DXd derivative, liposome preparation containing derivative and application of DXd derivative in drug delivery

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a DXd derivative, a liposome preparation containing the derivative and application of the DXd derivative in drug delivery. The derivative is a compound as shown in a formula I or a pharmaceutically acceptable salt thereof, which is obtained by modifying DXd through a linking group; the DXd derivative can be prepared into a liposome preparation through pH gradient. The liposome has the characteristics of high drug loading capacity, high encapsulation efficiency, good stability and the like. After injection administration, the circulation time of the medicine in a body can be greatly prolonged, the accumulation amount of the medicine at a tumor part is increased, the anti-tumor effect of the medicine is improved, and the purposes of effect enhancement and toxicity reduction are achieved. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a DXd derivative, a liposomal preparation containing the derivative, and applications in drug delivery. Background Art

[0002] DXd (exatecan derivative), as a new generation of topoisomerase I (TOPO-I) inhibitor, has shown significant advantages in the development of antibody-drug conjugates (ADCs) due to its unique molecular properties. Its high membrane permeability is conducive to transmembrane transport, and the prominent bystander effect can effectively kill adjacent tumor cells. In particular, it can avoid the P-glycoprotein (P-gp)-mediated multidrug resistance mechanism, making it one of the most promising cytotoxic warheads in the ADC drug delivery system. HER2-targeted ADC drugs represented by Trastuzumab deruxtecan ( approved by the FDA in 2019) precisely achieved the precise treatment of patients with unresectable or metastatic HER2-positive breast cancer by conjugating DXd with trastuzumab. However, due to problems such as poor water solubility and high toxicity, DXd is currently only applied to ADC drugs and has not been involved in other dosage forms, severely limiting the wider clinical application of this drug.

[0003] As a multifunctional and widely used drug delivery system, liposomes have advantages such as high biocompatibility, good stability, and low immunogenicity. They can encapsulate lipophilic and hydrophilic drugs simultaneously through the lipid bilayer or the internal aqueous phase, significantly improving drug solubility and enhancing targeting ability. The drug loading methods of liposomes include passive drug loading and active drug loading. Since the active drug loading method can achieve advantages such as high encapsulation efficiency, excellent stability, and good pharmacokinetic properties, most of the currently marketed liposomes use the active drug loading method to encapsulate drugs. However, this technology limits the application of non-ionized chemotherapeutic drugs. Therefore, researchers proposed to modify non-ionized drugs with groups to achieve high encapsulation efficiency and good stability. Although non-ionized drugs can be actively encapsulated by modifying groups, improving problems such as poor drug solubility and high toxicity, significantly increasing the maximum tolerated dose and safety of the drug. However, after drug derivatization, the activity of chemotherapeutic drugs is usually affected, limiting the maximization of efficacy.

[0004] DXd is an electrically neutral compound. If it is embedded in the phospholipid bilayer of liposomes by passive drug loading, it will face two major technical challenges: first, the sudden release of drugs in the blood circulation will lead to the failure of targeted delivery; second, nonspecific release may cause serious systemic toxic reactions. Therefore, it is necessary to derivatize it and load it into liposomes in an active manner. However, the group type and connecting chain length of the derivatized intermediate directly affect the hydrophobicity, chemical stability and drug loading capacity of the modified product, and also affect the drug activity of the derivative. At the same time, balancing the anti-tumor activity and biosafety of the derivative is still a key issue that needs to be optimized. Therefore, it is urgent to improve the safety of DXd and increase the drug loading of the preparation by optimizing the compound structure, so as to improve its potential for clinical application. Summary of the invention

[0005] In view of the above technical background, the present invention provides a DXd derivative and a liposome preparation containing the derivative and application of the DXd derivative in drug delivery.

[0006] To achieve the above purpose, the present invention adopts the technical solution as follows:

[0007] A DXd derivative, characterized in that the derivative is DXd modified by a linking group to obtain a compound represented by formula 1 or a pharmaceutically acceptable salt thereof;

[0008]

[0009] In the formula,

[0010] The linking group is an alkyl chain or olefin chain which is unsubstituted or substituted with at least one of the following heteroatoms, wherein the heteroatom is an O, S, N, Se, or Si heteroatom;

[0011] [N] The modifying group is selected from the following functional groups:

[0012] (1) Weakly basic groups with a pKa of 5.0-7.4;

[0013] (2) Weakly acidic groups with a pKa of 3.0-6.0;

[0014] or, (3) a chelating group capable of coordinating with a metal ion.

[0015] The above-mentioned linking group may contain an ester bond, a carbonate bond, an amide bond, an imine bond, a hydrazone bond, a carbamate bond, a borate bond, an oxidized oxalate bond, a monosulfide bond, a monoselenoether bond, a disulfide bond, a diselenide bond, a disulfide bond, a diselenide bond, a trisulfide bond or a thioketal bond.

[0016] The above-mentioned weakly acidic groups: carboxyl, etc.;

[0017] Weakly basic groups: N-methylpiperazine, morpholinyl, piperidinyl, tetrahydropyridinyl or other tertiary amine structures;

[0018] Metal ion coordination groups: phenolic acids, phenylboronic acids, aliphatic amino groups, aromatic amino groups, carboxyl groups, enol groups, alkoxy groups, carbonyl groups, thiol groups, phosphate groups, phosphonic acid groups, etc.

[0019] Preferably, in the formula of the derivative,

[0020] The linking group is an unsubstituted or C1-C substituted by at least one of the following heteroatoms 10 alkyl chain or C2-C 10 alkene chain, wherein the heteroatoms are O, S, N, Se, Si heteroatoms;

[0021] [N] modification groups are selected from the following functional groups:

[0022] (1) Weakly basic groups with pKa 5.0 - 7.4;

[0023] Or, (2) Chelating groups capable of coordinating with metal ions.

[0024] More preferably, in the formula of the derivative, the linking group is an unsubstituted or C1-C substituted by at least one of the following heteroatoms 10 alkyl chain, wherein the heteroatoms are O, S, N heteroatoms;

[0025] [N] modification groups are selected from weakly basic groups with pKa 5.0 - 7.4.

[0026] Even more preferably, in the formula of the derivative, the linking group is an unsubstituted or C1-C substituted by at least one of the following heteroatoms 10 alkyl chain, wherein the heteroatoms are O, S, N heteroatoms;

[0027] [N] modification groups are selected from N-methylpiperazinyl, morpholinyl, piperidinyl, tetrahydropyridyl or other tertiary amines.

[0028] Even more preferably, the derivative is

[0029]

[0030] An application of a said DXd derivative, the application of the DXd derivative in the preparation of liposomal formulations.

[0031] A liposome of DXd derivative, the liposome is composed of phospholipid, cholesterol, DXd derivative, inner aqueous solution and outer aqueous buffer; wherein, the total ratio of the DXd derivative and phospholipid is 1:5 - 20; the cholesterol content accounts for the molar ratio of lipid components (excluding drugs) of 95:5 - 50:50.

[0032] The said phospholipid is natural phospholipid and / or synthetic phospholipid, and their derivatives or functionalized phospholipids;

[0033] The above functionalized phospholipids are PEGylated phospholipids, and their content accounts for 0-20% of the lipid components.

[0034] The inner aqueous phase solution is one or more of citric acid solution, ammonium sulfate solution, triethylamine salt of sucrose octasulfate, and triethylamine solution of sulfobutyl ether-β-cyclodextrin;

[0035] The outer aqueous phase buffer is one or more of sucrose solution, HBS buffer, phosphate buffer, and carbonate buffer.

[0036] The particle size of the derivative liposomes is about 50-140 nm, preferably 60-85 nm.

[0037] A method for preparing the DXd derivative liposomes as described above,

[0038] (1) Form blank liposomes by the thin film dispersion method or ethanol injection method according to the above components;

[0039] (2) Establish a transmembrane pH gradient or ammonium ion gradient;

[0040] (3) Co-incubate the obtained blank liposomes with an organic solvent solution containing the derivative as claimed in claim 1 or a pharmaceutically acceptable salt thereof to obtain the final drug-loaded liposomes.

[0041] Furthermore,

[0042] (1) Dissolve phospholipids, cholesterol, and PEGylated phospholipids in an organic solvent in proportion, drop it into the inner aqueous phase solution, stir uniformly above the phospholipid phase transition temperature, and remove the organic solvent after stirring to obtain multi-chamber liposomes, and then obtain uniformly sized unilamellar liposomes after treatment;

[0043] (2) Replace the outer aqueous phase of the unilamellar liposomes obtained in step (1) with agarose gel Sepharose CL-4B to obtain blank liposomes with a gradient of inner and outer aqueous phases of the liposomes;

[0044] (3) Co-incubate the blank liposomes obtained in step (2) with an organic solvent solution containing the derivative as claimed in claim 1 or a pharmaceutically acceptable salt thereof to obtain the final drug-loaded liposomes.

[0045] In step (3), the organic solvent can be methanol, ethanol, acetone, tetrahydrofuran, acetonitrile, DMSO, DMF, etc.; the dosage of the drug-containing organic solvent is 2%-40% of the total volume of the drug-loaded blank liposomes, preferably 3%-25%.

[0046] Use of the derivative or an acceptable salt thereof or the DXd derivative liposomes as described above in the preparation of anti-tumor drugs.

[0047] The present invention prepares DXd into a derivative and then uses it to prepare an actively loaded liposome for use in the preparation of anti-tumor drugs, improving the bioavailability of the drug while reducing the drug toxicity, enriching the dosage form of DXd, and having great potential for clinical application.

[0048] The beneficial effects of the present invention compared with the prior art are as follows:

[0049] (1) By derivatizing DXd, the present invention endows it with certain binding properties, enabling the development of an actively loaded liposome preparation of the drug.

[0050] (2) The actively loaded liposome prepared by the present invention has the advantages of uniform particle size, good stability, high drug loading, etc., and is easy to industrialize.

[0051] (3) The DXd derivative liposome prepared by the present invention can prolong the half-life of the drug, increase the AUC, improve the bioavailability of DXd, give full play to the drug effect, and effectively overcome the technical defects of fast metabolism and poor tissue distribution of the original drug.

[0052] (4) The DXd derivative liposome prepared by the present invention takes into account the excellent anti-tumor activity and good biological safety of DXd, and successfully solves the problems such as single dosage form (only applicable to ADC drugs) and narrow therapeutic window existing in traditional DXd preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments will be briefly introduced below.

[0054] Figure 1 1H-NMR of the DXd derivative (DXdd-2PA) with 4-methyl-1-piperazineethyl as the base part in Example 1 of the present invention 1 1H-NMR;

[0055] Figure 2 1H-NMR of the DXd derivative (DXdd-4PA) with 4-methyl-1-piperazinebutyl as the base part in Example 2 of the present invention 1 1H-NMR;

[0056] Figure 3 1H-NMR of the DXd derivative (DXdd-5PA) with 4-methyl-1-piperazinepentyl as the base part in Example 3 of the present invention 1 1H-NMR;

[0057] Figure 4 1H-NMR of the DXd derivative (DXdd-7PA) with 4-methyl-1-piperazineheptyl as the base part in Example 4 of the present invention 1 1H-NMR;

[0058] Figure 5Effect of the type of internal aqueous phase on the encapsulation efficiency of four DXd derivatives in Example 5 of the present invention;

[0059] Figure 6 Effect of the cholesterol content in phospholipids on the encapsulation efficiency of four DXd derivatives in Example 6 of the present invention;

[0060] Figure 7 Effect of the drug-lipid ratio on the encapsulation efficiency of four DXd derivatives in Example 7 of the present invention;

[0061] Figure 8 Storage stability diagram of four kinds of DXd actively loaded liposomes in Example 8 of the present invention;

[0062] Figure 9 In vitro cytotoxicity diagram of four DXd derivatives and their actively loaded liposomes in 4T1 cells in Example 9 of the present invention;

[0063] Figure 10 In vivo antitumor effect of DXd derivative actively loaded liposomes in Example 10 of the present invention;

[0064] Figure 11 Survival curve of the efficacy of DXd derivative actively loaded liposomes in treating mice in Example 10 of the present invention;

[0065] Figure 12 Number of lung nodules diagram after treatment with DXd derivative actively loaded liposomes in Example 10 of the present invention;

[0066] Figure 13 Diagram of the change in the body weight of mice after treatment with DXd derivative actively loaded liposomes in Example 10 of the present invention;

[0067] Figure 14 Effect of the particle size of DXd derivative actively loaded liposomes on the in vivo antitumor effect and diagram of the change in the body weight of mice in Example 12 of the present invention.

[0068] Among them, DXdd2-lipo is DXd-2PA actively loaded liposome, DXdd4-lipo is DXd-4PA actively loaded liposome, DXdd5-lipo is DXd-5PA actively loaded liposome, and DXdd7-lipo is DXd-7PA actively loaded liposome. Detailed Description of the Invention

[0069] The present invention will be described in detail below with reference to the embodiments. However, the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only part of the embodiments of the present invention. For those skilled in the art, without creative efforts, obtaining other similar embodiments will fall within the protection scope of the present invention.

[0070] The DXd derivative of the present invention is prepared into a DXd derivative active drug-loaded liposome, and the drug is encapsulated to obtain a nanoliposome preparation with high drug loading, high encapsulation rate, good stability, and both anti-tumor activity and biosafety, thereby achieving effective drug delivery for the treatment of cancer and metastatic cancer.

[0071] Example 1: Synthesis of DXd derivative (DXdd-2PA) with 4-methyl-1-piperazineethyl as the basic part

[0072] 4-Methyl-1-piperazineacetic acid (48.1 mg, 0.3 mmol) was dissolved in dichloromethane. EDCI (171.9 mg, 0.9 mmol) and DMAP (108.9 mg, 0.9 mmol) were added in turn under ice bath. The mixture was stirred for 1 h under ice bath to activate the carboxyl group. After activation, DXd (50 mg, 0.1 mmol) was added and the activation was continued for 1 h. The mixture was then transferred to a 25°C water bath and stirred for 12 h. The reaction progress was detected by thin film chromatography. After DXd was completely consumed, the product was separated and purified using a preparative phase (47 mg, 75%).

[0073] The structure of the derivative was determined by hydrogen nuclear magnetic resonance spectroscopy, and the solvent used was deuterated DMSO. The results are as follows Figure 1 As shown, the results of H NMR spectrum analysis are as follows:

[0074] DXdd-4PA: 1 H NMR (400MHz, DMSO) δ8.75 (d, J=8.7Hz, 1H), 7.81

[0075] (d,J=11.0Hz,1H),7.33(d,J=3.3Hz,1H),5.60(dt,J=8.9,4.5Hz,1H),5.50-5.37(m,2H), 5.32-5.12(m,2H),4.66-4.51(m,2H),3.60-3.40(m,3H),3.18(s,3H),2.81(d,J=2.7Hz,2H ),2.73(d,J=35.4Hz,2H),2.41(s,3H),2.12(d,J=36.5Hz,3H),1.88(dq,J=11.5,5.9Hz,2H ), 1.76 (s, 3H), 1.38 (dd, J = 14.6, 7.0Hz, 1H), 1.24 (d, J = 3.2Hz, 1H), 0.89 (t, J = 7.2Hz, 3H).

[0076] Example 2 Synthesis of DXd derivative (DXdd-4PA) with 4-methyl-1-piperazinebutyl as the basic part

[0077] Similar to the synthesis method of Example 1, replace 4-methyl-1-piperazineacetic acid with 4-methyl-1-piperazinebutyric acid, and finally obtain DXdd-4PA. Nuclear magnetic resonance hydrogen spectroscopy was used to determine the structure of the derivative, and the solvent selected was deuterated DMSO. The results are as Figure 1 shown, and the nuclear magnetic resonance hydrogen spectrum analysis results are as follows:

[0078] DXdd-4PA: 1 H NMR(400MHz,DMSO)δ8.76(d,J=8.7Hz,1H),7.81

[0079] (d,J=11.0Hz,1H),7.33(d,J=1.7Hz,1H),5.42(s,2H),5.22(d,J=22.5Hz,4H),4.59-4.51(m,2H),2.89(t,J=4.5Hz,2H),2.42(s,4H),2.07(s,16H),1.23(d,J=3.2Hz,4H),0.89-0.84(m,3H).

[0080] Synthesis of DXd derivative (DXdd-5PA) with 4-methyl-1-piperazinepentyl as the basic part in Example 3

[0081] Similar to the synthesis method of Example 1, replace 4-methyl-1-piperazineacetic acid with 4-methyl-1-piperazinevaleric acid, and finally obtain DXdd-5PA. Nuclear magnetic resonance hydrogen spectroscopy was used to determine the structure of the derivative, and the solvent selected was deuterated DMSO. The results are as Figure 1 shown, and the nuclear magnetic resonance hydrogen spectrum analysis results are as follows:

[0082] DXdd-5PA: 1 H NMR(400MHz,DMSO)δ8.72(d,J=8.7Hz,1H),7.82

[0083] (d,J=10.9Hz,1H),7.33(s,1H),5.68-5.52(m,2H),5.43(s,2H),5.30-5.14(m,2H),4.60-4.46(m,2H),3.18(s,2H),2.85(s,2H),2.48-2.31(m,5H),2.07(s,8H),1.93-1.80(m,2H),1.75(s,5H),1.61(s,2H),1.37(dd,J=14.8,7.0Hz,2H),0.88(t,J=7.3Hz,3H).

[0084] Synthesis of DXd derivative (DXdd-7PA) with 4-methyl-1-piperazinepentyl as the basic part in Example 4

[0085] Similar to the synthesis method of Example 1, 4-methyl-1-piperazineacetic acid was replaced with 4-methyl-1-piperazineheptanoic acid, and finally DXdd-5PA was obtained. The structure of the derivative was determined by 1H NMR, and the solvent used was DMSO-d6. The results are as Figure 1 shown. The 1H NMR analysis results are as follows: DXdd-7PA: 1 H NMR(400MHz,DMSO)δ8.67(dd,J=8.7,4.6Hz,1H),7.81(d,J=11.0Hz,1H),7.32(d,J=2.1Hz,1H),5.57(dd,J=8.9,4.8Hz,2H),5.43(d,J=3.2Hz,2H),5.22(d,J=24.8Hz,2H),4.56-4.46(m,2H),3.13-3.07(m,2H),2.85(s,2H),2.54(s,2H),2.49-1.61(m,16H),1.60-1.38(m,4H),1.34-1.14(m,6H),0.87(t,J=7.4Hz,3H).

[0086] Effect of the type of inner aqueous phase on the encapsulation efficiency of DXd derivative-loaded active liposomes in Example 5

[0087] Hydrogenated soy phosphatidylcholine (HSPC) (85 mg), cholesterol (Chol) (15 mg), and DSPE-PEG2000 (5 mg) were weighed and dissolved in absolute ethanol (20 mg / ml). Under stirring at 65 °C, the solution was added dropwise to different inner aqueous phase solutions. The types of inner aqueous phase were 250 mM ammonium sulfate solution, triethylamine salt of sucrose octasulfate, or 250 mM citrate buffer. After stirring for 30 min, the remaining absolute ethanol was removed using a rotary evaporator. Then, the particle size of the liposomes was reduced by a pneumatic liposome basic device to obtain unilamellar liposomes with a particle size of about 130 nm. The liposomes were passed through an agarose gel chromatography column equilibrated with 20 mM HBS buffer solution to obtain blank liposomes. The blank liposomes were co-incubated with an ethanol solution containing the DXd derivative prepared in the above example at a phospholipid mass to drug mass ratio of 10:1 at 60 °C for 30 min. After drug loading, the drug loading was terminated by ice bath, and the particle size and encapsulation efficiency of the liposomes were measured (see Figure 5 ).

[0088] The results showed that when the triethylamine salt of sucrose octasulfate was used as the inner aqueous phase, the encapsulation efficiency of the four DXd derivatives was >90%, which was significantly better than other buffer salts. Therefore, the triethylamine salt of sucrose octasulfate was selected as the optimal inner aqueous phase for DXd derivative-loaded active liposomes.

[0089] Example 6: Effect of Cholesterol Content in the Phospholipid Composition on the Encapsulation Efficiency of Actively Loaded Liposomes of DXd Derivatives

[0090] HSPC, Chol, and DSPE-PEG2000 with different mass ratios of 95:5:0.5, 85:15:0.5, and 75:25:0.5 were weighed separately and dissolved in absolute ethanol (20 mg / ml). Under the condition of stirring at 65 °C, it was added dropwise to 250 mM triethylamine sucrose octasulfate. After stirring for 30 min, the remaining absolute ethanol was removed using a rotary evaporator. Then, the liposome particle size was reduced by a pneumatic liposome basic device to obtain unilamellar liposomes with a particle size of about 130 nm. The liposomes were passed through an agarose gel chromatography column equilibrated with 20 mM HBS buffer solution to obtain blank liposomes. The blank liposomes were co-incubated with the ethanol solution containing the DXd derivative prepared in the above example at a phospholipid mass to drug mass ratio of 10:1 at 60 °C for 30 min. After drug loading was completed, the drug loading was terminated by ice bath, and the liposome particle size and encapsulation efficiency were measured (see Figure 6 ).

[0091] The results showed that when the cholesterol content was 15% (mol) of the phospholipid concentration, all four DXdds could achieve a relatively high encapsulation. Therefore, a cholesterol content of 15% of the phospholipid concentration was selected as the optimal phospholipid composition for the actively loaded liposomes of DXd derivatives.

[0092] Example 7: Effect of Drug-Lipid Ratio on the Encapsulation Efficiency of Actively Loaded Liposomes of DXd Derivatives

[0093] HSPC, Chol, and DSPE-PEG2000 with a mass ratio of 85:15:0.5 were weighed separately and dissolved in absolute ethanol (20 mg / ml). Under the condition of stirring at 65 °C, it was added dropwise to 250 mM triethylamine sucrose octasulfate. After stirring for 30 min, the remaining absolute ethanol was removed using a rotary evaporator. Then, the liposome particle size was reduced by a pneumatic liposome basic device to obtain unilamellar liposomes with a particle size of about 130 nm. The liposomes were passed through an agarose gel chromatography column equilibrated with 20 mM HBS buffer solution to obtain blank liposomes. The blank liposomes were co-incubated with the ethanol solution containing the DXd derivative prepared in the above example at a phospholipid mass to drug mass ratio of 10:1 or 8:1 at 60 °C for 30 min. After drug loading was completed, the drug loading was terminated by ice bath, and the liposome particle size and encapsulation efficiency were measured (see Figure 7 ).

[0094] The results showed that when the drug-lipid ratio was 1:10 (w / w), the encapsulation efficiency of all derivatives reached the peak value (91.3% to 99.7%). However, when it was increased to 1:8, the encapsulation efficiency decreased to less than 90%. Among them, DXdd-4PA could maintain the encapsulation efficiency above 90% at the drug-lipid ratio of 8:1, while the encapsulation efficiencies of the other three DXd derivatives were less than 90%. Different linkers would affect the encapsulation efficiency. The drug-lipid ratio of 1:10 was selected for subsequent experiments.

[0095] Example 8 Preparation of Actively Loaded Liposomes of DXd Derivatives

[0096] Weigh HSPC (85 mg), Chol (15 mg), and DSPE-PEG2000 (5 mg) respectively and dissolve them in absolute ethanol (20 mg / ml). Under the condition of stirring at 65 °C, add them dropwise to 250 mM triethylamine sucrose octasulfate, stir for 30 min, and then remove the remaining absolute ethanol using a rotary evaporator. Then, reduce the particle size of the liposomes through a pneumatic basic liposome device to obtain unilamellar liposomes with a particle size of about 130 nm. Pass the liposomes through an agarose gel chromatography column equilibrated with 20 mM HBS buffer solution to obtain blank liposomes. Incubate the blank liposomes with the ethanol solution containing the DXd derivatives prepared in the above examples at a phospholipid mass to drug mass ratio of 10:1 at 60 °C for 30 min. After drug loading, terminate the drug loading by placing it in an ice bath, and measure the particle size and encapsulation efficiency of the liposomes.

[0097] Table 1 Characterization of Actively Loaded Liposomes of Four DXd Derivatives under the Optimal Formulation

[0098]

[0099] The results showed that the particle sizes of the four DXd derivatives were about 130 nm, the PDI was less than 0.2, the zeta potential was about -15 mV, and the encapsulation efficiency was greater than 90%.

[0100] Example 9 Storage Stability Experiment of Actively Loaded Liposomes of Four DXd Derivatives

[0101] Place the four kinds of liposomes of DXd derivatives prepared according to Examples 7, 8, and 9 in a 4 °C environment and store them in the dark. Measure the particle size and PDI every 5 days (see Figure 8 ).

[0102] The results showed that the particle sizes and PDI of the four kinds of liposomes of DXd derivatives did not change significantly within 15 days, indicating good stability.

[0103] Example 10 Cytotoxicity Evaluation Experiment of Actively Loaded Liposomes of Four DXd Derivatives

[0104] 4T1 cells in the logarithmic growth phase were seeded into 96-well plates (2000 cells / well) and allowed to adhere and grow for 12 hours. The culture medium was then discarded, and different concentrations of drug-containing culture media were added. The drug groups were DXd, DXdd-2PA, DXdd-4PA, DXdd-5PA, DXdd-7PA, DXdd2-lipo, DXdd4-lipo, DXdd5-lipo, and DXdd7-lipo. After co-incubating the cells with the drugs for 48 h, 20 μL of MTT was added and incubated at 37 °C for 4 h. The culture medium was then discarded, 200 μL of DMSO was added to dissolve the formazan crystals, and the absorbance of each group was measured at a wavelength of 570 nm using a microplate reader to calculate the cell viability (see Figure 9 ).

[0105] The results showed that due to the differences in the cleavage and release of active structures, DXdd derivatives with different alkyl chain lengths exhibited significant differences in drug activity. Compared with the DXd solution, the IC50 values of DXdd-2PA, DXdd-4PA, DXdd-5PA, and DXdd-7PA were reduced by 6.49, 3.66, 5.61, and 65.61 times, respectively. Among them, DXdd-4PA showed the strongest cytotoxicity. The cytotoxicity results of drug-loaded liposomes showed that there were significant differences in the anti-tumor efficacy of different derivatives: the drug-loaded liposomes of the short-chain derivative DXdd-2PA and the medium-chain derivative DXdd-4PA showed stronger cell killing effects than the long-chain derivatives (DXdd-5PA, DXdd-7PA).

[0106] Pharmacokinetic experiments of four DXd derivative active drug-loaded liposomes in Example 11

[0107] Pharmacokinetic experiments were carried out using 4T1 tumor-bearing mice. 4T1 cells were inoculated into the mammary pads of Balb / c mice to establish an orthotopic mouse breast cancer model. When the tumor volume reached 250 mm 3 , the mice were randomly divided into 5 groups: the DMSO solution of DXd, DXdd2-lipo, DXdd4-lipo, DXdd5-lipo, and DXdd7-lipo groups, and the dosing dose was the same as that of DXd at 5 mg / mL. At specific time points (10 min, 1 h, 6 h, 12 h, 24 h), blood was collected by eye puncture from 3 mice at each time point, and plasma samples were obtained by centrifugation. Plasma samples were extracted by the protein precipitation method. 50 μL of plasma sample was taken and 400 μL of acetonitrile containing 5% formic acid was added, vortexed for 3 min, centrifuged at 130000 rpm for 10 min, the supernatant was taken and centrifuged again, and HPLC was used to detect the DXd derivatives and the released parent drug DXd in the samples to calculate the pharmacokinetic parameters (see Figure 10 ). At the same time, the DMSO solution of DXd was used as the control group.

[0108] Table 2 Pharmacokinetic parameters of DXdd actively loaded liposomes

[0109]

[0110] Example 12 Pharmacodynamic experiments of four kinds of DXd derivative actively loaded liposomes

[0111] 4T1 cells were inoculated into the mammary pads of Balb / c mice to establish an orthotopic mouse breast cancer model. When the tumor volume grew to about 100 mm 3 ³, the tumor-bearing mice were randomly divided into 6 groups with 5 mice in each group. The groups were: normal saline group (control), DMSO solution of DXd, DXdd2-lipo, DXdd4-lipo, DXdd5-lipo, DXdd7-lipo. The equivalent dose of DXd was 5 mg / kg, administered via the tail vein once every three days for a total of 4 times. The tumor volume and body weight of the mice were measured every two days. The death of the mice was recorded and the survival period of the mice was plotted. On the 45th day, all the mice were sacrificed, the lungs of the mice were collected, fixed with picric acid, and the number of lung metastasis foci was recorded (see Figures 10 - 12 ).

[0112] The results showed that compared with the normal saline group, the DMSO solution of DXd had a certain inhibitory effect on the tumor growth of mice. Among the four kinds of DXd derivative actively loaded liposomes, DXdd2-lipo had the worst anti-tumor effect. In contrast, DXdd4-lipo, DXdd5-lipo, and DXdd7-lipo could significantly inhibit tumor growth. 4T1 orthotopic tumors were prone to form metastasis foci in the lungs. Compared with other formulation groups, the number of surface metastasis nodules in the lungs was significantly reduced after treatment with DXdd4-lipo, the survival period was significantly prolonged, and no mice died within 45 days after treatment, showing superior anti-tumor and anti-metastatic abilities. After a single administration, one tumor-bearing mouse in the DXd solution group died and the body weight decreased significantly, indicating that the free drug had severe acute toxicity. After each administration in the DXdd2-lipo group, the body weight decreased severely, showing significant toxic and side effects. With the increase of the alkyl chain length, there was no significant change in the body weight of the tumor-bearing mice in the DXdd4-lipo, DXdd5-lipo, and DXdd7-lipo groups, suggesting that the long-chain derivatives could reduce the systemic toxicity. In summary, among the four kinds of DXd derivative actively loaded liposomes, DXdd-4PA liposomes had stronger anti-tumor effects and good biosafety.

[0113] Example 13 Preparation of DXd derivative actively loaded liposomes with different particle sizes

[0114] Weigh HSPC (85 mg), Chol (15 mg), and DSPE-PEG2000 (5 mg) separately and dissolve them in absolute ethanol (20 mg / ml). Under the condition of stirring at 65 °C, drop the solution into 250 mM triethylamine sucrose octasulfate, stir for 30 min, and then remove the remaining absolute ethanol using a rotary evaporator. Then, reduce the particle size of the liposomes through a pneumatic liposome basic device to obtain unilamellar liposomes with particle sizes of about 80 nm and 130 nm. Pass the liposomes through an agarose gel chromatography column equilibrated with 20 mM HBS buffer solution to obtain blank liposomes with different particle sizes. Incubate the blank liposomes with the ethanol solution containing DXdd-4PA prepared in the above example at a phospholipid mass to drug mass ratio of 10:1 at 60 °C for 30 min. After drug loading is completed, terminate the drug loading by placing it in an ice bath, and measure the particle size and encapsulation efficiency of the liposomes.

[0115] Table 3 Characterization of actively loaded liposomes of DXd derivatives with two particle sizes

[0116]

[0117] Example 14 Pharmacodynamic experiments of actively loaded liposomes of DXd derivatives with two different particle sizes

[0118] Inoculate 4T1 cells into the mammary pads of Balb / c mice to establish an orthotopic mouse breast cancer model. When the tumor volume grows to about 100 mm 3 , randomly divide the tumor-bearing mice into 6 groups, with 5 mice in each group. The groups are: normal saline group (control), DXdd4-lipo (80 nm), DXdd4-lipo (130 nm), where the equivalent dose of DXd is 5 mg / kg. Administer the drug via the tail vein once every three days for a total of 4 times. Detect the tumor volume and body weight of the mice every two days. (See Figure 14 ).

[0119] The results show that compared with the normal saline control group, liposomes of DXd derivatives with both particle sizes can significantly inhibit tumor growth. Compared with the normal saline control group, the tumor volume in the DXdd4-lipo group with a particle size of 130 nm decreased by 4.9 times. The tumor in the DXdd4-lipo group with a particle size of 80 nm stopped growing during the treatment period, and the volume decreased by 15.6 times compared with the normal saline group and 3.18 times compared with the DXdd4-lipo treatment group with a particle size of 130 nm. There was no significant change in the body weight of tumor-bearing mice in the two groups of DXdd4-lipo with different particle sizes. In summary, liposomes of DXd derivatives with a particle size of 80 nm have better anti-tumor effects.

[0120] The DXd weakly basic derivative of the present invention is formed by connecting DXd with a weakly basic intermediate through an ester bond, such that the drug is cleaved under the action of esterase in vivo to release the active drug. Its general structural formula is as follows: wherein the linking group is a C1-C7 alkyl group; [N] is an N-methylpiperazinyl group, morpholine, or its tertiary amine structure. The DXd weakly basic derivative can be prepared into a liposomal formulation by a pH gradient. The liposome has the characteristics of high drug loading, high encapsulation efficiency, good stability, etc. After injection administration, it can greatly increase the circulation time of the drug in vivo, increase the accumulation amount of the drug at the tumor site, improve the anti-tumor effect of the drug, and achieve the purpose of enhancing efficacy and reducing toxicity.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent substitution on some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A DXd derivative, characterized in that The derivative is DXd modified by a linking group to obtain a compound of formula 1 or a pharmaceutically acceptable salt thereof; In the formula, The linking group is an alkyl chain or olefin chain which is unsubstituted or substituted with at least one of the following heteroatoms, wherein the heteroatom is an O, S, N, Se, or Si heteroatom; [N] The modifying group is selected from the following functional groups: (1) Weakly basic groups with a pKa of 5.0-7.4; (2) Weakly acidic groups with a pKa of 3.0-6.0; or, (3) a chelating group capable of coordinating with a metal ion.

2. The DXd derivative according to claim 1, characterized in that In the derivative formula, The linking group is a C1-C 10 Alkyl chain or C2-C 10 Olefin chains, wherein the heteroatoms are O, S, N, Se, Si heteroatoms; [N] The modifying group is selected from the following functional groups: (1) Weakly basic groups with a pKa of 5.0-7.4; Or, (2) a chelating group capable of coordinating with a metal ion.

3. The DXd derivative according to claim 2, characterized in that In the derivative formula, the linking group is a C1-C 10 Alkyl chain, wherein the heteroatom is O, S, or N heteroatom; The [N] modifying group is selected from weakly basic groups with a pKa of 5.0-7.

4.

4. The DXd derivative according to claim 3, characterized in that In the derivative formula, the linking group is a C1-C 10 Alkyl chain, wherein the heteroatom is O, S, or N heteroatom; [N] The modifying group is selected from N-methylpiperazinyl, morpholinyl, piperidinyl, tetrahydropyridinyl or other tertiary amines.

5. A use of the DXd derivative according to claim 1, characterized in that: The application of the DXd derivative in preparing liposome preparations.

6. A DXd derivative liposome, characterized in that: The liposomes are composed of phospholipids, cholesterol, DXd derivatives, inner aqueous phase solution and outer aqueous phase buffer; wherein the total ratio of the DXd derivatives to the phospholipids is 1:5-20; and the molar ratio of the cholesterol content to the lipid component (excluding drugs) is 95:5-50:

50.

7. The derivative liposome according to claim 6, characterized in that: The phospholipids are natural phospholipids and / or synthetic phospholipids, and their derivatives or functionalized phospholipids; the inner aqueous phase solution is one or more of citric acid solution, ammonium sulfate solution, sucrose octasulfate triethylamine salt, and sulfobutyl ether-β-cyclodextrin triethylamine solution; the outer aqueous phase buffer is one or more of sucrose solution, HBS buffer, phosphate buffer, and carbonate buffer.

8. The DXd derivative liposome according to claim 6, characterized in that: The particle size of the DXd derivative liposome is about 50-140 nm.

9. A method for preparing the DXd derivative liposome according to claim 6, characterized in that: (1) forming blank liposomes according to the above-mentioned components by a thin film dispersion method or an ethanol injection method; (2) establishing a transmembrane pH gradient or ammonium ion gradient; (3) The obtained blank liposomes are co-incubated with an organic solvent solution containing the derivative according to claim 1 or a pharmaceutically acceptable salt thereof to obtain the final drug-loaded liposomes.

10. The use according to claim 1 or claim 6, characterized in that: Use of the derivative or an acceptable salt thereof as claimed in claim 1 or the DXd derivative liposome as claimed in claim 6 in the preparation of anti-tumor drugs.

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