Interferon exosome composition, preparation as well as preparation method and application of interferon exosome composition

Abstract

The invention belongs to the technical field of medicines, and particularly discloses an interferon exosome composition, a preparation as well as a preparation method and application of the interferon exosome composition. The composition provided by the invention comprises interferon and exosome, wherein the dosage ratio of the interferon to the exosome is (50-300): (5-30). The composition has the technical effects that 1) by limiting the dosage ratio of the interferon to the exosome, the melanoma treatment effect of the composition is remarkably improved and is superior to the treatment effect of independently using the exosome and the interferon and the treatment effect of combining the interferon with the PD-1 monoclonal antibody; and 2) the preparation prepared from the composition has good physical, chemical and biological stability, the stability of the interferon and the exosome in the preparation is greatly improved through the synergistic cooperation of various auxiliary materials, so that the same volume can bear a higher dosage of the composition, the total injection amount of a patient is effectively reduced, and the injection cost is reduced. Therefore, the pain of a patient in the injection process is remarkably relieved, and the treatment compliance of the patient is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an interferon exosome composition, a preparation thereof, a preparation method and an application thereof. Background Art

[0002] Malignant melanoma is a tumor with a relatively high degree of malignancy, with a relatively low incidence rate but a relatively high fatality rate. The incidence rate of melanoma in China is nearly 0.9 per 100,000, and about 20,000 new melanoma patients are added every year, and the incidence rate is increasing year by year.

[0003] After interferon α (IFNα) entered clinical practice, a large number of clinical studies have shown that the median survival period of high-risk melanoma can be increased from more than 2 years to more than 3 years, and the 5-year survival rate can be increased from more than 30% to more than 40%. The IFNαs applied to the treatment of melanoma mainly include interferon α-1b (IFNα-1b) and interferon α-2b (IFNα-2b). It has been found that IFNα-1b is significantly superior to IFNα-2b in many aspects such as directly killing tumor cells. The 5-year survival rate of melanoma patients treated with IFNα-1b reaches 68.7%. It is publicly reported in the literature that the 5-year survival rate of 287 patients treated with IFNα-2b abroad is 46%. High-dose IFNα is the only drug approved by the US FDA for adjuvant treatment of high-risk melanoma, while IFNα-2b is difficult to benefit in clinical studies of low-risk and advanced melanoma due to its large side effects. IFNα-1b is a first-class new drug in China. It can not only be used for adjuvant treatment of stage II and III high-risk melanoma, but also has good curative effects on stage IV melanoma. The adverse reactions are much lower than those of IFNα-2b, and the tolerated dose of patients is at least 5 times that of IFNα-2b.

[0004] The inhibitory effect of IFNα-1b on melanoma cell proliferation is significantly dose-dependent. When treating melanoma, it is usually necessary to administer subcutaneous injection or intramuscular injection at a high dose (such as 300 μg / time or 600 μg / time) at the affected area of the patient. The injection volume is large. Since the dose that each injection site can withstand is limited, multiple injections are often required, the injection time is long, the affected area of the patient has severe pain, and the tolerance and compliance are extremely poor.

[0005] In addition, although the efficacy of IFNα-1b alone is superior to that of IFNα-2b alone, it is far from solving the clinical treatment problems of melanoma. Therefore, a combined treatment strategy based on IFNα-1b is formulated to explore its combined treatment regimens with programmed death ligand 1 inhibitors, targeted drugs, angiogenesis inhibitors, etc., which is bringing hope for saving melanoma patients. Some studies have shown that the combination of IFNα-1b and anti-PD-1 monoclonal antibody in the treatment of advanced unresectable melanoma has better efficacy and good safety than the use of either alone. However, there is still room for further improvement in the development of the combination of IFNα-1b and other drugs. Summary of the Invention

[0006] To solve the above problems, the present invention provides an interferon-exosome composition, a preparation thereof, and a preparation method and application for treating melanoma.

[0007] Among them, the composition for treating melanoma includes interferon and exosomes, and the dosage ratio of the interferon to the exosomes is 50-300:5-30.

[0008] Preferably, the exosomes are preferably NK cell-derived exosomes.

[0009] Furthermore, the interferon is interferon α-1b.

[0010] Furthermore, the dosage ratio of the interferon to the exosomes is 100-200:10-20.

[0011] The present invention forms a unique composition by formulating interferon and exosomes in a specific ratio. Experimental studies have shown that this composition exhibits a significant synergistic effect in the treatment of melanoma, can significantly reduce the tumor growth rate of mice, and its efficacy is not only significantly higher than the single therapy of using exosomes or interferon alone, but also exceeds the compositions prepared from the two in other ratios and the treatment regimens of anti-PD-1 monoclonal antibody and the combination of anti-PD-1 monoclonal antibody and interferon. This breakthrough result comes from the multi-dimensional synergistic effect of exosomes and interferon in the tumor microenvironment and the direct action of the effector molecules carried by exosomes themselves on tumor cells, forming a "two-pronged" targeted attack; at the same time, interferon activates immune cells, and exosomes further enhance the activity and infiltration of immune cells, forming a positive feedback loop of "interferon → immune activation → exosome-enhanced immunity → tumor killing", synergistically inhibiting the proliferation of tumor cells and inducing their apoptosis. Exosomes can also inhibit the expression of immune checkpoint molecules and reverse tumor immune escape. Finally, interferon and exosomes also reduce the possibility of tumor cells developing drug resistance through a single pathway through the synergistic effect of different mechanisms.

[0012] Among them, each 1000 mL of the interferon-exosome preparation contains the following components: Interferon: 50mg-300mg; Exosomes: 5mg-30mg; Osmotic pressure regulator: 5g-10g; pH adjuster: 1g-2g; Stabilizer: 10g-30g; The balance is sterile water for injection.

[0013] Furthermore, the weight of the stabilizer in each 1000 mL of the interferon exosome preparation is: 15g-25g.

[0014] Furthermore, the preparation is an injection.

[0015] Compared with the existing interferon aqueous solution preparations, the interferon content contained in each 1000 mL of the preparation of the present invention is significantly increased, that is, under the premise of the same interferon dosage, the corresponding volume of the preparation provided by the present invention is significantly reduced, which can significantly reduce the injection dose in clinical application, relieve the patient's pain and improve compliance.

[0016] Furthermore, the stabilizer includes human albumin, polyethylene glycol, polysorbate and sodium sulfite in a mass ratio of 10-20:1-5:1-1.5:0.5-1.

[0017] After experimental analysis, it was found that the stability of the preparations prepared by using any one of the above stabilizers alone or in combination, or in combination with other stabilizer ingredients, was far inferior to the preparations prepared by using the four ingredients in the above proportions as stabilizers. This shows that compounding the above four ingredients in a specific proportion can significantly improve the stability of the preparation.

[0018] Working principle: Human albumin, with its special molecular structure, tightly binds to specific sites on the surface of interferon and exosomes, forming a protective "barrier" on the surface of both. It can not only effectively reduce the nonspecific binding of interferon and exosomes with other substances in the surrounding environment, but also significantly reduce the impact of physical forces such as surface tension in the solution on proteins and exosomes. For example, it can buffer the induction of surface tension on the aggregation tendency of proteins and prevent protein molecules from approaching each other to form aggregates. At the same time, it can also protect the membrane structure of exosomes and prevent them from rupturing or deforming under the action of various forces, thereby maintaining the structural integrity and functional stability of interferon and exosomes. Human albumin has good compatibility with excipients such as polysorbate 80. Together, they construct a stable colloidal environment, which can further enhance the protective effect on proteins and exosomes, and effectively prevent them from denaturing or aggregating during the production, storage and use of the preparation.

[0019] Polyethylene glycol 8000 has a large steric hindrance effect, which enables it to form a steric hindrance around interferon and exosomes, preventing them from approaching and aggregating with each other. When the solution flows or is affected by other external factors, polyethylene glycol 8000 can adjust the viscosity of the solution, reducing the damage to the structures of interferon and exosomes caused by the shear force generated by the solution flow. It can also interact with interferon molecules, stabilizing the native conformation of interferon to a certain extent, preventing its unfolding, and maintaining the biological activity of the protein. Polyethylene glycol 8000 can work in concert with osmotic pressure regulators to jointly regulate the physical properties of the solution, making the overall environment of the preparation more suitable for the stable existence of interferon and exosomes.

[0020] As a non-ionic surfactant, polysorbate 80 has a unique amphiphilic structure and can adsorb on the surfaces of interferon and exosomes. By reducing the surface tension, it effectively prevents the hydrophobic regions of protein molecules from being exposed in the solution, avoiding protein aggregation caused by hydrophobic interactions. Polysorbate 80 is crucial for maintaining the integrity of exosomes. It can prevent the fusion and rupture of exosome membranes, ensuring that exosomes can maintain their normal structures and functions. Polysorbate 80 works closely with components such as human serum albumin to jointly prevent interferon and exosomes from being damaged due to interfacial phenomena during production, storage, and use.

[0021] As an antioxidant, sodium sulfite plays a key antioxidant role in the preparation. It can rapidly consume the oxygen in the preparation through its own oxidation-reduction reaction, thereby reducing the oxidation-reduction potential in the solution. It can effectively prevent the oxidation of easily oxidizable components in interferon and exosomes, avoiding the structural changes of bioactive components and the loss of biological activity caused by oxidation reactions. Sodium sulfite and components such as polyethylene glycol 8000 jointly create a stable chemical environment. It can not only prevent oxidation but also reduce a series of problems such as protein aggregation that may be caused by oxidation, comprehensively enhancing the stability of the entire preparation and ensuring that the preparation maintains good quality and efficacy within the validity period.

[0022] Furthermore, the stabilizer includes human serum albumin, polyethylene glycol, polysorbate, and sodium sulfite with a mass ratio of 12 - 15:3 - 5:1.1 - 1.3:0.6 - 0.8.

[0023] Furthermore, the stabilizer includes human serum albumin, polyethylene glycol, polysorbate, and sodium sulfite with a mass ratio of 15:5:1.3:0.8.

[0024] Furthermore, the stabilizer includes human serum albumin, polyethylene glycol, polysorbate, and sodium sulfite with a mass ratio of 12:3:1.1:0.6.

[0025] After experimental verification, the preparation obtained by adding the stabilizer in the above ratio has good stability. When the dosage of each component is further increased, the improvement in stability is relatively slow. Considering the stability gain and production cost comprehensively, the above stabilizer formulation and dosage are more optimal.

[0026] Furthermore, the preparation method of the NK-derived exosomes is as follows: 1) Collect peripheral blood from healthy individuals. Use lymphocyte separation medium and collect peripheral blood mononuclear cells (PBMC) by density gradient centrifugation; 2) Incubate the peripheral blood mononuclear cells with CD56 antibody, and then collect NK cells using magnetic bead separation; 3) Culture the cells using NK serum-free medium. Add IL-12 and IL-15 to the medium. When culturing to the fourth day, supplement the NK serum-free medium containing IL-12 and IL-15; 4) Collect the culture supernatant after 7 days of culture. Centrifuge at 300g for 10 min to remove NK cells, and take the supernatant; 5) Filter the centrifuged supernatant through a hollow fiber column, and then perform ultra-high speed centrifugation. The centrifugation conditions are 4°C, 100,000×g, 60 min. After centrifugation, collect the precipitate, and the obtained precipitate is the exosomes.

[0027] The second aspect of the present invention also provides a preparation method of an interferon exosome preparation, which specifically includes the following steps: a) Take a pH regulator and an osmotic pressure regulator according to the formula, add them to sterile injection water and mix evenly. After sterilization, cool and reserve to obtain a first mixed solution; b) Add the stabilizer in the formula amount to the first mixed solution and stir evenly to obtain a second mixed solution; c) Add the interferon in the formula amount to the second mixed solution and stir evenly to obtain a third mixed solution; d) Add the exosomes in the formula amount to the third mixed solution and stir at a low speed to obtain the interferon exosome preparation.

[0028] Among them, the stirring speed after adding exosomes ≤ 50 rpm, and the time is 1 - 5 min. The exosomes can be either commercially available products or self-made exosomes.

[0029] The third aspect of the present invention also provides the use of the composition or preparation in the preparation of a drug for treating melanoma.

[0030] The fourth aspect of the present invention also provides a drug prepared using the above composition or preparation.

[0031] In summary, the present invention includes at least one of the following beneficial technical effects:

[0032] 1) Synergistic therapeutic advantages: By formulating interferon and exosomes in a specific ratio to form a unique composition, experimental studies have shown that this composition exhibits significant synergistic effects in melanoma treatment. Its efficacy is not only significantly higher than that of single therapies using exosomes, interferon, or PD-1 alone, but also superior to the combination therapy of interferon and anti-PD-1 monoclonal antibody; 2) The preparation of the present invention has achieved a double breakthrough in drug stability and patient experience through scientific optimization of the excipient formula. The composite stabilizer system used in the formula plays a key role: human albumin can effectively inhibit protein aggregation and damage to the exosome membrane structure, and together with other excipients, form a protective colloidal environment, effectively preventing the denaturation or aggregation of exosomes and interferon during the production, storage, and use of the preparation; polyethylene glycol maintains the natural conformation of interferon by means of steric hindrance; polysorbate 80 acts as a surfactant to reduce the surface tension; sodium sulfite protects the active ingredients through antioxidant action. This synergistic effect enables the preparation to maintain more than 90% of the interferon activity after 6 months in the accelerated stability test at 40°C, with no significant changes in the morphology and particle size distribution of exosomes. On this basis, the drug loading capacity per unit volume of the preparation has increased by more than 5 times, and the single injection volume has been reduced to 1 / 5 of the traditional therapy, significantly reducing the pain caused by multiple-point and multiple injections for patients, and significantly improving patient tolerance.

[0033] 3) While maintaining high drug stability and bioavailability, the present invention significantly reduces the excipient cost. Compared with the existing interferon α-1b injection, the present invention uses less excipients, which reduces the procurement cost to a certain extent and significantly improves the drug accessibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0035] Figure 1 It is the electron micrograph of exosomes prepared by the method provided by the present invention; Figure 2 It is the Western blot analysis of the expression of CD81, TGS101, and GAPDH in NK-derived exosomes (left: NK-derived exosomes prepared in Example 1, right: commercially available NK-derived exosomes); Figure 3 It is the change trend of tumor volume in each group before and after administration in each example and control example; Figure 4 It is the tumor growth rate before and after administration in each example and control example. Detailed implementation mode

[0036] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention. For those not specified in the examples regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through regular channels.

[0037] Example 1 Preparation of NK-derived exosomes 1) Collect peripheral blood from healthy individuals. Use lymphocyte separation medium and collect peripheral blood mononuclear cells (PBMC) by density gradient centrifugation, and count them using a cell counter; 2) Resuspend the cells with buffer according to the instruction manual of the NK cell separation kit. Calculate the volume of buffer to be added according to adding 40 μL of buffer for every 1×10 7 . Add 10 μL of CD56 antibody for every 1×10 7 cells, mix well and incubate at 2 - 8°C for 5 min; then add 30 μL of buffer for every 1×10 7 cells; add the magnetic bead mixture and incubate at 2 - 8°C for 10 min, and collect CD56 + NK cells by magnetic bead separation method. After elution, NK cells with a purity > 90% are obtained; 3) Culture the cells using NK serum-free medium, and add cytokines IL-12 (200 IU / mL) and IL-15 (50 ng / mL) to the medium. When culturing to the fourth day, supplement the NK serum-free medium containing IL-12 and IL-15; 4) Collect the culture supernatant after culturing for 7 days, and remove NK cells by low-speed centrifugation. The centrifugation parameters are: centrifugal force 300g, centrifugation time 10 min. Remove NK cells and take the supernatant; 5) Filter the supernatant prepared in step 4) using a hollow fiber column with a membrane pore size of 0.65 μm by ultrafiltration concentration method, take the filtrate, then ultrafiltrate and concentrate the filtrate using a hollow fiber column with a molecular cut-off of 500 KD, replace it with physiological saline, and perform sterile filtration. Take the supernatant to obtain the ultrafiltrate; 6) Centrifuge the ultrafiltrate prepared in step 5) at high speed. The centrifugation conditions are: 4°C, 100000g, 60 min. After centrifugation, collect the precipitate to obtain NK-derived exosomes.

[0038] 7) Observe the size and structure of the collected exosomes using an electron microscope, and identify the exosomes using western Blot.

[0039] Interferon exosome compositions of Examples 2-6 and Control Examples 1-4 The components and dosages of the compositions in Examples 2-6 and Control Examples 1-4 are shown in Table 1. Among them, the interferon is interferon α-1b stock solution (from Beijing Sanyuan Gene Pharmaceutical Co., Ltd.), and the exosomes are NK cell-derived exosomes prepared in Example 1.

[0040] Table 1. Components and dosages of the compositions of Examples 2-6 and Control Examples 1-4

[0041] Interferon exosome preparations of Examples 7-10 and Control Examples 5-13 The dosages of each component in the preparations of Examples 7-10 and Control Examples 5-13 are shown in Table 2. Among them, the interferon is interferon α-1b stock solution (from Beijing Sanyuan Gene Pharmaceutical Co., Ltd.), the polyethylene glycol is polyethylene glycol 8000, the polysorbate is polysorbate 80, and the balance is sterile injection water. The preparation method is as follows: a) Take the pH regulator and osmotic pressure regulator according to the formula, add them to sterile injection water, mix well, sterilize and cool for later use to obtain the first mixed solution; b) Add the formulated amount of stabilizer to the first mixed solution and stir evenly to obtain the second mixed solution; c) Add the formulated amount of interferon α-1b to the second mixed solution and stir evenly to obtain the third mixed solution; d) Add the formulated amount of exosomes to the third mixed solution and stir at a stirring speed of 50 rpm for 5 min to obtain the interferon exosome preparation.

[0042] Table 2. Components and dosages of each component in the preparations of Examples 7-10 and Control Examples 5-13

[0043] Test Example 1 Characterization of NK cell-derived exosomes As Figure 1 - Figure 2 shown, observation of the collected exosomes by electron microscope showed that the exosomes had a cup-shaped or spherical vesicle structure, with a diameter between 50 - 130 nm, and had a typical lipid bilayer structure. Identification of NK-derived exosomes by WesternBlot showed CD81 + TGS101 + .

[0044] Test Example 2 Therapeutic effects of each group of compositions on melanoma Model construction and drug administration: B16-F10 cells in the logarithmic growth phase were collected, resuspended and counted with normal saline after washing with PBS, and the cell density was adjusted to 1×10 7 cells / mL. They were inoculated intradermally into the back of C57BL / 6 mice at a dose of 1*10 6 cells / mouse (100 μL per mouse), and a total of 90 mice were inoculated, with an equal number of males and females. From the 3rd day after inoculation of tumor cells, the major axis (L, mm) and minor axis (W, mm) of the tumor were measured with a vernier caliper every 2-3 days, and the tumor volume was calculated. The formula for calculating the tumor volume was L×W 2 / 2. On the 7th day after tumor inoculation, 72 mice with successful modeling were selected and evenly divided into 12 groups according to the tumor growth volume and mouse body weight, with 6 mice in each group and an equal number of males and females. The experiments were set up as a normal saline control group, Example 2-6 groups, interferon + PD-1 monoclonal antibody group, PD-1 monoclonal antibody group, and Control Example 1-4 groups. Before drug administration every week after grouping, the mice were weighed and the drug dosage was calculated. The drug administration volume for each animal was 50 μL. The formula for calculating the drug administration volume for each example, control example, and normal saline group was: 4 μL×mouse body weight (g) / 20 g (equivalent to 1 mL per human use per time). If it was less than 50 μL, the volume was made up to 50 μL with normal saline. Each example, control example, and normal saline group were given intratumoral injection at the tumor site, twice a week for a total of four weeks. In the PD-1 monoclonal antibody group and interferon + PD-1 monoclonal antibody group (PD-1 monoclonal antibody and interferon were administered separately), RMP1-14 was used for the PD-1 monoclonal antibody. The drug concentration of the PD-1 monoclonal antibody was 20 mg / mL, and the administration dose was 36 mg / kg. The administration volume of the PD-1 monoclonal antibody: administration dose×body weight (kg) / drug concentration. If it was less than 50 μL, the volume was made up to 50 μL with normal saline. The PD-1 monoclonal antibody group adopted the administration method of tail vein injection, once every 2 weeks for a total of 3 times (W0, W2, W4); interferon α-1b stock solution was used for interferon. The drug concentration was 400 μg / mL, and the administration dose was 80 μg / kg. The administration volume of interferon: administration dose×body weight (kg) / drug concentration. If it was less than 50 μL, the volume was made up to 50 μL with normal saline. Interferon was given intratumorally at the tumor site, twice a week for a total of four weeks. During drug administration, the tumor volume of each group of mice was measured and calculated before drug administration every week. The results of the volume growth trend of melanoma and the tumor growth rate of each group of drugs are as Figure 3 - 4 shown.

[0045] by Figure 3 - 4It can be seen that the composition obtained by the ratio of interferon and exosomes defined in this application has a significantly higher therapeutic effect on melanoma than the drugs in each control example. It is proved that the composition obtained by the ratio of interferon and exosomes defined in the present invention has a significant synergistic effect and has an unexpected effect on the treatment of melanoma.

[0046] Test Example 3 Quality inspection and test results of interferon exosome preparations in Examples 7-10 and Control Examples 5-13

[0047] 3.1 Drug stability detection Accelerated stability test: Place the preparations in each group at 25 °C for 6 months, and detect the following physical properties and drug activity indicators at 0 month, 3 months, and 6 months respectively. The evaluation criterion is that the changes in each index should be within an acceptable range. If obvious changes occur, it indicates that the stability of the formulation is poor under accelerated conditions and further investigation is required.

[0048] 1) Physical property detection Appearance: Observe the color and clarity of the preparation under natural light. The evaluation criterion is that the color should be uniform and clear. If turbidity or color change occurs, the formulation can be excluded.

[0049] 2) pH value determination: Use a pH meter to measure the pH value of the preparation. The evaluation criterion is within the pH range of 4 - 9. Deviation from this range may affect the drug stability and safety, and adjustment or exclusion is required.

[0050] 3) Osmolality determination: Use an osmometer to measure the osmolality of the preparation. The evaluation criterion is that it should conform to the range of human physiological osmolality (about 280 - 320 mOsm / L). Too high or too low osmolality may cause adverse effects on the body, and formulations that do not meet the requirements need to be further optimized or discarded.

[0051] 4) Interferon activity and exosome morphology detection Interferon activity detection: Use the enzyme-linked immunosorbent assay (ELISA) method to regularly detect the activity of interferon α-1b. The evaluation criterion is that within the specified storage period, the decline in the activity of interferon α-1b should not exceed 10%. Preparations with too rapid decline in activity have poor stability.

[0052] Exosome morphology and particle size detection: Transmission electron microscopy (TEM) was used to observe the morphology of exosomes. The evaluation criteria were that exosomes should exhibit a typical cup-shaped or spherical vesicle structure, and the particle size distribution should be relatively concentrated and within the expected range. If the exosome morphology changes significantly or the particle size distribution is abnormal, it may affect its function and efficacy. A laser particle size analyzer was used to detect the exosome particle size distribution. If the proportion of exosomes in the particle size range of 80 nm - 120 nm reached more than 70%, the exosomes were evaluated as having a concentrated particle size.

[0053] 3.2 Biocompatibility and safety detection Cytotoxicity test: The CCK-8 method was used to co-culture the preparations with different formulations with human skin fibroblasts, and the survival rate and growth of human skin fibroblasts were detected. The evaluation criterion was that the cell survival rate should not be lower than 80%. If the cytotoxicity was obvious, the formulation might have an adverse effect on the body and needed to be further optimized.

[0054] Allergy reaction test: An allergy reaction test was carried out on guinea pigs to observe whether the animals showed allergic symptoms (including dyspnea, rash, and blood pressure drop) after injection.

[0055] The experimental results are shown in Table 3-5.

[0056] Table 3. Determination results of stability - physical indicators

[0057] Table 4. Determination results of stability - interferon activity, exosome morphology and particle size

[0058] Table 5. Determination results of biocompatibility and safety

[0059] Result analysis As can be seen from Table 3, after the preparations of Examples 7 - 10 were placed under high temperature and high humidity conditions for 6 months, the appearance, pH value, and osmotic pressure all met the standards. However, the preparations of Control Examples 7 and 13 showed a light yellow color in the 3rd month and a yellow color in the 6th month, the preparations of Control Examples 5, 8, and 10 showed a slightly yellow color in the 3rd month, and the preparation of Control Example 11 showed a slightly yellow color in the 6th month; while the pH and osmotic pressure of each group of examples and control examples met the standards after being placed for 6 months.

[0060] As can be seen from Table 4, after 6 months of storage, the interferon activities of the preparations in Examples 7-10 were still higher than 90%, the exosomes had normal morphology and concentrated particle sizes. While for the preparations in Comparative Examples 7-10 and 12-13, although the interferon activities were as high as over 90% at 0 month, after 6 months of storage, their activities decreased by more than 10%. For the preparations in Comparative Examples 5-6 and 11, the interferon activities were lower than 90% at 0 month, and after 6 months of storage, their activities decreased by a large margin. For the preparations in Comparative Examples 5 and 10, the particle size concentration decreased after 3 months of storage. For Comparative Examples 7, 9, 11-12, the morphology became slightly irregular and the particle size concentration decreased after 3 months of storage. For Comparative Example 13, the morphology became slightly irregular and the particle size concentration decreased after 6 months of storage.

[0061] As can be seen from Table 5, in the cytotoxicity tests of the preparations in each group, the cell survival rates were all higher than 80%, and there were no allergic reactions in each group of preparations.

[0062] As can be seen from Tables 3-5, by using the stabilizer provided by the present invention, the components synergistically enhance the effect, and can significantly improve the stability of the preparation. When removing, replacing one or more of the components, the stability of the preparation will decrease to varying degrees.

Claims

1. An interferon exosome composition for treating melanoma, characterized in that, The composition comprises interferon and exosomes, and the dosage ratio of interferon to exosomes is 50 - 300:5 - 30.

2. The composition according to claim 1, characterized in that, The dosage ratio of interferon to exosomes is 100 - 200:10 - 20.

3. A preparation comprising the composition according to claim 1, characterized in that, Each 1000 mL of the interferon - exosome preparation contains the following components: Interferon: 50 mg - 300 mg; Exosomes: 5 mg - 30 mg; Osmotic pressure regulator: 5 g - 10 g; pH regulator: 1 g - 2 g; Stabilizer: 10 g - 30 g; The balance is sterile injection water.

4. The preparation according to claim 3, characterized in that, The weight of the stabilizer in each 1000 mL of the interferon - exosome preparation is: 15 g - 25 g.

5. The preparation according to claim 3, characterized in that, The stabilizer comprises human serum albumin, polyethylene glycol, polysorbate and sodium sulfite in a mass ratio of 10 - 20:1 - 5:1 - 1.5:0.5 - 1.

6. The preparation according to claim 3, wherein The preparation method of the interferon - exosome preparation comprises the following steps: 1) Take the pH regulator and osmotic pressure regulator according to the formula, add them to sterile injection water, mix well, sterilize and then cool for standby to obtain the first mixed solution; 2) Add the formula amount of stabilizer to the first mixed solution, stir evenly to obtain the second mixed solution; 3) Add the formula amount of interferon to the second mixed solution, stir evenly to obtain the third mixed solution; 4) Add the formula amount of exosomes to the third mixed solution, stir at low speed to obtain the interferon - exosome preparation.

7. The composition according to claim 1 or 2, or the preparation according to any one of claims 3-6, characterized in that, The exosomes are NK - derived exosomes.

8. The preparation according to claim 7, characterized in that, The preparation method of the NK - derived exosomes is as follows: Collect healthy human peripheral blood, use lymphocyte separation medium, and collect peripheral blood mononuclear cells (PBMC) by density gradient centrifugation; Incubate the peripheral blood mononuclear cells with CD56 antibody, and then collect NK cells by magnetic bead separation method; Culture the cells with NK serum - free medium, add IL - 12 and IL - 15 to the medium, and when culturing to the fourth day, supplement the NK serum - free medium containing IL - 12 and IL - 15; Collect the culture supernatant after culturing for 7 days, remove cells by low - speed centrifugation, filter through a filter membrane, and then perform ultra - high - speed centrifugation. The centrifugation conditions are 4°C, 100000×g, 60 min. After centrifugation, collect the precipitate, and the obtained precipitate is the exosomes.

9. Use of the composition according to claim 1 or 2 or the preparation according to any one of claims 3 - 6 in the preparation of a drug for treating melanoma.

10. A drug prepared using the composition according to claim 1 or 2 or the preparation according to any one of claims 3 - 6.

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