CN114681437A - Mitoxantrone composition and preparation method thereof - Google Patents

Abstract

The invention discloses a mitoxantrone pharmaceutical composition, which comprises a first composition and a second composition, wherein the first composition comprises composite particles formed by an immunologic adjuvant and a solubilizer, the second composition comprises mitoxantrone hydrochloride or mitoxantrone lactate, soluble alginate, a pH regulator and an excipient, and the compatibility problem of a chemotherapy medicament containing mitoxantrone and a soluble alginate auxiliary material is solved by improving the formulation combination.

Description

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 thereof.

Background

Chemotherapy, surgical resection or radiation therapy are the most common cancer treatment strategies. Chemotherapy is one of the main current clinical treatments for tumors, and is the main treatment for tumors that are prone to metastasis or have metastasized. However, the chemotherapy mode commonly used in clinic is systemic administration, has no good selectivity on pathological change parts, and has toxic and side effects on normal organs. How to inhibit tumor metastasis and prevent recurrence while local treatment is a problem all the time. Many drugs have many problems in the process of transferring from laboratory verification to batch production. Anthraquinone drugs are chemotherapy drugs with wide application, can induce Immunogenic Cell Death (ICD), include doxorubicin, epirubicin, pyrarubicin, mitoxantrone, and the like, generally exist in the form of hydrochloride, and are very ideal choices for chemotherapy immune cocktail therapy. Although there are many reports in the literature and patents that the drugs have good therapeutic effects in the laboratory stage, the related operability of the production and use of drugs, and the long-term stability of the sterilized and stored drug products still face a series of difficulties, such as compatibility conflict between drug components. These drugs cannot exert their combined therapeutic effects when they are not used in combination, but if they are used in combination, they have compatibility problems, sterilization problems and storage stability problems during the process of drug preparation. The failure to solve these pharmaceutical phase problems will result in the failure of many experimental drugs to be truly clinically applied.

Disclosure of Invention

The invention provides a mitoxantrone pharmaceutical composition, provides an anticancer pharmaceutical composition which can generate a synergistic anticancer effect and reduce the probability of cancer metastasis and recurrence, can effectively kill in-situ tumors, can inhibit the growth of distal metastatic tumors and reduce the probability of tumor recurrence through immunoreaction, reduces the side effect of chemotherapy through an in-situ chemotherapy mode, and provides a production and preparation process which can be massively produced, and has good product stability.

The research and development team of this application discovers in the experiment that mixing sodium alginate with the hydrochloric acid salt form of anthraquinone class medicine directly, will appear the agglomeration fast, can lead to pharmaceutical composition system unstable, also the compatibility can not be exactly. Such heterogeneous systems, which are injected directly into the tumor, although still achieving a comparable therapeutic effect, cannot be prepared as standardized pharmaceutical preparations and also have problems of uncertain dosage for direct use, whereas the individual pharmaceutical components will agglomerate after a certain storage time (more than about 6 hours) and cannot be dispersed in an aqueous solution, resulting in complete disuse. The unstable condition and uncertainty of the composition system can occur after long-term storage, so that the combined medicine can not completely meet the requirements of stable quality and long-term storage after the medicine is on the market; further research finds that anthraquinone drugs are generally prepared into preparations in the form of positively charged hydrochloride, sodium alginate is polyanionic macromolecule and has a large amount of negative charges, and aggregation phenomenon is caused by electrostatic interaction with anthraquinone drug molecules, and on the other hand, pi-pi stacking interaction exists between anthraquinone drug molecules (containing a plurality of aromatic ring structures), which is an important reason that the anthraquinone drugs are easy to aggregate under the condition that self charges are shielded.

The research and development team tries to overcome the compatibility problem of the mitoxantrone-containing chemotherapeutic drug and the soluble alginate auxiliary material by improving the formulation combination scheme and the preparation method.

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

a mitoxantrone pharmaceutical composition comprising a first composition comprising composite particles of a fat-soluble immunoadjuvant and a surfactant and a second composition comprising mitoxantrone or a soluble salt thereof, a lyotropic alginate, a pH adjusting agent and an excipient.

In particular, the immunoadjuvant of the first composition comprises one or more of imiquimod (R837), ranisimmod (R848), or glucopyranoside lipid a (mpla).

Specifically, the mitoxantrone or soluble salt thereof comprises mitoxantrone hydrochloride or mitoxantrone lactate.

Further, the mass ratio of the mitoxantrone to the imiquimod is 0.5: 1-1: 20, preferably 1: 2-1: 15.

Further, the hydrophobic moiety of the surfactant contains 20 or more oxypropylene units; specifically included are poloxamer 188, poloxamer 237, poloxamer 338 or poloxamer 407.

In a juxtaposition alternative, the hydrophobic moiety of the surfactant contains one or more hydrocarbon chains having a total number of carbon atoms of not less than 15; the oil-in-water emulsion concretely comprises at least one of sorbitan sesquioleate, soybean phospholipid, glycerin monostearate, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, sorbitan stearate (span 60), stearate, vitamin E polyethylene glycol succinate, polyoxyethylene alkyl ether, polyoxyethylene stearate, polyoxyl stearate (40), sucrose stearate, polyoxyethylene castor oil derivatives, cetostearyl alcohol 1000 or lecithin.

Further, the first composition is a microcomposite particle of the immunoadjuvant and the solubilizing agent. Preferably, the particle size of the imiquimod R837 and poloxamer 188 composite particles is 0.5-5 microns. When the particle size of the immunologic adjuvant is 0.5-5 microns, the immunologic adjuvant can be stably released after the tumor cells are killed by the chemical drug, so that the concentration of the immunologic adjuvant can reach a better proportion at a proper release time, and an immune system can better generate specific immunity.

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

Specifically, the excipient in the second composition comprises one or more of mannitol, lactose, sucrose, simple syrup, sorbitol, polyethylene glycol, sulfobutyl beta cyclodextrin, hydroxypropyl beta cyclodextrin or sodium carboxymethylcellulose.

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

Further, the mitoxantrone composition further comprises a third composition comprising an immune checkpoint inhibitor or an IDO inhibitor.

Specifically, the immune checkpoint inhibitor comprises an antibody immune checkpoint blocker, a small molecule inhibitor or a peptide inhibitor; preferably, the antibody-based immune checkpoint blocking agent comprises one or more of anti-CTLA-4, anti-PD-1 or anti-PD-L1; the small molecule inhibitor comprises one or more of CA-170, PM-327, BMS-8, BMS-37, BMS-202, BMS-230, BMS242, BMS-1001, BMS-1166 and JQ 1; the peptide inhibitor comprises one or more of DPPA-1.

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

Further optionally, the third composition further comprises a gelling auxiliary material, and the gelling auxiliary material comprises a compound of soluble alkaline earth metal ions. The gelling auxiliary materials comprise calcium chloride, calcium sulfate or magnesium chloride and the like.

Further optionally, the second composition further comprises 1% of tween-80 as a surfactant, so that the dissolution rate of the mitoxantrone can be increased.

The invention also provides a preparation method of the mitoxantrone composition, which comprises the following steps:

s1: the proportion is 1: (1-10) weighing mitoxantrone or soluble salt thereof and an excipient, adding a water solution, stirring, adding a pH regulator to regulate the pH of the solution to 7.8-9.2, and filtering and sterilizing the obtained solution by a micron filter membrane;

s2: the weight ratio of 1: (1-5) weighing soluble alginate and a protective filler, adding water, stirring, and filtering and sterilizing the obtained solution through a micron filter membrane;

s3: and (3) uniformly mixing the solutions S1 and S2 under the protection of nitrogen, filtering and sterilizing by using a 0.22 mu m filter membrane, bottling, precooling, freeze-drying, filling nitrogen and sealing.

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

By adopting the technical scheme of the invention, the following beneficial technical effects can be achieved:

the technical scheme of the invention further solves the problems of the feasibility of the treatment scheme and the long-term storage stability of the drug product in the medicament preparation stage. The fat-soluble immunologic adjuvant in the first composition can cause the instability of a suspension to generate obvious precipitates and particles after being subjected to moist heat sterilization at 121 ℃, the water dispersibility is greatly reduced, but the water dispersibility and the stability of the fat-soluble immunologic adjuvant can be ensured by the composite particles of the fat-soluble immunologic adjuvant and the surfactant after being subjected to sterilization.

The technical scheme of the invention solves the problem of compatibility of the mitoxantrone hydrochloride-containing chemotherapeutic drug and the alginate auxiliary material. Solves the problem of drug safety caused by unstable drug composition system due to the agglomeration of the existing mitoxantrone and the hydrochloride thereof and sodium alginate. The dosage form and the preparation method of the related embodiment of the patent overcome the compatibility problem of chemotherapy drug mitoxantrone and sodium alginate as an auxiliary material by simultaneously improving the preparation method of the dosage form, inhibit the interaction of pi electronic stacking between anthraquinone drug molecules, protect the molecular structure from being damaged, avoid the agglomeration problem of each component, simultaneously avoid the oxidation risk of mitoxantrone, greatly increase the feasibility of the finished drug stage, greatly accelerate the redissolution time after freeze-drying, and facilitate the clinical operation. The composition can form a porous reticular cross-linked gel structure after being injected into a tumor body, so that other components mixed in alginate colloid can be slowly released, the chemotherapeutic drug is locked at the tumor part in a limited mode, the systemic exposure level of the chemotherapeutic drug in normal organs in the body is reduced, the effect of the chemotherapeutic drug is enhanced, and the toxic and side effects of the chemotherapeutic drug are reduced.

In addition, the chemotherapeutic drug used in the invention is a chemotherapeutic drug capable of causing tumor immunogenic cell death, and can kill tumor cells and expose tumor-associated antigens, thereby providing targets for helping immune cells to recognize cancer cells, activating immune system and specifically eliminating cancer cells. However, this effect requires the uptake, processing and presentation of antigen to T cells in large numbers to further activate the immune response, and antigen presenting cells require the assistance of an immunoadjuvant to more effectively enrich the tumor site and act. Therefore, while ICD medicines are adopted, immunological adjuvants are required to be introduced to synergically enhance the anti-tumor immune response. The invention combines the two components together, and realizes the technical effects of eliminating tumor cells in situ, generating tumor vaccine in vivo and inhibiting tumor metastasis and recurrence by virtue of a slow-release system.

Drawings

FIG. 1 is a schematic diagram of the formulation steps;

FIG. 2 is a graph of in situ tumor (direct drug injection) growth on the H22 tumor model;

FIG. 3 is a graph of distal tumor (no drug direct injection) growth on the H22 tumor model;

FIG. 4 is a graph of in situ tumor (direct drug injection) growth on the CT26 tumor model;

FIG. 5 is a graph of distal tumor (no drug direct injection) growth on the CT26 tumor model;

FIG. 6 is a graph of the growth of tumors in situ treated with the present formulations at different doses on a CT26 tumor model;

FIG. 7 is a graph of the growth of distal tumors treated with different doses of the present formulation on a CT26 tumor model;

FIG. 8 is a graph of the change in body weight of mice treated with different doses of the present formulation on a CT26 tumor model.

Detailed Description

Example A: preparation of the formulations

FIG. 1 is a schematic representation of the steps for making and using a mitoxantrone pharmaceutical composition.

Example a 1: preparation of the first composition:

weighing a certain amount of immune adjuvant imiquimod R837 solid, and carrying out air flow crushing treatment under the crushing air pressure of 6-10bar to obtain the micron-sized imiquimod R837. The proportion is 1: (0.025-5) weighing a micron-sized immune adjuvant imiquimod R837 and a surfactant poloxamer 188, preferably 2g R837, adding a proper amount of poloxamer 188(0.05g, 0.3g, 0.6g, 1g, 2g, 4g, 6g, 8g and 10g), adding 100mL of water for injection, and stirring at 500rpm for 0.5-2 hours to obtain a suspension. The suspension was homogenized under high pressure for 2-4 times under 750 and 1200ba pressure, and the suspension was filled into 10mL ampoules, 6mL each, for a total of 30 vials, by peristaltic pump. And (3) carrying out heat sealing to obtain a micron suspension, and carrying out moist heat sterilization at 105-150 ℃ for 15-20 minutes.

Poloxamer 188 is a new class of polymeric nonionic surfactants, and has multiple uses including: as an emulsifier, a stabilizer and a solubilizer, the water dispersibility and stability of R837 can be further enhanced.

The hydrophobic structure part of the surfactant contains not less than 20 oxypropylene units; specifically, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407 are included. In a juxtaposition alternative, the hydrophobic moiety of the surfactant contains one or more hydrocarbon chains having a total number of carbon atoms of not less than 15; the oil-in-water emulsion concretely comprises at least one of sorbitan sesquioleate, soybean phospholipid, glycerin monostearate, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, sorbitan stearate (span 60), stearate, vitamin E polyethylene glycol succinate, polyoxyethylene alkyl ether, polyoxyethylene stearate, polyoxyl stearate (40), sucrose stearate, polyoxyethylene castor oil derivatives, cetostearyl alcohol 1000, or lecithin.

Poloxamer is a series of multipurpose pharmaceutic adjuvants, and has the advantages of no toxicity, no antigenicity, no sensitization, no irritation, no hemolysis and stable chemical properties. Poloxamer 188 is one of the series of adjuvants with good safety. Poloxamer 188 can enable micro-scale powder obtained after airflow crushing of imiquimod to be processed by a liquid-phase micro-nano process to obtain imiquimod micro-scale particle suspension with good size uniformity, and poloxamer 188 can also help the imiquimod micro-scale particle suspension (6.0mg/mL or less) to ensure water dispersibility and stability after autoclaving.

However, while poloxamer 188-coated imiquimod microparticle suspensions maintain good suspension stability after autoclaving at lower concentrations (6.0mg/mL), if the imiquimod concentration is too high during sterilization, the imiquimod agglomerates after sterilization and can no longer stabilize the suspension. Lecithin is a natural surfactant, and imiquimod micro-particles which are subjected to high-pressure homogenization treatment by using lecithin as a stabilizer have good stability, and even if the imiquimod micro-particles are sterilized at high temperature under high imiquimod concentration, the suspension of the imiquimod micro-particles still does not agglomerate but keeps stable suspension.

Table 1, imiquimod/poloxamer 188(P188) suspension preparation process and data:

a new technical route of combining air flow grinding with high-pressure homogenization or air flow grinding with a high-shear method is adopted to prepare micron-scale fat-soluble immunologic adjuvant micron particle suspension. The preparation method overcomes the technical prejudice and the actual technical problem in the preparation process of the micron particles, the high-pressure homogenization process or the high-shear process is a liquid-phase processing method, the fat-soluble immunologic adjuvant is a semi-solid medicament, and experiments show that if the high-pressure homogenization or the high-shear process is directly carried out on the fat-soluble immunologic adjuvant, the blockage of a homogenizing valve can be caused, so that the micron particles can not be obtained; although the high-shear method can partially obtain micron particles, the uniformity of the obtained particles is extremely poor, and most particles cannot achieve the expected granulation and pulverization effects and yield; according to the invention, the primary powder is obtained by an air flow crushing process, and then the high-pressure homogenization or high-shear method is carried out under the condition of adding a solution of a surfactant, so that the high-pressure homogenization or high-shear micron particles can be subjected to rapid surface modification and surface modification.

Table 2: adding the micro-particle powder of the imiquimod after airflow crushing into different surfactant aqueous solutions (the mass ratio of the imiquimod to the surfactant is 1: 3), and then carrying out high-pressure homogenization treatment on the obtained mixture to obtain the water dispersible property of the imiquimod

Table 3: redispersibility of imiquimod suspension (6.0mg/mL) with different surfactants as described above after autoclaving (imiquimod: surfactant mass ratio ═ 1: 3)

Because the micron-sized particle suspension needs to be subjected to standard autoclaving operation before being injected into a tumor to meet the requirement of sterility, the micron-sized particles are required to be ensured not to be significantly agglomerated under the condition of about 121 ℃, a surfactant is required to have enough strong adsorption capacity with the particle surface, and hydrophobic interaction is mainly relied on, the hydrophobic structure of the selected surfactant plays an important role in protecting the stability of the micron-sized suspension under autoclaving, and the hydrophobic structure part of the selected surfactant contains one or more hydrocarbon chains with the total number of not less than 15 carbon atoms or the hydrophobic structure part of the surfactant contains not less than 20 oxypropylene units. As in tables 2 and 3, poloxamer 124, due to insufficient hydrophobic structure, was unstable after autoclaving.

Table 4: suspension stability after autoclaving of different P188-dispersed imiquimod suspensions (R837 concentration 6.0mg/mL upon sterilization) added

Poloxamer 188R 837	Suspension stability after autoclaving
		0.5:1	A large amount of granular aggregates appear
1:1	Small amount of granular aggregates appeared
		2:1	Small amount of granular aggregates appeared
3:1	Uniformly dispersed without the appearance of granular aggregates
		4:1	Uniformly dispersed without the appearance of granular aggregates
5:1	Uniformly dispersed without the occurrence of granular aggregates

Although theoretically, the more dispersant the better the dispersion, the ratio is generally not more than 5:1, because: poloxamer 188(P188) is viscous, and has high viscosity when being excessively concentrated; and avoids excessive introduction of impurities into the dispersant.

Table 5: suspension stability of P188 dispersed imiquimod suspensions of different concentrations after autoclaving (P188: imiquimod R837 mass ratio 3: 1). The P188-coated imiquimod suspension maintained good stability when autoclaved at low R837 concentrations, but had significantly reduced autoclave stability at high R837 concentrations.

Concentration of R837 upon sterilization	Suspension stability after autoclaving
		3.0mg/mL	Uniformly dispersed without the occurrence of granular aggregates
6.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
		9.0mg/mL	The appearance of partially particulate aggregates
12.0mg/mL	A large amount of granular aggregates appear
		15.0mg/mL	A large amount of granular aggregates appear
18.0mg/mL	A large amount of granular aggregates appear

Table 6: suspension stability after autoclaving of imiquimod suspensions (R837 concentration: 6.0mg/mL or 18mg/mL at sterilization) dispersed with lecithin at different ratios was added. Lecithin can keep good suspension stability after high-concentration imiquimod suspension is autoclaved even at a lower proportion.

Lecithin R837	Concentration of R837 upon sterilization	Suspension stability after autoclaving
			0.025:1	6.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
0.05:1	6.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
			0.1:1	6.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
0.25:1	6.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
			0.5:1	6.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
1:1	6.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
			0.025:1	18.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
0.05:1	18.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
			0.1:1	18.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
0.25:1	18.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
			0.5:1	18.0mg/mL	Uniformly dispersed without the appearance of granular aggregates
1:1	18.0mg/mL	Uniformly dispersed without the appearance of granular aggregates

Example a 2:

weighing a certain amount of solid of the immune adjuvant Rasimethide (R848), and carrying out air flow crushing treatment under the crushing air pressure of 6-10bar to obtain micron-sized Rasimethide (R848).

The proportion is 1: (0.025-5) weighing the micron-sized immune adjuvant Rasimethide (R848) and poloxamer 407 surfactant, preferably 0.2g R848, adding a proper amount of poloxamer 407(0.005g, 0.01g, 0.2g, 0.4g, 0.8g, 1g), adding 200mL of water for injection, and stirring at 100-500rpm for 0.5-2 hours to obtain a suspension.

Homogenizing the suspension at 750-1200bar pressure for 2-4 times to obtain suspension, and filling the suspension into 10mL ampoules (6 mL each) with a peristaltic pump for 30 bottles. And (3) carrying out heat sealing to obtain a micron suspension, and carrying out moist heat sterilization at 105-150 ℃ for 15-20 minutes.

Poloxamer 407 is a novel class of polymeric nonionic surfactants, having a variety of uses including: as an emulsifier, stabilizer and solubilizer, the water dispersibility and stability of R848 can be further enhanced.

Example a 3:

weighing a certain amount of liposoluble immunologic adjuvant glucopyranoside lipid A (MPLA); the selected surfactant is a mixed surfactant of poloxamer 188 and lecithin in a mass ratio of 9:1, and the other preparation methods are the same as those in the embodiment A2.

Example a 4:

otherwise, as in example A1, a quantity of the lipid-soluble immunoadjuvant imiquimod (R837) is weighed out; the selected surfactant is a mixed surfactant of poloxamer 188 and lecithin in a mass ratio of 3: 1. The suspension stability after autoclaving of R837 was somewhat affected by the dosing concentration of different surfactants, and the results are shown in table 7. The long-term stability after autoclaving of R837 in the presence of lecithin is superior to the effect of P188 solubilisation of R837 alone, resulting in particles with smaller size and better homogeneity. And the influence of the feeding concentration can be enlarged in equal proportion, so that the technical effect of increasing the final concentration of R837 is achieved.

Table 7: suspension stability after R837 autoclaving with different concentrations of surfactant

R837 poloxamer 188 lecithin	Long term stability after autoclaving
		12mg/mL:36mg/mL:0mg/mL	A large amount of granular aggregates appear
12mg/mL:36mg/mL:12mg/mL	Uniformly dispersed without the occurrence of granular aggregates
		18mg/mL:54mg/mL:0mg/mL	A large amount of granular aggregates appear
18mg/mL:54mg/mL:18mg/mL	Uniformly dispersed without the appearance of granular aggregates

Therefore, the mixing of the two surfactants can further increase the suspension stability performance of the self-sustained-release immunologic adjuvant micron particles in autoclaving, and particularly the suspension stability performance is remarkable at higher surfactant concentration. Two or more surfactant combinations different in hydrophilic-lipophilic balance (HLB value) or two surfactants different in hydrophobic structure portion (for example, one surfactant containing not less than 20 oxypropylene units, or one surfactant containing one or more hydrocarbon chains not less than 15 carbon atoms in total) are used as the coating layer of the microparticles. The two surfactants with different solubilities are not completely and homogeneously dispersed with each other, but form a relatively uniform and locally gathered dispersion structure, after the formed coating composite particles enter a tumor body, the surfactant with a larger HLB value is firstly dissolved, so that a plurality of tiny openings or tiny defect regions are formed on the surface of the coating of the micrometer particles, the surface area of the inner layer immunologic adjuvant micrometer particles is gradually changed, effective components are gradually released, and a medicament combination scheme with various models can be obtained by adjusting the selection or proportioning relation of two or more surfactants according to the actual requirements of different tumor bodies and human bodies.

Table 8: variation in particle size after R837 autoclaving with addition of different proportions of surfactant

Meanwhile, as shown in table 8, the presence of both lecithin and P188 resulted in minimal changes in particle size before and after sterilization of R837 and a smaller particle size distribution range, i.e., the presence of both lecithin and P188 more contributed to the stability of the sample during the sterilization process. Wherein 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%, Dmax is the maximum particle size of the particles in the sample, and the smaller the difference between the three is, the higher the uniformity of the sample particles is. In the experiment, the suspension sample with the simultaneous existence of the P188 and the lecithin can not generate wall hanging after being stored for a long time. It is worth noting that microparticle size uniformity is an important parameter to ensure stable and reproducible drug release behavior in vivo.

Example B:

example B1: preparation of the second composition:

s1: preparing a sodium alginate/mannitol or sodium alginate/lactose solution according to the proportion 1 (1-5), wherein the concentration of the sodium alginate solution is 2.5mg/mL, 5mg/mL, 10mg/mL, 20mg/mL and 40mg/mL, the final concentration of excipient mannitol or lactose is 1-50 mg/mL, 20-100 mg/mL and 40-200 mg/mL, and the mannitol or lactose is added after the sodium alginate solution is uniformly stirred;

s2: preparing 1mg/mL, 2mg/mL and 3mg/mL mitoxantrone hydrochloride solutions, and adjusting the pH to 8.0-8.7 by using 20mg/mL sodium hydroxide solution;

s3: and (2) uniformly mixing the solutions S1 and S2 according to the volume ratio of 1:1 under the condition of nitrogen protection at the temperature of 2-8 ℃ or room temperature, filtering and sterilizing by using a 0.22 mu m filter membrane, then subpackaging in penicillin bottles, precooling, freeze-drying, filling nitrogen, and sealing the bottles.

Example B2: preparation of the second composition:

s1: preparing a sodium alginate/mannitol solution according to the proportion 1 (1-5), wherein the concentration of the sodium alginate solution is 5mg/mL, 10mg/mL, 20mg/mL and 40mg/mL, the mannitol is added after the sodium alginate solution is uniformly stirred, and the final concentration of the mannitol is 1-50 mg/mL, 20-100 mg/mL and 40-200 mg/mL;

s2: preparing a 6mg/mL mitoxantrone hydrochloride solution, and adjusting the pH value to 8.5-9.2 by using a 20mg/mL sodium hydroxide solution;

s3: and (2) dissolving the S1 and the S2 in nitrogen at 2-8 ℃ or room temperature, uniformly mixing according to a proper proportion, filtering and sterilizing by using a 0.22 mu m filter membrane, then subpackaging in penicillin bottles, precooling, freeze-drying, filling nitrogen and sealing the bottles.

Example B3: preparation of the second composition:

s1: preparing a sodium alginate/lactose solution according to the proportion 1 (1-5), wherein the concentration of the sodium alginate solution is 5mg/mL, 10mg/mL, 20mg/mL and 40mg/mL, the lactose is added after the sodium alginate solution is uniformly stirred, and the final concentration of the lactose is 1-50 mg/mL, 20-100 mg/mL and 40-200 mg/mL;

s2: preparing a 6mg/mL mitoxantrone hydrochloride solution, and adjusting the pH value to 7.8-8.5 by using a 20mg/mL sodium hydroxide solution;

s3: and (2) dissolving the S1 and the S2 in nitrogen at 2-8 ℃ or room temperature, uniformly mixing according to a proper proportion, filtering and sterilizing by using a 0.22 mu m filter membrane, then subpackaging in penicillin bottles, precooling, freeze-drying, filling nitrogen and sealing the bottles.

Table 9, influence of pH in the preparation of mitoxantrone/sodium alginate/mannitol.

Note: too high a pH may accelerate the oxidation of mitoxantrone. An overly basic environment can result in deprotonation of the phenolic hydroxyl group of mitoxantrone, and such molecules can be readily oxidized. Therefore, the pH should not be too high.

Further experimental verification shows that for anthraquinone chemicals such as doxorubicin hydrochloride, epirubicin hydrochloride, pirarubicin hydrochloride and the like, even if protons of the anthraquinones chemicals are neutralized by NaOH, the anthraquinones still agglomerate after being mixed with sodium alginate. The mitoxantrone is a special case, and can be combined with sodium alginate without agglomeration in a certain interval along with the increase of the pH value, so that the mitoxantrone is special for the mitoxantrone.

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

the effect of nitrogen conditions on the stability of mitoxantrone/sodium alginate was investigated. Mixing mitoxantrone and sodium alginate under nitrogen-filled condition, wherein the content of single impurity and total impurity of the prepared mitoxantrone/sodium alginate is slowly changed, and the content of single impurity and total impurity is still maintained within the limit at the acceleration condition of 60 ℃ for 30 days; if the nitrogen gas is not used for protection during the mixing of mitoxantrone and sodium alginate, the impurity content is exceeded at day 5. It is necessary to demonstrate that the nitrogen gas charging step of the present invention is advantageous for the stability of the formulation.

Example B4: research on sustained-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％

pH7.4 (+ 1% Tween-80)

4.8％

18.2％

36.6％

63.4％

70.4％

78.3％

pH4.0 (+ 1% Tween-80)

5.3％

19.4％

37.2％

60.7％

69.1％

75.5％

The release rate of mitoxantrone from the sodium alginate/calcium ion hydrogel varied with time. Putting the sodium alginate/calcium ions wrapping the mitoxantrone into a gauze belt, and dialyzing in buffer solutions with different pH values. The buffer solution with pH7.4 is added with 2mM CaCl₂The pH of the phosphate buffer solution of (3) is 4.0, and the buffer solution is acetic acid-sodium acetate buffer solution. In order to increase the dissolution rate, 1% tween-80 can be added to the buffer. The release of mitoxantrone from sodium alginate/calcium ion hydrogel is slower under both acidic and neutral conditions, due to the stronger interaction of mitoxantrone with sodium alginate after protonation. In order to increase the dissolution rate of mitoxantrone, 1% tween-80 was added to the solution in the last two groups of the experiment as a surfactant. The control of the release rate of mitoxantrone can be achieved very significantly by adjusting the amount of surfactant added to the composition.

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 rate of release of imiquimod from the sodium alginate/calcium ion hydrogel varies over time. Putting the sodium alginate/calcium ions wrapping imiquimod into a gauze belt, and dialyzing in buffer solutions with different pH values. The buffer solution with pH7.4 is added with 2mM CaCl₂The pH of the phosphate buffer solution of (3) is 4.0, and the buffer solution is acetic acid-sodium acetate buffer solution. Imiquimod released more rapidly under acidic conditions. Control of the rate of release of mitoxantrone can be achieved very significantly by adjusting the pH of the composition.

Example C:

instructions for mixing the drug solution with the lyophilized formulation:

the first use scheme is as follows: the second composition freeze-dried powder injection is dissolved in the suspension of the first composition, the composition solution is directly injected to the tumor part of a patient in a mode of clinical intervention administration and direct puncture administration, and the composition solution is ensured to be uniformly filled in the whole tumor by adopting a mode of multi-point injection during injection.

The use scheme II comprises the following steps: having a third composition, said third composition being administered by intravenous injection.

The use scheme is three: after the focus of a tumor patient is removed by normal operation, considering the problem that tumor cells at the focus of the tumor patient cannot be completely removed by the surgical removal, the freeze-dried powder injection of the second composition can be dissolved in the suspension of the first composition, then the first composition is sprayed on the wound site after the surgical removal by using a syringe or a spray bottle, then a proper amount of calcium chloride solution can be sprayed on the site to gelatinize the site, and finally the wound is sutured.

The use scheme is four: with the third composition, the third composition may be injected intravenously or sprayed onto the wound. The scheme helps to eliminate residual cancer cells and can inhibit tumor metastasis and recurrence.

After the composition is injected into a tumor, firstly, alginate in the second composition is utilized to encounter calcium ions in organism tissues or gelling auxiliary materials in the third composition to be quickly gelled to form a porous reticular cross-linked structure, so that other components mixed in the reticular cross-linked structure can be slowly released, and thus, mitoxantrone chemotherapeutic drugs are limited, the effect of the mitoxantrone chemotherapeutic drugs is enhanced, and the toxic and side effects of the mitoxantrone chemotherapeutic drugs are reduced; secondly, the ICD chemotherapeutic mitoxantrone in the second composition can not only effectively kill tumor cells, but also cause the tumor cells to generate immunogenicity and die, generate tumor-associated antigens and activate tumor specific immunoreaction; thirdly, the immune adjuvant in the first composition enhances the capacity of antigen presenting cells, and further amplifies the corresponding immune response; finally, the use of a third class of components immune checkpoint inhibitors or IDO inhibitors prevents the metastatic tumor from escaping the immune response, allowing immunotherapy to more effectively kill the tumor, thereby inhibiting metastasis and recurrence of the tumor.

Before use, the suspension of the first composition of the invention is added to the lyophilisate of the second composition, and then intratumoral injection is carried out. Killing in-situ tumor, promoting immunogenic cell death, activating tumor specific immunoreaction, and inhibiting the growth of far-end tumor by immunoreaction, and the recurrence of tumor can be inhibited by immunological memory effect.

Cancer treatment is a very complex result, because both the immune system of the body and the growth mechanisms of cancer cells are very complex. The experiment can achieve relatively excellent treatment effect, and except for the explanation of other parts of the patent, the experiment also can include the following reason that imiquimod R837 micron particles are adopted, and water-insoluble R837 powder is crushed in a liquid phase to obtain a particle size of 0.5-5 microns, so that the imiquimod R837 micron particles have both water phase dispersibility and a proper release period in an in-situ gel forming state, and can be better matched with other medicine components.

Poloxamer 188 is added into the R837 emulsion, and poloxamer 188 is a novel high-molecular nonionic solubilizer and has multiple purposes including: as an emulsifier, a stabilizer and a solubilizer, the water dispersibility and stability of the R837 emulsion can be further enhanced.

The composition can form a porous reticular cross-linked structure after being injected into a tumor body, so that other components mixed in alginate colloid can be slowly released, thereby carrying out limited-domain locking on the chemotherapeutic drug containing carboxylic acid ligand, enhancing the effect and reducing the toxic and side effects. In addition, when the particle size of the immunologic adjuvant is 0.5-5 micrometers, the immunologic adjuvant can be stably released after the tumor cells are killed by the chemical drug, so that the concentration for enhancing the immunologic effect reaches the optimal matching relation on the release time.

The hydrophobic structure part of the surfactant contains not less than 20 oxypropylene units; specifically, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407 are included. In a juxtaposition alternative, the hydrophobic moiety of the surfactant contains one or more hydrocarbon chains having a total number of carbon atoms of not less than 15; the oil-in-water emulsion concretely comprises at least one of sorbitan sesquioleate, soybean phospholipid, glycerin monostearate, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, sorbitan stearate (span 60), stearate, vitamin E polyethylene glycol succinate, polyoxyethylene alkyl ether, polyoxyethylene stearate, polyoxyl stearate (40), sucrose stearate, polyoxyethylene castor oil derivatives, cetostearyl alcohol 1000, or lecithin.

Poloxamers are a series of multipurpose pharmaceutical excipients, and are non-toxic, non-antigenic, non-allergenic, non-irritating, non-blood-soluble and chemically stable. Poloxamer 188 is one of the series of adjuvants with good safety.

The excipients used included: one or more of mannitol, lactose, sucrose, simple syrup, sorbitol, polyethylene glycol, sulfobutyl beta cyclodextrin, hydroxypropyl beta cyclodextrin or sodium carboxymethylcellulose has good protective effect on mitoxantrone and sodium alginate.

Example D: the therapeutic effects produced by the compositions of the present invention are as follows

Example D1: the preparation can be used for treating H22 hepatocarcinoma model (bilateral tumor model)

Mice H22 liver cancer tumors (right as in-situ tumors and left as distal tumors) were planted on the left and right ends of the back of the mice, and the tumor-bearing mice were divided into six groups of six mice each, and injected intratumorally.

A first group: physiological saline;

second group: vinorelbine (4 mg per kg body weight)/imiquimod/sodium alginate;

third group: vincristine (1 mg per kg body weight)/imiquimod/sodium alginate;

and a fourth group: paclitaxel (3mg per kg body weight)/imiquimod/sodium alginate;

and a fifth group: docetaxel (4 mg per kg body weight)/imiquimod/sodium alginate;

a sixth group: mitoxantrone (3mg per kg body weight)/imiquimod/sodium alginate.

After intratumoral injection of the left orthotopic tumor, the right distal tumor was not injected and the length and width of the orthotopic and distal tumors were measured every two days with a vernier caliper, the volume of the tumor being (length multiplied by (width squared)) divided by 2. The growth curves of the in situ tumor and the distal tumor are shown in fig. 2 and fig. 3.

In situ and distal tumor inhibition rates for each of the groups in Table 13, example D1

Grouping	Tumor inhibition in situ (%)	Tumor inhibition rate of distal tumors (%)
			First group	0	0
Second group	73	46
			Third group	77	42
Fourth group	53	8
			Fifth group	75	68
Sixth group	90	66

The treatment effect is as follows: from the in situ tumor growth curve (fig. 2), the injection site tumors of the mice in the group 6 were significantly inhibited, and the tumor inhibition rates were all over 90% (table 13), which are significantly better than those of the other groups. From the growth curve of the distal tumor (fig. 3), the mice in groups 5 and 6 have significantly inhibited distal tumors, and the tumor inhibition rate is over 60%, thus showing significant therapeutic effect. The present formulation proved to be the most effective of a variety of drugs.

Example D2: treatment experiment of the preparation on CT26 colon cancer tumor model (bilateral tumor model)

The left and right ends of the back of the mouse are respectively planted with mouse CT26 colon cancer tumor (the right side is regarded as the in situ tumor, and the left side is regarded as the distal tumor), and the tumor-bearing mice are divided into five groups, six of each group are respectively injected into the tumor.

A first group: physiological saline (reference example);

second group: vinorelbine (4 mg per kg body weight)/imiquimod/sodium alginate;

third group: docetaxel (4 mg per kg body weight)/imiquimod/sodium alginate;

and a fourth group: mitoxantrone (3mg per kg body weight)/sodium alginate;

and a fifth group: mitoxantrone (3mg per kg body weight)/imiquimod/sodium alginate.

After intratumoral injection of the left orthotopic tumor, the right distal tumor was not injected and the length and width of the orthotopic and distal tumors were measured every two days with a vernier caliper, the volume of the tumor being (length times (width squared)) divided by 2.

In situ and distal tumor inhibition rates for each of the groups in Table 14, example D2

Grouping	Tumor inhibition in situ (%)	Tumor inhibition rate of distal tumors (%)
			First group	0	0
Second group	79	33
			Third group	69	27
Fourth group	67	65
			Fifth group	97	96

The treatment effect is as follows: from the in situ tumor growth curve (fig. 4) and the distal tumor growth curve (fig. 5), the injection site tumor and the distal tumor of the mice in the group 5 were significantly inhibited, and hardly grew, and the tumor inhibition rate was over 90% (table 14), which is significantly better than that of the other groups. The present formulation proved to be most effective in a variety of drug combinations.

Example D3: treatment experiment of different doses of the preparation on mouse CT26 tumor model

And then, the left and right ends of the back of the mouse are respectively planted with mouse CT26 colon cancer tumor (the right side is regarded as the original tumor, and the left side is regarded as the distal tumor), and the tumor-bearing mice are divided into 8 groups, and 6 mice in each group are respectively injected into the tumor.

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

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

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

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

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

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

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

group eight: physiological saline (reference example);

after intratumoral injection of the left orthotopic tumor, the right distal tumor was not injected and the length and width of the orthotopic and distal tumors were measured every two days with a vernier caliper, the volume of the tumor being (length multiplied by (width squared)) divided by 2. The growth curves of the in situ tumor and the distal tumor are shown in FIG. 6 and FIG. 7, and the change of the body weight of the mouse is shown in FIG. 8.

Tumor inhibition rates for the groups in Table 15, example D3 for in situ and distant tumors

Grouping	Tumor inhibition in situ (%)	Tumor inhibition rate of distal tumors (%)
			First group	92	86
Second group	73	58
			Third group	88	72
Fourth group	69	70
			Fifth group	64	88
Sixth group	58	70
			Seventh group	73	6
Eighth group	0	0

The treatment effect is as follows: the formulations of the present invention can still achieve superior therapeutic effects over non-inventive compositions when the concentrations of the components are varied. From the in situ tumor growth curve (fig. 6), the mitoxantrone concentration in the composition is 3mg/mL, and the sodium alginate concentration is 10mg/mL, the composition can almost completely inhibit the tumor growth, meanwhile, according to the analysis of the far-end tumor growth curve (fig. 7), the composition can more effectively inhibit the far-end tumor growth when the component concentration is higher, and the higher component concentration can realize the higher tumor inhibition rate (table 15). In addition, the composition of mitoxantrone and imiquimod did not significantly inhibit tumor growth in the absence of sodium alginate, and the sodium alginate-deficient formulation resulted in a decrease in weight of mice according to the weight change curve of mice (fig. 8), whereas the three-component formulation described in the present invention had little effect on weight of mice, indicating that the sodium alginate formulation can significantly reduce the toxic side effects of the drug. Compared with the composition disclosed by the invention, the growth of the distal tumor of the mice in the group lacking imiquimod is hardly inhibited, which shows that the mitoxantrone composition disclosed by the invention can effectively inhibit the growth of the in-situ tumor and the growth of the distal tumor, and has certain dose dependence.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art. The present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and features disclosed herein.

Claims (10)

1. A mitoxantrone composition characterized by: a first composition and a second composition;

the first composition comprises a microparticle suspension formed by a fat-soluble immunologic adjuvant and a surfactant;

the second composition comprises mitoxantrone or a soluble salt thereof, a lyotropic alginate, a pH adjuster, and an excipient.

2. Mitoxantrone composition according to claim 1 characterized in that: the lipid soluble immune adjuvant in the first composition comprises one or more of imiquimod (R837), ranimod (R848), or glucopyranoside lipid a (mpla).

3. Mitoxantrone composition according to claim 1 characterized in that: the mitoxantrone soluble salt comprises mitoxantrone hydrochloride or mitoxantrone lactate.

4. Mitoxantrone composition according to claim 1 characterized in that: the hydrophobic structure part of the surfactant contains not less than 20 oxypropylene units; specifically, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407 are included.

5. Mitoxantrone composition according to claim 1 characterized in that: the hydrophobic moiety of the surfactant contains one or more hydrocarbon chains having a total number of carbon atoms of not less than 15; the compound is characterized by specifically comprising sorbitan sesquioleate, soybean phospholipid, glycerin monostearate, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, sorbitan stearate (span 60), stearate, vitamin E polyethylene glycol succinate, polyoxyethylene alkyl ether, polyoxyethylene stearate, polyoxyl stearate (40), sucrose stearate, polyoxyethylene castor oil derivatives, cetostearyl 1000 or lecithin.

6. Mitoxantrone composition according to claim 1 characterized in that: the excipients in the second composition include one or more of mannitol, lactose, sucrose, simple syrup, sorbitol, polyethylene glycol, sulfobutyl beta cyclodextrin, hydroxypropyl beta cyclodextrin, or sodium carboxymethylcellulose.

7. Mitoxantrone composition according to claim 1 characterized in that: the pH regulator comprises NaOH, KOH or ammonia water.

8. Mitoxantrone composition according to claim 1 characterized in that: the soluble alginate in the third composition comprises one or more of sodium alginate, potassium alginate or ammonium alginate.

9. A method for preparing a mitoxantrone composition, comprising the steps of:

s1: the proportion is 1: (1-10) weighing mitoxantrone or soluble salt thereof and an excipient, adding a water solution, stirring, adding a pH regulator to regulate the pH of the solution to 7.8-9.2, and filtering and sterilizing the obtained solution by a micron filter membrane;

s2: the weight ratio of 1: (1-5) weighing soluble alginate and a protective filler, adding water, stirring, and filtering and sterilizing the obtained solution through a micron filter membrane;

s3: and (3) uniformly mixing the solutions S1 and S2 under the protection of nitrogen, filtering and sterilizing by using a 0.22 mu m filter membrane, bottling, precooling, freeze-drying, filling nitrogen and sealing.

10. The method for preparing a mitoxantrone composition in accordance with claim 9 wherein in step S2 a pH adjusting agent is added to adjust the pH of the solution to 8.0 to 8.7.

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