WO2022087865A1 - 一种双氧水在实体肿瘤治疗中的应用 - Google Patents

一种双氧水在实体肿瘤治疗中的应用 Download PDF

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WO2022087865A1
WO2022087865A1 PCT/CN2020/124240 CN2020124240W WO2022087865A1 WO 2022087865 A1 WO2022087865 A1 WO 2022087865A1 CN 2020124240 W CN2020124240 W CN 2020124240W WO 2022087865 A1 WO2022087865 A1 WO 2022087865A1
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tumor
drug
hydrogen peroxide
injection
drugs
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于保法
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济南嘉佐医药科技有眼公司
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    • 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
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/40—Peroxides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • 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 invention belongs to the field of biomedicine, in particular to an adjuvant for solid tumor treatment.
  • Cancer is a disease that seriously threatens human health.
  • the commonly used clinical treatments are surgery, radiotherapy, and chemotherapy.
  • chemotherapy is part of the treatment plan, combined with surgery and radiation therapy.
  • chemotherapy is the only effective way to treat cancer, especially when a patient cannot have surgery.
  • Chemotherapy is a common method of treating cancer with one or more anticancer drugs, which are usually given by intravenous injection or by mouth. Chemotherapy drugs work by disrupting the DNA metabolism of rapidly growing cells in the hope of stopping or slowing the growth of rapidly growing and dividing cancer cells.
  • the efficacy of chemotherapy depends on the type of cancer and the stage of the cancer. It is very effective against some cancers, such as leukemia, but not some tumors, such as pancreatic and liver cancer.
  • Chemotherapy has serious side effects such as vomiting, leukopenia (WBC), hair loss, weight loss and other toxic effects. Due to the highly toxic side effects, many cancer patients cannot successfully complete the full course of chemotherapy, and some even die from the side effects of chemotherapy due to poor tolerance.
  • WBC leukopenia
  • anticancer drugs are often due to the poor targeting specificity of such drugs to tumors in patients.
  • the drug circulates through most normal organs of the patient as well as the intended target tumor (less than 5% of the drug reaches the tumor), while more than 95% of the drug passes through the patient's systemic circulation.
  • Poor target specificity which leads to side effects, also reduces the efficacy of chemotherapy, as only a small fraction of the drugs properly target the tumor.
  • the efficacy of chemotherapy is further reduced due to poor retention of anticancer drugs in the target tumor.
  • Drug delivery techniques include intratumoral injection or slow drug release to minimize drug-induced systemic toxic effects.
  • gels and nanoparticles such as liposomes, have been used for the delivery of anticancer therapeutics in order to improve the diffusion and retention of drugs in tumors after injection.
  • this class of drugs destroys tumor cells in the free state only after the drug dissociates from the carrier and enters the tumor or their carrier is degraded to release the drug in a free state within the tumor. Once off the carrier, the drug can circulate It kills tumors all over the body rather than locally, which can still cause side effects.
  • formulations of emulsions and gel vehicles are difficult to administer and unevenly distributed within tumors compared to water-soluble drugs, limiting the clinical efficacy and use of various drug delivery systems or formulations.
  • ITCT intracranial chemotherapy
  • ITCT intracranial chemotherapy
  • the direct injection of chemotherapeutic drugs into the tumor site is an attractive new option for systemic therapy due to its higher safety profile and more aggressiveness (higher doses).
  • the local retention provided by intratumoral administration in the ITCT protocol results in slowing and/or reducing drug entry into the systemic circulation, thereby minimizing the exposure of distant tissues to the drug, thereby reducing the incidence of systemic side effects.
  • ITCT can also provide higher local drug concentrations that are maintained in the tumor (ie, at or near the site of administration) for extended periods of time, and intratumoral concentrations that cannot be achieved with systemic administration.
  • the earliest clinical reports of direct intratumoral injection of chemotherapy drugs were proposed by Bateman in 1955 and 1958 more than 50 years ago.
  • ITCT has unique advantages for the tumor itself, regardless of local administration, tumor recurrence and metastasis are still major problems. Meanwhile, drug combination, distribution and retention time of intratumorally injected drugs are key factors affecting the efficacy of ITCT.
  • ROS reactive oxygen species
  • Cell membranes and organelle membranes contain a large amount of unsaturated lipids, which can be oxidized by lipoxygenases (LOXs) and ROS to form LPO.
  • LOXs lipoxygenases
  • the accumulation or excess of ROS in tissue cells will cause the peroxidation of intracellular unsaturated lipids, resulting in the accumulation of a large amount of LPO in tumor cells, eventually causing apoptosis.
  • ROS also have certain damaging effects on DNA and proteins. For example, hydrogen peroxide can denature proteins when the hydrogen peroxide concentration reaches 0.05% to 0.1%.
  • ROS-based drug carriers such as liposomes containing iron sources, high molecular polymers containing free radical generators, etc.
  • they are not water-soluble, and there will be the aforementioned difficulties in administration and The problem of uneven distribution in the tumor; and the use of water-soluble drugs will cause drug leakage (into the circulatory system, such as blood), causing side effects.
  • the purpose of the present invention is to use peroxide as a new anticancer drug adjuvant to prolong the retention time of water-soluble chemotherapeutic drugs in intratumoral chemotherapy.
  • the present invention provides an application of hydrogen peroxide in the treatment of solid tumors, which can effectively prolong the retention time of water-soluble chemotherapeutic drugs in intratumoral chemotherapy, increase cell membrane permeability, stimulate the release of intracellular proteins and interact with Tumor-associated antigens are coordinated.
  • the present invention adopts the following technical solutions.
  • the application is that the peroxide is mixed with the anticancer drug to form a composition.
  • the anticancer drug is one or several water-soluble drugs.
  • the water-soluble drug refers to that the solubility of the drug in water can reach or exceed the minimum concentration that causes biological effects.
  • the anticancer drug is selected from but not limited to chlorambucil, such as dichloromethyldiethylamine, adenosine cyclophosphate, milphalan, chlorambucil, bendamustine; nitrous Base ureas, such as chloroethyl nitrosourea; alkaloids, such as vincristine, vindesine, vinorelbine; podophyllotoxins, such as teniposide, etoposide; antibiotics, such as, Bleomycin, daunorubicin, glareomycin, mitomycin, doxorubicin; antimetabolites, such as fluorouracil, cytarabine, gemcitabine.
  • chlorambucil such as dichloromethyldiethylamine, adenosine cyclophosphate, milphalan, chlorambucil, bendamustine
  • nitrous Base ureas such as chloroethyl nitrosourea
  • alkaloids
  • the peroxide is selected from hydrogen peroxide.
  • the solid tumor can be primary or metastatic.
  • the solid tumor is selected from, but not limited to, myeloma, squamous cell carcinoma, such as pancreatic cancer, liver cancer, melanoma, cervical cancer, oral cancer, esophageal cancer, lung squamous cell carcinoma, breast cancer, ovarian cancer.
  • the mixed use of hydrogen peroxide and water-soluble anticancer drugs in the present invention has little side effects: according to the size of the tumor, a suitable dose of the compound solution is injected under the guidance of CT or ultrasound; even if the entire dose leaks to the whole body, there will not be many side effects, because The entire dose is only 1/20-1/30 of the dose of traditional chemotherapy drugs.
  • the invention can also keep the activity of the anticancer drug at the injection site continuously: the intratumoral hydrogen peroxide keeps the high concentration of the drug in the tumor for a long time, so as to achieve better curative effect.
  • the invention also has immune function, can induce immune response, control micropathological changes of tumor cells, and prevent tumor metastasis or recurrence.
  • the present invention can also trigger autologous neo-endogenous multiple tumor antigens: true neo-endogenous autoantigens that directly generate tumors in vivo without surgery can produce multiple autologous TAA (tumor-associated antigen) antibodies.
  • the technology of the present invention is simple in clinical use, easy to train, and can be used to treat primary or secondary solid tumors.
  • the technology of the invention is safe and effective, and provides a new feasible method for tumor treatment, especially for advanced tumor treatment.
  • the technical solution for the combined use of hydrogen peroxide and water-soluble anticancer drugs provided by the present invention can inject an appropriate dose of compound drug solution according to the size of the tumor under the guidance of medical imaging technology.
  • the occurrence of the reaction overcomes the shortcomings of uneven drug distribution in tumors and drug leakage to the whole body, and synergistically increases the concentration and residence time of drug action; it can make the drug stay for a long time to achieve better efficacy; it can also trigger autoimmunity response, control the micropathology of tumor cells, and prevent tumor metastasis or recurrence.
  • Figure 1 shows the SPECT scan images of the experimental group and the control group at different time points, where a is the control group and b is the experimental group;
  • Fig. 2 is the radioactivity-time curve of bleomycin in mouse tumor
  • Fig. 3 is the transmission electron microscope picture of tumor tissue of tumor-bearing mice at different times after injection
  • Figure 4 is a T/W-time curve of cytarabine in mouse tumors
  • Fig. 5 is the scanning electron microscope picture of mouse melanoma cell smear
  • Fig. 6 is the signal image of cytarabine labeled with 99m Tc at different time points
  • Fig. 7 is the change of the concentration of cytarabine and doxorubicin in plasma with time
  • Figure 8 shows the changes in the proportion of cytarabine and doxorubicin in plasma with time
  • Figure 9 shows the pathological changes of tumor tissues in different treatment groups at different time points after injection
  • Figure 10 shows the survival curves of tumor-bearing mice in different treatment groups.
  • Example 1 The effect of hydrogen peroxide on bleomycin metabolism and cell morphology
  • Control group add 0.2 mL of BLM-I 131 solution to 0.12 mL of normal saline;
  • Test group 0.2 mL of BLM-I 131 solution was added to 0.12 mL of hydrogen peroxide, and the final concentration of hydrogen peroxide was 7.23 mg/mL.
  • the prepared B16 melanoma tumor solution was injected subcutaneously into the left front leg of C57BL/6 male mice to establish a subcutaneous solid tumor model.
  • 6 mice were randomly divided into 2 groups (control group and treatment group), and intratumoral injection was performed until the maximum diameter of the tumor reached 5-8 mm, and the injection dose was 0.2 mL.
  • the radioactivity of mouse tumor tissue was measured by SPECT scanning imager, and the activity of I 131 was used to indicate the retention of bleomycin in tumor tissue. time, the results are shown in Figures 1 and 2.
  • the BLM-I 131 injection containing hydrogen peroxide in the test group could remain in the tumor for 168 hours, while the BLM-I 131 in the control group remained in the tumor for only 8 hours.
  • the radioactivity of BLM-I 131 in the control group dropped rapidly to 30% after 1.5 h and to the background value within 8 h.
  • the radioactivity of B1M-I 131 in the tumor continued to decrease within 8h, peaked around 24h, and then slowly decreased to the background level at 168h. It can be seen that the retention time of the BLM-I 131 injection containing hydrogen peroxide in the tumor was significantly longer than that of the injection without. This shows that intratumoral injection of H 2 O 2 combined with anticancer drugs can prolong the action time of the drugs and destroy tumor cells more effectively.
  • Tumor biopsies were taken from the experimental mice, and the specimens were completely immersed in an EP tube containing glutaraldehyde fixative, and stored at 4°C in the dark. Sections were stained in time. Under the transmission electron microscope (5000 ⁇ 14), the pictures of cell morphology at different time points are shown in Figure 3, among which, A is the tissue morphology of the control group after injection for 1 hour; B is the tissue morphology of the experimental group after the injection for 1 hour; C is the injection of the experimental group for 12 hours The tissue morphology after injection; D and E are the tissue morphology of the experimental group after injection for 48 hours; F is the tissue morphology of the experimental group after injection for 168 hours.
  • A is the tissue morphology of the control group after injection for 1 hour
  • B is the tissue morphology of the experimental group after the injection for 1 hour
  • C is the injection of the experimental group for 12 hours
  • D and E are the tissue morphology of the experimental group after injection for 48 hours
  • Example 2 The effect of hydrogen peroxide on the metabolism and intratumoral distribution of cytarabine
  • Cytarabine hydrochloride was labeled with 99m Tc ( 99m Tc Ara-C), and it was prepared into a solution with a concentration of 6.25 mg/mL with hydrogen peroxide solution and physiological saline solution, respectively, as the test group (the final concentration of hydrogen peroxide was 3.62 mg. /mL) and the control group.
  • the prepared B16 melanoma cell suspension was injected subcutaneously into the left front leg of C57BL/6 male mice to establish a subcutaneous solid tumor model.
  • 6 mice were randomly divided into 2 groups (control group and treatment group), and intratumoral injection was performed until the maximum diameter of the tumor reached 5-8 mm, and the injection dose was 0.1 mL.
  • each mouse was mounted in a row on the plate and placed under emission computed tomography (ECT). Images were taken with an ECT machine (model: spectapx-409) at 0.25h, 0.5h, 1h, 2h, and 19h, respectively.
  • the 99mTc -labeled Ara-C signal in tumor tissue was calculated, and the average curve of T/W was calculated (T: the signal of the isotope-labeled drug counted in the image in the bright area of the tumor, W: the isotope-labeled drug counted in the image in the entire tumor margin area signal), the results are shown in Figure 4, which shows the T/W versus time after injection. The results showed that H 2 O 2 could significantly prolong the maintenance time of labeled Ara-C in the tumor.
  • Example 3 The effect of hydrogen peroxide on the metabolism of cytarabine in humans
  • Cytarabine hydrochloride was labeled with 99m Tc ( 99m Tc Ara-C), and it was prepared into a solution with a concentration of 10 mg/mL with hydrogen peroxide solution and physiological saline solution, respectively, and intratumorally injected into two tumors of a patient with liver cancer. , with hydrogen peroxide marked A, without hydrogen peroxide marked B.
  • the 99mTc -labeled cytarabine signal was monitored with an ECT imaging system at different time points after intratumoral injection. The results are shown in Figure 6, where the upper part is the actual image and the lower part is the schematic diagram.
  • the radiation intensity of points A and B was recorded as 100% after 15 minutes of injection; after 4 hours of injection treatment, the radiation intensity of point A decreased to 82%, while that of point B was only 16%; after 24 hours of treatment, the radiation intensity of point A decreased to 82%. , the radiation intensity of point A decreased to 60%, while the radiation intensity of point B decreased to the background intensity (0%). This indicates that the retention time of cytarabine in tumors was significantly prolonged with hydrogen peroxide compared with no hydrogen peroxide.
  • Example 4 Effect of combined use of hydrogen peroxide and chemotherapeutic drugs on drug distribution in tumor-bearing mice
  • the prepared H22 mouse liver cancer cell suspension was injected subcutaneously into the inner foreleg of Balb/c mice with 5 ⁇ 10 5 cells/0.1 mL of H22 liver cancer cells to establish a subcutaneous solid tumor model.
  • the intratumoral injection was performed according to the following scheme, and the injection dose was 0.2mL: the saline contained doxorubicin hydrochloride 1mg/mL, cytarabine hydrochloride 10mg/mL, H 2 O 2 18 mg/mL.
  • the concentrations of cytarabine and doxorubicin in serum samples were at extremely low levels and gradually decreased over time.
  • the concentrations of cytarabine and doxorubicin in venous blood 1 h after injection were (19680.26 ⁇ 3302.49) ng/mL and (112.71 ⁇ 46.59) ng/mL, respectively, accounting for (0.974 ⁇ 0.163)% and ( 0.056 ⁇ 0.023)%.
  • cytarabine and doxorubicin detected in blood accounted for (0.002 ⁇ 0.001)% and (0.002 ⁇ 0.005)% of the total injected amount, respectively.
  • the leakage of intratumoral injection to blood circulation does not exceed 1.137% of the total dose.
  • the test also showed that the total amount of cytarabine in the tumor (119.957 ⁇ 77.814) ⁇ g was 6 times that of the total amount of cytarabine in the blood (19.483 ⁇ 3.269) ⁇ g, and the total amount of doxorubicin in the tumor was (7.285 ⁇ 4.707) ⁇ g It is about 65 times of the total amount of doxorubicin in blood (0.112 ⁇ 0.046) ⁇ g.
  • Example 5 The effect of hydrogen peroxide and chemotherapy drug tumor injection on the survival curve of tumor-bearing mice
  • the prepared H22 hepatoma cell suspension was subcutaneously injected into the inner side of the front legs of Balb/c mice, and 5 ⁇ 10 5 H22 hepatoma cells/0.1 mL were injected to establish a subcutaneous solid tumor model.
  • the intratumoral injection was carried out according to the following scheme, and the injection dose was 0.1 mL: (1) blank control group: normal saline; (2) positive control group: doxorubicin hydrochloride 1 mg /mL, cytarabine hydrochloride 10mg/mL; (3) Test group: doxorubicin hydrochloride 1mg/mL, cytarabine hydrochloride 10mg/mL, H 2 O 2 18mg/mL.
  • A1-3 is the pathological section of the positive control group injected at 1h, 24h, and 168h.
  • Figure (100 ⁇ ); B1 is the pathological section picture (100 ⁇ ) of the experimental group at 24 hours of injection, and B2 is the pathological section of the experimental group at 168 hours of injection (400 ⁇ ).
  • B1 is the pathological section picture (100 ⁇ ) of the experimental group at 24 hours of injection
  • B2 is the pathological section of the experimental group at 168 hours of injection (400 ⁇ ). It can be seen from the figure that the tumor cells in the positive control group grew well 1 hour after injection, the tumor cells infiltrated the muscle 24 hours after injection, and the tumor tissue was partially necrotic at 168 hours after injection. 168h obvious fibrosis hyperplasia. This indicates that more inflammatory cells appeared in the tumor after chemotherapy drugs combined with hydrogen peroxide injection, thereby forming fibrotic cells.

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Abstract

提供了一种双氧水在实体肿瘤治疗中的应用，其中过氧化氢与水溶性抗癌药物联合用于肿瘤内注射，抗癌药物为一种或者几种水溶性药物的组合。可在医学影像引导下，根据肿瘤大小，注射适当剂量的复方药剂溶液，操作简单、副作用小，能够有效降低副反应的发生，克服了肿瘤中药物分布不均和药物向全身泄漏的缺点，并协同增加了药物作用的浓度和停留时间；能够使药物长时间滞留，达到更好的疗效；还能够引发自体免疫反应，控制肿瘤细胞的微病变，防止肿瘤转移或复发。

Description

一种双氧水在实体肿瘤治疗中的应用 技术领域
本发明属于生物医药领域，具体涉及一种用于实体肿瘤治疗的助剂。
背景技术
癌症是一种严重威胁人类健康的疾病，目前临床常用的治疗手段为手术治疗，放射治疗，化学治疗。通常化学治疗是治疗计划的一部分，与手术和放射治疗相结合。但有时，化学疗法是治疗癌症的唯一有效方法，尤其是当患者无法进行手术时。
化学疗法是使用一种或多种抗癌药治疗癌症的常用方法，这些药物通常通过静脉内注射给药，也有口服给药。化疗药物的作用是破坏快速生长的细胞的DNA代谢，以期阻止或减慢快速生长和分裂的癌细胞的生长。化学疗法的功效取决于癌症的类型和癌症的阶段。它对某些癌症(如白血病)非常有效，但对某些肿瘤(如胰腺癌和肝癌)却无效。化学疗法具有严重的副作用，例如呕吐，白细胞低下(WBC)，脱发，体重减轻和其他毒性作用。由于毒性极高的副作用，许多癌症患者无法成功完成完整的化疗过程，甚至有一些患者由于耐受性差而死于化疗的副作用。抗癌药物的极端副作用通常是由于此类药物对患者体内肿瘤的靶向特异性差所致。药物通过患者的大多数正常器官以及预期的目标肿瘤循环(不到5％的药物到达肿瘤)，而超过95％的药物通过患者的全身循环。导致副作用的不良靶标特异性也降低了化学疗法的效力，因为只有一小部分药物可正确靶向肿瘤。由于抗癌药物在靶标肿瘤中的保留能力较差，因此化疗的功效进一步降低。
正因如此，药物的靶向性输送成为一个重要的研究内容，各种制剂的开发成功改善了患者的依从性和便利性。药物输送技术包括肿瘤内注射或药物缓慢释放，以最大程度地降低由药物引起的全身毒性作用。目前，为了改善药物在注射后在肿瘤中的扩散和保留，已将凝胶和纳米颗粒(如脂质体)用于抗癌治疗药物的输送。实际上，该类药物仅在药物从载体上解离并进入肿瘤或它们的载体降解以在肿瘤内将药物释放为游离状态后，才能破坏游离状态的肿瘤细胞，一旦脱离载体后，药物可以循环到全身而不是在局部杀死肿瘤，从而仍然会导致副作用。另外，与水溶性药物相比，乳剂和凝胶载剂的制剂给药困难并且在肿瘤内分布不均，限制了各种药物输送系统或制剂的临床效果和使用。
化学治疗药物直接注入肿瘤部位的方法，例如ITCT(肿瘤内化学疗法)，由于其更高的安全和更具攻击性(更高剂量)，成为一种极具吸引力的全身治疗的新选择。ITCT方案中肿瘤内给药所提供的局部滞留导致减慢和/或减少药物进入体循环，从而使远处组织暴露于药物的可能性降至最低，从而降低了全身副作用的发生率。ITCT还可以提供较高的局部药物浓度，在肿瘤中(即，在给药部位或附近)长时间保持该浓度，而此肿瘤内浓度是全身性给药所不能达到的浓度。直接肿瘤内注射化疗药物的最早临床报道是50多年前Bateman在1955年和1958年提出的。在过去的几十年中，ITCT的临床研究越来越多，并取得了令人鼓舞的结果。虽然ITCT对肿瘤本身有独特的优势，但无论局部给药，肿瘤复发和转移仍是主要问题。同时，药物的组合、分布和肿瘤内注射药物的保留时间是影响ITCT疗效的关键因素。
半个多世纪以来的广泛研究表明，活性氧(ROS)在癌症中起着重要 作用。细胞膜及细胞器膜上含有大量的不饱和脂质，可以被脂氧合酶(lipoxygenase,LOXs)和ROS等氧化形成LPO。组织细胞内的ROS积累或过量，将引起细胞内不饱和脂质发生过氧化，造成大量的LPO堆积在肿瘤细胞内，最终引起凋亡。ROS对DNA和蛋白质也都有一定的破坏作用。如，当过氧化氢浓度达到0.05％到0.1％时，过氧化氢可以使蛋白质变性。
目前有多项基于ROS的药物载体，如包含铁源的脂质体，包含自由基生成物的高分子多聚物等，但是其并非水溶性的，会存在前面所述及的给药困难和瘤内分布不均问题；而采用水溶性的药物会造成药物外漏(进入循环系统，如血液)，引起副反应的发生。现阶段，尚无关于过氧化物与其他抗癌药物联合使用对瘤内药物代谢和持续药物库形成的报道。本发明的目的在于将过氧化物作为一种新的抗癌药物佐剂从而延长肿瘤内化学疗法中水溶性化疗药物留存时间。
发明内容
针对肿瘤内化学疗法中问题，本发明提供一种双氧水在实体肿瘤治疗中的应用，可以有效延长肿瘤内化学疗法中水溶性化疗药物留存时间、增加细胞膜通透性并刺激释放细胞内蛋白并与肿瘤相关抗原发生配位。
为实现上述目的，本发明采用如下技术方案。
一种过氧化物在制备实体肿瘤治疗药物中的应用。
所述应用为过氧化物与抗癌药物混合成为组合物。所述抗癌药物为一种或者几种水溶性药物。本发明中，水溶性药物是指药物在水中的溶解度能够达到或超过引起生物效应的最低浓度。
进一步的，所述抗癌药物选自但不限于氮芥类，如，二氯甲基二乙胺、 环磷腺苷、米尔法兰、苯丁酸氮芥、苯达莫司汀；亚硝基脲类，如，氯乙亚硝脲；生物碱，如，长春新碱、长春地辛、长春瑞宾；鬼臼毒素类，如，替尼泊苷、依托泊苷；抗生素类，如，博来霉素、道诺霉素、光辉霉素、丝裂霉素、阿霉素；抗代谢类，如，氟尿嘧啶、阿糖胞苷、吉西他滨。
进一步的，所述过氧化物选自过氧化氢。
进一步的，所述实体肿瘤可以是原发性的，也可以是转移瘤。所述实体肿瘤选自但不限于骨髓瘤、鳞状细胞癌，如胰腺癌、肝癌、黑色素瘤、宫颈癌、口腔癌、食管癌、肺鳞癌、乳腺癌、卵巢癌。
本发明具有以下优点：
本发明中过氧化氢与水溶性抗癌药物混合使用的副作用小：根据肿瘤大小，在CT或超声引导下注射合适剂量的复方溶液；即使整个剂量泄漏到全身，也不会产生很多副作用，因为整个剂量仅为传统化疗药物剂量的1/20-1/30。本发明还能够持续保持抗癌药物在注射部位的活性：肿瘤内过氧化氢使药物在肿瘤内的高浓度维持较长时间，以达到更好的疗效。本发明还具有免疫功能，能够诱导免疫反应，控制肿瘤细胞的微病变，防止肿瘤转移或复发。本发明还能够引发自体新内源性多种肿瘤抗原：不经手术直接在体内生成肿瘤的真新内源性自体抗原，可产生多种自体TAA(肿瘤相关抗原)抗体。本发明的技术临床使用简单、易于训练，可用于治疗原发性或继发性实体瘤。本发明的技术安全有效，为肿瘤治疗，特别是晚期肿瘤治疗提供了一种新的可行方法。
本发明提供的过氧化氢与水溶性抗癌药物联合使用的技术方案，可在医学影像技术的引导下，根据肿瘤大小，注射适当剂量的复方药剂溶液， 操作简单、副作用小，能够有效降低副反应的发生，克服了肿瘤中药物分布不均和药物向全身泄漏的缺点，并协同增加了药物作用的浓度和停留时间；能够使药物长时间滞留，达到更好的疗效；还能够引发自体免疫反应，控制肿瘤细胞的微病变，防止肿瘤转移或复发。
附图说明
图1为不同时间点试验组和对照组的SPECT扫描图像，其中a为对照组，b为试验组；
图2为博莱霉素在小鼠肿瘤中的放射性-时间曲线；
图3为注射后不同时间荷瘤小鼠肿瘤组织的透射电镜图片；
图4为阿糖胞苷在小鼠肿瘤中的T/W-时间曲线；
图5为小鼠黑色素瘤细胞涂片的扫描电镜图片；
图6为不同时间点 99mTc标记的阿糖胞苷信号图像；
图7为血浆中阿糖胞苷和阿霉素的浓度随时间的变化；
图8为血浆中阿糖胞苷和阿霉素的占比随时间的变化；
图9为注射后不同时间点不同处理组肿瘤组织病理变化；
图10为不同处理组荷瘤小鼠的生存曲线。
具体实施方式
下面结合实施例和附图对本发明做进一步说明，但本发明不受下述实施例的限制。
实施例1 过氧化氢对博来霉素代谢和细胞形态的影响
将盐酸博来霉素以I 131进行放射性标记，以Sephadex G250色谱柱纯化，获得I 131标记博来霉素(BLM-I 131)，将其配制成放射强度为1.002mCi/mL的BLM-I 131溶液(以γ-粒子计)备用，其中0.2mCi＝49.67μg博来霉素。
按照以下比例制备两份I 131标记博来霉素注射液：
(1)对照组：0.12mL生理盐水中加入0.2mL BLM-I 131溶液；
(2)试验组：0.12mL双氧水中加入0.2mL BLM-I 131溶液，双氧水的终浓度7.23mg/mL。
在无菌条件下，将制备的B16黑色素瘤瘤液注射于C57BL/6雄性小鼠左前腿皮下，建立皮下实体瘤模型。肿瘤株接种后，将6只小鼠随机分为2组(对照组和治疗组)，直到肿瘤最大直径达5-8mm，进行瘤内注射，注射剂量为0.2mL。于注射后0.5h、2h、4h、8h、24h、48h、96h、120h、144h、168h采用SPECT扫描成像仪测定小鼠肿瘤组织的放射性，以I 131的活性表示肿瘤组织中博来霉素的留存时间，结果如图1和图2所示。
试验组含有过氧化氢的BLM-I 131注射液在肿瘤中可以保持168h，而对照组的BLM-I 131在肿瘤中留存时间仅8h。对照组中BLM-I 131的放射性在1.5h后迅速降至30％，在8h内迅速降至背景值。试验组中BlM-I 131在肿瘤中的放射性在8h内持续下降，在24h前后出现峰值，然后缓慢下降，至168h降低至背景水平。可见，含有过氧化氢的BLM-I 131注射剂在肿瘤中的留存时间显著长于不含的注射液。这说明，H 2O 2与抗癌药物联用肿瘤内注射，能够延长药物作用时间，更有效地破坏肿瘤细胞。
对试验小鼠取肿瘤活检，标本完全浸入含有戊二醛固定剂的EP管中， 4℃避光保存，及时进行切片染色。透射电镜下(5000×14)，不同时间点细胞形态图片如图3所示，其中，A为对照组肿瘤注射1h后组织形态；B为试验组注射1h后组织形态；C为试验组注射12h后组织形态；D和E为试验组注射48h后组织形态；F为试验组注射168h后组织形态。
通过图3可知，对照组注射1h内未观察到明显的细胞形态改变，细胞状态正常。而在肿瘤内注射含过氧化氢的药剂后，试验组1h内即观察到大量白细胞出现，细胞膜上出现微孔；12h时可观察到较多的浓缩异染色质和细胞碎片，肿瘤细胞萎缩；48h时观察到肿瘤细胞排列松散，出现较多的间质和胶原，出现大量白细胞和脱落细胞；168h时肿瘤细胞细胞收缩，异染色质增多、聚集，呈现凋亡状态。这说明，过氧化氢能够快速引起细胞凋亡、改变细胞形态，并在细胞膜上造成孔洞，有利于药物进一步进入和肿瘤相关抗原的释放。
实施例2 过氧化氢对阿糖胞苷代谢及瘤内分布的影响
将盐酸阿糖胞苷以 99mTc标记( 99mTc Ara-C)，将其分别用双氧水溶液和生理盐水溶液配制成浓度为6.25mg/mL的溶液，分别作为试验组(双氧水的终浓度3.62mg/mL)和对照组。
在无菌条件下，将制备的B16黑色素瘤细胞悬浮液注射于C57BL/6雄性小鼠左前腿皮下，建立皮下实体瘤模型。肿瘤株接种后，将6只小鼠随机分为2组(对照组和治疗组)，直到肿瘤最大直径达5-8mm，进行瘤内注射，注射剂量为0.1mL。注射后，将每只小鼠固定在板上成一排，并置于发射计算机断层扫描(ECT)下。分别于0.25h、0.5h、1h、2h、19h用ECT机(型号：spectapx-409)拍摄图像。计算肿瘤组织中 99mTc标记的Ara-C 信号，并计算T/W的平均曲线(T：肿瘤内明亮区域内图像计数的同位素标记药物信号，W：整个肿瘤边缘区域内图像计数的同位素标记药物信号)，结果如图4，该图显示了注射后的T/W与时间的关系。结果表明，H 2O 2可明显延长标记Ara-C在肿瘤内的维持时间。
取肿瘤活检，标本完全浸入含有戊二醛固定剂的EP管中，4℃避光保存，及时送电镜室进行切片。FEI Nova Nano SEM 450扫描电镜下(100×1)荷瘤小鼠黑色素瘤细胞图片如图5所示，其中，A为对照组肿瘤注射1h后状态；B为试验组注射1h后状态；C为试验组注射4h后状态；D为试验组注射24h后状态。
通过图5可知，对照组注射1h可观察到细胞碎片和少量晶体析出。而在肿瘤内注射含过氧化氢的药剂后，试验组1h内有大量晶体析出，4h时出现更多的晶体，24h时出现较多的细胞碎片和更多的晶体。这说明，过氧化氢与抗癌药物联用有利于药物进入肿瘤细胞。
实施例3 过氧化氢对人体阿糖胞苷代谢的影响
将盐酸阿糖胞苷以 99mTc标记( 99mTc Ara-C)，将其分别用双氧水溶液和生理盐水溶液配制成浓度为10mg/mL的溶液，对一例肝癌患者的两个肿瘤进行瘤内注射，使用过氧化氢的标记为A，不使用过氧化氢的标记为B。肿瘤内注射后不同时间点用ECT成像系统监测 99mTc标记的阿糖胞苷信号。结果如图6所示，其中，上部分是实际成像图，下部分是示意图。
如图所示，注射15min后A、B两点的放射强度记为100％；注射治疗4h后，A点的放射强度下降为82％，而B点的放射强度仅为16％；治疗24h后，A点的放射强度下降为60％，而B点的放射强度以降至背景强度(0％)。 这说明，使用过氧化氢与不使用过氧化氢相比，阿糖胞苷在肿瘤中的保留时间显著延长。
实施例4 荷瘤小鼠中过氧化氢和化疗药物联用对药物分布影响
在无菌条件下，将制备的H22小鼠肝癌细胞悬浮液注射于Balb/c小鼠前腿内侧皮下注射H22肝癌细胞5×10 5个/0.1mL，建立皮下实体瘤模型。肿瘤株接种后，直到肿瘤最大直径达5-8mm，按照以下方案进行瘤内注射，注射剂量为0.2mL：生理盐水中包含盐酸阿霉素1mg/mL，盐酸阿糖胞苷10mg/mL，H 2O 2 18mg/mL。
注射后1h、3h、7h、12h、24h、48h，肝素管取静脉血，离心获取血浆，肿瘤组织以生理盐水均浆后离心获得上清液，以血浆和上清液为样品进行HPLC-MS检测分析，检测阿糖胞苷和阿霉素的浓度。肿瘤注射后，阿糖胞苷和阿霉素在血浆中的浓度随时间的变化如图7所示；血浆中药剂含量占肿瘤注射总量百分比随时间的变化如图8所示。
根据图7和图8可知，血清样品中阿糖胞苷和阿霉素浓度处于极低水平，而且随着时间的推移逐渐降低。注射后1h静脉血中阿糖胞苷和阿霉素浓度分别为(19680.26±3302.49)ng/mL和(112.71±46.59)ng/mL，分别占肿瘤注射总剂量的(0.974±0.163)％和(0.056±0.023)％。注射后48小时，血液中检测到的阿糖胞苷和阿霉素分别占总注射量的(0.002±0.001)％和(0.002±0.005)％。
通过对注射1-48h后肿瘤组织的检测发现：各肿瘤中阿糖胞苷和阿霉素的浓度均呈现出很高的水平且随时间逐渐降低，1h时肿瘤内阿糖胞苷和阿霉素的浓度分别为(84405.68±42499.23)ng/mL和(5504.23±3417.06)ng/mL， 分别占肿瘤注射总剂量的(5.998±3.891)％和(3.642±2.353)％。阿糖胞苷浓度从1小时到7小时迅速下降，而阿霉素浓度随着时间的推移下降得更慢。
根据以上试验结果，瘤内注射对血液循环的泄漏量不超过总剂量的1.137％。试验还表明，肿瘤中阿糖胞苷总量(119.957±77.814)μg是血液中阿糖胞苷总量(19.483±3.269)μg的6倍，肿瘤中阿霉素总量(7.285±4.707)μg是血液中阿霉素总量(0.112±0.046)μg的65倍左右。
为了研究为什么血液和肿瘤药物的总和不等于肿瘤注射药物的总量，对荷瘤小鼠肿瘤进行体外PK研究。在玻璃器皿中向每个肿瘤注射0.2mL前述阿糖胞苷、阿霉素和过氧化氢的混合注射液，然后采用与体内研究相同的方法制备检测样品以用于进行HPLC-MS检测分析。结果发现：检测到的阿霉素占总剂量的3.414％，与瘤内注射后1h体内PK研究中阿霉素的剂量比(3.642±2.353)％非常接近。结果表明，95％-97％的阿霉素与肿瘤蛋白粘附，注射后立即处于非游离状态，无瘤外渗漏。在肿瘤内注射药物中，阿霉素的检出率约为3％。根据这一事实和逆向计算，肿瘤在1h内的残留率应为98％-99％。类似的，检测到阿糖胞苷占总剂量的约5％，93％-95％的阿糖胞苷与肿瘤蛋白粘附，处于非游离状态。
综合以上结果可知，荷瘤小鼠注射过氧化氢和水溶性化疗药物后，阿霉素释放到血液中的量不超过总剂量的1％左右，而超过98％的药物残留在肿瘤内；血液中释放的阿糖胞苷量不超过给药总剂量的5％，超过93％的药物残留在肿瘤内，因此，过氧化氢与水溶性化疗药物进行肿瘤内注射在临床是安全的。过氧化氢有助于水溶性化疗药物成分在肿瘤中的停留时间更 长，并使进入外周血的量降至最低，从而将副作用降至最低。
实施例5 过氧化氢和化疗药物肿瘤注射对荷瘤小鼠生存曲线的影响
在无菌条件下，将制备的H22肝癌细胞悬浮液注射于Balb/c小鼠前腿内侧皮下，注射H22肝癌细胞5×10 5个/0.1mL，建立皮下实体瘤模型。肿瘤株接种后，直到肿瘤最大直径达5-8mm，按照以下方案进行瘤内注射，注射剂量为0.1mL：(1)空白对照组：生理盐水；(2)阳性对照组：盐酸阿霉素1mg/mL，盐酸阿糖胞苷10mg/mL；(3)试验组：盐酸阿霉素1mg/mL，盐酸阿糖胞苷10mg/mL，H 2O 2 18mg/mL。
于注射后1h、24h、168h对阳性对照和试验组小鼠的肿瘤进行组织切片病理观察，结果如图9所示，其中，A1-3为阳性对照组注射1h、24h、168h时的病理切片图(100×)；B1为试验组注射24h的病理切片图(100×)，B2为试验组注射168h时的病理切片图(400×)。由该图可知，阳性对照组注射后1小时肿瘤细胞生长良好，注射后24小时肿瘤细胞浸润肌肉，注射后168h肿瘤组织部分坏死；而试验组注射后24小时有炎性细胞浸润区，注射后168h明显纤维化增生。这说明，化疗药物联合双氧水注射后肿瘤中出现更多的炎症细胞，从而形成纤维化细胞。
对三组小鼠进行为期100天的生存分析，绘制生存曲线，结果如图10所示：试验组小鼠的中位生存期为75天，阳性对照组和空白对照组分别为56天和46天。这说明，试验组显著延长了生存期，与阳性对照组和空白对照组的p值分别为0.022和0.015，差异显著。

Claims (8)

  1. 一种过氧化物在制备实体肿瘤治疗药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，过氧化物与抗癌药物混合成为组合物。
  3. 根据权利要求2所述的应用，其特征在于，所述抗癌药物为一种或者几种水溶性药物。
  4. 根据权利要求2所述的应用，其特征在于，所述抗癌药物选自氮芥类，亚硝基脲类，生物碱，鬼臼毒素，类抗生素类，抗代谢类抗癌药物。
  5. 根据权利要求4所述的应用，其特征在于，所述氮芥类抗癌药物选自二氯甲基二乙胺、环磷腺苷、米尔法兰、苯丁酸氮芥或苯达莫司汀；亚硝基脲类抗癌药物为氯乙亚硝脲；生物碱抗癌药物选自长春新碱、长春地辛或长春瑞宾；鬼臼毒素类抗癌药物选自替尼泊苷或依托泊苷；抗生素类抗癌药物选自博来霉素、道诺霉素、光辉霉素、丝裂霉素或阿霉素；抗代谢类抗癌药物选自氟尿嘧啶、阿糖胞苷或吉西他滨。
  6. 根据权利要求1所述的应用，其特征在于，所述过氧化物选自过氧化氢。
  7. 根据权利要求1所述的应用，其特征在于，所述实体肿瘤为原发性肿瘤或转移瘤。
  8. 根据权利要求1所述的应用，其特征在于，所述实体肿瘤选自骨髓瘤或鳞状细胞癌。
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