Pharmaceutical composition and application thereof

Abstract

The invention provides a pharmaceutical composition and application thereof, and relates to the technical field of medicine preparation. The pharmaceutical composition comprises S-(-)-cotinine or a pharmaceutically acceptable salt of the S-(-)-cotinine, and rivastigmine or a pharmaceutically acceptable salt of the rivastigmine. Compared with a single drug, the pharmaceutical composition containing S-(-)-cotinine and rivastigmine provided by the invention shows remarkable synergistic interaction and multi-target comprehensive intervention advantages, provides a new choice with better curative effect and more comprehensive action mechanism for treating neurodegenerative diseases such as Alzheimer's disease, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation technology, and in particular to a pharmaceutical composition and its application. Background Technology

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease, characterized by senile plaques formed by the deposition of β-amyloid (Aβ) protein in the brain, neurofibrillary tangles (NFTs) caused by hyperphosphorylated Tau protein, loss of cholinergic neurons, and persistent neuroinflammation and oxidative stress. These pathological processes are interconnected and collectively lead to progressive cognitive decline.

[0003] Currently, the first-line drugs for treating Alzheimer's disease (AD) in clinical practice are mainly cholinesterase inhibitors, such as donepezil, galantamine, and rivastigmine. Among them, rivastigmine is a reversible dual inhibitor of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). It enhances cholinergic neurotransmission by inhibiting the degradation of acetylcholine in the synaptic cleft, thereby improving cognitive function in patients with mild to moderate AD. However, rivastigmine monotherapy has significant limitations: its efficacy decreases significantly as the disease progresses; and it primarily targets the cholinergic system, offering insufficient intervention for the complex multifactorial pathological mechanisms of AD (such as neuroinflammation, oxidative stress, and metal ion imbalances), making it difficult to effectively curb disease progression.

[0004] Therefore, there is an urgent need to develop a new treatment strategy that can intervene in the complex pathological process of AD through multiple targets, thereby providing a more comprehensive and effective solution for AD treatment. Summary of the Invention

[0005] In view of this, the present invention proposes a pharmaceutical composition and its application. By proposing a novel pharmaceutical composition comprising rivastigmine and S-(-)-cotinine, the present invention utilizes the complementarity and synergy of the two in their different mechanisms of action to provide a more effective new strategy for the treatment of Alzheimer's disease and other related neurodegenerative diseases.

[0006] In a first aspect, the present invention provides a pharmaceutical composition comprising S-(-)-cotinine or a pharmaceutically acceptable salt thereof, and rivastigmine or a pharmaceutically acceptable salt thereof.

[0007] S-(-)-cotinine, also known as (5S)-1-methyl-5-pyridin-3-ylpyrrolidone-2-one, has the following molecular structure: ; Cabernetin, also known as N-ethyl-N-methyl-carbamate 3-[(S)-1-(dimethylamino)ethyl]phenyl ester, has the following molecular structure: .

[0008] Furthermore, the mass ratio of S-(-)-cotinine to rivastigmine is 1:1 to 1:5.

[0009] Furthermore, the mass ratio of S-(-)-cotinine to rivastigmine is 1:1.

[0010] Furthermore, the pharmaceutically acceptable salts of the S-(-)-cotinine include its hydrochloride, tartrate, citrate, or fumarate; the pharmaceutically acceptable salts of the rivastigmine are its tartrate.

[0011] Furthermore, the pharmaceutical composition further includes one or more pharmaceutically acceptable excipients.

[0012] Secondly, the present invention relates to the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for treating neurodegenerative diseases.

[0013] Thirdly, the present invention provides a medicament for treating neurodegenerative diseases, the medicament comprising the above-described pharmaceutical composition.

[0014] Furthermore, the treatment of neurodegenerative diseases includes, but is not limited to, Alzheimer's disease.

[0015] The pharmaceutical composition of the present invention and its application have the following advantages over the prior art: (1) The pharmaceutical composition of the present invention, when combined with S-(-)-cotinine, produces a significant synergistic enhancement effect on the inhibitory activity of butyrylcholinesterase (BuChE). In vitro experimental results show that the IC50 of the combination is significantly enhanced. 50 The value was significantly lower than that of rivastigmine monotherapy, and the inhibitory efficacy was greatly improved, indicating that the combination of the two can greatly enhance the inhibitory effect on BuChE, thereby more effectively enhancing central cholinergic neurotransmission and providing stronger pharmacological support for improving the cognitive function of Alzheimer's patients.

[0016] (2) The pharmaceutical composition provided by this invention achieves synergistic intervention across multiple targets and pathways. Cabavirin primarily enhances cognitive function by inhibiting acetylcholinesterase and butyrylcholinesterase to increase acetylcholine levels; S-(-)-cotinine exerts anti-inflammatory, antioxidant, and neuroprotective effects by activating α7 nicotinic acetylcholine receptors (α7-nAChR), effectively inhibiting the release of inflammatory factors such as nitric oxide (NO) and interleukin-6 (IL-6), and scavenging reactive oxygen species (ROS) to reduce oxidative stress damage. Simultaneously, S-(-)-cotinine also exhibits synergistic effects with Cu 2+ Fe 2+ Zn2+ The chelating ability of metal ions helps to inhibit Aβ protein aggregation and Tau protein hyperphosphorylation, thereby comprehensively regulating the pathological process of Alzheimer's disease from multiple key aspects.

[0017] (3) The present invention uses S-(-)-cotinine to prepare a pharmaceutical composition, which not only has low toxicity and good safety, but also has significantly better pharmacological activity than its R-(+)-enantiomer and racemic mixture. In addition, the composition also shows a synergistic trend in antioxidant properties, with stronger and more efficient free radical scavenging ability, further enhancing the overall neuroprotective effect, and providing a new option with better efficacy and broader application prospects for the treatment of neurodegenerative diseases such as Alzheimer's disease. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 ABTS of different concentrations of the test compound solutions prepared in Example 4 and Comparative Examples 1-8 of this invention + Schematic diagram of free radical scavenging activity and its corresponding kinetic fitting curve, and schematic diagram of antioxidant effect at the same concentration; wherein, Figure AB is the ABTS of Comparative Example 1. + Schematic diagram of free radical scavenging activity and its corresponding kinetic fitting curve. Figures C and D show the ABTS in Comparative Example 2. + Schematic diagram of free radical scavenging activity and its corresponding kinetic fitting curve. Figure EF shows the ABTS in Comparative Example 3. + Schematic diagram of free radical scavenging activity and its corresponding kinetic fitting curve. Figure GH shows the ABTS in Comparative Example 4. + Schematic diagram of free radical scavenging activity and its corresponding kinetic fitting curve. Figure IJ shows the ABTS in Comparative Example 5. + Schematic diagram of free radical scavenging activity and its corresponding kinetic fitting curve. Figure KL shows the ABTS in Example 4. + Schematic diagram of free radical scavenging activity and its corresponding kinetic fitting curve. Figure MN shows the ABTS in Comparative Example 6. + Schematic diagram of free radical scavenging activity and its corresponding kinetic fitting curve. Figure OP shows the ABTS in Comparative Example 7. + Schematic diagram of free radical scavenging activity and its corresponding kinetic fitting curve. Figure QS is the ABTS of Comparative Example 1. +Schematic diagram of free radical scavenging activity and its corresponding kinetic fitting curve; Figure T is a schematic diagram of the antioxidant effect at the same concentration. Figure 2 The absorbance standard curves and NO concentrations in different culture media were plotted for Example 6 and Comparative Examples 16-19 of the present invention; in the figure, A is the plotted absorbance standard curve, and B is the NO concentration in different culture media. Figure 3 The figures show the IL-6 standard curves and the concentrations of IL-6 in different culture media for Example 7 and Comparative Examples 20-23 of the present invention; in the figures, A is the IL-6 standard curve and B is the concentration of IL-6 in different culture media. Figure 4 This is a schematic diagram of the fluorescence intensity of the culture media prepared in Example 7 and Comparative Examples 20-23 of the present invention. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0021] The pathological process of Alzheimer's disease (AD) involves multiple interconnected pathways. In recent years, research has gradually shifted towards multi-target treatment strategies, aiming to simultaneously intervene in multiple pathological stages of AD. Nicotinic acetylcholine receptors (nAChRs), especially the α7 subtype, are considered highly promising therapeutic targets. Activation of α7-nAChRs not only improves cognitive function but also provides multiple benefits such as neuroprotection, anti-Aβ toxicity, and inhibition of neuroinflammation. S-(-)-cotinine is the main metabolite of nicotine in the human body, with significantly lower toxicity than nicotine and good safety. Studies have shown that S-(-)-cotinine possesses multiple biological activities, including neuroprotection, anti-inflammation, antioxidant effects, and regulation of neurotransmitters. It can activate α7-nicotinic acetylcholine receptors (α7-nAChRs), which play an important role in regulating neuroinflammation, inhibiting Aβ toxicity, improving synaptic plasticity, and enhancing cognitive function. Furthermore, S-(-)-cotinine has been shown to inhibit Aβ aggregation, reduce Tau protein phosphorylation, and scavenge free radicals, demonstrating great potential for treating neurodegenerative diseases. However, S-(-)-cotinine, as a single drug, has weak cholinesterase inhibitory activity and is insufficient to completely reverse the cholinergic deficiency in AD.

[0022] However, to date, no studies have publicly reported or suggested the use of the combination of S-(-)-cotinine and rivastigmine in the treatment of Alzheimer's disease, nor is there any evidence that this particular combination can produce unexpected synergistic effects, especially in inhibiting butyrylcholinesterase (BuChE), scavenging free radicals, chelating metal ions, and synergistically resisting neuroinflammation.

[0023] Therefore, based on this unmet clinical need and technological gap, the inventors developed a novel pharmaceutical composition containing rivastigmine and S-(-)-cotinine, which utilizes the complementarity and synergy of the two in different mechanisms of action to provide a more comprehensive and effective treatment option for AD, especially suitable for patients who do not respond well to existing monotherapy.

[0024] The following are embodiments of the present invention. Unless otherwise specified, the materials involved in the following examples are all conventional commercial products or raw materials that can be prepared by existing known chemical methods.

[0025] Example 1 This embodiment provides a pharmaceutical composition comprising the following components: S-(-)-cotinine 1.0 g, rivastigmine tartrate 1.0 g, citric acid (pH adjuster) 0.5 g, sorbitol (sweetener) 150.0 g, sodium benzoate (preservative) 1.0 g, and an appropriate amount of purified water; The pharmaceutical composition is prepared by the following method: Citric acid, sorbitol, and sodium benzoate were dissolved in approximately 800 mL of purified water and stirred until completely dissolved. S-(-)-cotinine and rivastigmine tartrate were added and stirred until completely dissolved. The volume was brought to 1000 mL with purified water, stirred thoroughly, filtered, and bottled to obtain an oral solution containing 10 mg of S-(-)-cotinine and 10 mg of rivastigmine per 10 mL of oral solution.

[0026] Example 2 This embodiment provides a pharmaceutical composition comprising the following components: S-(-)-cotinine 0.5 g, rivastigmine tartrate 1.0 g, citric acid (pH adjuster) 0.5 g, sorbitol (sweetener) 150.0 g, sodium benzoate (preservative) 1.0 g, and an appropriate amount of purified water; The pharmaceutical composition is prepared by the following method: Citric acid, sorbitol, and sodium benzoate were dissolved in approximately 800 mL of purified water and stirred until completely dissolved. S-(-)-cotinine and rivastigmine tartrate were added and stirred until completely dissolved. The volume was brought to 1000 mL with purified water, stirred thoroughly, filtered, and bottled to obtain an oral solution containing 5 mg of S-(-)-cotinine and 10 mg of rivastigmine per 10 mL of oral solution.

[0027] Example 3 This embodiment provides a pharmaceutical composition comprising the following components: S-(-)-cotinine 0.33 g, rivastigmine tartrate 1.67 g, citric acid (pH adjuster) 0.5 g, sorbitol (sweetener) 150.0 g, sodium benzoate (preservative) 1.0 g, and an appropriate amount of purified water; The pharmaceutical composition is prepared by the following method: Citric acid, sorbitol, and sodium benzoate were dissolved in approximately 800 mL of purified water and stirred until completely dissolved. S-(-)-cotinine and rivastigmine tartrate were added and stirred until completely dissolved. The volume was brought to 1000 mL with purified water, stirred thoroughly, filtered, and bottled to obtain an oral solution containing 3.3 mg of S-(-)-cotinine and 16.7 mg of rivastigmine per 10 mL.

[0028] The following examples are used to investigate the effect of the pharmaceutical compositions provided by this invention on ABTS. + The effect of [the study] is used to evaluate the antioxidant effect of the pharmaceutical compositions of the present invention.

[0029] Example 4 This embodiment provides the application of a group of pharmaceutical compositions, including: Preparation of phosphate buffered saline (PBS) at pH 7.4: Accurately weigh 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 and dissolve them in 800 mL distilled water. Adjust the pH of the solution to 7.4 with dilute hydrochloric acid, sonicate for 20 min, and store at room temperature for later use.

[0030] Preparation of 7 mM 2,2'-adiazon-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) stock solution: Weigh 3.84 mg ABTS and dissolve it in 900 μL of deionized water.

[0031] Preparation of 2.45 mM K2S8O8 stock solution: Weigh 6.63 mg K2S8O8 and add it to 100 μL 50 mM PBS buffer (pH=7.4, 100 mM NaCl).

[0032] ABTS + Preparation of the free radical solution: Add 100 μL of the above K₂S₈O₈ solution to 900 μL of ABTS solution and react at room temperature in the dark for at least 18 h. Then, add 700 μL of the above 7 mM ABTS solution. +The free radical solution was added to 50 mM PBS buffer (pH=7.4, 100 mM NaCl), and after mixing, it was allowed to stand for 10 min. The absorbance value at 734 nm was measured by UV spectrophotometry. The absorbance value was 0.7±0.2. It was stored in the dark for later use.

[0033] Preparation of test compound solution: Mix S-(-)-cotinine (COT) and rivastigmine tartrate (RIV) at a mass ratio of 1:1 and dissolve them in DMSO to prepare a 10 mM stock solution for later use.

[0034] Comparative Example 1 The difference from Example 4 is that the solution of the test compound was prepared as follows: S-(-)-cotinine (COT) was dissolved in DMSO to prepare a 10 mM stock solution for later use. The rest remained unchanged.

[0035] Comparative Example 2 The difference from Example 4 is that the solution of the test compound was prepared as follows: Cabavirin tartrate (RIV) was dissolved in DMSO to prepare a 10 mM stock solution for later use. The rest remained unchanged.

[0036] Comparative Example 3 The difference from Example 4 is that the solution of the test compound was prepared as follows: S-(-)-nicotine (NIC) was dissolved in DMSO to prepare a 10 mM stock solution for later use. The rest remained unchanged.

[0037] Comparative Example 4 The difference from Example 4 is that the solution of the test compound was prepared as follows: Racemic cotinine (RAC) was dissolved in DMSO to prepare a 10 mM stock solution for later use. The rest remained unchanged.

[0038] Comparative Example 5 The difference from Example 4 is that the solution of the test compound was prepared as follows: R-(+)-cotinine (R-COT) was dissolved in DMSO to prepare a 10 mM stock solution for later use. The rest remained unchanged.

[0039] Comparative Example 6 The difference from Example 4 is that the solution of the test compound was prepared as follows: Dissolve COT+NIC in DMSO to prepare a 10 mM stock solution for later use. All other conditions remain unchanged.

[0040] Comparative Example 7 The difference from Example 4 is that the solution of the test compound was prepared as follows: Dissolve NIC+RIV in DMSO to prepare a 10 mM stock solution for later use. All other conditions remain unchanged.

[0041] Comparative Example 8 The difference from Example 4 is that the solution of the test compound was prepared as follows: 6-Hydroxy-2,5,7,8-Tetramethyltryptane-2-carboxylic acid (Trolox) was dissolved in DMSO to prepare a 10 mM stock solution for later use. The rest remained unchanged.

[0042] The test compound solutions prepared in Examples 4 and Comparative Examples 1-8 were diluted with ethanol to a concentration of 10 mM to 0.04-1.6 mM to serve as working solutions for subsequent experiments.

[0043] Take 0.4 mL of the compound working solution of different concentrations and transfer it into several sets of 5 mL EP tubes, then add 3.0 mL of diluted ABTS. + The absorbance of the free radical stock solution was 0.7 ± 0.2 nm. The reaction was carried out at room temperature for 6 min, and the absorbance at 734 nm was measured using a UV spectrophotometer. This absorbance is denoted as A1. 3.0 mL of K2S8O8 solution was used instead of ABTS. + The absorbance of the free radical stock solution is recorded as A2. 0.4 mL of ethanol is used to replace the sample; the absorbance is recorded as A3. ABTS + The free radical scavenging activity was calculated as follows: ABTS + Free radical scavenging rate (%) = (A3 - A1 + A2) / A3 × 100%; Test results are as follows Figure 1 As shown, the curves were fitted using the dynamic model reported in the literature (Munoz-Munoz et al.) to obtain key dynamic parameters.

[0044] Depend on Figure 1 As shown in (A, C, E, G, I, K, M, O, Q), ABTS⁺ free radicals exhibit a characteristic absorption peak at 734 nm. When an antioxidant compound is added, the absorbance of this peak decreases, and the higher the compound concentration or the stronger the antioxidant activity, the more significant the decrease in absorbance. This figure visually confirms that all tested compounds can scavenge ABTS⁺ free radicals in a concentration-dependent manner.

[0045] Depend on Figure 1As shown in (B, D, F, H, J, L, N, P, S), the curve of Comparative Example 3 is the steepest, indicating the highest scavenging efficiency, but its antioxidant capacity is slightly lower than that of Comparative Example 8. The curves of Comparative Examples 1 and 2 are flatter, indicating weaker antioxidant capacity. The curves of Comparative Examples 4, 5, 6, and 7, i.e., the three different configurations of cotinine, almost overlap, indicating that its antioxidant capacity is independent of optical configuration and depends only on molecular structure. The curve of Example 4 is above the single-drug curves of COT and RIV in the high-concentration range, indicating that the combination of the two has a synergistic antioxidant effect at high concentrations.

[0046] Depend on Figure 1 As shown in (T), at a fixed concentration, the faster the curve decreases, the faster the reaction rate of the compound. The fitting data are shown in Table 1. Table 1. n of the target compound's ability to capture ABTS+ radicals app and T EC50

[0047] Combination Figure 1 As shown in T and Table 1, S-(-)-cotinine possesses certain antioxidant capacity, but its strength and rate are far lower than those of S-(-)-nicotine. This may be due to the inhibition of the NH bond in the pyrrole ring by the carbonyl group in its molecular structure. However, its antioxidant effect is significantly enhanced when used in combination with rivastigmine.

[0048] The following examples are used to investigate the effect of the pharmaceutical composition provided by the present invention on butyrylcholinesterase (BuChE) and to evaluate the BuChE inhibitory activity of the pharmaceutical composition of the present invention.

[0049] Example 5 This embodiment provides the application of a group of pharmaceutical compositions, including: Tris-HCl buffer stock solution: Tris-HCl pH=8.0, NaCl 0.1 M, MgCl2·6H2O 50 mM 0.02 M.

[0050] Take 25 mL of 1M Tris-HCl buffer, add 2.924 g NaCl, then add 2.033 g MgCl2·6H2O, mix, and add distilled water to 500 mL for subsequent experiments.

[0051] Preparation of 20 U / mL BuChE stock solution: Dissolve 55 mg of BuChE in 30 mL of 0.1% BSA enzyme buffer solution and shake well. Aliquot the prepared solution into 5 mL EP tubes and store at -80 ℃.

[0052] Preparation of 1.5 mM DTNB (5,5'-dithiobis(2-nitrobenzoic acid) solution: Weigh 55.4025 mg of DTNB and dissolve it in 100 mL of Tris-HCl buffer solution. Shake well. Store at -20 °C.

[0053] Preparation of 15 mM BTCl (thioiodide butyrylcholine) solution: Weigh 0.475845 g of BTCl and dissolve it in 100 mL of Tris-HCl buffer solution, then shake well. Store at -20 ℃.

[0054] Preparation of sample and positive control solutions: The initial concentration of the sample to be tested is 10 mM, and it is diluted to the corresponding concentration with Tris-HCl buffer solution during testing.

[0055] Preparation of the test compound: Weigh equal masses of COT and RIV and dissolve them separately in DMSO to prepare 10 mM stock solutions, then mix thoroughly.

[0056] Comparative Example 9 The difference from Example 5 is that the test compound solution was prepared as S-(-)-cotinine (COT), while the rest remained unchanged.

[0057] Comparative Example 10 The difference from Example 5 is that the solution of the test compound was prepared as follows: Racemic cotinine (RAC) remains unchanged.

[0058] Comparative Example 11 The difference from Example 5 is that the solution of the test compound was prepared as follows: R-(+)-cotinine (R-COT), the rest remain unchanged.

[0059] Comparative Example 12 The difference from Example 5 is that the solution of the test compound was prepared as follows: S-(-)-nicotine (NIC), the rest remain unchanged.

[0060] Comparative Example 13 The difference from Example 5 is that the solution of the test compound was prepared as follows: Cabernet tartrate (RIV), the rest remain unchanged.

[0061] Comparative Example 14 The difference from Example 5 is that the solution of the test compound was prepared as follows: NIC+RIV, the rest remain unchanged.

[0062] Comparative Example 15 The difference from Example 5 is that the solution of the test compound was prepared as follows: COT+NIC, the rest remain unchanged.

[0063] The test compound solutions prepared in Examples 5 and Comparative Examples 9-15 were diluted with ethanol to a concentration of 10 mM to 0.04-1.6 mM to prepare working solutions for subsequent experiments, including the following steps: First, add 100 μL of buffer solution to each well of the 96-well plate. Then, add 20 μL of different concentrations of the analyte compound (6 gradients, each concentration set to 3 replicates) to the sample wells sequentially. Add equal volumes to the standard group (containing the aforementioned Tris-HCl buffer stock solution + BuChE stock solution) and the blank group (containing only BuChE stock solution). Next, add 20 μL of BuChE stock solution to the sample and standard wells, and add 20 μL of Tris-HCl buffer to the blank group. Incubate at 37 °C for 5 min in the dark to equilibrate the reaction system. After adding 20 μL of BTCI solution to all wells, immediately measure the absorbance at 412 nm (denoted as A0). Subsequently, add 40 μL of DTNB chromogenic reagent to each well, incubate at 37 °C for 5 min in the dark, and measure the absorbance at 412 nm again (denoted as A1~A3). The inhibition rate (%) was calculated using the formula: [(A1-A0)-(A2-A3)] / (A1-A0)×100% (where A1 is the final value of the standard group, A0 is the final value of the blank group, A2 is the final value of the sample group, and A3 is the initial value of the blank group). Finally, the concentration-inhibition rate curve was fitted using Origin, and the IC50 was calculated. 50 The test results are shown in Table 2.

[0064] Table 2 Butyrylcholinesterase activity of the target compounds I C50 (n=3)

[0065] Table 2 shows that rivastigmine monotherapy exhibits extremely strong inhibitory activity against butyrylcholinesterase (BuChE), while S-(-)-cotinine monotherapy shows relatively weak inhibitory activity. However, when the two are used in combination at a 1:1 ratio, their IC50 values ​​are significantly lower. 50 The concentration of S-(-)-cotinine was significantly lower than that of rivastigmine monotherapy, and the inhibitory activity was further greatly enhanced, exhibiting an effect far exceeding that of monotherapy or simple additive therapy. This result clearly demonstrates that the combination of S-(-)-cotinine and rivastigmine produces a very strong synergistic inhibitory effect, effectively enhancing the inhibitory capacity against BuChE, and providing key pharmacological evidence for the significant efficacy of the composition of this invention in the treatment of neurodegenerative diseases.

[0066] Example 6 This embodiment provides the application of a group of pharmaceutical compositions, including: (1) Cell culture and plating Remove BV2 microglia from the culture flask, discard the supernatant, and wash twice with pre-warmed 50 mM PBS buffer. Add 2 mL of complete culture medium and gently pipette for 30 seconds to detach the cells. Transfer the cell suspension to a 5 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add 1 mL of complete culture medium to resuspend the cells, and pipette thoroughly. Add a small amount of cell suspension to a cell counting chamber, and count the cells in five large squares under a microscope. Multiply the sum by 5 to obtain the total cell count (in ten thousand units), and adjust the cell density to 4 × 10⁻⁶ cells / cells using complete culture medium. 4 Cells / mL. Take a 24-well plate, add 200 μL of the above cell suspension to each well (mix continuously during sample addition), and then add 800 μL of complete culture medium to make the total volume of each well 1 mL. Set up a control group and a blank group, and incubate the 24-well plate in a 37℃, 5% CO2 incubator overnight for 12 hours to allow the cells to adhere.

[0067] (2) Remove the 24-well plate and observe the cell growth status under a microscope. When the cell confluence reaches 80%-90%, perform the following operations: Add 980 μL of complete culture medium + 10 μL of a mixed solution of S-(-)-cotinine and rivastigmine (20 μM S-(-)-cotinine + 20 μM rivastigmine).

[0068] Comparative Example 16 The difference from Example 6 is that: (2) the 24-well plate is removed, and the cell growth status is observed under a microscope. When the cell confluence reaches 80%-90%, the following operations are performed, including: Add 980 μL of complete culture medium + 10 μL of S-(-)-cotinine solution (40 μM S-(-)-cotinine) to it.

[0069] Comparative Example 17 The difference from Example 6 is that: (2) the 24-well plate is removed, and the cell growth status is observed under a microscope. When the cell confluence reaches 80%-90%, the following operations are performed, including: Add 980 μL of complete culture medium + 10 μL of rivastigmine solution (40 μM rivastigmine) to it.

[0070] Comparative Example 18 (Blank Group) The difference from Example 6 is that: (2) the 24-well plate is removed, and the cell growth status is observed under a microscope. When the cell confluence reaches 80%-90%, the following operations are performed, including: Add 990 μL of complete culture medium + 10 μL of complete culture medium to it.

[0071] Comparative Example 19 (Standard Group) The difference from Example 6 is that: (2) the 24-well plate is removed, and the cell growth status is observed under a microscope. When the cell confluence reaches 80%-90%, the following operations are performed, including: Add 990 μL of complete culture medium + 10 μL of LPS solution (lipopolysaccharide, purchased from Beijing Ita Biotechnology Co., Ltd.) (1.5 μg / mL).

[0072] The 24-well plates obtained in Example 6 and Comparative Examples 16-19 were incubated at 37°C with 5% CO2 for 24 hours. After incubation, the 24-well plates were removed, and 50 μL of supernatant was transferred from each well to a new 96-well plate for subsequent NO content determination.

[0073] The NO content was determined using the Griess method. The Griess reagent was prepared as follows: Reagent A: Weigh 1 g of anhydrous p-aminobenzenesulfonic acid and dissolve it in 6 mL of 85% concentrated phosphoric acid, then add deionized water to make up to 100 mL; Reagent B: Weigh 0.1 g of N-(1-naphthyl)-ethylenediamine dihydrochloride and dilute to 100 mL with deionized water.

[0074] Standard curve preparation: Take 1 M sodium nitrite standard and dilute it sequentially with complete culture medium to prepare standard solutions of 100, 60, 40, 20, 10, 5, 2, 1, and 0 μM / mL. Add 50 μL of each concentration of standard or the supernatant to be tested to a 96-well plate, then add 50 μL of Griess reagent A and 50 μL of Griess reagent B sequentially. Mix well and incubate at 37°C in the dark for 5 minutes. Measure the absorbance (OD) of each well at 540 nm using a microplate reader. 540 A standard curve was plotted with sodium nitrite concentration on the x-axis and absorbance values ​​on the y-axis, as shown below. Figure 2 As shown in A in the diagram.

[0075] Sample detection: Add 50 μL of Griess reagent A and 50 μL of Griess reagent B to a 96-well plate containing the test supernatant, mix well, react under the conditions described above, and measure the OD. 540 Value. Calculate the NO concentration in the supernatant of each sample based on the standard curve.

[0076] Inhibition rate calculation: The inhibition rate of LPS-induced NO release in each group was calculated using the following formula: Inhibition rate (%) = [(Comparative Example 19 OD) 540- Example 6 OD 540 ) / (Comparative Example 19 OD 540 - Comparative Example 18 OD 540 ) ] × 100%; All experiments were performed in triplicate, and the results are expressed as mean ± standard deviation (Mean ± SD).

[0077] Test results are as follows Figure 2 As shown in B in the diagram.

[0078] Depend on Figure 2 As indicated by B in the diagram, S-(-)-cotinine, compared to racemic cotinine and R-(+)-cotinine, exhibits superior ability to inhibit LPS-induced NO release from BV2 cells. Compared to its prodrugs S-(-)-nicotinine and rivastigmine, it significantly reduces LPS-induced NO release from BV2 cells. The combination therapy was also examined. In the combination therapy group, S-(-)-cotinine, co-incubated with rivastigmine, significantly reduced NO production. This suggests that S-(-)-cotinine has a protective effect against NO-induced neuronal apoptosis and damage. When used in combination with other drugs, it can enhance this NO-inhibiting effect, providing better protection for nerve cells, reducing neuroinflammation, and thus making it more suitable for the treatment of Alzheimer's disease (AD).

[0079] Example 7 This embodiment provides the application of a group of pharmaceutical compositions, including: (1) Cell culture and plating Same as Example 6.

[0080] (2) Culture medium 980 μL complete culture medium + 10 μL mixed solution of S-(-)-cotinine and rivastigmine (concentration 400 nM).

[0081] Comparative Example 20 The difference from Example 7 is that the culture medium consists of 980 μL of complete culture medium + 10 μL of S-(-)-cotinine solution (concentration 400 nM).

[0082] Comparative Example 21 The difference from Example 7 is that the culture medium consists of 980 μL of complete culture medium + 10 μL of rivastigmine solution (concentration 400 nM).

[0083] Comparative Example 22 (Blank Group) The difference from Example 7 is that the culture medium consists of 990 μL of complete culture medium + 10 μL of complete culture medium.

[0084] Comparative Example 23 (Standard Group) The difference from Example 7 is that the culture medium consists of 990 μL of complete culture medium + 10 μL of LPS solution.

[0085] Test 1 The 24-well plates prepared in Example 7 and Comparative Examples 20-23 were incubated at 37°C in a 5% CO2 incubator for 4 hours. Subsequently, 10 μL of LPS solution (final concentration 1.5 μg / mL) was added to each well of Comparative Examples 20, 21, 7, and 23, while 10 μL of complete culture medium was added to Comparative Example 22. After incubation for another 24 hours, the cell supernatant from each well was collected and stored at -80°C for later analysis.

[0086] The concentration of IL-6 in the supernatant was detected using the Mouse IL-6 ELISA kit. The specific operating steps are as follows: (1) Reagent preparation: Washing solution: Take 1 mL of 30× concentrated washing solution and dilute it with 29 mL of distilled water to make 1× washing solution.

[0087] Standards: The IL-6 standard was diluted with standard diluent to eight concentration gradients: 1000.0, 500.0, 250.0, 125.0, 62.5, 31.25, 15.6, and 0 pg / mL.

[0088] Biotinylated antibody working solution: Dilute 100× biotinylated detection antibody at a ratio of 1:100 with biotinylated antibody diluent.

[0089] SABC working solution: Dilute 100× SABC with SABC diluent at a ratio of 1:100.

[0090] TMB developer: Mix TMB developer solution A and solution B in a 1:1 ratio until homogeneous, and store in the dark.

[0091] (2) Detection steps: Sample loading: Take a pre-coated antibody-labeled ELISA plate, add 50 μL of standard diluent to the blank wells, and add 50 μL of standard or sample supernatant to the remaining wells. After sealing the plate, incubate at 37°C for 50 minutes.

[0092] Washing: Discard the liquid in the wells, add 300 μL of 1× washing solution to each well, soak for 1-2 minutes, repeat washing 3 times, and pat dry.

[0093] Add biotinylated antibody: Add 100 μL of biotinylated antibody diluent to the blank wells and 100 μL of biotinylated antibody working solution to the remaining wells. Incubate at 37°C in the dark for 50 minutes.

[0094] Washing the plate: Same as step 2 above.

[0095] Add SABC: Add 100 μL of SABC working solution to each well and incubate at 37°C in the dark for 30 minutes.

[0096] Washing the plate: Same as step 2 above.

[0097] Color development: Add 100 μL of freshly prepared TMB mixture to each well and react at 37°C in the dark for 10-20 minutes.

[0098] Termination and detection: Add 50 μL of stop solution to each well, mix well, and measure the absorbance (OD) of each well at 450 nm using a microplate reader within 30 minutes. 540 ).

[0099] (3) Data Analysis Plotting the standard concentration on the x-axis, corresponding to OD 540 The ordinate is used to plot the IL-6 standard curve using a four-parameter fitting method, as shown below. Figure 3 As shown in A, the IL-6 concentration in the supernatant of each test sample was calculated based on the standard curve. The IL-6 concentration (pg / mL) in the sample was calculated using the following formula: IL-6 concentration (pg / mL) = Standard concentration × (sample tube OD) 540 - Blank tube OD 540 ) / (Standard pipe OD 540 - Blank tube OD 540 ) All experiments were performed in triplicate, and the results are expressed as mean ± standard deviation (Mean ± SD).

[0100] Test results are as follows Figure 3 As shown in B in the diagram.

[0101] Depend on Figure 3As indicated by B in the diagram, S-(-)-cotinine, compared to racemic cotinine and R-(+)-cotinine, exhibits superior ability to inhibit LPS-induced IL-6 release from BV2 cells. Compared to its prodrug S-(-)-nicotinine and the positive control group rivastigmine, it significantly reduces LPS-induced IL-6 release from BV2 cells. The combination therapy was also examined. In the combination therapy group, S-(-)-cotinine, co-incubated with rivastigmine, significantly reduced IL-6 production. This suggests that S-(-)-cotinine has a protective effect against neuronal apoptosis and damage induced by the inflammatory factor IL-6. When used in combination with other drugs, it can enhance this ability to inhibit IL-6, providing better protection for nerve cells, reducing neuroinflammation, and thus making it more suitable for the treatment of Alzheimer's disease (AD).

[0102] Test 2 The 24-well plates prepared in Example 7 and Comparative Examples 20-23 were incubated at 37°C in a 5% CO2 incubator for 4 hours. Subsequently, 10 μL of LPS solution (final concentration 1.5 μg / mL, for BV2 cells) was added to each well of Comparative Examples 20, 21, 7, and 23, while 10 μL of complete culture medium was added to Comparative Example 22. Incubation continued for 6 hours.

[0103] Discard the culture medium from each well and dilute the DCFH-DA probe to 10 μM with serum-free medium. Add 500 μL of DCFH-DA solution to each well and incubate at 37°C in the dark for 30 minutes. After incubation, discard the probe solution and gently wash the cells three times with serum-free medium to remove any probe that has not entered the cells. Finally, add 200 μL of PBS buffer to each well to disperse the cells.

[0104] The 24-well plate was placed under an inverted fluorescence microscope, and the intensity of green fluorescence in the cells was detected using 488 nm excitation light and 525 nm emission light. Higher fluorescence intensity indicates higher intracellular ROS levels.

[0105] Test results are as follows Figure 4 As shown.

[0106] Depend on Figure 4The results showed that, regarding LPS-induced ROS production in BV2 cells, the intensity of the green fluorescence indicated that the S-(-)-cotinine and S-(-)-nicotinic acid groups had a better ROS scavenging ability than rivastigmine. Specifically, the expression of S-(-)-cotinine was superior to that of racemic cotinine and R-(+)-cotinine, indicating that the levorotatory form of cotinine is more active in cells than the dextrorotatory and racemic forms. Co-incubation of S-(-)-cotinine with rivastigmine further aided in ROS scavenging. This suggests that combined drug therapy is more effective in eliminating the abnormally high levels of ROS in neuroinflammation.

[0107] In summary, the S-(-)-cotinine and rivastigmine pharmaceutical composition provided by this invention exhibits significantly superior overall efficacy compared to monotherapy through multi-target synergistic effects. This composition not only demonstrates synergistic enhancement in inhibiting butyrylcholinesterase (BuChE), with activity far exceeding that of rivastigmine monotherapy, effectively enhancing cholinergic neurotransmission; simultaneously, S-(-)-cotinine exerts multiple effects including anti-inflammatory, antioxidant, metal ion chelation, and neuroprotective properties, synergistically inhibiting the release of inflammatory factors such as nitric oxide (NO) and interleukin-6 (IL-6), scavenging reactive oxygen species (ROS), and intervening in core pathological processes such as Aβ aggregation and Tau protein phosphorylation. Experiments have confirmed that the S-(-) configuration of S-(-)-cotinine exhibits optimal activity, and its combination with rivastigmine also shows a synergistic trend in antioxidant kinetics, laying a solid foundation for the treatment of neurodegenerative diseases such as Alzheimer's disease.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, It contains S-(-)-cotinine or a pharmaceutically acceptable salt thereof, and rivastigmine or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of S-(-)-cotinine to rivastigmine is 1:1 to 1:

5.

3. The pharmaceutical composition according to claim 2, characterized in that, The mass ratio of S-(-)-cotinine to rivastigmine is 1:

1.

4. The pharmaceutical composition according to claim 1, characterized in that, Pharmaceutically acceptable salts of the S-(-)-cotinine include its hydrochloride, tartrate, citrate, or fumarate; pharmaceutically acceptable salts of the rivastigmine are its tartrate.

5. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition further includes one or more pharmaceutically acceptable excipients.

6. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a medicament for treating neurodegenerative diseases.

7. A drug for treating neurodegenerative diseases, characterized in that, The drug comprises the pharmaceutical composition as described in any one of claims 1-4.

8. The medicament for treating neurodegenerative diseases as described in claim 6, characterized in that, The treatment of neurodegenerative diseases includes, but is not limited to, Alzheimer's disease.

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