Conjugates of apo a1 short peptides and tubulin inhibitors and their self-assembled nanoformulations

Abstract

The present application belongs to the technical field of medicine, and mainly relates to a conjugate of an ApoA1 short peptide and a tubulin inhibitor and a self-assembled nano preparation thereof, wherein the ApoA1 short peptide and the tubulin inhibitor are coupled through a linker. The self-assembled nano preparation comprises the following components: 1-100 parts of the conjugate of the ApoA1 short peptide and the tubulin inhibitor; 1-100 parts of phospholipid; 1-100 parts of cholesteryl ester; and 1-100 parts of water for injection or phosphate buffer. The conjugate of the ApoA1 short peptide and the tubulin inhibitor improves the targeting of the tubulin inhibitor on tumor cells and reduces the toxic side effects of the tubulin inhibitor; the self-assembled nano preparation has the advantages of low cost, few operation steps, simple process, good repeatability, can realize efficient mass production at low cost, and is conducive to storage and drug administration.

Description

Technical Field

[0001] This invention relates to conjugates of ApoA1 short peptides and microtubule inhibitors and their self-assembled nanoformulations, belonging to the field of pharmaceutical technology. Background Technology

[0002] Tubulin inhibitors (D) are chemical substances that kill specific cells. They are more toxic than typical chemotherapy drugs and are used clinically to inhibit tumor cell growth, thereby treating cancer. However, in addition to their high toxicity, tubulin inhibitors also have poor water solubility, and their non-specific distribution in the body leads to strong toxic side effects on normal cells, thus greatly limiting their clinical application. In recent years, structural modifications to D to improve its targeted distribution have become a new research direction. Summary of the Invention

[0003] To address the aforementioned deficiencies in the prior art, this invention provides a conjugate of ApoA1 short peptide and a microtubule inhibitor, and its self-assembled nanoformulation.

[0004] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0005] One objective of this invention is to provide a conjugate of an ApoA1 short peptide and a tubulin inhibitor, wherein the ApoA1 short peptide and the tubulin inhibitor are coupled via a linker, and the structure of the conjugate is shown in Formula I:

[0006]

[0007] Based on the above technical solution, the present invention can also be improved as follows:

[0008] Furthermore, the microtubule inhibitors include at least one of maytansine DM1, maytansine DM4, methylaurestatin E (MMAE), methylaurestatin F (MMAF), monomethylaurestatin D (MMAD), compressoritine, vincristine, and vindesine.

[0009] Furthermore, the linker includes at least one of the following: reduction-sensitive disulfide bond, pH-sensitive hydrazone bond, thioether bond, amide bond, and peptide bond.

[0010] Furthermore, the peptide bond is an enzyme-responsive peptide bond or its related site, such as a lysosomal protease, phosphatase, or sulfatase.

[0011] Furthermore, the sequence of the ApoA1 short peptide includes at least the Ac-CDDGFAEKFKEAVKDYFAKFWD fragment or the GFAEKFKEAVKDYFAKFWD fragment.

[0012] The preparation method of the conjugate of ApoA1 short peptide and microtubule inhibitor provided by the present invention is as follows:

[0013] Taking a disulfide bond as the linker as an example, the steps include:

[0014] ①Preparation of D-mercaptopyridine intermediates

[0015] Weigh the tubulin inhibitor and dithiopyridine, mix and dissolve them, stir at a constant temperature until fully reacted, collect the reaction solution, remove impurities, separate the product, remove the solvent to obtain the D-mercaptopyridine intermediate, and the reaction equation is as follows:

[0016]

[0017] ②Preparation of coupling compounds

[0018] Weigh the ApoA1 short peptide and D-mercaptopyridine intermediate, mix and dissolve them, stir at a constant temperature until fully reacted, collect the reaction solution, remove impurities, separate the product, remove the solvent to obtain the conjugate, and the reaction equation is as follows:

[0019]

[0020] In the preparation process of the above-mentioned conjugates of ApoA1 short peptide and tubulin inhibitor:

[0021] In step ①, a is any one of 2,2'-dithiodipyridine, acyl chloride, succinic acid, and 2-(2-pyridyldithio)ethylamine hydrochloride.

[0022] In steps ① and ②, the solution is any one of methanol, acetonitrile, dichloromethane, dimethyl sulfoxide, tetrahydrofuran, acetone, N,N-dimethylformamide, and N,N-dimethylacetamide.

[0023] In steps ① and ②, the method for removing impurities and separating products is any one of extraction, dialysis, ultrafiltration, preparative liquid chromatography, or chromatography.

[0024] In steps ① and ②, the solvent in the product can be removed using any of the following instruments: rotary evaporator, freeze dryer, or vacuum drying oven.

[0025] The second objective of this invention is to provide a self-assembled nano-formulation that possesses characteristics such as tumor targeting, reduced toxic biotoxicity, activation of anti-tumor immune responses, uniform particle size, high drug loading, good stability, suitability for intravenous injection, long circulation time, low toxicity, and simple preparation process. It comprises the following components in parts by weight:

[0026] 1-100 parts of the conjugate of the ApoA1 short peptide and the microtubule inhibitor as described above; 1-100 parts of phospholipid; 1-100 parts of cholesterol ester; 1-100 parts of water for injection or phosphate buffer.

[0027] Furthermore, the phospholipid is at least one of dimyristoyl phosphatidylcholine, hydrogenated soybean phosphatidylcholine, distearyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, distearyl phosphatidylglycerol, and dimyristoyl phosphatidylglycerol.

[0028] Furthermore, the cholesterol ester is oleic cholesterol ester.

[0029] Furthermore, its particle size is 5-200 nm; its zeta potential is -50-50 mV.

[0030] Furthermore, the above-mentioned self-assembled nano-formulations are prepared by the following method:

[0031] I. Phospholipids and cholesterol esters are dissolved in an organic phase, and the solvent is removed by rotary evaporation to obtain a lipid film;

[0032] II. Add water for injection or phosphate buffer to the lipid film obtained in step I and hydrate by shaking.

[0033] III. Dissolve the conjugate of ApoA1 short peptide and microtubule inhibitor in water for injection, then add it to the hydration mixture obtained in step II, and homogenize by shaking under high shear force.

[0034] IV. Remove uncoated impurities to obtain self-assembled nano-formulations.

[0035] Furthermore, in step I, the organic phase includes at least one of dichloromethane, methanol, and acetonitrile.

[0036] Furthermore, in step II, the hydration process is carried out by using any of the following instruments: an ultrasonic cleaner, a vortex oscillator, or a water bath, to achieve full hydration through external force vibration and temperature changes.

[0037] Furthermore, in step III, during the process of dissolving the conjugate of ApoA1 short peptide and tubulin inhibitor in water for injection, the pH can be adjusted to ensure complete dissolution.

[0038] Furthermore, in step III, the high shear force condition is: homogenization is performed under the action of an ultrasonic cell disruptor.

[0039] Furthermore, in step IV, at least one of centrifugation, dialysis, and ultrafiltration is used to remove impurity precipitates.

[0040] The beneficial effects of this invention are as follows:

[0041] I. The conjugate of ApoA1 short peptide and microtubule inhibitor of the present invention improves the targeting of microtubule inhibitor to tumor cells, reduces the toxicity to bone marrow cells and immune cells, and greatly reduces toxic side effects such as bone marrow toxicity and liver toxicity.

[0042] II. The conjugate of the ApoA1 short peptide and the microtubule inhibitor of the present invention can induce immunogenic death of tumor cells and trigger anti-tumor immune response and immune memory effect.

[0043] Third, the self-assembled nano-formulation of the present invention has the characteristics of high drug loading capacity and good structural stability, and also has the advantages of tumor targeting, high tumor inhibition rate, low toxicity and side effects, activation of anti-tumor immune response, and good biocompatibility.

[0044] Fourth, the self-assembled nano-formulation of the present invention has low cost, few operation steps, simple process, good repeatability, and can achieve low-cost and high-efficiency mass production, which is beneficial for storage and administration to the body. Attached Figure Description

[0045] Figure 1 The 1H NMR spectrum of intermediate DM1 mercaptopyridine from Example 1;

[0046] Figure 2 Low-resolution electrospray ionization mass spectrometry of conjugate AD from Example 1;

[0047] Figure 3 High performance liquid chromatography of the conjugate AD from Example 1;

[0048] Figure 4 This is a transmission electron microscope image of the nano-formulation AD-HDL from Example 2;

[0049] Figure 5 The particle size distribution of the nano-formulation AD-HDL in Example 2;

[0050] Figure 6 The zeta potential distribution of the nano-formulation AD-HDL in Example 2;

[0051] Figure 7 To detect the ATP content released from cells into the culture medium using an ELISA kit.

[0052] Figure 8 The ELISA kit was used to detect the HMGB1 content released from cells into the culture medium;

[0053] Figure 9 Results of mouse weight monitoring;

[0054] Figure 10 A mouse bone marrow smear under a microscope;

[0055] Figure 11 A pathological scan of a mouse liver;

[0056] Figure 12 The curve shows the relative tumor volume growth of mice. Detailed Implementation

[0057] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0058] Example 1

[0059] Synthesis and purification of AD conjugate

[0060] Weigh 80 mg of DM1 and 246 mg of 2,2'-dithiopyridine, and dissolve them separately in 10 mL of methanol. Then, add the methanol solution of DM1 dropwise to the methanol solution of 2,2'-dithiopyridine and stir overnight at room temperature. Drop the reaction mixture onto the bottom of a normal-phase thin-layer chromatography plate. After the liquid has air-dried, use a 20 mL mixture of ethyl acetate and petroleum ether (v / v) as the developing solvent for chromatography separation. Then, scrape off the plate containing the target band and dissolve the separated product using a 50 mL mixture of methanol and dichloromethane (v / v) as the dissolving agent. Remove the solvent by rotary evaporation and lyophilization to obtain the intermediate DM1 mercaptopyridine. The 1H NMR spectrum is shown below. Figure 1 As shown, this demonstrates the successful synthesis of the DM1 mercaptopyridine intermediate.

[0061] Weigh 15 mg of DM1 mercaptopyridine intermediate and 45 mg of ApoA1 short peptide, dissolve them in 2 mL of ultra-dry dimethyl sulfoxide, and stir at room temperature for 48 hours. Collect the reaction solution, and use a preparative liquid chromatography column with a C18 column. The mobile phase is uniformly increased from acetonitrile to water at a volume ratio of 10:90 to 90:10 within 30 minutes, with a flow rate of 1-50 mL / min and detection wavelengths of 254 nm and 220 nm. Separate the product peaks, freeze-dry to remove the solvent, and obtain the conjugate AD (i.e., the conjugate of ApoA1 short peptide and microtubule inhibitor).

[0062] The results of low-resolution electrospray ionization source mass spectrometry characterization and high-performance liquid chromatography are shown in the figure. Figure 2-3 This demonstrates the successful synthesis of the coupling compound AD.

[0063] Example 2

[0064] Preparation of self-assembled nanoformulation AD-HDL

[0065] ① Dissolve 5 mg of DMPC (myristoyl phosphatidylcholine) and 0.2 mg of oleic acid cholesterol ester in 10 mL of a mixture of dichloromethane and methanol with a volume ratio of 3:1, and remove the solvent by rotary evaporation to obtain a uniform film.

[0066] ② Add 10 mL of phosphate buffer to the membrane from step ① and shake to hydrate it, thus obtaining the hydrated solution;

[0067] ③ Dissolve 10 mg of the coupling compound AD prepared in Example 1 in 10 mL of phosphate buffer (adjust the pH to weakly alkaline with NaOH beforehand), and then add the coupling compound AD solution to the hydration solution in step ②; shake thoroughly to homogenize at 10% power with a frequency of 30 seconds on and 30 seconds off;

[0068] ④ Centrifuge the system at 7000g for 10 minutes, take the supernatant, and obtain the self-assembled nano-formulation AD-HDL.

[0069] Transmission electron microscopy images and particle size distribution are shown in Figure 4-5 The average particle size was measured to be 17.6 nm, and the Zeta potential distribution is shown in the figure. Figure 6 The measured Zeta potential was -13.3 mV, indicating the successful preparation of the nano-formulation AD-HDL.

[0070] The efficacy and safety of the nano-formulation AD-HDL prepared in the above examples were tested in the following experiments.

[0071] I. Evaluation of the immunogenicity of AD-HDL nanoparticle-induced cell death in CT26 tumor cells

[0072] CT26 tumor cells were seeded at a density of 4 x 10⁴ cells per well in 24-well plates and cultured in a CO₂ incubator for 12 hours. Then, AD-HDL aqueous solution, liposome blank control (HDL), and phosphate-buffered saline (PBS) were added and incubated for 4 hours each. Fresh medium was then added, and the cells were cultured for another 12 hours. The supernatant was then aspirated, and the levels of ATP and HMGB1 released into the medium by the cells were detected using an ELISA kit and compared. Results are shown below. Figure 7-8 The results showed that AD-HDL stimulation of CT26 cells led to a significant increase in ATP and HMGB1 in the culture medium, while no such phenomenon was observed after treatment with liposome blank control and phosphate buffer, indicating that AD-HDL induced immunogenic cell death in CT26 cells.

[0073] II. Preliminary Safety Evaluation of Nano-Formulation AD-HDL

[0074] Fifty-four healthy BALB / c mice weighing 20±2g were randomly divided into 9 groups. Each group was administered PBS (one group, 0.2mL / kg), free DM1 (three groups, 0.5, 1, and 2mg / kg each), or AD-HDL solution (five groups, 1, 2, 3, 4, and 5mg / kg each) via tail vein injection. Mice were weighed daily, and a 15% decrease in body weight was considered the lower limit of safety. Systemic toxicity was monitored, and the monitoring statistics are shown in the graph below. Figure 9 The results showed that mice in the free DM1 group exhibited significant toxicity at a dose of 1 mg / kg, with a sharp weight loss of 85%; while the AD-HDL group did not show significant weight loss at doses below 4 mg / kg, demonstrating good biocompatibility.

[0075] III. Evaluation of the effects of nano-formulation AD-HDL on bone marrow toxicity

[0076] Fifteen healthy BALB / c mice weighing 20±2g were randomly divided into three groups. Each group was administered PBS (0.2mL / kg), free DM1 (1mg / kg), or AD-HDL solution (1mg / kg) via tail vein injection, respectively. Three days after administration, the mice were sacrificed, and two femoral vertebrae were harvested. The bone marrow contents were flushed out with phosphate buffer, and bone marrow smears were prepared and stained with Wright-Giemsa stain. Microscopic examination of the smears is shown in the image below. Figure 10 The results showed that in the free DM1 group, the proportion of nucleated cells in bone marrow cells decreased significantly at a dose of 1 mg / Kg, indicating significant bone marrow suppression; while in the AD-HDL group, no significant change in the proportion of bone marrow cells was observed after a dose of 1 mg / Kg, indicating that AD-HDL significantly alleviated the bone marrow toxicity caused by free DM1 and had good safety.

[0077] IV. Evaluation of the hepatotoxicity relief achieved by the nano-formulation AD-HDL

[0078] Fifteen healthy BALB / c mice weighing 20±2g were randomly divided into three groups. Each group was administered PBS (0.2mL / kg), free DM1 (1mg / kg), or AD-HDL solution (1mg / kg) via tail vein injection, respectively. Three days after administration, the mice were sacrificed, and their livers were dissected, fixed with paraformaldehyde, and sectioned for hematoxylin-eosin (HE) staining. Pathological scan images are shown below. Figure 11 The results showed that when the dose of free DM1 was 1 mg / kg, vacuolar damage occurred in the liver tissue; while when the dose of AD-HDL was 1 mg / kg, no significant changes in liver structure were observed, indicating that AD-HDL significantly alleviated liver damage caused by free DM1 and had good safety.

[0079] IV. Evaluation of the inhibitory effect of the nano-formulation AD-HDL on tumor growth in CT26-bearing mice

[0080] Twenty-four healthy BALB / c mice weighing 20±2g were randomly divided into three groups. Each group received a subcutaneous injection of 100μL of CT26 tumor cell suspension (2 x 10⁷ cells / mL) in the buttocks until the tumor volume reached 100mm². 3 Subsequently, mice were administered PBS (0.2 mL / kg) and AD-HDL solution (0.5 mg / kg and 3 mg / kg), respectively, via tail vein injection, once every 3 days for a total of 3 administrations. Tumor volume was recorded daily, and relative tumor volume growth curves were plotted. Results are shown below. Figure 12 The results showed that in the AD-HDL-treated group, the tumor volume growth rate was slowed down and tumor growth was significantly inhibited after the administration of AD-HDL at a dose of 0.5 mg / Kg; at a dose of 3 mg / Kg, the inhibition of tumor growth was further enhanced, demonstrating a strong anti-tumor effect.

[0081] 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 conjugate of ApoA1 short peptide and a microtubule inhibitor, characterized in that, The ApoA1 short peptide and the microtubule inhibitor are coupled via a linker; The microtubule inhibitors include at least one of maytansin DM1 and maytansin DM4; The linker includes disulfide bonds; The sequence of the ApoA1 short peptide is the Ac-CDDGFAEKFKEAVKDYFAKFWD fragment.

2. A self-assembled nanoformulation, characterized in that, Based on parts by weight, it comprises the following components: 1-100 parts of the conjugate of the ApoA1 short peptide and the microtubule inhibitor as described in claim 1; 1-100 parts of phospholipid; 1-100 parts of cholesterol ester; 1-100 parts of water for injection or phosphate buffer.

3. The self-assembled nanoformulation according to claim 2, characterized in that, The phospholipid is at least one of dimyristoyl phosphatidylcholine, hydrogenated soybean phosphatidylcholine, distearyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, distearyl phosphatidylglycerol, and dimyristoyl phosphatidylglycerol.

4. The self-assembled nanoformulation according to claim 2, characterized in that, The cholesterol ester is oleic cholesterol ester.

5. The self-assembled nanoformulation according to claim 2, characterized in that, Its particle size is 5-200 nm; its zeta potential is -50-50 mV.

6. The self-assembled nanoformulation according to claim 2, characterized in that, It is prepared by the following method: I. Phospholipids and cholesterol esters are dissolved in an organic phase, and the solvent is removed by rotary evaporation to obtain a lipid film; II. Add water for injection or phosphate buffer to the lipid film obtained in step I and hydrate by shaking. III. Dissolve the conjugate of ApoA1 short peptide and microtubule inhibitor in water for injection, then add it to the hydration mixture obtained in step II, and homogenize by shaking under high shear force. IV. Remove uncoated impurities to obtain self-assembled nano-formulations.

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