Drug for treating neurodegenerative disease

Abstract

Provided are a pharmaceutical composition for preventing and / or treating neurodegenerative diseases and use thereof. The pharmaceutical composition comprises rivastigmine, liraglutide, and derivatives thereof; the pharmaceutical composition further comprises a sustained-release system, the sustained-release system can interact with the rivastigmine, the liraglutide, and the derivatives thereof to form a copolymer that enables sustained drug release, and the sustained-release system is a triblock hydrogel sustained-release system. The combination of the liraglutide and the rivastigmine can ameliorate the learning and memory impairments associated with Alzheimer's disease, with the effect superior to monotherapy and exhibiting a synergistic effect.

Description

A drug for treating neurodegenerative diseases Technical Field

[0001] The present 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 conditions characterized by the loss of neurons, the cells that line the brain and spinal cord. The brain and spinal cord are composed of neurons, which perform diverse functions, such as controlling movement, processing sensory information, and making decisions. Brain and spinal cord cells 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, worsening over time to lead to 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, and myasthenia gravis.

[0003] Alzheimer's disease (AD) is a neurodegenerative disorder with unknown etiology. Clinical manifestations include progressive cognitive decline, accompanied by decreased social function, personality and behavioral changes, and psychiatric abnormalities. Cognitive decline primarily manifests as memory impairment, aphasia, apraxia, agnosia, and executive dysfunction, while psychiatric abnormalities primarily include agitation, delusions, hallucinations, depression, and apathy. Pathological changes include cortical atrophy, widening of sulci and gyri, enlarged ventricles, a significant decrease in neurons, aggregation of amyloid-β proteins to form senile plaques, hyperphosphorylation of tau proteins to form neurofibrillary tangles, and significant decreases in choline acetylase and acetylcholine levels.

[0004] Alzheimer's disease primarily affects people over 65 years old. The resulting decline in functional ability and changes in personality and behavior place a significant burden on society and their families. Since the disease was first discovered in 1906, scientists have conducted extensive research. In particular, in the past decade, the development of amyloid-β inhibitors has been highly sought after. Unfortunately, the underlying cause of Alzheimer's disease remains unclear, and the failure of several amyloid-β inhibitors has cast a shadow over the development of subsequent drugs. Currently, there are only a few approved drugs for the treatment of Alzheimer's disease, and their efficacy is limited. Currently marketed drugs primarily include cholinesterase inhibitors, N-methyl-D-aspartate receptor antagonists, brain metabolism enhancers, and gut microbiota modulators. Other drugs under development include amyloid-β inhibitors and tau aggregation inhibitors. Some of these drugs are symptomatic, while others only improve a few pathological markers. Few can truly improve the cognitive impairment caused by Alzheimer's disease, and they are associated with numerous adverse reactions.

[0005] Rivastigmine Tartrate (Riv), chemically known as (S)-N-ethyl-N-methyl-3-[1-(dimethylamino)-ethyl]phenylcarbamate-2R,3R-tartrate, is a second-generation cholinesterase inhibitor used to treat mild to moderate Alzheimer's dementia. It is highly selective for the hippocampus and cortex, increasing the function of cholinergic neurons in the brain by inhibiting acetylcholinesterase, thereby improving cognitive function in Alzheimer's patients. It can also slow the formation of amyloid-β fragments of the amyloid precursor protein, slowing the progression of Alzheimer's disease. Rivastigmine exists as a liquid at room temperature and cannot be used as a solid formulation. Clinically, its use as a transdermal agent suffers from the problem of easy penetration but short duration, preventing stable, long-term administration. Rivastigmine salts also suffer from poor penetration efficiency. CN117045646A discloses the use of a co-amorphous compound of rivastigmine and valsartan as an active 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. It was approved by the FDA for marketing in the United States on January 25, 2010, as a new drug for the treatment of type 2 diabetes. Liraglutide shares 97% homology with natural human GLP-1, with only two structural modifications: arginine substitution at position 34 for lysine, and the addition of a 16-carbon palmitic acid side chain mediated by glutamic acid at position 26. This structural modification not only preserves the biological activity of GLP-1 but also protects it from degradation by the DPP-4 enzyme. Compared to traditional insulin injections, liraglutide offers a longer-lasting effect. Liraglutide has the following pharmacological effects: (1) glucose concentration-dependent insulin secretion; (2) inhibition of postprandial glucagon secretion and reduction of glycogen release; (3) enhanced insulin sensitivity; (4) slowing gastric emptying; (5) appetite suppression and weight loss. (6) repair of pancreatic β cells; (7) cardiovascular protection. Liraglutide's insulin secretion and glucagon secretion inhibition effects are glucose concentration-dependent. Therefore, when blood glucose levels are normal, continuous administration of liraglutide does not cause hypoglycemia. This shows 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 sustained-release system using a PLGA-PEG-PLGA triblock copolymer encapsulating LRT, which can stably maintain drug release. Further research using this system revealed that the combination of liraglutide and rivastigmine has a synergistic effect in improving Alzheimer's disease-related learning and memory impairment. Based on this, the present invention was 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 contains 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 pharmaceutically acceptable excipients.

[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, wherein the pharmaceutical composition has the effect of preventing and / or treating neurodegenerative diseases.

[0019] Furthermore, the other active ingredient is any one that can alleviate symptoms caused by or associated with neurodegenerative diseases, but is different from rivastigmine, 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 preparations, buccal tablets, suppositories and / or lyophilized powder injections.

[0022] Furthermore, the various preparations may also contain colorants, preservatives, spices, flavorings, sweeteners or other materials 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; 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 preparing 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 further 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 further comprises 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, or the like 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, 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. Beneficial effects

[0037] The PLGA-PEG-PLGA triblock copolymer encapsulated with LRT developed in 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 Alzheimer's disease-related learning and memory impairment better than using either drug alone, and has a synergistic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 shows the characterization of PLGA-PEG-PLGA triblock copolymers by H NMR and GPC.

[0039] Figure 2 shows the interaction between LRT and a PLGA-PEG-PLGA triblock copolymer as measured by circular dichroism. (A. Structural formula of the PLGA-PEG-PLGA triblock copolymer and LRT; B. Circular dichroism analysis of the structural changes in the interaction between LRT and the polymer.)

[0040] Figure 3 shows the co-assembly of LRT and a PLGA-PEG-PLGA triblock copolymer. A. Changes in critical micelle concentration after LRT incorporation into the polymer; B. Temperature-dependent dynamic light scattering analysis of the phase transition after LRT incorporation into the polymer; C. Rheological modulus measurement after LRT incorporation into the polymer; D. Changes in the gelation temperature window after LRT incorporation into the polymer.

[0041] Figure 4 shows the solubilization effect of Riv micelles. Statistical analysis of the effect of different drug-loaded gel preparation methods on Riv drug loading concentration.

[0042] Figure 5 shows the in vivo and in vitro drug release experiments of LRT / Riv@gel. A. In vitro drug release patterns of LRT and Riv from the drug-loaded LRT / Riv@gel hydrogel; B. In vivo drug release experiment of LRT@Gel in mice following subcutaneous injection; C. Time series of plasma drug concentrations after subcutaneous injection of LRT@Gel in mice with a gradient dose; D. Statistical analysis of plasma drug concentrations after subcutaneous injection of LRT@Gel in mice with a gradient dose; E. Subcutaneous degradation curve of the hydrogel after a single subcutaneous injection of 0.2 ml of LRT / Riv@gel.

[0043] Figure 6 shows that LRT*Riv@Gel synergistically improves Alzheimer's disease-related learning and memory. A. Experimental flow chart; B. Statistical analysis of the latency of the Barnes maze learning phase for each group of mice; C. Statistical analysis of the percentage of time spent in the target quadrant of the Barnes maze for 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

[0044] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below may be combined with each other as long as they do not conflict with each other.

[0045] 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.

[0046] As described herein, rivastigmine (Riv) is a brain-selective acetylcholinesterase inhibitor of the amino acid class that promotes cholinergic neurotransmission by delaying the degradation of released acetylcholine by intact cholinergic neurons. The recommended starting dose of rivastigmine is 3 mg / day (1.5 mg twice daily). Based on individual differences, the dose should be increased at least every two weeks to reach the maximum tolerable dose, but should not exceed 12 mg per day. The target dose for most patients should be between 6 and 12 mg per day.

[0047] Rivastigmine Pharmacokinetics: Rivastigmine is completely and rapidly absorbed, reaching peak plasma concentrations approximately 1 hour after oral administration. Rivastigmine is rapidly and extensively metabolized (plasma half-life approximately 1 hour), primarily through cholinesterase-mediated hydrolysis. It is primarily excreted through the kidneys as metabolites, with only <1% of the drug excreted in the feces.

[0048] Pharmacodynamics of rivastigmine: The plasma half-life of rivastigmine is approximately 1 hour, and the acetylcholinesterase inhibition period is approximately 9 hours. Therefore, it is recommended that rivastigmine should not be continued in patients with asymptomatic overdose within the next 24 hours.

[0049] The term "liraglutide injection" (LRT) as used herein is a synthetic, acylated human glucagon-like peptide-1 (GLP-1) analog. The recommended dose of liraglutide is: a starting dose of 0.6 mg subcutaneously daily. After at least one 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 clinical response, the dose can be increased to 1.8 mg after at least one week to further improve the glucose-lowering effect. The recommended daily dose should not exceed 1.8 mg.

[0050] 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, with no specific organ identified as the primary elimination pathway. The elimination half-life is approximately 13 hours.

[0051] 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.

[0052] The term "Barnes maze" as used herein is a common paradigm for testing spatial memory in animals. It is established by exploiting the fact that rodents avoid light, prefer darkness, and are exploratory. The animals are reinforced by escaping from a lighted, open platform to a dark, narrow box below the platform, called a target box. After training, the animals learn and remember the location of the target box. This model is less stressful for animals and is particularly suitable for stress-related memory research and behavioral phenotyping studies in knockout mice.

[0053] The term "PLGA" as used herein refers to polylactic-co-glycolic acid, a copolymer formed by the polymerization reaction of lactic acid and glycolic acid. It is a degradable functional polymer organic compound that is non-toxic and has good biocompatibility, capsule-forming and film-forming properties. In the present invention, the PLGA-PEG-PLGA triblock copolymer is formed by the polymerization of PLGA and polyethylene glycol (PEG).

[0054] The term "storage modulus" as used in the present invention is essentially Young's modulus, which is an indicator of the material's rebound after deformation and represents the material's ability to store elastic deformation energy, and is represented as G' in this article.

[0055] The term "loss modulus" in the present invention describes the phenomenon that energy is dissipated (transformed) into heat when the material generates deformation. It is a measure of energy loss and is a damping attenuation term, denoted as G" in this article. When the storage modulus G' > the loss modulus G", it indicates that the bulk phase is more inclined to the characteristics of an elastic solid, and the characteristics of a viscous fluid are weaker than those of an elastic solid. At this time, the structure of the "X system" can have two explanations: (1) It indicates the gradual gelation or the gradual formation of the internal structure in 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 been significantly improved compared with other systems. When the storage modulus G' < the loss modulus G", the 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.

[0056] Example 1 Sustained-release system of LRT encapsulated by PLGA-PEG-PLGA triblock copolymer

[0057] 1. Synthesis of PLGA-PEG-PLGA triblock copolymer

[0058] The triblock copolymer PLGA-PEG-PLGA was synthesized by ring-opening polymerization of LA and GA monomers using PEG1500 as a macromolecular initiator and Sn(Oct)2 as a catalyst. 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, it was continuously stirred and reacted at 150 °C under an argon atmosphere. After the reaction ended, the product was placed in hot water and stirred for washing. Finally, the final product was obtained by freeze-drying the mixture for 48 h.

[0059] The number-average molecular weight (Mn) of the synthesized PLGA-PEG-PLGA copolymer and the molar ratio of LA to GA (LA / GA) were characterized by 1H NMR (Bruker, AVANCE III, 400 MHz) (Figure 1). Deuterated chloroform (CDCl3) was used as the solvent and tetramethylsilane (TMS) as the internal standard. The molecular weight distribution index (DM) of the copolymer was determined using an Agilent 1260 GPC system. Tetrahydrofuran (THF) was used as the mobile phase with a flow rate of 1 mL / min, and polystyrene standards were used for calibration.

[0060] 2. Interaction between LRT and PLGA-PEG-PLGA triblock copolymer

[0061] 2.1 Preparation of hydrogel

[0062] PLGA-PEG-PLGA spontaneously forms a micellar structure in aqueous solution, with the hydrophobic PLGA as the core and the hydrophilic PEG as the shell. LRT, on the other hand, has a hydrophilic peptide chain and a hydrophobic saturated C-16 chain. Therefore, when LRT is incorporated into the system, it co-assembles with the polymer rather than remaining free in the medium.

[0063] A 25 wt% copolymer / water solution was prepared by mixing PLGA-PEG-PLGA with saline at a mass ratio of 1:3, and then transferred to a sample vial. The mixture was magnetically stirred for several days. When the system reached uniformity and no bubbles were present, the hydrogel system, termed T-gel, was successfully prepared. To obtain liraglutide-loaded T-gel, powdered liraglutide was added to the polymer solution at a specific mass ratio and stirred until the system was completely clear and homogeneous. This resulted in the preparation of liraglutide-loaded T-gel (LRT@gel). For the rivastigmine-loaded hydrogel (Riva@gel), a specific mass of rivastigmine and polymer was thoroughly dissolved in acetone. After complete removal of the solvent by rotary evaporation and freeze-drying, the mixture was added to saline (also containing 25 wt% polymer) to prepare the final solution. Subsequently, liraglutide powder was dissolved in Riv@T-gel to obtain a hydrogel capable of co-delivering liraglutide and rivastigmine, designated LRT / Riv@gel.

[0064] 2.2 Circular dichroism detection of the interaction between LRT and PLGA-PEG-PLGA triblock copolymer

[0065] Circular dichroism (CD) spectroscopy was used to determine changes in the peptide's secondary structure before and after interaction with the polymer, further elucidating the potential interaction between the two. The secondary structure of Lira in different solutions (with and without PLGA-PEG-PLGA) was analyzed using CD spectroscopy. CD measurements were performed over a wavelength range of 200 to 280 nm. Each spectrum was baseline corrected by subtracting the corresponding ultrapure water signal.

[0066] As shown in Figure 2, the CD spectrum of the LRT aqueous solution exhibits two distinct negative peaks at 208nm and 222nm, suggesting 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 the aqueous solution changes, as evidenced by the disappearance of the original negative peak at 208nm and the appearance of a larger negative peak at 225nm. This suggests that a certain degree of interaction occurs between the polypeptide and the polymer, leading to changes in the polypeptide's secondary structure, most likely due to the dissolution of the α-helix of the hydrophilic polypeptide segment during co-assembly in the micelles.

[0067] 2.3 Co-assembly of LRT and PLGA-PEG-PLGA triblock copolymer

[0068] 2.3.1 Critical micelle concentration determination

[0069] 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 0.4mM DPH methanol solution was added to each sample to make the final DPH concentration in the system reach 4μM / L. After equilibration of the system at 25°C for 12 hours, 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 micelles, resulting in a significant increase in absorbance. The CMC value was determined by identifying the inflection point of the polymer logarithmic concentration curve based on the difference in absorbance at 377nm and 400nm.

[0070] 2.3.2 Phase diagram

[0071] The phase diagram of the polymer aqueous solution system was determined using 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 solutions were 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 occurred within 30 seconds after inversion, the system was considered to be in a gel state.

[0072] 2.3.3 Rheological studies

[0073] The storage modulus (G') and loss modulus (G") of the polymer solution (25 wt%) were measured using a rheometer. The cone-plate parameters were 60 mm (diameter) and 1°. The prepared polymer solution was equilibrated at 4°C for 12 h 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 plate. In the temperature sweep experiment, the heating rate was set to 0.2°C / min and the angular frequency was set to 10 rad / s. The rheological properties of the hydrogel loaded with two drugs were also measured in the same manner.

[0074] Dynamic light scattering (DLS) at variable temperatures further verified the interaction between LRT and the PLGA-PEG-PLGA triblock copolymer. Addition of LRT to the system significantly decreased the critical micelle concentration (CMC), indicating that the amphiphilic nature of LRT promoted micelle formation. DLS analysis also revealed that micelles aggregated with increasing temperature. The micelles containing LRT aggregated at a lower temperature, formed to a greater extent, and formed more rapidly. These results demonstrate that LRT is not simply incorporated into the gel but rather co-assembles with the polymer. Rheological experiments show that the system undergoes a sol-gel phase transition when the storage modulus (G') exceeds the loss modulus (G") of the material. The addition of LRT to the material decreases the gelation temperature, increases the viscosity before gelation, and increases the storage modulus (G') after gelation. This macroscopically confirms the co-assembly of LRT and PLGA-PEG-PLGA. Phase diagrams of the system reveal that the addition of LRT maintains the gelation window width while decreasing the temperature range (Figure 3).

[0075] 2.4 Effect of micellar solubilization on rivastigmine

[0076] 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 into 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, ultrasonically disperse it, and then vortex for 10 seconds to thoroughly wash away 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.

[0077] As shown in Figure 4, the drug-loaded gel system prepared by the organic solvent dispersion (OSP) method had a higher Riv solubility content than that prepared by the mechanical mixing (MM) method, and the difference was statistically significant (P<0.05).

[0078] Example 2 LRT / Riv@gel in vivo and in vitro release experiments

[0079] 1. In vitro drug release: sustained release effect of LRT / Riv@gel

[0080] 0.5 mL of LRT / Riv@gel was added to the release tube, and the tube was then placed in a water bath shaker for equilibrium. Next, preheated PBS containing 0.025% NaN3 was added as the release medium. At each sampling time point, the release solution was withdrawn 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).

[0081] The in vitro results show that the two drugs exhibit different release patterns: hydrophobic rivastigmine exhibits an almost linear release pattern, while amphiphilic liraglutide exhibits a faster initial release pattern and a sustained release pattern over a period of up to 30 days (Figure 5A).

[0082] 2. Hydrogel degradation in vivo

[0083] ICR mice were anesthetized with isoflurane, and 0.2 mL of the gel formulation (LRT / Riv@gel) was injected into the subcutaneous tissue on both sides of the back. At specific time intervals, some mice were euthanized for necropsy. Subsequently, optical images of the remaining hydrogel were captured, and the mass of this residual gel was quantitatively analyzed. In addition, tissue samples containing the residual gel were collected for histological analysis.

[0084] In vivo, the hydrogel gradually degraded over a period of one month. Due to the effects of erosion, the hydrophilic portion of the hydrogel was lost more rapidly in the early stages, resulting in a faster release of liraglutide than in vitro. Pharmacokinetic results indicate that the system can achieve long-term sustained release in vivo for up to 21 days. Hydrophobic rivastigmine is less affected by erosion, and its release behavior in vitro and in vivo is more consistent. Overall, the system can achieve sustained drug delivery in mice for about two weeks (Figures 5B-E).

[0085] Example 3 Mouse maze experiment under the action of LRT*Riv@Gel

[0086] Sixteen-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 test learning and memory abilities.

[0087] The Barnes maze test is a behavioral test that assesses spatial learning and memory in animals. Before the experiment, mice must be acclimated to the experimental environment. They are handled daily to prevent stress and allowed to acclimate to the experimental room several days in advance. The experimental apparatus must be cleaned with 75% alcohol and water, and visual markers are affixed to the walls to guide the mice. The positions of the markers must not be changed during the experiment. The experiment consists of an acclimation phase, an acquisition training phase, and a test phase. During the acclimation phase, a safety box is attached to the platform. After acclimating for 1 minute, the mice are placed in the center of the apparatus and are free to explore until they enter the safety box or until 5 minutes have passed. The apparatus is then cleaned. The acquisition training phase lasts four days, three times a day. Mice begin in a start chamber in a different quadrant and explore the maze, stimulating white noise to find a fixed safety box. Each trial lasts three minutes. If the mouse fails to find the safety box within the specified time, the researcher will place it back in the maze and keep it there for 30 seconds. From the second training session onward, the maze rotates randomly, but the target box is fixed to prevent the mice from relying on scent rather than memory to find the target. During the experiment, data such as the latency to explore the hole, latency to reach the target box, and number of errors were recorded. During the test phase, the safety box was removed, and mice began exploring the maze from the start chamber in different quadrants, stimulating white noise to find the fixed safety box. This test lasted for 3 minutes, and data such as the time spent in the target quadrant was recorded.

[0088] As shown in Figure 6, the results show that LRT / Riv@Gel improves the learning and memory impairment of 5*FAD mice better than LRT@Gel and Riv@Gel alone, as manifested by shortened learning latency and increased percentage of time spent in the target quadrant in the Barnes maze test. The differences are 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, wherein The pharmaceutical composition further comprises 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, wherein 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, wherein 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, 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 further comprises 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, rivastigmine, liraglutide and derivatives thereof.

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