WO2024113752A1 - 黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用 - Google Patents

黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用 Download PDF

Info

Publication number
WO2024113752A1
WO2024113752A1 PCT/CN2023/099383 CN2023099383W WO2024113752A1 WO 2024113752 A1 WO2024113752 A1 WO 2024113752A1 CN 2023099383 W CN2023099383 W CN 2023099383W WO 2024113752 A1 WO2024113752 A1 WO 2024113752A1
Authority
WO
WIPO (PCT)
Prior art keywords
immune checkpoint
unresponsive
hyperprogressive
baicalin
tumor
Prior art date
Application number
PCT/CN2023/099383
Other languages
English (en)
French (fr)
Inventor
鄢丹
张瑜
赵薇
曹邦伟
王爱婷
龙江兰
Original Assignee
首都医科大学附属北京友谊医院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 首都医科大学附属北京友谊医院 filed Critical 首都医科大学附属北京友谊医院
Priority to EP23772397.8A priority Critical patent/EP4400109A1/en
Priority to US18/373,980 priority patent/US12029747B2/en
Publication of WO2024113752A1 publication Critical patent/WO2024113752A1/zh

Links

Classifications

    • 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
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention belongs to the field of biomedicine, and in particular to the application of baicalin in the preparation of tumor drugs targeting immune checkpoint inhibitors for tumors, specifically the application of baicalin in the preparation of drugs for treating tumors that are unresponsive/hyperprogressive to immune checkpoint inhibitors.
  • Immune checkpoint inhibitors are the first-line or second-line treatment for most cancer patients.
  • HPD hyperprogressive disease
  • traditional treatment methods usually use measures such as changing treatment plans to prevent further tumor progression (The Lancet Oncology, 21(10), e463–e476.). Since the reselection of treatment plans may lead to the loss of the best treatment time window, it also brings multiple physical, mental, and economic pressures to patients. How to restore the patient's immune response without changing the original treatment plan when immune checkpoint inhibitors produce no response or hyperprogression is the key to solving the above problems and is also the most feasible research direction for clinical treatment.
  • Foxp3 + Treg cells are a type of immunosuppressive cells, and their excessive proliferation inhibits the effects of immune checkpoint inhibitors. Therefore, inhibiting the number of Foxp3 + Tregs in tumors may prevent or treat the occurrence of no response to immune checkpoint inhibitors and HPD.
  • Baicalin has a regulatory effect on immune cells, but there are no reports on the role and efficacy of baicalin in synergizing with immune checkpoint inhibitors to treat unresponsiveness and hyperprogression caused by excessive Foxp3 + Treg cells.
  • the technical solution of this application is to study the defects of unresponsiveness and hyperprogression caused by excessive Foxp3 + Treg cells treated with immune checkpoint inhibitors, and propose a technical solution to overcome the above defects through the synergistic effect of baicalin.
  • This application proposes the use of baicalin to prepare a drug for treating tumors that are unresponsive to immune checkpoint inhibitors and have hyperprogression caused by excessive Foxp3 + Treg cells, in response to the defects of unresponsiveness and hyperprogression caused by excessive Foxp3 + Treg cells in the treatment of immune checkpoint inhibitors.
  • This drug can effectively overcome the above-mentioned treatment defects of unresponsiveness/hyperprogression.
  • the present invention provides the use of baicalin in preparing a drug for treating tumors that are unresponsive/hyperprogressive to immune checkpoint inhibitors due to an increase in Foxp3 + Treg cells.
  • the tumor that is unresponsive/hyperprogressive to immune checkpoint inhibitors due to excessive Foxp3 + Treg cells is at least one of melanoma, non-small cell lung cancer, renal cell carcinoma, liver cancer, colorectal cancer, urothelial bladder cancer and pancreatic cancer.
  • the immune checkpoint inhibitors in the above applications include one or a mixture of two or more of PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4.
  • the mass percentage of baicalin in the medicine prepared by the above application is 0.05%-99.5%, and the daily dosage is 4mg/day to 1500mg/day.
  • the medicine is administered orally or by the intestine.
  • pharmaceutically acceptable excipients may be used as additives in the drug, and the drug may be prepared into a solid preparation or a liquid preparation.
  • the above-mentioned drugs are usually administered before or simultaneously with the immune checkpoint inhibitor.
  • the baicalin described in the present invention is used in combination with an immune checkpoint inhibitor to exert an anti-tumor sensitization effect by inhibiting the number of Foxp3 + Treg cells in tumors.
  • the baicalin described in the present application can cooperate with immune checkpoint inhibitors to effectively treat the phenomenon of tumor-bearing mice not responding to immune checkpoint inhibitors and hyperprogression caused by excessive Foxp3 + Treg cells, and no obvious adverse reactions were found, with good safety and broad prospects.
  • Figure 1 shows the tumor volume of R-FMT and NR-FMT mice before and after PD-1 mAb intervention treatment
  • Figure 2 shows the tumor weights of R-FMT and NR-FMT mice before and after PD-1 mAb intervention treatment
  • Figure 3 shows the tumor volume of NR-FMT mice before and after treatment with PD-1mAb, HQG, and HQG+PD-1mAb;
  • FIG4 shows the tumor weight of NR-FMT mice before and after treatment with PD-1 mAb, HQG, and HQG+PD-1 mAb;
  • Figure 5 shows the number of Foxp3 + Treg cells in the tumors of NR-FMT mice before and after intervention with PD-1mAb, HQG, and HQG+PD-1mAb.
  • HQG and PD-1mAb indicate: baicalin and PD-1 inhibitor, respectively;
  • the R-Control group and NR-Control group represent: the blank group of the response model and the blank group of the non-response/hyperprogression model, respectively;
  • the R-PD-1mAb group and the NR-PD-1mAb group represent: the response model PD-1 inhibitor administration group and the non-response/hyperprogression model PD-1 inhibitor administration group, respectively;
  • NR-HQG group and NR-HQG+PD-1mAb group represent: baicalin administration group and baicalin and PD-1 inhibitor combined administration group in the non-response/hyperprogression model, respectively;
  • *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001 indicate that the difference between the two groups is statistically significant; ns indicates that there is no statistically significant difference between the two groups.
  • the applicant used the tumor-bearing mouse model with no response and hyperprogression to immune checkpoint inhibitors as the research object, and gave immune checkpoint inhibitors, baicalin (HQG), and HQG combined with immune checkpoint inhibitors (HQG and immune checkpoint inhibitors were given at the same time; or HQG was given first, and then immune checkpoint inhibitors were given) to study the changes in mouse tumor volume and tumor weight.
  • the results showed that the tumor volume of the immune checkpoint inhibitor alone treatment group was significantly larger than that of the control group on the 3rd and 6th days of administration, indicating that the hyperprogression phenomenon occurred. From the 9th day to the end of the experiment, the tumor volume was compared with the control group.
  • the applicant also studied the number of Foxp3 + Treg cells in the above-mentioned tumor-bearing mouse model.
  • the results showed that the number of Foxp3 + Treg cells in the control group of tumor-bearing mice that did not respond to immune checkpoint inhibitors increased compared with the control group of tumor-bearing mice that responded to immune checkpoint inhibitors, indicating that the phenomenon of no response and hyperprogression to immune checkpoint inhibitors may be related to the immunosuppressive effect of the increase in Foxp3 + Treg cells in the tumor.
  • HQG alone did not downregulate the number of Foxp3 + Treg cells in the tumor, but the number of Foxp3 + Treg cells in the HQG combined with immune checkpoint inhibitor group was significantly downregulated after treatment. It shows that HQG combined with immune checkpoint inhibitors can reduce the number of Foxp3 + Treg cells in the tumor, promote the immune checkpoint inhibitors to exert immunoregulatory effects, and improve the phenomenon of no response and hyperprogression of tumor-bearing mice to immune checkpoint inhibitors.
  • Table 1 Main materials and sources used in the examples of this application
  • ABX broad-spectrum antibiotics
  • Preparation of fecal suspension of clinical patients Take the pre-frozen fecal samples of clinical non-small cell lung cancer PD-1mAb responding patients (R) and non-responding patients (NR) and resuspend them in sterile sodium chloride solution (0.9%). The dilution ratio is 1g of feces diluted into 10mL volume and the feces is stirred until there are no obvious large particles. Filter with a 200-mesh sterile mesh filter to remove large particles in the feces, and collect the filtrate in a sterile centrifuge tube. Vortex for 5 minutes to obtain a resuspension. Centrifuge at 600 ⁇ g for 5 minutes to remove insoluble matter. Immediately package the fecal suspension obtained by centrifugation in a clean workbench and freeze at -20°C for later use.
  • HQG solution for intragastric administration Weigh an appropriate amount of HQG, prepare a 20 mg/mL suspension with physiological saline, and store at -20°C for later use.
  • Preparation of PD-1 mAb solution Take an appropriate amount of PD-1 mAb and prepare it into a 2.5 mg/mL suspension with normal saline for immediate use.
  • Example 2 Establishment of a PD-1 mAb-unresponsive Lewis lung cancer (LLC) tumor-bearing mouse model
  • Pseudo-germ-free mouse model Mice were treated with broad-spectrum antibiotics to construct a pseudo-germ-free mouse model.
  • 36 female C57BL/6J mice aged 1 week (experimental animal license number: SCXK (Beijing) 2019-0009) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. Housing conditions: SPF cleanliness, room temperature 20-22°C, humidity 60 ⁇ 5%, 12-hour light and dark alternation, free food and water. All animal protocols in this experiment were approved by the Beijing Medconna Animal Ethics Committee, and animal experimental operations were performed under the guidance of the Laboratory Animal Protection Association.
  • mice After one week of adaptive feeding, the mice were gavaged with 100 ⁇ L ABX every day for 3 consecutive days.
  • Fecal microbiota transplantation LLC tumor-bearing mouse model Fecal microbiota transplantation LLC tumor-bearing mouse model:
  • Fecal microbiota transplantation Two days after ABX was discontinued, the frozen fecal suspensions of clinical non-small cell lung cancer PD-1mAb responders (R) and non-responders (NR) were taken out (1 g/10 mL), heated in a 37°C water bath, and gavaged into ABX-treated mice, 100 ⁇ L per mouse, for 7 consecutive days.
  • R clinical non-small cell lung cancer PD-1mAb responders
  • NR non-responders
  • R-FMT is fecal microbiota transplantation into LLC tumor-bearing mice that respond to PD-1 mAb
  • NR-FMT is fecal microbiota transplantation into LLC tumor-bearing mice that do not respond to PD-1 mAb.
  • Example 3 Therapeutic effect of HQG on LLC tumor-bearing mice without response to PD-1 mAb
  • the R-FMT group mice were divided into the R-Control group and the R-PD-1mAb group, and the NR-FMT group was divided into the NR-Control group and the NR-PD-1mAb group, the NR-HQG group and the NR-HQG+PD-1mAb group, with 6 mice in each group.
  • the administration method and dosage are as follows:
  • R-Control and NR-Control groups Normal saline was gavaged daily until the end of the experiment (200 ⁇ L/d), and normal saline was injected intraperitoneally once every 3 days (100 ⁇ L/time), for a total of 5 injections.
  • R-PD-1mAb and NR-PD-1mAb groups Normal saline was gavaged every day until the end of the experiment (200 ⁇ L/d), and PD-1mAb was injected intraperitoneally once every 3 days (100 ⁇ L/time), for a total of 5 injections.
  • NR-HQG group HQG was gavaged every day until the end of the experiment (200 ⁇ L/d), and normal saline was injected intraperitoneally once every 3 days (100 ⁇ L/time), for a total of 5 injections.
  • NR-HQG+PD-1mAb group HQG was gavaged every day until the end of the experiment (200 ⁇ L/d), and PD-1mAb was injected intraperitoneally once every 3 days (100 ⁇ L/time), for a total of 5 injections.
  • mice The body weight of mice was monitored and the tumor volume was measured every 3 days during the administration period.
  • the tumor volume and weight of the mice were not reduced after treatment with the NR-HQG group, but the NR-HQG+PD-1mAb group was able to significantly reduce the increase in tumor volume and weight in the NR-PD-1mAb-unresponsive and hyperprogressive phenomena, as shown in Figures 3 and 4.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Endocrinology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

公开了黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用，上述肿瘤对免疫检查点抑制剂无应答/超进展是由于Foxp3 +Treg细胞过多导致，所针对的肿瘤为黑素瘤、非小细胞肺癌、肾细胞癌、肝癌、结直肠癌、尿路上皮膀胱癌和胰腺癌中的一种，所用的免疫检查点抑制剂包含PD-1抑制剂、PD-L1抑制剂、CTLA-4抑制剂中的一种或两种及以上的混合物。通过黄芩苷协同免疫检查点抑制剂实施肿瘤中Foxp3 +Treg细胞数量抑制，发挥抗肿瘤增敏作用，能够有效治疗Foxp3 +Treg细胞过多导致的多种荷瘤对免疫检查点抑制剂无应答及超进展现象，且未发现明显的不良反应，安全性好，具有广阔的应用前景。

Description

黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用 技术领域
本发明属于生物医药领域，特别是针对肿瘤的免疫检查点抑制剂的肿瘤药物制备中提出的黄芩苷应用，具体为黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用。
背景技术
免疫检查点抑制剂是临床多数肿瘤患者的一线或二线治疗方案。然而，临床观察到超过80％的肿瘤患者无法对免疫检查点抑制剂产生应答，还有15％的肿瘤患者会出现超进展现象，称为超进展性疾病(HPD)；在临床治疗中，如果发生上述两种情况，传统的处理方式通常会采用更换治疗方案等措施来避免肿瘤继续进展(The Lancet Oncology,21(10),e463–e476.)。由于治疗方案的重新选择可能会导致最佳治疗时间窗口的错失，也给患者带来身体、精神、及经济物质上的多重压力。如何在免疫检查点抑制剂产生无应答或超进展的情况下，在不改变原有治疗方案的情况下恢复患者的免疫应答，是解决上述问题的关键，也是临床治疗最为切实可行的研究方向。
研究表明，对免疫检查点抑制剂无应答或者HPD与肿瘤内高度增殖的Foxp3+Treg细胞有关，(Proceedings of the National Academy of Sciences,2019,116(20):9999-10008.)。Foxp3+Treg细胞是一类免疫抑制细胞，其过度增殖会抑制免疫检查点抑制剂的作用。因此，抑制肿瘤内Foxp3+Treg数量有可能会预防或治疗对免疫检查点抑制剂无应答及HPD的发生。
黄芩苷对免疫细胞具有调节作用，但是黄芩苷在协同免疫检查点抑制剂治疗Foxp3+Treg细胞过多引起的无应答及超进展方面的作用和功效，尚未见相关报道。本申请的技术方案恰是针对免疫检查点抑制剂治疗Foxp3+Treg细胞过多引起的无应答及超进展的缺陷展开研究，提出一种通过黄芩苷协同作用以克服上述缺陷的技术方案。
发明内容
本申请就是针对免疫检查点抑制剂治疗Foxp3+Treg细胞过多引起的无应答及超进展的缺陷提出利用黄芩苷制备用于治疗Foxp3+Treg细胞过多导致的对免疫检查点抑制剂无应答及超进展的肿瘤药物，该药物可以有效克服上述无应答/超进展的治疗缺陷。
为实现上述目的，采用以下技术方案：
本发明提供了黄芩苷在制备用于治疗Foxp3+Treg细胞增多导致对免疫检查点抑制剂无应答/超进展的肿瘤药物中的应用。
在黄芩苷的上述应用中，Foxp3+Treg细胞过多导致的对免疫检查点抑制剂无应答/超进展的肿瘤为黑素瘤、非小细胞肺癌、肾细胞癌、肝癌、结直肠癌、尿路上皮膀胱癌和胰腺癌中的至少一种。
上述应用中的免疫检查点抑制剂包含PD-1抑制剂、PD-L1抑制剂、CTLA-4中的一种或两种及以上的混合物。
利用上述应用所制备的药物中的黄芩苷质量百分含量为0.05％-99.5％，每天给药4mg/天至1500mg/天，药物采用口服或肠道给药方式。
为了方便药物的制备，可以将药学上可接受的辅料作为添加剂用于该药物中，并将药物制作为固体制剂或液体制剂的剂型。
上述药物给药时间通常选择在免疫检查点抑制剂之前给药或与所述免疫检查点抑制剂同时给药。
本发明所述的黄芩苷与免疫检查点抑制剂联合应用，通过抑制肿瘤中Foxp3+Treg细胞数量，发挥抗肿瘤增敏作用。本申请所述的黄芩苷能够协同免疫检查点抑制剂有效治疗Foxp3+Treg细胞过多导致的荷瘤小鼠对免疫检查点抑制剂无应答及超进展现象，且未发现明显的不良反应，安全性好，具有广阔的前景。
附图说明
图1是R-FMT和NR-FMT小鼠经PD-1mAb干预治疗前后的肿瘤体积；
图2是R-FMT和NR-FMT小鼠经PD-1mAb干预治疗前后的肿瘤重量；
图3是NR-FMT小鼠经PD-1mAb、HQG和HQG+PD-1mAb干预治疗前后的肿瘤体积；
图4是NR-FMT小鼠经PD-1mAb、HQG和HQG+PD-1mAb干预治疗前后的肿瘤重量；
图5是NR-FMT小鼠经PD-1mAb、HQG和HQG+PD-1mAb干预治疗前后小鼠的肿瘤内Foxp3+Treg细胞数量。
其中，图1-图5中，
HQG、PD-1mAb分别表示：黄芩苷、PD-1抑制剂；
R-Control组和NR-Control组分别表示：应答模型空白组和无应答/超进展模型空白组；
R-PD-1mAb组和NR-PD-1mAb组分别表示：应答模型PD-1抑制剂给药组和无应答/超进展模型PD-1抑制剂给药组；
NR-HQG组和NR-HQG+PD-1mAb组分别表示：无应答/超进展模型的黄芩苷给药组和黄芩苷和PD-1抑制剂抑制剂联合给药组；
*P<0.05、**P<0.01、***P<0.001、****P<0.0001表示两组间相比差异具有统计学意义；ns表示两组间相比差异无统计学意义。
具体实施方式
为了更清楚阐述本申请的黄芩苷的应用效果和作用，申请人以免疫检查点抑制剂无应答及超进展的荷瘤小鼠模型为研究对象，分别给予免疫检查点抑制剂、黄芩苷(HQG)、HQG与免疫检查点抑制剂联合治疗(同时给予HQG和免疫检查点抑制剂；或先给予HQG，再给予免疫检查点抑制剂)，研究小鼠肿瘤体积和肿瘤重量的变化。结果显示：免疫检查点抑制剂单独治疗组瘤体积在给药第3天和第6天瘤体积明显大于control组，说明出现了超进展现象，第9天开始到实验终点，瘤体积与control组相比，有增大趋势，但无显著性差异；HQG与免疫检查点抑制剂联合治疗组的小鼠肿瘤体积从第12天开始，与control组相比有降低趋势，第15天降低有显著性差异；HQG单独治疗组小鼠肿瘤的体积和重量与control组相比无显著性差异。结果表明HQG协同免疫检查点抑制剂能改善荷瘤小鼠对免疫检查点抑制剂无应答及超进展的现象，但HQG单独治疗组无效。
采用本申请的方案，申请人还研究了上述荷瘤小鼠模型中的Foxp3+Treg细胞数量，结果免疫检查点抑制剂无应答的荷瘤小鼠control组较免疫检查点抑制剂应答的荷瘤小鼠control组Foxp3+Treg细胞数量增多，说明对免疫检查点抑制剂出现无应答及超进展现象可能与瘤内Foxp3+Treg细胞的增多产生的免疫抑制作用有关。另外，HQG单独治疗后没有下调瘤内Foxp3+Treg细胞数量，但HQG联合免疫检查点抑制剂组治疗后Foxp3+Treg细胞数量出现显著性下调。说明HQG联合免疫检查点抑制剂可通过降低瘤内Foxp3+Treg细胞数量，促进免疫检查点抑制剂发挥免疫调节作用，改善荷瘤小鼠对免疫检查点抑制剂无应答及超进展的现象。
上述研究中，HQG单独治疗组和联合治疗组未出现明显的不良反应。
为验证上述结果的准确性，下面对具体实验过程中的结果和数据进行介绍和分析。为了清楚、明确将实验测试中所使用的药物和试剂予以介绍，表1给出了不同试剂的名称、货号及生产制作方信息。
表1：本申请实施例选用的主要材料及来源
实施例1：实验溶液的制备
广谱抗生素(ABX)的制备：称取硫酸新霉素、甲硝唑、氨苄西林，万古霉素适量，加生理盐水得40mg/mL的硫酸新霉素、甲硝唑、氨苄西林和20mg/mL的万古霉素。混匀，分装至15mL离心管，置于-20℃存储备用。
临床患者粪便悬液的制备：取提前冷冻的临床非小细胞肺癌PD-1mAb应答患者(R)和无应答患者(NR)粪便样品重新悬浮在无菌氯化钠溶液(0.9％)中，稀释比例均为1g粪便稀释成10mL体积并将粪便搅匀至无明显大颗粒。用200目无菌网滤筛过滤以除去粪便中的大颗粒，所得滤液收集在无菌离心管中。通过涡旋5分钟得到重悬液。600×g离心5分钟去除不溶物。立即将离心所得粪便悬液在洁净工作台内进行分装，-20℃冻存备用。
灌胃用HQG溶液的制备：称取HQG适量，用生理盐水配成20mg/mL混悬液，-20℃存储备用。
PD-1mAb溶液的制备：吸取PD-1mAb适量，用生理盐水配成2.5mg/mL混悬液，现配现用。
实施例2：PD-1mAb无应答Lewis肺癌(LLC)荷瘤小鼠模型的建立
伪无菌小鼠造模：采用广谱抗生素处理小鼠以构建伪无菌小鼠模型。5 周龄的36只雌性C57BL/6J小鼠(实验动物许可证号：SCXK(京)2019-0009)购自北京维通利华实验动物技术有限公司。饲养条件：SPF级洁净度，室温20～22℃，湿度60±5％，12小时明暗交替，自由饮食饮水。本实验中所有动物方案经北京迈德康纳动物伦理委员会批准，并在实验动物保护协会规定的指导下进行动物实验操作。
小鼠适应性饲养一周后，每天灌胃100μL ABX，连续灌胃3天。
粪菌移植LLC荷瘤小鼠造模：
粪菌移植：ABX停用2天后，将冻存好的临床非小细胞肺癌PD-1mAb应答患者(R)和无应答患者(NR)粪便悬液取出(1g/10mL)，37℃水浴加热，ABX处理的小鼠进行灌胃，每只100μL，连续灌胃7天。
LLC细胞皮下接种：将大约1×107cells/mL LLC细胞分别接种于粪菌移植小鼠右侧腋下,接种量为0.2mL/小鼠。游标卡尺测量肿瘤的最长径及最短径，体积＝1/2长径×短径2。
R-FMT为粪菌移植PD-1mAb应答LLC荷瘤小鼠；NR-FMT为粪菌移植PD-1mAb无应答LLC荷瘤小鼠。
实施例3：HQG对PD-1mAb无应答LLC荷瘤小鼠的治疗作用
小鼠肿瘤长到20-50mm3后，将R-FMT组小鼠分为R-Control组和R-PD-1mAb组，NR-FMT组分为NR-Control组和NR-PD-1mAb组，NR-HQG组和NR-HQG+PD-1mAb组，每组6只小鼠。给药方式及给药量如下：
R-Control和NR-Control组：每天分别生理盐水灌胃至实验结束(200μL/d)，每3天腹腔注射一次生理盐水(100μL/次)，共注射5次。
R-PD-1mAb和NR-PD-1mAb组：每天分别生理盐水灌胃至实验结束(200μL/d)，每3天腹腔注射一次PD-1mAb(100μL/次)，共注射5次。
NR-HQG组：每天进行HQG灌胃至实验结束(200μL/d)，每3天腹腔注射一次生理盐水(100μL/次)，共注射5次。
NR-HQG+PD-1mAb组：每天进行HQG灌胃至实验结束(200μL/d)，每3天腹腔注射一次PD-1mAb(100μL/次)，共注射5次。
给药期间每3天监测小鼠体重，测量瘤体积。
结果如图1-2，NR-FMT和R-FMT处理后，NR-Control组荷瘤小鼠瘤体积和瘤重大于R-Control组，经PD-1mAb干预治疗后，R-PD-1mAb组与R-Control组相比，瘤体积和瘤重降低，说明小鼠出现免疫应答；而NR-PD-1组小鼠瘤体积和瘤重大于NR-Control组，说明PD-1mAb干预NR-FMT组荷瘤小鼠后出现无应答及超进展现象。
与NR-Control组相比，NR-HQG组治疗后，没有降低小鼠瘤体积和瘤重，但NR-HQG+PD-1mAb组能够显著降低NR-PD-1mAb无应答及超进展现象出现的瘤体积和瘤重增大，如图3、图4。
实施例4：流式检测肿瘤组织Foxp3+Treg细胞
收集R-Control组和R-PD-1mAb组，NR-Control组和NR-PD-1mAb组，NR-HQG组和NR-HQG+PD-1mAb组小鼠的新鲜EDTA抗凝血0.2mL，每组5只，与PBS以1:1混合，取一洁净离心管加入1mL小鼠淋巴细胞分离液，将血液-PBS混合液缓慢加于1mL淋巴细胞分离液液面上，以800g，30min，升速7降速3离心，收集中间淋巴细胞层。肿瘤组织加入3mL PBS轻轻研磨后经200目滤网过滤至离心管中，取一洁净离心管加入3mL小鼠淋巴细胞分离液，将2mL肿瘤单细胞悬液缓慢加于3mL淋巴细胞分离液液面上，以800g，30min，升速7降速3离心，收集中间淋巴细胞层。加入2mL PBS洗涤细胞，500g，离心5min，弃上清。(若有明显红细胞可加入2mL红细胞裂解液洗涤细胞，500g，离心5min，弃上清，再用2mL PBS洗涤细胞)。用RPMI 1640完全培养基重悬细胞，取2×106个细胞转移至尖底96孔板中。每孔加入Cell Activation Cocktail(with Brefeldin A)0.2uL(1:1000)，37℃，5％CO2孵育5h。孵育结束后，收集细胞，离心弃上清，加入抗体CD4、CD25各1uL，室温避光孵育15min。加入0.2mL细胞固定液，室温避光固定30min，500g离心5min弃上清留细胞沉淀。加入0.2mL 1×细胞破膜液，离心弃上清。再次加入0.2mL1×细胞破膜液，离心弃上清。样本加入Foxp3抗体1uL，室温避光孵育。加入0.2mL细胞破膜液，离心弃上清。用0.1mL cell staining buffer重悬细胞，上机检测。
结果如图5所示，与R-Control组相比，NR-Control组肿瘤中Foxp3+Treg细胞有明显上调，PD-1mAb干预治疗前后，两组Foxp3+Treg细胞数量无显著差异。说明肿瘤出现超进展现象可能与瘤内Foxp3+Treg细胞的增多产生的免疫抑制作用有关。另外，HQG单独治疗后没有下调瘤内Foxp3+Treg细胞数量，但HQG联合PD-1mAb组Foxp3+Treg细胞数量出现显著性下调。说明HQG联合PD-1mAb可通过降低瘤内Foxp3+Treg细胞数量，减少其引起的免疫抑制作用，进而促进PD-1mAb发挥免疫调节作用。
本发明中的具体实施例仅是对本发明的解释，其并不对本发明限制，本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改，但只要在本发明的权利要求范围内都受到专利法的保护。

Claims (9)

  1. 黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用，其特征在于，所述肿瘤对免疫检查点抑制剂无应答/超进展是Foxp3+Treg细胞过多导致。
  2. 根据权利要求1所述的黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用，其特征在于，所述肿瘤为黑素瘤、非小细胞肺癌、肾细胞癌、肝癌、结直肠癌、尿路上皮膀胱癌和胰腺癌中的至少一种。
  3. 根据权利要求1所述的黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用，其特征在于，所述免疫检查点抑制剂包含PD-1抑制剂、PD-L1抑制剂、CTLA-4抑制剂中的一种或两种及以上的混合物。
  4. 根据权利要求1所述的黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用，其特征在于，所述药物给药量为4mg/天～1500mg/天。
  5. 根据权利要求1所述的黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用，其特征在于，黄芩苷在所述药物中的质量百分含量为0.05％-99.5％。
  6. 根据权利要求1所述的黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用，其特征在于，所述药物采用口服或肠道给药。
  7. 根据权利要求6所述的黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用，其特征在于，所述药物中还包括药学上可接受的辅料。
  8. 根据权利要求1所述的黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用，其特征在于，所述药物为固体制剂或液体制剂。
  9. 根据权利要求1或3所述的黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用，其特征在于，所述药物在免疫检查点抑制剂之前或同时给药。
PCT/CN2023/099383 2022-12-02 2023-06-09 黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用 WO2024113752A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP23772397.8A EP4400109A1 (en) 2022-12-02 2023-06-09 Use of baicalin in preparing medicament for treating tumor unresponsive/hyperprogressive to immune checkpoint inhibitor
US18/373,980 US12029747B2 (en) 2022-12-02 2023-09-28 Use of baicalin in preparation of drug for treating tumor irresponsive to immune checkpoint inhibitors (ICIs)/undergoing hyperprogression

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211537380.3 2022-12-02
CN202211537380.3A CN115624562B (zh) 2022-12-02 2022-12-02 黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US18/373,980 Continuation US12029747B2 (en) 2022-12-02 2023-09-28 Use of baicalin in preparation of drug for treating tumor irresponsive to immune checkpoint inhibitors (ICIs)/undergoing hyperprogression

Publications (1)

Publication Number Publication Date
WO2024113752A1 true WO2024113752A1 (zh) 2024-06-06

Family

ID=84910319

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/099383 WO2024113752A1 (zh) 2022-12-02 2023-06-09 黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用

Country Status (2)

Country Link
CN (1) CN115624562B (zh)
WO (1) WO2024113752A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115624562B (zh) * 2022-12-02 2023-05-09 首都医科大学附属北京友谊医院 黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190192602A1 (en) * 2016-05-23 2019-06-27 Yale University Improved Therapeutic Index of Anti-Immune Checkpoint Inhibitors Using Combination Therapy Comprising A PHY906 Extract, A Scutellaria baicalensis Georgi (S) Extract or A Compound From Such Extracts
CN111000860A (zh) * 2019-10-28 2020-04-14 江苏省中医院 黄芩苷联合白术多糖上调Treg细胞治疗免疫性复发性流产的用途
CN114642732A (zh) * 2020-12-21 2022-06-21 苏州普瑞森生物科技有限公司 一种组合物及其在制备治疗肿瘤的药物中的应用
CN114870009A (zh) * 2022-06-10 2022-08-09 菏泽学院 一种抗肿瘤联合组合物及其应用和抗肿瘤药物
CN115624562A (zh) * 2022-12-02 2023-01-20 首都医科大学附属北京友谊医院 黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101491533B (zh) * 2008-12-18 2011-05-04 复旦大学附属中山医院 黄芩苷在制药中的应用
CN109953996A (zh) * 2019-03-05 2019-07-02 澳门大学 野黄芩苷在预防或治疗疾病的药物及调节剂中的应用、药物和调节剂
CN112138024A (zh) * 2019-06-26 2020-12-29 里弗斯Pah有限责任公司 治疗严重形式的肺动脉高压的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190192602A1 (en) * 2016-05-23 2019-06-27 Yale University Improved Therapeutic Index of Anti-Immune Checkpoint Inhibitors Using Combination Therapy Comprising A PHY906 Extract, A Scutellaria baicalensis Georgi (S) Extract or A Compound From Such Extracts
CN111000860A (zh) * 2019-10-28 2020-04-14 江苏省中医院 黄芩苷联合白术多糖上调Treg细胞治疗免疫性复发性流产的用途
CN114642732A (zh) * 2020-12-21 2022-06-21 苏州普瑞森生物科技有限公司 一种组合物及其在制备治疗肿瘤的药物中的应用
CN114870009A (zh) * 2022-06-10 2022-08-09 菏泽学院 一种抗肿瘤联合组合物及其应用和抗肿瘤药物
CN115624562A (zh) * 2022-12-02 2023-01-20 首都医科大学附属北京友谊医院 黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KAMADA TAKAHIRO, TOGASHI YOSUKE, TAY CHRISTOPHER, HA DANBEE, SASAKI AKINORI, NAKAMURA YOSHIAKI, SATO EIICHI, FUKUOKA SHOTA, TADA Y: "PD-1 + regulatory T cells amplified by PD-1 blockade promote hyperprogression of cancer", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 116, no. 20, 14 May 2019 (2019-05-14), pages 9999 - 10008, XP093108590, ISSN: 0027-8424, DOI: 10.1073/pnas.1822001116 *
KE, M. ET AL.: "Baicalein and baicalin promote antitumor immunity by suppressing PD-L1 expression in hepatocellular carcinoma cells", INTERNATIONAL IMMUNOPHARMACOLOGY, vol. 75, 19 August 2019 (2019-08-19), pages 1 - 10, XP085816198, DOI: 10.1016/j.intimp.2019.105824 *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 116, no. 20, 2019, pages 9999 - 10008

Also Published As

Publication number Publication date
CN115624562B (zh) 2023-05-09
CN115624562A (zh) 2023-01-20

Similar Documents

Publication Publication Date Title
Sato et al. Treatment of neuromyelitis optica: an evidence based review
Chen et al. The role of Th17 cells in inflammatory bowel disease and the research progress
CN112203669A (zh) 微生物组相关的免疫疗法
Borghi et al. Targeting the aryl hydrocarbon receptor with indole-3-aldehyde protects from vulvovaginal candidiasis via the IL-22-IL-18 cross-talk
WO2024113752A1 (zh) 黄芩苷在制备治疗对免疫检查点抑制剂无应答/超进展的肿瘤的药物中的应用
Pastille et al. Inhibition of TLR4 signaling impedes tumor growth in colitis-associated colon cancer
WO2023134204A1 (zh) 脆弱拟杆菌荚膜多糖a与pd-1及pd-l1抗体联合治疗呼吸系统肿瘤的应用
Liu et al. Propofol promotes activity and tumor-killing ability of natural killer cells in peripheral blood of patients with colon cancer
EP2969010A1 (en) Use of levocetirizine and montelukast in the treatment of autoimmune disorders
Peng et al. Effects of rhein on intestinal epithelial tight junction in IgA nephropathy
WO2023134210A1 (zh) 脆弱拟杆菌荚膜多糖a与免疫检查点抑制剂的新应用
Wang et al. Arsenic trioxide inhibits lung metastasis of mouse colon cancer via reducing the infiltration of regulatory T cells
CN114224889A (zh) 西奥罗尼联合免疫检查点抑制剂在抗肿瘤治疗中的应用
Li et al. Alternol triggers immunogenic cell death via reactive oxygen species generation
WO2023134201A1 (zh) 脆弱拟杆菌与免疫检查点抑制剂联用在治疗消化系统肿瘤中的应用
JP2024009886A (ja) 好中球調節物質と免疫チェックポイントの調節物質の組み合わせを使用したがん処置
WO2023134202A1 (zh) 脆弱拟杆菌荚膜多糖a与免疫检查点抑制剂联用在制备治疗消化系统肿瘤药物中的应用
Zhang et al. Inhibition of galectin‐3 augments the antitumor efficacy of PD‐L1 blockade in non‐small‐cell lung cancer
Matasar et al. Mosunetuzumab Safety Profile in Patients With Relapsed/Refractory B-cell Non-Hodgkin Lymphoma: Clinical Management Experience From a Pivotal Phase I/II Trial
CN117045801A (zh) m6A RNA甲基化酶抑制剂与免疫检查点抑制剂联合治疗肿瘤
Miyata et al. Rebamipide enemas–new effective treatment for patients with corticosteroid dependent or resistant ulcerative colitis
US12029747B2 (en) Use of baicalin in preparation of drug for treating tumor irresponsive to immune checkpoint inhibitors (ICIs)/undergoing hyperprogression
US20240180943A1 (en) USE OF BAICALIN IN PREPARATION OF DRUG FOR TREATING TUMOR IRRESPONSIVE TO IMMUNE CHECKPOINT INHIBITORS (ICIs)/UNDERGOING HYPERPROGRESSION
CN113769096B (zh) 6-磷酸葡萄糖脱氢酶抑制剂的医药用途
Shypulin et al. Study of the Exogenous Peptide Effect on the TGF-β1 Expression-A Risk Factor for the Hepatocellular Carcinoma Recurrence

Legal Events

Date Code Title Description
ENP Entry into the national phase

Ref document number: 2023772397

Country of ref document: EP

Effective date: 20230927