Medicine for treating neurodegenerative diseases

Abstract

The invention belongs to the field of medicine application, and particularly relates to a medicine for treating neurodegenerative diseases. The invention particularly provides a pharmaceutical composition for preventing and / or treating neurodegenerative diseases and application of the pharmaceutical composition. The pharmaceutical composition comprises rivastigmine, liraglutide and derivatives of rivastigmine and liraglutide. The pharmaceutical composition further comprises a sustained-release system, the sustained-release system can interact with rivastigmine, liraglutide and derivatives thereof to form a drug sustained-release system, and the sustained-release system is a triblock hydrogel sustained-release system. The combination of liraglutide and rivastigmine can improve learning and memory impairment related to Alzheimer's disease, the effect of the composition is superior to that of single medication, and the composition has a synergistic effect.

Description

Technical Field

[0001] The invention belongs to the field of drug application, and in particular, relates to a drug for treating neurodegenerative diseases. Background Art

[0002] Neurodegenerative diseases, also known as neurodegenerative disorders, are disease states in which there is a loss of cells, neurons, in the brain and spinal cord. The brain and spinal cord are made up of neurons, which have different functions, such as controlling movement, processing sensory information, and making decisions. Cells in the brain and spinal cord generally do not regenerate, so excessive damage can be devastating and irreversible. Neurodegenerative diseases are caused by the loss of neurons or their myelin sheaths, which worsen over time to cause functional impairment. Neurodegenerative diseases include Alzheimer's disease, amyotrophic lateral sclerosis, ataxia-telangiectasia, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, Huntington's disease, cerebellar atrophy, multiple sclerosis, Parkinson's disease, primary lateral sclerosis, spinal muscular atrophy, cerebral ischemia, spastic paraplegia, myasthenia gravis, etc.

[0003] Alzheimer's disease (AD) is a neurodegenerative disease with unknown etiology. Its clinical manifestations are mainly persistent and progressive cognitive impairment and the accompanying social life function impairment, personality and behavioral changes, and mental abnormalities. Cognitive impairment is mainly manifested as memory impairment, aphasia, apraxia, agnosia, and executive dysfunction, while mental abnormalities are mainly manifested as agitation, delusion, hallucination, depression, and apathy. The main pathological changes include cerebral cortical atrophy, widening of sulci, enlargement of ventricles, a large number of neurons reduced, β-amyloid protein aggregation to form senile plaques, Tau protein hyperphosphorylation to form neurofibrillary tangles, and a significant decrease in choline acetylase and acetylcholine content.

[0004] The main patients with Alzheimer's disease are elderly people over 65 years old. The decline of patients' living functions and changes in personality and behavior have brought a very heavy burden to society and patients' families. Since the discovery of Alzheimer's disease in 1906, scientists have conducted a lot of related research. Especially in the past 10 years, the development of β-amyloid protein inhibitors has been very popular. Unfortunately, the cause of Alzheimer's disease is still unclear, and the failure of many β-amyloid protein inhibitors has cast a shadow on the development of subsequent drugs. The number of drugs currently approved for the treatment of Alzheimer's disease is small and the therapeutic effect is limited. The existing drugs on the market mainly include cholinesterase inhibitors, N-methyl-D-aspartate receptor antagonists, brain metabolism promoters, and intestinal flora regulators; the drugs under development mainly include β-amyloid protein inhibitors, Tau protein aggregation inhibitors, etc. Some of these drugs are symptomatic treatments, while others can only improve a few pathological indicators. Few of them can really improve cognitive dysfunction caused by Alzheimer's disease, and there are many adverse reactions.

[0005] Rivastigmine Tartrate (Riv), chemical name (S)-N-ethyl-N-methyl-3-[1-(dimethylamino)-ethyl]phenyl carbamate-2R,3R-tartrate], is a second-generation cholinesterase inhibitor and can be used to treat mild and moderate Alzheimer's dementia. It is highly selective in the hippocampus and cortical areas of the brain, and increases the function of cholinergic neurons in the brain by inhibiting acetylcholinesterase, thereby improving the cognitive effects of Alzheimer's patients. In addition, it can also slow down the formation of amyloid β-amyloid precursor protein fragments and slow down the deterioration of Alzheimer's disease. Rivastigmine exists in liquid form at room temperature and cannot be used as a solid preparation; when used as a transdermal agent in clinical practice, it has the problem of easy penetration but short duration, and cannot be administered stably for a long time, while rivastigmine salt has the disadvantage of poor penetration efficiency. CN117045646A discloses the use of a co-amorphous compound of rivastigmine and valsartan as an effective pharmaceutical ingredient in the preparation of a drug for preventing or treating cardiovascular diseases or neurodegenerative diseases or complications of the above diseases.

[0006] Liraglutide (LRT) is a glucagon GLP-1 (7-37) analog developed by Novo Nordisk of Denmark. As a new drug for the treatment of type 2 diabetes, it was approved by the FDA for marketing in the United States on January 25, 2010. Liraglutide has 97% homology with the natural GLP-1 in the human body. Its structure has only been modified in two parts: lysine at position 34 is replaced by arginine, and a 16-carbon palmitic acid side chain mediated by glutamic acid is added to lysine at position 26. This structural modification not only allows liraglutide to retain the biological activity of GLP-1, but also avoids degradation by the DPP-4 enzyme. Compared with traditional insulin injections, liraglutide has a long-lasting effect. Liraglutide has the following pharmacological effects: (1) glucose concentration-dependent insulin secretion; (2) inhibiting postprandial glucagon secretion and reducing glycogen release; (3) enhancing insulin sensitivity; (4) slowing gastric emptying; (5) suppressing appetite and reducing weight. (6) repairing pancreatic β cells; (7) protecting cardiovascular system. Liraglutide's insulin secretion and glucagon secretion inhibition effects are dependent on blood glucose concentration. Therefore, when blood glucose levels are normal, continuous administration of liraglutide will not cause hypoglycemia. It can be seen that liraglutide can not only maintain normal blood glucose levels in patients, but also repair pancreatic β cells to a certain extent. It also has a certain effect on diabetic complications and is currently an advanced diabetes treatment drug. Studies have shown that type 2 diabetes (T2DM) is considered a risk factor for AD and PD (Holscher, 2014), indicating that damage to insulin signaling can be a factor in initiating or accelerating the development of AD. Patent CN110312520B discloses a GIP / GLP-1 co-agonist peptide for the treatment of Alzheimer's disease and Parkinson's disease. In Alzheimer's disease, small-scale trials have demonstrated that liraglutide can reduce inflammation, inhibit apoptosis, prevent toxic protein aggregation, enhance long-term potentiation and autophagy, and restore dysfunctional insulin signaling, thereby improving brain glucose metabolism and functional connectivity. Summary of the invention

[0007] The present invention has developed a PLGA-PEG-PLGA triblock copolymer-encapsulated LRT sustained-release system, which can stably maintain drug release. Further research using this system found that the combination of liraglutide and rivastigmine has a synergistic effect on improving Alzheimer's disease-related learning and memory impairment. Based on this, the present invention is completed.

[0008] In a first aspect, the present invention provides a pharmaceutical composition for preventing and / or treating neurodegenerative diseases, wherein the pharmaceutical composition comprises rivastigmine and liraglutide and derivatives thereof.

[0009] Furthermore, the pharmaceutical composition also includes a sustained-release system, which can interact with rivastigmine, liraglutide and their derivatives to form a drug sustained-release system, and the sustained-release system is a triblock hydrogel sustained-release system.

[0010] In one embodiment of the present invention, the sustained-release system is a PLGA-PEG-PLGA triblock hydrogel system.

[0011] Preferably, the rivastigmine is rivastigmine bitartrate.

[0012] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0013] Furthermore, the neurodegenerative disease is selected from one or more of Alzheimer's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, Huntington's disease, cerebellar atrophy, multiple sclerosis, Parkinson's disease, primary lateral sclerosis, spinal muscular atrophy, cerebral ischemia, spastic paraplegia and / or myasthenia gravis.

[0014] In a second aspect, the present invention provides a preparation for preventing and / or treating neurodegenerative diseases, wherein the preparation comprises the pharmaceutical composition described in the first aspect, and a pharmaceutically acceptable excipient.

[0015] Furthermore, the dosage form of the preparation includes but is not limited to solution, injection, infusion, oral solution, tablet, capsule, powder, granule, paste or gel.

[0016] Furthermore, the preparation can be taken orally, injected, or the like to prevent and / or treat neurodegenerative diseases.

[0017] Furthermore, the neurodegenerative disease is selected from one or more of Alzheimer's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, Huntington's disease, cerebellar atrophy, multiple sclerosis, Parkinson's disease, primary lateral sclerosis, spinal muscular atrophy, cerebral ischemia, spastic paraplegia and / or myasthenia gravis.

[0018] In a third aspect, the present invention provides a pharmaceutical composition, comprising the pharmaceutical composition described in the first aspect or the preparation described in the second aspect and other active ingredients, and the pharmaceutical composition has the effect of preventing and / or treating neurodegenerative diseases.

[0019] Furthermore, the other active ingredients are any one that can alleviate symptoms caused by or associated with neurodegenerative diseases, but are different from rivastigmine and liraglutide and their derivatives.

[0020] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the pharmaceutical composition.

[0021] Furthermore, the pharmaceutical composition can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

[0022] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0023] Furthermore, the drug can be administered via injection, cavity administration, respiratory tract administration or mucosal administration.

[0024] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; and the respiratory tract administration includes oral and nasal administration.

[0025] Furthermore, the neurodegenerative disease is selected from one or more of Alzheimer's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, Huntington's disease, cerebellar atrophy, multiple sclerosis, Parkinson's disease, primary lateral sclerosis, spinal muscular atrophy, cerebral ischemia, spastic paraplegia and / or myasthenia gravis.

[0026] In a fourth aspect, the present invention provides a use of a pharmaceutical composition in the preparation of a preparation for preventing and / or treating neurodegenerative diseases.

[0027] Furthermore, the pharmaceutical composition comprises a therapeutically effective amount of rivastigmine and liraglutide and derivatives thereof.

[0028] Furthermore, the pharmaceutical composition also comprises a sustained-release system, which is a copolymer that can interact with rivastigmine, liraglutide and their derivatives to form a sustained-release drug.

[0029] Preferably, the rivastigmine is rivastigmine bitartrate.

[0030] Furthermore, the preparation also contains pharmaceutically acceptable excipients.

[0031] Furthermore, the dosage form of the preparation includes but is not limited to solution, injection, infusion, oral solution, tablet, capsule, powder, granule, paste or gel.

[0032] Furthermore, the preparation can be taken orally, injected, etc. to prevent and / or treat neurodegenerative diseases.

[0033] Furthermore, the neurodegenerative disease is selected from one or more of Alzheimer's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, Huntington's disease, cerebellar atrophy, multiple sclerosis, Parkinson's disease, primary lateral sclerosis, spinal muscular atrophy, cerebral ischemia, spastic paraplegia and / or myasthenia gravis.

[0034] In a fifth aspect, the present invention provides a sustained-release preparation for treating neurodegenerative diseases, wherein the sustained-release preparation contains PLGA-PEG-PLGA triblock hydrogel, rivastigmine and liraglutide and derivatives thereof.

[0035] Furthermore, the rivastigmine is rivastigmine bitartrate.

[0036] Furthermore, the neurodegenerative disease is selected from one or more of Alzheimer's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, Huntington's disease, cerebellar atrophy, multiple sclerosis, Parkinson's disease, primary lateral sclerosis, spinal muscular atrophy, cerebral ischemia, spastic paraplegia and / or myasthenia gravis.

[0037] Beneficial Effects

[0038] The PLGA-PEG-PLGA triblock copolymer prepared by the present invention can stably maintain drug release for up to 2 weeks; the combination of liraglutide and rivastigmine in the present invention can improve the learning and memory impairment associated with Alzheimer's disease better than the use of either drug alone, and has a synergistic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The PLGA-PEG-PLGA triblock copolymers were characterized by H NMR and Gel Permeation Chromatography.

[0040] Figure 2 Circular dichroism detection of the interaction between LRT and PLGA-PEG-PLGA triblock copolymer. (A. Structural formula of PLGA-PEG-PLGA triblock copolymer and LRT; B. Circular dichroism detection of the structural changes of LRT and polymer interaction.)

[0041] Figure 3 Co-assembly of LRT and PLGA-PEG-PLGA triblock copolymer. A. Changes in critical micelle concentration after LRT is mixed into polymer; B. Variable temperature dynamic light scattering detection of phase change after LRT is mixed into polymer; C. Rheology-modulus measurement after LRT is mixed into polymer; D. Changes in gelation temperature window after LRT is mixed into polymer.

[0042] Figure 4The solubilization effect of Riv micelles. Statistical analysis of the effect of different drug-loaded gel preparation methods on Riv drug concentration.

[0043] Figure 5 The in vivo and in vitro release experiments of LRT / Riv@gel. A. In vitro drug release pattern of LRT / Riv@gel drug-loaded hydrogels LRT and Riv; B. In vivo release experiment of LRT@Gel subcutaneously injected into mice; C. Time series of blood drug concentration after subcutaneous injection of gradient-dose LRT@Gel mice; D. Statistics of blood drug concentration after subcutaneous injection of gradient-dose LRT@Gel mice; E. Skin degradation curve of hydrogel after a single subcutaneous injection of 0.2 ml of LRT / Riv@gel drug-loaded hydrogel.

[0044] Figure 6 LRT*Riv@Gel synergistically improves learning and memory related to Alzheimer's disease. A. Experimental flow chart; B. Statistical analysis of the latency of the Barnes maze learning period of each group of mice; C. Statistical analysis of the percentage of time spent in the target quadrant of the Barnes maze of each group of mice. Compared with NS@Gel, *P<0.05, ***P<0.001; compared with NS@Gel-5*FAD, ##P<0.01, ###P<0.001; compared with LRT@Gel-5*FAD, $P<0.05; compared with Riv@Gel-5*FAD, &P<0.05. DETAILED DESCRIPTION

[0045] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0046] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0047] As described herein, "Rivastigmine" (Riv) is an amino acid brain selective acetylcholinesterase inhibitor that promotes cholinergic nerve conduction by delaying the degradation of released acetylcholine by fully functional cholinergic neurons. The recommended dose of rivastigmine is 3 mg / day (1.5 mg twice daily). Depending on individual differences, the dose should be increased at least every 2 weeks to reach the maximum tolerable dose, but it should not exceed 12 mg per day. The target dose value for most patients should be set within the range of 6-12 mg per day.

[0048] Pharmacokinetics of rivastigmine: Rivastigmine is completely and rapidly absorbed, reaching peak plasma concentrations in about 1 hour after oral administration. Rivastigmine is rapidly and extensively metabolized (plasma half-life of about 1 hour) primarily through cholinesterase-mediated hydrolysis, and is excreted primarily through the kidneys as metabolites, with only <1% of the drug excreted in the feces.

[0049] Pharmacodynamics of rivastigmine: The plasma half-life of rivastigmine is about 1 hour, and the acetylcholinesterase inhibition period is about 9 hours. Therefore, it is recommended that rivastigmine should not be continued in patients with asymptomatic overdose within the next 24 hours.

[0050] The term "liraglutide" (Liraglutide injection, LRT) described herein is a synthetic acylated human glucagon-like peptide-1 (GLP-1) analog. The recommended dose of liraglutide is: the starting dose is 0.6 mg subcutaneous injection per day. After at least 1 week, the dose should be increased to 1.2 mg. It is expected that some patients will benefit from increasing the dose from 1.2 mg to 1.8 mg. Based on the clinical response, in order to further improve the hypoglycemic effect, the dose can be increased to 1.8 mg after at least one week. The recommended daily dose does not exceed 1.8 mg.

[0051] Liraglutide pharmacokinetics: Liraglutide is slowly absorbed after subcutaneous injection, reaching maximum concentration 8-12 hours after administration. Liraglutide is metabolized in a manner similar to large molecular proteins, and no specific organ has been identified as the main elimination pathway, with an elimination half-life of approximately 13 hours.

[0052] Pharmacodynamics of liraglutide: The duration of action of liraglutide is 24 hours, and it can improve blood sugar control by lowering fasting and postprandial blood sugar in patients with type 2 diabetes.

[0053] The term "Barnes maze" as described herein is the main commonly used paradigm for detecting animal spatial memory, which is established by taking advantage of the characteristics of rodents that they avoid light, prefer darkness and love to explore. The reinforcement obtained by the animal is to escape from a bright, open platform to a dark, narrow box located below the platform, which is called a target box. After training, the animal learns and remembers the location of the target box. This model has less stress stimulation on animals and is particularly suitable for stress-related memory research and behavioral phenotype research of gene knockout mice.

[0054] The term "PLGA" described herein is polylactic-co-glycolic acid, which is a copolymer formed by the polymerization reaction of lactic acid and glycolic acid. It is a degradable functional polymer organic compound, non-toxic and has good biocompatibility, capsule-forming and film-forming properties; in the present invention, PLGA and polyethylene glycol (PEG) are polymerized to form a PLGA-PEG-PLGA triblock copolymer.

[0055] The term "storage modulus" in the present invention: namely storage modulus; essentially it is the Young's modulus, which is an index for the rebound of a deformed material, representing the ability of the material to store elastic deformation energy, and is denoted as G' in this text.

[0056] The term "loss modulus" in the present invention describes the phenomenon that energy is dissipated (transformed) into heat when the material deforms, and it is a measure of energy loss, being a damping attenuation term; it is denoted as G'' in this text. When the storage modulus G' > the loss modulus G'', it indicates that this bulk phase is more inclined to the characteristics of an elastic solid, and the characteristics of a viscous fluid are weaker than those of the elastic solid. At this time, the structure of the "X system" can have two explanations: (1) It indicates the gradual formation of a gel, or the gradual formation of the internal structure within the bulk phase; (2) It indicates that the oscillation at this moment does not destroy the bulk phase structure, which to some extent shows whether the strength of this structure has a significant improvement compared with other systems; when the storage modulus G' < the loss modulus G'', this bulk phase is more inclined to a viscoelastic liquid, which means that the colloid or the internal structure of the sample partially collapses or completely collapses, and gradually transforms from a viscoelastic solid to a liquid.

[0057] Example 1: Sustained-release system of LRT encapsulated by PLGA-PEG-PLGA triblock copolymer

[0058] 1. Synthesis of PLGA-PEG-PLGA triblock copolymer

[0059] By using PEG1500 as a macromolecular initiator and Sn(Oct)2 as a catalyst, ring-opening polymerization of LA and GA monomers was carried out to synthesize the triblock copolymer PLGA-PEG-PLGA. During the synthesis process, first, PEG1500 was vacuum-dried at 120 °C for 3 h. When the system cooled to 80 °C, an anhydrous toluene solution of LA and GA monomers (molar ratio 3:1) was added, and at the same time, Sn(Oct)2 (accounting for 0.4% of the total mass of LA and GA) was added. The mixture was kept under vacuum to remove toluene. Then, under an argon atmosphere, the reaction was continuously stirred at a temperature of 150 °C. After the reaction ended, the product was placed in hot water and stirred for washing. Finally, the mixture was freeze-dried for 48 h to obtain the final product.

[0060] 1H NMR (Bruker, AVANCE III, 400 MHz) was used to characterize the number-average molecular weight (Mn) of the synthesized PLGA-PEG-PLGA copolymer and the molar ratio of LA to GA (LA / GA) ( Figure 1 ). Deuterated chloroform (CDCl3) was used as the solvent, and tetramethylsilane (TMS) was used as the internal standard. The molecular weight distribution index of the copolymer was measured using an Agilent 1260 GPC system Tetrahydrofuran (THF) was used as the mobile phase at a flow rate of 1 mL / min, and polystyrene standards were used for calibration.

[0061] 2. Interaction between LRT and PLGA-PEG-PLGA triblock copolymer

[0062] 2.1 Hydrogel configuration

[0063] For PLGA-PEG-PLGA, a micellar structure with hydrophobic PLGA as the core and hydrophilic PEG as the shell will spontaneously form in aqueous solution. The peptide chain of LRT is hydrophilic, and the saturated C-16 chain is hydrophobic. Therefore, when LRT is mixed into the system, it will be co-assembled with the polymer instead of being free in the medium.

[0064] A 25 wt% copolymer / water solution was prepared by mixing PLGA-PEG-PLGA with saline in a mass ratio of 1:3, and then transferred to a sample bottle. The mixture was magnetically stirred for several days. When the system reached uniformity and no bubbles were present, the hydrogel system called T-gel was considered to have been successfully prepared. In order to obtain T-gel loaded with liraglutide, powdered liraglutide was added to the above polymer aqueous solution in a specific mass ratio and stirred until the system was completely clear and uniform, that is, T-gel loaded with liraglutide (LRT@gel) was considered to have been prepared. For the drug-loaded hydrogel of rivastigmine (Riva@gel), a certain mass of rivastigmine and polymer were thoroughly dissolved in acetone. After the solvent was completely removed by rotary evaporation and freeze drying, the mixture was added to saline (polymer mass fraction was also 25 wt%) to prepare the final solution. Subsequently, a hydrogel capable of co-delivering liraglutide and rivastigmine was obtained by dissolving liraglutide powder in Riv@T-gel, named LRT / Riv@gel.

[0065] 2.2 Circular dichroism detection of the interaction between LRT and PLGA-PEG-PLGA triblock copolymer

[0066] Circular dichroism was used to determine the changes in the secondary structure of the peptide before and after the interaction between LRT and the polymer, and further speculate on the possible interaction behavior between LRT and the polymer. That is, the secondary structure of Lira in different solutions (with or without PLGA-PEG-PLGA) was analyzed using circular dichroism spectroscopy. The CD was measured in the wavelength range of 200 to 280nm. Each spectrum was corrected by subtracting the corresponding ultrapure water signal as the baseline.

[0067] like Figure 2The results showed that the CD spectrum of LRT aqueous solution showed two obvious negative peaks at 208nm and 222nm, indicating that LRT exists in a typical α-helix conformation in water. When mixed with a polymer at a concentration of 0.5wt%, the conformation of LRT in aqueous solution changed, as shown by the disappearance of the original negative peak at 208nm and the appearance of a larger negative peak at 225nm, indicating that the polypeptide and the polymer had a certain degree of interaction, resulting in changes in the secondary structure of the polypeptide, and the most likely reason was that the co-assembly in the micelles digested the α-helix of the hydrophilic polypeptide segment.

[0068] 2.3 Co-assembly of LRT and PLGA-PEG-PLGA triblock copolymer

[0069] 2.3.1 Determination of critical micelle concentration

[0070] The hydrophobic probe DPH was used to determine the critical micelle concentration (CMC) of polymers in water. In the experiment, a series of polymer aqueous solutions with concentrations ranging from 0.001wt% to 0.5wt% were first prepared. Then, a DPH methanol solution with a concentration of 0.4mM was added to each sample to make the final DPH concentration in the system reach 4μM / L. After equilibrating the system at 25°C for 12h, the absorption spectrum of the system in the range of 320-420nm was recorded using a UV-visible spectrophotometer. Due to the sensitivity of the hydrophobic probe DPH to the microenvironment, when the polymer concentration exceeds the CMC and micelles are formed, the probe enters the hydrophobic core of the micelle, resulting in a significant increase in absorbance. The CMC value is determined by identifying the inflection point of the polymer logarithmic concentration curve based on the difference in absorbance at 377nm and 400nm.

[0071] 2.3.2 Phase diagram

[0072] The phase diagram of the polymer aqueous solution system was determined by the tubular inversion method. First, a series of polymer aqueous solutions with concentrations of 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 and 25 wt% were prepared by dissolving the polymer in deionized water. The solution was stored to ensure complete equilibrium. Subsequently, 0.5 mL of each solution was transferred to a vial and sealed. The vial was placed in a water bath and heated to 60 ° C at a rate of 1 ° C per step. After equilibration at each temperature for 15 minutes, the vial was inverted to determine the state of the system. If no obvious flow occurs within 30 seconds after inversion, the system is considered to be in a gel state.

[0073] 2.3.3 Rheological studies

[0074] The storage modulus (G') and loss modulus (G") of the polymer solution (25wt%) were measured using a rheometer. The cone-plate parameters were 60mm (diameter) and 1°. The prepared polymer solution was equilibrated at 4°C for 12h and then transferred to the lower plate in a liquid state. To prevent water evaporation during the measurement, a layer of low-viscosity silicone oil was added to the edges of the cone and the plate. In the temperature scanning experiment, the heating rate was set to 0.2°C / min and the angular frequency was set to 10rad / s. The rheological properties of the hydrogel loaded with two drugs were also measured in the same way.

[0075] Variable temperature dynamic light scattering was used to further verify the interaction between LRT and PLGA-PEG-PLGA triblock copolymer. After adding LRT to the system, the critical micelle concentration (CMC) decreased significantly, indicating that the amphiphilicity of LRT promoted the formation of micelles by the polymer; variable temperature dynamic light scattering suggested that micelles would aggregate with the increase of temperature, among which the micelles containing LRT had a lower aggregation temperature, a greater degree of aggregate formation, and a faster speed. The above results all prove that LRT is not simply mixed into the gel, but is coupled with the polymer in the form of co-assembly. In the rheological experiment, when the storage modulus (G') of the material exceeds the loss modulus (G"), the system undergoes a sol-gel phase transition. It can be seen that when LRT is added to the material, the gelation temperature of the system decreases, the viscosity increases before gelation, and the storage modulus (G') increases after gelation. This verifies the co-assembly of LRT and PLGA-PEG-PLGA from a macroscopic perspective. In the phase diagram of the measured system, it was found that the gel window width of the system remained unchanged after the addition of LRT, while the temperature range decreased (such as Figure 3 shown).

[0076] 2.4 Effect of micellar solubilization on rivastigmine

[0077] The effects of normal saline (NS) and Riv@gel gel systems prepared by different methods on Riv concentration were compared. Accurately weigh 0.5 g of Riv@gel in a centrifuge tube (n=5) and centrifuge at high speed at 4°C. Carefully separate the supernatant and precipitate. Add 1 mL of saturated rivastigmine solution to the precipitate, disperse it by ultrasonication, and then vortex for 10 seconds to thoroughly wash the residual polymer solution. Centrifuge to obtain a layered system and discard the supernatant. Repeat the washing under the same conditions. Collect the precipitate, freeze-dry it, and redissolve it in acetonitrile. Dilute 200 μL of the redissolved solution to 10 mL to keep it within the linear range of the curve. The concentration of rivastigmine was determined by high-performance liquid chromatography (HPLC) and compared with the saturated solubility of rivastigmine in deionized water.

[0078] like Figure 4The results showed that compared with the mechanical mixing method (MM), the drug-loaded gel system prepared by the organic solvent dispersion method (OSP) had a higher Riv solubility content, and the difference was statistically significant (P<0.05).

[0079] Example 2 LRT / Riv@gel in vivo and in vitro release experiments

[0080] 1. In vitro drug release: sustained release effect of LRT / Riv@gel

[0081] 0.5 mL of LRT / Riv@gel was added to the release tube, and the tube was then balanced in a water bath oscillator. Next, preheated PBS containing 0.025% NaN3 was added as the release medium. At each sampling time point, the release solution was extracted and replaced with an equal volume of PBS. The collected samples were stored at -20°C until analysis. After the release experiment was completed, the concentrations of liraglutide and rivastigmine in the collected medium were quantitatively analyzed by high performance liquid chromatography (HPLC).

[0082] From the in vitro results, it can be seen that the two drugs show different release patterns. The hydrophobic rivastigmine shows an almost linear release. The amphiphilic liraglutide has a faster release in the early stage and a sustained release in the later stage, which can reach 30 days ( Figure 5 A).

[0083] 2. Hydrogel degradation in vivo

[0084] ICR mice were anesthetized with isoflurane, and 0.2 mL of the gel preparation (LRT / Riv@gel) was injected into the subcutaneous tissue on both sides of the back of the mice. At specific time intervals, some mice were euthanized for anatomical examination. Subsequently, optical images of the remaining hydrogel were taken, and the mass of these residual gels was quantitatively analyzed. In addition, tissue samples containing the remaining gel were collected for histological analysis.

[0085] In vivo, the hydrogel gradually degraded over a period of one month. Due to the influence of dissolution, the hydrophilic part of the hydrogel was lost faster in the early stage, which resulted in a faster release of liraglutide than in vitro. From the pharmacokinetic results, it can be seen that the system can achieve long-term sustained release for up to 21 days in vivo. The hydrophobic rivastigmine is less affected by dissolution, and its release behavior in vivo and in vitro is more consistent. Overall, the system can achieve sustained drug delivery in mice for about two weeks ( Figure 5 BE).

[0086] Example 3 Mouse maze experiment under the action of LRT*Riv@Gel

[0087] 16-week-old 5*FAD mice were treated with NS@Gel (blank control), 5.0 mg / kg LRT@Gel (liraglutide and PLGA-PEG-PLGA triblock hydrogel copolymer), 5.0 mg / kg Riv@Gel (rivastigmine and PLGA-PEG-PLGA triblock hydrogel copolymer) or 2.5 mg / kg LRT*2.5 mg / kg Riv@Gel (liraglutide mixed with rivastigmine and PLGA-PEG-PLGA triblock hydrogel copolymer) for 8 weeks. The treatment dose was 0.2 mL / time, injected subcutaneously, once every 2 weeks, for a total of 4 treatments. After 8 weeks, the Barnes maze was used to detect learning and memory abilities.

[0088] The Barnes maze test is a behavioral experiment to assess the spatial learning and memory ability of animals. Before the experiment, the mice need to be adapted to the experimental environment, stroked every day to prevent stress, and adapted to the experimental room several days in advance. The experimental equipment needs to be cleaned with 75% alcohol and clean water, and visual markers are pasted on the walls to guide the mice. The position of the markers cannot be changed during the experiment. The experiment is divided into an adaptation phase, an acquisition training phase, and a test phase. In the adaptation phase, the safety box is connected to the platform. After the mice adapt to it for 1 minute, they are placed in the center of the instrument to explore freely until they enter the safety box or 5 minutes later, and the equipment is cleaned. The acquisition training phase lasts for 4 days, 3 times a day. The mice start from the starting room in different quadrants, explore the maze and find a fixed safety box under white noise stimulation. Each test lasts 3 minutes. If the mouse does not find the safety box within the specified time, the researchers will put it in and stay for 30 seconds. From the second training, the maze will rotate randomly, but the position of the target box is fixed to prevent the mice from relying on smell rather than memory to find the target. The latency to explore the hole, the latency to reach the target box, the number of errors, and other data were recorded during the experiment. In the test phase, the safety box was removed, and the mice started from the starting room in different quadrants, explored the maze, and searched for the safety box in a fixed position under the stimulation of white noise. The test was conducted once for 3 minutes, and the data such as the time spent in the target quadrant was recorded.

[0089] like Figure 6 The results showed that LRT / Riv@Gel was superior to LRT@Gel and Riv@Gel alone in improving the learning and memory impairment of 5*FAD mice, as manifested by a shortened learning latency and an increased percentage of time spent in the target quadrant in the Barnes maze test, and the differences were statistically significant (P<0.05).

Claims

1. A pharmaceutical composition for preventing and / or treating neurodegenerative diseases, comprising rivastigmine and liraglutide and derivatives thereof.

2. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition also contains a sustained-release system, which can interact with rivastigmine, liraglutide and their derivatives to form a drug-sensitive sustained-release system, and the sustained-release system is a triblock hydrogel sustained-release system.

3. The pharmaceutical composition according to claim 1, characterized in that The neurodegenerative disease is selected from one or more of Alzheimer's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, Huntington's disease, cerebellar atrophy, multiple sclerosis, Parkinson's disease, primary lateral sclerosis, spinal muscular atrophy, cerebral ischemia, spastic paraplegia, myasthenia gravis, etc.

4. A preparation for preventing and / or treating neurodegenerative diseases, comprising the pharmaceutical composition according to claim 1 and pharmaceutically acceptable excipients.

5. The preparation according to claim 4, characterized in that The dosage form of the preparation includes, but is not limited to, solution, injection, infusion, oral solution, tablet, capsule, powder, granule, paste or gel.

6. A pharmaceutical composition, comprising the pharmaceutical composition according to claim 1 or the preparation according to claim 2 and other active ingredients, wherein the pharmaceutical composition has the effect of preventing and / or treating neurodegenerative diseases.

7. The pharmaceutical composition according to claim 6, characterized in that The other active ingredients are any one that can alleviate symptoms caused by or associated with neurodegenerative diseases, but are different from rivastigmine and liraglutide and their derivatives.

8. Use of a pharmaceutical composition in the preparation of a preparation for preventing and / or treating neurodegenerative diseases, wherein the pharmaceutical composition comprises a therapeutically effective amount of rivastigmine and liraglutide and derivatives thereof.

9. The use according to claim 8, characterized in that The pharmaceutical composition also contains a sustained-release system, which is a copolymer that can interact with rivastigmine, liraglutide and their derivatives to form a sustained-release drug.

10. A sustained-release preparation for treating neurodegenerative diseases, comprising PLGA-PEG-PLGA triblock hydrogel and rivastigmine and liraglutide and derivatives thereof.

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