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WO2019034042A1 - 用于预测吉西他滨药物敏感性的生物标记物及其用途 - Google Patents

用于预测吉西他滨药物敏感性的生物标记物及其用途 Download PDF

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WO2019034042A1
WO2019034042A1 PCT/CN2018/100371 CN2018100371W WO2019034042A1 WO 2019034042 A1 WO2019034042 A1 WO 2019034042A1 CN 2018100371 W CN2018100371 W CN 2018100371W WO 2019034042 A1 WO2019034042 A1 WO 2019034042A1
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acid
cancer
gemcitabine
akr1c1
pharmaceutically acceptable
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杨岚
王凤
郭殿武
单佳祺
冯飞玉
郭文广
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Hangzhou Yuanchang Medical Sci-Tech Co Ltd
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Hangzhou Yuanchang Medical Sci-Tech Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the present invention relates to the field of molecular biology and medicine, and in particular to biomarkers for predicting drug sensitivity of gemcitabine and uses thereof.
  • Gemcitabine is a commonly used anti-tumor chemotherapy drug in clinical practice.
  • Gemcitabine Hydrochloride is indicated for the treatment of inoperable advanced or metastatic pancreatic cancer and for the treatment of locally advanced or metastatic non-small cell lung cancer, in combination with paclitaxel, for the treatment of relapse after unassisted/neoadjuvant chemotherapy, unresectable, local recurrence or Metastatic breast cancer.
  • gemcitabine Even in the current emergence of targeted drugs and new immunotherapeutics, gemcitabine still has a wide range of clinical applications, especially in the field of pancreatic cancer, gemcitabine is the standard of treatment.
  • different patients have different sensitivity or effectiveness to gemcitabine, and there are primary and acquired drug resistance problems. Therefore, the search for effective gemcitabine drug sensitivity markers to guide the use of drugs, not only can avoid the patient's treatment due to improper drug selection delay, but also can monitor the drug treatment effect during the treatment process, to achieve rational drug use and individualized treatment purposes.
  • gemcitabine resistance is mainly related to its uptake and transport process disorders, intracytoplasmic activation, enzyme breakdown and DNA repair.
  • the research includes hENT1 (human balanced nucleoside transporter 1) and RRM1 (ribonucleotide reductase M1). ), RRM2 (ribonucleotide reductase M2), dCK (deoxycytidine kinase) and other genes or proteins.
  • the present invention seeks to find new and more accurate gemcitabine sensitivity Sexual predictive indicators.
  • the aldol.keto reductase (AKR) superfamily has 15 families, of which 15 are human AKR members. Most AKR members can catalyze a simple redox reaction with nicotinamide adenine dinucleotide phosphate NADP as a cofactor. They have a wide range of substrates, including sugars, fatty aldehydes, steroid hormones, prostaglandins and carcinogens. Therefore, members of the AKR superfamily may be enzymes that play important roles in life activities such as hormone synthesis, drug metabolism, inflammatory reactions, and carcinogen detoxification. Studies have shown that human AKR superfamily members are associated with a variety of tumor development, and their activity is closely related to tumor progression and therapeutic effects.
  • AKRlB10 is an early diagnostic marker for smoking-related non-small cell lung cancer, especially lung squamous cell carcinoma, and the role of AKR1C1 ⁇ 3 in the tolerance of multiple chemotherapeutic drugs in lung cancer makes it an important predictor of prognosis and also for the improvement of lung cancer drugs.
  • Sensitivity provides an effective target for action.
  • the present invention firstly discovered that the AKR1C1, AKR1C2, and AKR1C3 families of the aldosterone reductase (AKR) family members are associated with gemcitabine resistance.
  • the present invention investigated the correlation between gemcitabine drug sensitivity and AKR1C1 to C3 expression in 38 nude mouse tumor xenograft models. The results showed that among the 15 models with high expression of AKR1C1 ⁇ C3, 13 models showed poor efficacy and insensitivity of gemcitabine. Among the 23 models with low expression of AKR1C1 ⁇ C3, 19 models showed the efficacy of gemcitabine. Good, performance sensitivity.
  • biomarkers AKR1C1, AKR1C2, AKR1C3 for predicting drug sensitivity in the treatment of cancer with gemcitabine or a pharmaceutically acceptable salt thereof, and uses thereof.
  • the invention provides the use of AKR1C1, AKR1C2 or AKR1C3 as a biomarker for predicting drug sensitivity in the treatment of cancer with gemcitabine or a pharmaceutically acceptable salt thereof.
  • the pharmaceutically acceptable salt comprises an acid addition salt of gemcitabine with an acid, for example, an acid addition salt of gemcitabine with an acid selected from the group consisting of inorganic acids such as hydrochloric acid, hydrofluoric acid, Hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid or nitric acid; organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid , lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, Niacin, pamoic acid, pectic acid, persulf
  • inorganic acids such as
  • the pharmaceutically acceptable salt is gemcitabine hydrochloride.
  • the cancer is colon cancer, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, melanoma, liver cancer, breast cancer, cervical cancer.
  • the lung cancer includes non-small cell lung cancer including squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, large cell carcinoma, and the like, and the non-small cell lung cancer is preferably lung squamous cell carcinoma.
  • the present invention provides a method of predicting drug sensitivity in the treatment of cancer comprising a drug comprising gemcitabine or a pharmaceutically acceptable salt thereof.
  • the method comprises applying AKR1C1, AKR1C2 or AKR1C3 as a biomarker.
  • the method can also include detecting the amount of expression of AKR1C1, AKR1C2 or AKR1C3 in the patient or in an ex vivo sample.
  • a therapeutically effective amount of gemcitabine or a pharmaceutically acceptable salt thereof can be administered as a single agent or in combination with one or more other agents, wherein the combination does not cause unacceptable Adverse reactions.
  • Suitable active substances in the combination include conventional drugs suitable for chemotherapy or targeted therapy, such as alkylating agents, platinum compounds, DNA modifiers, topoisomerase inhibitors, microtubule modifiers, antimetabolites , anticancer antibiotics, hormones/antagonists, aromatase inhibitors, small molecule kinase inhibitors, photosensitizers, antibodies, cytokines, drug conjugates, vaccines or other drugs.
  • the pharmaceutically acceptable carrier can be a carrier that is relatively non-toxic and harmless to the patient at a concentration consistent with the effective activity of the active ingredient, such that any side effects caused by the carrier do not destroy the The beneficial effects of the active ingredients.
  • the pharmaceutically effective amount of a compound, or a pharmaceutically acceptable salt thereof is preferably an amount that produces a result or affects the particular condition being treated.
  • Gemcitabine or a pharmaceutically acceptable salt thereof can be administered in the following manner together with a pharmaceutically acceptable carrier well known in the art using any effective conventional dosage unit form including immediate release, sustained release and timed release formulations. : oral, parenteral, topical, nasal, ocular, sublingual, rectal, vaginal administration, etc.
  • the present invention provides a kit for predicting drug sensitivity in the treatment of cancer comprising gemcitabine or a pharmaceutically acceptable salt thereof, the kit comprising:
  • An agent that detects at least one of the expression levels of AKR1C1, AKR1C2, and AKR1C3 in an individual sample is an agent that detects at least one of the expression levels of AKR1C1, AKR1C2, and AKR1C3 in an individual sample.
  • the individual sample is selected from the group consisting of human cancer tissues, and specifically, may be selected from the group consisting of colon cancer, lung cancer, stomach cancer, pancreatic cancer, ovarian cancer, melanoma, liver cancer, breast cancer, and cervical cancer.
  • the present invention has the beneficial effects of using the aldosterone reductase (AKR) family member AKR1C1, AKR1C2 or AKR1C3 as a biomarker for predicting the drug sensitivity of gemcitabine in the treatment of cancer, thereby guiding clinical drug use, not only avoiding drug selection
  • the blindness avoids delaying the patient's condition due to improper drug selection, and can also monitor the therapeutic effect of the drug during the treatment process, and realize the rationality of the drug and the purpose of individualized treatment.
  • AKR1C1-C3 in clinical cancer cases further validates the high correlation between AKR1C1-C3 expression and gemcitabine efficacy, indicating the rationality, feasibility and height of using AKR1C1, AKR1C2 or AKR1C3 to predict the efficacy of gemcitabine in the treatment of cancer. Effectiveness.
  • Figure 1 shows the expression levels of AKR1C1 to C3 in different cells. GAPDH or ⁇ -tubulin is used as an internal reference. The higher the gray value of the strip, the higher the expression level of the corresponding protein.
  • Figure 9 Antitumor effect of gemcitabine in a MDA-MB-435 melanoma xenograft model.
  • a solid support such as a nitrocellulose membrane, a difluorinated resin membrane, etc.
  • the polypeptide acts as an antigen, and the corresponding antibody recognizes and binds to the antibody, and then reacts with the enzyme or the isotope-labeled secondary antibody, and develops the color of the substrate or autoradiography to detect the expression amount of the target protein.
  • AKR1C1 ⁇ C3 protein The expression of AKR1C1 ⁇ C3 protein in different cells was examined.
  • Cells human colon cancer cells CW-2, LoVo, HCT116, SW620, COLO 205, HT-29, RKO, DLD-1, NCI-H716, SW1116, SW948; human lung cancer cells NCI-H292, A549, NCI-H460, NCI-H1650, HCC827, NCI-H1299, NCI-H1975, SK-MES-1, NCI-H1437, NCI-H1944; human gastric cancer cells BGC-823, HGC-27, SGC-7901, KATO III, NCI-N87, SNU-1; human pancreatic cancer cells AsPC-1, BxPC-3, PANC-1, Capan-2, CFPAC-1, MIA PaCa-2, HPAF-II; human breast cancer cells BT-474, SK-BR-3 , MCF-7, T-47D, MDA-MB-231, MDA-MB-468; human ovarian cancer cell line SK-OV-3, NIH: OVCAR-3
  • AKR1C1 mouse monoclonal antibody (MAB6529) was purchased from R&D Systems, AKR1C2 rabbit polyclonal antibody (13035) from Cell Signaling Technology, AKR1C3 mouse monoclonal antibody (MAB7678) from R&D, GAPDH rabbit monoclonal antibody (2118) Purchased from Cell Signaling Technology, HRP- ⁇ -tubulin mouse monoclonal antibody (ab012) was purchased from Lianke Bio.
  • HRP-labeled goat anti-rabbit IgG and HRP-labeled horse anti-mouse IgG were purchased from Cell Signaling Technology.
  • the cells were cultured in a 25 cm 2 flask, and cells were collected at 80-90% coverage, and 200-300 ⁇ l of RIPA lysate (50 mM Tris ⁇ HCl, 150 mM NaCl, 1% NP-40, 1% SDS, was added according to the number of cells. 1 ⁇ protease inhibitor Cocktail), vortexed and placed on ice for 30 min. After centrifugation at 14,000 rpm for 10 min at 4 ° C, the supernatant was taken and quantified using a BSA protein quantitative assay kit. Add 5 ⁇ loading buffer and bring to a boil.
  • RIPA lysate 50 mM Tris ⁇ HCl, 150 mM NaCl, 1% NP-40, 1% SDS
  • AKR1C1 ⁇ C3 were highly expressed in COLO 205 cells; AKR1C1 and AKR1C2 were moderately high expression in NCI-H716 cells, AKR1C3 was highly expressed; and three proteins in HCT 116, RKO and DLD-1 cells were observed. Low expression; AKR1C1 and AKR1C2 were underexpressed in CW-2, LoVo, SW620, HT-29, SW1116 and SW948 cells, and AKR1C3 was moderately or highly expressed.
  • AKR1C1 ⁇ C3 were highly expressed in A549, NCI-H460, SK-MES-1, NCI-H1437 and NCI-H1944 cells; NCI-H292, NCI-H1650, NCI-H1299 and NCI-H1975 cells AKR1C1 ⁇ C3 were all expressed low; in HCC827 cells, AKR1C1 ⁇ C3 were all expressed low.
  • AKR1C1 ⁇ C3 was highly expressed in SGC-7901 cells; AKR1C1 ⁇ C3 was down-regulated in HGC-27 and SNU-1; AKR1C1 was moderately expressed in BGC-823 cells, low to moderate expression of AKR1C2, and high expression of AKR1C3.
  • AKR1C1 and AKR1C2 proteins were underexpressed and AKR1C3 was moderately expressed.
  • AKR1C1 ⁇ C3 were highly expressed in AsPC-1 and BxPC-3 cells; AKR1C1 ⁇ C3 were down-regulated in PANC-1 and Capan-2; low to moderate expression of AKR1C1 and AKR1C2 in CFPAC-1 cells AKR1C3 is highly expressed; in MIA PaCa-2 cells, AKR1C1 is moderately expressed, AKR1C2 and AKR1C3 are underexpressed; in HPAF-II cells, AKR1C1 is underexpressed, AKR1C2 is low to moderately expressed, and AKR1C3 is highly expressed.
  • AKR1C1 ⁇ C3 were down-regulated in BT-474, MCF-7, T-47D, MDA-MB-231 and MCF7 cells; AKR1C1 and AKR1C2 were underexpressed in SK-BR-3 cells, and AKR1C3 was Moderate expression; AKR1C1 and AKR1C2 are highly expressed in MDA-MB-468 cells, and AKR1C3 is moderately expressed.
  • AKR1C1 ⁇ C3 were all expressed in NIH:OVCAR-3 and A2780 cells; AKR1C1 ⁇ C3 were highly expressed in SK-OV-3 cells.
  • AKR1C1 ⁇ C3 were down-regulated in BEL-7402 cells; AKR1C1 and AKR1C2 were low to moderately expressed in Hep G2 cells, and AKR1C3 was highly expressed.
  • AKR1C1 and AKR1C2 were both lowly expressed and AKR1C3 was highly expressed.
  • Gemcitabine hydrochloride was purchased from Meilun Bio, matrigel was purchased from Corning, USA (Cat. No. 354262), and BALB/c nude mice were purchased from Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.
  • tumors were grown to 100-200 mm3, they were divided into two groups, namely, control group and gemcitabine-administered group, with 6 animals in each group.
  • Gemcitabine hydrochloride was administered at a dose of 120 mg/kg, administered intraperitoneally, on the first and fourth days of each week.
  • V0 is the measured tumor volume at the time of sub-cage administration (ie, d0)
  • Vt is the tumor volume at each measurement.
  • T/C% TRTV/CRTV*100%
  • TRTV average RTV of the treatment group
  • CRTV mean RTV of the vehicle control group.
  • Gemcitabine showed differences in its efficacy in different xenograft models.
  • the anti-tumor effect of gemcitabine in different nude mice xenograft models was significantly different.
  • gemcitabine significantly inhibited the growth of HCT-116, HT-29, CW-2, LoVo, RKO, and DLD-1 xenografts, T/C% ⁇ 20 %;
  • the inhibitory effect of gemcitabine was poor, T/C%>40%; in the NCI-H716 model, the inhibitory effect of gemcitabine was intermediate, and the T/C% was about 38%. .
  • gemcitabine has a significant inhibitory effect on NCI-H1650, NCI-H1975 and NCI-H1299 xenografts in nude mice, T/C ⁇ 20%; whereas for A549, NCI- H460, NCI-H1437 and NCI-H1944 were poorly inhibited (see Figure 3) with T/C > 40%.
  • gemcitabine has a significant inhibitory effect on the growth of NCI-N87, HGC-27 and SNU-1 xenografts, T/C ⁇ 20%; but inhibition of BGC-823 and SGC-7901 xenografts Poor effect, T / C > 50%.
  • gemcitabine has a significant inhibitory effect on Capan-2, PANC-1, and CFPAC-1 xenografts, T/C ⁇ 20%; inhibition of BxPC-3 and AsPC-1 xenografts Poor, T/C>40%.
  • gemcitabine has a poor inhibitory effect on HeLa xenograft tumors, with a T/C >50%.
  • Gemcitabine also showed strong antitumor activity in the MDA-MB-435 melanoma model (see Figure 9) with T/C ⁇ 20%.
  • AKR1C1 to C3 can be used as biomarkers for predicting drug sensitivity in the treatment of cancer with gemcitabine or a pharmaceutically acceptable salt thereof.
  • AKR1C1 rabbit polyclonal antibody (ab192785) was purchased from abcam
  • AKR1C2 antibody (AM05295PU-N) was purchased from Origene
  • AKR1C3 antibody (ab49680) was purchased from abcam
  • BOND III automatic dyeing machine was purchased from Leica (Germany).
  • the paraffin sample was sliced to a thickness of about 4 ⁇ m. After drying in an oven, it was placed in a BOND III automatic dyeing machine for immunohistochemistry.
  • the primary antibody was diluted 1:200, the incubation time was 15 min, and the BOND Polymer Refine incubation time was 8 min.
  • the expression level of AKR1C1 ⁇ C3 in different samples was determined by scoring the tissue sections under the light microscope.
  • AKR1C1 ⁇ C3 The expression of AKR1C1 ⁇ C3 in lung squamous cell carcinoma is correlated with the efficacy of gemcitabine (Table 1).

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Abstract

本发明提供了AKR1C1、AKR1C2或AKR1C3作为预测吉西他滨或其药学上可接受的盐治疗癌症时的药物敏感性的生物标记物的应用。所述癌症为结肠癌、肺癌、胃癌、胰腺癌、卵巢癌、黑色素瘤、肝癌、乳腺癌、宫颈癌。利用本发明所述技术方案来指导临床用药，不但可以避免药物选择的盲目性，避免由于药物选择不当而延误病人病情，而且还能在治疗过程中监控药物治疗效果，实现用药的合理性和个体化治疗的目的。

Description

用于预测吉西他滨药物敏感性的生物标记物及其用途 技术领域
本发明涉及分子生物学及医学领域，具体涉及用于预测吉西他滨药物敏感性的生物标记物及其用途。
背景技术
吉西他滨是目前临床上常用的抗肿瘤化疗药物。盐酸吉西他滨适用于治疗不能手术的晚期或转移性胰腺癌及治疗局部进展性或转移性非小细胞肺癌，与紫杉醇联合，适用于治疗经辅助/新辅助化疗后复发，不能切除的、局部复发或转移性乳腺癌。即使在目前靶向药物和新型免疫治疗药物不断涌现的情况下，吉西他滨仍然具有广泛的临床应用，特别是在胰腺癌领域，吉西他滨是标准治疗药物。但是在临床治疗过程中，不同病人对吉西他滨的敏感性或有效性差别较大，存在原发性和获得性耐药问题。因此，寻找有效的吉西他滨药物敏感性的标记物来指导用药，不但可以避免由于药物选择不当延误病人治疗，而且还能在治疗过程中监控药物治疗效果，实现用药合理性和个体化治疗的目的。
目前研究认为吉西他滨耐药主要与其摄取转运过程障碍、胞浆内活化、分解酶异常及DNA修复有关，研究内容包括hENT1(人平衡型核苷转运蛋白1)、RRM1(核糖核苷酸还原酶M1)、RRM2(核糖核苷酸还原酶M2)、dCK(脱氧胞苷激酶)等基因或蛋白。
针对这些基因或蛋白的研究也很多，但是不同研究之间的研究结果有差异。例如RolandAndersson(World J Gastroenterol.2014；20:8482–8490)等研究者对10个临床研究进行了系统回顾，涉及855名患者，分为高和低的hENT1组，研究结果显示高表达hENT病人的总生存期显著长于低表达者。但是在一项随机III期临床研究——CONKO-001临床试验(Eur J Cancer.2015；51:1546–1554)中接受辅助吉西他滨化疗的患者的组织样本中采用SP120兔单抗通过免疫组化检测hENT1表达，结果证实hENT1没有任何预测作用。而且另一项临床研究(NCT01124786，J Clin Oncol.2013；31:4453-4461)中同样显示hENT1没有预测吉西他滨敏感性的作用。Fujit等人(Neoplasia.2010；12:807–817)在70例切除的PDAC病人中检测了hENT1、dCK、RRM1、RRM2的mRNA，其中40例病人接受了吉西他滨为基础的辅助化疗。研究结果显示高表达dCK和低RRM2病人的无疾病进展生存期更长。Mar é chal等人(Gastroenterology.2012；143:664–674.e1-6)进行的另一项研究显示高表达hENT1和dCK的病人接受吉西他滨辅助治疗时可以预测有更长的生存期。这些研究结果差异较大的原因包括研究者采用的检测方法不同、病人用药情况复杂、肿瘤异质性、样本数量少等。除此之外有可能是没有找到更为精确的预测指标。
发明内容
针对目前不同病人对吉西他滨的敏感性或有效性差别较大，存在原发性和获得性耐药问题以及吉西他滨敏感性预测指标的不确定性问题，本发明试图寻找到新的更精确的吉西他滨敏感性预测指标。
醛酮还原酶(aldo.keto reductase，AKR)超家族共有15个家族，其中人源AKR成员共有15个。大多数AKR成员均能以烟酰胺腺嘌呤二核苷磷酸NADP为辅因子催化简单的氧化还原反应。它们作用底物范围广泛，包括糖类、脂肪醛、甾体类激素、前列腺素和致癌物等。因此，AKR超家族成员可能是在激素合成、药物代谢、炎症反应、致癌物解毒等生命活动中发挥重要作用的酶类。研究表明，人源AKR超家族成员与多种肿瘤发生发展具有相关性，其活性高低与肿瘤的进展及治疗效果密切相关。越来越多的证据表明(国际肿瘤学.2013；40(1):43-46)，AKR超家族部分成员与肿瘤抗药性相关。AKRlB10成为吸烟相关非小细胞肺癌，尤其肺鳞癌的早期诊断标志物，而AKR1C1～3在肺癌多种化疗药物耐受过程中的作用特征使其成为预后预测的重要因子，也为提高肺癌药物敏感性提供了有效的作用靶点。但是关于AKR家族与吉西他滨耐药性的关系从未有研究或报道。
本发明首次发现了醛酮还原酶(aldo.keto reductase，AKR)家族成员AKR1C1、AKR1C2、AKR1C3家族与吉西他滨耐药性有关。本发明在38个裸鼠肿瘤移植瘤模型中研究了吉西他滨药物敏感性与AKR1C1～C3表达的相关性。研究结果表明在15个AKR1C1～C3高表达的模型中，13个模型结果显示吉西他滨药效差，表现不敏感性；在23个AKR1C1～C3低表达的模型中，19个模型结果显示吉西他滨药效好，表现敏感性。
因此，本发明的目的在于提供用于预测吉西他滨或其药学上可接受的盐治疗癌症时的药物敏感性的生物标记物AKR1C1、AKR1C2、AKR1C3及其用途。
一方面，本发明提供了AKR1C1、AKR1C2或AKR1C3作为预测吉西他滨或其药学上可接受的盐治疗癌症时的药物敏感性的生物标记物的应用。
根据本发明的实施方案，所述药学上可接受的盐包括吉西他滨与酸形成的酸加成盐，例如吉西他滨与选自下列的酸形成的酸加成盐：无机酸如盐酸、氢氟酸、氢溴酸、氢碘酸、硫 酸、焦硫酸、磷酸或硝酸；有机酸如甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一烷酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡糖酸、3-羟基-2-萘甲酸、烟酸、扑酸、果胶酯酸、过硫酸、3-苯基丙酸、苦味酸、特戊酸、2-羟基乙磺酸、衣康酸、氨基磺酸、三氟甲磺酸、十二烷基硫酸、乙磺酸、苯磺酸、对甲苯磺酸、甲磺酸、2-萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、己二酸、藻酸、马来酸、富马酸、D-葡糖酸、扁桃酸、抗坏血酸、葡庚酸、甘油磷酸、天冬氨酸、磺基水杨酸、半硫酸或硫氰酸。
作为实例，所述药学上可接受的盐为盐酸吉西他滨。
根据本发明的实施方案，所述的癌症为结肠癌、肺癌、胃癌、胰腺癌、卵巢癌、黑色素瘤、肝癌、乳腺癌、宫颈癌。
所述的肺癌包括非小细胞肺癌，所述非小细胞肺癌包括鳞癌、腺癌、腺鳞癌、大细胞癌等，所述非小细胞肺癌优选为肺鳞癌。
另一方面，本发明提供了一种预测含有吉西他滨或其药学上可接受的盐的药物治疗癌症时的药物敏感性的方法。
根据本发明的实施方案，所述方法包括，应用AKR1C1、AKR1C2或AKR1C3作为生物标记物。
所述方法还可以包括，检测患者体内或离体样品中的AKR1C1、AKR1C2或AKR1C3的表达量。
根据本发明的实施方案，可将治疗有效量的吉西他滨或其药学上可接受的盐作为单一药剂给药或者与一种或多种其它药剂组合给药，其中所述组合不会引起不可接受的不良反应。所述组合中合适的活性物质包括常规的适用于化疗或靶向治疗的药物，如烷化剂、铂化合物、DNA改性剂、拓扑异构酶抑制剂、微管改性剂、抗代谢药物、抗癌抗生素、激素/拮抗剂、芳香酶抑制剂、小分子激酶抑制剂、光敏剂、抗体、细胞因子、药物轭合物、疫苗或其他药物等。
所述药学上可接受的载体可以是这样的载体，其在与活性成分的有效活性一致的浓度下对患者相对无毒且无害，以至于由所述载体引起的任何副作用不会破坏所述活性成分的有益作用。化合物或其药学上可接受的盐的药学有效量优选是对正在治疗的具体病况产生结果或者产生影响的量。可使用包括速释、缓释和定时释放制剂在内的任意有效的常规剂量单位形式，将吉西他滨或其药学上可接受的盐与本领域公知的药学上可接受的载体一起以如下方式给药：口服、肠胃外、局部、鼻腔、眼部、舌下、直肠、阴道给药等。
另一方面，本发明还提供了一种用于预测含有吉西他滨或其药学上可接受的盐的药物治疗癌症时的药物敏感性的试剂盒，所述试剂盒包括：
检测个体样本中，AKR1C1、AKR1C2、AKR1C3中至少一种表达水平的试剂。
所述个体样本选自人癌症组织，具体的，可选自结肠癌、肺癌、胃癌、胰腺癌、卵巢癌、黑色素瘤、肝癌、乳腺癌、宫颈癌。
本发明的有益效果：利用醛酮还原酶(aldo.keto reductase，AKR)家族成员AKR1C1、AKR1C2或AKR1C3作为预测吉西他滨治疗癌症时的药物敏感性的生物标记物来指导临床用药，不但可以避免药物选择的盲目性，避免由于药物选择不当而延误病人病情，而且还能在治疗过程中监控药物治疗效果，实现用药的合理性和个体化治疗的目的。临床癌症病例AKR1C1-C3的表达检测结果进一步验证了AKR1C1-C3表达与吉西他滨疗效之间的高度关联性，说明了应用AKR1C1、AKR1C2或AKR1C3来预测吉西他滨治疗癌症的疗效的合理性，可行性以及高度有效性。
附图说明
图1AKR1C1～C3在不同细胞中的表达量分析。GAPDH或β-tubulin作为内参。条带灰度值越高表示对应蛋白的表达量越高。
图2吉西他滨在不同结肠癌裸鼠移植瘤模型中的抗肿瘤作用。
图3吉西他滨在不同肺癌裸鼠移植瘤模型中的抗肿瘤作用。
图4吉西他滨在不同胃癌裸鼠移植瘤模型中的抗肿瘤作用。
图5吉西他滨在不同胰腺癌裸鼠移植瘤模型中的抗肿瘤作用。
图6吉西他滨在不同卵巢癌裸鼠移植瘤模型中的抗肿瘤作用。
图7吉西他滨在HepG2肝癌裸鼠移植瘤模型中的抗肿瘤作用。
图8吉西他滨在HeLa宫颈癌移植瘤模型中的抗肿瘤作用。
图9吉西他滨在MDA-MB-435黑色素瘤裸鼠移植瘤模型中的抗肿瘤作用。
具体实施方式
以下内容将进一步说明本发明，但不应被理解为限制本发明的主旨和保护范围。
实验一：蛋白质免疫印迹(Western blot,WB)
实验原理：
经过聚丙烯酰胺凝胶电泳(polyacryamide gel electrophoresis,PAGE)分离的蛋白质样品，转移并结合到固相载体上(如硝酸纤维素膜,二氟化树脂膜等)，以固相载体上的蛋白质或多肽作为抗原，其对应的抗体能识别并与之结合，再与酶或同位素标记的第二抗体起反应，经过底物显色或放射自显影以检测目的蛋白的表达量。
实验目的：
检测AKR1C1～C3蛋白在不同细胞中的表达。
实验材料：
细胞：人结肠癌细胞CW-2，LoVo，HCT116，SW620，COLO 205，HT-29，RKO，DLD-1,NCI-H716，SW1116,SW948；人肺癌细胞NCI-H292，A549，NCI-H460，NCI-H1650，HCC827，NCI-H1299，NCI-H1975，SK-MES-1，NCI-H1437，NCI-H1944；人胃癌细胞BGC-823，HGC-27，SGC-7901，KATO III，NCI-N87，SNU-1；人胰腺癌细胞AsPC-1，BxPC-3，PANC-1，Capan-2，CFPAC-1，MIA PaCa-2，HPAF-II；人乳腺癌细胞BT-474，SK-BR-3，MCF-7，T-47D，MDA-MB-231，MDA-MB-468；人卵巢癌细胞SK-OV-3，NIH:OVCAR-3，A2780；人肝癌细胞BEL-7402，Hep G2；人宫颈癌细胞HeLa；人黑色素瘤细胞MDA-MB-435均购买自中国科学院典型培养物保藏委员会细胞库，并按该网站提供的细胞培养条件进行培养。
一抗：AKR1C1鼠单克隆抗体(MAB6529)购买自R&D Systems，AKR1C2兔多克隆抗体(13035)购买自Cell Signaling Technology，AKR1C3鼠单克隆抗体(MAB7678)购买自R&D，GAPDH兔单克隆抗体(2118)购买自Cell Signaling Technology，HRP-β-tubulin鼠单克隆抗体(ab012)购买自联科生物。
二抗：HRP标记的山羊抗兔IgG和HRP标记的马抗鼠IgG均购买自Cell Signaling Technology。
实验方法：
细胞于25cm 2培养瓶中进行培养，待80～90％覆盖率时收集细胞，根据细胞数量加入200-300μl RIPA裂解液(50mM Tris·HCl,150mM NaCl,1％NP-40,1％SDS,1×蛋白酶 抑制剂Cocktail)，涡旋混匀后置于冰上裂解30min。14000rpm，4℃离心10min后取上清，用BSA蛋白定量检测试剂盒进行定量。并加入5×loading buffer，煮沸。
每个样品30μg总蛋白经10％SDS-PAGE电泳分离后，250mA电转至PVDF膜上，用TBST配制的5％脱脂奶粉室温封闭1h，然后在4℃用一抗孵育过夜，AKR1C1抗体用5％脱脂奶粉稀释1000倍，AKR1C2抗体稀释500倍，AKR1C3抗体稀释2000倍，GAPDH或β-tubulin抗体稀释10000倍。用TBST彻底洗涤后经二抗(5％BSA稀释5000倍)室温孵育1h，再次用TBST彻底洗涤后加入ECL化学发光底物显色，曝光。检测β-tubulin的膜经一抗孵育后，不经二抗孵育，洗涤后直接加入ECL化学发光底物进行曝光。实验以GAPDH或β-tubulin作为内参。
实验结果：
AKR1C1～C3三个蛋白在不同细胞中的表达量具体如图1所示，从图1可知：
人结肠癌细胞中，COLO 205细胞中AKR1C1～C3均高表达；NCI-H716细胞中AKR1C1和AKR1C2为中等到高表达，AKR1C3为高表达；HCT 116、RKO、DLD-1细胞中三个蛋白均低表达；CW-2、LoVo、SW620、HT-29、SW1116和SW948细胞中AKR1C1和AKR1C2低表达，AKR1C3中等或高表达。
人肺癌细胞中，A549、NCI-H460、SK-MES-1、NCI-H1437、NCI-H1944细胞中AKR1C1～C3均高表达；NCI-H292、NCI-H1650、NCI-H1299和NCI-H1975细胞中AKR1C1～C3均低表达；HCC827细胞中，AKR1C1～C3均低表达。
人胃癌细胞中，SGC-7901细胞中AKR1C1～C3高表达；HGC-27和SNU-1中AKR1C1～C3均低表达；BGC-823细胞中AKR1C1中等表达，AKR1C2为低到中等表达，AKR1C3高表达；KATOIII和NCI-N87细胞中，AKR1C1和AKR1C2蛋白低表达，AKR1C3中等表达。
人胰腺癌细胞中，AsPC-1和BxPC-3细胞中AKR1C1～C3均高表达；PANC-1和Capan-2中AKR1C1～C3均低表达；CFPAC-1细胞中，AKR1C1和AKR1C2低到中等表达，AKR1C3高表达；MIA PaCa-2细胞中，AKR1C1中到高等表达，AKR1C2和AKR1C3低表达；HPAF-II细胞中，AKR1C1低表达，AKR1C2低到中等表达，AKR1C3高表达。
人乳腺癌细胞中，BT-474、MCF-7、T-47D、MDA-MB-231和MCF7细胞中AKR1C1～C3均低表达；SK-BR-3细胞中AKR1C1和AKR1C2为低表达，AKR1C3为中等表达；MDA-MB-468细胞中AKR1C1和AKR1C2高表达，AKR1C3中等表达。
人卵巢癌细胞中，NIH:OVCAR-3和A2780细胞中AKR1C1～C3均低表达；SK-OV-3 细胞中AKR1C1～C3均高表达。
人肝癌细胞中，BEL-7402细胞中AKR1C1～C3均低表达；Hep G2细胞中AKR1C1和AKR1C2低到中等表达，AKR1C3高表达。
人宫颈癌细胞HeLa中，AKR1C1和AKR1C2均低表达，AKR1C3高表达。
人黑色素瘤细胞MDA-MB-435中，AKR1C1～C3均低表达。
实验二：裸鼠体内移植瘤药效试验
实验目的：研究吉西他滨对不同肿瘤裸鼠移植瘤的药效。
实验材料：
盐酸吉西他滨购买自美仑生物，matrigel购买自美国Corning公司(货号：354262)，BALB/c裸小鼠购买自上海西普尔-必凯实验动物有限公司。
实验方法：将结肠癌细胞CW-2、LoVo、HCT116、SW620、COLO 205、HT-29、RKO、DLD-1、NCI-H716；人肺癌细胞NCI-H292、A549、NCI-H460、NCI-H1650、HCC827、NCI-H1299、NCI-H1975、SK-MES-1、NCI-H1944、NCI-H1437；人胃癌细胞BGC-823、HGC-27、SGC-7901、NCI-N87、SNU-1；人胰腺癌细胞AsPC-1、BxPC-3、PANC-1、Capan-2、CPFAC-1、MIA PaCa-2、HPAF-II；人乳腺癌细胞MDA-MB-231；人卵巢癌细胞SK-OV-3、A2780；人肝癌细胞BEL-7402、HepG2；人宫颈癌细胞HeLa；人黑色素瘤细胞MDA-MB-435在体外培养后，收集指数生长期的细胞悬液至适合浓度并与matrigel 1:1混合后用于BALB/c裸小鼠皮下肿瘤接种，建立各种移植瘤模型，待肿瘤生长至100～200mm3后分为2组，即control组和吉西他滨给药组，每组6只动物。盐酸吉西他滨给药剂量为120mg/kg，腹腔注射给药，每周第1天和第4天给药。每周2次测量肿瘤长径a(mm)、短径b(mm)，根据以下公式计算肿瘤体积(tumor volume,V)：V＝1/2×a×b2(mm3)；根据肿瘤体积结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为：RTV＝Vt/V0。其中V0为分笼给药时(即d0)测量所得肿瘤体积，Vt为每一次测量时的肿瘤体积。抗肿瘤活性的评价指标为相对肿瘤增殖率T/C(％)，计算公式如下：T/C％＝TRTV/CRTV*100％；其中TRTV：治疗组平均RTV；CRTV：溶媒对照组平均RTV。采用Excel表统计，T.Test检验。
实验结果：
吉西他滨在不同移植瘤模型中其药效表现出差异性。
在相同给药剂量和给药方案下，吉西他滨在不同裸鼠移植瘤模型中的抗肿瘤作用表现差异较大。在9个结肠癌移植瘤模型中(见图2)，吉西他滨对HCT-116、HT-29、CW-2、LoVo、RKO、DLD-1移植瘤生长具有显著抑制作用，T/C％<20％；但在COLO205、SW620移植瘤模型中，吉西他滨的抑制作用较差，T/C％>40％；在NCI-H716模型中，吉西他滨的抑制作用介于中间，T/C％约为38％。
同样地，在肺癌移植瘤模型中(见图3)，吉西他滨对NCI-H1650、NCI-H1975和NCI-H1299裸鼠移植瘤具有显著抑制作用，T/C<20％；而对A549、NCI-H460、NCI-H1437和NCI-H1944移植瘤抑制效果差(见图3)，T/C>40％。
在胃癌模型中(见图4)，吉西他滨对NCI-N87、HGC-27和SNU-1移植瘤生长具有显著抑制作用，T/C<20％；但是对BGC-823和SGC-7901移植瘤抑制作用差，T/C>50％。
在胰腺癌模型中(见图5)，吉西他滨对Capan-2、PANC-1、CFPAC-1移植瘤具有显著抑制作用，T/C<20％；对BxPC-3和AsPC-1移植瘤抑制作用差，T/C>40％。
在卵巢癌模型中也观察到类似的现象(见图6)，吉西他滨对A2780和SK-OV-3移植瘤抑制作用差异较大，对A2780抑制作用明显，而对SK-OV-3模型作用较差。在肝癌模型中(见图7)，吉西他滨对HepG2移植瘤抑制效果差，T/C>50％。
在宫颈癌模型中(见图8)，吉西他滨对HeLa移植瘤移植瘤抑制效果差，T/C>50％。
吉西他滨在MDA-MB-435黑色素瘤模型中也表现出较强的抗肿瘤活性(见图9)，T/C<20％。
实验一和实验二的研究结果表明：在吉西他滨敏感性与AKR1C1～C3表达之间存在强相关性，具体而言，在AKR1C1～C3高表达的模型中，吉西他滨药效差，表现不敏感性；AKR1C1～C3低表达的模型中，吉西他滨药效好，表现敏感性。因此，AKR1C1～C3可以作为生物标记物用于预测吉西他滨或其药学上可接受的盐治疗癌症时的药物敏感性。
实验三：检测AKR1C1～C3蛋白在吉西他滨治疗的肺鳞癌病例中的表达
实验目的：通过临床肺鳞癌病例回顾性验证AKR1C1～C3表达量与吉西他滨疗效之间的相关性。
实验材料：
36例使用过吉西他滨的肺鳞癌病人的石蜡包埋病理组织，由浙江省肿瘤医院提供。AKR1C1兔多克隆抗体(ab192785)购买自abcam公司，AKR1C2抗体(AM05295PU-N)购买自Origene公司；AKR1C3抗体(ab49680)购买自abcam公司；BOND III全自动染色机购买自Leica公司(德国)。
实验方法：
对石蜡样本进行切片，厚度约为4μm，烤箱干燥后，放置于BOND III全自动染色机进行免疫组化检测。一抗1:200稀释，孵育时间为15min，BOND Polymer Refine孵育时间8min。最后通过在光学显微镜下对组织切片按染色程度进行评分，判断AKR1C1～C3在不同样本中的表达量。
实验结果：
肺鳞癌中AKR1C1～C3表达量与吉西他滨的疗效具有相关性(表1)
在36例使用过吉西他滨的肺鳞癌中，有15例疗效评估为部分有效(Partial response，PR),15例疗效评估为病情稳定(Stable disease，SD)，6例疗效评估为疾病进展(Progressive disease，PD)，因此客观有效率为41.7％(15/36)。
在这36例病人中，27例检测出AKR1C1～C3高表达，疗效评估分别为：8例PR，13例SD，6例PD，因此客观有效率为29.6％(8/27)。9例检测出AKR1C1～C3低表达，疗效评估分别为：7例PR，2例SD，因此客观有效率为77.8％(7/9)。
统计结果显示：在肺鳞癌中，AKR1C1～C3低表达的病人较高表达的病人使用吉西他滨后有更好的疗效(客观有效率分别为77.8％和29.6％)。36例病人客观有效率为41.7％，而AKR1C1～C3低表达的病人客观有效率为77.8％，所以通过检测AKR1C1～C3表达量筛选AKR1C1～C3低表达的肺鳞癌病人可提高吉西他滨的有效率。综上，通过临床病例回顾性研究发现AKR1C1～C3可以作为吉西他滨临床疗效的生物标志物，用于预测吉西他滨的疗效。
表1.肺鳞癌中AKR1C1～C3表达量与吉西他滨疗效相关性分析
Figure PCTCN2018100371-appb-000001
Figure PCTCN2018100371-appb-000002

Claims (10)

  1. AKR1C1、AKR1C2或AKR1C3作为预测吉西他滨或其药学上可接受的盐治疗癌症时的药物敏感性的生物标记物的应用。
  2. 如权利要求1所述的应用，其中所述药学上可接受的盐为吉西他滨与选自下列的酸形成的酸加成盐：盐酸、氢氟酸、氢溴酸、氢碘酸、硫酸、焦硫酸、磷酸或硝酸；甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一烷酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡糖酸、3-羟基-2-萘甲酸、烟酸、扑酸、果胶酯酸、过硫酸、3-苯基丙酸、苦味酸、特戊酸、2-羟基乙磺酸、衣康酸、氨基磺酸、三氟甲磺酸、十二烷基硫酸、乙磺酸、苯磺酸、对甲苯磺酸、甲磺酸、2-萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、己二酸、藻酸、马来酸、富马酸、D-葡糖酸、扁桃酸、抗坏血酸、葡庚酸、甘油磷酸、天冬氨酸、磺基水杨酸、半硫酸或硫氰酸。
  3. 如权利要求1或2所述的应用，其中所述药学上可接受的盐为吉西他滨盐酸盐。
  4. 如权利要求1-3任一项所述的应用，其中所述癌症为结肠癌、肺癌、胃癌、胰腺癌、卵巢癌、黑色素瘤、肝癌、乳腺癌、宫颈癌，所述肺癌优选为非小细胞肺癌，更优选为鳞癌、腺癌、腺鳞癌、大细胞癌。
  5. 一种预测含有吉西他滨或其药学上可接受的盐的药物治疗癌症时的药物敏感性的方法，其中所述方法包括应用AKR1C1、AKR1C2或AKR1C3作为生物标记物。
  6. 一种预测含有吉西他滨或其药学上可接受的盐的药物治疗癌症时的药物敏感性的方法，其中所述方法包括，检测患者体内或离体样品中的AKR1C1、AKR1C2或AKR1C3的表达量。
  7. 如权利要求5或6所述的方法，其中所述药学上可接受的盐为吉西他滨与选自下列的酸形成的酸加成盐：盐酸、氢氟酸、氢溴酸、氢碘酸、硫酸、焦硫酸、磷酸或硝酸；甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一烷酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡糖酸、3-羟基-2-萘甲酸、烟酸、扑酸、果胶酯酸、过硫酸、3-苯基丙酸、苦味酸、特戊酸、2-羟基乙磺酸、衣康酸、氨基磺酸、三氟甲磺酸、十二烷基硫酸、乙磺酸、苯磺酸、对甲苯磺酸、甲磺酸、2-萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、己二酸、藻酸、马来酸、富马酸、D-葡糖酸、扁桃酸、抗坏血酸、葡庚酸、甘油磷 酸、天冬氨酸、磺基水杨酸、半硫酸或硫氰酸。
  8. 如权利要求5或6所述的方法，其中所述癌症为结肠癌、肺癌、胃癌、胰腺癌、卵巢癌、黑色素瘤、肝癌、乳腺癌、宫颈癌，所述肺癌优选为非小细胞肺癌，更优选为鳞癌、腺癌、腺鳞癌、大细胞癌。
  9. 一种用于预测含有吉西他滨或其药学上可接受的盐的药物治疗癌症时的药物敏感性的试剂盒，所述试剂盒包括：
    检测个体样本中，AKR1C1、AKR1C2、AKR1C3中至少一种表达水平的试剂。
  10. 如权利要求9所述的试剂盒，所述个体样本选自人癌症细胞组织，具体的，所述癌症细胞组织可选自结肠癌、肺癌、胃癌、胰腺癌、卵巢癌、黑色素瘤、肝癌、乳腺癌、宫颈癌细胞，优选为非小细胞肺癌细胞，更优选为鳞癌、腺癌、腺鳞癌、大细胞癌细胞。
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