JPH0365356B2 - - Google Patents
Info
- Publication number
- JPH0365356B2 JPH0365356B2 JP14339682A JP14339682A JPH0365356B2 JP H0365356 B2 JPH0365356 B2 JP H0365356B2 JP 14339682 A JP14339682 A JP 14339682A JP 14339682 A JP14339682 A JP 14339682A JP H0365356 B2 JPH0365356 B2 JP H0365356B2
- Authority
- JP
- Japan
- Prior art keywords
- substance
- heat
- nucleic acid
- present
- fraction
- 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.)
- Expired
Links
- 239000000126 substance Substances 0.000 claims description 88
- 102000039446 nucleic acids Human genes 0.000 claims description 43
- 108020004707 nucleic acids Proteins 0.000 claims description 43
- 150000007523 nucleic acids Chemical class 0.000 claims description 43
- 238000000034 method Methods 0.000 claims description 42
- 230000000259 anti-tumor effect Effects 0.000 claims description 39
- 108020004414 DNA Proteins 0.000 claims description 38
- 102000053602 DNA Human genes 0.000 claims description 36
- 239000000061 acid fraction Substances 0.000 claims description 29
- 238000010438 heat treatment Methods 0.000 claims description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 239000000284 extract Substances 0.000 claims description 16
- 238000004519 manufacturing process Methods 0.000 claims description 14
- 239000002244 precipitate Substances 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 13
- 238000011282 treatment Methods 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 11
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- 238000004440 column chromatography Methods 0.000 claims description 8
- 238000003505 heat denaturation Methods 0.000 claims description 8
- 239000002246 antineoplastic agent Substances 0.000 claims description 7
- 150000003839 salts Chemical class 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 238000010521 absorption reaction Methods 0.000 claims description 6
- 238000000862 absorption spectrum Methods 0.000 claims description 6
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 6
- DMBHHRLKUKUOEG-UHFFFAOYSA-N diphenylamine Chemical compound C=1C=CC=CC=1NC1=CC=CC=C1 DMBHHRLKUKUOEG-UHFFFAOYSA-N 0.000 claims description 6
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 6
- 231100000053 low toxicity Toxicity 0.000 claims description 6
- 238000002844 melting Methods 0.000 claims description 6
- 230000008018 melting Effects 0.000 claims description 6
- 230000007935 neutral effect Effects 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 6
- 102000007260 Deoxyribonuclease I Human genes 0.000 claims description 5
- 108010008532 Deoxyribonuclease I Proteins 0.000 claims description 5
- 230000007704 transition Effects 0.000 claims description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 4
- 229920002684 Sepharose Polymers 0.000 claims description 4
- 230000002378 acidificating effect Effects 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- OIPPWFOQEKKFEE-UHFFFAOYSA-N orcinol Chemical compound CC1=CC(O)=CC(O)=C1 OIPPWFOQEKKFEE-UHFFFAOYSA-N 0.000 claims description 4
- RPACBEVZENYWOL-XFULWGLBSA-M sodium;(2r)-2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate Chemical compound [Na+].C=1C=C(Cl)C=CC=1OCCCCCC[C@]1(C(=O)[O-])CO1 RPACBEVZENYWOL-XFULWGLBSA-M 0.000 claims description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 3
- 229940104302 cytosine Drugs 0.000 claims description 3
- 108010079502 exoribonuclease T Proteins 0.000 claims description 3
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 3
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 229930024421 Adenine Natural products 0.000 claims description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 239000004480 active ingredient Substances 0.000 claims description 2
- 229960000643 adenine Drugs 0.000 claims description 2
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 claims description 2
- 238000000921 elemental analysis Methods 0.000 claims description 2
- 238000002523 gelfiltration Methods 0.000 claims description 2
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 229940113082 thymine Drugs 0.000 claims description 2
- 239000012266 salt solution Substances 0.000 claims 2
- 239000003550 marker Substances 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 32
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 22
- 238000012360 testing method Methods 0.000 description 18
- 235000002639 sodium chloride Nutrition 0.000 description 17
- 206010028980 Neoplasm Diseases 0.000 description 14
- 238000005119 centrifugation Methods 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- 239000000725 suspension Substances 0.000 description 14
- 230000001580 bacterial effect Effects 0.000 description 13
- 239000002504 physiological saline solution Substances 0.000 description 11
- 239000011780 sodium chloride Substances 0.000 description 11
- 239000003125 aqueous solvent Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- 201000009030 Carcinoma Diseases 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 6
- 241000700198 Cavia Species 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 239000012535 impurity Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 244000063299 Bacillus subtilis Species 0.000 description 4
- 235000014469 Bacillus subtilis Nutrition 0.000 description 4
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 4
- 231100000111 LD50 Toxicity 0.000 description 4
- 101000981253 Mus musculus GPI-linked NAD(P)(+)-arginine ADP-ribosyltransferase 1 Proteins 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 108010083644 Ribonucleases Proteins 0.000 description 4
- 102000006382 Ribonucleases Human genes 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- 239000001569 carbon dioxide Substances 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- JDRAOGVAQOVDEB-KTKRTIGZSA-N (3-hydroxy-2,3,3a,5,6,6a-hexahydrofuro[3,2-b]furan-6-yl) (z)-octadec-9-enoate Chemical compound OC1COC2C(OC(=O)CCCCCCC\C=C/CCCCCCCC)COC21 JDRAOGVAQOVDEB-KTKRTIGZSA-N 0.000 description 2
- 102000016911 Deoxyribonucleases Human genes 0.000 description 2
- 108010053770 Deoxyribonucleases Proteins 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 2
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 230000007059 acute toxicity Effects 0.000 description 2
- 231100000403 acute toxicity Toxicity 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 208000003455 anaphylaxis Diseases 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 210000000416 exudates and transudate Anatomy 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000002601 intratumoral effect Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 239000011565 manganese chloride Substances 0.000 description 2
- 229940099607 manganese chloride Drugs 0.000 description 2
- 235000002867 manganese chloride Nutrition 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 229920002477 rna polymer Polymers 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 239000007762 w/o emulsion Substances 0.000 description 2
- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 description 1
- WFIYPADYPQQLNN-UHFFFAOYSA-N 2-[2-(4-bromopyrazol-1-yl)ethyl]isoindole-1,3-dione Chemical compound C1=C(Br)C=NN1CCN1C(=O)C2=CC=CC=C2C1=O WFIYPADYPQQLNN-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 206010002199 Anaphylactic shock Diseases 0.000 description 1
- 101100005765 Arabidopsis thaliana CDF1 gene Proteins 0.000 description 1
- 101100007579 Arabidopsis thaliana CPP1 gene Proteins 0.000 description 1
- 241001037822 Bacillus bacterium Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 210000001239 CD8-positive, alpha-beta cytotoxic T lymphocyte Anatomy 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 229920000298 Cellophane Polymers 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 208000006552 Lewis Lung Carcinoma Diseases 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 229920002433 Vinyl chloride-vinyl acetate copolymer Polymers 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- DIZPMCHEQGEION-UHFFFAOYSA-H aluminium sulfate (anhydrous) Chemical compound [Al+3].[Al+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O DIZPMCHEQGEION-UHFFFAOYSA-H 0.000 description 1
- 230000036783 anaphylactic response Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 229960000190 bacillus calmette–guérin vaccine Drugs 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
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- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 239000012931 lyophilized formulation Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
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- 235000019786 weight gain Nutrition 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Compounds Of Unknown Constitution (AREA)
Description
本発明は、バチルス属細菌から得られる抗腫瘍
活性を有する加熱変性デオキシリボ核酸BD−01
に関する。
本発明者等は先に結核菌の変性デオキシリボ核
酸が抗腫瘍性を有し、かつ毒性が極めて低いこと
を見い出し、特許出願（特開昭57−139096号）し
たが、更に結核菌以外の他の菌種由来のデオキシ
リボ核酸についても研究を進めた。その結果、培
養が容易で、かつ安全性の高いバチルス属細菌か
ら製造した変性デオキシリボ核酸にも高い抗腫瘍
活性があり、かつ毒性が極めて低いことを見出
し、本物質を加熱変性デオキシリボ核酸、BD−
01と命名した。以下本物質を単に加熱変性デオキ
シリボ核酸、変性デオキシリボ核酸、デオキシリ
ボ核酸、本物質と称することが多い。
本発明によつて得られるデオキシリボ核酸は宿
主介在性の高い抗腫瘍性を有し、毒性も極めて低
く、かつ水性溶媒に可溶であることから医薬品と
して好適である。
すなわち本発明は、加熱変性によつて抗腫瘍活
性を有せしめてなる加熱変性デオキシリボ核酸
BD−01及びその塩に関し、更に詳細には、バチ
ルス属細菌より得られる抗腫瘍活性を有する加熱
変性デオキシリボ核酸BD−01に関し、そして、
その製法及び抗腫瘍剤に関するものである。
さらに本発明はバチルス属細菌破砕物を遠心分
離して得られる核酸画分を加熱することを特徴と
する抗腫瘍活性を有する加熱変性デオキシリボ核
酸BD−01の製造法に関するものである。
さらに本発明は核酸画分の分離法として無細胞
抽出液に核酸凝集剤を加え、得られる沈澱の可溶
画分から分離することを特徴とする抗腫瘍活性を
有する加熱変性デオキシリボ核酸BD−01の製造
法に関するものである。
さらに本発明は核酸画分の分離法として無細胞
抽出液に核酸凝集剤を加え、得られる沈澱を水ま
たは塩類溶液に対して透析した後加熱し、その上
清から分離することを特徴とする抗腫瘍活性を有
する加熱変性デオキシリボ核酸BD−01の製造法
に関するものである。
さらに本発明は、デオキシリボ核酸含有物を約
80〜120℃に加熱して、抗腫瘍性を有せしめるこ
とを特徴とする抗腫瘍活性を有する加熱変性デオ
キシリボ核酸BD−01の製造法である。
さらに本発明は、加熱変性によつて抗腫瘍活性
を有せしめてなる加熱変性デオキシリボ核酸BD
−01を有効成分とする抗腫瘍剤である。
本発明に用いられる微生物は、バチルス属に属
する細菌であるが、好適なものとしては、枯草菌
バチルスズブチリス（Bacillus subtilis）ATCC
−6633株あるいはバチルスナトー（Bacillus
natto）IAM−1071株などが挙げられる。
微生物の培養方法や菌体の破砕方法は、通常の
方法に従つてよい。たとえばバチルスズブチリス
（Bacillus subtilis）の場合は普通ブイヨン
（Nutrient Broth）培地を用いて37℃程度の温度
で４〜12時間振盪培養することにより良好な培養
物が得られ、これを遠心分離または過して菌体
を得ることが出来る。得られる菌体を水、好まし
くは適当な緩衝液と充分混合し、氷冷しながらダ
イノミル（Dyno Mill）あるいはフレンチ
（French）プレスなどにより菌体を破砕して破砕
菌体懸濁液を得る。この懸濁液を遠心して無細胞
抽出液を得るが、このときの遠心分離の条件は、
未破砕菌体、部分的に破砕された菌体、細胞壁画
分等を沈渣として除去出来る通常の遠心力で良
く、たとえば5000xg、10分間以上遠心してその
上清を取得する。
本発明における無細胞抽出液とは、破砕菌体懸
濁液から遠心分離により、未破砕菌体、部分的に
破砕された菌体、細胞壁画分等を出来るだけ除い
た画分のことである。
無細胞抽出液から核酸画分を得るには、無細胞
抽出液を直接有機溶剤処理して核酸画分を得る方
法（例えばマーマー（Marmur）法やフエノール
法、以下直接有機溶剤法と称する）と無細胞抽出
液に核酸凝集剤を加えて核酸画分を含む沈澱を得
る方法が適用可能である。
直接有機溶剤法は小量規模（通常1g以下）の
本発明物質を得るのに適した方法であり、得られ
る核酸画分（以下BD核酸画分と称する）は、通
常抗原性を有する多糖や蛋白等の不純物を含有す
るので密度勾配遠心法等により不純物を出来るだ
け除去した後得られる精製デオキシリボ核酸画分
を加熱処理して本発明物質を得るか、あるいは
BD核酸画分をまず加熱処理した後遠心法や沈澱
法等によつて不純物を除去して本発明物質を得
る。
BD核酸画分や精製デオキシリボ核酸画分の加
熱処理条件は、後述の調製法と共通するので、後
に詳しく述べる。
本発明における加熱の目的は本発明物質の分子
量分布を調節し、２本鎖構造を１本鎖構造にかえ
るとともに不純物の除去効率を高め、製剤化を容
易にし、用時の溶解性を高め、抗腫瘍活性が高
く、かつ、毒性、副作用の低い本発明物質を得る
ことである。
無細胞抽出液に核酸凝集剤を加えて核酸画分を
沈澱させる方法は、核酸画分を濃縮出来るので大
量規模の本発明物質を得るのに適した方法であ
る。
この方法において用いられる核酸凝集剤は通常
の核酸凝集剤のいずれも使用可能であるが、後の
工程で用いた凝集剤を簡便に除去出来るという意
味で、低分子凝集剤が好ましい。その好適な例と
しては、塩化カルシウム、塩化マンガン、塩化マ
グネシウム、硫酸アルミニウム等の多価金属陽イ
オンあるいはストレプトマイシン、カナマイシン
等の水溶性塩基性抗生物質またはその塩等が挙げ
られる。核酸凝集剤の使用量はその種類により適
宜選択出来るが、たとえば多価金属陽イオンでは
0.1〜10％、好ましくは0.1〜３％、抗生物質では
0.01〜10％、好ましくは0.1〜１％を抽出液に対
して使用するのが適当である。
操作としては無細胞抽出液に核酸凝集剤を加
え、生成した沈澱を遠心あるいは過等の方法に
より分離して核酸画分を含む懸濁液を得る。この
懸濁液より核酸凝集剤を除くには透析が適当であ
る。その際に使用する水性溶媒は、核酸凝集剤の
除去効率を高めるために適当なイオン強度を有す
る、PHが中性附近の緩衝液が好ましい。たとえば
好適なものとしては0.1〜1M NaCl含有リン酸緩
衝液、0.1〜1M KClクエン酸緩衝液、0.1〜1M
NaCl含有炭酸ナトリウム緩衝液、0.1〜1M
NaCl含有トリス塩酸緩衝液等が挙げられる。
核酸画分を含む懸濁液を含塩緩衝液に対して透
析した後要すればさらに水に対して透析して塩類
を除く。透析済みの核酸画分を含む懸濁液を以下
BN−１画分と称する。BN−１画分を得る操作
は、全て冷却下で行ない、核酸画分の不必要な分
解、変性を防止する。冷却温度は０ないし10℃が
好ましい。
BN−１画分から本発明物質を得るには２つの
方法が可能である。１つの方法は、BN−１画分
から核酸画分を分離した後、得られた核酸画分を
加熱して本発明物質を得る方法であり、もう一つ
の方法はBN−１画分を加熱処理した後本発明物
質を分離する方法である。
前者の方法においては、まず、BN−１画分の
含有物濃度と水性溶媒のPHや塩濃度等を調節した
後、遠心分離または、過等の方法によりBN−
１画分からデオキシリボ核酸を含む可溶画分を分
離する。この可溶画分から沈澱法あるいはカラム
クロマト法あるいは電気泳動法さらに要すればリ
ボヌクレアーゼ処理法等の方法によりデオキシリ
ボ核酸画分を分離して、加熱処理すれば本発明物
質が得られる。BN−１画分から可溶画分を分離
する際の好適なBN−１画分の濃度は１〜15mg／
mlである。水性溶媒のPHは中性附近が好ましく、
通常酸あるいは塩基あるいは緩衝液でPH５から８
の範囲に調節する。水性溶媒のイオン強度は、遠
心分離等の際の沈澱の除去効率に影響を与え、通
常0.1以上が好ましく、要すれば食塩あるいは塩
化カリウム等を加えてイオン強度を調節しても良
い。
このように調節したBN−１画分を通常
20000xgで５分間以上遠心分離すると、核酸画分
を含む清澄な可溶画分がその上清として得られ
る。この上清からストレプトマイシンあるいは塩
化マンガンあるいはセチルトリメチルアンモニウ
ムブロミド等を用いる沈澱法あるいはセフアロー
ズ等を用いるカラムクロマト法あるいは塩化ビニ
ル−酢酸ビニル共重合体等を用いる電気泳動法等
によりデオキシリボ核酸画分を分離することが出
来る。
以上の操作は全て０〜10℃で行うのが好まし
い。このようにして得られたデオキシリボ核酸画
分をさらにリボヌクレアーゼ処理後加熱処理する
かあるいは加熱処理後リボヌクレアーゼ処理すれ
ば精製された本発明物質が得られるが、加熱条件
は後者の方法と共通するので後に詳述する。
後者の方法では、まずBN−１画分を加熱する
が、この加熱操作は核酸画分の適切な変性を行な
うことおよび不純物を変性させ以後の本発明物質
の分離操作を容易にすることを目的とする。加熱
条件は後に示すように本発明物質の抗腫瘍活性に
も密接に関連しており、適切な条件範囲が存在す
る。加熱の要因としては、加熱時の溶質の濃度、
水性溶媒の種類とPH、イオン強度、加熱温度、加
熱時間が挙げられ、適切な条件範囲は次のとおり
である。
溶質の濃度は１〜15mg／mlが適切である。水性
溶媒が水あるいは食塩等の含塩水（イオン強度が
0.1〜0.5）の場合は80℃ないし120℃で５分間な
いし120分間、水性溶媒が緩衝液の場合は緩衝液
のPHによつて大きく影響され、中性附近のPHでは
加熱温度は高く、加熱時間も長くし、酸性あるい
はアルカリ性では、加熱温度は低く、加熱時間も
短くするのが好ましい。
例えば水性溶媒が強酸性あるいは強アルカリ性
の場合は、加熱温度を80〜120℃に限定するもの
ではなく、室温付近でも充分である。また、水性
溶媒がアルカリ性の場合はPH、加熱温度および時
間を適宜選択すれば本発明物質からリボ核酸を除
去しうる点で有利な場合もある。
以上のごとく、適切な加熱条件を選ぶことによ
り後に示すように本発明の目的とする抗腫瘍活性
が高く、かつ抗原性の低い加熱変性デオキシリボ
核酸を含む懸濁液を得ることが出来る。
ここに示した加熱条件は本発明物質調製法の重
要な要因であり、本明細書で示したいずれの調製
法にも適用可能である。
このようにしてBN−１画分を加熱した後冷却
し、遠心または過等の方法により沈渣を除け
ば、本発明物質を含む清澄な溶液を得る。この溶
液から本発明物質を分離するのは容易であり、通
常の方法によつて可能である。その適切な例とし
ては、核酸凝集剤による沈澱法、有機溶媒による
分画法、カラムクロマト法あるいは電気泳動法等
とリボヌクレアーゼ処理法との組み合わせが挙げ
られる。これらの方法によつて得られた本発明物
質は、そのまま製剤の原体として使用するか、あ
るいは凍結乾燥して乾燥粉末にすることも可能で
ある。
本発明物質中のデオキシリボ核酸の分子量は約
３万から100万の間に分布し、グアニン、シトシ
ン（GC）含量は約41％である。本発明物質の転
移温度Tmは、明確な値を示さず、さらに、ヒド
ロキシアパタイトのカラムクロマトグラフイーに
より分析すると本発明物質が変性された１本鎖の
デオキシリボ核酸であることを意味する。
本明細書に示した方法によつて得られる物質の
なかには、加熱変性デオキシリボ核酸以外に、微
量のリボ核酸、蛋白、糖等を含むものもあるが、
いずれも混入物と言える程度であり、要すればこ
れらはさらに精製して除くことが出来る。
次に実施例１において精製して得られたBD−
01（以下BD−A1と表示する）について理化学的
性質を調べ、その結果を示す。
なお、実施例２で得られたBD−01（以下BD−
A2と表示する）の精製品についても同様の理化
学的性質を示している。
本物質、加熱変性デオキシリボ核酸BD−A1の
理化学的性質（Na塩による）：(1) 元素分析
（％）：
Ｃ：26.20〜30.68 H4.01〜4.92 Ｎ：12.56〜
13.80
Ｐ：8.03〜8.98 Na：2.81〜4.52
(2) 分子量：３万〜100万
蛋白質をマーカーとし、セフアロースCL−
6Bを用いたゲル濾過法によれば20万付近をピ
ークとして３万〜100万に分布する（分子量分
布図は第１図に示すとおり）。
(3) 融 点：明確な融点を示さない。
(4) 紫外線吸収スペクトル：中性水溶液は260nm
付近に吸収極大波長を、また230nm付近に吸収
極小波長を有する（第２図に示すとおり）。
(5) 赤外線吸収スペクトル：780cm-1付近、1090
cm-1付近、1230cm-1付近、1700cm-1付近に核酸
の特性吸収帯を有する（第３図に示すとおり）。
(6) 溶剤に対する溶解性：水に可溶、エタノー
ル、メタノール、エーテル、アセトンに不溶。
(7) 呈色反応
Ａ） オルシノール反応：STS法により分画
したRNA画分に対して陰性。
Ｂ） ジフエニルアミン反応：STS法により
分画したDNA画分に対して陽性。
Ｃ） ニンヒドリン反応：本物質10μg／mlの
濃度で陰性。
Ｄ） アンスロン反応：本物質10μg／mlの濃
度で陰性。
(8) 塩基性、酸性、中性の区別：本物質の水溶液
のPHは6.5〜7.5である。
(9) 物質の色：白色粉末
(10) 特性：加熱処理によつて可溶性を付与すると
ともに、高い抗腫瘍活性を有せしめ、かつ毒性
および抗原性が極めて低くなつている。
(11) 塩基組成（％）：グアニン：21.0アデニン：
26.8
シトシン：19.7チミン：
32.5
（方法は化学分析によ
る）
(12) 酵素処理：本物質をデオキシリボヌクレアー
ゼＩ（DNaseI）で処理すると抗腫瘍性がなく
なるが、リボヌクレアーゼT2（RNaseT2）の
処理では抗腫瘍性は変化しない。
(13) ヒドロキシアパタイトのカラムクロマトグ
ラフイーにより分析すると１本鎖構造である。
(14) 転移温度（Tm）：測定された融解曲線から
は明確な転移温度Tmは求められない。
本発明物質が示す抗腫瘍活性の本体が、加熱変
性デオキシリボ核酸であることは、本発明物質を
デオキシリボ核酸分解酵素（DNaseI）で処理す
ると抗腫瘍活性がなくなること、本発明物質をリ
ボ核酸分解酵素（RNaseT2）で処理しても抗腫
瘍活性が変化しないことから明らかである。
本発明物質を抗腫瘍剤として用いる場合は、注
射剤の型で用いるのが好ましい。本発明物質は、
単独であるいは通常用いられる添加剤、賦型剤を
加えて液剤、あるいは同時溶解型の凍結乾燥製剤
として適用可能である。
また本発明物質は水中油滴型あるいは油中水滴
型のエマルジヨンとしても適適用可能である。
本発明物質の使用量、投与経路は適宜選択され
るが使用量は体重Kgあたり0.01ないし100mgが好
ましく、投与経路は皮内、皮下、静脈内、腹腔内
投与や腫瘍内投与が行なわれる。さらに、本物質
は経口投与も可能である。
本発明物質はモルモツトやマウスの種々の腫瘍
系に対して高い抗腫瘍作用を示す。例えばマウス
の同系腫瘍であるIMCカルチノーマ、あるいは
ルイス ラング カルチノーマに対して本発明物
質は生理食塩液に溶解した型で腫瘍細胞と接触さ
せた後、動物体内に移植することによる腫瘍の生
着抑制（サプレツシヨン活性）あるいは動物体内
に生着した腫瘍組織内に投与することによる抗腫
瘍活性（リグレツシヨン活性）において原料であ
る菌体と同等か、あるいはそれよりも高い抗腫瘍
活性を示した。また、モルモツトの同系腫瘍であ
るライン１０に対しては、油中水滴型の本発明物
質の腫瘍内投与により、本発明物質は、原発腫瘍
の増殖抑制だけでなく、所属リンパ節への腫瘍の
転移も抑制した。
さらに本発明物質は、生理食塩液に溶解して腫
瘍組織とは異る場所に投与することによつても
IMCカルチノーマに対して抗腫瘍作用を示した。
本発明物質の急性毒性はマウスに対する静脈内
投与による体重Kgあたりの50％致死量LD50値が
500mg以上であることから、極めて低い。
また本発明物質の抗原性についても、モルモツ
トを用いたアナフイラキシー試験、遅延型アレル
ギー試験によつて、本発明物質が極めて安全であ
ることが判明した。
その他本発明物質の発熱性、疼痛性、起炎性、
肉芽形成性等も原料菌体に比較し、極めて低く、
通常の医薬としての適用には問題にならない程度
であることが、種々の試験により判明した。
各種の腫瘍細胞に対する本発明物質の細胞増殖
抑制作用を調べたところ、本発明物質は、ほとん
ど細胞増殖抑制作用を示さなかつた。このことか
ら本発明物質は宿主の免疫反応を介して抗腫瘍作
用を示すと考えられるので本発明物質の免疫学的
活性を種々検討した。その結果、本発明物質はマ
ウスのキラーＴ細胞増強効果、およびマクロフア
ージ活性化作用の他にナチユラルキラー細胞活性
増強作用を示した。
以上の種々の知見から本発明物質は抗腫瘍剤と
して極めて有用なものと考えられる。
以下に本発明物質の製造法を実施例により、ま
た、本発明物質の抗腫瘍剤としての有用性を試験
例により示す。
実施例 １
ストレプトマイシンを用いて得られる懸濁液の
可溶画分から本発明物質の製造
ペプトン 10g
イーストエキス 10g
NaCl 5g
水を加えて１にし、PH6.0〜7.0に調節する。
Bacillus subtilis，ATCC6633株を上記組成の
ペプトン培地で37℃、６時間振盪培養し、培養液
を遠心分離処理して得た湿菌体98gを100mlの
10mMリン酸緩衝液（PH7.0）に懸濁した後、氷
冷下ダイノミルで破砕し、20000xgで20分間遠心
分離して上清の無細胞抽出液を得た。この抽出液
にストレプトマイシン硫酸塩を終濃度が0.3％に
なるように添加し、充分撹拌した後、４℃で一晩
静置し、生成した沈澱を遠心分離により集め、
0.5MNacl含有10mMリン酸緩衝液（PH7.0）に懸
濁した。この懸濁液をセロハンチユーブに詰めて
同じ緩衝液に対して透析し、続いて蒸留水に対し
て透析して核酸画分を含む懸濁液175ml（以下こ
の懸濁液をBN−１と称する）を得た。この懸濁
液の固形物濃度は４mg／mlであり、湿菌体あたり
0.7％の収率であつた。
次にBN−１全量にNaCl1.58gを加えて撹拌し、
加熱変性処理（100℃、60分）した後、20000xg、
20分間遠心分離して上清を得た。この上清に
NaClを最終濃度が0.4Mになるように加えて溶解
した後、セチルトリメチルアンモニウムブロミド
（以下このものをCTABと称す。東京化成会社
製）を最終濃度が0.2％（Ｗ／Ｖ）になるように
加えて充分撹拌し、室温に30分間静置した。生成
した沈澱を遠心分離により集め、1MNaCl液33ml
に溶解した後、等量のクロロホルム−イソアミル
アルコール（24：１）の混液を加えて振盪、遠心
分離して水層部分を得た。この操作をさらに２回
くり返した後、得られた水層に３倍量の99.5％エ
タノールを加えて撹拌し、４℃で一晩静置した。
生成した沈澱を遠心分離により集め、蒸留水20ml
に溶解した後、蒸留水に対して透析し、精製核酸
溶液を得た。この溶液を1N NaOHで中和後、凍
結乾燥して乾燥標品158mgを得た。本品90mgを
0.05M酢酸緩衝液（PH4.5）10mlに溶解した後、
同緩衝液２mlに溶解したリボヌクレアーゼT2（以
下RNaseT2と略記、三共会社製）200Uを添加
し、37℃で22時間反応させた。反応液に等量のク
ロロホルム−イソアミルアルコール（24：１）の
混液を加えて振盪後、遠心分離して水層部分を得
た。この操作をさらにくり返した後、水層の全量
を、あらかじめ0.5M炭酸水素アンモニウム液で
洗浄した径2.5cm、長さ90cmのセフアデツクスＧ
−100（Pharmacia Fine Chemicals社製）カラム
に負荷し、同液で溶出した後、最初に溶出する画
分としてデオキシリボ核酸を含む画分を得た。こ
の画分を水に対して透析した後、NaOHで中和
後、凍結乾燥して本発明物質72mgを得た。以下こ
のものをBD−A1と称す。
BD−A1のデオキシリボ核酸の含量（純度）は
５％過塩素酸で加水分解した後、ジフエニルアミ
ン法（Biochemical Journal 62，315，1956）
により定量した時98.5％であつた。
実施例 ２
核酸含有懸濁液から酸処理して得られる本発明
物質の製造
実施例１と同様の方法で得たNB−１、100ml
に対して98％蟻酸、80mlを加え室温で１時間撹拌
した後、20000xg、15分間遠心分離して上清を得
た。この上清をロータリーエバポレーターにて濃
縮、乾固した後、蒸留水に溶解し、凍結乾燥し
た。この乾燥品360mgに対し、0.4M NaCl40mlを
加えて室温で１時間撹拌した後、1N NaOHでPH
5.0に調整後、室温で30分撹拌した。この溶液を
20000xg、15分間遠心分離して上清を得た後、
0.4MNaClに溶解した５％CTAB液を1.6ml加え
て充分撹拌し、室温に30分間静置した。生成した
沈澱を遠心分離により集め、以下実施例１と同様
に有機溶剤処理、透析、凍結乾燥処理、
RNaseT2処理、Sephadex、Ｇ−100カラムクロ
マトグラフイー、透析および凍結乾燥して本発明
物質37mgを得た。以下このものをBD−A2と称
す。
BD−A2のデオキシリボ核酸含量は実施例１と
同様の方法で分析すると98.1％であつた。
実施例 ３
液剤
BD−A1、10mgを10mlのPBSマイナス液（栄研
化学社製）に溶解し、ニユクリポアーNo.20
（Nuclepore社製）を用いて無菌過した。得ら
れた液を1.5mlずつバイアル瓶に無菌的に分注
して本発明物質の液剤を得た。
実施例 ４
凍結乾燥製剤
BD−A1、10mgの注射用蒸留水に溶解し、次に
500mgのマンニトールを加えて溶解した後、ニユ
クリポアーNo.20を用いて無菌過した。得られた
液を1.5mlずつ無菌的にバイアル瓶に分注した
後、凍結乾燥して本発明物質の凍結乾燥製剤を得
た。
実施例 ５
エマルジヨン剤
BD−A1、４mgを0.5mlの生理食塩液に溶解し、
次にドラケオール６−VR（Drakeol ６−VR，
Pensilvania Retining Company製）とアラシー
ルＡ（Arlacel Ａ，Atlas Chemical Industries
製）の8.5：1.5の混液0.5mlを加えて連結注射針を
用いて油中水型のエマルジヨンを得た。
試験例 １
マウスIMCカルチノーマに対する抗腫瘍作用
本発明物質のマウスIMCカルチノーマに対す
る抗腫瘍性を調べた。
試験系は次のとおりである。
CDF1、雌性マウスの皮内に５×105個のIMC
カルチノーマ細胞を移植し、移植後１日目から隔
日に計６回、実施例３の方法で調製した製剤を１
回あたり0.1mlずつ、腫瘍内に投与した。移植後
35日目に腫瘍を摘出し、その重量を測定した結果
は第１表のとおりであつた。
The present invention provides heat-denatured deoxyribonucleic acid BD-01 having antitumor activity obtained from Bacillus bacteria.
Regarding. The present inventors previously discovered that the modified deoxyribonucleic acid of Mycobacterium tuberculosis has antitumor properties and extremely low toxicity, and filed a patent application (Japanese Patent Application Laid-open No. 139096/1983). We also conducted research on deoxyribonucleic acid derived from bacterial species. As a result, we found that denatured deoxyribonucleic acid produced from Bacillus bacteria, which is easy to culture and highly safe, also has high antitumor activity and extremely low toxicity.
It was named 01. Hereinafter, this substance is often simply referred to as heat-denatured deoxyribonucleic acid, modified deoxyribonucleic acid, deoxyribonucleic acid, or this substance. The deoxyribonucleic acid obtained by the present invention has highly host-mediated antitumor properties, has extremely low toxicity, and is soluble in aqueous solvents, so it is suitable as a pharmaceutical. That is, the present invention provides heat-denatured deoxyribonucleic acid which has antitumor activity through heat denaturation.
Regarding BD-01 and its salts, more specifically regarding heat-denatured deoxyribonucleic acid BD-01 having antitumor activity obtained from Bacillus bacteria, and
The present invention relates to its production method and antitumor agent. Furthermore, the present invention relates to a method for producing heat-denatured deoxyribonucleic acid BD-01 having antitumor activity, which comprises heating a nucleic acid fraction obtained by centrifuging a crushed Bacillus bacterium. Furthermore, the present invention provides a method for separating the nucleic acid fraction by adding a nucleic acid flocculant to a cell-free extract and separating it from the soluble fraction of the resulting precipitate. It concerns the manufacturing method. Furthermore, the present invention is characterized in that, as a method for separating nucleic acid fractions, a nucleic acid flocculant is added to a cell-free extract, the resulting precipitate is dialyzed against water or a saline solution, heated, and separated from the supernatant. The present invention relates to a method for producing heat-denatured deoxyribonucleic acid BD-01 having antitumor activity. Furthermore, the present invention provides deoxyribonucleic acid-containing substances about
This is a method for producing heat-denatured deoxyribonucleic acid BD-01 having antitumor activity, which is characterized by heating to 80 to 120°C to impart antitumor properties. Furthermore, the present invention provides heat-denatured deoxyribonucleic acid BD which has antitumor activity through heat-denaturation.
It is an antitumor agent containing -01 as an active ingredient. The microorganism used in the present invention is a bacterium belonging to the genus Bacillus, and preferred examples include Bacillus subtilis ATCC
−6633 strain or Bacillus nato (Bacillus
natto) IAM-1071 strain. A method for culturing microorganisms and a method for disrupting bacterial cells may be carried out according to conventional methods. For example, in the case of Bacillus subtilis, a good culture can be obtained by culturing in Nutrient Broth medium at a temperature of about 37°C for 4 to 12 hours with shaking, which is then centrifuged or filtered. The bacterial cells can be obtained by The resulting bacterial cells are thoroughly mixed with water, preferably an appropriate buffer solution, and crushed with a Dyno Mill or French press while cooling on ice to obtain a suspension of crushed bacterial cells. This suspension is centrifuged to obtain a cell-free extract, and the centrifugation conditions at this time are as follows:
Ordinary centrifugal force that can remove unbroken bacterial cells, partially crushed bacterial cells, cell wall fractions, etc. as a precipitate may be used, for example, by centrifuging at 5000xg for 10 minutes or more to obtain the supernatant. In the present invention, the cell-free extract is a fraction obtained by removing as much of unbroken bacterial cells, partially crushed bacterial cells, cell wall fractions, etc. as possible from a suspension of crushed bacterial cells by centrifugation. . To obtain a nucleic acid fraction from a cell-free extract, there are two methods: directly treating the cell-free extract with an organic solvent to obtain a nucleic acid fraction (for example, the Marmur method or the phenol method, hereinafter referred to as the direct organic solvent method); An applicable method is to add a nucleic acid flocculant to a cell-free extract to obtain a precipitate containing a nucleic acid fraction. The direct organic solvent method is suitable for obtaining the substance of the present invention in small quantities (usually 1 g or less), and the resulting nucleic acid fraction (hereinafter referred to as BD nucleic acid fraction) usually contains antigenic polysaccharides and Since it contains impurities such as proteins, the purified deoxyribonucleic acid fraction obtained after removing as many impurities as possible by density gradient centrifugation or the like is heat-treated to obtain the substance of the present invention, or
The BD nucleic acid fraction is first heat-treated, and then impurities are removed by centrifugation, precipitation, etc. to obtain the substance of the present invention. The heat treatment conditions for the BD nucleic acid fraction and the purified deoxyribonucleic acid fraction are the same as those for the preparation method described below, so they will be described in detail later. The purpose of heating in the present invention is to adjust the molecular weight distribution of the substance of the present invention, change the double-stranded structure to a single-stranded structure, increase the efficiency of removing impurities, facilitate formulation, increase solubility during use, It is an object of the present invention to obtain a substance of the present invention that has high antitumor activity and low toxicity and side effects. A method in which a nucleic acid flocculant is added to a cell-free extract to precipitate a nucleic acid fraction is a method suitable for obtaining the substance of the present invention on a large scale because the nucleic acid fraction can be concentrated. As the nucleic acid flocculant used in this method, any ordinary nucleic acid flocculant can be used, but a low molecular flocculant is preferable in the sense that the flocculant used in a later step can be easily removed. Suitable examples thereof include polyvalent metal cations such as calcium chloride, manganese chloride, magnesium chloride, and aluminum sulfate, water-soluble basic antibiotics such as streptomycin and kanamycin, or salts thereof. The amount of nucleic acid flocculant used can be selected appropriately depending on the type of nucleic acid flocculant, but for example, for polyvalent metal cations,
0.1-10%, preferably 0.1-3%, for antibiotics
It is appropriate to use 0.01 to 10%, preferably 0.1 to 1%, based on the extract. As for the operation, a nucleic acid flocculant is added to the cell-free extract, and the resulting precipitate is separated by centrifugation or other methods to obtain a suspension containing the nucleic acid fraction. Dialysis is suitable for removing the nucleic acid flocculant from this suspension. The aqueous solvent used in this case is preferably a buffer solution with an appropriate ionic strength and a pH around neutrality in order to increase the removal efficiency of the nucleic acid flocculant. For example, suitable ones include phosphate buffer containing 0.1-1M NaCl, 0.1-1M KCl citrate buffer, 0.1-1M
Sodium carbonate buffer containing NaCl, 0.1-1M
Examples include NaCl-containing Tris-HCl buffer. After the suspension containing the nucleic acid fraction is dialyzed against a salt-containing buffer, it is further dialyzed against water to remove salts, if necessary. The suspension containing the dialyzed nucleic acid fraction is
It is called the BN-1 fraction. All operations for obtaining the BN-1 fraction are performed under cooling to prevent unnecessary decomposition and denaturation of the nucleic acid fraction. The cooling temperature is preferably 0 to 10°C. Two methods are possible to obtain the substance of the invention from the BN-1 fraction. One method is to separate the nucleic acid fraction from the BN-1 fraction and then heat the obtained nucleic acid fraction to obtain the substance of the present invention.The other method is to heat-treat the BN-1 fraction. In this method, the substance of the present invention is separated after In the former method, first, after adjusting the content concentration of the BN-1 fraction and the pH and salt concentration of the aqueous solvent, BN-1 is separated by centrifugation or filtration.
A soluble fraction containing deoxyribonucleic acid is separated from one fraction. The substance of the present invention can be obtained by separating a deoxyribonucleic acid fraction from this soluble fraction by precipitation, column chromatography, electrophoresis, and if necessary, ribonuclease treatment, followed by heat treatment. The preferred concentration of the BN-1 fraction when separating the soluble fraction from the BN-1 fraction is 1 to 15 mg/
ml. The pH of the aqueous solvent is preferably around neutral;
Usually acid or base or buffer solution with pH 5 to 8
Adjust within the range. The ionic strength of the aqueous solvent affects the removal efficiency of precipitates during centrifugation, etc., and is usually preferably 0.1 or more, and if necessary, the ionic strength may be adjusted by adding salt, potassium chloride, etc. The BN-1 fraction adjusted in this way is usually
When centrifuged at 20,000xg for 5 minutes or more, a clear soluble fraction containing a nucleic acid fraction is obtained as the supernatant. From this supernatant, the deoxyribonucleic acid fraction is separated by precipitation using streptomycin, manganese chloride, cetyltrimethylammonium bromide, etc., column chromatography using Sepharose, etc., or electrophoresis using vinyl chloride-vinyl acetate copolymer, etc. I can do it. All of the above operations are preferably carried out at 0 to 10°C. The purified substance of the present invention can be obtained by further treating the deoxyribonucleic acid fraction thus obtained with ribonuclease followed by heat treatment, or by heat treatment followed by ribonuclease treatment, but since the heating conditions are the same as in the latter method, Explain in detail. In the latter method, the BN-1 fraction is first heated, and the purpose of this heating operation is to appropriately denature the nucleic acid fraction and to denature impurities to facilitate the subsequent separation of the substance of the present invention. shall be. As will be shown later, the heating conditions are closely related to the antitumor activity of the substance of the present invention, and there is an appropriate range of conditions. Heating factors include solute concentration during heating,
The type of aqueous solvent, PH, ionic strength, heating temperature, and heating time are listed, and the appropriate condition range is as follows. A suitable concentration of solute is 1 to 15 mg/ml. The aqueous solvent is water or saline water such as common salt (ionic strength is
0.1 to 0.5) at 80℃ to 120℃ for 5 minutes to 120 minutes.If the aqueous solvent is a buffer solution, it will be greatly affected by the pH of the buffer solution; if the pH is around neutral, the heating temperature will be high; In acidic or alkaline conditions, it is preferable to set the heating temperature to a low temperature and shorten the heating time. For example, when the aqueous solvent is strongly acidic or alkaline, the heating temperature is not limited to 80 to 120°C, and a temperature around room temperature is sufficient. Furthermore, when the aqueous solvent is alkaline, it may be advantageous in that ribonucleic acid can be removed from the substance of the present invention by appropriately selecting the pH, heating temperature, and time. As described above, by selecting appropriate heating conditions, it is possible to obtain a suspension containing heat-denatured deoxyribonucleic acid with high antitumor activity and low antigenicity, which is the objective of the present invention, as will be shown later. The heating conditions shown here are important factors in the method of preparing the substance of the present invention, and are applicable to any of the preparation methods shown herein. After heating the BN-1 fraction in this manner, the fraction is cooled and the precipitate is removed by centrifugation or filtration to obtain a clear solution containing the substance of the present invention. The substance of the present invention can be easily separated from this solution using conventional methods. Suitable examples include a precipitation method using a nucleic acid flocculant, a fractionation method using an organic solvent, a column chromatography method, or a combination of electrophoresis method and a ribonuclease treatment method. The substances of the present invention obtained by these methods can be used as they are as raw materials for pharmaceutical preparations, or can be freeze-dried to form dry powders. The molecular weight of deoxyribonucleic acid in the substance of the present invention ranges from about 30,000 to 1,000,000, and the guanine and cytosine (GC) content is about 41%. The transition temperature Tm of the substance of the present invention does not show a clear value, and furthermore, analysis by hydroxyapatite column chromatography indicates that the substance of the present invention is a denatured single-stranded deoxyribonucleic acid. Some of the substances obtained by the method shown in this specification contain trace amounts of ribonucleic acid, protein, sugar, etc. in addition to heat-denatured deoxyribonucleic acid.
All of these can be considered to be contaminants, and if necessary, they can be removed by further purification. Next, BD- obtained by purification in Example 1
We investigated the physical and chemical properties of 01 (hereinafter referred to as BD-A1) and present the results. Note that BD-01 obtained in Example 2 (hereinafter referred to as BD-01)
The purified product of A2) also shows similar physical and chemical properties. Physical and chemical properties of this substance, heat-denatured deoxyribonucleic acid BD-A1 (by Na salt): (1) Elemental analysis (%): C: 26.20~30.68 H4.01~4.92 N: 12.56~
13.80 P: 8.03-8.98 Na: 2.81-4.52 (2) Molecular weight: 30,000-1,000,000 Sepharose CL-
According to the gel filtration method using 6B, it is distributed between 30,000 and 1,000,000 with a peak around 200,000 (the molecular weight distribution map is shown in Figure 1). (3) Melting point: Does not show a clear melting point. (4) Ultraviolet absorption spectrum: 260nm for neutral aqueous solution
It has a maximum absorption wavelength near 230 nm and a minimum absorption wavelength near 230 nm (as shown in Figure 2). (5) Infrared absorption spectrum: around 780cm -1 , 1090
It has characteristic absorption bands of nucleic acids around cm -1 , around 1230 cm -1 , and around 1700 cm -1 (as shown in Figure 3). (6) Solubility in solvents: Soluble in water, insoluble in ethanol, methanol, ether, and acetone. (7) Color reaction A) Orcinol reaction: Negative for RNA fractions fractionated by STS method. B) Diphenylamine reaction: Positive for DNA fraction fractionated by STS method. C) Ninhydrin reaction: Negative at a concentration of 10μg/ml of this substance. D) Anthrone reaction: Negative at a concentration of 10μg/ml of this substance. (8) Basic, acidic, and neutral: The pH of an aqueous solution of this substance is 6.5 to 7.5. (9) Color of the substance: White powder (10) Characteristics: It is made soluble through heat treatment, has high antitumor activity, and has extremely low toxicity and antigenicity. (11) Base composition (%): Guanine: 21.0 Adenine:
26.8 Cytosine: 19.7 Thymine:
32.5 (The method is based on chemical analysis) (12) Enzyme treatment: Treatment of this substance with deoxyribonuclease I (DNase I) eliminates antitumor properties, but treatment with ribonuclease T 2 (RNaseT 2 ) does not change the antitumor properties. (13) When analyzed by hydroxyapatite column chromatography, it has a single-stranded structure. (14) Transition temperature (Tm): A clear transition temperature Tm cannot be determined from the measured melting curve. The fact that the main body of the antitumor activity exhibited by the substance of the present invention is heat-denatured deoxyribonucleic acid means that when the substance of the present invention is treated with deoxyribonuclease (DNaseI), the antitumor activity disappears; This is clear from the fact that the antitumor activity does not change even after treatment with (RNaseT 2 ). When the substance of the present invention is used as an antitumor agent, it is preferably used in the form of an injection. The substance of the present invention is
It can be applied alone or with commonly used additives and excipients as a liquid preparation, or a co-dissolved freeze-dried preparation. The substance of the present invention can also be applied as an oil-in-water or water-in-oil emulsion. The amount and route of administration of the substance of the present invention are selected as appropriate, but the amount used is preferably 0.01 to 100 mg per kg of body weight, and administration routes include intradermal, subcutaneous, intravenous, intraperitoneal, and intratumoral administration. Furthermore, this substance can also be administered orally. The substance of the present invention exhibits high antitumor activity against various tumor types in guinea pigs and mice. For example, for IMC carcinoma or Lewis Lang carcinoma, which are syngeneic tumors in mice, the substance of the present invention is dissolved in physiological saline and brought into contact with the tumor cells, and then implanted into the animal body to inhibit tumor engraftment ( The antitumor activity (suppression activity) or antitumor activity (regression activity) when administered into tumor tissue engrafted in an animal body was equivalent to or higher than that of the bacterial cells used as the raw material. Furthermore, for line 10, which is a syngeneic tumor in guinea pigs, by intratumoral administration of the substance of the present invention in the form of water-in-oil droplets, the substance of the present invention not only suppresses the growth of the primary tumor, but also suppresses tumor growth in the regional lymph nodes. Metastasis was also suppressed. Furthermore, the substance of the present invention can also be administered by dissolving it in physiological saline and administering it to a site different from the tumor tissue.
It showed antitumor activity against IMC carcinoma. The acute toxicity of the substance of the present invention is determined by the 50% lethal dose LD50 value per kg body weight when administered intravenously to mice.
It is extremely low as it is over 500mg. Regarding the antigenicity of the substance of the present invention, an anaphylaxis test and a delayed allergy test using guinea pigs revealed that the substance of the present invention is extremely safe. Other pyrogenic, painful, and inflammatory properties of the substance of the present invention,
Granulation-forming properties are also extremely low compared to the raw bacterial cells.
It has been found through various tests that this level is not a problem for ordinary pharmaceutical applications. When the cell growth inhibitory effect of the substance of the present invention on various tumor cells was investigated, the substance of the present invention showed almost no cell growth inhibitory effect. From this, it is thought that the substance of the present invention exhibits an antitumor effect through the immune response of the host, and therefore various immunological activities of the substance of the present invention were investigated. As a result, the substance of the present invention showed an effect of enhancing killer T cells in mice, an effect of activating macrophages, and an effect of enhancing natural killer cell activity. Based on the above various findings, the substance of the present invention is considered to be extremely useful as an antitumor agent. EXAMPLES The method for producing the substance of the present invention will be illustrated below by Examples, and the usefulness of the substance of the present invention as an antitumor agent will be illustrated by Test Examples. Example 1 Production of the substance of the present invention from the soluble fraction of a suspension obtained using streptomycin Peptone 10g Yeast extract 10g NaCl 5g Add water to 1 and adjust the pH to 6.0 to 7.0. Bacillus subtilis, ATCC6633 strain, was cultured with shaking at 37°C for 6 hours in a peptone medium with the above composition, and 98 g of wet bacterial cells obtained by centrifuging the culture solution was added to 100 ml of
After suspending in 10mM phosphate buffer (PH7.0), the cells were crushed with a Dynomill under ice cooling, and centrifuged at 20,000xg for 20 minutes to obtain a supernatant cell-free extract. Streptomycin sulfate was added to this extract to a final concentration of 0.3%, stirred thoroughly, and allowed to stand overnight at 4°C. The precipitate formed was collected by centrifugation.
It was suspended in 10mM phosphate buffer (PH7.0) containing 0.5M Nacl. This suspension was packed in cellophane tubes and dialyzed against the same buffer, and then dialyzed against distilled water to obtain 175 ml of a suspension containing the nucleic acid fraction (hereinafter this suspension is referred to as BN-1). ) was obtained. The solid concentration of this suspension was 4 mg/ml, and
The yield was 0.7%. Next, 1.58g of NaCl was added to the entire amount of BN-1 and stirred.
After heat denaturation treatment (100℃, 60 minutes), 20000xg,
The supernatant was obtained by centrifugation for 20 minutes. In this supernatant
After adding and dissolving NaCl to a final concentration of 0.4M, cetyltrimethylammonium bromide (hereinafter referred to as CTAB, manufactured by Tokyo Kasei Co., Ltd.) was added to a final concentration of 0.2% (W/V). The mixture was added, thoroughly stirred, and allowed to stand at room temperature for 30 minutes. Collect the generated precipitate by centrifugation and add 33ml of 1M NaCl solution.
After dissolving in the solution, an equal amount of a mixture of chloroform and isoamyl alcohol (24:1) was added, followed by shaking and centrifugation to obtain an aqueous layer. After repeating this operation two more times, 3 times the amount of 99.5% ethanol was added to the resulting aqueous layer, stirred, and allowed to stand overnight at 4°C.
Collect the generated precipitate by centrifugation and add 20ml of distilled water.
After dissolving the nucleic acid in water, it was dialyzed against distilled water to obtain a purified nucleic acid solution. This solution was neutralized with 1N NaOH and then lyophilized to obtain 158 mg of a dry sample. 90mg of this product
After dissolving in 10ml of 0.05M acetate buffer (PH4.5),
200 U of ribonuclease T 2 (hereinafter abbreviated as RNaseT 2 , manufactured by Sankyo Co., Ltd.) dissolved in 2 ml of the same buffer was added, and the mixture was reacted at 37° C. for 22 hours. An equal amount of a mixture of chloroform and isoamyl alcohol (24:1) was added to the reaction solution, which was then shaken and centrifuged to obtain an aqueous layer. After repeating this operation, the entire amount of the aqueous layer was washed with a 2.5 cm diameter and 90 cm long Cefdex G
-100 (manufactured by Pharmacia Fine Chemicals) column and eluted with the same solution, a fraction containing deoxyribonucleic acid was obtained as the first eluted fraction. This fraction was dialyzed against water, neutralized with NaOH, and lyophilized to obtain 72 mg of the substance of the present invention. Hereinafter, this product will be referred to as BD-A1. The content (purity) of deoxyribonucleic acid in BD-A1 was determined by the diphenylamine method (Biochemical Journal 62 , 315, 1956) after hydrolysis with 5% perchloric acid.
It was 98.5% when quantified by Example 2 Production of the substance of the present invention obtained by acid treatment from a nucleic acid-containing suspension 100 ml of NB-1 obtained by the same method as Example 1
After adding 80 ml of 98% formic acid to the mixture and stirring at room temperature for 1 hour, centrifugation was performed at 20,000 xg for 15 minutes to obtain a supernatant. This supernatant was concentrated and dried using a rotary evaporator, then dissolved in distilled water and freeze-dried. To 360 mg of this dry product, add 40 ml of 0.4 M NaCl, stir at room temperature for 1 hour, and then PH with 1 N NaOH.
After adjusting to 5.0, the mixture was stirred at room temperature for 30 minutes. This solution
After centrifuging at 20,000xg for 15 minutes to obtain the supernatant,
1.6 ml of 5% CTAB solution dissolved in 0.4 M NaCl was added, thoroughly stirred, and left at room temperature for 30 minutes. The generated precipitate was collected by centrifugation, and then treated with an organic solvent, dialysis, freeze-drying, and the like as in Example 1.
RNaseT 2 treatment, Sephadex, G-100 column chromatography, dialysis and freeze drying yielded 37 mg of the substance of the present invention. Hereinafter, this product will be referred to as BD-A2. The deoxyribonucleic acid content of BD-A2 was analyzed in the same manner as in Example 1 and was found to be 98.1%. Example 3 Dissolve 10 mg of liquid BD-A1 in 10 ml of PBS minus solution (manufactured by Eiken Kagaku Co., Ltd.), and add Nuclepore No. 20.
(manufactured by Nuclepore). The obtained liquid was aseptically dispensed into vials in 1.5 ml portions to obtain a liquid preparation of the substance of the present invention. Example 4 Lyophilized formulation BD-A1 was dissolved in 10 mg of distilled water for injection, and then
After adding and dissolving 500 mg of mannitol, the mixture was sterilized using Nuclepore No. 20. The obtained liquid was aseptically dispensed into vials in 1.5 ml portions and then lyophilized to obtain a lyophilized preparation of the substance of the present invention. Example 5 Emulsion agent BD-A1, 4 mg was dissolved in 0.5 ml of physiological saline,
Next, Drakeol 6-VR (Drakeol 6-VR,
Arlacel A (manufactured by Pennsylvania Retining Company) and Arlacel A (manufactured by Atlas Chemical Industries)
A water-in-oil emulsion was obtained by adding 0.5 ml of a 8.5:1.5 mixture of the following products (manufactured by Kasei Chemical Co., Ltd.) and using a connected injection needle. Test Example 1 Antitumor activity against mouse IMC carcinoma The antitumor activity of the substance of the present invention against mouse IMC carcinoma was investigated. The test system is as follows. CDF1, 5 × 10 IMCs intradermally in female mice
Carcinoma cells were transplanted, and the preparation prepared by the method of Example 3 was administered once every other day for a total of 6 times starting from the 1st day after transplantation.
0.1 ml per dose was administered intratumorally. After transplant
The tumor was excised on the 35th day and its weight was measured, and the results are shown in Table 1.
【表】
後述の検定を含めて平均腫瘍重量および治癒例
数の出現頻度の有意差検定にはスチユーデントの
ｔ検定法とフイツシヤーの検定法をそれぞれ用い
た。Ｔ／Ｃは対照群の平均腫瘍重量に対する投与
群の平均腫瘍重量のパーセント比である。BCG
はBCGワクチン（日本ビーシージー会社製）を
用いた。対照としては生理食塩液を用いた。
試験例 ２
マウス、ルイス・ラング・カルチノーマに対す
る抗腫瘍作用
本発明物質のマウス、ルイス・ラング・カルチ
ノーマに対する抗腫瘍作用を調べた。
試験系は次のとおりである。
C57BL／６系雌性マウスの皮内に５×105個の
ルイス、ラング カルチノーーマ（Lewis Lung
Carcinoma）細胞を移植し、移植後１，４，８，
11，15，18日目に実施例３の方法で調製した製剤
を１回あたり0.1mlずつ腫瘍内に投与した。移植
後26日目に腫瘍を摘出し、その重量を測定すると
ともに、肺における転移巣の数も調べた。
結果は第２表に示した。
対照として生理食塩液を用い、BCGは、BCG
ワクチンを用いた。[Table] Student's t-test and Fitscher's test were used to test for significant differences in the frequency of occurrence of average tumor weight and number of cured cases, including the tests described below. T/C is the percentage ratio of the average tumor weight of the treated group to the average tumor weight of the control group. BCG
used BCG vaccine (manufactured by Nippon BCG Company). Physiological saline was used as a control. Test Example 2 Antitumor Effect on Lewis Lang Carcinoma in Mice The antitumor effect of the substance of the present invention on Lewis Lang Carcinoma in mice was investigated. The test system is as follows. 5 × 10 5 Lewis Lung carcinomas were found intradermally in a C57BL/6 female mouse.
Carcinoma) cells were transplanted, and after transplantation 1, 4, 8,
On the 11th, 15th, and 18th days, 0.1 ml of the preparation prepared by the method of Example 3 was administered intratumorally at each time. On the 26th day after transplantation, the tumor was removed, its weight was measured, and the number of metastatic foci in the lungs was also examined. The results are shown in Table 2. Using physiological saline as a control, BCG is BCG
A vaccine was used.
【表】
試験例 ３
核酸分解酵素処理物の抗腫瘍作用：
本発明物質を核酸分解酵素で処理することによ
り本発明物質の抗腫瘍活性の本体を調べた。
試験法は次のとおりである。
BD−A1をデオキシリボ核酸分解酵素DNaseI
（Sigma Ghemicals製）で処理した後得られた分
解物をクロロホルム処理してDNaseIを除き、次
にセフアデツクスG10（Pharmacia Fine
Chemicals製）カラムにより脱塩、凍結乾燥し
て、本発明物質の核酸分解酵素処理物を得た。得
られた処理物の抗腫瘍活性を試験例１と同様に試
験し、第３表に示す結果を得た。[Table] Test Example 3 Antitumor activity of a substance treated with a nuclease: The main antitumor activity of the substance of the present invention was investigated by treating the substance with a nuclease. The test method is as follows. BD-A1 is deoxyribonuclease DNaseI
(manufactured by Sigma Ghemicals), the resulting decomposition product was treated with chloroform to remove DNase I, and then Sephadex G10 (Pharmacia Fine
The product was desalted using a column (manufactured by Chemicals) and lyophilized to obtain a nuclease-treated product of the substance of the present invention. The antitumor activity of the obtained treated product was tested in the same manner as in Test Example 1, and the results shown in Table 3 were obtained.
【表】
Ｔ／Ｃは対照群の平均腫瘍重量に対する投与群
の平均腫瘍重量のパーセント比である。
対照としては生理食塩液を用い、BCGはBCG
ワクチンを用いた。
試験例 ４
ナチユラルキラー（NK）細胞の活性増強作
用：
本発明物質のNK細胞に対する活性増強作用を
調べた。
試験法は次のとおりである。
生理食塩液、BD−A1又はBD−A2の生理食塩
液溶解液（１mg／ml）を８週令のC57BL／６系雌
性マウスに一匹あたり0.3ml腹腔内投与してその
48時間後に腹腔浸出細胞を採取した。採取液中の
細胞数を10％牛胎児血清添加RPMI1640培地で２
×106個／mlに調整した後プラスチツクシヤーレ
に移して37℃、５％炭酸ガス大気下で90分間イン
キユベートし、シヤーレに付着性細胞、非付着性
の細胞を得た。各々の細胞５×105個と 51Crで標
識したマウス白血病細胞YAC−１、１×104個を
37℃、５％炭酸ガス大気下で10％牛胎児血清添加
RPMI1640培地を用いて４時間培養した。その培
養上清に遊離された 51Crの放射能をオートガン
マーカウンター（Packard製）で測定し、腹腔浸
出細胞のYAC−１細胞に対する細胞障害作用を
調べた。T/C is the percentage ratio of the average tumor weight of the treated group to the average tumor weight of the control group. Physiological saline was used as a control, and BCG was
A vaccine was used. Test Example 4 Activity-enhancing effect on natural killer (NK) cells: The activity-enhancing effect of the substance of the present invention on NK cells was investigated. The test method is as follows. Physiological saline, a solution of BD-A1 or BD-A2 in physiological saline (1 mg/ml) was intraperitoneally administered to 8-week-old C 57 BL/6 female mice in an amount of 0.3 ml per mouse.
Peritoneal exudate cells were collected 48 hours later. The number of cells in the collected solution was reduced to 2 with RPMI1640 medium supplemented with 10% fetal bovine serum.
After adjusting the concentration to ×10 6 cells/ml, the cells were transferred to a plastic shear and incubated at 37° C. for 90 minutes in a 5% carbon dioxide atmosphere to obtain adherent and non-adherent cells on the shear. 5 × 10 5 cells of each cell and 1 × 10 4 mouse leukemia cells YAC-1 labeled with 51 Cr.
Added 10% fetal bovine serum at 37℃ under 5% carbon dioxide atmosphere
Culture was performed for 4 hours using RPMI1640 medium. The radioactivity of 51 Cr released in the culture supernatant was measured using an autogamma counter (manufactured by Packard), and the cytotoxic effect of the peritoneal exudate cells on YAC-1 cells was investigated.
【表】
試験例 ５
細胞増殖直接阻害作用
本発明物質のYAC−１マウス白血病細胞と
FM3Aマウス乳癌細胞の細胞増殖に対する直接阻
害作用を調べた。
試験系は次のとおりである。
8.4×104個のYAC−１細胞と15.4×104個の
FM3A細胞をそれぞれ10％牛胎児血清添加
RPMI1640培地に浮遊せしめ、37℃、５％炭酸ガ
ス大気下で24時間培養後BD−A1を１mg／mlの最
終濃度に加え、同じ条件下でさらに培養を継続し
た。培養開始後24時間目毎にトリパンブルー染色
により生細胞数を測定した。結果は第５表に示し
た。[Table] Test Example 5 Direct inhibition of cell proliferation Effect of the present substance on YAC-1 mouse leukemia cells
The direct inhibitory effect on cell proliferation of FM3A mouse breast cancer cells was investigated. The test system is as follows. 8.4 × 10 4 YAC-1 cells and 15.4 × 10 4
Each FM3A cell was supplemented with 10% fetal bovine serum.
The cells were suspended in RPMI1640 medium and cultured for 24 hours at 37°C under a 5% carbon dioxide atmosphere. BD-A1 was added to a final concentration of 1 mg/ml, and the culture was continued under the same conditions. The number of living cells was measured by trypan blue staining every 24 hours after the start of culture. The results are shown in Table 5.
【表】
試験例 ６
急性毒性
１群10匹の５週令ddY系雄性マウス（平均体重
23g）にBD−A1を生理食塩液に溶解して体重１
Kgあたり500mgを静脈内投与した。本発明物質は
投与後１週間の観察期間中、体重増加の抑制を示
さず、死亡例もなかつた。この結果から本発明物
質の静脈内投与における50％致死用量LD50は500
mg／Kg以上と考えられる。
試験例 ７
抗原性：
生理食塩液に溶解したBD−A1を１群６匹のハ
ートレー系雌性モルモツト（平均体重354g）に
１匹あたり１mgずつ、週３回合計６回皮内投与し
て感作した。最終感作日から２週間経過後BD−
A1を生理食塩液に溶解して体重Kgあたり10mgあ
るいは２mgを静脈内投与した。チヤレンジ前後の
モルモツトの行動観察により本発明物質の抗原性
を調べたところ本発明物質は前記投与量では全く
アナフイラキシーシヨツクを誘発しなかつた。[Table] Test Example 6 Acute Toxicity 10 5-week-old ddY male mice per group (average weight
23g), dissolve BD-A1 in physiological saline and weigh 1
500 mg per kg was administered intravenously. The substance of the present invention did not inhibit body weight gain during the observation period of one week after administration, and there were no cases of death. From this result, the 50% lethal dose LD 50 for intravenous administration of the substance of the present invention is 500.
It is considered to be more than mg/Kg. Test Example 7 Antigenicity: BD-A1 dissolved in physiological saline was intradermally administered to 6 Hartley female guinea pigs (average weight 354 g) per group at a dose of 1 mg per animal, 3 times a week for a total of 6 times to sensitize them. did. 2 weeks after the last sensitization date BD-
A1 was dissolved in physiological saline and administered intravenously at a dose of 10 mg or 2 mg per kg body weight. The antigenicity of the substance of the present invention was investigated by observing the behavior of guinea pigs before and after the challenge, and it was found that the substance of the present invention did not induce anaphylactic shock at all at the above dose.
第１図は本発明物質のセフアローズCL−6Bカ
ラムクロマトによる溶出図を、第２図は同物質の
紫外線吸収スペクトルを、第３図は同物質の赤外
線吸収スペクトルを示す。
FIG. 1 shows the elution pattern of the substance of the present invention by Sepharose CL-6B column chromatography, FIG. 2 shows the ultraviolet absorption spectrum of the same substance, and FIG. 3 shows the infrared absorption spectrum of the same substance.
Claims (1)
り、下記の理化学的性質（Na塩による）を示す
加熱変性デオキシリボ核酸BD−01及びその塩。 (1) 元素分析（％）： Ｃ：26.20〜30.68 Ｈ：4.01〜4.92 Ｎ：12.56〜13.80 Ｐ：8.03〜8.98 Na：2.81〜4.52 (2) 分子量：３万〜100万 蛋白質をマーカーとし、セフアロースCL−
6Bを用いたゲル濾過法によれば20万付近をピ
ークとして３万〜100万に分布する。 (3) 融 点：明確な融点を示さない。 (4) 紫外線吸収スペクトル：中性水溶液は260nm
付近に吸収極大波長を、また230nm付近に吸収
極小波長を有する。 (5) 紫外線吸収スペクトル：780cm-1付近、1090
cm-1付近、1230cm-1付近、1700cm-1付近に核酸
の特性吸収帯を有する。 (6) 溶剤に対する溶解性：水に可溶、エタノー
ル、メタノール、エーテル、アセトンに不溶。 (7) 呈色反応 Ａ） オルシノール反応：STS法により分画
したRNA画分に対して陰性。 Ｂ） ジフエニルアミン反応：STS法により
分画したDNA画分に対して陽性。 Ｃ） ニンヒドリン反応：本物質10μg／mlの
濃度で陰性。 Ｄ） アンスロン反応：本物質10μg／mlの濃
度で陰性。 (8) 塩基性、酸性、中性の区別：本物質の水溶液
のPHは6.5〜7.5である。 (9) 物質の色：白色粉末 (10) 特性：加熱処理によつて可溶性を付与すると
ともに、高い抗腫瘍活性を有せしめ、かつ毒性
および抗原性が極めて低くなつている。 (11) 塩基組成（％）： グアニン：21.0 アデニン：26.8 シトシン：19.7 チミン：32.5 （方法は化学分析による） (12) 酵素処理：本物質をデオキシリボヌクレアー
ゼＩ（DNaseI）で処理すると抗腫瘍性がなく
なるが、リボヌクレアーゼT2（RNaseT2）の
処理では抗腫瘍性は変化しない。 (13) ヒドロキシアパタイトのカラムクロマトグ
ラフイーにより分析すると１本鎖構造である。 (14) 転移温度（Tm）：測定された融解曲線から
は明確な転移温度Tmは求められない。 ２ バチルス属菌より得られたものである特許請
求の範囲第１項記載の加熱変性デオキシリボ核酸
BD−01。 ３ その破砕物を遠心分離して得られる無細胞抽
出液から得られる核酸画分を加熱変性することに
よりBD−01が製造できるバチルス属細菌を使用
し、その破砕物にこれらの処理を行うことを特徴
とする抗腫瘍活性を有する加熱変性デオキシリボ
核酸BD−01の製造法。 ４ 核酸画分の分離法として無細胞抽出液を有機
溶剤処理して分離することを特徴とする特許請求
の範囲第３項記載の製造法。 ５ 核酸画分の分離法として無細胞抽出液に核酸
凝集剤を加え、得られる沈澱を水または塩類溶液
に対して透析した後、その可溶画分から分離する
ことを特徴とする特許請求の範囲第３項記載の製
造法。 ６ 加熱変性を約80゜−120℃で行うことを特徴と
する特許請求の範囲第３〜５項のいずれか１項に
記載の製造法。 ７ その破砕物を遠心分離して得られる無細胞抽
出液に核酸凝集剤を加え、得られる沈澱を水また
は塩類溶液に対して透析した後加熱変性してその
上清からBD−01を分離することができるバチル
ス属細菌を使用し、その破砕物にこれらの処理を
行うことを特徴とする抗腫瘍活性を有する加熱変
性デオキシリボ核酸BD−01の製造法。 ８ 加熱変性を約80゜−120℃で行うことを特徴と
する特許請求の範囲第７項に記載の製造法。 ９ 加熱変性によつて抗腫瘍活性を有せしめてな
る加熱変性デオキシリボ核酸BD−01を有効成分
とする抗腫瘍剤。 １０ 加熱変性デオキシリボ核酸BD−01がバチ
ルス属菌より得られたものである特許請求の範囲
第９項記載の抗腫瘍剤。[Scope of Claims] 1. Heat-denatured deoxyribonucleic acid BD-01 and its salts, which have antitumor activity through heat-denaturation and exhibit the following physicochemical properties (based on Na salt). (1) Elemental analysis (%): C: 26.20-30.68 H: 4.01-4.92 N: 12.56-13.80 P: 8.03-8.98 Na: 2.81-4.52 (2) Molecular weight: 30,000-1,000,000 Sepharose using protein as a marker CL−
According to the gel filtration method using 6B, it is distributed between 30,000 and 1,000,000 with a peak around 200,000. (3) Melting point: Does not show a clear melting point. (4) Ultraviolet absorption spectrum: 260nm for neutral aqueous solution
It has a maximum absorption wavelength near 230 nm and a minimum absorption wavelength near 230 nm. (5) Ultraviolet absorption spectrum: around 780cm -1 , 1090
It has characteristic absorption bands of nucleic acids around cm -1 , around 1230 cm -1 , and around 1700 cm -1 . (6) Solubility in solvents: Soluble in water, insoluble in ethanol, methanol, ether, and acetone. (7) Color reaction A) Orcinol reaction: Negative for RNA fractions fractionated by STS method. B) Diphenylamine reaction: Positive for DNA fraction fractionated by STS method. C) Ninhydrin reaction: Negative at a concentration of 10μg/ml of this substance. D) Anthrone reaction: Negative at a concentration of 10μg/ml of this substance. (8) Basic, acidic, and neutral: The pH of an aqueous solution of this substance is 6.5 to 7.5. (9) Color of the substance: White powder (10) Characteristics: It is made soluble through heat treatment, has high antitumor activity, and has extremely low toxicity and antigenicity. (11) Base composition (%): Guanine: 21.0 Adenine: 26.8 Cytosine: 19.7 Thymine: 32.5 (method is based on chemical analysis) (12) Enzyme treatment: When this substance is treated with deoxyribonuclease I (DNase I), it exhibits antitumor properties. However, treatment with ribonuclease T 2 (RNaseT 2 ) does not change the antitumor activity. (13) When analyzed by hydroxyapatite column chromatography, it has a single-stranded structure. (14) Transition temperature (Tm): A clear transition temperature Tm cannot be determined from the measured melting curve. 2. The heat-denatured deoxyribonucleic acid according to claim 1, which is obtained from Bacillus bacteria.
BD-01. 3 Using Bacillus bacteria that can produce BD-01 by heating and denaturing the nucleic acid fraction obtained from the cell-free extract obtained by centrifuging the disrupted product, and subjecting the disrupted product to these treatments. A method for producing heat-denatured deoxyribonucleic acid BD-01 having antitumor activity, characterized by: 4. The production method according to claim 3, wherein the nucleic acid fraction is separated by treating the cell-free extract with an organic solvent. 5. Claims characterized in that as a method for separating nucleic acid fractions, a nucleic acid flocculant is added to a cell-free extract, the resulting precipitate is dialyzed against water or a salt solution, and then separated from the soluble fraction. The manufacturing method described in paragraph 3. 6. The manufacturing method according to any one of claims 3 to 5, characterized in that the heat denaturation is carried out at about 80° to 120°C. 7 Add a nucleic acid flocculant to the cell-free extract obtained by centrifuging the crushed product, dialyze the resulting precipitate against water or a salt solution, heat denaturation, and separate BD-01 from the supernatant. 1. A method for producing heat-denatured deoxyribonucleic acid BD-01 having antitumor activity, which comprises using Bacillus bacteria capable of producing BD-01, and subjecting the crushed product to these treatments. 8. The manufacturing method according to claim 7, characterized in that the heat denaturation is carried out at about 80°-120°C. 9. An antitumor agent containing heat-denatured deoxyribonucleic acid BD-01, which has anti-tumor activity through heat denaturation, as an active ingredient. 10. The antitumor agent according to claim 9, wherein the heat-denatured deoxyribonucleic acid BD-01 is obtained from Bacillus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14339682A JPS5934887A (en) | 1982-08-20 | 1982-08-20 | Antitumor substance bd-01, its preparation and pharmaceutical preparation containing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14339682A JPS5934887A (en) | 1982-08-20 | 1982-08-20 | Antitumor substance bd-01, its preparation and pharmaceutical preparation containing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5934887A JPS5934887A (en) | 1984-02-25 |
| JPH0365356B2 true JPH0365356B2 (en) | 1991-10-11 |
Family
ID=15337786
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP14339682A Granted JPS5934887A (en) | 1982-08-20 | 1982-08-20 | Antitumor substance bd-01, its preparation and pharmaceutical preparation containing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5934887A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2517999Y2 (en) * | 1989-08-23 | 1996-11-20 | カヤバ工業株式会社 | Cushion device |
| JPH0380107U (en) * | 1989-12-08 | 1991-08-16 |
-
1982
- 1982-08-20 JP JP14339682A patent/JPS5934887A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5934887A (en) | 1984-02-25 |
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