WO2022142739A1 - Mitoxantrone composition and preparation method therefor - Google Patents

Abstract

Disclosed is a mitoxantrone pharmaceutical composition, comprising a first composition and a second composition, wherein the first composition comprises composite particles formed by an immune adjuvant and a solubilizer, and the second composition comprises mitoxantrone or a soluble salt thereof, a soluble alginate, a pH regulator, and an excipient. By means of improving the combination of the dosage forms, the compatibility problem of a chemotherapy drug containing mitoxantrone with soluble alginate excipients is overcome; and the present invention further provides a method for preparing a mitoxantrone composition.

Description

A kind of mitoxantrone composition and preparation method thereof technical field

The invention relates to the field of pharmaceutical preparations for treating tumors, in particular to a mitoxantrone composition and a preparation method.

Background technique

Chemotherapy, surgical resection, or radiation therapy are the most commonly used cancer treatment strategies. Chemotherapy is one of the main treatment methods for the clinical treatment of tumors at present. For those tumors that have the tendency to metastasize or have already metastasized, chemotherapy is the main treatment method. However, the commonly used chemotherapy modes in clinic are systemic administration, which does not have good selectivity to the lesion site, and also has toxic and side effects on normal organs. How to suppress tumor metastasis and prevent its recurrence at the same time of local treatment has always been a problem that plagues the world. However, there are many problems in the transformation of many drugs from laboratory verification to mass production. Anthraquinones are widely used chemotherapy drugs that can induce immunogenic cell death (ICD), including doxorubicin, epirubicin, piarubicin, mitoxantrone, etc. Available in salt form, it is ideal for chemoimmune cocktail therapy. Although many literatures and patents have reported that drugs in the laboratory stage have good therapeutic effects, the operability in the production and use of related drugs, as well as the sterilization and long-term storage stability of drug products still face a series of challenges. Difficulties, such as compatibility conflicts between drug components. When these drugs are not used in combination, they cannot exert their combined therapeutic effect, but if they want to use them in combination, they will face compatibility problems, sterilization problems and storage stability problems in the process of making medicines. If these problems in the pharmaceutical stage cannot be solved, many experimental drugs will not be able to truly go into clinical application.

SUMMARY OF THE INVENTION

The present invention provides a mitoxantrone pharmaceutical composition, which can produce a synergistic anticancer effect and reduce the probability of cancer metastasis and recurrence. It inhibits the growth of distant metastatic tumors and reduces the probability of tumor recurrence, reduces the side effects of chemotherapy through in situ chemotherapy, and provides a mass-produced production and preparation process with good product stability.

The research and development team of the present application found in the experiment that directly mixing sodium alginate with the hydrochloride form of anthraquinone drugs will cause agglomerates to appear rapidly, which will lead to the instability of the pharmaceutical composition system, that is, incompatibility. Such a heterogeneous system is directly injected into the tumor, although a considerable therapeutic effect can still be obtained, but it cannot be prepared into a standardized pharmaceutical preparation, and the direct use will also have the problem of uncertainty of dose, and storage for a period of time (more than about 6 hours), the drug components will agglomerate and fail to disperse in the aqueous solution, rendering them completely unusable. Long-term storage will lead to instability and uncertainty in the composition system, resulting in that the combination drug cannot fully meet the needs of stable quality and long-term storage after the drug is marketed; further research has found that anthraquinone drugs generally use positively charged salts. The salt form is prepared as a preparation, while sodium alginate is a polyanionic polymer with a large number of negative charges, which will cause electrostatic interaction with anthraquinone drug molecules to cause agglomeration. On the other hand, anthraquinone drug molecules (containing There is a pi-pi stacking (pi electron stacking) interaction between multiple aromatic ring structures, which is also an important reason why it is very easy to agglomerate under the condition that its own charge is shielded.

The research and development team tried to overcome the compatibility problem of mitoxantrone-containing chemotherapeutic drugs and soluble alginate excipients by improving the dosage form combination scheme and preparation method.

In order to solve the related technical problems, the present invention provides the following solutions:

A mitoxantrone pharmaceutical composition, comprising a first composition and a second composition, the first composition comprising a microparticle suspension formed by a fat-soluble immune adjuvant and a surfactant; the second composition Substances include mitoxantrone or its soluble salts, readily soluble alginates, pH adjusters and excipients.

Specifically, the immune adjuvant in the first composition includes one or more of imiquimod (R837), resiquimod (R848), or glucopyranoside lipid A (MPLA).

Specifically, the mitoxantrone or its soluble salt includes mitoxantrone hydrochloride or mitoxantrone lactate.

Further, the mass ratio of mitoxantrone to imiquimod ranges from 1:2 to 1:20, preferably 1:2 to 1:15.

Further, the hydrophobic structure part of the surfactant in the first composition contains no less than 20 oxypropylene units; specifically, including Poloxamer 188, Poloxamer 237, Poloxamer Poloxamer 338 or Poloxamer 407.

Optionally, the hydrophobic moiety of the surfactant in the first composition contains one or more hydrocarbon chains of not less than 15 carbon atoms; specifically, including sorbitan sesquioleate, Soy Lecithin, Glyceryl Monostearate, Polysorbate 40, Polysorbate 60, Polysorbate 65, Polysorbate 80, Polysorbate 85, Sorbitan Stearyl (Span 60), Stearate, Vitamin E polyethylene succinate, polyoxyethylene alkyl ether, polyoxyethylene stearate, polyoxy (40) stearate, sucrose stearate, polyoxyethylene castor oil derivatives, At least one of polycitrol 1000 or lecithin.

Further, the first composition is a micron composite particle of the immune adjuvant and the solubilizer. Preferably, the particle size of the composite particles of imiquimod R837 and poloxamer 188 is 0.5-5 microns. When the particle size of the immune adjuvant is 0.5 to 5 microns, it can maintain a steady release after the chemical drug kills the tumor cells, so that the concentration of the immune adjuvant can reach a better ratio at the right release time. Make the immune system better produce specific immunity.

Specifically, the readily soluble alginate in the second composition includes one or more of sodium alginate, potassium alginate or ammonium alginate.

Specifically, the excipients in the second composition include mannitol, lactose, sucrose, simple syrup, sorbitol, polyethylene glycol, sulfobutyl beta cyclodextrin, hydroxypropyl beta cyclodextrin or carboxymethyl one or more of sodium cellulose.

Specifically, in the second composition, the pH adjusting agent is preferably NaOH, KOH or ammonia water.

Further, the mitoxantrone composition further includes a third composition, and the third composition includes an immune checkpoint inhibitor or an IDO inhibitor.

Specifically, the immune checkpoint inhibitors include antibody immune checkpoint blockers, small molecule inhibitors or peptide inhibitors; preferably, the antibody immune checkpoint blockers include anti-CTLA-4 , one or more of anti-PD-1 or anti-PD-L1; the small molecule inhibitors include CA-170, PM-327, BMS-8, BMS-37, BMS-202, BMS- 230, one or more of BMS242, BMS-1001, BMS-1166, BMS-1001, BMS-1166 or JQ1; the peptide inhibitors include one or more of DPPA-1.

Specifically, the IDO inhibitor includes one or more of BMS-986205, IDO inhibitor 1, NLG919, NLG8189, PF-06840003, Epacadostat or 4-phenylimidazole.

Further optionally, the third composition further includes a gel-forming adjuvant, and the gel-forming adjuvant includes a compound of soluble alkaline earth metal ions. The specific gel-forming auxiliary materials include calcium chloride, calcium sulfate or magnesium chloride and the like.

Further optionally, the second composition further includes 1% Tween-80 as a surfactant, which can improve the dissolution rate of mitoxantrone.

The present invention also provides a preparation method of the mitoxantrone composition, comprising the following steps:

S1: Weigh mitoxantrone or its soluble salt and excipient in proportion 1: (1-10), add aqueous solution, stir, add pH adjuster to adjust the pH of the solution to 7.8-9.2, and pass the obtained solution through Micron membrane filtration and sterilization to obtain the first liquid;

S2: Weigh easily soluble alginate and protective filler in proportion 1:(1～5), add water, stir, filter and sterilize the obtained solution through a micron filter to obtain a second liquid;

S3: Mix the first liquid and the second liquid obtained in S1 and S2 evenly under the protection of nitrogen, filter and sterilize with a 0.22 μm filter membrane, then bottle, pre-cool, freeze-dry, and fill with nitrogen After sealing the bottle.

Further preferably, in step S2 of the preparation method, a pH adjuster is added to adjust the pH of the solution to 8.0-8.7.

Adopting the technical scheme of the present invention will have the following beneficial technical effects:

The technical solution of the present invention further solves the problems of the practicability of the treatment plan and the long-term storage stability of the drug product in the drug preparation stage. After the fat-soluble immune adjuvant in the first composition is sterilized by moist heat at 105°C to 150°C, the suspension will become unstable and produce obvious precipitation and particles, and the water dispersibility will be greatly reduced, but the fat-soluble immune adjuvant and The complex particles of surfactant can ensure the water dispersibility and stability of the fat-soluble immune adjuvant after sterilization.

The technical solution of the present invention overcomes the compatibility problem of mitoxantrone hydrochloride-containing chemotherapeutic drugs and alginate excipients. The invention solves the problem of agglomeration when existing mitoxantrone and its hydrochloride and sodium alginate coexist, and the resulting pharmaceutical composition system is unstable, thereby causing the problem of drug safety. The dosage form and preparation method of the relevant embodiments of the present patent, by simultaneously improving the preparation method of the dosage form, overcomes the compatibility problem of the chemotherapeutic drug mitoxantrone and the adjuvant sodium alginate, and suppresses the interaction of pi electron stacking between the anthraquinone drug molecules , protect the molecular structure from being destroyed, avoid the problem of agglomeration of each component, and avoid the oxidation risk of mitoxantrone, which greatly increases the feasibility of the drug preparation stage, and also greatly speeds up the reconstitution time after lyophilization, which is convenient for clinical operations. . The composition can form a porous network cross-linked gel structure after being injected into the tumor, so that the other components mixed in the alginate colloid can be slowly released, thereby locking the chemotherapeutic drugs at the tumor site, while reducing the chemotherapeutic drugs. systemic exposure levels in normal organs in the body, enhancing its efficacy while reducing its toxic side effects.

In addition, the chemotherapeutic drug used in the present invention is a chemotherapeutic drug that can cause tumor immunogenic cell death. While killing tumor cells, it exposes tumor-related antigens, provides a target that helps immune cells recognize cancer cells, activates the immune system, to specifically remove cancer cells. However, this effect requires a large number of antigen-presenting cells to ingest, process, and present antigens to T cells to further activate this immune response, and antigen-presenting cells need the help of immune adjuvants to be more effectively enriched in the tumor site and play a role. effect. Therefore, while using ICD drugs, it is also necessary to introduce immune adjuvants to synergistically enhance the anti-tumor immune response. The invention combines the two together, and with the aid of a sustained-release system, realizes the technical effect of eliminating tumor cells in situ while producing tumor vaccine in vivo and inhibiting tumor metastasis and recurrence.

Description of drawings

Fig. 1 is a schematic diagram of preparation steps;

Fig. 2 is the growth curve graph of orthotopic tumor (direct drug injection) on H22 tumor model;

Figure 3 is a graph showing the growth of distal tumors (direct injection without drug) on the H22 tumor model;

Fig. 4 is the growth curve graph of orthotopic tumor (direct drug injection) on CT26 tumor model;

Figure 5 is a graph of the growth of distal tumors (direct injection without drug) on the CT26 tumor model;

Figure 6 is a graph showing the growth curve of orthotopic tumors treated with different doses of this preparation on the CT26 tumor model;

Fig. 7 is the growth curve graph of the distal tumor treated with different doses of this preparation on the CT26 tumor model;

Figure 8 is a graph showing the body weight change of mice treated with different doses of this preparation on the CT26 tumor model.

Detailed ways

Example A: Preparation of Formulations

Figure 1 is a schematic diagram of the preparation and use steps of a mitoxantrone pharmaceutical composition.

Example A1: Preparation of the first composition:

A certain amount of the immune adjuvant imiquimod R837 solid is weighed and subjected to jet pulverization, and the pulverization pressure is 6-10 bar to obtain micron-scale imiquimod R837. Proportion 1: (0.025～5) Weigh the micron-scale immune adjuvant imiquimod R837 and the surfactant poloxamer 188, preferably 2g R837, add an appropriate amount of poloxamer 188 (0.05g, 0.3g, 0.6g, 1g, 2g, 4g, 6g, 8g, 10g), add 100mL of water for injection, and stir at 100-500rpm for 0.5-2 hours to obtain a suspension. Homogenize the above suspension under high pressure at 750-1200ba for 2-4 times, suck the suspension with a peristaltic pump and fill it into 10mL ampoule bottles, each bottle is 6mL, for a total of 30 bottles. After melting and sealing, a micron suspension is obtained, which is sterilized by moist heat at 105°C to 150°C for 15-20 minutes.

Poloxamer 188 is a new type of polymer nonionic surfactant, which has various uses, including: as emulsifier, stabilizer and solubilizer, which can further enhance the water dispersibility and stability of R837.

The hydrophobic structure part of the used surfactant contains no less than 20 oxypropylene units; specifically, it includes Poloxamer 188, Poloxamer 237, Poloxamer 338, and Poloxamer 407. Optionally, the hydrophobic structure part of the surfactant contains one or more hydrocarbon chains with a total of not less than 15 carbon atoms; specifically including sorbitan sesquioleate, soybean lecithin, glyceryl monostearate , polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, sorbitan stearyl (Span 60), stearate, vitamin E polyethylene succinate , polyoxyethylene alkyl ethers, polyoxyethylene stearate, polyoxy(40) stearate, sucrose stearate, polyoxyethylene castor oil derivatives, polycidol 1000, or lecithin at least one of them.

Poloxamer is a series of multi-purpose pharmaceutical excipients, which are non-toxic, non-antigenic, non-sensitizing, non-irritating, non-hemolytic and chemically stable. Poloxamer 188 is one of the series of excipients with better safety. Poloxamer 188 can make the micron-sized powder obtained after imiquimod jet pulverization can be processed by liquid phase micro-nano process to obtain imiquimod micron-sized particle suspension with good size uniformity, Poloxamer 188 can also help imiquimod micron-sized particle suspensions (6.0 mg/mL and below) ensure water dispersibility and stability after autoclaving.

However, although the suspension of imiquimod microparticles coated with Poloxamer 188 maintains better suspension stability after autoclaving at a lower concentration (6.0 mg/mL), if imiquimod is sterilized during sterilization If the concentration is too high, it will cause imiquimod to agglomerate and clump after sterilization and can no longer be stably suspended. Lecithin is a natural surfactant, and the imiquimod microparticles treated by high pressure homogenization with lecithin as a stabilizer have good stability. Still does not agglomerate but maintains a stable suspension.

Table 1. Imiquimod/Poloxamer 188 (P188) suspension preparation process and data:

The new technical route of jet milling combined with high pressure homogenization or jet milling combined with high shear method, prepared micron-scale lipid-soluble immune adjuvant microparticle suspension. The preparation method overcomes the technical prejudice and practical technical problems in the preparation process of micro-particles. The high-pressure homogenization process or the high-shear process is a liquid-phase processing method, while the fat-soluble immune adjuvant is a semi-solid state. The experiment found that if the fat-soluble immune adjuvant is directly subjected to high-pressure homogenization or high-shearing process, the homogenization valve will be blocked and micro-particles cannot be obtained; while direct high-shear method can partially obtain micro-particles, However, the uniformity of the obtained particles is extremely poor, and most of the particles cannot achieve the expected granulation and pulverization effect and yield; and in the present invention, the primary powder is first obtained through the jet pulverization process, and then the solution is carried out under the condition of adding a surfactant. High-pressure homogenization or high-shear method, which can perform rapid surface modification and surface modification of high-pressure homogenization or high-shear micro-particles, because of the presence of surfactants, lipid-soluble immune adjuvants can be discretized. Disperse in the liquid phase, so that the primary powder of the lipid-soluble immune adjuvant can be processed by the liquid-phase micro-nano technology and obtain the lipid-soluble immune adjuvant micron-sized particle suspension with good size uniformity.

Table 2: Add different surfactant aqueous solutions (imiquimod:surfactant mass ratio=1:3) to the imiquimod microparticle powder after jet pulverization, and then carry out high-pressure homogenization treatment. water dispersibility

Table 3: Redispersibility of the above-mentioned imiquimod suspensions (6.0 mg/mL) with different surfactants added after autoclaving (imiquimod:surfactant mass ratio=1:3)

Since the micron-sized particle suspension needs to be subjected to standard autoclaving before being injected into the tumor to meet the sterility requirements, it is necessary to ensure that the micron-sized particles do not undergo significant agglomeration at 105°C to 150°C. The surfactant and the particle surface have a strong enough adsorption capacity, mainly relying on the hydrophobic interaction, so the hydrophobic structure of the selected surfactant plays an important role in protecting the stability of the micron-scale suspension under high pressure sterilization. The hydrophobic structure part of the surfactant contains one or more hydrocarbon chains with a total of not less than 15 carbon atoms or the hydrophobic structure part of the surfactant contains not less than 20 oxypropylene units. As shown in Tables 2 and 3, Poloxamer 124, due to insufficient hydrophobic structure, was unstable after autoclaving.

Table 4: Suspension stability of imiquimod suspensions (R837 concentration = 6.0 mg/mL during sterilization) with different ratios of P188 dispersed after autoclaving

Poloxamer 188:R837	Suspension stability after autoclaving
0.5:1	A large number of granular aggregates appear
1:1	A small amount of granular aggregates appear
2:1	A small amount of granular aggregates appear
3:1	Homogeneous dispersion and no granular aggregates
4:1	Homogeneous dispersion and no granular aggregates
5:1	Homogeneous dispersion and no granular aggregates

Although theoretically, the more dispersant, the better the dispersibility, but the ratio is generally not more than 5:1. The reason is: Poloxamer 188 (P188) itself is viscous, and the concentration is too high, and the viscosity is large; Introduce more impurities.

Table 5: Suspension stability of different concentrations of imiquimod suspensions dispersed by P188 after autoclaving (P188: imiquimod R837 mass ratio=3:1). P188-coated imiquimod suspensions maintained good stability under autoclaving at low R837 concentrations, but decreased significantly at high R837 concentrations.

R837 concentration during sterilization	Suspension stability after autoclaving
3.0mg/mL	Homogeneous dispersion and no granular aggregates
6.0mg/mL	Homogeneous dispersion and no granular aggregates
9.0mg/mL	Partially granular aggregates appear
12.0mg/mL	A large number of granular aggregates appear
15.0mg/mL	A large number of granular aggregates appear

18.0mg/mL

A large number of granular aggregates appear

Table 6: Suspension stability of imiquimod suspensions (R837 concentration at sterilization = 6.0 mg/mL or 18.0 mg/mL) dispersed with different proportions of lecithin after autoclaving. Lecithin maintains good suspension stability after autoclaving of high-concentration imiquimod suspensions, even at low ratios.

Lecithin: R837	R837 concentration during sterilization	Suspension stability after autoclaving
0.025:1	6.0mg/mL	Homogeneous dispersion and no granular aggregates
0.05:1	6.0mg/mL	Homogeneous dispersion and no granular aggregates
0.1:1	6.0mg/mL	Homogeneous dispersion and no granular aggregates
0.25:1	6.0mg/mL	Homogeneous dispersion and no granular aggregates
0.5:1	6.0mg/mL	Homogeneous dispersion and no granular aggregates
1:1	6.0mg/mL	Homogeneous dispersion and no granular aggregates
0.025:1	18.0mg/mL	Homogeneous dispersion and no granular aggregates
0.05:1	18.0mg/mL	Homogeneous dispersion and no granular aggregates
0.1:1	18.0mg/mL	Homogeneous dispersion and no granular aggregates
0.25:1	18.0mg/mL	Homogeneous dispersion and no granular aggregates
0.5:1	18.0mg/mL	Homogeneous dispersion and no granular aggregates
1:1	18.0mg/mL	Homogeneous dispersion and no granular aggregates

Example A2:

A certain amount of immune adjuvant Resiquimod (R848) solid is weighed and subjected to jet pulverization, and the pulverization pressure is 6-10 bar to obtain micron-scale Resiquimod (R848).

Proportion 1: (0.025～5) Weigh micron-scale immune adjuvant Resiquimod (R848) and surfactant Poloxamer 407, preferably 0.2g R848, add an appropriate amount of Poloxamer 407 (0.005g, 0.01 g, 0.2 g, 0.4 g, 0.8 g, 1 g), add 200 mL of water for injection, and stir at 100-500 rpm for 0.5-2 hours to obtain a suspension.

The above suspension is homogenized under high pressure at 750-1200bar for 2-4 times to obtain the suspension, and the suspension is sucked by a peristaltic pump and filled into 10mL ampoule bottles, each bottle of 6mL, for a total of 30 bottles. After melting and sealing, a micron suspension is obtained, which is sterilized by moist heat at 105°C to 150°C for 15-20 minutes.

Poloxamer 407 is a new type of polymer nonionic surfactant, which has many uses including: as emulsifier, stabilizer and solubilizer, which can further enhance the water dispersibility and stability of R848.

Example A3:

Weigh a certain amount of fat-soluble immune adjuvant glucopyranoside lipid A (MPLA); the surfactant selected is a mixed surfactant with a mass ratio of Poloxamer 188 and lecithin of 9:1, and other preparation methods Same as Example A2.

Example A4:

Other preparation methods are the same as in Example A1, take by weighing a certain amount of fat-soluble immune adjuvant imiquimod (R837); the surfactant selected is the mixed surface of poloxamer 188 and the mass ratio of lecithin 3:1 active agent. The feeding concentration of different surfactants has a certain influence on the suspension stability of R837 after autoclaving, and the results are shown in Table 7. In the presence of lecithin, the long-term stability of R837 after autoclaving is better than the effect of solubilizing R837 with P188 alone, and the obtained particles have smaller particle size and better uniformity. And the influence of the feeding concentration can be expanded in equal proportion, so as to achieve the technical effect of increasing the final concentration of R837.

Table 7: Suspension stability after autoclaving of R837 with different concentrations of surfactants

R837: Poloxamer 188: Lecithin	Long-term stability after autoclaving
12mg/mL: 36mg/mL: 0mg/mL	A large number of granular aggregates appear
12mg/mL: 36mg/mL: 12mg/mL	Homogeneous dispersion and no granular aggregates
18mg/mL: 54mg/mL: 0mg/mL	A large number of granular aggregates appear
18mg/mL: 54mg/mL: 18mg/mL	Homogeneous dispersion and no granular aggregates

It can be seen that the mixing of the two surfactants can further increase the suspension stability performance of the self-sustained-release immune adjuvant microparticles in autoclaving, especially when the surfactant concentration is higher. Two or more surfactant combinations with different hydrophilic-lipophilic balance (HLB values) or two surfactants with different hydrophobic structural moieties (for example, one surfactant contains not less than 20 oxypropylene units) , or a surfactant containing one or more hydrocarbon chains with a total of not less than 15 carbon atoms) as the coating layer of the microparticles. The two surfactants with different solubility are not completely homogeneously dispersed with each other, but form a relatively uniform and locally aggregated dispersed structure. After the formed coating composite particles enter the tumor, the surfactant with a larger HLB value. First dissolve, so that some tiny openings or tiny defect areas are formed on the surface of the coating layer of the microparticles, so that the surface area of the inner layer of immune adjuvant microparticles gradually changes, and the active ingredients are gradually released. According to actual needs, through the selection of two or more surfactants or the preparation of the proportioning relationship, various types of drug combination schemes can be obtained.

Table 8: Particle size change after autoclaving of R837 with different ratios of surfactants

At the same time, as shown in Table 8, the R837 obtained by the coexistence of lecithin and P188 has the smallest particle size change before and after sterilization, and the particle size distribution range is smaller, that is, the coexistence of lecithin and P188 is more helpful for the sample to be sterilized. stability. Among them, D50 is the corresponding particle size when the cumulative particle size distribution in the sample reaches 50%, D90 is the corresponding particle size when the cumulative particle size distribution in the sample reaches 90%, and Dmax is the maximum particle size of the particles in the sample. The higher the homogeneity of the sample particles. It was also observed in the experiment that the suspension samples in which P188 and lecithin coexisted would not hang on the wall after being left for a long time. It is worth noting that the size uniformity of microparticles is an important parameter to ensure stable and reproducible drug release behavior in vivo.

Example B:

Example B1: Preparation of Second Composition:

S1: Prepare sodium alginate/mannitol or sodium alginate/lactose solution in proportion 1:(1～5), wherein the concentration of sodium alginate solution is 2.5mg/ml, 5mg/ml, 10mg/mL, 20mg/mL , 40mg/mL, the final concentration of excipient mannitol or lactose is 1~50mg/mL, 20~100mg/mL, 40~200mg/mL, and the sodium alginate solution is stirred well before adding mannitol or lactose;

S2: prepare mitoxantrone hydrochloride solutions with concentrations of 1 mg/ml, 2 mg/ml and 3 mg/mL, and adjust its pH to 8.0-8.7 with 20 mg/mL sodium hydroxide solution;

S3: Under the condition of 2～8℃ or room temperature, mix the solutions described in S1 and S2 according to the volume ratio of 1:1 under the protection of nitrogen gas, filter and sterilize with a 0.22μm filter membrane, and then pack them into vials. , after pre-cooling, freeze-drying, and seal the bottle after filling with nitrogen.

Example B2: Preparation of Second Composition:

S1: Prepare sodium alginate/mannitol solution in proportion 1:(1～5), wherein the sodium alginate solution concentration is 5mg/mL, 10mg/mL, 20mg/mL, 40mg/mL, and the sodium alginate solution is stirred evenly Then add mannitol, the final concentration of mannitol is 1～50mg/mL, 20～100mg/mL, 40～200mg/mL;

S2: prepare a mitoxantrone hydrochloride solution with a concentration of 6 mg/mL, and adjust its pH to 8.5-9.2 with a 20 mg/mL sodium hydroxide solution;

S3: At 2-8°C or room temperature, dissolve S1 and S2 in a nitrogen-filled protective condition and mix them evenly in an appropriate ratio, filter and sterilize with a 0.22 μm filter membrane, and then pack them into vials, pre-cooling After lyophilization, the bottle was sealed after nitrogen filling.

Example B3: Preparation of Second Composition:

S1: Prepare sodium alginate/lactose solution according to the ratio 1:(1～5), wherein the sodium alginate solution concentration is 5mg/mL, 10mg/mL, 20mg/mL, 40mg/mL, after the sodium alginate solution is stirred evenly Then add lactose, the final concentration of lactose is 1～50mg/mL, 20～100mg/mL, 40～200mg/mL;

S2: prepare a mitoxantrone hydrochloride solution with a concentration of 6 mg/mL, and adjust its pH to 7.8-8.5 with a 20 mg/mL sodium hydroxide solution;

S3: At 2-8°C or room temperature, dissolve S1 and S2 in a nitrogen-filled protective condition and mix them evenly in an appropriate ratio, filter and sterilize with a 0.22 μm filter membrane, and then pack them into vials, pre-cooling After lyophilization, the bottle was sealed after nitrogen filling.

Table 9. Influencing factors of pH in the preparation of mitoxantrone/sodium alginate/mannitol.

NOTE: An overly basic environment can cause deprotonation of the phenolic hydroxyl group of mitoxantrone, and such molecules may be susceptible to oxidation. Therefore, the pH value should not be too high.

Further experimental verification showed that for anthraquinones such as doxorubicin hydrochloride, epirubicin hydrochloride, and pirarubicin hydrochloride, even if NaOH was used to neutralize their protons, agglomeration still appeared after mixing with sodium alginate. It can be seen that mitoxantrone is a special case. As the pH value increases, within a certain range, it can ensure the solubility of mitoxantrone and achieve no agglomeration after compatibility with sodium alginate. This phenomenon is that mitoxantrone does not agglomerate. Anthraquinone is unique.

Table 10. Influence data of nitrogen-filled environment on the stability of mitoxantrone/sodium alginate:

The effect of nitrogen conditions on the stability of mitoxantrone/sodium alginate was investigated. Mixing mitoxantrone and sodium alginate under nitrogen-filled conditions, the single- and total-impurity contents of the prepared mitoxantrone/sodium alginate changed slowly, and the single- and total-impurity contents at 30 days under the accelerated condition of 60 °C It remained within limits; the impurity level was exceeded by day 5 if mitoxantrone and sodium alginate were mixed without nitrogen protection. It is necessary to illustrate that the step of filling nitrogen gas in the present invention is beneficial to the stability of the formulation.

Example B4: Study on the slow-release effect of alginate

Table 11. Release data of mitoxantrone from sodium alginate/calcium ion hydrogel:

	5min	1h	3h	6h	12h	24h
pH 7.4	3.4%	4.2%	6.5%	8.7%	12.2%	14.3%
pH 4.0	2.7%	5.6%	8.8%	10.7%	14.6%	18.3%
pH 7.4 (+1% Tween-80)	4.8%	18.2%	36.6%	63.4%	70.4%	78.3%
pH 4.0 (+1% Tween-80)	5.3%	19.4%	37.2%	60.7%	69.1%	75.5%

The ratio of mitoxantrone released from sodium alginate/calcium ion hydrogels as a function of time. The mitoxantrone-encapsulated sodium/calcium ions were put into gauze bags and dialyzed in buffer solutions of different pH. The buffer solution at pH 7.4 was phosphate buffer with 2 mM CaCl ₂ added, and the buffer solution at pH 4.0 was acetic acid-sodium acetate buffer solution. The release of mitoxantrone from sodium alginate/calcium ion hydrogels was slow under both acidic and neutral conditions, which was due to the strong interaction of mitoxantrone with sodium alginate after protonation. In order to improve the dissolution rate of mitoxantrone, 1% Tween-80 was added to the solution as a surfactant in the two groups after the above experiment. By adjusting the amount of surfactant added in the composition, the release rate of mitoxantrone can be significantly controlled.

Table 12. Release data of imiquimod from sodium alginate/calcium ion hydrogel:

	5min	1h	3h	6h	12h	24h
pH 7.4	0.0%	1.3%	2.7%	3.4%	4.2%	5.6%
pH 4.0	1.2%	12.3%	27.6%	50.1%	71.0%	85.4%

The ratio of imiquimod released from sodium alginate/calcium ion hydrogels as a function of time. The imiquimod-coated sodium alginate/calcium ions were put into gauze bags and dialyzed in buffer solutions of different pH. The buffer solution at pH 7.4 was phosphate buffer with 2 mM CaCl ₂ added, and the buffer solution at pH 4.0 was acetic acid-sodium acetate buffer solution. Imiquimod has a faster release rate under acidic conditions. By adjusting the pH of the composition, control of the release rate of imiquimod can be achieved very significantly.

Example C:

Instructions for the use of the mixed liquid and lyophilized preparations:

Use scheme 1: The above-mentioned second composition lyophilized powder for injection is dissolved in the suspension of the first composition, and the composition solution is directly injected into the tumor site of the patient by means of clinical interventional administration and direct puncture administration. The multi-point injection method is used to ensure that the composition solution fills the entire tumor evenly.

Use scheme two: with the third composition, the third composition is administered by intravenous injection.

Use scheme 3: After the tumor patient has undergone normal surgical resection of the lesion site, considering the problem that the tumor cells in the lesion site cannot be completely removed by surgical resection, the lyophilized powder injection of the second composition can be dissolved in the suspension of the first composition. Then use a syringe or spray bottle to spray the wound site after surgical resection, and then spray an appropriate amount of calcium chloride solution on the site to make it gel, and finally suture the wound.

Use scheme 4: With the third composition, the third composition can be injected intravenously or sprayed on the wound. The regimen helps to destroy remaining cancer cells and inhibits tumor metastasis and recurrence.

After the composition is injected into the tumor, firstly, when the alginate in the second composition encounters calcium ions in the organism tissue or the gel-forming excipients in the third composition, it will rapidly gel to form a porous network The linked structure enables the slow release of other components mixed in the network-like cross-linked structure, thereby confining the mitoxantrone chemotherapeutic drugs, enhancing their effects and reducing their toxic and side effects; secondly, the second composition The ICD chemotherapeutic drug mitoxantrone can not only effectively kill tumor cells, but also make them produce immunogenic death, generate tumor-related antigens, and activate tumor-specific immune responses; again, the immune adjuvant in the first composition The ability of antigen-presenting cells is enhanced, which further amplifies the corresponding immune response; finally, the use of the third type of component immune checkpoint inhibitor or IDO inhibitor makes the metastatic tumor unable to escape the immune response, so that immunotherapy can be more effective. Kill tumors, thereby inhibiting tumor metastasis and recurrence.

Prior to use, the suspension of the first composition of the present invention was added to the lyophilized powder of the second composition, followed by intratumoral injection. It kills in situ tumors, promotes immunogenic cell death, activates anti-tumor-specific immune responses, and uses immune responses to inhibit the growth of distant tumors. The immune memory effect can inhibit recurrence.

Cancer treatment is a very complex and comprehensive outcome, because both the body's immune system and the growth mechanism of cancer cells are very complex. In addition to the explanations in other parts of this patent, the reason why this experiment can achieve a relatively excellent therapeutic effect may include the following reasons. Using imiquimod R837 micron particles, the water-insoluble R837 powder was pulverized in the liquid phase to obtain 0.5 -5 micron particle size, so that it has both water-phase dispersibility and a suitable release cycle in the state of in-situ gel formation, and can be well coordinated with other drug components.

Adding Poloxamer 188 to R837 emulsion, Poloxamer 188 is a new type of polymer nonionic surfactant, which has many uses including: as emulsifier, stabilizer and solubilizer, which can further enhance Water dispersibility and stability of R837 emulsion.

The composition can form a porous network cross-linked structure after being injected into the tumor, so that other components mixed in the alginate colloid can be slowly released, so as to limit the locking of the chemotherapeutic drugs containing carboxylic acid ligands. Its effects also reduce its toxic side effects. In addition, when the particle size of the immune adjuvant is 0.5 to 5 microns, it can maintain a steady release after the chemical drug kills the tumor cells, so that the concentration of the immune-enhancing effect can be optimally matched with the release timing.

The hydrophobic structure part of the used surfactant contains no less than 20 oxypropylene units; specifically, it includes Poloxamer 188, Poloxamer 237, Poloxamer 338, and Poloxamer 407. Optionally, the hydrophobic structure part of the surfactant contains one or more hydrocarbon chains with a total of not less than 15 carbon atoms; specifically including sorbitan sesquioleate, soybean lecithin, glyceryl monostearate , polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, sorbitan stearyl (Span 60), stearate, vitamin E polyethylene succinate , polyoxyethylene alkyl ethers, polyoxyethylene stearate, polyoxy(40) stearate, sucrose stearate, polyoxyethylene castor oil derivatives, polycidol 1000, or lecithin at least one of them.

Poloxamer is a series of multi-purpose pharmaceutical excipients, which are non-toxic, non-antigenic, non-sensitizing, non-irritating, non-hemolytic, and chemically stable. Poloxamer 188 is one of the series of excipients with better safety.

Excipients used include: mannitol, lactose, sucrose, simple syrup, sorbitol, polyethylene glycol, sulfobutyl beta cyclodextrin, hydroxypropyl beta cyclodextrin, or one of sodium carboxymethyl cellulose. One or more of them have good protective effect on mitoxantrone and sodium alginate.

Example D: The therapeutic effect produced by the composition of the present invention is as follows

Example D1: Therapeutic experiment of this preparation on H22 liver cancer tumor model (bilateral tumor model)

Mouse H22 liver cancer tumors were implanted on the left and right ends of the back of the mice (the right side was regarded as the orthotopic tumor, and the left side was regarded as the distal tumor), and the tumor-bearing mice were divided into six groups, six in each group, and injected into the tumor respectively. .

The first group: saline;

Group 2: Vinorelbine (4 mg/kg body weight)/imiquimod/sodium alginate;

Group 3: vincristine (1 mg/kg body weight)/imiquimod/sodium alginate;

Group 4: Paclitaxel (3 mg/kg body weight)/imiquimod/sodium alginate;

Group 5: Docetaxel (4 mg/kg body weight)/imiquimod/sodium alginate;

Group 6: Mitoxantrone (3 mg/kg body weight)/imiquimod/sodium alginate.

After intratumoral injection to the left orthotopic tumor and no injection to the right distal tumor, the length and width of the orthotopic tumor and the distal tumor were measured with vernier calipers every two days, and the tumor volume was (length times ( Width squared)) divided by 2. Refer to Figure 2 and Figure 3 for the growth curves of orthotopic and distal tumors.

Table 13. Tumor inhibition rates of orthotopic and distal tumors in each group in Example D1

grouping	In situ tumor inhibition rate (%)	Distant tumor tumor suppression rate (%)
First group	0	0
Second Group	73	46
The third group	77	42
Fourth group	53	8
Group 5	75	68
The sixth group	90	66

Therapeutic effect: From the in situ tumor growth curve (Figure 2), the tumors at the injection site of the mice in group 6 were significantly inhibited, and the tumor inhibition rates were all over 90% (Table 13), which was significantly better than the other groups. From the distal tumor growth curve (Figure 3), it can be seen that the distal tumors of the mice in groups 5 and 6 were significantly inhibited, and the tumor inhibition rate exceeded 60%, showing a significant therapeutic effect. It has been proved that this preparation is the most effective among various drugs.

Example D2: Therapeutic experiment of this preparation on CT26 colon cancer tumor model (bilateral tumor model)

Mouse CT26 colon cancer tumors were implanted on the left and right ends of the back of the mice (the right side was regarded as the orthotopic tumor, and the left side was regarded as the distal tumor), and the tumor-bearing mice were divided into five groups, six in each group, respectively. injection.

The first group: normal saline (reference example);

Group 2: Vinorelbine (4 mg/kg body weight)/imiquimod/sodium alginate;

Group 3: Docetaxel (4 mg/kg body weight)/imiquimod/sodium alginate;

Group 4: Mitoxantrone (3 mg/kg body weight)/sodium alginate;

Group 5: Mitoxantrone (3 mg/kg body weight)/imiquimod/sodium alginate.

After intratumoral injection to the left orthotopic tumor and no injection to the right distal tumor, the length and width of the orthotopic tumor and the distal tumor were measured with vernier calipers every two days, and the tumor volume was (length times ( Width squared)) divided by 2.

Table 14. Tumor inhibition rates of orthotopic and distal tumors in each group in Example D2

grouping	In situ tumor inhibition rate (%)	Distant tumor tumor suppression rate (%)
First group	0	0
Second Group	79	33
The third group	69	27
Fourth group	67	65
Group 5	97	96

Therapeutic effect: From the orthotopic tumor growth curve (Figure 4) and the distal tumor growth curve (Figure 5), both the injection site tumor and the distal tumor in group 5 mice were significantly inhibited and almost no longer grew. , the tumor inhibition rate was more than 90% (Table 14), which was significantly better than the other groups. This formulation proved to be the most effective in a variety of drug combinations.

Example D3: Therapeutic experiment of different doses of this preparation on mouse CT26 tumor model

Mouse CT26 colon cancer tumors were implanted on the left and right ends of the back of the mice (the right side was regarded as the in situ tumor, and the left side was regarded as the distal tumor), and the tumor-bearing mice were divided into 8 groups, 6 in each group, respectively injection.

The first group: mitoxantrone (3mg/mL)/imiquimod/sodium alginate (10mg/mL);

The second group: mitoxantrone (2mg/mL)/imiquimod/sodium alginate (10mg/mL);

The third group: mitoxantrone (3mg/mL)/imiquimod/sodium alginate (5mg/mL);

The fourth group: mitoxantrone (2mg/mL)/imiquimod/sodium alginate (5mg/mL);

The fifth group: mitoxantrone (3mg/mL)/imiquimod;

The sixth group: mitoxantrone (2mg/mL)/imiquimod;

The seventh group: mitoxantrone (3mg/mL)/sodium alginate (10mg/mL);

The eighth group: normal saline (reference example);

After intratumoral injection of the orthotopic tumor and no injection to the distal tumor, the length and width of the orthotopic tumor and the distal tumor were measured with vernier calipers every two days, and the tumor volume was (length times (width squared) ) divided by 2. Refer to Figure 6 and Figure 7 for the growth curves of orthotopic and distal tumors, and Figure 8 for changes in the body weight of mice.

Table 15. Tumor inhibition rates of orthotopic and distal tumors in each group in Example D3

grouping	In situ tumor inhibition rate (%)	Distant tumor tumor suppression rate (%)
First group	92	86
Second Group	73	58
The third group	88	72
Fourth group	69	70
Group 5	64	88
The sixth group	58	70
Group 7	73	6
eighth group	0	0

Therapeutic effect: the preparation of the present invention can still achieve a therapeutic effect superior to the composition of the non-present invention when the concentration of each component is changed. From the in situ tumor growth curve (Figure 6), when the concentration of mitoxantrone in the composition was 3 mg/mL and the concentration of sodium alginate was 10 mg/mL, the tumor growth was almost completely inhibited. ( FIG. 7 ) analysis, higher concentrations of components can more effectively inhibit the growth of distal tumors, and higher concentrations of components can achieve higher tumor inhibition rates (Table 15). In addition to this, in the absence of sodium alginate, the combination of mitoxantrone and imiquimod did not significantly inhibit tumor growth and, according to the mouse body weight change curve (Figure 8), lacked sodium alginate The preparation of sodium alginate led to a decrease in the body weight of mice, while the three-component preparation of the present invention had little effect on the body weight of mice, indicating that the sodium alginate preparation could significantly reduce the toxic and side effects of the drug. Compared with the composition of the present invention, the distal tumor growth of mice in the group lacking imiquimod was hardly inhibited, indicating that the mitoxantrone composition of the present invention can effectively inhibit the growth of in situ tumors and simultaneously inhibit Distal tumor growth in a dose-dependent manner.

The above description of the disclosed embodiments enables any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art. The present invention is not to be limited to the embodiments shown herein, but only needs to be consistent with the principles and features disclosed herein.

Claims (13)

1. A mitoxantrone composition, characterized in that: comprising a first composition and a second composition;

   The first composition comprises a microparticle suspension formed by a fat-soluble immune adjuvant and a surfactant;

   The second composition includes mitoxantrone or a soluble salt thereof, a readily soluble alginate, a pH adjuster and an excipient.
2. The mitoxantrone composition according to claim 1, wherein the fat-soluble immune adjuvant in the first composition comprises imiquimod R837, resimod R848, or glucopyranoside one or more of A.
3. The mitoxantrone composition according to claim 1, wherein the mitoxantrone soluble salt comprises mitoxantrone hydrochloride or mitoxantrone lactate.
4. The mitoxantrone composition of claim 1, wherein the surfactant in the first composition has a hydrophobic moiety, and the hydrophobic moiety comprises not less than 20 oxypropylenes Base unit; specifically, includes Poloxamer 188, Poloxamer 237, Poloxamer 338 or Poloxamer 407.
5. The mitoxantrone composition of claim 1, wherein the surfactant in the first composition has a hydrophobic moiety, and the hydrophobic moiety contains not less than 15 carbon atoms one or more hydrocarbon chains; specifically, including sorbitan sesquioleate, soybean lecithin, glycerol monostearate, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, Polysorbate 85, Sorbitan Stearyl (Span 60), Stearate, Vitamin E Polyethylene Succinate, Polyoxyethylene Alkyl Ether, Polyoxyethylene Stearate, Polyhydrocarbon Stearate At least one of oxy(40) ester, sucrose stearate, polyoxyethylene castor oil derivative, polycitorol 1000 or lecithin.
6. The mitoxantrone composition according to claim 1, wherein the excipients in the second composition comprise mannitol, lactose, sucrose, simple syrup, sorbitol, polyethylene glycol, One or more of sulfobutyl beta cyclodextrin, hydroxypropyl beta cyclodextrin or sodium carboxymethyl cellulose.
7. The mitoxantrone composition according to claim 1, wherein the pH adjusting agent comprises NaOH, KOH or ammonia water.
8. The mitoxantrone composition according to claim 1, wherein the readily soluble alginate in the second composition comprises one or more of sodium alginate, potassium alginate or ammonium alginate kind.
9. The mitoxantrone composition of claim 1, further comprising a third composition comprising an immune checkpoint inhibitor or an IDO inhibitor.
10. The mitoxantrone composition according to claim 9, wherein the immune checkpoint inhibitor comprises an antibody immune checkpoint blocker, a small molecule inhibitor or a peptide inhibitor.
11. The mitoxantrone composition according to claim 10, wherein the antibody immune checkpoint blocker comprises one of anti-CTLA-4, anti-PD-1 or anti-PD-L1 or more; the small molecule inhibitors include CA-170, PM-327, BMS-8, BMS-37, BMS-202, BMS-230, BMS242, BMS-1001, BMS-1166, BMS-1001, One or more of BMS-1166 or JQ1; the peptide inhibitor includes one or more of DPPA-1.
12. A kind of preparation method of mitoxantrone composition, it is characterized in that comprising the steps:

    S1: Weigh mitoxantrone or its soluble salt and excipient in proportion 1: (1～10), add aqueous solution, stir, add pH adjuster to adjust the pH of the solution to 7.8～9.2, pass the obtained solution through Micron membrane filtration and sterilization to obtain the first liquid;

    S2: Weigh easily soluble alginate and protective filler in proportion 1:(1～5), add water, stir, filter and sterilize the obtained solution through a micron filter to obtain a second liquid;

    S3: Mix the first liquid and the second liquid obtained in S1 and S2 evenly under the protection of nitrogen, filter and sterilize with a 0.22 μm filter membrane, then bottle, pre-cool, freeze-dry, and fill with nitrogen After sealing the bottle.
13. The method for preparing the mitoxantrone composition according to claim 11, wherein in step S2, a pH adjuster is added to adjust the pH of the solution to 8.0-8.7.

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