CN116617222B - 小分子离子通道阻滞剂mk-801在制备治疗肿瘤或抗感染药物的应用 - Google Patents
小分子离子通道阻滞剂mk-801在制备治疗肿瘤或抗感染药物的应用 Download PDFInfo
- Publication number
- CN116617222B CN116617222B CN202310521213.8A CN202310521213A CN116617222B CN 116617222 B CN116617222 B CN 116617222B CN 202310521213 A CN202310521213 A CN 202310521213A CN 116617222 B CN116617222 B CN 116617222B
- Authority
- CN
- China
- Prior art keywords
- tumor
- macrophages
- nmdar
- ion channel
- channel blocker
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 57
- 102000004310 Ion Channels Human genes 0.000 title claims abstract description 23
- 150000001793 charged compounds Chemical class 0.000 title claims abstract description 17
- 239000003814 drug Substances 0.000 title claims description 6
- 229940079593 drug Drugs 0.000 title claims description 5
- 208000015181 infectious disease Diseases 0.000 title description 4
- 238000002360 preparation method Methods 0.000 title description 2
- 208000014018 liver neoplasm Diseases 0.000 claims abstract description 22
- 201000007270 liver cancer Diseases 0.000 claims abstract description 21
- 230000000694 effects Effects 0.000 claims abstract description 14
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 claims description 7
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 claims description 7
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 claims description 7
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 claims description 7
- 150000003384 small molecules Chemical class 0.000 claims description 3
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- 239000003623 enhancer Substances 0.000 claims 1
- 210000002540 macrophage Anatomy 0.000 abstract description 41
- 238000011282 treatment Methods 0.000 abstract description 34
- 102000004868 N-Methyl-D-Aspartate Receptors Human genes 0.000 abstract description 33
- 108090001041 N-Methyl-D-Aspartate Receptors Proteins 0.000 abstract description 33
- 108090000862 Ion Channels Proteins 0.000 abstract description 19
- 102100040678 Programmed cell death protein 1 Human genes 0.000 abstract description 12
- 101710089372 Programmed cell death protein 1 Proteins 0.000 abstract description 12
- 230000002401 inhibitory effect Effects 0.000 abstract description 10
- 210000001744 T-lymphocyte Anatomy 0.000 abstract description 9
- 230000000259 anti-tumor effect Effects 0.000 abstract description 9
- 230000001506 immunosuppresive effect Effects 0.000 abstract description 9
- 230000004614 tumor growth Effects 0.000 abstract description 9
- 210000000822 natural killer cell Anatomy 0.000 abstract description 8
- 230000001105 regulatory effect Effects 0.000 abstract description 8
- 230000006870 function Effects 0.000 abstract description 7
- 102000037982 Immune checkpoint proteins Human genes 0.000 abstract description 6
- 108091008036 Immune checkpoint proteins Proteins 0.000 abstract description 6
- 230000008685 targeting Effects 0.000 abstract description 6
- 101100260702 Mus musculus Tinagl1 gene Proteins 0.000 abstract description 3
- 230000003213 activating effect Effects 0.000 abstract description 3
- 101150088826 arg1 gene Proteins 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 3
- 101100167135 Mus musculus Chil3 gene Proteins 0.000 abstract description 2
- 230000004913 activation Effects 0.000 abstract description 2
- 230000028993 immune response Effects 0.000 abstract description 2
- 241000699670 Mus sp. Species 0.000 description 21
- 210000004979 bone marrow derived macrophage Anatomy 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 14
- 238000007920 subcutaneous administration Methods 0.000 description 12
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- 238000001514 detection method Methods 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- 238000002347 injection Methods 0.000 description 9
- 210000004881 tumor cell Anatomy 0.000 description 9
- 206010027476 Metastases Diseases 0.000 description 6
- 102100031925 NADPH-dependent diflavin oxidoreductase 1 Human genes 0.000 description 6
- 238000011529 RT qPCR Methods 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 6
- 238000009169 immunotherapy Methods 0.000 description 6
- 230000033228 biological regulation Effects 0.000 description 5
- 210000004981 tumor-associated macrophage Anatomy 0.000 description 5
- 239000011575 calcium Substances 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000053642 Catalytic RNA Human genes 0.000 description 3
- 108090000994 Catalytic RNA Proteins 0.000 description 3
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 3
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 3
- 230000005975 antitumor immune response Effects 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 229910001424 calcium ion Inorganic materials 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 230000009401 metastasis Effects 0.000 description 3
- 229910001414 potassium ion Inorganic materials 0.000 description 3
- 108091092562 ribozyme Proteins 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 206010062016 Immunosuppression Diseases 0.000 description 2
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 206010027457 Metastases to liver Diseases 0.000 description 2
- 206010061309 Neoplasm progression Diseases 0.000 description 2
- 102100029438 Nitric oxide synthase, inducible Human genes 0.000 description 2
- 101710089543 Nitric oxide synthase, inducible Proteins 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 238000002619 cancer immunotherapy Methods 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000007012 clinical effect Effects 0.000 description 2
- 238000003501 co-culture Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 229910001415 sodium ion Inorganic materials 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 210000002303 tibia Anatomy 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000005751 tumor progression Effects 0.000 description 2
- 210000000689 upper leg Anatomy 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 206010017533 Fungal infection Diseases 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 102000018899 Glutamate Receptors Human genes 0.000 description 1
- 108010027915 Glutamate Receptors Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101001023379 Homo sapiens Lysosome-associated membrane glycoprotein 1 Proteins 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 102000004086 Ligand-Gated Ion Channels Human genes 0.000 description 1
- 108090000543 Ligand-Gated Ion Channels Proteins 0.000 description 1
- 102100035133 Lysosome-associated membrane glycoprotein 1 Human genes 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- HOKKHZGPKSLGJE-GSVOUGTGSA-N N-Methyl-D-aspartic acid Chemical compound CN[C@@H](C(O)=O)CC(O)=O HOKKHZGPKSLGJE-GSVOUGTGSA-N 0.000 description 1
- 229940099433 NMDA receptor antagonist Drugs 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003556 anti-epileptic effect Effects 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 239000001961 anticonvulsive agent Substances 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000012830 cancer therapeutic Substances 0.000 description 1
- 230000005773 cancer-related death Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- ITVPBBDAZKBMRP-UHFFFAOYSA-N chloro-dioxido-oxo-$l^{5}-phosphane;hydron Chemical compound OP(O)(Cl)=O ITVPBBDAZKBMRP-UHFFFAOYSA-N 0.000 description 1
- DQTRYXANLKJLPK-UHFFFAOYSA-N chlorophosphonous acid Chemical compound OP(O)Cl DQTRYXANLKJLPK-UHFFFAOYSA-N 0.000 description 1
- ACSIXWWBWUQEHA-UHFFFAOYSA-N clodronic acid Chemical compound OP(O)(=O)C(Cl)(Cl)P(O)(O)=O ACSIXWWBWUQEHA-UHFFFAOYSA-N 0.000 description 1
- 229960002286 clodronic acid Drugs 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 238000011284 combination treatment Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000018732 detection of tumor cell Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- LBOJYSIDWZQNJS-CVEARBPZSA-N dizocilpine Chemical compound C12=CC=CC=C2[C@]2(C)C3=CC=CC=C3C[C@H]1N2 LBOJYSIDWZQNJS-CVEARBPZSA-N 0.000 description 1
- 229950004794 dizocilpine Drugs 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000005206 flow analysis Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229940126533 immune checkpoint blocker Drugs 0.000 description 1
- 230000008629 immune suppression Effects 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004941 influx Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 239000003703 n methyl dextro aspartic acid receptor blocking agent Substances 0.000 description 1
- 239000002539 nanocarrier Substances 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 230000036963 noncompetitive effect Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 229940001470 psychoactive drug Drugs 0.000 description 1
- 239000004089 psychotropic agent Substances 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/439—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom the ring forming part of a bridged ring system, e.g. quinuclidine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
本发明公开一种靶向NMDAR的小分子离子通道阻滞剂MK‑801在抑制肝癌进展中的应用。进一步，所述的NMDAR阻滞剂MK‑801通过抑制肿瘤微环境中巨噬细胞上NDMAR的过度激活，抑制巨噬细胞Chil3，Arg1等免疫抑制分子的表达，解除其免疫抑制表型，激活CD8+T细胞和NK细胞的抗肿瘤功能，抑制肝癌的进展。同时，MK‑801联合PD‑1抗体显著提高免疫检查点治疗的疗效。本发明的实施中通过实验验证了NMDAR阻滞剂MK‑801通过调节巨噬细胞表型和功能激活抗肿瘤体免疫反应，抑制肿瘤生长的作用。
Description
技术领域
本发明属于生物医疗技术领域，具体涉及的小分子离子通道阻滞剂MK-801在抑制肿瘤或抗感染进展方面的应用，以及小分子离子通道阻滞剂MK-801在调控肿瘤浸润巨噬细胞功能方面的应用。
背景技术
原发性肝癌目前作为全球第六大常见癌症以及全球癌症相关死亡的第三大原因，严重威胁着人类的生命健康。肝癌是世界上最常见的恶性肿瘤之一。肝癌的发生发展分子机制复杂，致病因素多，近年来，针对肝癌的治疗已经取得了显著的临床效果，目前治疗方式主要有手术切除、放疗或化疗以及联合治疗等方案。近年来，肿瘤免疫治疗已经成为继传统疗法后快速发展的新一代恶性肿瘤治疗方法，取得了显著的临床效果，具有巨大的临床应用前景。随着肿瘤生物学和免疫学的快速发展，免疫检查点阻断剂在多种类型肿瘤中取得显著的临床进展，通过激活体内免疫系统增强抗肿瘤免疫应答，抑制肿瘤的生长。靶向PD-1的抗体主要的肝癌免疫治疗药物，在肝癌治疗中的客观缓解率大约20％。依然有很多肝癌病人不响应PD-1抗体的治疗或复发。亟需开发新的肝癌治疗药物或提高PD-1抗体疗效的策略。
肿瘤免疫治疗的效果在很大程度上取决于肿瘤的微环境。肿瘤微环境(TME)组成复杂，通过多层次调控机制决定着肿瘤的进展和命运。其中肿瘤相关巨噬细胞(TAM)通过分泌免疫抑制性分子在肿瘤免疫逃逸中发挥关键作用。巨噬细胞主要分为M1型巨噬细胞(被称为经典型巨噬细胞)和M2型巨噬细胞(被称为替代性活化巨噬细胞)。在肿瘤发展过程中巨噬细胞逐渐由抗肿瘤的M1型转变为促肿瘤的M2型。肿瘤浸润巨噬细胞称为TAM(肿瘤相关巨噬细胞)，是免疫抑制性肿瘤微环境形成的关键免疫细胞群。诱导肿瘤微环境巨噬细胞由M2型转向M1型现已成为重要的肿瘤治疗策略。一般地，肿瘤患者体内免疫反应是被免疫抑制的状态，并且TAM在其中发挥了重要作用。因此，明确肿瘤微环境中TAM的调控方式和机制及其参与肿瘤免疫逃逸的作用机理，为靶向TAM的肿瘤免疫治疗提供新的潜在策略。
多种离子对巨噬细胞功能具有重要调节作用，包括钙离子、钾离子、镁离子等。调节不同离子在巨噬细胞内外以及其在细胞内不同细胞器中的分布，能够改变巨噬细胞表型和功能，实现对不同疾病的干预。
N-甲基-D-天冬氨酸受体(NMDAR)是一种离子型谷氨酸受体(iGluRs)，对Ca2+高度通透的配体门控离子通道。NMDAR是一种四聚体，包括两个NR1亚基和两个调节亚基(NR2A/B/C/D，NR3A/B)。谷氨酸、甘氨酸、多胺等等神经递质使NMDAR激活导致其构象改变离子通道开放，容许钙离子和钠离子大量流入细胞，钾离子流出细胞，改变细胞内多种信号和生物学过程。NMDAR对巨噬细胞功能具有重要的调节作用，开发靶向NMDAR离子通道的阻滞剂用于调节巨噬细胞，从而实现肝癌免疫治疗或增强现有免疫治疗药物的疗效。MK-801是NMDAR离子通道的特异阻滞剂，能够阻断NMDAR介导的钙离子和钠离子流入细胞，钾离子流出细胞。其化学结构式如下：
发明内容
本发明的主要目的就是针对现有技术中的问题，提供一种靶向NMDAR的小分子离子通道阻滞剂MK-801在制备抗肿瘤药物的应用。
为实现上述发明目的，本发明一方面提供了小分子离子通道阻滞剂MK-801在制备抗肝癌、抑制肝癌进展或抗感染药物的应用。
进一步地，所述的小分子离子通道阻滞剂MK-801抑制肝癌进展依赖于免疫系统，解除肿瘤浸润巨噬细胞的抑制表型，激活CD8+T细胞和NK细胞的抗肿瘤功能。
进一步地，所述的感染为细菌感染、真菌感染或病毒感染。
本发明的另一方面还提供了小分子离子通道阻滞剂MK-801在联合免疫检查点抑制剂提高免疫治疗效果方面的应用。
进一步地，所述的小分子离子通道阻滞剂MK-801联合PD-1抗体处理，提高免疫治疗的效果。
本发明还提供的一种药物组合物，其主要特点是，包括小分子离子通道阻滞剂MK-801和抗PD-1抗体。
本发明提供了靶向NMDAR的一种小分子离子通道阻滞剂MK-801，可以解除肿瘤微环境中巨噬细胞的免疫抑制表型，进一步激活CD8+T细胞和NK细胞的抗肿瘤功能，抑制肿瘤的进展。具体的，肿瘤细胞上清能够上调BMDM(骨髓衍生巨噬细胞)上NMDAR表达。小分子离子通道阻滞剂MK-801处理BMDM，巨噬细胞中Arg1、Chil3等免疫抑制基因被显著抑制，IL-6、TNFα、iNOS等炎症因子及相关蛋白表达上调。同时共刺激分子CD80和CD86显著上调，进一步增强了CD8+T细胞和NK细胞功能，抑制小鼠肝癌进展。
本发明还提供了该小分子离子通道阻滞剂MK-801在纳米载体靶向治疗中的应用。
本发明采用的小分子离子通道阻滞剂MK-801，能够增强免疫检查点治疗的疗效。具体的，MK-801单独处理抑制肿瘤生长，联合MK-801和PD-1抗体处理荷瘤小鼠，发现联合处理显著提高肿瘤抑制效果。对于许多肿瘤类型，免疫检查点抑制剂单药效果不佳，本发现为NDAMR阻滞剂联合免疫检查点阻断肿瘤治疗中提供可能性。
附图说明
图1为本发明的实施例1中通过流式细胞术检测肿瘤细胞上清处理能够上调BMDM上NMDAR NR1亚基的表达的结果示意图。
图2(a)至4(c)为本发明的实施例2中qPCR(实时定量PCR)和流式细胞术检测NMDAR阻滞剂MK-801处理对巨噬细胞的调节作用结果示意图。
图5(a)至8(c)为本发明的实施例3的结果示意图，NMDAR阻滞剂MK-801抑制荷瘤小鼠皮下移植瘤和转移瘤的生长依赖于巨噬细胞，同时增强免疫浸润的CD8+T和NK细胞功能抑制荷瘤小鼠皮下移植瘤和转移瘤的生长。
图9(a)至9(e)为本发明的实施例4中NMDAR阻滞剂MK-801联合PD-1抗体处理荷瘤小鼠、增强肿瘤抑制效果的结果示意图。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施例，进一步阐述本发明。
MK-801，又称地佐环平/地卓西平(dizocilpine)，是一种已知的中枢神经系统/抗癫痫/脑保护/精神类药物，是一种选择性非竞争性NMDA受体拮抗剂，Kd为37.2nM。MK-801通过与NMDA相关离子通道内的位点结合而起作用，通过阻滞离子通道，从而阻止Ca2+的流动。
本发明研究证实，NMDAR阻滞剂MK-801能够抑制C57BL/6J野生型小鼠肝癌皮下瘤的生长。进一步证实，小分子离子通道阻滞剂MK-801通过抑制肿瘤浸润巨噬细胞上NMDAR过度激活，解除巨噬细胞介导的免疫抑制，增强CD8+T和NK细胞抗肿瘤免疫反应，抑制肿瘤生长。同时，NMDAR阻滞剂MK-801显著提高PD-1抗体的肿瘤抑制效果，为开发合理的联合治疗提高包括肝癌在内的多种恶性肿瘤的临床疗效提供重要策略。
本发明的还提供了NMDAR阻滞剂MK-801抑制肝癌进展的检测方法，包括以下步骤：
1)构建C57BL/6J野生型小鼠Hepa1-6BL肝癌皮下瘤模型和肝癌转移模型，腹腔给予NMDAR阻滞剂MK-801，评估MK-801的抗肿瘤能力；
2)体外NMDAR阻滞剂MK-801处理C57BL/6J野生型小鼠骨髓衍生巨噬细胞(BMDM)，评估MK-801对巨噬细胞的调控作用；
3)小分子离子通道阻滞剂MK-801在联合免疫检查点抑制剂提高免疫治疗效果方面的应用。
实施例1
巨噬细胞上NMDAR表达
本实施例所采取的实验方法为：
体外诱导分化小鼠骨髓衍生巨噬细胞与肿瘤上清处理，流式检测NMDAR NR1亚基的表达。
1.1诱导分化小鼠骨髓衍生巨噬细胞：
1.1.1取C57BL/6J野生型成年小鼠，处死小鼠后将小鼠浸泡在75％的酒精中消毒2-3min。在腹部做一个切口，用剪刀分离骨骼上的肌肉，取小鼠胫骨和股骨随后将其浸泡在PBS缓冲液中。后续步骤在无菌操作台中完成。
1.1.2用无菌镊子取出胫骨和股骨后，剪刀在其两侧剪去小块，用装有PBS的20mL注射器重复上下交替冲洗骨髓，将冲出的骨髓细胞经过70μm细胞筛收集至50mL无菌离心管中，4℃条件下2000rpm离心4min。
1.1.3往细胞团块中加入1mL 1×红细胞裂解液悬浮细胞，静置2min后，加入10mLPBS进行洗涤，4℃，2000rpm离心4min。
1.1.4将细胞用RPMI 1640培养液重悬(含5％ L929上清)并且调整细胞密度至2x106/孔，铺至未处理的6孔细胞培养板中于37℃、5％ CO2条件下进行培养，细胞培养至第5天时，显微镜下观察骨髓分化来源的巨噬细胞。
1.2流式检测BMDM上NMDAR NR1亚基的表达
1.2.1第5天的BMDM细胞，用Hepa1-6BL细胞培养上清(CM)或者新鲜培养液(FM)处理24h后，消化重悬后转移至96孔U底板，分为对照组和肿瘤上清处理组。
1.2.2加入NMDAR NR1亚基抗体染色后加入FITC标记的二抗染色，BD Aria III仪器进行检测，FlowJo软件进行分析。
结果如图1所示，显示肿瘤上清能够上调BMDM上NMDAR NR1表达。
实施例2
NMDAR阻滞剂MK-801对巨噬细胞的调节作用
定量RT-PCR检测NMDAR阻滞剂MK-801处理对巨噬细胞的调节作用
2.1第5天的BMDM细胞，分为对照组和处理组，其中处理组加入MK-801 300μM处理6h，弃去培养液，PBS洗一次，每孔加入1mL RNA Trizol室温放置5min后，转移至1.5mL无核酶离心管。
2.2提取总RNA。加入200μL氯仿上下颠倒15次冰上静置5min，4℃ 12000rpm离心15min；取上清至新的无核酶离心管，等体积加入异丙醇冰上静置30min，4℃ 12000rpm离心10min；弃上清加入1mL无水乙醇轻轻混匀，4℃ 7500rpm离心5min；弃上清晾干无水乙醇后加入30μL无核酶水轻轻混匀，检测浓度。
2.3逆转录cDNA。按照HiScript II q RT SuperMix for qPCR(+gDNA wiper)(Vazyme R223-01)说明书逆转合成cDNA。
2.4实时定量PCR。按照ChamQ SYBR qPCR Master Mix(Vazyme Q311-02)进行实时定量PCR。
2.5实时定量PCR检测，引物信息为：
2.6数据分析遵循△△Ct算法。
使用MK-801处理BMDM后qPCR检测巨噬细胞极化相关基因变化，和肿瘤细胞共培养后检测表达共刺激分子CD86、CD80变化，BMDM和CD8 T细胞共培养实验检测MK-801抑制NMDAR时对巨噬细胞的抗原提呈能力的影响。
结果显示：如图2(a)至2(i)所示，与对照组相比，MK-801处理BMDM 6h后，巨噬细胞中Arg1、Chil3等免疫抑制基因被显著抑制，IL-6、TNFα、iNOS等炎症因子表达上调；如图3(a)至3(d)所示，肿瘤细胞共培养诱导巨噬细胞表面共刺激分子CD80和CD86显著上调；进一步地，将诱导第4天的BMDM细胞重新铺板至96孔平底板中，铺板24h后，分选所得WT小鼠CD8+T细胞经CellTraceTM Violet标记后加入BMDM细胞中，补充2ug/mL CD3抗体，将96孔板放至37℃、5％CO2的培养箱中培养，共培养3天后流式检测CD8+T细胞增殖。如图4(a)至4(c)所示，MK-801处理增强了巨噬细胞的抗原提呈能力和CD8+T增殖。
实施例3
MK-801抑制肝癌荷瘤小鼠皮下移植瘤和转移瘤的生长
3.1模型构建
3.1.1肝癌小鼠皮下移植瘤：C57BL/6J野生型成年小鼠皮下注射Hepa1-6BL细胞5x105每只鼠。高压肝转移瘤：尾静脉注射Hepa1-6BL细胞7×105每只鼠。分为两组，对照组和处理组。肿瘤细胞注射完成后第3天，每天对照组腹腔给予PBS，处理组腹腔注射MK-801(0.2mg/kg)。体内巨噬细胞清除：分别在注射皮下肿瘤前一天、第5、10天腹腔注射(1mg/mouse)氯磷酸脂质体(Clodronate Liposomes)去除巨噬细胞。
3.1.2小鼠皮下移植瘤肿瘤细胞注射完成后第7天开始测量小鼠皮下移植瘤体积，待对照组肿瘤体积长至～100mm2，剖杀小鼠。高压肝转移瘤观察小鼠生存期。
3.2流式检测肿瘤浸润免疫细胞功能
3.2.1剖杀荷瘤小鼠取出皮下肿瘤，选取200mg肿瘤组织至于12孔板中，加入消化液(RPMI 1640培养基+胶原酶+DNA酶)，剪碎后37℃摇床消化30min。
3.2.2消化好的肿瘤组织经尼龙布过滤至50mL离心管中，2000rpm离心4min，弃掉上清，加入PBS重悬，96孔U底板进行染色，BD Aria III仪器进行检测，FlowJo软件进行分析。
结果显示：构建肝癌小鼠皮下移植瘤和高压肝转移瘤模型，腹腔给予MK-801处理，如图5(a)至5(c)所示，皮下肿瘤生长受到显著抑制；如图6(a)至6(d)所示，肝转移数目显著减少；进一步地，使用氯磷酸脂质体去除巨噬细胞，如图7所示，发现在缺少巨噬细胞时MK-801无法有效抑制肿瘤生长，提示MK-801通过调控巨噬细胞影响肿瘤发展。
进一步流式分析，MK-801处理对肿瘤浸润免疫细胞的调控作用，结果显示，如图8(a)至8(c)所示，MK-801处理显著促进了肿瘤浸润的CD8+T和NK细胞分泌的CD107a、IFNγ，激活CD8+T和NK细胞抗肿瘤功能。
实施例4
MK-801联合免疫检查点治疗
进一步评估MK-801联合免疫检查点治疗对小鼠肝癌的作用：
如实施例3所述，构建肝癌小鼠皮下移植瘤模型，分为四组，分别为对照组Control，给予MK-801处理，PD-1抗体处理以及联合MK-801和PD-1抗体处理，其中，MK-801处理具体为：肿瘤细胞注射完成后第3天开始每天腹腔注射MK-801(0.2mg/kg)；抗PD-1抗体处理具体为：肿瘤细胞注射完成后第7天腹腔注射抗PD-1(100μg/mouse)抗体；联合MK-801和PD-1抗体处理具体为：肿瘤细胞注射完成后第3天开始每天腹腔注射MK-801(0.2mg/kg)，并在第7天腹腔注射抗PD-1(100μg/mouse)抗体。小鼠皮下移植瘤肿瘤细胞注射完成后第7天开始测量小鼠皮下移植瘤体积。
结果如图9(a)至9(e)所示，显示MK-801能够增强PD-1抗体的抗肿瘤疗效。
以上结果表明在肿瘤微环境中MK-801阻断NMDAR能够解除巨噬细胞介导的免疫抑制，增强抗肿瘤免疫反应，抑制肿瘤生长。同时增强免疫检查点治疗的抗肿瘤疗效。
在此说明书中，本发明已参照其特定的实施例作了描述。但是，很显然仍可以作出各种修改和变换而不背离本发明的精神和范围。因此，说明书和附图应被认为是说明性的而非限制性的。
Claims (6)
1.小分子离子通道阻滞剂MK-801联合免疫检查点抑制剂在制备治疗肿瘤的药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述的肿瘤为肝癌。
3.根据权利要求1所述的应用，其特征在于，所述的免疫检查点抑制剂为抗PD-1抗体。
4.小分子离子通道阻滞剂MK-801在制备免疫检查点抑制剂疗效增强剂的应用。
5.根据权利要求4所述的应用，其特征在于，所述的免疫检查点抑制剂为抗PD-1抗体。
6.一种药物组合物，其特征在于，包括小分子离子通道阻滞剂MK-801和抗PD-1抗体。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310521213.8A CN116617222B (zh) | 2023-05-10 | 2023-05-10 | 小分子离子通道阻滞剂mk-801在制备治疗肿瘤或抗感染药物的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310521213.8A CN116617222B (zh) | 2023-05-10 | 2023-05-10 | 小分子离子通道阻滞剂mk-801在制备治疗肿瘤或抗感染药物的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116617222A CN116617222A (zh) | 2023-08-22 |
| CN116617222B true CN116617222B (zh) | 2024-05-03 |
Family
ID=87635706
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310521213.8A Active CN116617222B (zh) | 2023-05-10 | 2023-05-10 | 小分子离子通道阻滞剂mk-801在制备治疗肿瘤或抗感染药物的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116617222B (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019154884A1 (en) * | 2018-02-07 | 2019-08-15 | Ecole Polytechnique Federale De Lausanne (Epfl) | Method for determining cancer invasiveness and patient prognosis |
| CN113597312A (zh) * | 2018-01-25 | 2021-11-02 | M·里维特 | 用于改善的免疫疗法的方法 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021046225A1 (en) * | 2019-09-03 | 2021-03-11 | Flagship Pioneering Innovations V, Inc. | Methods and compositions for treating cancer |
-
2023
- 2023-05-10 CN CN202310521213.8A patent/CN116617222B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113597312A (zh) * | 2018-01-25 | 2021-11-02 | M·里维特 | 用于改善的免疫疗法的方法 |
| WO2019154884A1 (en) * | 2018-02-07 | 2019-08-15 | Ecole Polytechnique Federale De Lausanne (Epfl) | Method for determining cancer invasiveness and patient prognosis |
Non-Patent Citations (1)
| Title |
|---|
| NR2B介导ERK信号通路对肝癌细胞增殖的影响;张薇等;江苏医药;20181231;第44卷(第11期);1257 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116617222A (zh) | 2023-08-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bosiljcic et al. | Targeting myeloid-derived suppressor cells in combination with primary mammary tumor resection reduces metastatic growth in the lungs | |
| Melichar et al. | Immunology of the peritoneal cavity: relevance for host-tumor relation | |
| CN106574241A (zh) | 癌症免疫疗法组合物和方法 | |
| EP3228701A1 (en) | Method for preparing lymphocytes capable of infiltrating a solid tumor, lymphocytes obtainable by said method and uses thereof | |
| US20190160099A1 (en) | Pharmaceutical composition and use thereof | |
| CN101626781A (zh) | 制备具有抗肿瘤免疫应答反应的细胞群的方法 | |
| CN116617222B (zh) | 小分子离子通道阻滞剂mk-801在制备治疗肿瘤或抗感染药物的应用 | |
| Okita et al. | Targeting of CD4+ CD25high cells while preserving CD4+ CD25low cells with low-dose chimeric anti-CD25 antibody in adoptive immunotherapy of cancer | |
| US20230059785A1 (en) | Methods for enhancing t cells using venetoclax | |
| CN112791106B (zh) | 药物组合物及其治疗疾病的用途 | |
| CN113117088B (zh) | 钙激活氯离子通道的抑制剂在肿瘤免疫疗法中的应用 | |
| KR101651171B1 (ko) | Il-12를 발현하는 간엽 줄기세포를 포함하는 간암 치료용 조성물 및 이를 이용한 치료방법 | |
| CN112870369A (zh) | 用于抑制黑素瘤细胞膜表面pd-l1表达的靶向药物及应用 | |
| US20210355221A1 (en) | Targeting the Non-Canonical NFkB Pathway in Cancer Immunotherapy | |
| CN110856717A (zh) | 一种肿瘤免疫调节剂及其用途 | |
| WO2019108750A1 (en) | Methods of mobilizing marrow infiltrating lymphocytes and uses thereof | |
| US20230346901A1 (en) | Methods and vaccine compositions to treat cancers | |
| US11890268B2 (en) | Dibenzothiophene derivatives and methods of treating cancer therewith | |
| Rodado et al. | TAMI-53. Cysteine IS a limiting factor for glioma proliferation and survival | |
| CN109762042B (zh) | 一种治疗癌症的药物、其合成方法和应用 | |
| Wang | Current progress of immunotherapy based on NK cell in cancer therapy | |
| CN116808036A (zh) | 小分子阻滞剂酒石酸艾芬地尔在制备治疗肿瘤药物的应用 | |
| Zhou | Future Trends in CAR-T Cellular Immunotherapy and the Pros and Cons in Cancer Treatment | |
| CN116139275A (zh) | Cd300ld抑制剂在制备预防、诊断或治疗肿瘤产品中的用途 | |
| Morató et al. | Laparoscopic vs Open pancreaticoduodenectomy: Preliminary results from a randomized controlled trial (PADULAP TRIAL) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant |