WO2022143893A1 - 一种膀胱灌注药物组合物及其制备方法和应用 - Google Patents

一种膀胱灌注药物组合物及其制备方法和应用 Download PDF

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WO2022143893A1
WO2022143893A1 PCT/CN2021/143056 CN2021143056W WO2022143893A1 WO 2022143893 A1 WO2022143893 A1 WO 2022143893A1 CN 2021143056 W CN2021143056 W CN 2021143056W WO 2022143893 A1 WO2022143893 A1 WO 2022143893A1
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pharmaceutical composition
intravesical infusion
hydrochloride
composition according
imiquimod
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PCT/CN2021/143056
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English (en)
French (fr)
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刘庄
邓中清
许欢
陶惠泉
吴宇辰
周炫坊
李广志
吴松
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苏州百迈生物医药有限公司
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Priority claimed from CN202011609565.1A external-priority patent/CN114681612A/zh
Priority claimed from CN202111499923.2A external-priority patent/CN116251191A/zh
Priority claimed from CN202111498069.8A external-priority patent/CN114053230B/zh
Application filed by 苏州百迈生物医药有限公司 filed Critical 苏州百迈生物医药有限公司
Priority to EP21914624.8A priority Critical patent/EP4257121A4/en
Priority to CN202180088555.4A priority patent/CN116710106A/zh
Publication of WO2022143893A1 publication Critical patent/WO2022143893A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the present application relates to the field of biomedicine, in particular to a pharmaceutical composition for intravesical instillation and a preparation method and application thereof.
  • Bladder cancer is a common urinary system tumor. Although compared with lung cancer and gastrointestinal tumors, bladder cancer has lower morbidity and mortality, but its high recurrence rate has made bladder cancer one of the most expensive cancers in clinical treatment. . Infusion chemotherapy after transurethral tumor resection for bladder cancer is the first choice for bladder cancer treatment, but the current clinical infusion therapy has low clinical absolute remission rate, high tumor recurrence rate, serious side effects, and poor patient prognosis. At the same time, in recent years, the incidence of bladder cancer in my country has been increasing year by year, which is higher than the world average. In the past 30 years, the research on new infusion small molecule drugs has been slow, and my country is facing severe clinical problems in the prevention and treatment of bladder cancer.
  • NMIBC non-muscle invasive bladder cancer
  • MIBC muscle invasive bladder cancer
  • TURBT transurethral resection of bladder tumor
  • chemotherapeutic drugs can induce the expression of immunogenic protein molecules on the cell surface by inducing apoptosis or other programmed death of tumor cells, and then stimulate the body's anti-tumor immune response, a phenomenon known as tumor immunogenic cells. death (immunogenic cell death, ICD). Therefore, it may be one of the ways to prepare and improve the chemotherapy-immunotherapy drug system. Currently, only a few clinically relevant drugs have been shown to induce the ICD phenomenon in clinical applications.
  • These drugs mainly include: (1) doxorubicin and anthracyclines, which have been used to treat cancers such as small cell lung cancer; (2) epirubicin, an anthracycline licensed for breast cancer patients (3) idarubicin, an anthracycline drug currently used to treat acute myeloid leukemia; (4) mitoxantrone, approved for breast cancer, non-Hodgkin lymphoma, and prostate cancer patients (5) bleomycin, a glycopeptide antibiotic used for the palliative treatment of testicular cancer, squamous cell carcinoma of the head and neck, cervix, and vulva; (6) Velcade bortezomib, with Proteasome inhibitor in patients with multiple myeloma and T-cell lymphoma; (7) Oxaliplatin in combination with 5-fluorouracil and folinic acid for the treatment of advanced colorectal cancer.
  • T cells need antigen-presenting cells (APCs) to ingest, process and present these antigens to themselves before they can be activated to truly exert their effects, while antigen-presenting cells need the help of immune adjuvants to more effectively present antigens.
  • APCs antigen-presenting cells
  • Anthracyclines have a high susceptibility to a variety of transitional epithelial cancers.
  • Epirubicin is a third-generation anthracycline semi-synthetic compound that can directly intercalate between DNA base pairs, interfere with the transcription process, and prevent the mRNA expression. formation and play an anti-tumor effect.
  • anthracycline chemotherapy drugs represented by epirubicin and pirarubicin are the first-line treatment drugs for bladder cancer perfusion.
  • tumors still have a high recurrence rate (over 50%) within one year after intravesical therapy with epirubicin or pirarubicin.
  • TLRs Toll-like receptors
  • PAMPs pathogen-associated molecular patterns
  • TLR7 and TLR8 activate TLR7 and TLR8.
  • R837 and the immunostimulatory oligonucleotide CpG recognize and stimulate TLR7 and TLR9, respectively
  • R848 synthetic immunomodulator R848
  • TLR7 and TLR8 activate TLR7 and TLR8.
  • TLRs agonists have the function of immune adjuvants, and have great potential for tumor therapy, but their effectiveness and applicability are limited by the side effects of their systemic release of pro-inflammatory factors and cytokines. Therefore, the in vivo therapy of TLR7 as a ligand mainly focuses on the application of TLR7 as an immune adjuvant target such as antiviral or antitumor local therapy.
  • TLR agonists for bladder cancer treatment.
  • TMX-101 directly instills a gel formulation containing R837 components into the bladder. It is reported that R837 has shown good safety in this clinical trial, and some patients have benefited.
  • monoclonal antibody There is no disclosure of the latest research results for the combination treatment of bladder cancer with monoclonal antibody.
  • Infusion chemotherapy alone is not effective in inhibiting the progression of bladder cancer, and is prone to toxic side effects and adverse reactions. The patients are easily tolerated and have an unsatisfactory prognosis. How to improve the therapeutic effect of intravesical infusion chemotherapy is a major topic in intravesical infusion therapy technology. How to combine water-soluble chemical drugs with fat-soluble immune adjuvants is also a key issue to further promote the combined chemotherapy and immunotherapy of bladder cancer.
  • the application provides a pharmaceutical composition for intravesical instillation, which can produce synergistic anti-cancer effects, reduce the probability of cancer metastasis and recurrence, and can effectively kill in situ tumors while inhibiting distant metastases through immune responses. growth and reduce the probability of tumor recurrence.
  • the present application provides a pharmaceutical composition for intravesical instillation, comprising: an immune adjuvant or a soluble salt thereof, and a chemical drug capable of causing immunogenic cell death.
  • the chemotherapeutic agent capable of causing immunogenic cell death comprises: anthracycline chemotherapeutics, platinum chemotherapeutics, fluorouracil or gemcitabine.
  • 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 , arubicin or its soluble salt, idarubicin or its soluble salt.
  • the soluble salt is the hydrochloride salt.
  • the anthracycline chemotherapeutic drugs include doxorubicin hydrochloride, epirubicin hydrochloride, pirarubicin hydrochloride, and mitoxantrone hydrochloride.
  • the platinum-based chemotherapeutic agent comprises nedaplatin, carboplatin, lobaplatin, or oxaliplatin.
  • the immunological adjuvant or a soluble salt thereof comprises a soluble salt of an immunological adjuvant.
  • the immune adjuvant or a soluble salt thereof comprises an imidazoquinoline immune adjuvant or a soluble salt thereof.
  • the imidazoquinoline immunoadjuvant comprises imiquimod and derivatives thereof, or resiquimod and derivatives thereof, or soluble salts of imiquimod and derivatives thereof, or Soluble salts of resiquimod and its derivatives.
  • the soluble salts of the immunoadjuvant include imiquimod R837 hydrochloride, resimod R848 hydrochloride or other pharmaceutically acceptable salts, CpG, polyIC, polyICLC, At least one of the STING stimulants.
  • the mass ratio of the immune adjuvant or its soluble salt to the chemical drug capable of causing immunogenic cell death is 1:100-6:1.
  • the mass ratio of the imiquimod R837 hydrochloride to the chemical that can cause immunogenic death is 1:20-1:1, wherein the immunogenic death can be caused
  • the chemicals used are fluorouracil or gemcitabine.
  • the mass ratio of the imiquimod R837 hydrochloride to the chemical drug capable of causing immunogenic death is 1:1 to 6:1, wherein the chemical drug comprises anthracene Cyclic chemotherapy drugs or platinum-based chemotherapy drugs, the anthracycline-based chemotherapy drugs include doxorubicin hydrochloride, epirubicin hydrochloride, pirarubicin hydrochloride, and mitoxantrone hydrochloride; the platinum-based chemotherapy drugs include Daplatin, carboplatin, lobaplatin, or oxaliplatin.
  • the mass ratio of the resiquimod R848 hydrochloride to the chemical drug capable of causing immunogenic cell death is 1:20-1:1, wherein the immunogenic
  • the chemical drugs for primary death include anthracycline chemotherapy drugs or platinum chemotherapy drugs, and the anthracycline chemotherapy drugs include doxorubicin hydrochloride, epirubicin hydrochloride, pirarubicin hydrochloride or mitoxantrone hydrochloride;
  • the platinum-based chemotherapy drugs include nedaplatin, carboplatin, lobaplatin or oxaliplatin.
  • the mass ratio of the resimod R848 hydrochloride to the chemical that can cause immunogenic death is 1:100-1:10, wherein the immunogenic death can be caused
  • the chemicals used include fluorouracil or gemcitabine.
  • the concentration of the immune adjuvant ranges from 0.5 mg/mL to 30 mg/mL.
  • the intravesical infusion pharmaceutical composition further includes a pH adjusting agent.
  • the pH of the reconstituted solution is 3.8 ⁇ 5.5.
  • the intravesical infusion pharmaceutical composition further includes a lyoprotectant.
  • the lyoprotectant includes at least one of sucrose, lactose, mannitol, and cyclodextrin.
  • the lyoprotectant is lactose.
  • the intravesical pharmaceutical composition is a lyophilized powder formulation.
  • the mass ratio of the anthracycline to the immune adjuvant is 1:0.1-1:10.
  • the mass ratio of the anthracycline to imiquimod and its derivatives is 1:1-1:10.
  • the mass ratio of the anthracycline to the soluble salt of imiquimod and its derivatives is 1:1-1:10.
  • the mass ratio of the anthracycline to resiquimod is 1:0.1-1:5.
  • the mass ratio of the anthracycline to the soluble salt of resiquimod and its derivatives is 1:0.1-1:5.
  • the intravesical infusion pharmaceutical composition comprises: epirubicin or a soluble salt thereof, imiquimod or a soluble salt thereof, and a pH adjusting agent.
  • the intravesical infusion pharmaceutical composition is a lyophilized powder formulation.
  • the intravesical infusion pharmaceutical composition further includes a lyoprotectant.
  • the pH of the lyophilized powder dosage form after reconstitution is 3.8-5.5 when the epirubicin concentration is 1-5 mg/mL.
  • the pH of the lyophilized powder dosage form after reconstitution is 4.0-5.0 when the concentration of epirubicin is 1-5 mg/mL.
  • the pH of the lyophilized powder dosage form after reconstitution is 4.0-4.2, 4.2-4.5 or 4.5-5.0 when the epirubicin concentration is 1-5 mg/mL.
  • the mass ratio of epirubicin or its hydrochloride to imiquimod or its soluble salt is 1:1-1:10.
  • the mass ratio of epirubicin or its hydrochloride to imiquimod or its soluble salt is 1:2-1:4.
  • the lyoprotectant includes sucrose, lactose, mannitol, cyclodextrin.
  • the lyoprotectant is lactose.
  • the mass fraction of the lyophilized protective agent in the lyophilized powder formulation is 65% to 96%.
  • the content of the lyophilized protective agent in the lyophilized powder formulation is 80% to 94%.
  • the intravesical infusion pharmaceutical composition further comprises a mucosal penetration enhancer.
  • the mucosal penetration enhancer includes at least one of azone, hyaluronidase, lauryl alcohol, and oleic acid.
  • the present application also provides a method for preparing a pharmaceutical composition for intravesical instillation, which includes the following steps: S1: dissolving the immune adjuvant with dilute acid to obtain a dilute salt solution of the immune adjuvant; S2: obtaining the immune adjuvant with S1 The dilute salt solution of S1 dissolves the chemical drug that can cause immunogenic cell death to obtain a mixed solution; S3: add a pH adjuster to the mixed solution of S1, and control the pH between 2.0-5.5.
  • the step S3 further includes the following steps: S31: adding a lyophilization protective agent to the mixed solution; and/or S32: adding a mucosal penetration enhancer to the mixed solution.
  • the preparation method further includes step S4: freeze-drying the final solution.
  • the pH is controlled between 3.8-5.5.
  • the chemical drug capable of causing immunogenic cell death includes anthracycline drugs, and in step S3, the final concentration of the anthracycline drugs is 1 ⁇ 5 mg/mL.
  • the present application also provides a method for preparing a pharmaceutical composition for intravesical instillation, which comprises the following steps: S1: combining immune adjuvants CpG, polyIC, polyICLC, water-soluble STING stimulator with an immunogenic cell death The chemical medicine of S1 is added into water for injection, and mixed uniformly until dissolved; S2: add a suitable freeze-drying protective agent to the solution of S1, and the solution is subjected to freeze-drying treatment.
  • the present application also provides the use of the intravesical instillation pharmaceutical composition in the preparation of intravesical instillation preparations.
  • Figure 1 is the ultrasound images of bladder parts at different times after combined infusion of doxorubicin hydrochloride (DOX) and imiquimod hydrochloride (R837) for the treatment of in situ bladder tumors;
  • DOX doxorubicin hydrochloride
  • R837 imiquimod hydrochloride
  • Figure 2 is a physical map of the final size of the tumor in different groups in the combined perfusion treatment of pirarubicin hydrochloride (THP) and imiquimod hydrochloride (R837) in the experiment of bladder in situ tumor;
  • THP pirarubicin hydrochloride
  • R837 imiquimod hydrochloride
  • Fig. 3 is the in vivo fluorescence imaging images of mice at different time points in the experiment of combined perfusion therapy of pirarubicin hydrochloride (THP) and imiquimod hydrochloride (R837) in different proportions of bladder orthotopic tumor;
  • Figure 4 is a statistical graph of the mean fluorescence intensity of the bladder site of mice at different time points in an experiment of combined perfusion therapy of pirarubicin hydrochloride (THP) and imiquimod hydrochloride (R837) in different proportions of bladder orthotopic tumors;
  • THP pirarubicin hydrochloride
  • R837 imiquimod hydrochloride
  • Figure 5 is a tumor fluorescence imaging image of an experiment of combined infusion of epirubicin hydrochloride (EPI) and imiquimod hydrochloride (R837) for the treatment of bladder orthotopic tumors;
  • EPI epirubicin hydrochloride
  • R837 imiquimod hydrochloride
  • Figure 6 is a graph of the fluorescence intensity statistics of the tumor site at the end of epirubicin hydrochloride (EPI) and imiquimod hydrochloride combined infusion therapy for bladder orthotopic tumors;
  • EPI epirubicin hydrochloride
  • imiquimod hydrochloride combined infusion therapy for bladder orthotopic tumors
  • Fig. 7 is the mouse tumor growth curve graph that EPI and R837 are used in combination for bladder cancer perfusion therapy
  • Figure 8 is a graph showing the change in body weight of mice within 1-9 days after perfusion administration in the toxicity experiment when EPI and R837 are used in combination (dose ratio is 1:6);
  • Figure 9 is a graph showing the change in body weight of mice within 1-4 days after perfusion administration in the toxicity experiment when EPI and R837 are used in combination (dose ratio is 1:10);
  • Figure 10 is a statistical diagram of EPI content in mouse blood
  • Figure 11 is a graph of fluorescence intensity statistics of EPI content in mouse bladder tissue
  • Figure 12 is a statistical graph of EPI fluorescence signal intensity in macrophages in mouse bladder tissue
  • Figure 13 is a graph showing the tumor mass statistics of different groups of mice after the mixed freeze-dried powder preparation of epirubicin and imiquimod is applied to bladder cancer mice and treated with bladder cancer by intravesical instillation;
  • Figure 14 is a graph showing the tumor mass in mice after the mixed freeze-dried powder preparation of pirarubicin and imiquimod is used for infusion therapy of mouse bladder cancer;
  • Figure 15 is a graph showing tumor mass statistics of different groups of mice after intravesical instillation of a mixed freeze-dried powder formulation of doxorubicin and imiquimod for bladder cancer;
  • Figure 16 is an anatomical diagram of the lesion site of mice in different groups on the 3rd day after the second administration of intravesical infusion therapy
  • Figure 17 is a graph showing the tissue quality statistics of different groups of mice on the 3rd day after the second administration of intravesical instillation
  • Figure 18 is a flow cytometric analysis chart of killer T cells infiltrating the tumor in different groups of mice on the 3rd day after the second administration of intravesical infusion therapy;
  • Figure 19 is a flow cytometric analysis chart of CD3 + /CD4 + T cells in tumor sites of different groups of mice on the 3rd day after the second administration of intravesical infusion therapy;
  • Figure 20 is a statistical graph of intracellular IFN- ⁇ content after the second administration of intravesical infusion therapy on the 3rd day after the CD8 + cells in the tumor sites of different groups of mice were stimulated by the cell stimulation mixture;
  • Figure 21 is a statistical graph of the relative content of tumor necrosis factor in the lesions of mice in different groups on the 3rd day after the second administration of intravesical infusion therapy;
  • Figure 22 is a statistical graph of the relative content of interferon-gamma in different groups of mice on the 3rd day after the second administration of intravesical infusion therapy;
  • Figure 23 is a statistical graph of the effect of mucosal penetration enhancers on the retention of epirubicin in the bladder;
  • Figure 24 is a statistical graph of the effect of mucosal penetration enhancers on imiquimod retention in the bladder.
  • the present application provides a pharmaceutical composition for intravesical instillation.
  • the research and development team has found through experiments that the soluble salts of immune adjuvants and the chemical drugs that can cause immunogenic cell death (ICD drugs, immunogenic cell death) ) when used in combination with intravesical instillation drugs, can produce synergistic anti-cancer effects, reduce the probability of cancer metastasis and recurrence of anti-cancer drug compositions, while effectively killing in situ tumors, it can also inhibit the growth and development of distant metastatic tumors through immune responses. Reduce the chance of tumor recurrence.
  • ICD drugs immunogenic cell death
  • the attenuating effect of the system of the present invention is different from the previous mechanism of reducing the toxicity of the drug system by changing the local and systemic absorption of the perfused drug in the dosage form.
  • it can significantly reduce the toxic and side effects of chemotherapy drugs on normal tissues by increasing the channel of macrophage phagocytosis of tumor free chemotherapeutic drugs in tissues. This drug entry method was first tried and used in perfusion studies. Observed.
  • the present invention provides a pharmaceutical composition for intravesical instillation, comprising a soluble salt of an immune adjuvant and a chemical drug capable of causing immunogenic cell death. Further, the mass ratio of the soluble salt of the immune adjuvant to the chemical drug capable of causing immunogenic cell death is 1:100-6:1.
  • the mass ratio of the soluble salt of the immune adjuvant to the chemical drug capable of causing immunogenic cell death is 2:1 to 4:1.
  • the chemical drugs that can cause immunogenic cell death include anthracycline chemotherapy drugs, platinum chemotherapy drugs, fluorouracil or gemcitabine; optionally, the anthracycline chemotherapy drugs include doxorubicin hydrochloride, hydrochloric acid Epirubicin, pirarubicin hydrochloride, mitoxantrone hydrochloride; the platinum-based chemotherapy drugs include nedaplatin, carboplatin, lobaplatin or oxaliplatin.
  • the soluble salts of the immune adjuvant include imiquimod R837 hydrochloride or other pharmaceutically acceptable soluble salts, Resiquimod R848 hydrochloride or other pharmaceutically acceptable immune adjuvant soluble salts , CpG, polyIC, polyICLC, STING stimulator (with can include at least one of IMSA-101, GSK-3745417, BMS-986301, SB-11285, MK-1454).
  • the mass ratio of the imiquimod R837 hydrochloride to the chemical that can cause immunogenic death is 1:20 to 1:1, and the chemical that can cause immunogenic death is Fluorouracil or gemcitabine.
  • the mass ratio of the imiquimod R837 hydrochloride to the chemical drug that can cause immunogenic death is 1:1 to 6:1, wherein the chemical drug includes anthracycline chemotherapy drugs or platinum chemotherapy drugs Medicines, the anthracycline chemotherapy drugs include doxorubicin hydrochloride, epirubicin hydrochloride, pirarubicin hydrochloride, and mitoxantrone hydrochloride; the platinum chemotherapy drugs include nedaplatin, carboplatin, and lobaplatin or oxaliplatin.
  • the mass ratio of the resimod R848 hydrochloride to the chemical drug that can cause immunogenic cell death is 1:10-1:1, wherein the chemical drug that can cause immunogenic cell death
  • the anthracycline chemotherapy drugs include doxorubicin hydrochloride, epirubicin hydrochloride, pirarubicin hydrochloride, mitoxantrone hydrochloride; the platinum chemotherapy drugs These include nedaplatin, carboplatin, lobaplatin, or oxaliplatin.
  • the mass ratio of the resiquimod R848 hydrochloride to the chemical that can cause immunogenic death is 1:100-1:10, wherein the chemical that can cause immunogenic death includes Fluorouracil or gemcitabine.
  • lyophilization co-solvents such as mannitol, lactose, etc.
  • pH adjusters such as mannitol, lactose, etc.
  • concentration of the soluble salt of the immune adjuvant ranges from 0.5 mg/mL to 30 mg/mL.
  • the dosage form is lyophilized powder.
  • the present invention also provides a preparation method of the intravesical infusion pharmaceutical composition, comprising the following steps:
  • S1 Dissolve the immune adjuvant imiquimod R837 or resimod R848 in a dilute acid solution (such as hydrochloric acid, lactic acid, acetic acid) or other pharmaceutically acceptable immune adjuvants (such as glucopyranoside lipid A ( MPLA)) soluble salt solution, add chemical drugs that can cause immunogenic cell death to the solution, and mix evenly until dissolved;
  • a dilute acid solution such as hydrochloric acid, lactic acid, acetic acid
  • other pharmaceutically acceptable immune adjuvants such as glucopyranoside lipid A ( MPLA)
  • MPLA glucopyranoside lipid A
  • S2 add a suitable freeze-drying co-solvent (such as mannitol, lactose, etc.), a pH adjuster to the solution of S1, and control the pH value between 2.0-5.5;
  • a suitable freeze-drying co-solvent such as mannitol, lactose, etc.
  • step S3 freeze-drying the solution obtained in step S2.
  • S2 Add a suitable freeze-drying auxiliary solvent (such as mannitol, lactose, etc.) to the solution of S1, and perform freeze-drying treatment on the solution.
  • a suitable freeze-drying auxiliary solvent such as mannitol, lactose, etc.
  • the present invention also provides the application of the combined medicine in the preparation of intravesical infusion medicine.
  • ICD-type chemical drugs can reduce the systemic side effects of ICD-type chemical drugs. According to the available evidence, it is preliminarily inferred that the mechanism of action of this phenomenon is that the combined immune adjuvant increases the phagocytosis of free chemotherapy drugs in tumor cells by macrophages. Thereby, the toxic and side effects of chemotherapeutic drugs on normal tissues can be significantly reduced.
  • ICD drugs can induce immunogenic cell death of tumor cells, thereby activating the immune system to specifically eliminate cancer cells. After antigen-presenting cells engulf dead cancer cells, they can direct the immune system to track, recognize and kill other cancer cells.
  • Soluble immune adjuvants activate a series of downstream signals after being recognized with TLRs, induce the secretion of inflammatory cytokines, chemokines and type I interferons, and promote tumor-associated antigens to be more effectively presented by antigen-presenting cells to have tumor-killing effects. of T cells.
  • immune adjuvants when ICD-type chemotherapy drugs make cancer cells die into tumor-associated antigens, the introduction of immune adjuvants can further stimulate the uptake and processing of these antigens by antigen-presenting cells and present them to T cells more effectively, thereby amplifying the An antitumor immune response.
  • the existence of immune adjuvant also promotes the accumulation of a large number of macrophages around the tumor tissue of the bladder.
  • the ICD drug exerts preliminary chemotherapy and immune stimulation, it engulfs a large number of free chemotherapy drugs outside the tumor cells, which can reduce the effect of chemotherapy drugs on normal cells. tissue damage, thereby reducing the systemic toxic and side effects of ICD drugs, and also increasing the dosage of ICD drugs within a certain range, or improving the overall therapeutic effect.
  • the local combined administration of soluble immune adjuvants and ICD chemotherapy drugs can significantly improve the effect of chemotherapy drugs in tumor treatment and prevention of metastasis and recurrence.
  • the introduction can further stimulate the uptake and processing of these antigens by APC cells and present them to T cells more effectively, thereby amplifying this anti-tumor immune response.
  • the combined infusion of immune adjuvants and ICD-type chemotherapeutic drugs is applied to the treatment of bladder cancer to prevent recurrence. Has a very good improvement effect.
  • the combination of the ICD chemotherapeutic drugs and the immune adjuvant according to the present invention has the technical effect of efficiently treating and preventing the metastasis and recurrence of bladder cancer.
  • the two types of drugs were directly mixed and then injected into the bladder to verify the efficacy. It was found that, taking the chemotherapy drugs epirubicin and imiquimod as an example, when the ratio of the chemotherapy drugs epirubicin and imiquimod was within a suitable range, the comparison between the chemotherapy drugs infusion and the chemotherapy drugs alone can be obtained. The treatment effect is significantly improved, and the toxic and side effects are not increased, and it is expected to become a new type of drug preparation for intravesical instillation with significant clinical value.
  • the present invention provides a compound preparation of intravesical instillation drugs, including anthracyclines, immunomodulators and pH regulators, wherein the immunomodulators include imidazoquinoline immunomodulators and soluble salts thereof.
  • anthracyclines include epirubicin or a soluble salt thereof, pirarubicin or a soluble salt thereof, mitoxantrone or a soluble salt thereof, doxorubicin or a soluble salt thereof, arubicin or a soluble salt thereof.
  • the soluble salt is hydrochloride.
  • the intravesical instillation drug freeze-dried powder formulation also includes a freeze-drying protective agent.
  • the lyoprotectant includes sucrose, lactose, mannitol, and cyclodextrin.
  • the lyoprotectant is lactose.
  • the mass fraction of the freeze-dried protective agent in the freeze-dried powder preparation is 65% to 96%.
  • the content of the lyophilized protective agent in the lyophilized powder formulation is 80% to 94%.
  • the pH adjusting agent is weak base, alkaline buffer, sodium hydroxide, hydrochloric acid.
  • the pH adjusting agent is sodium bicarbonate, hydrochloric acid.
  • the pH of the solution is 3.8-5.5. It should be noted that the purpose of defining the reconstituted concentration here is to define the range of the pH value after reconstituted, not to limit the reconstituted concentration.
  • the pH of the solution is 4.0-5.0. It should be noted that the purpose of defining the reconstituted concentration here is to define the range of the pH value after reconstituted, not to limit the reconstituted concentration.
  • imidazoquinoline immunomodulators include imiquimod and its derivatives, or resiquimod and its derivatives, or soluble salts of imiquimod and its derivatives, or resiquimod and its derivatives. Soluble salts of derivatives.
  • the mass ratio of the anthracycline chemical to the imidazoquinoline immunomodulator is 1:0.1-1:10.
  • the mass ratio of anthracycline to imiquimod is 1:1-1:10. Further optionally, the mass ratio of anthracycline to imiquimod is 1:2-1:4.
  • the mass ratio of the anthracycline and resiquimod is 1:0.1-1:5.
  • freeze-dried powder formulation also includes a mucosal penetration enhancer.
  • the mucosal penetration enhancer includes at least one of azone, hyaluronidase, lauryl alcohol, and oleic acid.
  • the mucosal penetration enhancer may be added during the preparation process, or may be mixed before use.
  • the compound preparation of the anthracycline and immune adjuvant of the present invention is mixed with a mucosal penetration enhancer and then perfused.
  • the mucosal penetration enhancer can be a commercially available medicinal mucosal penetration enhancer or a clinical A mucosal penetration enhancer approved for use in the experimental phase.
  • the present invention also proposes a preparation method of intravesical instillation drug freeze-dried powder preparation.
  • step S3 also includes:
  • step S3 also includes:
  • the dilute acid in S1 is hydrochloric acid, lactic acid or acetic acid.
  • a further optional range for adjusting pH in S3 is 4.0-5.0.
  • step S4 is also included: freeze-drying the final solution obtained in S3 to obtain a freeze-dried powder preparation.
  • the invention also provides the application of the freeze-dried powder mixed preparation in the preparation of intravesical infusion medicine.
  • Adopting the technical scheme of the present invention has the following beneficial effects:
  • the invention provides a compound preparation of intravesical instillation drugs, a mixed freeze-dried powder preparation of anthracycline drugs and imidazoquinoline immunomodulators.
  • the lyophilized powder dosage form is more helpful to protect the long-term chemical stability of anthracyclines in the mixed preparation.
  • the better tumor inhibitory effect can cause stronger anti-tumor immune response, and compared with the single anthracycline drug, the compound preparation of the present invention can reduce the toxic and side effects.
  • the mixed preparation of anthracycline and immunomodulator of the present invention can achieve clinical application through simple reconstitution operation, and can be directly perfused for treatment, or can be mixed with a mucosal penetration enhancer and perfused to effectively inhibit tumor growth and recurrence.
  • immunomodulators can further stimulate antigen-presenting cells to uptake, process and present antigens to T cells, further amplifying the anti-tumor immune response.
  • the long-term chemical stability of the mixed preparations of anthracyclines and immunomodulators is realized, the generation rate of impurities of anthracyclines in the presence of immunomodulators is significantly reduced, the long-term storage stability of the preparations is improved, and the preparations are increased. Ease of use and safety. In the future clinical use, the longer shelf life is beneficial to the production, storage and transportation of the drug, which will help to realize the real clinical application.
  • Adding a mucosal penetration enhancer to the preparation can enhance the retention time of the drug solution on the bladder wall after perfusion treatment, and is helpful to further improve the curative effect of this type of preparation.
  • the results showed that when the concentration ratio of the chemotherapeutic drug epirubicin and imiquimod was within an appropriate range, a significantly improved therapeutic effect could be obtained compared with the chemotherapy drug perfusion alone, without increasing the toxic and side effects.
  • a novel drug preparation for intravesical instillation with significant clinical value has shown that the anti-tumor mechanism of this new preparation is mainly through the immunogenic cell death of tumor cells induced by chemotherapy drugs such as epirubicin to expose tumor antigens.
  • chemotherapy drugs such as epirubicin to expose tumor antigens.
  • imiquimod can activate the immune system and recruit tumor cells. Antigen-presenting cells enter the tumor to recognize tumor antigens and activate T-cell immune responses against the tumor.
  • this compound lyophilized preparation has a stability "golden pH range", and the epirubicin/imiquimod compound preparation prepared in this pH range has very good stability .
  • This phenomenon is contrary to the conventional understanding of the stability trend of epirubicin single preparation solution state and our investigation results of the solution state stability of this compound preparation.
  • epirubicin hydrochloride is stable in both injection and lyophilized powder state.
  • epirubicin preparations on the market exist in two dosage forms: injection and lyophilized powder.
  • this reaction is phenomenologically accelerated by acid catalysis (protonation of oxygen atoms on glycosidic bonds at low pH is an intermediate step of the reaction), so an increase in pH will significantly slow down the production of impurity A.
  • too high pH would lead to a decrease in the solubility of imiquimod molecules (the precipitation of imiquimod from the solution above pH 5.0), and at the same time, too high pH would also lead to another An increase in an unknown impurity (presumed to be an oxidation product of epirubicin).
  • the compound preparation of epirubicin and imiquimod can obtain a dosage form that can be stably stored in the presence of a suitable pH range and a suitable content of excipients (lactose).
  • a mucosal penetration enhancer to the preparation can enhance the penetration and retention of the drug solution in the bladder mucosa after perfusion treatment, which is helpful to further improve the curative effect of the preparation.
  • the invention provides a mixed freeze-dried powder preparation of epirubicin and imiquimod.
  • the lyophilized powder dosage form is more helpful to protect the long-term chemical stability of epirubicin in the mixture than the traditional injection dosage form.
  • a good tumor suppressor effect can induce a stronger anti-tumor immune response.
  • the present invention provides a compound preparation of epirubicin, comprising epirubicin or its soluble salt, imiquimod or its soluble salt, pH adjusting agent and freeze-drying protection agent.
  • epirubicin compound preparation is in the form of freeze-dried powder.
  • the mass ratio of epirubicin or its soluble salt to imiquimod or its soluble salt is 1:1-1:10.
  • the mass ratio of epirubicin or its soluble salt to imiquimod or its soluble salt is 1:2-1:4.
  • the pH adjusting agent can be weak base, sodium hydroxide, hydrochloric acid.
  • the pH adjusting agent is sodium bicarbonate, hydrochloric acid.
  • the lyoprotectant includes sucrose, lactose, mannitol, and cyclodextrin.
  • the lyoprotectant is lactose.
  • the mass fraction of the freeze-dried protective agent in the freeze-dried powder preparation is 65% to 96%.
  • the mass fraction of the lyoprotectant is 80% to 94%.
  • the mixed lyophilized powder formulation of epirubicin and imiquimod also includes a mucosal penetration enhancer.
  • the mucosal penetration enhancer includes at least one of azone, hyaluronidase, lauryl alcohol, and oleic acid.
  • the mucosal penetration enhancer may be added during the preparation process, or may be mixed before use.
  • the pH of the epirubicin and imiquimod mixed preparation before lyophilization is 3.8-5.5. It should be noted that the purpose of defining the reconstituted concentration here is to define the range of the pH value after reconstituted, not to limit the reconstituted concentration.
  • the pH when the epirubicin concentration is 1-5 mg/mL is 3.8-5.5. It should be noted that the purpose of defining the reconstituted concentration here is to define the range of the pH value after reconstituted, not to limit the reconstituted concentration.
  • the pH of the lyophilized powder dosage form when the concentration of epirubicin is 1 mg/mL after reconstitution is 4.0-5.0.
  • the pH values of the lyophilized powder dosage form after reconstitution are 4.0-4.4, 4.4-5.0, 5.0-5.5 when the epirubicin concentration is 1 mg/mL.
  • the invention provides a preparation method of a mixed freeze-dried powder preparation of epirubicin and imiquimod.
  • step S3 also includes:
  • the dilute acid solution described in S1 is hydrochloric acid, lactic acid, and acetic acid.
  • the preferred range for pH adjustment in S3 is 4.0 to 5.0.
  • the pH value adjusted in S3 may be 3.8-4.0, 4.0-4.4, 4.4-5.0.
  • S4 freeze-drying the final solution obtained in S3 to obtain a freeze-dried powder preparation.
  • the invention also provides the application of the freeze-dried powder mixed preparation in the preparation of intravesical infusion medicine.
  • the mixed preparation of epirubicin and imiquimod of the present invention can realize clinical application through simple reconstitution operation, and can effectively inhibit tumor growth and recurrence through the method of perfusion.
  • imiquimod can further stimulate antigen uptake, processing and presentation to T cells by antigen-presenting cells, further amplifying the anti-tumor immune response.
  • the long-term chemical stability of the mixed preparation of epirubicin and imiquimod is realized, the generation rate of epirubicin impurities in the presence of imiquimod is significantly reduced, the long-term storage stability of the preparation is improved, and the preparation is increased. Ease of use and safety.
  • the terms “immunoadjuvant” and “immunomodulator” are often used interchangeably.
  • the immunoadjuvant itself may exist in the form of a soluble salt, or it may be converted into a soluble salt form by certain operations, therefore, the term “immune adjuvant” includes an immunoadjuvant or its soluble form. Salt.
  • lyoprotectant is often used interchangeably with “lyophilization co-solvent”.
  • the term “pharmaceutical composition” may generally refer to a composition comprising a pharmaceutically active ingredient.
  • the pharmaceutical composition may also contain other ingredients.
  • the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, eg, a pH adjusting agent, eg, a lyoprotectant.
  • a pharmaceutically acceptable carrier eg, a pH adjusting agent, eg, a lyoprotectant.
  • pharmaceutical composition also covers the case of combined preparations.
  • the intravesical drug composition may be a combined intravesical drug formulation, for example, the intravesical drug compound formulation may include an anthracycline, and an immune adjuvant (immunomodulator).
  • the intravesical drug combination formulation may also include a pH adjusting agent.
  • the intravesical infusion pharmaceutical composition can be a compound preparation of epirubicin.
  • the epirubicin compound formulation may include epirubicin or a soluble salt thereof, imiquimod or a soluble salt thereof, and a pH adjusting agent.
  • Example A1 Weigh 10 mg of R837, add 5 mL of hydrochloric acid with a concentration of 0.015 M, and shake until the solution is completely clear.
  • the ICD chemotherapeutic drug doxorubicin hydrochloride is added to the solution, and the mass ratio of imiquimod to doxorubicin hydrochloride is 1:1, 2:1, 4:1, 6:1 and 10:1.
  • Add a suitable freeze-drying auxiliary solvent such as mannitol
  • a pH adjuster such as sodium hydroxide
  • Embodiment A2 take by weighing 10mg R837, add the hydrochloric acid that 5mL concentration is 0.015M, shake until the solution is completely clear.
  • the ICD chemotherapeutic drug epirubicin hydrochloride was added to the solution, and the mass ratios of imiquimod and epirubicin hydrochloride were 1:1, 2:1, 4:1, 6:1 and 10:1.
  • Add a suitable freeze-drying auxiliary solvent such as mannitol
  • a pH adjuster such as sodium hydroxide
  • Embodiment A3 take by weighing 10mg R837, add the hydrochloric acid that 5mL concentration is 0.015M, shake until the solution is completely clarified.
  • the ICD chemotherapeutic drug pirarubicin hydrochloride was added to the solution, and the mass ratios of imiquimod and pirarubicin hydrochloride were 1:1, 2:1, 4:1, 6:1 and 10:1.
  • Add a suitable freeze-drying auxiliary solvent such as mannitol
  • a pH adjuster such as sodium hydroxide
  • Embodiment A4 take by weighing 10mg R837, add the hydrochloric acid that 5mL concentration is 0.015M, shake until the solution is completely clear.
  • the ICD chemotherapeutic drug mitoxantrone hydrochloride is added to the solution, and the mass ratio of imiquimod to mitoxantrone hydrochloride is 1:1, 2:1, 4:1, 6:1 and 10:1.
  • Add a suitable freeze-drying auxiliary solvent such as mannitol
  • a pH adjuster such as sodium hydroxide
  • Embodiment A5 take by weighing 10mg R837, add 5mL concentration to be 0.015M lactic acid or acetic acid (do not use hydrochloric acid), shake until the solution is completely clear.
  • the ICD chemotherapeutic drug oxaliplatin was added to the solution, and the mass ratios of imiquimod and oxaliplatin were 1:1, 2:1, 4:1, 6:1 and 10:1.
  • Add a suitable freeze-drying auxiliary solvent such as mannitol
  • a pH adjuster such as sodium hydroxide
  • Embodiment A6 take by weighing 2mg R837, add the hydrochloric acid that 5mL concentration is 0.005M, shake until the solution is completely clear.
  • the ICD chemotherapeutic drug fluorouracil was added to the solution, and the mass ratios of imiquimod and fluorouracil were 1:1, 1:2, 1:5, 1:10 and 1:20.
  • Add a suitable freeze-drying auxiliary solvent such as mannitol add a pH adjuster such as sodium hydroxide, control the pH value between 3.0-6.0, and freeze-dry to prepare a freeze-dried powder.
  • Embodiment A7 take by weighing 2mg R848, add the hydrochloric acid that 5mL concentration is 0.005M, shake until the solution is completely clear.
  • the ICD chemotherapeutic drug doxorubicin hydrochloride was added to the solution, and the mass ratios of R848 and doxorubicin hydrochloride were 1:1, 1:2, 1:5, 1:10 and 1:20.
  • Add a suitable freeze-drying auxiliary solvent such as mannitol
  • Add a pH adjuster such as sodium hydroxide, control the pH value between 2.0-5.5, and freeze-dry to prepare a freeze-dried powder.
  • Embodiment A8 take by weighing 2mg R848, add the hydrochloric acid that 5mL concentration is 0.005M, shake until the solution is completely clear.
  • the ICD chemotherapeutic drug epirubicin hydrochloride was added to the solution, and the mass ratios of R848 and epirubicin hydrochloride were 1:1, 1:2, 1:5, 1:10 and 1:20.
  • Add a suitable freeze-drying auxiliary solvent such as mannitol
  • Add a pH adjuster such as sodium hydroxide, control the pH value between 2.0-5.5, and freeze-dry to prepare a freeze-dried powder.
  • Embodiment A9 Weigh 1mg R848, add 2.5mL of hydrochloric acid with a concentration of 0.005M, then add 5-15mL of water for injection, and shake until the solution is completely clear.
  • the ICD chemotherapeutic drug fluorouracil was added to the solution, and the mass ratios of R848 and fluorouracil were 1:10, 1:25, 1:100 and 1:200.
  • Add a suitable freeze-drying auxiliary solvent such as mannitol
  • Add a pH adjuster such as sodium hydroxide, control the pH value between 3.0-6.0, and freeze-dry to prepare a freeze-dried powder.
  • Example A10 Gemcitabine hydrochloride and CpG were mixed in an aqueous solution at a mass ratio of 100:1, and suitable freeze-drying co-solvents mannitol and lactose were added, and the pH value was controlled between 2.5-4.0 to prepare a freeze-dried powder.
  • Example B1 The combined drug system is used for bladder cancer perfusion therapy, and the curative effect is significantly improved compared with a single chemotherapy drug or a single immune adjuvant (imiquimod);
  • Group B1-1 blank group (ultrapure water ddH 2 O, 100 ⁇ L);
  • Group B1-2 R837 (1.5mg/mL, 100 ⁇ L);
  • Group B1-3 DOX (1.5 mg/mL, 100 ⁇ L);
  • Groups B1-4 DOX+R837 (1.5 mg/mL, 100 ⁇ L, 1:1).
  • FIG. 1 shows the photoacoustic imaging of the bladder in situ tumor.
  • the area inside the outer dashed line is the mouse bladder area, and the middle area of the two dashed lines is the tumor tissue.
  • FIG. 2 is the image of the bladder orthotopic tumor body obtained from the mice dissected one week after the second perfusion treatment, the results show the volume of the bladder orthotopic tumor volume of the mice in the DOX and R837 perfusion combined group (B1-4) It is smaller and has a more significant tumor inhibitory effect, so it is inferred that the combination of DOX and R837 perfusion can improve the efficacy of chemotherapy drugs.
  • Example B2 Evaluation of R837 hydrochloride and pirarubicin hydrochloride (hereinafter referred to as THP) combined infusion treatment of bladder tumor in situ.
  • the aforementioned drugs are the hydrochloride of chemotherapeutic drugs and the hydrochloride of R837.
  • the clinical dosage form of R837 is an ointment and is not suitable for perfusion application.
  • its existing patented dosage form for intravesical perfusion is an emulsion (TMX-101). Therapeutic effect of R837 hydrochloride combined with THP perfusion.
  • mice The orthotopic bladder tumor in mice was established by a method similar to that described in Example B1. One week after the inoculation, in vivo imaging was performed to confirm the establishment of the tumor model. The mice were randomly divided into 3 groups, and then the mice were anesthetized by intraperitoneal injection of pentobarbital, respectively. Intravesical instillation of the following drug combinations:
  • Group B2-1 blank group (ultrapure water ddH 2 O, 100 ⁇ L);
  • Group B2-3 THP (0.6 mg/ml, 100 ⁇ L) + R837 (0.6 mg/mL, 100 ⁇ L).
  • the bladder was instilled for 1 hour, once a week, four times in total. Before each infusion treatment, the mouse bladder tumor was detected by a small animal imaging system, and the tumor size was characterized by the average fluorescence intensity of the tumor tissue.
  • the in vivo imaging data of mice at 0-35 days are shown in Figure 3, and Table 1 shows the average fluorescence intensity of the mouse bladder orthotopic tumor tissue in Figure 3 and the survival rate of each group.
  • the fluorescence intensity of the tumor in the bladder of the mice in the THP and R837 hydrochloride combined perfusion group (B2-3) was significantly lower than that in the other control groups, and the mice in this group had a good survival rate (100%) until 35 days, so it is inferred that Due to the good water solubility and high bioavailability of perfusion of R837 hydrochloride, the combined use of R837 hydrochloride with THP can improve the tumor-inhibiting effect of chemotherapeutic drugs and improve the survival rate of tumor-bearing mice.
  • Table 1 Mean fluorescence intensity of orthotopic tumor tissue of mouse bladder and survival rate of each group
  • Example B3 Evaluate the effect of combined perfusion therapy of pirarubicin hydrochloride (hereinafter referred to as THP) and R837 hydrochloride in different proportions in the treatment of bladder in situ tumors.
  • THP pirarubicin hydrochloride
  • R837 hydrochloride in different proportions in the treatment of bladder in situ tumors.
  • Orthotopic bladder tumors were established in mice by a method similar to that described in Example B1.
  • the experiment adopted the treatment plan of infusion therapy starting the day after inoculation.
  • the tumor growth was detected by the small animal in vivo imaging system, and the tumor inhibition level was evaluated by the mean fluorescence intensity of the tumor tissue.
  • the drug combination for intravesical instillation in the experiment is as follows:
  • Group B3-1 blank group (ultrapure water ddH 2 O, 100 ⁇ L);
  • Group B3-2 THP (0.25mg/mL, 100 ⁇ L);
  • Group B3-3 THP (0.25mg/mL, 100 ⁇ L) + R837 (0.8mg/mL, 100 ⁇ L);
  • Group B3-4 THP (0.25mg/mL, 100 ⁇ L) + R837 (0.4mg/mL, 100 ⁇ L);
  • Group B3-7 THP (0.25 mg/mL, 100 ⁇ L) + BCG (2.6 mg/mL, 100 ⁇ L).
  • Example B4 Evaluation of epirubicin hydrochloride (hereinafter referred to as EPI) combined with different R837 hydrochloride ratios by perfusion in the treatment of bladder in situ tumors.
  • EPI epirubicin hydrochloride
  • Group B4-1 blank group (ultrapure water dd H 2 O);
  • Group B4-2 R837 (240 ⁇ g);
  • Group B4-3 EPI (60 ⁇ g);
  • Group B4-4 EPI (60 ⁇ g) + R837 (60 ⁇ g);
  • Group B4-5 EPI (60 ⁇ g) + R837 (120 ⁇ g);
  • Group B4-6 EPI (60 ⁇ g) + R837 (240 ⁇ g);
  • Group B4-7 MMC (60 ⁇ g) + R837 (120 ⁇ g).
  • FIG. 5 shows the fluorescence in vivo imaging of small animals before the first infusion treatment and one week after the second infusion treatment. survival rate.
  • the mean fluorescence intensity of in situ tumors in mice was weakened, and the survival time of mice was improved. Death due to MMC toxicity occurred, so the combined application of EPI and R837 was significantly better than the combined use of MMC and R837.
  • Figure 6 shows the body weight of the mice in each group on the 9th day after the start of administration.
  • Table 2 Mean fluorescence intensity of orthotopic tumor tissue of mouse bladder and survival rate of each group
  • Example C1 ICD drug combined with R837 for bladder cancer perfusion therapy to inhibit the evaluation experiment of distal tumor growth.
  • mice in the EPI+R837 (360 ⁇ g) group and the mice in the healthy blank group (C57BL/6) were subcutaneously treated with MB49 cells (1 ⁇ 10 6 cells). ) were inoculated, and tumor growth was observed.
  • the data of the subcutaneous tumor volume of mice can show that the subcutaneous tumor volume of the mice in the EPI+R837 group is significantly smaller than that of the blank control group, which confirms that the combination of EPI and R837 has a tumor-specific immune distal tumor inhibition effect, which can reduce bladder cancer. Tumor recurrence and metastasis.
  • Example C2 Attenuating effect and mechanism evaluation of ICD drug combined with R837 in different mass ratios (1:6, 1:10).
  • mice in each group were perfused with equal doses of ultrapure water (ddH 2 O), R837 (600 ⁇ g), EPI (60 ⁇ g) and EPI (60 ⁇ g)+R837 (600 ⁇ g) in the blank group, respectively.
  • Body weight was recorded every day.
  • the weight of the mice in the EPI and R837 mass ratio (1:10) group was significantly lower than that of the other control groups. increased, offsetting the attenuation effect of the original combination.
  • the combined administration of R837 and ICD chemotherapeutic drugs within the appropriate combined dose range can significantly reduce the toxic and side effects of the chemotherapeutic drugs.
  • FIG 10 shows the EPI content in the blood of mice detected by fluorescence spectrophotometer 4 hours after perfusion administration.
  • the experimental data show that there is no significant difference in the concentration of EPI in the blood of the two groups of mice when EPI and EPI+R837 are combined.
  • the total phagocytosis of EPI in bladder tissue was evaluated by flow cytometry.
  • Figure 11 shows the data of EPI flow fluorescence analysis of mouse bladder tissue cells.
  • R837 has the effect of immune regulation. Although an appropriate increase in the proportion of R837 will reduce the toxic and side effects of chemical drugs, excessive R837 combined with chemical drugs will cause strong mucosal irritation, but it will affect small adverse effects on mice. Therefore, it is proved by this example that the preferred chemical drug/R837 ratio is not less than 1:10.
  • an immunoadjuvant when used in combination with a chemical drug that can cause immunogenic cell death as a pharmaceutical composition for intravesical instillation, there is a competition between the attenuation effect and the new toxic and side effects, so there is a certain proportional relationship between the pharmaceutical composition, although The difference in the properties of different drugs leads to the difference in the proportional relationship, but when the immune adjuvant is used in combination with the chemical drug that can cause immunogenic cell death as a pharmaceutical composition for intravesical instillation, the ICD class can be reduced by the technical solution claimed in the present invention.
  • the systemic side effects of chemical drugs can also increase the dosage of ICD drugs within a certain range, and significantly improve the therapeutic effect of the drugs.
  • the compound preparation of epirubicin and R837 was prepared without freeze-drying treatment, and the impurity changes of the injection dosage form under different pH and temperature conditions were studied to judge whether the injection dosage form met the actual production and use of the compound preparation.
  • Example D1 Chemical stability of intravesical infusion pharmaceutical composition injection formulations under different pH conditions.
  • imiquimod impurity B remained at about 0.05%
  • imiquimod impurity A was maintained at about 0.05%
  • imiquimod impurity C was not Detected
  • the maximum unknown single impurity is kept at about 0.18%
  • the total impurities are basically within 0.39%, and none of them exceeds the limit: impurity A content 0.15%, impurity B content 0.20%, impurity C content 0.15%, maximum unknown single impurity content 0.20 %, the total impurity content is 1.0%. Therefore, the dosage form of epirubicin/imiquimod compound injection does not have good stability, and the increase of main impurities comes from epirubicin, which cannot guarantee the safety and effectiveness of clinical medication.
  • Table 3 The initial impurity content of epirubicin in the dosage forms of epirubicin/imiquimod compound injection with different pH and the impurity content record table of epirubicin (EPI) left standing at 40°C for 5 days .
  • Embodiment D2 the influence of different storage conditions on the stability of injection dosage form
  • Example D1 Considering that the temperature at which the preparation was placed in Example D1 was 40°C, the generation of impurities may be accelerated, so the stability of the preparation at lower temperature was investigated.
  • the injection dosage form for intravesical instillation is most likely not Preferred dosage forms for this formulation.
  • Table 4 The initial impurity content of epirubicin (EPI) in the epirubicin/imiquimod compound injection preparation and the 10th day of standing at different storage temperatures, epirubicin/imiquimod compound injection Impurity content record table of epirubicin (EPI) in the preparation.
  • Example D show that the compound preparation is difficult to produce, store and use as an injection dosage form, so an experiment was designed to investigate the feasibility of the compound preparation as a lyophilized powder dosage form.
  • Embodiment E0 Research on the druggability of freeze-dried dosage forms:
  • the mass ratio of bicin and imiquimod is 1:1 to 1:10; secondly, try to adjust the pH of the solution to observe whether the pH change affects the stability of the solution; then add lyoprotectant sucrose, lactose, mannitol or cycloheximide Dextrin (concentration between 10mg/mL ⁇ 50mg/mL), observe the state of the sample, whether there is obvious instability, freeze-dry the sample with freeze-drying technology, the mass fraction of the freeze-drying protective agent after freeze-drying is 65% to 96%, observe the appearance of the lyophilized powder, whether there are any unshaped, agglomerated, agglomerated or uneven samples that are discernible to the naked eye; When the concentration of epirubicin in the solution is 1 mg/mL, the pH value is close to the value before freeze-drying, about 3.8-5.5.
  • the lyophilized powder formulation can be further studied for stability to detect changes in impurity content.
  • Example E1 Preliminary attempt to add lyoprotectant to formulation stability
  • the liquid preparation was prepared according to the method of Example D1.
  • the initial pH was about 3.0.
  • the pH was adjusted to 3.5 with sodium bicarbonate, and the final concentrations of epirubicin and imiquimod were 1 mg/mL, respectively. and 4mg/mL, and add different contents of lactose or mannitol before freeze-drying (concentrations are 10mg/mL, 25mg/mL, 50mg/mL during preparation, and the converted mass fractions are 67%, 83%, 91%),
  • concentrations are 10mg/mL, 25mg/mL, 50mg/mL during preparation, and the converted mass fractions are 67%, 83%, 91%)
  • the impurity content on the 0th day after lyophilization was investigated. The results are shown in Table 5.
  • Example E2 Lactose as a lyoprotectant, the stability of the lyophilized powder formulation standing at 25°C for 5 days.
  • Lactose two ponds
  • mannitol non-saccharides
  • cyclodextrins cyclic oligosaccharides
  • glucose monosaccharides
  • a liquid preparation was prepared according to the method of Example D1, the pH of the solution was adjusted to 3.5 or 4.0, and different freeze-drying protective agents were added respectively.
  • the final concentration of imiquimod was 4 mg/mL, and the concentration of epirubicin was 1 mg/mL.
  • the final content of the freeze-dried protective agent was 91%, and the changes in the impurity content of epirubicin hydrochloride placed under the condition of 40° C. for 5 days were detected. The results are shown in Table 5.
  • Table 7 adds epirubicin/imiquimod compound lyophilized powder formulations with different initial pHs of different lyophilized protective agents.
  • Example E4 Effect of lactose content on the stability of freeze-dried powder formulations
  • Example D1 the mixed injection of EPI and R837 was prepared, after adding different contents of lactose protective agent, the pH was adjusted to 4.0 with sodium bicarbonate, and the final imiquimod concentration was 4 mg/mL, and the epirubicin ratio was 4 mg/mL.
  • Star concentration is 1mg/mL
  • the concentration of lyophilized protective agent is 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL
  • the corresponding lyophilized protective agent mass fractions after lyophilization are 80%, 86%, 89%, respectively. , 91%, and the impurity content was detected on the day after lyophilization, placed at 40°C on the 5th day and the 13th day.
  • Example E5 Preparation samples under different pH conditions were prepared to investigate the stability of the preparation after lyophilization.
  • Liquid preparations with pH values of 3.5, 4.0, 4.5 and 5.0 were prepared by the method of Example D1, the final concentration of imiquimod was 4 mg/mL, and the concentration of epirubicin was 1 mg/mL. After being placed for a certain period of time under different temperature conditions, the lyophilized powder was reconstituted, and the impurity content was detected. The experimental conditions and results are shown in Table 9. Different from the change trend of total impurities in epirubicin hydrochloride in injection dosage form, when the initial pH value increases, the content of total impurities increases slowly.
  • pH of the preparation before lyophilization was 3.5
  • the impurities in the sample exceeded the limit specified by the Pharmacopoeia on the 5th day at 40°C. Therefore, under this condition, the sample was not subjected to 30-day impurity analysis.
  • Example E6 Change the ratio of the two components to study the change of the impurity content of the lyophilized preparation.
  • the liquid preparations with pH values of 3.5, 4.0, and 4.5 were prepared by the method of Example D1, and lactose was added.
  • the final imiquimod concentration was 2 mg/mL
  • the epirubicin concentration was 1 mg/mL
  • the lactose content was as shown in Table 10. shown.
  • the impurity content of epirubicin on the day after lyophilization and stored at 40° C. for 27 days was recorded, and the records are shown in Table 10. The results showed that with the increase of the initial pH value of the sample and the increase of the lactose content, the impurity content of the sample decreased when placed at 40 °C for 27 days.
  • Table 10 Record table of the impurity content of epirubicin (EPI) when the epirubicin/imiquimod compound lyophilized powder preparation was placed in an environment of 40° C. for 27 days under different initial pH values.
  • Example E7 Further Screening for Optimal pH
  • the mixed solution of epirubicin and imiquimod was prepared according to the method of Example D1, the pH of the solution was adjusted to 3.8, 4.0, 4.2, 4.4, and the lyophilized protective agent lactose was added.
  • the ratio of imiquimod was 1:2 or 1:4, the final concentration of lyoprotectant was 91%, and the corresponding mass fractions in the two specifications of lyophilized samples were 94% and 91%, respectively.
  • Example F1 Stability of the mixed lyophilized powder formulation of epirubicin hydrochloride and resiquimod (R848)
  • a mixed preparation was prepared, specifically, R848 was dissolved with hydrochloric acid to obtain a hydrochloride solution of R848, and epirubicin hydrochloride was further added, and the mass ratio of R848 to epirubicin was 4:1.
  • the lyophilized protective agent lactose was added, and the final content of lactose was 91%. Freeze-drying was performed, and the freeze-dried powder was placed at different ambient temperatures for stability investigation, and samples were taken on the 5th and 10th days to detect changes in the impurity content of the samples.
  • the impurity detection results of epirubicin are shown in Table 13.
  • the maximum unknown single impurity content of epirubicin increases after mixing. It has exceeded the limit specified in the standard.
  • the content of the largest unknown single impurity is still slowly increasing, which does not meet the quality requirements of the preparation. Adjusting the initial pH value to 4.0, the initial content of the largest unknown single impurity is greatly reduced.
  • Table 13 The initial impurity content of epirubicin and R848 mixed lyophilized powder preparation under different pH conditions and the impurity content record table of epirubicin when standing for 5 days and 10 days under different temperature conditions .
  • Example F2 Stability study of mixed lyophilized powder formulation of doxorubicin (ADM) and imiquimod.
  • the mixed liquid preparation of doxorubicin and imiquimod was prepared by the method of Example D1.
  • the initial pH of doxorubicin hydrochloride and imiquimod after mixing was 3.2, and the pH after adjustment was 4.0.
  • Two pH values were explored.
  • the difference in chemical stability of the freeze-dried powder preparations obtained after freeze-drying, the freeze-drying protective agent is lactose, and the content is 91%.
  • imiquimod was very stable in the mixed preparation, and its impurity content was basically unchanged. kept at around 0.05%, impurities kept at around 0.05%, impurities Undetected, the maximum unknown single impurity is kept at about 0.18%, and the total impurities are basically within 0.39%.
  • the impurity content record of doxorubicin in the stability investigation is as shown in Table 14, according to the guidance of the US Pharmacopoeia, the impurity A of doxorubicin is Impurity C is The impurities of doxorubicin will also increase rapidly under lower pH conditions. When the pH is adjusted to 4.0, the impurities can be kept at a safer level and have better stability.
  • Embodiment F3 investigate the stability investigation of mitoxantrone hydrochloride and imiquimod compound freeze-dried powder preparation
  • the mixed liquid preparation of mitoxantrone hydrochloride and imiquimod was prepared by the method of Example D1, and the freeze-dried powder preparation was obtained by freeze-drying after adding the freeze-drying protectant, wherein the content of the freeze-drying protectant lactose was 91%.
  • the stability of imiquimod is consistent with the phenomenon of the aforementioned mixed preparation, and there is no significant change in imiquimod-related impurities, and they are all within the limit.
  • 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 US Pharmacopoeia, impurity B and impurity C were not detected. The maximum unknown single impurity content of mitoxantrone hydrochloride should not exceed 1.0%, and the total impurity content should not exceed 1.0%.
  • Embodiment F4 investigate the stability of mixed lyophilized powder formulation of pirarubicin and imiquimod
  • a mixed freeze-dried powder preparation of pirarubicin and imiquimod was prepared, the pH of the preparation before freeze-drying was 4.0 or 5.0, the freeze-drying protective agent was lactose, and the content was 91%.
  • the impurity content of imiquimod and pirarubicin after lyophilization was detected, and the results are shown in Table 16 and Table 17.
  • Table 16 The initial impurity content of imiquimod in the mixed lyophilized powder formulation of pirarubicin hydrochloride and imiquimod at different initial pH values and the impurity content of imiquimod when placed at 40°C for 5 days and 15 days recording sheet.
  • Table 17 The initial impurity content of pirarubicin hydrochloride under different initial pH values of pirarubicin hydrochloride and imiquimod mixed lyophilized powder formulation and pirarubicin hydrochloride when placed at 40°C for 5 days and 15 days Star impurity content record sheet.
  • Example G1 Application of epirubicin in combination with R837 in the treatment of bladder cancer
  • a mouse MB49 bladder carcinoma in situ tumor model was established and randomly divided into 4 groups:
  • EPI perfuse epirubicin solution dissolved in 5% glucose solution
  • R837 Perfusion of imiquimod suspension dispersed in 5% dextrose solution
  • EPI+R837 mixed lyophilized powder preparation of epirubicin and imiquimod dissolved in 5% glucose solution
  • the first intravesical administration was recorded as the 0th day, the second administration was performed on the 7th day, and the mice were sacrificed on the 12th day.
  • the tumor mass was significantly lower than that of the other groups, indicating that the mixed lyophilized powder preparation of epirubicin and imiquimod has the effect of inhibiting the growth of bladder cancer.
  • Example G2 Application of pirarubicin (THP) combined with R837 in the treatment of bladder cancer
  • mice were randomly divided into groups:
  • THP perfuse pirarubicin solution dissolved in 5% glucose solution
  • R837 Perfusion of imiquimod suspension dispersed in 5% dextrose solution
  • THP+R837 mixed lyophilized powder preparation of pirarubicin and imiquimod dissolved in 5% glucose solution
  • Example 14 And carry out intravesical infusion therapy, the same as the treatment frequency and termination time in Example D1, after two intravesical infusion treatments, the mouse bladder cancer was dissected to record the tumor mass, the statistical chart is shown in Figure 14, 5% glucose solution
  • the compound preparation obtained by dissolving has obvious anti-tumor effect.
  • the tumor inhibition rates are 28.23% and 38.33%, respectively, while in mice treated with the compound preparation, the tumor inhibition rate is as high as 81.6%.
  • E(A+B) is the tumor inhibition rate of the compound preparation treatment group
  • EA and EB are two types, respectively.
  • the tumor inhibition rate when the components are used alone, when q ⁇ 1, indicates that the two components have a synergistic effect. It can be obtained by formula calculation, q>1, indicating that the form of compound preparation can achieve the synergistic effect of anthracycline and immune adjuvant.
  • Example G3 Application of Doxorubicin (ADM) in combination with Requimod (R848) in the treatment of bladder tumors
  • a mouse bladder cancer in situ model was established by the same method as in Example G1, a mixed freeze-dried powder preparation of doxorubicin and imiquimod was prepared for use, and the mice were randomly divided into groups (Blank: perfused with 5% glucose solution; ADM : perfusion with pirarubicin solution dissolved in normal saline; R848: perfusion with 5% glucose solution-dispersed resiquimod suspension; ADM+R848: perfusion with 5% glucose solution-dissolved pirarubicin mixed with resiquimod Freeze-dried powder preparation), wherein, before the compound preparation is used, hydroxypropyl methylcellulose is added as a penetration enhancer component for intravesical instillation therapy.
  • mice bladder cancer was dissected and the tumor mass was recorded.
  • the statistical chart is shown in Figure 15.
  • the compound preparation obtained by redissolving in normal saline was in the same range as the mucosa.
  • the penetration enhancer it has a more obvious anti-tumor effect.
  • the tumor inhibition rate of mice was 85.1%, while in the group treated with R848 or doxorubicin alone, the tumor inhibition rates of mice were 21.8% and 8.6%, respectively. obvious improvement.
  • the compound freeze-dried powder preparation of the anthracycline and imidazoquinoline immune adjuvant according to the present invention can be used to treat bladder tumors in the form of intravesical instillation.
  • Example H1 Treatment of bladder cancer in situ
  • mice with relatively uniform tumor growth were selected and grouped. There are 9 mice in each group, there are four groups in total, and the groupings are as follows:
  • the dose of epirubicin was 60 ⁇ g/a, and the dose of R837 was 240 ⁇ g/a.
  • the mice were sacrificed, the tumors in the bladder of the mice were taken, and the tumors were photographed and recorded.
  • the photo of mouse bladder tumor is shown in Figure 16. It can be seen with the naked eye that the diameter of the bladder of the mice in the epirubicin + R837 group is the smallest.
  • the tumor was weighed, and the bladder tumor mass of different groups of mice was counted. The results are shown in Figure 17. The smaller the tumor mass of the fourth group of mice, that is, the freeze-dried powder preparation has the best therapeutic effect, which is consistent with the naked eye in Figure 16. The observations are consistent.
  • Example H2 Flow analysis of immune cells in lymph nodes and bladder.
  • Example H1 The proportion and activity of immune cells in different samples in Example H1 were detected and analyzed by flow cytometry.
  • FIG. 18 shows the infiltration of CD3 + and CD8 + T cells in the bladder.
  • CD8 + T cells are killer T cells that have a direct killing effect on tumors. The existence of these cells indicates that immunotherapy plays a role in tumor treatment, and , the more infiltration of CD8 + T cells, the stronger the immunotherapy effect.
  • Figure 19 shows the infiltration of CD3 + and CD4 + T cells in the tumor.
  • CD4 + T cells can stimulate the activation of CD8 + T cells, maintain and strengthen the anti-tumor immune response, and on the other hand, play an anti-tumor effect through the IFN- ⁇ mechanism. Tumor effect, therefore, the increase in the content of CD4 + T cells at the initial stage of treatment contributes to the anti-tumor immune response.
  • the CD4 + T cells at the tumor site in the treatment group treated with the preparation of the present invention the content of An increase of more than 2 times indicates that the epirubicin compound preparation of the present invention can induce a stronger anti-tumor immune response.
  • Figure 20 shows the proportion of functional CD8 + T cells in the tumor site after being stimulated by the cell stimulation mixture.
  • the cells with tumor-killing potential in CD8 + T cells will secrete IFN- ⁇ after being stimulated by the cell stimulation mixture, and by flow antibody staining, CD8 and intracellular IFN- ⁇ are labeled, and the cells with double positive signals are accounted for CD8 monocytogenes.
  • the proportion of positive cells show that about 30% of the CD8 + T cells infiltrated in the tumor site of the mice treated with the freeze-dried powder preparation of the present invention can accumulate IFN- ⁇ in the cells after being stimulated, which is the one-component treatment group.
  • the proportion of corresponding cells is 1.5-6 times, indicating that the proportion of functional CD8 + T cells in the tumor site is higher and has greater immunotherapy potential, that is, the freeze-dried powder preparation of the present invention has a stronger effect on bladder cancer immunotherapy.
  • Example H3 Cytokine analysis was performed on the supernatant obtained after centrifugation in Example C2.
  • the detection results are shown in Figure 21 and Figure 22 .
  • the content of two cytokines, especially the content of TNF- ⁇ was higher in the compound preparation group.
  • the content of TNF- ⁇ in the compound preparation group was 2-3 times higher than that in the other groups, which proved that the mixed preparation had the effect of significantly enhancing the anti-tumor immune response.
  • the content of IFN- ⁇ in the compound preparation group was higher than that in any of the groups, and was about 1.5 to 2 times that of the other groups.
  • Embodiment 1 Mucosal penetration enhancer promotes the verification of drug retention effect at bladder site
  • Compound preparation 100 ⁇ L of the compound preparation of epirubicin and imiquimod is injected through the bladder;
  • Azone+compound preparation prepare a compound lyophilized powder preparation containing 2% (w/w) azone, and administer 100 ⁇ L to mice by intravesical instillation after reconstitution;
  • the above mixture was directly perfused into the mouse bladder, the perfusion process was carried out during the mouse anesthesia, the perfusion liquid was kept in the bladder for 1 hour, and then the liquid was discharged, the mouse was released from anesthesia, and the mouse was given 500 ⁇ L of water by gavage.
  • the mouse bladder samples were obtained by dissection, and each sample was weighed and recorded. The samples were placed in an equal volume of buffer solution for grinding, and the active ingredients were extracted with methanol and the extract, and the supernatant was obtained by centrifugation. Relative to the content per gram of tissue, the results are shown in Figure 23 and Figure 24.
  • Figure 23 is a statistical graph of the effect of mucosal penetration enhancers on the retention of epirubicin in the bladder, and it can be seen that the tissue relative content of epirubicin;
  • Figure 24 is the effect of mucosal penetration enhancers on the retention of imiquimod in the bladder Statistical chart of the effect of the amount, we can see the relative tissue content of imiquimod.
  • the compound preparation with the addition of azone components can significantly improve the retention of the drug in the bladder tissue, and its retention amount is more than 2 times, indicating that the addition of the penetration enhancer can help the retention of the active ingredients, and further has the potential to increase the curative effect. Effect.

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Abstract

本申请涉及一种膀胱灌注药物组合物，其包括免疫佐剂或其可溶性盐，以及能引起免疫原性细胞死亡的化药。所述免疫佐剂及其可溶性盐与能引起免疫原性细胞死亡的化药做为膀胱灌注药物联用时，能够在减少化药副作用的同时，产生协同抗癌作用，降低癌症转移及复发的概率。本申请还提供了所述膀胱灌注药物组合物的制备方法及其用途。

Description

一种膀胱灌注药物组合物及其制备方法和应用 技术领域
本申请涉及生物医药领域，具体的涉及一种膀胱灌注药物组合物及其制备方法和用途。
背景技术
膀胱癌是常见的泌尿系统肿瘤，虽然与肺癌和胃肠道肿瘤相比，膀胱癌具有较低的发病率和死亡率，但其高复发率已使膀胱癌成为临床治疗费用最高的癌症之一。膀胱癌经尿道肿瘤切除术后灌注化疗为膀胱癌治疗的首选方案，但目前灌注治疗的临床绝对缓解率低、肿瘤复发率高、毒副作用大、病人预后差。同时，近年来我国膀胱癌发病率呈逐年上升趋势，高于世界平均水平，且近30年来新型灌注小分子药物研究缓慢，我国面临严峻的膀胱癌预防治疗临床难题。
临床上常见的膀胱癌主要有两种，非肌层浸润膀胱癌(non-muscle invasive bladder cancer,NMIBC)和肌层浸润膀胱癌(muscle invasive bladder cancer,MIBC)。其中，经尿道膀胱肿瘤切除术(TURBT)辅以灌注药物治疗是非肌层浸润膀胱癌的一线标准治疗方案。有研究表明，非肌层浸润膀胱癌患者在接受TURBT膀胱肿瘤切除手术后，5年内有50％～70％的复发概率。膀胱灌注是在TURBT手术切除肿瘤后通过将化疗药物等肿瘤治疗制剂注入膀胱内部，直接杀伤肿瘤细胞或诱导体内非特异性免疫反应，达到抑制膀胱癌转移复发的效果。但是目前临床上膀胱灌注治疗的绝对缓解率低、肿瘤复发率较高，并且毒副作用大，病人预后差。研究表明一些化疗药物可以通过诱导肿瘤细胞发生凋亡或其他程序性死亡诱导具有免疫原性的蛋白分子在细胞表面表达，随后激发机体抗肿瘤免疫反应，这一现象被称为肿瘤免疫原性细胞死亡(immunogenic cell death，ICD)。因而有可能是制备和改良化疗-免疫联合治疗药物体系的途径之一。目前，在临床应用中只有少数临床相关药物已被证明能够引发ICD现象。这些药物主要包括：(1)阿霉素和蒽环类药物，这些药物已用于治疗小细胞肺癌等癌症；(2)表阿霉素，一种许可用于乳腺癌患者的蒽环类药物；(3)伊达比星，一种目前用于治疗急性髓细胞白血病的蒽环类药物；(4)米托蒽醌，被批准用于乳腺癌、非霍奇金淋巴瘤和前列腺癌患者的蒽环类药物；(5)博来霉素，被用于治疗睾丸癌、头颈部、宫颈和外阴的鳞状癌姑息性治疗的糖肽抗生素；(6)万珂硼替佐米，用于多发性骨髓瘤和T细胞淋巴瘤患者的蛋白酶体抑制剂；(7)奥沙利铂与5-氟尿嘧啶和亚叶酸组合使用，用于晚期结直肠癌的治疗。然而目前发现绝大多数ICD药物并不能高效持续的对肿瘤细胞发挥促免疫杀伤作用，因为并不是有 了肿瘤相关抗原免疫细胞就能有效的识别并杀死癌细胞。真正发挥作用，能追击消灭癌细胞的是免疫细胞中的杀伤性T细胞，所谓杀伤性T细胞是被激活的T细胞。T细胞需要抗原呈递细胞(APC)摄取、处理并将这些抗原呈递给自己之后才能被激活真正发挥效应，而抗原呈递细胞则需要免疫佐剂的帮助，才能更有效的将抗原呈递出去。
蒽环类药物对多种移行上皮细胞癌具有较高的易感性，表柔比星是第三代蒽环类半合成化合物，能够直接嵌入DNA碱基对之间，干扰转录过程，阻止mRNA的形成而起到抗肿瘤作用。目前，以表柔比星、吡柔比星为代表的蒽环类化疗药物是膀胱癌灌注治疗的一线治疗药物。但是，对于高危非肌层浸润膀胱癌患者，在施用表柔比星或吡柔比星进行膀胱灌注治疗后，肿瘤依然具有很高的一年内复发率(超过50％)。
肿瘤的免疫治疗在近年来备受关注，免疫治疗的关键步骤分为充分的抗原暴露，有效的抗原呈递以及随后的瘤内浸润与杀伤。而免疫激动剂作为呈上启下的关键步骤能够实现抗原呈递，其中代表性免疫激动剂如Toll样受体(Toll-like receptors,TLRs)是一种模式识别受体，作为固有免疫系统的重要组成部分，其作用是感知和识别病原体相关分子模式(PAMPs)，该受体在免疫细胞中广泛表达，受到相应的刺激后会促进针对肿瘤的T细胞应答。例如，咪喹莫特(R837)和免疫刺激剂寡核苷酸CpG分别识别、刺激TLR7和TLR9，合成免疫调节剂R848(Resiquimod)可激活TLR7和TLR8。大量研究报道TLRs激动剂具有免疫佐剂的作用，肿瘤治疗应用潜力巨大，但其有效性和应用性受到其促炎症因子及细胞因子的系统性释放副作用的限制。因此，TLR7作为配体的体内治疗主要集中在抗病毒或抗肿瘤局部治疗等TLR7作为免疫佐剂靶点的应用中。
在临床试验中，有将TLR激动剂用于膀胱癌治疗的研究。例如TMX-101直接将含有R837组分的凝胶制剂进行膀胱灌注，据报道，R837在该临床试验中表现出较好的安全性，部分患者受益，该项目被收购后继续开展研究，用于和单抗的联合治疗膀胱癌，暂无最新研究结果的披露。这些临床试验提示了TLR激动剂在膀胱灌注治疗中的应用潜力，但是均缺乏抗原暴露的过程，不能够完全发挥TLR激动剂的免疫增效作用，还有进一步优化的空间。
单纯的灌注化疗对膀胱癌进展抑制效果不好、易产生毒副作用和不良反应，病人易耐受且预后不理想。如何提高膀胱灌注化疗的治疗效果是膀胱灌注治疗技术中的一大课题。如何将水溶性化药和脂溶性免疫佐剂联合也是进一步推动膀胱癌化疗免疫联合治疗的关键问题。
发明内容
本申请提供了一种膀胱灌注药物组合物，所述药物组合物能够产生协同抗癌作用，降低癌症转移及复发概率，在有效杀灭原位肿瘤的同时还可以通过免疫反应抑制远端转移肿瘤的 生长和降低肿瘤复发的概率。
一方面，本申请提供了一种膀胱灌注药物组合物，其包括：免疫佐剂或其可溶性盐，以及能引起免疫原性细胞死亡的化药。
在某些实施方式中，所述能引起免疫原性细胞死亡的化药包括：蒽环类化疗药物、铂类化疗药物、氟尿嘧啶或吉西他滨。
在某些实施方式中，所述蒽环类化药包括表柔比星或其可溶性盐、吡柔比星或其可溶性盐、米托蒽醌或其可溶性盐、多柔比星或其可溶性盐、阿柔比星或其可溶性盐、伊达比星或其可溶性盐。
在某些实施方式中，所述可溶性盐为盐酸盐。
在某些实施方式中，所述蒽环类化疗药物包括盐酸多柔比星、盐酸表柔比星、盐酸吡柔比星、盐酸米托蒽醌。
在某些实施方式中，所述铂类化疗药物包括奈达铂、卡铂、洛铂或奥沙利铂。
在某些实施方式中，所述免疫佐剂或其可溶性盐包括免疫佐剂的可溶性盐。
在某些实施方式中，所述免疫佐剂或其可溶性盐包括咪唑喹啉类免疫佐剂或其可溶性盐。
在某些实施方式中，所述咪唑喹啉类免疫佐剂包括咪喹莫特及其衍生物，或瑞喹莫德及其衍生物，或咪喹莫特及其衍生物的可溶性盐，或瑞喹莫德及其衍生物的可溶性盐。
在某些实施方式中，所述所述免疫佐剂的可溶性盐包括咪喹莫特R837盐酸盐、雷西莫特R848盐酸盐或其他药学上可接受的盐、CpG、polyIC、polyICLC、STING刺激剂中的至少一种。
在某些实施方式中，所述免疫佐剂或其可溶性盐与所述能引起免疫原性细胞死亡的化药的质量比例为1:100～6:1。
在某些实施方式中，所述咪喹莫特R837盐酸盐与所述能引起免疫原性死亡的化药的质量比为1:20～1:1，其中所述能引起免疫原性死亡的化药为氟尿嘧啶或吉西他滨。
在某些实施方式中，所述所述咪喹莫特R837盐酸盐与所述能引起免疫原性死亡的化药的质量比为1:1～6:1，其中所述化药包括蒽环类化疗药物或铂类化疗药物，所述蒽环类化疗药物包括盐酸多柔比星、盐酸表柔比星、盐酸吡柔比星、盐酸米托蒽醌；所述铂类化疗药物包括奈达铂、卡铂、洛铂或奥沙利铂。
在某些实施方式中，所述所述雷西莫特R848盐酸盐与所述能引起免疫原性细胞死亡的化药的质量比例为1:20～1:1，其中所述能引起免疫原性死亡的化药包括蒽环类化疗药物或铂类化疗药物，所述蒽环类化疗药物包括盐酸多柔比星、盐酸表柔比星、盐酸吡柔比星或盐酸 米托蒽醌；所述铂类化疗药物包括奈达铂、卡铂、洛铂或奥沙利铂。
在某些实施方式中，所述雷西莫特R848盐酸盐与所述能引起免疫原性死亡的化药的质量比为1:100～1:10，其中所述能引起免疫原性死亡的化药包括氟尿嘧啶或吉西他滨。
在某些实施方式中，所述免疫佐剂的浓度范围在0.5mg/mL～30mg/mL。
在某些实施方式中，所述膀胱灌注药物组合物还包括pH调节剂。
在某些实施方式中，所述pH调节剂使得所述膀胱灌注药物组合物复溶后，当蒽环类化药浓度为1～5mg/mL时，所述复溶后的溶液的pH为3.8～5.5。
在某些实施方式中，所述膀胱灌注药物组合物还包括冻干保护剂。
在某些实施方式中，所述冻干保护剂包括蔗糖、乳糖、甘露醇和环糊精中的至少一种。
在某些实施方式中，所述冻干保护剂为乳糖。
在某些实施方式中，所述膀胱灌注药物组合物为冻干粉制剂。
在某些实施方式中，所述蒽环类化药与所述免疫佐剂的质量比为1:0.1～1:10。
在某些实施方式中，所述蒽环类化药与咪喹莫特及其衍生物的质量比为1:1～1:10。
在某些实施方式中，所述蒽环类化药与咪喹莫特及其衍生物的可溶性盐的质量比为1：1～1：10。
在某些实施方式中，所述蒽环类化药与瑞喹莫德的质量比为1:0.1～1:5。
在某些实施方式中，所述蒽环类化药与瑞喹莫德及其衍生物的可溶性盐的质量比为1:0.1～1:5。
在某些实施方式中，所述膀胱灌注药物组合物包括：表柔比星或其可溶性盐，咪喹莫特或其可溶性盐和pH调节剂。
在某些实施方式中，所述所述膀胱灌注药物组合物为冻干粉制剂。
在某些实施方式中，所述膀胱灌注药物组合物还包括冻干保护剂。
在某些实施方式中，所述冻干粉剂型的复溶后表柔比星浓度为1～5mg/mL时的pH为3.8～5.5。
在某些实施方式中，所述冻干粉剂型的复溶后表柔比星的浓度为1～5mg/mL时的pH为4.0～5.0。
在某些实施方式中，所述冻干粉剂型在复溶后表柔比星浓度为1～5mg/mL时的pH为4.0～4.2，4.2～4.5或4.5～5.0。
在某些实施方式中，所述表柔比星或其盐酸盐与咪喹莫特或其可溶性盐的质量比为1:1～1:10。
在某些实施方式中，所述表柔比星或其盐酸盐与咪喹莫特或其可溶性盐的质量比为1:2～1:4。
在某些实施方式中，所述冻干保护剂包括蔗糖、乳糖、甘露醇、环糊精。
在某些实施方式中，所述冻干保护剂为乳糖。
在某些实施方式中，所述冻干保护剂在冻干粉制剂中的质量分数为65％～96％。
在某些实施方式中，所述冻干保护剂在冻干粉制剂中的含量为80％～94％。
在某些实施方式中，所述膀胱灌注药物组合物还包括粘膜促渗剂。
在某些实施方式中，所述粘膜促渗剂包括氮酮、透明质酸酶、月桂醇、油酸中的至少一种。
另一方面，本申请还提供了一种膀胱灌注药物组合物的制备方法，其包括如下步骤：S1：用稀酸溶解免疫佐剂，得到免疫佐剂的稀酸盐溶液；S2：用S1获得的所述稀酸盐溶液溶解能引起免疫原性细胞死亡的化药，得到混合溶液；S3：向S1的混合溶液中加入pH调节剂，并将pH控制在2.0-5.5之间。
在某些实施方式中，所述步骤S3还包括如下步骤：S31：向所述混合溶液中加入冻干保护剂；和/或S32：向所述混合溶液中加入粘膜促渗剂。
在某些实施方式中，所述制备方法还包括步骤S4：对所述最终溶液进行冻干处理。
在某些实施方式中，所述步骤S3中将pH控制在3.8-5.5之间。
在某些实施方式中，在所述制备方法中，所述能引起免疫原性细胞死亡的化药包括蒽环类化药，在步骤S3中，所述蒽环类化药的最终浓度为1～5mg/mL。
另一方面，本申请还提供了一种膀胱灌注药物组合物的制备方法，其包括如下步骤：S1：将免疫佐剂CpG、polyIC、polyICLC、水溶性STING刺激剂与能引起免疫原性细胞死亡的化药加入注射用水，混合均匀至溶解；S2：向S1的溶液中加入合适的冻干保护剂，将溶液进行冻干处理。
另一方面，本申请还提供了所述膀胱灌注药物组合物在制备膀胱灌注制剂中的用途。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1是盐酸阿霉素(DOX)与咪喹莫特盐酸盐(R837)联合灌注治疗膀胱原位肿瘤后不同时间的膀胱部位超声影像图；
图2是盐酸吡柔比星(THP)与咪喹莫特盐酸盐(R837)联合灌注治疗膀胱原位肿瘤实验中不同组别瘤体最终大小实物图；
图3是盐酸吡柔比星(THP)与咪喹莫特盐酸盐(R837)不同配比联合灌注治疗膀胱原位肿瘤实验中不同时间点小鼠的活体荧光成像图；
图4是盐酸吡柔比星(THP)与咪喹莫特盐酸盐(R837)不同配比联合灌注治疗膀胱原位肿瘤实验中不同时间点小鼠的膀胱部位平均荧光强度统计图；
图5是盐酸表柔比星(EPI)与咪喹莫特盐酸盐(R837)联合灌注治疗膀胱原位肿瘤实验的肿瘤荧光成像图；
图6是盐酸表柔比星(EPI)与咪喹莫特盐酸盐联合灌注治疗膀胱原位肿瘤结束时肿瘤部位的荧光强度统计数据图；
图7是EPI与R837联用用于膀胱癌灌注治疗的小鼠肿瘤生长曲线图；
图8是EPI和R837联用(剂量比为1:6)时毒性实验中灌注给药后1-9天内小鼠体重变化曲线图；
图9是EPI和R837联用(剂量比为1:10)时毒性实验中灌注给药后1-4天内小鼠体重变化曲线图；
图10是小鼠血液中EPI含量统计图；
图11是小鼠膀胱组织中EPI含量的荧光强度统计图；
图12是小鼠膀胱组织中巨噬细胞中EPI荧光信号强度统计图；
图13是表柔比星和咪喹莫特混合冻干粉制剂应用于膀胱癌小鼠，通过膀胱灌注治疗膀胱癌后不同组别小鼠的肿瘤质量统计图；
图14是吡柔比星和咪喹莫特混合冻干粉制剂用于小鼠膀胱癌灌注治疗后，小鼠肿瘤质量统计图；
图15是多柔比星和咪喹莫特混合冻干粉制剂经过膀胱灌注治疗膀胱癌后不同组别小鼠的肿瘤质量统计图；
图16是膀胱灌注治疗第二次给药后第3天，不同组别小鼠病灶部位解剖图；
图17是膀胱灌注治疗第二次给药后第3天，不同组别小鼠病灶部位组织质量统计图；
图18是膀胱灌注治疗第二次给药后第3天，不同组别小鼠肿瘤部浸润的杀伤性T细胞流式分析统计图；
图19是膀胱灌注治疗第二次给药后第3天，不同组别小鼠肿瘤部位CD3 +/CD4 +T细胞的流式分析统计图；
图20是膀胱灌注治疗第二次给药后第3天，不同组别小鼠肿瘤部位CD8 +细胞经细胞刺激混合物刺激后，胞内IFN-γ含量统计图；
图21是膀胱灌注治疗第二次给药后第3天，不同组别小鼠病灶部位肿瘤坏死因子相对含量统计图；
图22是膀胱灌注治疗第二次给药后第3天，不同组别小鼠病灶部位γ干扰素的相对含量统计图；
图23是粘膜促渗剂对表柔比星在膀胱中滞留量影响的统计图；
图24是粘膜促渗剂对咪喹莫特在膀胱中滞留量影响的统计图。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
发明详述
为解决相关技术问题，本申请提供一种膀胱灌注药物组合物，本研发团队经过实验发现，免疫佐剂的可溶性盐与能引起免疫原性细胞死亡的化药(ICD类化药，immunogenic cell death)作为膀胱灌注药物联用时，能够产生协同抗癌作用，降低癌症转移及复发概率的抗癌药物组合物，在有效杀灭原位肿瘤的同时还可以通过免疫反应抑制远端转移肿瘤的生长和降低肿瘤复发的概率。同时，本发明体系的减毒作用不同于以往通过剂型改变灌注药物局部及系统吸收以降低药物系统毒性的作用机制，本发明通过药物组合的灌注给药，在化药发挥免疫原性作用的基础上，通过提高巨噬细胞对组织中肿瘤细胞外游离化疗药物的吞噬作用的通道进入病灶，从而显著降低化疗药物对正常组织的毒副作用，该药物进入方式是在灌注研究中被首 先尝试并被观察到的。
本发明提供一种膀胱灌注药物组合物：包括免疫佐剂的可溶性盐和能引起免疫原性细胞死亡的化药。进一步地，所述免疫佐剂的可溶性盐与所述能引起免疫原性细胞死亡的化药的质量比例为1:100～6:1。
进一步地，所述免疫佐剂的可溶性盐与所述能引起免疫原性细胞死亡的化药的质量比例为2:1～4:1。
进一步地，所述能引起免疫原性细胞死亡的化药包括蒽环类化疗药物、铂类化疗药物、氟尿嘧啶或吉西他滨；可选的，所述蒽环类化疗药物包括盐酸多柔比星、盐酸表柔比星、盐酸吡柔比星、盐酸米托蒽醌；所述铂类化疗药物包括奈达铂、卡铂、洛铂或奥沙利铂。
进一步地，所述免疫佐剂的可溶性盐包括咪喹莫特R837盐酸盐或其他药学上可接受的可溶性盐、雷西莫特R848盐酸盐或其他药学上可接受的免疫佐剂可溶性盐、CpG、polyIC、polyICLC、STING刺激剂(具有可包括IMSA-101、GSK-3745417、BMS-986301、SB-11285、MK-1454)中的至少一种。
进一步地，所述咪喹莫特R837盐酸盐与所述能引起免疫原性死亡的化药的质量比为1:20～1:1，其中所述能引起免疫原性死亡的化药为氟尿嘧啶或吉西他滨。
进一步地，所述咪喹莫特R837盐酸盐与所述能引起免疫原性死亡的化药的质量比为1:1～6:1，其中化药包括蒽环类化疗药物或铂类化疗药物，所述蒽环类化疗药物包括盐酸多柔比星、盐酸表柔比星、盐酸吡柔比星、盐酸米托蒽醌；所述铂类化疗药物包括奈达铂、卡铂、洛铂或奥沙利铂。
进一步地，所述雷西莫特R848盐酸盐与所述能引起免疫原性细胞死亡的化药的质量比例为1:10～1:1，其中所述能引起免疫原性死亡的化药包括蒽环类化疗药物或铂类化疗药物，所述蒽环类化疗药物包括盐酸多柔比星、盐酸表柔比星、盐酸吡柔比星、盐酸米托蒽醌；所述铂类化疗药物包括奈达铂、卡铂、洛铂或奥沙利铂。
进一步地，所述雷西莫特R848盐酸盐与所述能引起免疫原性死亡的化药的质量比为1:100～1:10，其中所述能引起免疫原性死亡的化药包括氟尿嘧啶或吉西他滨。
进一步地，还包括冻干助溶剂(如甘露醇、乳糖等)，pH调节剂。
进一步地，所述免疫佐剂的可溶性盐的浓度范围在0.5mg/mL～30mg/mL。
进一步地，所述剂型为冻干粉。
本发明还提供一种膀胱灌注药物组合物的制备方法，包括如下步骤：
方案1：
S1：将免疫佐剂咪喹莫特R837或雷西莫特R848溶于稀酸溶液(如盐酸、乳酸、醋酸)或其他药学上可接受的免疫佐剂(如吡喃葡糖苷脂质A(MPLA))可溶性盐溶液，往溶液中加入能引起免疫原性细胞死亡的化药，混合均匀至溶解；
S2：向S1的溶液中加入合适的冻干助溶剂(如甘露醇、乳糖等)，pH调节剂，并将pH值控制在2.0-5.5之间；
S3：将S2步骤获得溶液进行冻干处理。
方案2：
一种膀胱灌注药物组合物的制备方法，其特征在于包括如下步骤：
S1：将免疫佐剂CpG、polyIC、polyICLC、水溶性STING刺激剂与能引起免疫原性细胞死亡的化药加入注射用水，混合均匀至溶解；
S2：向S1的溶液中加入合适的冻干助溶剂(如甘露醇、乳糖等)，将溶液进行冻干处理。
本发明还提供了所述组合药物在制备膀胱灌注药物中的应用。
采用本发明的技术方案，具有如下的有益技术效果：
可降低ICD类化药的系统性毒副作用，根据现已掌握的证据初步推断该现象的作用机制为联用的免疫佐剂提高了巨噬细胞对组织中肿瘤细胞外游离化疗药物的吞噬作用，从而显著降低化疗药物对正常组织的毒副作用。膀胱癌经尿道肿瘤切除术后进行灌注治疗，ICD类药物可以引发肿瘤细胞的免疫原性细胞死亡，从而激活免疫系统，使其特异性地清除癌细胞。抗原呈递细胞吞噬死亡癌细胞之后，能引导免疫系统跟踪、识别和杀死其他癌细胞。因此，ICD类化疗药物在使得癌细胞免疫原性死亡之后，癌细胞残渣中的肿瘤相关抗原将被暴露给免疫细胞，提供了帮助免疫细胞识别癌细胞的靶标，帮助免疫系统建立肿瘤细胞特异性的免疫反应。可溶性免疫佐剂在与TLR识别后激活一系列下游信号，诱导炎症细胞因子、趋化因子和I型干扰素的分泌，促使肿瘤相关抗原能更有效的被抗原呈递细胞提呈给具有肿瘤杀伤作用的T细胞。因此当ICD类化疗药物在使癌细胞死亡变成肿瘤相关抗原之后，免疫佐剂的引入可以进一步刺激抗原呈递细胞对这些抗原的摄取和处理，并更有效提呈给T细胞，从而放大了这一抗肿瘤免疫反应。同时，免疫佐剂的存在也促使大量巨噬细胞在膀胱的肿瘤组织周围的富集，在ICD药物发挥初步化疗及免疫激发作用后吞噬大量肿瘤细胞外游离的化疗药物，能够减少化疗药物对正常组织的损伤，从而降低ICD类化药的系统性毒副作用，亦可在一定范围内提高ICD药物的用量，或提高整体的治疗效果。
可溶性免疫佐剂与ICD类化疗药物局部联用给药可显著提高化疗药物的肿瘤治疗及预防转移复发的效果，当ICD类化疗药物在使癌细胞死亡变成肿瘤相关抗原之后，免疫佐剂的引入可以进一步刺激APC细胞对这些抗原的摄取和处理，并更有效呈递给T细胞，从而放大了这一抗肿瘤免疫反应，免疫佐剂与ICD类化疗药物联合灌注应用对膀胱癌的治疗预防复发具有很好的改善作用。就像是一个在体内产生的肿瘤疫苗，在ICD类化疗药物和免疫佐剂的联合作用下，原位肿瘤被杀死并变成了肿瘤疫苗。因而，本发明所述ICD类化疗药物与免疫佐剂联用的药物体系具备高效治疗和预防膀胱癌转移复发的技术效果。
申请人尝试将蒽环类化药与咪喹莫特联合使用，并应用于膀胱癌的灌注治疗，将两类药物直接混合后进行膀胱灌注，验证疗效。结果发现，以化疗药物表柔比星与咪喹莫特为例，当化疗药物表柔比星与咪喹莫特的比例在一个合适的范围之间时，可以获得与单独化疗药物灌注相比显著提高的治疗效果，且不增加毒副作用，有望成为一种具有显著临床价值的膀胱灌注新型药物制剂。进一步研究表明，这一新型制剂抗肿瘤机理，主要是通过表柔比星等化疗药物诱导肿瘤细胞的免疫原性死亡暴露肿瘤相关抗原，同时咪喹莫特作为TLR7激动剂可以激活免疫系统，招募抗原呈递细胞到肿瘤内来识别肿瘤抗原，进而激活针对肿瘤的T细胞免疫反应。基于该原理，我们又尝试了不同蒽环类化药与咪喹莫特(R837)或瑞喹莫德(R848)联用治疗肿瘤的效果，均取得了理想的疗效。
但是，在进一步地制剂研究中发现，蒽环类化疗药物如表柔比星与免疫佐剂咪喹莫特的简单混合制剂中，咪喹莫特分子的存在会导致表柔比星等蒽环类化疗药物分子结构的不稳定，不论是以注射液的溶液形态保存、还是以混合后冻干粉的形态保存，该制剂中杂质的含量都会明显增加，并在5天时间内突破药典规定的药物的杂质限度，无法成为可以稳定保存的便于临床应用的药物制剂，二者的简单混合不利于药物分子的化学稳定性。因此，如何提高蒽环类化药与咪唑喹啉类免疫调节剂的复方制剂的化学稳定性，是设计制备二者联合用药的复方制剂的关键问题。为了解决上述衍生问题，我们进一步对蒽环类化药与咪唑喹啉类免疫调节剂复方制剂的稳定性进行了系统研究。
首先，我们发现表柔比星与咪喹莫特在溶液中低pH值(如pH 3.0)条件下更为稳定，但蒽环类化药仍然会逐渐降解，该条件下的混合制剂无法长期储存，因此注射液剂型对于本制剂不可行。对于冻干制剂而言，我们意外的发现在常见的冻干保护剂中，乳糖对该制剂的稳定性具有显著的保护效果，同时乳糖的含量增加会有利于制剂稳定性的提升。尽管如此，在pH 3.0的条件下制的冻干制剂依然无法长期保存，无法通过稳定性加速实验的考察。我们发现这一蒽环类化药免疫佐剂的复方冻干制剂有一个稳定性的“pH值黄金区间”，在这个pH范 围内制备得到的表柔比星/咪喹莫特复合制剂具有非常好的稳定性！该现象与本领域技术人员对表柔比星单一制剂的常规理解(常规情况下，pH越低越稳定)以及我们对该复方制剂溶液状态稳定性趋势(pH越低越稳定)的预测结果相反。
进一步地，我们考察了其他蒽环类药物如米托蒽醌、多柔比星和咪唑喹啉类免疫调节剂(R837或R848)复方冻干粉制剂的稳定性，基于表柔比星和R837的详细考察结果，尝试了类似的pH条件下不同化合物的稳定性。结果发现，这些化合物化学结构相似或相同，验证了复方制剂最佳的配方，使得两类物质之间任意组合均具有较好的稳定性。
本发明提出了一种膀胱灌注药物复方制剂，包括蒽环类化药，免疫调节剂和pH调节剂，其中，所述免疫调节剂包括咪唑喹啉类免疫调节剂及其可溶性盐。
进一步地，蒽环类化药包括表柔比星或其可溶性盐、吡柔比星或其可溶性盐、米托蒽醌或其可溶性盐、多柔比星或其可溶性盐、阿柔比星或其可溶性盐、伊达比星或其可溶性盐。
进一步可选地，所述可溶性盐为盐酸盐。
进一步地，所述膀胱灌注药物冻干粉制剂还包括冻干保护剂。
进一步地，所述冻干保护剂包括蔗糖、乳糖、甘露醇、环糊精。
进一步可选地，冻干保护剂为乳糖。
进一步地，所述冻干保护剂在冻干粉制剂中的质量分数为65％～96％。
可选地，冻干保护剂在冻干粉制剂中的含量为80％～94％。
进一步地，所述pH调节剂为弱碱，碱性缓冲液，氢氧化钠，盐酸。
进一步可选地，所述pH调节剂为碳酸氢钠，盐酸。
进一步地，所述膀胱灌注药物复方制剂在复溶后，当蒽环类化药浓度为1～5mg/mL时，溶液的pH为3.8～5.5。需要说明的是，这里定义复溶浓度的目的是为了定义复溶后的pH值的范围，并不是对复溶浓度的限定。
进一步可选地，所述膀胱灌注药物冻干粉制剂在复溶后，当蒽环类化药浓度为1～5mg/mL时，溶液的pH为4.0～5.0。需要说明的是，这里定义复溶浓度的目的是为了定义复溶后的pH值的范围，并不是对复溶浓度的限定。
进一步地，咪唑喹啉类免疫调节剂包括咪喹莫特及其衍生物，或瑞喹莫德及其衍生物，或咪喹莫特及其衍生物的可溶性盐，或瑞喹莫德及其衍生物的可溶性盐。
进一步地，蒽环类化药与咪唑喹啉类免疫调节剂的质量比为1:0.1～1:10。
进一步可选地，蒽环类化药与咪喹莫特的质量比为1:1～1:10。进一步可选地，蒽环类化药与咪喹莫特的质量比为1:2～1:4。
进一步可选地，蒽环类化药与瑞喹莫德的质量比为1:0.1～1:5。
进一步地，所述冻干粉制剂还包括粘膜促渗剂。
进一步地，所述粘膜促渗剂包括氮酮、透明质酸酶、月桂醇、油酸中的至少一种。
进一步地，所述粘膜促渗剂可以在制备过程中加入，也可以在使用前混合。
本发明所述蒽环类化药和免疫佐剂的复方制剂在使用前，与粘膜促渗剂混合后灌注，所述粘膜促渗剂可以是市面上已有的药用粘膜促渗剂或临床试验阶段批准使用的粘膜促渗剂。
本发明还提出了一种膀胱灌注药物冻干粉制剂的制备方法。
包括如下步骤：
S1：用稀酸溶解免疫调节剂，得到免疫调节剂的盐溶液；
S2：用S1获得的所述稀酸盐溶液溶解蒽环类药物，得到混合溶液；
S3：用酸性或碱性缓冲液或溶液调节所述混合溶液的pH至3.8～5.5得到最终溶液，此时药物的终浓度为1～5mg/mL。
进一步地：所述步骤S3还包括：
S31：向所述混合溶液中加入冻干保护剂；
进一步地：所述步骤S3还包括：
S32：。向所述混合溶液中加入粘膜促渗剂
进一步地，S1中所述稀酸为盐酸、乳酸或醋酸。
进一步可选地，S3中调节pH的进一步可选范围是4.0～5.0。
进一步地，还包括步骤S4：对S3中得到的最终溶液进行冻干处理得到冻干粉制剂。
本发明还提供所述冻干粉混合制剂在制备膀胱灌注药物中的应用。采用本发明的技术方案，具有如下的有益效果：
本发明提供一种膀胱灌注药物复方制剂，蒽环类药物和咪唑喹啉类免疫调节剂混合冻干粉制剂。在大量实验分析中发现，冻干粉剂型相较于传统的注射剂型，更有助于保护蒽环类药物在混合制剂中的长期化学稳定性，同时，混合制剂在膀胱灌注肿瘤治疗中，具有较好的肿瘤抑制效果，能够引起更强烈的抗肿瘤免疫反应，并且相较于单独的蒽环类化药，本发明所述复方制剂能够降低毒副作用。
本发明所述蒽环类化药和免疫调节剂混合制剂能够通过简单的复溶操作实现临床应用，可以直接灌注治疗，也可以加入粘膜促渗剂混合后灌注来有效抑制肿瘤生长和复发。蒽环类化药在引起肿瘤免疫原性死亡后，免疫调节剂能够进一步刺激抗原呈递细胞对抗原的摄取、处理以及呈递给T细胞，进一步放大抗肿瘤免疫反应。
可降低蒽环类化药或免疫调节剂的系统性毒副作用。
实现了蒽环类化药和免疫调节剂混合制剂的长期化学稳定性，显著降低了免疫调节剂存在的情况下蒽环类化药杂质的产生速率，提高了制剂长期储存稳定性，增加了制剂使用的便利性和安全性。未来的临床使用，较长的货架期有利于药物的生产、储存和运输，有助于实现真正的临床应用。
在本制剂中加入粘膜促渗剂，可以增强药物溶液灌注治疗后在膀胱壁上滞留时间，有助于进一步提升本类制剂的疗效。
申请人尝试将化疗药物表柔比星与咪喹莫特直接混合后用于膀胱癌的灌注治疗。结果发现，当化疗药物表柔比星与咪喹莫特的浓度比例在一个合适的范围之间时，可以获得与单独化疗药物灌注相比显著提高的治疗效果，且不增加毒副作用，有望成为一种具有显著临床价值的膀胱灌注新型药物制剂。进一步研究表明，这一新型制剂抗肿瘤机理，主要是通过表柔比星等化疗药物诱导肿瘤细胞的免疫原性细胞死亡暴露肿瘤抗原，同时咪喹莫特作为TLR7激动剂可以激活免疫系统，招募抗原呈递细胞到肿瘤内来识别肿瘤抗原，进而激活针对肿瘤的T细胞免疫反应。
但是，我们在进一步地制剂研究中发现，化疗药物如表柔比星与免疫佐剂咪喹莫特的简单混合制剂中，咪喹莫特分子的存在会导致表柔比星等化疗药物分子结构的不稳定，不论是以注射液的溶液形态保存、还是以混合后冻干粉的形态保存，该制剂中杂质的含量都会明显增加，并在很短时间内突破药典规定的药物的杂质限度，无法成为可以稳定保存便于临床应用的药物制剂，二者的简单混合不利于药物分子的化学稳定性。因此，如何提高表柔比星与咪喹莫特复方制剂的化学稳定性，是设计制备二者联合用药的复方制剂的关键问题。
为了解决上述问题，我们对各种条件下表柔比星与咪喹莫特复方制剂的稳定性进行了系统研究。我们发现表柔比星与咪喹莫特在溶液中低pH值(如pH＝3.0)条件下更为稳定(远远优于pH 4.0的条件)，但仍然会逐渐降解导致制剂无法长期储存，因此注射液剂型对于本制剂不可行。对于冻干粉制剂而言，我们发现乳糖对该制剂的稳定性具有显著的保护效果，同时乳糖的含量增加会有利于制剂稳定性的提升。尽管如此，在pH 3.0的条件下制得的冻干制剂依然无法长期保存，无法通过稳定性加速实验的考察。出乎意料的是，我们发现这一复方冻干制剂有一个稳定性的“pH黄金区间”，在这个pH范围内制备得到的表柔比星/咪喹莫特复方制剂具有非常好的稳定性。该现象与人们对表柔比星单一制剂溶液状态的稳定性趋势的常规理解以及我们对该复方制剂溶液状态稳定性考察结果相反。
通过进一步深入分析表柔比星的杂质谱图和化学结构，我们发现表柔比星与咪喹莫特的复方体系在水溶液和冻干制剂状态下的降解机理是不同的。首先，盐酸表柔比星不论在注射液还是冻干粉状态下都是稳定的，目前上市的表柔比星制剂存在注射液和冻干粉两种剂型。但对于复方制剂而言，在溶液相，咪喹莫特的存在会导致表柔比星一个未知单杂组分的快速上升，这个反应的速率随pH的升高而升高，在pH 3.0这一低pH值下，该反应依然会缓慢发生(注：更低的pH值的药液虽然对稳定性有利，但是会在使用时对膀胱造成很大的刺激和损害)。
在冻干的固态下，咪喹莫特的存在则会导致表柔比星杂质A(脱去糖环结构的降解产物)的快速上升，其原因猜测是咪喹莫特分子结构中芳香环上氮原子的孤对电子作为亲核基团进攻表柔比星的糖苷键，导致该化学键的断裂和降解杂质A的产生。通过加入乳糖作为冻干保护剂，可以一定程度上阻隔表柔比星分子和咪喹莫特分子的直接接触，从而减缓这一反应的发生。同时，这一反应从现象来看是酸催化加速的反应(低pH下糖苷键上氧原子的质子化是反应的中间步骤)，因此pH的升高会显著减缓杂质A的产生。另一方面，我们发现过高的pH则会导致咪喹莫特分子溶解性的下降(pH 5.0以上咪喹莫特从溶液中析出)，同时过高的pH还会导致表柔比星另外一个未知杂质(推断为表柔比星的氧化产物)的增加。综上，表柔比星与咪喹莫特的复方制剂在一个合适的pH区间、合适含量的辅料(乳糖)存在下，可以获得一个可以稳定保存的剂型。
此外，我们还发现加入在本制剂中加入粘膜促渗剂，可以增强药物溶液灌注治疗后在膀胱粘膜中的渗透和滞留，有助于进一步提升本制剂的疗效。
本发明提供一种表柔比星和咪喹莫特混合冻干粉制剂。在大量实验分析中发现，冻干粉剂型相较于传统的注射剂型，更有助于保护表柔比星在混合物中的长期化学稳定性，同时，混合制剂在膀胱灌注肿瘤治疗中，具有较好的肿瘤抑制效果，能够引起更强烈的抗肿瘤免疫反应。
本发明提出了一种表柔比星复方制剂，包括表柔比星或其可溶性盐，咪喹莫特或其可溶性盐，pH调节剂和冻干保护剂。
进一步地，所述表柔比星复方制剂为冻干粉剂型。
进一步地，表柔比星或其可溶性盐与咪喹莫特或其可溶性盐的质量比为1:1～1:10。
进一步可选地，表柔比星或其可溶性盐与咪喹莫特或其可溶性盐的质量比为1:2～1:4。
进一步地，所述pH调节剂可以是弱碱、氢氧化钠、盐酸。
进一步可选地，所述pH调节剂是碳酸氢钠、盐酸。
进一步地，所述冻干保护剂包括蔗糖、乳糖、甘露醇、环糊精。
进一步地，所述冻干保护剂为乳糖。
进一步地，所述冻干保护剂在冻干粉制剂中的质量分数为65％～96％。
进一步可选地，冻干保护剂的质量分数为80％～94％。
进一步地，所述表柔比星和咪喹莫特混合冻干粉制剂还包括粘膜促渗剂。
进一步地，所述粘膜促渗剂包括氮酮、透明质酸酶、月桂醇、油酸中的至少一种。
进一步地，所述粘膜促渗剂可以在制备过程中加入，也可以在使用前混合。
进一步地，所述表柔比星和咪喹莫特混合制剂冻干前的pH为3.8～5.5。需要说明的是，这里定义复溶浓度的目的是为了定义复溶后的pH值的范围，并不是对复溶浓度的限定。
进一步地，所述表柔比星和咪喹莫特混合冻干粉制剂复溶后，表柔比星浓度为1～5mg/mL时的pH为3.8～5.5。需要说明的是，这里定义复溶浓度的目的是为了定义复溶后的pH值的范围，并不是对复溶浓度的限定。
进一步地，所述冻干粉剂型的复溶后表柔比星的浓度为1mg/mL时的pH为4.0～5.0。
进一步可选地，所述冻干粉剂型复溶后表柔比星浓度为1mg/mL时pH值为4.0～4.4，4.4～5.0，5.0～5.5。
本发明提出了一种表柔比星和咪喹莫特混合冻干粉制剂的制备方法。
包括如下步骤：
S1：用稀酸溶解咪喹莫特或其可溶性盐，得到咪喹莫特稀酸盐溶液；
S2：用S1的溶液溶解表柔比星，得到混合溶液；
S3：用碱性缓冲液或碱性溶液调节混合溶液的pH至3.8～5.5；
进一步地：所述步骤S3还包括：
S31：向所述混合溶液中加入粘膜促渗剂；
进一步地，S1中所述稀酸溶液为盐酸、乳酸、醋酸。
进一步地，S3中调节pH的优选范围是4.0～5.0。
进一步可选地，S3中调节pH值可以是3.8～4.0，4.0～4.4，4.4～5.0。
进一步地，还包括S4：对S3中得到的最终溶液进行冻干处理得到冻干粉制剂。
本发明还提供所述冻干粉混合制剂在制备膀胱灌注药物中的应用。
采用本发明的技术方案，具有如下的有益效果：
本发明所述表柔比星和咪喹莫特混合制剂能够通过简单的复溶操作实现临床应用，通过灌注的方法有效抑制肿瘤生长和复发。表柔比星在引起肿瘤免疫原性死亡后，咪喹莫特能够 进一步刺激抗原呈递细胞对抗原的摄取、处理以及呈递给T细胞，进一步放大抗肿瘤免疫反应。
实现了表柔比星和咪喹莫特混合制剂的长期化学稳定性，显著降低了咪喹莫特存在的情况下表柔比星杂质的产生速率，提高了制剂长期储存稳定性，增加了制剂使用的便利性和安全性。
在本申请中，术语“免疫佐剂”和“免疫调节剂”通常可以互换使用。在某些实施方式中，所述免疫佐剂本身可以以可溶性盐形式存在，也可以通过某些操作将其转化为可溶性盐的形式，因此，术语“免疫佐剂”包含免疫佐剂或其可溶性盐。
在本申请中，术语“冻干保护剂”通常可以与“冻干助溶剂”互换使用。
在本申请中，术语“药物组合物”通常可以指包含药物活性成分的组合物。所述药物组合物还可以包含其他成分。例如，所述药物组合物还可以包含药学上可接受的载剂，例如，pH调节剂，例如，冻干保护剂。在本申请中，术语“药物组合物”还涵盖复方制剂的情况。
例如，所述膀胱灌注药物组合物可以为膀胱灌注药物复方制剂，例如，所述膀胱灌注药物复方制剂可以包括蒽环类化药，和免疫佐剂(免疫调节剂)。例如，所述膀胱灌注药物复方制剂还可以包括pH调节剂。
例如，所述膀胱灌注药物组合物可以为表柔比星复方制剂。例如，所述表柔比星复方制剂可以包括表柔比星或其可溶性盐，咪喹莫特或其可溶性盐和pH调节剂。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的膀胱灌注药物组合物、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例中涉及到的杂质，其名称、具体结构及对照品CAS如下：
Figure PCTCN2021143056-appb-000001
Figure PCTCN2021143056-appb-000002
实施例A制备配伍
实施例A1：称取10mg R837，加入5mL浓度为0.015M的盐酸，振荡直至溶液完全澄清。往该溶液中加入ICD化疗药物盐酸多柔比星，咪喹莫特与盐酸多柔比星的质量比为1:1、2:1、4:1、6:1以及10:1。加入合适的冻干助溶剂如甘露醇，加入pH调节剂如氢氧化钠，将pH值控制在2.0-5.5之间，冻干制备为冻干粉剂。
实施例A2:称取10mg R837，加入5mL浓度为0.015M的盐酸，振荡直至溶液完全澄清。往该溶液中加入ICD化疗药物盐酸表柔比星，咪喹莫特与盐酸表柔比星的质量比为1:1、2:1、4:1、6:1以及10:1。加入合适的冻干助溶剂如甘露醇，加入pH调节剂如氢氧化钠，将pH值控制在2.0-5.5之间，冻干制备为冻干粉剂。
实施例A3:称取10mg R837，加入5mL浓度为0.015M的盐酸，振荡直至溶液完全澄 清。往该溶液中加入ICD化疗药物盐酸吡柔比星，咪喹莫特与盐酸吡柔比星的质量比为1:1、2:1、4:1、6：1以及10:1。加入合适的冻干助溶剂如甘露醇，加入pH调节剂如氢氧化钠，将pH值控制在2.0-5.5之间，冻干制备为冻干粉剂。
实施例A4:称取10mg R837，加入5mL浓度为0.015M的盐酸，振荡直至溶液完全澄清。往该溶液中加入ICD化疗药物盐酸米托蒽醌，咪喹莫特与盐酸米托蒽醌的质量比为1:1、2:1、4:1、6:1以及10:1。加入合适的冻干助溶剂如甘露醇，加入pH调节剂如氢氧化钠，将pH值控制在2.0-5.5之间，冻干制备为冻干粉剂。
实施例A5:称取10mg R837，加入5mL浓度为0.015M的乳酸或醋酸(不得使用盐酸)，振荡直至溶液完全澄清。往该溶液中加入ICD化疗药物奥沙利铂，咪喹莫特与奥沙利铂的质量比为1:1、2:1、4:1、6:1以及10:1。加入合适的冻干助溶剂如甘露醇，加入pH调节剂如氢氧化钠，将pH值控制在4.5-6.0之间，冻干制备为冻干粉剂。
实施例A6:称取2mg R837，加入5mL浓度为0.005M的盐酸，震荡直至溶液完全澄清。往该溶液中加入ICD化疗药物氟尿嘧啶，咪喹莫特与氟尿嘧啶的质量比为1:1、1:2、1:5、1:10以及1:20。加入合适的冻干助溶剂如甘露醇，加入pH调节剂如氢氧化钠，将pH值控制在3.0-6.0之间，冻干制备为冻干粉剂。
实施例A7:称取2mg R848，加入5mL浓度为0.005M的盐酸，振荡直至溶液完全澄清。往该溶液中加入ICD化疗药物盐酸多柔比星，R848与盐酸多柔比星的质量比为1:1、1:2、1:5、1:10以及1:20。加入合适的冻干助溶剂如甘露醇，加入pH调节剂如氢氧化钠，将pH值控制在2.0-5.5之间，冻干制备为冻干粉剂。
实施例A8:称取2mg R848，加入5mL浓度为0.005M的盐酸，振荡直至溶液完全澄清。往该溶液中加入ICD化疗药物盐酸表柔比星，R848与盐酸表柔比星的质量比为1:1、1:2、1:5、1:10以及1:20。加入合适的冻干助溶剂如甘露醇，加入pH调节剂如氢氧化钠，将pH值控制在2.0-5.5之间，冻干制备为冻干粉剂。
实施例A9:称取1mg R848，加入2.5mL浓度为0.005M的盐酸，再加入注射用水5-15mL，振荡直至溶液完全澄清。往该溶液中加入ICD化疗药物氟尿嘧啶，R848与氟尿嘧啶的质量比为1：10、1:25、1:100以及1:200。加入合适的冻干助溶剂如甘露醇，加入pH调节剂如氢氧化钠，将pH值控制在3.0-6.0之间，冻干制备为冻干粉剂。
实施例A10：盐酸吉西他滨与CpG按质量比100：1在水溶液中混合，加入合适的冻干助溶剂甘露醇、乳糖，pH值控制在2.5-4.0之间，制备为冻干粉剂。
实施例B对比实验设计及实施应用
实施例B1：联合药物体系用于膀胱癌灌注治疗，与单独化疗药物或者单独免疫佐剂(咪喹莫特)相比，疗效显著提升；
结合图1评价盐酸阿霉素(以下简称DOX)与咪喹莫特盐酸盐(以下简称R837)联合灌注治疗膀胱原位肿瘤的效果。将健康C57BL/6雌性小鼠(18-20g)分为四组，每组4只，通过膀胱壁注射fLuc-MB49细胞(1×10 5个)建立膀胱癌原位模型，接种一周后进行小鼠活体成像观察确认肿瘤模型的建立，随后小鼠腹腔注射戊巴比妥麻醉，分别膀胱灌注以下药物组合：
第B1-1组：空白组(超纯水ddH 2O，100μL)；
第B1-2组：R837(1.5mg/mL，100μL)；
第B1-3组：DOX(1.5mg/mL，100μL)；
第B1-4组：DOX+R837(1.5mg/mL，100μL，1:1)。
灌注时间为1小时，每周一次，共两次，每周治疗前通过光声成像系统对其原位肿瘤大小进行检测。图1为膀胱原位肿瘤光声成像，图中外侧虚线内区域为小鼠膀胱区域，两条虚线中间区域为肿瘤组织。通过对比两条虚线之间区域的面积可以判断R837与DOX联合灌注组(B1-4)对膀胱原位肿瘤的抑制作用均显著强于单纯的R837组(B1-2)与单纯DOX组(B1-3)；图2为第二次灌注治疗后一周解剖的小鼠得到的膀胱原位肿瘤瘤体的图像，结果显示DOX与R837灌注联用组(B1-4)小鼠膀胱原位肿瘤体积更小，具有更显著的抑瘤效果，因此推断DOX与R837灌注联用可提高化疗药物疗效。
实施例B2：评价R837盐酸盐与盐酸吡柔比星(以下简称THP)联用灌注治疗膀胱原位肿瘤实验。前述中的药物为化疗药物的盐酸盐及R837的盐酸盐，R837临床剂型为软膏不适合灌注应用，目前其已有的膀胱灌注专利剂型为乳剂(TMX-101)，在此我们考察了R837盐酸盐与THP联用灌注的治疗效果。
采用实施例B1中所述类似的方法建立小鼠原位膀胱肿瘤，接种一周后进行小鼠活体成像观察确认肿瘤模型的建立，随机分3组，随后小鼠腹腔注射戊巴比妥麻醉，分别膀胱灌注以下药物组合：
第B2-1组：空白组(超纯水ddH 2O，100μL)；
第B2-2组：THP(0.6mg/mL，100μL)；
第B2-3组：THP(0.6mg/ml，100μL)+R837(0.6mg/mL，100μL)。
膀胱灌注1小时，每周灌注一次，共四次，每次灌注治疗前采用小动物成像系统对小鼠膀胱肿瘤进行检测，通过肿瘤组织的平均荧光强度表征肿瘤大小。0-35天小鼠活体成像数据如图3中所示，表1中为图3中小鼠膀胱原位肿瘤组织的平均荧光强度及各组的存活率。THP与R837的盐酸盐联合灌注组(B2-3)小鼠膀胱部位的肿瘤荧光强度明显小于其它对照组，并且该组小鼠直到35天具有很好的生存率(100％)，因此推断由于R837盐酸盐的水溶性好，灌注生物利用度高，R837的盐酸盐与THP联用能改善化疗药物的抑瘤效果，提高荷瘤小鼠生存率。
表1小鼠膀胱原位肿瘤组织的平均荧光强度及各组的生存率
Figure PCTCN2021143056-appb-000003
实施例B3：评价盐酸吡柔比星(以下简称THP)与R837盐酸盐不同配比联合灌注治疗膀胱原位肿瘤的作用效果。
采用实施例B1中所述类似的方法建立小鼠原位膀胱肿瘤，为了模拟临床上膀胱肿瘤切除术后的灌注治疗，本次实验采取接种后第二天开始灌注治疗的治疗方案，治疗前通过小动物活体成像系统对肿瘤生长情况进行检测，通过肿瘤组织平均荧光强度评价肿瘤的抑制水平。实验中膀胱灌注药物组合如下：
第B3-1组：空白组(超纯水ddH 2O，100μL)；
第B3-2组：THP(0.25mg/mL，100μL)；
第B3-3组：THP(0.25mg/mL，100μL)+R837(0.8mg/mL，100μL)；
第B3-4组：THP(0.25mg/mL，100μL)+R837(0.4mg/mL，100μL)；
第B3-5组：THP(0.25mg/mL，100μL)+R837(0.2mg/mL，100μL)；
第B3-6组：R837(0.8mg/mL，100μL)；
第B3-7组：THP(0.25mg/mL，100μL)+BCG(2.6mg/mL，100μL)。
每周灌注1小时，治疗三次，第16天各组别小鼠膀胱原位肿瘤荧光统计数据如图3所示，其中平均荧光强度越高代表肿瘤组织越大。图3中R837组(B3-6)与空白组(B3-1)的平均荧光强度无显著差异，表明单纯R837在此剂量下无显著的抑瘤效果，而THP与R837联合使用组(B3-3～5)的肿瘤荧光强度明显低于THP单独使用组及BCG阳性对照组(B3-7)，表明THP与R837联合使用具有显著的协同治疗效果，且随着R837配比的增加治疗效果也会提高，治疗效果明显优于BCG阳性对照组。因次，以上数据再次验证了R837与ICD药物联用具有协同抑瘤作用的推断。
实施例B4：评价盐酸表柔比星(以下简称EPI)与不同R837盐酸盐配比联用灌注治疗膀胱原位肿瘤实验。
为了进一步验证ICD药物与TLR7激动剂联合灌注可提高化疗药物治疗效果并降低化疗药物毒副作用的推断，我们评价EPI(60μg)与R837盐酸盐(60-240μg)不同配比联合灌注的小鼠膀胱原位肿瘤治疗效果，以非ICD药物丝裂霉素C(MMC)作为对照，结果如图4所示。以实施例B1中的方法建立小鼠原位肿瘤模型，小鼠接种细胞后5天开始灌注治疗(设为第0天)，小鼠随机分组如下：
第B4-1组：空白组(超纯水dd H 2O)；
第B4-2组：R837(240μg)；
第B4-3组：EPI(60μg)；
第B4-4组：EPI(60μg)+R837(60μg)；
第B4-5组：EPI(60μg)+R837(120μg)；
第B4-6组：EPI(60μg)+R837(240μg)；
第B4-7组：MMC(60μg)+R837(120μg)。
每组小鼠每次灌注体积100μL，灌注1小时，每周灌注一次，共2次。图5为小鼠第一次灌注治疗前及第二次灌注治疗后一周的小动物荧光活体成像，表2为小鼠膀胱原位肿瘤的平均荧光强度及各组别小鼠在第14天的生存率。根据本实验结果，随着R837盐酸盐与EPI联用投料比例的不断增加，小鼠原位肿瘤平均荧光强度减弱，小鼠生存时间改善，且MMC与R837联用组小鼠在实验早期即出现由于MMC毒性引起的死亡，因此EPI与R837的联合应用效果明显优于MMC与R837联用。图6为各组别小鼠开始给药后第9天时的体重情况，对比柱形图中数据，单纯EPI组小鼠体重(B4-2)较空白组(B4-1)出现明显的体重降低，而与R837联用组小鼠体重(B4-4～6)随着R837配比增加体重减少降低，因此提示ICD药物与R837联用的减毒作用。
表2小鼠膀胱原位肿瘤组织的平均荧光强度及各组的存活率
Figure PCTCN2021143056-appb-000004
Figure PCTCN2021143056-appb-000005
实施例C膀胱灌注药物组合物的效果评价
实施例C1：ICD药物与R837联用用于膀胱癌灌注治疗，抑制远端肿瘤生长的评价实验。
上述实验证实了ICD药物EPI与免疫佐剂R837联用对膀胱原位肿瘤的生长抑制及化疗减毒作用，且在一定剂量范围内随着免疫佐剂R837剂量的增加联合治疗效果越显著。在第14天时EPI+R837(360μg)组表现出十分显著的小鼠原位肿瘤治疗效果，为了进一步评价ICD药物与R837联用产生的特异性免疫作用对远端肿瘤生长的抑制作用，在第14天完成灌注治疗(第三次)后两周(第28天)时对EPI+R837(360μg)组小鼠以及健康空白组小鼠(C57BL/6)进行皮下MB49细胞(1×10 6个)接种，观察肿瘤的生长情况。皮下肿瘤接种后第4天开始测量记录皮下肿瘤大小，每隔一天测量一次，肿瘤体积计算公式为V＝(长×宽 2)/2。图7小鼠皮下肿瘤体积的数据可以说明EPI+R837组小鼠皮下肿瘤体积明显小于空白对照组，即证实EPI与R837联用产生了肿瘤特异性免疫远端肿瘤抑制作用，此作用可降低膀胱肿瘤的复发转移。
实施例C2：ICD药物与R837不同质量配比联用(1：6、1：10)的减毒作用及机制评价。
为了进一步确认R837与ICD药物联用后可减弱化疗药物自身毒副作用的推论及二者联用最大剂量范围，我们选用健康C57BL/6雌性小鼠进行进一步的毒性评价。四组小鼠每组6只，分别灌注等剂量的空白组超纯水(ddH 2O)、R837(360μg)、EPI(60μg)以及EPI(60μg)+R837(360μg)，灌注1小时，每周一次，灌注2次，隔天记录体重。图8所示，经两次灌注给药后(1-9天)，EPI(60μg)组小鼠体重较空白组及R837(360μg)组出现明显的 降低，而同样给药剂量的EPI与R837质量比(1：6)联用组小鼠无明显体重降低，后面巨噬细胞吞噬的实验证明了该效果。接下来我们采用类似的方法评价EPI与R837质量比(1：10)联用时是否存在减毒作用的用量范围。四组小鼠每组6只，分别灌注等剂量的空白组超纯水(ddH 2O)、R837(600μg)、EPI(60μg)以及EPI(60μg)+R837(600μg)，灌注1小时，隔天记录体重。图9所示，EPI与R837质量比(1:10)联用组小鼠体重较其它对照组反而出现明显的降低，推测是R837质量占比继续增加时，R837用量增加导致新的毒副作用的增加，抵消了原本联用时的减毒作用。同时结合实例B部分的实验结果可以推断R837与ICD化疗药物在适当联用剂量范围内联合灌注给药可显著降低化疗药物的毒副作用。
为了解释联用减毒的原因，首先考察膀胱灌注给药后4小时小鼠血液及膀胱组织中化疗药物的含量差异。图10为通过荧光分光光度计检测到灌注给药4小时后小鼠血液中EPI含量，实验数据表明EPI和EPI+R837联用这两组小鼠血液中化药EPI浓度无显著性差异。同时经流式对膀胱组织中EPI总吞噬量进行评价，图11为小鼠膀胱组织细胞的EPI流式荧光分析数据，实验结果表明EPI单独使用与其与R837联合使用在小鼠膀胱组织中的摄取率无明显差异，即与R837联用不会影响化疗药物EPI的系统及局部吸收。然而，对小鼠膀胱组织中巨噬细胞中EPI阳性比例流式分析检测实验中发现，EPI+R837联用组膀胱组织巨噬细胞对化疗药物的吞噬能力强于单纯EPI组(图12)，结合上述治疗效果改善的现象，可以推断在化疗药物发生免疫原性细胞死亡后，巨噬细胞将大量胞外游离的化疗药物吞噬，因此降低了胞外化疗药物对正常组织的毒副作用。但是，R837作为一种免疫刺激剂，有免疫调控的功效，虽然适量增加R837的比例会降低化药的毒副作用，但是过量的R837与化药联用会造成较强的粘膜刺激，反而对小鼠产生不良影响。因此，通过该实施例证明优选的化药/R837比例不低于1:10。因此当免疫佐剂与能引起免疫原性细胞死亡的化药联用作为膀胱灌注药物组合物时，存在减毒作用和新的毒副作用的竞争，因此存在一定的药物组合物的比例关系，虽然不同药物的性质的差异导致比例关系的差异，但是当免疫佐剂与能引起免疫原性细胞死亡的化药联用作为膀胱灌注药物组合物时，可通过本发明所主张的技术方案降低ICD类化药的系统性毒副作用，亦可在一定范围内提高ICD药物的用量，明显提高药物的治疗效果。
实施例D对注射液剂型稳定性的研究
制备表柔比星和R837复方制剂，不做冻干处理，研究注射剂型在不同pH条件、温度条件下的杂质变化情况，判断注射剂型是否满足该复方制剂的实际生产使用情况。
实施例D1：不同药液pH条件下膀胱灌注药物组合物注射制剂的化学稳定性。
将咪喹莫特用盐酸溶解后加入盐酸表柔比星进行溶解，溶液的pH值较低，灌注时会对组织产生较大刺激，为了使用时减少对组织的刺激，需要选择兼顾制剂稳定性与使用安全性的酸碱值。
用盐酸溶解R837得到浓度为4mg/mL的R837盐酸盐溶液，再加入盐酸表柔比星(EPI)溶解，EPI浓度为1mg/mL，然后使用碳酸氢钠调节溶液pH至3.0、3.5、4.0，分别标记为注射液1、注射液2、注射液3，用液相色谱检测配制当天以及放置于40℃环境下第5天的药物制剂中表柔比星、咪喹莫特杂质的含量，结果如表3所示。杂质主要来自于盐酸表柔比星，杂质A(EPI-A)的结构为
Figure PCTCN2021143056-appb-000006
杂质C(EPI-C)的结构为
Figure PCTCN2021143056-appb-000007
结果显示，在三种pH条件下，相较于原料药，制剂的总杂质含量随着时间而增长，并且，在40℃环境下放置第5天，总杂质含量是原料药总杂质含量的3倍以上。即使在pH＝3.0的最稳定条件，EPI的未知单杂在40℃存放仅5天后，也有显著增加，预计很快就会超过药典规定的最大允许限度。同时发现咪喹莫特的杂质含量相对稳定，在多次检测中，咪喹莫特杂质B保持在0.05％左右，咪喹莫特杂质A保持在0.05％左右，咪喹莫特杂质C均未检出，最大未知单杂保持在0.18％左右，总杂质基本在0.39％以内，均未超出限度：杂质A含量0.15％、杂质B含量0.20％、杂质C含量0.15％、最大未知单杂含量0.20％，总杂质含量1.0％。因此，表柔比星/咪喹莫特复方注射液剂型并不具有良好的稳定性，且主要杂质增长来自于表柔比星，不能保证临床用药的安全性、有效性。
表3不同pH下表柔比星/咪喹莫特复方注射液剂型中表柔比星的起始杂质含量以及在40℃下静置5天的表柔比星(EPI)的杂质含量记录表。
Figure PCTCN2021143056-appb-000008
实施例D2：不同储藏条件对注射液剂型稳定性的影响
考虑到实施例D1中制剂放置的温度为40℃，可能会加速杂质产生，因此探究较低温度下制剂的稳定性。采用实施例D1中的方法制备pH＝3.0和pH＝3.5的膀胱灌注注射剂，置于冷藏条件、15℃、25℃下，于样品制备完成当天以及在不同温度下放置第10天，检测制剂样品中杂质含量，结果如表4所示。随着存储温度的升高，两个pH条件下的注射液的总杂含量均有升高，印证了注射液剂型的稳定性与温度相关。同时发现，即便在冷藏条件下，最大未知单杂仍有增长趋势，最大未知单杂的升高同样不利于制剂的总体稳定性和安全性，因此，膀胱灌注的注射液剂型，极有可能不是该制剂的优选剂型。
表4表柔比星/咪喹莫特复方注射制剂中表柔比星(EPI)的起始杂质含量以及在不同储藏温度下静置第10天，表柔比星/咪喹莫特复方注射制剂中表柔比星(EPI)的杂质含量记录表。
Figure PCTCN2021143056-appb-000009
Figure PCTCN2021143056-appb-000010
实施例E冻干剂型稳定性考察
实施例D中的结果表明该复方制剂很难作为注射剂型生产、储存和使用，因此设计实验考察复方制剂的冻干粉剂型可行性。
实施例E0：冻干剂型成药性研究：
为了初步研究探究冻干粉制剂的成药可能性，对辅料、pH等可能对成药性产生影响的因素进行初筛。
将咪喹莫特使用盐酸溶解或使用纯水溶解咪喹莫特盐酸盐得到咪喹莫特盐酸盐的水溶液，然后溶解盐酸表柔比星，得到含有有效成分的溶液，有效成分表柔比星和咪喹莫特的质量比为1：1～1：10；其次，尝试调节溶液pH，观察pH值变化是否影响溶液稳定性；随后加入冻干保护剂蔗糖、乳糖、甘露醇或环糊精(浓度在10mg/mL～50mg/mL之间)，观察样品的状态，是否出现明显不稳定的情况，用冷冻干燥技术对样品进行冻干，冻干后冻干保护剂的质量分数为65％～96％，观察冻干粉外观，是否出现肉眼可辨的不成型、结团、结块或不均匀的样品；最后，将冻干样品加溶媒复溶，观察是否出现不溶解、不均匀的现象，并且复测溶液pH，当溶液中表柔比星浓度为1mg/mL时，pH值均与冻干前数值接近，约为3.8～5.5。
在制剂制备过程中，pH调节过程、冻干保护剂的加入未导致溶液出现不均匀或沉淀等明显不稳定的情况，冻干后样品均保持较好的形貌，并且复溶顺利，未出现不溶解或溶解不均匀的现象，因此该冻干粉制剂配方可以做进一步的稳定性研究，检测杂质含量变化。
实施例E1：初步尝试加入冻干保护剂对制剂稳定性的影响
按照实施例D1的方法制备液体制剂，未调节pH值时，初始pH约为3.0，此外，使用碳酸氢钠调节pH至3.5，表柔比星和咪喹莫特的终浓度分别为1mg/mL和4mg/mL，并于冻干前加入不同含量的乳糖或甘露醇(制备时浓度为10mg/mL、25mg/mL、50mg/mL，换算得到质量分数为67％，83％，91％)，考察其冻干后第0天的杂质含量。结果如表5所示。结果显 示，不加入任何冻干保护剂时，复方制剂中起始的最大未知单杂含量已超出限度，加入合适含量的冻干保护剂后，起始杂质含量有所降低。乳糖对冻干制剂的保护效果优于甘露醇，pH的升高以及乳糖含量的增加，均有利于产品的稳定性。
表5加入不同含量的乳糖或甘露醇后，表柔比星/咪喹莫特冻干粉制剂中表柔比星(EPI)的起始杂质含量记录表。
Figure PCTCN2021143056-appb-000011
实施例E2：乳糖作为冻干保护剂，25℃下冻干粉制剂静置5天的稳定性。
为了进一步探究冻干保护剂含量及起始pH对冻干粉稳定性的影响，将实施例E1中的4-1、5-1、6-1号样品放置于25℃条件下，于第5天复溶后检测样品的pH值及杂质含量，与0天的数据列表如表6所示。从杂质含量变化来看，起始pH值为3.5的冻干粉制剂，其杂质含量变化优于pH值为3.0的冻干粉制剂，同时，乳糖含量为91％时，杂质含量增长最少。
表6加入乳糖的表柔比星/咪喹莫特复方冻干粉制剂中表柔比星(EPI)的起始杂质含量以及在25℃下放置5天表柔比星(EPI)的杂质含量记录表。
Figure PCTCN2021143056-appb-000012
Figure PCTCN2021143056-appb-000013
实施例E3：不同冻干保护剂的筛选
基于对冻干保护剂效果的初步探索及保护剂含量的尝试，进一步探究相同含量下，不同冻干保护剂对冻干粉制剂的稳定性的影响。并基于对pH值变化趋势和杂质变化趋势的关系，进一步尝试更高pH值下，冻干后的制剂稳定性。
从注射剂常用冻干保护剂中选取乳糖(二塘)、甘露醇(非糖类)、环糊精(环状低聚糖)、葡萄糖(单糖)进行考察，验证其在91％的冻干保护剂用量下，对冻干粉制剂的保护效果。
首先，按照实施例D1的方法制备液体制剂，调节溶液pH至3.5或4.0，分别加入不同的冻干保护剂，最终咪喹莫特浓度为4mg/mL，表柔比星浓度为1mg/mL，冻干后冻干保护剂的最终含量为91％，检测其在40℃条件下放置5天的盐酸表柔比星杂质含量变化，结果如表5所示。表7中，起始pH为4.0的冻干粉制剂，当冻干保护剂为乳糖时，其稳定性最好，40℃下放置5天的杂质含量仍然保持较低水平，杂质无显著增长，pH＝3.5条件下杂质增长显著；其他冻干保护剂在pH3.5和pH4.0两个条件下，杂质均增长明显。因此，冻干保护剂优选乳糖。
表7加入不同冻干保护剂的不同起始pH的表柔比星/咪喹莫特复方冻干粉制剂。表柔比星
的起始杂质含量以及在40℃下放置第5天时表柔比星(EPI)的杂质含量记录表。
Figure PCTCN2021143056-appb-000014
Figure PCTCN2021143056-appb-000015
实施例E4：乳糖含量对冻干粉制剂稳定性的影响
基于前面实施例的结果，考察pH值为4.0时，加入不同含量乳糖对冻干粉制剂杂质含量的影响。
按照实施例D1所述的制备方法，配制EPI与R837混合注射液，加入不同含量的乳糖保护剂后，用碳酸氢钠调节pH至4.0，最终咪喹莫特浓度为4mg/mL，表柔比星浓度为1mg/mL，冻干保护剂浓度为20mg/mL，30mg/mL，40mg/mL，50mg/mL，冻干后对应的冻干保护剂质量分数分别为80％、86％、89％、91％，并于冻干后当天、40℃下放置第5天、第13天检测杂质含量。结果如表8所示。随着冻干保护剂起始含量的增加，相同pH条件、相同温度下放置的不同复方制剂，其杂质含量增幅变缓，说明其化学稳定性增加。冻干保护剂含量在80％、86％、89％和91％的条件下，杂质含量均未超出限度冻干保护剂起始含量为91％时，化学稳定性最好。
表8加入不同含量的乳糖的表柔比星/咪喹莫特复方冻干粉制剂的pH为4.0的复溶液中，表柔比星(EPI)的起始杂质含量以及在40℃下放置第5天、第13天的杂质含量记录表。
Figure PCTCN2021143056-appb-000016
实施例E5：制备不同pH条件下的制剂样品，考察冻干后制剂的稳定性。
用实施例D1的方法制备pH值分别为3.5、4.0、4.5和5.0的液体制剂，最终咪喹莫特浓度为4mg/mL，表柔比星浓度为1mg/mL，经预冷冻处理后进行冻干，在不同温度条件下放置 一定时间后，复溶冻干粉，检测其中杂质含量，实验条件及结果如表9所示。与注射剂型中盐酸表柔比星的总杂变化趋势不同，当起始pH值升高，总杂质的含量增幅变缓，在冻干保护剂为乳糖、含量为91％的情况下，未知单杂、总杂的变化均低于注射液剂型的杂质含量增幅，即冻干粉制剂在相同的pH(pH＝5.0)和温度条件(25℃)下，较长期放置(30天)的杂质含量远低于注射液剂型短期放置(5天)的杂质含量，并符合现行各国药典标准。其中制剂冻干前pH＝3.5时，样品杂质在40℃下第5天已超过药典规定的限度，因此，该条件下样品不做30天杂质分析。
表9不同起始pH值下，表柔比星/咪喹莫特冻干粉制剂在不同温度环境中放置不同时间后表柔比星(EPI)的杂质含量记录表。
Figure PCTCN2021143056-appb-000017
实施例E6：改变两种组分的比例，研究冻干制剂的杂质含量变化。
用实施例D1的方法制备pH值分别为3.5、4.0、4.5的液体制剂，加入乳糖，最终咪喹莫特浓度为2mg/mL，表柔比星浓度为1mg/mL，乳糖含量如表10中所示。记录冻干之后当天以及在40℃下存放27天的表柔比星杂质含量，记录如表10所示。结果显示，随着样品起始pH值的增加和乳糖含量的增加，在40℃下放置27天时样品的杂质含量减少，起始pH＝4.0 时，样品中乳糖含量为67％时，总杂含量在第27天超出限度，其余条件下杂质含量均低于限度。pH＝4.5时，几种乳糖含量均能减少冻干后样品中杂质含量的增加。
表10不同起始pH值下，表柔比星/咪喹莫特复合复方冻干粉制剂在40℃环境中放置27天时表柔比星(EPI)的杂质含量记录表。
Figure PCTCN2021143056-appb-000018
实施例E7：对最优pH进行进一步筛选
按照实施例D1的方法配制表柔比星和咪喹莫特的混合溶液，调节溶液pH为3.8、4.0、4.2、4.4，加入冻干保护剂乳糖，两种规格的样品中表柔比星和咪喹莫特的比例为1：2或1：4，冻干保护剂的最终浓度为91％，在两种规格的冻干样品中对应的质量分数分别为94％和91％。冻干后在40℃条件下放置样品，不同时间点检测样品中的杂质含量，结果如表11所示。
结果显示，不管何种浓度比例下，表柔比星杂质增长趋势与起始pH值相关，pH值越高，制剂越稳定，当pH值为3.8时，最大未知单杂的含量超出限度，pH在3.8以上时，均满足杂质含量稳定性要求。
表11不同起始pH值下，表柔比星/咪喹莫特复合复方冻干粉制剂中的表柔比星起始杂质含量以及在40℃环境中放置19天、30天时表柔比星(EPI)的杂质含量记录表。
Figure PCTCN2021143056-appb-000019
实施例F不同蒽环类药物与咪唑喹啉类免疫调节剂混合冻干粉制剂在pH＝4.0时的稳定性考察
实施例F1：盐酸表柔比星与瑞喹莫德(R848)混合冻干粉制剂的稳定性
首先制备混合制剂，具体的，用盐酸溶解R848得到R848的盐酸盐溶液，进一步地加入盐酸表柔比星，R848与表柔比星的质量比为4:1。加入冻干保护剂乳糖，乳糖终含量为91％。进行冻干，并将冻干粉置于不同环境温度下进行稳定性考察，在第5天和第10天取样检测样品中杂质含量变化。
与R837混合表柔比星不同的是，R848用盐酸溶解并加入表柔比星后，溶液体系的起始pH为5.5左右，高于盐酸咪喹莫特的pH，因此，使用酸性溶液将体系pH调节至4.0左右，检测原pH(pH＝5.5)条件和pH＝4.0条件下的杂质变化情况，R848的杂质检测结果如表12所示。由于现暂无R848上市药品，因此无法从杂质含量的限度判断R848的稳定性，但从表12的数据中可以看出，无论是在25℃条件还是40℃条件下，R848的杂质含量几乎没有随着时间的延长而发生变化，因此，可以判断R848在混合冻干粉制剂中稳定性良好。
表12表柔比星与瑞喹莫德(R848)混合冻干粉制剂在不同pH条件下的R848的起始杂质含量及温度条件下静置5天和10天时，样品中R848的杂质含量记录表。
Figure PCTCN2021143056-appb-000020
表柔比星的杂质检测结果如表13所示，当体系的冻干前pH不经调节时，即起始pH＝5.5，混合后便引起表柔比星的最大未知单杂含量的增加，已超过标准规定的限度，后续的稳定性实验中，最大未知单杂的含量还在缓慢增加，不符合制剂质量要求。调节起始pH值至4.0，最大未知单杂的起始含量便大大降低，随着时间延长，在不同温度条件下，杂质含量均未出现较大增长，并一直处于限度以内，说明pH＝4.0的条件下，该混合物冻干粉制剂具有较好的稳定性。
表13表柔比星与R848混合冻干粉制剂在不同pH条件下的表柔比星起始杂质含量及在不同温度条件下静置5天和10天时的表柔比星的杂质含量记录表。
Figure PCTCN2021143056-appb-000021
Figure PCTCN2021143056-appb-000022
实施例F2：多柔比星(ADM)与咪喹莫特混合冻干粉制剂的稳定性考察。
用实施例D1的方法制备多柔比星与咪喹莫特混合液体制剂，盐酸多柔比星与咪喹莫特混合后的起始pH＝3.2，调节后pH＝4.0，探究2种pH值下冻干后得到的冻干粉制剂的化学稳定性的差异，冻干保护剂为乳糖，含量为91％。在检测中发现，咪喹莫特在混合制剂中十分稳定，其杂质含量基本不变，杂质
Figure PCTCN2021143056-appb-000023
保持在0.05％左右，杂质
Figure PCTCN2021143056-appb-000024
保持在0.05％左右，杂质
Figure PCTCN2021143056-appb-000025
未检出，最大未知单杂保持在0.18％左右，总杂质基本在0.39％以内，不同温度下放置不同时间仅会导致咪喹莫特杂质的微小波动，均未超出R837制剂标准中规定的限度：杂质A 0.15％、杂质B 0.20％、杂质C 0.15％、最大未知单杂0.20％，总杂质1.0％。
稳定性考察中多柔比星的杂质含量记录如表14所示，根据美国药典指导，多柔比星的杂质A为
Figure PCTCN2021143056-appb-000026
杂质C为
Figure PCTCN2021143056-appb-000027
多柔比星的杂质在较低pH条件下，也会出现快速增长的情况，当调节pH至4.0后，杂质能保持在较安全的水平，稳定性更佳。
表14盐酸多柔比星(ADM)与咪喹莫特(R837)复方冻干粉制剂在不同pH条件下的起始杂质含量及在不同温度条件下静置5天和10天时多柔比星的杂质含量记录表
Figure PCTCN2021143056-appb-000028
实施例F3：考察盐酸米托蒽醌与咪喹莫特复方冻干粉制剂的稳定性考察
用实施例D1的方法制备盐酸米托蒽醌和咪喹莫特混合液体制剂，在加入冻干保护剂后冷冻干燥制得冻干粉制剂，其中冻干保护剂乳糖的含量为91％，测试制剂冻干前pH＝3.2(未经过酸碱度调节)和pH＝4.0(调节后pH)时，冻干粉的化学稳定性。其中咪喹莫特的稳定性与前述混合制剂的现象一致，咪喹莫特相关杂质均无明显变化，并均处于限度以内。盐酸米托蒽醌的杂质A为
Figure PCTCN2021143056-appb-000029
杂质D为
Figure PCTCN2021143056-appb-000030
盐酸米托蒽醌的杂质含量如表11所示，根据美国药典的指导，未检出杂质B和杂质C，盐酸米托蒽醌的最大未知单杂含量应不超过1.0％，总杂含量不超过3.0％，杂质含量记录表15中，无论在低pH值还是高pH值下，均未出现超出杂质限度的情况，但是相较于原始溶解后得到的pH，调节pH值为4.0后，杂质增幅要更缓慢，基本在起始杂质含量的值附近波动，说明pH＝4.0时，冻干粉制剂更加稳定。
表15盐酸米托蒽醌与R837混合冻干粉制剂在不同pH条件下的盐酸米托蒽醌的起始杂质含量及不同温度条件下的盐酸米托蒽醌的杂质含量记录表
Figure PCTCN2021143056-appb-000031
Figure PCTCN2021143056-appb-000032
实施例F4：考察吡柔比星与咪喹莫特混合冻干粉制剂的稳定性
制备吡柔比星和咪喹莫特混合冻干粉制剂，冻干前制剂pH＝4.0或5.0，冻干保护剂为乳糖，含量为91％。检测冻干后咪喹莫特和吡柔比星的杂质含量，结果如表16和表17所示。结果显示，咪喹莫特杂质含量在不同pH条件下均无较大变化，说明咪喹莫特在体系中较为稳定。在pH＝4.0的条件下，吡柔比星的杂质增长大于pH＝5.0(实测pH＝4.99)条件下的杂质变化，药典中没有规定吡柔比星药物杂质检测限度，因此仅能通过杂质增长趋势判断，pH＝5.0的条件更有利于制剂稳定性。
表16盐酸吡柔比星和咪喹莫特混合冻干粉制剂在不同起始pH值下的咪喹莫特起始杂质含量以及在40℃下放置5天和15天时咪喹莫特杂质含量记录表。
Figure PCTCN2021143056-appb-000033
表17盐酸吡柔比星和咪喹莫特混合冻干粉制剂的在不同起始pH值下的盐酸吡柔比星起始杂质含量以及在40℃下放置5天和15天时盐酸吡柔比星杂质含量记录表。
Figure PCTCN2021143056-appb-000034
Figure PCTCN2021143056-appb-000035
实施例G动物实验
实施例G1：表柔比星联用R837在膀胱癌治疗中的应用
制备表柔比星与R837混合冻干粉制剂备用。
建立小鼠MB49膀胱原位癌肿瘤模型，随机分为4组:
Blank：灌注5％葡萄糖溶液；
EPI：灌注5％葡萄糖溶液溶解的表柔比星溶液；
R837：灌注5％葡萄糖溶液分散的咪喹莫特混悬液；
EPI+R837：灌注5％葡萄糖溶液溶解的表柔比星与咪喹莫特混合冻干粉制剂；
第一次进行膀胱灌注给药记为第0天，在第7天进行第二次给药，第12天处死小鼠，取膀胱肿瘤记录质量，并统计不同组别肿瘤质量的均值，结果如图13所示，其中，使用复方制剂治疗的小鼠，肿瘤质量明显低于其他组别，说明表柔比星和咪喹莫特的混合冻干粉制剂具有抑制膀胱癌生长的效果。
实施例G2：吡柔比星(THP)联用R837在膀胱癌治疗中的应用
与实施例G1中相同的方法建立小鼠膀胱癌原位模型，制备吡柔比星与咪喹莫特的混合冻干粉制剂备用，将小鼠随机分组：
Blank：灌注5％葡萄糖溶液；
THP：灌注5％葡萄糖溶液溶解的吡柔比星溶液；
R837：灌注5％葡萄糖溶液分散的咪喹莫特混悬液；
THP+R837：灌注5％葡萄糖溶液溶解的吡柔比星与咪喹莫特混合冻干粉制剂，
并进行膀胱灌注治疗，同实施例D1中的治疗频次和终止时间，经过两次膀胱灌注治疗之后，解剖得到小鼠膀胱癌记录瘤体质量，统计图如图14所示，5％葡萄糖溶液复溶得到的复方制剂具有明显的抗肿瘤效果，单独使用吡柔比星或R837时，抑瘤率分别为28.23％、38.33％，而使用复方制剂治疗的小鼠，抑瘤率高达81.6％。使用金氏公式q＝E(A+B)/(EA+EB-EA*EB)计算药物协同作用，E(A+B)为复方制剂治疗组的抑瘤率，EA和EB分别为两种组分单独使用时的抑瘤率，当q≥1时，说明两种成分具有协同效果。通过公式计算可得，q＞1，说明复方制剂的形式能够达到蒽环类化药和免疫佐剂协同作用的效果。
实施例G3：多柔比星(ADM)联用瑞喹莫德(R848)在膀胱肿瘤治疗中的应用
与实施例G1中相同的方法建立小鼠膀胱癌原位模型，制备多柔比星与咪喹莫特的混合冻干粉制剂备用，将小鼠随机分组(Blank：灌注5％葡萄糖溶液；ADM：灌注生理盐水溶解的吡柔比星溶液；R848：灌注5％葡萄糖溶液分散的瑞喹莫德混悬液；ADM+R848：灌注5％葡萄糖溶液溶解的吡柔比星与瑞喹莫德混合冻干粉制剂)，其中，复方制剂使用前，加入羟丙基甲基纤维素作为促渗剂组分，进行膀胱灌注治疗。各组经过两次膀胱灌注之后，解剖得到小鼠膀胱癌记录瘤体质量，统计图如图15所示，较实施例G1和G2的效果相比，生理盐水复溶得到的复方制剂在与粘膜促渗剂联用后，具有更加明显的抗肿瘤效果。其中复方制剂治疗的组别，小鼠抑瘤率为85.1％，而单独使用R848或多柔比星治疗的组别中，小鼠的抑瘤率分别为21.8％和8.6％，复方制剂的疗效显著提升。
综合实施例G中的多组数据，本发明所述蒽环类化药和咪唑喹啉类免疫佐剂的复方冻干粉制剂，可以通过膀胱灌注的形式治疗膀胱肿瘤。
实施例H肿瘤治疗实验
实施例H1：治疗原位膀胱癌
建立C57BL/6小鼠膀胱癌原位肿瘤模型，具体的，在第0天，对雌性C57BL/6小鼠接种2*10 5个Luc-MB49细胞。接种后第4天，通过活体荧光成像判断肿瘤大小，挑选肿瘤生长较为均一的小鼠，进行分组。每组9只小鼠，共四组，其分组情况为：
1：通过膀胱灌注100μL的5％葡萄糖溶液；
2：通过膀胱灌注100μL的5％葡萄糖溶液溶解的表柔比星溶液；
3：通过膀胱灌注100μL的5％葡萄糖溶液溶解的盐酸咪喹莫特冻干粉剂；
4：通过膀胱灌注100μL的5％葡萄糖溶液溶解的本发明所述冻干粉制剂；
其中，表柔比星的给药剂量为60μg/只，R837的剂量为240μg/只。在接种肿瘤的第4天和第11天分别进行膀胱灌注给药，第二次治疗后第3天，牺牲小鼠，取小鼠膀胱内的肿瘤，对肿瘤进行拍照记录，不同组别的小鼠膀胱肿瘤照片如图16所示，肉眼可见的，表柔比星+R837组小鼠膀胱直径最小，其余组别肿瘤大小不均一，且大多数肿瘤大于该组小鼠。对肿瘤进行称重，统计不同组别小鼠膀胱肿瘤质量，结果如图17所示，第4组小鼠肿瘤质量越小，即冻干粉制剂具有最好的治疗效果，与图16中肉眼观测结果一致。
实施例H2：对淋巴结和膀胱中的免疫细胞进行流式分析。
用流式细胞仪检测分析实施例H1中不同样品的免疫细胞比例及活性。
对膀胱和淋巴结样品分别进行匀浆处理，经1800rpm，5min离心处理后，分离上清和沉淀，并在沉淀中加入消化酶，制备组织单细胞悬液。使用流式抗体对不同免疫细胞进行标记，并用流式细胞仪对不同免疫细胞的含量进行分析，结果如图18-图20所示。图18为膀胱中CD3 +、CD8 +T细胞的浸润情况，CD8 +T细胞为杀伤性T细胞，对肿瘤具有直接的杀伤作用，该细胞的存在说明免疫治疗在肿瘤治疗中发挥了作用，并且，CD8 +T细胞浸润越多，免疫治疗效果越强，从统计图可以看出，表柔比星通过免疫原性死亡导致肿瘤部位免疫反应的产生，联合R837后，免疫杀伤作用进一步增强，说明本发明的膀胱灌注制剂，具有更好的免疫治疗潜力。
图19为肿瘤中CD3 +、CD4 +T细胞的浸润情况，CD4 +T细胞一方面可刺激CD8 +T细胞的激活，维持和加强抗肿瘤免疫反应，另一方面可通过IFN-γ机制发挥抗肿瘤效果，因此，在治疗初期CD4 +T细胞含量的增加有助于抗肿瘤免疫反应，从图19数据可以看出，肿瘤部位CD4 +T细胞在膀胱灌注本发明所述制剂治疗组中，含量增加2倍以上，说明本发明所述表柔比星复方制剂能够引起更强烈的抗肿瘤免疫反应。
图20为肿瘤部位CD8 +T细胞经细胞刺激混合物刺激后，其中功能性CD8 +T细胞的比例。CD8 +T细胞中具有肿瘤杀伤潜力的细胞会在经过细胞刺激混合物刺激后，胞内分泌IFN-γ，通过流式抗体染色，标记CD8和胞内IFN-γ，检测双阳性信号的细胞占CD8单阳性细胞的比例。结果显示，经过本发明所述冻干粉制剂治疗的小鼠，其肿瘤部位浸润的CD8 +T细胞中有30％左右能够在受到刺激后在胞内蓄积IFN-γ，是单组份治疗组相应细胞比例的1.5～6倍，说明肿瘤部位功能性CD8 +T细胞比例更高，有更大的免疫治疗潜力，即本发明所述冻干粉制剂具有更强的膀胱癌免疫治疗的效果。
综合几种免疫相关细胞的流式分析，说明膀胱灌注用冻干粉制剂具有良好的激活抗肿瘤免疫反应的效果。
实施例H3：对实施例C2中离心后获得的上清液进行细胞因子分析。使用细胞因子检测试剂盒检测不同组别小鼠中肿瘤组织部位肿瘤坏死因子(TNF-α)和γ干扰素(IFN-γ)的含量，检测结果如图21、图22所示。TNF-α和IFN-γ的含量越高，说明肿瘤部位的抗肿瘤免疫反应越强。相比之下，复方制剂组中两种细胞因子含量都比较高，尤其是TNF-α的含量。如图21所示，复方制剂组中，TNF-α的含量为其它组别的2～3倍，证明混合制剂具有显著增强抗肿瘤免疫反应的作用。图22中，复方制剂组的IFN-γ含量高于任一组，约为其他组别的1.5～2倍。
实施例I粘膜促渗剂促进药物在膀胱部位滞留效果的验证
1.复合制剂：通过膀胱灌注100μL的表柔比星与咪喹莫特的复方制剂；
2.氮酮+复合制剂：制备含有2％(w/w)氮酮的复方冻干粉制剂，复溶后通过膀胱灌注向小鼠施用100μL的量；
将上述混合液直接进行小鼠膀胱灌注，灌注过程在小鼠麻醉过程进行，灌注液在膀胱中保留1小时，随后排出液体，小鼠脱离麻醉状态，以灌胃的方式帮助小鼠饮水500μL，促进排便，待药物排出6小时后，解剖获得小鼠膀胱样本，对每个样本进行称重记录。将样本置等体积的缓冲液中进行研磨处理，并用甲醇及萃取液对有效成分进行萃取，离心获得上清液，然后用高效液相色谱检测不同样本中萃取得到的有效成分的含量，计算其相对每克组织的含量，结果如图23、图24所示。图23是粘膜促渗剂对表柔比星在膀胱中滞留量影响的统计图，可以看出表柔比星的组织相对含量；图24是粘膜促渗剂对咪喹莫特在膀胱中滞留量影响的统计图，可以看出咪喹莫特的组织相对含量。其中增加氮酮组分的复方制剂，能够显著提高药物在膀胱组织的滞留，其滞留量达到2倍以上，说明促渗剂的加入能够有助于有效成分的滞留，进一步有潜在的增加疗效的作用效果。
对所公开的实施例的上述说明，使得本技术领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对于本领域技术人员而言将是显而易见的。本发明不会被限制于本文所示的这些实施例，只需要符合与本文所公开的原理与特点一致即可。

Claims (48)

  1. 一种膀胱灌注药物组合物，其包括：免疫佐剂或其可溶性盐，以及能引起免疫原性细胞死亡的化药。
  2. 根据权利要求1所述的膀胱灌注药物组合物，其中所述能引起免疫原性细胞死亡的化药包括：蒽环类化疗药物、铂类化疗药物、氟尿嘧啶或吉西他滨。
  3. 根据权利要求2所述的膀胱灌注药物组合物，其中所述蒽环类化药包括表柔比星或其可溶性盐、吡柔比星或其可溶性盐、米托蒽醌或其可溶性盐、多柔比星或其可溶性盐、阿柔比星或其可溶性盐、伊达比星或其可溶性盐。
  4. 根据权利要求3所述的膀胱灌注药物组合物，其中所述可溶性盐为盐酸盐。
  5. 根据权利要求2-4中任一项所述的膀胱灌注药物组合物，其中所述蒽环类化疗药物包括盐酸多柔比星、盐酸表柔比星、盐酸吡柔比星、盐酸米托蒽醌。
  6. 根据权利要求2-5中任一项所述的膀胱灌注药物组合物，其中所述铂类化疗药物包括奈达铂、卡铂、洛铂或奥沙利铂。
  7. 根据权利要求1-6中任一项所述的膀胱灌注药物组合物，其中所述免疫佐剂或其可溶性盐包括免疫佐剂的可溶性盐。
  8. 根据权利要求1-7中任一项所述的膀胱灌注药物组合物，其中所述免疫佐剂或其可溶性盐包括咪唑喹啉类免疫佐剂或其可溶性盐。
  9. 根据权利要求8所述的膀胱灌注药物组合物，其中所述咪唑喹啉类免疫佐剂包括咪喹莫特及其衍生物，或瑞喹莫德及其衍生物，或咪喹莫特及其衍生物的可溶性盐，或瑞喹莫德及其衍生物的可溶性盐。
  10. 根据权利要求7-9中任一项所述的膀胱灌注药物组合物，其中所述免疫佐剂的可溶性盐包括咪喹莫特R837盐酸盐、雷西莫特R848盐酸盐或其他药学上可接受的盐、CpG、polyIC、polyICLC、STING刺激剂中的至少一种。
  11. 根据权利要求1-10中任一项所述的膀胱灌注药物组合物，其中所述免疫佐剂或其可溶性盐与所述能引起免疫原性细胞死亡的化药的质量比例为1:100～6:1。
  12. 根据权利要求10-11中任一项所述的膀胱灌注药物组合物，其中所述咪喹莫特R837盐酸盐与能引起免疫原性死亡的化药的质量比为1:20～1:1，其中所述能引起免疫原性死亡的化药为氟尿嘧啶或吉西他滨。
  13. 根据权利要求10-11中任一项所述的膀胱灌注药物组合物，其中所述咪喹莫特R837盐酸盐与能引起免疫原性死亡的化药的质量比为1:1～6:1，其中所述化药包括蒽环类化疗药物或铂类化疗药物，所述蒽环类化疗药物包括盐酸多柔比星、盐酸表柔比星、盐酸吡柔比星、盐酸米托蒽醌；所述铂类化疗药物包括奈达铂、卡铂、洛铂或奥沙利铂。
  14. 根据权利要求10-11中任一项所述的膀胱灌注药物组合物，其中所述雷西莫特R848盐酸盐与所述能引起免疫原性细胞死亡的化药的质量比例为1:20～1:1，其中所述能引起免疫原性死亡的化药包括蒽环类化疗药物或铂类化疗药物，所述蒽环类化疗药物包括盐酸多柔比星、盐酸表柔比星、盐酸吡柔比星或盐酸米托蒽醌；所述铂类化疗药物包括奈达铂、卡铂、洛铂或奥沙利铂。
  15. 根据权利要求10-11中任一项所述的膀胱灌注药物组合物，其中所述雷西莫特R848盐酸盐与所述能引起免疫原性死亡的化药的质量比为1:100～1:10，其中所述能引起免疫原性死亡的化药包括氟尿嘧啶或吉西他滨。
  16. 根据权利要求1-15中任一项所述的膀胱灌注药物组合物，其中所述免疫佐剂的浓度范围在0.5mg/mL～30mg/mL。
  17. 根据权利要求1-16中任一项所述的膀胱灌注药物组合物，其还包括pH调节剂。
  18. 根据权利要求17所述的膀胱灌注药物组合物，其中所述pH调节剂使得所述膀胱灌注药物组合物复溶后，当蒽环类化药浓度为1～5mg/mL时，所述复溶后的溶液的pH为3.8～5.5。
  19. 根据权利要求1-18中任一项所述的膀胱灌注药物组合物，其还包括冻干保护剂。
  20. 根据权利要求19所述的膀胱灌注药物组合物，其中所述冻干保护剂包括蔗糖、乳糖、甘露醇和环糊精中的至少一种。
  21. 根据权利要求20所述的膀胱灌注药物组合物，其中所述冻干保护剂为乳糖。
  22. 根据权利要求1-21中任一项所述的膀胱灌注药物组合物，所述膀胱灌注药物组合物为冻干粉制剂。
  23. 根据权利要求1-22中任一项所述的膀胱灌注药物组合物，其中蒽环类化药与免疫佐剂的质量比为1:0.1～1:10。
  24. 根据权利要求23所述的膀胱灌注药物组合物，所述蒽环类化药与咪喹莫特及其衍生物的质量比为1:1～1:10。
  25. 根据权利要求23所述的膀胱灌注药物组合物，所述蒽环类化药与咪喹莫特及其衍生物的可溶性盐的质量比为1：1～1：10。
  26. 根据权利要求23所述的膀胱灌注药物组合物，所述蒽环类化药与瑞喹莫德的质量比为1:0.1～1:5。
  27. 根据权利要求23所述的膀胱灌注药物组合物，所述蒽环类化药与瑞喹莫德及其衍生物的可溶性盐的质量比为1:0.1～1:5。
  28. 根据权利要求1-27中任一项所述的膀胱灌注药物组合物，其包括：表柔比星或其可溶性 盐，咪喹莫特或其可溶性盐和pH调节剂。
  29. 根据权利要求28所述的膀胱灌注药物组合物，其为冻干粉剂型。
  30. 根据权利要求28所述的膀胱灌注药物组合物，其还包括冻干保护剂。
  31. 根据权利要求29-30中任一项所述的膀胱灌注药物组合物，其中所述冻干粉剂型的复溶后表柔比星浓度为1～5mg/mL时的pH为3.8～5.5。
  32. 根据权利要求29-31中任一项所述的膀胱灌注药物组合物，所述冻干粉剂型的复溶后表柔比星的浓度为1～5mg/mL时的pH为4.0～5.0。
  33. 根据权利要求29-32中任一项所述的膀胱灌注药物组合物，所述冻干粉剂型在复溶后表柔比星浓度为1～5mg/mL时的pH为4.0～4.2，4.2～4.5或4.5～5.0。
  34. 根据权利要求28-33中任一项所述的膀胱灌注药物组合物，所述表柔比星或其盐酸盐与咪喹莫特或其可溶性盐的质量比为1:1～1:10。
  35. 根据权利要求28-34中任一项所述的膀胱灌注药物组合物，所述表柔比星或其盐酸盐与咪喹莫特或其可溶性盐的质量比为1:2～1:4。
  36. 根据权利要求30-35中任一项所述的膀胱灌注药物组合物，其中所述冻干保护剂包括蔗糖、乳糖、甘露醇、环糊精。
  37. 根据权利要求30-36中任一项所述的膀胱灌注药物组合物，其中所述冻干保护剂为乳糖。
  38. 根据权利要求30-37中任一项所述的膀胱灌注药物组合物，其中所述冻干保护剂在冻干粉制剂中的质量分数为65％～96％。
  39. 根据权利要求30-38中任一项所述的膀胱灌注药物组合物，其中所述冻干保护剂在冻干粉制剂中的含量为80％～94％。
  40. 根据权利要求1-39中任一项所述的膀胱灌注药物组合物，其还包括粘膜促渗剂。
  41. 根据权利要求40所述的膀胱灌注药物组合物，其中所述粘膜促渗剂包括氮酮、透明质酸酶、月桂醇、油酸中的至少一种。
  42. 一种膀胱灌注药物组合物的制备方法，其包括如下步骤：S1：用稀酸溶解免疫佐剂，得到免疫佐剂的稀酸盐溶液；S2：用S1获得的所述稀酸盐溶液溶解能引起免疫原性细胞死亡的化药，得到混合溶液；S3：向S1的混合溶液中加入pH调节剂，并将pH控制在2.0-5.5之间。
  43. 根据权利要求42所述的制备方法，其中S3还包括如下步骤：S31：向所述混合溶液中加入冻干保护剂；和/或S32：向所述混合溶液中加入粘膜促渗剂。
  44. 根据权利要求42所述的制备方法，其还包括步骤S4：对所述最终溶液进行冻干处理。
  45. 根据权利要求42-44中任一项所述的制备方法，其中所述步骤S3中将pH控制在3.8-5.5之间。
  46. 根据权利要求42-45中任一项所述的制备方法，其中所述能引起免疫原性细胞死亡的化药包括蒽环类化药，在步骤S3中，所述蒽环类化药的最终浓度为1～5mg/mL。
  47. 一种膀胱灌注药物组合物的制备方法，其包括如下步骤：S1：将免疫佐剂CpG、polyIC、polyICLC、水溶性STING刺激剂与能引起免疫原性细胞死亡的化药加入注射用水，混合均匀至溶解；S2：向S1的溶液中加入合适的冻干保护剂，将溶液进行冻干处理。
  48. 权利要求1-41中任一项所述的膀胱灌注药物组合物在制备膀胱灌注制剂中的用途。
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CN202111499923.2A CN116251191A (zh) 2021-12-09 2021-12-09 一种膀胱灌注药物复方制剂及其制备方法和应用
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