WO2017114215A1 - 重组人钙调磷酸酶b亚基的应用 - Google Patents

重组人钙调磷酸酶b亚基的应用 Download PDF

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WO2017114215A1
WO2017114215A1 PCT/CN2016/110744 CN2016110744W WO2017114215A1 WO 2017114215 A1 WO2017114215 A1 WO 2017114215A1 CN 2016110744 W CN2016110744 W CN 2016110744W WO 2017114215 A1 WO2017114215 A1 WO 2017114215A1
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dose
group
rhcnb
administration
injection
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PCT/CN2016/110744
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French (fr)
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韩克胜
符健
黄宗文
田树红
韩丽芳
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海口奇力制药股份有限公司
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Publication of WO2017114215A1 publication Critical patent/WO2017114215A1/zh
Priority to US16/020,363 priority Critical patent/US10646553B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03016—Phosphoprotein phosphatase (3.1.3.16), i.e. calcineurin

Definitions

  • the present invention relates to the field of proteins, and in particular to the use of recombinant human calmodulin phosphatase B subunit.
  • Calcineurin is the only known protein phosphatase that relies on Ca 2+ /CaM (calmodulin) and consists of a 1:1 ratio of A and B subunits.
  • the A subunit (CNA) is a catalytic subunit and the B subunit (CNB) is a regulatory subunit.
  • CN plays an important role in the immune activation pathway and is a key enzyme for T cell activation.
  • CN is easily inactivated and unstable, and CNB, as a regulatory subunit of the enzyme, can promote the activity of CNA, and has relatively small molecular mass and stable properties.
  • CNB can stimulate T cell and NK cell proliferation, enhance NK cell killing activity, enhance macrophage phagocytosis and other immune functions, and recombinant human calcineurin B subunit (rhCNB) is innovative oncology drugs for genetic engineering in development.
  • the present invention provides the use of a recombinant human calmodulin phosphatase B subunit.
  • the invention adopts IVIS Kinetics small animal living body imaging system to study the therapeutic effect of rhCNB for injection on BALB/c nude mice bearing human gastric cancer cell MGC-803 carcinoma in situ, and further investigates the injection of rhCNB for specific types of tumor cells. Killing and/or inhibition, exploring the time-dependent and dose-effect relationship of the pharmacodynamic effects of the test substance, providing a basis for drug efficacy and other toxicological related tests.
  • the present invention provides the following technical solutions:
  • the present invention provides the use of rhCNB in the preparation of a medicament for killing and/or inhibiting gastric cancer cell MGC-803.
  • the rhCNB is administered at a dose of not less than 10 mg/kg in the preparation of a medicament for killing and/or inhibiting gastric cancer cell MGC-803.
  • the rhCNB is administered in an amount of from 10 mg/kg to 160 mg/kg in the use of rhCNB in the manufacture of a medicament for killing and/or inhibiting gastric cancer cell MGC-803.
  • the rhCNB is administered at a concentration of 0.5 mg/mL to 2 mg/mL in the preparation of a medicament for killing and/or inhibiting gastric cancer cell MGC-803.
  • the rhCNB is administered intravenously in the preparation of a drug for killing and/or inhibiting gastric cancer cell MGC-803; administered once a day, continuously
  • the drug was administered for 15 days or the first dose was 10 mg/kg, and the dose was increased by 1 time every 3 days, followed by 10 mg/kg, 20 mg/kg, 40 mg/kg, 80 mg/kg, and 160 mg/kg.
  • the invention also provides the application of rhCNB in the preparation of a medicament for treating a human gastric cancer cell MGC-803 carcinoma in situ in a BALB/c nude mouse.
  • the rhCNB is administered in a medicament for treating a BALB/c nude mouse-bearing human gastric cancer cell MGC-803 carcinoma in situ cancer, wherein the dose of rhCNB is not less than 10 mg. /kg.
  • the rhCNB is administered at a dose of 10 mg/kg in the preparation of a medicament for treating a BALB/c nude mouse-bearing human gastric cancer cell MGC-803 carcinoma in situ cancer. 160 mg/kg.
  • the rhCNB is administered at a concentration of 0.5 mg/mL in the preparation of a medicament for treating a BALB/c nude mouse-bearing human gastric cancer cell MGC-803 carcinoma in situ cancer. ⁇ 2mg/mL.
  • the rhCNB is administered intravenously in the preparation of a medicament for treating a BALB/c nude mouse-bearing human gastric cancer cell MGC-803 carcinoma in situ cancer.
  • 1 day administration continuous administration for 15 days or the first dose is 10 mg/kg, and the dose is increased by 1 time every 3 days, followed by 10 mg/kg, 20 mg/kg, 40 mg/kg, 80 mg/ Kg, 160mg/kg.
  • the results showed: injection of rhCNB G (high dose group), Z (medium dose group), D (low dose group), DZ (increasing dose group) administration group and positive control SD, X administration.
  • the control of tumor cell proliferation was significantly statistically significant compared with the vehicle control group, p ⁇ 0.01.
  • the positive control B administration group and the vehicle control group were statistically significant, p ⁇ 0.05.
  • the results showed that compared with the vehicle control group, the injection was administered with rhCNB G (high dose group), Z (medium dose group), D (low dose group), DZ (incremental dose group).
  • the control group and the positive control group B, SD and X had significant statistical significance for the control of tumor cell proliferation, p ⁇ 0.001.
  • the results of d7 after the last administration showed that the rhCNB G (high dose group), Z (middle dose group), D (low dose group), DZ (incremental dose group) administration group and the positive group were compared with the vehicle control group.
  • Control of tumor cell proliferation was significantly statistically significant in the control B-administered group, p ⁇ 0.001.
  • the SD and X administration groups were statistically significant, p ⁇ 0.01.
  • Figure 1 shows the experimental bioluminescence detection data of each group of experiments.
  • the abscissa "1 ⁇ 7" respectively means “pre-dose, d3, d6, d9, d12, d15 after the first dose and recovery period d7 after the last administration”;
  • G-rhCNB high-dose group for injection (40mg/ Kg) "Z-injection rhCB medium dose group (20mg/kg)", “D-injection rhCNB low dose group (10mg/kg)", DZ-injection rhCNB dose escalation group (10mg/kg ⁇ 20mg/ Kg ⁇ 40mg/kg ⁇ 80mg/kg ⁇ 160mg/kg, 10mg/kg is the initial dose, continuous administration for 3d to increase one dose, and so on;)”; the ordinate is the intensity of bioluminescence detection;
  • Figure 2 shows a living body image; wherein "X" indicates that the animal has died before the end of the recovery period;
  • Figure 3 shows a necropsy diagram
  • the invention discloses the application of the recombinant human calmodulin phosphatase B subunit, and those skilled in the art can learn from the contents of the present article and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • IVIS Kinetics Small Animal Live Imaging System was used to dynamically analyze the injection of rhCNB to BALB/c nude mice.
  • Therapeutic effect of gastric cancer cell MGC-803 in situ cancer Firstly, a subcutaneous xenograft model of BALB/c nude mice bearing human gastric cancer cells MGC-803 was established, and then a BALB/c nude mouse human gastric cancer cell MGC-803 orthotopic transplantation tumor model was established.
  • rhCNB medium dose group for injection administered at a dose of 20 mg/kg, the concentration is 1 mg/mL
  • Low-dose group for injection of rhCNB administered dose: 10 mg/kg, concentration of 0.5 mg/mL
  • rhCNB dose-increasing group for injection first dose was 10 mg/kg, increased by 1 time every 3 days, in turn
  • the positive control group was injected with recombinant human interleukin-2 (administered at a dose of 164,000 IU/kg, administered at a concentration of 8200 IU).
  • the Caliper IVIS Kinetics Small Animal In Vivo Imaging System was used to detect the body weight before the first dose (on the day), every 3 days after the first dose, and on the 7th day after the last dose, and the body weight of the animals was weighed before each test. At the end of the last test, the animals were sacrificed, gross necropsy, tumor inoculated organs (including tumor tissue) were taken out, and immunohistochemical examination of pathological examination and apoptosis was performed.
  • results During the experiment, except for the positive control group, the animals in the high-dose cisplatin group were observed, and the general symptoms were observed and the body weight was not significantly different from the vehicle control group. After 3 and 6 consecutive administrations, there was no statistically significant difference between the administration groups and the vehicle control group, and the p values were all >0.05. The results of 6 consecutive administrations showed that the proliferation of tumor cells in the dose-administered group and the positive control group were well controlled, but there was no statistical significance compared with the vehicle control group. Values are >0.05.
  • the results showed: injection of rhCNB G (high dose group), Z (medium dose group), D (low dose group), DZ (increasing dose group) administration group and positive control SD, X administration.
  • the control of tumor cell proliferation was significantly statistically significant compared with the vehicle control group, p ⁇ 0.01.
  • the positive control B administration group and the vehicle control group were statistically significant, p ⁇ 0.05.
  • the results showed that compared with the vehicle control group, the injection was administered with rhCNB G (high dose group), Z (medium dose group), D (low dose group), DZ (incremental dose group).
  • control group and the positive control group B, SD and X had significant statistical significance for the control of tumor cell proliferation, p ⁇ 0.001.
  • the results of d7 after the last administration showed that the rhCNB G (high dose group), Z (middle dose group), D (low dose group), DZ (incremental dose group) administration group and the positive group were compared with the vehicle control group.
  • Control B The control group had significant statistical significance for the control of tumor cell proliferation, p ⁇ 0.001.
  • the SD and X administration groups were statistically significant, p ⁇ 0.01.
  • Tumor cell line luc bioluminescent labeled gastric cancer cell line MGC-803 was purchased from Shanghai Lechen Biotechnology Co., Ltd.
  • Cell culture medium gibco RPMI1640 cell culture medium (500 mL/bottle)
  • Fetal bovine serum MP fetal bovine serum (500mL / bottle)
  • mice BALB/c nude mice were purchased from Guangdong Medical Laboratory Animal Center, and the certificate number was No4400720001367.
  • Example 1 Establishment of subcutaneous xenograft model of BALB/c nude mice bearing human gastric cancer cell MGC-803
  • Human gastric cancer cell MGC-803 was routinely cultured in vitro, digested and centrifuged to prepare a cell suspension of about 1 ⁇ 10 7 cells/mL, and 0.2 mL/only inoculated subcutaneously in the right axilla of mice.
  • the well-growing subcutaneous xenografts were removed and cut into tumor tissue pieces of about 2 x 2 x 1 mm size for use.
  • the animals were inoculated with isoflurane for gas anesthesia, and the animals were fed for 12 hours. After the animal is anesthetized, take the supine position, and cut the skin layer by layer on the left side 2 to 5 mm under the xiphoid process. The opening is about 1 cm.
  • the small scorpion gently pulls out the stomach and exposes it.
  • the tumor tissue block is placed on the stomach of the nude mouse near the side of the door to make a “pocket suture”, and the tumor tissue block is wrapped therein. The layer is sutured to the skin, and An Erdian eliminates it and puts it back into the cage for normal feeding. Seven days after surgery, detection, grouping and administration were performed using a small animal living imaging system.
  • mice Ninety-nine nude mice were randomly divided into high-dose rhCNB group for injection, rhCNB medium-dose group for injection, low-dose group for injection, low-dose group for injection, rhCNB dose-increasing group for injection, and positive control for injection.
  • the rhCNB administration group for the injection of test samples is provided with a high dose group for the test sample, a medium dose group for the test sample, a low dose group for the test sample, and a dose group for the test sample. The dose and concentration are shown. Table 1.
  • the clinically administered dose is 100,000 to 800,000 IU/time, and is administered once a day.
  • the average human body weight of 60kg it is calculated as 800,000 IU ⁇ 60 ⁇ 13,300 IU/kg, which is equivalent to the mouse equivalent dose of 13,300 IU/kg ⁇ 12.33 ⁇ 164,000 IU/kg, and the concentration is 0.82 million IU/kg. mL.
  • the general clinical daily dose is 15-20 mg/m 2 body surface area, and continuous administration is 5 days per week.
  • the dose was 0.405 mg/kg x 12.33 ⁇ 5 mg/kg, and as a high dose, the concentration was 0.25 mg/mL. Since the administration time is 15d continuous administration, the animal may die. Therefore, another low-dose group is administered at a dose of 1 mg/kg at a concentration of 0.05 mg/mL.
  • the general clinical daily dose is 4-6 mg/time, 1 time/day, and 15-30 days is a course of treatment.
  • the experiment is set to 2mg/kg and the concentration is 0.1. Mg/mL.
  • the route of administration is tail vein injection.
  • the time and frequency of administration of rhCNB for injection of the test sample were designed according to the time and frequency of clinical trial use.
  • the time and frequency of administration of the positive control group were designed according to the time and frequency of clinical use.
  • the high, medium, low and dose-increasing groups of the test samples were administered once a day for 15 days;
  • the vehicle control group was administered once a day for 15 days;
  • the positive drug injection was administered once daily with the interleukin-2 administration group for 15 days;
  • the positive drug injection was administered once a day in the high-dose cisplatin group for 5 days;
  • the positive drug injection was administered once daily in the low-dose administration group of cisplatin for 15 days;
  • the positive drug injection was administered once daily by the oxycamptothecin administration group, and the administration was continued for 15 days;
  • the general symptoms of the animals were observed before and after administration.
  • the indicators mainly included whether the food was normal, whether the drinking water was normal, whether the appearance of the stool was normal, whether the appearance of the urine was normal, and the day. Whether the normal activity is normal, whether the breathing is normal, whether the heartbeat is normal, whether there is secretion and weight in the nose and mouth.
  • the Caliper IVIS Kinetics small animal live imaging system was used for the test, and the body weight of the animals was weighed before each test. Bioluminescence detection was performed 10 mL to 15 min after injection of 0.1 mL/10 g body weight of the intraperitoneal injection solution (concentration: 15 mg/mL).
  • the data obtained from the experiment were statistically analyzed by SPSS analysis software, and the difference between the drug-administered group and the vehicle control group and between the drug-administered groups was compared.
  • the general symptoms of the animals were observed before and after the administration, except for the high-dose administration group of the cisplatin in the positive control group, and the drinking water, stool appearance, urine appearance, daily activities, and breathing of the animals in the other administration groups. There were no significant differences between the symptoms of heartbeat, eye and nose secretion and the vehicle control group. In addition, the experimental foods of each group of animals were reduced, the main reason may be related to the tumor's digestive function in the stomach.
  • Body weights were measured before the first dose (on the day), every 3 days after the first dose, and on the 7th day after the last dose, except for the weight of the positive control cisplatin high dose group and the vehicle control group. There were significant differences (p ⁇ 0.05), and there was no significant difference in body weight between the other groups and the vehicle control group, p>0.05.
  • Bioluminescence detection was performed using a Caliper IVIS small animal in vivo imaging system before the first administration (the day), every 3 days after the first administration, and on the 7th day after the last administration. The test results are shown in Figure 1, Figure 2 and Table 2.
  • the number in the bracket "()" is the number of surviving animals; G - high dose group for rhCNB injection, Z-injection group for rhCNB, D-injection group for low dose of rhCNB, DZ- Injecting rhCNB dose escalation group, SG-positive control drug injection cisplatin high dose group, SD-positive control drug injection cisplatin low dose group, B-positive control drug injection interleukin-2 Drug group, X-positive control drug injection with oxycamptothecin group, Y-vehicle control group; d0—before administration, d3—d3 after the first dose, d6—d6 after the first dose, d9—first time After drug d9, d12—d12 after the first dose, d15—d15 after the first dose, d21—d7 after the last dose; “ * ” p ⁇ 0.05, “ ** ” p ⁇ 0.01, “ ⁇ ”p ⁇ 0.001.
  • rhCNB In the therapeutic effect of rhCNB for injection on BALB/c nude mice bearing human gastric cancer cell MGC-803 carcinoma in situ, after continuous administration for 3-6 times, rhCNB 10mg/kg, 20mg/kg, 40mg/ The kg dose group had a certain inhibitory effect on the growth of human gastric cancer cell MGC-803 solid tumor, but there was no statistically significant difference compared with the vehicle control group (p>0.05).
  • the results of the subsequent administration showed that the effects of high, medium, low and dose escalation of rhCNB on the inhibition of tumor cell proliferation were significantly different from those of the vehicle control group (p ⁇ 0.001), and the effect was compared with injection.

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Abstract

提供了一种重组人钙调蛋白磷酸酶B亚基（rhCNB）在制备杀伤和/或抑制胃癌细胞MGC-803的药物中的应用。

Description

重组人钙调磷酸酶B亚基的应用
本申请要求于2015年12月30日提交中国专利局、申请号为201511021735.3、发明名称为“重组人钙调磷酸酶B亚基的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及蛋白领域，特别涉及重组人钙调蛋白磷酸酶B亚基的应用。
背景技术
钙调磷酸酶(Calcineurin,CN)是目前所知唯一一种依赖Ca2+/CaM(钙调素)的蛋白磷酸酶，由A，B亚基以1：1的比例组成。A亚基(CNA)是催化亚基，B亚基(CNB)是调节亚基。CN在免疫激活通路中发挥重要作用，是T细胞活化的关键酶。CN作为大分子酶蛋白，易失活，不稳定，而CNB作为该酶的调节亚基，能促进CNA的活性，且相对分子质量小，性质稳定。由研究表明CNB能刺激T细胞和NK细胞增殖以及增强NK细胞的杀伤活性，增强巨噬细胞吞噬能力等免疫功能，且重组人钙调磷酸酶B亚基(Recombinant human Calcineurin B subunit,rhCNB)是开发中的基因工程创新肿瘤药物。
但目前未见rhCNB对胃癌细胞MGC-803杀伤或抑制作用的相关报道。
发明内容
有鉴于此，本发明提供重组人钙调蛋白磷酸酶B亚基的应用。本发明采用IVIS Kinetics小动物活体成像系统，研究注射用rhCNB对BALB/c裸小鼠荷人胃癌细胞MGC-803原位癌的治疗作用，进一步考察受试物注射用rhCNB对特定类型肿瘤细胞的杀伤和(或)抑制作用，探索受试物产生药效作用的时效和量效关系，为药物的有效性和其它毒理学相关试验提供依据。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了rhCNB在制备杀伤和/或抑制胃癌细胞MGC-803的药物中的应用。
在本发明的一些具体实施方案中，rhCNB在制备杀伤和/或抑制胃癌细胞MGC-803的药物中的应用中所述rhCNB的给药剂量为不少于10mg/kg。
在本发明的一些具体实施方案中，rhCNB在制备杀伤和/或抑制胃癌细胞MGC-803的药物中的应用中所述rhCNB的给药剂量为10mg/kg～160mg/kg。
在本发明的一些具体实施方案中，rhCNB在制备杀伤和/或抑制胃癌细胞MGC-803的药物中的应用中所述rhCNB的给药浓度为0.5mg/mL～2mg/mL。
在本发明的一些具体实施方案中，rhCNB在制备杀伤和/或抑制胃癌细胞MGC-803的药物中的应用中所述rhCNB的给药方式为静脉注射给药；每日给药1次，连续给药15日或者首次给药剂量为10mg/kg，每3日提高1倍剂量递增给药，依次为10mg/kg、20mg/kg、40mg/kg、80mg/kg、160mg/kg。
本发明还提供了rhCNB在制备治疗BALB/c裸小鼠荷人胃癌细胞MGC-803原位癌移植瘤的药物中的应用。
在本发明的一些具体实施方案中，rhCNB在制备治疗BALB/c裸小鼠荷人胃癌细胞MGC-803原位癌移植瘤的药物中的应用中所述rhCNB的给药剂量为不少于10mg/kg。
在本发明的一些具体实施方案中，rhCNB在制备治疗BALB/c裸小鼠荷人胃癌细胞MGC-803原位癌移植瘤的药物中的应用中所述rhCNB的给药剂量为10mg/kg～160mg/kg。
在本发明的一些具体实施方案中，rhCNB在制备治疗BALB/c裸小鼠荷人胃癌细胞MGC-803原位癌移植瘤的药物中的应用中所述rhCNB的给药浓度为0.5mg/mL～2mg/mL。
在本发明的一些具体实施方案中，rhCNB在制备治疗BALB/c裸小鼠荷人胃癌细胞MGC-803原位癌移植瘤的药物中的应用中所述rhCNB的给药方式为静脉注射给药；每日给药1次，连续给药15日或者首次给药剂量为10mg/kg，每3日提高1倍剂量递增给药，依次为10mg/kg、20mg/kg、40mg/kg、80mg/kg、160mg/kg。
在实验期间，除阳性对照注射用顺铂高剂量给药组动物以外，其余动物的一般症状观察和体重与溶媒对照组比较无明显差异。连续给药3次和6次后，各给药组与溶媒对照组比较无统计学意义，p值均＞0.05。连续给药6次后的检测结果显示：注射用rhCNB各剂量给药组和阳性对照给药组的肿瘤细胞增殖情况均得到了较好的控制，但与溶媒对照组比较无统计学意义，p值均＞0.05。连续给药9d后检测结果显示：注射用rhCNB G(高剂量组)、Z(中剂量组)、D(低剂量组)、DZ(递增剂量组)给药组和阳性对照SD、X给药组对肿瘤细胞增殖的控制与溶媒对照组比较均具有显著统计学意义，p〈0.01，阳性对照B给药组与溶媒对照组比较均具有统计学意义，p〈0.05。连续给药d12和d15次后检测结果显示：与溶媒对照组比较，注射用rhCNB G(高剂量组)、Z(中剂量组)、D(低剂量组)、DZ(递增剂量组)给药组和阳性对照B、SD、X给药组对肿瘤细胞增殖的控制均具有极显著统计学意义，p〈0.001。末次给药后d7检测结果显示：与溶媒对照组比较，注射用rhCNB G(高剂量组)、Z(中剂量组)、D(低剂量组)、DZ(递增剂量组)给药组和阳性对照B给药组对肿瘤细胞增殖的控制均具有极显著统计学意义，p〈0.001。SD、X给药组具有显著统计学意义，p〈0.01。
在注射用rhCNB对BALB/c裸小鼠荷人胃癌细胞MGC-803原位癌移植瘤的治疗作用中，对肿瘤的有效治疗(与溶媒对照组比较，p〈0.05)时间在连续给药6次以后，在整个给药期间，肿瘤能得到有效的控制。停药1周后，对肿瘤仍然具有持续有效的治疗效果(与溶媒对照组比较，p〈0.001)，优于阳性对照注射用顺铂和注射用羟喜树碱，与注射用重组人白介素-2相当。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示实验各组肿瘤生物发光检测数据
Figure PCTCN2016110744-appb-000001
其中横坐标“1～7”分别表示“给药前、首次给药后d3、d6、d9、d12、d15和末次给药后恢复期d7”；“G—注射用rhCNB高剂量组(40mg/kg)”“Z—注射用rhCNB中剂量组(20mg/kg)”、“D—注射用rhCNB低剂量组(10mg/kg)”、DZ—注射用rhCNB剂量递增组(10mg/kg→20mg/kg→40mg/kg→80mg/kg→160mg/kg，10mg/kg为初始剂量，连续给药3d提高一个剂量，以此类推；)”；纵坐标为生物发光检测强度；
图2示活体成像图；其中，“×”表示该动物在恢复期结束前已经死亡；
图3示剖检图。
具体实施方式
本发明公开了重组人钙调蛋白磷酸酶B亚基的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
依据“细胞毒类抗肿瘤药物非临床研究技术指导原则”和“抗肿瘤药物药效学指导原则”，采用IVIS Kinetics小动物活体成像系统，动态分析注射用rhCNB对BALB/c裸小鼠荷人胃癌细胞MGC-803原位癌的治疗作用。首先建立BALB/c裸小鼠荷人胃癌细胞MGC-803皮下移植瘤模型，然后建立BALB/c裸小鼠荷人胃癌细胞MGC-803原位移植瘤模型。取建模成功的裸小鼠90只，按生物发光数值平均分为9个给药组，包括：注射用rhCNB高剂量组(给药剂量为40mg/kg，给药浓度为2mg/mL)、注射用rhCNB中剂量组(给药剂量为20mg/kg，给药浓度为1mg/mL)、 注射用rhCNB低剂量组(给药剂量为10mg/kg，给药浓度为0.5mg/mL)、注射用rhCNB剂量递增组(首次给药剂量为10mg/kg，每3日提高1倍剂量，依次为10mg/kg、20mg/kg、40mg/kg、80mg/kg、160mg/kg)、阳性对照注射用重组人白介素-2给药组(给药剂量为16.4万IU/kg，给药浓度为8200IU/mL)、阳性对照注射用顺铂高剂量给药组(5mg/kg，给药浓度为0.25mg/mL)、阳性对照注射用顺铂低剂量给药组(1mg/kg，给药浓度为0.05mg/mL)、阳性对照注射用羟喜树碱给药组(2mg/kg，给药浓度为0.1mg/mL)和溶媒对照组。实验各组每日给药1次，除阳性对照注射用顺铂高剂量给药组连续给药5日外，其余各组连续给药15日。每次给药前和给药后对动物进行一般症状观察。首次给药前(当日)、首次给药后每隔3日和末次给药后第7日分别用Caliper IVIS Kinetics小动物活体成像系统检测1次，每次检测前对动物体重进行称量。末次检测结束后处死动物，大体剖检，取出肿瘤接种器官(包括肿瘤组织)，进行病理检查和细胞凋亡的免疫组化检测。
结果 在实验期间，除阳性对照注射用顺铂高剂量给药组动物以外，其余动物的一般症状观察和体重与溶媒对照组比较无明显差异。连续给药3次和6次后，各给药组与溶媒对照组比较无统计学意义，p值均＞0.05。连续给药6次后的检测结果显示：注射用rhCNB各剂量给药组和阳性对照给药组的肿瘤细胞增殖情况均得到了较好的控制，但与溶媒对照组比较无统计学意义，p值均＞0.05。连续给药9d后检测结果显示：注射用rhCNB G(高剂量组)、Z(中剂量组)、D(低剂量组)、DZ(递增剂量组)给药组和阳性对照SD、X给药组对肿瘤细胞增殖的控制与溶媒对照组比较均具有显著统计学意义，p〈0.01，阳性对照B给药组与溶媒对照组比较均具有统计学意义，p〈0.05。连续给药d12和d15次后检测结果显示：与溶媒对照组比较，注射用rhCNB G(高剂量组)、Z(中剂量组)、D(低剂量组)、DZ(递增剂量组)给药组和阳性对照B、SD、X给药组对肿瘤细胞增殖的控制均具有极显著统计学意义，p〈0.001。末次给药后d7检测结果显示：与溶媒对照组比较，注射用rhCNB G(高剂量组)、Z(中剂量组)、D(低剂量组)、DZ(递增剂量组)给药组和阳性对照B 给药组对肿瘤细胞增殖的控制均具有极显著统计学意义，p〈0.001。SD、X给药组具有显著统计学意义，p〈0.01。
结论 在注射用rhCNB对BALB/c裸小鼠荷人胃癌细胞MGC-803原位癌移植瘤的治疗作用中，对肿瘤的有效治疗(与溶媒对照组比较，p〈0.05)时间在连续给药6次以后，在整个给药期间，肿瘤能得到有效的控制。停药1周后，对肿瘤仍然具有持续有效的治疗效果(与溶媒对照组比较，p〈0.001)，优于阳性对照注射用顺铂和注射用羟喜树碱，与注射用重组人白介素-2相当。
本发明提供的重组人钙调磷酸酶B亚基的应用中所用原料及试剂均可由市场购得。
肿瘤细胞株：luc生物发光标记的胃癌细胞株MGC-803购于上海乐晨生物科技有限公司。
细胞培养液：gibco RPMI1640细胞培养液(500mL/瓶)
胎牛血清：MP胎牛血清(500mL/瓶)
细胞消化液：gibco细胞消化液
底物：Caliper萤火虫D-荧光素钾盐
麻醉剂：异氟烷
实验动物：BALB/c裸小鼠，购于广东省医学实验动物中心，合格证编号为№4400720001367。
下面结合实施例，进一步阐述本发明：
实施例1BALB/c裸小鼠荷人胃癌细胞MGC-803皮下移植瘤模型的建立
人胃癌细胞MGC-803体外常规培养，消化离心后制成浓度约为1×107个细胞/mL的细胞混悬液，0.2mL/只接种于小鼠右侧腋窝皮下。
实施例2BALB/c裸小鼠荷人胃癌细胞MGC-803原位移植瘤模型的建立
取出生长良好的皮下移植瘤，切成约2×2×1mm大小的肿瘤组织块备用。用异氟烷对待接种动物进行气体麻醉，术前进食12h。动物麻醉后取仰卧位，剑突下2～5mm处靠左侧横向逐层剪开皮肤，开口约1cm，用 小镊子将胃轻柔得拉出，使其暴露，用1#手术线将肿瘤组织块于裸小鼠胃大弯靠近有门一侧上方做“荷包缝合”，将肿瘤组织块包在其中，逐层缝合皮肤，安尔典消，放回笼内正常饲养。手术后7d，用小动物活体成像系统进行检测、分组和给药。
实施例3动物分组
取接种好的裸小鼠90只，按生物发光检测数据随机分为注射用rhCNB高剂量组、注射用rhCNB中剂量组、注射用rhCNB低剂量组、注射用rhCNB剂量递增组、阳性对照注射用重组人白介素-2给药组、阳性对照注射用顺铂高剂量给药组、阳性对照注射用顺铂低剂量给药组、阳性对照注射用羟喜树碱给药组和溶媒对照组9个组，每组10只。
实施例4剂量设计
1.2.4.1供试品注射用rhCNB剂量设计
参照“抗肿瘤药物药效学指导原则”中的“治疗组设高、中、低剂量治疗组，一般按4：2：1设置”、“细胞毒类抗肿瘤药物非临床研究技术指导原则”中的“高剂量不宜超过受试物的最大耐受量”，临床第一个疗程拟用量为60mg/日以及临床第二、第三、第四疗程拟采用递增剂量。因此，供试品注射用rhCNB给药组共设供试品高剂量组、供试品中剂量组、供试品低剂量组和供试品剂量递增组4个组，给药剂量和浓度见表1。
1.2.4.2阳性药的剂量设计
1.2.4.2.1阳性药注射用重组人白介素-2的剂量设计
根据注射用重组人白介素-2说明书中临床常用给药剂量为10～80万IU/次，每日给药1次。按人体平均体重为60kg计算为80万IU÷60≈1.33万IU/kg，换算为小鼠等效剂量为1.33万IU/kg×12.33≈16.4万IU/kg，给药浓度为0.82万IU/mL。
1.2.4.2.2阳性药注射用顺铂的剂量设计
根据注射用顺铂说明书中说明一般临床日给药剂量为15-20mg/m2体表面积，每周连续给药5d。人体平均体表面积为1.62m2，即临床给药 剂量为15mg/m2×1.62m2＝24.3mg，按人体平均体重为60kg计算为24.3mg÷60＝0.405mg/kg，换算为小鼠等效剂量为0.405mg/kg×12.33≈5mg/kg，作为高剂量，给药浓度为0.25mg/mL。由于给药时间为15d连续给药，动物可能会出现死亡，因此，再另设一个低剂量组，给药剂量为1mg/kg，给药浓度为0.05mg/mL。
1.2.4.2.3阳性药注射用羟喜树碱的剂量设计
根据注射用羟喜树碱说明书中说明一般临床日给药剂量为4-6mg/次，1次/日，15-30d为1疗程。按人体平均体重为60kg计算为6mg÷60＝0.1mg/kg，换算为小鼠等效剂量为0.1mg/kg×12.33≈1.23mg/kg，本实验设为2mg/kg，给药浓度为0.1mg/mL。
实施例5
1.2.5给药途径
给药途径为尾静脉注射。
1.2.6给药体积：20mL/kg
1.2.7给药时间/频率
供试品注射用rhCNB给药给药时间和频率依据临床拟用时间和频率来设计，阳性对照组给药时间和频率依据临床用药时间和频率来设计。
供试品高、中、低及剂量递增组均每日给药1次，连续给药15d；
溶媒对照组每日给药1次，连续给药15d；
阳性药注射用白介素-2给药组每日给药1次，连续给药15d；
阳性药注射用顺铂高剂量给药组每日给药1次，连续给药5d；
阳性药注射用顺铂低剂量给药组每日给药1次，连续给药15d；
阳性药注射用羟喜树碱给药组每日给药1次，连续给药15d；
1.2.8检测指标
1.2.8.1症状观察
给药前和给药后对动物的一般症状进行观察，观察指标主要包括：进食是否正常、饮水是否正常、粪便外观是否正常、尿液外观是否正常、日 常活动是否正常、呼吸是否正常、心跳是否正常、眼鼻口是否有分泌物和体重等。
1.2.8.2体重和生物发光检测
首次给药前(当日)、首次给药后每隔3日和末次给药后第7日分别用Caliper IVIS Kinetics小动物活体成像系统检测1次，每次检测前对动物体重进行称量，按0.1mL/10g体重腹腔注射底物溶液(浓度为15mg/mL)注射后10～15min进行生物发光检测。
表1实验各组给药剂量及给药日程
Figure PCTCN2016110744-appb-000002
Figure PCTCN2016110744-appb-000003
Figure PCTCN2016110744-appb-000004
Figure PCTCN2016110744-appb-000005
1.2.8.3统计分析
实验所得数据用SPSS分析软件进行统计分析，比较给药组与溶媒对照组之间以及各给药组之间的差异显著性。
实施例6实验结果
2.1一般症状观察
给药前和给药后对动物的一般症状进行观察，除阳性对照注射用顺铂高剂量给药组外，其余各给药组动物动物饮水、粪便外观、尿液外观、日常活动、呼吸、心跳、眼鼻口分泌物等症状与溶媒对照组比较未见明显差异。此外，实验各组动物的进食量均减少，主要原因可能与胃部的肿瘤影响了动物的消化功能。
2.2体重
首次给药前(当日)、首次给药后每隔3日和末次给药后第7日分别对动物进行体重测量，除阳性对照注射用顺铂高剂量给药组动物体重与溶媒对照组比较存在明显差异(p＜0.05)外，其余各给药组体重与溶媒对照组比较未见明显差异，p＞0.05。
2.3生物发光检测
首次给药前(当日)、首次给药后每隔3日和末次给药后第7日，用Caliper IVIS小动物活体成像系统分别进行生物发光检测。检测结果见图1、图2和表2。
根据图1所示数据，可以看出：在给药期间及恢复期，除阳性对照注射用顺铂高剂量给药组和溶媒对照组外，其余各给药组存活肿瘤细胞数量均在一个较小地范围内(＜1E+10)波动。其中阳性对照注射用顺铂高剂量给药组在d6次给药后检测数据出现急剧上升，在对人肝癌细胞Bel-7402原位癌实验中所出现的情况较为类似，但用触摸其实体瘤时发现其体积并未明显增大。在连续给药9次后，除阳性对照注射用顺铂高剂量给药组和溶媒对照组外，其余各给药组肿瘤细胞增殖数量明显小于溶媒对照组。
表2实验各组不同给药时期的p值及差异显著性分析
Figure PCTCN2016110744-appb-000006
Figure PCTCN2016110744-appb-000007
注：括号“()”内的数为存活动物数；G—注射用rhCNB高剂量给药组，Z—注射用rhCNB中剂量给药组，D—注射用rhCNB低剂量给药组，DZ—注射用rhCNB剂量递增给药组，SG—阳性对照药注射用顺铂高剂量给药组，SD—阳性对照药注射用顺铂低剂量给药组，B—阳性对照药注射用白介素-2给药组，X—阳性对照药注射用羟喜树碱给药组，Y—溶媒对照组；d0—给药前，d3—首次给药后d3，d6—首次给药后d6，d9—首次给药后d9，d12—首次给药后d12，d15—首次给药后d15，d21—末次给药后d7；“*”p〈0.05，“**”p〈0.01，“Δ”p〈0.001。
表2结果表明：连续给药3次和6次后，各给药组与溶媒对照组比较无统计学意义，p值均＞0.05。
连续给药6次后的检测结果显示：注射用rhCNB各剂量给药组和阳性对照给药组的肿瘤细胞增殖情况均得到了较好的控制，但与溶媒对照组比较无统计学意义，p值均＞0.05。
连续给药9d后检测结果显示：注射用rhCNB G、Z、D、DZ给药组和阳性对照SD、X给药组对肿瘤细胞增殖的控制与溶媒对照组比较均具有 显著统计学意义，p〈0.01，阳性对照B给药组与溶媒对照组比较均具有统计学意义，p〈0.05。
连续给药d12和d15次后检测结果显示：与溶媒对照组比较，注射用rhCNB G、Z、D、DZ给药组和阳性对照B、SD、X给药组对肿瘤细胞增殖的控制均具有极显著统计学意义，p〈0.001。
末次给药后d7检测结果显示：与溶媒对照组比较，注射用rhCNB G、Z、D、DZ给药组和阳性对照B给药组对肿瘤细胞增殖的控制均具有极显著统计学意义，p〈0.001。SD、X给药组具有显著统计学意义，p〈0.01。
值得关注的是，在实验期间，注射用rhCNB高、中、低剂量组均有动物在不同的给药期间无法检测到肿瘤细胞的存在(见表3、图2、图3)，结合剖检结果看，这些动物的移植瘤已经完全消失。连续给药后9～12次后检测结果显示高、低剂量均为1只；连续给药后15次后高剂量为2只，低剂量仍为1只；末次给药后d7检测结果表明高剂量组有4只，中剂量组有2只，低剂量组有1只。
此外，试验期间部分动物出现了死亡，结合肝癌原位癌实验结果，初步认为注射用顺铂给药组动物死亡主要是由于药物毒性所致，其余各给药组动物死亡主要由于胃癌严重影响其进食所致。
表3实验各组未检测及观察到肿瘤的动物数(只)
Figure PCTCN2016110744-appb-000008
3结论
在注射用rhCNB对BALB/c裸小鼠荷人胃癌细胞MGC-803原位癌移植瘤的治疗作用中，连续给药3～6次后，注射用rhCNB 10mg/kg、20mg/kg、40mg/kg剂量给药组对人胃癌细胞MGC-803实体瘤的生长均具有一定的抑制作用，但与溶媒对照组比较无显著统计学意义(p＞0.05)。在此后的给药过程中检测结果显示，注射用rhCNB高、中、低和剂量递增组对抑制肿瘤细胞增殖的作用与溶媒对照组比较均具有极显著差异(p〈0.001)，效果与注射用重组人白介素-2、羟喜树碱、注射用顺铂阳性对照组相当。末次给药后d7检测结果表明，注射用rhCNB高、中、低和剂量递增组对抑制肿瘤细胞增殖的作用与溶媒对照组比较仍都具有极显著差异(p〈0.001)，对肿瘤的治疗效果优于阳性对照组。提示，停药7d后，注射用rhCNB高、中、低和剂量递增各给药组对照组肿瘤细胞的增殖仍然具有较好的抑制作用(p〈0.001)。
综合以上分析，可初步得出如下结论：
在注射用rhCNB对BALB/c裸小鼠荷人胃癌细胞MGC-803原位癌移植瘤的治疗作用中，对肿瘤的有效治疗(与溶媒对照组比较，p〈0.05)时间在连续给药6次以后，在整个给药期间，肿瘤能得到有效的控制。停药1周后，对肿瘤仍然具有持续有效的治疗效果(与溶媒对照组比较，p〈0.001)，优于阳性对照注射用顺铂和注射用羟喜树碱，与注射用重组人白介素-2相当。
以上对本发明所提供的重组人钙调蛋白磷酸酶B亚基的应用进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (10)

  1. rhCNB在制备杀伤和/或抑制胃癌细胞MGC-803的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述rhCNB的给药剂量为不少于10mg/kg。
  3. 根据权利要求1或2所述的应用，其特征在于，所述rhCNB的给药剂量为10mg/kg～160mg/kg。
  4. 根据权利要求1至3任一项所述的应用，其特征在于，所述rhCNB的给药浓度为0.5mg/mL～2mg/mL。
  5. 根据权利要求1至4任一项所述的应用，其特征在于，所述rhCNB的给药方式为静脉注射给药；每日给药1次，连续给药15日或者首次给药剂量为10mg/kg，每3日提高1倍剂量递增给药，依次为10mg/kg、20mg/kg、40mg/kg、80mg/kg、160mg/kg。
  6. rhCNB在制备治疗BALB/c裸小鼠荷人胃癌细胞MGC-803原位癌移植瘤的药物中的应用。
  7. 根据权利要求6所述的应用，其特征在于，所述rhCNB的给药剂量为不少于10mg/kg。
  8. 根据权利要求6或7所述的应用，其特征在于，所述rhCNB的给药剂量为10mg/kg～160mg/kg。
  9. 根据权利要求6至8任一项所述的应用，其特征在于，所述rhCNB的给药浓度为0.5mg/mL～2mg/mL。
  10. 根据权利要求6至9任一项所述的应用，其特征在于，所述rhCNB的给药方式为静脉注射给药；每日给药1次，连续给药15日或者首次给药剂量为10mg/kg，每3日提高1倍剂量递增给药，依次为10mg/kg、20mg/kg、40mg/kg、80mg/kg、160mg/kg。
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