CN116869938A

CN116869938A - 1,2, 4-oxadiazole-pyridine liposome and preparation method and application thereof

Info

Publication number: CN116869938A
Application number: CN202310922580.9A
Authority: CN
Inventors: 李增; 夏居程; 董双宏; 杨丽丽; 王芳
Original assignee: Anhui Medical University
Current assignee: Anhui Medical University
Priority date: 2023-07-26
Filing date: 2023-07-26
Publication date: 2023-10-13

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a 1,2, 4-oxadiazole-pyridine liposome and a preparation method and application thereof. The liposome is formed by encapsulating 1,2, 4-oxadiazole-pyridine compound 10b by phosphatidylethanolamine, fucosterol and triethanolamine to form a lipid microcapsule, wherein the structural formula of the 1,2, 4-oxadiazole-pyridine compound 10b is shown as formula A:the invention provides a liposome based on a 1,2, 4-oxadiazole-pyridine compound 10b and a preparation method thereof, wherein the 1,2, 4-oxadiazole-pyridine compound 10b can be used for intravenous injection administration through the arrangement of the liposome, and the bioavailability of an effective ingredient can be improved.

Description

1,2, 4-oxadiazole-pyridine liposome and preparation method and application thereof

Technical Field

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a 1,2, 4-oxadiazole-pyridine liposome and a preparation method and application thereof.

Background

Alzheimer's disease is one of the most common neurodegenerative diseases and is clinically characterized by cognitive dysfunction, particularly memory loss, in patients. In recent years, as the global population ages, the prevalence increases year by year, and AD worldwide is expected to reach 1.3 billion by 2050. Current FDA approved drugs for the treatment of AD often provide temporary or incomplete relief from symptoms with serious side effects. Thus, there is an urgent need to develop more effective Alzheimer's disease therapeutic drugs.

Glycogen synthase kinase 3 beta (GSK-3 beta) plays a key and central role in the pathogenesis of AD. GSK-3 beta is one of the main participants in Tau (a microtubule-associated protein) phosphorylation, stabilization of microtubules, and promotion of microtubule assembly. GSK-3β overactivation can cause excessive phosphorylation of Tau protein, rendering Tau protein unable to bind to microtubules, thus destabilizing microtubules; subsequently, free Tau protein accumulates and aggregates in neurons, leading to neuronal fiber tangles and neuronal death. Automated high-throughput fluorescence imaging system studies have shown that abnormal increases in GSK-3 beta activity lead to localization/transport of beta-secretase and a number of aβ secretion disorders. In a mouse model over-expressing amyloid precursor protein, GSK-3 beta is able to reduce aβ and its associated toxicity, improve aβ -induced performance, and reduce neuronal loss. Oxidative stress and neuroinflammation are also associated with abnormal activation of GSK-3β. Thus, the kinase is involved in almost all pathways leading to the onset of AD. Despite these preliminary findings, high-intensity and selective GSK-3 β inhibitors developed to the clinical stage failed to exhibit expected/desired therapeutic effects in AD, cognitive enhancement, improvement of behavioral disorders, and the like.

In this case, the use of a multi-target ligand strategy to enhance the performance of GSK-3β inhibitors by synergism to other important targets of AD (e.g., improving glucose metabolism and anti-neuroinflammation) is an effective strategy to treat AD.

The applicant has previously studied and found that 1,2, 4-oxadiazole-pyridine compounds (CN 111362930A, CN 113651805A) show good effects on inhibiting GSK-3 beta and resisting neuroinflammation, and have potential to develop into GSK-3 beta inhibitors and medicines for treating AD. However, 1,2, 4-oxadiazole-pyridine compounds are used as novel GSK-3 beta inhibitors, and the administration stability has an influence on the curative effect. The targeting delayed release drug, reducing the in vivo elimination rate is a clinically ideal mode, so the formulation development is particularly important.

Disclosure of Invention

In order to solve the above technical problem, one of the objects of the present invention is to provide a 1,2, 4-oxadiazole-pyridine liposome, wherein the liposome is formed by encapsulating 1,2, 4-oxadiazole-pyridine compound 10b with phosphatidylethanolamine, fucosterol and triethanolamine to form a lipid microcapsule, and the structural formula of the 1,2, 4-oxadiazole-pyridine compound 10b is shown in formula (a):

the second purpose of the invention is to provide a preparation method of the 1,2, 4-oxadiazole-pyridine liposome, which specifically comprises the following steps: dissolving 1-4 parts by weight of phosphatidylethanolamine and 0.5-2 parts by weight of fucosterol in an organic solvent, freeze-drying to obtain freeze-dried powder, hydrating the freeze-dried powder by using an aqueous solution containing 1 part by weight of 1,2, 4-oxadiazole-pyridine compound 10b and 0.1-0.6 part by weight of triethanolamine, and then sequentially carrying out ultrasonic treatment, filtration and concentration to obtain the product, namely the required 1,2, 4-oxadiazole-pyridine liposome.

Preferably, the phosphatidylethanolamine, the fucosterol and the triethanolamine are used in an amount of 2 parts by weight, 1 part by weight and 0.3 part by weight, respectively.

Preferably, the organic solvent is any one or a combination of more than one of n-butanol, chloroform and isopropanol.

Preferably, the hydration conditions are specifically: hydrating at 55-60 deg.c for 10-25 min; the power of the ultrasonic wave is 50-100W, and the ultrasonic wave is 5-10 min.

The invention further aims to provide an application of the 1,2, 4-oxadiazole-pyridine liposome in preparation of a medicine for treating Alzheimer's disease.

Preferably, the medicament further comprises a pharmaceutically acceptable carrier, wherein the carrier is any one or more of a stabilizer, a colorant, a diluent, a slow release agent, a filler and a flavoring agent.

The beneficial effects of the invention are as follows:

previous studies by the applicant indicate that 1,2, 4-oxadiazole-pyridine compound 10b is a novel glycogen synthase kinase 3 beta (GSK-3 beta) inhibitor and has excellent GSK-3 beta inhibition and anti-neuroinflammation dual effects. The physicochemical properties are represented by white solid compounds, which are readily soluble in organic solvents such as methanol, ethanol, chloroform, etc., and insoluble in water (Wang M, liu T, chen S, et al design and synthesis of 3- (4-pyridyl) -5- (4-sulfamido-phenyl) -1,2,4-oxadiazolederivatives as novel GSK-3β inhibitors and evaluation of their potential as multifunctional anti-Alzheimer' S reagent-science direct [ J ]. European Journal of Medicinal Chemistry,2020,209.DOI:10.1016/J. Ejmech.2020.112874.).

The invention provides a liposome based on a 1,2, 4-oxadiazole-pyridine compound 10b and a preparation method thereof, wherein the liposome can be embedded to realize targeted delayed release of the drug, improve the drug effect and reduce the in vivo elimination speed on the one hand; on the other hand, the medicine can reduce the toxicity in vivo and lighten allergic reaction and immune reaction.

By the arrangement of the liposome, the 1,2, 4-oxadiazole-pyridine compound 10b can be used for intravenous administration, and the bioavailability of the active ingredient can be improved.

The liposome adopts the fucosterol, the triethanolamine and the phosphatidylethanolamine to form a closed lipid vesicle, so that the drug loading capacity and the encapsulation rate of the main drug 1,2, 4-oxadiazole-pyridine compound 10b can be improved.

Drawings

FIG. 1 is a graph showing the particle size distribution of 1,2, 4-oxadiazole-pyridine liposomes prepared in example 3;

FIG. 2 is an in vitro release rate of liposomes prepared in examples 1-5;

FIG. 3 is a graph showing the change in body weight of each group of mice in example 9;

FIG. 4 is a section HE staining of viscera of each group of mice in example 9;

FIG. 5 is a graph showing the results of the individual data obtained in the Morris water maze test for the mice of example 10; wherein A is escape latency during 4-day training of mice of different groups, B is swimming speed of mice of different groups, C is number of times mice of different groups cross the virtual platform, and D is time that mice of different groups stay in the target quadrant.

FIG. 6 is a representative trace plot of mice in example 10 in the Morris water maze test.

Detailed Description

The following describes the technical scheme of the present invention in more detail with reference to examples:

unless otherwise indicated, terms used herein have meanings conventionally understood by those skilled in the art.

The 1,2, 4-oxadiazole-pyridine compound 10b used in the present invention was synthesized by the method disclosed in article Design and synthesis of- (4-pyridyl) -5- (4-sulfamido-phenyl) -1,2,4-oxadiazolederivatives as novel GSK-3β inhibitors and evaluation of their potential as multifunctional anti-Alzheimer's agents-science direct.

Intermediate 6 is obtained by reacting 2-chloro-4-cyanopyridine with hydroxylamine hydrochloride, intermediate 7 is generated by reacting intermediate 6 with 4-nitrobenzoyl chloride, intermediate 8 is obtained by reducing intermediate 7 with Pd/C and hydrazine hydrate, intermediate 9 is generated by reacting intermediate 8 with 4-tert-butylbenzoyl chloride, and finally, the intermediate 9 is reacted with 2-chloroaniline to obtain the required 1,2, 4-oxadiazole-pyridine compound 10b.

Example 1

Dissolving 2 parts by weight of phosphatidylethanolamine and 1 part by weight of fucosterol in n-butanol, freeze-drying (freezing to-10 ℃ C., vacuum drying) to obtain freeze-dried powder, hydrating the freeze-dried powder with an aqueous solution containing 1 part by weight of 1,2, 4-oxadiazole-pyridine compound 10b and 0.3 part by weight of triethanolamine, performing ultrasonic treatment for 10min, performing microfiltration, and concentrating to obtain 1,2, 4-oxadiazole-pyridine liposome.

Example 2

Dissolving 1 part by weight of phosphatidylethanolamine and 0.5 part by weight of fucosterol in chloroform, freeze-drying (freezing to-10 ℃ C., vacuum drying) to obtain freeze-dried powder, hydrating the freeze-dried powder with an aqueous solution containing 1 part by weight of 1,2, 4-oxadiazole-pyridine compound 10b and 0.1 part by weight of triethanolamine, performing ultrasonic treatment for 10min, performing microfiltration, and concentrating to obtain the 1,2, 4-oxadiazole-pyridine liposome.

Example 3

Dissolving 4 parts by weight of phosphatidylethanolamine and 2 parts by weight of fucosterol in isopropanol, freeze-drying (freezing to-10 ℃ C., vacuum drying) to obtain freeze-dried powder, hydrating the freeze-dried powder with an aqueous solution containing 1 part by weight of 1,2, 4-oxadiazole-pyridine compound 10b and 0.6 part by weight of triethanolamine, performing ultrasonic treatment for 10min, performing microfiltration, and concentrating to obtain the 1,2, 4-oxadiazole-pyridine liposome.

Example 4

Dissolving 3 parts by weight of phosphatidylethanolamine and 1.5 parts by weight of fucosterol in n-butanol, freeze-drying (freezing to-10 ℃ C., vacuum drying) to obtain freeze-dried powder, hydrating the freeze-dried powder with an aqueous solution containing 1 part by weight of 1,2, 4-oxadiazole-pyridine compound 10b and 0.4 part by weight of triethanolamine, performing ultrasonic treatment for 10min, performing microfiltration, and concentrating to obtain 1,2, 4-oxadiazole-pyridine liposome.

Example 5

Example 6

Drug loading and encapsulation efficiency evaluation of 1,2, 4-oxadiazole-pyridine liposome

The entrapment rate and drug loading rate of the liposome were measured by dialysis, the liposome prepared in examples 1 to 5 was collected, diluted ten times with methanol, sonicated and centrifuged to extract the supernatant, the content of the main drug 1,2, 4-oxadiazole-pyridine compound 10b was measured by ultraviolet spectrophotometry, the entrapment rate (Drug encapsulation efficiency, EE%) and drug loading rate (Drug loading content, DL%) were calculated according to the formula, and the calculation results are shown in table 1.

Table 1 drug loading and encapsulation efficiency of 1,2, 4-oxadiazole-pyridine liposomes

The high and low drug loading directly affects the clinical application dosage of the drug, the larger the drug loading is, the easier the clinical requirement is met, but the too high drug loading can also cause the drug leakage. The test result shows that the drug loading rate of the liposome prepared by the invention is between 28 and 32 percent, and the encapsulation rate is more than 88 percent, so that the closed lipid vesicle is formed by adopting the fucosterol, the triethanolamine and the phosphatidylethanolamine, the encapsulation rate is high, and the drug loading rate is proper.

The particle size of the 1,2, 4-oxadiazole-pyridine liposome prepared in example 3 was measured using a Nano S900 laser particle sizer, and the result is shown in fig. 1, and it can be seen that the liposome was dispersed and uniform in size.

Example 7

Evaluation of Release Rate of 1,2, 4-oxadiazole-pyridine Liposome

1mL of the 1,2, 4-oxadiazole-pyridine liposome prepared in the examples 1-5 is respectively sucked into a dialysis bag, the dialysis bag is placed into 50mL of 0.01M PBS solution containing 1% Tween 80, the temperature is kept constant for shaking for 1,2,4, 8, 12, 24, 36, 48 and 72 hours at 37 ℃, samples in the dialysis bag at each time point are collected, 10 times of methanol is used for diluting and demulsification, the supernatant is sucked by centrifugation, the absorbance of the supernatant is measured at a wavelength of 270nm, and the in vitro release rates of the 1,2, 4-oxadiazole-pyridine liposome at different time points are calculated according to the standard curve of the 1,2, 4-oxadiazole-pyridine compound 10b in the methanol solution.

The results are shown in FIG. 2. It can be seen that the in vitro release rate of the 1,2, 4-oxadiazole-pyridine liposome is better than that of the 1,2, 4-oxadiazole-pyridine compound 10b, and the effect is better.

Example 8

1,2, 4-oxadiazole-pyridine liposome permeability assay

MDCKII-MDR1 cells (1X 10) ⁵ Individual cells/cm ² ) Inoculated into a polyester 12-well Transwell (pore size: 0.4 μm, diameter: 12mm, apical volume: 0.5mL, basolateral: volume 1.5 mL) of the insert. The medium was discarded from the AP (apical) and BL (basolateral) chambers of each insert, and then the monolayer cells were washed 3 times with DPBS (Dulbecco's phosphate buffered saline). Verifying the formation of MDCKII-MDR1 single-layer fusion membrane by dynamic observation and measurement of transmembrane resistance (TEER) value; diazepam and FD4 (isothiocyanate-dextran fluorescein) were used as internal controls to label extracellular and bystander pathways to confirm the integrity of the tight junctions during the assay.

AP and BL chambers were added to a liposome solution (prepared in example 3, 75. Mu.M) dissolved in the test medium and incubated for 2 hours, and then the flux from AP to BL or BL to AP was analyzed.

Apparent permeability coefficient (Papp) is calculated as follows in cm/s:

where Va is the receptor pore volume, time is the total transit time, [ Drug ] receptor is the concentration of Drug in the receptor pore, [ Drug ] initial is the initial Drug concentration in the AP or BL cavity.

The jet ratio (ER) is calculated as follows:

wherein Papp, BL-AP is the apparent permeability of the substrate transported to the top, and Papp, AP-BL is the apparent permeability of the substrate transported to the top. Jet ratios greater than 2 indicate that the test compound may be a substrate for P-gp delivery.

The results are shown in Table 2.

TABLE 2 bidirectional transport of 1,2, 4-oxadiazole-pyridine liposomes in MDCKII-MDR1 cells

^a Data are means±SD of three determination

^b Efflux ratio(ER)was calculated using the following equation:ER PappBL/PappAP.

It can be seen that the 1,2, 4-oxadiazole-pyridine liposome prepared in example 3 shows high permeability superior to diazepam and 1,2, 4-oxadiazole-pyridine compound 10b, and has an ER value of less than 2.

Example 9

1,2, 4-oxadiazole-pyridine liposome acute toxicity detection

After selecting C57BL male mice for 14 days, observing whether each group of mice develop normally, die or not and abnormal behaviors, and whether water intake is normal or not. Specifically, 1,2, 4-oxadiazole-pyridine liposome prepared in example 3 was administered by intraperitoneal injection 1 time a day, and after 14 days of treatment, detection was performed with empty carriers without drug as empty groups; the low dose group was treated with 10mg/kg of 1,2, 4-oxadiazole-pyridine liposomes; the high dose group was treated with 20mg/kg of 1,2, 4-oxadiazole-pyridine liposome (e.g., mice weighing 20g were intraperitoneally injected with 0.2 mL).

The change in body weight (mean ± standard error) of each group of mice is shown in fig. 3, and the body weight of the mice increases smoothly with increasing days. After 14 days, mice were sacrificed and frozen sections of heart, liver, spleen, lung, kidney were HE stained for each group of mice.

The staining results are shown in fig. 4, in which the structures of heart, liver, spleen, lung and kidney tissues of each group of mice are clear and complete, the cells are orderly arranged, the cell nuclei are normal, inflammatory cell infiltration and abnormal secretion are avoided, and the fact that the 1,2, 4-oxadiazole-pyridine liposome is injected into the abdominal cavity does not cause damage to organs of the mice is shown.

Example 10

Improvement of spatial learning and memory capacity of male C57BL mice by 1,2, 4-oxadiazole-pyridine liposome

Test materials: male C57BL mice (18-23 g), 8 week old, were purchased from the university of Anhui traditional Chinese medicine laboratory center. The mice are raised under standard conditions and in an environment with controllable temperature and humidity (23-25 ℃, 40-60 percent and 12 hours).

Treatment and modeling: 30 mice were randomly divided into 5 groups, and the normal and model groups were perfused with 0.5% sodium carboxymethylcellulose (CMC-Na) solution for 7 days, and the drug group and the positive control group were perfused with different concentrations of 1,2, 4-oxadiazole-pyridine liposomes (example 3, 30mg/kg, 10 mg/kg) and donepezil (10 mg/kg), respectively. On day 7, each group of mice was injected with scopolamine except for the normal group (normal saline) to establish an AD model.

Morris water maze test:

the Morris water maze (Morris water maze, MWM) device mainly comprises a circular pool (with the diameter of 150 cm and the height of 60 cm), an escape platform with the diameter of 10 cm, a data acquisition system and an analysis system. The circular pool was first filled with water (1.0 cm above the escape platform) and titanium dioxide (0.25 g/L) was added to cloudy the water. The platform was fixed in the third quadrant (four quadrants) of the circular pool and all mice were trained for 4 days, four training trials per day.

One mouse (head facing the pool wall) was randomly placed in four quadrants and allowed to rest on the platform for 15 seconds if the mouse successfully found the platform within 60 seconds, otherwise, it was brought to the platform and left for 15 seconds. After 4 days, the platform was removed from its position, the mouse was placed in the first quadrant furthest from the platform quadrant to find the platform (60 s), and the time spent in the platform quadrant and the number of entries into the zone within the mouse 60s were recorded.

The results are shown in fig. 5 and 6, and the date is expressed as mean ± SD (n=8). Statistical significance by two-way ANOVA analysis: ^ns p>0.5， ^* p<0.05， ^** p<0.01 and ^*** p<0.001 compared to model set. The escape latency during 4-day training of the different groups of mice is shown in fig. 5a, B in fig. 5 is the swimming speed of the different groups of mice, C is the number of times the different groups of mice traverse the virtual platform, and D is the time the different groups of mice stay in the target quadrant. Compared with the model group, the drug group reduces the escape latency of the mice in a dose-dependent manner, namely reduces the time for the mice to find a platform; when the platform was removed, the residence time in the target quadrant and the number of crossing the virtual platform for mice treated with a 30mg/kg dose of 1,2, 4-oxadiazole-pyridine liposome were significantly better than the 10mg/kg dose.

Fig. 6 shows representative trajectories of mice in the Morris water maze test, from which it can be seen that after removal of the platform, the time taken for mice treated at a dose of 30mg/kg to enter the quadrant of the platform and the number of times to enter this zone are significantly better than the other groups, except for the normal group.

The above is merely a preferred practical example of the present invention, and is not intended to limit the invention; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The 1,2, 4-oxadiazole-pyridine liposome is characterized in that a lipid microcapsule is formed by phosphatidylethanolamine, fucosterol and triethanolamine to encapsulate a 1,2, 4-oxadiazole-pyridine compound 10b, and the structural formula of the 1,2, 4-oxadiazole-pyridine compound 10b is shown as a formula (A):

2. a method for preparing the 1,2, 4-oxadiazole-pyridine liposome according to claim 1, which comprises the following steps: dissolving 1-4 parts by weight of phosphatidylethanolamine and 0.5-2 parts by weight of fucosterol in an organic solvent, freeze-drying to obtain freeze-dried powder, hydrating the freeze-dried powder by using an aqueous solution containing 1 part by weight of 1,2, 4-oxadiazole-pyridine compound 10b and 0.1-0.6 part by weight of triethanolamine, and then sequentially carrying out ultrasonic treatment, filtration and concentration to obtain the product, namely the required 1,2, 4-oxadiazole-pyridine liposome.

3. The method for preparing 1,2, 4-oxadiazole-pyridine liposome according to claim 2, wherein the amounts of phosphatidylethanolamine, fucosterol and triethanolamine are 2 parts by weight, 1 part by weight and 0.3 part by weight, respectively.

4. The method for preparing 1,2, 4-oxadiazole-pyridine liposome according to claim 2 or 3, wherein the organic solvent is any one or a combination of more than one of n-butanol, chloroform and isopropanol.

5. A method for the preparation of 1,2, 4-oxadiazole-pyridine liposomes according to claim 2 or 3, characterized in that the hydration conditions are in particular: hydrating at 55-60 deg.c for 10-25 min; the power of the ultrasonic wave is 50-100W, and the ultrasonic wave is 5-10 min.

6. Use of the 1,2, 4-oxadiazole-pyridine liposome according to claim 1 in the preparation of a medicament for treating alzheimer's disease.

7. The medicament of claim 6, further comprising a pharmaceutically acceptable carrier, wherein the carrier is any one or more of a stabilizer, a colorant, a diluent, a slow release agent, a filler, and a flavoring agent.