A pharmaceutical combination product for enhancing the therapeutic effect of nanomedicines

Abstract

The present application relates to a kind of drug combination products for enhancing the therapeutic effect of nanomedicine, belong to nanomedicine technical field.The drug combination product includes following (1), (2) and (3): (1) stimulus-responsive nanomedicine;(2) mononuclear phagocyte system blocker;(3) exogenous reducing substance.The drug combination product provided by the present application carries out the regulation in time sequence to the in vivo delivery and release process of nanomedicine by "first block then loose" delivery strategy, realizes the effective accumulation and intelligent release of nanomedicine in target tissue, to improve the therapeutic effect.

Description

Technical Field

[0001] This invention relates to a drug combination product that enhances the therapeutic effect of nanomedicines, belonging to the field of nanomedicine technology. Background Technology

[0002] Nanomedicines have shown great potential in the treatment of diseases such as cancer. However, in practical applications, the in vivo delivery efficiency and drug release efficiency of nanomedicines remain limited, thus affecting their therapeutic effects and clinical translation. The main reason for this predicament lies in the complex and unpredictable in vivo fate of nanomedicines. Specifically, the delivery of nanomedicines in vivo typically involves multiple continuous processes, including blood circulation, organ distribution, target tissue accumulation, and drug release. Based on the process of nanomedicine delivery and the exertion of its therapeutic effect in vivo, it can be divided into two main stages: the circulation and distribution stage before the nanomedicine reaches the target tissue, and the drug release stage after the nanomedicine reaches the target tissue.

[0003] In the first stage, after entering the systemic circulation, nanomedicines are easily recognized and cleared by the mononuclear phagocyte system (MPS) in organs such as the liver and spleen. This shortens the blood circulation time, reduces the amount of drug reaching the target tissue, and affects the accumulation of nanomedicines in the target tissue. In the second stage, even if some nanomedicines can accumulate in the target tissue, the release efficiency of the active drug components they carry in the local environment may still be insufficient, making it difficult to form a sustained and effective drug concentration at the lesion site, thus affecting the therapeutic effect.

[0004] Existing technologies typically optimize single steps in the nanomedicine delivery process, such as using polyethylene glycol (PEG) modification to prolong cycle time or constructing stimulus-responsive nanocarriers to promote drug release. However, these strategies generally focus on regulating a single stage and do not adequately consider the dynamic changes throughout the entire nanomedicine delivery process in vivo or the synergistic relationships between different stages. Therefore, how to systematically regulate the delivery and release of nanomedicines in vivo to simultaneously improve target tissue accumulation and drug release efficiency is a problem that needs further research in this field. Summary of the Invention

[0005] To address the technical problem of poor in vivo delivery and release efficiency leading to suboptimal therapeutic effects in existing nanomedicine technologies, this invention provides a drug combination product that enhances the therapeutic efficacy of nanomedicines. The drug combination product provided by this invention employs a "block-then-release" delivery strategy to regulate the temporal sequence of the in vivo delivery and release process of nanomedicines, achieving effective accumulation and intelligent release of nanomedicines in target tissues, thereby improving therapeutic efficacy.

[0006] A pharmaceutical combination product for enhancing the therapeutic effect of nanomedicine, the pharmaceutical combination product comprising the following (1), (2) and (3):

[0007] (1) Stimulus-responsive nanomedicines; (2) Mononuclear phagocyte system blockers; (3) Exogenous reducing substances; The stimulus-responsive nanomedicine is a nanoformulation formed by the self-assembly of a prodrug formed by coupling an active drug molecule with a fatty alcohol via a disulfide bond or by assembly with a pharmaceutically acceptable nanocarrier excipient; the mononuclear phagocyte system blocker is one or more of phospholipid liposomes or phospholipid liposomes with chemically modified surfaces; the exogenous reducing substance is one or more of vitamin C, cysteine, N-acetylcysteine, reduced glutathione, or lipoic acid.

[0008] Furthermore, the mononuclear phagocyte system blocker (MPS blocker) refers to a class of substances that can temporarily inhibit or saturate the function of the mononuclear phagocyte system, and they typically have similar or identical physicochemical properties (such as carrier composition, particle size, and surface charge) to therapeutic nanomedicines. This invention preferably reduces the in vivo clearance of nanomedicines by saturating the phagocytic activity of the mononuclear phagocyte system.

[0009] Furthermore, the phospholipid-type liposomes or chemically modified phospholipid-type liposomes described in this invention can be obtained through existing technologies or purchased.

[0010] In the drug combination product for enhancing the therapeutic effect of nanomedicine described in this invention, a mononuclear phagocyte system blocker is first used to reduce the clearance rate of nanomedicine, enabling the stimulus-responsive nanomedicine to circulate in the body and accumulate in the target tissue. Then, an exogenous reducing substance is used to release the active drug components of the nanomedicine, thereby enhancing the therapeutic effect.

[0011] Preferably, the exogenous reducing substance is N-acetylcysteine ​​(NAC) or reduced glutathione (GSH).

[0012] In the drug combination product for enhancing the therapeutic effect of nanomedicines described in this invention, (1), (2) and (3) exist in a form that is isolated from each other.

[0013] In the drug combination product for enhancing the therapeutic effect of nanomedicine described in this invention, the drug combination product is administered in a simultaneous or sequential order.

[0014] Furthermore, the sequential administration order is as follows: first, administer a mononuclear phagocyte system blocker; second, administer a stimulus-responsive nanomedicine; and finally, administer an exogenous reducing agent.

[0015] Furthermore, a stimulus-responsive nanomedicine was administered 0.5–3 h after the administration of a mononuclear phagocyte system blocker, and an exogenous reducing agent was administered 12–24 h after the administration of the nanomedicine.

[0016] The method for enhancing the therapeutic effect of nanomedicines through drug combination products of the present invention includes a blocking phase and a clearing phase. By constructing a temporal regulation strategy of "blocking first and then clearing," the delivery and release process of nanomedicines in vivo is regulated. The method includes the following steps: (1) Blocking phase: Before or simultaneously with the administration of therapeutic stimulus-responsive nanomedicine, an effective amount of mononuclear phagocyte system blocker is administered to temporarily inhibit or saturate the function of the mononuclear phagocyte system, reduce the clearance rate of nanomedicine, and create a time window for efficient enrichment of target tissue. (2) Unblocking stage: After the nanomedicine accumulates in the target tissue to the peak or near the peak, an effective amount of exogenous stimulant is given to trigger the release of active drug components from the nanomedicine, thereby achieving a high local drug concentration and enhancing the therapeutic effect.

[0017] In the drug combination product for enhancing the therapeutic effect of nanomedicines according to the present invention, the dosage of the stimulus-responsive nanomedicine is 1~20 μmol / kg based on the active drug concentration; the dosage of the mononuclear phagocyte system blocker is 20~1000 μmol / kg based on the phospholipid concentration; and the dosage of the exogenous reducing substance is 200~10000 μmol / kg.

[0018] In the drug combination product for enhancing the therapeutic effect of nanomedicines according to the present invention, the active drug is one or more of antitumor drugs, anti-inflammatory drugs, antibacterial drugs, or antifungal drugs.

[0019] Preferably, the drug is one or more of the antitumor drugs.

[0020] More preferably, the antitumor drug is one or more of taxanes or anthraquinones.

[0021] More preferably, the taxane compound is one or more of paclitaxel, docetaxel, cabazitaxel, lalotaxel, salitaxel, comotaxel, or miratataxel; and the anthraquinone compound is one or more of daunorubicin, doxorubicin, epirubicin, pirarubicin, or aclarubicin.

[0022] In the drug combination product for enhancing the therapeutic effect of nanomedicines according to the present invention, the stimulus-responsive nanomedicine is a nanoformulation formed by thin-film hydration and ultrasonic treatment of a prodrug, phospholipid, cholesterol, and a PEG modifier. The average particle size of the nanoformulation is 50-200 nm. The structural formula of the prodrug is shown below: .

[0023] Preferably, the nano-formulation is prepared by the following method: (1) Paclitaxel The prodrug of decanoic acid, phospholipids, cholesterol and PEG modifier were dissolved in an organic solvent, and the organic solvent was removed by rotary evaporation to obtain a thin film on the bottle wall. (2) Paclitaxel is obtained by adding a water-soluble medium and hydrating at a constant temperature. Crude liposomes of ecstasylate prodrug were obtained; after sonication, paclitaxel particles with uniform particle size were obtained. Liposomes of dodecyl alcohol prodrug are nano-formulations.

[0024] Furthermore, the paclitaxel Docetaxel prodrug is prepared by the following method:

[0025] (1) Place 2,2' Dithiodiacetic acid is dehydrated by acetic anhydride to obtain a dicarboxylic acid anhydride; wherein the dehydration reaction takes 0.5 h to 4 h; the dehydration reaction temperature is 15℃ to 37℃; and the 2,2' The molar ratio of dithiodiacetic acid to the acetic anhydride is 1: (1~50); (2) Under nitrogen protection, 11-cubecool was in 4 Under the action of dimethylaminopyridine, a ring-opening esterification reaction occurs with the diacid anhydride, and the intermediate product 11-eicosyl ester is obtained by column chromatography; wherein the ring-opening esterification reaction takes 6 h to 24 h and the ring-opening esterification reaction takes 15 °C to 37 °C; the 4 The molar ratio of dimethylaminopyridine, the fatty alcohol, and the diacid anhydride is 1 : (1~10) : (1~20); (3) The intermediate product 11-tetracohol monoester was dissolved in anhydrous dichloromethane, and the catalyst dissolved in dichloromethane was added dropwise. After activation in an ice bath, paclitaxel was added and reacted under nitrogen protection. The reaction product was purified by liquid phase separation to obtain the paclitaxel. 11-Cecidool prodrug; wherein the intermediate product 11-cecidool monoester; and the catalyst is 1 Hydroxybenzotriazole, carbodiimide and 4 The molar ratio of dimethylaminopyridine to paclitaxel is 1 : (1~10) : (2~6) : (0.2~5) : (0.5~10); the reaction temperature is 25℃~37℃; and the reaction time is 24h~48h.

[0026] In the drug combination product for enhancing the therapeutic effect of nanomedicines according to the present invention, the average particle size of the mononuclear phagocyte system blocker is 50~500 nm; the surface is chemically modified by using polyethylene glycol for surface modification, and / or the surface is connected with a ligand targeting a macrophage receptor; the receptor is one or more of mannose receptor, scavenger receptor or immunoglobulin Fc receptor.

[0027] The modulation strategy of the mononuclear phagocyte system blocker described in this invention is a mononuclear phagocyte system saturation strategy, and its blocking effect is jointly influenced by factors such as the particle size, dosage, and administration time of the blocker. In one embodiment, by combining a larger particle size with a lower dosage, the interference of the blocker in the target tissue can be reduced while achieving mononuclear phagocyte system saturation.

[0028] Preferably, the particle size of the blocking agent is 400 nm, and the dosage is 50 μmol / kg.

[0029] Another object of the present invention is to provide the application of a combination of stimulus-responsive nanomedicines, mononuclear phagocyte system blockers and exogenous reducing substances in the preparation of the above-mentioned pharmaceutical combination products that enhance the therapeutic effect of nanomedicines.

[0030] Furthermore, the enhanced therapeutic effect of the nanomedicine is to enhance its anti-tumor effect.

[0031] Furthermore, the tumor is one or more of breast cancer, lung cancer, prostate cancer, ovarian cancer, or melanoma.

[0032] The beneficial effects of this invention are: The drug combination product provided by this invention can temporally regulate the in vivo fate of nanomedicines, achieving synergistic regulation in both the in vivo delivery and drug release stages. Firstly, by blocking the clearance of nanomedicines by the mononuclear cell phagocytic system during the blocking stage, the accumulation of nanomedicines in target tissues is increased. Secondly, by unblocking the process, the release of the active ingredient from the nanomedicines is triggered, increasing the concentration of active drug in the target tissue. The drug combination product described in this invention is applicable to various stimulus-responsive nanomedicine systems and can provide a new technical solution for the design of nanomedicine delivery systems. Attached Figure Description

[0033] Figure 1 This is the mass spectrum of the paclitaxel-dodecyl alcohol prodrug obtained in Example 1 of the present invention.

[0034] Figure 2 The paclitaxel-dodecyl alcohol prodrug obtained in Example 1 of this invention 1 H-NMR spectrum.

[0035] Figure 3This is a purity diagram of the paclitaxel-dodecyl alcohol prodrug obtained in Example 1 of the present invention.

[0036] Figure 4 This is a stability study of blank liposome blockers with different particle sizes in Example 3 of the present invention. Figures a, b, c, and d show particle size changes at 4°C, 25°C, 10% FBS, and rat plasma, respectively.

[0037] Figure 5 This is a pharmacokinetic diagram of the paclitaxel-dodecyl alcohol prodrug liposome in Example 4 of the present invention, and the plasma concentration-time curve of the prodrug and parent drug.

[0038] Figure 6 This figure illustrates the chemical stability of the paclitaxel-dodecanoic acid prodrug liposomes in rat plasma in Example 4 of this invention. In the figure, a represents prodrug degradation and parent drug release rate; b represents particle size and PDI changes.

[0039] Figure 7 This diagram illustrates the tissue distribution of paclitaxel-ticodobalamin prodrug liposomes before and after blockade in Example 5 of this invention. Specifically, a, b, and c represent the accumulation of paclitaxel-ticodobalamin prodrug liposomes in the liver, spleen, and tumor before and after blockade with different particle size / dose inhibitors, expressed as the area under the drug-time curve (AUC); d represents the AUC ratio of the target organ to the MPS-phagocytic organ; and e represents the accumulation of prodrug and parent drug within the tumor before and after blockade in different groups.

[0040] ns: P≥0.05; P < 0.05; P < 0.01; P<0.001; P < 0.0001 (all two-tailed t-tests) Figure 8 The pharmacokinetic behavior of the paclitaxel-dodecanoic acid prodrug liposome before and after blockade in Example 6 of this invention, as well as the plasma concentration-time curve of the prodrug and parent drug (tumor-bearing mouse model).

[0041] Figure 9 The pharmacokinetic behavior of the paclitaxel-dodecanoic acid prodrug liposome before and after blockade in Example 6 of this invention, as well as the plasma concentration-time curve of the prodrug and parent drug (in a healthy rat model without tumor).

[0042] ns: P≥0.05; P < 0.05 (all two-tailed t-tests) Figure 10 This study investigates the blocking effect of the paclitaxel-dodecanoic acid prodrug liposomes of Example 7 of the present invention in in vitro cell experiments. Specifically, a) represents the blocking efficiency (BE) of different doses / particle sizes of the blocking agent on coumarin-6 FLs in RAW 264.7 cells; b) represents the blocking efficiency (BE) in 4T1 cells; and c) represents the dual blocking assessment in a transwell co-culture model.

[0043] ns: P≥0.05; P < 0.05; P < 0.01; P<0.001; P < 0.0001 (all two-tailed t-tests) Figure 11 The in vitro release curves of the paclitaxel-dococol prodrug liposomes of Example 9 of the present invention under different NAC molar ratios (paclitaxel-dococol prodrug liposomes: NAC).

[0044] Figure 12 This is an in vivo validation of the exogenous reducing stimulant used in Example 10 of the present invention. Wherein, a represents the percentage of prodrug activation and parent drug release under different dosing regimens; b and c represent the distribution of prodrug and parent drug within the tumor 1 hour and 4 hours after NAC stimulation at 12-hour and 24-hour intervals, respectively.

[0045] ns: P≥0.05; P < 0.05; P < 0.01 (all two-tailed t-tests) Figure 13 This diagram illustrates the in vivo antitumor activity of the paclitaxel-dodecanoic acid prodrug liposomes of Example 11 of this invention. In the diagram, a represents the tumor volume change during the in vivo antitumor experiment; b represents the tumor burden during the in vivo antitumor experiment; c represents the tumor image during the in vivo antitumor experiment; and d represents the body weight change during the in vivo antitumor experiment.

[0046] ns: P≥0.05; P < 0.05; P < 0.01; P<0.001; P < 0.0001 (all two-tailed t-tests) Figure 14 The pharmacokinetic behavior of the paclitaxel-dodecanoic acid prodrug liposome in Example 12 of this invention under the regulation of the "blocking then dispersing" strategy, and the blood concentration-time curve of the prodrug and parent drug.

[0047] Figure 15 This diagram illustrates the tissue distribution of the paclitaxel-ticodone prodrug liposomes of Example 12 of this invention under the regulation of a "block-then-release" strategy. Specifically, a, b, and c represent the drug-time curves and AUC analyses of the paclitaxel-ticodone prodrug liposomes in the liver, spleen, and tumor with and without the "block-then-release" strategy, respectively; d represents the accumulation of the prodrug and parent drug within the tumor at various time points with and without the "block-then-release" strategy; e represents the drug-time curve and AUC analysis of the release of the parent drug from the tumor with and without the "block-then-release" strategy; and f represents the ratio of tumor accumulation to intratumoral release AUC with and without the "block-then-release" strategy.

[0048] ns: P≥0.05; P < 0.05; P<0.001; P < 0.0001 (all two-tailed t-tests) Detailed Implementation The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the invention, but do not limit the invention in any way.

[0049] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0050] Example 1 Synthesis of paclitaxel-dodecyl alcohol prodrug Weigh 1.45 g (8 mmol) of 2,2'-dithiodiacetic acid into a round-bottom flask, then add 10 mL of acetic anhydride to dissolve it completely, and stir at room temperature for 2 h. Add 30 mL of toluene to the system in three portions, and dry the mixture under reduced pressure. Dissolve the resulting product in 30 mL of dichloromethane, and add 4 mmol of 11-hexaenoic acid and DMAP (85.4 mg, 0.7 mmol). Stir the reaction under nitrogen protection at room temperature. Monitor the reaction process by thin-layer chromatography, and purify the intermediate product, hexaenoic acid-dithiodiacetic acid monoester, by silica gel column chromatography. The intermediate (0.3 mmol), EDCI (115 mg, 0.6 mmol), HOBT (63.6 mg, 0.3 mmol), and DMAP (14.7 mg, 0.12 mmol) were dissolved in 100 mL of dichloromethane and activated in an ice bath for 2 h. Paclitaxel (213.48 mg, 0.25 mmol) was then added, and the mixture was stirred for another 36 h under nitrogen protection at room temperature. The reaction was monitored by thin-layer chromatography. The target product was purified by preparative liquid chromatography to obtain the paclitaxel-cobalamin prodrug.

[0051] The molecular weight of the product was confirmed by mass spectrometry, and the mass spectrum is shown below. Figure 1 As shown.

[0052] use 1 H-NMR confirmed the structure of the product. Figure 2 The spectral analysis results are as follows: 1 H NMR (600 MHz, Chloroform- d, δ): 8.15 (d, J = 7.7 Hz, 2H, Ar-H), 7.77(d, J = 7.7 Hz, 2H, Ar-H), 7.61 (t, J = 7.4 Hz, 1H, Ar-H), 7.52 (t, J = 7.7 Hz, 2H,Ar-H), 7.50-7.36 (m, 8H, Ar-H, -CONH-), 7.32 (t, J = 7.1 Hz, 1H, Ar-H), 6.29(s, 1H, 10-H), 6.26 (t, J = 8.9 Hz, 1H, 2'-H), 6.01 (dd, J = 9.2, 3.4 Hz, 1H, 3'-H), 5.69 (d, J= 7.1 Hz, 1H, 13-H), 5.51 (d, J = 3.4 Hz, 1H, -OH), 4.98 (d, J = 9.5Hz, 1H, 2-H), 4.90-4.83 (m, 1H, -COO CH -), 4.45 (dd, J = 11.0, 6.6 Hz, 1H, 5-H),4.32 (d, J = 8.6 Hz, 1H, 20α-H), 4.21 (d, J = 8.6 Hz, 1H, 20β-H), 3.82 (d, J = 7.0Hz, 1H, 7-H), 3.65-3.53 (m, 4H, -OC CH 2SS CH 2CO-), 2.84 (s, 1H, -OH), 2.57 (td, J = 9.7, 5.1 Hz, 1H, 3-H), 2.46 (s, 3H, -OCO CH 3), 2.36 (dd, J = 15.5, 9.3 Hz, 1H,14α-H), 2.23 (s, 3H, -COCH3), 2.14 (dd, J = 15.4, 8.8 Hz, 1H, 14β-H), 1.94 (s,3H, 18- CH 3), 1.89 (t, J = 13.1 Hz, 1H, 6α-H), 1.68 (s, 3H, 19- CH 3), 1.56-1.51(m, 5H, 6β-H, -COOCH(CH2)2-), 1.27-1.22 (m, 35H, 16- CH 3, -CH2 16), 1.13 (s,3H, 17- CH 3), 0.88-0.86 (m, 6H, -CH2 CH 3 2). The purity of the product was determined using high-performance liquid chromatography (HPLC), and the purity chromatogram is shown below. Figure 3 As shown.

[0053] Example 2 Preparation of paclitaxel-dodecyl alcohol prodrug liposomes Accurately weigh paclitaxel-dodecyl alcohol prodrug (2.66 mg), HSPC (17.46 mg), cholesterol (1.11 mg), and DSPE-mPEG. 2K (8.03 mg) was fully dissolved in 10 mL of chloroform, and the chloroform was removed under reduced pressure using a rotary evaporator to obtain a thin film. Deionized water was preheated to 60 °C and added to a round-bottom flask. Hydration was carried out at 60 °C for 30 min to obtain crude paclitaxel liposomes. After treatment with an ultrasonic cell disruptor (150 W, 5 min), paclitaxel-cobalamin prodrug liposomes were obtained. As shown in Table 1, the particle size was 101.50 nm, the PDI was 0.19, and the encapsulation efficiency was 99.69%.

[0054] Table 1. Particle size, particle size distribution, zeta potential, encapsulation efficiency, and drug loading of paclitaxel-dodecanoic acid prodrug liposomes

[0055] Example 3 Preparation of blank liposomes (MPS blocker) Accurately weigh HSPC (69.84 mg), cholesterol (4.44 mg), and DSPE-mPEG. 2K (32.12 mg) was fully dissolved in 10 mL of chloroform, and the chloroform was removed under reduced pressure using a rotary evaporator to obtain a thin film. Deionized water was preheated to 60°C and added to a gai-shaped flask. Hydration was carried out at 60°C for 30 min to obtain crude blank liposomes. These crude liposomes were then treated with an ultrasonic cell disruptor under different ultrasonic conditions (150 W, 0-5 min) to obtain blank liposomes of different particle sizes, as shown in Table 2, with particle sizes of approximately 200 nm and 400 nm, respectively. After one month of storage at 4°C, the particle size of the blank liposomes did not change significantly. However, in 10% FBS and rat plasma stability tests, the particle size of the larger blank liposomes increased significantly, indicating instability. Figure 4 ).

[0056] Table 2. Particle size, particle size distribution, and zeta potential of blank liposome blockers with different particle sizes

[0057] Example 4 Pharmacokinetic study of paclitaxel-dodecyl alcohol prodrug liposomes Twelve male SD rats (180-220 g) were randomly divided into three groups. Rats were fasted for 12 hours before drug administration but had free access to water. The three groups of rats were intravenously injected with paclitaxel solution, paclitaxel albumin nanoparticles, and the paclitaxel-dodecanoic acid prodrug liposomes prepared in Example 3, respectively, at a dose of 5 mg / kg (equivalent to the paclitaxel dose). Blood was collected from the orbital sinus at specified time points, and plasma was obtained by centrifugation. The drug concentration in the plasma was determined by liquid chromatography-mass spectrometry.

[0058] Experimental results are as follows Figure 5 As shown, paclitaxel solution has a short half-life, and the paclitaxel in it is rapidly eliminated from the bloodstream. In contrast, the circulation time of paclitaxel-dodecanoate prodrug liposomes is significantly prolonged, with a significantly longer total drug-time area under the curve (AUC). 0-24 h The concentrations of paclitaxel solution and paclitaxel albumin nanoparticles were 100 and 150 times higher, respectively (Table 3). Meanwhile, the paclitaxel-dodecanoic acid prodrug liposomes exhibited a significantly increased maximum drug concentration (C0). max Its clearance rate (Cl) was only 1% of that of paclitaxel solution. These results provide a prerequisite for the efficient delivery of paclitaxel to tumor sites. Furthermore, during blood circulation, the paclitaxel-dodecanoate prodrug liposomes release relatively little paclitaxel, indicating good stability. Figure 6 ).

[0059] Table 3. Pharmacokinetic parameters of paclitaxel-dodecanoic acid prodrug liposomes

[0060] Example 5 Effect of MPS blockade on the in vivo biodistribution of paclitaxel-dodecanoate prodrug liposomes 4T1 cell suspension was inoculated into BALB / c mice. When the tumor volume reached 150-200 mm, the cells were allowed to grow. 3 At that time, tumor-bearing mice were randomly divided into the following four groups (n = 3) and subjected to corresponding interventions: (1) Control group G1: Paclitaxel-dodecyl alcohol prodrug liposomes prepared in Example 2 were injected via tail vein; (2) Standard blocking group G2: Small particle size blank liposomes (50 μmol HSPC / kg, 200 nm) prepared in Example 3 were injected via tail vein, followed by injection of paclitaxel-dodecyl alcohol prodrug liposomes 1.5 h later; (3) Large particle size blocking group G3: Large particle size blank liposomes (50 μmol HSPC / kg, 400 nm) prepared in Example 3 were injected via tail vein, followed by injection of paclitaxel-dodecyl alcohol prodrug liposomes 1.5 h later; (4) High-dose blocking group G4: Small particle size blank liposomes (800 μmol HSPC / kg, 200 nm) prepared in Example 3 were injected via tail vein, followed by injection of paclitaxel-dodecyl alcohol prodrug liposomes 1.5 h later; The dosage of paclitaxel was uniformly 11.7 μmol / kg in all groups. Mice were sacrificed at 1, 4, 8, 12 and 24 h after administration, and major organs (heart, liver, spleen, lung, kidney) and tumors were isolated. The drug concentration in each tissue was determined by liquid chromatography-mass spectrometry.

[0061] The results are as follows Figure 7 The results showed that, compared with the untreated control group, all pretreatment groups (G2-G4) significantly reduced the distribution of paclitaxel-cobalamin prodrug liposomes in key MPS organs, such as the liver, spleen, and the elimination organ, the kidney, with corresponding organ-drug-time area under the curve (AUC) significantly lower than the control group. Simultaneously, pretreatment significantly promoted its accumulation in tumor tissue. Among them, group G3 (large particle size, low dose) exhibited the strongest tumor-targeting accumulation ability, with its intratumoral AUC significantly higher than that of groups G2 and G4. The AUC ratio of tumor tissue to MPS tissue was calculated. 瘤 / AUC MPS The higher the ratio, the stronger the ability of the nanomedicine to evade MPS capture and accumulate in the tumor. Group G3 had the highest targeting ratio, reaching 0.248. In contrast, despite a higher lipid dose, group G4 did not show a significant improvement in targeting efficacy compared to group G2. These results indicate that, among the factors examined, the particle size of the blocker is the key factor affecting MPS saturation and tumor targeting efficiency, rather than simply relying on increasing the dose.

[0062] Example 6 Effects of MPS blockade on the pharmacokinetics of paclitaxel-dopechol prodrug liposomes (1) In the tumor-bearing mouse model described in Example 5, blood was collected from the orbital cavity at 1, 4, 8, 12 and 24 h after administration, and the drug concentration in the plasma was determined by LC-MS / MS. The drug concentration in the plasma was determined by liquid chromatography-mass spectrometry.

[0063] (2) Eight male SD rats (180-220 g) were randomly divided into two groups. Before administration, the rats were fasted for 12 h but allowed free access to water. The experimental group received a pre-treatment injection via tail vein into large-particle-size blank liposomes (50 μmol HSPC / kg, 400 nm) selected in Example 5, followed by a paclitaxel-dodecanoate prodrug liposome injection 1.5 h later at a dose of 5 mg / kg (equivalent to paclitaxel). The control group received no pretreatment but received a direct tail vein injection of the same dose of prodrug liposomes. Blood was collected from the orbital cavity at specified time points, and plasma was obtained by centrifugation. The drug concentration in the plasma was determined by liquid chromatography-mass spectrometry.

[0064] Experimental results are as follows Figure 8-9 As shown in Example 5, although the distribution of nanomedicines in tissues was significantly altered in the blood of tumor-bearing mice, no significant difference in drug concentration was observed in the blood of each group. In a healthy rat model, compared with the control group, the experimental group pretreated with blank liposomes showed a 1.6-fold increase in the total AUC of paclitaxel-dodecanoate prodrug liposomes, along with a higher peak concentration and a lower clearance rate. The experiments demonstrated that the MPS blocking strategy could prolong the systemic circulation time of nanomedicines in tumor-free models (Table 3); in tumor-bearing models, this effect was further manifested in promoting the redistribution of drugs from the circulatory system to tumor tissues, thereby significantly enhancing drug accumulation at the tumor site without significantly affecting systemic exposure levels (Table 4).

[0065] Table 3. Pharmacokinetic parameters of paclitaxel-dodecanoate prodrug liposomes before and after MPS blockade in tumor-free rats

[0066] Table 4. Pharmacokinetic parameters of paclitaxel-dodecanoate prodrug liposomes before and after MPS blockade in tumor-bearing mice.

[0067] Example 7 Validation of the effect of MPS blockade on paclitaxel-dodecanoate prodrug liposomes in cells (1) Uptake of coumarin-6 liposomes in a monolayer cell culture model Flow cytometry was used to determine the uptake of self-assembled nanoparticles of small molecule prodrugs in RAW 264.7 macrophages and mouse breast cancer (4T1) cells. RAW 264.7 and 4T1 cells were cultured at 2 × 10⁻⁶ cells per cell line. 5 cells / well and 1×10 5Cells were seeded at different densities into 12-well plates and incubated for 24 h to allow for cell adhesion. After cell adhesion, cells were pretreated with blank liposomes of different concentrations (0.1-2.0 mM) and particle sizes (200 nm or 400 nm) for 1.5 h, followed by incubation at 37°C for 1 h or 12 h with coumarin-6-labeled liposomes (250 ng / mL). The control group received only coumarin-6-labeled liposomes. After incubation, cells were washed, collected, and dispersed in PBS. Flow cytometry was used to assess cell uptake of coumarin-6 liposomes.

[0068] (2) Uptake of coumarin-6 liposomes in the Transwell co-culture model An MPS model was established using the Transwell system. Cells were cultured in 12-well Transwell plates separated by 0.4 μm porous membranes, with 4 T1 cells (1 × 10⁻⁶) seeded in the lower chamber. 5 RAW 264.7 cells (5 × 10⁻⁶ cells) were seeded in the upper chamber. 4 Cells). After 24 hours, blank liposomes under different conditions were added to the upper chamber for pretreatment, followed by the addition of coumarin-6-labeled liposomes 1.5 hours later. The group without any pretreatment served as a control. After co-culturing for another 12 hours, cells from both the upper and lower chambers were collected, washed with PBS, and their uptake of fluorescently labeled liposomes was analyzed by flow cytometry.

[0069] The results are as follows Figure 10 As shown, in macrophages, the blocking effect of blank liposomes exhibited a dose- and particle size-dependent pattern: the blocking efficiency increased with increasing dose, reaching saturation at ≥1 mM; at the same dose, large-diameter blank liposomes (400 nm) induced a more significant and sustained phagocytic inhibition effect. In 4T1 tumor cells, the addition of blank liposomes also caused a dose-dependent decrease in coumarin-6 liposome uptake, with a blocking efficiency exceeding 30% at doses ≥1 mM, indicating that high-dose blank liposomes also blocked tumor cells. Furthermore, unlike the particle size-dependent trend observed in macrophages, small-diameter liposomes were more easily internalized by tumor cells and induced stronger blocking, while large-diameter liposomes had a weak effect on tumor cell uptake behavior. These phenomena were further validated in a co-culture model. Although high-dose inhibitors effectively inhibited macrophage uptake, they also led to a significant decrease in fluorescence signals in tumor cells; while treatment with low-dose, large-diameter inhibitors selectively inhibited macrophage uptake while minimizing the impact on tumor cells, resulting in the highest accumulated fluorescence levels in tumor cells. The results of the cell and in vivo tissue distribution experiments in Example 5 are consistent with the results of the experiments, indicating that low-dose, large-particle-size blank liposomes can achieve selective MPS blockade, enhancing tumor accumulation while minimizing interference with tumor cell uptake.

[0070] Example 8 cytotoxicity analysis of paclitaxel-dodecanoate prodrug liposomes and after supplementation with exogenous stimulants (1) Toxicity evaluation of N-acetylcysteine ​​(NAC) combined with prodrug liposomes To investigate the universality of the described exogenous stimulation strategy in different tumor cell types, the MTT assay was used to examine the toxicity of NAC combined with paclitaxel-dodecanoate prodrug liposomes to mouse breast cancer (4T1) cells and human non-small cell lung cancer (A549) cells. 4T1 cells and A549 cells were cultured at 2 × 10⁻⁶ cells per cell line. 3 Cells were seeded into 96-well plates and incubated for 12 hours to allow them to adhere. After cell adhesion, paclitaxel solution and the paclitaxel-dodecyl alcohol prodrug liposomes prepared in Example 2 were added. The plates were then serially diluted with cell culture medium, with 200 μL of the test solution added to each well, and three parallel wells were used for each concentration. Four hours after drug addition, NAC was serially diluted with cell culture medium, with 50 μL of the test solution added to each well, and three parallel wells were used for each concentration. After 48 hours, the 96-well plates were removed, the old culture medium was discarded, and 135 μL of MTT solution diluted with fresh culture medium (100 μL fresh culture medium + 35 μL 5 mg / mL MTT solution) was added to each well. The plates were incubated for 4 hours, the culture medium was discarded, and the 96-well plates were inverted on filter paper to thoroughly absorb any remaining liquid. Then, 200 μL of DMSO was added to each well and the plates were shaken for 10 minutes to dissolve the blue-purple crystals. The absorbance was measured at 490 nm using an ELISA reader.

[0071] (2) Comparison of the toxicity of different stimulants combined with prodrug liposomes to 4T1 cells To compare the effects of different exogenous stimulants in combination therapy, the above method was used to compare the toxicity of NAC and reduced glutathione (GSH) combined with paclitaxel-dodecyl alcohol prodrug liposomes on 4T1 cells. The cell treatment procedure was the same as (1), except that gradient dilutions of NAC or GSH were added 4 h after drug administration, and the subsequent steps were the same.

[0072] As shown in Tables 5, 6, and 7, the cytotoxicity of the paclitaxel-cobalamin prodrug liposomes in Example 2 was weaker than that of the paclitaxel solution because the prodrug requires a smart drug release process to exert its effect in cells. As shown in Tables 2-4, the addition of a reducing agent accelerated the drug release rate, leading to enhanced cytotoxicity of the paclitaxel-cobalamin prodrug liposomes. Specifically, cytotoxicity gradually increased with increasing NAC concentration, reaching its peak at molar ratios of 1:200 and 1:500. However, when the reducing agent ratio increased to 1:1000, the cytotoxicity of the prodrug liposomes decreased. Therefore, 1:200 and 1:500 are the optimal ratios for combining the reducing agent with the paclitaxel-cobalamin prodrug liposomes. These trends were consistently observed in 4T1 and A549 cells, supporting broad applicability to various tumor types. Furthermore, GSH also exhibited a consistent synergistic effect, indicating that this method has broader applicability and application potential.

[0073] Table 5. Half-maximal inhibitory concentrations (IC50) of paclitaxel-dodecanoic acid prodrug liposomes and paclitaxel solution combined with NAC in 4T1 cells. 50 (nmol / L)

[0074] Table 6. Half-maximal inhibitory concentrations (IC50) of paclitaxel-dodecanoic acid prodrug liposomes and paclitaxel solution combined with NAC in A549 cells. 50 (nmol / L)

[0075] Table 7. Half-maximal inhibitory concentrations (IC50) of paclitaxel-dodecanoic acid prodrug liposomes and paclitaxel solution combined with GSH in 4T1 cells. 50 (nmol / L)

[0076] Example 9 Paclitaxel-dodecanoate prodrug liposomes supplement in vitro release following exogenous stimulation. 200 nmol of the paclitaxel-dodecanoic acid prodrug liposome prepared in Example 2 was added to 30 mL of PBS (pH 7.4) containing 30% ethanol (v / v). To investigate its reduction-responsive release behavior, different amounts of NAC were added to the medium, with the molar ratio of prodrug to NAC ranging from 1:0 to 1:1000. Each sample was placed in a 37°C constant-temperature shaker, and samples were taken at predetermined time points.

[0077] The results are as follows Figure 11As shown, paclitaxel-dodecanoate prodrug liposomes all exhibited significant stimulus-responsive drug release behavior in the presence of NAC. NAC significantly promoted drug release in a molar ratio range of 1:200 to 1:500. When the molar ratio was increased to 1:1000, the drug release rate was inhibited, and the cumulative release was lower than that of the control group without the addition of reducing agent. This release behavior corresponds to the cytotoxic trend described in Example 8.

[0078] Example 10 Effects of exogenous stimulants on the biodistribution of paclitaxel-dodecanoate prodrug liposomes in vivo 4T1 cell suspension was inoculated into BALB / c mice. When the tumor volume reached 150-200 mm, the cells were allowed to grow. 3 At that time, tumor-bearing mice were randomly divided into the following three groups (n = 3) and subjected to corresponding interventions: (1) Control group: only the same dose of paclitaxel-dodecyl alcohol prodrug liposomes were injected via the tail vein, without any stimulation treatment; (2) 24 h stimulation group: Paclitaxel-dodecanoic acid prodrug liposomes were injected via tail vein, followed by NAC injection via tail vein 24 h later; (3) 12 h stimulation group: Paclitaxel-dodecyl alcohol prodrug liposome was injected into the tail vein, and NAC was injected into the tail vein 12 h later; The stimulation time points (12 h and 24 h) were set based on the peak window of intratumoral drug accumulation determined by the in vivo biodistribution in Example 5. Figure 7 e). The dosage of paclitaxel was uniformly 11.7 μmol / kg in all groups. Mice were sacrificed 1 h and 4 h after NAC administration, tumor tissue was dissected, and the drug concentration in the tumor was determined by liquid chromatography-mass spectrometry.

[0079] The results are as follows Figure 12 As shown, supplementing with NAC 12 h or 24 h after injection of paclitaxel-dodecanoate prodrug liposomes effectively promoted the activation of the prodrug in tumor tissue. The intervention strategy of administering NAC 24 h later was more effective, inducing a 25%-30% increase in paclitaxel release compared to the 12 h group, and achieving 2.4 times the intratumoral drug activation efficiency of the control group without NAC supplementation. This indicates that prolonging the intratumoral accumulation time is beneficial for subsequent drug release efficiency.

[0080] Example 11 In vivo pharmacodynamic evaluation of the "block first, then divert" sequential strategy 4T1 cells (3 × 10⁻⁶) were subcutaneously injected into the right back of female BALB / c mice. 6 (One tumor per animal) was used to establish a xenograft model. The tumor was allowed to grow to approximately 100 mm in volume. 3Mice bearing tumors were randomly divided into 10 groups (n = 5). These groups included a saline group, a standard paclitaxel injection group, an albumin-bound paclitaxel group, an MPS blocking combination group, a prodrug liposome monotherapy group and its combination with MPS blocking and exogenous stimulation, respectively, as well as a complete "block-then-drain" standard group (large particle size blocker) and a control group (small particle size blocker). The standard blocker was a 400 nm blank liposome, and the small particle size blocker was a 200 nm blank liposome. The dose of both blockers was 50 μmol HSPC / kg. All groups were administered intravenously in the following sequence, twice a week for a total of four times: first, the blocker or saline was injected via tail vein; 1.5 h later, paclitaxel-dodecanoate prodrug liposomes, paclitaxel injection, or paclitaxel albumin nanoparticles (11.7 μmol / kg) were injected; and 24 h later, NAC (2340 μmol / kg) or saline was injected. Tumor volume (long axis) of the tumor-bearing mice was measured and recorded daily. Short diameter The tumor volume and body weight were plotted as a function of the minor axis ( / 2) and body weight, and curves showing the changes in tumor volume and body weight over time were generated. After the experiment, the mice were euthanized, the tumor tissue was dissected, and the tumor bearing rate (tumor weight / mouse body weight) was calculated. 100%).

[0081] The results are as follows Figure 13 As shown, the paclitaxel-dodecanoate prodrug liposome monotherapy group has demonstrated tumor-suppressive effects comparable to clinical formulations paclitaxel solution and paclitaxel albumin nanoparticles. Both MPS blockade and NAC stimulation alone effectively enhanced the efficacy of the prodrug liposomes, reducing tumor burden by 1.45-fold and 1.53-fold, respectively, compared to the monotherapy groups, with no statistically significant difference between the two. This result confirms that accumulation and release are two independent and key tunable processes. The crucial synergistic effect was evident in the "block-then-release" combined intervention group, which exhibited the strongest tumor-suppressive effect, reducing tumor burden by 2.46-fold and inducing the strongest apoptosis and proliferation inhibition. Its effect was significantly superior to either single intervention, demonstrating the necessity and superiority of the sequential synergistic strategy. Furthermore, while the efficacy of the combination group using small-particle-size blockers was better than that of the prodrug liposome monotherapy, it was lower than that of the standard combination group. This is consistent with the superior mechanism of large-particle-size blockers in MPS blockade revealed in Examples 5 and 7, further confirming the importance of rational blocker design. Meanwhile, the MPS blocking strategy also improved the efficacy of paclitaxel albumin nanoparticles, which are also nanomedicines, but had no effect on solution-form paclitaxel injections, indicating that this strategy is applicable to nanomedicines that are easily cleared by MPS and has a certain degree of universality.

[0082] Example 12 The "block-then-redirect" sequential strategy for reconstructing the in vivo fate of paclitaxel-dodecanoate prodrug liposomes (1) Tissue distribution: 4T1 tumor-bearing BALB / c mice (tumor volume 150-200 mm) were used. 3 Mice were randomly assigned to groups (n = 3). The experimental group received intravenous injections following a "block-then-reactive" strategy: a standard blocker (400 nm blank liposomes, 50 μmol HSPC / kg), followed by paclitaxel-dodecanoate prodrug liposomes (11.7 μmol paclitaxel / kg, denoted as T0) 1.5 h later, and NAC (2340 μmol / kg) 24 h after T0. The control group received only paclitaxel-dodecanoate prodrug liposomes, with saline used instead of either the blocker or NAC. Mice were sacrificed at 8, 12, 25, 28, and 32 h after T0, and major organs (heart, liver, spleen, lung, kidney) and tumors were isolated. Drug concentrations in each tissue were determined using liquid chromatography-mass spectrometry.

[0083] (2) Pharmacokinetic study: Male SD rats (180-200 g, n = 4) were divided into two groups. The experimental group received the above-mentioned blocking agent 1.5 h before intravenous injection of paclitaxel-dococol prodrug liposome (5.9 μmol paclitaxel / kg), and NAC (1170 μmol / kg) 24 h later; the control group received only paclitaxel-dococol prodrug liposome. Blood was collected at predetermined time points, plasma was separated, and samples were processed and analyzed by UPLC-MS / MS according to the established procedure.

[0084] Pharmacokinetic and tissue distribution results are as follows Figure 14 and Figure 15 As shown, under the "block-then-release" strategy, the in vivo fate of the paclitaxel-dodecanoate prodrug liposomes was effectively regulated. MPS blockade significantly prolonged the systemic circulation time of the prodrug liposomes, with its area under the plasma concentration-time curve (AUC) increasing by approximately two times compared to the control group. Subsequent administration of NAC did not cause significant fluctuations in plasma free paclitaxel levels, indicating that this strategy effectively avoided systemic premature drug release. Benefiting from prolonged blood circulation, the accumulation of the prodrug in tumor tissue increased by approximately 1.43 times, and its retention in organs of the mononuclear phagocyte system was significantly reduced, with a tumor-targeted AUC ratio increasing by 2.40 times. Furthermore, the prodrug accumulated in the tumor efficiently released the active ingredient under NAC triggering, manifested as a sustained increase in intratumoral paclitaxel concentration and a total release increase of approximately 2.14 times. These results confirm that the "block-then-release" strategy, through temporal regulation, synergistically enhances the tumor-targeted accumulation and release of nanomedicines, thereby significantly improving therapeutic efficacy.

Claims

1. A drug combination product for enhancing the therapeutic effect of nanomedicine, characterized in that: The drug combination product comprises the following (1), (2) and (3): (1) Stimulus-responsive nanomedicines; (2) Mononuclear phagocyte system blockers; (3) Exogenous reducing substances; The stimulus-responsive nanomedicine is a nanoformulation formed by the self-assembly of a prodrug formed by coupling an active drug molecule with a fatty alcohol via a disulfide bond or by assembly with a pharmaceutically acceptable nanocarrier excipient; the mononuclear phagocyte system blocker is one or more of phospholipid liposomes or phospholipid liposomes with chemically modified surfaces; the exogenous reducing substance is one or more of vitamin C, cysteine, N-acetylcysteine, reduced glutathione, or lipoic acid.

2. The pharmaceutical combination product according to claim 1, characterized in that: (1), (2) and (3) exist in a form that is isolated from each other.

3. The pharmaceutical combination product according to claim 1, characterized in that: The drug combination product can be administered simultaneously or sequentially.

4. The pharmaceutical combination product according to claim 3, characterized in that: The sequential administration order is as follows: first, administer a mononuclear phagocyte system blocker; second, administer a stimulus-responsive nanomedicine; and finally, administer an exogenous reducing agent.

5. The pharmaceutical combination product according to claim 4, characterized in that: The mononuclear phagocyte system blocker was administered 0.5–3 h later, followed by the administration of a stimulus-responsive nanomedicine, and then an exogenous reducing agent was administered 12–24 h after the administration of the nanomedicine.

6. The pharmaceutical combination product according to claim 1, characterized in that: The dosage of the stimulus-responsive nanomedicine is 1-20 μmol / kg based on the active drug concentration; the dosage of the mononuclear phagocyte system blocker is 20-1000 μmol / kg based on the phospholipid concentration; and the dosage of the exogenous reducing agent is 200-10000 μmol / kg.

7. The pharmaceutical combination product according to claim 1, characterized in that: The stimulus-responsive nanomedicine is a nanoformulation formed by thin-film hydration and ultrasonic treatment of a prodrug, phospholipids, cholesterol, and a PEG modifier. The average particle size of the nanoformulation is 50-200 nm. The structural formula of the prodrug is shown below: 。 8. The pharmaceutical combination product according to claim 1, characterized in that: The average particle size of the mononuclear phagocyte system blocker is 50-500 nm; the surface is chemically modified with polyethylene glycol and / or has a ligand attached to a macrophage receptor; the receptor is one or more of mannose receptor, scavenger receptor or immunoglobulin Fc receptor.

9. The use of a combination of stimulus-responsive nanomedicine, mononuclear phagocyte system blocker and exogenous reducing agent in the preparation of a pharmaceutical combination product according to any one of claims 1 to 8 that enhances the therapeutic effect of nanomedicine.

10. The application according to claim 9, characterized in that: The enhanced nanomedicine therapeutic effect is to enhance the anti-tumor effect; the tumor is one or more of breast cancer, lung cancer, prostate cancer, ovarian cancer, or melanoma.

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