Tropane alkaloid composition for treating COPD and its application

Abstract

The present invention relates to the technical field of heterocyclic compound drugs and their preparation and application, and specifically to a tropane alkaloid composition for the treatment of COPD and its application. The composition combines tropane alkaloids, mannose-6-phosphate and polylactic acid-glycolic acid copolymer, and is constructed into a nanocomposite by nanoparticle preparation technology for aerosol inhalation administration. The composition aims to solve the problems of low bioavailability and lack of targeting in conventional administration of tropane alkaloids. Targeted delivery is achieved by introducing mannose-6-phosphate into the surface of nanoparticles, promoting drug enrichment in the lungs, and using polylactic acid-glycolic acid copolymer to control drug release, thereby increasing local drug concentration and therapeutic efficacy in the lungs.

Description

Technical Field

[0001] The present invention relates to the technical field of heterocyclic compound drugs and their preparation and application, and in particular to a tropane alkaloid composition for treating COPD and its application. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease characterized by persistent airway inflammation and irreversible airflow limitation, with pathological manifestations including small airway remodeling and alveolar destruction. In the lungs of COPD patients, inflammatory cells such as alveolar macrophages increase significantly and secrete a variety of inflammatory mediators, promoting disease progression. Studies have shown that alveolar macrophages play a key role in the pathogenesis of COPD and are an important target for anti-inflammatory therapy. However, because alveolar macrophages in COPD patients respond poorly to traditional glucocorticoid anti-inflammatory therapy, new strategies are urgently needed to effectively control chronic lung inflammation.

[0003] The first-line treatment for COPD is combined inhalation therapy of bronchodilators and anti-inflammatory drugs. Currently, commonly used bronchodilators in clinical practice include beta-receptor agonists and anticholinergics. Anticholinergics achieve bronchial smooth muscle relaxation and reduced mucus secretion by blocking vagus nerve-mediated bronchoconstriction. Tropane alkaloids are a typical class of anticholinergic drugs with pharmacological effects such as inhibiting glandular secretion and relieving bronchospasm. However, the bioavailability of the active ingredients of tropane alkaloids in traditional dosage forms is low and the duration of action is short. The bioavailability of tropane alkaloids is only about 10-48% when administered orally, and the peak blood level is reached about 0.5 hours after administration, indicating that the drug is absorbed quickly but has a strong transient effect, and frequent administration is required to maintain efficacy. In COPD inhalation therapy, if a tropane alkaloid solution is directly inhaled through atomization, the drug may also have a limited residence time in the lungs due to factors such as mucociliary clearance and rapid lung absorption, making it difficult to maintain sufficient concentration at the lesion site. In addition, free small molecule drugs lack selectivity for the lesion site and may still partially enter the systemic circulation after inhalation, causing systemic side effects. Prior art patent CN101669917A discloses a scopolamine hydrobromide orally disintegrating microcapsule tablet based on the "NiMS" system and a preparation method thereof, which is made from the "NiMS" system containing scopolamine hydrobromide and excipients. Excipients include microcrystalline cellulose, sodium carboxymethyl starch, mannitol, lactose, xylitol, magnesium stearate, micropowdered silica gel, etc. The microcapsule tablets prepared therefrom dissolve rapidly in the mouth through saliva, and the "NiMS" system has a sustained-release effect, but the scopolamine hydrobromide component contained therein does not have a targeted effect and still affects the whole body.

[0004] In summary, a new technical solution is urgently needed to use tropane alkaloids for targeted aerosol inhalation treatment of COPD, to solve the technical pain points of low bioavailability and lack of targeting of tropane alkaloid drugs in existing technologies, and to improve the efficacy and safety of pulmonary drug delivery. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of existing tropane alkaloid drugs in the treatment of COPD, such as low bioavailability and poor targeting, and to provide a tropane alkaloid composition and its use in the treatment of COPD. The composition uses a combination of tropane alkaloids, mannose-6-phosphate, and polylactic acid-glycolic acid copolymer to construct nanoparticles for aerosol inhalation administration, thereby achieving targeted and efficient drug delivery to the lungs. Tropane alkaloids, as the active ingredient of the present invention, achieve improved drug delivery performance through nanocarriers; M6P, as a targeting ligand, can be specifically recognized by receptors on the surface of alveolar macrophages and mediate phagocytosis of nanoparticles, thereby enriching the drug in the lesion area of ​​the lung; PLGA polymer, as a biodegradable carrier matrix, encapsulates the tropane alkaloids to form nanoparticles, achieving sustained and controlled release of the drug, reducing rapid clearance of the drug in the lungs and systemic absorption.

[0006] The specific technical solutions are as follows:

[0007] A tropane alkaloid composition for treating COPD, comprising tropane alkaloids, mannose-6-phosphate and polylactic acid-glycolic acid copolymer, wherein the tropane alkaloids, mannose-6-phosphate and polylactic acid-glycolic acid copolymer are combined to form a nanoparticle-shaped composition.

[0008] Furthermore, the mass ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 50:50 to 75:25.

[0009] Furthermore, the mass ratio of the tropane alkaloids to the polylactic acid-glycolic acid copolymer is 1:5 to 1:20.

[0010] Furthermore, the mannose-6-phosphate is used as a targeting ligand and is connected to the surface of the poly(lactic acid-co-glycolic acid) copolymer via a covalent bond.

[0011] Furthermore, the encapsulation efficiency of the polylactic acid-glycolic acid copolymer for the tropane alkaloids is ≥50%, and the drug loading amount of the tropane alkaloids in the nanoparticle composition is ≥5%.

[0012] Furthermore, the nanoparticle composition can be formulated into a suspension for administration in the form of aerosolization.

[0013] Furthermore, the tropane alkaloid is hyoscyamine.

[0014] The present invention also provides a method for preparing a tropane alkaloid composition for treating COPD, comprising the following steps:

[0015] S1: preparing the tropane alkaloids to form an inner aqueous phase, and preparing the polylactic acid-co-glycolic acid to form an organic phase;

[0016] S2: injecting the inner aqueous phase prepared in S1 into the organic phase while emulsifying in an ice bath to obtain W / O colostrum;

[0017] S3: transferring the W / O colostrum prepared in S2 into a polyvinyl alcohol solution and emulsifying again to obtain a W / O / W emulsion;

[0018] S4: The W / O / W emulsion prepared in S3 was magnetically stirred at room temperature to evaporate the solvent and centrifuged to recover the nanoparticles, which were washed with distilled water to obtain wet nanoparticles, and then the wet nanoparticles were resuspended in a buffer solution;

[0019] S5: adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to the wet nanoparticle suspension prepared in S4 for activation, then adding mannose-6-phosphate, reacting at room temperature, and purifying by ultrafiltration to obtain a tropane alkaloid composition.

[0020] Furthermore, the re-emulsification described in S3 specifically includes using a high-speed homogenizer to emulsify at 5000-10000 rpm for 60 seconds.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The nanoparticles of the present invention can penetrate deep into the bronchi and alveoli after aerosol inhalation. Their average particle size is in the micrometer / nanometer range, which facilitates bypassing upper respiratory tract deposition and directly reaching the lower respiratory tract. Compared with traditional solution aerosol administration, the nanocomposition of the present invention significantly improves the drug deposition rate in the lungs, allowing a larger effective dose to be delivered to the lungs.

[0023] (2) The M6P ligand on the surface of the nanoparticles of the present invention enables them to be preferentially taken up by target cells such as alveolar macrophages. The distribution of the drug in the lungs is more concentrated in the diseased tissue, reducing the proportion of drug escaping into the blood circulation, thereby reducing the risk of systemic adverse reactions. Compared with non-targeted nanoparticles or free drugs, the composition of the present invention has a higher drug concentration in the target tissue and a lower concentration in non-target sites such as plasma.

[0024] (3) After the nanocomposition of the present invention enters the lungs, the tropane alkaloids released exert an anticholinergic effect, effectively relieving bronchospasm and improving ventilation. At the same time, after the alveolar macrophages take up the drug, the inflammatory mediators they release are inhibited, which helps to alleviate the chronic inflammation associated with COPD. By targeting these cells, the present invention achieves the dual therapeutic effects of bronchodilator and local anti-inflammatory. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a flow chart of a method for preparing a tropane alkaloid composition for treating COPD according to the present invention;

[0026] Figure 2 This is the Fourier transform infrared spectrum in Experimental Example 1 of the present invention;

[0027] Figure 3 This is a comparison chart of the mean and standard deviation of the tropane alkaloid concentrations in lung tissue and plasma tested in Experimental Example 4 of the present invention;

[0028] Figure 4 This is a comparison chart of the mean and standard deviation of interleukin-8 and tumor necrosis factor-α concentrations in bronchoalveolar lavage fluid tested in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0029] The following examples further illustrate and describe the technical solutions of the present invention. It is particularly noted that each specific embodiment is intended to be a concretization and explanation of the technical solutions and should not be construed as limiting the scope of protection of the present invention. Persons of ordinary skill in the art are entitled to modify the technical solutions of these embodiments and to substitute equivalent features for some or all of the technical features. Such modifications or substitutions do not alter the essence of the corresponding technical solutions and do not deviate from the scope of the technical solutions described in the present invention.

[0030] The present invention provides a tropane alkaloid composition for treating COPD and its application, as shown in the attached Figure 1 The figure shows a flow chart of the preparation method of the tropane alkaloid composition of the present invention. The preparation of the tropane alkaloid composition of the present invention utilizes nanoemulsification-solvent evaporation technology. Preferably, a water-oil-water (W / O / W) double emulsification process is used to encapsulate the tropane alkaloid (hydrophilic) in poly(lactic-co-glycolic acid) (PLGA) nanoparticles. Mannose-6-phosphate (M6P) targeting ligands are then introduced onto the nanoparticle surface. By adjusting the raw material ratio and process parameters, the nanoparticle size, encapsulation efficiency, and surface ligand density can be controlled. The detailed preparation steps are as follows:

[0031] 1. Selection of main raw materials

[0032] Medical grade PLGA polymer is selected as the carrier material, preferably a copolymer with a lactic acid: glycolic acid mass ratio of 50:50 to 75:25, a molecular weight range of 10,000 to 50,000, and a carboxyl end group to facilitate subsequent coupling with the ligand. The active drug is a tropane alkaloid, preferably hyoscyamine, and hyoscyamine hydrobromide is used when preparing the solution; the targeting ligand is mannose-6-phosphate (M6P) or a soluble salt thereof, preferably M6P disodium salt; the emulsifier is a hydrophilic colloid stabilizer such as polyvinyl alcohol (PVA); the coupling reagent is a water-soluble carbodiimide, preferably 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), the combination of which can better couple M6P to the surface of the nanoparticles; other auxiliary reagents such as the buffer solution are preferably 2-morpholineethanesulfonic acid buffer (MES) or phosphate buffered saline (PBS), and the organic solvent is selected from dichloromethane or ethyl acetate.

[0033] 2. Preparation of the Inner Aqueous Phase

[0034] A certain amount of tropane alkaloids is dissolved in deionized water to prepare an inner aqueous phase solution. Subsequently, depending on the required drug loading, the mass ratio of tropane alkaloids to PLGA is in the range of 5:1 to 20:1. The preferred process is to weigh the tropane alkaloids, dissolve them in ultrapure water to form an inner aqueous phase, and add buffer salt to adjust the pH to neutral, which can improve drug stability.

[0035] 3. Organic Phase Preparation

[0036] The PLGA polymer is dissolved in an organic solvent as the oil phase. The organic solvent used is low-boiling, water-immiscible dichloromethane or ethyl acetate. The preferred process is to weigh the PLGA, place it in dichloromethane, and stir thoroughly until it is completely dissolved to prepare a polymer organic phase solution. 0.5 wt% of the hydrophobic additive D-α-tocopheryl polyethylene glycol succinate is added to the prepared oil phase to enhance emulsification.

[0037] 4. Colostrum Preparation (W / O)

[0038] The aqueous phase solution is slowly added to the PLGA oil phase and emulsified using a high-speed homogenizer or ultrasonic probe in an ice bath to form a water-in-oil primary emulsion. Emulsification parameters can be adjusted according to the target particle size. A preferred process includes emulsification at 15,000 rpm for 60 seconds using a homogenizer, or intermittently using a 100W ultrasonic probe for 30 seconds to produce a uniform and fine colostrum. This step produces droplets containing tropane alkaloids that are encapsulated by the polymer organic phase, forming a W / O emulsion.

[0039] 5. Secondary emulsification (W / O / W)

[0040] The obtained colostrum is quickly transferred to the pre-cooled external aqueous phase and emulsified again to form a water-in-oil-in-water double emulsion, wherein the volume ratio of the inner and outer aqueous phases is preferably 1:30 to 1:50; the external aqueous phase is an aqueous solution containing a stabilizer, preferably 20 mL of a 2% w / v PVA solution. During preparation, the colostrum is added dropwise to the PVA solution under stirring conditions and emulsified using a high-speed homogenizer at 5000 to 10000 rpm for 60 seconds to form a uniform W / O / W secondary emulsion. This preparation process further thins the polymer interface and locks the inner aqueous phase droplets to prevent polymerization leakage.

[0041] 6. Solvent evaporation and nanoparticle solidification

[0042] The prepared W / O / W emulsion was magnetically stirred at room temperature for 2 hours to allow the organic solvent to fully evaporate. As the organic solvent, such as dichloromethane, evaporated, the PLGA dissolved therein gradually precipitated and solidified, encapsulating the inner aqueous phase to form solid nanoparticles. After the organic solvent was completely removed, the emulsion was observed to change from milky white to a translucent suspension. At this time, the nanoparticle precipitate was recovered by centrifugation. The centrifugation parameters were set at a speed of 12,000 rpm and a centrifugation time of 10 minutes. After discarding the supernatant, the mixture was washed with distilled water 3 to 5 times to remove residual PVA and free drug. Finally, the nanoparticles were resuspended in MES or PBS buffer for later use.

[0043] Particle Size Control Based on the Above-mentioned Steps: The average particle size of the nanoparticles of the present invention can be controlled by adjusting the process parameters in the above-mentioned steps. Increasing the emulsification shear rate and decreasing the polymer concentration will produce nanoparticles with smaller particle sizes and narrower particle size distribution, while decreasing the shear strength or increasing the polymer dosage will increase the particle size. By controlling the rotation speed and time of the primary and secondary emulsifications, as well as the volume ratio of the internal and external aqueous phases, the average particle size of the nanoparticles can be controlled within the range of 100-500 nm, preferably 180-220 nm. The particle size distribution of the nanoparticles of the present invention is preferably PDI < 0.3. Nanoparticles of this preferred size are more suitable for deposition in the alveoli.

[0044] 7.M6P coupling

[0045] The PLGA drug-loaded nanoparticles obtained above were surface-coupled with an M6P ligand to impart alveolar macrophage targeting. This coupling method employed a water-soluble carbodiimide activation-esterification coupling reaction: EDC and NHS were added to the nanoparticles suspended in a weakly acidic MES or PBS buffer at a molar ratio of EDC:NHS:PLGA carboxyl group of 2:2:1. The nanoparticles were activated at room temperature for 15 minutes. Then, M6P disodium salt solution was added at a mass ratio of M6P:PLGA of 5:95, and the mixture was allowed to react for 2 hours.

[0046] Through the above reaction, the carboxyl groups at the ends of the PLGA chains and on the surface of the nanoparticles form covalent bonds with the hydroxyl / phosphate groups on the M6P molecules, fixing M6P on the surface of the nanoparticles. After the reaction is completed, ammonium chloride is added to the system to quench the residual active esters, and the coupled nanoparticles are separated from the free reaction reagents by ultrafiltration to purify the tropane alkaloid nanoparticles with surface-bound M6P.

[0047] 8. Storage Form of Nanoparticle Composition

[0048] To facilitate clinical use and long-term storage, the nanoparticle composition of the present invention can be prepared as a lyophilized powder. Preparation: Add an appropriate amount of a cryoprotectant, preferably 5wt% mannitol or 6wt% sucrose, to the nanoparticle suspension. After thorough mixing, freeze-dry the suspension using a freeze dryer to obtain a loose nanoparticle powder. The addition of a cryoprotectant effectively prevents aggregation of the nanoparticles during the lyophilization process, maintaining the stability of the particle size and active ingredient.

[0049] The lyophilized powder preparation, when stored sealed and protected from light at room temperature, remains stable for over six months without significant change in particle size. Upon use, the lyophilized powder is aseptically resuspended in normal saline to prepare a nanoparticle suspension suitable for aerosol inhalation. After lyophilization and reconstitution, the nanoparticle morphology and size remain essentially the same as before lyophilization, and the active tropane alkaloid content remains above 95% of the initial dose, meeting quality requirements for aerosol administration.

[0050] Example 1

[0051] A tropane alkaloid composition for treating COPD and its application are as follows:

[0052] S1: Dissolve 10 mg of L-anisidine hydrobromide in 0.5 mL of pure water to form the inner aqueous phase; take PLGA (lactic acid: glycolic acid = 50:50, carboxyl end-capped, M w ≈30 kDa) 100 mg was dissolved in 5 mL of dichloromethane and 0.5 wt% of the hydrophobic auxiliary agent D-α-tocopheryl polyethylene glycol succinate was added to form an organic phase.

[0053] S2: Use a step-by-step injection device to slowly inject the inner aqueous phase into the PLGA dichloromethane solution, and use a probe-type ultrasonic instrument to emulsify while injecting. Place it in an ice bath during emulsification, set the ultrasonic power to 100W, and emulsify in gap mode for 30 seconds to obtain W / O colostrum.

[0054] S3: Immediately transfer the W / O colostrum into 20 mL of 2% w / v PVA solution and homogenize at 10,000 rpm for 60 seconds to form a W / O / W emulsion.

[0055] S4: The W / O / W emulsion was magnetically stirred at room temperature for 2 hours to evaporate the solvent, and the nanoparticles were recovered by centrifugation at 12,000 rpm for 10 minutes and washed 3-5 times with distilled water. Subsequently, the wet nanoparticles were resuspended in 5 mL of MES buffer.

[0056] S5: EDC / NHS was added to the wet nanoparticle suspension for activation, with the molar ratio of EDC:NHS:PLGA carboxyl set to 2:2:1, and then M6P disodium salt solution was added in an amount such that the mass ratio of M6P:PLGA was 5:95. The reaction was carried out at room temperature for 2 hours. Ammonium chloride was then added to quench the residual active esters, and tropane alkaloid composition nanoparticles were obtained after purification by ultrafiltration.

[0057] Example 2

[0058] Referring to the preparation method of Example 1, this example examined the changes in nanoparticle properties at higher drug loadings. 50 mg of PLGA was dissolved in 4 mL of dichloromethane, and 10 mg of L-anisidine hydrobromide was dissolved in 0.5 mL of water. W / O / W emulsification and solvent evaporation were performed as in Example 1 to obtain drug-loaded nanoparticles. Subsequently, EDC / NHS activation was performed, and 5 mg of M6P was added as a surface ligand.

[0059] Example 3

[0060] Referring to the preparation method of Example 1, this example examined the effect of reducing drug loading (increasing the polymer ratio) on nanoparticle performance. 200 mg of PLGA was dissolved in 8 mL of dichloromethane, and 10 mg of L-anisidine hydrobromide was dissolved in 1 mL of water. Nanoparticles were prepared using the same emulsification process as in Example 1 and coupled with 5 mg of M6P ligand.

[0061] Example 4

[0062] Referring to the preparation method of Example 1, this example increased the ligand density on the nanoparticle surface to evaluate changes in targeting efficiency. L-anisopamine-loaded PLGA nanoparticles were prepared according to the method of Example 1, and then the amount of M6P disodium salt was doubled in the coupling step, while other conditions remained unchanged.

[0063] Example 5

[0064] This example, based on Example 1, prepared nanoparticles with larger particle sizes by reducing the emulsification shear strength and investigated their drug delivery efficacy. Specifically, the homogenization speed in the secondary emulsification step in Example 1 was reduced from 10,000 rpm to 5,000 rpm, while the remaining steps and formulations remained the same.

[0065] Comparative Example 1

[0066] PLGA-loaded L-anisidine nanoparticles were prepared according to the method of Example 1, but without the M6P coupling step. 100 mg of PLGA and 10 mg of L-anisidine hydrobromide were emulsified according to the emulsification process of Example 1 to prepare drug-loaded nanoparticles, which were directly used for evaluation.

[0067] Comparative Example 2

[0068] A conventional nebulized inhalation formulation of L-anisopamine was prepared as a control. L-anisopamine hydrobromide was dissolved in 0.9% saline to create a clear solution at a concentration of 0.5 mg / mL. This solution was used directly for nebulized administration.

[0069] Experimental Example 1

[0070] The sample of the composite nanoparticles prepared in Example 1 was subjected to Fourier transform infrared spectroscopy (FTIR) testing. The testing method was based on the national standard GB / T 32199-2015: "General Rules for Qualitative Analysis Techniques of Infrared Spectroscopy". The test results are shown in the attached figure. Figure 2 As shown in the figure, it can be seen that the characteristic peak of the sample PLGA C = O stretching vibration (ester carbonyl) is at; COC stretching vibration (ether bond) at The inner superposition shows the COC asymmetric stretching vibration attributed to the ester bond in the PLGA skeleton; the P=O stretching vibration (phosphate) The internal superposition is attributed to the P=O bond of the M6P phosphate group, and the POC stretching vibration is The internal superposition shows that the POC bond belongs to the connection between the phosphate and the sugar ring, but it partially overlaps with the CO peak of PLGA; the CN stretching vibration (tertiary amine) The weak peak in the inner part is the tertiary amine group in the tropane ring of hyoscyamine, which is not obvious because it is covered by the strong peak of PLGA / M6P. The CO stretching vibration (ester bond) is The overlap with the carbonyl group of PLGA belongs to the ester group in the molecule of L-anisodamine. The peak is broadened and slightly shifted; the broad peak at 3400 cm⁻¹ contains the OH group of M6P, the terminal hydroxyl group of PLGA and the residual moisture of the sample; as mentioned above, the position of the characteristic absorption peak is basically consistent with the expected characterization of the nanoparticle complex, indicating that the composition of the preparation is in line with expectations.

[0071] Experimental Example 2

[0072] The nanoparticles finally prepared in Examples 1 to 5 and Comparative Example 1 were sampled, and their particle size parameters and Zeta potential were measured respectively. The test method was as follows: 1 mL of the nanoparticle sample dispersion suspension was taken into a 5 mL centrifuge tube, diluted to 5 mL with pure water, and the particle size and Zeta potential of the nanoparticle sample were measured using a laser particle size analyzer. The test results are shown in Table 1.

[0073] Table 1 Comparison of experimental results of Experimental Example 2

[0074]

[0075] From the above results, it can be seen that the particle size of Example 2 is slightly smaller than that of Example 1. When the mass ratio of polymer to drug is reduced from 10:1 to 5:1, the particle size of the nanoparticles formed will be slightly reduced; Example 3 is slightly larger than Example 1, and the high polymer content will increase the particle size accordingly; Example 4 is similar to Example 1, and the slight increase is due to the increase in surface-modified ligands; Example 5 greatly increases the particle size by reducing the emulsification shear strength, and the particle size distribution is also widened; the particle size and dispersity of Comparative Example 1 are similar to those of Example 1, indicating that the ligand modification has little effect on these parameters; the overall change in the Zeta potential of the Examples and Comparative Examples is small.

[0076] Experimental Example 3

[0077] The nanoparticles finally prepared in Examples 1 to 5 and Comparative Example 1 were sampled and their encapsulation efficiency and drug loading were measured using a high performance liquid chromatography (HPLC) instrument. The specific parameters were set as follows:

[0078] Free drug separation: Using ultrafiltration centrifugation, take 1 mL of the nanoparticle sample suspension, place it in an ultracentrifuge tube, and centrifuge it at 10,000 rpm for 10 minutes. The supernatant (containing free drug) is aspirated and precipitated as drug-loaded nanoparticles;

[0079] Total drug release: Using the organic solvent demulsification method, take another 1 mL of the nanoparticle sample suspension, add acetonitrile at a ratio of 1:2 v / v, vortex for 5 minutes, and ultrasonicate for 10 minutes (in an ice bath) to completely dissolve the PLGA carrier and release the drug completely;

[0080] Sample processing after separation and release: the supernatant (free drug) and total drug release solution were filtered with a 0.22 μm filter membrane and prepared for HPLC injection; the precipitate (drug-loaded nanoparticles) was washed with distilled water, freeze-dried, and weighed (for drug loading calculation);

[0081] HPLC analysis conditions were set as follows: the chromatographic column was a C18 reverse-phase column (250 × 4.6 mm, 5 μm); the mobile phase was acetonitrile / 0.1% TFA aqueous buffer salt, gradient elution; the flow rate was set to 1.0 mL / min; the detection wavelength was the characteristic absorption wavelength of L-anisidine; the column temperature was set to 30°C, the injection volume was set to 20 μL, and the running time was set to 15-20 minutes.

[0082] The encapsulation efficiency calculation formula is: EE =[( C T -C F ) / C T ]×100%; the drug loading calculation formula is: drug loading DL =[( C T - C F ) / W np ]×100%; among which, C T is the total amount of drug in the nanoparticles, C F is the amount of free drug, W np is the dry weight of drug-loaded nanoparticles. The test results are shown in Table 2.

[0083] Table 2 Comparison of experimental results of Experimental Example 3

[0084]

[0085] It can be seen from the above results that the encapsulation efficiency of Example 2 is greatly reduced and the drug loading is increased, which indicates that when the mass ratio of polymer to drug is reduced from 10:1 to 5:1, part of the drug is not effectively encapsulated due to the increase in the relative content of the drug; the encapsulation efficiency of Example 3 is improved, but the drug loading is reduced, which shows that a higher polymer content helps to completely encapsulate the drug and form a thicker carrier matrix, but the corresponding increase in particle size will cause a decrease in drug loading; the encapsulation efficiency and drug loading of Example 4 are similar to those of Example 1, indicating that the increase of the modified ligand has little effect on the encapsulation efficiency and drug loading; although the particle size of Example 5 is greatly increased, it has little effect on the encapsulation efficiency and drug loading; the encapsulation efficiency and drug loading of Comparative Example 1 are similar to those of Example 1, which also shows that the modified ligand itself has little effect on such parameters.

[0086] Experimental Example 4

[0087] The drugs finally prepared in Examples 1 to 5 and Comparative Examples 1 to 2 were subjected to animal experiments to evaluate their medicinal effects. The specific experimental settings are as follows:

[0088] (1) Construction of COPD model: The experimental animals were obtained from the breeding farm of the Sichuan Provincial Experimental Animal Committee, license number: SCXK (Sichuan) 2018-14. Healthy experimental rabbits (male or female, weighing 2.0-2.5 kg) were selected, and a chronic obstructive pulmonary disease (COPD) model was induced by intratracheal instillation of lipopolysaccharide (LPS). The specific process was as follows: on the first day, 2 mL of LPS solution (containing approximately 2 mg of LPS) was slowly instilled into the lungs of the anesthetized rabbit through the trachea; on the 15th day, the same LPS intratracheal administration was repeated, and after the LPS was instilled, the chest was gently patted to assist distribution; during the modeling period, the general condition of the animals, such as changes in respiratory rate, coughing, and activity, was closely observed; through the above two intratracheal LPS stimulations, continuous airway inflammation and tissue damage in the rabbit lungs can be induced in a relatively short period of time, gradually forming a COPD model. This type of LPS induction method will cause chronic inflammatory changes similar to COPD in the bronchial and lung tissues of the model animals, with significantly increased levels of inflammatory mediators such as IL-8 and TNF-α, and decreased lung function indicators.

[0089] (2) Animal grouping: After the COPD model was successfully established, the model rabbits were evenly divided into 7 groups according to body weight and lesion severity, with 6 animals in each group. Each group was given different treatments:

[0090] Group Example 1: administered with the composition described in Example 1; Group Example 2: administered with the composition described in Example 2; Group Example 3: administered with the composition described in Example 3; Group Example 4: administered with the composition described in Example 4; Group Example 5: administered with the composition described in Example 5; Group Comparative Example 1: administered with the treatment regimen of Comparative Example 1; Group Comparative Example 2: administered with the treatment regimen of Comparative Example 2. The active ingredients in each treatment regimen were all tropane alkaloid active ingredients at equimolar doses to ensure comparability; except for the differences in treatment, all other modeling and housing conditions were identical.

[0091] (3) Administration and dosage: After the model was established, drug intervention began. The Example 1, 2, 3, 4, and 5 groups were administered with the tropane alkaloid composition of the present invention, while the Comparative Example 1 and Comparative Example 2 were administered with the drug-loaded nanoparticles and drug-containing solution prepared at the end. The preferred route of administration was transbronchial atomization inhalation, so that the composition acted directly on the lungs. The dosage was calculated based on the tropane alkaloid equivalent, with each rabbit receiving a daily dose of the composition equivalent to 0.5 mg / kg of tropane alkaloid. All administrations were performed once daily for 2 consecutive weeks. Specifically, the Example Group and the Comparative Example Group began administering the drug on the 16th day after the model was established, and the drug was administered once a day at a fixed time until the 28th day. The vital signs and behavior of the rabbits were strictly monitored during the administration process to ensure safety.

[0092] (4) Observation and sampling time points: During the drug intervention period, observation and sampling time points were set before, during, and after drug administration. First, before the first dose, that is, between the 15th and 16th days after the model was established, baseline lung function tests were performed on each group of model rabbits to record the degree of lung function decline caused by modeling.

[0093] Symptoms, such as relief of dyspnea and cough, were regularly observed during dosing. Any serious adverse reactions were promptly recorded and addressed. One hour after the final dose, endpoint assessments were performed, including pulmonary function testing and biological sample collection. For endpoint sampling, rabbits were anesthetized and pulmonary function tests were performed. They were then sacrificed by carotid exsanguination. Arterial blood was immediately collected for plasma separation, and lung tissue and bronchoalveolar lavage fluid (BALF) were obtained for subsequent analysis.

[0094] (5) Pulmonary function test: The respiratory function of each group of rabbits was evaluated using a pulmonary function test device. For each test, the animals were first anesthetized intravenously (50 mg / kg sodium pentobarbital), fixed in the supine position, and intubated. The rabbits were then connected to a pulmonary function test system (FlexiVent pulmonary function tester). Mechanical ventilation and pressure-volume testing were used to measure a number of pulmonary function parameters, including dynamic lung compliance (C_dyn), airway resistance (R_aw), residual volume, and peak expiratory flow rate. In addition, forced exhalation was simulated to measure the forced expiratory volume in one second (FEV_1) and forced vital capacity (FVC), and the FEV_1 / FVC ratio was calculated.

[0095] To quantify the therapeutic effect, the "pulmonary function improvement rate (%)" was calculated for each animal. This percentage represents the percentage improvement in the post-treatment parameter relative to the pre-treatment baseline value. The improvement rate is the average combined improvement rate for each parameter. The specific formula for calculating the improvement rate of each parameter is: Pulmonary function improvement rate = [(post-treatment parameter – pre-treatment parameter) ÷ pre-treatment parameter] × 100%. The average improvement rate for each group is used to compare the efficacy of different treatments. It is expected that the group treated with the combination will see significant improvement in pulmonary function parameters, while the control group, which did not receive effective treatment, will see limited improvement. Adequate anesthesia and accurate instrument calibration are essential during testing.

[0096] (6) Determination of tropane alkaloid concentration in lung tissue and plasma: The concentration of tropane alkaloids was quantitatively determined in lung tissue and plasma samples collected after the last administration by high performance liquid chromatography-mass spectrometry (HPLC-MS). Lung tissue samples were taken from the middle lobe of the right lung of each rabbit, minced, accurately weighed, and added with physiological saline to prepare a 10% tissue homogenate; the homogenate was extracted with an organic solvent, centrifuged to remove impurities, and the supernatant was taken for testing; plasma samples were obtained by anticoagulation and centrifugation of arterial blood. The processed lung homogenate extract and plasma samples were injected into a liquid chromatography-tandem mass spectrometer to determine their concentrations; each group of samples was measured in parallel 3 times and the average value was taken to calculate the average concentration of tropane alkaloids in lung tissue (in μg / g wet weight) and the average concentration in plasma (in ng / mL) of each group of rabbits. It is expected that the composition of the present invention can form a higher concentration in lung tissue and a lower concentration in plasma after pulmonary administration, reflecting the lung targeted enrichment characteristics.

[0097] (7) Analysis of inflammatory indicators in alveolar lavage fluid: The degree of airway inflammation is assessed by the level of cytokines in bronchoalveolar lavage fluid (BALF). Immediately after the animals are sacrificed, alveolar lavage is performed: sterile saline is slowly injected into the tracheal cannula of each rabbit, with a total volume of 30 mL per kg of body weight, in 5 injections. After each injection, the chest is gently massaged for a few seconds and the lavage fluid is withdrawn; the collected BALF is combined and the total volume is measured and centrifuged at 1000 rpm for 10 minutes, and the supernatant is collected for analysis. The content of proinflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) in BALF is determined by enzyme-linked immunosorbent assay (ELISA); preferably, commercial rabbit IL-8 and TNF-α ELISA kits are used, and the standard curve is prepared according to the instructions and the sample concentration is detected in pg / mL; each sample is tested in parallel 3 times and the average is taken. This indicator reflects the level of local airway inflammation: COPD models typically show elevated levels of IL-8 and TNF-α in the BALF, indicating significant infiltration and activation of inflammatory cells such as neutrophils. Comparing the concentrations of inflammatory factors in the BALF after treatment in each group can be used to assess the anti-inflammatory effect of the composition. It is expected that treatment in each of the Example groups will significantly reduce IL-8 and TNF-α levels.

[0098] The experimental results are shown in Table 3 and Appendix Figure 3 , Attachment Figure 4 shown.

[0099] Table 3 Comparison of experimental results of Experimental Example 4

[0100]

[0101] Note: BALF = bronchoalveolar lavage fluid, IL-8 = interleukin-8, TNF-α = tumor necrosis factor-α.

[0102] Combining Table 3 and the accompanying drawings, it can be seen that the Example 1, 2, 3, 4, and 5 groups are superior to the comparative example group in terms of the main pharmacodynamic indicators: the concentration of tropane alkaloids in the lung tissue of each example group is significantly higher than that of the comparative example group, while the drug concentration in the plasma is lower, indicating that the delivery of the composition allows the drug to be more distributed and retained in the lung tissue, reducing systemic exposure; however, the parameters of Experimental Example 4 have decreased because the excessive ligand affects the surface properties of the nanoparticles, resulting in slight agglomeration during the suspension atomization stage. Correspondingly, the lung function improvement rate of the Example group is much higher than that of the comparative example group, but the various parameters of the Example 5 group are relatively low, mainly because the drug-loaded nanoparticles have a larger particle size, and some nanoparticles, although guided by the targeting effect, are easily retained and cannot reach the lungs; in contrast, the comparative example 1 group only showed a moderate improvement in lung function, and the comparative example 2 group had basically no significant improvement. In terms of inflammatory indicators, IL-8 and TNF-α levels in the BALF of the Example group decreased significantly compared to the model baseline, and the post-treatment levels were significantly lower than those in the Control Group; this indicates that the composition effectively alleviated airway inflammation in COPD model rabbits, inhibited the release of inflammatory mediators, and thus improved lung function. Overall, the tropane alkaloid composition described in the Example demonstrated excellent efficacy in lung-targeted drug delivery and COPD treatment, with high drug concentrations in the lungs and low systemic side effects, significantly improving lung function and reducing airway inflammation, offering significant advantages over traditional regimens.

Claims

1. A tropane alkaloid composition for treating COPD, characterized in that: The composition comprises tropane alkaloids, mannose-6-phosphate and polylactic acid-glycolic acid copolymer, wherein the tropane alkaloids, mannose-6-phosphate and polylactic acid-glycolic acid copolymer are combined to form a nanoparticle-shaped composition. The preparation of the composition comprises the following steps: S1: preparing the tropane alkaloids to form an inner aqueous phase, and preparing the polylactic acid-co-glycolic acid to form an organic phase; S2: injecting the inner aqueous phase prepared in S1 into the organic phase while emulsifying in an ice bath to obtain W / O colostrum; S3: The W / O colostrum prepared in S2 is transferred into a polyvinyl alcohol solution and re-emulsified at a high speed of 5000-10000 rpm for 60 seconds to obtain a W / O / W emulsion; S4: The W / O / W emulsion prepared in S3 was magnetically stirred at room temperature to evaporate the solvent and centrifuged to recover the nanoparticles, which were washed with distilled water to obtain wet nanoparticles, and then the wet nanoparticles were resuspended in a buffer solution; S5: 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added to the wet nanoparticle suspension prepared in S4 for activation, and the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide:N-hydroxysuccinimide:polylactic acid-glycolic acid copolymer carboxyl group is set to 2:2:

1. Mannose-6-phosphate is then added in an amount such that the mass ratio of mannose-6-phosphate:polylactic acid-glycolic acid copolymer is 5:

95. After reaction at room temperature, the tropane alkaloid composition is obtained by ultrafiltration purification, and the added mannose-6-phosphate is covalently bonded to the surface of the polylactic acid-glycolic acid copolymer as a targeting ligand.

2. The tropane alkaloid composition for treating COPD according to claim 1, wherein: The mass ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 50:50 to 75:

25.

3. The tropane alkaloid composition for treating COPD according to claim 1, wherein: The mass ratio of the tropane alkaloids to the polylactic acid-glycolic acid copolymer is 1:5 to 1:

20.

4. The tropane alkaloid composition for treating COPD according to claim 1, wherein: The encapsulation rate of the polylactic acid-glycolic acid copolymer for the tropane alkaloids is ≥50%, and the drug loading amount of the tropane alkaloids in the nanoparticle composition is ≥5%.

5. The tropane alkaloid composition for treating COPD according to claim 1, wherein: The nanoparticle composition is formulated into a suspension for administration in the form of aerosolization.

6. The tropane alkaloid composition for treating COPD according to claim 1, wherein: The tropane alkaloid is L-anisidine.

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