WO2021012886A1 - 一种抗肿瘤的联合用药物组合物及其应用 - Google Patents

一种抗肿瘤的联合用药物组合物及其应用 Download PDF

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WO2021012886A1
WO2021012886A1 PCT/CN2020/098692 CN2020098692W WO2021012886A1 WO 2021012886 A1 WO2021012886 A1 WO 2021012886A1 CN 2020098692 W CN2020098692 W CN 2020098692W WO 2021012886 A1 WO2021012886 A1 WO 2021012886A1
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plasmodium
tumor
pharmaceutical composition
treatment group
combination
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French (fr)
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陶铸
亢中奎
秦莉
陈小平
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广州中科蓝华生物科技有限公司
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Priority to AU2020317239A priority Critical patent/AU2020317239A1/en
Priority to EP20843839.0A priority patent/EP4005591A4/en
Priority to US17/629,673 priority patent/US20220249643A1/en
Publication of WO2021012886A1 publication Critical patent/WO2021012886A1/zh

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    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • A—HUMAN NECESSITIES
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    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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    • 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
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    • 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
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    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/68—Protozoa, e.g. flagella, amoebas, sporozoans, plasmodium or toxoplasma
    • A—HUMAN NECESSITIES
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
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    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the application belongs to the field of biomedicine, and relates to an anti-tumor combined pharmaceutical composition and its application.
  • Tumor treatment methods include three traditional modes: surgery, radiation, and chemotherapy (including targeted therapy), as well as new biological treatments (including immunotherapy).
  • Clinically, comprehensive treatment is the mainstay.
  • Surgery combined with radiotherapy and chemotherapy has a better effect on the treatment of early-detected tumors, but the effect is not good for advanced tumors.
  • various biological therapies provide more options.
  • Targeted drugs, antibodies, immunotherapy drugs (such as PD-1) and cell therapy such as CAR-T, TIL cells) have made some progress, but It has not stopped the rapid development of cancer.
  • Malaria is mainly a vector-borne infectious disease transmitted by the bite of Anopheles mosquitoes and caused by Plasmodium infection. It is listed by the World Health Organization as one of the world's three major infectious diseases (AIDS, tuberculosis and malaria).
  • AIDS HIV, tuberculosis and malaria.
  • Plasmodium falciparum Plasmodium falciparum
  • Plasmodium vivax Plasmodium vivax
  • Plasmodium ovale Plasmodium nori. The most common are Plasmodium falciparum and Plasmodium vivax.
  • Plasmodium charlie Plasmodium yoelii
  • Plasmodium berghei Plasmodium venturia. It was proved in mice that Plasmodium infection significantly inhibited tumor growth and metastasis in tumor-bearing mice with tumor models such as lung cancer, liver cancer, colon cancer, and breast cancer, and significantly prolonged the survival time of tumor-bearing mice.
  • chemotherapy mainly uses cytotoxic drugs to kill tumor cells, but it does not specifically recognize tumor cells, and there are toxic side effects of drugs.
  • Existing chemotherapy is increasingly difficult to control tumor development.
  • Combining chemotherapy and biological therapies to treat tumors has become a promising treatment strategy, combining chemotherapy and biological therapies to exert a synergistic effect.
  • CN107929231A discloses an in-situ thermosensitive gel drug delivery system for combined chemotherapy and immunity.
  • the delivery system is composed of drug carrier peptide dendrimers, anti-tumor drugs and temperature-sensitive gel matrix material PLGA-PEG-PLGA.
  • the anti-tumor drugs are encapsulated in the cavity of peptide dendrimers to form drug-loaded peptide trees
  • the drug-loaded peptide dendrimer is wrapped in a temperature-sensitive gel matrix through a swelling process to form an injectable sol at room temperature, and an in-situ drug delivery system that turns into a gel at body temperature.
  • the in situ delivery system of the invention can form a gel near the tumor, slowly release drug-loaded peptide dendrimer molecules to act on tumor tissues, and peptide dendrimer molecules act on macrophages in tumor tissues to produce NO , It acts on tumors together with anti-tumor drugs to form a combined chemotherapy and immune therapy to inhibit tumor development.
  • CN103347521A discloses a method for treating cancer, which includes the administration of a metabolically targeted chemotherapy-immunotherapy regimen.
  • a metabolically targeted chemotherapy-immunotherapy regimen may include the following steps: administering a therapeutically effective dose of one or more immunological agents (eg, therapeutic antibodies) in a subject suffering from cancer to stimulate immunity Response, and administration of a therapeutically effective dose of one or more chemotherapeutic agents.
  • the method of the invention can be used to treat any type of cancer, especially malignant and metastatic advanced cancer.
  • CN102481365A discloses a method for treating various types of cancers/tumors by administering a combination of a dll4 antagonist and a chemotherapeutic agent, wherein the dll4 antagonist, especially a dll4 antibody and fragments thereof that specifically bind to dll4.
  • a combination therapy shows a synergistic effect compared to treatment with either therapeutic agent alone. Therefore, the method of the present invention is particularly beneficial for cancer patients who have low tolerance to side effects caused by the high dose required for treatment with either therapeutic agent alone, because the effective dose can be reduced.
  • a pharmaceutical composition and a kit containing a dll4 antagonist and a chemotherapeutic agent are also provided.
  • This application provides an anti-tumor combined pharmaceutical composition and its application.
  • the combined pharmaceutical composition successfully combines chemotherapy and malaria parasite immunotherapy (a biological therapy), and has high biological safety , Has stronger anti-tumor activity than single chemotherapy or single Plasmodium immunotherapy, can prolong the survival time of tumor mouse models, and provide new strategies and ideas for cancer treatment; at the same time, it can reduce the dosage of chemotherapy drugs , Reduce the side effects of chemotherapy drugs and reduce the cost of treatment for cancer patients; in addition, chemotherapy drugs can block tumor cell proliferation or directly kill tumor cells to cause tumor cell death, and promote tumor cells to be phagocytosed and processed by macrophages to release tumor antigens.
  • the induction of protozoal immunotherapy promotes the enhancement of the antigen presentation ability of macrophages and dendritic cells, thereby inducing a stronger anti-tumor specific immune response and exerting the synergistic effect of continuous immunotherapy and chemotherapy.
  • the present application provides an anti-tumor combined pharmaceutical composition, which includes Plasmodium and chemotherapeutics.
  • the Plasmodium in the combined pharmaceutical composition exerts anti-tumor effects mainly by activating natural immunity and inducing a certain specific immunity.
  • the dangerous signal molecules released by the Plasmodium infection include glycosylphosphatidyl Inositol anchors, heme, immunostimulatory nucleic acid motifs, and other unknown molecules, these pathogen-related pattern recognition molecules can be recognized by the pattern recognition receptors of the host's immune cells.
  • Pattern recognition receptors activated by Plasmodium pattern recognition molecules trigger different transcriptional programs and stimulate multiple downstream signal pathways to induce systemic immune responses, including the release of pro-inflammatory factors and Th1 cytokines, and activation of NK cells, NKT cells, and macrophages Cells and dendritic cells further activate CD4 + and CD8 + T cells, inhibit the production of cytokines such as TGF- ⁇ and IL-10, inhibit regulatory T cells (Tregs), bone marrow-derived suppressor cells (MDSCs) and tumors Cellular activities such as related macrophages (TAMs) improve the tumor immunosuppressive microenvironment and transform the immunosuppressive microenvironment in tumor patients into an immune activated state, thereby transforming the tumor into an effective tumor vaccine.
  • Plasmodium infection activates immunity through damage-related molecular patterns. Red blood cells infected by Plasmodium and known endogenous uric acid and microvesicles can induce similar immune activity.
  • chemotherapeutic drugs mainly include alkylating chemotherapy drugs, antimetabolites chemotherapeutics, antibiotic chemotherapeutics, hormone chemotherapeutics, animal and plant chemotherapeutics and miscellaneous chemotherapeutics drug.
  • Alkylating agent chemotherapeutic drugs directly act on DNA to prevent cancer cells from regenerating.
  • Common alkylating agent drugs include cyclophosphamide, ifosfamide, and cetepa.
  • Anti-metabolic chemotherapy drugs prevent cell division and proliferation by interfering with the synthesis of DNA and RNA, mainly gemcitabine, pemetrexed, fluorouracil, cytarabine, methotrexate, mercaptopurine, hydroxyurea, etc.
  • Antibiotic chemotherapeutic drugs interfere with DNA by inhibiting the action of enzymes and mitosis or changing cell membranes. They are mainly cell cycle non-specific drugs, including mitomycin, daunorubicin, pingyangmycin, doxorubicin, and radioactive drugs. Nematoxin D, Mitoxantrone, etc.
  • Animal and plant chemotherapeutics are mainly plant alkaloids and natural products, which can prevent the synthesis of proteins necessary for cell regeneration by inhibiting mitosis or enzyme action, mainly including vincristine, etoposide, teniposide, paclitaxel, and doxyl They are usually used in combination with other chemotherapy drugs to treat tumors.
  • Hormonal chemotherapy drugs kill or slow down the growth of hormone-dependent tumor cells through the application of certain hormones or antagonists, mainly including tamoxifen, megestrol, triptorelin, and medroxyprogesterone.
  • Miscellaneous chemotherapy drugs mainly including cisplatin, carboplatin, oxaliplatin, asparaginase, etc.
  • HIV protease inhibitors are a class of chemotherapeutic drugs for the treatment of HIV infection. They inhibit the protease activity of the virus, thereby inhibiting the cleavage of aspartyl protein precursors into its functional form to prevent the maturation of HIV virus particles.
  • HIV PIs mainly include nelfinavir (nelfinavir), saquinavir (saquinavir), indinavir (indinavir), and ritonavir (ritonavir). Research by Gills et al.
  • HIV PIs has anti-cancer activity on more than 60 cell lines, and explained mechanisms including endoplasmic reticulum stress, autophagy, apoptosis, and inhibition of the Akt pathway.
  • nelfinavir combined with gemcitabine, cisplatin or radiotherapy can enhance Akt inhibition and enhance the sensitivity of radiotherapy, and no side effects caused by Nelfinavir have been found.
  • Rengan et al. found that in patients with unresectable non-small cell lung cancer, nelfinavir combined with etoposide, cisplatin, or radiotherapy also found no dose-dependent toxicity of nelfinavir.
  • Plasmodium infection can systemically activate the body's natural immunity and enhance the body's specific immunity against tumors.
  • Chemotherapy drugs including HIV PIs, can directly kill tumor cells or inhibit the proliferation of tumor cells.
  • the combined pharmaceutical composition successfully combines chemotherapy and malaria parasite immunotherapy (a biological therapy), and has high biological safety, which is stronger than single chemotherapy and single malaria parasite immunotherapy.
  • Anti-tumor activity can prolong the survival time of cancer-bearing mice and provide new strategies and ideas for cancer treatment; at the same time, it can reduce the dosage of chemotherapy drugs, reduce the side effects of chemotherapy drugs, and reduce the treatment cost of tumor patients;
  • the combined pharmaceutical composition can promote tumor cells to release tumor antigens, induce a stronger anti-tumor specific immune response, and exert the synergistic effect of continuous immunotherapy and chemotherapy.
  • the chemotherapeutic drugs include alkylating agent chemotherapeutics, antimetabolites chemotherapeutics, antibiotic chemotherapeutics, animal and plant chemotherapeutics, miscellaneous chemotherapeutics, or HIV protease inhibitors.
  • the alkylating agent chemotherapeutic agent includes cyclophosphamide or ifosfamide.
  • the antimetabolite chemotherapeutic drug includes gemcitabine, pemetrexed, 5-fluorouracil, cytarabine or methotrexate.
  • the antibiotic chemotherapeutic agent includes mitomycin, doxorubicin or actinomycin D.
  • the animal and plant chemotherapeutic drugs include etoposide, docetaxel, paclitaxel, vincristine or irinotecan.
  • the miscellaneous chemotherapeutic drugs include cisplatin, carboplatin, oxaliplatin or asparaginase.
  • the HIV protease inhibitor chemotherapeutic drugs include nelfinavir, saquinavir, indinavir or ritonavir.
  • the dosage form of the combined pharmaceutical composition includes any pharmaceutically acceptable dosage form.
  • any pharmaceutically acceptable dosage form for example, tablets, powders, suspensions, granules, capsules, injections, sprays, solutions, enemas, emulsions, films, suppositories, patches, nasal drops or pills, etc.
  • the combination pharmaceutical composition further includes any one or a combination of at least two of the pharmaceutically acceptable pharmaceutical excipients.
  • the combination pharmaceutical composition described in the present application can be administered alone or in combination with adjuvants to form an appropriate dosage form for administration.
  • the adjuvants include diluents, excipients, fillers, binders, wetting agents, and disintegrants. Any one or a combination of at least two of desolvents, emulsifiers, solubilizers, solubilizers, osmotic pressure regulators, surfactants, pH regulators, antioxidants, bacteriostatic agents, or buffers.
  • the combination of the at least two kinds such as a combination of a diluent and an excipient, a combination of an emulsifier and a cosolvent, a combination of a filler and a binder and a wetting agent, and the like.
  • the combined pharmaceutical composition is a single compound preparation.
  • the combined pharmaceutical composition is a combination of two separate formulations.
  • the two separate formulations are administered simultaneously.
  • the two separate formulations are administered sequentially.
  • the combined pharmaceutical composition can be in the form of a single compound preparation, or a combination of two separate preparations; when it is a combination of two separate preparations, the mode of administration can be simultaneous administration or sequential administration
  • the plasmodium can be administered first and then the chemotherapy drugs can be administered at intervals, or the chemotherapeutic drugs can be administered first and then the malaria parasites can be administered at a certain interval, or the plasmodium and the chemotherapy drugs can be administered alternately.
  • the administration route of the combination pharmaceutical composition includes intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration, preferably intraperitoneal injection .
  • the combined pharmaceutical composition is a combined pharmaceutical composition loaded on a pharmaceutical carrier.
  • the pharmaceutical carrier includes liposomes, micelles, dendrimers, microspheres or microcapsules.
  • the present application provides an application of the above-mentioned combination pharmaceutical composition in the preparation of antineoplastic drugs.
  • the tumor includes lung cancer, stomach cancer, colon cancer, liver cancer, breast cancer or pancreatic cancer.
  • the present application provides a novel anti-tumor combination therapy, which is a combination therapy of chemotherapy and malaria parasite therapy.
  • the combination of chemotherapy and malaria parasite immunotherapy has higher biological safety, and has stronger anti-tumor activity than single chemotherapy and single malaria parasite immunotherapy, and can more effectively prolong the survival of tumor-bearing mice It provides new strategies and ideas for cancer treatment; at the same time, it can reduce the dosage of chemotherapy drugs, reduce the side effects of chemotherapy drugs, and reduce the treatment cost of tumor patients; in addition, the combination therapy can promote tumor cells to release tumor antigens. Induce a stronger anti-tumor specific response and exert the synergistic effect of continuous immunotherapy and chemotherapy.
  • the tumor includes lung cancer, stomach cancer, colon cancer, liver cancer, breast cancer or pancreatic cancer.
  • the chemotherapeutics used in the chemotherapy include alkylating agent chemotherapeutics, antimetabolites chemotherapeutics, antibiotic chemotherapeutics, animal and plant chemotherapeutics, miscellaneous chemotherapeutics, or HIV protease inhibitors.
  • the alkylating agent chemotherapeutic agent includes cyclophosphamide or ifosfamide.
  • the antimetabolite chemotherapeutic drug includes gemcitabine, pemetrexed, 5-fluorouracil, cytarabine or methotrexate.
  • the antibiotic chemotherapeutic agent includes mitomycin, doxorubicin or actinomycin D.
  • the animal and plant chemotherapeutic drugs include etoposide, docetaxel, paclitaxel, vincristine or irinotecan.
  • the miscellaneous chemotherapeutic drugs include cisplatin, carboplatin, oxaliplatin, asparaginase and the like.
  • the HIV protease inhibitor chemotherapeutic drugs include nelfinavir, saquinavir, indinavir, and ritonavir.
  • the administration route of the chemotherapy includes intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration, preferably intravenous injection Or oral administration.
  • the route of administration of the malaria parasite therapy includes intravenous injection.
  • the anti-tumor combined pharmaceutical composition involved in this application combines chemotherapy and Plasmodium immunotherapy (a kind of biological therapy), and has higher biological safety than single chemotherapy and single Plasmodium immunotherapy. Stronger anti-tumor activity can more effectively prolong the survival period of cancer patients, and provide new strategies and ideas for cancer treatment; at the same time, it can reduce the dosage of chemotherapy drugs, reduce the side effects of chemotherapy drugs, and reduce tumor patients.
  • the combined pharmaceutical composition can promote tumor cells to release tumor antigens, induce a stronger anti-tumor specific response, and exert the synergistic effect of continuous immunotherapy and chemotherapy.
  • Figure 1 is a graph of the tumor growth curve in Example 1;
  • Figure 2 is a graph of survival curves of tumor-bearing mice in Example 1;
  • Figure 3 is a graph showing the infection rate of Plasmodium in Example 1;
  • Figure 4 is a graph showing the body weight growth curve of tumor-bearing mice in Example 1.
  • Figure 5 is a graph of tumor growth curve in Example 2.
  • Figure 6 is a graph of survival curves of tumor-bearing mice in Example 2.
  • Figure 7 is a graph showing the infection rate of Plasmodium in Example 2.
  • Figure 8 is a graph showing the body weight growth curve of tumor-bearing mice in Example 2.
  • Figure 9 is a graph of the tumor growth curve in Example 3.
  • Figure 10 is a graph of survival curves of tumor-bearing mice in Example 3.
  • Fig. 11 A graph of the infection rate of Plasmodium in Example 3.
  • Figure 12 is a graph showing the body weight growth curve of tumor-bearing mice in Example 3.
  • Figure 13 is a graph of the tumor growth curve in Example 4.
  • Figure 14 is a graph of survival curves of tumor-bearing mice in Example 4.
  • Example 15 is a graph of the infection rate of Plasmodium in Example 4.
  • Figure 16 is a graph showing the body weight growth curve of tumor-bearing mice in Example 4.
  • Figure 17 is a graph of the tumor growth curve in Example 5.
  • Figure 18 is a graph of survival curves of tumor-bearing mice in Example 5.
  • Figure 19 is a graph showing the infection rate of Plasmodium in Example 5.
  • Figure 21 is a graph of tumor growth curve in Example 6.
  • Figure 22 is a graph of survival curves of tumor-bearing mice in Example 6.
  • Figure 23 is a graph showing the infection rate of Plasmodium in Example 6.
  • Figure 24 is a graph showing the body weight growth curve of tumor-bearing mice in Example 6.
  • Figure 25 is a graph of tumor growth curve in Example 7.
  • Figure 26 is a graph of survival curves of tumor-bearing mice in Example 7.
  • Figure 27 is a graph of the infection rate of Plasmodium in Example 7.
  • Figure 28 is a graph showing the body weight growth curve of tumor-bearing mice in Example 7.
  • Figure 29 is a graph of tumor growth curve in Example 8.
  • Figure 30 is a graph of survival curves of tumor-bearing mice in Example 8.
  • Figure 31 is a graph showing the infection rate of Plasmodium in Example 8.
  • Figure 32 is a graph showing the body weight growth curve of tumor-bearing mice in Example 8.
  • Figure 33 is a graph of the tumor growth curve in Example 9.
  • Figure 34 is a graph of survival curves of tumor-bearing mice in Example 9;
  • Figure 36 is a graph showing the body weight growth curve of tumor-bearing mice in Example 9;
  • Figure 37 is a graph of tumor growth curve in Example 10.
  • Figure 38 is a graph of survival curves of tumor-bearing mice in Example 10.
  • Figure 39 is a graph showing the infection rate of Plasmodium in Example 10.
  • Example 40 is a graph showing the body weight growth curve of the tumor-bearing mice in Example 10.
  • Figure 41 is a graph of the tumor growth curve in Example 11.
  • Figure 42 is a graph of survival curves of tumor-bearing mice in Example 11.
  • Figure 43 is a graph showing the infection rate of Plasmodium in Example 11.
  • Figure 44 is a graph showing the body weight growth curve of tumor-bearing mice in Example 11.
  • Figure 45 is a graph of the tumor growth curve in Example 12.
  • Figure 46 is a graph showing the survival curve of tumor-bearing mice in Example 12.
  • Figure 47 is a graph of the infection rate of Plasmodium in Example 12.
  • Fig. 48 is a graph showing the body weight growth curve of tumor-bearing mice in Example 12.
  • This embodiment provides an anti-tumor combined pharmaceutical composition, which includes Plasmodium yoelii and the chemotherapy drug gemcitabine, and observe the effect of gemcitabine combined with Plasmodium yoelii in treating lung cancer at different administration times.
  • Plasmodium Mouse Plasmodium yoelii (P.yoelii 17XNL, MRA-593, Py), a free gift from Malaria Research and Reference Reagent Resource Center (MR4);
  • Chemotherapeutic drugs Gemcitabine (Gemcitabine, abbreviated as GEM), purchased from Sigma-Aldrich;
  • Giemsa dye powder purchased from Sigma-Aldrich Company.
  • (4) Testing indicators include:
  • Tumor volume measurement The tumor is measured every 3 days, and the tumor volume (in cubic millimeters) is calculated using the ellipse volume calculation formula: (D ⁇ d ⁇ d)/2, where "D” represents the major diameter of the tumor, " d” means short diameter.
  • Tumor size was expressed as mean tumor volume ⁇ standard mean error (SEM), and a tumor growth curve was made.
  • SEM standard mean error
  • Statistical analysis between groups was performed by TWO-WAY ANOVA analysis of variance. When p ⁇ 0.05, it is represented by "*”, and when p ⁇ 0.01, it is represented by "**”, both of which indicate that the differences between groups have significant statistical significance.
  • the infection rate of Plasmodium is evaluated by the percentage of mouse red blood cells infected with Plasmodium. The calculation formula is: (number of red blood cells infected with Plasmodium/total red blood cells) ⁇ 100%; the specific operation is to take blood from the tail vein The slices were fixed with methanol and stained with Giemsa stain. The number of Plasmodium infecting red blood cells and the total number of red blood cells were observed under a microscope. The total number of red blood cells was about 1000. The infection rate of Plasmodium was calculated. The infection rate was average infection rate ⁇ standard average Error (SEM) representation, and draw the Plasmodium infection cycle curve to observe whether the chemotherapy drugs have an impact on the Plasmodium infection.
  • SEM standard average Error
  • Plasmodium yoelii treatment group (Py), gemcitabine treatment group administered on day 3 (GEM(d3)), gemcitabine treatment group administered on day 6 (GEM( d6), both the gemcitabine combination therapy group (Py+GEM(d3)) administered on day 3 and the gemcitabine combination therapy group (Py+GEM(d6)) administered on day 6 significantly inhibited the growth of lung cancer.
  • the combined treatment group administered on day 6 may have a better effect on suppressing lung cancer than the combined treatment group administered on day 3, but it is not statistically significant.
  • the combined treatment group administered on day 6 is better than a single Plasmodium yoelii treatment Group, single gemcitabine treatment group administered on the 6th day has a better effect of inhibiting lung cancer, with significant statistical significance.
  • the median survival time of the control tumor group is 30.5 days
  • the Plasmodium yoelii treatment group is 38.5 days
  • the gemcitabine treatment group administered on the 3rd day is 34.5 days
  • the 6th day The gemcitabine treatment group was administered for 33 days
  • the combination treatment group administered on day 3 was 42.5 days
  • the combination treatment group administered on day 6 was 45 days.
  • Plasmodium yoelii treatment group (Py), gemcitabine treatment group administered on day 3 (GEM(d3)), gemcitabine treatment group administered on day 6 (GEM(d6), gemcitabine treatment group administered on day 3) Both group (Py+GEM(d3)) and the gemcitabine combination therapy group (Py+GEM(d6)) administered on day 6 significantly prolonged the survival time of tumor-bearing mice.
  • the combination therapy group administered on day 6 The median survival of the combined treatment group administered on day 3 was long, but it was not statistically significant.
  • the median survival of the combined treatment administered on day 6, combined with a single treatment of Plasmodium yoelii and a single gemcitabine treatment The period is long, but the difference in survival is not statistically significant.
  • the gemcitabine combination group administered on the 6th day can significantly inhibit the growth of lung cancer and prolong the median survival time of tumor-bearing mice.
  • Gemcitabine has an inhibitory effect on the malaria parasite, but it does not eliminate the parasite.
  • the combination of gemcitabine and plasmodium has a superimposed effect on weight loss in tumor-bearing mice.
  • the total dose of 100mg/kg, gemcitabine administered on the 6th day and Plasmodium yoelii inoculated on the 7th day is a promising combination drug composition for the treatment of lung cancer.
  • This embodiment provides an anti-tumor combined pharmaceutical composition, the combined pharmaceutical composition comprising Plasmodium yoelii and the chemotherapy drug gemcitabine, observe that the total dose is the same, single or divided gemcitabine administration combined with Joel's The effect of malaria parasite therapy on lung cancer.
  • Steps (I) (II) (III) are the same as in Example 1.
  • mice randomly divided into six groups according to tumor size: control tumor group (Con), Plasmodium yoelii treatment group (Py), single-dose gemcitabine treatment group (GEM (single)), divided Gemcitabine treatment group (GEM (divided), single-dose combination therapy group (Py+GEM (single)), divided-dose combination therapy group (Py+GEM (divided)).
  • Group 10 mice 60 mice in total.
  • GEM (single) treatment group and Py+GEM (single) treatment group are administered on the 6th day of tumor inoculation, the dosage is 100 mg/kg, and the drug concentration is 10 mg/mL.
  • the GEM (fractionated) treatment group and the Py+GEM (fractionated) group were administered on the 6th and 13th day of tumor inoculation, respectively, at a dose of 50 mg/kg and a drug concentration of 5 mg/kg.
  • the split-dose gemcitabine combined treatment group is better than the single-dose gemcitabine combined treatment group to inhibit the growth of lung cancer.
  • the two combined treatments are better than the single Plasmodium yoelii treatment group and the single gemcitabine treatment group.
  • the effect of inhibiting the growth of lung cancer is good.
  • the median survival time of the control tumor group is 30.5 days
  • the Plasmodium yoelii treatment group is 38.5 days
  • the single-dose gemcitabine treatment group is 33 days
  • divided doses of gemcitabine The treatment group was 38.5 days
  • the single-dose gemcitabine combined with Plasmodium yoelii treatment group was 45 days
  • the divided dose of gemcitabine combined with Plasmodium yoelii treatment group was 52.5 days.
  • the two combined treatment groups significantly prolonged the median survival time of tumor-bearing mice. Both the two combination treatment groups were more effective than the corresponding single gemcitabine treatment group to prolong the median survival of tumor-bearing mice.
  • the split-dose combination therapy group is more effective than the single-dose combination therapy group to prolong the survival time of tumor-bearing mice.
  • the survival difference between single-dose combination therapy and single malaria parasite treatment is not statistically significant, and split-dose combination therapy is more effective than single malaria parasite therapy to prolong the median survival of tumor-bearing mice .
  • the combined treatment with gemcitabine administered in divided doses has the effect of inhibiting the growth of lung cancer and prolonging the median survival of tumor-bearing mice more effectively than the combined treatment with single-dose gemcitabine.
  • Single and divided administrations of gemcitabine have inhibitory effects on Plasmodium yoelii.
  • Divided administration of gemcitabine has less effect on the body weight of tumor-bearing mice than single administration.
  • the total dose of 100 mg/kg, gemcitabine administered in divided doses on the 6th and 13th days and the Plasmodium yoelii inoculated on the 7th day is a promising combination drug composition for the treatment of lung cancer.
  • This embodiment provides an anti-tumor combined pharmaceutical composition, which includes Plasmodium yoelii and the chemotherapeutic gemcitabine, to observe the effect of the optimized regimen of gemcitabine administration combined with Plasmodium immunotherapy in treating lung cancer.
  • Steps (I) (II) (III) are the same as in Example 1.
  • mice randomly divided into four groups according to tumor size: control tumor group (Con), malaria treatment group (Py), gemcitabine treatment group (GEM), gemcitabine combined with malaria treatment group (Py+GEM). There are 15 mice in each group, 60 mice in total.
  • the median survival time of the control tumor group is 26 days
  • the Plasmodium yoelii treatment group is 36 days
  • the gemcitabine treatment group is 36 days
  • the combination treatment group is 55 days.
  • Combination therapy is more effective than single Plasmodium yoelii treatment and single gemcitabine treatment to prolong the survival of tumor-bearing mice. It shows that the combined pharmaceutical composition of gemcitabine and Plasmodium yoelii can significantly prolong the survival of tumor-bearing mice.
  • the combined pharmaceutical composition of gemcitabine and Plasmodium yoelii has the effect of more effectively inhibiting the growth of lung cancer and prolonging the median survival period of tumor-bearing mice, but does not increase the toxic side effects of tumor-bearing mice.
  • the total dose of 100 mg/kg, gemcitabine administered in divided doses on the 6th and 13th days and the Plasmodium yoelii inoculated on the 7th day are a promising combination medicine composition for the treatment of lung cancer.
  • This embodiment provides an anti-tumor combined pharmaceutical composition, the combined pharmaceutical composition comprising Plasmodium chrysalis and chemotherapeutic gemcitabine, to observe the effect of gemcitabine administration combined with Plasmodium chrysii immunotherapy in the treatment of lung cancer with an optimized plan .
  • Example 3 The experimental materials and reagents required in this example are different from those in Example 3 only in that the malaria parasite uses the mouse Plasmodium chabaudi (MRA-429, Pc), from Malaria Research and Reference Reagent Resource Center (MR4) free of charge Gift.
  • MRA-429, Pc Mouse Plasmodium chabaudi
  • MR4 Malaria Research and Reference Reagent Resource Center
  • Step (I) (II) (III) is the same as in Example 3.
  • the control tumor group (LLC), the Plasmodium chara treatment group (Pc), the gemcitabine treatment group (GEM), the gemcitabine combined with the Plasmodium chara treatment group (Pc+GEM) are all Significantly inhibit the growth of lung cancer.
  • the combination therapy is more effective in inhibiting tumor growth than the single Plasmodium Charlie treatment group and the single gemcitabine treatment group. It shows that the combination medicine composition of gemcitabine and Plasmodium charlie has a more effective effect of inhibiting the growth of lung cancer.
  • the combined pharmaceutical composition of gemcitabine and Plasmodium chardii has the effect of more effectively inhibiting the growth of lung cancer and prolonging the median survival period of tumor-bearing mice, but does not increase the toxic side effects of tumor-bearing mice.
  • the total dose of 100 mg/kg, gemcitabine administered in divided doses on the 6th and 13th days and the Plasmodium chardii inoculated on the 7th day are a promising combination medicine composition for the treatment of lung cancer.
  • This embodiment provides an anti-tumor combined pharmaceutical composition.
  • the combined pharmaceutical composition includes Plasmodium yoelii and the chemotherapeutic drug cyclophosphamide. It is observed that the administration of cyclophosphamide combined with Plasmodium yoelii immunotherapy treats lung cancer. effect.
  • Example 2 The experimental materials and reagents required in this example are different from those in Example 1 only in that the chemotherapeutic drug is Cyclophosphamide (CTX), which is purchased from Sigma-Aldrich Company.
  • CTX Cyclophosphamide
  • Step (I) (II) (III) is the same as in Example 3.
  • Control tumor group (Con), Plasmodium treatment group (Py), Cyclophosphamide treatment group (CTX), Cyclophosphamide combined with Plasmodium treatment group (Py+CTX) Significantly inhibit the growth of lung cancer.
  • the combination therapy is more effective in inhibiting tumor growth than the single cyclophosphamide treatment group and the single Plasmodium yoelii treatment group. It shows that the combined pharmaceutical composition of cyclophosphamide and Plasmodium yoelii has the effect of inhibiting the growth of lung cancer more effectively.
  • the combined pharmaceutical composition of cyclophosphamide and Plasmodium yoelii has the effect of more effectively inhibiting the growth of lung cancer and prolonging the median survival period of tumor-bearing mice, and will not significantly increase the toxicity of tumor-bearing mice side effect.
  • the total dose of 60 mg/kg, cyclophosphamide administered in three doses on 6, 13, and 20 days, and Plasmodium yoelii inoculated on day 7, is a promising combination drug composition for the treatment of lung cancer.
  • This embodiment provides an anti-tumor combined pharmaceutical composition.
  • the combined pharmaceutical composition includes Plasmodium charlie and the chemotherapeutic drug cyclophosphamide. Observe the optimized plan of cyclophosphamide administration combined with the treatment of Plasmodium charlie Lung cancer effect.
  • Example 5 The difference between the experimental materials and reagent kits required in this example and Example 5 is only that the plasmodium is the mouse Plasmodium chabaudi (MRA-429, Pc), from Malaria Research and Reference Reagent Resource Center (MR4) Free gift.
  • MRA-429, Pc mouse Plasmodium chabaudi
  • MR4 Malaria Research and Reference Reagent Resource Center
  • Steps (I) (II) (III) are the same as in Example 5.
  • control tumor group (Con) plasmodium treatment group (Pc), cyclophosphamide treatment group (CTX), cyclophosphamide combined with plasmodium treatment group (Pc+CTX) are all Significantly inhibit the growth of lung cancer.
  • Combination therapy is more effective in inhibiting tumor growth than single Plasmodium chardii treatment and single cyclophosphamide treatment. It shows that the combined medicine composition of cyclophosphamide and Plasmodium chardii has the effect of inhibiting the growth of lung cancer more effectively.
  • This embodiment provides an anti-tumor combined pharmaceutical composition.
  • the combined pharmaceutical composition includes Plasmodium yoelii and the chemotherapeutic drug pemetrexed. Observe the optimized plan of pemetrexed administration combined with Plasmodium yoelii The effect of therapy on lung cancer.
  • the experimental materials and reagents required in this example are different from those in Example 3 only in that the chemotherapeutic drug is pemetrexed (abbreviated as PEM), purchased from Sigma-Aldrich Company.
  • PEM pemetrexed
  • Step (I) (II) (III) is the same as in Example 3.
  • mice randomly divided into four groups according to tumor size: control tumor group (Con), plasmodium treatment group (Py), pemetrexed treatment group (PEM), pemetrexed combined with plasmodium treatment group ( Py+PEM). There are 10 mice in each group, 40 mice in total.
  • control tumor group Con
  • plasmodium treatment group Py
  • pemetrexed treatment group PEM
  • pemetrexed combined with plasmodium treatment group Py+PEM
  • Combination therapy is more effective in inhibiting tumor growth than single pemetrexed treatment and single Plasmodium yoelii treatment. It shows that the combined pharmaceutical composition of pemetrexed and Plasmodium yoelii has the effect of inhibiting the growth of lung cancer more effectively.
  • the median survival time of the control tumor group is 28 days
  • the Plasmodium yoelii treatment group is 37 days
  • the pemetrexed treatment group is 32.5 days
  • the combination treatment group is 43 days .
  • the median survival time of combination therapy is longer than that of single malaria parasite treatment and single malaria parasite treatment, and the survival difference with single pemetrexed treatment is statistically significant, and there is no significant difference in survival from single malaria parasite treatment. The statistical significance. It is suggested that the combined pharmaceutical composition of pemetrexed and Plasmodium yoelii may be more effective than single treatment to prolong the survival period of tumor-bearing mice.
  • This embodiment provides an anti-tumor combined pharmaceutical composition, which includes Plasmodium yoelii and cisplatin, a chemotherapy drug, to observe the effect of cisplatin combined with Plasmodium yoelii therapy on lung cancer.
  • Example 3 The difference between the experimental materials required in this example and Example 3 is only that the chemotherapeutic drug is cis-platinum (DDP), which is purchased from Sigma-Aldrich.
  • DDP cis-platinum
  • Step (I) (II) (III) is the same as in Example 3.
  • Cisplatin administration the specific method is:
  • Combination therapy is more effective in inhibiting tumor growth than single cisplatin treatment and single Plasmodium yoelii treatment. It shows that the combined pharmaceutical composition of cisplatin and Plasmodium yoelii has a more effective effect of inhibiting the growth of lung cancer.
  • This embodiment provides an anti-tumor combined pharmaceutical composition.
  • the combined pharmaceutical composition includes Plasmodium yoelii and mitomycin. It is observed that different doses of mitomycin combined with Plasmodium yoelii immunotherapy are used to treat lung cancer. Effect.
  • the experimental materials and reagents required in this example are different from those in Example 3 only in that the chemotherapeutic drug is mitomycin (Mitomycin, abbreviated as MMC), which is purchased from Sigma-Aldrich.
  • MMC mitomycin
  • Step (I) (II) (III) is the same as in Example 3.
  • mice randomly divided into five groups according to tumor size: control tumor group (Con), plasmodium treatment group (Py), low-dose mitomycin combined with plasmodium treatment group (Py+MMC (low dose)) , Medium-dose mitomycin combined with Plasmodium treatment group (Py+MMC (medium dose)), high-dose mitomycin combined with Plasmodium treatment group (Py+MMC (high dose)). There are 10 mice in each group, 50 mice in total.
  • control tumor group (Con) plasmodium treatment group (Py), low-dose mitomycin combined with plasmodium treatment group (Py+MMC (low dose)), medium dose Mitomycin combined with Plasmodium treatment group (Py+MMC (medium dose)), high-dose mitomycin combined with Plasmodium treatment group (Py+MMC (high dose)) significantly inhibited the growth of lung cancer.
  • Combination therapy shows a dose-dependent effect of mitomycin, the larger the dose, the better the effect of inhibiting the growth of lung cancer. It shows that the combination of Plasmodium yoelii and mitomycin has a more effective effect on inhibiting the growth of lung cancer.
  • the tumor group is 28 days
  • the single Plasmodium yoelii treatment group is 36 days
  • the low-dose mitomycin combination treatment group is 44 days
  • the medium-dose mitosis The combined treatment group of mitomycin was 34 days
  • the combination treatment group of high-dose mitomycin was 31 days.
  • the low-dose mitomycin combination therapy group is more effective than the medium and high-dose mitomycin combination therapy group to prolong the survival of tumor-bearing mice, indicating the combination of low-dose mitomycin and Plasmodium yoelii Medication can more effectively prolong the survival period of tumor-bearing mice.
  • Medium and high doses of mitomycin may have certain side effects.
  • This embodiment provides an anti-tumor combined pharmaceutical composition.
  • the combined pharmaceutical composition includes Plasmodium yoelii and the chemotherapy drug docetaxel. It is observed that the administration of different doses of docetaxel combined with Plasmodium yoelii The effect of immunotherapy on lung cancer.
  • Example 9 The experimental materials and reagents required in this example are different from those in Example 9 only in that the chemotherapeutic drug is Docetaxel (DTX), which is purchased from Sanofi.
  • DTX Docetaxel
  • Step (I) (II) (III) is the same as in Example 9.
  • Control tumor group (Con), Plasmodium treatment group (Py), low-dose docetaxel combined with Plasmodium treatment group (Py+DTX (low dose)), medium Docetaxel combined with Plasmodium treatment group (Py+MMC (medium dose)), high dose docetaxel combined with Plasmodium treatment group (Py+MMC (high dose)) can significantly inhibit the growth of lung cancer.
  • the three combined treatments are better than the single malaria parasite treatment in inhibiting the growth of lung cancer.
  • the medium-dose docetaxel has the best anti-tumor effect, followed by high-dose and low-dose the worst. It shows that the combination of docetaxel and Plasmodium yoelii can more effectively inhibit the growth of lung cancer, but it does not show a dose-dependent relationship with docetaxel.
  • the tumor group is 28 days
  • the single Plasmodium yoelii treatment group is 36 days
  • the low-dose docetaxel combination treatment group is 38 days
  • the combination treatment group of sitoxel is 41 days
  • the combination treatment group of high-dose docetaxel is 30.5 days.
  • a single treatment of Plasmodium yoelii and a combination of medium and low doses of docetaxel can more effectively prolong the survival of tumor-bearing mice, while the combination of high-dose docetaxel cannot prolong the tumor-bearing mice Lifetime. There was no significant difference in survival between the two groups of combined treatments with medium and low doses and single Plasmodium yoelii treatment. Medium and high doses of docetaxel may have certain side effects.
  • This embodiment provides an anti-tumor combined pharmaceutical composition.
  • the combined pharmaceutical composition includes Plasmodium yoelii and the chemotherapeutic drug etoposide. It is observed that the administration of different doses of etoposide combined with Plasmodium yoelii immunotherapy The effect of treating lung cancer.
  • Example 9 The experimental materials and reagents required in this example are different from those in Example 9 only in that the chemotherapeutic drug is Etoposide (abbreviated as VP16), which is purchased from Bristol-Myers Squibb.
  • Etoposide abbreviated as VP16
  • Step (I) (II) (III) is the same as in Example 9.
  • mice randomly divided into five groups according to tumor size: control tumor group (Con), plasmodium treatment group (Py), low-dose etoposide combined with plasmodium treatment group (Py+VP16 (low dose)) , High-dose docetaxel combined with Plasmodium treatment group (Py+VP16 (high dose)). There are 10 mice in each group, 40 mice in total.
  • control tumor group (Con) plasmodium treatment group (Py), low-dose etoposide combined with plasmodium treatment group (Py+VP16 (low dose)), high dose
  • Docetaxel combined with Plasmodium treatment group (Py+VP16 (high dose)) can significantly inhibit the growth of lung cancer.
  • Low-dose etoposide combination therapy is more effective in inhibiting lung cancer growth than high-dose combination therapy.
  • Low-dose etoposide combination therapy is more effective in inhibiting the growth of lung cancer than single malaria parasite therapy. It shows that the combined pharmaceutical composition of low-dose etoposide and Plasmodium yoelii has the effect of inhibiting the growth of lung cancer more effectively.
  • the tumor group is 28 days
  • the Plasmodium yoelii treatment group is 36 days
  • the low-dose etoposide treatment group is 44 days
  • the high-dose etoposide treatment group is 44 days It was 40.5 days.
  • a single treatment of Plasmodium yoelii and a combination of high and low doses of etoposide can prolong the survival time of tumor-bearing mice.
  • the combination therapy of low-dose etoposide and the combination therapy of high-dose etoposide has a long median survival time, but the difference in survival is not statistically significant. High doses of etoposide may have certain side effects.
  • the combined treatment of low-dose etoposide and Plasmodium yoelii may effectively prolong the survival period of tumor-bearing mice.
  • This embodiment provides an anti-tumor combined pharmaceutical composition.
  • the combined pharmaceutical composition includes Plasmodium yoelii and HIV protease inhibitor nelfinavir. Observation of nelfinavir administration combined with Plasmodium yoelii immunity The effect of therapy on lung cancer.
  • NFV Nelfinavir
  • Step (I) (II) (III) is the same as in Example 9.
  • mice randomly divided into four groups according to tumor size: control tumor group (Con), plasmodium treatment group (Py), nelfinavir treatment group (NFV), nelfinavir combined with plasmodium treatment group ( Py+CTX). There are 10 mice in each group, 40 mice in total.
  • control tumor group Con
  • plasmodium treatment group Py
  • NFV nelfinavir treatment group
  • Py+CTX plasmodium treatment group
  • Combination therapy is more effective in inhibiting tumor growth than single nelfinavir treatment and single Plasmodium yoelii. It shows that the combined medicine composition of nelfinavir and Plasmodium yoelii has the effect of inhibiting the growth of lung cancer more effectively.
  • nelfinavir The combined treatment of nelfinavir and Plasmodium yoelii can more effectively inhibit the growth of lung cancer and better prolong the median survival of tumor-bearing mice.
  • Nefinavir does not increase the side effects of tumor-bearing mice.
  • the total dose of 4g/kg, nelfinavir administered on the 10th day and continued administration for 10 days, and the Plasmodium yoelii inoculated on the 7th day are promising combination pharmaceutical compositions for the treatment of lung cancer.

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Abstract

一种抗肿瘤的联合用药物组合物及其应用，所述联合用药物组合物包括疟原虫和化疗药物。该联合用药物组合物将化学疗法和疟原虫免疫疗法结合起来，具有较高的生物安全性，具有比单一化学疗法或单一疟原虫免疫疗法更强的抗肿瘤活性，能够延长癌症患者的生存期，为癌症的治疗提供了新的策略和思路；同时能够降低化疗药物的使用剂量，降低化疗药物产生的毒副作用，降低肿瘤患者的治疗成本；另外该联合用药物组合物能够促使释放肿瘤抗原，诱导更强的抗肿瘤特异性免疫反应，发挥持续的免疫疗法和化疗的协同效应。

Description

一种抗肿瘤的联合用药物组合物及其应用 技术领域
本申请属于生物医药领域，涉及一种抗肿瘤的联合用药物组合物及其应用。
背景技术
肿瘤治疗方法有手术、放射、化疗(含靶向治疗)三大传统模式，还有新型的生物治疗(含免疫治疗)。临床上以综合治疗为主，手术结合放化疗方式对于早期发现的肿瘤治疗效果较好，但对于进展到晚期的肿瘤，疗效欠佳。现在治疗晚期恶性实体瘤，各种生物治疗提供更多的选择，靶向药物、抗体、免疫治疗药物(如PD-1)和细胞治疗(如CAR-T、TIL细胞)，取得一些进展，但是还未能阻止癌症快速发展的形势。
疟疾主要是经按蚊叮咬传播而由疟原虫感染引起的虫媒传染病，被世界卫生组织列为世界三大传染病(艾滋病、结核病和疟疾)之一。感染人类的疟原虫主要有五种类型：恶性疟原虫、间日疟原虫、三日疟原虫、卵形疟原虫和诺氏疟原虫，最常见的是恶性疟原虫和间日疟原虫。小鼠疟原虫也主要有四种类型：夏氏疟原虫、约氏疟原虫、伯氏疟原虫和文氏疟原虫。在小鼠中证明了疟原虫感染显著抑制肺癌、肝癌、结肠癌、乳腺癌等肿瘤模型的荷瘤小鼠的肿瘤生长和转移，显著延长荷瘤小鼠的生存期。
化疗在肿瘤的临床治疗中主要通过细胞毒药物杀伤肿瘤细胞，但是并不能特异性识别肿瘤细胞，而且存在药物毒副作用，现有化疗越来越难以控制肿瘤发展的形势。将化疗与生物疗法联合治疗肿瘤成为一项具有广阔前景的治疗策略，综合利用化疗和生物疗法发挥协同效应。现有技术中也对其有相关报道。
CN107929231A公开了一种化疗免疫联合治疗的原位温敏凝胶药物递送系统。该递送系统由药物载体肽类树状大分子、抗肿瘤药物和温敏凝胶基质材料PLGA-PEG-PLGA组成，将抗肿瘤药物包载于肽类树状大分子空腔内形成载药肽类树状大分子，通过溶胀过程将载药肽类树状大分子包裹于温敏凝胶基质中，形成室温下为可注射型溶胶，体温下转变为凝胶的原位药物递送系统。该发明的原位递送系统能在肿瘤附近形成凝胶，缓慢释放出载药肽类树状聚合物分子作用于肿瘤组织，肽类树状聚合物分子作用于肿瘤组织内的巨噬细胞生成NO，与抗肿瘤药物共同作用于肿瘤，形成化疗免疫联合治疗，抑制肿瘤的发展。
CN103347521A公开了一种用于治疗癌症的方法，其包括施用代谢靶向的化疗-免疫治疗方案。在一个实施例中，代谢靶向的化疗-免疫治疗方案可包括以下步骤：在患有癌症的对象中施用治疗有效剂量的一种或多种免疫剂(例如，治疗性抗体)，以刺激免疫应答，以及施用治疗有效剂量的一种或多种化疗剂。该发明所述的方法可用于治疗任何类型的癌症，特别是恶性和转移的晚期癌症。
CN102481365A公开了一种通过施用dll4拮抗剂和化疗剂的组合从而治疗各种类型的癌症/肿瘤的方法，其中所述dll4拮抗剂，尤其是特异性结合dll4的dll4抗体和其片段。此类联 合治疗与单独用任一治疗剂治疗相比表现出协同作用。因此，该发明的方法对于由单独用任一治疗剂治疗所需的高剂量引起的副作用具有低耐受性的癌症患者尤其有益，这是因为能够降低有效剂量。还提供了含有dll4拮抗剂和化疗剂的药物组合物及药盒。
综上，现有技术中关于将化疗与生物疗法联合治疗肿瘤且能实现显著的抗肿瘤效果的策略还很有限，因此，开发出一种新的能获得显著抗肿瘤效果的生物化疗治疗策略是非常有意义的，其可以提供一种新的癌症治疗思路。
发明内容
本申请提供了一种抗肿瘤的联合用药物组合物及其应用，该联合用药物组合物成功地将化学疗法和疟原虫免疫疗法(一种生物疗法)结合起来，具有较高的生物安全性，具有比单一化学疗法或单一的疟原虫免疫疗法更强的抗肿瘤活性，能够延长肿瘤小鼠模型的生存期，为癌症的治疗提供了新的策略和思路；同时能够降低化疗药物的使用剂量，降低化疗药物产生的毒副作用，降低肿瘤患者的治疗成本；另外化疗药物通过阻断肿瘤细胞增殖或者直接杀伤肿瘤细胞导致肿瘤细胞死亡，促使肿瘤细胞被巨噬细胞吞噬加工释放肿瘤抗原，在疟原虫免疫疗法的诱导下促进巨噬细胞和树突状细胞的抗原呈递能力增强，从而诱导出更强的抗肿瘤特异性免疫反应，发挥持续的免疫疗法和化疗的协同效应。
一方面，本申请提供一种抗肿瘤的联合用药物组合物，所述联合用药物组合物包括疟原虫和化疗药物。
所述联合用药物组合物中的疟原虫主要通过激活天然免疫和诱导一定的特异性免疫发挥抗肿瘤的效应，具体来说：一方面，疟原虫感染释放的危险信号分子，包括糖基磷脂酰肌醇锚、血红素、免疫刺激核酸基序和其他未知分子，这些病原体相关的模式识别分子，能被宿主的免疫细胞的模式识别受体识别。由疟原虫模式识别分子激活的模式识别受体触发不同的转录程序和刺激多个下游信号途径诱导产生全身免疫反应，包括释放促炎因子和Th1型细胞因子，激活NK细胞、NKT细胞、巨噬细胞和树突状细胞，进一步激活CD4 +和CD8 +T细胞，抑制TGF-β、IL-10等细胞因子产生，抑制调节性T细胞(Tregs)、骨髓衍生的抑制性细胞(MDSCs)和肿瘤相关巨噬细胞(TAMs)等细胞活性，改善肿瘤免疫抑制微环境，将肿瘤患者的瘤内的免疫抑制微环境转变为免疫激活状态，从而将肿瘤转化为有效的肿瘤疫苗。另一方面，疟原虫感染通过损伤相关分子模式激活免疫，疟原虫感染的红细胞和已知的内源性尿酸、微泡都能诱导相似的免疫活性。
化疗药物种类繁多，根据化疗药物的来源和化学结构，化疗药物主要包括烷化剂类化疗药物、抗代谢类化疗药物、抗生素类化疗药物、激素类化疗药物、动植物类化疗药物和杂类化疗药物。烷化剂类化疗药物直接作用于DNA上，防止癌细胞再生，常见的烷化剂药物有环磷酰胺、异环磷酰胺、塞替派。抗代谢化疗药物，是通过干扰DNA和RNA的合成，阻止细胞分裂和增殖，主要是吉西他滨、培美曲塞、氟尿嘧啶、阿糖胞苷、甲氨蝶呤、巯嘌呤、 羟基脲等。抗生素类化疗药物是通过抑制酶的作用和有丝分裂或者改变细胞膜来干扰DNA，主要是细胞周期非特异性的药物，主要有丝裂霉素、柔红霉素、平阳霉素、多柔比星、放线菌素D、米托蒽醌等。动植物类化疗药物主要是植物碱和天然产物，可以通过抑制有丝分裂或酶的作用，从而防止细胞再生必需的蛋白质合成，主要有长春新碱、依托泊苷、替尼泊苷、紫杉醇、多西他赛，它们通常与其他类化疗药物联合用于治疗肿瘤。激素类化疗药物通过应用某些激素或者拮抗剂杀伤或者减缓激素依赖的肿瘤细胞的生长，主要有他莫昔芬、甲地孕酮、曲普瑞林、甲羟孕酮。杂类化疗药物，主要包括顺铂、卡铂、奥沙利铂、门冬酰胺酶等。
HIV蛋白酶抑制剂(HIV PIs)是一类治疗HIV感染的化疗药物，它们通过抑制病毒的蛋白酶活性，从而抑制天冬氨酰蛋白前体裂解成其功能形式以阻止HIV病毒颗粒的成熟。HIV PIs主要有nelfinavir(奈非那韦)、saquinavir(沙奎那韦)、indinavir(茚地那韦)、ritonavir(利托那韦)。Gills等人研究发现HIV PIs对60多个细胞系具有抗癌活性，阐述了包括内质网应激、自噬、凋亡以及抑制Akt通路等机制。在胰腺癌患者，nelfinavir联合吉西他滨、顺铂或放疗，可以增强Akt抑制水平和增强放疗的敏感性，而且并没有发现Nelfinavir引起的毒副作用。Rengan等发现在无法手术切除的非小细胞肺癌患者，nelfinavir联合依托泊苷、顺铂或放疗，也未发现nelfinavir剂量依赖的毒性反应。
疟原虫感染能系统性激活机体的天然免疫并能增强机体抗肿瘤的特异性免疫。而化疗药物，包括HIV PIs，能直接杀伤肿瘤细胞或者抑制肿瘤细胞的增殖。将疟原虫感染(疟原虫免疫疗法)和化疗药物联合起来，从激活免疫和降低肿瘤负荷两个方面入手，起到协同治疗肿瘤的作用，比单一的疟原虫免疫疗法或单一的化学疗法具有更显著的治疗效果。
综上，该联合用药物组合物成功地将化学疗法和疟原虫免疫疗法(一种生物疗法)结合起来，具有较高的生物安全性，比单一化学疗法和单一疟原虫免疫疗法具有更强的抗肿瘤活性，能够延长患癌小鼠的生存期，为癌症的治疗提供了新的策略和思路；同时能够降低化疗药物的使用剂量，降低化疗药物产生的毒副作用，降低肿瘤患者的治疗成本；另外该联合用药物组合物能够促使肿瘤细胞释放肿瘤抗原，诱导更强的抗肿瘤特异性免疫反应，发挥持续的免疫疗法和化疗的协同效应。
在本申请中，所述化疗药物包括烷化剂类化疗药物、抗代谢类化疗药物、抗生素类化疗药物、动植物类化疗药物、杂类化疗药物或HIV蛋白酶抑制剂。
在一个优选的实施方案中，所述烷化剂类化疗药物包括环磷酰胺或异环磷酰胺。
在一个优选的实施方案中，所述抗代谢类化疗药物包括吉西他滨、培美曲塞、5-氟尿嘧啶、阿糖胞苷或甲氨蝶呤。
在一个优选的实施方案中，所述抗生素类化疗药物包括丝裂霉素、多柔比星或放线菌素D。
在一个优选的实施方案中，所述动植物类化疗药物包括依托泊苷、多西他赛、紫杉醇、长春新碱或伊立替康。
在一个优选的实施方案中，所述杂类化疗药物包括顺铂、卡铂、奥沙利铂或门冬酰胺酶。
在一个优选的实施方案中，所述HIV蛋白酶抑制剂化疗药物包括奈非那韦、沙奎那韦、茚地那韦或利托那韦。
在本申请中，所述联合用药物组合物的剂型包括药剂学上可接受的任意一种剂型。例如片剂、散剂、混悬剂、颗粒剂、胶囊剂、注射剂、喷雾剂、溶液剂、灌肠剂、乳剂、膜剂、栓剂、贴剂、滴鼻剂或滴丸剂等。
在一个优选的实施方案中，所述联合用药物组合物还包括药剂学上可接受药用辅料中的任意一种或至少两种的组合。
本申请所述联合用药物组合物可单独给药也可以与辅料搭配做成适当的剂型进行给药，所述辅料包括稀释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、乳化剂、助溶剂、增溶剂、渗透压调节剂、表面活性剂、pH调节剂、抗氧剂、抑菌剂或缓冲剂中的任意一种或至少两种的组合。所述至少两种的组合例如稀释剂和赋形剂的组合、乳化剂和助溶剂的组合、填充剂和粘合剂和润湿剂的组合等。
在一个优选的实施方案中，所述联合用药物组合物为单一的复方制剂。
在另一个优选的实施方案中，所述联合用药物组合物为两种单独的制剂的组合。
在一个实施方案中，所述两种单独的制剂同时施用。
在一个实施方案中，所述两种单独的制剂依次施用。
所述联合用药物组合物可以为单一的复方制剂形式，也可以为两种单独的制剂的组合；当为两种单独的制剂的组合时，其用药方式可以为同时施用，也可以为依次施用，例如可以先施用疟原虫，间隔一段时间再施用化疗药物，也可以先施用化疗药物，间隔一段时间再施用疟原虫，或者疟原虫和化疗药物交替施用。
在本申请中，所述联合用药物组合物的给药途径包括静脉注射、腹腔注射、肌肉注射、皮下注射、口服给药、舌下给药、鼻腔给药或经皮给药，优选腹腔注射。
在一个优选的实施方案中，所述联合用药物组合物为负载于药用载体上的联合用药物组合物。
在一个实施方案中，所述药用载体包括脂质体、胶束、树枝状大分子、微球或微囊。
另一方面，本申请提供一种如上所述的联合用药物组合物在制备抗肿瘤药物中的应用。
在一个优选的实施方案中，所述肿瘤包括肺癌、胃癌、结肠癌、肝癌、乳腺癌或胰腺癌。
再一方面，本申请提供一种新型的抗肿瘤联合疗法，所述抗肿瘤联合疗法为化学疗法与疟原虫疗法的联合疗法。将化学疗法和疟原虫免疫疗法结合起来，具有较高的生物安全性，比单一化学疗法和单一的疟原虫免疫疗法具有更强的抗肿瘤活性，能够更有效地地延长荷瘤小鼠的生存期，为癌症的治疗提供了新的策略和思路；同时能够降低化疗药物的使用剂量，降低化疗药物产生的毒副作用，降低肿瘤患者的治疗成本；另外该联合疗法能够促使肿瘤细胞释放肿瘤抗原，诱导更强的抗肿瘤特异性反应，发挥持续的免疫疗法和化疗的协同效应。
在一个优选的实施方案中，所述肿瘤包括肺癌、胃癌、结肠癌、肝癌、乳腺癌或胰腺癌。
在一个实施方案中，所述化学疗法所使用的化疗药物包括烷化剂类化疗药物、抗代谢类 化疗药物、抗生素类化疗药物、动植物类化疗药物、杂类化疗药物或HIV蛋白酶抑制剂。
在一个优选的实施方案中，所述烷化剂类化疗药物包括环磷酰胺或异环磷酰胺。
在一个优选的实施方案中，所述抗代谢类化疗药物包括吉西他滨、培美曲塞、5-氟尿嘧啶、阿糖胞苷或甲氨蝶呤。
在一个优选的实施方案中，所述抗生素类化疗药物包括丝裂霉素、多柔比星或放线菌素D。
在一个优选的实施方案中，所述动植物类化疗药物包括依托泊苷、多西他赛、紫杉醇、长春新碱或伊立替康。
在一个优选的实施方案中，所述杂类化疗药物包括顺铂、卡铂、奥沙利铂、门冬酰胺酶等。
在一个优选的实施方案中，所述HIV蛋白酶抑制剂化疗药物包括奈非那韦、沙奎那韦、茚地那韦、利托那韦。
在一个优选的实施方案中，所述化学疗法的给药途径包括静脉注射、腹腔注射、肌肉注射、皮下注射、口服给药、舌下给药、鼻腔给药或经皮给药，优选静脉注射或口服给药。
在一个优选的实施方案中，所述疟原虫疗法的给药途径包括静脉注射。
相对于现有技术，本申请具有以下有益效果：
本申请所涉及的抗肿瘤的联合用药物组合物将化学疗法和疟原虫免疫疗法(一种生物疗法)结合起来，具有较高的生物安全性，比单一化学疗法和单一的疟原虫免疫疗法具有更强的抗肿瘤活性，能够更有效地延长癌症患者的生存期，为癌症的治疗提供了新的策略和思路；同时能够降低化疗药物的使用剂量，降低化疗药物产生的毒副作用，降低肿瘤患者的治疗成本；另外该联合用药物组合物能够促使肿瘤细胞释放肿瘤抗原，诱导更强的抗肿瘤特异性反应，发挥持续的免疫疗法和化疗的协同效应。
附图说明
图1是实施例1中肿瘤生长曲线图；
图2是实施例1中荷瘤小鼠的生存曲线图；
图3是实施例1中疟原虫感染率曲线图；
图4是实施例1中荷瘤小鼠的体重生长曲线图；
图5是实施例2中肿瘤生长曲线图；
图6是实施例2中荷瘤小鼠的生存曲线图；
图7是实施例2中疟原虫感染率曲线图；
图8是实施例2中荷瘤小鼠的体重生长曲线图；
图9是实施例3中肿瘤生长曲线图；
图10是实施例3中荷瘤小鼠的生存曲线图；
图11实施例3中疟原虫感染率曲线图；
图12是实施例3中荷瘤小鼠的体重生长曲线图；
图13是实施例4中肿瘤生长曲线图；
图14是实施例4中荷瘤小鼠的生存曲线图；
图15是实施例4中疟原虫感染率曲线图；
图16是实施例4中荷瘤小鼠的体重生长曲线图；
图17是实施例5中肿瘤生长曲线图；
图18是实施例5中荷瘤小鼠的生存曲线图；
图19是实施例5中疟原虫感染率曲线图；
图20是实施例5中荷瘤小鼠的体重生长曲线图；
图21是实施例6中肿瘤生长曲线图；
图22是实施例6中荷瘤小鼠的生存曲线图；
图23是实施例6中疟原虫感染率曲线图；
图24是实施例6中荷瘤小鼠的体重生长曲线图；
图25是实施例7中肿瘤生长曲线图；
图26是实施例7中荷瘤小鼠的生存曲线图；
图27是实施例7中疟原虫感染率曲线图；
图28是实施例7中荷瘤小鼠的体重生长曲线图；
图29是实施例8中肿瘤生长曲线图；
图30是实施例8中荷瘤小鼠的生存曲线图；
图31是实施例8中疟原虫感染率曲线图；
图32是实施例8中荷瘤小鼠的体重生长曲线图；
图33是实施例9中肿瘤生长曲线图；
图34是实施例9中荷瘤小鼠的生存曲线图；
图35是实施例9中疟原虫感染率曲线图；
图36是实施例9中荷瘤小鼠的体重生长曲线图；
图37是实施例10中肿瘤生长曲线图；
图38是实施例10中荷瘤小鼠的生存曲线图；
图39是实施例10中疟原虫感染率曲线图；
图40是实施例10中荷瘤小鼠的体重生长曲线图；
图41是实施例11中肿瘤生长曲线图；
图42是实施例11中荷瘤小鼠的生存曲线图；
图43是实施例11中疟原虫感染率曲线图；
图44是实施例11中荷瘤小鼠的体重生长曲线图；
图45是实施例12中肿瘤生长曲线图；
图46是实施例12中荷瘤小鼠的生存曲线图；
图47是实施例12中疟原虫感染率曲线图；
图48是实施例12中荷瘤小鼠的体重生长曲线图。
具体实施方式
下面通过具体实施方式来进一步说明本申请的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本申请，不应视为对本申请的具体限制。
实施例1
本实施例提供一种抗肿瘤的联合用药物组合物，所述联合用药物组合物包括约氏疟原虫和化疗药物吉西他滨，观察不同给药时间的吉西他滨联合约氏疟原虫治疗肺癌效果。
本实施例所需的实验材料和试剂包括：
动物：C57BL/6小鼠，雌性，6-8周龄，来源于上海斯莱克实验动物有限责任公司或北京维通利华实验动物有限公司；
疟原虫：小鼠约氏疟原虫(P.yoelii 17XNL，MRA-593，Py)，来自Malaria Research and Reference Reagent Resource Center(MR4)免费馈赠；
化学治疗药物：吉西他滨(Gemcitabine，缩写GEM)，购自Sigma-Aldrich公司；
Giemsa染剂粉：购自Sigma-Aldrich公司。
(1)建立动物模型，具体方法为：
(Ⅰ)细胞复苏：复苏小鼠Lewis肺癌细胞系LLC，在5％CO 2、37℃恒温培养箱中静置培养；
(Ⅱ)细胞扩培：每2-3天传代一次，当细胞长满培养皿底部的80％时，用0.25％胰酶-EDTA消化液消化，按照1:3稀释传代；
(Ⅲ)单细胞制备及细胞接种：取对数生长期的细胞，经胰酶消化后，用PBS洗涤3次，用无血清1640培养基重悬细胞，小鼠右侧肩胛区皮下接种，每只注射细胞悬液体积为0.1mL，LLC细胞接种量为5×10 5个/只(接种C57BL/6小鼠)；
(Ⅳ)实验分组：按照肿瘤大小随机分为六组：对照肿瘤组(Con)，约氏疟原虫治疗组(Py)，第3天给药的吉西他滨治疗组(GEM(d3))，第6天给药的吉西他滨治疗组(GEM(d6)，第3天给药的吉西他滨联合治疗组(Py+GEM(d3))，第6天给药的吉西他滨联合治疗组(Py+GEM(d6))。每组10只小鼠，共60只。
(2)疟原虫接种荷瘤小鼠，具体方法为：
(Ⅰ)疟原虫复苏：将冻存于液氮罐中的鼠疟原虫血(1.0mL/支)，于37℃水浴锅中快速震荡，使其混匀融化，保持疟原虫活性。
(Ⅱ)疟原虫接种：混匀后0.2mL/只腹腔注射接种C57BL/6小鼠，每次接种两只。
(Ⅲ)薄血膜制作和镜检：小鼠剪尾采血取约1-1.5μL，涂于载玻片，制作2.5cm长舌状薄血膜，吹风机吹干。甲醇浸润血膜1min，1×Giemsa染液染色30min，用自来水冲洗干净，吹干。用油镜100×观察疟原虫感染率。观察疟原虫感染率变化。
(Ⅳ)疟原虫溶液制备：当感染率达到3％-10％时，先红细胞计数，剪尾巴取血5μL重悬于995μL PBS中，红细胞计数。计算出每mL体积感染疟原虫的红细胞数。用0.2mL体积的3.8％枸橼酸钠抗凝剂润湿EP管，摘眼球采血，计算所需要的接种疟原虫的浓度和总量，用PBS配制浓度2.5×10 6/mL。
(V)接种荷瘤小鼠：肿瘤皮下接种第7天后，每只小鼠接种0.2mL，即接种5×10 5个疟原虫。
(3)吉西他滨给药，具体方法为：
(Ⅰ)给药方式：腹腔注射给药；
(Ⅱ)给药剂量：总剂量100mg/kg；
(Ⅲ)药物制备：溶解在生理盐水，配置浓度为10mg/mL；
(Ⅳ)给药时间：GEM(d3)治疗组和Py+GEM(d3)治疗组在肿瘤接种第3天给药，GEM(d6)治疗和Py+GEM(d6)治疗组在肿瘤接种第6天给药。
(4)检测指标包括：
(Ⅰ)肿瘤体积大小测量：每3天进行肿瘤测量，用椭圆体积计算公式计算肿瘤体积(单位立方毫米)：(D×d×d)/2，其中“D”表示肿瘤的长径，“d”表示短径。肿瘤大小以平均肿瘤体积±标准平均误差(SEM)表示，制作肿瘤生长曲线。通过TWO-WAY ANOVA方差分析进行组间统计分析，当p≤0.05时，用“*”表示，当p≤0.01时，用“**”表示，均说明组间差异具有显著的统计学意义。
(Ⅱ)小鼠生存统计：存活以中位生存期和延长的存活期的百分数来评定，采用Kaplan-Meier法估计生存率，做生存曲线图，计算中位生存时间。当p≤0.05时，用“*”表示，当p≤0.01时，用“**”表示，均说明组间差异具有显著的统计学意义。
(Ⅲ)疟原虫感染率统计：疟原虫感染率以小鼠红细胞感染疟原虫百分数来评定，计算公式为：(感染疟原虫红细胞数目/总红细胞)×100％；具体操作为尾静脉取血涂片，用甲醇固定，并用吉姆萨染液染色，显微镜下观察感染红细胞的疟原虫数量和总红细胞数，总红细胞数在1000个左右，计算疟原虫感染率，感染率以平均感染率±标准平均误差(SEM)表示，并绘制疟原虫感染周期曲线，观察化疗药物是否对疟原虫感染有影响。
(Ⅳ)小鼠体重称重：每3天进行称量，体重生长以平均体重±标准平均误差(SEM)来表示，观察化疗药物和疟原虫感染对荷瘤小鼠体重的影响。
(5)实验结果
(Ⅰ)如图1和表1所示：约氏疟原虫治疗组(Py)，第3天给药的吉西他滨治疗组(GEM(d3))，第6天给药的吉西他滨治疗组(GEM(d6)，第3天给药的吉西他滨联合治疗组(Py+GEM(d3))和第6天给药的吉西他滨联合治疗组(Py+GEM(d6))均显著地抑制肺癌的生长。第6天给药的联合治疗组可能比第3天给药的联合治疗组抑制肺癌的效果好，但不具有显著的统计学意义。第6天给药的联合治疗组比单一的约氏疟原虫治疗组、单一的第6天给药的吉西他滨治疗组抑制肺癌的效果更好，具有显著的统计学意义。
表1
组间比较 显著性
Con vs Py **
Con vs GEM(d3) **
Con vs GEM(d6) **
Con vs Py+GEM(d3) **
Con vs Py+GEM(d6) **
Py vs Py+GEM(d3) **
Py vs Py+GEM(d6) **
GEM(d6)vs Py+GEM(d6) **
(Ⅱ)如图2和表2所示：对照肿瘤组的中位生存期是30.5天，约氏疟原虫治疗组是38.5天，第3天给药的吉西他滨治疗组是34.5天，第6天给药的吉西他滨治疗组是33天，第3天给药的联合治疗组是42.5天，第6天给药的联合治疗组是45天。约氏疟原虫治疗组(Py)，第3天给药的吉西他滨治疗组(GEM(d3))，第6天给药的吉西他滨治疗组(GEM(d6)，第3天给药的吉西他滨联合治疗组(Py+GEM(d3))和第6天给药的吉西他滨联合治疗组(Py+GEM(d6))都显著地延长荷瘤小鼠的生存期。第6天给药的联合治疗组比第3天给药的联合治疗组的中位生存期长，但不具有显著的统计学意义。第6天给药的联合治疗与单一的约氏疟原虫治疗、单一的吉西他滨治疗的中位生存期长，但生存差异不具有显著的统计学意义。
表2
组间比较 显著性
Con vs GEM(d3) *
Con vs GEM(d6) **
GEM(d6)vs Py+GEM(d6) *
(Ⅲ)如图3所示：约氏疟原虫治疗组与两个联合治疗组的荷瘤小鼠其原虫血症时程周期比较一致，感染周期都在一个月左右。第3天给药的联合治疗组约氏疟原虫感染高峰期提前，第6天给药的联合治疗组的约氏疟原虫感染高峰期延后。说明吉西他滨对约氏疟原虫有一定的抑制作用，但不会清除疟原虫。
(Ⅳ)如图4所示：联合治疗组的荷瘤小鼠的体重主要受到疟原虫的影响，受到吉西他滨的影响较小，吉西他滨联合疟原虫使荷瘤小鼠的体重减轻作用叠加。
(6)小结：第6天给药的吉西他滨联合组能显著抑制肺癌的生长，延长荷瘤小鼠的中位生存期。吉西他滨对疟原虫有抑制作用，但不清除疟原虫。吉西他滨和疟原虫联合用药会对荷瘤小鼠的体重减轻作用叠加。总剂量100mg/kg、第6天给药的吉西他滨与第7天接种的约氏疟原虫，是很有潜力的用于治疗肺癌的联合用药组合物。
实施例2
本实施例提供一种抗肿瘤的联合用药物组合物，所述联合用药物组合物包括约氏疟原虫和化疗药物吉西他滨，观察总剂量相同情况下，单次或者分次吉西他滨给药联合约氏疟原虫疗法治疗肺癌效果。
本实施例所使用的实验材料和试剂与实施例1相同。
(1)建立动物模型，具体方法为：
步骤(Ⅰ)(Ⅱ)(Ⅲ)与实施例1相同。
(Ⅳ)实验分组：按照肿瘤大小随机分为六组：对照肿瘤组(Con)，约氏疟原虫治疗组(Py)，单次给药的吉西他滨治疗组(GEM(单次))，分次给药的吉西他滨治疗组(GEM(分次)，单次给药的联合治疗组(Py+GEM(单次))，分次给药的联合治疗组(Py+GEM(分次))。每组10只小鼠，共60只。
(2)疟原虫接种荷瘤小鼠，具体方法为：
步骤(Ⅰ)(Ⅱ)(Ⅲ)(Ⅳ)(V)与实施例1相同。
(3)吉西他滨给药，具体方法为：
(Ⅰ)给药方式：腹腔注射给药；
(Ⅱ)给药剂量：总剂量为100mg/kg；
(Ⅲ)药物制备：溶解在生理盐水，配置浓度为5mg/mL和10mg/mL两个浓度；
(Ⅳ)给药时间：GEM(单次)治疗组和Py+GEM(单次)治疗组在肿瘤接种第6天给药，给药剂量为100mg/kg，药物浓度为10mg/mL。GEM(分次)治疗组和Py+GEM(分次)组分别在肿瘤接种第6、13天给药，每次给药剂量为50mg/kg，药物浓度为5mg/kg。
(4)检测指标与实施例1一致。
(5)实验结果:
(Ⅰ)如图5和表3所示：对照肿瘤组(Con)，约氏疟原虫治疗组(Py)，单次给药的吉西他滨治疗组(GEM(单次))，分次给药的吉西他滨治疗组(GEM(分次)，单次给药的联合治疗组(Py+GEM(单次))，分次给药的联合治疗组(Py+GEM(分次))都显著抑制肺癌的生长。分次给药的吉西他滨联合治疗组比单次给药的吉西他滨给药联合治疗组抑制肺癌生长效果好。两组联合治疗分别都比单一的约氏疟原虫治疗组、单一的吉西他滨治疗组抑制肺癌生长的效果好。
表3
组间比较 显著性
Con vs Py **
Con vs GEM(单次) **
Con vs GEM(分次) **
Con vs Py+GEM(单次) **
Con vs Py+GEM(分次) **
Py vs Py+GEM(单次) **
Py vs Py+GEM(分次) **
GEM(单次)vs Py+GEM(单次) **
GEM(分次)vs Py+GEM(分次) **
Py+GEM(单次)vs Py+GEM(分次) **
(Ⅱ)如图6和表4所示：对照肿瘤组的中位生存期是30.5天，约氏疟原虫治疗组是38.5天，单次给药吉西他滨治疗组是33天，分次给药吉西他滨治疗组是38.5天，单次给药吉西他滨联合约氏疟原虫治疗组是45天，分次给药吉西他滨联合约氏疟原虫治疗组是52.5天。两个联合治疗组显著地延长荷瘤小鼠的中位生存期。两个联合治疗组都分别比对应的单一吉西他滨治疗组更加有效地延长荷瘤小鼠的中位生存期。分次给药的联合治疗组比单次给药的联合治疗组更加有效地延长荷瘤小鼠生存期。单次给药的联合治疗与单一的疟原虫治疗的生存差异不具显著的统计学意义，而分次给药的联合治疗比单一的疟原虫治疗更加有效地延长荷瘤小鼠的中位生存期。
表4
组间比较 显著性
Con vs Py+GEM(单次) *
Con vs Py+GEM(分次) **
Py vs Py+GEM(分次) **
GEM(单次)vs Py+GEM(单次) *
GEM(分次)vs Py+GEM(分次) **
Py+GEM(单次)vs Py+GEM(分次) *
(Ⅲ)如图7所示：约氏疟原虫治疗组与两个吉西他滨联合治疗组的荷瘤小鼠的原虫血症时程周期比较一致，感染周期在一个月左右。单次和分次吉西他滨给药，都推迟疟原虫高峰期出现时间，分次给药推迟的时间更长。说明吉西他滨对疟原虫有一定的抑制作用。
(Ⅳ)如图8所示：联合治疗组的荷瘤小鼠的体重主要受到疟原虫的影响，受到吉西他的影响较小，吉西他滨联合疟原虫使荷瘤小鼠的体重减轻作用轻微地叠加，但分次给药比单次给药对荷瘤小鼠的体重影响更小。
(6)小结：分次给药吉西他滨联合治疗比单次给药吉西他滨联合治疗具有更有效地抑制肺癌生长和延长荷瘤小鼠的中位生存期的作用。单次和分次的吉西他滨给药对约氏疟原虫都有抑制作用。吉西他滨分次给药比单次给药对荷瘤小鼠的体重影响更小。总剂量100mg/kg、第6与13天分次给药的吉西他滨与第7天接种的约氏疟原虫，是很有潜力的用于治疗肺癌的联合用药组合物。
实施例3
本实施例提供一种抗肿瘤的联合用药物组合物，所述联合用药物组合物包括约氏疟原虫和化疗药物吉西他滨，观察优化方案的吉西他滨给药联合疟原虫免疫疗法治疗肺癌的效果。
本实施例所需的实验材料和试剂与实施例1相同。
(1)建立动物模型，具体方法为：
步骤(Ⅰ)(Ⅱ)(Ⅲ)与实施例1相同。
(Ⅳ)实验分组：按照肿瘤大小随机分为四组：对照肿瘤组(Con)，疟原虫治疗组(Py)，吉西他滨治疗组(GEM)，吉西他滨联合疟原虫治疗组(Py+GEM)。每组15只小鼠，共60只。
(2)疟原虫接种荷瘤小鼠，具体方法为：
步骤(Ⅰ)(Ⅱ)(Ⅲ)(Ⅳ)(V)与实施例1相同。
(3)吉西他滨给药，具体方法为：
(Ⅰ)给药方式：腹腔注射给药；
(Ⅱ)给药剂量：100mg/kg；
(Ⅲ)药物制备：溶解在生理盐水，配置浓度为5mg/mL；
(Ⅳ)给药时间：GEM治疗组和Py+GEM治疗组在肿瘤接种第6、13天给药，每次给药剂量为50mg/kg。
(4)检测指标与实施例1一致。
(5)实验结果:
(Ⅰ)如图9和表5所示：对照肿瘤组(Con)，疟原虫治疗组(Py)，吉西他滨治疗组(GEM)，吉西他滨联合疟原虫治疗组(Py+GEM)都显著抑制肺癌的生长。吉西他滨与疟原虫的联合治疗比单一的吉西他滨治疗、单一的约氏疟原虫治疗更加有效地抑制肿瘤生长。说明吉西他滨和约氏疟原虫的联合用药物组合物具有更有效地抑制肺癌生长的效果。
表5
组间比较 显著性
Con vs Py **
Con vs GEM **
Con vs Py+GEM **
Py vs Py+GEM **
GEM vs Py+GEM **
(Ⅱ)如图10和表6所示：对照肿瘤组的中位生存期是26天，约氏疟原虫治疗组是36天，吉西他滨治疗组是36天，联合治疗组是55天。联合治疗比单一的约氏疟原虫治疗、单一的吉西他滨治疗更有效地延长荷瘤小鼠的生存。说明吉西他滨和约氏疟原虫的联合用药物组合物能够显著地延长荷瘤小鼠的生存。
表6
组间比较 显著性
Con vs Py **
Con vs GEM **
Con vs Py+GEM **
Py vs Py+GEM **
GEM vs Py+GEM **
(Ⅲ)如图11所示：吉西他滨使疟原虫高峰期推迟，原虫血症时程周期不变。吉西他滨对约氏疟原虫有一定的抑制作用，但是不会清除约氏疟原虫。说明吉西他滨可以与约氏疟原虫联合治疗肺癌，两种疗法干扰较小。
(Ⅳ)如图12所示：联合治疗组的荷瘤小鼠的体重主要受到疟原虫的影响，受到吉西他的影响较小，吉西他滨与疟原虫联合使荷瘤小鼠的体重减轻作用轻微地叠加，但不会显著地增加荷瘤小鼠的毒副作用。
(5)小结:吉西他滨和约氏疟原虫的联合用药物组合物具有更有效地抑制肺癌生长和延长荷瘤小鼠的中位生存期的效果，但不会增加荷瘤小鼠的毒副作用。总剂量100mg/kg、在第6、13天分次给药的吉西他滨与第7天接种的约氏疟原虫，是很有潜力的用于治疗肺癌的联合用药组合物。
实施例4
本实施例提供一种抗肿瘤的联合用药物组合物，所述联合用药物组合物包括夏氏疟原虫和化疗药物吉西他滨，观察优化方案的吉西他滨给药联合夏氏疟原虫免疫疗法治疗肺癌的效果。
本实施例所需的实验材料和试剂与实施例3的区别仅在于疟原虫使用小鼠夏氏疟原虫(Plasmodium chabaudi，MRA-429，Pc)，来自Malaria Research and Reference Reagent Resource Center(MR4)免费馈赠。
(1)建立动物模型，具体方法为：
步骤(Ⅰ)(Ⅱ)(Ⅲ)与实施例3相同。
(Ⅳ)实验分组：按照肿瘤大小随机分为四组：对照肿瘤组(LLC)，夏氏疟原虫治疗组(Pc)，吉西他滨治疗组(GEM)，吉西他滨联合夏氏疟原虫治疗组(Pc+GEM)。每组10只小鼠，共40只。
(2)疟原虫接种荷瘤小鼠，具体方法与实施例3相同。
(3)吉西他滨给药，具体方法为：
(Ⅰ)给药方式：腹腔注射给药；
(Ⅱ)给药剂量：总剂量为100mg/kg；
(Ⅲ)药物制备：溶解在生理盐水，配置浓度为5mg/mL；
(Ⅳ)给药时间：GEM治疗组和Pc+GEM治疗组在肿瘤接种第6、13天给药，每次给药剂量为50mg/kg。
(4)检测指标与实施例3一致。
(5)实验结果:
(Ⅰ)如图13和表7所示：对照肿瘤组(LLC)，夏氏疟原虫治疗组(Pc)，吉西他滨治疗组(GEM)，吉西他滨联合夏氏疟原虫治疗组(Pc+GEM)都显著抑制肺癌的生长。联合治疗比单一的夏氏疟原虫治疗组、单一的吉西他滨治疗组更加有效地抑制肿瘤的生长。说明吉西他滨和夏氏疟原虫的联合用药组合物具有更有效地抑制肺癌生长的效果。
表7
组间比较 显著性
Con vs Pc **
Con vs GEM **
Con vs Pc+GEM **
Pc vs Pc+GEM *
GEM vs Pc+GEM **
(Ⅱ)如图14和表8所示：比较荷瘤小鼠中位生存期，肿瘤组是27.5天，夏氏疟原虫治疗组是34天，吉西他滨治疗组是35天，联合治疗组是41天。联合治疗显著延长荷瘤小鼠的生存，但与单一的夏氏疟原虫治疗、单一的吉西他滨治疗的生存差异没有显著的统计学意义。说明吉西他滨与夏氏疟原虫的联合用药组合物能够显著地延长荷瘤小鼠的生存。
表8
组间比较 显著性
Con vs Pc+GEM **
(Ⅲ)如图15所示：夏氏疟原虫治疗组和联合治疗组的荷瘤小鼠的原虫血症时程周期一致。吉西他滨对夏氏疟原虫有抑制作用，但是不会清除疟原虫。说明吉西他滨可以与夏氏疟原虫联合治疗肺癌，两种疗法干扰较小。
(Ⅳ)如图16所示：联合治疗组的荷瘤小鼠的体重主要受到夏氏疟原虫的影响，受到吉西他滨的影响较小，吉西他滨与疟原虫联合使荷瘤小鼠的体重减轻作用轻微地叠加，但不会显著地增加荷瘤小鼠的毒副作用。
(6)小结:吉西他滨和夏氏疟原虫的联合用药物组合物具有更有效地抑制肺癌生长和延长荷瘤小鼠的中位生存期的效果，但不会增加荷瘤小鼠的毒副作用。总剂量100mg/kg、第6、13天分次给药的吉西他滨与第7天接种的夏氏疟原虫，是很有潜力的用于治疗肺癌的联合用药组合物。
实施例5
本实施例提供一种抗肿瘤的联合用药物组合物，所述联合用药物组合物包括约氏疟原虫和化疗药物环磷酰胺，观察环磷酰胺给药联合约氏疟原虫免疫疗法治疗肺癌的效果。
本实施例所需的实验材料和试剂与实施例1的区别仅在于化学治疗药物为环磷酰胺(Cyclophosphamide，缩写CTX)，购自Sigma-Aldrich公司。
(1)建立动物模型，具体方法为：
步骤(Ⅰ)(Ⅱ)(Ⅲ)与实施例3相同。
(Ⅳ)实验分组：按照肿瘤大小随机分为四组：对照肿瘤组(Con)，疟原虫治疗组(Py)，环磷酰胺治疗组(CTX)，环磷酰胺联合疟原虫治疗组(Py+CTX)。每组11只小鼠，共44只。
(2)疟原虫接种荷瘤小鼠的具体方法与实施例3相同。
(3)环磷酰胺给药，具体方法为：
(Ⅰ)给药方式：腹腔注射给药；
(Ⅱ)给药剂量：20mg/kg×3；
(Ⅲ)药物制备：溶解在生理盐水，配置浓度为2mg/mL；
(Ⅳ)给药时间：CTX治疗组和Py+CTX治疗组在肿瘤接种第6、13、20天给药，每次给药剂量为20mg/kg。
(4)检测指标与实施例3相同。
(5)实验结果:
(Ⅰ)如图17和表9所示：对照肿瘤组(Con)，疟原虫治疗组(Py)，环磷酰胺治疗组(CTX)，环磷酰胺联合疟原虫治疗组(Py+CTX)都显著地抑制肺癌的生长。联合治疗比单一的环磷酰胺治疗组、单一的约氏疟原虫治疗组更有效地抑制肿瘤的生长。说明环磷酰胺和约氏疟原虫的联合用药物组合物具有更有效地抑制肺癌生长的效果。
表9
组间比较 显著性
Con vs Py **
Con vs CTX *
Con vs Py+CTX **
Py vs Py+CTX *
CTX vs Py+CTX **
(Ⅱ)如图18和表10所示：比较荷瘤小鼠中位生存期，肿瘤组是28.5天，约氏疟原虫治疗组是37.5天，环磷酰胺治疗组是37天，联合治疗组是42.5天。单一的疟原虫治疗组、单一的环磷酰胺治疗组和联合治疗组都显著延长荷瘤小鼠的生存。联合治疗与单一的疟原虫治疗组、单一的环磷酰胺治疗组的生存差异不具有显著的统计学意义，但仍提示环磷酰胺和约氏疟原虫的联合用药物组合物可能延长荷瘤小鼠的生存。
表10
组间比较 显著性
Con vs Py *
Con vs CTX **
Con vs Py+CTX **
(Ⅲ)如图19所示：环磷酰胺对疟原虫的影响较小，约氏疟原虫治疗组和联合治疗组的 荷瘤小鼠的原虫血症感染时程周期比较一致。说明环磷酰胺可以与约氏疟原虫疗法联合用于治疗肺癌，两种疗法干扰较小。
(Ⅳ)如图20所示：联合治疗组的荷瘤小鼠的体重主要受到夏氏疟原虫的影响，受到环磷酰胺的影响较小，环磷酰胺与疟原虫联合使荷瘤小鼠的体重减轻作用轻微的叠加，但不会显著地增加荷瘤小鼠的毒副作用。
(5)小结:环磷酰胺和约氏疟原虫的联合用药物组合物具有更有效地抑制肺癌生长和延长荷瘤小鼠的中位生存期的效果，不会显著地增加荷瘤小鼠的毒副作用。总剂量60mg/kg、第6、13和20天分三次给药的环磷酰胺和第7天接种的约氏疟原虫，是很有潜力的用于治疗肺癌的联合用药组合物。
实施例6
本实施例提供一种抗肿瘤的联合用药物组合物，所述联合用药物组合物包括夏氏疟原虫和化疗药物环磷酰胺，观察优化方案的环磷酰胺给药联合夏氏疟原虫疗法治疗肺的癌效果。
本实施例所需的实验材料和试剂包与实施例5的区别仅在于疟原虫为小鼠夏氏疟原虫(Plasmodium chabaudi，MRA-429，Pc)，来自Malaria Research and Reference Reagent Resource Center(MR4)免费馈赠。
(1)建立动物模型，具体方法为：
步骤(Ⅰ)(Ⅱ)(Ⅲ)与实施例5相同。
(Ⅳ)实验分组：按照肿瘤大小随机分为四组：对照肿瘤组(Con)，疟原虫治疗组(Pc)，环磷酰胺治疗组(CTX)，环磷酰胺联合疟原虫治疗组(Pc+CTX)。每组10只小鼠，共40只。
(2)疟原虫接种荷瘤小鼠的具体方法与实施例5相同。
(3)环磷酰胺给药的具体方法与实施例5相同。
(4)检测指标与实施例5相同。
(5)实验结果:
(Ⅰ)如图21和表11所示：对照肿瘤组(Con)，疟原虫治疗组(Pc)，环磷酰胺治疗组(CTX)，环磷酰胺联合疟原虫治疗组(Pc+CTX)都显著地抑制肺癌的生长。联合治疗比单一的夏氏疟原虫治疗、单一的环磷酰胺治疗更有效地抑制肿瘤的生长。说明环磷酰胺和夏氏疟原虫的联合用药组合物具有更有效地抑制肺癌生长的效果。
表11
组间比较 显著性
Con vs Pc **
Con vs CTX *
Con vs Pc+CTX **
Pcvs Pc+CTX *
CTX vs Pc+CTX **
(Ⅱ)如图22和表12所示：肿瘤组是27.5天，夏氏疟原虫治疗组是34.5天，环磷酰胺治疗组是39.5天，联合治疗组是42.5天。联合治疗与单一的疟原虫治疗、环磷酰胺治疗的中位生存期长，但生存差异不具有显著的统计学意义，但仍提示夏氏疟原虫和环磷酰胺的联合用药物组合物可能比单一治疗更能延长荷瘤小鼠的生存。
表12
组间比较 显著性
Con vs CTX *
Con vs Pc+CTX **
(Ⅲ)如图23所示：夏氏疟原虫治疗组和联合治疗组的荷瘤小鼠的原虫血症感染时程周期比较一致，环磷酰胺对夏氏疟原虫的影响较小。说明环磷酰胺可以与夏氏疟原虫疗法联合用于治疗肺癌，两种疗法干扰较小。
(Ⅳ)如图24所示：联合治疗组的荷瘤小鼠的体重主要受到夏氏疟原虫的影响，受到环磷酰胺的影响较小，环磷酰胺与疟原虫联合使荷瘤小鼠的体重减轻作用轻微的叠加，但不会显著地增加荷瘤小鼠的毒副作用。
(6)小结:环磷酰胺与夏氏疟原虫的联合治疗比单一环磷酰胺、单一夏氏疟原虫更有效地抑制肺癌的生长，延长荷瘤小鼠的中位生存期，不会增加荷瘤小鼠的毒副作用。总剂量60mg/kg、第6、13和20天分三次给药的环磷酰胺和第7天接种的夏氏疟原虫，是很有潜力的用于治疗肺癌的联合用药组合物。
实施例7
本实施例提供一种抗肿瘤的联合用药物组合物，所述联合用药物组合物包括约氏疟原虫和化疗药物培美曲塞，观察优化方案的培美曲塞给药联合约氏疟原虫疗法治疗肺癌的效果。
本实施例所需的实验材料和试剂与实施例3的区别仅在于化学治疗药物为培美曲塞(缩写PEM)，购自Sigma-Aldrich公司。
(1)建立动物模型，具体方法为：
步骤(Ⅰ)(Ⅱ)(Ⅲ)与实施例3相同。
(Ⅳ)实验分组：按照肿瘤大小随机分为四组：对照肿瘤组(Con)，疟原虫治疗组(Py)，培美曲塞治疗组(PEM)，培美曲塞联合疟原虫治疗组(Py+PEM)。每组10只小鼠，共40只。
(2)疟原虫接种荷瘤小鼠的具体方法与实施例3相同。
(3)培美曲塞给药，具体方法为：
(Ⅰ)给药方式：腹腔注射给药；
(Ⅱ)给药剂量：20mg/kg×6；
(Ⅲ)药物制备：溶解在生理盐水，配置浓度为2mg/mL；
(Ⅳ)给药时间：PEM组和Py+PEM组在肿瘤接种第3、7、10、14、17、21天给药， 每次给药剂量为20mg/kg。
(4)检测指标与实施例3相同。
(5)实验结果:
(Ⅰ)如图25和表13所示：对照肿瘤组(Con)，疟原虫治疗组(Py)，培美曲塞治疗组(PEM)，培美曲塞联合疟原虫治疗组(Py+PEM)都显著抑制肺癌的生长。联合治疗比单一的培美曲塞治疗、单一的约氏疟原虫治疗更有效地抑制肿瘤的生长。说明培美曲塞和约氏疟原虫的联合用药物组合物具有更有效地抑制肺癌生长的效果。
表13
组间比较 显著性
Con vs Py **
Con vs PEM **
Con vs Py+PEM **
Py vs Py+PEM *
PEM vs Py+PEM **
(Ⅱ)如图26和表14所示：对照肿瘤组的中位生存期是28天，约氏疟原虫治疗组是37天，培美曲塞治疗组是32.5天，联合治疗组是43天。联合治疗比单一的疟原虫治疗、单一的疟原虫治疗的中位生存期长，与单一的培美曲塞治疗的生存差异具有显著的统计学意义，与单一的疟原虫治疗的生存差异没有显著的统计学意义。提示培美曲塞和约氏疟原虫的联合用药物组合物可能比单一的治疗更加有效地延长荷瘤小鼠的生存期。
表14
组间比较 显著性
Con vs Py *
Con vs Py+PEM **
PEM vs Py+PEM *
(Ⅲ)如图27所示：约氏疟原虫治疗组和联合治疗组的荷瘤小鼠的原虫血症时程周期比较一致。培美曲塞对疟原虫的影响较小。
(Ⅳ)如图28所示：联合治疗组的荷瘤小鼠的体重受到夏氏疟原虫和培美曲塞的双重影响，培美曲塞与疟原虫联合使荷瘤小鼠的体重减轻作用叠加，影响荷瘤小鼠的生存。
(6)小结:培美曲塞和约氏疟原虫的联合治疗具有更加有效地抑制肺癌的生长和延长荷瘤小鼠的中位生存期的效果。培美曲塞对约氏疟原虫没有抑制作用，但联合用药会使荷瘤小鼠体重更轻，影响荷瘤小鼠的生存。总剂量120mg/kg、第3、7、10、13、17、20天分六次给药的培美曲塞给药和第7天接种约氏疟原虫，是很有潜力的用于治疗肺癌的联合用药组合物。
实施例8
本实施例提供一种抗肿瘤的联合用药物组合物，所述联合用药物组合物包括约氏疟原虫 和化疗药物顺铂，观察顺铂联合约氏疟原虫疗法治疗肺癌的效果。
本实施例所需的实验材料与实施例3的区别仅在于化学治疗药物为顺铂(cis-platinum，缩写DDP)，购自Sigma-Aldrich公司。
(1)建立动物模型，具体方法为：
步骤(Ⅰ)(Ⅱ)(Ⅲ)与实施例3相同。
(Ⅳ)实验分组：按照肿瘤大小随机分为四组：对照肿瘤组(Con)，疟原虫治疗组(Py)，顺铂治疗组(DDP)，顺铂联合疟原虫治疗组(Py+DDP)。每组11只小鼠，共44只。
(2)疟原虫接种荷瘤小鼠的具体方法与实施例3相同。
(3)顺铂给药，具体方法为：
(Ⅰ)给药方式：腹腔注射给药；
(Ⅱ)给药剂量：1mg/kg×7；
(Ⅲ)药物制备：溶解在生理盐水，配置浓度为0.1mg/mL；
(Ⅳ)给药时间：DDP治疗组和Py+DDP治疗组在肿瘤接种第7天开始给药，每两天给一次药，给药两周，每次给药剂量为1mg/kg。
(4)检测指标与实施例3相同。
(5)实验结果:
(Ⅰ)如图29和表15所示：对照肿瘤组(Con)，疟原虫治疗组(Py)，顺铂治疗组(DDP)，顺铂联合疟原虫治疗组(Py+DDP)都显著抑制肺癌的生长。联合治疗比单一的顺铂治疗、单一的约氏疟原虫治疗更有效地抑制肿瘤的生长。说明顺铂和约氏疟原虫的联合用药物组合物具有更有效地的抑制肺癌生长的效果。
表15
组间比较 显著性
Con vs Py **
Con vs DDP **
Con vs Py+DDP **
Py vs Py+DDP *
DDP vs Py+DDP *
(Ⅱ)如图30和表16所示：比较荷瘤小鼠中位生存期，肿瘤组是27天，约氏疟原虫治疗组是37天，顺铂治疗组是30天，联合治疗组是38天。疟原虫治疗和联合治疗都延长荷瘤小鼠的生存。联合治疗组与单一的疟原虫治疗组相比并没有显著延长荷瘤小鼠的中位生存期，生存差异没有显著的统计学意义。
表16
组间比较 显著性
Con vs Py *
Con vs Py+DDP *
DDP vs Py+DDP *
(Ⅲ)如图31所示：联合治疗组比单一的约氏疟原虫治疗组的荷瘤小鼠的原虫血症时程周期短其原虫血症峰值也较低，提示顺铂对疟原虫有抑制作用。
(Ⅳ)如图32所示：顺铂联合约氏疟原虫治疗组的荷瘤小鼠要受到顺铂和疟原虫的双重影响，顺铂治疗和疟原虫都使荷瘤小鼠体重降低。
(6)小结:顺铂与约氏疟原虫的联合治疗比单一的顺铂治疗、单一的约氏疟原虫治疗更有效地抑制肺癌的生长，但并没有显著延长中位生存期。顺铂对约氏疟原虫感染周期有显著影响，顺铂和约氏疟原虫联合用药会使荷瘤小鼠体重更轻。总剂量7mg/kg、第7天开始给药两周的顺铂和第7天接种的约氏疟原虫，是很有潜力的治疗肺癌的联合用药组合物。
实施例9
本实施例提供一种抗肿瘤的联合用药物组合物，所述联合用药物组合物包括约氏疟原虫和丝裂霉素，观察不同剂量的丝裂霉素联合约氏疟原虫免疫疗法治疗肺癌的效果。
本实施例所需的实验材料和试剂与实施例3的区别仅在于化学治疗药物为丝裂霉素(Mitomycin，缩写MMC)，购自Sigma-Aldrich公司。
(1)建立动物模型，具体方法为：
步骤(Ⅰ)(Ⅱ)(Ⅲ)与实施例3相同。
(Ⅳ)实验分组：按照肿瘤大小随机分为五组：对照肿瘤组(Con)，疟原虫疗组(Py)，低剂量丝裂霉素联合疟原虫治疗组(Py+MMC(低剂量))，中剂量丝裂霉素联合疟原虫治疗组(Py+MMC(中剂量))，高剂量丝裂霉素联合疟原虫治疗组(Py+MMC(高剂量))。每组10只小鼠，共50只。
(2)疟原虫接种荷瘤小鼠的具体方法与实施例3相同。
(3)丝裂霉素给药，具体方法为：
(Ⅰ)给药方式：腹腔注射给药；
(Ⅱ)给药剂量：低剂量为0.027mg/kg×2，中剂量为0.083mg/kg×2，高剂量为0.25mg/kg×2；
(Ⅲ)药物制备：溶解在生理盐水，配置浓度为0.027、0.083和0.25mg/mL等三个浓度；
(Ⅳ)给药时间：(Py+MMC(低剂量))、(Py+MMC(中剂量))和(LLC+Py+MMC(高剂量))组在肿瘤接种第11、18天给药。
(4)检测指标与实施例3相同。
(5)实验结果:
(Ⅰ)如图33和表17所示：对照肿瘤组(Con)，疟原虫疗组(Py)，低剂量丝裂霉素联合疟原虫治疗组(Py+MMC(低剂量))，中剂量丝裂霉素联合疟原虫治疗组(Py+MMC(中剂量))，高剂量丝裂霉素联合疟原虫治疗组(Py+MMC(高剂量))都显著抑制肺癌的生长。联合治疗表现出丝裂霉素的剂量依赖效应，剂量越大，抑制肺癌的生长的效果越好。说明约 氏疟原虫和丝裂霉素的联合用药具有更有效地抑制肺癌生长的效果。
表17
组间比较 显著性
Con vs Py **
Con vs Py+MMC(低剂量) **
Con vs Py+MMC(中剂量) **
Con vs Py+MMC(高剂量) **
Py vs Py+MMC(低剂量) *
Py vs Py+MMC(中剂量) **
Py vs Py+MMC(高剂量) **
Py+MMC(低剂量)vs Py+MMC(高剂量) **
Py+MMC(中剂量)vs Py+MMC(高剂量) **
(Ⅱ)如图34和表18所示：肿瘤组是28天，单一的约氏疟原虫治疗组是36天，低剂量的丝裂霉素的联合治疗组是44天，中剂量的丝裂霉素的联合治疗组是34天，高剂量的丝裂霉素的联合治疗组是31天。低剂量的丝裂霉素联合治疗组比中、高剂量的丝裂霉素的联合治疗组更有效地延长荷瘤小鼠的生存期，说明低剂量的丝裂霉素和约氏疟原虫的联合用药能更有效地延长荷瘤小鼠的生存期。中、高剂量的丝裂霉素可能存在一定的毒副作用。
表18
组间比较 显著性
Con vs Py *
Con vs Py+MMC(低剂量) **
Py+MMC(低剂量)vs Py+MMC(高剂量) *
Py+MMC(中剂量)vs Py+MMC(高剂量) **
(Ⅲ)如图35所示：三个联合治疗的荷瘤小鼠的原虫血症高峰期出现的时间并不一致，丝裂霉素浓度越高，高峰期出现时间越推后。丝裂霉素对疟原虫有一定程度的抑制作用，浓度越高，抑制越明显，但不会清除疟原虫。
(Ⅳ)如图36所示：三个联合治疗组的荷瘤小鼠的体重主要受到疟原虫的影响，受到丝裂霉素的影响较小。
(6)小结:低剂量的丝裂霉素给药与约氏疟原虫的联合治疗能更有效地抑制肺癌的生长和延长荷瘤小鼠的生存期。低丝裂霉素对约氏疟原虫有轻微的抑制作用，但不会清除疟原虫。中、高剂量有一定的毒副作用，不适合与疟原虫联合治疗肺癌。总剂量0.054mg/kg、第11、18天分次给药的丝裂霉素和第7天接种的约氏疟原虫，是很有潜力的用于治疗肺癌的联合用药组合物。
实施例10
本实施例提供一种抗肿瘤的联合用药物组合物，所述联合用药物组合物包括约氏疟原虫和化疗药物多西他赛，观察不同剂量的多西他赛给药联合约氏疟原虫免疫疗法治疗肺癌的效果。
本实施例所需的实验材料和试剂与实施例9的区别仅在于化学治疗药物为多西他赛(Docetaxel，缩写DTX)，购自赛诺菲公司。
(1)建立动物模型，具体方法为：
步骤(Ⅰ)(Ⅱ)(Ⅲ)与实施例9相同。
(Ⅳ)实验分组：按照肿瘤大小随机分为五组：对照肿瘤组(Con)，疟原虫治疗组(Py)，低剂量的多西他赛联合疟原虫治疗组(Py+DTX(低剂量))，中剂量的多西他赛联合疟原虫治疗组(Py+MMC(中剂量))，高剂量的多西他赛联合疟原虫治疗组(Py+MMC(高剂量))。每组10只小鼠，共50只。
(2)疟原虫接种荷瘤小鼠的具体方法与实施例9相同。
(3)多西他赛给药，具体方法为：
(Ⅰ)给药方式：静脉注射给药；
(Ⅱ)给药剂量：低剂量为2.2mg/kg×3，中剂量为6.7mg/kg×3，高剂量为2mg/kg×3；
(Ⅲ)药物制备：溶解在生理盐水，配置浓度为0.22、0.673和2mg/mL等三个浓度；
(Ⅳ)给药时间：(Py+DTX(低剂量))、(Py+DTX(中剂量))和(Py+DTX(高剂量))组在肿瘤接种第3、10、17天给药。
(4)检测指标与实施例9相同。
(5)实验结果:
(Ⅰ)如图37和表19所示：对照肿瘤组(Con)，疟原虫治疗组(Py)，低剂量的多西他赛联合疟原虫治疗组(Py+DTX(低剂量))，中剂量的多西他赛联合疟原虫治疗组(Py+MMC(中剂量))，高剂量的多西他赛联合疟原虫治疗组(Py+MMC(高剂量))都能显著抑制肺癌的生长。三个联合治疗都比单一的疟原虫治疗的抑制肺癌生长的效果好。在联合治疗中，中剂量多西他赛的联合治疗抑瘤效果最好，高剂量次之，低剂量最差。说明多西他赛与约氏疟原虫的联合用药能更加有效地抑制肺癌的生长，但并不表现为多西他赛的剂量依赖关系。
表19
组间比较 显著性
Con vs Py **
Con vs Py+DTX(低剂量) **
Con vs Py+DTX(中剂量) **
Con vs Py+DTX(高剂量) **
Py vs Py+DTX(低剂量) *
Py vs Py+DTX(中剂量) **
Py vs Py+DTX(高剂量) **
Py+DTX(低剂量)vs Py+DTX(高剂量) **
Py+DTX(中剂量)vs Py+DTX(高剂量) *
(Ⅱ)如图38和表20所示：肿瘤组是28天，单一的约氏疟原虫治疗组是36天，低剂量的多西他赛的联合治疗组是38天，中剂量剂量的多西他赛的联合治疗组是41天，高剂量的多西他赛的联合治疗组是30.5天。单一的约氏疟原虫治疗和中、低剂量的多西他赛的联合治疗能更有效地延长荷瘤小鼠的生存期，而高剂量的多西他赛的联合治疗不能延长荷瘤小鼠的生存期。中、低剂量的两组联合治疗与单一的约氏疟原虫治疗的生存差异不具有显著的统计学意义。中、高剂量的多西他赛可能存在一定的毒副作用。
表20
组间比较 显著性
Con vs Py *
Con vs Py+DTX(低剂量) **
Con vs Py+DTX(中剂量) **
Py+DTX(中剂量)vs Py+DTX(高剂量) *
(Ⅲ)如图39所示：单一的约氏疟原虫治疗组和三个联合治疗组的荷瘤小鼠的原虫血症时程周期出现不一致。说明中、高剂量的多西他赛显著抑制疟原虫，导致感染周期异常，低剂量对疟原虫影响较小。
(Ⅳ)如图40所示：单一的约氏疟原虫治疗组和联合治疗组的荷瘤小鼠的体重受到疟原虫和多西他赛双重影响。联合治疗中，高剂量的多西他赛对荷瘤小鼠体重下降作用非常明显，中、低剂量的多西他赛则影响较小。
(6)小结:中剂量的多西他赛与约氏疟原虫的联合治疗能更有效地抑制肺癌的生长和延长荷瘤小鼠的中位生存期。总剂量6.7mg/kg、第3、10、17天分次给药的多西他赛和第7天接种的约氏疟原虫，是很有潜力的用于治疗肺癌的联合用药物组合物。
实施例11
本实施例提供一种抗肿瘤的联合用药物组合物，所述联合用药物组合物包括约氏疟原虫和化疗药物依托泊苷，观察不同剂量的依托泊苷给药联合约氏疟原虫免疫疗法治疗肺癌的效果。
本实施例所需的实验材料和试剂与实施例9的区别仅在于化学治疗药物为依托泊苷(Etoposide，缩写VP16)，购自百时美施贵宝公司。
(1)建立动物模型，具体方法为：
步骤(Ⅰ)(Ⅱ)(Ⅲ)与实施例9相同。
(Ⅳ)实验分组：按照肿瘤大小随机分为五组：对照肿瘤组(Con)，疟原虫治疗组(Py)，低剂量的依托泊苷联合疟原虫治疗组(Py+VP16(低剂量))，高剂量的多西他赛联合疟原虫治疗组(Py+VP16(高剂量))。每组10只小鼠，共40只。
(2)疟原虫接种荷瘤小鼠的具体方法与实施例9相同。
(3)依托泊苷给药，具体方法为：
(Ⅰ)给药方式：腹腔注射给药；
(Ⅱ)给药剂量：低剂量为10mg/kg×3，高剂量为30mg/kg×3；
(Ⅲ)药物制备：溶解在生理盐水，配置浓度为1和3mg/mL等三个浓度；
(Ⅳ)给药时间：(Py+VP16(低剂量))和(Py+VP16(高剂量))组，在肿瘤接种第11、14、17天给药。
(4)检测指标与实施例9相同。
(5)实验结果:
(Ⅰ)如图41和表21所示：对照肿瘤组(Con)，疟原虫治疗组(Py)，低剂量的依托泊苷联合疟原虫治疗组(Py+VP16(低剂量))，高剂量的多西他赛联合疟原虫治疗组(Py+VP16(高剂量))都能显著抑制肺癌的生长。低剂量的依托泊苷联合治疗与高剂量的联合治疗相比抑制肺癌生长的效果好。低剂量的依托泊苷联合治疗与单一的疟原虫治疗相比抑制肺癌生长的效果好。说明低剂量的依托泊苷和约氏疟原虫的联合用药物组合物具有更有效地抑制肺癌生长的效果。
表21
组间比较 显著性
Con vs Py **
Con vs Py+VP16(低剂量) **
Con vs Py+VP16(高剂量) **
Py vs Py+VP16(低剂量) *
Py+VP16(低剂量)vs Py+VP16(高剂量) *
(Ⅱ)如图42和表22所示：肿瘤组是28天，约氏疟原虫治疗组是36天，低剂量的依托泊苷联合治疗组是44天，高剂量的依托泊苷联合治疗组是40.5天。单一的约氏疟原虫治疗和高、低剂量的依托泊苷的联合治疗都能延长荷瘤小鼠的生存期。低剂量的依托泊苷的联合治疗与高剂量的依托泊苷的联合治疗的中位生存期长，但生存差异不具有显著的统计学意义。高剂量的依托泊苷可能存在一定的毒副作用。低剂量的依托泊苷与约氏疟原虫的联合治疗可能有效地延长荷瘤小鼠的生存期。
表22
组间比较 显著性
Con vs Py *
Con vs Py+VP16(低剂量) **
Con vs Py+VP16(高剂量) **
(Ⅲ)如图43所示：约氏疟原虫治疗组和两个联合约氏疟原虫治疗组的荷瘤小鼠的原虫血症时程周期不一致。说明依托泊苷对疟原虫有明显的抑制作用。
(Ⅳ)如图44所示：高、低剂量的依托泊苷联合约氏疟原虫治疗组的荷瘤小鼠的体重受到疟原虫和依托泊苷双重影响，使得荷瘤小鼠体重更轻。
(6)小结:低剂量的依托泊苷联合约氏疟原虫能更有效地抑制肺癌的生长和延长荷瘤小鼠的中位生存期。高、低依托泊苷对约氏疟原虫感染均有抑制作用，但不会清除疟原虫。总剂量30mg/kg、第11、14、17天分次给药的依托泊苷和在第7天接种的约氏疟原虫，是很有潜力的用于治疗肺癌的联合用药物组合物。
实施例12
本实施例提供一种抗肿瘤的联合用药物组合物，所述联合用药物组合物包括约氏疟原虫和HIV蛋白酶抑制剂奈非那韦，观察奈非那韦给药联合约氏疟原虫免疫疗法治疗肺癌的效果。
本实施例所需的实验材料和试剂与实施例9的区别仅在于化学治疗药物为奈非那韦(Nelfinavir，缩写NFV)，购自阿古龙医药制品公司。
(1)建立动物模型，具体方法为：
步骤(Ⅰ)(Ⅱ)(Ⅲ)与实施例9相同。
(Ⅳ)实验分组：按照肿瘤大小随机分为四组：对照肿瘤组(Con)，疟原虫治疗组(Py)，奈非那韦治疗组(NFV)，奈非那韦联合疟原虫治疗组(Py+CTX)。每组10只小鼠，共40只。
(2)疟原虫接种荷瘤小鼠的具体方法与实施例9相同。
(3)奈非那韦给药，具体方法为：
(Ⅰ)给药方式：腹腔注射给药；
(Ⅱ)给药剂量：400mg/kg×10；
(Ⅲ)药物制备：溶解在生理盐水，配置浓度为40mg/mL；
(Ⅳ)给药时间：NFV和Py+NFV在肿瘤接种第10天开始给药，连续给药10天，每次给药剂量为400mg/kg。
(4)检测指标与实施例9相同。
(5)实验结果:
(Ⅰ)如图45和表23所示：对照肿瘤组(Con)，疟原虫治疗组(Py)，奈非那韦治疗组(NFV)，奈非那韦联合疟原虫治疗组(Py+CTX)都有效地抑制肺癌的生长。联合治疗比单一的奈非那韦治疗、单一的约氏疟原虫能更有效地抑制肿瘤的生长。说明奈非那韦和约氏疟原虫的联合用药组合物具有更有效地抑制肺癌生长的效果。
表23
组间比较 显著性
Con vs Py **
Con vs NFV **
Con vs Py+NFV **
Py vs Py+NFV **
NFV vs Py+NFV **
(Ⅱ)如图46和表24所示：比较荷瘤小鼠中位生存期，肿瘤组是29天，约氏疟原虫治疗组是38天，奈非那韦治疗组是34天，奈非那韦联合约氏疟原虫治疗组是44天。单一的疟原虫治疗和联合治疗组都更有效地延长荷瘤小鼠的中位生存期。联合治疗比单一的奈非那韦治疗更有效地延长荷瘤小鼠的中位生存期，但与单一的疟原虫治疗的生存差异不具有显著的统计学意义。提示奈非那韦和约氏疟原虫的联合用药组合物可能更有效地延长荷瘤小鼠的中位生存期。
表24
组间比较 显著性
Con vs Py *
Con vs Py+NFV **
NFV vs Py+NFV *
(Ⅲ)如图47所示：单一的约氏疟原虫治疗组和联合治疗组的荷瘤小鼠的原虫血症时程周期比较一致。奈非那韦对疟原虫的影响较小，奈非那韦可以与约氏疟原虫疗法联合用于治疗肺癌。
(Ⅳ)如图48所示：单一的奈非那韦持续灌胃给药对荷瘤小鼠体重影响比较小，但联合治疗组的荷瘤小鼠的体重主要受疟原虫和奈非那韦持续灌胃给药的影响。
(6)小结:奈非那韦与约氏疟原虫的联合治疗能更有效地抑制肺癌的生长，更好的延长荷瘤小鼠的中位生存期。奈非那韦不会增加荷瘤小鼠的毒副作用。总剂量4g/kg、第10天开始给药持续10天给药的奈非那韦和第7天接种的约氏疟原虫，是很有潜力的用于治疗肺癌的联合用药物组合物。
申请人声明，本申请通过上述实施例来说明本申请的疟原虫免疫疗法联合化疗的多种组合方法及其在癌症治疗方面的应用，但本申请并不局限于上述实施例，即不意味着本申请必须依赖上述实施例才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。
以上详细描述了本申请的优选实施方式，但是，本申请并不限于上述实施方式中的具体细节，在本申请的技术构思范围内，可以对本申请的技术方案进行多种简单变型，这些简单变型均属于本申请的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本申请对各种可能的组合方式不再另行说明。

Claims (15)

  1. 一种抗肿瘤的联合用药物组合物，其包括疟原虫和化疗药物。
  2. 如权利要求1所述的联合用药物组合物，其中，所述疟原虫包括恶性疟原虫、间日疟原虫、三日疟原虫、卵形疟原虫或诺氏疟原虫中的任意一种或至少两种的混合，优选为恶性疟原虫或间日疟原虫。
  3. 如权利要求1或2所述的联合用药物组合物，其中，所述化疗药物包括烷化剂类化疗药物、抗代谢类化疗药物、抗生素类化疗药物、动植物类化疗药物、杂类化疗药物或HIV蛋白酶抑制剂。
  4. 如权利要求3所述的联合用药物组合物，其中，所述烷化剂类化疗药物包括环磷酰胺或异环磷酰胺；
    优选地，所述抗代谢类化疗药物包括吉西他滨、培美曲塞、5-氟尿嘧啶、阿糖胞苷或甲氨蝶呤；
    优选地，所述抗生素类化疗药物包括丝裂霉素、多柔比星或放线菌素D；
    优选地，所述动植物类化疗药物包括依托泊苷、多西他赛、紫杉醇、长春新碱或伊立替康；
    优选地，所述杂类化疗药物包括顺铂、卡铂、奥沙利铂或门冬酰胺酶；
    优选地，所述HIV蛋白酶抑制剂化疗药物包括奈非那韦、沙奎那韦、茚地那韦或利托那韦。
  5. 如权利要求1所述的联合用药物组合物，其中，所述联合用药物组合物包括：疟原虫和吉西他滨；疟原虫和环磷酰胺；疟原虫和环磷酰胺；疟原虫和培美曲塞；疟原虫和顺铂；疟原虫和丝裂霉素；疟原虫和多西他赛；疟原虫和依托泊苷；或者，疟原虫和奈非那韦。
  6. 如权利要求1-5中任一项所述的联合用药物组合物，其中，所述联合用药物组合物的剂型包括药剂学上可接受的任意一种剂型。
  7. 如权利要求1-6中任一项所述的联合用药物组合物，其中，所述联合用药物组合物还包括药剂学上可接受药用辅料中的任意一种或至少两种的组合。
  8. 如权利要求1-7中任一项所述的联合用药物组合物，其中，所述联合用药物组合物为单一的复方制剂。
  9. 如权利要求1-7中任一项所述的联合用药物组合物，其中，所述联合用药物组合物为单独的疟原虫制剂和单独的化疗药物制剂的组合。
  10. 如权利要求9所述的联合用药物组合物，其中，所述单独的疟原虫制剂和所述单独的化疗药物制剂同时施用或者依次施用。
  11. 如权利要求1-10中任一项所述的联合用药物组合物，其中，所述联合用药物组合物的给药途径包括静脉注射、腹腔注射、肌肉注射、皮下注射、口服给药、舌下给药、鼻腔给药或经皮给药，优选静脉注射或口服给药。
  12. 如权利要求1-11中任一项所述的联合用药物组合物，其中，所述联合用药物组合物 为负载于药用载体上的联合用药物组合物。
  13. 如权利要求12所述的联合用药物组合物，其中，所述药用载体包括脂质体、胶束、树枝状大分子、微球或微囊。
  14. 如权利要求1-13中任一项所述的联合用药物组合物在制备抗肿瘤药物中的应用。
  15. 如权利要求14所述的应用，其中，所述肿瘤包括肺癌、胃癌、结肠癌、肝癌、乳腺癌或胰腺癌。
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