CN115192721A - Bladder perfusion medicine compound preparation and preparation method and application thereof - Google Patents
Bladder perfusion medicine compound preparation and preparation method and application thereof Download PDFInfo
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- CN115192721A CN115192721A CN202210627784.5A CN202210627784A CN115192721A CN 115192721 A CN115192721 A CN 115192721A CN 202210627784 A CN202210627784 A CN 202210627784A CN 115192721 A CN115192721 A CN 115192721A
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- bladder
- preparation
- imiquimod
- epirubicin
- bladder perfusion
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Abstract
The invention discloses a bladder perfusion medicine compound preparation, which comprises an anthracycline chemotherapeutic medicine capable of causing immunogenic death of tumor cells, an immunomodulator and a pH regulator. The compound preparation has good chemical stability, and solves the problem of impurity growth caused by the influence of immunological adjuvant on anthracycline drugs.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a compound preparation of an anthracycline and an immunologic adjuvant, a preparation method and application in preparation of a bladder perfusion medicine.
Background
Bladder cancer is the most common malignant tumor of the urinary system, and the incidence rate is the ninth of the global malignant tumor; about 54.9 million new bladder cancer patients are suffered from the bladder cancer in 2020, and the number of dead people is nearly 20 ten thousand. The global bladder cancer market size has grown to $ 39 million in 2019, and is expected to reach $ 71 million in 2024, with bladder cancer being one of the most costly tumors to treat in the united states. The expensive treatment costs mainly result from the high recurrence rate and high metastasis rate of bladder cancer, second-line treatment regimens are lacking, and the clinical needs of bladder cancer are far from being met.
There are two major types of bladder cancer that are clinically common, non-muscle invasive bladder cancer and muscle invasive bladder cancer. chemotherapy/BCG instillation plus transurethral cystectomy (TURBT) is currently used clinically for non-muscle invasive bladder cancer. Studies have shown that this therapy is effective in low risk patients: the one-year recurrence rate is 15 percent, and the five-year recurrence rate is 30 percent; for intermediate-risk patients: the one-year recurrence rate is 38 percent, and the five-year recurrence rate is 62 percent; for high risk patients: the one-year recurrence rate is 61 percent, the five-year recurrence rate is 78 percent, and the BCG perfusion can solve the tumor recurrence problem to a certain extent, but the BCG perfusion can cause systemic reactions, such as long-time fever, the bladder perfusion BCG can have symptoms of frequent micturition, urgent micturition, hematuria and the like, and the immune-deficient patients can be at risk of suffering from the systemic BCG diseases by using the BCG.
The bladder perfusion is to inject chemotherapeutic medicine or tumor treating preparation capable of killing tumor cells directly into bladder to kill tumor cells directly or induce in vivo nonspecific immunoreaction to reach the effect of inhibiting the metastasis and recurrence of bladder cancer. The patients treated by the bladder perfusion adjuvant therapy have lower recurrence probability, but have low absolute remission rate, higher tumor recurrence rate, great toxic and side effects and poor patient prognosis.
Anthracyclines have high susceptibility to various transitional epithelial cell cancers, and currently, anthracyclines represented by epirubicin and pirarubicin are first-line treatment drugs for bladder cancer perfusion treatment. However, after the drug is singly applied to bladder perfusion treatment, the tumor still has high recurrence rate (more than 50%) within one year.
Toll-like receptors (TLRs) are a component of innate immunity, and in recent years have occupied a niche in the field of immunotherapy, and these receptors are widely expressed in immune cells and, after being stimulated accordingly, promote T cell responses against tumors. For example, imidazoquinoline based immunomodulators, a representative of which is imiquimod and its derivatives, are TLR-7 stimulators and have been used in the immunotherapy of superficial tumors.
Disclosure of Invention
The applicant tries to combine the anthracycline and imiquimod for the perfusion treatment of the bladder cancer, directly mixes the two medicines and then perfuses the bladder to verify the curative effect. The results show that when the ratio of the chemotherapeutic epirubicin to the imiquimod is in a proper range, the chemotherapeutic epirubicin to the imiquimod can obtain a remarkably improved treatment effect compared with the independent chemotherapeutic perfusion, does not increase toxic or side effect, and is expected to become a novel bladder perfusion medicinal preparation with remarkable clinical value. Further research shows that the novel preparation has an anti-tumor mechanism, and mainly induces immunogenic death of tumor cells to expose tumor-associated antigens through therapeutic drugs such as epirubicin, and simultaneously imiquimod serving as a TLR7 agonist can activate an immune system, recruit antigen presenting cells into tumors to identify the tumor antigens, and further activate T cell immune response aiming at the tumors. Based on the principle, the effect of combining different anthracyclines with imiquimod (R837) or resiquimod (R848) for treating tumors is tried, and ideal curative effect is achieved.
However, in further preparation studies, it was found that in a simple mixed preparation of an anthracycline chemotherapeutic drug such as epirubicin and an immune adjuvant imiquimod, the existence of imiquimod molecules can cause instability of the molecular structure of the anthracycline chemotherapeutic drug such as epirubicin, and the content of impurities in the preparation can be obviously increased no matter the preparation is stored in the form of solution of injection or in the form of mixed freeze-dried powder, the impurity limit of the drug specified by pharmacopoeia is broken through within 5 days, the preparation cannot be a clinically applicable pharmaceutical preparation which can be stably stored, and the simple mixing of the two is not favorable for the chemical stability of the drug molecules. Therefore, how to improve the chemical stability of the compound preparation of the anthracycline drug and the imidazoquinoline immunomodulator is a key problem for designing and preparing the compound preparation of the anthracycline drug and the imidazoquinoline immunomodulator. In order to solve the derivation problem, the stability of the anthracycline and imidazoquinoline immunomodulator compound preparation is further researched systematically.
First, we have found that epirubicin and imiquimod are more stable at low pH values (e.g., pH 3.0) in solution, but that anthracyclines are still gradually degraded, and that mixed formulations cannot be stored for long periods of time under these conditions, and thus, injection formulations are not feasible for this formulation. In the case of a freeze-dried preparation, we unexpectedly found that, in a common freeze-drying protective agent, lactose has a significant protective effect on the stability of the preparation, and meanwhile, the increase of the content of the lactose is beneficial to the improvement of the stability of the preparation. However, the lyophilized preparation prepared under the condition of pH3.0 still could not be stored for a long period of time, and it was not examined by the stability acceleration experiment. We find that the compound freeze-dried preparation of the anthracycline immunoadjuvant has a stable 'pH value golden interval', and the epirubicin/imiquimod compound preparation prepared in the pH range has very good stability! This phenomenon is contrary to the conventional understanding of epirubicin single formulations by those skilled in the art (the lower the pH is, the more stable in the conventional case) and our predicted results on the trend of stability of the compound formulation in solution state (the lower the pH is, the more stable).
Further, we examined the stability of other anthracycline drugs such as mitoxantrone, doxorubicin, and imidazoquinoline immunomodulators (R837 or R848) compound lyophilized powder formulations, and based on the detailed examination of epirubicin and R837, attempted the stability of different compounds under similar pH conditions. The results show 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 better 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 salt thereof.
Further, the anthracycline includes epirubicin or its soluble salt, pirarubicin or its soluble salt, mitoxantrone or its soluble salt, doxorubicin or its soluble salt, aclarubicin or its soluble salt, and idarubicin or its soluble salt.
Further optionally, the soluble salt is a hydrochloride salt.
Further, the lyophilized powder preparation of the bladder perfusion drug also comprises a freeze-drying protective agent.
Further, the freeze-drying protective agent comprises sucrose, lactose, mannitol and cyclodextrin.
Further optionally, the lyoprotectant is lactose.
Further, the mass fraction of the freeze-drying protective agent in the freeze-dried powder preparation is 65-96%.
Preferably, the content of the freeze-drying protective agent in the freeze-dried powder preparation is 80-94%.
Further, the pH regulator is weak base, alkaline buffer solution, sodium hydroxide and hydrochloric acid.
Further optionally, the pH adjusting agent is sodium bicarbonate, hydrochloric acid.
Further, after the bladder perfusion medicine compound preparation is redissolved, when the concentration of the anthracycline is 1-5 mg/mL, the pH value of the solution is 3.8-5.5. The purpose of the definition of the reconstitution concentration is to define the range of the pH value after reconstitution, and the reconstitution concentration is not limited.
Further optionally, after the lyophilized powder preparation for the bladder perfusion drug is redissolved, when the concentration of the anthracycline drug is 1-5 mg/mL, the pH of the solution is 4.0-5.0. The purpose of the definition of the reconstitution concentration is to define the range of the pH value after reconstitution, and the reconstitution concentration is not limited.
Furthermore, the imidazole quinoline immunomodulator comprises imiquimod and derivatives thereof, or resiquimod and derivatives thereof, or soluble salts of imiquimod and derivatives thereof, or soluble salts of resiquimod and derivatives thereof.
Furthermore, the mass ratio of the anthracycline drug to the imidazoquinoline immunomodulator is 1.
Further optionally, the mass ratio of the anthracycline drug to the imiquimod is 1.
Further optionally, the mass ratio of the anthracycline drug to the imiquimod is 1.
Further optionally, the mass ratio of the anthracycline drug to the resiquimod is 1.
Further, the freeze-dried powder preparation also comprises a mucosa penetration enhancer.
Further, the mucosa penetration enhancer comprises at least one of azone, hyaluronidase, lauryl alcohol and oleic acid.
Further, the mucosal permeation enhancer may be added during the preparation process, or may be mixed before use.
The compound preparation of the anthracycline and the immunologic adjuvant is mixed with a mucosa penetration enhancer and then is infused before use, and the mucosa penetration enhancer can be a medicinal mucosa penetration enhancer on the market or a mucosa penetration 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 using dilute acid to obtain a salt solution of the immunomodulator;
s2: dissolving the anthracycline in the dilute acid salt solution obtained in the step S1 to obtain a mixed solution;
s3: and (3) 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 method comprises the following steps: the step S3 further includes:
s31: adding a lyoprotectant to the mixed solution;
further, the method comprises the following steps: the step S3 further includes:
s32: adding a mucosa penetration enhancer to the mixed solution
Further, the dilute acid in S1 is hydrochloric acid, lactic acid or acetic acid.
Further optionally, a further optional range for adjusting the pH in S3 is 4.0 to 5.0.
Further, the method also comprises the step S4: and (4) performing 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 medicament. By adopting the technical scheme of the invention, the invention has the following beneficial effects:
the invention provides a bladder perfusion medicine compound preparation, which is a freeze-dried powder preparation mixed by an anthracycline medicine and an imidazoquinoline immunomodulator. In a large number of experimental analyses, the freeze-dried powder preparation is more beneficial to protecting the long-term chemical stability of the anthracycline in the mixed preparation compared with the traditional injection preparation, meanwhile, the mixed preparation has better tumor inhibition effect in the treatment of bladder perfusion tumor, can cause stronger antitumor immune response, and can reduce toxic and side effects compared with the single anthracycline medicine.
The anthracycline and immunomodulator mixed preparation can be clinically applied through simple redissolution operation, can be directly used for perfusion treatment, and can also be added with a mucosa permeation promoter for perfusion after mixing so as to effectively inhibit the growth and the recurrence of tumors. After the anthracyclines die due to tumor immunogenicity, the immunomodulators can further stimulate the antigen uptake, processing and presentation of antigen to T cells by antigen presenting cells, further amplifying the anti-tumor immune response.
Can reduce systemic side effects of anthracycline drugs or immunomodulators.
The long-term chemical stability of the mixed preparation of the anthracycline drug and the immunomodulator is realized, the generation rate of impurities of the anthracycline drug is obviously reduced in the presence of the immunomodulator, the long-term storage stability of the preparation is improved, and the convenience and the safety of the use of the preparation are improved. In future clinical use, the long shelf life is beneficial to the production, storage and transportation of the medicine, 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 infusion treatment can be prolonged, and the preparation is favorable for further improving the curative effect of the preparation.
Drawings
FIG. 1 is a statistical plot of tumor mass of different groups of mice treated for bladder cancer by bladder perfusion after administration of a mixed lyophilized powder formulation of epirubicin and imiquimod to the mice;
FIG. 2 is a statistical plot of mouse tumor mass after the mixed lyophilized powder formulation of pirarubicin and imiquimod was used for perfusion treatment of mouse bladder cancer;
FIG. 3 is a statistical plot of tumor mass of different groups of mice treated with doxorubicin and imiquimod mixed lyophilized powder formulation via bladder perfusion for bladder cancer;
FIG. 4 is a statistical plot of the effect of mucosal permeation enhancers on the retention of epirubicin in the bladder;
figure 5 is a statistical plot of the effect of mucosal permeation enhancers on imiquimod retention in the bladder.
Detailed Description
In order to more thoroughly express the technical scheme of the invention, the following specific examples are listed to demonstrate the technical effect; it is emphasized that these examples are intended to illustrate the invention and are not to be construed as limiting the scope of the invention.
As used in this specification and the appended claims, the terms "a," "an," "the," and/or "the" are not intended to be inclusive in the singular, but rather are intended to be inclusive in the plural, unless the context clearly dictates otherwise. The terms "comprises" and "comprising" merely indicate that elements are included that are specifically identified and do not constitute an exclusive list, as other elements may be included in a device. The term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment". Relevant definitions for other terms will be given in the following description.
Test method and test apparatus:
moist heat sterilization: shanghai Bo News medical Bio-instrumentation, inc. model YXQ-50A;
a freeze dryer: shanghai Dongfulong science and technology Co., ltd, model number LY0-0.5;
high performance liquid chromatography: waters e2695&2489;
a stability box: the manufacturer is Shanghai Suyun testing apparatus Co., ltd, and the model is YP-TGSD400.
Example A: study on the stability of the formulation of the injection
In example a, a compound preparation of epirubicin and R837 was prepared without lyophilization, and the change in impurities of the injection formulation under different pH conditions and temperature conditions was studied to determine whether the injection formulation satisfied the actual production and use conditions of the compound preparation.
Example A1: chemical stability of the bladder perfusion medicine composition injection preparation under different medicine liquid pH conditions.
The imiquimod is dissolved by hydrochloric acid and then dissolved by adding epirubicin hydrochloride, the pH value of the solution is low, great stimulation is generated to tissues during perfusion, and in order to reduce the stimulation to the tissues during use, the pH value which gives consideration to both the stability and the use safety of the preparation needs to be selected.
Dissolving R837 with hydrochloric acid to obtain R with concentration of 4mg/mL837 and epirubicin hydrochloride (EPI) is added to dissolve, the concentration of EPI is 1mg/mL, then sodium bicarbonate is used to adjust the pH of the solution to 3.0, 3.5 and 4.0, which are respectively marked as injection 1, injection 2 and injection 3, and the content of epirubicin and imiquimod impurities in the pharmaceutical preparation on the preparation day and on the 5 th day in the environment of 40 ℃ is detected by liquid chromatography, and the result is shown in Table 1. The impurities mainly come from epirubicin hydrochloride, and the structure of the impurity A (EPI-A) is
The structure of impurity C (EPI-C) is
The results show that the total impurity content of the formulation increases with time compared to the bulk drug at three pH conditions, and that the total impurity content is more than 3 times the total impurity content of the bulk drug on day 5 when left in a 40 ℃ environment. Even at the most stable conditions of pH =3.0, the unknown impurity of EPI increases significantly after only 5 days of storage at 40 ℃, and is expected to quickly exceed the maximum allowable limits set by the pharmacopoeia. Meanwhile, the impurity content of the imiquimod is relatively stable, in multiple detections, the imiquimod impurity B is kept at about 0.05%, the imiquimod impurity A is kept at about 0.05%, the imiquimod impurity C is not detected, the maximum unknown single impurity is kept at about 0.18%, the total impurities are basically within 0.39%, and the maximum impurity content is not exceeded: 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 epirubicin/imiquimod compound injection liquid type does not have good stability, and the main impurity growth comes from epirubicin, so that the safety and the effectiveness of clinical medication cannot be ensured.
Table 1: the initial impurity content of epirubicin in the epirubicin/imiquimod compound injection type at different pH and the impurity content of Epirubicin (EPI) left at 40 ℃ for 5 days are reported.
Example A2: effect of different storage conditions on the stability of injectable formulations
Considering that the temperature at which the formulation of example A1 was left to stand was 40 ℃, the generation of impurities might be accelerated, and therefore the stability of the formulation at lower temperatures was investigated. Bladder perfusion injections of pH =3.0 and pH =3.5 were prepared by the method of example A1, placed under refrigeration at 15 ℃,25 ℃, and tested for impurity content in the formulation samples on the day of sample preparation completion and on the 10 th day of standing at different temperatures, with the results 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, which proves that the stability of the injection formulation is related to the temperature. It was also found that even under refrigerated conditions there is a growing trend for the maximum unknown impurity, which is also detrimental to the overall stability and safety of the formulation, and therefore, the dosage form of bladder instillation injection is most likely not the preferred dosage form of the formulation.
Table 2: the initial impurity content of Epirubicin (EPI) in the epirubicin/imiquimod compound injection formulation and the impurity content of Epirubicin (EPI) in the epirubicin/imiquimod compound injection formulation when left to stand at different storage temperatures for 10 days are recorded in the table.
Example B: freeze-dried dosage form stability study
The results in example A show that the compound preparation is difficult to produce, store and use as an injection dosage form, so that design experiments are carried out to investigate the feasibility of the freeze-dried powder dosage form of the compound preparation.
Example B0: research on drug properties of freeze-dried dosage forms:
in order to preliminarily research and explore the possibility of the medicament formation of the freeze-dried powder preparation, the factors such as auxiliary materials, pH and the like which possibly influence the medicament formation are preliminarily screened.
Dissolving imiquimod hydrochloride by using hydrochloric acid or dissolving imiquimod hydrochloride by using pure water to obtain an aqueous solution of the imiquimod hydrochloride, and then dissolving epirubicin hydrochloride to obtain a solution containing effective components, wherein the mass ratio of the effective components epirubicin to the imiquimod is 1:1 to 1:10; secondly, trying to adjust the pH value of the solution and observing whether the change of the pH value influences the stability of the solution; 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 a sample, and freeze-drying the sample by using 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 the freeze-dried powder to see whether unformed, agglomerated, caked or uneven samples which are distinguishable by naked eyes appear; and finally, adding a solvent into the freeze-dried sample for redissolving, observing whether the phenomena of insolubility and nonuniformity occur, and retesting the pH value of the solution, wherein when the concentration of the epirubicin in the solution is 1mg/mL, the pH value is close to the value before freeze-drying and is about 3.8-5.5.
In the preparation process of the freeze-dried powder preparation, the conditions of unevenness or obvious instability such as precipitation and the like of the solution are not caused in the pH adjusting process and the addition of the freeze-drying protective agent, the sample after freeze-drying keeps a better appearance and is smoothly redissolved, and the phenomenon of insolubility or unevenness of dissolution does not occur, so that the formula of the freeze-dried powder preparation can be used for further stability research and detecting the change of impurity content.
Example B1: initial attempts to add lyoprotectants on the stability of the formulations
Liquid preparations were prepared according to the method of example A1, with an initial pH of about 3.0 when pH was not adjusted, and further, with pH adjusted to 3.5 using sodium bicarbonate, with epirubicin and imiquimod at final concentrations of 1mg/mL and 4mg/mL, respectively, and with lactose or mannitol at various contents (prepared at concentrations of 10mg/mL, 25mg/mL, 50mg/mL, converted to mass fractions of 67%,83%, 91%) added before lyophilization, and the impurity contents were examined on day 0 after lyophilization. The results are shown in Table 3. The result shows that when no freeze-drying protective agent is added, the initial maximum unknown single impurity content in the compound preparation exceeds the limit, and the initial impurity content is reduced after the freeze-drying protective agent with proper content is added. The protective effect of lactose on the freeze-dried preparation is better than that of mannitol, and the increase of pH and the increase of lactose content are both beneficial to the stability of the product.
Table 3: the initial impurity content of Epirubicin (EPI) in the epirubicin/imiquimod lyophilized powder formulation after addition of different amounts of lactose or mannitol is reported in the table.
Example B2: lactose is used as freeze-drying protective agent, and the freeze-dried powder preparation is kept standing for 5 days at 25 ℃.
To further investigate the effect of lyoprotectant content and initial pH on the stability of lyophilized powder, samples No. 4-1, 5-1, and 6-1 from example B4 were placed at 25 ℃ and tested for pH and impurity content after reconstitution on day 5, as shown in Table 4 together with the data for day 0. From the change of impurity content, the change of impurity content of the lyophilized powder preparation with the initial pH value of 3.5 is better than that of the lyophilized powder preparation with the pH value of 3.0, and meanwhile, when the lactose content is 91%, the increase of impurity content is minimum.
Table 4: the initial impurity content of Epirubicin (EPI) in the lactose-added epirubicin/imiquimod compound lyophilized powder formulation and the impurity content of Epirubicin (EPI) after 5 days at 25 ℃ are recorded.
Example B3: screening of different lyoprotectants
Based on the initial exploration on 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-dried powder preparation under the same content is further explored. And further trying the stability of the lyophilized preparation at a higher pH value based on the relationship between the pH value variation tendency and the impurity variation tendency.
Lactose (glucose), mannitol (non-saccharide), cyclodextrin (cyclic oligosaccharide) and glucose (monosaccharide) are selected from common freeze-drying protective agents for injection and are examined, and the protective effect of the freeze-drying protective agent on freeze-dried powder preparation is verified under the condition that the dosage of the freeze-drying protective agent is 91%.
First, a liquid preparation is prepared according to the method of example A1, the pH of the solution is adjusted to 3.5 or 4.0, different lyoprotectants are respectively added, the final concentration of imiquimod is 4mg/mL, the concentration of epirubicin is 1mg/mL, the final content of the lyoprotectants after freeze-drying is 91%, and the content change of epirubicin hydrochloride impurities after the lyoprotectant is placed at 40 ℃ for 5 days is detected, and the results are shown in table 5. In table 5, when the lyophilized powder preparation with an initial pH of 4.0 is lactose, the lyophilized powder preparation has the best stability, the impurity content remains low after standing at 40 ℃ for 5 days, the impurity does not increase significantly, and the impurity increases significantly under the condition of pH = 3.5; the impurities of other freeze-drying protective agents obviously grow under the conditions of pH3.5 and pH4.0. Therefore, lactose is preferred as the lyoprotectant.
Table 5: epirubicin/imiquimod compound freeze-dried powder preparations with different initial pH values and different freeze-drying protective agents are added. The table is recorded for the initial impurity level of epirubicin and the impurity level of Epirubicin (EPI) at day 5 when left at 40 ℃.
Example B4: effect of lactose content on the stability of lyophilized powder formulations
Based on the results of the previous examples, the effect of adding lactose at different levels on the impurity content of the lyophilized powder formulation at pH4.0 was examined.
According to the preparation method described in example A1, mixed injection of EPI and R837 is prepared, after different contents of lactose protective agent are added, sodium bicarbonate is used for adjusting pH to 4.0, the final concentration of imiquimod is 4mg/mL, the concentration of epirubicin is 1mg/mL, the concentration of freeze-drying protective agent is 20mg/mL,30mg/mL,40mg/mL and 50mg/mL, the mass fractions of the freeze-drying protective agent after freeze-drying are respectively 80%, 86%, 89% and 91%, and the impurity contents are detected on the day of freeze-drying, at 40 ℃, and on the 5 th day and the 13 th day. 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 and slowed down, which indicates that the chemical stability of the compound preparations is increased. The chemical stability is best when the content of impurities does not exceed the limit of the initial content of the lyoprotectant to 91% under the conditions of 80%, 86%, 89% and 91% of the lyoprotectant content.
Table 6: the table records the initial impurity content of Epirubicin (EPI) and the impurity content of 5 days and 13 days of standing at 40 ℃ in complex solutions with pH4.0 of epirubicin/imiquimod compound lyophilized powder formulations added with different lactose contents.
Example B5: preparation samples under different pH conditions were prepared and the stability of the lyophilized preparation was investigated.
Liquid preparations having pH values of 3.5, 4.0, 4.5 and 5.0 were prepared by the method of example A1 to obtain a final imiquimod concentration of 4mg/mL and an epirubicin concentration of 1mg/mL, and were lyophilized after a pre-freezing treatment, and after standing for a certain time under different temperature conditions, the lyophilized powder was redissolved to examine the impurity content therein, and the experimental conditions and results are shown in Table 7. Different from the trend of the total impurities of epirubicin hydrochloride in the injection dosage form, when the initial pH value is increased, the content of the total impurities is increased and slowed, and under the condition that the freeze-drying protective agent is lactose and the content is 91 percent, the changes of unknown single impurities and total impurities are lower than the content of the impurities of the injection dosage form, 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 dosage form which is placed for a short time (5 days) under the same pH (pH = 5.0) and temperature condition (25 ℃), and the freeze-dried powder preparation conforms to the current pharmacopoeia standards of various countries. Wherein the sample impurities have exceeded pharmacopoeia-specified limits on day 5 at 40 ℃ at pH =3.5 before lyophilization of the formulation, and therefore the sample is not analyzed for impurities for 30 days under these conditions.
Table 7: the impurity levels of Epirubicin (EPI) after the epirubicin/imiquimod lyophilized powder formulations were left in different temperature environments for different periods of time at different initial pH values are reported in the table.
Example B6: the ratio of the two components was varied and the change in impurity content of the lyophilized formulation was investigated.
Liquid formulations having pH values of 3.5, 4.0, 4.5, respectively, were prepared by the method of example A1, and lactose was added to give a final imiquimod concentration of 2mg/mL, an epirubicin concentration of 1mg/mL, and lactose content as shown in Table 8. Epirubicin impurity levels were recorded on the day following lyophilization and stored at 40 ℃ for 27 days, as shown in table 8. The results show that the impurity content of the samples decreased when placed at 40 ℃ for 27 days with increasing initial pH and increasing lactose content of the samples, with the total impurity content exceeding the limit on day 27 for an initial pH =4.0 and a lactose content of 67% in the samples, and the impurity content was below the limit for the remaining conditions. At pH =4.5, several lactose contents all reduced the increase in impurity levels in the lyophilized samples.
Table 8: the table shows the impurity content of Epirubicin (EPI) when the epirubicin/imiquimod compound freeze-dried powder preparation is placed in an environment of 40 ℃ for 27 days under different initial pH values.
Example B7: further screening for optimum 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, the lyoprotectant lactose was added, and the ratio of epirubicin and imiquimod was 1:2 or 1:4, the final concentration of lyoprotectant was 91%, with the corresponding mass fractions in the two specifications of lyophilized samples being 94% and 91%, respectively. After freeze-drying, the samples were placed at 40 ℃ and the content of impurities in the samples was measured at different time points, the results of which are shown in table 9.
The result shows that the epirubicin impurity growth trend is related to the initial pH value under any concentration ratio, 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 is out of limit, and when the pH value is above 3.8, the impurity content stability requirement is met.
Table 9: the initial impurity content of epirubicin in the epirubicin/imiquimod compound freeze-dried powder preparation and the impurity content of Epirubicin (EPI) when the epirubicin/imiquimod compound freeze-dried powder preparation is placed in an environment at 40 ℃ for 19 days and 30 days are recorded in a table.
Example C: stability study of mixed freeze-dried powder preparation of different anthracyclines and imidazoquinoline immunomodulators at pH =4.0
Example C1: stability of epirubicin hydrochloride and resiquimod (R848) mixed lyophilized powder preparation
Firstly, preparing a mixed preparation, specifically, dissolving R848 by hydrochloric acid to obtain a hydrochloride solution of R848, and further adding epirubicin hydrochloride, wherein the mass ratio of R848 to epirubicin is 4. Adding lactose as freeze-drying protective agent, wherein the final content of lactose is 91%. Freeze-drying, placing the freeze-dried powder at different environmental temperatures for stability investigation, and sampling on 5 th and 10 th days to detect the impurity content change in the sample.
Unlike the case of the mixture of epirubicin R837, after R848 is dissolved by hydrochloric acid and epirubicin is added, the initial pH of the solution system is about 5.5 and higher than that of imiquimod hydrochloride, so that the pH of the system is adjusted to about 4.0 by using an acidic solution, the impurity change of the original pH (pH = 5.5) condition and the impurity change of the pH =4.0 condition are detected, and the impurity detection result of R848 is shown in Table 8. Since R848 is not available on the market, the stability of R848 cannot be judged from the limit of the impurity content, but as can be seen from the data in table 8, the impurity content of R848 hardly changes with time regardless of the 25 ℃ or 40 ℃ condition, and therefore, it can be judged that R848 has good stability in the mixed lyophilized powder preparation.
Table 8: the impurity content of R848 in samples is reported when the lyophilized powder formulation of epirubicin mixed with resiquimod (R848) is allowed to stand for 5 days and 10 days at the initial impurity content of R848 at different pH conditions and temperatures.
The impurity detection results of epirubicin are shown in table 9, when the pH of the system before lyophilization is not adjusted, i.e. the initial pH =5.5, the maximum unknown single impurity content of epirubicin after mixing is increased and exceeds the limit specified by the standard, and in the subsequent stability experiment, the maximum unknown single impurity content is slowly increased and does not meet the quality requirements of the preparation. Adjusting the initial pH value to 4.0, the maximum unknown initial content of the single impurity is greatly reduced, and the impurity content is not greatly increased under different temperature conditions along with the prolonging of time and is always within the limit, which indicates that the mixture freeze-dried powder preparation has better stability under the condition of pH = 4.0.
Table 9: the table records the initial impurity content of epirubicin at different pH and the impurity content of epirubicin when left to stand for 5 days and 10 days at different temperature for the mixed lyophilized powder of epirubicin and R848.
Example C2: stability study of doxorubicin (ADM) and imiquimod mixed lyophilized powder formulation.
A mixed doxorubicin and imiquimod liquid formulation, the initial pH after mixing doxorubicin hydrochloride and imiquimod, was prepared as in example A1=3.2, pH =4.0 after adjustment, and the difference in chemical stability of lyophilized powder formulations obtained after lyophilization at 2 pH values was studied, and the lyoprotectant was lactose, with a content of 91%. In detection, the imiquimod is found to be quite stable in a mixed preparation, the impurity content of the imiquimod is basically unchanged, and the impurity B is
Keeping the content of the impurity A at about 0.05 percent
Keeping about 0.05 percent, and impurity C
The maximum unknown single impurity is kept to be about 0.18 percent and the total impurity is basically within 0.39 percent, and the minimum fluctuation of the imiquimod impurity can be caused only when the imiquimod is placed at different temperatures for different time, and the maximum unknown single impurity does not exceed the limit specified in the R837 preparation standard: 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 of doxorubicin in the stability study is reported in Table 10, and impurity A of doxorubicin is shown in accordance with the guidelines of the United states pharmacopoeia
Impurity C is
The impurities of doxorubicin can also grow rapidly under the condition of lower pH, and after the pH is adjusted to 4.0, the impurities can be kept at a safer level, so that the stability is better.
Table 10: the initial impurity content of the doxorubicin hydrochloride (ADM) and imiquimod (R837) compound lyophilized powder preparation under different pH conditions and the impurity content of doxorubicin when left standing for 5 days and 10 days under different temperature conditions are recorded in the table.
Example C3: investigation of stability of mitoxantrone hydrochloride and imiquimod Compound lyophilized powder preparation
A mixed solution preparation of mitoxantrone hydrochloride and imiquimod was prepared as in example A1, and lyophilized powder was prepared by lyophilization after addition of lyoprotectant, wherein the lyoprotectant lactose content was 91%, and the chemical stability of the lyophilized powder was tested when pH =3.2 (without pH adjustment) and pH =4.0 (pH after adjustment) before lyophilization. The stability of the imiquimod is consistent with the phenomenon of the mixed preparation, and related impurities of the imiquimod have no obvious change and are within the limit. The impurity A of mitoxantrone hydrochloride is
Impurity D is
The impurity content of mitoxantrone hydrochloride is shown in table 11, according to the guidance of the united states pharmacopoeia, impurity B and impurity C are not detected, the maximum unknown single impurity content of mitoxantrone hydrochloride should not exceed 1.0%, the total impurity content does not exceed 3.0%, in table 11, no matter under low pH value or high pH value, the impurity limit is not exceeded, but compared with the pH obtained after the original dissolution, after the pH value is adjusted to 4.0, the impurity amplitude is more slowly increased, basically fluctuates around the value of the initial impurity content, which indicates that when the pH =4.0, the freeze-dried powder preparation is more stable.
Table 11: the initial impurity contents of mitoxantrone hydrochloride and R837 mixed lyophilized powder formulations at different pH and temperature conditions are reported in the table.
Example C4: investigating the stability of the Pirarubicin and imiquimod mixed freeze-dried powder preparation
And preparing a mixed lyophilized powder preparation of the pirarubicin and the imiquimod, wherein the pH of the preparation is =4.0 or 5.0 before lyophilization, and the lyophilized protective agent 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 content of imiquimod impurity has no great change under different pH conditions, which indicates that the imiquimod is stable in the system. Under the condition of pH =4.0, the impurity increase of the pirarubicin is larger than the impurity change under the condition of pH =5.0 (the actually measured pH = 4.99), and the detection limit of impurities 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 increase trend of the impurities.
Table 12: the initial impurity levels of imiquimod at different initial pH values for the mixed lyophilized powder formulations of pirarubicin hydrochloride and imiquimod and the impurity levels of imiquimod when left at 40 ℃ for 5 days and 15 days are reported.
Table 13: tables are recorded for initial impurity levels of pirarubicin hydrochloride at different initial pH values for the mixed lyophilized powder formulations of pirarubicin hydrochloride and imiquimod and impurity levels of pirarubicin hydrochloride when left at 40 ℃ for 5 days and 15 days.
Example D: animal experiments
Example D1: application of epirubicin combined R837 in treatment of bladder cancer
And preparing a freeze-dried powder preparation of epirubicin and R837 for later use.
Establishing a mouse MB49 bladder orthotopic cancer tumor model, and randomly dividing the model into 4 groups:
blank: perfusing 5% glucose solution;
EPI: infusing epirubicin solution dissolved by 5% glucose solution;
r837: perfusing 5% glucose solution dispersed imiquimod suspension;
EPI + R837: infusing a 5% glucose solution dissolved epirubicin and imiquimod mixed freeze-dried powder preparation;
the first bladder perfusion administration was recorded as day 0, the second administration was performed on day 7, mice were sacrificed on day 12, bladder tumors were taken for recording the mass, and the mean of the masses of tumors of different groups was counted. The results are shown in fig. 1, wherein the tumor mass of the mice treated by the compound preparation is obviously lower than that of other groups, which indicates that the mixed lyophilized powder preparation of epirubicin and imiquimod has the effect of inhibiting the growth of bladder cancer.
Example D2: use of pirarubicin (THP) in combination with R837 in the treatment of bladder cancer
Establishing a mouse bladder cancer in-situ model by the same method as in the embodiment D1, preparing a mixed lyophilized powder preparation of pirarubicin and imiquimod for later use, and randomly grouping mice:
blank: perfusing 5% glucose solution;
THP: perfusing a pirarubicin solution dissolved in a 5% glucose solution;
r837: perfusing imiquimod suspension dispersed in 5% glucose solution;
THP + R837: injecting 5% glucose solution dissolved pirarubicin and imiquimod mixed freeze-dried powder preparation;
and bladder perfusion treatment is carried out, the treatment frequency and the termination time in the embodiment D1 are the same, after two bladder perfusion treatments, the tumor mass of the mouse bladder cancer record is obtained by dissection, the statistical chart is shown in figure 2, the compound preparation obtained by 5% glucose solution redissolving has obvious anti-tumor effect, the tumor inhibition rate is 28.23% and 38.33% when the pirarubicin or R837 is singly used, and the tumor inhibition rate of the mouse treated by the compound preparation is up to 81.6%. The synergistic effect of the medicines is calculated by using a King's formula q = E (A + B)/(EA + EB-EA-EB), E (A + B) is the tumor inhibition rate of a treatment group of the compound preparation, EA and EB are the tumor inhibition rates of two components when the two components are used independently respectively, and when q is more than or equal to 1, the two components have the synergistic effect. The formula calculation can be used, q is more than 1, and the formula shows that the form of the compound preparation can achieve the synergistic effect of the anthracycline and the immunologic adjuvant.
Example D3: application of Doxorubicin (ADM) in combination with resiquimod (R848) in bladder tumor treatment
Establishing a mouse bladder cancer in-situ model by the same method as that in the example D1, preparing a mixed freeze-dried powder preparation of doxorubicin and imiquimod for later use, and randomly grouping the mice (Blank: filling 5% glucose solution; ADM: filling a doxorubicin solution dissolved by normal saline, R848: filling a resiquimod suspension dispersed by 5% glucose solution, ADM + R848: filling a mixed freeze-dried powder preparation of doxorubicin and resiquimod dissolved by 5% glucose solution), wherein before using the compound preparation, hydroxypropyl methyl cellulose is added as a permeation enhancer component to carry out bladder perfusion treatment. After two bladder infusions, the groups were dissected to obtain mouse bladder cancer recorded tumor mass, and the statistical chart is shown in fig. 3, compared with the effects of examples D1 and D2, the compound preparation obtained by redissolving normal saline has more obvious anti-tumor effect after being combined with a mucosa penetration enhancer. In the group treated by the compound preparation, the tumor inhibition rate of the mouse is 85.1 percent, while in the group treated by R848 or doxorubicin alone, the tumor inhibition rate of the mouse is 21.8 percent and 8.6 percent respectively, and the curative effect of the compound preparation is obviously improved.
By combining the data in example D, the compound lyophilized powder preparation of the anthracycline and the imidazoquinoline immunoadjuvant can be used for treating bladder tumors in a bladder perfusion mode.
Example E: the mucosa penetration promoter helps retention of effective component in bladder
Example E1: influence of azone on retention effect of effective components of bladder perfusion preparation in bladder.
Grouping:
1. compound preparation: 100 μ L of a combination of epirubicin and imiquimod was instilled through the bladder;
2. azone + complex formulation: preparing a compound lyophilized powder formulation containing 2% (w/w) azone, and administering to mice an amount of 100 μ L by bladder instillation after reconstitution;
directly perfusing the bladder of the mouse with the mixed solution, wherein the perfusion process is carried out in the mouse anesthesia process, the perfusate is kept in the bladder for 1 hour, then discharging the liquid, the mouse is separated from the anesthesia state, and the mouse is assisted to drink 500 mu L of water in a stomach perfusion mode to promote defecation, dissecting to obtain mouse bladder samples after the medicine is discharged for 6 hours, and weighing and recording each sample. Grinding the sample in buffer solution with the same volume, extracting effective components with methanol and extractive solution, centrifuging to obtain supernatant, detecting the content of the effective components extracted from different samples with high performance liquid chromatography, and calculating the content of the effective components relative to each gram of tissue, with the results shown in fig. 4 and 5. FIG. 4 is a statistical plot of the effect of the mucosal permeation enhancer on epirubicin retention in the bladder, showing the relative tissue content of epirubicin; figure 5 is a statistical plot of the effect of mucosal permeation enhancer on imiquimod retention in the bladder, and it can be seen that the relative tissue content of imiquimod. The compound preparation added with the azone component can obviously improve the retention of the medicine in bladder tissues, the retention amount of the compound preparation reaches more than 2 times, and the addition of the penetration enhancer can help the retention of effective components and further has the potential effect of improving the curative effect.
The above examples are additionally described as follows:
all stability experiments in examples a-C samples were stored in a stability box under the conditions of the stability box accelerated stability testing: temperature 40 ℃/, humidity 75%; the stability box parameters of the long-term stability investigation experiment are: temperature 25 ℃ humidity 60% RH, protected from light.
The ' epirubicin and imiquimod mixed lyophilized powder preparation ' in the example D1 is the epirubicin and imiquimod mixed lyophilized powder preparation ' in the example B7, wherein the mass ratio of the epirubicin to the imiquimod is 4:1, the freeze-drying protective agent is lactose, the mass fraction of the lactose is about 91%, the pH value is about 4.2-4.4, and the administration doses of epirubicin and imiquimod are respectively 60 mu g/unit and 240 mu g/unit.
The 'pirarubicin and imiquimod mixed lyophilized powder preparation' in the example D2 is the mixture of the pirarubicin and the imiquimod in the example C4 in the mass ratio of 1:1, the freeze-drying protective agent is lactose, the mass fraction of the lactose is about 91%, the pH value is about 4.5-5.0, and the administration dose is 60 mu g per unit.
The 'doxorubicin and resiquimod mixed lyophilized powder preparation' described in example D3 is the doxorubicin and resiquimod mixed lyophilized powder preparation described in example C4, wherein the mass ratio of doxorubicin to resiquimod is 1:1, the freeze-drying protective agent is lactose, the mass fraction of the lactose is about 91%, the pH value is about 4.8, and the administration dosage is 150 mu g per capsule.
By combining the experimental data in example D, the compound lyophilized powder preparation of anthracyclines and imidazoquinolines immunoadjuvants is used to treat mouse bladder cancer by bladder instillation, and compared with single chemical or immunoadjuvant, the compound lyophilized powder preparation of anthracyclines and imidazoquinolines has significantly improved therapeutic effect, and the chemical and immunoadjuvants have synergistic therapeutic effect.
The compound epirubicin and imiquimod preparation described in example E1 is prepared by the preparation method described in example B0, wherein the initial pH of the lyophilized powder is 4.0 to 4.2, the mass fraction of the lyoprotectant is 91%, the administration dose of epirubicin is 60 μ g/piece, and the administration dose of imiquimod is 240 μ g/piece. The "compound lyophilized powder preparation containing azone" described in example E1 was prepared by the following steps: s1: dissolving imiquimod or soluble salt thereof by using dilute acid to obtain an imiquimod dilute acid salt solution; s2: dissolving epirubicin by using the solution of S1 to obtain a mixed solution; s3: adjusting the pH value of the mixed solution to 4.0-4.2 by using an alkaline buffer solution or an alkaline solution; s31: adding azone to the mixed solution; s32: and (3) adding a freeze-drying protective agent lactose into the solution obtained in the step (S3) and carrying out freeze-drying treatment to obtain a freeze-dried powder preparation, wherein the mass fraction of azone is 2%, the mass fraction of the freeze-drying protective agent is 89%, and the mass ratio of epirubicin to imiquimod is 1:4, example E1 two groups of mice were dosed with the same dose of epirubicin, imiquimod.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art. The present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and features disclosed herein.
Similarly, it should be noted that in the preceding description of embodiments of the application, various features are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure aiding in the understanding of one or more of the embodiments. This method of disclosure, however, is not intended to imply that more features are required than are expressly recited in the claims. Indeed, the embodiments may be characterized as having less than all of the features of a single embodiment disclosed above.
Numerals describing the number of components, attributes, etc. are used in some embodiments, it being understood that such numerals used in the description of the embodiments are modified in some instances by the use of the modifier "about", "approximately" or "substantially". Unless otherwise indicated, "about", "approximately" or "substantially" indicates that the numbers are allowed to vary by ± factor. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximations that may vary depending upon the desired properties of the individual embodiments.
Finally, it should be understood that the embodiments described herein are merely illustrative of the principles of the embodiments of the present application. Other variations are also possible within the scope of the present application. Thus, by way of example, and not limitation, alternative configurations of the embodiments of the present application can be viewed as being consistent with the teachings of the present application. Accordingly, the embodiments of the present application are not limited to only those embodiments explicitly described and depicted herein.
Claims (22)
1. The bladder perfusion medicine compound preparation is characterized by comprising an anthracycline, an immunomodulator and a pH regulator.
2. The bladder perfusion drug compound preparation as claimed in claim 1, wherein the mass ratio of the anthracycline drug to the immunomodulator is 1.
3. The compound bladder perfusion pharmaceutical formulation as claimed in claim 1, wherein the immunomodulator comprises an imidazoquinoline immunomodulator and a soluble salt thereof.
4. The bladder perfusion pharmaceutical compound preparation of claim 3, wherein the imidazole quinoline immunomodulator is selected from at least one of imiquimod, a derivative of imiquimod, resiquimod, or a derivative of resiquimod.
5. The bladder perfusion pharmaceutical combination of claim 3, wherein the soluble salt of the imidazoquinoline immunomodulator is selected from at least one of a soluble salt of imiquimod, a soluble salt of an imiquimod derivative, a soluble salt of resiquimod, or a soluble salt of a resiquimod derivative.
6. The bladder perfusion drug combination of claim 1, wherein the anthracycline is selected from the group consisting of epirubicin, soluble salts of epirubicin, pirarubicin, soluble salts of pirarubicin, mitoxantrone, soluble salts of mitoxantrone, doxorubicin, soluble salts of doxorubicin, aclarubicin, soluble salts of aclarubicin, idarubicin, and soluble salts of idarubicin.
7. The compound preparation of bladder perfusion medicines according to claim 6, wherein the soluble salts of epirubicin, pirarubicin, mitoxantrone, doxorubicin, aclarubicin and idarubicin are all hydrochlorides.
8. The bladder perfusion pharmaceutical compound preparation of claim 1, further comprising a mucosal permeation enhancer.
9. The bladder perfusion pharmaceutical compound preparation of claim 8, wherein the mucosa penetration enhancer is at least one selected from azone, hyaluronidase, lauryl alcohol and oleic acid.
10. A lyophilized powder formulation, comprising the bladder perfusion drug compound formulation of any one of claims 1-9.
11. Lyophilized powder formulation according to claim 10, further comprising a lyoprotectant.
12. Lyophilized powder formulation according to claim 11, wherein the lyoprotectant is selected from at least one of sucrose, lactose, mannitol, and cyclodextrin.
13. The lyophilized powder preparation of claim 10, wherein after reconstitution, when the anthracycline concentration is 1-5 mg/mL, the pH is 3.8-5.5.
14. A method for preparing a compound pharmaceutical formulation for bladder irrigation as defined in any one of claims 1 to 9, comprising the step of dissolving said immunomodulator with a dilute acid to obtain a dilute acid salt solution of said immunomodulator.
15. The method for preparing a compound preparation of a bladder perfusion drug according to claim 14, further comprising the step of dissolving the anthracycline with the dilute acid salt solution to obtain a mixed solution.
16. The method for preparing a compound preparation of a bladder perfusion medicament according to claim 15, comprising the step of adjusting the pH of the mixed solution.
17. The method for preparing a compound preparation of a bladder perfusion drug according to claim 16, wherein the pH value of the mixed solution is adjusted by using an acidic or alkaline buffer or solution.
18. The method for preparing a compound preparation of a bladder perfusion drug according to claim 17, further comprising the step of adding a lyoprotectant to the mixed solution and performing a lyophilization process.
19. The method for preparing a compound preparation of a bladder irrigation medicine according to any one of claims 17 or 18, further comprising the step of adding a mucosal permeation enhancer to the mixed solution.
20. A bladder perfusion drug comprising the bladder perfusion drug complex formulation of any one of claims 1-9.
21. A bladder perfusion drug comprising the lyophilized powder formulation of any one of claims 10-13.
22. A method of treating bladder cancer, comprising treating bladder cancer with the bladder perfusion agent of any one of claims 20 or 21.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009118754A2 (en) * | 2008-03-28 | 2009-10-01 | Astron Research Limited | A process for preparing a stable lyophilized composition |
| CN111375062A (en) * | 2018-12-29 | 2020-07-07 | 苏州百迈生物医药有限公司 | Biopolymer pharmaceutical composition for in-situ gel-forming chemotherapy and immune combination therapy |
| WO2021056815A1 (en) * | 2019-09-26 | 2021-04-01 | 苏州百迈生物医药有限公司 | Chemotherapeutic immune drug composition and preparation method thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009118754A2 (en) * | 2008-03-28 | 2009-10-01 | Astron Research Limited | A process for preparing a stable lyophilized composition |
| CN111375062A (en) * | 2018-12-29 | 2020-07-07 | 苏州百迈生物医药有限公司 | Biopolymer pharmaceutical composition for in-situ gel-forming chemotherapy and immune combination therapy |
| WO2021056815A1 (en) * | 2019-09-26 | 2021-04-01 | 苏州百迈生物医药有限公司 | Chemotherapeutic immune drug composition and preparation method thereof |
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| JIAOJIE WEI ET AL.: "Multifunctional polymeric micelle-based chemo-immunotherapy with immune checkpoint blockade for efficient treatment of orthotopic and metastatic breast cancer", 《ACTA PHARMACEUTICA SINICA B》 * |
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