: 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 axon growth rate of nerve cells), and Ea and Eb represent the inhibition rates of drug a (citicoline) and drug b (NMN), respectively (here refers to the axon growth rate of nerve cells). Judgment of drug synergistic index results: 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 nerve cell axon growth experiment data, E(Citicoline) = 449.76%, E(NMN) = 20.30%, E(Citicoline + NMN) = 671.04%, and finally calculated Q = 1.772>1.15, indicating that Citicoline and NMN have good pharmacodynamic synergy.
根据体内实验结果，优选的，胞磷胆碱每日用药量是0.1～2mmol/kg，NMN每日用药量是0.1～2mmol/kg。According to the results of in vivo experiments, preferably, the daily dosage of citicoline is 0.1-2 mmol/kg, and the daily dosage of NMN is 0.1-2 mmol/kg.
在以上方案的基础上，最优选的是，胞磷胆碱每日腹腔注射剂量是0.2～0.4mmol/kg，NMN每日腹腔注射剂量是0.1～0.4mmol/kg。Based on the above scheme, it is most preferred that 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.
实施例1Example 1
一种药物组合物，由胞磷胆碱钠盐与烟酰胺单核苷酸NMN组成；所述胞磷胆碱钠盐与烟酰胺单核苷酸NMN的摩尔比为1:1。A pharmaceutical composition, consisting of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 1:1.
实施例2Example 2
一种药物组合物，由胞磷胆碱钠盐与烟酰胺单核苷酸NMN组成；所述胞磷胆碱钠盐与烟酰胺单核苷酸NMN的摩尔比为2:1。A pharmaceutical composition, consisting of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 2:1.
实施例3Example 3
一种药物组合物，由胞磷胆碱钠盐与烟酰胺单核苷酸NMN组成；所述胞磷胆碱钠盐与烟酰胺单核苷酸NMN的摩尔比为3.33:1。A pharmaceutical composition, consisting of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 3.33:1.
实施例4Example 4
一种药物组合物，由胞磷胆碱钠盐与烟酰胺单核苷酸NMN组成；所述胞磷胆碱钠盐与烟酰胺单核苷酸NMN的摩尔比为4:1。A pharmaceutical composition, consisting of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 4:1.
实施例5 Example 5
一种药物组合物，由胞磷胆碱钠盐与烟酰胺单核苷酸NMN组成；所述胞磷胆碱钠盐与烟酰胺单核苷酸NMN的摩尔比为6:1。A pharmaceutical composition, consisting of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 6:1.
实施例6Example 6
一种药物组合物，由胞磷胆碱钠盐与烟酰胺单核苷酸NMN组成；所述胞磷胆碱钠盐与烟酰胺单核苷酸NMN的摩尔比为8:1。A pharmaceutical composition, consisting of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 8:1.
实施例7Example 7
一种药物组合物，由胞磷胆碱钠盐与烟酰胺单核苷酸NMN组成；所述胞磷胆碱钠盐与烟酰胺单核苷酸NMN的摩尔比为10:1。A pharmaceutical composition, consisting of citicoline sodium salt and nicotinamide mononucleotide NMN; the molar ratio of citicoline sodium salt to nicotinamide mononucleotide NMN is 10:1.
实施例8.胞磷胆碱和NMN单给药对低血清诱导Neuro-2a神经细胞轴突再生模型的作用。Example 8. Effects of single administration of citicoline and NMN on the low serum-induced Neuro-2a neuronal axon regeneration model.
实验方法：将Neuro-2a细胞接种在培养皿中，用含有0.1％FBS的MEM培养基诱导其轴突再生。然后将不同浓度(0.2mM、0.6mM、2mM)的胞磷胆碱和NMN分别作用于Neuro-2a细胞12、24及48h，对其进行明场拍摄，检测轴突长度以及有轴突细胞的数目是否显著增加。Experimental method: Neuro-2a cells were inoculated in a culture dish and induced to regenerate axons using MEM medium containing 0.1% FBS. Then, different concentrations (0.2mM, 0.6mM, 2mM) of citicoline and NMN were applied to Neuro-2a cells for 12, 24 and 48h, respectively, and bright field images were taken to detect whether the axon length and the number of cells with axons increased significantly.
结果如表1、表2和图1所示，作用时间为24h时，胞磷胆碱(2mM)能显著促进小鼠脑神经瘤细胞Neuro-2a轴突长度明显增长，有轴突细胞数目明显增加。*P<0.05，**P<0.01和***P<0.001与空白组相比；#P<0.05，##P<0.01和###P<0.001与模型组相比，N＝10。
The results are shown in Table 1, Table 2 and Figure 1. When the action time is 24h, citicoline (2mM) can significantly promote the axon length of mouse brain neuroma cells Neuro-2a and the number of axon cells. * 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.
表1
Table 1
表2 Table 2
实施例9.胞磷胆碱和NMN联合给药对低血清诱导Neuro-2a神经细胞轴突再生模型的作用。Example 9. Effect of combined administration of citicoline and NMN on the low serum-induced axon regeneration model of Neuro-2a neurons.
实验方法：将Neuro-2a神经细胞接种在培养皿中，用含有0.1％FBS的MEM培养基诱导其轴突再生。然后将不同比例(C1 or N1：2mM；C2 or N2：0.6mM；C3 or N3：0.2mM；C4 or N4：0.06mM；C5 or N5：0.02mM)的胞磷胆碱和NMN分别作用于Neuro-2a细胞12、24及48h，对其进行明场拍摄，检测轴突长度以及有轴突细胞的数目是否显著增加。Experimental method: Neuro-2a neurons were seeded in culture dishes and MEM medium containing 0.1% FBS was used to induce axon regeneration. Then, different ratios of citicoline and NMN (C1 or N1: 2mM; C2 or N2: 0.6mM; C3 or N3: 0.2mM; C4 or N4: 0.06mM; C5 or N5: 0.02mM) were applied to Neuro-2a cells for 12, 24 and 48h, and bright field images were taken to detect whether the axon length and the number of cells with axons increased significantly.
结果如表3和图2所示，作用时间为24h时，胞磷胆碱(2mM)+NMN(0.6mM)能显著促进小鼠脑神经瘤细胞Neuro-2a轴突长度明显增长，有轴突细胞数目明显增加。*P<0.05，**P<0.01和***P<0.001与空白组相比；#P<0.05，##P<0.01和###P<0.001与模型组相比，N＝10。
The results are shown in Table 3 and Figure 2. When the action time is 24h, citicoline (2mM) + NMN (0.6mM) can significantly promote the axon length of mouse brain neuroma cells Neuro-2a, and the number of axon cells is significantly increased. * 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.
表3table 3
实施例10.胞磷胆碱和NMN联合给药对低血清诱导小鼠原代皮层神经元轴突再生模型的作用。Example 10. Effect of combined administration of citicoline and NMN on the low serum-induced axon regeneration model of primary cortical neurons in mice.
实验方法：取怀孕15d的胎鼠，断头取脑，剥离脑膜。去掉除皮层以外的白质，用 0.25％胰酶消化，滤网过筛，离心重悬，以1×106/ml的密度接种于L-多聚赖氨酸包被的24孔板上，然后将不同比例(1:1、2:1、4:1，总剂量为2mM)的胞磷胆碱和NMN分别作用于细胞12、24及48h，对其进行明场拍摄，检测神经细胞轴突长度以及有轴突细胞的数目是否显著增加。Experimental method: Take fetal mice at 15 days of gestation, decapitate them, remove the brain, and peel off the meninges. Remove the white matter except the cortex, and use The cells were digested with 0.25% trypsin, sieved through a filter, resuspended by centrifugation, and seeded on a 24-well plate coated with L-polylysine at a density of 1×10 6 /ml. Then, different ratios of citicoline and NMN (1:1, 2:1, 4:1, with a total dose of 2 mM) were applied to the cells for 12, 24, and 48 h, respectively. Bright field images were taken to detect whether the length of the axons of the nerve cells and the number of cells with axons increased significantly.
结果如表4和图3所示，作用时间为24h时，Citicoline(1.6mM)+NMN(0.4mM)能显著促进小鼠原代皮层神经元轴突长度明显增长，有轴突细胞数目明显增加。#P<0.05，##P<0.01和###P<0.001与空白组相比；&P<0.05，&&P<0.01和&&&P<0.001与Citicoline(2mM)组相比，N＝10。
The results are shown in Table 4 and Figure 3. When the action time is 24h, Citicoline (1.6mM) + NMN (0.4mM) can significantly promote the significant growth of axon length of primary cortical neurons in mice, and the number of axon cells is significantly increased. # 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 (2mM) group, N=10.
表4Table 4
实施例11.胞磷胆碱和NMN单给药与联合给药对大鼠血管性痴呆模型的学习记忆能力的影响。Example 11. Effects of single and combined administration of citicoline and NMN on the learning and memory abilities of a rat vascular dementia model.
实施材料：雄性SD大鼠，体重220-250g，购自杭州子源实验动物科技有限公司。Materials: Male SD rats, weighing 220-250 g, were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.
动物分组：雄性SD大鼠90只，将大鼠随机分为6组，每组15只，分为空白组、模型组、Citicoline(200mg/kg)组、NMN(500mg/kg)组、Citicoline(133.33mg/kg)+NMN(66.67mg/kg)组、Citicoline(160mg/kg)+NMN(40mg/kg)组。除空白组，其他5组以无菌器械沿颈部正中切开皮肤及皮下组织，在一侧颈前肌肉和侧方肌肉间隙钝性分离该侧颈总动脉结扎，另一侧操作同上，缝合皮肤。空白组只钝性分离肌肉后缝合皮肤。手术7天后，大鼠脑血流趋于平稳，减少至术前的65％，开始给药。空白组和模型组每日腹腔注射等量的生理盐水，其他4组分别注射对应剂量的药物，连续28天。并于手术28天后开始进行水迷宫测试和新旧物体识别实验，于手术35天处死大鼠取脑。Animal grouping: 90 male SD rats were randomly divided into 6 groups, 15 rats in each group, including blank group, model group, Citicoline (200 mg/kg) group, NMN (500 mg/kg) group, Citicoline (133.33 mg/kg) + NMN (66.67 mg/kg) group, and Citicoline (160 mg/kg) + NMN (40 mg/kg) group. Except for the blank group, the skin and subcutaneous tissue were cut along the midline of the neck with sterile instruments in the other 5 groups, and the common carotid artery on one side was bluntly separated and ligated between the anterior neck muscle and the lateral muscle on one side. The operation on the other side was the same as above, and the skin was sutured. The blank group only bluntly separated the muscles and then sutured the skin. Seven days after the operation, the cerebral blood flow of the rats tended to be stable and decreased to 65% of the preoperative level, and the drug was 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 corresponding doses of drugs for 28 consecutive days. Water maze test and new and old object recognition experiment were performed 28 days after the operation, and the rats were killed and their brains were removed 35 days after the operation.
结果如表5、表6、表7和图4所示，手术造模后雄鼠的认知能力下降，而Citicoline(160mg/kg)+NMN(40mg/kg)能显著缩短Morris水迷宫大鼠的逃避潜伏期，增加平 台位置穿越次数、平台象限游泳距离和时间，增加对新物体的探索的次数和时间，且与Citicoline(200mg/kg)或NMN(500mg/kg)单独给药相比具有显著性优势，对血管性痴呆的治疗效果更加显著。*P<0.05，**P<0.01和***P<0.001与空白组相比；#P<0.05，##P<0.01和###P<0.001与模型组相比；&P<0.05，&&P<0.01和&&&P<0.001与Citicoline(200mg/kg)组相比，N＝6-15。
The results are shown in Table 5, Table 6, Table 7 and Figure 4. After surgical modeling, the cognitive ability of male rats decreased, while Citicoline (160 mg/kg) + NMN (40 mg/kg) could significantly shorten the escape latency of Morris water maze rats and increase the average The number of platform crossings, platform quadrant swimming distance and time, increased the number and time of exploring new objects, and had significant advantages compared with 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.
表5
table 5
表6
Table 6
表7Table 7
实施例12.胞磷胆碱和NMN单给药与联合给药对脑组织TNF-α、IL-1β、IL-6、CD206和IL-10基因表达的影响。Example 12. Effects of single and combined administration of citicoline and NMN on the gene expressions of TNF-α, IL-1β, IL-6, CD206 and IL-10 in brain tissue.
动物造模及给药方法同实施例7。取脑组织，在电子天平上称20mg左右，加入Trizol进行裂解，提取脑组织总的RNA，逆转录成cDNA，配制反应体系，进行扩增，用来检测脑组织TNF-α、IL-1β、IL-6、CD206和IL-10基因表达的变化。Animal modeling and administration methods were the same as in Example 7. Brain tissue was taken and weighed on an electronic balance to about 20 mg, and Trizol was added for lysis, and total RNA of brain tissue was extracted, reverse transcribed into cDNA, and a reaction system was prepared for amplification to detect changes in the gene expression of TNF-α, IL-1β, IL-6, CD206 and IL-10 in brain tissue.
结果如表8、表9和图5所示，手术造模后大鼠脑组织TNF-α、IL-1β、IL-6的mRNA表达水平升高，胞磷胆碱(160mg/kg)和NMN(40mg/kg)联合给药能显著降低脑组织的TNF-α、IL-1β、IL-6的mRNA表达水平，且与胞磷胆碱(200mg/kg)或NMN(500mg/kg)单独给药相比具有显著性优势；手术造模后给药，胞磷胆碱(160mg/kg)和NMN(40mg/kg)联合给药可以显著升高脑组织的CD206和IL-10的mRNA表达水平，且与胞磷胆碱(200mg/kg)或NMN(500mg/kg)单独给药相比具有显著性优势。这些结果提示，与胞磷胆碱或NMN单独给药相比，胞磷胆碱和NMN联合给药后可以更显著地改善大鼠脑内免疫微环境，对血管性痴呆的治疗效果更加显著。*P<0.05，**P<0.01和***P<0.001与空白组相比；#P<0.05，##P<0.01和###P<0.001与模型组相比；&P<0.05，&&P<0.01和&&&P<0.001与胞磷胆碱(200mg/kg)组相比；$P<0.05，$$P<0.01和$$$P<0.001与NMN(500mg/kg)组相比，N＝4。
As shown in Table 8, Table 9 and Figure 5, the mRNA expression levels of TNF-α, IL-1β and IL-6 in rat brain tissue increased after surgical modeling. The combined administration of citicoline (160 mg/kg) and NMN (40 mg/kg) can significantly reduce the mRNA expression levels of TNF-α, IL-1β and IL-6 in brain tissue, and has significant advantages over the single administration of citicoline (200 mg/kg) or NMN (500 mg/kg); after surgical modeling, the combined administration of citicoline (160 mg/kg) and NMN (40 mg/kg) can significantly increase the mRNA expression levels of CD206 and IL-10 in brain tissue, and has significant advantages over 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 the therapeutic effect on vascular dementia is 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; $ P<0.05, $$ P<0.01 and $$$ P<0.001 compared with the NMN (500 mg/kg) group, N=4.
表8
Table 8
表9Table 9
实施例13.胞磷胆碱和NMN单给药与联合给药对大鼠毒性考察。Example 13. Study on the toxicity of single and combined administration of citicoline and NMN to rats.
动物造模及给药方法同实施例7。每日统计各组大鼠体重，动物处死后，称量各只大鼠的脾重和肝重，计算各脏器系数并统计。The animal modeling and drug administration methods were the same as those in Example 7. The body weight of rats in each group was counted daily. After the animals were sacrificed, the spleen and liver weights of each rat were weighed, and the coefficients of each organ were calculated and counted.
结果如表10和图6所示，各组体重均根据天数缓慢增长，各脏器系数统计没有显著性差异，表明胞磷胆碱和NMN各剂量均无毒性，符合临床安全用药标准。

The results are shown in Table 10 and Figure 6. The body weight of each group increased slowly over the days, and there was no significant difference in the coefficients of each organ, indicating that all doses of citicoline and NMN were non-toxic and met the clinical safety drug use standards.

表10Table 10
综上所述，本发明采用大鼠颈部双侧颈总动脉结扎的方式造成脑持续性低灌注，缺血缺氧性脑损伤，制作血管性痴呆模型，考察胞磷胆碱和NMN联合给药对血管性痴呆模型的治疗作用。结果显示，在体外胞磷胆碱和NMN联合给药可以显著促进神经细胞系轴突再生，促进小鼠脑神经瘤细胞Neuro-2a轴突长度明显增长，有轴突数目细胞明显增多，促进小鼠原代皮层神经元突起的延长及再生，与胞磷胆碱或NMN单独给药相比具有显著性优势。在体内胞磷胆碱(160mg/kg)和NMN(40mg/kg)联合给药能够显著缩短Morris水迷宫大鼠的逃避潜伏期，增加平台位置穿越次数、平台象限游泳距离和时间，增加对新物体的探索的次数和时间；除此之外，胞磷胆碱(160mg/kg)和NMN(40mg/kg)能够显著改善大鼠脑内的神经免疫炎性微环境，对治疗血管性痴呆具有一定的参考意义。这些结果都说明胞磷胆碱和NMN联合用药在治疗血管性痴呆疾病上具有一定的药理药效协同作用。胞磷胆碱和NMN联合使用可进一步增加药效，减少不良反应，为治疗血管性痴呆疾病提供经济、稳定、安全、可靠的解决办法，具有1+1>2的意想不到的效果。In summary, the present invention uses bilateral common carotid artery ligation in the rat neck to cause persistent cerebral hypoperfusion and hypoxic-ischemic brain damage, to prepare a vascular dementia model, and to investigate the therapeutic effect of combined administration of citicoline and NMN on the vascular dementia model. The results showed that combined administration of citicoline and NMN in vitro can significantly promote axon regeneration in the neural cell system, promote a significant increase in the axon length of mouse brain neuroma cells Neuro-2a, significantly increase the number of axon cells, and promote the extension and regeneration of primary cortical neuron processes in mice, which has 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) can significantly shorten the escape latency of Morris water maze rats, increase the number of platform position crossings, platform quadrant swimming distance and time, and increase the number and time of exploring new objects; in addition, citicoline (160 mg/kg) and NMN (40 mg/kg) can significantly improve the neuroimmune inflammatory microenvironment in the rat brain, which has a certain reference significance for the treatment of vascular dementia. These results all indicate that the combined use of citicoline and NMN has a certain pharmacological and pharmacodynamic synergistic effect in the treatment of vascular dementia. The combined use of citicoline and NMN can further increase the efficacy and reduce adverse reactions, providing an economical, stable, safe and reliable solution for the treatment of vascular dementia, with an unexpected effect of 1+1>2.
最后说明的是，以上实施例仅用于说明本发明的技术方案而非限制，尽管通过参照本发明的优选实施例已经对本发明进行了描述，但本领域普通技术人员应当理解，可以在形式上和细节上对其做出各种各样的改变，而不偏离所附权利要求书所限定的本发明的精神和范围。 Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described with reference to the preferred embodiments of the present invention, it should be understood by those skilled in the art that various changes may be made in form and details without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims (10)

  1. 一种药物组合物，其特征在于，所述药物组合物由胞磷胆碱或其药学上可接受的盐与烟酰胺单核苷酸NMN/烟酰胺腺嘌呤二核苷酸NAD+或其药学上可接受的盐组成；所述胞磷胆碱或其药学上可接受的盐与NMN/NAD+或其药学上可接受的盐的摩尔比为1:1至10:1，其中胞磷胆碱或其药学上可接受的盐以胞磷胆碱计，NMN/NAD+或其药学上可接受的盐以NMN/NAD+计。A pharmaceutical composition, characterized in that the pharmaceutical composition consists 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 + .
  2. 根据权利要求1所述的药物组合物，其特征在于，所述胞磷胆碱或其药学上可接受的盐与NMN/NAD+或其药学上可接受的盐的摩尔比为1:1～8:1。The pharmaceutical composition according to claim 1, characterized in that the molar ratio of citicoline or a pharmaceutically acceptable salt thereof to NMN/NAD + or a pharmaceutically acceptable salt thereof is 1:1 to 8:1.
  3. 根据权利要求1所述的药物组合物，其特征在于，所述胞磷胆碱或其药学上可接受的盐与NMN/NAD+或其药学上可接受的盐的摩尔比为2:1～6:1.The pharmaceutical composition according to claim 1, characterized in that the molar ratio of citicoline or a pharmaceutically acceptable salt thereof to NMN/NAD + or a pharmaceutically acceptable salt thereof is 2:1 to 6:1.
  4. 根据权利要求1所述的药物组合物，其特征在于，所述胞磷胆碱或其药学上可接受的盐与NMN/NAD+或其药学上可接受的盐的摩尔比为2:1～4:1，更优选为3.3：1。The pharmaceutical composition according to claim 1, characterized in that the molar ratio of citicoline or a pharmaceutically acceptable salt thereof to NMN/NAD + or a pharmaceutically acceptable salt thereof is 2:1 to 4:1, more preferably 3.3:1.
  5. 根据权利要求1所述的药物组合物，其特征在于，胞磷胆碱药学上可接受的盐选自胞磷胆碱钠盐。The pharmaceutical composition according to claim 1, characterized in that the pharmaceutically acceptable salt of citicoline is selected from citicoline sodium salt.
  6. 权利要求1-5任一项所述的药物组合物在制备预防和/或治疗血管性痴呆的药物中的应用。Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a medicament for preventing and/or treating vascular dementia.
  7. 根据权利要求6的应用，其特征在于，所述药物组合物能够促进神经细胞系轴突再生，促进小鼠脑神经瘤细胞Neuro-2a轴突长度明显增长，有轴突细胞数目明显增加；The use according to claim 6 is characterized in that the pharmaceutical composition can promote axon regeneration in nerve cell lines, promote the significant growth of axon length in mouse brain neuroma cells Neuro-2a, and significantly increase the number of axon cells;
    和/或，所述药物组合物能够促进神经细胞轴突再生，促进小鼠原代皮层神经元突起的延长及再生；And/or, the pharmaceutical composition can promote the regeneration of nerve cell axons and promote the extension and regeneration of primary cortical neuron processes in mice;
    和/或，所述药物组合物能够显著缩短Morris水迷宫大鼠的逃避潜伏期，增加平台位置穿越次数、平台象限游泳距离和时间，增加对新物体的探索的次数和时间；And/or, the pharmaceutical composition can significantly shorten the escape latency of Morris water maze rats, increase the number of platform position crossings, platform quadrant swimming distance and time, and increase the number and time of exploration of new objects;
    和/或，所述药物组合物能够显著改善大鼠脑内的免疫炎性微环境，发挥治疗血管性痴呆的作用。And/or, the pharmaceutical composition can significantly improve the immune inflammatory microenvironment in the rat brain and play a role in treating vascular dementia.
  8. 一种药物制剂，其中含有治疗有效量的权利要求1-5任一项所述的药物组合物及药学上可接受的载体、佐剂或媒剂。A pharmaceutical preparation comprising a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier, adjuvant or vehicle.
  9. 根据权利要求8所述的药物制剂，其特征在于，所述药物制剂的剂型为胃肠道给药剂型、注射给药剂型或皮肤给药剂型，包括胶囊剂、散剂、片剂、颗粒剂、丸剂、注射剂、糖浆剂、口服液、吸入剂、霜剂、软膏剂、栓剂或贴剂。The pharmaceutical preparation according to claim 8, characterized in that the dosage form of the pharmaceutical preparation is a gastrointestinal dosage form, an injection dosage form or a skin dosage form, including capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, creams, ointments, suppositories or patches.
  10. 权利要求8或9所述的药物制剂在制备预防和/或治疗血管性痴呆的药物中的应用。 Use of the pharmaceutical preparation according to claim 8 or 9 in the preparation of a medicament for preventing and/or treating vascular dementia.
PCT/CN2023/111202 2022-11-11 2023-08-04 Citicoline pharmaceutical composition and use thereof WO2024098854A1 (en)

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