CN110152011B - Covalent link of immune regulatory factor and taxane and albumin nano preparation thereof - Google Patents

Abstract

The invention belongs to the field of pharmaceutical chemistry and pharmaceutical preparations, and relates to a covalent linker of an immune regulatory factor and taxane and a preparation method of an albumin nano preparation thereof. The linker is a compound obtained by performing ester bond condensation reaction on carboxyl of an immunomodulatory factor indoleamine 2, 3-dioxygenase (IDO) inhibitor, 1-methyl-D-tryptophan (D-1MT) and hydroxyl of a taxane compound, is prepared into an albumin nano preparation, and is delivered to tumors by utilizing the enhanced penetration and retention effects of the nano preparation on the tumors; the linker delivered to the tumor cells is used as a prodrug of D-1MT and taxane compounds, and is hydrolyzed into D-1MT and taxane compounds by esterase, so that the purpose of jointly delivering the two drugs to the tumor cells is realized, and the D-1MT realizes the effect of synergistically treating tumors by inhibiting IDO and taxane chemotherapy.

Description

A covalent linker of immunomodulatory factor and taxane and its albumin nanoparticle preparation

technical field

The invention belongs to the field of medicinal chemistry and pharmaceutical preparations, and relates to a covalent linker of an immunoregulatory factor and a taxane and a preparation method of an albumin nanometer preparation. The linker is composed of the immunomodulatory factor indoleamine 2,3-dioxygenase (IDO) inhibitor, 1-methyl-D-tryptophan (D-1MT), with taxanes via esters The compound obtained by the bond condensation reaction; the linker is prepared into an albumin nano-formulation, and the enhanced penetration and retention effects of the nano-formulation on the tumor are used to deliver the linker to the tumor to achieve the effect of synergistically treating the tumor.

Background technique

According to reports, in recent years, the incidence of malignant tumors has risen sharply, and the mortality rate has been high. According to the data of the World Health Organization, 36% of new cancer patients are Chinese every year, and malignant tumor has become a major stubborn disease affecting the life expectancy of Chinese people. Traditional anti-tumor drugs (such as paclitaxel, doxorubicin, etc.) have strong toxic effects. Said chemotherapeutic drugs inhibit tumor growth and are also accompanied by tumor resistance and recurrence.

Studies have shown that albumin exists in large amounts in plasma, and its isoelectric point is 4.7-4.9. It is an acidic protein, easily soluble in water, and stable between pH 4-9. Human serum albumin is the most abundant protein in the human body. Due to its natural structure, biodegradability, non-toxicity, and high biocompatibility, it has been approved for clinical use by the U.S. Food and Drug Administration (FDA). regarded as an ideal drug delivery vehicle.

Taxanes, including paclitaxel and docetaxel, have strong anti-tumor activity against a variety of tumors, and lead to the formation of stable non-functional microtubule bundles by enhancing tubulin polymerization and inhibiting microtubule depolymerization. Inhibits the mitosis and proliferation of cells; but due to its poor water solubility, its application in tumor therapy is greatly limited; taxane compounds have high affinity with serum albumin, and paclitaxel albumin nanoparticles prepared with human serum albumin as a carrier The suspension (trade name Abraxane) was approved by the FDA in 2005.

Indole 2,3-dioxygenase (IDO) is an important regulatory protein that participates in the formation of tumor immunosuppressive microenvironment and promotes tumor cell growth; studies have shown that IDO has a high level in various tumor tissues. Expression is the rate-limiting enzyme for the catabolism of tryptophan through the kynuric acid pathway, catalyzing the degradation of the essential amino acid tryptophan and causing the accumulation of its metabolites, resulting in cell cycle arrest and effector T cell death, increasing the number of regulatory T cells. The immunomodulator 1-methyl-D-tryptophan (D-1MT) is an IDO inhibitor that reduces the metabolism of tryptophan, allowing effector T cells to perform their normal functions and kill tumor cells.

Based on the current situation and basis of the prior art, the inventors of the present application intend to provide a covalent linker of an immunomodulatory factor and a taxane and an albumin nano-formulation thereof, which will produce the effect of synergistically treating tumors with the two drugs.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a covalent linker of an immunomodulatory factor and a taxane and an albumin nano-formulation based on the current situation and basis of the prior art.

The present invention designs and synthesizes a covalent linker of D-1MT and taxane. The linker is a compound obtained by the condensation reaction of D-1MT and taxane compounds through ester bond. The linker was prepared into an albumin nano-formulation, and the enhanced penetration and retention effects of the nano-formulation on the tumor were used to deliver the linker to the tumor; the linker delivered to the tumor cells was used as a precursor for D-1MT and taxane compounds. D-1MT is hydrolyzed into D-1MT and taxanes by esterase to achieve the purpose of co-delivery of the two drugs to tumor cells; D-1MT reduces its inhibition of effector T cells by inhibiting IDO, which is similar to taxane. Alkane chemotherapy achieves the effect of synergistic treatment of tumors.

The covalent linker of D-1MT and paclitaxel described in the present invention is a compound obtained by the condensation reaction between the carboxyl group in the molecular structure of D-1MT and the hydroxyl group in the molecular structure of paclitaxel through ester bond condensation reaction.

Specifically, the linker is compound 1 obtained by the condensation reaction of the carboxyl group of D-1MT and the 2'-hydroxyl group of paclitaxel through an ester bond, and its molecular formula is C ₅₉ H ₆₃ N ₃ O ₁₅ ,

Described compound 1 is synthesized by the following methods and steps:

Protection of -NH in aD _- 1MT (compound 11):

The protective agent selected for use is di-tert-butyl dicarbonate, and the introduction condition has di-tert-butyl dicarbonate/sodium hydroxide/dioxane/water, di-tert-butyl dicarbonate/sodium hydroxide/tetrahydrofuran/water, dicarbonic acid Di-tert-butyl ester/sodium bicarbonate/tetrahydrofuran/water etc., preferably, described introduction condition is di-tert-butyl dicarbonate/sodium bicarbonate/tetrahydrofuran/water;

b. Condensation reaction of compound 12 with paclitaxel:

The condensing agents used are dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2-(7-benzotriazole oxide)- N,N,N',N'-tetramethylurea hexafluorophosphate, etc.; preferably, the condensing agent is dicyclohexylcarbodiimide;

c. De-NH ₂ protection of compound 13.

Commonly used conditions are trifluoroacetic acid/chloroform/ice bath, trifluoroacetic acid/dichloromethane/ice bath, trifluoroacetic acid/dichloromethane/room temperature, etc.; preferably, the described conditions are trifluoroacetic acid/dichloromethane /Ice bath.

Specifically, the linker described in the present invention is compound 2 obtained by the condensation reaction of the carboxyl group of D-1MT and the hydroxyl group at the 7-position of paclitaxel through an ester bond, and its molecular formula is C ₅₉ H ₆₃ N ₃ O ₁₅ ,

The compound 2 was synthesized by the following methods and steps:

a. Protection of the 2' hydroxyl group of paclitaxel (compound 14):

The hydroxyl protective agent is selected to include triisopropylsilyl, triethylsilyl, diphenyl-tert-butylsilyl, tert-butyldimethylchloromethane, etc.; preferably, the protective agent is tert-butyldimethylsilyl methyl chloride;

b. Condensation of compound 15 and compound 12:

The condensing agents used are dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2-(7-benzotriazole oxide)- N,N,N',N'-tetramethylurea hexafluorophosphate, etc.; preferably, the condensing agent is dicyclohexylcarbodiimide;

c. Selectively remove the protecting group of the 2'-hydroxyl group in the structure of compound 16:

Reaction conditions, preferably, tetrabutylammonium fluoride/tetrahydrofuran/room temperature;

d. De- _NH2 protection of compound 17:

The conditions used are trifluoroacetic acid/chloroform/ice bath, trifluoroacetic acid/dichloromethane/ice bath, trifluoroacetic acid/dichloromethane/room temperature, etc.; preferably, the conditions are trifluoroacetic acid/dichloromethane /Ice bath.

Specifically, the linker described in the present invention is compound 3 obtained by the condensation reaction of the carboxyl group of D-1MT and the 2' and 7-position hydroxyl groups of paclitaxel through an ester bond, and its molecular formula is C ₇₁ H ₇₅ N ₅ O ₁₆ ,

Described compound 3 is synthesized by the following methods and steps:

a. Condensation of compound 2 and compound 12:

The condensing agents used are dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2-(7-benzotriazole oxide)- N,N,N',N'-tetramethylurea hexafluorophosphate, etc.; preferably, the condensing agent is dicyclohexylcarbodiimide;

b. De- _NH2 protection of compound 18:

The conditions used are trifluoroacetic acid/chloroform/ice bath, trifluoroacetic acid/dichloromethane/ice bath, trifluoroacetic acid/dichloromethane/room temperature, etc.; preferably, the conditions are trifluoroacetic acid/dichloromethane /Ice bath.

as well as,

The covalent linker of D-1MT and docetaxel designed and synthesized by the present invention is a compound obtained by the condensation reaction of the carboxyl group in the molecular structure of D-1MT and the hydroxyl group in the molecular structure of docetaxel through an ester bond condensation reaction;

The method for synthesizing the covalent linker of D-1MT and docetaxel is similar to the method for synthesizing the covalent linker between D-1MT and paclitaxel;

Specifically, the linker is compound 4 obtained by ester bond condensation reaction between the carboxyl group of D-1MT and the 2'-hydroxyl group of docetaxel, and its molecular formula is C ₅₅ H ₆₅ N ₃ O ₁₅ ,

Specifically, the linker is compound 5 obtained by the selective condensation reaction between the carboxyl group of D-1MT and the 7-position hydroxyl group of docetaxel, and its molecular formula is C ₅₅ H ₆₅ N ₃ O ₁₅ ,

Specifically, the linker is compound 6 obtained by the selective condensation reaction between the carboxyl group of D-1MT and the 10-position hydroxyl group of docetaxel, and its molecular formula is C ₅₅ H ₆₅ N ₃ O ₁₅ ,

Specifically, the linker is compound 7 obtained by the selective condensation reaction between the carboxyl group of D-1MT and the 10-position hydroxyl group of docetaxel, and its molecular formula is C ₅₅ H ₆₅ N ₃ O ₁₅ ,

Specifically, the linker is compound 8 obtained by the condensation reaction between the carboxyl group of D-1MT and the hydroxyl groups at the 2' and 7 positions of docetaxel through an ester bond, and its molecular formula is C ₆₇ H ₇₇ N ₅ O ₁₆ ,

Specifically, the linker is compound 9 obtained by ester bond condensation reaction between the carboxyl group of D-1MT and the 2' and 10-position hydroxyl groups of docetaxel, and its molecular formula is C67H77N5O16,

Specifically, the linker is compound 10 obtained by ester bond condensation reaction between the carboxyl group of D-1MT and the 2', 7 and 10 hydroxyl groups of docetaxel, and its molecular formula is C79H89N7O17,

The present invention adopts the above-mentioned covalent linker of D-1MT and taxane, specifically one of compounds 1-10, to prepare an albumin nano preparation.

Described albumin nano preparation is prepared by following technical scheme:

The preparation contains the linker, that is, one of compounds 1-10, and albumin; the mass ratio of the linker to albumin is 1:6-1:20; preferably, the mass ratio 1:9-1:15.

The albumin is one or a mixture of human serum albumin, recombinant human serum albumin and bovine serum albumin, preferably, the albumin is human serum albumin.

In the present invention, the above-mentioned preparation method of the albumin nano-formulation containing the linker comprises the following steps:

(1) Dissolve albumin in water;

(2) dissolving the linker (a kind of compound 1-10) in an organic solvent;

(3) mixing the aqueous solution of step (1) and the organic solution of step (2) uniformly to obtain a white emulsion;

(4) high pressure homogenization of the white emulsion, so that the particle size is controlled at 50-1000nm;

(5) The organic solvent of the emulsion (4) is removed under reduced pressure.

Preferably, the above preparation method further includes the step of dehydrating the nano-formulation solution obtained in step (5); preferably, the dehydration treatment is freeze-drying.

Preferably, the concentration of the albumin aqueous solution in step (1) is 0.5%-5% (w/v), preferably 1%-2% (w/v).

Preferably, the ratio of the linker in step (2) to the total protein in step (2) is 1:9-1:15.

Preferably, the ratio of the organic solvent in step (2) to the water in step (1) is 1:20-1:50.

Preferably, the organic solvent in step (2) is formed by mixing a hydrophobic organic solvent and a hydrophilic organic solvent. The hydrophobic organic solvent can be chloroform, dichloromethane or a mixed solvent of the two; the hydrophilic organic solvent can be anhydrous ethanol, methanol, propylene glycol or a mixed solvent of two or three of them. The ratio of the organic solvent to the aqueous solution in step (1) is 1:20-1:50.

Preferably, in the high-pressure homogenization described in step (4), the pressure range is 10000psi--20000psi, and the number of cycles is 10-20 times.

The present invention provides a covalent linker of D-1MT and taxane, the linker is a compound obtained by ester bond condensation reaction of D-1MT and taxane compounds; the linker is prepared into albumin The nano-formulation, utilizing the enhanced penetration and retention effect of the nano-formulation on the tumor, delivers the linker to the tumor, and can achieve the co-delivery of the two drugs to the tumor cells to achieve the effect of synergistically treating the tumor.

Embodiments of the present invention are described in detail below in conjunction with the accompanying drawings,

Description of drawings

Fig. 1. The intratumoral D-1MT concentration changes with time in female melanoma-bearing C57 mice by intravenous injection of compound 1 albumin nano-formulation at a dose of 12.34 mg of compound 1/kg, n=6.

Figure 2. The change curve of intratumoral paclitaxel concentration over time by intravenous injection of compound 1 albumin nano-formulation in female melanoma C57 mice at a dose containing 12.34 mg of compound 1/kg, n=6.

Figure 3. The compound 1 albumin nano-formulation was injected into the tail vein of the female melanoma-bearing C57 mice, and the administration dose was 12.34 mg of the compound 1/kg, and the curve of the intratumoral compound 1 concentration with time, n=6.

Figure 4. Survival curve of melanoma-bearing C57 female mice after intravenous administration, wherein, ^** p<0.01, ^*** p<0.001 (each treatment group: control group). ^## p<0.01 (paclitaxel albumin nano preparation group: compound 1 albumin nano preparation group), ^&& p<0.01 (paclitaxel albumin nano preparation and D-1MT mixed solution group: compound 1 albumin nano preparation group), n=10. Tumor cells were inoculated on day 0, and treatment was started on day 6.

FIG. 5 is a graph of body weight versus time after intravenous administration of melanoma-bearing C57 female mice, n=10, which shows that tumor cells were inoculated on day 0 and treatment was started on day 6. FIG.

Detailed ways

The following examples are used to further illustrate and explain the present invention, but not as a limitation of the present invention.

Example 1: Synthesis of Compound 1

Synthetic route 1

Reaction conditions and reagents:

a. sodium bicarbonate, di-tert-butyl dicarbonate, tetrahydrofuran/water, room temperature for 24 hours;

b. Paclitaxel, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, N,N-dimethylformamide, overnight at room temperature;

c. Trifluoroacetic acid, dichloromethane, ice bath for 30 minutes.

Synthesis of compound 12 in Example 1:

To the 100ml eggplant-shaped bottle, add 10ml double distilled water and 10ml tetrahydrofuran respectively, stir and mix, and then add 250mg (1.14mmol) of 1-methyl-D-tryptophan, 288.7mg (3.44mmol) of sodium bicarbonate and two 300 mg (1.37 mmol) of di-tert-butyl carbonate was stirred in an ice bath for 10 minutes and then moved to room temperature to continue the reaction for 24 hours. After completion of the reaction, spin dry tetrahydrofuran to obtain an aqueous layer. Under stirring in an ice bath, the pH of the aqueous layer was adjusted to 1 with 1 mol/L hydrochloric acid solution, extracted with ethyl acetate three times (15 ml × 3) and dried over anhydrous sodium sulfate, filtered, and rotated to dryness. Methane:methanol=30:1) yielded 270 mg of white solid. Yield: 270 mg, 74%;

Nuclear magnetic analysis: 1H NMR (600MHz, CD2Cl2, ppm): δ7.58 (d, J=9.0Hz, 1H), 7.32 (d, J=9.0Hz, 1H), 7.25-7.19 (m, 1H), 7.12- 7.07(m, 1H), 6.94(s, 1H), 5.09(s, 1H), 4.60(s, 1H), 3.70(s, 3H), 3.31(s, 2H), 1.41(s, 9H).

13C NMR (75MHz, CD2Cl2, ppm): δ174.6, 156.1, 137.2, 121.7, 119.1, 110.2, 78.7, 55.3, 32.9, 28.8.

Mass spectrometry ESI-MS m/z[M+H]+ Calculated: C17H22N2O4659.2; Found: 659.2;

Synthesis of compound 13 in Example 1:

Into a 25 ml eggplant-shaped bottle, 200 mg (0.63 mmol) of compound 12, 536.9 mg (0.63 mmol) of paclitaxel, 4.7 mg of 4-dimethylaminopyridine and 6 ml of anhydrous N,N-dimethylformamide were sequentially added. Under stirring in an ice bath, 155.7 mg (0.75 mmol) of dicyclohexylcarbodiimide was added, and the mixture was stirred for 5 minutes, then moved to room temperature and reacted overnight. After completion of the reaction, spin dry N,N-dimethylformamide, add 10 ml of dichloromethane, filter, spin dry, and perform silica gel column chromatography (dichloromethane:ethyl acetate=5:2) to obtain 346 mg of white solid. Yield: 346 mg, 47.7%;

Nuclear magnetic analysis: 1H NMR (600MHz, CDCl3) δ8.16 (d, J=7.6Hz, 2H), 7.75 (d, J=7.4Hz, 2H), 7.64 (t, J=7.3Hz, 1H), 7.60 ( d, J=7.8Hz, 1H), 7.55 (d, J=7.7Hz, 2H), 7.51 (t, J=7.4Hz, 1H), 7.39 (dd, J=16.2, 8.4Hz, 4H), 7.35– 7.29(m,4H),7.27–7.25(m,1H),7.14(t,J=7.0Hz,1H),6.98(dd,J=11.3,4.1Hz,2H),6.32(s,1H),6.26 (t, J=8.5Hz, 1H), 5.92 (d, J=5.6Hz, 1H), 5.71 (d, J=7.0Hz, 1H), 5.47 (s, 1H), 5.03–4.93 (m, 2H) ,4.75(d,J＝5.9Hz,1H),4.47(dd,J＝10.4,6.7Hz,1H),4.34(d,J＝8.5Hz,1H),4.22(d,J＝8.5Hz,1H) ,3.83(d,J＝6.6Hz,1H),3.67(s,3H),3.41(dd,J＝14.6,4.9Hz,1H),3.26(dd,J＝15.2,5.8Hz,1H),2.57( dd,J＝16.1,7.8Hz,1H),2.41(s,3H),2.32(d,J＝9.3Hz,1H),2.26(s,3H),2.17(dd,J＝15.4,5.8Hz,1H ),1.95(s,3H),1.89(d,J=12.2Hz,1H),1.70(s,3H),1.41(s,9H),1.27(s,3H),1.17(s,3H).

13C NMR(150MHz,CDCl3)δ203.84,171.78,171.25,169.79,167.93,167.14,167.05,155.32,142.84,136.92,136.85,133.72,133.60,132.80,131.95,130.23,129.18,129.08,128.70,128.57,128.11,127.96 ,127.17,126.77,121.85,119.27,118.73,109.37,107.94,84.45,81.05,80.18,79.15,76.43,75.63,75.09,74.59,72.14,71.89,66.90,58.54,53.78,53.14,45.57,43.18,35.52,32.53 ,28.28,27.45,26.75,22.55,22.11,20.83,14.72,9.60.

Mass spectrometry ESI-MS m/z[M+H]+ Calculated: C64H71N3O171154.6; Found: 1154.5;

Synthesis of Compound 1 in Example 1:

To a 25 ml eggplant bottle, 200 mg of compound 13 and 4 ml of dichloromethane were added. 4 ml of trifluoroacetic acid was added under stirring in an ice bath, and the reaction was carried out for 30 minutes. After the reaction was completed, 20 ml of saturated sodium carbonate solution was added to neutralize the trifluoroacetic acid, extracted with dichloromethane 3 times (30 ml × 3) and dried over anhydrous sodium sulfate, filtered, and rotated to dryness, and subjected to silica gel column chromatography (dichloromethane:acetic acid). ethyl ester = 1:1 and dichloromethane:methanol = 40:1) to give 80 mg of white solid. Yield: 80 mg, 43.8%;

Nuclear magnetic analysis: 1H NMR (600MHz, CDCl3) δ8.16 (d, J=7.1Hz, 2H), 7.83 (d, J=7.2Hz, 2H), 7.64 (t, J=7.4Hz, 1H), 7.57 ( d, J=7.9Hz, 1H), 7.56–7.53 (m, 2H), 7.53–7.51 (m, 1H), 7.50 (d, J=7.4Hz, 1H), 7.42–7.36 (m, 6H), 7.34 –7.30(m,1H),7.28-7.23(m,2H),7.14-7.10(m,1H),6.90(s,1H),6.31(s,1H),6.24(t,J=8.5Hz,1H ), 6.02(dd, J＝9.0, 3.8Hz, 1H), 5.69(d, J＝7.1Hz, 1H), 5.58(d, J＝3.8Hz, 1H), 4.98(d, J＝9.0Hz, 1H) ),4.45(dd,J=10.9,6.7Hz,1H),4.33(d,J=8.4Hz,1H),4.22(d,J=8.4Hz,1H),3.88–3.84(m,1H),3.82 (d, J=7.0Hz, 1H), 3.67 (s, 3H), 3.31 (dd, J=14.6, 4.8Hz, 1H), 2.99 (dd, J=14.7, 8.0Hz, 1H), 2.60–2.54 ( m,1H), 2.48(s,3H), 2.37–2.31(m,1H), 2.24(s,3H), 2.14–2.09(m,1H), 1.94(s,3H), 1.89(d,J= 14.3Hz, 1H), 1.70(s, 3H), 1.24(s, 3H), 1.15(s, 3H).

13C NMR(150MHz,CDCl3)δ203.16,170.62,169.42,167.55,166.61,166.42,142.00,136.39,136.24,133.09,133.03,132.25,131.35,129.61,128.56,128.41,128.11,128.05,127.89,127.41,127.17,126.70 ,126.42,126.11,121.27,118.60,117.99,108.86,108.06,83.79,80.44,78.50,75.80,74.96,74.44,73.97,71.48,71.30,57.89,54.37,52.50,44.97,42.54,34.93,34.84,31.98,29.63 ,26.15,22.14,21.46,20.20,14.21,8.98.

Mass Spectrometry ESI-MS calculated for m/z [M+H]+: C59H63N3O151054.4; found: 1054.4.

Example 2: Preparation and Evaluation of Albumin Nanoformulations Containing Compound 1

Dissolve human serum albumin in water and mix well, the protein concentration is 1.5% (w/v), and weigh 30g of 1.5% albumin aqueous solution (w/v) into a 50ml beaker. Weigh 30.0 mg of compound 1, dissolve it in an organic solvent (chloroform: anhydrous ethanol=85:15), the drug dosage is 1:15 to the protein dosage, and the ratio of the organic solvent to the water in the protein aqueous solution is 1:44. Stir, prepare crude milk, and obtain a white emulsion; homogenize the white emulsion under a pressure of 20,000 psi, so that the particle size is controlled at 50-200 nm; rotate the obtained compound 1 albumin nanoparticle solution, remove the organic solvent, and use 0.22 μm of sterile membrane filtration. Freeze-drying to obtain a freeze-dried powder of albumin nanoparticles of compound 1, and relevant parameters of the prepared nano-formulations are shown in Table 1.

Table 1

Particle size (nm) Zeta potential (mV) PDI Drug loading Encapsulation rate 116.5±0.520 -18.733±0.802 0.177±0.006 5.40%±0.13% 89.57%±5.30%

Detection of intratumoral drug concentration: 18 6-8 week old C57 female mice bearing B16-OVA melanoma (tumor volume of 400-600 mm3) were randomly divided into 3 groups, and the compound 1 albumin nano preparation was injected into the tail vein. , the administration dose was 12.34 mg/kg, after tail vein injection, the mice were sacrificed at 0.5 hours, 2 hours, and 12 hours, respectively, and the contents of compound 1, D-1MT and paclitaxel in tumor tissues were determined by high-performance liquid chromatography. ; Figures 1-3 show that compound 1, as the prodrug form of D-1MT and paclitaxel, was hydrolyzed into D-1MT and paclitaxel after reaching the tumor site, and 12 hours after administration, there were still some compounds 1 that were not completely degraded , can act on the tumor site for a long time;

Pharmacodynamic evaluation in vivo: Prepare compound 1 albumin nano-preparation according to the method described in this example; tumor model establishment: 6-8 weeks old C57 female mice, subcutaneously inoculated with B16-OVA cells (5×105 cells/mouse) , when the tumor volume reached 50 mm3, they were randomly divided into four groups (compound 1 albumin nano preparation group, paclitaxel albumin nano preparation and D-1MT mixed solution group, paclitaxel albumin nano preparation group and control group), Each group of 10 mice was intravenously injected with compound 1 albumin nano-preparation, the mixture of paclitaxel albumin nano-preparation and D-1MT, paclitaxel albumin nano-preparation and phosphate buffered saline (PBS), respectively, at a dose of 10 mg paclitaxel/ kg, containing 2.56 mg D-1MT/kg. Administered once every 3 days for a total of 5 times, the survival curve and the change curve of body weight versus time were drawn; Figure 4 shows the compound 1 albumin nano-preparation group, the mixed solution group of paclitaxel albumin nano-preparation and D-1MT The median survival time of the paclitaxel albumin nano-preparation group and the control group were 32.5 days, 27 days, 25 days and 21 days, respectively, indicating that the compound 1 albumin nano-preparation group was better than the pure paclitaxel albumin nano-preparation, paclitaxel white The mixture of protein nano-formulation and D-1MT has better anti-tumor effect; Figure 5 shows that the mice did not lose weight after administration for 5 times, confirming the low toxicity of compound 1 albumin nano-formulation.

Example 3: Preparation and Evaluation of Albumin Nanoformulations Containing Compound 3

Dissolve bovine serum albumin in water and mix well, the protein concentration is 2.0% (w/v), weigh 30g of 2.0% albumin aqueous solution (w/v) in a 50ml beaker, weigh 45.0mg of compound 3, dissolve in In an organic solvent (dichloromethane: anhydrous ethanol=90:10), the ratio of the drug dosage to the protein dosage=1:13, the ratio of the organic solvent to the water in the protein aqueous solution is 1:50, stirring at high speed to prepare crude milk, and obtain White emulsion; homogenize the white emulsion under a pressure of 18000 psi, so that the particle size is controlled at 50-500 nm; the obtained compound 3 albumin nanoparticle solution is rotary evaporated, and after removing the organic solvent, it is filtered with a 0.22 μm sterile membrane. Freeze-drying to obtain the freeze-dried powder of the albumin nanoparticles of compound 3, and the relevant parameters of the prepared nano-formulations are shown in Table 2;

Table 2

Particle size (nm) Zeta potential (mV) PDI Drug loading Encapsulation rate 250.5±0.510 -18.354±0.567 0.180±0.012 7.20%±0.14% 88.34%±4.35%

In vivo pharmacodynamic evaluation: Compound 3 albumin nano-formulation was prepared according to the method described in this example. Tumor model establishment: 6-8 weeks old C57 female mice were subcutaneously inoculated with B16-OVA cells (5×10 ⁵ cells/mouse), and when the tumor volume reached 50 mm, they were randomly divided into four groups (compound ³ white Protein nano preparation group, mixture of paclitaxel albumin nano preparation and D-1MT group, paclitaxel albumin nano preparation group and control group), 10 mice in each group were intravenously injected with compound 3 albumin nano preparation, paclitaxel albumin nano preparation The mixed solution with D-1MT, paclitaxel albumin nano-formulation and phosphate buffered saline, the administration dose is 10 mg paclitaxel/kg and 5.11 mg D-1MT/kg. Administered once every 3 days for a total of 5 times; the results showed that the compound 3 albumin nano-preparation group, the mixture of paclitaxel albumin nano-preparation and D-1MT group, the paclitaxel albumin nano-preparation group and the control group The median survival times were: 35.5 days, 27.5 days, 26 days and 20 days, respectively, indicating that the compound 3 albumin nano preparation group had better performance than the pure paclitaxel albumin nano preparation, the mixture of paclitaxel albumin nano preparation and D-1MT. Good anti-tumor effect, after 5 administrations, the compound 3 albumin nano-formulation did not cause weight loss in mice, which confirmed that the compound 3 albumin nano-formulation had low toxicity.

Example 4: Preparation and Evaluation of Albumin Nanoformulations Containing Compound 2

Dissolve recombinant human serum albumin in water and mix evenly, the protein concentration is 1.0% (w/v), and weigh 30g of 1.0% albumin aqueous solution (w/v) into a 50ml beaker. Weigh 30.0 mg of compound 2, dissolve it in an organic solvent (chloroform: propylene glycol=85:15), the drug dosage is greater than the protein dosage=1:10, and the ratio of the organic solvent to the water in the protein aqueous solution is 1:40, stirring at high speed, Colostrum was prepared to obtain a white emulsion; the white emulsion was homogenized under a pressure of 18000 psi, so that the particle size was controlled at 50-200 nm; the obtained compound 2 albumin nanoparticle solution was rotary evaporated, and after removing the organic solvent, 0.22 μm Bacterial membrane filtration. Freeze-drying to obtain a freeze-dried powder of albumin nanoparticles of compound 2; the relevant parameters of the prepared nano-formulations are shown in Table 3.

table 3

Example 5: Preparation and Evaluation of Albumin Nanoformulations Containing Compound 8

Dissolve human serum albumin in water and mix well, the protein concentration is 1.5% (w/v), weigh 30g of 1.5% albumin aqueous solution (w/v) in a 50ml beaker, weigh 30.0mg of compound 8, dissolve in In an organic solvent (chloroform: anhydrous ethanol=85:15), the ratio of the drug dosage to the protein dosage = 1:15, the ratio of the organic solvent to the water in the protein aqueous solution is 1:45, stirring at high speed to prepare colostrum to obtain a white emulsion ; Homogenize the white emulsion under a pressure of 18000 psi, so that the particle size is controlled at 50-200 nm; The obtained compound 8 albumin nanoparticle solution is rotary evaporated, and after removing the organic solvent, it is filtered with a 0.22 μm sterile filter and freeze-dried. , to obtain the lyophilized powder of the albumin nanoparticles of compound 8; the relevant parameters of the prepared nano-formulations are shown in Table 4;

Table 4

Particle size (nm) Zeta potential (mV) PDI Drug loading Encapsulation rate 147.3±0.310 -18.542±0.177 0.180±0.011 5.24%±0.12% 89.96%±3.55%

In vivo pharmacodynamic evaluation: prepare compound 8 albumin nano-formulation according to the method described in this example; tumor model establishment: 6-8 weeks old C57 female mice, subcutaneously inoculated with B16-OVA cells (5×10 ⁵ cells/mouse ), when the tumor volume reached 50 ^mm3 , they were randomly divided into four groups (compound 8 albumin nano preparation group, docetaxel albumin nano preparation and D-1MT mixed solution group, docetaxel albumin nano preparation group and control group), 10 mice in each group were intravenously injected with compound 8 albumin nano-preparation, the mixture of docetaxel albumin nano-preparation and D-1MT, docetaxel albumin nano-preparation and phosphate buffered saline, respectively. The dose is 10mg docetaxel/kg, 5.40mg D-1MT/kg, administered once every 3 days, for a total of 5 times, the results show that the compound 8 albumin nano preparation group, the docetaxel albumin nanometer The median survival time of the mixed solution group of the preparation and D-1MT, the docetaxel albumin nano preparation group and the control group were 36 days, 27.5 days, 25 days and 21 days, respectively. It shows that the compound 8 albumin nano preparation group has better anti-tumor effect than the simple docetaxel albumin nano preparation, the mixture of docetaxel albumin nano preparation and D-1MT; Protein nanoformulation did not cause weight loss in mice, confirming the low toxicity of compound 8 albumin nanoformulation.

Example 6: Preparation and Evaluation of Albumin Nanoformulations Containing Compound 4

Dissolve human serum albumin in water and mix well, the protein concentration is 1.5% (w/v), and weigh 30g of 1.5% albumin aqueous solution (w/v) into a 50ml beaker. Weigh 45.0 mg of compound 4, dissolve it in an organic solvent (chloroform: anhydrous ethanol=86:14), the drug dosage is greater than the protein dosage=1:10, and the ratio of the organic solvent to the water in the protein aqueous solution is 1:44. Stir to prepare colostrum to obtain a white emulsion; homogenize the white emulsion under a pressure of 18000 psi to control the particle size to 50-300 nm; rotate the obtained compound 4 albumin nanoparticle solution, remove the organic solvent, and use 0.22 μm The sterile filter membrane filter, freeze-drying, obtains the freeze-dried rice of the albumin nanoparticle of compound 4; The relevant parameters of the prepared nano-formulation are as shown in Table 5;

table 5

Particle size (nm) Zeta potential (mV) PDI Drug loading Encapsulation rate 200.5±0.330 -18.556±0.167 0.180±0.011 5.94%±0.23% 88.97%±3.13%

Pharmacodynamic evaluation in vivo: Prepare compound 4 albumin nano-preparation according to the method described in this example; tumor model establishment: 6-8 weeks old C57 female mice, subcutaneously inoculated with B16-OVA cells (5×10 ⁵ cells/mouse ), when the tumor volume reached 50 mm, they ^were randomly divided into four groups (compound 4 albumin nano-preparation group, docetaxel albumin nano-preparation and D-1MT mixed solution group, docetaxel albumin nano-preparation group and Control group), 10 mice in each group were intravenously injected with compound 4 albumin nano-preparation, the mixture of docetaxel albumin nano-preparation and D-1MT, polyenol albumin nano-preparation and phosphate buffered saline, respectively, at the dosage To contain 10 mg of docetaxel/kg, to contain 2.70 mg of D-1MT/kg. It was administered once every 3 days for a total of 5 administrations. The results showed that the compound 4 albumin nano-preparation group, the mixed solution group of docetaxel albumin nano-preparation and D-1MT, the docetaxel albumin nano-preparation group and the The median survival time of the control group was 33 days, 26.5 days, 25 days and 20 days, respectively. It shows that the compound 4 albumin nano preparation group has better anti-tumor effect than the simple docetaxel albumin nano preparation, the mixture of docetaxel albumin nano preparation and D-1MT; Protein nanoformulation did not cause weight loss in mice, confirming the low toxicity of compound 4 albumin nanoformulation.

Example 7: Preparation and Evaluation of Albumin Nanoformulations Containing Compound 10

Dissolve human serum albumin in water and mix well, the protein concentration is 1.5% (w/v), weigh 30g of 1.5% albumin aqueous solution (w/v) in a 50ml beaker, weigh 30.0mg of compound 10, dissolve in In the organic solvent (chloroform: anhydrous ethanol=87:13), the ratio of the drug dosage to the protein dosage = 1:15, and the ratio of the organic solvent to the water in the protein aqueous solution is 1:44. Stir at high speed to prepare colostrum to obtain a white emulsion; homogenize the white emulsion under a pressure of 20,000 psi, so that the particle size is controlled at 50-200 nm; rotate the obtained compound 10 albumin nanoparticle solution, remove the organic solvent, and use 0.22 μm sterile filter. Freeze-drying to obtain the freeze-dried powder of the albumin nanoparticles of compound 10; the relevant parameters of the prepared nano-formulations are shown in Table 6;

Table 6

Particle size (nm) Zeta potential (mV) PDI Drug loading Encapsulation rate 154.5±0.246 -18.339±0.185 0.157±0.021 5.04%±0.13% 89.23%±3.46%

In vivo pharmacodynamic evaluation: prepare compound 10 albumin nano-formulation according to the method described in this example; tumor model establishment: 6-8 weeks old C57 female mice, subcutaneously inoculated with B16-OVA cells (5×10 ⁵ cells/mouse ), when the tumor volume reached 50 ^mm3 , they were randomly divided into four groups (compound 10 albumin nano preparation group, docetaxel albumin nano preparation and D-1MT mixed solution group, docetaxel albumin nano preparation group) and control group), 10 mice in each group were intravenously injected with compound 10 albumin nano preparation, the mixture of docetaxel albumin nano preparation and D-1MT, docetaxel albumin nano preparation and phosphate buffered saline, respectively. The dose was 10 mg of docetaxel/kg, 8.10 mg of D-1MT/kg, administered once every 3 days, for a total of 5 administrations. The median survival time of the mixed solution group of the preparation and D-1MT, the docetaxel albumin nano preparation group and the control group were 39 days, 27.5 days, 25 days and 21 days, respectively. It shows that the compound 10 albumin nano preparation group has better anti-tumor effect than the simple docetaxel albumin nano preparation, the mixture of docetaxel albumin nano preparation and D-1MT; Protein nanoformulations did not cause weight loss in mice, demonstrating the low toxicity of compound 10 albumin nanoformulations.

Claims (7)

1. a covalent linker of an immunomodulatory factor and a taxane, wherein the immunomodulatory factor is 1-methyl-D-tryptophan (D-1MT); the taxane The compound is paclitaxel or docetaxel; the covalent linker is prepared by the condensation reaction between the carboxyl group of D-1MT and the hydroxyl group of the taxane compound through ester bond; The carboxyl group of described D-1MT and the hydroxyl group of paclitaxel 2' position, or 7 position, or 2' position and 7 position make one of the following structural compounds through ester bond condensation reaction:

The carboxyl group of the D-1MT and the hydroxyl group of docetaxel are 2', or 7, or 10, or 7 and 10, or 2' and 7, or 2' and 10, or The 2' position and the 7th position and the 10th position are prepared by ester bond condensation reaction to obtain one of the following structural compounds:

2 . The covalent linker of an immunomodulatory factor and a taxane according to claim 1 , wherein the albumin nano-formulation prepared by the linker comprises the linker and albumin. 3 . 3. by the covalent linker of immunomodulatory factor according to claim 2 and taxane, it is characterized in that, in the described albumin nano preparation, the mass ratio of linker and albumin is 1:6-1: 20. 4 . The covalent linker of immunomodulatory factor and taxane according to claim 2 , wherein the albumin nano-preparation has a particle size of 50-1000 nm. 5 . 5. according to the covalent linker of the described immunomodulatory factor of claim 2 and taxane, it is characterized in that, in described albumin nano preparation, albumin is selected from human serum albumin, recombinant human serum albumin and bovine serum albumin One or a mixture of serum albumins. 6. by the covalent linker of the described immunomodulatory factor of claim 2 and taxane, it is characterized in that, described albumin nano preparation is prepared by following method, comprises the following steps: (1) described albumin is dissolved in water; (2) the described linker is dissolved in the organic solvent; (3) mixing the aqueous solution of step (1) and the organic solution of step (2) uniformly; (4) high pressure homogenization of the mixed solution obtained in step (3); (5) The organic solvent is removed under reduced pressure from the product obtained in step (4). 7 . The covalent linker of an immunomodulatory factor and a taxane according to claim 6 , wherein, in the preparation method of the albumin nano-formulation, the concentration of the albumin aqueous solution is 0.5%-5%. 8 .

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