CN115192721B

CN115192721B - Bladder perfusion medicine compound preparation and preparation method and application thereof

Info

Publication number: CN115192721B
Application number: CN202210627784.5A
Authority: CN
Inventors: 刘庄; 邓中清; 陶惠泉; 吴宇辰; 周炫坊
Original assignee: Suzhou Baimai Biomedical Co ltd
Current assignee: Suzhou Baimai Biomedical Co ltd
Priority date: 2021-12-09
Filing date: 2021-12-09
Publication date: 2023-11-07
Anticipated expiration: 2041-12-09
Also published as: CN115192721A; CN116251191A

Abstract

The invention discloses a bladder perfusion medicine compound preparation, which comprises anthracycline chemotherapeutic drugs, an immune regulator and a pH regulator, wherein the anthracycline chemotherapeutic drugs can cause immunogenic death of tumor cells. The compound preparation has good chemical stability, and solves the problem of impurity growth caused by the influence of an immune adjuvant on an anthracycline.

Description

Bladder perfusion medicine compound preparation and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a compound preparation of anthracycline and immune adjuvant, a preparation method and application thereof in preparing bladder perfusion medicines.

Background

Bladder cancer is the most common malignancy of the urinary system, with the incidence listed as the ninth of global malignancy; the global bladder cancer new onset patients in 2020 are about 54.9 ten thousand people, and the death number is nearly 20 ten thousand people. Global bladder cancer market size has increased to $39 billion in 2019, estimated to reach $71 billion in 2024, and bladder cancer is one of the most costly tumors in the united states. The high cost of treatment comes mainly from the high recurrence rate and high metastasis rate of bladder cancer, and the lack of two-line treatment regimen, the clinical need for bladder cancer is far from being met.

There are two main types of clinically common bladder cancers, non-myogenic invasive bladder cancers and myogenic invasive bladder cancers. At present, chemo-therapy/BCG perfusion and transurethral bladder tumor resection (TURBT) are mainly adopted for non-myogenic invasive bladder cancer clinically. Studies have shown that this therapy is effective for low risk patients: 15% of recurrence rate in one year and 30% of recurrence rate in five years; for medium-risk patients: one-year recurrence rate is 38%, five-year recurrence rate is 62%; for high-risk patients: the recurrence rate of one year is 61%, the recurrence rate of five years is 78%, and BCG vaccine perfusion can solve the problem of tumor recurrence to a certain extent, but BCG vaccine perfusion can cause systemic reaction, such as long-time fever, and bladder perfusion BCG vaccine can show symptoms of frequent urination, urgent urination, hematuria and the like, and patients with immunodeficiency can risk suffering from systemic BCG vaccine diseases by using BCG vaccine.

The bladder perfusion is to inject a chemotherapeutic drug or a tumor therapeutic preparation with the effect of directly killing tumor cells into the bladder, directly killing the tumor cells or inducing in-vivo nonspecific immune response, so as to achieve the effect of inhibiting bladder cancer metastasis and recurrence. Patients treated by bladder perfusion assistance have reduced recurrence probability, but have low absolute remission rate, higher tumor recurrence rate, large toxic and side effects and poor prognosis.

Anthracyclines have high susceptibility to various transitional epithelial cell cancers, and currently, anthracycline chemotherapeutics represented by epirubicin and pirarubicin are first-line therapeutic drugs for bladder cancer perfusion treatment. However, tumors still have a high recurrence rate (over 50%) within one year following bladder infusion therapy with such drugs alone.

Toll-like receptors (TLRs) are components of innate immunity, and in recent years, they are widely expressed in immune cells, promote T cell responses against tumors after corresponding stimulation, and in the field of anti-tumor immunotherapy, more and more people are focusing attention and researching TLR pathways, and developing tumor immunotherapy drugs. For example, imidazoquinolines immunomodulators, of which imiquimod and its derivatives are representative, are TLR-7 stimulators, and have been used for immunotherapy of superficial tumors.

Disclosure of Invention

The applicant tries to combine anthracycline drug with imiquimod and apply the anthracycline drug to the perfusion treatment of bladder cancer, and the two drugs are directly mixed and then perfused into the bladder to verify the curative effect. As a result, it was found that, taking the chemotherapeutic agent epirubicin and imiquimod as examples, when the ratio of the chemotherapeutic agent epirubicin to imiquimod is in a suitable range, a significantly improved therapeutic effect can be obtained compared with the infusion of the chemotherapeutic agent alone, without increasing the toxic or side effects, and it is expected to become a novel drug preparation for bladder infusion having significant clinical value. Further research shows that the novel preparation has an anti-tumor mechanism that the tumor cell is mainly induced to die by chemotherapy drugs such as epirubicin to expose tumor-related antigens, and imiquimod serving as a TLR7 agonist can activate an immune system and recruit antigen presenting cells into tumors to identify tumor antigens so as to activate T cell immune response aiming at tumors. Based on the principle, we have tried the effect of combining different anthracyclines with imiquimod (R837) or resiquimod (R848) to treat tumors, and all the effects obtain ideal curative effects.

However, in further formulation studies, it was found that in a simple mixed formulation of an anthracycline chemotherapeutic drug such as epirubicin and imiquimod as an immunological adjuvant, the presence of imiquimod molecules can cause instability of the molecular structure of the anthracycline chemotherapeutic drug such as epirubicin, whether stored in the form of an injection solution or as a lyophilized powder after mixing, the impurity content in the formulation can be significantly increased, and the impurity limit of the drug prescribed in pharmacopoeia is broken through within 5 days, so that the drug formulation which can be stably stored and is convenient for clinical application cannot be obtained, and the simple mixing of the two is unfavorable for the chemical stability of the drug molecules. Therefore, how to improve the chemical stability of the compound preparation of the anthracycline and the imidazoquinoline immunomodulator is a key problem in designing and preparing the compound preparation for combined use of the anthracycline and the imidazoquinoline immunomodulator. In order to solve the above-mentioned derivative problems, we have further performed systematic studies on the stability of the anthracycline and imidazoquinoline immunomodulator compound formulation.

First, we found that epirubicin and imiquimod were more stable at low pH (e.g., pH 3.0) in solution, but anthracyclines were still gradually degraded, and the mixed formulation under this condition was not stored for a long period of time, and thus the injection formulation was not viable for the present formulation. For a freeze-dried preparation, it is surprisingly found that lactose has a remarkable protection effect on the stability of the preparation in a common freeze-dried protective agent, and the increase of the content of lactose is beneficial to the improvement of the stability of the preparation. Nevertheless, the lyophilized preparation prepared at pH 3.0 still cannot be stored for a long period of time and cannot be examined by a stability acceleration experiment. We find that the compound freeze-dried preparation of the anthracycline immunoadjuvant has a stable 'pH value golden section', and the epirubicin/imiquimod compound preparation prepared in the pH range has very good stability-! This phenomenon is contrary to our conventional understanding of a single formulation of epirubicin by those skilled in the art (the lower the pH, the more stable it is conventionally) and our prediction of the trend of the stability of the solution state of this compound formulation (the lower the pH, the more stable it is).

Further, we examined the stability of other anthracyclines such as mitoxantrone, doxorubicin and imidazoquinoline immunomodulators (R837 or R848) as a compound lyophilized powder formulation, and tried the stability of different compounds under similar pH conditions based on detailed examination results of epirubicin and R837. The result shows that the chemical structures of the compounds are similar or identical, and the optimal formula of the compound preparation is verified, so that any combination of the two substances has good stability.

The invention provides a bladder perfusion medicine compound preparation which comprises an anthracycline, an immunomodulator and a pH regulator, wherein the immunomodulator comprises an imidazoquinoline immunomodulator and soluble salts thereof.

Further, the anthracycline includes epirubicin or a soluble salt thereof, pirarubicin or a soluble salt thereof, mitoxantrone or a soluble salt thereof, doxorubicin or a soluble salt thereof, idarubicin or a soluble salt thereof.

Further alternatively, the soluble salt is a hydrochloride salt.

Further, the freeze-dried powder preparation of the bladder perfusion medicine also comprises a freeze-drying protective agent.

Further, the lyoprotectant comprises sucrose, lactose, mannitol and cyclodextrin.

Further alternatively, the lyoprotectant is lactose.

Further, the mass fraction of the freeze-drying protective agent in the freeze-drying powder preparation is 65% -96%.

Preferably, the content of the freeze-drying protective agent in the freeze-drying powder preparation is 80-94%.

Further, the pH regulator is weak base, alkaline buffer solution, sodium hydroxide and hydrochloric acid.

Further alternatively, the pH adjuster is sodium bicarbonate, hydrochloric acid.

Further, after the bladder perfusion medicine compound preparation is redissolved, when the concentration of anthracycline is 1-5 mg/mL, the pH of the solution is 3.8-5.5. The term "reconstitution concentration" is used herein to define the range of pH values after reconstitution, and is not limited to the reconstitution concentration.

Further alternatively, after the bladder perfusion medicine freeze-dried powder preparation is redissolved, when the concentration of the anthracycline is 1-5 mg/mL, the pH of the solution is 4.0-5.0. The term "reconstitution concentration" is used herein to define the range of pH values after reconstitution, and is not limited to the reconstitution concentration.

Further, the imidazoquinoline immunomodulator comprises imiquimod and its derivatives, or remiquimod and its derivatives, or soluble salts of imiquimod and its derivatives, or soluble salts of remiquimod and its derivatives.

Further, the mass ratio of the anthracycline to the imidazoquinoline immunomodulator is 1:0.1-1:10.

Further alternatively, the mass ratio of anthracycline to imiquimod is 1:1 to 1:10. Further alternatively, the mass ratio of anthracycline to imiquimod is 1:2-1:4.

Further alternatively, the mass ratio of anthracycline to resiquimod is 1:0.1-1:5.

Further, the freeze-dried powder preparation also comprises a mucous membrane penetration enhancer.

Further, the mucous membrane penetration enhancer comprises at least one of azone, hyaluronidase, lauryl alcohol and oleic acid.

Further, the mucous membrane penetration enhancer may be added during the preparation process or may be mixed prior to use.

The compound preparation of the anthracycline and the immune adjuvant is mixed with a mucous membrane permeation enhancer before use, and then is infused, wherein the mucous membrane permeation enhancer can be a medical mucous membrane permeation enhancer which is available in the market or a mucous membrane permeation enhancer approved for use in a clinical test stage.

The invention also provides a preparation method of the freeze-dried powder preparation of the bladder perfusion medicine.

The method comprises the following steps:

s1: dissolving an immunomodulator by dilute acid to obtain a salt solution of the immunomodulator;

s2: dissolving anthracycline by using the dilute acid salt solution obtained in the step S1 to obtain a mixed solution;

s3: and adjusting the pH value of the mixed solution to 3.8-5.5 by using an acidic or alkaline buffer solution or solution to obtain a final solution, wherein the final concentration of the medicine is 1-5 mg/mL.

Further: the step S3 further includes:

s31: adding a lyoprotectant into the mixed solution;

further: the step S3 further includes:

s32: adding a mucous membrane permeation enhancer to the mixed solution

Further, the dilute acid in S1 is hydrochloric acid, lactic acid or acetic acid.

Further alternatively, a further alternative range of the pH adjustment in S3 is 4.0 to 5.0.

Further, the method further comprises the step S4: and (3) carrying out freeze-drying treatment on the final solution obtained in the step (S3) to obtain a freeze-dried powder preparation.

The invention also provides application of the freeze-dried powder mixed preparation in preparation of a bladder perfusion medicine. The technical scheme of the invention has the following beneficial effects:

the invention provides a bladder perfusion medicine compound preparation, an anthracycline and imidazoquinoline immunomodulator mixed freeze-dried powder preparation. In a large number of experimental analysis, the freeze-dried powder preparation is more beneficial to protecting the long-term chemical stability of anthracycline drugs in mixed preparations compared with the traditional injection preparation, meanwhile, the mixed preparation has better tumor inhibition effect in bladder perfusion tumor treatment, can cause stronger anti-tumor immune response, and can reduce toxic and side effects compared with single anthracycline drugs.

The anthracycline and immunomodulator mixed preparation can realize clinical application through simple redissolution operation, can be directly used for perfusion treatment, and can also be added with a mucosa penetration enhancer for perfusion after mixing so as to effectively inhibit tumor growth and recurrence. After causing immunogenic death of the tumor, the immunomodulator is able to further stimulate antigen uptake, processing and presentation to T cells by antigen presenting cells, further amplifying the anti-tumor immune response.

Can reduce systemic toxic and side effects of anthracycline or immunomodulator.

The long-term chemical stability of the mixed preparation of the anthracycline and the immunomodulator is realized, the generation rate of the anthracycline impurity in the presence of the immunomodulator is obviously reduced, the long-term storage stability of the preparation is improved, and the convenience and safety of the use of the preparation are improved. In future clinical use, the longer shelf life is beneficial to the production, storage and transportation of medicines and is beneficial to realizing real clinical application.

The mucosa penetration enhancer is added into the preparation, so that the retention time of the drug solution on the bladder wall after perfusion treatment can be enhanced, and the curative effect of the preparation can be further improved.

Drawings

FIG. 1 is a statistical graph of tumor mass of mice of different groups after treatment of bladder cancer by bladder infusion, using a combination lyophilized powder formulation of epirubicin and imiquimod in bladder cancer mice;

FIG. 2 is a statistical graph of tumor mass in mice after administration of a mixed lyophilized powder formulation of pirarubicin and imiquimod for infusion treatment of bladder cancer in mice;

FIG. 3 is a graph showing tumor mass statistics of mice of different groups after bladder cancer treatment by bladder infusion with a doxorubicin and imiquimod mixed lyophilized powder formulation;

FIG. 4 is a statistical plot of the effect of a mucolytic agent on the retention of epirubicin in the bladder;

figure 5 is a statistical plot of the effect of a mucolytic on imiquimod retention in the bladder.

Detailed Description

Example a: study on stability of injection dosage form

In the embodiment A, the compound preparation of epirubicin and R837 is prepared, freeze-drying treatment is not carried out, the impurity change condition of the injection preparation under different pH conditions and temperature conditions is researched, and whether the injection preparation meets the actual production use condition of the compound preparation is judged.

Example A1: chemical stability of the injection preparation of the bladder perfusion pharmaceutical composition under different liquid medicine pH conditions.

The imiquimod is dissolved by hydrochloric acid and then added with epirubicin hydrochloride for dissolution, the pH value of the solution is low, and the solution can generate larger stimulation to tissues during perfusion, so that the stimulation to the tissues during use is reduced, and the acid-base value which has both preparation stability and use safety needs to be selected.

Dissolving R837 with hydrochloric acid to obtain R837 hydrochloride solution with concentration of 4mg/mL, and adding saltAcid Epirubicin (EPI) was dissolved at an EPI concentration of 1mg/mL, and then the pH of the solution was adjusted to 3.0, 3.5, 4.0 using sodium bicarbonate, labeled as injection 1, injection 2, and injection 3, respectively, and the contents of epirubicin and imiquimod impurities in the pharmaceutical preparations on the day of preparation and on day 5 placed in an environment of 40 ℃ were detected by liquid chromatography, and the results are shown in table 1. The impurity is mainly from epirubicin hydrochloride, and the structure of the impurity A (EPI-A) isThe impurity C (EPI-C) has the structure +.>

The results show that the total impurity content of the preparation increases with time under three pH conditions compared with the bulk drug, and the total impurity content is more than 3 times of the total impurity content of the bulk drug when the preparation is left for 5 days at 40 ℃. Even at the most stable condition of ph=3.0, there is a significant increase in unknown single impurities of EPI after storage at 40 ℃ for only 5 days, which is expected to soon exceed the maximum allowable limit specified in the pharmacopoeia. Meanwhile, the impurity content of imiquimod is found to be relatively stable, in the multiple detection, the imiquimod impurity B is kept at about 0.05%, the imiquimod impurity A is kept at about 0.05%, all the imiquimod impurity C is not detected, the maximum unknown single impurity is kept at about 0.18%, the total impurity is basically within 0.39%, and all the impurities are not beyond the limit: 0.15% of impurity A, 0.20% of impurity B, 0.15% of impurity C, 0.20% of maximum unknown single impurity content and 1.0% of total impurity content. Therefore, the compound injection of epirubicin/imiquimod has no good stability, and the main impurity growth comes from the epirubicin, so that the safety and effectiveness of clinical medication cannot be ensured.

Table 1: table of the initial impurity content of epirubicin/imiquimod compound injection formulations at different pH, impurity content record of Epirubicin (EPI) at 40 ℃ for 5 days.

Example A2: influence of different storage conditions on stability of injection dosage form

Considering that the formulation of example A1 was placed at a temperature of 40 ℃, it was possible to accelerate the generation of impurities, and thus the stability of the formulation at lower temperatures was investigated. The preparation of the bladder perfusion injections at ph=3.0 and ph=3.5 by the method of example A1 was carried out under refrigeration conditions at 15 ℃ and 25 ℃ on the day of completion of sample preparation and on the 10 th day of standing at different temperatures, and the impurity contents of the preparation samples were measured, and the results are shown in table 2. With the increase of the storage temperature, the total impurity content of the injection under the two pH conditions is increased, and the stability of the injection dosage form is proved to be related to the temperature. It has also been found that even under refrigerated conditions, the maximum unknown single has a growing tendency, and that the increase in the maximum unknown single is also detrimental to the overall stability and safety of the formulation, and therefore, the injectable formulation for bladder infusion is most likely not the preferred formulation for the formulation.

Table 2: table of the initial impurity content of Epirubicin (EPI) in the epirubicin/imiquimod compound injection and the impurity content of Epirubicin (EPI) in the epirubicin/imiquimod compound injection at different storage temperatures on day 10.

Example B: investigation of the stability of lyophilized dosage forms

The results in the example A show that the compound preparation is difficult to produce, store and use as an injection, so that the feasibility of the freeze-dried powder preparation of the compound preparation is investigated by design experiments.

Example B0: study of the patentability of lyophilized dosage forms:

in order to explore the possibility of patent medicine of the freeze-dried powder preparation in preliminary research, factors which may influence the patent medicine, such as auxiliary materials, pH and the like, are subjected to primary screening.

Dissolving imiquimod by using hydrochloric acid or dissolving imiquimod hydrochloride by using pure water to obtain an aqueous solution of imiquimod hydrochloride, and then dissolving epirubicin hydrochloride to obtain a solution containing active ingredients, wherein the mass ratio of the active ingredients of epirubicin to imiquimod is 1:1 to 1:10; secondly, attempting to adjust the pH of the solution, and observing whether the pH value change affects the stability of the solution; then adding a freeze-drying protective agent sucrose, lactose, mannitol or cyclodextrin (the concentration is between 10mg/mL and 50 mg/mL), observing the state of the sample, and judging whether obvious instability occurs or not, freeze-drying the sample by a freeze-drying technology, wherein the mass fraction of the freeze-drying protective agent after freeze-drying is 65-96%, and observing the appearance of freeze-dried powder, and judging whether naked-eye discernable non-formed, agglomerated or uneven samples occur or not; finally, the freeze-dried sample is re-dissolved by adding a solvent, whether the phenomenon of non-dissolution and non-uniformity occurs is observed, the pH value of the solution is re-measured, and when the concentration of the epirubicin in the solution is 1mg/mL, the pH value is close to the value before freeze-drying, and the pH value is about 3.8-5.5.

In the preparation process of the preparation, the pH adjustment process and the addition of the freeze-drying protective agent do not cause the conditions of uneven solution or obvious instability such as precipitation, the freeze-dried sample keeps good morphology, and the re-dissolution is smooth, and the phenomenon of undissolved or uneven dissolution does not occur, so that the freeze-dried powder preparation formula can be used for further stability research, and the impurity content change is detected.

Example B1: preliminary attempts to incorporate lyoprotectants to affect formulation stability

A liquid preparation was prepared as in example A1, with the pH not adjusted, and with the initial pH adjusted to about 3.0, and with sodium bicarbonate adjusted to a pH of 3.5, the final concentrations of epirubicin and imiquimod were 1mg/mL and 4mg/mL, respectively, and lactose or mannitol was added at various levels (concentrations of 10mg/mL, 25mg/mL, 50mg/mL at the time of preparation, and converted to a mass fraction of 67%,83%, 91%) before lyophilization, and the impurity content on day 0 after lyophilization was examined. The results are shown in Table 3. The results show that when no lyoprotectant is added, the maximum unknown single impurity content in the compound preparation is beyond the limit, and after the lyoprotectant with proper content is added, the initial impurity content is reduced. The protection effect of lactose on the freeze-dried preparation is superior to mannitol, and the increase of pH and lactose content are beneficial to the stability of the product.

Table 3: table of initial impurity levels of Epirubicin (EPI) in epirubicin/imiquimod lyophilized powder formulations after addition of varying levels of lactose or mannitol.

Example B2: lactose is used as a freeze-drying protective agent, and the freeze-dried powder preparation is stable for 5 days at 25 ℃.

To further investigate the effect of lyoprotectant content and initial pH on the stability of lyophilized powder, samples Nos. 4-1, 5-1, and 6-1 of example B4 were placed at 25deg.C, and the pH and impurity content of the samples were measured after reconstitution on day 5, and the data list for day 0 was shown in Table 4. From the viewpoint of impurity content change, the initial pH value of the freeze-dried powder preparation is 3.5, the impurity content change is superior to that of the freeze-dried powder preparation with the pH value of 3.0, and meanwhile, the impurity content is least increased when the lactose content is 91%.

Table 4: initial impurity content of Epirubicin (EPI) in lactose added epirubicin/imiquimod compound lyophilized powder formulation table of impurity content of Epirubicin (EPI) left at 25 ℃ for 5 days.

Example B3: screening of different lyoprotectants

Based on preliminary exploration of the effect of the freeze-drying protective agent and the attempt of the content of the protective agent, the influence of different freeze-drying protective agents on the stability of the freeze-drying powder preparation under the same content is further explored. And further trying to obtain the stability of the freeze-dried preparation at a higher pH value based on the relationship between the pH value change trend and the impurity change trend.

Lactose (two ponds), mannitol (non-sugar), cyclodextrin (cyclic oligosaccharide) and glucose (monosaccharide) are selected from the common freeze-drying protective agent for injection for investigation, and the protective effect of the freeze-drying protective agent on the freeze-drying powder preparation is verified under the condition that the consumption of the freeze-drying protective agent is 91 percent.

First, a liquid preparation was prepared according to the method of example A1, the pH of the solution was adjusted to 3.5 or 4.0, different lyoprotectants were added respectively, the final imiquimod concentration was 4mg/mL, the epirubicin concentration was 1mg/mL, the final content of the lyoprotectant after lyophilization was 91%, and the change in the impurity content of epirubicin hydrochloride was detected after 5 days of standing at 40 ℃ and the results are shown in table 5. In table 5, when the lyoprotectant is lactose, the lyoprotectant has the best stability, the impurity content is still kept at a low level after being placed at 40 ℃ for 5 days, the impurity is not obviously increased, and the impurity is obviously increased under the condition of ph=3.5; other lyoprotectants showed significant impurity growth at both pH3.5 and pH 4.0. Thus, the lyoprotectant is preferably lactose.

Table 5: and adding the epirubicin/imiquimod compound freeze-dried powder preparation with different initial pH values of different freeze-dried protective agents. Table of impurity content record of initial impurity content of Epirubicin (EPI) at day 5 when placed at 40 ℃.

Example B4: influence of lactose content on stability of lyophilized powder preparation

Based on the results of the previous examples, the effect of adding lactose with different content on the impurity content of the freeze-dried powder preparation is examined when the pH value is 4.0.

According to the preparation method described in the embodiment A1, preparing an EPI and R837 mixed injection, adding lactose protective agents with different contents, adjusting pH to 4.0 with sodium bicarbonate, wherein the final imiquimod concentration is 4mg/mL, the epirubicin concentration is 1mg/mL, the lyoprotectant concentration is 20mg/mL,30mg/mL,40mg/mL,50mg/mL, the mass fractions of the lyoprotectants corresponding to the lyoprotectants after lyophilization are respectively 80%, 86%, 89% and 91%, and detecting impurity contents on the day after lyophilization and on the day 5 and 13 after the lyophilization at 40 ℃. The results are shown in Table 6. With the increase of the initial content of the freeze-drying protective agent, the impurity content of different compound preparations placed under the same pH condition and the same temperature is increased slowly, which shows that the chemical stability of the compound preparation is increased. The chemical stability is best when the initial content of the lyoprotectant is 91% and the impurity content is not beyond the limit under the conditions that the lyoprotectant content is 80%, 86%, 89% and 91%.

Table 6: the initial impurity content of Epirubicin (EPI) and the impurity content record table of the 5 th day and 13 th day of standing at 40 ℃ are added into the compound solution of the lactose epirubicin/imiquimod compound freeze-dried powder preparation with different contents, wherein the pH value of the compound solution is 4.0.

Example B5: preparation samples under different pH conditions are prepared, and stability of the lyophilized preparation is examined.

Liquid preparations with pH values of 3.5, 4.0, 4.5 and 5.0 respectively were prepared by the method of example A1, the final imiquimod concentration was 4mg/mL, the epirubicin concentration was 1mg/mL, the preparation was subjected to pre-freezing treatment and then freeze-dried, after standing for a certain period of time under different temperature conditions, the freeze-dried powder was re-dissolved, and the impurity content was detected, and the experimental conditions and results were shown in Table 7. Different from the total impurity change trend of epirubicin hydrochloride in the injection, when the initial pH value is increased, the increase of the total impurity content is slowed, under the condition that the freeze-drying protective agent is lactose and the content is 91 percent, the unknown single impurity and the change of the total impurity are both lower than the increase of the impurity content of the injection, namely the impurity content of the freeze-dried powder preparation which is placed for a long time (30 days) is far lower than the impurity content of the injection which is placed for a short time (5 days) under the same pH value (pH=5.0) and temperature condition (25 ℃), and the freeze-dried powder preparation accords with the current national pharmacopoeia standard. Where ph=3.5 of the formulation before lyophilization, the sample impurity had exceeded the pharmacopoeia specified limit on day 5 at 40 ℃, and therefore, the sample was not subjected to impurity analysis for 30 days under this condition.

Table 7: table of impurity content recordings of Epirubicin (EPI) after various times of standing epirubicin/imiquimod lyophilized powder formulations in different temperature environments at different initial pH values.

Example B6: the impurity content change of the freeze-dried preparation is studied by changing the proportion of the two components.

Liquid formulations having pH values of 3.5, 4.0, and 4.5, respectively, were prepared as in example A1, lactose was added, the final imiquimod concentration was 2mg/mL, the epirubicin concentration was 1mg/mL, and the lactose content was as shown in Table 8. The impurity content of epirubicin was recorded the day after lyophilization and for 27 days at 40 ℃ and the record is shown in table 8. The results showed that as the initial pH of the sample increased and the lactose content increased, the impurity content of the sample decreased when left at 40 ℃ for 27 days, and at an initial ph=4.0, the total impurity content was out of limit at day 27, and the impurity content was below the limit under the remaining conditions, at a lactose content of 67% in the sample. At ph=4.5, several lactose levels reduced the increase in impurity levels in the lyophilized samples.

Table 8: table of impurity content record of Epirubicin (EPI) when epirubicin/imiquimod composite compound lyophilized powder formulations were placed in an environment of 40 ℃ for 27 days at different initial pH values.

Example B7: further screening for optimal pH

A mixed solution of epirubicin and imiquimod was prepared as in example A1, the pH of the solution was adjusted to 3.8, 4.0, 4.2, 4.4, and the lyoprotectant lactose was added, the ratio of epirubicin to imiquimod was 1 in two samples: 2 or 1:4, the final concentration of the lyoprotectant is 91%, and the corresponding mass fractions in the two specification lyophilized samples are 94% and 91%, respectively. Samples were placed at 40 ℃ after lyophilization and tested for impurity levels at various time points and the results are shown in table 9.

The results show that the increasing trend of the epirubicin impurity is related to the initial pH value no matter what concentration ratio is, the higher the pH value is, the more stable the preparation is, when the pH value is 3.8, the maximum unknown single impurity content exceeds the limit, and when the pH value is above 3.8, the impurity content stability requirement is met.

Table 9: table of impurity content record of Epirubicin (EPI) at 19 days and 30 days in environment of 40 ℃ for initial impurity content of epirubicin/imiquimod compound freeze-dried powder preparation at different initial pH values.

Example C: stability investigation of different anthracycline drugs and imidazoquinoline immunomodulators mixed freeze-dried powder preparation at pH=4.0

Example C1: stability of epirubicin hydrochloride and resiquimod (R848) mixed lyophilized powder formulation

Firstly, preparing a mixed preparation, specifically, dissolving R848 by using hydrochloric acid to obtain a hydrochloride solution of R848, and further adding epirubicin hydrochloride, wherein the mass ratio of R848 to epirubicin is 4:1. Adding lactose as freeze-drying protective agent, wherein the lactose content is 91%. Freeze-drying is carried out, the freeze-dried powder is placed at different environmental temperatures for stability investigation, and samples are sampled and detected on the 5 th day and the 10 th day for impurity content change.

Unlike R837 mixed epirubicin, when R848 was dissolved in hydrochloric acid and epirubicin was added, the initial pH of the solution system was about 5.5, which was higher than the pH of imiquimod hydrochloride, and therefore, the pH of the system was adjusted to about 4.0 using an acidic solution, and the change in impurities under the conditions of the original pH (ph=5.5) and ph=4.0 was detected, and the impurity detection results of R848 are shown in table 8. Since there is no pharmaceutical product on the market of R848 at present, the stability of R848 cannot be judged from the limit of the impurity content, but it can be seen from the data of table 8 that the impurity content of R848 hardly changes with the extension of time, regardless of the conditions of 25 ℃ or 40 ℃, and thus it can be judged that the stability of R848 in the mixed lyophilized powder formulation is good.

Table 8: table of impurity content of R848 in samples when the epirubicin and resiquimod (R848) mixed lyophilized powder formulations were left to stand for 5 days and 10 days at different pH conditions for the initial impurity content of R848 and temperature conditions.

The impurity detection results of epirubicin are shown in table 9, when the pH of the system is not adjusted before lyophilization, i.e., the initial ph=5.5, the maximum unknown single impurity content of epirubicin after mixing increases beyond the limit specified by the standard, and in the subsequent stability experiments, the maximum unknown single impurity content is slowly increased, which does not meet the quality requirements of the preparation. The initial pH value is regulated to 4.0, the initial content of the maximum unknown single impurity is greatly reduced, the impurity content is not greatly increased under different temperature conditions along with the time extension, and the impurity content is always within the limit, so that the mixture freeze-dried powder preparation has better stability under the condition of pH=4.0.

Table 9: table of impurity content record of epirubicin at different pH conditions and at 5 days and 10 days of standing for epirubicin and R848 mixed freeze-dried powder formulation.

Example C2: stability study of doxorubicin (ADM) and imiquimod mixed lyophilized powder formulations.

A liquid formulation of doxorubicin and imiquimod was prepared by the method of example A1, with an initial ph=3.2 after mixing doxorubicin hydrochloride and imiquimod, with ph=4.0 after adjustment,the difference of chemical stability of the freeze-dried powder preparation obtained after freeze-drying at 2 pH values is explored, and the content of the freeze-dried protective agent is 91 percent. In the detection, imiquimod is very stable in the mixed preparation, the impurity content of the imiquimod is basically unchanged, and the impurity BIs kept at about 0.05%, impurity A->Is kept at about 0.05%, impurity C->The maximum unknown single impurity is kept at about 0.18%, the total impurity is basically within 0.39%, and the micro fluctuation of imiquimod impurity can be caused only when the imiquimod impurity is placed at different temperatures for different times, and the limits specified in the R837 preparation standard are not exceeded: 0.15% of impurity A, 0.20% of impurity B, 0.15% of impurity C, 0.20% of maximum unknown single impurity and 1.0% of total impurities.

The impurity content record of doxorubicin in stability study is shown in Table 10, and impurity A of doxorubicin is according to the United states pharmacopoeia guidanceImpurity C is->The impurity of doxorubicin also can appear the condition of growing rapidly under lower pH condition, and when adjusting pH to 4.0, impurity can keep at safer level, and stability is better.

Table 10: table of impurity content record of doxorubicin hydrochloride (ADM) and imiquimod (R837) compound lyophilized powder formulations at different pH conditions for 5 days and 10 days at different temperatures.

Example C3: investigation of stability of mitoxantrone hydrochloride and imiquimod Compound lyophilized powder formulation

The method of example A1 was used to prepare a liquid mixture of mitoxantrone hydrochloride and imiquimod, and the mixture was lyophilized after the addition of a lyoprotectant to prepare a lyophilized powder formulation, wherein the lyoprotectant had a lactose content of 91%, and the chemical stability of the lyophilized powder formulation was tested at ph=3.2 (without pH adjustment) and ph=4.0 (pH after adjustment) before lyophilization. Wherein the stability of the imiquimod is consistent with the phenomenon of the mixed preparation, and the related impurities of the imiquimod have no obvious change and are all within limits. The impurity A of mitoxantrone hydrochloride isImpurity D is->The impurity contents of mitoxantrone hydrochloride are shown in table 11, according to the guidance of united states pharmacopeia, no impurity B and no impurity C are detected, the maximum unknown single impurity content of mitoxantrone hydrochloride should not exceed 1.0%, the total impurity content does not exceed 3.0%, and the impurity contents are recorded in table 11, no impurity limit is exceeded, no matter at low pH or high pH, but the impurity amplification is slower after adjusting pH to 4.0 compared to the pH obtained after the original dissolution, and basically fluctuates around the value of the initial impurity content, indicating that the lyophilized powder formulation is more stable at ph=4.0.

Table 11: the initial impurity content of mitoxantrone hydrochloride under different pH conditions and the impurity content of mitoxantrone hydrochloride under different temperature conditions of the mitoxantrone hydrochloride and R837 mixed freeze-dried powder preparation are recorded in the table.

Example C4: stability of mixed freeze-dried powder preparation of pirarubicin and imiquimod is examined

The preparation method comprises the steps of preparing a mixed lyophilized powder preparation of the pirarubicin and the imiquimod, wherein the pH=4.0 or 5.0 of the lyophilized preparation before lyophilization, and the lyoprotectant is lactose with the content of 91%. The impurity levels of imiquimod and pirarubicin after lyophilization were measured and the results are shown in tables 12 and 13. The results show that the impurity content of imiquimod has no large change under different pH conditions, which indicates that imiquimod is stable in the system. At a ph=4.0, the impurity increase of pirarubicin is greater than the impurity change at a ph=5.0 (measured ph=4.99), and the impurity detection limit of the pirarubicin drug is not specified in pharmacopoeia, so that the condition of ph=5.0 is more favorable for the stability of the preparation only by judging the impurity increase trend.

Table 12: imiquimod impurity content record table of imiquimod impurity content of the mixed lyophilized powder formulation of pirubicin hydrochloride and imiquimod at different initial pH values and at 40 ℃ for 5 days and 15 days.

Table 13: the initial impurity content of the pirarubicin hydrochloride and imiquimod mixed freeze-dried powder preparation at different initial pH values is recorded in table of the impurity content of the pirarubicin hydrochloride when the pirarubicin hydrochloride and imiquimod mixed freeze-dried powder preparation is placed at 40 ℃ for 5 days and 15 days.

Example D: animal experiment

Example D1: application of epirubicin combined R837 in bladder cancer treatment

And preparing the epirubicin and R837 mixed freeze-dried powder preparation for standby.

Mouse MB49 bladder carcinoma in situ tumor models were established, randomly divided into 4 groups:

blank: pouring a 5% glucose solution;

EPI: pouring a 5% dextrose solution-dissolved epirubicin solution;

r837: perfusing a 5% dextrose solution dispersed imiquimod suspension;

epi+r837: pouring a 5% dextrose solution dissolved epirubicin and imiquimod mixed freeze-dried powder preparation;

the first bladder perfusion dose was recorded as day 0, the second dose was performed on day 7, mice were sacrificed on day 12, bladder tumor recorded mass was taken, and the mean of the tumor masses from the different groups was counted. The results are shown in figure 1, wherein the tumor quality of mice treated with the compound preparation is obviously lower than that of other groups, which indicates that the mixed freeze-dried powder preparation of epirubicin and imiquimod has the effect of inhibiting the growth of bladder cancer.

Example D2: application of pirarubicin (THP) combined with R837 in bladder cancer treatment

A mouse bladder cancer in situ model was established in the same manner as in example D1, and a mixed lyophilized powder formulation of pirarubicin and imiquimod was prepared for use, and the mice were randomly grouped:

blank: pouring a 5% glucose solution;

THP: pouring a 5% dextrose solution-dissolved pirarubicin solution;

r837: perfusing a 5% dextrose solution dispersed imiquimod suspension;

thp+r837: pouring 5% glucose solution dissolved pirarubicin and imiquimod mixed freeze-dried powder preparation;

and bladder perfusion treatment is carried out, after two bladder perfusion treatments and the treatment frequency and the termination time are carried out, the recorded tumor mass quality of the bladder cancer of the mice is obtained through dissection, a statistical chart is shown in fig. 2, the compound preparation obtained by re-dissolving 5% glucose solution has obvious anti-tumor effect, the tumor inhibition rates are 28.23% and 38.33% respectively when the pirarubicin or R837 is singly used, and the tumor inhibition rate of the mice treated by the compound preparation is as high as 81.6%. The pharmaceutical synergy is calculated by using the golden formula q=e (a+b)/(ea+eb-EA), wherein E (a+b) is the tumor inhibition rate of the compound preparation treatment group, EA and EB are the tumor inhibition rates of the two components when the two components are used independently, and when q is more than or equal to 1, the two components are proved to have the synergistic effect. The q is more than 1, which is calculated by a formula, and shows that the form of the compound preparation can achieve the synergistic effect of the anthracycline and the immunoadjuvant.

Example D3: use of doxorubicin (ADM) in combination with resiquimod (R848) in bladder tumor treatment

A mouse bladder cancer in-situ model was established in the same manner as in example D1, and a mixed lyophilized powder formulation of doxorubicin and imiquimod was prepared for use, and the mice were randomly grouped (Blank: perfused with 5% dextrose solution; ADM: perfused with physiological saline-dissolved doxorubicin solution; R848: perfused with 5% dextrose solution-dispersed resiquimod suspension; ADM+R848: perfused with 5% dextrose solution-dissolved doxorubicin and resiquimod mixed lyophilized powder formulation), wherein hydroxypropyl methylcellulose was added as a permeation enhancer component prior to use of the compound formulation, for bladder perfusion treatment. After two times of bladder perfusion, the mice bladder cancer record tumor mass quality is obtained through dissection, the statistical chart is shown in figure 3, and compared with the effects of the embodiment D1 and the embodiment D2, the compound preparation obtained through physiological saline reconstitution has more obvious anti-tumor effect after being combined with a mucous membrane permeation promoter. The tumor inhibition rate of the compound preparation is 85.1% in the group treated by the compound preparation, and the tumor inhibition rate of the mice is 21.8% and 8.6% in the group treated by the R848 or doxorubicin alone, so that the curative effect of the compound preparation is obviously improved.

The compound freeze-dried powder preparation of the anthracycline and the imidazoquinoline immunoadjuvant can treat bladder tumors in a bladder perfusion mode by combining multiple groups of data in the embodiment D.

Example E: the mucosa penetration enhancer helps the bladder retention effect of the active ingredient in the preparation

Example E1: effect of azone on retention effect of active ingredient of bladder perfusion preparation in bladder.

Grouping:

1. composite preparation: the compound preparation of epirubicin and imiquimod is infused into the bladder by 100 mu L;

2. azone + complex formulation: preparing a compound freeze-dried powder preparation containing 2% (w/w) azone, and administering 100 mu L of the compound freeze-dried powder preparation to a mouse through bladder perfusion after reconstitution;

the mixed solution is directly poured into the urinary bladder of the mouse, the pouring process is carried out in the anesthetic process of the mouse, the poured solution is reserved in the urinary bladder for 1 hour, then the liquid is discharged, the mouse is separated from the anesthetic state, the mouse is helped to drink 500 mu L of water in a gastric lavage mode, defecation is promoted, after the medicine is discharged for 6 hours, the urinary bladder of the mouse is obtained through dissection, and weighing and recording are carried out on each sample. Grinding the sample in buffer solution with equal volume, extracting the effective components with methanol and extractive solution, centrifuging to obtain supernatant, detecting the content of the extracted effective components in different samples by high performance liquid chromatography, and calculating the content of the extracted effective components per gram of tissue, wherein the results are shown in fig. 4 and 5. FIG. 4 is a statistical plot of the effect of a mucolytic agent on the retention of epirubicin in the bladder, showing the relative tissue content of epirubicin; fig. 5 is a statistical plot of the effect of a mucolytic agent on imiquimod retention in the bladder, and the relative tissue content of imiquimod can be seen. The compound preparation added with the azone component can obviously improve the retention of the medicine in bladder tissues, the retention amount of the medicine reaches more than 2 times, and the addition of the permeation enhancer can help the retention of active ingredients, so that the compound preparation further has the effect of potentially increasing the curative effect.

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. The bladder perfusion medicine compound preparation is characterized by comprising an anthracycline, an immunomodulator and a pH regulator;

the mass ratio of the anthracycline to the immunomodulator is 1: 1-1: 4, a step of;

the anthracycline is selected from epirubicin, a soluble salt of epirubicin, pirarubicin, a soluble salt of pirarubicin, mitoxantrone, a soluble salt of mitoxantrone, doxorubicin, a soluble salt of doxorubicin;

the immunomodulator is imiquimod or a soluble salt of imiquimod; the pH of the bladder perfusion drug compound preparation is 4.0-5.0;

the bladder perfusion drug compound preparation further comprises a lyoprotectant, wherein the lyoprotectant is lactose, and the mass fraction of the lyoprotectant is 67% -94%.

2. The bladder perfusion pharmaceutical compound preparation according to claim 1, wherein the mass ratio of anthracycline to immunomodulator is 1: 2-1: 4.

3. the bladder perfusion medicine compound preparation is characterized by comprising an anthracycline, an immunomodulator and a pH regulator; the mass ratio of the anthracycline to the immunomodulator is 1:4, a step of;

the immunomodulator is resiquimod;

the anthracycline is selected from the group consisting of epirubicin and a soluble salt of epirubicin; the pH value of the bladder perfusion medicine compound preparation is 4.0;

the bladder perfusion drug compound preparation also comprises a lyoprotectant, wherein the lyoprotectant is lactose, and the mass fraction of the lyoprotectant is 91%.

4. A bladder perfusion pharmaceutical compound formulation according to claims 1-3, wherein the soluble salt is the hydrochloride salt.

5. The bladder perfusion pharmaceutical compound formulation according to any one of claims 1-4, further comprising a mucosal permeation enhancer.

6. The drug compound preparation for bladder perfusion according to claim 5, wherein the mucous membrane permeation promoter is at least one selected from azone, hyaluronidase, lauryl alcohol and oleic acid.

7. A lyophilized powder formulation comprising the bladder perfusion pharmaceutical compound formulation of any one of claims 1-6.

8. A method for preparing a bladder perfusion pharmaceutical compound formulation according to any one of claims 1 to 6, wherein the immunomodulator is dissolved with dilute acid to obtain a dilute acid salt solution of the immunomodulator; dissolving the anthracycline with the dilute acid salt solution to obtain a mixed solution, and regulating the pH value of the mixed solution; and the method also comprises the step of adding a freeze-drying protective agent into the mixed solution and performing freeze-drying treatment.

9. The method for preparing a compound preparation of a bladder irrigation drug according to claim 8, wherein the pH value of the mixed solution is adjusted by an acidic or basic buffer or solution.

10. The method for preparing a compound preparation for bladder perfusion according to any one of claims 8 or 9, further comprising the step of adding a mucous membrane permeation enhancer to the mixed solution.

11. A bladder perfusion drug, comprising the bladder perfusion drug compound preparation of any one of claims 1-6.

12. A bladder perfusion medicament comprising the lyophilized powder formulation of claim 7.