CN114010592B

CN114010592B - Imiquimod suspension preparation capable of being injected in tumor or around tumor as well as preparation method and application thereof

Info

Publication number: CN114010592B
Application number: CN202111307908.3A
Authority: CN
Inventors: 刘庄; 陶惠泉; 邓中清; 柏上; 周炫坊
Original assignee: Suzhou Baimai Biomedical Co ltd
Current assignee: Suzhou Baimai Biomedical Co ltd
Priority date: 2021-11-05
Filing date: 2021-11-05
Publication date: 2024-02-06
Anticipated expiration: 2041-11-05
Also published as: CN114010592A; CN116942609A

Abstract

The invention discloses an imiquimod suspension preparation capable of being injected in or around tumor, and a preparation method and application thereof. The imiquimod suspension preparation can realize stable dispersion effect after high-temperature high-pressure sterilization.

Description

Imiquimod suspension preparation capable of being injected in tumor or around tumor as well as preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to imiquimod suspension preparation capable of being injected in or around tumor, and a preparation method and application thereof.

Background

Imiquimod (R837) is an imidazole quinine amine small molecule immunomodulator, and the molecule is not a cytotoxic drug, and has no obvious effect of directly killing viruses or tumor cells. Imiquimod is a ligand of Toll-like receptor 7 (TLR 7) and can stimulate macrophages, monocytes and dendritic cells, induce the production of interferon alpha (IFN-alpha) and tumor necrosis factor alpha (TNF-alpha), and simultaneously stimulate the production of cytokines such as interleukin-2 (IL-2), IL-6, IL-8 and the like, thereby further stimulating the activation of cellular immunity, recognizing viruses or other tumor antigens, stimulating related immune responses and eliminating pathogenic factors. Imiquimod is applied to the treatment of condyloma acuminatum and is mainly used for external application.

Based on the stimulatory effect of imiquimod on the immune system, more and more researches clarify the mechanism of imiquimod in anti-tumor immunotherapy, which shows that imiquimod can be applied to anti-tumor immunotherapy as an immune adjuvant.

Currently, imiquimod has been approved for the treatment of head and neck actinic keratosis and superficial basal cell carcinoma. In addition, a plurality of clinical experiments prove that imiquimod plays an immune adjuvant effect in the treatment of superficial tumors such as squamous cell carcinoma, metastatic melanoma, intraepithelial neoplasia of vulva and the like, and has certain application potential.

However, imiquimod itself is a small fat-soluble molecule, poorly soluble in water, and at the same time, imiquimod has a strong skin irritation, and by applying 5% imiquimod cream to the naked skin of mice, a model of psoriasis-like skin lesions in mice can be established, which is sufficient to demonstrate the irritation of imiquimod to normal tissues. The external administration is advantageous and disadvantageous, and although it has a better immunopotentiating effect on the immunotherapy of individual superficial lesions, it also limits the immunotherapeutic applications of imiquimod in other tumors.

Currently, there are two main approaches to preparing injection solutions containing imiquimod, one is to directly dissolve imiquimod with an acid, for example, dissolving imiquimod in a hydrochloride form, and dispersing the imiquimod in an aqueous phase. However, the solution obtained by this method has a low pH, generally about 3.0 to 4.0, and the solution at this pH is used for living organisms and has a certain irritation. In addition, imiquimod hydrochloride as a small molecule can rapidly ooze out of the tumor and enter blood after being injected into the tumor, so that the imiquimod hydrochloride has higher acute exposure (brings safety risk) in the blood after being injected, and meanwhile, the half-life of the imiquimod hydrochloride in the tumor is very short and can be rapidly cleared, so that the immune activation effect after the intratumoral administration cannot be maintained for a long time.

Another way to prepare imiquimod injection is to load R837 with amphiphilic macromolecules or other nanostructures that can load hydrophobic drugs. However, the preparation process of the nano particles is often complex, which is unfavorable for process amplification and standardized mass production. In addition, these nanoparticle formulations tend to be difficult to stabilize under terminal autoclaving conditions (terminal autoclaving is the preferred sterilization strategy for injection).

Therefore, the development of injectable imiquimod formulations as immunoadjuvants for use in immunotherapy of non-superficial tumors is of great importance. The preparation can realize long-time retention and slow release of imiquimod in tumors, and reduce the exposure of imiquimod in blood and normal tissues so as to ensure the safety of clinical use of imiquimod; in addition, in order to meet the demands of industrial transformation, the preparation method of the preparation needs to be capable of realizing scale-up, and the stability of the preparation needs to meet the demands of terminal high-temperature high-pressure sterilization.

Disclosure of Invention

In order to solve the related technical problems, the invention provides an imiquimod suspension preparation which can be injected in or around tumor, comprising imiquimod micron particles, a surfactant containing a higher fatty acid chain and a dispersion medium. Wherein the dispersion medium is water, physiological saline or glucose solution.

Specifically, the average particle size of imiquimod microparticles is 0.5-5.0 μm.

Wherein the surfactant containing a higher fatty acid chain is an ionic surfactant containing a higher fatty acid chain.

Specifically, the surfactant containing a higher fatty acid chain includes anionic surfactants and amphiphilic ionic surfactants.

Specifically, the surfactant containing a higher fatty acid chain includes linear alkyl carboxylate, linear alkyl sulfonate, linear alkyl sulfate, linear alkanol sulfate, and the like.

Specifically, the anionic surfactant containing higher fatty acid chains is sodium oleate, sodium dodecyl sulfate, sodium stearate, sodium N-lauroyl sarcosinate, sodium cocoyl methyl taurate, sodium N-lauroyl glutamate, sodium laureth carboxylate, and dodecyl phosphate.

Preferably, the surfactant containing a higher fatty acid chain is a phospholipid ionic surfactant.

Specifically, the phospholipid ionic surfactant is lecithin, soybean phospholipid, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol.

Preferably, the dispersion medium is water.

Preferably, the mass ratio of the surfactant containing the higher fatty acid chain to imiquimod is 0.025-3: 1.

preferably, the mass ratio of the surfactant containing the higher fatty acid chain to imiquimod is 0.1-1: 1.

the imiquimod suspension preparation which can be injected in or around tumor can be retained and slowly released in the tumor for a long time, and is further combined with therapies of tumor cell immunogenicity death caused by chemotherapy, radiotherapy, alcohol ablation and the like, so that the anti-tumor immune response is obviously enhanced, the in-situ tumor is effectively eliminated, the systemic anti-tumor immune response is induced, and the tumor metastasis and the growth of the far-end tumor are inhibited. Meanwhile, the micro imiquimod suspension preparation has better stability, can realize high-temperature high-pressure sterilization and achieves the preparation safety standard of clinical application. The micron-sized imiquimod suspension capable of terminal sterilization has the characteristics of simple components, simple and convenient preparation, stable finished product and sterile low pyrogen.

The invention provides a preparation method of imiquimod suspension preparation capable of being injected in or around tumor.

The method comprises the following steps:

s1: dispersing a surfactant containing a higher fatty acid chain and imiquimod micron particles in the same dispersion system, and stirring to obtain a suspension;

s2: homogenizing the suspension;

s3: filling the homogenized suspension, sealing, and sterilizing at high temperature and high pressure.

Wherein the conditions of high-temperature high-pressure sterilization are 110-145 ℃ for 5-30 min.

Specifically, the micro imiquimod suspension preparation is subjected to high-temperature high-pressure sterilization treatment, and the micro imiquimod suspension preparation is free of coagulation or caking, or can be redispersed into uniform suspension through simple shaking after caking/coagulation.

The invention also provides application of the imiquimod suspension preparation in preparing an anti-tumor combined immunotherapy preparation.

Specifically, the imiquimod suspension preparation can realize slow release of chemical drugs after being mixed with the platinum chemical drugs.

Specifically, the imiquimod suspension preparation can realize slow release of chemicals after being mixed with anthracyclines.

In particular, the imiquimod suspension preparation is used for preparing the preparation for enhancing anti-tumor immunotherapy. In particular embodiments, an effective dose of a micronized imiquimod suspension formulation may be administered to a patient in need thereof, wherein the micronized imiquimod suspension formulation is used in a intratumoral or peritumoral injection.

The technical scheme of the invention has the following technical effects:

the terminally sterilizable micro-sized imiquimod suspension of the present invention provides injectable imiquimod suspension formulations capable of applying imiquimod in the immunotherapy of non-superficial tumors. With the help of the surfactant containing the higher fatty acid chain, the sterile and pyrogen-free stable dosage form can be obtained by high-temperature high-pressure sterilization, and has good uniformity and stability. Compared with the imiquimod hydrochloride small molecule injection, the micro-sized imiquimod suspension has longer half-life in tumor; compared with imiquimod nano-particle preparation, the preparation process of the micro-sized imiquimod suspension has stronger feasibility of amplified production, can keep the long-term stability of the dosage form after high-temperature high-pressure sterilization, and can meet the clinical use requirement.

The imiquimod suspension can be applied to enhanced anti-tumor immunotherapy by combining therapy means such as radiotherapy and chemical ablation in an intratumoral or peritumoral injection mode, and can be injected after being premixed with platinum drugs or anthracyclines, so that slow release of the drugs can be caused, the acting time of the drugs at focus positions can be prolonged, the anti-tumor immune response of the combined drugs can be enhanced, the growth of remote tumors can be effectively inhibited, and tumor metastasis and recurrence can be prevented.

Drawings

FIG. 1 is a photograph of a suspension of imiquimod of micron size sterilized with the addition of different surfactants containing higher fatty acid chains, after shaking;

FIG. 2 is an in situ tumor growth curve of mice of different groups in a micrometer-sized imiquimod suspension combined with radiotherapy experiment;

FIG. 3 is a graph showing distal tumor growth in mice of different groups in a micrometer-sized imiquimod suspension in combination with radiotherapy experiment;

FIG. 4 is a graph of tumor growth in mice of different groups in a micrometer-sized imiquimod suspension in combination with alcohol ablation therapy experiment;

FIG. 5 is a graph showing the relative amounts of platinum in major tissues and organs before and after oxaliplatin is mixed with a suspension of imiquimod in the form of a micrometer scale, 72 hours after intratumoral injection;

FIG. 6 is a graph showing the concentration of drug in blood over time after intratumoral injection, before and after mixing oxaliplatin with a suspension of imiquimod in the micrometer scale;

FIG. 7 is a graph of tumor growth in a bilateral mouse tumor model with in situ tumors, in the vehicle control group, oxaliplatin single agent treatment group, imiquimod suspension formulation treatment group, oxaliplatin and imiquimod suspension formulation combination treatment group, respectively;

FIG. 8 is a graph of tumor growth curves for a bilateral tumor model and a distant tumor in mice, with groups of vehicle control, oxaliplatin single agent treatment, imiquimod suspension formulation treatment, oxaliplatin and imiquimod suspension formulation combination treatment, respectively;

FIG. 9 is a graph of the in vitro release profile of doxorubicin following mixing with a micronized imiquimod suspension formulation wherein the suspending agent in the imiquimod suspension formulation is lecithin;

FIG. 10 is a graph of the in vitro release profile of doxorubicin following mixing with various concentrations of a suspension formulation of microscale imiquimod wherein the suspending agent in the imiquimod suspension formulation is lecithin;

FIG. 11 is a graph of epirubicin release in vitro following mixing with a micrometer sized imiquimod suspension formulation wherein the suspending agent in the imiquimod suspension formulation is lecithin;

fig. 12 is a graph of epirubicin release in vitro following mixing with a micrometer sized imiquimod suspension formulation wherein the suspending agent in the imiquimod suspension formulation is phosphatidylglycerol.

Detailed Description

Example a: examination of suspension aid

Example A1: preparation of imiquimod suspension

Preparation of imiquimod suspension formulations of various suspending agents.

Imiquimod suspensions were prepared using the example of the surfactant lecithin containing higher fatty acid chains.

S1: preparing lecithin into uniform suspensions with different concentrations, adding imiquimod powder to enable the concentration of the imiquimod to be 1-18 mg/mL, and stirring the suspension;

s2: homogenizing the suspension obtained in the step S1;

s3: filling the homogenized suspension, sealing, and sterilizing at high temperature and high pressure. The conditions of high temperature and high pressure sterilization are as follows: sterilizing at high temperature of 110-145 ℃ for 5-30 minutes.

Example A2: influence of the stability of different suspending agents imiquimod suspensions

The choice of the suspending agent is based on several factors, firstly, as the suspending agent applied to the injection formulation, the approved injection-grade pharmaceutic adjuvant is selected, so that the potential safety hazard of the suspending agent is avoided; second, the suspending agent itself cannot chemically react with the drug molecule to alter the drug activity or increase the toxicity.

It is primarily determined from the three aspects whether the suspending agent contributes to stabilization of the imiquimod suspension after terminal sterilization.

First, the appearance changes of the suspensions before and after autoclaving were observed and the samples were defined as stable, general, unstable based on the appearance changes. Specifically, whether macroscopic particles or agglomerates, sticking to the wall, and no redispersion occurred or not was observed, and the corresponding situation was recorded. When the sample does not have the phenomenon, the sample is considered to have better stability after sterilization; when the sample is sterilized and the sample is subjected to the phenomenon, but can be redispersed to obtain uniform suspension after shaking or jolt, the sample is regarded as a general state after sterilization; a sample is considered unstable when it is subjected to the above phenomena after sterilization and no redispersed suspension is obtained after varying degrees of shaking or jolt.

Secondly, the particle size distribution in the imiquimod suspension preparation before and after the step S3 in the example A1 is detected, and the detection means is dynamic light scattering. The key parameters in the detection are D50 and D90. Wherein D50 is the median particle size of the particles in the suspension, meaning that 50% of the particles in the suspension have a particle size below this value, a classical value representing the size of the particle, commonly used to represent the average particle size of the particles; d90 means that 90% of the particles in the system have a particle size below this value. The difference between D50 and D90 can account for the span of the particle size distribution, as well as the size uniformity. When analyzing the detection data, the magnitudes of the D50 and D90 values of the samples and the changes of the D50 and D90 before and after sterilization are mainly judged: the larger the D50 and D90 values, the less well dispersed the particles; the greater the increase in D50 and D90 values, the poorer the stability of the sample; thus, the larger the D50 and D90 values and the larger the increase in both values indicate that the suspending agent used in the sample is not able to effectively suspend to yield a wet heat sterilizable formulation product.

Thirdly, placing the sterilized sample for a long time, observing the state of the sample and detecting the average particle size of the sample, and if the sample can still be resuspended and D50 and D90 are not obviously increased or the difference between D90 and D50 is smaller, the suspension adjuvant can be regarded as being helpful for increasing the stability of the micro-imiquimod suspension. In the scheme, the long-term placing condition is 2-8 ℃ and the time is 12 months.

Based on the above two criteria, particle diameter values and phenomena before and after sterilization of the different samples, which were left for a long period of time, were recorded as shown in table 1, and states of the different samples were photographed and recorded as shown in fig. 1.

The different types of surfactants added to the 1-9 DEG samples in FIG. 1 are respectively as follows: lecithin, tween-80, tween-20, poloxamer 188, poloxamer 407, polyoxyethylene castor oil, vitamin E polyethylene glycol succinate, sodium oleate, and phosphatidylglycerol.

In fig. 1, 1 degree, 8 degree and 9 degree samples are uniformly dispersed suspension, and the rest samples have the phenomena of wall sticking, caking and even precipitation in different degrees.

Table 1: particle size change and phenomenon recording table of imiquimod suspension added with different types of suspending agents before and after sterilization and placed for a long time after sterilization (wherein, after sterilization, 1 week after sterilization is finished; long-term placement means placement for 12 months)

9 kinds of surfactants are selected, wherein polyoxyethylene nonionic surfactants such as Tween-80, tween-20, polyoxyethylene castor oil and the like can generate a clouding phenomenon when the temperature of the solution is raised to a certain degree, namely acting force between the surfactant and water is destroyed by high temperature, the solution becomes unstable, and when the temperature of the system is lowered below a clouding point, some solutions can restore to be transparent again, and some solutions cannot be recovered. As a polyoxyethylene surfactant, poloxamers are generally considered to have good water solubility, and do not show a cloud point when heated at normal pressure. However, it was found in experiments that when autoclaving using poloxamer 188 or poloxamer 407 as the surfactant, the short-term stability was generally not easily controlled and the long-term stability was not achieved to the desired stabilizing effect. In combination, all nonionic surfactants do not achieve the desired effect of stabilizing the suspension, i.e., they do not help the sterilized suspension disperse uniformly.

Compared with non-ionic surfactants, including anionic surfactants and zwitterionic surfactants, can be added into the system as suspending agents, so that the long-term stability of imiquimod suspension after sterilization can be ensured. Further analysis of the structure of suspending agents that stabilize suspensions has found that these ionic surfactants each contain a higher fatty chain structure and that the molecular weight of the hydrophobic end is much greater than the hydrophilic end. Therefore, the ionic surfactant containing the higher fatty chain can effectively help the micro imiquimod suspension to maintain the stability after terminal sterilization.

Example A3: stabilization of imiquimod by other proportions of surfactants

Generally, the less inactive ingredients of a pharmaceutical formulation, the lower the safety risk of use and storage, while ensuring drug formation. Thus, we further take lecithin as an example, and try to verify the suspending effect using a lower proportion of surfactant containing higher fatty acid chains. Suspensions of different lecithin to R837 micron particle mass ratios were prepared as in example A1, the concentration of R837 was 15 mg/mL, the particle size of the micron particles in the suspension was measured after autoclaving at high temperature and the stability status of the suspension was observed and recorded as shown in table 2.

Table 2: evaluation of suspension effect of low proportion of lecithin on R837 suspension

From the results, it can be seen that the low proportion of lecithin can still ensure the stability of the suspension after high temperature and high pressure sterilization, and the particle size of imiquimod micrometer particles does not change much, even compared with the sample added with the ionic surfactant with higher proportion, the particle size distribution is more concentrated, i.e. the particle size is more uniform. Therefore, the mass ratio of the surfactant containing the higher fatty acid chain to imiquimod can be 0.025-3: 1. preferably, the mass ratio of the phospholipid ionic surfactant to the imiquimod can be 0.025-1: 1.

example A4: investigation of the types of dispersion media of imiquimod suspension formulations

When large-volume injection is clinically administered, an isotonic regulator is usually added to avoid local tissue injury or microenvironment disturbance caused by osmotic pressure change, so that the influence of the common isotonic regulator on the sterilization stability of the imiquimod suspension preparation is examined.

Imiquimod suspension formulations were prepared using the procedure of example A1 at a concentration of 1 mg/mL, except that: in step S1, physiological saline or 5% glucose is used to prepare a solution, and the solution is mixed with imiquimod microparticles for homogenization. No caking was observed before and after autoclaving, indicating that the dispersion medium of the formulation can be used directly with physiological saline or 5% dextrose solution.

Furthermore, the preparation scale is enlarged, the stability of the preparation product is inspected, the agglomeration condition does not occur after the wet heat sterilization, the preparation can be well dispersed after long-term placement, the particle size change is small, and the feasibility of the conditions is further verified.

Example B: animal experiment

Example B1: biodistribution of living beings

Inoculating CT26 tumor cells on the back of the mice, establishing a mouse CT26 subcutaneous tumor model, and equally dividing the mice with consistent tumor sizes into 3 groups of 3 mice, wherein the grouping conditions are as follows:

PLGA-R837: PLGA nanoparticles loaded with R837;

r837 HCl: an aqueous solution of R837 hydrochloride, wherein the dispersibility of R837 in the system is excellent;

imiquimod suspension formulation: the suspension of imiquimod with terminal sterilization in micron order (the suspending agent is lecithin).

According to the grouping, different preparations containing the same dose of R837 are injected into the tumors of each group of mice, the pharmacokinetic characteristics of the mice within 72 hours after injection are studied according to the general method, and the peak time (T _max ) Peak concentration (C) _max ) Half-life (t) _1/2 ) The results are shown in Table 3.

C _max And T _max Reflecting the rate of absorption of the drug from a formulation into the systemic blood circulation, the time to peak (T) _max ) In agreement, however, PLGA nanoparticle-encapsulated R837 was exposed to significant amounts of blood soon, whereas no significant exposure of drug occurred in a short period of time after intratumoral injection of the micro-imiquimod suspension formulation; in addition, the half-life period of blood circulation of the three dosage forms is far different, and compared with a nano preparation and a small molecule preparation, the half-life period of the micro-sized imiquimod suspension is obviously prolonged, namely, the micro-sized imiquimod suspension is applied in a mode of intratumoral administration, and the micro-sized imiquimod suspension has obvious slow release effect.

Table 3: pharmacokinetic parameter mean statistics for different groups of mice

The long retention of the immune adjuvant in the tumor can theoretically stimulate the anti-tumor immune response more effectively, and the application of the micro-sized imiquimod suspension in the combined immunotherapy of the external radiation therapy or the alcohol ablation therapy is proved by design experiments to prove the anti-tumor immune enhancement effect of the slow-release dosage form.

Example C: synergistic radiotherapy and alcohol ablation therapy for micro imiquimod suspension

Example C1: therapeutic experiments of micrometer imiquimod suspension preparation combined with radiotherapy

Inoculating CT26 colon cancer tumor cells on the back of the mouse, and establishing a subcutaneous dual-tumor model of the colon cancer of the mouseIn-situ tumor and distal tumor, respectively, when the in-situ tumor volume is about 100 mm ³ At this time, mice were randomly divided into 6 groups. The grouping is as follows:

vehicle: a vehicle control group, wherein in-situ tumor is subjected to intratumoral injection of dispersion medium, and the injection volume is 25 mu L;

RT: an external radiation treatment group, wherein in-situ tumor is subjected to X-ray irradiation treatment, the radiation dose is 4 Gy, and the treatment is carried out on the day of starting treatment and the day 3 respectively;

r837: the imiquimod micron-sized suspension preparation provided by the invention has the concentration of R837 of 6 mg/mL and the injection dosage of 25 mu L;

r837+ RT: in situ tumors were subjected to X-ray irradiation treatment similar to RT groups after intratumoral injection of 25. Mu.L of 6 mg/mL imiquimod micro-scale suspension.

In the R837+ RT group, the in situ tumor was treated with external radiation therapy for half an hour after intratumoral injection of imiquimod suspension formulation, the in situ tumor of each mouse was not treated with any treatment for the distal tumor, the volumes of the in situ tumor and the distal tumor of the mice were monitored, and tumor growth curves were prepared, the results are shown in fig. 3 (in situ tumor growth curve) and fig. 4 (distal tumor growth curve), and tumor inhibition rates were calculated, and the results are shown in table 4. Table 4 is a statistical table of tumor inhibition rates for in situ tumors and distant tumors. The drug synergy is calculated according to the golden formula q=e (a+b)/(ea+eb-EA, wherein E (a+b) is the tumor inhibition rate of the combination treatment group, EA and EB are the tumor inhibition rates of the two means when used alone, and when q is greater than or equal to 1, the two means are shown to have synergistic effects. Calculated, the q value of the in-situ tumor is 1.17, and the q value of the distant tumor is 1.63, which have synergistic effect.

Meanwhile, the image shows that the tumor growth can be inhibited to a certain extent by multiple times of radiotherapy, and the effect of tumor radiotherapy can be further improved by injecting imiquimod into tumors. The micrometer imiquimod suspension preparation stimulates the strongest systemic anti-tumor immune response due to the long retention of the imiquimod suspension preparation in tumor parts and the high bioavailability in vivo, and the growth of the far-end tumor is inhibited, thereby achieving a synergistic effect with external radiotherapy means.

Table 4: treatment of mouse subcutaneous tumor model by imiquimod suspension preparation and combined radiotherapy, and tumor inhibition rate of treatment end point

In conclusion, the micrometer imiquimod suspension preparation can be combined with external radiotherapy to enhance in-vivo anti-tumor immune response, especially amplify the far-end effect in radiotherapy and inhibit the growth of far-end tumors.

Example C2: treatment experiments with micro-sized imiquimod suspension formulations in combination with alcohol ablation.

Alcohol ablation is one of the local tumor chemoablative therapies, and the treatment purpose is achieved by injecting absolute ethyl alcohol into the tumor to coagulate and necrotize tumor tissues. However, it is difficult to completely remove the tumor without affecting the dosage of surrounding normal tissues by simply injecting chemical ablation means such as alcohol or hydrochloric acid. In the embodiment, the anti-tumor effect of the micro-imiquimod suspension preparation combined with the chemotherapy is proved by combining the micro-imiquimod suspension preparation with the alcohol ablation therapy.

A mouse subcutaneous tumor model was first established. Specifically, tumor cells were inoculated into the backs of mice until tumor volumes grew to 100 mm ³ At this time, the mice were randomly divided into 5 groups of 5 mice each, the grouping being as follows:

control: blank control group;

r837: injecting a micrometer-sized imiquimod suspension around the tumor;

ETOH: intratumoral injection of absolute ethanol;

etoh+r837 (25): injecting 25 mu L of micrometer-sized imiquimod suspension around tumor, and injecting absolute ethyl alcohol into tumor;

etoh+r837 (50): the tumor was injected with 50 μl of imiquimod suspension and the tumor was injected with absolute ethanol.

Wherein the concentration of the micro-imiquimod suspension is 12 mg/mL, and the injection dosage of the absolute ethyl alcohol is 30 mu L. In the combined treatment group, the micrometer-sized imiquimod suspension preparation is injected into subcutaneous parts around tumors at intervals of about 10 minutes, and then the absolute ethyl alcohol is injected into the tumors for administration. The tumor volume change of the mice was monitored, and a tumor growth curve was prepared, and the results are shown in fig. 4.

In experiments, it is found that the mice only use the absolute ethyl alcohol group, the tumor fibrosis and crusting appear in the center of the tumor, but the peripheral tumor tissues are not completely eliminated, the peripheral tumor tissues gradually develop, the outer diameter continues to increase, and the difference between the number of the tumor volumes of the group and the control group is not large, so that the tumor growth curves of the alcohol ablation group and the blank control group almost coincide. Except for the special case, as can be seen from fig. 4, the tumor growth of mice combined with the micro-imiquimod suspension and the alcohol ablation group is obviously inhibited, and the application of different doses of micro-imiquimod can improve the curative effect of alcohol ablation, achieve better tumor treatment effect, inhibit tumor growth, and show that the micro-imiquimod suspension can enhance the alcohol ablation treatment effect of tumors.

Example D: the imiquimod suspension can help to realize the slow release of platinum drugs and enhance the effect of anti-tumor immune response caused by immunogenic cell death

Example D1: verifying the sustained release effect of oxaliplatin when mixed with imiquimod suspension

Inoculating colon cancer (CT 26) tumor cells on the back of a mouse tumor, establishing a subcutaneous tumor model of the mouse, forming tumors at the part to be inoculated for about one week, and randomly dividing the mouse into two groups, namely:

OXA: oxaliplatin solution

OXA-R837: oxaliplatin solution after mixing with micrometer imiquimod suspension

Correspondingly, oxaliplatin solution or oxaliplatin solution mixed with micrometer-sized imiquimod suspension preparation is injected into each group of mice, then blood samples of the mice are taken at different time points (10 min, 30min, 1 h, 3 h, 6 h, 9 h, 12 h, 24 h, 48 h and 72 h), the mice are killed at the end points, main organs and tumors are obtained, and the relative content of platinum ions in the blood samples and organs is detected by inductively coupled plasma mass spectrometry (ICP-MS) and is used for making statistical graphs. The results are shown in fig. 5 and 6.

Fig. 5 shows the biological distribution of oxaliplatin, compared with free oxaliplatin, the injection of the oxaliplatin after premixing with the imiquimod micron-sized suspension can significantly increase the retention of platinum drugs at tumor sites, and after 72-h drug injection, the platinum content at the tumor sites of mice in the mixed injection group is tens times that in the free group, which indicates that the imiquimod micron-sized suspension can increase the retention of platinum drugs at tumor sites and slow down the release of oxaliplatin.

Fig. 6 is a graph showing the change in the concentration of oxaliplatin in blood over time. Compared with free oxaliplatin drug solution, oxaliplatin mixed with the micrometer-sized imiquimod suspension has more obvious slow release effect, and is particularly characterized by lower peak concentration, later peak time and longer time in blood. The specific time to peak (Tmax), peak concentration (Cmax) and area under the curve (AUC) are shown in table 3, which increases the exposure time of oxaliplatin in the circulatory system and increases the bioavailability of oxaliplatin after mixing with a suspension of imiquimod in the micrometer scale.

Table 3: oxaliplatin pharmacokinetic data

Example D2: synergistic antitumor effect of oxaliplatin and micron-sized imiquimod suspension preparation combined use

Different numbers of colon cancer (CT 26) tumor cells (1/5 of the inoculation amount on the left side and the right side) are respectively inoculated on the left side and the right side of the back of the mouse, a subcutaneous bilateral tumor model of the mouse is built, the right side view is in-situ tumor, and the left side view is far-end tumor. Tumor to be in situ about 100. 100 mm ³ Mice were randomized into 4 groups for treatment individually. The grouping situation is as follows:

VEHICAL: solvent group, injection of physiological saline 30. Mu.L

OXA: injection of oxaliplatin solution 30 μl

R837: injection of 30. Mu.L of imiquimod suspension

Oxa+r837: injection of 30. Mu.L of suspension of imiquimod and oxaliplatin after mixing

Intratumoral injection administration of in situ tumors was performed on the first day of treatment, wherein oxaliplatin concentration was 4 mg/mL and imiquimod concentration was 6 mg/mL, and after administration, the tumor volumes of mice were recorded and tumor growth curves were prepared, as shown in fig. 7 (in situ tumor growth curve), fig. 8 (distal tumor growth curve). FIG. 7 is an in situ tumor growth curve of mice, calculated tumor inhibition rates for the treatment groups are shown in Table 4.

Table 4: tumor inhibition rates of different groups on day 15 after treatment

The drug synergy is calculated according to the golden formula q=e (a+b)/(ea+eb-EA, EB), wherein E (a+b) is the tumor inhibition rate of the combination treatment group, EA and EB are the tumor inhibition rates of the two components when used alone, and when q is greater than or equal to 1, the two components are shown to have synergistic effects. The calculated q of the in-situ tumor inhibition rate is about 1.1, and the q of the distant tumor is about 1.27, which indicates that the micro-imiquimod suspension has the synergistic oxaliplatin chemotherapy effect.

Meanwhile, oxaliplatin can cause immunogenic death of tumors, the addition of imiquimod can enhance the anti-tumor immune effect, and the systemic anti-tumor immune response is caused, so that the growth of remote tumors is inhibited, compared with the case that each component is used alone, the oxaliplatin and imiquimod can effectively inhibit the growth of the remote tumors after being subjected to intratumoral injection simultaneously, and the slower the tumor growth and the smaller the intra-group difference of mice in the combined treatment group shown in fig. 8 are.

Example E: investigation of the sustained-release effect of imiquimod micro-scale suspension formulations on anthracyclines.

Example E1: in vitro release experiments of doxorubicin after mixing imiquimod micron-sized suspension formulations with Doxorubicin (DOX).

Grouping and sample preparation:

1) DOX: an aqueous solution of doxorubicin at a concentration of 3 mg/mL;

2) DOX+R837: 3 mg doxorubicin was dissolved with 1 mL imiquimod micron-sized suspension formulation (lecithin-containing and sterilized samples, where imiquimod concentration was 12 mg/mL).

The specific experimental steps are as follows: the prepared solutions of each group are respectively added into a dialysis bag (the molecular weight cut-off is 3500D), and are placed into a PBS solution of 500 mL for dialysis, the concentration of the drug in the dialysate is detected at different time points, the detection means is to detect the ultraviolet absorbance of the wave band of the drug, the drug content is calculated, the ratio is made with the initial drug content, and the relative drug content change curve of the dialysate of different samples is manufactured, and the result is shown in figure 9. The drug release curve trend of the micro-sized imiquimod suspension is obviously slowed down after the micro-sized imiquimod suspension is mixed with doxorubicin, which indicates that the micro-sized imiquimod suspension can obviously reduce the drug release rate and achieve the slow release effect. The in vitro simulated release experiment can reflect the in vivo behavior of the drug to a certain extent, and shows that the injection after the micro-imiquimod suspension preparation and the doxorubicin are premixed can slow down the release rate of the drug after in-situ injection, and can prolong the residence time of the chemotherapeutic drug at the tumor part and increase the retention quantity of the drug at the tumor part, thereby enhancing the effect of the drug at the tumor part and reducing the toxic and side effects of the drug on the system.

Example E2: the mixing ratio of the imiquimod micron-sized suspension preparation and the doxorubicin is changed, and the slow release effect is verified.

Grouping and sample preparation:

1) DOX: an aqueous solution of doxorubicin at a concentration of 3 mg/mL;

2) DOX+R837: dissolving doxorubicin with a micrometer-sized imiquimod suspension formulation having a concentration of 12 mg/mL and a final concentration of 3 mg/mL;

3) DOX+R837 (1/3): the doxorubicin was dissolved with a micron-sized imiquimod suspension formulation having a concentration of 4 mg/mL and a final concentration of 3 mg/mL.

Three sets of drug release curves were plotted using the same test method and data processing as in example E1, and the results are shown in fig. 10. After the dosage of the imiquimod micro-scale suspension is reduced, the release rate of the medicine is increased, but compared with a pure aqueous solution of doxorubicin, the release rate is still slowed down, and further the fact that the micro-scale imiquimod suspension preparation can slow down the release of doxorubicin, the slow-release effect is related to the proportion of the two, the higher the concentration of the micro-scale imiquimod suspension preparation is, the better the slow-release effect is, and the concentration proportion of the doxorubicin to the imiquimod is 1: 1-1: 18.

example E3: in vitro release experiments after mixing Epirubicin (EPI) with a suspension formulation of imiquimod in the micrometer scale.

Grouping and sample preparation:

1) EPI: an aqueous solution of epirubicin at a concentration of 2 mg/mL;

2) Epi+r837: dissolving epirubicin with a micron-sized imiquimod suspension preparation, wherein the final concentration of the epirubicin is 2 mg/mL;

the same as in example E1, samples were placed in dialysis bags (molecular weight cut-off 3500D), a slow-release system of 500 mL PBS solution, the drug release amounts at different time points were examined, the release percentages were calculated and the drug release curves were plotted, and the results are shown in FIG. 11. The results show that early in the in vitro release experiments (before 6 h), the drug release profiles of the different groups were similar, but with time, the epirubicin release was slowed in combination with the micrometer-sized imiquimod suspension formulation.

The inventors speculate that the anthracycline in the mixed solution may form a pi-pi stacking force with imiquimod microparticles, whereas doxorubicin and epirubicin are isomers, and from the analysis structure, doxorubicin and imiquimod form a more stable pi-pi stacking force, thus exhibiting a stronger sustained release effect.

When the relative concentration of the drug and the suspension is too high, the excessive drug is in a free state, the part of the drug can be released rapidly in a short time, and the change of the drug metabolism is related to the micro-imiquimod particles through the pi-pi interaction stabilization of the drug and the imiquimod particles, so that the drug retention is enhanced and the release is slowed down after the injection of the mixed solution in tumor.

Example E4: sustained release effect of micro imiquimod suspension preparation on epirubicin obtained by taking phosphatidylglycerol as suspending agent

Grouping and sample preparation:

EPI: an aqueous solution of epirubicin at a concentration of 2 mg/mL;

epi+r837 (0.25 PG): the micro imiquimod suspension preparation obtained by suspending with phosphatidylglycerol dissolves epirubicin, wherein the mass ratio of the phosphatidylglycerol to the imiquimod is 0.25:1, final epirubicin concentration of 2 mg/mL;

epi+r837 (3 PG): the micro imiquimod suspension preparation obtained by suspending with phosphatidylglycerol dissolves epirubicin, wherein the mass ratio of the phosphatidylglycerol to the imiquimod is 3:1, the final epirubicin concentration was 2 mg/mL.

In a similar way to example E1, the release condition of the drug is detected by a dialysis experiment, and the result is shown in FIG. 12, the dissolution rate of the drug is obviously slowed down, the release amount of the drug is only about 1/4 of that of the control group after 24 hours, the proportion of the phosphatidylglycerol is increased, the release of the drug is slower, and the electrostatic acting force is formed by the negative charge on the surface of the phosphatidylglycerol and the positive charge on the surface of the epirubicin, so that the sustained release effect is stronger.

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 limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and features disclosed herein.

Claims

1. An imiquimod suspension preparation capable of being injected in or around tumor, which is characterized by comprising imiquimod micron particles, a surfactant containing higher fatty acid chains and a dispersion medium; the surfactant containing the higher fatty acid chain is lecithin; the mass ratio of the lecithin to the imiquimod micron particles is 0.025-0.5: 1, a step of; homogenizing the imiquimod suspension preparation; the concentration of the imiquimod micron particles is 1-18 mg/mL.

2. The intratumoral or peritumoral injectable imiquimod suspension formulation according to claim 1, characterized in that the imiquimod microparticles have an average particle size of 0.5 to 5 μm.

3. The imiquimod suspension formulation for intratumoral or peritumoral injection according to claim 1, wherein the mass ratio of lecithin to imiquimod microparticles is 0.1-0.5: 1.

4. a method of preparing an intratumorally or peritumorally injectable imiquimod suspension formulation according to any one of claims 1 to 3, comprising the steps of:

s2: homogenizing the suspension;

5. The method according to claim 4, wherein the conditions of high temperature and high pressure sterilization are: 110-145 ℃ for 5-30 min.

6. An intratumoral or peritumoral injectable imiquimod suspension formulation according to any one of claims 1 to 3, wherein the imiquimod suspension formulation is premixed with a platinating agent to assist in the slow release of the platinating agent.

7. An intratumoral or peritumoral injectable imiquimod suspension formulation according to any one of claims 1 to 3, wherein the imiquimod suspension formulation is premixed with an anthracycline to assist in the slow release of the anthracycline.