WO2022095610A1 - 芬戈莫德在防治鞘脂紊乱引起的神经退行性疾病中的医药用途 - Google Patents

芬戈莫德在防治鞘脂紊乱引起的神经退行性疾病中的医药用途 Download PDF

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WO2022095610A1
WO2022095610A1 PCT/CN2021/118796 CN2021118796W WO2022095610A1 WO 2022095610 A1 WO2022095610 A1 WO 2022095610A1 CN 2021118796 W CN2021118796 W CN 2021118796W WO 2022095610 A1 WO2022095610 A1 WO 2022095610A1
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sphingolipid
disorders
fingolimod
mice
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刘俊平
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杭州端丽生物技术有限公司
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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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • 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
    • A61K31/13—Amines
    • A61K31/133—Amines having hydroxy groups, e.g. sphingosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Definitions

  • the invention belongs to the field of medical biotechnology, and relates to the medicinal use of fingolimod (FTY720) in preventing and treating neurodegenerative diseases caused by sphingolipid disorders.
  • fingolimod fingolimod
  • FTY720 fingolimod or fingolimod in Chinese
  • FTY720 fingolimod or fingolimod in Chinese
  • the chemical name is 2-amino-2-[2-(4-octylphenyl)]-1,3-propanediol hydrochloride
  • the molecular weight is 343.94g/ mol.
  • Phosphorylated fingolimod exerts its effect by binding to the S1P receptor [Hla T, et al. Science, 2001], thereby restricting lymphocytes in the lymph nodes, preventing lymphocytes from entering the central nervous system, thereby protecting the central nervous system effect.
  • fingolimod has also been used to suppress post-transplant immune rejection because of its good immunosuppressive effect [Park SI, et al. Braz J Med Biol Res, 2005], which has been approved by the US Food and Drug Administration (FDA). Became the first oral drug to treat relapsing forms of multiple sclerosis (MS). It can be seen that the currently known effect of fingolimod is to improve neurological deficits by regulating inflammatory and immune processes, and its therapeutic effect does not directly act on nerve cells.
  • ATP13A2 mutations in the ATP13A2 gene are closely associated with the development of Parkinson's disease, especially early-onset Parkinsonism and dementia (Kufor-Rakeb syndrome (KRS); MIM606693) [Fleming, S.M., et al. 2018].
  • ATP13A2 mutations are associated with hereditary spastic paraplegia (SPG) [Estrada-Cuzcano, A., Brain, 2017] and neuronal ceroid lipofuscinoses (NCL) [Schultheis, P.J., et al.
  • SPG hereditary spastic paraplegia
  • NCL neuronal ceroid lipofuscinoses
  • Spastic paraplegia is a group of neurodegenerative diseases, often manifested as pyramidal syndrome, with progressive limb paralysis and lower extremity spasticity. In addition to limb spasticity and weakness, symptoms include epilepsy, deafness, cerebellar dysfunction, cognitive impairment, visual impairment, and peripheral neuropathy. Muscle tone of both lower extremities increased, tendon reflexes were hyperactive, pathological reflexes were positive, and scissors gait was present. More than 70 different types are known, including all modes of inheritance, including autosomal dominant, autosomal recessive, X-linked, or non-Mendelian mitochondrial inheritance. ATP13A2 gene mutation is an important pathogenic gene of SPG at home and abroad.
  • HSP herniated regional pain syndrome
  • the progressive aggravation of HSP will seriously affect the patient's ability to work and self-care, and there is currently no effective method to prevent, terminate or reverse the disease, only through drug, physical or surgical treatment to relieve the patient's symptoms.
  • the pathological changes are mainly in the axonal degeneration and/or demyelination of the bilateral corticospinal tracts in the spinal cord, usually in the thoracic segment.
  • ALS Amyotrophic Lateral Sclerosis
  • ATP13A2 mutations were found to be an important pathogenic lesion in ALS. It causes muscle paralysis after injury including upper motor neuron and lower motor neuron, including bulb (the so-called bulb, which refers to the muscle innervated by the medulla oblongata), limbs, trunk, chest and abdomen. Muscles gradually weaken and atrophy. The etiology of ALS is still unclear. Some patients progress slowly, but some patients progress rapidly. Within a few months, the muscle weakness of the extremities can progress to the weakness of the thoracic respiratory muscles, and then respiratory failure occurs, which threatens the patient. s life. At present, there is no specific treatment for this disease. Riluzole, as a specific drug for this disease, has different therapeutic effects and is not ideal. Some patients have better results, but some patients are basically ineffective in oral drug treatment.
  • Corticobasal degeneration is a chronic progressive neurodegenerative disease characterized by asymmetrical onset of akinetic-rigid syndrome, apraxia, dystonia and postural abnormalities. Clinically, progressive Parkinsonism is seen, with markedly asymmetrical signs and symptoms of damage to the cerebral cortex and basal ganglia, and its pathological changes are accumulation of abnormal tau protein in neurons and glial cells.
  • Spinocerebellar ataxia is a hereditary disease with ataxia as the main symptom. In terms of pathology, its manifestations are various. Common atrophy and degeneration of nerve cells, loss of myelin sheath, and mild hyperplasia of glial cells. , resulting in extensive degeneration of the cerebellar hemisphere, vermis, and middle and lower cerebellar peduncle, and disappearance of Purkinje cells; neuronal cells in the posterior column of the spinal cord and Clarke's column atrophy or disappear, secondary to glial proliferation, posterior root and spinal ganglion Degeneration, myelin Loss, especially in the lumbar, sacral spinal cord is more obvious. There is currently no complete cure, and it is an incurable disease.
  • NCL Neuronal ceroid lipofuscinoses
  • CLN9 and CLN5 are a group of at least 13 distinct, progressive neurodegenerative diseases that are associated with damage to CLN genes 1-14 (CLN9 and CLN5). mutation). ATP13A2 mutation is a recently discovered causative gene.
  • Features of NCL include brain degeneration and deposition of lysosomal autofluorescent storage substances called lipofuscin.
  • NCLs are common childhood-onset autosomal recessive disorders characterized by muscle twitching episodes, visual impairment, intellectual decline, and early death.
  • Adult-onset NCL is not always CLN4 (autosomal dominant Kufs disease or Parry disease), but recessive-onset CLN6 (MIM 601780) and CLN5 mutations are also present.
  • Sphingomyelin is the main component of nerve myelin sheath and cell membrane structure, and is the main component of synthesizing glycosphingolipids such as cerebroside and ganglioside, and producing other sphingolipids (such as ceramide, sphingosine). Phospholipids.
  • glycosphingolipids such as cerebroside and ganglioside
  • other sphingolipids such as ceramide, sphingosine.
  • Phospholipids Phospholipids.
  • sphingomyelin is synthesized by the endoplasmic reticulum and transported to the cell membrane by the Golgi apparatus, and then degraded by the lysosome.
  • impairment of sphingomyelin degradation due to lysosomal sphingomyelinase deficiency causes neurodegenerative diseases.
  • the purpose of the present invention is to provide the medicinal use of fingolimod (FTY720) in the prevention and treatment of neurodegenerative diseases caused by sphingolipid disorders, that is, to provide fingolimod (FTY720) in the preparation and prevention of neurodegenerative diseases caused by sphingolipid disorders Application in disease medicine. It is found in the experiments of the present invention that FTY720 has the effect of regulating sphingolipidase, that is, FTY720 can directly regulate the sphingolipid metabolism of nerve cells, thereby protecting nerve cell apoptosis caused by sphingolipid metabolism disorder. Further research found that FTY720 can effectively reduce lipofuscin deposition and myelin tear in brain tissue, that is, it has a direct protective effect on nerve cells.
  • Spastic paraplegia, ALS, corticobasal degeneration, spinocerebellar ataxia, and neuronal ceroid lipofuscinosis are neurodegenerative diseases associated with sphingomyelin deficiency, which are related to sphingolipid metabolism disorders.
  • the FTY720 medicine provided by the invention has a relieving effect on the above-mentioned neurodegenerative diseases through the regulation of sphingolipid metabolism.
  • neurodegenerative diseases include spastic paraplegia, ALS, corticobasal degeneration, spinocerebellar ataxia, and neuronal ceroid lipofuscinosis, as well as reduced or absent sphingomyelin, or impaired recycling, resulting in of neuromotor diseases with sphingolipid catabolism disorders.
  • the route of administration is oral administration, intraperitoneal injection administration, and intracerebroventricular injection administration.
  • the medicine is prepared from an effective dose of FTY720 and pharmaceutically acceptable excipients, and the total mass fraction of FTY720 in the medicine is 0.01-10%.
  • the FTY720 drug powder was dissolved in 0.9% normal saline, and the dosage range was 0.05-5 mg/kg.
  • the present invention provides a novel prevention and treatment of neurodegenerative diseases such as spastic paraplegia, ALS, corticobasal degeneration, spinocerebellar ataxia and neuronal ceroid lipofuscin
  • This drug was originally used as a first-line immunosuppressive drug in clinical practice, and there are a few reports of anticancer treatment, but it has not been used for other drugs at present.
  • the present invention through different routes and different doses of drug treatment experimental research shows that FTY720 can effectively relieve and significantly improve the motor function of spastic paraplegic mice, FTY720 can make old paraplegic mice stand again, and increase the number of standing paraplegic mice.
  • FTY720 can effectively reduce/reduce lipofuscin deposition and myelin tear in mouse brain tissue.
  • FTY720 can significantly alleviate the reduction or deficiency of sphingomyelin, promote the recycling of sphingomyelin, and correct the sphingolipid disorder caused by sphingomyelin deficiency. of neurological dysfunction.
  • the left picture of Figure 1 shows the comparison of the content of various lipid components in wild-type (Ctrl) and pathological model (KD) SH-SY5Y cells.
  • the abscissa is the value of log2(KD/Ctrl), which represents the multiple difference.
  • the ordinate is -log10 (statistical p value).
  • the lipids with significant differences between the two groups can be directly reflected.
  • the right panel of Figure 1 is a heat map of different SM content in wild-type (Ctrl) and pathological model (KD) SH-SY5Y cells. It can also be seen that the SM content in the KD group was significantly increased compared to Ctrl.
  • Figure 2 shows the contents of sphingomyelin SM-C16, SM-C18, ceramide Cer-C16, Cer-C18 and sphingosine Sph in lysosomes in wild-type (Ctrl) and pathological model (KD) SH-SY5Y cells. It can be seen that sphingomyelin significantly increased in pathological model cells and was stored in lysosomes.
  • Figure 3 shows the lipid content in the substantia nigra of wild-type and pathological model mice.
  • Figure 4 shows the lipid content in the prefrontal cortex of wild-type and pathological model mice.
  • Figure 5 shows the lipid content in the hypothalamus of wild-type and pathological model mice.
  • Figure 6 is a graph showing the comparison of the content of various lipid components in wild-type (Ctrl) and pathological model (KO) MEF cells.
  • the abscissa is the value of log2 (KO/Ctrl), and the ordinate is -log10 (statistical p-value).
  • the graph visually reflects the lipids that are significantly different between the two groups. It can be seen that in the two groups of cells, the content of glycosphingolipids in the pathological model group was significantly reduced.
  • Figure 7 shows that under the condition of the same substrate concentration, adding different concentrations of sphingolipidase, the absorbance value is proportional to the enzyme activity.
  • Blue line sphingolipidase activity value under normal conditions
  • red line sphingolipidase activity value after adding FTY720.
  • Figure 8 shows the effects of different sphingomyelinase inhibitor candidates on the content of intracellular C18 sphingomyelin in MEF cells.
  • Figure 9 shows the effects of different sphingomyelinase inhibitor candidate drugs on the functioning of disease model mice on a rotarod.
  • Figure 10 shows the differentially expressed genes related to sphingolipid metabolism in the prefrontal cortex of the brain tissue of FTY720 mice obtained by single-cell gene analysis. Red indicates high expression and blue indicates low expression.
  • Figure 11 shows the lipidomics detection results obtained by LC-MS method, blue is the ratio of disease group/wild group; red is the ratio of FTY treatment group/disease group.
  • Figure 12 shows the standing times of disease model mice after oral administration of different doses of FTY720.
  • Figure 13 shows the standing times of disease model mice before and after oral administration of FTY720. 1-7 days, the disease model mice were treated with drug administration, 7-14 days with drug withdrawal treatment, and 15-21 days with secondary administration treatment.
  • Figure 14 shows that after 5 days of intraperitoneal injection of FTY720 (0.25mg/kg/day, i.p.) in mice, the rotarod test detected C57BL/6 normal + normal saline group (WT+C group), neurodegenerative disease model mice + normal saline group (KO+C group), normal+FTY720 group (WT+FTY group), neurodegenerative disease model mice+FTY720 group (KO+FTY group) and other four groups.
  • Figure 15 shows that after 7 days of oral administration to mice (0.5mg/kg/day, o.p.), the rotarod test detected C57BL/6 normal + normal saline group (WT+C group), neurodegenerative disease model mice + normal saline group (KO+C group), normal+FTY720 group (WT+FTY group), neurodegenerative disease model mice+FTY720 group (KO+FTY group) and other four groups.
  • Figure 16 shows that after 7 days of intraventricular injection in mice (10 ⁇ g, i.c.v.), the C57BL/6 normal + normal saline group (WT+C group) and the neurodegenerative disease model mice + normal saline group ( Motor function of four groups: KO+C group), normal+FTY720 group (WT+FTY group), and neurodegenerative disease model mice+FTY720 group (KO+FTY group).
  • Figure 17 shows that after 14 days of oral administration to the ventricle of aged mice, standing test detected C57BL/6 normal + normal saline group (WT+C group), neurodegenerative disease model mice + normal saline group (KO+C group), normal Motor function of four groups including +FTY720 group (WT+FTY group), neurodegenerative disease model mice +FTY720 group (KO+FTY group).
  • Figure 18 shows that after 14 days of oral administration to the ventricle of aged mice, the rotarod test detected C57BL/6 normal + normal saline group (WT+C group), neurodegenerative disease model mice + normal saline group (KO+C group), Motor function of four groups including normal+FTY720 group (WT+FTY group) and neurodegenerative disease model mice+FTY720 group (KO+FTY group).
  • Figure 19 is a comparison diagram of the standing of the same elderly paraplegic mouse before and after administration.
  • Fig. 20 is the comparison photos of the hindlimb and the ground (FBA) of the same aged paraplegic mouse before and after administration.
  • Figure 21 shows C57BL/6 normal + normal saline group (WT group), neurodegenerative disease model mice + normal saline group (KO+Vehicle group), neurodegenerative disease model mice + FTY720 group (KO+FTY group) After two weeks of administration, the brain tissues of the three groups of mice were taken for transmission electron microscopy to observe the lipofuscin deposition maps (a-c) and statistical maps (d) in their neurons.
  • Figure 22 shows C57BL/6 normal + normal saline group (WT group), neurodegenerative disease model mice + normal saline group (KO+Vehicle group), neurodegenerative disease model mice + FTY720 group (KO+FTY group) After two weeks of administration, the brain tissue of the three groups of mice was taken for transmission electron microscopy to observe the myelin tear (a-c) and statistical graph (d).
  • Figure 23 shows the normal+physiological saline group (WT+C group), the neurodegenerative disease model mice+physiological saline group (KO+C group), the normal+FTY720 group (WT+FTY group), the neurodegenerative disease model mice Figures of neurotransmitter content in different brain tissue parts of mice in four groups including mouse+FTY720 group (KO+FTY group).
  • Example 1 The neurodegenerative diseases of movement disorders have the following characteristic indicators: sphingomyelin storage in lysosomes Experimental method: using human neuroblastoma cells (SH-SY5Y) cells to transfect pathogenic gene-related lentiviruses 48 hours later, the neurodegenerative disease cell model was constructed. Lipids were extracted from SH-SY5Y cell lysosomes using the fat extraction method and appropriate internal standards were added. All lipid analyses were performed in electrospray ionization (ESI) mode using LC/MS.
  • ESI electrospray ionization
  • Example 2 Neurodegenerative diseases with movement disorders with special pathological changes Index 2: Decreased sphingomyelin, increased ceramide
  • mice brain tissue substantially nigra, prefrontal cortex, hypothalamus
  • lipids were extracted from the brain tissue using the fat extraction method, and appropriate internal standards were added. All lipid analyses were performed in electrospray ionization (ESI) mode using LC/MS.
  • ESI electrospray ionization
  • FTY720 was purchased from Sigma Company, Cas No. 162359-56-0.
  • the activity of sphingomyelinase was measured using the sphingolipidase detection kit (ab138876).
  • the experiment was divided into two groups: the normal sphingolipidase activity measurement group and the sphingolipidase activity measurement group after FTY720 treatment.
  • the results are shown in Figure 7. It can be seen that under different sphingolipidase concentrations, compared with the control group, the sphingolipidase activities were significantly decreased after FTY treatment.
  • Example 5 Screening of candidate drugs for sphingomyelinase inhibitors at the cellular level
  • mice were dosed orally (0.5 mg/kg). The effect of the drug on the motor function of the mice was examined after one week of administration.
  • Example 7 FTY720 regulates genes related to intracellular sphingolipid metabolism
  • mice were intraperitoneally injected with normal saline FTY720 (0.5mg/kg), samples of the prefrontal cortex of the mouse brain tissue were extracted and analyzed for Smpd1, Smpd2, Smpd3, Smpd4, Enpp7, Asah1, Asah2, Acer1, Acer2, Acer3, Cers1, Cers2, Cers3, Cers4, Cers5, Cers6, Col4a3bp, Cerk, Gba, Ugcg, Degs1.
  • Example 8 FTY720 regulates intracellular sphingolipid content
  • mice C57BL/6 normal + normal saline group (WT+C group), neurodegenerative disease model mice + normal saline group (KO+C group), normal + FTY720 group (WT+FTY group), neurodegenerative disease model mice+FTY720 group (KO+FTY group).
  • WT+C group C57BL/6 normal + normal saline group
  • K+C group normal + FTY720 group
  • WT+FTY group normal + FTY720 group
  • lipids were extracted from the brain tissue samples using the fat extraction method, and appropriate internal standards were added. All lipid analyses were performed in electrospray ionization (ESI) mode using LC/MS. The results showed ( FIG.
  • mice The spastic paraplegic mice (KO mice) were divided into four groups, and three doses of normal saline or FTY720 0.05, 0.1, and 0.5 mg/kg/day were administered orally, and the mice standing experiment was performed 7 days after oral administration. detection.
  • Example 10 FTY720 increases the standing ability of paraplegic model mice in a time-dependent manner
  • mice C57BL/6 wild normal mice (WT) and spastic paraplegia model mice (KO).
  • WT wild normal mice
  • KO spastic paraplegia model mice
  • mice C57BL/6 normal + normal saline group (WT+C group), neurodegenerative disease model mice + normal saline group (KO+C group), normal + FTY720 group (WT+FTY group), neurodegenerative disease model mice+FTY720 group (KO+FTY group).
  • Mice were injected intraperitoneally with FTY720 (0.25 mg/kg/day, i.p.). After 5 days, the rotarod test was used to detect the motor function of the mice.
  • mice C57BL/6 normal + normal saline group (WT+C group), neurodegenerative disease model mice + normal saline group (KO+C group), normal + FTY720 group (WT+FTY group), neurodegenerative disease model mice+FTY720 group (KO+FTY group).
  • FTY720 was dissolved in water and administered to mice orally (0.5mg/kg/day). One week later, the motor function of mice was detected by rotarod test.
  • mice C57BL/6 normal + normal saline group (WT+C group), neurodegenerative disease model mice + normal saline group (KO+C group), normal + FTY720 group (WT+FTY group), neurodegenerative disease model mice+FTY720 group (KO+FTY group).
  • WT+C group C57BL/6 normal + normal saline group
  • K+C group neurodegenerative disease model mice + normal saline group
  • FTY720 group WT+FTY group
  • neurodegenerative disease model mice+FTY720 group K+FTY group
  • mice C57BL/6 normal + normal saline group (WT+C group), senile disease model mice + normal saline group (KO+C group), normal + FTY720 group (WT+FTY group) , Neurodegenerative disease model mice+FTY720 group (KO+FTY group). Mice were administered orally (0.5 mg/kg/day), and after two weeks, the motor function of the mice was detected by standing test and rotarod test.
  • Example 15 FTY720 attenuates the phenotype of lipofuscin deposition and myelin tear in mouse brain tissue
  • mice C57BL/6 wild normal mice (WT), disease model mice + saline (KO + Vehicle) and FTY720 administration (KO + FTY720). Mice were sacrificed two weeks after oral administration, and brain tissue was collected and fixed for transmission electron microscopy analysis.
  • Result 1 As shown in Figure 21, compared with the control mice (picture a), the model mice had a large amount of lipofuscin deposition in their brain tissue (picture b, indicated by red arrows). lipofuscin was significantly reduced (panel c).
  • Figure d shows the results of counting the number of intracellular lipofuscin in more than 100 cells. It can be seen that the number of lipofuscin was significantly reduced after FTY720 treatment.
  • Example 16 FTY720 alleviates abnormal changes of neurotransmitters in mouse brain tissue
  • mice C57BL/6 normal + normal saline group (WT+C group), neurodegenerative disease model mice + normal saline group (KO+C group), normal + FTY720 group (WT+FTY group), neurodegenerative disease model mice+FTY720 group (KO+FTY group).
  • WT+C group C57BL/6 normal + normal saline group
  • K+C group normal + FTY720 group
  • WT+FTY group normal + FTY720 group
  • neurodegenerative disease model mice+FTY720 group K+FTY group

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Abstract

本发明提供芬戈莫德在防治鞘脂紊乱引起的神经退行性疾病中的医药用途。研究显示芬戈莫德可有效缓解并显著提高痉挛性截瘫小鼠的运动功能，可使老年截瘫小鼠重新站立，增加截瘫小鼠站立次数。同时，可有效减少/降低小鼠脑组织中的脂褐素沉积和髓鞘撕裂，可显著缓解鞘磷脂减少或缺乏，促进鞘磷脂再循环利用，纠正鞘磷脂缺乏引起鞘脂紊乱导致的神经功能障碍。芬戈莫德可在制备防治鞘脂紊乱引起的神经退行性疾病药物中的应用。芬戈莫德药物可用于预防和治疗鞘脂紊乱引起的神经退行性疾病如痉挛性截瘫、渐冻症、皮质基底节变性、脊髓小脑共济失调以及神经元蜡样脂褐素沉积病范畴内的疾病。

Description

芬戈莫德在防治鞘脂紊乱引起的神经退行性疾病中的医药用途 技术领域
本发明属于医药生物技术领域，涉及芬戈莫德(FTY720)在防治鞘脂紊乱引起的神经退行性疾病中的医药用途，是指FTY720(fingolimod)在制备对鞘脂紊乱引起的神经退行性疾病如痉挛性截瘫、渐冻症、皮质基底节变性、脊髓小脑共济失调以及神经元蜡样脂褐素沉积病等的医药用途。
技术背景
FTY720(fingolimod或中文翻译称芬戈莫德)的分子式为C19H33NO2.HCl，化学名称为2-amino-2-[2-(4-octylphenyl)]-1,3-propanediol hydrochloride，分子量为343.94g/mol。磷酸化的芬戈莫德通过与S1P受体结合发挥效应[Hla T,et al.Science,2001]，进而将淋巴细胞限制在淋巴结内，防止淋巴细胞进入中枢神经系统，从而起到保护中枢神经的作用。同时，芬戈莫德也因其良好的免疫抑制作用被运用于抑制移植后免疫排斥反应[Park SI,et al.Braz J Med Biol Res,2005]，已获美国食品药品管理局(FDA)批准成为首个治疗复发型多发性硬化(MS)的口服药物。由此可知，芬戈莫德目前已知的作用是通过调节炎症和免疫过程改善神经功能缺失，其治疗作用并不直接作用于神经细胞。
最近，基因与疾病关联性研究表明，ATP13A2基因突变与帕金森氏病、尤其是早发性帕金森综合征和痴呆症(Kufor-Rakeb综合征(KRS)；MIM606693)发生发展关系密切[Fleming,S.M.,et al.2018]。此外，ATP13A2突变与遗传性痉挛性截瘫(spastic paraplegia,SPG)有关[Estrada-Cuzcano,A.,Brain,2017]，与神经元蜡样脂褐素病(neuronal ceroid lipofuscinoses,NCL)有关[Schultheis,P.J.,et al.Human molecular genetics,2013]，还与肌萎缩侧索硬化症或渐冻症(amyotrophic lateral sclerosis，ALS)有关[Spataro,R.,et al.Hum Genomics,2019]。但是，ATP13A2基因突变与以上这些运动性疾病的因果关系、以及如何靶向干预在防治策略方面尚不清楚。
痉挛性截瘫(spastic paraplegia，SPG)一组神经退行性疾病，常表现为锥体综合征状，伴有进行性肢体瘫痪和下肢痉挛。除肢体痉挛、无力症状，还有包括癫痫、耳聋、小脑功能障碍、认知障碍、视力障碍、以及周围神经病变。双下肢肌张力增高，腱反射活跃亢进，病理反射阳性，呈剪刀步态。已知有70多种不同的类型，包括常染色体显性遗传、常染色体隐性遗传、X连锁或非孟德尔线粒体母系遗传的所有遗传模式。ATP13A2基因突变是国内外SPG的一个重要致病基因。HSP进行性加重会严重影响病人的劳动能力和生活自理能力，并且目前尚无有效的方法预防、终止或逆转该疾病，只能通过药物、物理或手术治疗来缓解病人的 症状。其病理改变主要在脊髓中双侧皮质脊髓束的轴索变性和(或)脱髓鞘，常以胸段最重。
渐冻症(amyotrophic Lateral Sclerosis，ALS)，中文译为肌萎缩侧索硬化，是一种无法治愈并且致命的神经退行性疾病。最近发现，ATP13A2突变是ALS的重要致病损伤。引起包括上运动神经元和下运动神经元损伤后导致肌肉麻痹，包括球部(所谓球部，就是指的是延髓支配的这部分肌肉)、四肢、躯干、胸部腹部的肌肉逐渐无力和萎缩。渐冻症的病因目前尚不明确，有些患者病情进展缓慢，但也有部分患者病情进展比较迅速，数月内可从肢端肌肉乏力发展至胸廓呼吸肌乏力，进而出现呼吸衰竭，从而威胁到患者的生命。目前此病尚无特效治疗，利鲁唑作为该病的特效药物，治疗效果因人而异，且并不理想，部分患者效果较好，但也有部分患者口服药物治疗基本无效。
皮质基底节变性(corticobasaldegeneration，CBD)是一种慢性进展性神经变性疾病，以不对称发作的无动性强直综合征、失用、肌张力障碍及姿势异常为其临床特征。临床上可见进行性帕金森综合征，明显不对称的大脑皮质和基底节受损症状和体征，其病理改变为神经元和胶质细胞中异常tau蛋白的蓄积。
脊髓小脑共济失调是以运动失调为主要症状的一种遗传性疾病，病理方面，其表现多种多样，常见的有神经细胞的萎缩、变性，髓鞘的脱失，胶质细胞轻度增生，从而出现小脑半球及蚓部、小脑中下脚广泛变性，浦肯野细胞消失；脊髓后柱及克拉克柱的神经细胞萎缩或消失，继发胶质细胞增生，后根与脊神经节变性、髓鞘脱失，尤其在腰、骶段脊髓更为明显。目前没有彻底根治的办法，属于不治之症。
神经元蜡样脂褐素沉积病(neuronal ceroid lipofuscinoses,NCL)是一组至少有13种不同的、进行性神经退行性疾病，这些疾病类型是根据CLN基因1-14损伤有关(CLN9也是CLN5基因突变)。ATP13A2突变是最近发现的致病基因。NCL的特征包括大脑退化和溶酶体自身荧光储存物质(称为脂褐素)沉积。NCLs常见是儿童期发病的常染色体隐性疾病，表现为肌肉抽搐发作、视力障碍、智力衰退和早期死亡。成人发病的NCL并不总是CLN4型(常染色体显性Kufs病或Parry病)，还出现隐性发病CLN6(MIM 601780)及CLN5突变。成年发病患者大多年龄在20岁到40岁之间，表现为肌肉抽搐痉挛发作。较温和的特征包括行为改变、进行性震颤、肌阵挛、记忆力丧失和频繁跌倒。发作后，出现缓慢的、进行性的认知衰退、共济失调、记忆丧失和言语障碍。有些病人表现出抑郁的迹象。死亡年龄一般在30多岁到60岁之间，死亡原因是严重的神经系统损害和多器官功能衰竭。
鞘磷脂是神经髓鞘及细胞膜结构的主要成分，是合成鞘糖脂如脑苷脂(cerebroside)和神经节苷脂(ganglioside)、及产生其它鞘脂(如神经酰胺、鞘氨醇)的主要磷脂。目前，已知鞘磷脂由内质网合成并经高尔基体运输到细胞膜，再经溶酶体降解。但是，因为溶酶体鞘 磷脂酶缺乏而导致的鞘磷脂降解障碍，则引起神经退行性疾病。
发明内容
本发明的目的是提供芬戈莫德(FTY720)在防治鞘脂紊乱引起的神经退行性疾病中的医药用途，也即提供芬戈莫德(FTY720)在制备防治鞘脂紊乱引起的神经退行性疾病药物中的应用。本发明实验发现，FTY720具有调控鞘脂酶作用，即FTY720可直接调节神经细胞的鞘脂代谢，从而保护有鞘脂代谢紊乱引发的神经细胞凋亡。进一步研究发现，FTY720可有效减少脑组织脂褐素沉积和髓鞘撕裂，即对神经细胞有直接保护作用。而痉挛性截瘫、渐冻症、皮质基底节变性、脊髓小脑共济失调以及神经元蜡样脂褐素沉积病作为一种鞘磷脂缺乏相关的神经退行性病变，与鞘脂代谢紊乱有关，本发明提供的FTY720药物通过鞘脂代谢调节对上述神经退行性疾病有缓解效果。
所述神经退行性疾病包括痉挛性截瘫、渐冻症、皮质基底节变性、脊髓小脑共济失调以及神经元蜡样脂褐素沉积病，以及鞘磷脂减少或缺乏，或再循环利用障碍，导致的伴有鞘脂分解代谢紊乱的神经运动性疾病。
给药途径为口服给药，腹腔注射给药，和脑室注射给药。
所述药物由有效剂量的FTY720和药学上允许的辅料制成，所述药物中FTY720的总质量分数为0.01～10％。
本发明制备的。将FTY720药物粉末溶解于0.9％的生理盐水中，给药浓度范围为0.05-5mg/kg。
为了解决现有技术中存在的问题，本发明提供一种新的预防及治疗神经退行性疾病如痉挛性截瘫、渐冻症、皮质基底节变性、脊髓小脑共济失调以及神经元蜡样脂褐素沉积病等的药物，该药物原用于临床一线免疫抑制药物，有少量抗癌治疗报道，目前尚未用于其他。本发明通过不同途径和不同剂量的给药治疗实验研究，显示FTY720可有效缓解并显著提高痉挛性截瘫小鼠的运动功能，FTY720可使老年截瘫小鼠重新站立，增加截瘫小鼠站立次数。同时，FTY720可有效减少/降低小鼠脑组织中的脂褐素沉积和髓鞘撕裂，FTY720可显著缓解鞘磷脂减少或缺乏，促进鞘磷脂再循环利用，纠正鞘磷脂缺乏引起鞘脂紊乱导致的神经功能障碍。
附图说明
图1左图为野生型(Ctrl)与病理模型(KD)SH-SY5Y细胞各脂类成分含量的比较图。横坐标为log2(KD/Ctrl)的数值，表示倍数差别。纵坐标为-log10(统计学p值)，根据这个图，可直观反映出两组具有显著差异的脂类。由此可知，两组细胞中，病理模型组中不同碳链的SM显著增加。图1右图为野生型(Ctrl)与病理模型(KD)SH-SY5Y细胞中不同SM 含量的热图。也可以看出，KD组中SM含量比Ctrl显著增加。
图2为野生型(Ctrl)与病理模型(KD)SH-SY5Y细胞中溶酶体中鞘磷脂SM-C16,SM-C18,神经酰胺Cer-C16，Cer-C18以及鞘氨醇Sph的含量。由此可知，病理模型细胞中鞘磷脂显著增加，贮积于溶酶体中。
图3为野生型和病理模型小鼠黑质部位各脂质含量。
图4为野生型和病理模型小鼠前额皮层部位各脂质含量。
图5为野生型和病理模型小鼠下丘脑部位各脂质含量。
图6为野生型(Ctrl)与病理模型(KO)MEF细胞各脂类成分含量的比较图。横坐标为log2(KO/Ctrl)的数值，纵坐标为-log10(统计学p值)。该图可直观反映出两组具有显著差异的脂类。由此可知，两组细胞中，病理模型组中鞘糖脂含量显著减少。
图7为在相同底物浓度条件下，加入不同浓度的鞘脂酶，吸光度的值与酶活性成正比。蓝线：正常条件下鞘脂酶活性值，红线：添加FTY720后鞘脂酶的活性值。
图8为不同鞘磷脂酶抑制剂候选药物对MEF细胞胞内C18鞘磷脂含量的影响。
图9为不同鞘磷脂酶抑制剂候选药物对疾病模型小鼠在转棒上运功能力的影响。
图10为根据单细胞基因分析法得到的FTY720小鼠脑组织前额皮层部位与鞘脂代谢相关的差异表达基因。红色表示高表达，蓝色表示低表达。
图11为根据液质联用方法得到的脂质组学检测结果，蓝色为疾病组/野生组的比例；红色为FTY治疗组/疾病组的比例。
图12为不同剂量FTY720口服给药后疾病模型小鼠的站立次数。
图13为FTY720口服给药前后疾病模型小鼠的站立次数。1-7天，疾病模型小鼠给药处理，7-14天停药处理，15-21天二次给药处理。
图14为小鼠腹腔注射FTY720(0.25mg/kg/day,i.p.)5天后，转棒实验检测C57BL/6正常+生理盐水组(WT+C组)、神经退行性疾病模型小鼠+生理盐水组(KO+C组)、正常+FTY720组(WT+FTY组)、神经退行性疾病模型小鼠+FTY720组(KO+FTY组)等四组的运动功能。
图15为小鼠口服给药7天后(0.5mg/kg/day,o.p.)，转棒实验检测C57BL/6正常+生理盐水组(WT+C组)、神经退行性疾病模型小鼠+生理盐水组(KO+C组)、正常+FTY720组(WT+FTY组)、神经退行性疾病模型小鼠+FTY720组(KO+FTY组)等四组的运动功能。
图16为小鼠脑室注射一次性给药7天后(10μg,i.c.v.)，转棒实验检测C57BL/6正常+生理盐水组(WT+C组)、神经退行性疾病模型小鼠+生理盐水组(KO+C组)、正常+FTY720组(WT+FTY组)、神经退行性疾病模型小鼠+FTY720组(KO+FTY组)等四组的运动功能。
图17为老年小鼠脑室口服给药14天后，站立实验检测C57BL/6正常+生理盐水组(WT+C组)、神经退行性疾病模型小鼠+生理盐水组(KO+C组)、正常+FTY720组(WT+FTY组)、神经退行性疾病模型小鼠+FTY720组(KO+FTY组)等四组的运动功能。
图18为老年小鼠脑室口服给药14天后，转棒实验检测C57BL/6正常+生理盐水组(WT+C组)、神经退行性疾病模型小鼠+生理盐水组(KO+C组)、正常+FTY720组(WT+FTY组)、神经退行性疾病模型小鼠+FTY720组(KO+FTY组)等四组的运动功能。
图19为给药前后同一只老年截瘫小鼠站立情况前后对比图。
图20为给药前后同一只老年截瘫小鼠后肢与地面所成角度(FBA)前后对比照片。
图21为C57BL/6正常+生理盐水组(WT组)、神经退行性疾病模型小鼠+生理盐水组(KO+Vehicle组)、神经退行性疾病模型小鼠+FTY720组(KO+FTY组)等三组小鼠给药两周后取脑组织进行透射电镜观察其神经元中脂褐素沉积图(a-c)及统计图(d)。
图22为C57BL/6正常+生理盐水组(WT组)、神经退行性疾病模型小鼠+生理盐水组(KO+Vehicle组)、神经退行性疾病模型小鼠+FTY720组(KO+FTY组)等三组小鼠给药两周后取脑组织进行透射电镜观察其髓鞘撕裂情况(a-c)及统计图(d)。
图23为正常+生理盐水组(WT+C组)、神经退行性疾病模型小鼠+生理盐水组(KO+C组)、正常+FTY720组(WT+FTY组)、神经退行性疾病模型小鼠+FTY720组(KO+FTY组)等四组的小鼠不同脑组织部位中神经递质含量图。
具体实施方式：
下面结合附图和实施例对本发明作进一步的说明。所述的实施例可使本专业技术人员更全面地理解本发明，但不以任何方式限制本发明。
实施例1：运动障碍的神经退行性类疾病具有如下特征指标：鞘磷脂在溶酶体中贮积实验方法：利用人神经母细胞瘤细胞(SH-SY5Y)细胞转染致病基因相关慢病毒48小时，构建神经退行性疾病细胞模型。使用脂肪提取方法从SH-SY5Y细胞溶酶体中提取脂质，并加入适当的内标。使用液质联用仪，以电喷雾电离(ESI)模式进行所有脂质分析。
结果显示：如图1所示，我们对200多种脂类进行了分析，结果表明(图1左火山图)，只有鞘磷脂在该疾病模型中显著增加。用热图对不同碳链和饱和度的鞘磷脂进行进一步比较可知(图1右热图)，该神经退行性疾病细胞模型的溶酶体内很多鞘磷脂都有不同程度的贮积。接下来，又对体内几种主要的鞘磷脂和神经酰胺进行测定。结果显示(图2)，在病理模型中溶酶体内SM-C16,SM-C18显著增加，而神经酰胺变化不明显。
实施例2：具有运动障碍的神经退行性类疾病具有特殊病变指标2：鞘磷脂减少，神经酰胺增加
实验对象：神经退行性疾病模型小鼠脑组织。
实验方法：取小鼠脑组织(黑质，前额皮质，下丘脑)，使用脂肪提取方法从脑组织中提取脂质，并加入适当的内标。使用液质联用仪，以电喷雾电离(ESI)模式进行所有脂质分析。
结果显示：疾病小鼠脑组织中鞘脂代谢异常，如图3(黑质)，图4(前额皮质)，图5(下丘脑)所示，与正常对照小鼠相比，ATP13A2 KO小鼠脑组织(黑质，前额皮质，下丘脑)中鞘磷脂SM显著性减少，而神经酰胺含量(Cer-C24:1,Cer-C20,Cer-C18)则显著性升高。
实施例3：具有运动障碍的神经退行性类疾病具有特殊病变指标3：鞘糖脂分解
实验方法：分离野生型和神经退行性疾病模型小鼠胎鼠(15天)的成纤维细胞MEF，使用脂肪提取方法从MEF细胞溶酶体中提取脂质，并加入适当的内标。使用液质联用仪，以电喷雾电离(ESI)模式进行所有脂质分析。结果显示(图6)：ATP13A2敲除MEF细胞溶酶体内鞘糖脂(GM1,GM2,GM3，蓝色系点表示)等含量显著下降，由此可知在病理模型中神经细胞中的鞘糖脂可能参与脂代谢分解从而含量减少。
实施例4：FTY720对鞘磷脂酶的抑制作用
实验方法：FTY720购自Sigma公司，Cas号162359-56-0。利用鞘脂酶检测试剂盒(ab138876)试剂盒测定鞘磷脂酶的活性。实验分为两组：正常鞘脂酶活性测定组和FTY720处理后鞘脂酶活性测定组。结果见图7可知：在不同鞘脂酶浓度下，与对照组相比，FTY处理后鞘脂酶活性均有显著下降。
实施例5：细胞水平鞘磷脂酶抑制剂候选药物筛选
实验方法：我们挑选文献报道的十种鞘磷脂酶抑制剂药物对胞内鞘磷脂的调节作用进行筛选，分别为芬戈莫德(Fingolimod)，氟西汀(Fluoxetine)，阿米替林(Amitriptyline)，氯米帕明(Clomipramine)，地昔帕明(Desipramine)，西尼莫德(Siponimod)，KRP-203，氨氯地平(Amlodipine)，舍曲林(Sertraline)曲米帕明(Trimipramine)等。利用疾病模型小鼠胚胎分离获得带有致病基因的MEF细胞，并进行培养。将上述十种药物处理MEF细胞48小时后，检测细胞内C18鞘磷脂的含量。
结果显示：见图8，几种鞘脂酶抑制剂对胞内鞘磷脂的含量均有不同程度的上调效果，其中，芬戈莫德、氟西汀、阿米替林和氯米帕明对胞内鞘C18磷磷脂含量的提升最明显。
实施例6：动物水平鞘磷脂酶抑制剂候选药物筛选
实验方法：我们选取芬戈莫德(Fingolimod)，氟西汀(Fluoxetine)，氯米帕明(Clomipramine)，地昔帕明(Desipramine)，曲米帕明(Trimipramine)等五种药物对疾病小 鼠进行口服给药(0.5mg/kg)。给药一周后检测药物对小鼠运动功能的影响。
结果显示：见图9，在五种药物中FTY720能增加疾病模型小鼠的运动功能，且效果最明显。
实施例7：FTY720调节细胞内鞘脂代谢相关基因
实验方法：实验分为4组：C57BL/6正常+生理盐水组(WT+C组)、神经退行性疾病模型小鼠+生理盐水组(KO+C组)、正常+FTY720组(WT+FTY组)、神经退行性疾病模型小鼠+FTY720组(KO+FTY组)。小鼠腹腔注射生理盐水FTY720(0.5mg/kg)一周后，提取小鼠脑组织前额皮层部位样本，并对Smpd1 Smpd2 Smpd3 Smpd4 Enpp7 Asah1 Asah2 Acer1 Acer2 Acer3 Cers1 Cers2 Cers3 Cers4 Cers5 Cers6 Col4a3bp Cerk Gba Ugcg Degs1 Degs2 Sgpl1 Sptlc1 Sptlc2 Sptlc3 Abca1 Abca2 Abca7 Abca12 Abcc1 Abcg1 Abcg2 Sgms1 Sgms2 Cftr Sphk1 Sphk2 S1pr1 Kit Hexa Hexb Gal3st1 Gaphdh TERC TERT Ki67等47个鞘脂代谢相关基因进行比较分析。结果显示(图10)：经过FTY处理后，KO小鼠的smpd1，enpp7等基因上调而cers4，cerk，sphk1等基因下调。
实施例8：FTY720调节细胞内鞘脂含量
实验方法：实验分为4组：C57BL/6正常+生理盐水组(WT+C组)、神经退行性疾病模型小鼠+生理盐水组(KO+C组)、正常+FTY720组(WT+FTY组)、神经退行性疾病模型小鼠+FTY720组(KO+FTY组)。小鼠腹腔注射生理盐水FTY720(0.5mg/kg)一周后，取小鼠脑组织纹状体部位样本，使用脂肪提取方法从脑组织样品中提取脂质，并加入适当的内标。使用液质联用仪，以电喷雾电离(ESI)模式进行所有脂质分析。结果显示(图11)：在疾病模型小鼠纹状体部位的鞘磷脂含量下降，而神经酰胺、葡萄糖神经酰胺、半乳糖神经酰胺的含量上升。经过FTY处理后，疾病模型组小鼠的鞘磷脂和神经酰胺的含量大部分都恢复到正常组水平。
实施例9：FTY720呈剂量依赖性增加截瘫模型小鼠的站立能力
实验方法：将痉挛性截瘫小鼠(KO小鼠)分成四组，予以生理盐水或FTY720 0.05，0.1，和0.5mg/kg/day三个剂量进行口服给药，口服7天后进行小鼠站立实验检测。
结果显示：见图12，截瘫小鼠与对照组小鼠相比，其每30分钟的站立次数显著减少，而小鼠在不同剂量FTY处理后站立次数有不同程度提高，呈剂量依赖型。
实施例10：FTY720呈时间依赖性增加截瘫模型小鼠的站立能力
实验方法：实验分为C57BL/6野生正常小鼠(WT)和痉挛性截瘫模型小鼠(KO)两组，同一时间予以FTY720 0.5mg/k/day口服给药，给药0-7天后停药，停药一周后恢复给药，期间每隔一天检测小鼠的站立情况。
结果显示：见图13，截瘫小鼠与对照组小鼠相比，其每30分钟的站立次数随着给药时间的增加显著增加，而停药后小鼠的站立次数有逐渐降低，到第15-21天恢复给药后，其站立次数又逐渐增加。
实施例11：腹腔注射FTY720增加疾病模型小鼠的运动功能
实验方法：实验分为4组：C57BL/6正常+生理盐水组(WT+C组)、神经退行性疾病模型小鼠+生理盐水组(KO+C组)、正常+FTY720组(WT+FTY组)、神经退行性疾病模型小鼠+FTY720组(KO+FTY组)。小鼠腹腔注射FTY720(0.25mg/kg/day,i.p.)。5天后，利用转棒实验检测小鼠运动功能。
实验结果：见图14，与对照组相比，FTY处理后KO模型小鼠在转棒中停留的时间显著延长，表明给药处理后小鼠的运动能力显著提高。
实施例12：口服给药FTY720增加疾病模型小鼠的运动功能
实验方法：实验分为4组：C57BL/6正常+生理盐水组(WT+C组)、神经退行性疾病模型小鼠+生理盐水组(KO+C组)、正常+FTY720组(WT+FTY组)、神经退行性疾病模型小鼠+FTY720组(KO+FTY组)。FTY720溶于水中对小鼠进行口服给药(0.5mg/kg/day)，一周后，利用转棒实验检测小鼠运动功能。
实验结果：见图15，与对照组相比，FTY处理后KO模型小鼠在转棒中停留的时间显著延长，表明给药处理后小鼠的运动能力显著提高。
实施例13：脑室注射FTY720增加疾病模型小鼠的运动功能
实验方法：实验分为4组：C57BL/6正常+生理盐水组(WT+C组)、神经退行性疾病模型小鼠+生理盐水组(KO+C组)、正常+FTY720组(WT+FTY组)、神经退行性疾病模型小鼠+FTY720组(KO+FTY组)。对小鼠进行脑室注射一次性给药(10μg,i.c.v.)，一周后，利用转棒实验检测小鼠运动功能。
实验结果：见图16，与对照组相比，FTY处理后KO模型小鼠在转棒中停留的时间显著延长，表明给药处理后小鼠的运动能力显著提高。
实施例14：口服给药FTY720增加老年疾病模型小鼠的运动功能
实验方法：实验分为4组：C57BL/6正常+生理盐水组(WT+C组)、老年疾病模型小鼠+生理盐水组(KO+C组)、正常+FTY720组(WT+FTY组)、神经退行性疾病模型小鼠+FTY720组(KO+FTY组)。对小鼠进行口服给药(0.5mg/kg/day)，两周后，利用站立实验和转棒实验检测小鼠运动功能。
实验结果：与对照组相比，FTY处理后KO模型小鼠站立次数有显著增加(图17)，而在转棒中停留的时间显著延长(图18)，表明给药处理后小鼠的运动能力显著提高。同时， 将同一只老年小鼠放在直立的玻璃烧杯中，观察它的站立情况(图19)，给药前，小鼠几乎都趴在烧杯底部很少直立，给药后可以拍摄到老年小鼠能依附在烧杯壁中站立起来。我们也对老年截瘫小鼠进行了后肢与地面所成角度的测定(FBA,foot-based angle)，根据图片可以观察到老年小鼠给药后，其FBA角度明显增加，表示药物可以部分恢复小鼠截瘫后肢拖尾的表型(图20)。
实施例15：FTY720减轻小鼠脑组织脂褐素沉积和髓鞘撕裂的表型
实验方法：实验分为C57BL/6野生正常小鼠(WT)，疾病模型小鼠+生理盐水(KO+Vehicle)以及FTY720给药处理(KO+FTY720)三组。口服给药两周后处死小鼠，取脑组织固定后进行透射电镜分析。
结果1：见图21，与对照组小鼠相比(a图)，模型小鼠其脑组织中有大量的脂褐素沉积(b图，红色箭头指示)，经过FTY720处理后神经元细胞内的脂褐素显著减少(c图)。图d为对100个以上细胞统计胞内脂褐素个数的结果，可见脂褐素的数量经过FTY720处理后显著减少。
结果2：见图22，与对照组小鼠相比(a图)，模型小鼠其脑组织中的髓鞘也呈明显的撕裂状(b图)，经过FTY720处理后髓鞘撕裂的情况有明显改善，髓鞘层变得更为致密(c图)。图d为对100个以上髓鞘统计撕裂情况的结果，可见经过FTY720处理后撕裂髓鞘的数量显著减少。
实施例16：FTY720减轻小鼠脑组织神经递质的异常变化
实验方法：实验分为4组：C57BL/6正常+生理盐水组(WT+C组)、神经退行性疾病模型小鼠+生理盐水组(KO+C组)、正常+FTY720组(WT+FTY组)、神经退行性疾病模型小鼠+FTY720组(KO+FTY组)。小鼠腹腔注射生理盐水FTY720(0.5mg/kg)一周后，取小鼠脑组织纹状体、下丘脑、黑质等部位样本，提取脑内神经递质。使用液质联用仪，对多巴胺、血清素以及γ氨基丁酸等神经递质进行分析。结果显示(图23)：在疾病模型小鼠纹状体、黑质部位的多巴胺、血清素和γ氨基丁酸含量下降。经过FTY处理后，疾病模型组小鼠的神经递质含量大部分都恢复到正常组水平。

Claims (4)

  1. 芬戈莫德在防治鞘脂紊乱引起的神经退行性疾病中的医药用途，所述芬戈莫德其化学名称为2-amino-2-[2-(4-octylphenyl)]-1,3-propanediol hydrochloride，其特征在于，所述医药用途是在制备防治鞘脂紊乱引起的神经退行性疾病药物中的应用。
  2. 根据权利要求1所述的芬戈莫德在防治鞘脂紊乱引起的神经退行性疾病中的医药用途，其特征在于，所述神经退行性疾病包括痉挛性截瘫、渐冻症、皮质基底节变性、脊髓小脑共济失调以及神经元蜡样脂褐素沉积病，以及鞘磷脂减少或缺乏，或再循环利用障碍，导致的伴有鞘脂分解代谢紊乱的神经运动性疾病。
  3. 根据权利要求1所述的芬戈莫德在防治鞘脂紊乱引起的神经退行性疾病中的医药用途，其特征在于，所述药物由有效剂量的FTY720和药学上允许的辅料制成。
  4. 根据权利要求1所述的芬戈莫德在防治鞘脂紊乱引起的神经退行性疾病中的医药用途，其特征在于，所述药物的给药途径为口服给药、腹腔注射给药、脑室注射给药。
PCT/CN2021/118796 2020-11-09 2021-09-16 芬戈莫德在防治鞘脂紊乱引起的神经退行性疾病中的医药用途 WO2022095610A1 (zh)

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