Precursor nano self-assembly immune composition as well as preparation method and application thereof
Through the nanoimmune composition self-assembled in physiological aqueous media, the problems of low loading efficiency and complex preparation of nanocarrier systems are solved, and the efficient loading and bioavailability of chemotherapy drugs are achieved, and the anti-tumor effect is enhanced.
Patent Information
- Application Number
- CN202411444629.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-16
- Publication Date
- 2025-01-03
AI Technical Summary
The existing nanocarrier systems have limited loading efficiency for active drugs, complex preparation process, lack repetition and controllability, and it is difficult to achieve large-scale production and clinical transformation. Chemotherapy drugs cause damage to the immune system, resulting in reduced efficacy and worsening of the disease.
The precursor nano-self-assembled immune composition consisting of covalent coupling of 1-methyltryptophan and indole-3-methanol, consisting of precursor nano-self-assembled immune compositions composed of covalent coupling of 1-methyltryptophan and indole-3-methanol, and is constructed by simple and controllable Methods Self-assembled into nanoscale ultramicro particles in physiological aqueous media to improve the bioavailability of drugs and anti-tumor efficacy.
It realizes efficient loading of chemotherapy drugs and a simple and controllable preparation process, facilitates industrial production, improves the bioavailability of drugs and anti-tumor efficacy, and enhances the inhibitory ability of tumor cells.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomedicine, in particular to a precursor nanometer self-assembly immune composition and a preparation method and application thereof. Background Art
[0002] Cytotoxic chemotherapy drugs represented by paclitaxel and docetaxel are one of the main means of tumor treatment at present. They inhibit the growth and spread of cancer by directly killing tumor cells or inhibiting their growth and proliferation. However, while killing tumor cells, this type of therapy will also cause serious damage to the patient's immune system and induce tumor resistance and recurrence, resulting in reduced efficacy and worsening of the disease.
[0003] 1-Methyltryptophan (1-MT) is an effective inhibitor of the immune checkpoint indoleamine-2,3-dioxygenase 1 (IDO1). IDO1 is highly expressed in tumor tissues and tumor-draining lymph nodes, and can catalyze the degradation of tryptophan along the kynurenine pathway. Its metabolites inhibit the function of T cells and induce the expansion of regulatory T cells, thereby mediating the immunosuppression of the tumor microenvironment.
[0004] With the widespread application of nanotechnology in the field of biomedicine, nano-delivery systems constructed based on various polymers, inorganic or lipid materials have shown great potential in the application value and development prospects in achieving effective in vivo drug delivery. Compared with free drug solutions, nano-delivery systems have the advantages of improving the in vivo process of drugs, regulating the drug release rate, enhancing the targeting of lesion tissues, improving bioavailability and reducing drug toxicity and side effects. However, nano-delivery systems constructed based on traditional materials have limited loading efficiency for active drugs, and the preparation process is complex, lacking repeatability and controllability, making it difficult to achieve large-scale production and clinical transformation.
[0005] Therefore, the existing technology still cannot effectively solve the defects of such drugs in the clinical application process, and there is an urgent need to develop new safe, efficient and accessible drug delivery systems. Summary of the invention
[0006] The purpose of the present invention is to provide a precursor nano self-assembling immune composition and its preparation method and application in view of the deficiencies of the prior art. The immune composition with the characteristics of the present invention can realize efficient loading of chemotherapeutic drugs, and the preparation process is simple and controllable, which is convenient for industrial production; it can quickly self-assemble into nano-scale ultrafine particles in physiological aqueous media, thereby improving the bioavailability of such drugs and enhancing their anti-tumor efficacy.
[0007] To achieve the above object, the technical solution adopted by the present invention is: to provide a precursor nano self-assembly immune composition, which is composed of immune carrier molecules, active drugs and solvents.
[0008] Preferably, the immune carrier molecule is formed by covalent coupling of 1-methyltryptophan and indole-3-methanol, and the structural formula is shown in Formula 1:
[0009] Formula 1
[0010] Preferably, the active drug is a small molecule drug that can interact with the immune carrier molecule.
[0011] Preferably, the chemical small molecule drug is any one or more combinations of paclitaxel, docetaxel, cabazitaxel, doxorubicin, epirubicin, mitoxantrone, camptothecin, 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, irinotecan, topotecan, belotecan, vincristine, vindesine, vinorelbine, cisplatin, oxaliplatin, carboplatin, methotrexate, pemetrexed, cytarabine, capecitabine, gemcitabine, fluorouracil, teniposide, etoposide, ritonavir, homoharringtonine, indirubin, shikonin, curcumin, gambogic acid, ginsenoside, norcantharidin, arsenite, rapamycin, chlorin, and indocyanine green.
[0012] Preferably, the vehicle is a physiologically compatible vehicle.
[0013] Preferably, the physiologically compatible solvent is any one or more combinations of 1,2-propylene glycol, glycerol, isopropanol, ethanol, benzyl alcohol, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, and polyethylene glycol, wherein the molecular weight of the polyethylene glycol ranges from 200 to 20,000.
[0014] Preferably, the weight percentages of the components are: immune carrier molecule: 1-99%, active drug: 1-99%, solvent: 1-99%.
[0015] Preferably, the particle size of the immune composition self-assembled in a physiological aqueous medium is 10 to 1000 nm.
[0016] The preparation method of immune carrier molecules comprises the following steps: dissolving 1-methyl-N-Fmoc-tryptophan in N, N-dimethylformamide, incubating in the dark for 1-2 hours under the catalytic action of 4-dimethylaminopyridine, carbodiimide hydrochloride and 1-hydroxybenzotriazole, then adding indole-3-methanol and reacting in the dark at 5-40° C. for 24-48 hours under nitrogen protection; adding piperidine to remove the Fmoc protecting group, and separating and purifying to obtain the carrier molecule of formula 1.
[0017] The preparation method of the precursor nanometer self-assembly immune composition is as follows: the immune carrier molecules, active drugs and solvents are mixed in a certain ratio, and stirred to make the mixture uniform to form a clear and transparent solution.
[0018] The precursor nanometer self-assembly immune composition can be further encapsulated in a soft capsule or a liquid-fillable hard capsule; it can also be made into a concentrated solution for injection.
[0019] Application of precursor nano self-assembled immune composition in the preparation of anti-tumor drugs.
[0020] Compared with the prior art, the advantages and positive effects of the present invention are: it can achieve efficient loading of chemotherapy drugs, and the preparation process is simple and controllable, which is convenient for industrial production; it can quickly self-assemble into nano-scale ultrafine particles in physiological aqueous media, thereby improving the bioavailability of such drugs and enhancing their anti-tumor efficacy. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 This is a particle size distribution diagram of the precursor nano self-assembling immune composition prepared in Example 2 of the present invention self-assembled in physiological saline;
[0022] Figure 2 This is a particle size distribution diagram of the precursor nano self-assembling immune composition prepared in Example 3 of the present invention self-assembled in physiological saline;
[0023] Figure 3 This is a particle size distribution diagram of the precursor nano self-assembling immune composition prepared in Example 4 of the present invention self-assembled in physiological saline; DETAILED DESCRIPTION
[0024] The present invention is further illustrated by the following examples.
[0025] The present invention provides a precursor nanometer self-assembly immune composition, which is composed of immune carrier molecules, active drugs and solvents; the particle size of the immune composition self-assembled in a physiological aqueous medium is 10-1000 nm.
[0026] The immune carrier molecule in the nanocomposition is formed by covalently coupling 1-methyltryptophan and indole-3-methanol as shown in formula 1.
[0027] Formula 1
[0028] The preparation method of immune carrier molecules comprises the following steps: dissolving 1-methyl-N-Fmoc-tryptophan in N, N-dimethylformamide, incubating in the dark for 1-2 hours under the catalytic action of 4-dimethylaminopyridine, carbodiimide hydrochloride and 1-hydroxybenzotriazole, then adding indole-3-methanol and reacting in the dark at 5-40° C. for 24-48 hours under nitrogen protection; adding piperidine to remove the Fmoc protecting group, and separating and purifying to obtain the carrier molecule of formula 1.
[0029] The active drug is a chemical small molecule drug that can interact with the immune carrier molecule, wherein the chemical small molecule drug is any one or more combinations of paclitaxel, docetaxel, cabazitaxel, doxorubicin, epirubicin, mitoxantrone, camptothecin, 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, irinotecan, topotecan, belotecan, vincristine, vindesine, vinorelbine, cisplatin, oxaliplatin, carboplatin, methotrexate, pemetrexed, cytarabine, capecitabine, gemcitabine, fluorouracil, teniposide, etoposide, ritonavir, homoharringtonine, indirubin, shikonin, curcumin, gambogic acid, ginsenoside, norcantharidin, arsenite, rapamycin, dihydrochlorin, and indocyanine green.
[0030] The solvent is a physiologically compatible solvent, wherein the physiologically compatible solvent is any one or more combinations of 1,2-propylene glycol, glycerol, isopropanol, ethanol, benzyl alcohol, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, and polyethylene glycol, wherein the molecular weight range of the polyethylene glycol is 200-20000.
[0031] The preparation method of the precursor nanometer self-assembly immune composition is as follows: the immune carrier molecules, active drugs and solvents are mixed in a certain ratio, and stirred to make the mixture uniform to form a clear and transparent solution.
[0032] The precursor nanometer self-assembly immune composition can be further encapsulated in a soft capsule or a liquid-fillable hard capsule; it can also be made into a concentrated solution for injection.
[0033] The prepared precursor nano self-assembled immune composition can achieve efficient loading of chemotherapy drugs, and the preparation process is simple and controllable, which is convenient for industrial production; it can quickly self-assemble into nano-scale ultrafine particles in physiological aqueous media, thereby improving the bioavailability of such drugs and enhancing their anti-tumor efficacy.
[0034] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.
[0035] Embodiment 1:
[0036] This embodiment provides an immune carrier molecule with a structure as shown in Formula 1 and a preparation method thereof. Specifically, 1.8 mmol 1-methyl-tryptophan hydrochloride is dissolved in 3 mL of dichloromethane, 2.7 mmol carbodiimide hydrochloride, 3.2 mmol 1-hydroxybenzotriazole, and 0.18 mmol 4-dimethylaminopyridine are added, and incubated at 4°C for 1.5 hours; then 3.6 mmol indole-3-methanol is added and reacted at room temperature for 24 to 48 hours under nitrogen protection; after removing the solvent by evaporation under reduced pressure, the precipitate is washed with water and ether in turn, and vacuum dried to obtain a carrier molecule with a structure as shown in Formula 1. The obtained product is 1 HNMR data are as follows: 1 H NMR (600MHz, Chloroform-d) δ 8.87 (d, J=7.0 Hz, 1H), 7.77-7.71 (m, 1H), 7.62-7.57 (m,1H), 7.38 (d, J=6.9 Hz, 1H), 7.37-7.31 (m, 2H), 7.24 (ddd, J=8.4, 7.1, 1.3Hz, 1H), 7.15 (td, J=7.6, 1.3 Hz, 1H), 7.12-7.04 (m, 2H), 6.92 (s, 1H), 5.44(d, J=12.6 Hz, 1H), 5.18 (d, J=12.6 Hz, 1H), 4.00 (tt, J=6.8, 5.9 Hz, 1H), 3.81 (s, 3H), 3.15 (qd, J=15.2, 6.0 Hz, 2H), 2.97 (t, J=7.1 Hz, 1H), 2.72 (t,J=7.1 Hz, 1H).
[0037] Embodiment 2:
[0038] This example provides a method for preparing a precursor nano self-assembled immune composition containing paclitaxel. Specifically, the immune carrier molecule (120 mg) obtained in Example 1 and paclitaxel (80 mg) are added to polyethylene glycol 200 (0.8 g), and stirred to mix evenly to form a clear and transparent solution.
[0039] Embodiment 3:
[0040] This example provides a method for preparing a precursor nano self-assembled immune composition containing docetaxel. Specifically, the immune carrier molecule (150 mg) obtained in Example 1 and docetaxel (100 mg) are added to polyethylene glycol 400 (0.75 g), and stirred to mix evenly to form a clear and transparent solution. The soft capsules are directly filled.
[0041] Embodiment 4:
[0042] This embodiment provides a method for preparing a precursor nano self-assembled immune composition containing irinotecan. Specifically, the immune carrier molecule (200 mg) obtained in Example 1 and irinotecan (100 mg) are added to polyethylene glycol 400 (0.6 g) and 1,2-propylene glycol (0.1 g), and stirred to mix them evenly to form a clear and transparent solution. After sterilization, a concentrated solution for injection is prepared.
[0043] Embodiment 5:
[0044] This example evaluates the self-assembly performance of the precursor nanometer self-assembly immune composition in physiological aqueous medium. The particle size distribution of the immune composition after self-assembly in physiological saline was measured using a laser particle size analyzer. The results are as follows: Figures 1 to 3 The precursor nanometer self-assembling immune composition of the present invention can self-assemble into ultrafine particles with an average particle size of 50-100 nm in a physiological aqueous medium.
[0045] Embodiment 6:
[0046] This example evaluates the drug leakage rate of the precursor nanometer self-assembled immune composition in the physiological aqueous medium during self-assembly, and the results are shown in Table 1. The precursor nanometer self-assembled immune composition of the present invention has a low drug leakage rate during self-assembly in the physiological aqueous medium.
[0047] Table 1. Drug leakage rate of immune composition during self-assembly process
[0048] Test samples Leakage rate (%) Example 2 1.8±0.5 Example 3 2.4±0.6 Example 4 5.3±1.2
[0049] Embodiment 7:
[0050] This example investigates the ability of the precursor nano self-assembled immune composition to inhibit tumor cell proliferation in vitro. The immune composition prepared in Examples 2 to 4 was used as an example to evaluate its in vitro cytotoxicity. Specifically, HCT116 cells in the logarithmic growth phase were inoculated into a 96-well plate (5×10 3 / well), after the cells adhered to the wall, the culture medium was replaced with the culture medium containing the immune composition, and incubated at 37°C for 48 hours. Then, 10 μL of CCK8 reagent was added to each well. After incubation for 4 hours, the OD value of each well was measured at a wavelength of 450 nm. The cell survival rate was calculated to obtain the half inhibitory concentration (IC) of the drug on cell growth. 50 ). The results of the in vitro proliferation inhibition of tumor cells by the prepared immune composition are shown in Table 2. The half inhibition concentration of the immune composition provided by the present invention is significantly lower than that of the free drug control group, and the ability to inhibit the proliferation of tumor cells is significantly improved.
[0051] Table 2. Half inhibitory concentration of immune composition on tumor cells
[0052] Test samples <![CDATA[IC 50 (μM)]]> Test samples <![CDATA[IC 50 (μM)]]> Paclitaxel >100 Example 2 51.3±3.6 Docetaxel 39.4±2.4 Example 3 27.9±2.1 Irinotecan 26.4±1.5 Example 4 15.2±1.8
[0053] Embodiment 8:
[0054] This example evaluates the in vivo oral bioavailability of the precursor nano self-assembled immune composition. Specifically, healthy male SD rats (6 to 8 weeks) were randomly divided into groups (6 in each group), and were orally gavaged with docetaxel solution and soft capsules prepared in Example 3 (both at a dose of 10 mg / kg), and the same dose of docetaxel solution was injected into the tail vein as a control group. 0.3 mL of blood was collected from the rat's orbit at different time points after administration, and the plasma was collected by heparin sodium anticoagulation and centrifugation, and stored at -80°C for testing. The results of the pharmacokinetic test are shown in Table 3. The immune composition provided by the present invention can significantly improve the oral bioavailability of the loaded drug.
[0055] Table 3. Results of oral pharmacokinetic studies
[0056] Test samples Docetaxel solution (IV) Docetaxel solution (PO) Example 3 (PO) <![CDATA[T max (h)]]> － 1.0 3.0 <![CDATA[C max (ng / mL)]]> 324.3±29.4 15.0±4.2 57.8±10.3 <![CDATA[AUC 0-∞ (ng*h / mL)]]> 1041.7±120.1 82.6±11.9 665.2±83.5 Fa (%) － 7.9 63.8
[0057] Embodiment 9:
[0058] This example evaluates the in vivo anti-tumor efficacy of the precursor nano self-assembled immune composition. Specifically, 100 μL of HCT116 cell suspension (1×10 6 ) was subcutaneously inoculated into the ventral skin of Balb / c nude mice until the tumor volume reached 100-150mm 3 The tumor-bearing mice were randomly divided into three groups (5 mice in each group) and injected with physiological saline, irinotecan injection and the injection concentrate prepared in Example 4 (calculated according to irinotecan, the dose was 5 mg / kg) through the tail vein, respectively. The drugs were administered once every three days for 4 consecutive times. The mice were killed 7 days after administration, the tumors were separated and weighed, and the tumor inhibition rate was calculated: tumor inhibition rate = (average weight of the control group - average weight of the experimental group) / average weight of the control group × 100%. The immune composition provided by the present invention can effectively inhibit the tumor growth of tumor-bearing mice, and its tumor growth inhibition rate is significantly different from that of the control group.
[0059] Table 4. Tumor inhibition rate of nanocomposites
[0060] Test samples Tumor weight (g) Tumor inhibition rate (%) Normal saline 1.82±0.36 – Irinotecan 1.03±0.25 43.4 Example 4 0.47±0.31 74.2
[0061] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.
Claims
1. A precursor nano self-assembling immune composition, characterized in that: It is composed of immune carrier molecules, active drugs and solvents.
2. The precursor nano self-assembling immune composition according to claim 1, characterized in that: The immune carrier molecule is formed by covalent coupling of 1-methyltryptophan and indole-3-methanol, and the structural formula is shown in Formula 1: Formula 1.
3. The precursor nano self-assembling immune composition according to claim 1, characterized in that: The active drug is a small chemical molecule drug that can interact with the immune carrier molecule; Preferably, the chemical small molecule drug is any one or more combinations of paclitaxel, docetaxel, cabazitaxel, doxorubicin, epirubicin, mitoxantrone, camptothecin, 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, irinotecan, topotecan, belotecan, vincristine, vindesine, vinorelbine, cisplatin, oxaliplatin, carboplatin, methotrexate, pemetrexed, cytarabine, capecitabine, gemcitabine, fluorouracil, teniposide, etoposide, ritonavir, homoharringtonine, indirubin, shikonin, curcumin, gambogic acid, ginsenoside, norcantharidin, arsenite, rapamycin, chlorin, and indocyanine green.
4. The precursor nano self-assembling immune composition according to claim 1, characterized in that: The solvent is a physiologically compatible solvent; Preferably, the physiologically compatible solvent is any one or more combinations of 1,2-propylene glycol, glycerol, isopropanol, ethanol, benzyl alcohol, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, and polyethylene glycol, wherein the molecular weight range of polyethylene glycol is 200-20000.
5. The precursor nano self-assembling immune composition according to claims 1 to 4, characterized in that: The weight percentages of the components are: immune carrier molecule: 1-99%, active drug: 1-99%, solvent: 1-99%.
6. The precursor nano self-assembling immune composition according to claims 1 to 5, characterized in that: The particle size of the immune composition self-assembled in a physiological aqueous medium is 10 to 1000 nm.
7. The method for preparing the immune carrier molecule according to any one of claims 1 to 6, characterized in that: The preparation method is prepared by the following steps: 1-methyl-N-Fmoc-tryptophan is dissolved in N, N-dimethylformamide, incubated in the dark for 1 to 2 hours under the catalytic action of 4-dimethylaminopyridine, carbodiimide hydrochloride and 1-hydroxybenzotriazole, then indole-3-methanol is added to react at 5 to 40° C. in the dark for 24 to 48 hours under nitrogen protection; piperidine is added to remove the Fmoc protecting group, and then the carrier molecule of formula 1 is separated and purified.
8. The method for preparing the precursor nano self-assembling immune composition according to any one of claims 1 to 6, characterized in that: The immune carrier molecule, active drug and solvent are mixed according to the ratio of claim 5, and stirred to make the mixture uniform to form a clear and transparent solution.
9. The method for preparing the precursor nano self-assembling immune composition according to claim 8, further comprising: The clear and transparent solution is further encapsulated in a soft capsule or a liquid-fillable hard capsule; Or further made into a concentrated solution for injection.
10. Use of the precursor nano self-assembling immune composition according to any one of claims 1 to 6 in the preparation of anti-tumor drugs.