WO2020001640A1 - 甘露糖醛二酸的组合物在治疗帕金森氏症中的应用 - Google Patents

甘露糖醛二酸的组合物在治疗帕金森氏症中的应用 Download PDF

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WO2020001640A1
WO2020001640A1 PCT/CN2019/093803 CN2019093803W WO2020001640A1 WO 2020001640 A1 WO2020001640 A1 WO 2020001640A1 CN 2019093803 W CN2019093803 W CN 2019093803W WO 2020001640 A1 WO2020001640 A1 WO 2020001640A1
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mannuronic acid
total weight
acid
use according
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PCT/CN2019/093803
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French (fr)
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耿美玉
辛现良
杜晓光
张真庆
丁健
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上海绿谷制药有限公司
中国科学院上海药物研究所
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Priority to EP19825144.9A priority Critical patent/EP3815691A4/en
Priority to JP2020572820A priority patent/JP2021528460A/ja
Priority to KR1020217001708A priority patent/KR20210038874A/ko
Priority to US17/256,807 priority patent/US11406654B2/en
Priority to AU2019296851A priority patent/AU2019296851A1/en
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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
    • A61K31/7016—Disaccharides, e.g. lactose, lactulose
    • 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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
    • 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
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • 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
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • 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
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs

Definitions

  • the present invention relates to the application of the optimal composition of mannanuronic acid obtained by biological activity screening method in the treatment of Parkinson's disease.
  • Parkinson's disease is a common neurological degenerative disease that occurs in middle-aged and older people aged 50-60 years.
  • the main lesions are in the substantia nigra and striatum pathways, leading to resting tremor, increased muscle tone, and sluggish movement.
  • Parkinson's disease is the fourth most common neurodegenerative disease in the elderly, with 1% of people over 65 years old and 0.4% of people over 40 years old.
  • the drugs for clinical treatment of Parkinson's disease are mainly compound levodopa preparations, dopamine receptor agonists, monoamine oxidase inhibitors, anticholinergic preparations and amantadine, etc., but there are shortcomings such as large side effects and reduced long-term application effects.
  • Mannuronic acid has been widely valued for its potential medicinal value.
  • Mannuronic acid is usually prepared from alginic acid through multiple steps.
  • M and G sections can be separated from the raw alginic acid.
  • the general method can be simply described as: after the initial degradation of alginic acid, a mixed polysaccharide of polymannuronic acid and polyguluronic acid can be obtained, and after the mixed polysaccharide is precipitated by the acid method, the polyguluraldehyde can be removed therefrom Acid and further purification to obtain a homopolymannuronic acid (hereinafter also referred to as "M-stage intermediate") having a purity of 90% or more.
  • M-stage intermediate homopolymannuronic acid
  • the method for preparing oligomannuronic acid is as follows: the M-stage intermediate obtained above is heated under acidic conditions and further acidolyzed to obtain small fragments of mannuronic acid polymers in a desired molecular weight range.
  • the reducing terminal can be oxidized to a ring-opened sugar diacid.
  • Patent Documents 1 and 2 are hereinafter collectively referred to as prior patents, which are all incorporated herein by reference.
  • reaction process of the mannuronic acid disclosed in the prior patent can be expressed by the following reaction equation (II), that is, the mannuronic acid C1-position aldehyde group at the reducing end of the oligomannuronic polysaccharide is oxidized to a carboxyl group.
  • a common oxidant is a basic copper sulfate solution, that is, a film reagent.
  • This oxidation method has been adopted in a prior patent. Specifically, under basic conditions, the reaction substrate is polymannuronic acid, namely The above M-stage intermediate is added to the copper sulfate solution and reacted in a boiling water bath for 15 minutes to 2 hours.
  • This method uses Cu2 + ions as an oxidant to oxidize aldehyde groups, and a brick red cuprous oxide precipitate is generated in the reaction. This reaction is often used to identify reducing sugars.
  • mannan-oligosaccharides have anti-Alzheimer's disease (AD) and anti-diabetic effects.
  • the pathogenesis of Alzheimer's disease and type 2 diabetes is closely related to amyloid ( ⁇ -amyloid and amylin).
  • ⁇ -amyloid and amylin amyloid
  • protein oligomers are produced, which further aggregate to form fibers.
  • These protein aggregates are cytotoxic, induce oxidative damage to mitochondria in cells, and trigger cascades of inflammatory responses, causing a large number of neurons and ⁇ -cell damage, eventually leading to Alzheimer's disease and type 2 diabetes.
  • Mannan oligosaccharic acid targets amyloid and antagonizes its cascade response, thereby preventing and treating Alzheimer's disease and type 2 diabetes.
  • An earlier patent CN106344592A discloses the application of mannuronic acid oligosaccharide with a carboxyl group at the reducing end and its derivative in the treatment of Parkinson's disease, and discloses a drug of a tetrasaccharide-decasaccharide mixture in the treatment of Parkinson's disease. Effective activity.
  • the invention relates to the use of a mannuronic acid oligosaccharide composition in the treatment of Parkinson's disease.
  • the invention also relates to a method for treating Parkinson's disease, which comprises administering to a patient in need of treatment a therapeutically effective amount of the mannuronic acid oligosaccharide composition according to the invention.
  • the mannuronic acid oligosaccharide composition according to the present invention comprises a mannuronic acid having the formula (III) or a pharmaceutically acceptable salt thereof:
  • n is an integer selected from 1-9
  • m is selected from 0, 1 or 2
  • m ' is selected from 0 or 1
  • the total weight of n 1-5 of the mannuronic acid accounts for more than 60% of the total weight of the composition
  • a specific proportion of the mannuronic acid composition shows a beneficial effect on the treatment of Parkinson's disease, while reducing the production cost, and because it is derived from the safety of natural products, it is beneficial to improve the quality of life of patients.
  • Figure 1 is the mass spectrum of disaccharide, trisaccharide and tetrasaccharide in product A.
  • FIG. 2 is a mass spectrum of pentasaccharide, hexasaccharide, and heptose in product A.
  • FIG. 2 is a mass spectrum of pentasaccharide, hexasaccharide, and heptose in product A.
  • Figure 3 is a mass spectrum of octaose, nonasaccharide, and decasaccharide in product A.
  • Figure 4 shows the effect of different oligosaccharide compositions and hexasaccharides on the climbing time of PD animals on day 11.
  • the samples on the horizontal axis of the figure are: i: control group; ii: model group; iii: product A; iv: Product B; v: Product C; vi: Product D; vii: Comparative experimental sample; viii: Hexose.
  • Figure 5 shows the effects of different oligosaccharide compositions and hexasaccharides on the incubation period of PD animals on day 11; the abscissa reference numerals are the same as in Figure 4.
  • FIG. 6 shows the effect of different oligosaccharide compositions and hexasaccharides on the climbing time of PD animals on the 14th day; the abscissa reference numerals are the same as those in FIG. 4.
  • FIG. 7 shows the effects of different oligosaccharide compositions and hexasaccharides on the incubation period of PD animals on the 14th day; the abscissa reference numerals are the same as those in FIG. 4.
  • FIG. 8 shows the effects of different oligosaccharide compositions and hexasaccharides on the crawling time of PD animals on the 17th day; wherein the abscissa reference numerals are the same as those in FIG. 4.
  • FIG. 9 shows the effects of different oligosaccharide compositions and hexasaccharides on the incubation period of the 17th day of PD animals; the abscissa reference numerals are the same as those in FIG. 4.
  • the invention relates to the use of a mannuronic acid oligosaccharide composition in the treatment of Parkinson's disease.
  • the mannuronic acid oligosaccharide composition used comprises a mannuronic acid having the formula (III) or a pharmaceutically acceptable salt thereof:
  • n is an integer selected from 1-9
  • m is selected from 0, 1 or 2
  • m ' is selected from 0 or 1
  • the total weight of n 1-5 of the mannuronic acid accounts for more than 60% of the total weight of the composition
  • the mannuronic acid oligosaccharide composition according to the present invention is a mixture of mannuronic acid with different polymerization degrees, and its main component is a mannuronic acid oligosaccharide having a polymerization degree of 2 to 10.
  • the most active sugars in mannuronic acid are 4-10 sugars, especially 6 sugars.
  • the inventors found that adding a certain proportion of the less active 2-3 sugars to the 4-10 sugars with the highest activity, the biological activity does not decrease or even at the same quality of the administered dose There is still improvement.
  • composition activity can reach or even be better than the composition disclosed in the earlier application, and because the 2-3 sugars are not removed as impurities, the yield of the product is theoretically significantly higher than that of the product disclosed in the earlier application, which greatly reduces Production costs and reduction of waste emissions are easier to achieve in actual production and easier to achieve large-scale industrial production.
  • the ratio is between 1.0 and 3.5, preferably between 1.0 and 3.0.
  • the weight percent content of each degree of polymerization of the mannuronic acid oligosaccharide in the mannuronic acid oligosaccharide composition of the present invention is: 5-25% disaccharide, 15-30% trisaccharide, Tetrasaccharide 15-28%, pentasaccharide 5-25%, hexasaccharide 2-20%, heptose 2-20%, octose 2-20%, nonaose 2-20%, and decaose 2-20%.
  • the weight percentage content of oligosaccharides in the composition is: 5-25% disaccharides, 15-30% trisaccharides, 15-28% tetrasaccharides, 10-20% pentasaccharides, 5-15% hexasaccharides, Heptasaccharide is 3-10%, octose is 2-5%, nonaose is 1-5%, and decaose is 1-5%. More preferably, the weight percentage of oligosaccharides in the composition is: 10-20% disaccharides, 18-30% trisaccharides, 15-28% tetrasaccharides, 15-20% pentasaccharides, and 5-10% hexasaccharides. , Heptasaccharide 3-5%, octaose 2-5%, nonaose 1-3%, and ten sugar 1-3%.
  • the pharmaceutically acceptable salt is a sodium salt or a potassium salt.
  • the inventor of the present patent application found that when the above-prepared oligosaccharides were compounded at a certain ratio, a highly active oligosaccharide composition could be obtained, the activity of which was higher than that of the most active hexasaccharide; especially the addition of specific Compositions with a ratio of disaccharides and trisaccharides are more active than compositions without disaccharides and trisaccharides.
  • the ratio of each oligosaccharide in the high-activity oligosaccharide composition needs to be combined according to the following proportional relationship:
  • the medicament for treating Parkinson's disease comprises a mannuronic acid oligosaccharide composition comprising a mannuronic acid having the formula (III) or a pharmaceutically acceptable salt thereof, and one or A variety of pharmaceutically acceptable carriers.
  • the medicament according to the invention may be tablets, hard capsules, soft capsules, enteric capsules, microcapsules, granules, syrups, injections, granules, emulsions, suspensions, solutions and for oral or parenteral administration In the form of a slow-release preparation.
  • the pharmaceutically acceptable carrier in the present invention refers to a pharmaceutically acceptable carrier well known to those skilled in the art.
  • the pharmaceutically acceptable carrier in the present invention includes, but is not limited to, fillers, wetting agents, adhesives, and disintegrating agents. , Lubricants, adhesives, glidants, taste-masking agents, surfactants, preservatives, etc.
  • Fillers include, but are not limited to, lactose, microcrystalline cellulose, starch, powdered sugar, dextrin, mannitol, calcium sulfate, and the like.
  • Wetting agents and binders include, but are not limited to, sodium carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, gelatin, sucrose, polyvinylpyrrolidone, and the like.
  • Disintegrating agents include, but are not limited to, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, croscarmellose sodium, low-substituted hydroxypropyl cellulose, and the like.
  • Lubricants include, but are not limited to, magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol, magnesium lauryl sulfate, and the like.
  • Binders include, but are not limited to, gum arabic, alginic acid, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, glucose binding agents, dextrin, dextrose, ethyl cellulose, gelatin, liquid glucose, guar Gum, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, magnesium aluminum silicate, maltodextrin, methyl cellulose, polymethacrylate, polyvinylpyrrolidone, pregelatinized starch , Sodium alginate, sorbitol, starch, syrup and tragacanth.
  • Glidants include, but are not limited to, colloidal silica, powdered cellulose, magnesium trisilicate, silica, and talc.
  • Taste-masking agents include, but are not limited to, aspartame, stevioside, fructose, glucose, syrup, honey, xylitol, mannitol, lactose, sorbitol, maltitol, glycyrrhizin.
  • Surfactants include, but are not limited to, Tween-80, Poloxamer.
  • Preservatives include, but are not limited to, paraben, sodium benzoate, potassium sorbate, and the like.
  • treatment generally refers to obtaining the desired pharmacological and / or physiological effect.
  • the effect may be preventive based on the complete or partial prevention of the disease or its symptoms; and / or based on the partial or complete stabilization or cure of the disease and / or side effects due to the disease, which may be therapeutic.
  • Treatment encompasses any treatment of a patient's disease, including: (a) preventing a disease or symptom that occurs in a patient who is susceptible to a disease or condition that has not yet been diagnosed; (b) suppressing the symptoms of the disease, That is, preventing its development; or (c) alleviating the symptoms of the disease, that is, causing the disease or the symptoms to deteriorate.
  • the mannuronic acid oligosaccharide composition for treating Parkinson's disease in the present invention comprises a mannuronic acid having the formula (III) or a pharmaceutically acceptable salt thereof:
  • n is an integer selected from 1-9
  • m is selected from 0, 1 or 2
  • m ' is selected from 0 or 1
  • the total weight of n 1-5 of the mannuronic acid accounts for more than 60% of the total weight of the composition
  • the method for preparing the mannuronic acid oligosaccharide composition for treating Parkinson's disease includes the following steps:
  • the M-stage intermediate of the raw materials used in the present invention can be prepared by methods known in the art. For example, the methods disclosed in Chinese Patent Application No.98806637.8 and CN02823707.2.
  • the general method can be simply described as: after the initial degradation of alginic acid, a mixed polysaccharide of polymannuronic acid and polyguluronic acid can be obtained, and after the mixed polysaccharide is precipitated by the acid method, the polyguluraldehyde can be removed Acid, further purification can obtain a homopolymannuronic acid with a purity of more than 90%, that is, an M-stage intermediate.
  • the M-stage intermediate is dissolved in an appropriate amount of water at room temperature or under heating conditions, stirred, and ozone is continuously introduced, and the reaction starts.
  • the reaction pH can be adjusted to between 3-13, preferably 4-10, more preferably 6-8, by adding dilute hydrochloric acid or dilute NaOH solution.
  • the temperature is preferably 0-70 ° C, and more preferably 10-45 ° C.
  • the reaction product obtained above was prepared into a solution having a concentration of about 10%, and was separated by a molecular cut-off membrane to remove degradation products below monosaccharides, and the impermeable liquid was collected.
  • the molecular retention membrane MWCO used has a specification of 1000 Da to 3000 Da, preferably 2000 Da.
  • the collected solution was concentrated on a rotary evaporator and vacuum dried to obtain an oligomannuronic acid mixture. After analysis, it was found that these products are all disaccharide-decasaccharide oligosaccharides whose composition is in a certain ratio range.
  • Examples 1-3 in which three compositions A, B, and C are separately prepared according to the foregoing method.
  • the oligosaccharide mixture obtained in step (1) is dissolved, formulated to a concentration of about 10%, separated by a P6 gel chromatography column, and detected by UV, and each effluent component is collected, and components with the same degree of polymerization are combined.
  • Nine components of 2-10 sugars were collected, desalted by G10 gel column chromatography, concentrated on a rotary evaporator, and dried under vacuum.
  • a specific purification preparation process is shown in Example 4. These column chromatography, desalting and drying operations are known to those skilled in the art.
  • the pharmacological activity of the prepared composition was compared with that of the hexasaccharide purified in step (2).
  • the results show that the oligosaccharide composition of the present invention is significantly better than the most active hexasaccharide in a single degree of polymerization oligosaccharide, and Compositions that do not contain disaccharides and trisaccharides are slightly less active than hexasaccharides. It can be seen that the combination of oligosaccharides with different polymerization degrees can play a synergistic effect.
  • mice were randomly divided into 8 groups: a blank control group, an MPTP model group, and an administration group, with 14 animals in each group. Animals were dosed on the same day. The blank control group and the MPTP model group were perfused with saline. The other groups were given the corresponding drugs, once a day for 17 consecutive days. From the 6th day, modeling drugs were given. The animals in the blank control group were subcutaneously given 10ml / kg of saline, and the remaining animals were given subcutaneously MPTP 25mg / kg once a day for five days.
  • MPTP selectively destroys dopaminergic neurons in the brain substantia nigra.
  • MPTP-induced PD animal model is the most classic animal model similar to the pathological changes and clinical characteristics of human Parkinson's disease.
  • the main symptoms of PD are resting tremor, increased muscle tone, and decreased exercise.
  • the head-turning time and climbing-down time of the pole climbing experiment can represent the overall activity coordination ability of mice.
  • Step 1) Preparation of mannuronic acid oligosaccharide mixture
  • the method of preparing the M-stage intermediate as disclosed in the previous patent is briefly described as follows: 5Kg of sodium alginate is prepared into a solution of about 10%, and diluted hydrochloric acid is added to adjust the pH to about 3.0, and the temperature is raised to 80 ° C, stirred, and reacted. 10hr, stop heating, cool to room temperature, add NaOH to adjust pH to 9.0, add dilute hydrochloric acid to adjust pH to 2.85, centrifuge at 5000rpm for 10min, collect supernatant, add HCl to adjust pH to 1.0, centrifuge, collect precipitate, rotate The evaporator was concentrated and dried under vacuum to obtain 1500 g of M-stage intermediate.
  • Step 2) Proportion and structure analysis of oligosaccharides with various polymerization degrees in mannuronic acid product A
  • disaccharides-decasaccharides are represented by dp2-dp10, respectively, dp2 is 19%, dp3 is 25%, dp4 is 22%, dp5 is 13%, dp6 is 9%, dp7 is 6%, and dp8 is 3 %, Dp9 is 2%, and dp10 is 1%.
  • Step 3) LC-MS analysis of the structure of oligosaccharides with various degrees of polymerization in Mannuronic acid product A
  • Mass spectrometry conditions Agilent 6540QTOF; ion source: ESI collision voltage 120V; negative ion mode.
  • the acquisition signal (m / z) width is 100-1000.
  • Example 1 100 g of the M-stage intermediate in Example 1 was weighed, dissolved in distilled water, and prepared into a volume of 0.8 L. The solution was adjusted to pH 4.0 with NaOH and reacted at room temperature at 25 ° C. The gas flow at the outlet of the oxygen cylinder and the power of the ozone generator were adjusted so that the ozone mass concentration flow reached 1 g / hr and passed into the reaction solution. After 10 hours of reaction, stop introducing ozone, add appropriate amount of water to adjust the solution concentration to about 15%, and filter with an ultrafiltration membrane with a molecular weight cut off of 1000 Da. Collect the impervious liquid, concentrate on a rotary evaporator, and dry in vacuo to obtain 80 g of mannaldehyde Acid Product B.
  • GE Superdex peptide
  • MALS multi-angle laser scattering
  • dp2-dp10 Disaccharides-decasaccharides are represented by dp2-dp10, respectively, dp2 is 20%, dp3 is 25%, dp4 is 19%, dp5 is 12%, dp6 is 9%, dp7 is 5%, and dp8 is 5 %, Dp9 is 3%, and dp10 is 2%.
  • Example 1 100 g of the M-stage intermediate in Example 1 was weighed, dissolved in distilled water, and then a 1.5 L volume solution was prepared. The pH was adjusted to 9.0 with NaOH, and the reaction was performed at 45 ° C in a water bath. The gas flow at the outlet of the oxygen cylinder and the power of the ozone generator were adjusted so that the ozone mass concentration flow reached 3 g / hr and passed into the reaction solution. After reacting for 2 hours, stop introducing ozone, add an appropriate amount of water to adjust the solution concentration to about 5%, and filter through an ultrafiltration membrane with a molecular weight cut-off of 3000 Da. Collect the impervious liquid, concentrate on a rotary evaporator, and dry in vacuo to obtain 60 g of mannaldehyde. Acid Product C.
  • GE Superdexpeptide
  • MALS multi-angle laser scattering
  • dp2-dp10 disaccharides-decasaccharides are represented by dp2-dp10, respectively, dp2 is 8%, dp3 is 20%, dp4 is 28%, dp5 is 19%, dp6 is 13%, dp7 is 6%, and dp8 is 3 %, Dp9 is 2%, and dp10 is 1%.
  • a method for preparing a single degree of mannanuronic acid oligosaccharide is as follows:
  • Sample preparation Take 300g from the mannuronic acid product A prepared in Example 1, dissolve it with water, configure it into a 1000mL concentrated solution, and place it in a 4 ° C refrigerator for later use. After each use, 50 mL was taken out and diluted with water, and then filtered with 0.22um ultrafiltration membrane.
  • Chromatographic separation conditions The chromatograph is AKTA pure 150 (purchased from GE), equipped with UV detector and automatic collector. Separation chromatographic column: 1.2kg BioGel P6 (purchased from Bole Company) mixed with deionized water, vacuum degassed, manually packed into a glass column (10cm inner diameter), washed with pure water 10 times the column volume, the column bed is stable , The height is 1.0m. Then use 0.02M NaCl solution as the mobile phase. After equilibrating 10 times the column volume, start loading.
  • the flow rate of the pump is set to 1 mL / min. After 100 mL of the sample solution is pumped to the top of the column by the pump that comes with the chromatograph, switch to the mobile phase and elute at a flow rate of 5 mL / min. After the volume of the stagnant water flowed out, automatic collection was started, and 50 mL was collected per tube.
  • Composition product D Mannuronic acid oligosaccharide having a single degree of polymerization prepared in Example 4 was accurately weighed according to the degree of polymerization from disaccharide to decasaccharide, and the weight of each sugar was as follows: 3.0 g of disaccharide , 3.0g of trisaccharide, 1.5g of tetrasaccharide, 1.5g of pentasaccharide, 0.4g of hexasaccharide, 0.2g of heptasaccharide, 0.2g of octose, 0.1g of nonaperose, 0.1g of decasuose, and mix to obtain 10g of composition product D.
  • the neutral liquid was slowly added to 4 times the volume of ethanol in the liquid, alcohol-precipitated, and left to stand overnight.
  • the solid matter obtained by alcohol precipitation was separated by filtration, and the solid matter obtained by filtration and washing was separated by filtration during the filtration and separation to obtain a white filter cake.
  • the filter cake was dried in an oven at 60 ° C to obtain a crude alginate oligosaccharide.
  • a fresh oxidant copper hydroxide was prepared by adding 25 ml of a 5% (weight percent) copper sulfate solution to 50 ml of a 10% (weight percent) sodium hydroxide solution and immediately mixing.
  • the fresh oxidizing agent copper hydroxide was immediately added to 40 ml of the above-mentioned 5% (wt%) algin gum oligosaccharide solution, and simultaneously heated by a boiling water bath until no red brick precipitate was generated.
  • the reaction system was centrifuged to remove a precipitate to obtain a supernatant.
  • the eluent as a mobile phase was 0.2 mol ⁇ L-1NH 4 HCO. 3 .
  • the eluate was collected from the column chromatography using a plurality of 5 ml test tubes in order, and then the sugar content of the eluate in each of the headers was detected by the sulfuric acid-carbazole method. According to the detection results, eluates containing alginate oligosaccharide components with different molecular weights were collected. The eluates containing alginate oligosaccharide components with different molecular weights were respectively concentrated under reduced pressure and freeze-dried, and component 1 was discarded to obtain alginate oligosaccharide components 2-12 having different molecular weights respectively.
  • the experimental results show that compared with the blank control group, the model group has a significantly longer incubation period and climbing time. Compared with the model group, the incubation period and climbing time of each administration group were shortened to different degrees. Among them, the product A, B, and C had better pharmacological activity than the comparative experimental samples, and better than the previously expected single-degree polymerization hexasaccharide with the highest activity, but the product D had weaker activity than hexasaccharide. Without being bound by any theory, it is speculated that the ratio between oligosaccharides in the composition has a significant effect on the activity of the product. The addition of a certain proportion of disaccharides and trisaccharides has a synergistic effect, but when the proportion of disaccharides and trisaccharides is too high At higher levels, the activity of the composition is reduced. See Figure 4-9.

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Abstract

本发明涉及一种甘露糖醛二酸寡糖组合在治疗帕金森氏症方面的应用。

Description

甘露糖醛二酸的组合物在治疗帕金森氏症中的应用 技术领域
本发明涉及通过生物活性筛选的方法得到甘露糖醛二酸的最佳组合物在治疗帕金森氏症方面的应用。
背景技术
帕金森氏症(Parkinson's disease,PD)是发生在50－60岁以上中老年人的常见神经系统变性疾病。主要病变在黑质和纹状体通路，导致静止性震颤、肌张力增高、运动迟缓。帕金森病是老年人中第四位最常见的神经变性疾病，在65岁以上人群中，1％患有此病；在40岁以上人群中为0.4％。临床治疗帕金森氏症的药物主要为复方左旋多巴制剂、多巴胺受体激动剂、单胺氧化酶抑制剂、抗胆碱能制剂和金刚烷胺等，但存在副作用大和长期应用效果下降等缺点。
甘露糖醛二酸由于其潜在的药用价值已经受到广泛的重视。甘露糖醛二酸通常以海藻酸为原料经过多步骤制得。
在原料海藻酸的多糖分子中，有由甘露糖醛酸(D-mannuronic acid)通过β-1,4-糖苷键连接形成的M段、古罗糖醛酸(L-guluronic acid)通过α-1,4-糖苷键连接形成的G段，以及由这两种糖杂合形成的MG段。甘露糖醛酸和古罗糖醛酸的结构式如下(Ⅰ)式所示：
Figure PCTCN2019093803-appb-000001
M段和G段可以从原料海藻酸中分离。通常的方法可以简单描述为：将海藻酸初步降解后可得到聚甘露糖醛酸和聚古罗糖醛酸的混合多糖，混合多糖再经酸法沉淀后，可以除去其中的聚古罗糖醛 酸，进一步精制可以得到纯度在90％以上的均聚甘露糖醛酸(下文中也称“M段中间体”)。例如，可参见中国专利申请No.98806637.8以及CN02823707.2所披露的方法。
制备寡聚甘露糖醛酸的做法如下：将上述得到的M段中间体在酸性条件下加热进一步酸解得到所需分子量范围的小片段甘露糖醛酸聚合物。另外，也有通过氧化降解的办法提升降解效率，同时可以将还原末端氧化为开环的糖二酸，详见耿美玉等人的中国专利申请200580009396.5(专利文献1)及美国专利US 8835403B2(专利文献2)。为了方便叙述，专利文献1和2在下文中统称为在先专利，它们以引证的方式全部并入本文。
在先专利披露的甘露糖醛二酸的反应过程可通过如下反应方程式(Ⅱ)表示，即寡聚甘露糖醛酸多糖还原端的甘露糖醛酸C1-位醛基氧化成羧基。
Figure PCTCN2019093803-appb-000002
在上述氧化转化过程中，常用的氧化剂有碱性硫酸铜溶液，即菲林试剂，在先专利即采用了该氧化方法，具体为：在碱性条件下，将反应底物聚甘露糖醛酸即上文的M段中间体加入硫酸铜溶液中，在沸水浴中反应15分钟至2小时。该法是以Cu2+离子为氧化剂氧化醛基，反应中产生砖红色的氧化亚铜沉淀，这个反应常用于鉴定还原性糖。
在先专利公开了甘露寡糖二酸具有抗阿尔茨海默病(Alzheimer’s disease,AD)和抗糖尿病的作用。阿尔茨海默病与Ⅱ型糖尿病的发病过程与淀粉样蛋白(β-amyloid及amylin)密切相关。淀粉样蛋白聚集以后产生蛋白寡聚体，进一步聚集形成纤维。这些蛋白聚集物有细胞毒性，在细胞内诱导氧化反应损伤线粒体以及引发炎症反应等 级联反应，造成大量的神经元和β细胞损伤，最终导致阿尔茨海默病与Ⅱ型糖尿病的发生。甘露寡糖二酸靶向淀粉样蛋白并拮抗其诱导的级联反应，由此具有预防和治疗阿尔茨海默病与Ⅱ型糖尿病的作用。
在先专利CN106344592A公开了还原端1位为羧基的甘露糖醛酸寡糖及其衍生物在治疗帕金森氏症方面的应用，并公开了四糖-十糖混合物在治疗帕金森病方面的药效活性情况。
发明内容
本发明涉及一种甘露糖醛二酸寡糖组合物在治疗帕金森氏症中的用途。本发明还涉及一种治疗帕金森氏症的方法，包括给予需要治疗的患者治疗有效量的本发明所述的甘露糖醛二酸寡糖组合物。
本发明涉及的甘露糖醛二酸寡糖组合物包含具有式(Ⅲ)的甘露糖醛二酸或其药学上可接受的盐：
Figure PCTCN2019093803-appb-000003
其中n为选自1-9的整数，m选自0，1或2，m’选自0或1，
并且其中，
n＝1-5的甘露糖醛二酸的重量总和占所述组合物总重量的60％以上；
n＝1-2的甘露糖醛二酸的重量总和占所述组合物总重量的低于60％。
申请人发现，特定比例的甘露糖醛二酸组合物对于治疗帕金森症表现出有利的效果，同时降低了生产成本，并且由于其源于天然产物的安全性，有利于提高患者生活质量。
附图说明
图1是产品A中二糖、三糖和四糖的质谱图。
图2是产品A中五糖、六糖和七糖的质谱图。
图3是产品A中八糖、九糖和十糖的质谱图。
图4表示不同寡糖组合物及六糖对PD动物第11天爬下时间的影响；图中横坐标的编号分别对应的样品为：i:对照组；ii:模型组；iii:产品A；iv:产品B；v:产品C；vi:产品D；vii:对比实验样品；viii:六糖。
图5表示不同寡糖组合物及六糖对PD动物第11天潜伏期的影响；其中横坐标附图标记同图4。
图6表示不同寡糖组合物及六糖对PD动物第14天爬下时间的影响；其中横坐标附图标记同图4。
图7表示不同寡糖组合物及六糖对PD动物第14天潜伏期的影响；其中横坐标附图标记同图4。
图8表示不同寡糖组合物及六糖对PD动物第17天爬下时间的影响；其中横坐标附图标记同图4。
图9表示不同寡糖组合物及六糖对PD动物第17天潜伏期的影响；其中横坐标附图标记同图4。
具体实施方式
下文将对本发明的各个方面进行具体说明，但本发明并不限于这些具体的实施方式。本领域技术人员可以根据下文公开内容的实质对本发明进行一些修改和调整，这些调整也属于本发明的范围。
本发明涉及一种甘露糖醛二酸寡糖组合物在治疗帕金森氏症中的用途。所使用的甘露糖醛二酸寡糖组合物包含具有式(Ⅲ)的甘露糖醛二酸或其药学上可接受的盐：
Figure PCTCN2019093803-appb-000004
其中n为选自1-9的整数，m选自0，1或2，m’选自0或1，
并且其中，
n＝1-5的甘露糖醛二酸的重量总和占所述组合物总重量的60％以上；
n＝1-2的甘露糖醛二酸的重量总和占所述组合物总重量的低于60％。
本发明涉及的甘露糖醛二酸寡糖组合物是不同聚合度的甘露糖醛二酸的混合物，其主要成分是聚合度为2至10的甘露糖醛二酸寡糖。根据在先申请已知，在甘露糖醛二酸中，活性最高的糖为4-10糖，特别是6糖。但是，与已知的现有技术不同，发明人发现，在活性最高的4-10糖基础上添加一定比例活性较低的2-3糖，同等质量的给药剂量下，生物活性不降低甚至还有提高。不囿于任何理论，推测这可能是由于分子量较小的2-3糖虽然不能单独起效，但跟其他寡糖混合后能起到协同增效的作用。但当2-3糖的比例过高时，组合物的整体活性还是会因2-3糖本身活性较低而降低。因此，组合物中2-3糖的比例必须控制在一定的范围之内。
在实际制备过程中，氧化降解反应中会产生一定量的2-3糖，通常会因其活性低，为避免影响到产品的药效，而将从产物中分离后去除。而基于发明人的上述发现，不需要将氧化降解产物中的2-3糖分离除去，而只需控制氧化降解反应的条件，将2-3糖的比例控制在一定的范围之内，获得的组合物活性能达到甚至优于在先申请所公开的组合物，且因不用将2-3糖作为杂质去除，产品得率理论上也显著高于在先申请所公开的产品得率，大大降低生产成本，减少废弃物的排放，在实际生产中更容易实现，更易于实现工业化大生产。
根据一个优选的实施方案，本发明的甘露糖醛二酸寡糖组合物中m+m’＝1或2的甘露糖醛二酸的重量总和不低于所述组合物总重量的50％以上，优选60％-90％，更优选70％-90％。特别地，本发明的甘露糖醛二酸寡糖组合物中m+m’＝1的甘露糖醛二酸的重量总和不低于所述组合物总重量的10％，优选30-40％。在另一个优选实施方案中，本发明的甘露糖醛二酸寡糖组合物中m+m’＝2的甘露糖醛二酸的重量总和不低于所述组合物总重量的10％，优选30-50％。
根据一个优选实施方案，本发明的甘露糖醛二酸寡糖组合物中n＝1-5的甘露糖醛二酸寡糖的重量总和占所述组合物总重量的80-95％。
根据一个优选实施方案，本发明的甘露糖醛二酸寡糖组合物中n＝1-2的甘露糖醛二酸寡糖的重量总和所述组合物总重量的10-50％，优选，优选25-50％。
根据一个优选实施方案，本发明的甘露糖醛二酸寡糖组合物中n＝1-3的甘露糖醛二酸寡糖的重量总和所述组合物总重量的20-70％。
根据一个优选实施方案，本发明的甘露糖醛二酸寡糖组合物中n＝1-3的甘露糖醛二酸的重量总和与n＝4-7的甘露糖醛二酸寡糖重量总和的比例在1.0-3.5之间，优选在1.0-3.0之间。
根据一个优选实施方案，本发明的甘露糖醛二酸寡糖组合物中各聚合度甘露糖醛二酸寡糖的重量百分含量为：二糖5-25％，三糖15-30％，四糖15-28％，五糖5-25％，六糖2-20％，七糖2-20％，八糖2-20％，九糖2-20％，十糖2-20％。特别地，组合物中寡糖的重量百分含量为：二糖5-25％，三糖15-30％，四糖15-28％，五糖10-20％，六糖5-15％，七糖3-10％，八糖2-5％，九糖1-5％，十糖1-5％。更优地，组合物中寡糖的重量百分含量为：二糖10-20％，三糖18-30％，四糖15-28％，五糖15-20％，六糖5-10％，七糖3-5％，八糖2-5％，九糖1-3％，十糖1-3％。
本发明的甘露糖醛二酸寡糖组合中，其中所述药学上可接受的盐是钠盐或钾盐。
本专利申请的发明人发现，当上述制备的寡糖按照一定的比例进行复配，可以得到高活性的寡糖组合物，其活性比活性最好的六糖还要高；尤其是添加了特定比例二糖和三糖的组合物，其活性高于不含二糖和三糖的组合物。高活性寡糖组合物中的各寡糖比例需要按照如下的比例关系进行组合：
组合物中n＝1-5的甘露糖醛二酸的重量总和占所述组合总重量 的60％以上，优选80-95％。n＝1-2的甘露糖醛二酸的重量总和占所述组合总重量的低于60％，优选10-50％，更优选25-50％。n＝1-3的甘露糖醛二酸寡糖的重量总和所述组合总重量的20-70％。其中n＝1-3的甘露糖醛二酸寡糖的重量总和与n＝4-7的甘露糖醛二酸寡糖重量总和的比例在1.0-3.5之间，优选在1.0-3.0之间。
本发明所述用于治疗帕金森氏症的药物包含甘露糖醛二酸寡糖组合物，其包含具有式(Ⅲ)的甘露糖醛二酸或其药学上可接受的盐，以及一种或多种药学上可接受载体。本发明所述药物可以是片剂、硬胶囊、软胶囊、肠溶胶囊、微囊剂、颗粒剂、糖浆剂、注射剂、颗粒剂、乳剂、悬浮液、溶液和用于口服或非口服给药的缓释制剂的形式。
本发明所述药学上可接受载体是指本领域技术人员熟知的药学上可接受的载体，本发明的药学上可接受载体包括但不限于：填充剂、润湿剂、黏合剂、崩解剂、润滑剂、粘合剂、助流剂、掩味剂、表面活性剂、防腐剂等。填充剂包括但不限于乳糖、微晶纤维素、淀粉、糖粉、糊精、甘露醇、硫酸钙等。润湿剂与黏合剂包括但不限于羧甲基纤维素钠、羟丙基纤维素、羟丙基甲基纤维素、明胶、蔗糖、聚乙烯吡咯烷酮等。崩解剂包括但不限于羧甲基淀粉钠、交联聚乙烯吡咯烷酮、交联羧甲基纤维素钠、低取代羟丙基纤维素等。润滑剂包括但不限于硬脂酸镁、微粉硅胶、滑石粉、氢化植物油、聚乙二醇、月桂醇硫酸镁等。粘合剂包括但不限于阿拉伯胶、藻酸、羧甲基纤维素钙、羧甲基纤维素钠、葡萄糖结合剂、糊精、右旋糖、乙基纤维素、明胶、液体葡萄糖、瓜尔胶、羟乙基纤维素、羟丙基纤维素、羟丙基甲基纤维素、硅酸铝镁、麦芽糖糊精、甲基纤维素、聚甲基丙烯酸酯、聚乙烯吡咯烷酮、预明胶化淀粉、藻酸钠、山梨醇、淀粉、糖浆和黄蓍胶。助流剂包括但不限于胶体二氧化硅、粉状纤维素、三硅酸镁、二氧化硅和滑石粉。掩味剂包括但不限于阿斯巴坦、甜菊苷、果糖、葡萄糖、糖浆、蜂蜜、木糖醇、甘露醇、乳糖、山梨醇、麦芽糖醇、甘草甜素。表面活性剂包括但不限于吐 温-80、泊洛沙姆。防腐剂包括但不限于尼泊金酯、苯甲酸钠、山梨酸钾等。
本文使用的术语“治疗”一般是指获得需要的药理和/或生理效应。该效应根据完全或部分地预防疾病或其症状，可以是预防性的；和/或根据部分或完全稳定或治愈疾病和/或由于疾病产生的副作用，可以是治疗性的。本文使用的“治疗”涵盖了对患者疾病的任何治疗，包括：(a)预防易感染疾病或症状但还没诊断出患病的患者所发生的疾病或症状；(b)抑制疾病的症状，即阻止其发展；或(c)缓解疾病的症状，即，导致疾病或症状退化。
甘露糖醛二酸寡糖组合物
本发明中用于治疗帕金森氏症的甘露糖醛二酸寡糖组合物，其包含具有式(Ⅲ)的甘露糖醛二酸或其药学上可接受的盐：
Figure PCTCN2019093803-appb-000005
其中n为选自1-9的整数，m选自0，1或2，m’选自0或1，
并且其中，
n＝1-5的甘露糖醛二酸的重量总和占所述组合物总重量的60％以上；
n＝1-2的甘露糖醛二酸的重量总和占所述组合物总重量的低于60％。
在一个示例性的实施方案中，本发明所述用于治疗帕金森氏症的甘露糖醛二酸寡糖组合物的制备方法包括如下几个步骤：
(1)甘露糖醛二酸产品的制备：
M段中间体的制备。如前文所述，本发明中采用的原料M段中间体可以通过现有技术中已知的方法制备。例如中国专利申请 No.98806637.8以及CN02823707.2所披露的方法。通常的方法可以简单描述为：将海藻酸初步降解后可得到聚甘露糖醛酸和聚古罗糖醛酸的混合多糖，混合多糖再经酸法沉淀后，可以除去其中的聚古罗糖醛酸，进一步精制可以得到纯度在90％以上的均聚甘露糖醛酸，即M段中间体。
臭氧氧化降解。在室温或者加热条件下使M段中间体溶解于适量的水中，搅拌，持续通入臭氧，反应开始进行。反应pH值可以通过滴加稀盐酸或者稀NaOH溶液调节至3-13之间，优选4-10，更优选6-8。温度优选为0-70℃，更优选10-45℃。反应完成以后，停止通入臭氧，调节pH至中性。
膜分离纯化。将上述所得的反应产物配成约10％浓度的溶液，通过分子截留膜分离，去除单糖以下的降解产物，收集未透过液。所采用的分子截留膜MWCO规格为1000Da-3000Da，优选2000Da。收集液经旋转蒸发仪浓缩、真空干燥即得寡聚甘露糖醛二酸混合物。经分析发现这些产品均是以二糖-十糖的寡糖且其含量是在一定比例范围的组合物。该制备方法的具体实例可参见实施例1-3，其中按照前述方法，分别制备了A、B、C三个组合物。
(2)单一聚合度寡糖的制备
将步骤(1)所得的寡糖混合物溶解，配成约10％左右的浓度，经P6凝胶色谱柱分离，紫外检测，收集各流出组分，合并相同聚合度的组分。收集到2-10糖的9个组分，分别经G10凝胶柱层析脱盐，旋转蒸发仪浓缩，真空干燥即得。一个具体的纯化制备过程见实施例4。这些柱层析、脱盐和干燥等操作是本领域技术人员所已知的。
(3)寡糖组合物的活性比较
将所制备的组合物与步骤(2)中纯化获得的六糖同时比较药理活性，结果表明本发明的寡糖组合物要明显比单一聚合度寡糖中活性最好的六糖还要好，而不包含二糖和三糖的组合物的活性略低于六糖。由此看出，不同聚合度的寡糖组合后能发挥协同增效的作用，当组合物中二-六糖的比例高于60％以上，且二、三糖的比例不高于 60％时，组合物的活性最高；但二、三糖的比例超过60％时，组合物的活性也会降低。
动物模型及药效活性评价步骤
抗帕金森症(PD)的药效评价动物模型
取小鼠随机分为8组：空白对照组、MPTP模型组、给药组，每组14只动物。动物分组当天开始给药，空白对照组和MPTP模型组灌胃生理盐水，其余各组均给予相应药物，每天给药1次，连续给药17天。从第6天起给予造模药物，空白对照组动物皮下给予生理盐水10ml/kg，其余动物皮下给予MPTP 25mg/kg，每天一次，共五天。
分别于实验第11、14、17天进行行为学检测。将小鼠头向上轻柔的放在粗糙的杆顶(直径8毫米，高55厘米)。小鼠从头向上调整至头完全向下的时间记录为潜伏期(T-turn)，小鼠从向下运动至四肢全部到达杆底的时间记录为爬下时间(T-LA)，超过30秒按照30秒记录。每只小鼠重复检测5次取平均值。
MPTP对脑黑质多巴胺能神经元有选择性的破坏作用，MPTP导致的PD动物模型是最为经典的类似人类帕金森病病理变化及临床特征的动物模型。PD的主要症状表现为为静止性震颤、肌张力增高、运动减少等，爬杆实验的调头时间和爬下时间可以代表小鼠的整体活动协调能力。
本发明的优点在以下非限制性的实施例中进一步进行说明。但实施例中采用的具体材料及其用量，以及其他实验条件并不应理解为对本发明的限制。除非特别指明，本发明中份数、比例、百分比等均以质量计。
实施例
实施例1：
步骤1)：甘露糖醛二酸寡糖混合物的制备
如在先专利所披露的方法制备M段中间体，具体操作简述如下：将5Kg海藻酸钠配成约10％的溶液，加稀盐酸调pH至3.0左右，升温至80℃，搅拌，反应10hr，停止加热，冷却至室温后，加NaOH调pH值至9.0，再加稀盐酸回调pH至2.85，离心机5000rpm离心10min，收集上清，加HCl调pH至1.0，离心，收集沉淀，旋转蒸发仪浓缩，真空干燥得M段中间体1500g。称取500g M-段中间体，加蒸馏水溶解后，配成5L体积的溶液，NaOH调pH至6.5，水浴加热，控制反应温度到75℃。调节氧气钢瓶出口的气流量和臭氧发生器的功率，使得臭氧质量浓度流量达到8g/hr，通入反应液中。反应4hr后停止通入臭氧，加适量水调整溶液浓度至10％左右，以截留分子量为2000Da的超滤膜过滤，收集未透过液，旋转蒸发仪浓缩，真空干燥，得350g甘露糖醛二酸产品A。
步骤2)：甘露糖醛二酸产品A中各聚合度寡糖的比例和结构分析
精密称取100mg上述干燥的甘露糖醛二酸产品A，加水溶解配制成10mg/mL的浓度，过0.22um滤膜，做为供试样品溶液。采用Superdex peptide(GE公司)分子排阻色谱联用多角度激光散射(MALS，怀雅特公司)测定组合物中不同聚合度寡糖的比例。实验方法如下：
色谱柱：Superdex peptide 10/300Gl
流动相：0.1mol/L NaCl
进样量：10uL
流速：0.3mL/min
测试结果：二糖-十糖分别以dp2-dp10表示，分别为dp2为19％，dp3为25％，dp4为22％，dp5为13％，dp6为9％，dp7为6％，dp8为3％，dp9为2％，dp10为1％。
步骤3)：LC-MS分析甘露糖醛二酸产品A中各聚合度寡糖的 结构
实验条件：
色谱柱：Superdex peptide 10/300Gl
流动相：20％甲醇+80％80mmol/L NH4Ac
流速：0.1mL/min
柱温：25℃±0.8℃。
质谱条件：Agilent 6540QTOF；离子源：ESI碰撞电压120V；负离子模式。采集信号(m/z)宽度为100-1000。
各聚合度寡糖的质谱图见附图1-3所示。对质谱图中各信号峰进行归属，验证了产品A中所有寡糖的分子结构，即通式(Ⅲ)所示的结构。信号归属及该信号所对应的结构见下表1。
Figure PCTCN2019093803-appb-000006
由上述质谱结构解析发现，产品A中糖链还原末端的甘露糖醛酸氧化为糖二酸结构(结构见通式Ⅲ)，该糖二酸可以是含6个碳(m+m’＝3)的甘露糖二酸结构，含量约为10％～30％，也可以是甘露糖二酸的脱羧产物，即5个碳(m+m’＝2)的糖二酸(30～50％)和4个碳(m+m’＝1)的糖二酸(30％～40％)。
实施例2：
称取100g实施例1中的M-段中间体，加蒸馏水溶解后，配成0.8L体积的溶液，NaOH调pH至4.0，室温25℃反应。调节氧气钢瓶出口的气流量和臭氧发生器的功率，使得臭氧质量浓度流量达到1g/hr，通入反应液中。反应10hr后停止通入臭氧，加适量水调整溶液浓度至15％左右，以截留分子量为1000Da的超滤膜过滤，收集未透过液，旋转蒸发仪浓缩，真空干燥，得80g甘露糖醛二酸产品B。
采用Superdex peptide(GE公司)分子排阻色谱联用多角度激光散射(MALS，怀雅特公司)测定B中各聚合度寡糖组分的比例。测定方法同实施例1中相关部分。测试结果：二糖-十糖分别以dp2-dp10表示，分别为dp2为20％，dp3为25％，dp4为19％，dp5为12％，dp6为9％，dp7为5％，dp8为5％，dp9为3％，dp10为2％。
实施例3：
称取100g实施例1中的M-段中间体，加蒸馏水溶解后，配成1.5L体积的溶液，NaOH调pH至9.0，水浴45℃反应。调节氧气钢瓶出口的气流量和臭氧发生器的功率，使得臭氧质量浓度流量达到3g/hr，通入反应液中。反应2hr后停止通入臭氧，加适量水调整溶液浓度至5％左右，以截留分子量为3000Da的超滤膜过滤，收集未透过液，旋转蒸发仪浓缩，真空干燥，得60g甘露糖醛二酸产品C。
采用Superdex peptide(GE公司)分子排阻色谱联用多角度激光 散射(MALS，怀雅特公司)测定C中各聚合度寡糖组分的比例。测定方法同实施例1中相关部分。测试结果：二糖-十糖分别以dp2-dp10表示，分别为dp2为8％，dp3为20％，dp4为28％，dp5为19％，dp6为13％，dp7为6％，dp8为3％，dp9为2％，dp10为1％。
实施例4：
单一聚合度的甘露糖醛二酸寡糖的制备，方法如下：
1、样品准备：由实施例1中制备得到的甘露糖醛二酸产品A中取出300g，加水溶解，配置成1000mL的浓溶液，放置在4℃冰箱备用。每次使用时取出50mL加水稀释1倍后，用0.22um超滤膜抽滤。
2、色谱分离条件：色谱仪为AKTA pure 150(购置于GE公司)，配UV检测器和自动收集器。分离色谱柱：1.2kg BioGel P6(购于伯乐公司)用去离子水混合，真空脱气以后，手动填装到玻璃柱(10cm内径)中，纯水冲洗10倍柱体积以后，色谱柱床稳定，高度为1.0m。然后改用0.02M的NaCl溶液为流动相，平衡10倍柱体积以后，开始上样。
3、上样和分离：泵的流速设置为1mL/min，将100mL的样品溶液通过色谱仪自带的泵抽到色谱柱顶端后，切换到流动相，以5mL/min的流速洗脱，待死水体积部分流出以后，开始自动收集，每管收集50mL。
4、重复上样，20次重复制备以后，合并相同组分，旋转蒸发仪浓缩，冷冻干燥，得到二糖至十糖共9个单一聚合度的寡糖。
实施例5
组合物与六糖之间的药理活性评价，考察组合物中不同聚合度寡糖之间的协同增效作用及寡糖比例范围。
样品准备：
组合物产品D：实施例4中制备得到的单一聚合度的甘露糖醛二酸寡糖，按照聚合度的大小从二糖到十糖准确称量，各糖取出的重量如下：二糖3.0g，三糖3.0g，四糖1.5g，五糖1.5g，六糖0.4g，七糖0.2g，八糖0.2g，九糖0.1g，十糖0.1g，混匀得10g组合物产品D。
对比实验样品制备
参照在先专利CN106344592A实施例1和2披露的方法制备含四糖-十糖的混合物
称取1g多聚甘露糖醛酸钠盐(重均分子量8235Da，上海绿谷制药有限公司提供)，加入适量蒸馏水配成1％(重量百分比)的多聚甘露糖醛酸钠水溶液。用盐酸将所述1％的多聚甘露糖醛酸钠水溶液的pH值调节为4，然后将该水溶液置于高压釜中。在110℃温度下加热反应4小时。从高压釜中取出该反应后的溶液并使其冷却。冷却后，用NaOH溶液调节该反应后溶液的pH值得到中性液体。在搅拌条件下，将所述中性液体缓慢加入到该液体体积4倍体积量的乙醇中，进行醇沉并静置过夜。过滤分离醇沉所得固体物质，并在过滤分离时用无水乙醇洗涤过滤分离所得固体物质，最终得到白色滤饼。将该滤饼置于60℃烘箱中干燥，得褐藻胶寡糖粗品。
取5g褐藻胶寡糖粗品配成5％(重量百分比)的水溶液。通过向50ml的10％(重量百分比)氢氧化钠溶液中加入25ml的5％(重量百分比)的硫酸铜溶液并立即混匀制备得到新鲜氧化剂氢氧化铜。将该新鲜氧化剂氢氧化铜立即加入到40ml上述5％(重量百分比)的褐藻胶寡糖溶液中，同时通过沸水浴进行加热，直至不再有砖红色沉淀产生。将该反应体系进行离心处理以去除沉淀从而得到上清液。取少许上清液再次加入所述氧化剂，检查是否还有砖红色沉淀产生。若还有砖红色沉淀产生，则将上述离心所得全部上清液与另外部分的所述氧化剂继续进行反应，直至检验不再有砖红色沉淀产生为止。将最后得到的反应体系离心分离获得上清液。向上清液中 加入4倍体积量的95％乙醇进行醇沉，并静置过夜。过滤分离醇沉所得固体物质，并用无水乙醇洗涤该固体物质。将所得固体物质置于60℃烘箱中烘干，得到式(II)所示褐藻胶寡糖粗品。
取上述褐藻胶寡糖粗品1g，配成10％(重量百分比)的水溶液，用95％乙醇溶液再次进行醇沉，过滤分离再次醇沉所得沉淀物并任选地用无水乙醇洗涤。分离该沉淀物并干燥，得到固体物质。将该固体物质配成5％(重量百分比)的水溶液，用3μm孔径膜过滤该水溶液并收集滤液。将该滤液在分子排阻色谱Bio-Gel-P6凝胶柱(1.6×180cm，购自Bio-Rad公司)上进行洗脱分离，作为流动相的洗脱液为0.2mol·L-1NH 4HCO 3。依次使用多个5毫升试管从该柱色谱收集洗脱液，然后用硫酸-咔唑法检测所述各集液管中洗脱液的糖含量。根据该检测结果分别收集含有不同分子量褐藻胶寡糖组分的洗脱液。将含有不同分子量褐藻胶寡糖组分的洗脱液各自分别减压浓缩并冷冻干燥，弃去组分1，得到分别具有不同分子量的式(II)所示褐藻胶寡糖组分2-12(n分别具有0-10的值)收集并合并n＝2-8的式(II)所示褐藻胶寡糖洗脱液并干燥，然后得到n＝2-8的式(II)所示褐藻胶寡糖混合物(四-十糖混合物)，作为对比实验样品。
采用Superdex peptide(GE公司)分子排阻色谱联用多角度激光散射(MALS，怀雅特公司)测定对比实验样品中各聚合度寡糖组分的比例。测定方法同实施例1中相关部分。测试结果：四糖-十糖分别以dp4-dp10表示，分别为dp4为10％，dp5为12％，dp6为13％，dp7为14％，dp8为15％，dp9为19％，dp10为17％。
实施例1、2、3中分别制备得到的产品A、B、C，本实施例中的产品D以及对比实验样品的寡糖比例如下表2所示。
表2 甘露糖醛二酸寡糖组合物产品及对比试验样品中的寡糖百分比
Figure PCTCN2019093803-appb-000007
Figure PCTCN2019093803-appb-000008
以上A、B、C、D四个样品各取10g，按照“抗PD的药效评价动物模型”所描述的方法，比较这些组合物与六糖(6T)及对比实验样品的药理活性。
实验结果显示，模型组与空白对照组相比，前者潜伏期和爬下时间明显延长。与模型组相比，各给药组潜伏期和爬下时间有不同程度缩短。其中产品A、B、C的药效活性均好于对比实验样品，且好于之前预期的活性最高的单一聚合度的六糖，但产品D的活性弱于六糖。不囿于任何理论，推测组合物中各寡糖之间的比例对于产品的活性有显著影响，添加一定比例的二糖、三糖有协同增效作用，但当二糖、三糖的比例过高时则会降低组合物的活性。见附图4-9。

Claims (14)

  1. 一种甘露糖醛二酸寡糖组合物用于制备治疗帕金森氏症药物的用途；其中所述甘露糖醛二酸寡糖组合物包含具有式(Ⅲ)的甘露糖醛二酸或其药学上可接受的盐：
    Figure PCTCN2019093803-appb-100001
    其中n为选自1-9的整数，m选自0，1或2，m’选自0或1，
    并且其中，
    n＝1-5的甘露糖醛二酸的重量总和占所述组合物总重量的60％以上；
    n＝1-2的甘露糖醛二酸的重量总和占所述组合物总重量的低于60％。
  2. 根据权利要求1所述的用途，其中所述的甘露糖醛二酸寡糖组合物中，n＝1-2的甘露糖醛二酸的重量总和占所述组合物总重量的10-50％，优选25-50％。
  3. 根据权利要求1所述的用途，其中所述的甘露糖醛二酸寡糖组合物中，n＝1-3的甘露糖醛二酸的重量总和与n＝4-7的甘露糖醛二酸重量总和的比例在1.0-3.5之间。
  4. 根据权利要求1所述的用途，其中所述的甘露糖醛二酸寡糖组合物中，m+m’＝1或2的甘露糖醛二酸的重量总和不低于所述组合物总重量的50％以上，优选60％-90％，更优选70％-90％。
  5. 根据权利要求4所述的用途，其中m+m’＝1的甘露糖醛二酸的重量总和不低于所述组合物总重量的10％，优选30-40％。
  6. 根据权利要求4所述的用途，其中m+m’＝2的甘露糖醛二酸的重量总和不低于所述组合物总重量的10％，优选30-50％。
  7. 根据权利要求1所述的用途，其中n＝1-5的甘露糖醛二酸的重量总和占所述组合物总重量的80-95％。
  8. 根据权利要求1所述的用途，其中n＝1-3的甘露糖醛二酸的重量总和占所述组合物总重量的20-70％。
  9. 根据权利要求3所述的用途，其中n＝1-3的甘露糖醛二酸的重量总和与n＝4-7的甘露糖醛二酸重量总和的比例在1.0-3.0之间。
  10. 根据权利要求1-8任一项所述的用途，其中各聚合度甘露糖醛二酸在所述组合物中的重量百分含量为：二糖5-25％，三糖15-30％，四糖15-28％，五糖5-25％，六糖2-20％，七糖2-20％，八糖2-20％，九糖2-20％，十糖2-20％。
  11. 据权利要求10所述的用途，其中各聚合度甘露糖醛二酸在所述组合物中的重量百分含量为：二糖5-25％，三糖15-30％，四糖15-28％，五糖10-20％，六糖5-15％，七糖3-10％，八糖2-5％，九糖1-5％，十糖1-5％。
  12. 根据权利要求11所述的用途，其中各聚合度甘露糖醛二酸在所述组合物中的重量百分含量为：二糖10-20％，三糖18-30％，四糖15-28％，五糖10-20％，六糖5-10％，七糖3-5％，八糖2-5％，九糖1-3％，十糖1-3％。
  13. 根据权利要求1-11所述的用途，其中所述药学上可接受的 盐是钠盐或钾盐。
  14. 一种治疗患有帕金森氏症的患者的方法，其包括给予需要的患者有效量的根据权利要求1-13任一项所述的甘露糖醛二酸寡糖组合物。
PCT/CN2019/093803 2018-06-29 2019-06-28 甘露糖醛二酸的组合物在治疗帕金森氏症中的应用 WO2020001640A1 (zh)

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