WO2023000263A1 - 一种褐藻寡糖的用途 - Google Patents

一种褐藻寡糖的用途 Download PDF

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WO2023000263A1
WO2023000263A1 PCT/CN2021/107883 CN2021107883W WO2023000263A1 WO 2023000263 A1 WO2023000263 A1 WO 2023000263A1 CN 2021107883 W CN2021107883 W CN 2021107883W WO 2023000263 A1 WO2023000263 A1 WO 2023000263A1
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mice
brown algae
group
aga
oligosaccharide
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PCT/CN2021/107883
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French (fr)
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高河勇
刘振德
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海糖(江苏)生物医药科技有限公司
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/734Alginic acid
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/06Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88Lyases (4.)

Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to an application of fucoidan oligosaccharide.
  • Alginate is mainly found in the cell walls of kelp, sargassum and macroalgae, and is a kind of linear, unbranched, negatively charged polysaccharide compound.
  • Alginate is a binary compound composed of ⁇ -D-(1,4)-mannuronic acid (M) and ⁇ -L-(1,4)-guluronic acid (G). linear block compounds.
  • alginate oligosaccharides In recent years, due to the unique structure of alginate oligosaccharides, its activity research has become a hot spot in the research of sugar drugs, and its biological activity research has made important progress. Studies have found that alginate oligosaccharides and their derivatives have a variety of biological activities, such as antioxidant, anti-tumor, anti-coagulation, immune regulation, neuroprotection, anti-inflammatory activity, anti-viral activity, anti-senile dementia, anti-uria Road stones, anti-diabetes, etc.
  • biological activities such as antioxidant, anti-tumor, anti-coagulation, immune regulation, neuroprotection, anti-inflammatory activity, anti-viral activity, anti-senile dementia, anti-uria Road stones, anti-diabetes, etc.
  • Carbohydrates are a class of highly complex and varied biomacromolecules. Regardless of chemical cleavage or enzymatic cleavage, it is difficult to separate oligosaccharides or polysaccharides with a uniform degree of polymerization. So far, almost all studies have used oligosaccharides or polysaccharides as a mixture of a series of sugars with close polymerization degrees, which brings great difficulties to their activity research, metabolism, toxicology and drug quality research.
  • the fucobiose has two structures of ⁇ G and/or ⁇ M and a combination of any ratio thereof; fucotriose has four structures of ⁇ GG, ⁇ GM, ⁇ MM and ⁇ MG and a combination of any ratio thereof; fucotetraose has two structures of ⁇ GGG, ⁇ GGM, Eight structures of ⁇ GMG, ⁇ GMM, ⁇ MMG, ⁇ MMM, ⁇ MGG and ⁇ MGM and their combinations in any ratio; all oligosaccharides are linked by glycosidic bonds at positions 1 and 4 of monosaccharides; G stands for ⁇ -L-guluronic acid; M stands for ⁇ -D-mannuronic acid; ⁇ means that ⁇ -elimination occurs at the 4,5 positions of ⁇ -L-guluronic acid and/or ⁇ -D-mannuronic acid, and the 4,5 positions of the non-reducing end are generated as common Unsaturated monosaccharides with yoke double bonds; the structure of each monosacchari
  • HUA hyperuricemia
  • MSU monosodium urate
  • hyperuricemia has become another common metabolic disease after diabetes.
  • hyperuricemia and gout are a continuous pathological process.
  • the 2020 "Practice Guidelines for Hyperuricemia/Gout Patients” also pointed out that hyperuricemia and gouty arthritis are different states of the same disease, and they are divided into asymptomatic hyperuricemia, asymptomatic urate deposition, asymptomatic Hyperuricemia with urate deposition, gout, tophi gout, erosive gout, initial gout attack, and recurrent gout attack.
  • anti-inflammatory and analgesic drugs can be used to improve the quality of life of patients, such as targeted use within 24 hours
  • Non-steroidal anti-inflammatory drugs, colchicine and corticosteroids to help patients relieve pain.
  • uric acid-lowering drugs used to help regulate uric acid levels.
  • Uric acid-lowering drugs are mainly divided into three categories: 1) xanthine oxidase inhibitors, such as allopurinol, febuxostat and topirostat, are used to inhibit uric acid synthesis and maintain uric acid at a normal level; 2) promote Uric acid excretion drugs, such as probenecid, benzbromarone, recinad, and dotinoler, can accelerate the excretion of uric acid in the body.
  • Sodium bicarbonate is also beneficial to the excretion of uric acid, and can be used together with other urate-lowering drugs, or used alone for patients with mildly elevated uric acid.
  • uricase can degrade uric acid into water-soluble allantoin, which is more easily excreted by the kidneys.
  • rasburicase and prekexi also known as pegoloxase
  • uricase can degrade uric acid into water-soluble allantoin, which is more easily excreted by the kidneys.
  • Gout patients are prone to liver damage due to long-term use of drugs, so they are prone to some liver diseases, such as high blood pressure and high blood fat.
  • liver diseases such as high blood pressure and high blood fat.
  • joint deformities, uric acid nephropathy, type 2 diabetes, cardiovascular disease, and lipid metabolism disorders are also more important hazards of gout, which have brought considerable economic burdens to individuals, families, and society.
  • the present invention conducts further research on the fucoidan oligosaccharide, and provides a use of the fucoidan oligosaccharide.
  • the purpose of the present invention is to provide a use of fucoidan oligosaccharides.
  • the present invention provides a use of a fucoidan oligosaccharide or a pharmaceutically acceptable salt thereof in the preparation of a drug for the treatment of gout, wherein the fucoidan oligosaccharide is fucobiose, fucotriose and/or fucoidan sugar.
  • the fucoidan oligosaccharides are composed of monosaccharides G, M and/or ⁇ linked by glycosidic bonds at positions 1 and 4; wherein, G represents ⁇ -L-guluronic acid , M means ⁇ -D-mannuronic acid, ⁇ means ⁇ -elimination occurs at the 4,5 positions of ⁇ -L-guluronic acid or ⁇ -D-mannuronic acid, and the 4,5 positions are conjugated Unsaturated monosaccharides with double bonds.
  • the fucobiose is selected from ⁇ G, ⁇ M or a combination thereof.
  • the fucoidose is selected from one or more of ⁇ GG, ⁇ GM, ⁇ MM and ⁇ MG.
  • the fucotetraose is selected from one or more of ⁇ GGG, ⁇ GGM, ⁇ GMG, ⁇ GMM, ⁇ MMG, ⁇ MMM, ⁇ MGG and ⁇ MGM.
  • the pharmaceutically acceptable salt is sodium salt, potassium salt, calcium salt, magnesium salt and/or ammonium salt.
  • the present invention provides a fucoidan oligosaccharide or a pharmaceutically acceptable salt thereof for use in the treatment of gout, wherein the fucoidan oligosaccharide is fucobiose, fucotriose and/or fucotetraose.
  • the present invention provides a method for treating gout, comprising administering a therapeutically effective amount of fucoidan oligosaccharide or a pharmaceutically acceptable salt thereof to a patient in need, wherein the fucoidan oligosaccharide is fucobiose, fucotriose and / or Fucotetraose.
  • the fucodiose, trisaccharide and tetrasaccharide with a uniform polymerization degree of the present invention have revolutionary progress in the quality control, pharmacology, toxicology and other analysis and research of carbohydrate raw materials.
  • the fucobiose, trisaccharide and tetrasaccharide of the present invention can significantly improve the symptoms of joint swelling and mechanical hyperalgesia in mice.
  • the gait behavior study of mice also shows that the fucobiose, trisaccharide and tetrasaccharide of the present invention can significantly improve the abnormal gait behavior of mice. Therefore, the fucobiose, trisaccharide and tetrasaccharide of the present invention have strong efficacy in treating acute gout.
  • the fucoidan oligosaccharide drugs provided by the present invention are derived from marine algae, have no toxic and side effects, and can be used for a long time.
  • Figure 1 shows the high performance liquid chromatogram of fucobiose at a wavelength of 230nm
  • Figure 2 shows the hydrogen nuclear magnetic spectrum ( 1 HNMR, solvent D 2 O) of fucobiose
  • FIG. 3 shows the high resolution mass spectrum (HRMS (ESI)) of fucobiose
  • Figure 4 shows the high performance liquid chromatogram of fucoidan at a wavelength of 230nm
  • FIG. 5 shows the hydrogen nuclear magnetic spectrum ( 1 HNMR, the solvent is D 2 O) of fucoidan
  • Figure 6 shows the high resolution mass spectrum (HRMS (ESI)) of fucoidan
  • Figure 7 shows a high performance liquid chromatogram of fucoidose at a wavelength of 230nm
  • Figure 8 shows the hydrogen nuclear magnetic spectrum ( 1 HNMR, the solvent is D 2 O) of fucotetraose
  • Figure 9 shows the high resolution mass spectrum (HRMS (ESI)) of fucotetraose
  • Figure 10 shows the establishment and evaluation of the acute gouty arthritis mouse model
  • Figure 11 shows the effect of fucobiose on inhibiting the symptoms of joint pain in AGA model mice
  • Figure 12 shows the effect of fucoidan on inhibiting the symptoms of joint pain in AGA model mice
  • Figure 13 shows the effect diagram of fucotetraose inhibiting the symptoms of joint pain in AGA model mice
  • Figure 14 shows the mass spectrum of mixed fucoidan oligosaccharides with a degree of polymerization of 2-8;
  • Figure 15 shows the research on the effect of fucoidan, mixed sugar and indomethacin on inhibiting joint pain in AGA model mice;
  • Figure 16 shows the effect of fucoidan on the gait behavior of AGA model mice.
  • Fucobiose sodium salt if the two carboxyl groups in the molecule are both sodium salts, the theoretical sodium ion content is 11.58%; the actual ion chromatography test shows that the sodium ion content is 10.3%. If detected by the residue on ignition method, the sodium ion exists in the form of sodium sulfate, and the theoretical residue ratio should be 35.77%; the actual residue on ignition detection, the residue is 34.3%; the results obtained by the two detection methods are relatively close, indicating that the compound carboxylate The acid functionality is indeed in the sodium salt form. However, the measured values are slightly smaller than the theoretical value, probably because the sodium salt is a weak acid and strong base salt, and a small part of carboxylic acid is still in a free state.
  • Fucotriose sodium salt if the three carboxyl groups in the molecule are all sodium salts, the theoretical content of sodium ions is 11.59%; the actual ion chromatography test shows that the content of sodium ions is 9.9%. If detected by the residue on ignition method, the sodium ion exists in the form of sodium sulfate, and the theoretical residue ratio should be 35.80%; the measured residue on ignition is 33.01%. The results obtained by the two detection methods are relatively close, indicating that the carboxylic acid functional group of the compound is indeed in the form of a sodium salt. However, the measured values are slightly smaller than the theoretical value, probably because the sodium salt is a weak acid and strong base salt, and a small part of carboxylic acid is still in a free state.
  • the obtained fucotetraose sodium salt was tested for purity by high-performance liquid chromatography (HPLC, 230 nm), and its structure was identified by hydrogen nuclear magnetic spectrum ( 1 HNMR) and high-resolution mass spectrometry (HRMS-ESI).
  • Healthy clean-grade male C57BL/6 mice were divided into control group (Veh) and model group (MSU), with 6 mice in each group.
  • the model group was injected with 1 mg/20 ⁇ l sodium urate (MSU) solution at the posterior side of the right ankle joint of the mouse along the medial side of the Achilles tendon at 30-40° to the ankle joint cavity, and the control group was injected in the same way, etc. amount of phosphate buffered saline (PBS). After the injection, the degree of swelling of the ankle joint of the mice and the change of the mechanical pain threshold were observed.
  • MSU sodium urate
  • Determination of ankle joint swelling Use a vernier caliper to detect the diameter of the ankle joint on the affected side of the mouse at four time points before modeling, 2h, 6h, 24h and 48h after modeling, and measure three times in a row, and take the average as the final reading.
  • Ankle joint swelling current measurement diameter - pre-model measurement diameter.
  • Mechanical pain behavior measurement mice were placed in a transparent plastic box on an elevated barbed wire, covered with transparent plexiglass, and adapted to the environment for 45 minutes. Then, according to the "Up and Down" method, at four time points before the cast, 2h, 6h, 24h and 48h after the cast, von Frey wires of different specifications were used to detect the pain threshold of the mouse's right hind paw near the ankle. Using the formula, the paw withdrawal threshold was calculated.
  • FIG. 10 shows the establishment and effect evaluation of acute gouty arthritis mouse model.
  • Fig. 10A is a graph showing the swelling of the right ankle joint of mice in the AGA model group after injection of urate (MSU) crystals. After injection of urate (MSU) crystals in the right ankle joint of the mice in the AGA model group for 24 hours, the ankle joints of the mice in the control group injected with the solvent phosphate buffered saline (PBS) were significantly swollen.
  • Figure 10B is the comparison of the slices after H&E staining of the ankle joints of mice in the control group and the model group.
  • FIG. 10C shows the change of ankle joint diameter between mice injected with urate crystals at the right ankle joint and mice injected with PBS at the ankle joint. It can be seen that the ankle joints of mice in the control group had only slight swelling within a few hours after the injection of PBS, but quickly subsided, and basically remained unchanged after returning to normal. The ankle joints of mice injected with urate crystals swelled rapidly, and there was still obvious swelling after 48 hours.
  • Figure 10D shows the comparison of mechanical hyperalgesia in the right hind paw of the two groups of mice, and the results show that the mechanical pain threshold of the mice injected with MSU was significantly reduced. The above results are consistent with previous reports, thus suggesting that the AGA mouse model was established successfully.
  • AOS2 fucobiose is abbreviated as "AOS2”.
  • AGA model mice were prepared as described in Example 4. The mice were randomly divided into 5 groups: control group (Veh+Veh), model group (MSU+Veh), MSU+AOS2 dose groups (400mg/kg, 200mg/kg, 100mg/kg). Different concentrations of AOS2 were injected intraperitoneally in each dose group of MSU+AOS2, 1 hour before casting, 5 hours, 23 hours and 47 hours after casting; the control group and model group were injected intraperitoneally with the same volume of control solvent PBS (Fig. 11A). Mechanical pain and swelling were detected before modeling and 2, 6, 24 and 48 hours after modeling. The specific detection method is consistent with Example 4.
  • Figure 11 shows the effect of fucobiose AOS2 on inhibiting joint pain in AGA model mice.
  • Figure 11A is a schematic diagram of dosing time points.
  • Figure 11B is a time-course chart of the effect of different doses of AOS2 (100mg/kg, 200mg/kg, 400mg/kg, i.p.) on mechanical pain in the ankle joint of AGA model mice. After administration of different doses of AOS2, 200 and 400 mg/kg AOS2 can relieve mechanical pain in AGA model mice to varying degrees.
  • Fig. 11C is a statistical diagram of the area under the curve of 11B.
  • Figure 11D shows the time course of the effect of different doses of AOS2 (100mg/kg, 200mg/kg, 400mg/kg, i.p.) on the swelling of the ankle joints of AGA model mice. After giving different doses of AOS2, the swelling of the ankle joint was significantly reduced, and the higher the dose, the smaller the swelling.
  • Figure E is a statistical chart of the area under the curve in Figure D. The above experimental results show that AOS2 can effectively inhibit the pain and swelling of ankle joints in AGA model mice, and this effect is dose-dependent.
  • AGA model mice were prepared as described in Example 4.
  • fucoidan is referred to as "AOS3" for short.
  • the mice were randomly divided into 5 groups: control group (Veh+Veh), model group (MSU+Veh), MSU+AOS3 dose groups (400mg/kg, 200mg/kg, 100mg/kg).
  • Different concentrations of AOS3 were injected intraperitoneally in each dose group of MSU+AOS3, 1 hour before casting, 5 hours, 23 hours and 47 hours after casting respectively; the control group and model group were injected with the same volume of control solvent PBS (Figure 12A). Mechanical pain and swelling were detected before modeling and 2, 6, 24 and 48 hours after modeling. The specific detection method is consistent with Example 4.
  • Fig. 12 shows the effect of fucoidan AOS3 on inhibiting joint pain in AGA model mice.
  • Fig. 12A is a schematic diagram of administration time points.
  • Fig. 12B is a time-course chart of the effect of different doses of AOS3 (100mg/kg, 200mg/kg, 400mg/kg, i.p.) on mechanical pain in ankle joints of AGA model mice.
  • Fig. 12C is a statistical diagram of the area under the curve of 12B.
  • FIG. 12D is a time-course chart of the effect of different doses of AOS3 (100mg/kg, 200mg/kg, 400mg/kg, i.p.) on the swelling of the ankle joints of AGA model mice.
  • Fig. 12E is a statistical diagram of the area under the curve in Fig. 12D. Figures 12D and 12E show the changes in swelling of ankle joints in mice over time.
  • AGA model mice were prepared as described in Example 4.
  • fucotetraose is abbreviated as "AOS4".
  • the mice were randomly divided into 5 groups: control group (Veh+Veh), model group (MSU+Veh), MSU+AOS4 dose groups (400mg/kg, 200mg/kg, 100mg/kg).
  • Each dose group of MSU+AOS4 was intraperitoneally injected with different concentrations of AOS4, and administered 1 h before, 5, 23 and 47 h after the molding; the control group and the model group were intraperitoneally injected with the same volume of control solvent PBS (Fig. 13A). Mechanical pain and swelling were detected before modeling and 2, 6, 24 and 48 hours after modeling. The specific detection method is consistent with Example 4.
  • FIG. 13 shows the effect of fucotetraose AOS4 on inhibiting joint pain symptoms in AGA model mice.
  • Figure 13A is a schematic diagram of dosing time points.
  • Fig. 13B is a time-course chart of the effect of different doses of AOS4 (100mg/kg, 200mg/kg, 400mg/kg, i.p.) on mechanical pain in ankle joints of AGA model mice.
  • Fig. 13C is a statistical diagram of the area under the curve in Fig. 13B.
  • FIG. 13D is a time-course chart of the effect of different doses of AOS4 (100mg/kg, 200mg/kg, 400mg/kg, i.p.) on the swelling of the ankle joints of AGA model mice.
  • Fig. 13E is a statistical diagram of the area under the curve in Fig. 13D.
  • Figures 13D and 13E show the changes in mouse ankle swelling over time. After giving different doses of AOS4, the swelling of the ankle joint was significantly reduced, and the higher the dose, the smaller the swelling. The above experimental results show that AOS4 can effectively inhibit the pain and swelling of ankle joints in AGA model mice, and this effect is dose-dependent.
  • AOS2, AOS3 and AOS4 all show better analgesic activity on AGA mouse model when the dosage is 200mg/kg. Therefore, in the next experiment, the inventors selected AOS3 with a concentration of 200 mg/kg for further in vivo pharmacological activity research.
  • AGA model mice were prepared as described in Example 4.
  • Mix AOS3 with mixed sugars mixed fucoidan oligosaccharides with a polymerization degree of 2-8, whose mass spectrum is shown in Figure 14, obtained from Ocean University of China, referred to as “AOS mixed” in the present invention
  • indomethacin in the present invention referred to as "Indo"
  • mice were randomly divided into 6 groups: control group (Veh+Veh), model group (MSU+Veh), MSU+AOS3 group (200mg/kg), MSU+AOS3 group (100mg/kg), MSU+AOS mixed group (200mg/kg), MSU+indomethacin group (MSU+Indo, 10mg/kg).
  • Drugs in each dosage group were intraperitoneally injected with the specified amount of drugs, and administered 1 hour before the injection, 5, 23 and 47 hours after the injection; the control group and the model group were injected with the same volume of control solvent PBS (Figure 15A). Mechanical pain and swelling were detected before modeling and 2, 6, 24 and 48 hours after modeling. The specific detection method is consistent with Example 4.
  • Figure 15 shows the effect of AOS3, mixed sugar and contrast drug indomethacin on inhibiting joint pain symptoms in AGA model mice.
  • Figure 15A is a schematic diagram of dosing time points.
  • Figure 15B is AOS3 (100mg/kg, 200mg/kg, i.p.), mixed sugar (200mg/kg, i.p.) and indomethacin (10mg/kg, i.p., the effective dose in mice) on AGA model mouse ankle Time-course diagram of mechanical pain effects in joints.
  • Fig. 15C is a statistical diagram of the area under the curve in Fig. 15B.
  • Figure 15D is a time-course chart of the effects of different drugs on the swelling of the ankle joints of AGA model mice.
  • Fig. 15E is a statistical diagram of the area under the curve in Fig. 15D.
  • Figures 15D and 15E show the changes in swelling of ankle joints in mice over time.
  • the AGA mouse model was prepared as described in Example 4.
  • the mice were randomly divided into 3 groups: control group (Veh+Veh), model group+vehicle group (MSU+Veh), model group+AOS3 group (MSU+AOS3).
  • the MSU+AOS3 group was intraperitoneally injected with 200mg/kg of AOS3, 1h before, 5h and 23h after the molding, respectively.
  • the control group and the model group+solvent group were injected with the same volume of control solvent PBS.
  • FIG. 16A firstly, the experimental mice were pre-adapted to the test environment for a period of time.
  • the gait behavior test was carried out at two time points of 8h and 24h after the mold, that is, within 10-15 seconds, the mice in each group passed the track at a uniform speed without stopping, and the camera below collected the gait images in real time, and then used DigiGait (Mouse Specifics, Inc. ., USA) software to analyze the gait behavior parameters of mouse hind paw footprint area and swing duration.
  • DigiGait Mae Specifics, Inc. ., USA
  • Figure 16 shows the effect of fucoidan AOS3 on the gait behavior of AGA model mice.
  • Fig. 16A is a schematic diagram of Catwalk mouse gait recording and analysis.
  • Figure 16B is a representative diagram of the area of the paw prints of the mice in each group after passing the track within 5 seconds. The paw prints showed that the area of the right hind paw of the mice in the model group + solvent group was significantly reduced compared with that of the healthy side at 24 hours after the model, and a clear limp-like gait appeared. However, the grounding area of the right hind paw of the mice in the model group + AOS3 group was improved.
  • Fig. 16A is a schematic diagram of Catwalk mouse gait recording and analysis.
  • Figure 16B is a representative diagram of the area of the paw prints of the mice in each group after passing the track within 5 seconds. The paw prints showed that the area of the right hind paw of the mice in the model group + solvent group was significantly reduced compared with that of the healthy side at 24 hours after
  • 16C is a statistical graph of the relative percentage of the area of the paw of the affected side and the area of the healthy side of the mice in each group at 8 and 24 hours after modeling. Compared with the healthy side, the area of the affected paw of the mice in the model group + solvent group was significantly reduced, while the area of the affected paw of the mice in the model group + AOS3 group was significantly improved.
  • Fig. 16D is a statistical graph of the relative percentage of the swinging time of the paw of the affected side in the air and the swinging time of the healthy side of the paw in each group at 8 and 24 hours after modeling.
  • AOS3 can effectively improve the abnormal changes in gait behavior of AGA model mice.
  • AOS2, AOS3 and AOS4 can dose-dependently relieve joint swelling and mechanical hyperalgesia symptoms of acute gouty arthritis (AGA) model mice; AOS3 can significantly improve the abnormal gait behavior of AGA model mice, 200mg
  • the analgesic effect of AOS3 at a dose of 10 mg/kg was better than that of indomethacin (10 mg/kg dose), and also better than that of mixed sugar at the same dose.

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Abstract

本发明提供一种褐藻寡糖或其药学上可接受的盐在制备用于治疗痛风的药物中的用途,其中所述褐藻寡糖为褐藻二糖、褐藻三糖或褐藻四糖。研究发现,在急性痛风性关节炎(AGA)小鼠模型上,本发明的褐藻二糖、三糖和四糖可明显改善小鼠关节肿胀与机械痛觉过敏症状。小鼠的步态行为学研究也表明,本发明的褐藻二糖、三糖和四糖可显著改善小鼠的步态行为学异常。本发明的褐藻寡糖具有较强的治疗急性痛风的功效。本发明所提供的褐藻寡糖类药物,来源于海洋藻类,无任何毒副作用,可长期使用。

Description

一种褐藻寡糖的用途 技术领域
本发明属于生物医药技术领域,具体涉及一种褐藻寡糖的用途。
背景技术
糖类(Carbohydrate)与核酸、蛋白质一起并称为三大生命物质。褐藻胶主要存在于海带、马尾藻以及巨藻的细胞壁中,是一类直链、无分枝,具负电荷的多糖类化合物。褐藻胶是由β-D-(1,4)-甘露糖醛酸(Mannuronic acid,M)和α-L-(1,4)-古洛糖醛酸(Guluronic acid,G)组成的二元线型嵌段化合物。
近年来,褐藻胶寡糖因结构独特,其活性研究成为了糖类药物研究中的热点,其生物活性研究取得了重要进展。人们研究发现,褐藻胶寡糖及其衍生物具有多种生物活性,例如抗氧化性、抗肿瘤、抗凝血、免疫调节、神经保护、抗炎活性、抗病毒活性、抗老年痴呆、抗尿路结石、抗糖尿病等。
糖类是一类高度复杂同时又变化多端的生物大分子。不论化学裂解或酶法裂解,均难以分离得到均一聚合度的寡聚糖或多聚糖。迄今为止,几乎所有的研究采用的寡糖或多聚糖均为聚合度接近的一系列糖的混合物,这为其活性研究、代谢、毒理以及药物的质量研究等带来非常大的困难。
发明人前期的研究针对目前糖类研究的难点,开发了一系列专一性较强的褐藻胶裂解酶,可以分别将褐藻胶分解为纯度较高,非还原性末端具有共轭双键,聚合度均一的褐藻二糖、三糖或四糖;经过酶灭活,离心取上清液,浓缩后,再以凝胶柱或离子交换树脂进一步纯化为均一聚合度的褐藻二糖、三糖或四糖。所述褐藻二糖为ΔG和/或ΔM两种结构及其任意比例的组合;褐藻三糖为ΔGG、ΔGM、ΔMM和ΔMG四种结构及其任意比例的组合;褐藻四糖为ΔGGG、ΔGGM、ΔGMG、ΔGMM、ΔMMG、ΔMMM、ΔMGG和ΔMGM八种结构及其任意比例的组合;所有寡糖均为单糖1,4位糖苷键连接;G代表α-L-古洛糖醛酸;M代表β-D-甘露糖醛酸;Δ表示α-L-古洛糖醛酸和/或β-D-甘露糖醛酸4,5位发生β-消除,生成非还原端4,5位为共轭双键的不饱和单糖;各单糖的结构如下图所示:
Figure PCTCN2021107883-appb-000001
以ΔGM为例,相应褐藻三糖的结构如下:
Figure PCTCN2021107883-appb-000002
无论男性还是女性,非同日2次血尿酸(Serum Uric Acid,SUA)水平超过420μmol/L,称之为高尿酸血症(Hyper-Uricemia,HUA)。血尿酸超过其在血液或组织液中的饱和度可在关节局部形成尿酸单钠晶体(Monosodium Urate,MSU)并沉积,诱发局部炎症反应和组织破坏,即痛风(gout);可在肾脏沉积引发急性肾病、慢性间质性肾炎或肾结石,称之为尿酸性肾病。
高尿酸血症在不同种族患病率为2.6%~36%,痛风为0.03%~15.3%,中老年男性与绝经后女性是其高发人群,近年呈现明显上升和年轻化趋势。当前流行病学研究显示大约有10%的高尿酸血症发展为痛风,痛风患者大约有80~90%是高尿酸血症。Meta分析显示,中国高尿酸血症的总体患病率为13.3%,痛风为1.1%。全球高尿酸血症已经成为继糖尿病之后的又一常见代谢性疾病。
随着认识的深入,越来越多的学者意识到高尿酸血症与痛风是一个连续的病理过程。2020年《高尿酸血症/痛风患者实践指南》亦提出高血尿酸与痛风性关节炎是同一疾病的不同状态,将其分为无症状高尿酸血症、无症状尿酸盐沉积、无症状高尿酸血症伴尿酸盐沉积、痛风、痛风石性痛风、侵蚀性痛风、初次痛风发作、复发型痛风发作8个状态。
痛风的药物治疗分为两大类,一种是消炎镇痛治疗,用于痛风突然急性发作时,可以采用抗炎镇痛的药物来提高患者的生活质量,如在24小时之内针对性使用非甾体抗炎药、秋水仙碱和糖皮质激素来帮助患者减轻病痛。第二种是用于辅助调节尿酸含量的降低尿酸药物。
降尿酸药物主要分为3类:1)黄嘌呤氧化酶抑制剂,例如别嘌醇、 非布司他和托匹司他,用于抑制尿酸合成,维持尿酸处于一个正常的水平;2)促进尿酸排泄药物,如丙磺舒、苯溴马隆、雷西纳德和多替诺雷等,能够起到加快体内尿酸排出的作用。碳酸氢钠也利于尿酸排出,可以与其他降尿酸药一同使用,或者单用于轻度尿酸升高的患者。3)重组尿酸酶,例如拉布立酶和普瑞凯希(又称为培戈洛酶);尿酸酶可以将尿酸降解为水溶性的尿囊素,更易经肾脏排出。也有研究发现,痛风患者肠道菌群的变化可通过免疫调节作用在痛风的发生、发展中发挥重要作用。
痛风患者由于长期服用药物,容易对肝脏造成损伤,所以容易出现一些肝脏疾病,例如高血压、高血脂等。除了痛风性关节炎之外,关节畸形、尿酸性肾病、2型糖尿病、心血管疾病和脂质代谢紊乱也是痛风较为重要的危害,给个人、家庭、社会都带来了不小的经济负担。
本发明针对褐藻寡糖进行了进一步研究,并提供一种褐藻寡糖的用途。
发明内容
因此,本发明的目的是提供一种褐藻寡糖的用途。
本发明的目的是通过以下技术方案来实现的:
一方面,本发明提供一种褐藻寡糖或其药学上可接受的盐在制备用于治疗痛风的药物中的用途,其中所述褐藻寡糖为褐藻二糖、褐藻三糖和/或褐藻四糖。
在本发明的某些实施方案中,所述褐藻寡糖是由单糖G、M和/或Δ通过1,4位糖苷键连接构成的;其中,G表示α-L-古洛糖醛酸,M表示β-D-甘露糖醛酸,Δ表示α-L-古洛糖醛酸或β-D-甘露糖醛酸的4,5位发生β-消除,生成4,5位为共轭双键的不饱和单糖。
在本发明的某些实施方案中,所述褐藻二糖选自ΔG、ΔM或其组合。
在本发明的某些实施方案中,所述褐藻三糖选自ΔGG、ΔGM、ΔMM和ΔMG中的一种或多种。
在本发明的某些实施方案中,所述褐藻四糖选自ΔGGG、ΔGGM、ΔGMG、ΔGMM、ΔMMG、ΔMMM、ΔMGG和ΔMGM中的一种或多种。
在本发明的某些实施方案中,所述药学上可接受的盐为钠盐、钾盐、钙盐、镁盐和/或铵盐。
另一方面,本发明提供一种褐藻寡糖或其药学上可接受的盐,其用于治疗痛风,其中所述褐藻寡糖为褐藻二糖、褐藻三糖和/或褐藻四糖。
又一方面,本发明提供一种治疗痛风的方法,包括给予需要的患者治疗有效量的褐藻寡糖或其药学上可接受的盐,其中所述褐藻寡糖为褐藻二糖、褐藻三糖和/或褐藻四糖。
本发明的均一聚合度的褐藻二糖、三糖和四糖,对于糖类原料药的质量控制、药理、毒理等分析研究,均具有革命性进步。
研究发现,在急性痛风性关节炎(AGA)小鼠模型上,本发明的褐藻二糖、三糖和四糖可明显改善小鼠关节肿胀与机械痛觉过敏症状。小鼠的步态行为学研究也表明,本发明的褐藻二糖、三糖和四糖可显著改善小鼠的步态行为学异常。因此,本发明的褐藻二糖、三糖和四糖具有较强的治疗急性痛风的功效。本发明所提供的褐藻寡糖类药物,来源于海洋藻类,无任何毒副作用,可长期使用。
附图说明
以下,结合附图来详细说明本发明的实施方案,其中:
图1示出了褐藻二糖在230nm波长下的高效液相色谱图;
图2示出了褐藻二糖的核磁氢谱图( 1HNMR,溶剂D 2O);
图3示出了褐藻二糖的高分辨质谱图(HRMS(ESI));
图4示出了褐藻三糖在230nm波长下的高效液相色谱图;
图5示出了褐藻三糖的核磁氢谱图( 1HNMR,溶剂为D 2O);
图6示出了褐藻三糖的高分辨质谱图(HRMS(ESI));
图7示出了褐藻四糖在230nm波长下的高效液相色谱图;
图8示出了褐藻四糖的核磁氢谱图( 1HNMR,溶剂为D 2O);
图9示出了褐藻四糖的高分辨质谱图(HRMS(ESI));
图10示出了急性痛风性关节炎小鼠模型的建立及评价;
图11示出了褐藻二糖抑制AGA模型小鼠关节疼痛症状的效果图;
图12示出了褐藻三糖抑制AGA模型小鼠关节疼痛症状的效果图;
图13示出了褐藻四糖抑制AGA模型小鼠关节疼痛症状的效果图;
图14示出了聚合度为2-8的混合褐藻寡糖的质谱图;
图15示出了褐藻三糖、混合糖和吲哚美辛抑制AGA模型小鼠关节疼痛效果的研究;
图16示出了褐藻三糖对AGA模型小鼠步态行为学的影响。
具体实施方式
下面结合实施例对本发明做进一步的说明,实施例仅为解释和说明性的,绝不意味着以任何方式限制本发明的范围。
实施例1 均一聚合度褐藻二糖的制备及其结构鉴定
将100g采购的褐藻胶(购自青岛明月海藻集团有限公司)溶于水,在一定温度下,加入褐藻糖裂解酶(获得自中国海洋大学),经过一定时间的裂解后,高速离心机离心,取上层清液。将清液再以凝胶柱纯化,去除少量的杂质寡糖和多糖及非糖类杂质,得到褐藻二糖钠盐60g。将所得褐藻二糖钠盐用高效液相色谱法(HPLC,230nm)进行纯度检测,并用核磁氢谱( 1HNMR)和高分辨质谱(HRMS-ESI)进行结构鉴定。
HPLC:纯度99.06%,RT=13.6min(相关谱图见图1);
1HNMR谱图见图2;
HRMS(ESI)m/z:C 12H 15O 12{(M-H) -},计算值为351.0569,实测值为351.0572(M-H) -(相关谱图见图3);
褐藻二糖钠盐,如果分子中两个羧基均为钠盐,则钠离子理论含量为11.58%;实际离子色谱法检测,钠离子含量为10.3%。如用炽灼残渣法检测,则钠离子以硫酸钠的形式存在,理论残渣比例应该为35.77%;实际炽灼残渣检测,残渣为34.3%;两种检测方法所得结果均比较接近,说明化合物羧酸官能团确为钠盐形式。但实测值均略小于理论值,可能是因为其钠盐为弱酸强碱盐,有少部分羧酸仍呈游离状态。
实施例2 均一聚合度褐藻三糖的制备及其结构鉴定
将100g采购的褐藻胶溶于水,在一定温度下,加入褐藻糖裂解酶(获得自中国海洋大学),经过一定时间的裂解后,高速离心机离心,取上层清液。将清液再以凝胶柱纯化,去除少量的杂质寡糖和多糖及非糖类杂质,得到褐藻三糖钠盐70g。将所得褐藻三糖钠盐用高效液相色谱法(HPLC,230nm)进行纯度检测,并用核磁氢谱( 1HNMR)和高分辨质谱(HRMS-ESI)进行结构鉴定。
HPLC:纯度100%,RT=17.43min(相关谱图见图4);
1HNMR谱图见图5;
HRMS(ESI)m/z:C 18H 23O 18{(M-H) -},计算值为527.0890,实测值为527.0891(M-H) -(相关谱图见图6);
褐藻三糖钠盐,如果分子中三个羧基均为钠盐,则钠离子理论含量为 11.59%;实际离子色谱法检测,钠离子含量为9.9%。如用炽灼残渣法检测,则钠离子以硫酸钠的形式存在,理论残渣比例应该为35.80%;实测炽灼残渣为33.01%。两种检测方式,所得结果均比较接近,说明化合物羧酸官能团确为钠盐形式。但实测值均略小于理论值,可能是因为其钠盐为弱酸强碱盐,有少部分羧酸仍呈游离状态。
实施例3 均一聚合度褐藻四糖的制备及其结构鉴定
将100g采购的褐藻胶溶于水,在一定温度下,加入褐藻糖裂解酶(获得自中国海洋大学),经过一定时间的裂解后,高速离心机离心,取上层清液。将清液再以凝胶柱纯化,去除少量的杂质寡糖和多糖及非糖类杂质,得到褐藻四糖钠盐55g。将所得褐藻四糖钠盐用高效液相色谱法(HPLC,230nm)进行纯度检测,并用核磁氢谱( 1HNMR)和高分辨质谱(HRMS-ESI)进行结构鉴定。
HPLC:纯度99.71%,RT=18.71min(相关谱图见图7);
1HNMR谱图见图8;
HRMS(ESI)m/z:C 24H 31O 24{(M-H) -},计算值为703.1211,实测值为703.1207(M-H) -(相关谱图见图9);
褐藻四糖钠盐,如果分子中四个羧基均为钠盐,则钠离子理论含量为11.59%;实际离子色谱法检测,钠离子含量为9.8%。如用炽灼残渣法检测,则钠离子以硫酸钠的形式存在,理论残渣比例应该为35.80%;实测炽灼残渣,残渣为32.5%。两种检测方式,所得结果均比较接近,说明化合物羧酸官能团确为钠盐形式。但实测值均略小于理论值,可能是因为其钠盐为弱酸强碱盐,有少部分羧酸仍呈游离状态。
实施例4 急性痛风性关节炎(AGA)模型小鼠的建立
将健康清洁级雄性C57BL/6小鼠分为对照组(Veh)、模型组(MSU),每组6只。经医用酒精消毒后,模型组在小鼠右踝关节后侧沿跟腱内侧以30-40°方向刺入踝关节腔,注射1mg/20μl尿酸钠(MSU)溶液,对照组采用同样方式注射等量磷酸盐缓冲液(PBS)。注射完毕后,观察小鼠踝关节肿胀程度以及机械痛阈值变化情况。踝关节肿胀测定:使用游标卡尺,分别于造模前,造模后2h、6h、24h和48h,四个时间点检测小鼠患侧踝关节直径,连续测取三次,取其均值作为最终读数。踝关节肿胀=当前测量直径-模前测量直径。机械痛行为测定:将小鼠放置于高架铁丝网上的透 明塑料盒中,并盖上透明有机玻璃,适应环境45min。随后按照“Up and Down”方法,在模前,模后2h、6h和24h和48h,四个时间点,采用不同规格von Frey丝检测小鼠右侧后爪近踝部的疼痛阈值。利用公式,计算得到缩爪阈值。
图10示出了急性痛风性关节炎小鼠模型的建立及效果评价。图10A为AGA模型组小鼠右踝关节注射尿酸盐(MSU)结晶后肿胀情况图。AGA模型组小鼠右踝关节注射尿酸盐(MSU)结晶24h后,与注射溶剂磷酸盐缓冲液(PBS)的对照组小鼠踝关节相比,出现明显肿胀。图10B为对照组和模型组小鼠踝关节H&E染色后,切片进行比对。可以观察到,相比对照组,AGA模型组小鼠踝关节切片中,出现大量炎性细胞浸润。图10C为右踝关节注射尿酸盐结晶后的小鼠与踝关节注射PBS的小鼠相比,踝关节直径的变化情况。可以看出,对照组小鼠踝关节在注射完PBS后几小时内仅有轻微肿胀,但迅速消退,恢复正常后基本未再变化。而注射尿酸盐结晶的小鼠踝关节则迅速肿大,直至48h后,仍有明显肿胀现象。图10D表示两组小鼠右后爪机械痛过敏现象比较,结果显示注射MSU后的小鼠,其机械痛阈值显著降低。上述结果与以往报道一致,因此提示AGA小鼠模型建立成功。
实施例5 褐藻二糖(AOS2)对AGA模型小鼠镇痛效应的研究
在本发明中,褐藻二糖简称为“AOS2”。如实施例4所述制备AGA模型小鼠。将小鼠随机分为5组:对照组(Veh+Veh)、模型组(MSU+Veh)、MSU+AOS2各剂量组(400mg/kg,200mg/kg,100mg/kg)。MSU+AOS2各剂量组腹腔注射不同浓度AOS2,分别于模前1h、模后5h、23h和47h给药;对照组与模型组腹腔注射同等体积对照溶剂PBS(图11A)。于造模前与模后2、6、24和48h进行机械痛与肿胀程度检测。具体检测方法与实施例4一致。
图11示出了褐藻二糖AOS2抑制AGA模型小鼠关节疼痛症状的效果。图11A为给药时间点示意图。图11B为不同剂量AOS2(100mg/kg,200mg/kg,400mg/kg,i.p.)对AGA模型小鼠踝关节机械痛作用的时程图。给予不同剂量AOS2后,200及400mg/kg AOS2可不同程度缓解AGA模型小鼠机械痛。图11C为11B曲线下面积图统计图。图11D显示的是不同剂量AOS2(100mg/kg,200mg/kg,400mg/kg,i.p.)对AGA模型小鼠踝关节肿胀作用的时程图。给予不同剂量AOS2后,踝关节肿胀明显降 低,且剂量越大,肿胀越小。图E为图D曲线下面积统计图。上述实验结果说明,AOS2可有效抑制AGA模型小鼠踝关节疼痛与肿胀,且该作用呈现一定剂量依赖性。
实施例6 褐藻三糖(AOS3)对AGA模型小鼠镇痛效应的研究
如实施例4所述制备AGA模型小鼠。在本发明中,褐藻三糖简称为“AOS3”。将小鼠随机分为5组:对照组(Veh+Veh)、模型组(MSU+Veh)、MSU+AOS3各剂量组(400mg/kg,200mg/kg,100mg/kg)。MSU+AOS3各剂量组腹腔注射不同浓度AOS3,分别于模前1h、模后5、23和47h给药;对照组与模型组腹腔注射同等体积对照溶剂PBS(图12A)。于造模前与模后2、6、24和48h进行机械痛与肿胀程度检测。具体检测方法与实施例4一致。
图12示出了褐藻三糖AOS3抑制AGA模型小鼠关节疼痛症状的效果。图12A为给药时间点示意图。图12B为不同剂量AOS3(100mg/kg,200mg/kg,400mg/kg,i.p.)对AGA模型小鼠踝关节机械痛作用的时程图。图12C为12B曲线下面积统计图。如图12B和12C所示,给予不同剂量AOS3后,200mg/kg及400mg/kg AOS3明显缓解AGA模型小鼠机械痛,100mg/kg剂量的AOS3对机械痛也有部分缓解作用,可见明显的剂量依赖关系。图12D为不同剂量AOS3(100mg/kg,200mg/kg,400mg/kg,i.p.)对AGA模型小鼠踝关节肿胀作用的时程图。图12E为图12D曲线下面积统计图。图12D和12E显示小鼠踝关节肿胀情况随时间变化情况。给予不同剂量AOS3后,踝关节肿胀明显降低,且剂量越大,肿胀越小。上述实验结果说明,AOS3可有效抑制AGA模型小鼠踝关节疼痛与肿胀,且该作用呈现一定剂量依赖性。
实施例7 褐藻四糖(AOS4)对AGA模型小鼠镇痛效应的研究
如实施例4所述制备AGA模型小鼠。在本发明中,褐藻四糖简称为“AOS4”。将小鼠随机分为5组:对照组(Veh+Veh)、模型组(MSU+Veh)、MSU+AOS4各剂量组(400mg/kg,200mg/kg,100mg/kg)。MSU+AOS4各剂量组腹腔注射不同浓度AOS4,分别于模前1h、模后5、23和47h给药;对照组与模型组腹腔注射同等体积对照溶剂PBS(图13A)。于造模前与模后2、6、24和48h进行机械痛与肿胀程度检测。具体检测方法与实施例4一致。
图13示出了褐藻四糖AOS4抑制AGA模型小鼠关节疼痛症状的效果。图13A为给药时间点示意图。图13B为不同剂量AOS4(100mg/kg,200mg/kg,400mg/kg,i.p.)对AGA模型小鼠踝关节机械痛作用的时程图。图13C为图13B曲线下面积统计图。如图13B和13C所示,AGA模型小鼠给予不同剂量AOS4后,各剂量水平均可不同程度缓解AGA模型小鼠机械痛,200mg/kg及400mg/kg效果尤其明显。图13D为不同剂量AOS4(100mg/kg,200mg/kg,400mg/kg,i.p.)对AGA模型小鼠踝关节肿胀作用的时程图。图13E为图13D曲线下面积统计图。图13D和13E显示小鼠踝关节肿胀情况随时间变化情况。给予不同剂量AOS4后,踝关节肿胀明显降低,且剂量越大,肿胀越小。上述实验结果说明,AOS4可有效抑制AGA模型小鼠踝关节疼痛与肿胀,且该作用呈现一定剂量依赖性。
由于AOS2、AOS3和AOS4在200mg/kg的剂量时,均显示出较好的对AGA小鼠模型镇痛的活性。因此在接下来的实验中,发明人选用浓度为200mg/kg的AOS3进行进一步体内药理学活性研究。
实施例8 AOS3与混合糖和对比药物的AGA模型小鼠镇痛效应的研究
如实施例4所述制备AGA模型小鼠。将AOS3与混合糖(聚合度为2-8的混合褐藻寡糖,其质谱图参见图14,获得自中国海洋大学,本发明中简称为“AOS混”)和吲哚美辛(本发明中简称为“Indo”)进行实验对比。将小鼠随机分为6组:对照组(Veh+Veh)、模型组(MSU+Veh)、MSU+AOS3组(200mg/kg)、MSU+AOS3组(100mg/kg)、MSU+AOS混组(200mg/kg)、MSU+吲哚美辛组(MSU+Indo,10mg/kg)。给药各剂量组腹腔注射规定量的药品,分别于模前1h、模后5、23和47h给药;对照组与模型组腹腔注射同等体积对照溶剂PBS(图15A)。于造模前与模后2、6、24和48h进行机械痛与肿胀程度检测。具体检测方法与实施例4一致。
图15示出了AOS3、混合糖和对比药物吲哚美辛抑制AGA模型小鼠关节疼痛症状的效果。图15A为给药时间点示意图。图15B为AOS3(100mg/kg,200mg/kg,i.p.)、混合糖(200mg/kg,i.p.)和吲哚美辛(10mg/kg,i.p.,小鼠体内的有效剂量)对AGA模型小鼠踝关节机械痛作用的时程图。图15C为图15B曲线下面积统计图。如图15B和15C所示,AGA模型小鼠给药后,均可不同程度缓解AGA模型小鼠机械痛,200mg/kg的AOS3 效果最明显,好于100mg/kg的AOS3和对比药物吲哚美辛(10mg/kg),200mg/kg的混合糖效果最差。图15D为不同药品对AGA模型小鼠踝关节肿胀作用的时程图。图15E为图15D曲线下面积统计图。图15D和15E显示小鼠踝关节肿胀情况随时间变化情况。给药后,踝关节肿胀情况均有所改善,尤其AOS3(200mg/kg)组肿胀明显降低;AOS3剂量减小(100mg/kg)后,肿胀改善效果变差,与吲哚美辛效果接近;混合糖效果最差。上述实验结果说明,AOS3可有效抑制AGA模型小鼠踝关节疼痛与肿胀,该作用呈现一定的剂量依赖性,并且其效果好于混合糖和吲哚美辛。
实施例9 AOS3对AGA小鼠步态行为学异常的改善作用研究
如实施例4所述制备AGA小鼠模型。将小鼠随机分为3组:对照组(Veh+Veh)、模型组+溶剂组(MSU+Veh)、模型组+AOS3组(MSU+AOS3)。MSU+AOS3组腹腔注射200mg/kg剂量的AOS3,分别于模前1h、模后5h和23h给药,对照组与模型组+溶剂组则腹腔注射同等体积对照溶剂PBS。如图16A所示,首先将实验小鼠进行一段时间的测试环境预适应。于模后8h、24h两个时间点进行步态行为学测试,即在10-15秒内各组小鼠匀速不停顿通过跑道,下方摄像机实时采集步态影像,随后使用DigiGait(Mouse Specifics,Inc.,USA)软件对小鼠后爪进行足印面积和摆动持续时间的步态行为学参数进行分析。
图16示出了褐藻三糖AOS3对AGA模型小鼠步态行为学的影响。图16A为Catwalk小鼠步态仪记录和分析示意图。图16B为各组小鼠5秒内通过跑道后足爪印面积的代表图。足爪印迹显示,模型组+溶剂组小鼠在模后24h右后爪面积相比健侧足爪显著减少,出现明显跛行样步态。而模型组+AOS3组小鼠患侧右后爪着地面积出现改善。图16C为各组小鼠于造模后8和24h患侧足爪面积同健侧足爪面积的相对百分比统计图。模型组+溶剂组小鼠患侧足爪面积相比于健侧出现显著降低,而模型组+AOS3组小鼠患侧足爪面积出现显著改善。图16D为各组小鼠于造模后8和24h患侧足爪在空中摆动时间同健侧足爪摆动时间的相对百分比统计图。数据显示,在模后24h,模型组+溶剂组小鼠患侧足爪空中摆动时间显著延长,而模型组+AOS3组小鼠患肢空中摆动时间相比模型组+溶剂组显著减少。上述结果提示,AGA模型小鼠在步态行为学中出现患侧足爪着地面积减少以及足爪空中摆动时间延长等跛行样行为。这与痛风患者跛行样行为一致。而AOS3可有效改善AGA模型小鼠步态行为学的异常改变。
实施例10 大鼠单次AOS3灌胃急性经口毒性试验
取SD大鼠雌雄各40只,体重160-180g左右,随机分成4组,分别为阴性对照组(生理盐水)、褐藻三糖AOS3低剂量组(0.25g/kg体重)、中剂量组(1.0g/kg体重)和高剂量组(2.0g/kg体重),剂量分别相当于大鼠药效学起效剂量的25倍、100倍、200倍,每组雌雄各10只,实验前隔夜禁食,不禁水。连续14天观察动物的状况,各组动物无明显差异,精神状态良好,呼吸正常,行为正常,活动量正常,行走步态未观察到任何异常。未出现中毒症状和死亡情况。各组大鼠的初始体重与终末体重无明显差异。肝脏、胰腺、肾脏、胃、卵巢、脑部的病理切片显示各器官无病变产生。血液检查指标正常。以上结果说明,AOS3为实际无毒级。
实验结论:AOS2、AOS3和AOS4可剂量依赖性缓解急性痛风性关节炎(AGA)模型小鼠关节肿胀与机械痛觉过敏症状;AOS3可显著改善AGA模型小鼠所表现的步态行为学异常,200mg/kg剂量下的AOS3镇痛效果优于吲哚美辛(10mg/kg剂量),也优于同样剂量下的混合糖。
最后需要在此指出的是:以上仅是本发明的部分优选实施例,不能理解为对本发明保护范围的限制,本领域的技术人员根据本发明的上述内容做出的一些非本质的改进和调整均属于本发明的保护范围。

Claims (8)

  1. 一种褐藻寡糖或其药学上可接受的盐在制备用于治疗痛风的药物中的用途,其中所述褐藻寡糖为褐藻二糖、褐藻三糖和/或褐藻四糖。
  2. 根据权利要求1所述的用途,其中所述褐藻寡糖是由单糖G、M和/或Δ通过1,4位糖苷键连接构成的;其中,G表示α-L-古洛糖醛酸,M表示β-D-甘露糖醛酸,Δ表示α-L-古洛糖醛酸或β-D-甘露糖醛酸的4,5位发生β-消除,生成4,5位为共轭双键的不饱和单糖。
  3. 根据权利要求2所述的用途,其中所述褐藻二糖选自ΔG、ΔM或其组合。
  4. 根据权利要求2所述的用途,其中所述褐藻三糖选自ΔGG、ΔGM、ΔMM和ΔMG中的一种或多种。
  5. 根据权利要求2所述的用途,其中所述褐藻四糖选自ΔGGG、ΔGGM、ΔGMG、ΔGMM、ΔMMG、ΔMMM、ΔMGG和ΔMGM中的一种或多种。
  6. 根据权利要求1所述的用途,其中所述药学上可接受的盐为钠盐、钾盐、钙盐、镁盐和/或铵盐。
  7. 一种褐藻寡糖或其药学上可接受的盐,其用于治疗痛风,其中所述褐藻寡糖为褐藻二糖、褐藻三糖和/或褐藻四糖。
  8. 一种治疗痛风的方法,包括给予需要的患者治疗有效量的褐藻寡糖或其药学上可接受的盐,其中所述褐藻寡糖为褐藻二糖、褐藻三糖和/或褐藻四糖。
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