CN106317067A - Anti-tumor medicine conjugate, preparation method, preparation and application - Google Patents

Abstract

The invention discloses an antitumor drug conjugate, a preparation method and the preparation and application of nano micelles. The structural formula of the conjugate provided by the present invention is shown in formula (I), which is formed by connecting 7-ethyl-10-hydroxycamptothecin and paclitaxel antineoplastic drugs through a link, wherein L is a link; R ₁ is phenyl or tert _- butoxy, R2 is acetyl, H or methyl, _R3 is H or methyl, the steps are simple, the preparation cost is low, the stability is high, the safety is good, and it meets the requirements of clinical medicine. Suitable for large-scale industrial production. The invention also discloses nanomicelles composed of antitumor drug coupling and amphiphilic polymers as shown in formula (I) and its antitumor application. In vivo tests show that nanomicelles have good antitumor effects and have better antitumor effects. Market prospect and value.

Description

A kind of antitumor drug conjugate, preparation method, preparation and application

technical field

The invention belongs to the technical field of research and development of anti-tumor drugs, and in particular relates to an anti-tumor drug conjugate, a preparation method, a preparation and an application.

Background technique

Cancer is a common disease that seriously threatens the safety of human life. In the current tumor treatment, drug-based chemotherapy plays an irreplaceable role, but long-term monotherapy can easily make the human body resistant to specific drugs. The combined drug regimen can improve the anti-tumor efficacy of the drug, delay the generation of drug resistance in the body, and reduce adverse reactions and side effects (Ma L, Kohli M, Smith A. Nanoparticles for Combination Drug Therapy. ACSnano.2013, 7: 9518 -25.) At present, the treatment of tumors has changed from the initial single drug to combination drug.

7-Ethyl-10-hydroxycamptothecin (SN38) is the active ingredient of the clinical antitumor drug irinotecan (CPT-11), and its antitumor activity in vitro is 100-1000 times that of CPT-11. However, because it is extremely difficult to dissolve in water and can only be dissolved in a few organic solvents, its use in biomedicine is greatly limited. Currently, SN38 delivery systems reported at home and abroad mainly include physical embedding and chemical bonding. However, the drug-loading capacity and drug-loading efficiency of the physically embedded SN38 drug-loading system are mostly unstable, and the drug release is uncontrollable, and there is a phenomenon of burst release. The chemically bonded SN38 drug-loading system has problems such as low drug-loading capacity, difficult synthesis, and complex molecular structure.

Paclitaxel is originally a natural anti-tumor drug extracted from the trunks and barks of various plants of the genus Taxus. It has obvious curative effects on many cancers. So far, paclitaxel and its semi-synthetic analogue docetaxel have become the most sold anticancer drugs in history and are widely used in antitumor therapy. However, the water solubility of paclitaxel is extremely low, and its application is also subject to certain restrictions.

Polymer micelle is a new type of nanocarrier that has developed rapidly in recent years. It is formed by self-assembly of amphiphilic block copolymers in water. It has a core-shell structure with a hydrophobic core and a hydrophilic shell. The hydrophobic core can Load insoluble drugs and improve the stability of insoluble drugs. The hydrophilic shell can effectively avoid the phagocytosis of the reticuloendothelial system, prolong the circulation time of drugs in the body, improve the pharmacokinetic behavior of drugs in vivo, and improve drug targeting . The difficulty in the research of anti-tumor combined drug system is how to effectively encapsulate two or more drugs in the same liposome to make them stable in blood circulation and ensure that the anti-tumor active ingredients act on tumor cells according to their synergistic ratio .

Our previous research found that the unique chemical structures of camptothecin and paclitaxel make it difficult to directly encapsulate them in the same nanocarrier. The chemical modification method can ensure the anti-tumor activity of the drugs, so that the drugs can be co-encapsulated in the same nanocarrier. in nanocarriers.

So far, there are no reports on the combination of camptothecins and paclitaxel antineoplastic drugs and the preparation and application of nanomicelles.

Contents of the invention

The invention provides an antineoplastic drug conjugate, which is formed by linking 7-ethyl-10-hydroxycamptothecin and paclitaxel antineoplastic drugs through connecting bonds, with simple steps and low preparation cost.

The invention also provides an anti-tumor drug conjugate nano-micelle preparation, which has a simple preparation method, high stability and good safety, meets the requirements of clinical medication, and is suitable for large-scale industrial production.

The invention also provides the application of an anti-tumor drug conjugate nano-micelle preparation in anti-tumor, which has relatively moderate release speed, high safety and no side effects.

An antitumor drug conjugate, the antitumor drug conjugate has the following structure:

A—L—B

Among them: the structure of A is:

The structure of B is:

The position of the zigzag line is the connection position with L;

That is, the general structural formula of the antitumor drug conjugate is as (I):

Wherein, L is a connecting segment, R ₁ is phenyl or tert-butoxy, R ₂ is acetyl, H or methyl, R ₃ is H or methyl.

As preferably, in structural formula (I), when R ₁ is phenyl, R ₂ is acetyl, R ₃ is H; R ₁ is tert-butoxy, R ₂ is H, R ₃ is H; or, R When ₁ is tert-butoxy, R ₂ is methyl, R ₃ is methyl;

Preferably, the connecting section has the following structure:

n=2-6;

As a further preference, said n=2.

That is, the structures of the preferred antitumor drug conjugates are as follows:

The present invention also provides a preparation method of the above-mentioned antineoplastic drug conjugate, comprising: reacting the precursor raw material of A structure with the dianhydride corresponding to L to prepare intermediate compound (II), and then intermediate compound (II) React with the precursor material of B structure to obtain the final anti-tumor drug conjugate;

The structure of the intermediate compound (II) is as follows:

The precursor raw material structure of the A structure is as follows:

The precursor raw material structure of the B structure is as follows:

As a preference, when n=2, the structure of the intermediate compound (II) is as follows:

Correspondingly, the dianhydride corresponding to L is succinic anhydride; when n takes other values, the corresponding dianhydride can be used and prepared according to the above method.

Preferably, the precursor material of the A structure is selected from paclitaxel (PTX), docetaxel (DTX), cabazitaxel (DTX) and the like. The precursor material of the B structure is 7-ethyl-10-hydroxycamptothecin.

As a preference, in the reaction of the precursor material of the A structure with succinic anhydride or other dianhydrides corresponding to L, DMAP is generally added as a catalyst; the reaction can be completed at room temperature. The molar ratio of the precursor material of the structure A to succinic anhydride is 1: (1-4).

As a preference, when the intermediate compound (II) reacts with the precursor raw material of B structure, a condensing agent and a catalyst are generally added, the condensing agent generally selects EDC HCl, the catalyst generally selects DMAP, and simultaneously neutralizes EDC by DIEA. Hydrochloric acid in HCl. The reaction can be carried out at room temperature, and the reaction conditions are mild. Preferably, the molar ratio of the intermediate compound (II) to the precursor material of structure B is 1:(1-1.5), more preferably 1:1.

An anti-tumor drug conjugate nanomicelle preparation, the nano-micelle is composed of an anti-tumor drug conjugate and an amphiphilic macromolecule, and the mass ratio of the amphiphilic macromolecule to the drug is 5:1～ 1:10.

Preferably, the amphiphilic polymer is selected from any one of DSPE-PEG, PEG-PLA, PEG-PLGA, mPEG-PLA, and mPEG-PLGA. As a further preference, the amphiphilic polymer is DSPE-PEG ₂₀₀₀ .

Preferably, the preparation is powder or tablet, injection, pill and the like.

A preparation method of the above-mentioned anti-tumor drug conjugate nanomicelle preparation, comprising: dissolving the anti-tumor drug conjugate and amphiphilic polymer material in an organic solvent, and then adding it to water to obtain Compuri Tin nanoformulations.

An application of the antitumor drug conjugate nanomicelle in antitumor.

The invention is formed by linking 7-ethyl-10-hydroxycamptothecin and paclitaxel antineoplastic drugs through connecting bonds, has simple steps, low preparation cost, high stability and good safety, meets the requirements of clinical medication, and is suitable for large-scale industrial production. The invention couples the anti-tumor drug with the amphiphilic polymer to make nano micelles, and in vivo tests show that the nano micelles have good anti-tumor effect and have good market prospect and value.

Description of drawings

Fig. 1 is the synthetic route of SN38-PTX conjugate 1;

Fig. 2 is the synthetic route of SN38-DTX conjugate 2;

Figure 3 is the synthetic route of SN38-CTX conjugate 3;

Fig. 4 is the electron microscope particle size figure of nanomicelle 1-NM in embodiment 4;

Fig. 5 is the electron microscope particle size figure of nanomicelle 2-NM in embodiment 5;

Fig. 6 is the electron microscope particle size figure of nanomicelle 3-NM in embodiment 6;

Fig. 7 is the in vitro stability test result of nanomicelle in embodiment 5,7,8,9;

Fig. 8 is the in vitro release of nano micelles in embodiment 5;

Fig. 9 is the in vitro release of nano micelles in embodiment 7;

Figure 10 is the effect of inhibiting tumor cell proliferation in vitro of the conjugate in Example 2;

Fig. 11 is the tumor targeting test result of nanomicelle in embodiment 5;

Fig. 12 is the in vivo antitumor effect of nanomicelle in embodiment 4,5,6;

In the figure, SN38 means 7-ethyl-10-hydroxycamptothecin, PTX means paclitaxel, DTX means docetaxel, CTX means cabazitaxel, CPT-11 means irinotecan, DMF means dimethylformamide, DMAP means dimethylaminopyridine, EDC·HCl means 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, HOBT means 1-hydroxybenzotriazole, HBTU means benzo Triazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, DSPE-PEG means distearoylphosphatidylethanolamine-polyethylene glycol, DIEA means N,N-diisopropyl Ethylamine.

detailed description

The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited thereto.

Example 1

The synthetic route of the SN38-PTX conjugate is shown in Figure 1, and the specific steps are as follows:

PTX (500mg, 0.59mmol) and succinic anhydride (176mg, 1.77mmol) were added to a 100mL round bottom flask, dissolved in 8mL of anhydrous pyridine, then DMAP (7.2mg, 0.06mmol) was added, stirred at 25°C for 3h, and the oil pump Remove pyridine, wash once with 0.1N HCl and saturated saline; dry the organic phase with anhydrous sodium sulfate, filter, collect the filtrate and remove the solvent under reduced pressure; separate and purify the solid by column chromatography (DCM:MeOH=80:1) The product 5 (550 mg, yield 98%) was obtained.

The ¹ H NMR nuclear magnetic data and mass spectral data of product 5 are as follows:

¹ H NMR (400MHz, CDCl ₃ ): δ1.14(s, 3H), 1.23(s, 3H), 1.69(s, 3H), 1.89-1.92(d, 4H, J=11.2), 2.20-2.22( d,4H,J＝9.6),2.44(s,3H),2.54-2.64(m,4H),2.67-2.70(t,2H),3.68(s,2H),3.80-3.82(d,2H,J =6.8),4.20-4.23(d,1H,J=8.8),4.30-4.32(d,1H,J=8.4),4.42-4.46(m,1H),4.97-5.00(d,1H,J=8.8 ),5.53-5.54(d,1H,J=3.2),5.69-5.71(d,1H,J=7.6),5.98-6.01(m,1H),6.30(s,1H),7.13-7.15(d, 1H, J=9.2), 7.40-7.45(m, 6H), 7.50-7.54(t, 3H), 7.60-7.62(t, 1H), 7.76-7.77(d, 1H, J=7.2), 8.10-8.12 (d,2H,J=7.2).

HRMS: Calcd. for _C51H55NO17 ] ⁺ [M ⁺ H] ⁺ = _954.3548 ; observed: _954.3534 .

Add product 5 (550mg, 0.58mmol) and SN38 (226mg, 0.58mmol) in a 100mL round bottom flask, dissolve in 18mL of anhydrous DMF, then add EDC·HCl (122mg, 0.64mmol), DMAP (7.2mg, 0.06 mmol) and DIEA (144 μL, 0.87 mmol), stirred overnight at 25°C, removed the solvent with an oil pump, added DCM, and washed with 5% citric acid, saturated NaHCO ₃ , and saturated brine in turn; the organic phase was washed with anhydrous sodium sulfate After drying and filtering, the filtrate was collected and the solvent was removed under reduced pressure; the solid was separated and purified by column chromatography (DCM:MeOH=100:1) to obtain the product SN38-PTX conjugate 1 (320 mg, yield 42%).

The ¹ H NMR nuclear magnetic data and mass spectral data of the product SN38-PTX conjugate 1 are as follows:

¹ H NMR (400MHz, CDCl ₃ ): δ0.84-0.90(m,3H),1.04-1.07(t,3H),1.17(s,3H),1.70(s,3H),1.89-1.93(m, 5H),1.99(s,4H),2.24(s,4H),2.50-2.53(m,4H),2.89-2.95(m,4H),2.99-3.08(m,3H),3.77(s,1H) ,3.84-3.85(d,1H,J=6.8),4.21-4.23(d,1H,J=8.4),4.32-4.35(d,1H,J=8.8),4.46-4.49(m,1H),4.98 -5.01(d,1H,J=9.2),5.17-5.21(m,2H),5.30(s,1H),5.34(s,1H),5.53-5.54(m,1H),5.71-5.77(m, 2H),6.05-6.07(m,1H),6.32(s,2H),7.00-7.04(m,3H),7.11-7.14(t,3H),7.42-7.43(d,4H,J＝4), 7.49-7.52 (m, 3H), 7.59-7.61 (d, 3H), 7.64 (s, 1H), 7.86 (s, 1H), 8.16-8.19 (m, 3H).

HRMS: Calcd. for [ _C73H73N3O21 ] ⁺ [ _M +H] ⁺ = _1328.4815 ; observed: _1328.4798 .

Example 2

The synthetic route of the SN38-DTX conjugate is shown in Figure 2, and the specific steps are as follows:

Add DTX (500mg, 0.62mmol) and succinic anhydride (186mg, 1.86mmol) into a 100mL round-bottomed flask, dissolve in 8mL of anhydrous pyridine, then add DMAP (7.2mg, 0.06mmol), stir at 25°C for 3h, oil pump Remove pyridine, wash once with 0.1N HCl and saturated saline; dry the organic phase with anhydrous sodium sulfate, filter, collect the filtrate and remove the solvent under reduced pressure; separate and purify the solid by column chromatography (DCM:MeOH=80:1) The product 6 (500 mg, yield 91%) was obtained.

The ¹ H NMR nuclear magnetic data and mass spectral data of product 5 are as follows:

¹ H NMR (400MHz, CDCl ₃ ): δ1.08-1.13(t,3H),1.20(s,3H),1.32-1.34(d,9H,J＝7.2),1.74-1.76(d,3H,J =6.4),1.83-1.86(d,2H,J=10.0),1.89-1.91(d,3H,J=7.2),2.27-2.28(t,2H),2.37-2.39(d,2H,J=8.4 )2.53-2.64(m,5H),2.77-2.83(m,2H),3.89-3.92(t,1H),4.17-4.20(t,1H),4.23-4.32(m,2H),4.95-4.97( d,1H,J=8.4),5.26(s,1H),5.39-5.48(m,2H),5.66-5.67(d,1H,J=7.2),6.18-6.21(t,1H),7.29-7.31 (d, 1H, J = 7.6), 7.37-7.40 (m, 2H), 7.49-7.54 (q, 2H), 7.61-7.63 (t, 1H), 8.09-8.10 (d, 2H, J = 6.8).

HRMS: Calcd. for [ _C47H57NO17 ] ⁺ [M ⁺ H] ⁺ = _908.3704 ; observed: _908.3680 .

Added product 6 (420mg, 0.46mmol) and SN38 (180mg, 0.46mmol) in a 100mL round bottom flask, dissolved in 18mL of anhydrous DMF, then added HOBT (68mg, 0.51mmol), HBTU (193mg, 0.51mmol) and DIEA (69 μL, 0.42 mmol), stirred overnight at 25°C, removed the solvent with an oil pump, added DCM, and washed with 5% citric acid, saturated NaHCO ₃ , and saturated saline in sequence; the organic phase was dried with anhydrous sodium sulfate, filtered After collecting the filtrate, the solvent was removed under reduced pressure; the solid was separated and purified by column chromatography (DCM:MeOH=100:1) to obtain the product SN38-DTX conjugate 2 (220 mg, yield 37%).

The ¹ H NMR data and mass spectrometry data of the product SN38-DTX conjugate 2 are as follows:

¹ H NMR (400MHz, CDCl ₃ ): δ1.02-1.06(t,3H),1.11(s,3H),1.21(s,3H),1.26(s,2H),1.29(s,2H),1.31 (s,9H),1.37-1.41(t,3H),1.76(s,3H),1.82-1.97(m,7H),2.43(s,3H),2.58-2.65(m,1H),2.81-2.88 (m,1H),2.93-2.99(m,3H),3.11-3.18(m,2H),3.82-3.84(m,1H),3.91-3.93(d,1H,J＝6.8),4.18-4.20( d,1H,J=7.6),4.26-4.34(m,2H),4.97-4.99(d,1H,J=8.0),5.15(s,1H),5.25(s,2H),5.33(s,1H ),5.39-5.48(m,3H),5.67-5.71(m,1H),5.73-5.77(t,1H),6.22(m,1H),7.28-7.32(t,3H),7.36-7.40(t ,2H),7.48-7.52(t,2H),7.53-7.56(d,1H,J＝8.8),7.59-7.63(m,1H)7.64(s,1H),7.82-7.83(d,1H,J =2.4), 8.10-8.12 (d, 2H, J=7.2), 8.21-8.23 (d, 1H, J=9.2).

HRMS: Calcd. for [ _C69H75N3O21 ] ⁺ [ _M +H] ⁺ = _1282.4971 ; observed: _1282.4947 .

Example 3

The synthetic route of the SN38-CTX conjugate is shown in Figure 3, and the specific steps are as follows:

Add CTX (500mg, 0.60mmol) and succinic anhydride (180mg, 1.80mmol) into a 100mL round-bottom flask, dissolve in 8mL of anhydrous pyridine, then add DMAP (7.2mg, 0.06mmol), stir at 25°C for 3h, oil pump Remove pyridine, add DCM to dissolve, and wash with 0.1N HCl and saturated brine successively; the organic phase is dried with anhydrous sodium sulfate, filtered, and the solvent is removed under reduced pressure after collecting the filtrate; the solid is separated and purified by column chromatography (DCM :MeOH=80:1) to obtain product 7 (549 mg, yield 98%).

The ¹ H NMR nuclear magnetic data and mass spectral data of product 7 are as follows:

¹ H NMR (400MHz, CDCl ₃ ): δ1.21-1.22(s,6H),1.36(s,9H),1.57(s,7H),1.72(s,3H),1.87-1.88(d,3H, J=1.2),2.25-2.27(d,1H,J=9.2),2.66-2.73(m,5H),3.30(s,3H),3.45(s,4H),3.80-3.88(m,2H), 4.16-4.18(d, 1H, J=8.4), 4.29-4.31(d, 1H, J=8.4), 4.80(s, 1H), 4.96-4.98(d, 1H, J=8.0), 5.62-5.64( d,1H,J=7.2),6.19-6.23(t,1H),7.31-7.33(m,1H),7.38-7.42(m,4H),7.47-7.50(t,2H),7.59-7.63(t , 1H), 8.08-8.10 (d, 2H, J=7.2).

HRMS: Calcd. for [ _C49H61NO17 ] ⁺ [M ⁺ H] ⁺ = _936.4017 ; observed: _936.4026 .

Add product 7 (548 mg, 0.59 mmol) and SN38 (229 mg, 0.59 mmol) to a 100 mL round bottom flask, dissolve in 18 mL of anhydrous DMF, then add EDC·HCl (125 mg, 0.65 mmol), DMAP (79 mg, 0.65 mmol ) and DIEA (146 μL, 0.89 mmol), stirred overnight at 25°C, removed the solvent with an oil pump, added DCM, and washed with 5% citric acid, saturated NaHCO ₃ , and saturated saline in turn; the organic phase was dried with anhydrous sodium sulfate , filtered, the filtrate was collected and the solvent was removed under reduced pressure; the solid was separated and purified by column chromatography (DCM:MeOH=100:1) to obtain the product SN38-CTX conjugate 3 (321 mg, yield 42%).

The ¹ H NMR data and mass spectrometry data of the product SN38-CTX conjugate 1 are as follows: ¹ H NMR (400MHz, CDCl3): δ1.03-1.06(t, 3H), 1.20(s, 3H), 1.22(s, 3H),1.25(s,2H),1.33(s,9H),1.38-1.41(t,3H),1.72(s,3H),1.86-1.94(m,2H),2.00(s,3H),2.44 (s,3H),2.67-2.75(m,1H),2.85-2.94(m,2H),2.96(s,2H),3.13-3.18(q,2H),3.30(s,3H),3.42(s ,3H),3.74(s,1H),3.84-3.86(d,1H,J=7.2),3.88-3.93(m,1H),4.16-4.18(d,1H,J=8.4),4.30-4.32( d,1H,J=8.4),4.81(s,1H),4.99-5.01(d,1H,J=9.2),5.26(s,2H),5.30(s,2H),5.42(s,1H), 5.64-5.66(d, 1H, J=7.2), 5.74-5.78(d, 1H, J=16.0), 6.25-6.29(t, 1H), 7.29-7.33(t, 3H), 7.37-7.40(t, 2H),7.47-7.53(m,3H),7.58-7.62(m,1H),7.65(s,1H),7.84-7.85(d,1H,J＝2.4),8.10-8.12(d,2H,J =7.2), 8.21-8.23 (d, 1H, J=9.2).

HRMS: Calculated for [ _C71H79N3O21 ] ⁺ [ _M ⁺ H] ⁺ = _1310.5284 ; Observed: _1310.5251 .

Example 4

Take an appropriate amount of the SN38-PTX conjugate 1 prepared in Example 1 and DSPE-PEG ₂₀₀₀ (the mass ratio of the conjugate to DSPE-PEG ₂₀₀₀ is 1:5), dissolve it in DMSO (40mg/mL), and dilute it into water (conjugated The final concentration of the conjugate was 0.5 mg/mL), and nanomicelles (referred to as 1-NM) loaded with SN38-PTX conjugate 1 were obtained. The electron microscope particle size is shown in Figure 4, and the results show that the obtained nanomicelles are uniform in size and regular in shape.

Example 5

Take an appropriate amount of SN38-DTX conjugate 2 prepared in Example 2 and DSPE-PEG ₂₀₀₀ (the mass ratio of the conjugate to DSPE-PEG ₂₀₀₀ is 1:5), dissolve it in DMSO (40mg/mL), and dilute it into water (drug final concentration of 0.5 mg/mL), to obtain nanomicelles (referred to as 2-NM) encapsulating SN38-DTX conjugate 2, with an encapsulation efficiency of 99.0% and a drug loading capacity of 19.3%. The electron microscope particle size is shown in Figure 5, and the results show that the obtained nanomicelles are uniform in size and regular in shape.

Example 6

Take an appropriate amount of the SN38-CTX conjugate 3 prepared in Example 3 and DSPE-PEG ₂₀₀₀ (the mass ratio of the conjugate to DSPE-PEG ₂₀₀₀ is 1:5), dissolve it in DMSO (40mg/mL), and dilute it into water (drug final concentration of 0.5 mg/mL), to obtain nanomicelles (referred to as 3-NM) loaded with SN38-CTX conjugate 3. The electron microscope particle size is shown in Figure 6, and the results show that the obtained nanomicelles are uniform in size and regular in shape.

Example 7

Take an appropriate amount of SN38-DTX conjugate 2 prepared in Example 2 and DSPE-PEG ₂₀₀₀ (the mass ratio of the conjugate to DSPE-PEG ₂₀₀₀ is 10:1), dissolve it in DMSO (40mg/mL), and dilute it into water (drug final concentration of 0.1 mg/mL), to obtain nanomicelles encapsulating SN38-DTX conjugate 2, with an encapsulation efficiency of 99.4% and a drug loading capacity of 92.3%. The measured particle size is 79±18nm, and PDI (0.237) shows that the particle size distribution is uniform.

Example 8

Take an appropriate amount of SN38-DTX conjugate 2 prepared in Example 2 and DSPE-PEG ₂₀₀₀ (the mass ratio of the conjugate to DSPE-PEG ₂₀₀₀ is 5:1), dissolve it in DMSO (40mg/mL), and dilute it into water (drug final concentration of 0.1 mg/mL), to obtain nanomicelles encapsulating SN38-DTX conjugate 2, with an encapsulation efficiency of 99.6% and a drug loading capacity of 85.6%. The measured particle size is 86±18nm, and PDI (0.208) shows that the particle size distribution is uniform.

Example 9

Take an appropriate amount of DTX-SN38 conjugate 2 prepared in Example 2 and DSPE-PEG ₂₀₀₀ (the mass ratio of the conjugate to DSPE-PEG ₂₀₀₀ is 1:1), dissolve it in DMSO (40mg/mL), and dilute it into water (drug final concentration of 0.1 mg/mL), the nanomicelles loaded with DTX-SN38 conjugate 2 were obtained, with an encapsulation efficiency of 99.3% and a drug loading capacity of 54.6%. The measured particle size is 74±15nm, and PDI (0.138) shows that the particle size distribution is uniform.

The following further illustrates the characteristics of the nanomicelles of the present invention and their therapeutic effect on tumors by test examples:

test case 1

Place the nano micelles prepared in Examples 5, 7, 8, and 9 at room temperature, and pass through a particle size analyzer at regular intervals to detect the in vitro stability of the nano micelles. The test results are shown in Figure 7. The results show that each group of nano micelles Under the long-term room temperature environment for 14 days, the particle size is relatively stable, showing that the nanoparticles prepared in Examples 5, 7, 8, and 9 have good stability.

test case 2

The nanomicelles (0.1mg/mL) prepared in Example 5 were placed in dialysis bags with a molecular weight of 7000kDa, and the external 25mL pH was 7.4 (containing 0.2% Tween) in phosphate buffer, 37°C, and the rotation speed was 150r /min environment, the external phosphate buffer solution was taken out at regular intervals, and the SN38 content was measured with an ultraviolet spectrophotometer, so as to obtain the in vitro release of nanomedicine. From the experimental results shown in Figure 8, it can be seen that the nanomicelles prepared in Example 5 can be slowly released in an in vitro environment for a long time, and there is no obvious burst release phenomenon, which is conducive to the long-term circulation of drugs in the body.

Test case 3

The nano-micelles (0.1mg/mL) prepared in Example 5 were respectively placed in dialysis bags with a molecular weight of 7000kDa, in 25mL of phosphate buffer solution with a pH of 7.4 or 4.6 (containing 0.2% Tween) outside, at 37°C, at a speed of In an environment of 150r/min, take out the external phosphate buffer solution at 2h, 4h, 8h, 12h, 24h, 48h, 72h and 120h respectively, and measure the SN38 content with a UV spectrophotometer, thereby obtaining the nanomedicine prepared in Example 5 In vitro release at two pH environments. From the experimental results shown in Figure 9, it can be seen that the nanomicelle prepared in Example 5 can be slowly released in an in vitro environment for a long time, and there is no obvious burst release phenomenon, which is beneficial to the long-term circulation of the drug in the body.

Test case 4

Cells in the logarithmic growth phase (HCT-116, LoVo, A549) were taken, digested with trypsin, seeded on a plate (96-well plate, 5× ¹⁰³ cells/well), incubated at 37°C for 24 hours to allow the cells to adhere to the wall, and SN38, DTX was used as the control group, and different concentrations of DTX-SN38 conjugate 2 (100 μL/well) prepared in Example 2 were added. After 48 hours of culture, 30 μL of thiazolyl blue (MTT, 5 mg/mL) was added to each well, and removed after 4 hours of continuous culture. For the supernatant, add 100 μL dimethyl sulfoxide (DMSO) to each well, oscillate to fully dissolve the crystallized product, measure the absorbance of each well on a microplate reader at 492 nm, draw the cell survival curve, and calculate the effect of each group of nanoparticles on the cells. Half inhibitory concentration (IC ₅₀ ) values. The in vitro toxicity results of antitumor drug liposomes to various tumor cells are shown in Figure 10 and Table 1.

Table 1: IC ₅₀ (μM) of DTX-SN38 conjugate 2 on tumor cells in Example 2

It can be seen from Figure 10 and Table 1 that the DTX-SN38 conjugate 2 prepared in Example 2 has similar cytotoxicity to free SN38, and has a strong anti-tumor cell proliferation effect.

Test case 5

Balb/c nude mice (5 weeks old) were subcutaneously inoculated with HCT-116 intestinal cancer cells and divided into three groups: free Cy 5.5 (fluorescent dye), nanomicelles loaded with Cy 5.5 (denoted as Cy 5.5-NM) and Cy 5.5-NM loaded simultaneously 5. 5-DTX conjugate 4 and nanomicelles in Example 5 (denoted as 2/4-NM). Single administration of the tail vein (Cy 5.5 dose 20μg/mouse), after a certain interval after the administration, the distribution of the drug in the nude mice was analyzed by an in vivo imager, and the hearts, liver, spleen, lungs, kidneys, and tumor tissues of the nude mice in each group were collected for 24 hours to analyze the fluorescence of the drug. strength. The result is shown in Figure 11. It shows that 2/4-NM has a strong distribution in the tumor tissue after 24 hours, confirming that the nanomicelle prepared in Example 5 has strong tumor targeting and retention.

Test case 6

The tumor inhibition evaluation of nanomicelles 1-NM, 2-NM and 3-NM in Examples 4 and 5 on animal subcutaneous intestinal cancer HCT-116 model was carried out. Two weeks after tumor transplantation in Balb/c nude mice, the tail vein was administered every two days for a total of three times: normal saline, CPT-11 (11.5 mg/kg), 1-NM, 2-NM and 3-NM (SN38 Equivalent dose 10mg/kg), a total of 6 groups. Taking the first administration as day 0, the changes in tumor volume were measured for statistical results. The drug efficacy evaluation results on subcutaneous tumors are shown in (a) and (b) in Figure 12 . As can be seen from (a) in Figure 12, nanomicelles 1-NM, 2-NM and 3-NM in Examples 4 and 5 have a significant effect on inhibiting tumor growth relative to clinical CPT-11, and in Example 5 2 The effect of -NM is better than that of the nanomedicine in 1-NM in Example 4. It can be seen from Figure 12(b) that during one month of treatment, nanomicelles 1-NM, 2-NM and 3-NM in Examples 4 and 5 had no significant effect on the body weight of nude mice, and the trend of change was similar to that of the normal saline group , showing that the drug has good in vivo compatibility and has no obvious toxic and side effects on nude mice.

Claims (10)

1. an antitumor medicine conjugate, it is characterised in that described antitumor medicine conjugate has a following structure:

A—L—B

Wherein: the structure of A is:

The structure of B is:

Wherein, L is linkage section, R₁For phenyl or tert-butoxy, R₂For acetyl group, H or methyl, R₃For H or methyl；For A Connection position with B Yu L.

Antitumor medicine conjugate the most according to claim 1, it is characterised in that in A structure: work as R₁During for phenyl, R₂For Acetyl group, R₃For H；R₁During for tert-butoxy, R₂For H, R₃For H；Or R₁During for tert-butoxy, R₂For methyl, R₃For methyl.

Antitumor medicine conjugate the most according to claim 1, it is characterised in that described linkage section has a following structure:

N=2-6.

4. the preparation method of antitumor medicine conjugate described in an any one of claims 1 to 3, it is characterised in that including: A The dicarboxylic anhydride that precursor raw material is corresponding with L reacts, and prepares midbody compound (II), and midbody compound (II) is again with B's Precursor raw material reacts, and obtains final antitumor medicine conjugate, and above-mentioned reaction is the most at room temperature carried out；

Described midbody compound (II) structure is as follows:

The precursor raw material structure of described A structure is as follows:

The precursor raw material structure of described B structure is as follows:

5. antitumor medicine conjugate nano-micelle preparations described in an any one of claims 1 to 3, it is characterised in that described Nano-micelle is made up of antitumor medicine conjugate and amphipathy macromolecule, and described amphipathy macromolecule with the mass ratio of medicine is 5:1～1:10.

Antitumor medicine conjugate nano-micelle preparations the most according to claim 5, it is characterised in that described is amphipathic Macromolecule is selected from any one in DSPE-PEG, PEG-PLA, PEG-PLGA, mPEG-PLA, mPEG-PLGA.

Antitumor medicine conjugate nano-micelle preparations the most according to claim 6, it is characterised in that described is amphipathic Macromolecule is DSPE-PEG₂₀₀₀。

8. according to the antitumor medicine conjugate nano-micelle preparations described in any one of claim 5～7, it is characterised in that institute The preparation stated is powder or tablet, injection, pill.

9. a preparation method for antitumor medicine conjugate nano-micelle preparations described in any one of claim 5～8, its feature It is, including, antitumor medicine conjugate and amphipathy macromolecule material are dissolved in organic solvent, is then added to water In, i.e. can get medicament-carried nano preparation.

10. antitumor medicine conjugate nano-micelle application in antitumor described in an any one of claim 5～8.