Boron-containing drug-polyhydroxy drug conjugate, nano-drug and prodrug

Abstract

The invention relates to a boron-containing drug-polyhydroxy drug conjugate, a nano-drug and a prodrug, the boron-containing drug-polyhydroxy drug conjugate comprises a boron-containing drug and a polyhydroxy drug, the boron-containing drug contains a boric acid group, and the polyhydroxy drug at least contains two hydroxyls. The invention initiates a kind of ADDC (nano-drug) which does not need to be purified and is derived by click chemistry, and the improvement of drug economy, biocompatibility and cancer treatment effect is realized.

Description

Technical Field

[0001] This application belongs to the field of medicine, and particularly relates to a boron-containing drug-polyhydroxy drug conjugate, a nano-drug, and a prodrug. Background Art

[0002] Since the 1940s, when it was realized that single-agent cancer chemotherapy could only provide temporary relief, there has been an ongoing search for rational combination drug / chemotherapy regimens. The progress of nanomedicine has accelerated this process, and the combination of combination chemotherapy and nanomedicine has become the forefront of modern cancer treatment. In particular, carrier-free drug delivery systems (DDSs) have eliminated many obstacles (such as targeted drug delivery, tissue penetration, drug resistance, toxicity, etc.), simplifying the development of drug formulations and the evaluation of biological safety. To ensure that the active parent drug can be selectively activated and released in the targeted tumor, a stimulus-responsive or biodegradable linker unit should be synthesized when preparing a prodrug delivery system. Among them, chemical conjugation is superior to physical mixing in preparing low-toxic prodrugs and precisely co-delivering drugs for combination chemotherapy, such as amphiphilic drug-drug conjugates (ADDCs).

[0003] The main strategies for chemical drug conjugation are to utilize their inherent functional groups or to customize biodegradable linker units, but both methods have low coupling efficiency (e.g., esterification, amidation, etherification, Schiff base reaction, etc.) and require purification (such as precipitation, column chromatography, semi-preparative high-performance liquid chromatography, dialysis, etc.). In addition, the former strategy has limitations, and the continuous conversion efficiency from the free drug to the conjugate and then to the parent drug is relatively low. For the latter, the stimulus concentration in the physiological environment is usually much lower than that in in vitro simulation experiments, resulting in unsatisfactory drug release and poor in vivo efficacy. Taking the most commonly used esterification reaction as an example, ADDCs usually obtain a medium chemical conversion rate (50%-70%) when fed in a non-equimolar ratio. Subsequently, the degradation of the ester bond is usually very slow. Unless additional esterase is supplemented, the in vitro parent drug release rate of ADDCs in phosphate-buffered saline (PBS, pH 5) is usually less than 45% within 48 hours. To sum up, the overall utilization rate of drugs currently using the conjugation strategy (from the free drug to the conjugate and then to the parent drug) is about 20%-30%, showing limited drug economy.

[0004] Therefore, screening clinical drugs with efficient chemical bond conversion ability is crucial for promoting the progress of ADDCs. This can not only identify available drug-drug conjugation pathways, screen potential drug combinations and customizable linker units, but also regulate the physicochemical properties of the corresponding self-assembled nano-drugs. With the development of carbon-based drugs, boron-containing molecules have expanded the diversity of treatments and have evolved into important pharmacophores due to their reversible electrophilicity. They promote pharmacological activity by generating bio-reversible covalent bonds within the active sites of target proteins. Compared with esterification and amidation reactions, boric acid chemical reactions are spontaneously reversible and can be optimized as click chemistry methods to respond more sensitively to physiological stimuli from the tumor microenvironment. Nevertheless, the research on small molecule boron-containing drug-drug conjugates is still in its infancy, and the efficacy is only slightly enhanced compared with free drugs, and it is still difficult to achieve synergistic effects. Summary of the Invention

[0005] To address the above deficiencies in the prior art, the present invention provides a boron-containing drug-polyhydroxy drug conjugate, a nano-drug, and a prodrug.

[0006] In the first aspect of the present invention, a boron-containing drug-polyhydroxy drug conjugate is provided, which includes a boron-containing drug and a polyhydroxy drug. The boron-containing drug contains a boric acid group, and the polyhydroxy drug contains at least 2 hydroxyl groups.

[0007] Preferably, the boron-containing drug is bortezomib (BTZ).

[0008] Preferably, the polyhydroxy drug contains a cis-diol structure, and the polyhydroxy drug is further preferably at least one of azacitidine (AZA), capecitabine (CAP), deoxyfluorouridine (DFUR), piceatannol (PIC), dopamine, and its analogs. The dopamine analog is preferably acrylamide dopamine (DPA).

[0009] In the second aspect of the present invention, a preparation method of a boron-containing drug-polyhydroxy drug conjugate is provided, which is prepared by subjecting the boron-containing drug and the polyhydroxy drug to a boric acid esterification reaction.

[0010] Preferably, the molar ratio of the boron-containing drug to the polyhydroxy drug in the feed is 1-10:1-10, and more preferably 1:1.

[0011] Preferably, the boron-containing drug and the polyhydroxy drug are dissolved in a first solvent, and under the action of an organic base, the boron-containing drug-polyhydroxy drug conjugate is prepared.

[0012] Preferably, the first solvent is selected from one or more of THF, DMF, DMA, benzene, toluene, or halogenated methane.

[0013] Preferably, the first solvents are all anhydrous solvents.

[0014] Preferably, the solvents in which the boron-containing drug and the polyhydroxy drug are dissolved can be the same or different.

[0015] Preferably, the reaction temperature of the click chemistry is 10-50 °C, more preferably 20-40 °C.

[0016] Preferably, the reaction time of the click chemistry is 0.1-12 h, more preferably 0.5-5 h.

[0017] Preferably, the organic base is an organic amine, more preferably triethylamine, and most preferably anhydrous triethylamine.

[0018] The third aspect of the present invention provides a nano-drug of a boron-containing drug-polyhydroxy drug conjugate, which is prepared by a nano-precipitation method from the boron-containing drug-polyhydroxy drug conjugate described in the first aspect of the present invention.

[0019] Preferably, the nano-precipitation method is as follows: adding the boron-containing drug-polyhydroxy drug conjugate to a second solvent for mixing, and then adding the mixed solution to a third solvent, adjusting the pH of the solution to weakly alkaline and mixing,

[0020] removing the solvent to obtain the boron-containing drug-polyhydroxy drug conjugate nano-drug.

[0021] Preferably, the second solvent is selected from one or more of THF, DMF, DMA, benzene, toluene or halogenated methane. Preferably, the third solvent is selected from one or two of water or alcohol, and more preferably deionized water.

[0022] Preferably, the removal of the solvent is to remove at least the second solvent.

[0023] The fourth aspect of the present invention provides a prodrug of a boron-containing drug-polyhydroxy drug conjugate, and the structure of the prodrug at least includes a boron-containing drug-polyhydroxy drug conjugate and a ketone-terminated hyperbranched polyamide-amine (HPAP).

[0024] The fifth aspect of the present invention provides a preparation method of a prodrug of a boron-containing drug-polyhydroxy drug conjugate, comprising the following steps:

[0025] (1) Coupling hyperbranched polyamide-amine (HPAA) with piperidone to obtain ketone-terminated hyperbranched polyamide-amine (HPAP);

[0026] (2) Reacting the polyhydroxy drug with HPAP through a Schiff base reaction to obtain the HPAP prodrug HPAP-A;

[0027] (3) The boron-containing drug is reacted with HPAP-A through boronate esterification to obtain the prodrug HPAP-AB of the boron-containing drug-polyhydroxy drug conjugate.

[0028] Preferably, the conditions for the Schiff base reaction are heating, condensing, reflux and stirring, and the conditions for the borate esterification reaction are preferably heating under the action of an organic base.

[0029] Preferably, the molar ratio of the boron-containing drug, the polyhydroxy drug, and HPAP is

[0030] 1:1~50:0.01~0.5, preferably 1:1~5:0.01~0.05.

[0031] The sixth aspect of the present invention provides a use of a boron-containing drug-polyhydroxy drug conjugate, a boron-containing drug-polyhydroxy drug conjugate nanodrug, and a prodrug of a boron-containing drug-polyhydroxy drug conjugate in the preparation of anticancer drugs.

[0032] The beneficial effects of the present invention are:

[0033] The present invention provides an ADDC strategy based on boron-containing drugs and polyhydroxy drugs without purification. Specifically, the present invention finds that boron-containing drugs (such as bortezomib (BTZ) and ixazomib (Ixazomib)) containing boronic acid groups or boronic acid ester groups can be coupled with cis-diol-containing drugs (such as azacitidine (AZA), capecitabine (CAP), doxifluridine (DFUR), piceatannol (PIC), dopamine and its analogs (such as acrylamide dopamine-DPA) in a short time, which meets the characteristics of click reaction. The boron-containing drug-polyhydroxy drug conjugate prepared by the present invention has an efficacy significantly higher than that of the physical mixture of boron-containing drugs and polyhydroxy drugs, and the prodrug of the boron-containing drug-polyhydroxy drug conjugate prepared by the present invention reduces drug toxicity while ensuring efficacy. For example, the potency of BTZ-AZA conjugates significantly exceeds that of BTZ & AZA mixtures that have been studied in clinical trials; BTZ and AZA are further constructed into polymer prodrugs with controllable size and feed ratio, which improves the synergistic effect of BTZ-AZA conjugates on A549 adenocarcinoma cells, and the IC50 value of HPAP-AB (AZA and BTZ have a molar ratio of 1:1) is 22 times lower than that of BTZ-AZA conjugates, while reducing organ toxicity. This invention also pioneered a class of click chemistry-derived ADDC nanomedicines that do not require purification, achieving improved drug economy, biocompatibility, and cancer treatment efficacy. This strategy can provide more insights for the development of smart combination drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The technical solution of the present application is further described below in conjunction with the accompanying drawings and embodiments.

[0035] Figure 1 This is a partial roadmap for preparing boron-containing drug - polyhydroxy drug conjugates in this application.

[0036] Figure 2 This is the 1H NMR spectrum diagram described in Example 1 of this application.

[0037] Figure 3 This is the 13C NMR spectrum diagram described in Example 1 of this application.

[0038] Figure 4 This is the 1H NMR spectrum diagram described in Example 2 of this application.

[0039] Figure 5 This is the 13C NMR spectrum diagram described in Example 2 of this application.

[0040] Figure 6 This is the 1H NMR spectrum diagram described in Example 3 of this application.

[0041] Figure 7 This is the 13C NMR spectrum diagram described in Example 3 of this application.

[0042] Figure 8 This is the 1H NMR spectrum diagram described in Example 4 of this application.

[0043] Figure 9 This is the 1H NMR spectrum diagram described in Example 5 of this application.

[0044] Figure 10 This is the hydrodynamic size (D h ), polydispersity index (PDI), and surface potential (ζ) of different BTZ-drug SAN drugs measured under different pH conditions in Example 6 of this application.

[0045] Figure 11 This is the TEM diagram of different BTZ-drug SAN drugs measured under different pH conditions in Example 6 of this application.

[0046] Figure 12 This is the 1H NMR spectrum diagram described in Example 7 of this application.

[0047] Figure 13 This is the GPC result diagram described in Example 7 of this application.

[0048] Figure 14 This is the FTIR spectrum diagram described in Example 7 of this application.

[0049] Figure 15 This is the in vitro cytotoxicity test data diagram described in Example 10 of this application.

[0050] Figure 16It is a data graph showing the detailed uptake of the BTZ-AZA conjugate and HPAP-A-B in organelles described in Example 10 of this application.

[0051] Figure 17 It is the WB result of important signals related to lysosome permeability and apoptosis after treatment with drugs in different ratios described in Example 10 of this application. Detailed implementation mode

[0052] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other.

[0053] In the description of the present invention, the relevant definitions are as follows:

[0054] ADDC: Amphiphilic drug-drug conjugate

[0055] BTZ: Bortezomib

[0056] AZA: Azacitidine

[0057] CAP: Capecitabine

[0058] DFUR: Doxifluridine

[0059] PIC: Piceatannol

[0060] DPA: Dopamine analogue-acrylamide dopamine

[0061] SAN: Self-assembled nanodrug

[0062] THF: Tetrahydrofuran

[0063] DMF; Dimethylformamide

[0064] DMA: Dimethylacetamide

[0065] The technical solutions of this application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0066] Example 1 Preparation of BTZ-AZA conjugate drug

[0067] Dissolve BTZ (38.4 mg, 0.1 mmol) and AZA (24.4 mg, 0.1 mmol) separately in a mixed solvent of anhydrous tetrahydrofuran (3 mL) and anhydrous DMF (1 mL), add anhydrous sodium sulfate (100.0 mg) and anhydrous triethylamine (30 μL) to remove moisture and adjust the pH value of the reaction solution respectively. Stir the reaction mixture at 40 °C for 30 min, concentrate the filtrate after filtration, and dry it under vacuum to obtain the raw material product. Yield: 59.1 mg, Yield rate: 100%. MS m / z [M+Na] +Calculated 615.41, actual 615.41 [M+Na] + .

[0068] BTZ and AZA were completely consumed within 30 min in a mixed solvent of anhydrous tetrahydrofuran (3 mL) and anhydrous DMF (1 mL). As shown by nuclear magnetic resonance hydrogen spectrum ( 1 H NMR) (see Figure 2 ), the proton signals of the diol in AZA disappeared at 5.12 and 5.43 ppm. The corresponding nuclear magnetic resonance carbon spectrum ( 13 C) showed (see Figure 3 ) that the carbon signal peaks of the diol methylene completely shifted from 69.5 and 74.4 ppm to 51.1 and 79.0 ppm. The molecular weight signals detected by mass spectrometry were 615.41 and 1207.83 respectively, which were consistent with the theoretical molecular weights of the BTZ-AZA conjugate and its dimer. The ultraviolet-visible spectrophotometry (UV-Vis) spectrum of BTZ-AZA showed obvious characteristic peak overlap between BTZ and AZA, and there was a slight peak red shift compared with the mixture of BTZ and AZA. In Fourier transform infrared spectroscopy (FTIR), AZA showed two characteristic out-of-plane vibration bands of boric acid at 598 and 628 cm -1 and had a typical O-H stretching vibration band at 3199 cm -1 . In contrast, the single characteristic out-of-plane vibration band of BTZ-AZA shifted to 657 cm -1 , and a new B-O stretching vibration peak appeared at 1452 cm -1 , indicating the formation of borate ester.

[0069] Example 2 Preparation of BTZ-CAP Conjugate Drug

[0070] BTZ (38.4 mg, 0.1 mmol) and CAP (35.9 mg, 0.1 mmol) were separately dissolved in a mixed solvent of anhydrous tetrahydrofuran (3 mL) and anhydrous DMF (1 mL), and anhydrous sodium sulfate (100.0 mg) and anhydrous triethylamine (30 μL) were added to remove moisture and adjust the pH value of the reaction solution respectively. After stirring at 40 °C for 30 min, the mixture was filtered after the reaction, the filtrate was concentrated, and vacuum dried for 3 h. The raw material product could be obtained without further purification. Yield: 70.5 mg, yield: 100%. MS m / z [M+TEA+H] + Calculated 809.77, actual 809.79 [M+TEA+H] + .

[0071] 1 H NMR (see Figure 4 ), 13 C NMR (seeFigure 5 ) and mass spectrometry identified the quantitative and complete conversion from free CAP to the BTZ-CAP conjugate.

[0072] Example 3 Preparation of BTZ-DFUR Conjugated Drug

[0073] BTZ (38.4 mg, 0.1 mmol) and DFUR (24.6 mg, 0.1 mmol) were separately dissolved in anhydrous tetrahydrofuran (3 mL) and anhydrous DMF (1 mL). Anhydrous sodium sulfate (100.0 mg) and anhydrous triethylamine (30 μL) were added to remove water and adjust the pH of the reaction solution, respectively. The mixture was stirred at 40 °C for 30 min, the reaction mixture was filtered, the filtrate was concentrated, and dried under vacuum to obtain the raw material product. Yield: 59.2 mg, Yield rate: 100%. 1 1H NMR (see Figure 6 ), 13 13C NMR (see Figure 7 ), MS m / z [M+Na] + Calculated 617.23, Actual 617.24 [M+Na] + .

[0074] Example 4 Preparation of BTZ-PIC Conjugated Drug

[0075] BTZ (38.4 mg, 0.1 mmol) and PIC (24.4 mg, 0.1 mmol) were separately dissolved in a mixed solvent of anhydrous tetrahydrofuran (3 mL) and anhydrous DMF (1 mL). Anhydrous sodium sulfate (100.0 mg) and anhydrous triethylamine (30 μL) were added to remove moisture and adjust the pH of the reaction solution, respectively. The mixture was stirred at 40 °C for 30 min, the reaction mixture was filtered, the filtrate was concentrated, and dried under vacuum to obtain the raw material product. Yield rate: 100%, and the product was analyzed by 1 1H NMR (see Figure 8 ).

[0076] According to the 1 1H NMR nuclear magnetic resonance hydrogen spectrum integration of the BTZ&PIC reaction mixture, a new multiplet peak appeared at 8.45 - 8.72 ppm, and the proton signal of the methyl alkyne in BTZ was split into two groups of peaks at 4.67 - 4.86 ppm.

[0077] Example 5 Preparation of BTZ-DPA Conjugated Drug

[0078] BTZ (38.4 mg, 0.1 mmol) and acrylamide dopamine (DPA) (20.1 mg, 0.1 mmol) were separately dissolved in a mixed solvent of anhydrous tetrahydrofuran (3 mL) and anhydrous DMF (1 mL). Anhydrous sodium sulfate (100.0 mg) and anhydrous triethylamine (30 μL) were added to remove moisture and adjust the pH of the reaction solution, respectively. After stirring at 40 °C for 30 min, the reaction mixture was filtered, the filtrate was concentrated, and vacuum dried to obtain the raw material product. Yield: 100%, analyzed by 1 1H NMR (see Figure 9 ).

[0079] Example 6 Preparation of Boron-Containing Drug - Polyhydroxy Drug Conjugate Nanodrug BTZ-drug SAN

[0080] The preparation of BTZ-drug SAN was basically carried out by the nanoprecipitation method. Taking BTZ-AZA SAN as an example, 2.0 mg of BTZ-AZA prepared in Example 1 was dissolved in 400 μL of THF and stirred in the dark for 5 min. Then, the solution was dropped into 2 mL of deionized water, the pH of the solution was adjusted using PBS buffer, and then stirred vigorously for 30 min to gradually form a stable turbid emulsion. By using the typical nanoprecipitation method, after vigorous stirring and subsequent evaporation of THF, a nanodrug stock solution containing BTZ-AZA conjugate without obvious precipitation was obtained (1 mg·mL -1 ). As Figure 10 shown, when the pH was 7.4, the hydrodynamic diameter D h of the BTZ-AZA solution was 224.5 ± 2.3 nm, the dispersity index (PDI) was 0.086, and the surface potential (ζ) was -21.4 ± 2.5 mV. After incubation under acidic conditions (pH 5.0), the cleavage of the BTZ-AZA conjugate led to a decrease in D h to 194.4 ± 1.2 nm, an increase in PDI to 0.258, and a surface potential ζ of -26.3 ± 0.12 mV. The results of transmission electron microscopy (TEM) measurement showed (see Figure 11 ), at pH 5.0 and pH 7.4, the diameters of the BTZ-AZA conjugate were 187.9 ± 15.7 and 85.9 ± 41.3 nm, respectively. Compared with DLS, the smaller size obtained by TEM was due to the dry state of the sample.

[0081] For conjugates such as BTZ-CAP, BTZ-DFUR, BTZ-PIC, and BTZ-DPA, the changing trends of their size and surface potential were similar to those of the BTZ-AZA conjugate.

[0082] Example 7 Preparation of Prodrug

[0083] In the Michael addition reaction of HPAA, piperidone was introduced to couple with the amide end group to generate keto-end group HPAA (HPAP). Secondly, AZA was linked to HPAP through a Schiff base reaction to obtain the HPAP prodrug carrying AZA (HPAP-A). Finally, BTZ was linked to HPAP-A through a borate esterification reaction to prepare the HPAP prodrug carrying the AZA-BTZ conjugate (HPAP-A-B). The specific reaction process is as follows.

[0084] HPAP: N,N'-methylenebisacrylamide (MBA) (616.68 mg, 4 mmol) was dissolved in 8 ml of a mixed solvent of MeOH and water (7 / 3, v / v), and then 1-(2-aminoethyl)piperazine - AEPZ (258.4 mg, 2 mmol) was slowly added to the above solution. After deoxygenation with argon, it was kept in the dark at 50 °C for 4 days. Then, the reaction mixture was vigorously shaken in acetone to precipitate. The dried white powdery solid HPAA (150 mg) was dissolved in MeOH / H2O (5.6 mL / 2.4 mL). In addition, below 37 °C, an ethyl acetate solution of piperidone (300 mg, 2 g of piperidone dissolved in 20 ml of ethyl acetate) was added for reaction for 7 days. The reaction mixture was vigorously shaken in acetone to precipitate and dried in vacuo to obtain HPAP. The weight-average molecular weight of HPAP was 25,100, and the molecular weight distribution width was 1.74.

[0085] HPAP-AZA (HPAP-A): HPAP (100 mg) was dissolved in 5 mL of H2O. Azacitidine (200 mg) was dissolved in 50 mL of ethanol. Then, HPAP (100 mg) dissolved in 5 mL of H2O was dropped into the above azacitidine solution, and the mixture was stirred under reflux with condensation at 90 °C for 4 days. The yellow powder crystals were collected by rotary evaporation. Then, a small amount of pure water was added to dissolve, and the product was collected by ultrafiltration at 4000 rpm for 4 times.

[0086] HPAP-BTZ-AZA (HPAP-A-B): BTZ (15.4 mg) and HPAP-AZA (30 mg) were respectively dissolved in anhydrous tetrahydrofuran (10 mL) and anhydrous DMF (5 mL). Anhydrous sodium sulfate (20 mg) and anhydrous triethylamine (50 μL) were added to the mixed solvent, and the mixture was stirred overnight at 40 °C. Subsequently, the mixture was filtered to remove anhydrous sodium sulfate. The yellow viscous product was obtained directly by rotary evaporation and drying.

[0087] See Figure 12 , for the 1The ¹H NMR spectrum showed BTZ (7.05 - 7.36 ppm), AZA (3.48 - 4.11 ppm), and HPAP (2.11 - 2.76 ppm). Based on the proton integration areas of BTZ and AZA, the coupling efficiency of BTZ, AZA, and HPAP was over 95%. Gel permeation chromatography (GPC) indicated (see Figure 13 ), after AZA coupling, the average molecular weight (M w ) of the conjugate increased from 25,100 to 31,600 compared to HPAP. However, due to the competitive interaction between the HPAP prodrug HPAP - A - B of the boron - containing drug - polyhydroxy drug conjugate and the stationary phase of the column, the average molecular weight of HPAP - A - B could not be accurately obtained by GPC. As shown by the FTIR spectrum (see Figure 14 ), after AZA grafting, the typical C=O stretching vibration band at 1718 cm -1 disappeared, indicating that the piperidone terminal group of HPAP was completely consumed by AZA. Subsequently, the coupling of BTZ produced a single characteristic out - of - plane vibration band of borate ester at 660 cm -1 . In addition, after the coupling of the diol of the BTZ and AZA groups, the typical O - H and N - H stretching vibration bands (3200 - 3450 cm -1 ) became significantly narrower. These results together verified the successful synthesis of HPAP - A - B, and the ratio of AZA to BTZ was approximately 1:1. Additionally, referring to the preparation process of the BTZ - AZA conjugate, an in vitro drug release experiment of HPAP - A - B was conducted. After incubation for 24 h under acidic conditions (pH = 5), the cumulative release amounts of BTZ and AZA increased to 77.6% and 77.3% respectively.

[0088] Example 8 In vitro performance of BTZ - AZA SAN

[0089] To study the cellular uptake of BTZ-AZA SAN, Cyanine 5.5 (Cy 5.5) was encapsulated during the self-assembly process of the BTZ-AZA conjugate. After 4 h of cellular internalization, confocal laser scanning microscopy (CLSM) images showed that Cy5.5 co-incubated with BTZ-AZA SAN (BTZ-AZA@Cy5.5 SAN) exhibited stronger red fluorescence in A549 cells than free Cy5.5, and its cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI). Compared with BTZ, AZA, and the BTZ&AZA mixture, BTZ-AZA SAN had good cellular uptake ability. Subsequently, the in vitro anti-cancer activity of BTZ-AZA SAN was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. After 48 h of incubation, the half-maximal inhibitory concentration (IC50) of BTZ-AZA SAN against A549 cells was 13.3 nM, significantly lower than that of the BTZ&AZA mixture (21.1 nM) and free BTZ (552.4 nM). The curves of the combination index (CI) versus the cell killing rate (fraction affected, Fa) after 48 h of treatment with different drug formulations of BTZ and AZA showed that most of the CI values of BTZ-AZA SAN and the BTZ&AZA mixture were below 1, and the CI values of BTZ-AZA SAN at the corresponding Fa were much lower than those of the BTZ&AZA mixture. These results indicated that the synergistic effect of BTZ-AZA SAN on A549 cells was significantly stronger than that of the BTZ&AZA mixture.

[0090] Furthermore, the activation of caspase-3 protein was detected by Western blotting (WB). Typical double bands of cleaved caspase-3 with molecular weights of 17 kDa and 19 kDa appeared in all BTZ-containing formulations. Quantitative analysis showed that the expression of cleaved caspase-3 in cells treated with BTZ-AZA SAN was significantly higher than that in other groups. A549 cells were co-incubated with BTZ-AZA SAN, the BTZ&AZA mixture, AZA, and BTZ for 48 h (at the same concentration of BTZ or AZA), and then stained with Annexin V-FITC / PI for flow cytometry detection. The results showed that the apoptosis rates (Q2+Q3) induced by AZA, BTZ, and the BTZ&AZA mixture were 14.5%, 34.1%, and 38.9%, respectively, while the apoptosis rate induced by BTZ-AZA SAN increased to 55.1%. Therefore, compared with the free drugs and mixtures at the same dose, the synergistic effect endows BTZ-AZA SAN with the best pro-apoptotic ability.

[0091] Example 9 In Vivo Anti-Tumor Activity of BTZ-AZA SAN

[0092] Before in vivo treatment, the pharmacokinetics and biodistribution of BTZ-AZA SAN were investigated using an A549 xenograft mouse model. Compared with free BTZ, the nanoform significantly extended the half-life (T 1 / 2 ) of BTZ-AZA SAN to 28.8 h. To understand the dynamic distribution of BTZ-AZA SAN in more detail, we acquired in vivo and ex vivo imaging of Cy5.5-labeled BTZ-AZA SAN at different time points (0.5, 1, 3, 6, 12 h post-injection) using the IVIS system. Cy5.5 rapidly accumulated in the liver at 1 h post-injection, while the retention of BTZ-AZA SAN in the liver reached its maximum at 6 h post-injection. Similar metabolic differences were also observed in organs such as the spleen, lung, and tumor. Due to the enhanced permeability and retention (EPR) effect, BTZ-AZA SAN showed a higher tumor uptake rate than free Cy5.5 within 6 h post-injection. Subsequently, the in vivo therapeutic efficacy of BTZ-AZA SAN was evaluated by comparison with BTZ&AZA mixture, AZA, BTZ, and PBS control groups. All administrations were performed intravenously every three to four days (days 0, 3, 7, 10). To distinguish the efficacy of different drugs, in vivo treatment was initiated in A549 tumor-bearing mice with a relatively large initial tumor volume of approximately 200 mm 3 . In this case, the BTZ&AZA mixture, free AZA, and BTZ hardly inhibited tumor growth, while BTZ-AZA SAN showed significant therapeutic efficacy.

[0093] Example 10 Anticancer Activity of HPAP-A-B In Vitro

[0094] The MTT assay showed that the HPAP carrier significantly altered the in vitro cytotoxicity of BTZ-based drugs against A549 cells. Figure 15It can be seen that when the molar ratio of AZA / BTZ in HPAP-A-B is equal to 2, the IC50 value of HPAP-A-B(2:1) is 421.14 nM, which is between that of BTZ and the BTZ-AZA conjugate. Surprisingly, when the molar ratio of AZA to BTZ in HPAP-A-B is equal, the corresponding HPAP-A-B(1:1) exhibits stronger cytotoxicity, with an IC50 value of 0.57 nM, which is 22 times lower than that of the BTZ-AZA conjugate. In addition, at the corresponding Fa values, all CI values of HPAP-A-B(1:1) are lower than those of the BTZ-AZA conjugate, indicating that the synergistic effect of the BTZ-AZA conjugate is enhanced when using the HPAP macromolecular carrier. However, the HPAP carrier itself does not have obvious intrinsic cytotoxicity. HPAP-A-B shows a faster cellular internalization rate (0.5 h), and the mean fluorescence intensity (MFI) analysis shows that HPAP-A-B has better tumor accumulation than the BTZ-AZA conjugate, which may be attributed to the positive potential of the HPAP carrier and the EPR effect.

[0095] As an emerging targeted strategy for precise cancer therapy, the present invention further investigated the detailed uptake of the nano-drugs BTZ-AZA SAN and HPAP-A-B(1:1) prepared in Example 6 in organelles to explain their enhanced synergistic effect. Figure 16 It can be seen that the BTZ-AZA conjugate mainly accumulates in cellular mitochondria, and its co-localization analysis shows that the Pearson correlation coefficients (PCC) of mitochondria and lysosomes are 0.69 and 0.35, respectively. In contrast, HPAP-A-B shows selective uptake in lysosomes, with a PCC as high as 0.87, while the PCC in mitochondria is only 0.51. After using bafilomycin A1 (BafA1) and mitochondrial fission inhibitor 1 (mdivi-1), the uptake of HPAP-A-B(1:1) in lysosomes and mitochondria is inhibited to 0.37 and 0.16, respectively, while the uptake of the BTZ-AZA conjugate in lysosomes shows no obvious inhibitory effect. Recently, lysosomes have been exploited as a target for cancer therapy. The accumulation of chemotherapeutic drugs in lysosomes may sensitize the membrane, increase its permeability, and increase protease leakage into the cytoplasm, thereby promoting lysosomal and apoptotic death and improving multidrug resistance. Through the SA-β-galactosidase staining experiment, the staining signal of A549 cells treated with HPAP-A-B(1:1) is stronger than that of cells treated with BTZ and the BTZ-AZA conjugate, indicating that HPAP-A-B has the strongest ability to induce cellular senescence.

[0096] The present invention further investigated some important signals related to lysosomal permeability and apoptosis after treatment with different ratios of drugs, such as B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X regulator (Bax), and nuclear factor κB (NF-κB). The WB results showed (see Figure 17 ), the A549 cells treated with HPAP-A-B (1:1) had the most significant changes in reducing the expression of NF-κB, Bcl-2, and Bax, and the highest activated caspase-3 and Bax / Bcl-2 ratios. Overall, the conjugation of the BTZ-AZA conjugate with the HPAP carrier significantly enhanced apoptosis in A549 cells.

[0097] Example 11 Attenuating and synergistic effects of HPAP-A-B in vivo

[0098] Similar to BTZ-AZA SAN, the biodistribution of HPAP-A-B (1:1) was investigated by in vivo and in vitro IVIS imaging in the present invention. The use of the HPAP carrier reduced the T 1 / 2 of HPAP-A-B (1:1) to 18.8 h. Generally speaking, the biodistribution of HPAP-A-B was similar to that of BTZ-AZA SAN prepared in Example 6. The positive surface potential (12.7 ± 0.46 mV) enhanced the uptake of HPAP-A-B in the liver, kidney, and intestine, but there was no significant enhancement in the uptake and cumulative retention in tumors. To ensure the biosafety of long-term treatment, multiple administrations of HPAP-A-B (1:1) (administered once every three to four days, at a dose of 0.8 mg / kg BTZ, for a total of seven times) were carried out to evaluate its toxicity. The blood biochemical results showed that the treatment with BTZ alone led to a significant increase in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), creatine kinase isoenzyme MB (CK-MB), and lactate dehydrogenase (LDH), respectively indicating possible damage to the liver, heart, and cell membrane. At the same time, no abnormal renal function was detected through the indicators of creatinine (CREA) and blood urea nitrogen (BUN). Although the BTZ-AZA conjugate eliminated the risk to liver function, it might still cause potential damage to the heart. In contrast, after treatment with HPAP-A-B, the indicators of the main organs were all within the normal range. In addition, no BTZ-induced inflammatory response was detected in the HPAP-A-B treatment group by enzyme-linked immunosorbent assay (ELISA), and the expressions of immunoglobulin G (IgG) and IgM were close to normal levels. To ensure the efficacy between different drugs and balance the efficacy difference and toxicity, we set the tumor volume of the initially treated mice at 100 mm 3 as follows. All drugs (at a dose of 0.8 mg·kg -1For BTZ), as described previously, it was administered once every three to four days. The drug treatments of BTZ, BTZ-AZA SAN, and HPAP-A-B were all effective, while HPAP and HPAP-A could hardly inhibit tumor growth. However, all the mice treated with BTZ died due to high toxicity 48 days after treatment, while HPAP-A-B showed a significant advantage in reducing toxicity and enhancing efficacy compared to BTZ-AZA SAN starting from day 32. Except for the groups treated with BTZ and HPAP, the mice in other groups did not show obvious weight loss. During the 72-day treatment period, all the mice in the HPAP-A-B treatment group survived, while the survival rates of other groups were all lower than 40% on day 56. In addition, the results of pathological sections showed that the drugs containing free BTZ caused signs of liver necrosis and renal edema, which was consistent with the previous reports, while HPAP-A-B treatment did not cause obvious organ damage.

[0099] In summary, the present invention uses boronic acid click chemistry to convert combination drugs into ADDCs. The quantitative conversion and synthesis process without purification not only simplifies the application scenarios of combination chemotherapy, but also enhances drug economy, biocompatibility, and the chemotherapy effect of lung cancer. In addition, the utilization of the polymer HPAP carrier can further change the uptake of the HPAP-A-B prodrug by cell organelles and significantly enhance the synergistic effect of the BTZ-AZA conjugate. Due to the characteristics of click chemistry, this novel ADDC conjugation strategy can expand the scope of responsive prodrugs and accelerate the development of combination chemotherapy.

[0100] Inspired by the above ideal embodiments according to the present application, through the above description, relevant staff can completely make various changes and modifications without departing from the technical idea of this application. The technical scope of this application is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A boron-containing drug-polyhydroxy drug conjugate, characterized in that: It comprises a boron-containing drug and a polyhydroxy drug, wherein the boron-containing drug contains a boric acid group and the polyhydroxy drug contains at least two hydroxyl groups.

2. The boron-containing drug-polyhydroxy drug conjugate according to claim 1, characterized in that: The boron-containing drug is bortezomib; the polyhydroxy drug preferably contains a cis-diol structure, and is further preferably at least one selected from azacitidine, capecitabine, doxifluridine, piceatannol, and dopamine and its analogs, and the dopamine analog is preferably acrylamide dopamine.

3. A method for preparing the boron-containing drug-polyhydroxy drug conjugate according to claim 1 or 2, characterized in that: The boron-containing drug and the polyhydroxy drug are prepared by borate esterification reaction; preferably, the molar feed ratio of the boron-containing drug to the polyhydroxy drug is 1-10:1-10, more preferably 1:

1.

4. The method according to claim 3, characterized in that The boron-containing drug and the polyhydroxy drug are dissolved in a first solvent, and under the action of an organic base, the boron-containing drug-polyhydroxy drug conjugate is prepared; preferably, the first solvent is selected from one or more of THF, DMF, DMA, benzene, toluene or methyl halides; more preferably, the first solvent is an anhydrous solvent; further preferably, the solvents in which the boron-containing drug and the polyhydroxy drug are dissolved can be the same or different.

5. The method according to claim 3 or 4, characterized in that: The reaction temperature is 10 to 50° C., preferably 20 to 40° C.; the reaction time is preferably 0.1 to 12 h, more preferably 0.5 to 5 h; the organic base is preferably an organic amine, more preferably triethylamine.

6. A boron-containing drug-polyhydroxy drug conjugate nanodrug, characterized in that: The boron-containing drug-polyhydroxy drug conjugate according to claim 1 or 2 is prepared by a nanoprecipitation method.

7. The boron-containing drug-polyhydroxy drug conjugate nanodrug according to claim 7, characterized in that: The nanoprecipitation method is as follows: adding the boron-containing drug-polyhydroxy drug conjugate according to claim 1 or 2 to a second solvent for mixing, then adding the mixed solution to a third solvent, adjusting the pH of the solution to weak alkalinity and mixing, removing the solvent, and obtaining the boron-containing drug-polyhydroxy drug conjugate nanomedicine; preferably, the second solvent is selected from one or more of THF, DMF, DMA, benzene, toluene or methyl halides; preferably, the third solvent is selected from one or both of water or alcohol, more preferably deionized water; preferably, removing the solvent is removing at least the second solvent.

8. A prodrug of a boron-containing drug-polyhydroxy drug conjugate, characterized in that: The structure of the prodrug at least includes the boron-containing drug-polyhydroxy drug conjugate according to claim 1 or 2 and the keto-terminated hyperbranched polyamide-amine.

9. A method for preparing a prodrug of the boron-containing drug-polyhydroxy drug conjugate according to claim 8, characterized in that: The steps include: (1) coupling a hyperbranched polyamide-amine (HPAA) with piperidone to obtain a keto-terminated hyperbranched polyamide-amine (HPAP); (2) reacting the polyhydroxy drug with HPAP via a Schiff base to obtain the HPAP prodrug HPAP-A; (3) reacting the boron-containing drug with HPAP-A through boronate esterification to obtain a prodrug HPAP-AB of the boron-containing drug-polyhydroxy drug conjugate; Preferably, the conditions for the Schiff base reaction are heating, condensing, reflux and stirring, and the conditions for the borate esterification reaction are preferably heating under the action of an organic base;. Preferably, the molar ratio of the boron-containing drug, the polyhydroxy drug and HPAP is 1:1-50:0.01-0.5, preferably 1:1-5:0.01-0.

05.

10. Use of the boron-containing drug-polyhydroxy drug conjugate according to claim 1 or 2, the boron-containing drug-polyhydroxy drug conjugate nanodrug according to claim 6 or 7, and the prodrug of the boron-containing drug-polyhydroxy drug conjugate according to claim 8 in the preparation of anticancer drugs.

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