Solamargine and osimertinib co-loaded liposome nano preparation as well as preparation and application thereof
By preparing a liposome nanoformation co-loaded with solanine and osimertinib, the problem of osimertinib resistance in NSCLC was solved, achieving effective treatment and tumor-targeted delivery for drug-resistant lung cancer, while reducing drug toxicity.
Patent Information
- Application Number
- CN202511827685.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-05
- Publication Date
- 2026-02-27
AI Technical Summary
In the current technology, osimertinib has drug resistance problems when treating non-small cell lung cancer (NSCLC), especially EGFR-dependent resistance caused by C797S mutation and other complex non-EGFR-dependent resistance mechanisms, and there is a lack of effective treatment methods.
We developed a liposomal nanoformulation co-loaded with lysodium tretinoin and osimertinib. By combining lysodium tretinoin and osimertinib in a specific molar ratio, we prepared a nanoscale liposomal nanoformulation. By utilizing their tumor targeting and synergistic effects, we reversed the osimertinib resistance effect.
It significantly improved the treatment efficacy for drug-resistant lung cancer, enhanced the tumor targeting of the drug, reduced toxic side effects, and reversed osimertinib resistance, providing a new treatment approach.
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Figure CN121570484A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of medicine, and particularly relates to an australine and osimertinib co-loaded liposome nano-preparation as well as preparation and application thereof. BACKGROUND
[0002] Lung cancer has high morbidity and mortality, especially non-small cell lung cancer (NSCLC). At the same time, lung cancer faces challenges such as low early diagnosis rate and drug resistance to targeted therapy, and has low 5-year survival rate.
[0003] With the development of precision medicine, EGFR-TKI targeted drugs have become the standard treatment for patients with EGFR mutation-positive NSCLC, significantly prolonging the survival of patients. However, the problem of EGFR-TKI drug resistance seriously restricts the clinical efficacy.
[0004] Osimertinib, as the third-generation EGFR-TKI, has become the first-line treatment choice for patients with EGFR mutation-positive NSCLC, but its drug resistance problem has become a major bottleneck in clinical treatment. The mechanism of osimertinib resistance is complex and diverse, mainly including EGFR-dependent resistance and non-EGFR-dependent resistance.
[0005] In EGFR-dependent resistance, C797S mutation is the most common acquired drug resistance mutation, accounting for about 15-40% of drug-resistant patients, which causes osimertinib to be unable to effectively bind to the EGFR kinase domain; the mechanism of non-EGFR-dependent resistance is more complex, including bypass signal activation (such as MET amplification accounting for about 15-20%, HER2 amplification accounting for about 5-10%), abnormal downstream signaling pathways (such as KRAS mutation, BRAF mutation, etc.), histological transformation (about 5-15% of patients transform into small cell lung cancer), and epigenetic changes. It is worth noting that about 30-40% of osimertinib-resistant patients have not found a clear mechanism of drug resistance, which brings great challenges to clinical treatment.
[0006] Australine (australine) is a natural steroidal alkaloid with significant anticancer potential, and important progress has been made in its antitumor mechanism and application research in recent years.
[0007] Nanomaterials are a new class of functional materials with nanoscale characteristics. Their unique properties stem from a special size range between macroscopic matter and microscopic atoms. These materials break through the limitations of traditional materials in terms of structure, exhibiting many surprising properties. Nanomaterials show significant application prospects in the field of tumor therapy: as intelligent delivery systems, they can simultaneously encapsulate osimertinib and other adjuvant drugs, overcoming multiple drug resistance mechanisms through synergistic effects; after surface modification, they can achieve tumor-targeted delivery, improving drug bioavailability and reducing toxic side effects; multifunctional nanoprobes can monitor treatment response in real time and provide early warning of drug resistance; novel nanosensors help reveal unknown drug resistance mechanisms, providing a basis for precision treatment. These characteristics make nanotechnology a new strategy for overcoming the bottleneck of osimertinib resistance.
[0008] Currently, there are no reports on liposomal nanoformations of Australian solanine or liposomal nanoformations co-loaded with Australian solanine and osimertinib. Summary of the Invention
[0009] The purpose of this invention is to provide a co-loaded liposome nanoformulation of solanine and osimertinib, and its preparation and application.
[0010] The objective of this invention is achieved through the following technical solution: The liposome nanoformulation of solanine and osimertinib co-loaded contains solanine (SM) and osimertinib (OS) in a molar ratio of 1:(0.25~4.34). The dosage of solanine is 0.5~32 μM.
[0011] For sensitive lung cancer, the nano-formulation preferably contains a combination of solanine and osimertinib in a molar ratio of 1:4, which provides a synergistic effect in treatment. When the molar ratio of solanine to osimertinib is 1:4, the dose of solanine is 0.5~4 μM. For drug-resistant lung cancer, the nano-formulation preferably contains a combination of solanine and osimertinib in a molar ratio of 1:(0.25~1) or 1:(4~4.34), which can achieve a synergistic effect in treatment. More preferably, the composition comprises solanine and osimertinib in a molar ratio of 1:0.25, 1:0.5, 1:1, 1:4 or 1:4.34; When the molar ratio of solanine to osimertinib is 1:0.25, the dose of solanine is 0.5~2.0 μM. When the molar ratio of solanine to osimertinib is 1:0.5, the dose of solanine is 1 or 16 μM. The dosage of solasodine is 0.5-1 or 16 μM when the molar ratio of solasodine to osimertinib is 1:1; The dosage of solasodine is 0.5-16 μM, preferably 0.5-8 μM when the molar ratio of solasodine to osimertinib is 1:4; The dosage of solasodine is 2 μM when the molar ratio of solasodine to osimertinib is 1:4.34; For experimental animals, the nano-preparation preferably contains drugs consisting of solasodine and osimertinib at a molar ratio of 1:4.34, under which ratio, a synergistic effect can be achieved in treatment; The dosage of solasodine is 3 mg / kg when the molar ratio of solasodine to osimertinib is 1:4.34, and the drug is administered continuously for 20 days.
[0012] The mass ratio of the membrane material to the drug in the nano-preparation is (10-15):1, preferably 13.6:1; The membrane material preferably consists of egg yolk lecithin, cholesterol and polyethylene glycol; Further preferably, the membrane material consists of egg yolk lecithin, cholesterol and polyethylene glycol at a mass ratio of (12-18):1:1, preferably at a mass ratio of 15:1:1.
[0013] The preparation method of the solasodine and osimertinib co-loaded liposome nano-preparation comprises the following steps: (1) Dissolve the membrane material and the drug in organic solvents respectively, mix the obtained membrane material solution and drug solution, and remove the organic solvent by distillation under reduced pressure to form a layer of white film; (2) Add a hydrating agent to the product of step (1) and shake to obtain the solasodine and osimertinib co-loaded liposome nano-preparation; The organic solvent in step (1) is preferably chloroform and / or methanol; The shaking in step (2) can be rotary shaking and / or ultrasonic.
[0014] The solasodine and osimertinib co-loaded liposome nano-preparation can be used for preparing a drug for treating lung cancer.
[0015] The lung cancer includes sensitive lung cancer and drug-resistant lung cancer; The drug-resistant lung cancer particularly refers to osimertinib-resistant lung cancer; The sensitive lung cancer particularly refers to H1975 cell strains and PC9 cell strains; The drug-resistant lung cancer particularly refers to H1975OR cell strains and PC9OR cell strains.
[0016] The present application has the following advantages and effects over the prior art: 1. The present application first uses solasodine combined with osimertinib to treat lung cancer, and unexpectedly finds that the two have obvious synergistic anticancer effects at multiple ratios and multiple doses; and this coordination effect is more sufficient on drug-resistant lung cancer cells, bringing a new way for the treatment of drug-resistant lung cancer in clinic.
[0017] 2. The present application first prepares a solasodine and osimertinib co-loaded liposome nanofomulation, which has significant tumor targeting, can better accumulate in tumor tissue sites, and play an anti-tumor growth effect, and to some extent, reduce the toxic side effects of free drugs; at the same time, the nanofomulation can significantly reverse the osimertinib resistance effect of lung cancer. BRIEF DESCRIPTION OF DRAWINGS
[0018] Figure 1 is the best combined ratio of solasodine and osimertinib for inhibiting H1975 OR cells; Figure 2 is the best combined ratio of solasodine and osimertinib for inhibiting PC9 OR cells; Figure 3 is the 1:4 combined ratio of solasodine and osimertinib for synergistically inhibiting the growth of H1975 and PC9 cells; wherein, when solasodine or osimertinib is used alone, the dose on the horizontal axis is the respective dose; when the two drugs are used in combination, the corresponding horizontal axis is the dose of solasodine.
[0019] Figure 4 is the appearance, particle size, zeta potential and stability detection of S / O NPs nanofomulation.
[0020] Figure 5 is the in vivo distribution and tumor targeting of S / O NPs nanofomulation.
[0021] Figure 6 is that S / O NPs nanofomulation significantly inhibits tumor cell growth.
[0022] Figure 7 is that S / O NPs nanofomulation significantly promotes tumor cell apoptosis.
[0023] Figure 8 is that S / O NPs significantly reverse the osimertinib resistance of lung cancer.
[0024] Figure 9 is that S / O NPs significantly inhibit the growth of lung cancer xenografts. DETAILED DESCRIPTION
[0025] The present application will be further described in detail below in conjunction with the examples and drawings, but the embodiments of the present application are not limited thereto.
[0026] Example 1 Exploring the optimal combination ratio of solasodine and osimertinib to inhibit the growth of H1975 OR cells H1975 OR cells were treated with solasodine (SM) and osimertinib (OS) at different molar ratios (SM:OS at 1:0.25, 1:0.5, 1:1, 1:2 and 1:4) for 24 hours, and CCK8 assay was used to detect the growth of cells.
[0027] It was found that the growth inhibition of H1975 OR cells by solasodine and osimertinib was dose-dependent (P<0.05) Figure 1 ).
[0028] Subsequently, the CompuSyn software was used to calculate the synergy index (CI), and CI value <1 indicated that the two drugs had a synergistic effect. The results showed that at the combination ratio of 1:4, 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM and 16 μM of solasodine combined with 2 μM, 4 μM, 8 μM, 16 μM, 32 μM and 64 μM of osimertinib had a significant synergistic anticancer effect, and the CI combination index was less than 1. Among them, the combination effect of solasodine at 4 μM was the strongest, as shown in Tables 1-5.
[0029] At the combination ratio of 1:0.25, 0.5 μM, 1 μM and 2 μM of solasodine combined with 0.125 μM, 0.25 μM and 0.5 μM of osimertinib also had a significant synergistic anticancer effect.
[0030] Table 1: Combination index analysis when SM:OS is 1:0.25
[0031] Table 2: Combination index analysis when SM:OS is 1:0.5
[0032] Table 3: Combination index analysis when SM:OS is 1:1
[0033] Table 4: Combination index analysis when SM:OS is 1:2
[0034] Table 5: Combination index analysis when SM:OS is 1:4
[0035] Example 2 Exploring the optimal combination ratio of solasodine and osimertinib to inhibit the growth of PC9 OR cells PC9OR cells were treated with solamargine and osimertinib at different ratios (SM:OS 1:0.25, 1:0.5, 1:1, 1:2 and 1:4) for 24 hours, and CCK8 assay was used to detect the growth of cells. The results showed that the growth inhibition of PC9OR cells by solamargine and osimertinib was dose-dependent (Fig. 1). Figure 2 ).
[0036] The CompuSyn software was used to calculate the synergy index (CI). The results showed that at the ratio of 1:4, 0.5 μM, 1 μM, 2 μM, 4 μM and 8 μM of solamargine combined with 2 μM, 4 μM, 8 μM, 16 μM and 32 μM of osimertinib respectively had significant synergistic anticancer effect, and the CI combination index was less than 1. Among them, the combination of 4 μM of solamargine was the strongest, as shown in Tables 6-10.
[0037] At the ratios of 1:0.25, 1:0.5 and 1:1, some dose combinations also showed significant synergistic anticancer effect.
[0038] Table 6: Combination index analysis when SM:OS is 1:0.25
[0039] Table 7: Combination index analysis when SM:OS is 1:0.5
[0040] Table 8: Combination index analysis when SM:OS is 1:1
[0041] Table 9: Combination index analysis when SM:OS is 1:2
[0042] Table 10: Combination index analysis when SM:OS is 1:4
[0043] Example 3 Optimal combination ratio of solamargine and osimertinib for combination inhibition effect on osimertinib-sensitive cell lines From the experimental results of Examples 1 and 2, the optimal synergistic anticancer effect of solamargine and osimertinib was achieved at the ratio of 1:4. In order to verify whether this combination ratio is applicable to sensitive cell lines (H1975 cells and PC9 cells), the following experiments were conducted to explore: H1975 and PC9 cells were treated with solamargine and osimertinib alone or in a ratio of 1:4 for 24 hours, and CCK8 assay was used to detect the growth of cells.
[0044] Results showed that solamargine and osimertinib had obvious growth inhibition effect on H1975 and PC9 cells. And solamargine and osimertinib in a ratio of 1:4 (molar ratio) had significant synergistic anticancer effect, as shown below. Figure 3
[0045] The CompuSyn software was used to calculate the synergistic index (CI). Results showed that solamargine at 0.5 μM, 1 μM, 2 μM and 4 μM combined with osimertinib at 2 μM, 4 μM, 8 μM and 16 μM respectively had significant synergistic anticancer effect in a ratio of 1:4, and the CI combination index was less than 1. In H1975 cells, the strongest combination effect was observed when solamargine was at 4 μM, as shown in Table 11 below. In PC9 cells, the strongest combination effect was observed when solamargine was at 2 μM, as shown in Table 12 below.
[0046] Table 11: Analysis of the combination index of SM to OS at a ratio of 1:4 on H1975 cells
[0047] Table 12: Analysis of the combination index of SM to OS at a ratio of 1:4 on PC9 cells
[0048] Example 4 The preparation of solamargine and osimertinib co-loaded liposome nanofomulation included the following steps: (1) Take 30 mg of egg yolk lecithin, dissolve in 3 mL of chloroform; take 2 mg of cholesterol, dissolve in 2 mL of chloroform; take 2 mg of polyethylene glycol, dissolve in 2 mL of chloroform; take 0.76 mg of solamargine, dissolve in 760 μL of methanol; take 1.74 mg of osimertinib, dissolve in 1.74 mL of methanol; place the above components in an ultrasonic instrument for ultrasonic assisted dissolution; (2) Turn on the condenser and rotary evaporator, set the condensation temperature to 4°C and the heating temperature to 60°C; (3) Prepare a clean and dry 50 mL flask, and after the instrument temperature reaches the required temperature, pour the above solution into the flask and mix well, then fix the flask on the rotary evaporator; (4) Turn on the rotary evaporator, set the air pressure to about 168 mbar and the rotation speed to 3-4, and rotary evaporate for 5 minutes.
[0049] (5) After the rotary evaporation, a thin white film will be formed at the bottom of the flask. At this time, turn off the water bath and the condenser, and add 1.5 mL of pure water into the flask.
[0050] (6) Run the flask empty, and set the speed to 3 for 15 mins.
[0051] (7) Collect the solution into a 2 mL centrifuge tube, and use the insert ultrasonic instrument to ultrasonicate for 3 minutes, with the power set to 20% (total power of the instrument is 900 W), and ultrasonic for 3 s and stop for 3 s. The obtained solution is the S / O NPs nano-preparation.
[0052] Example 5 Characterization of the S / O NPs nano-preparation (1) Particle size and stability detection of the S / O NPs nano-preparation The S / O NPs nano-preparation prepared in Example 3 was detected for characterization. Transmission electron microscopy was used to detect the morphology, a laser particle size analyzer was used to detect the particle size and potential. The S / O NPs nano-particle suspension was placed in a 4°C refrigerator, or in a PBS solution containing 10% fetal bovine serum, and at a predetermined time point, the sample was taken out, the particle size change of the nano-particle was detected, and the stability was evaluated.
[0053] The results show that the S / O NPs nano-preparation is a spherical structure with smooth surface ( Figure 4 A), the particle size is about 91.09 nm ( Figure 4 B), and the charge is positive (+30.68) ( Figure 4 C). The stability detection results ( Figure 4 D) show that the S / O NPs nano-preparation maintains a relatively stable particle size within 1-7 days, is uniformly dispersed (PDI is about 0.0518), and has good stability and dispersity.
[0054] (2) In vivo distribution and tumor targeting of the S / O NPs nano-preparation 100 μL of free Cy5.5 and Cy5.5-labeled S / O NPs liposome nanoparticles were respectively injected into tumor-bearing mice (1x10 6 LLC cells were inoculated subcutaneously in mice, and a transplanted tumor mouse model was established 5-7 days later) in vivo, and the fluorescence signal in the mice was detected by a small animal live imaging system, as shown in Figure 5 A-B. At 8 h after injection, the nanoparticles were obviously accumulated in the tumor tissue, and at 12 h, the fluorescence signal was the strongest in the tumor site. The fluorescence signal of free Cy5.5 was the strongest at 4 h, and then gradually weakened. At 8 h, 12 h and 24 h, the fluorescence signal of the Cy5.5-labeled S / O NPs nanoparticles in the tumor site was stronger than that of the free Cy5.5.
[0055] Subsequently, the fluorescence intensity of heart, liver, spleen, lung and kidney was semi-quantitatively analyzed, and the fluorescence signal intensity of the two groups of mice in the above organs had no statistical difference, as shown in the following Figure 5 C-D.
[0056] The results of ex vivo experiments after 24h showed that the average fluorescence signal of Cy5.5-labeled S / O NPs nanoparticles at the tumor site was about 2.8 times that of free Cy5.5, as shown in the following Figure 5 E-F.
[0057] After preparing the tumor tissue into a single cell suspension, the fluorescence signal intensity was quantitatively analyzed using a flow cytometer, and the results were consistent with the semi-quantitative analysis of the in vivo imaging system, as shown in the following Figure 5 G-H.
[0058] Example 6 S / O NPs has better anti-cancer effect than free single drug (1) S / O NPs has better inhibitory effect on cell growth than free drug alone and combination H1975OR and PC9OR cells were treated with free solasodine (2 μM) and osimertinib (8.68 μM), combination (SM 2 μM+OS 8.68 μM), and S / O NPs nanoforum (containing SM 2 μM, OS 8.68 μM), respectively, and CCK8 was used to detect cell growth.
[0059] The results showed that the anti-cancer effect of S / O NPs nanoforum was significantly better than that of solasodine and osimertinib free drugs alone and their combination, as shown in the following Figure 6
[0060] (2) S / O NPs has better induction effect on cell apoptosis than free drug alone and combination The dosages of step (2) were the same as those of step (1); H1975OR and PC9OR cells were treated with free solasodine and osimertinib, combination, and S / O NPs nanoforum for 24 hours, and flow cytometry was used to detect cell apoptosis. The results showed that the anti-cancer effect of S / O NPs nanoforum was significantly better than that of solasodine and osimertinib free drugs alone and their combination, as shown in the following Figure 7
[0061] Example 7 S / O NPs significantly reverses lung cancer osimertinib resistance H1975OR cells and PC9OR cells were treated with S / O NPs (containing SM 2 μM and OS 8.68 μM) and soly-marine (SM) (dose 2 μM) for 2 weeks, respectively. During the treatment, the growth of the cells was observed, and the cells were passaged and the fresh culture medium was replaced in time. After 2 weeks, the H1975OR-S / O NPs pretreated cells and PC9OR-S / O NPs pretreated cells were obtained, and the H1975OR-SM pretreated cells and PC9OR-SM pretreated cells were obtained.
[0062] Subsequently, the above cell lines and the parent H1975OR and PC9OR cells were treated with osimertinib, and the half maximal inhibitory concentration (IC50) of osimertinib on each cell line was detected.
[0063] The results showed that the IC50 value of osimertinib on the cell lines pretreated with S / O NPs and soly-marine was significantly reduced, and the pretreatment with S / O NPs had a better effect than the pretreatment with soly-marine, as shown in the following table. Figure 8
[0064] In summary, S / O NPs can significantly reverse the osimertinib resistance effect of lung cancer, and the reversing effect is better than that of soly-marine alone.
[0065] Example 8 S / O NPs significantly inhibit the growth of lung cancer xenografts Construction of mouse lung cancer xenograft model: mouse lung cancer cells (Lewis Lung Carcinoma Cells, LLC, 1x10 6 The mice were inoculated with the lung cancer cells subcutaneously in the armpit, and obvious tumor tissues were observed 5-7 days later, indicating that the xenograft model was successfully constructed.
[0066] Soly-marine (SM, 3 mg / kg), osimertinib (OS, 7.5 mg / kg) and S / O NPs (containing 3 mg / kg of SM and 7.5 mg / kg of OS) were administered for 20 days, and all were administered by tail vein injection.
[0067] The results showed that soly-marine and osimertinib could inhibit the growth of the xenografts to a certain extent, including the tumor volume and tumor weight, as shown in the following table. Figure 9 A-C. Soly-marine combined with osimertinib had a certain synergistic effect, as shown in the following table. Figure 9 A-C.
[0068] More importantly, S / O NPs showed stronger tumor growth inhibition effect compared with solanigrine and osimertinib alone or in combination, as shown below Figure 9 A-C.
[0069] In addition, the results showed that the body weight of mice in each group had no significant difference, indicating that the drugs had no obvious toxic side effects. Even the body weight of mice in the S / O NPs treatment group increased slightly, indicating that S / O NPs had the advantage of less toxicity and effectively improved the survival of mice, as shown below Figure 9 D-E.
[0070] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.
Claims
1. A liposome nanoformulation co-loaded with solanine and osimertinib, characterized in that... The drug consists of solanine and osimertinib in a molar ratio of 1:(0.25~4.34).
2. The nano-formulation according to claim 1, characterized in that: The dosage of the solanine is 0.5~32 μM.
3. The nano-formulation according to claim 1, characterized in that: For sensitive lung cancer, the nanoformulation contains a drug consisting of solanine and osimertinib in a molar ratio of 1:
4.
4. The nano-formulation according to claim 3, characterized in that: When the molar ratio of lysimachia alafenamide to osimertinib is 1:4, the dose of lysimachia alafenamide is 0.5~4 μM.
5. The nano-formulation according to claim 1, characterized in that: For drug-resistant lung cancer, the nano-formulation contains a drug composed of solanine and osimertinib in a molar ratio of 1:(0.25~1) or 1:(4~4.34).
6. The nano-formulation according to claim 1, characterized in that: The composition consists of solanine and osimertinib in a molar ratio of 1:0.25, 1:0.5, 1:1, 1:4 or 1:4.
34.
7. The nano-formulation according to claim 6, characterized in that: When the molar ratio of solanine to osimertinib is 1:0.25, the dose of solanine is 0.5~2.0 μM; When the molar ratio of solanine to osimertinib is 1:0.5, the dose of solanine is 1 or 16 μM. When the molar ratio of solanine to osimertinib is 1:1, the dose of solanine is 0.5-1 or 16 μM. When the molar ratio of solanine to osimertinib is 1:4, the dosage of solanine is 0.5~16 μM, preferably 0.5~8 μM; When the molar ratio of lysimachia alafenamide to osimertinib is 1:4.34, the dose of lysimachia alafenamide is 2 μM.
8. The nano-formulation according to claim 1, characterized in that: The nano-formulation wherein the mass ratio of membrane material to drug is (10~15):
1.
9. The method for preparing the liposome nanoformation co-loaded with solanine and osimertinib according to any one of claims 1 to 8, characterized in that... Includes the following steps: (1) Dissolve the membrane material and the drug separately with an organic solvent. After mixing the resulting membrane material solution and drug solution, remove the organic solvent by vacuum distillation to form a white thin film. (2) Add a hydrating agent to the product of step (1) and shake to obtain a co-loaded liposome nanoformulation of lycopene and osimertinib.
10. The use of the liposome nanoformulation of solanine and osimertinib co-loaded according to any one of claims 1 to 8 in the preparation of a drug for treating lung cancer.