JPH0369917B2 - - Google Patents
Info
- Publication number
- JPH0369917B2 JPH0369917B2 JP57142662A JP14266282A JPH0369917B2 JP H0369917 B2 JPH0369917 B2 JP H0369917B2 JP 57142662 A JP57142662 A JP 57142662A JP 14266282 A JP14266282 A JP 14266282A JP H0369917 B2 JPH0369917 B2 JP H0369917B2
- Authority
- JP
- Japan
- Prior art keywords
- substance
- heat
- present
- nucleic acid
- 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 - Lifetime
Links
- 239000000126 substance Substances 0.000 claims description 94
- 238000000034 method Methods 0.000 claims description 46
- 230000000259 anti-tumor effect Effects 0.000 claims description 45
- 102000039446 nucleic acids Human genes 0.000 claims description 41
- 108020004707 nucleic acids Proteins 0.000 claims description 41
- 150000007523 nucleic acids Chemical class 0.000 claims description 41
- 108020004414 DNA Proteins 0.000 claims description 39
- 102000053602 DNA Human genes 0.000 claims description 37
- 239000000061 acid fraction Substances 0.000 claims description 30
- 238000010438 heat treatment Methods 0.000 claims description 25
- 239000000284 extract Substances 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 238000004519 manufacturing process Methods 0.000 claims description 15
- 239000000047 product Substances 0.000 claims description 15
- 241000589516 Pseudomonas Species 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 238000011282 treatment Methods 0.000 claims description 12
- 239000002244 precipitate Substances 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 8
- 239000002246 antineoplastic agent Substances 0.000 claims description 7
- 238000004440 column chromatography Methods 0.000 claims description 7
- 238000003505 heat denaturation Methods 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
- 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
- 238000002844 melting Methods 0.000 claims description 6
- 230000008018 melting Effects 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 230000007935 neutral effect Effects 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- 231100000053 low toxicity Toxicity 0.000 claims description 5
- 239000003960 organic solvent Substances 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 230000007704 transition Effects 0.000 claims description 5
- 102000007260 Deoxyribonuclease I Human genes 0.000 claims description 4
- 108010008532 Deoxyribonuclease I Proteins 0.000 claims description 4
- 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
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 4
- 108010079502 exoribonuclease T Proteins 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
- 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
- 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
- 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 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
- 229940104302 cytosine Drugs 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 37
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 20
- 238000012360 testing method Methods 0.000 description 19
- 206010028980 Neoplasm Diseases 0.000 description 17
- 230000001580 bacterial effect Effects 0.000 description 16
- 235000002639 sodium chloride Nutrition 0.000 description 15
- 239000000725 suspension Substances 0.000 description 13
- 230000000694 effects Effects 0.000 description 12
- 239000002504 physiological saline solution Substances 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 238000005119 centrifugation Methods 0.000 description 11
- 239000011780 sodium chloride Substances 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 9
- 239000003125 aqueous solvent Substances 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 7
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- 241000700198 Cavia Species 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 238000001816 cooling Methods 0.000 description 6
- 239000002158 endotoxin Substances 0.000 description 6
- 230000001404 mediated effect Effects 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
- 201000009030 Carcinoma Diseases 0.000 description 5
- 241000187479 Mycobacterium tuberculosis Species 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
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 102000016911 Deoxyribonucleases Human genes 0.000 description 4
- 108010053770 Deoxyribonucleases Proteins 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
- 239000013543 active substance Substances 0.000 description 4
- 230000036760 body temperature Effects 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 229920006008 lipopolysaccharide Polymers 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 3
- 241001465754 Metazoa Species 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
- 238000012258 culturing Methods 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
- 239000002510 pyrogen Substances 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 210000004881 tumor cell Anatomy 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
- 241000700199 Cavia porcellus Species 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
- 206010027476 Metastases Diseases 0.000 description 2
- 101710163270 Nuclease Proteins 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
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 2
- 241000589776 Pseudomonas putida Species 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
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 230000015556 catabolic process Effects 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
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- 210000000416 exudates and transudate Anatomy 0.000 description 2
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- 238000004108 freeze drying Methods 0.000 description 2
- 230000002601 intratumoral effect Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- GZQKNULLWNGMCW-PWQABINMSA-N lipid A (E. coli) Chemical compound O1[C@H](CO)[C@@H](OP(O)(O)=O)[C@H](OC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCCCC)[C@@H](NC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCC)[C@@H]1OC[C@@H]1[C@@H](O)[C@H](OC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](NC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](OP(O)(O)=O)O1 GZQKNULLWNGMCW-PWQABINMSA-N 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- 239000011565 manganese chloride Substances 0.000 description 2
- 229940099607 manganese chloride Drugs 0.000 description 2
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- 229910052751 metal Inorganic materials 0.000 description 2
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- 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
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- 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
- XMTQQYYKAHVGBJ-UHFFFAOYSA-N 3-(3,4-DICHLOROPHENYL)-1,1-DIMETHYLUREA Chemical compound CN(C)C(=O)NC1=CC=C(Cl)C(Cl)=C1 XMTQQYYKAHVGBJ-UHFFFAOYSA-N 0.000 description 1
- WTFUTSCZYYCBAY-SXBRIOAWSA-N 6-[(E)-C-[[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]methyl]-N-hydroxycarbonimidoyl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C/C(=N/O)/C1=CC2=C(NC(O2)=O)C=C1 WTFUTSCZYYCBAY-SXBRIOAWSA-N 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
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- 101100005765 Arabidopsis thaliana CDF1 gene Proteins 0.000 description 1
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- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 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
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
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- 229920005654 Sephadex Polymers 0.000 description 1
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- 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
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- 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
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
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- 201000011510 cancer Diseases 0.000 description 1
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- BKHZIBWEHPHYAI-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol Chemical compound ClC(Cl)Cl.CC(C)CCO BKHZIBWEHPHYAI-UHFFFAOYSA-N 0.000 description 1
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- 230000001900 immune effect Effects 0.000 description 1
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- 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
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 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
- 239000012528 membrane Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000035407 negative regulation of cell proliferation Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000001293 nucleolytic effect Effects 0.000 description 1
- 239000007764 o/w emulsion Substances 0.000 description 1
- 230000001354 painful effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
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- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 230000001698 pyrogenic effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 229960002385 streptomycin sulfate Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
【発明の詳細な説明】
本発明はシユードモナス属に属する細菌から得
られる抗腫瘍活性を有する加熱変性デオキシリボ
核酸PD−01に関する。
ブドウ糖非発酵性のグラム陰性桿菌であるシユ
ードモナス属に属する細菌から得られる宿主介在
性の抗腫瘍活性物質については他のグラム陰性菌
類と同様に菌体の外膜に存在するリポ多糖体（内
毒素）が知られている。
しかし、シユードモナス属の細菌を含めたグラ
ム陰性桿菌が産生するリポ多糖体は、宿主介在性
の抗腫瘍活性以外に、それ自体が生体に対して同
時に発熱原性を示すばかりでなく、重篤な致死毒
性あるいはシヨツクを誘発することが知られてお
り、医薬としては安全性の面で問題が多い。この
リポ多糖体から化学的処理によつて毒性作用を切
り離して得られる無毒化リピドＡについての研究
の動きもみられるが無毒化リピドＡ単独の抗腫瘍
活性は十分な水準とは言い難い（Cancer
Immunol，Immunother，７，43，1979）。
また、近年シユドモナス属に属する緑膿菌の感
染防御抗原の解析研究を通じて得られた緑膿菌の
感染防御共通抗原であるOEP（Original
endotoxin protein）に宿主介在性の抗腫瘍活性
がみられることが指摘されているが（J.
Biochemistry，83，711，1978.，GANN，63，
503，1972.，GANN，64，523，1973），OEPは
約80％の蛋白質と約20％のリポ多糖体との複合体
であることから抗原性を含めた副作用などに問題
があり実用に供するまでに至つていない。
本発明者らは先に結核菌から得られる抗腫瘍活
性物質について広範な検討を加えた結果、結核菌
の無細胞抽出液か得られる加熱変性デオキシリボ
核酸が宿主介在性の強力な抗腫瘍活性を有し、か
つ毒性および抗原性が極めて低いことを初めて見
い出し、特許（特開昭57−139096）を出願した。
さらに、本発明者らは、シユードモナス属に属
する細菌の菌体抽出物についても新規な抗腫瘍活
性物質を見い出すべく、結核菌の経験に基づいて
活性物質の検索を鋭意進めた結果、シユードモナ
ス属に属する細菌から結核菌の場合に用いたのと
同様の方法で調製した加熱変性デオキシリボ核酸
も高い抗腫瘍活性を示し、かつ毒性が極めて低
く、発熱性や抗原性がほとんどないことを明らか
にし、本発明物質を加熱変性デオキシリボ核酸
PD−01と命名し本発明を完成するに至つた。
以下、本発明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
あるいは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結
核菌由来の該物質と同様に宿主介在性の強い抗腫
瘍活性を有し、その作用はこれまでに知られてい
る動物細胞由来のデオキシリボ核酸等の腫瘍細胞
への直接障害作用に基づく抗腫瘍作用（Science
149，997，1965，Cancer Research 27，
2342，1967，Proceedings of the National
Academy of Science 61，207，1968）に比べ
はるかにに強力である。
本発明物質とその製造法およびその宿主介在性
の強力な抗腫瘍活性は本発明者らによつて初めて
明らかにされたものである。
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
PD−01及びその塩に関し、更に詳細には、シユ
ードモナ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PD−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。
さらに本発明はシユードモナス属細菌破砕物を
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PD−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PD−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PD−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PD−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PD
−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、シユードモナス
属に属する細菌であるが、好適なものとしては、
シユードモナスプテイダ（Pseudomonas
putida）IFO12996株やシユードモナスアエルギ
ノーサ（Pseudomonas aeruginosa）ATCC−
10145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。たとえばシユードモナスプテ
イダ（Pseudomonas putida）の場合ペプトン酵
母エキス培地あるいは普通ブイヨン培地を用いて
30〜37℃程度の温度で24時間通気あるいは静置培
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。得られた菌体を水、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
法等、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PD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
PD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。
PD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、同時の溶解性を高め、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PN
−１画分と称する。PN−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。
PN−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、PN−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PN−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、PN−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PN−
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。PN−１画分から可溶画分を分
離する際の好適なPN−１画分の濃度は１〜15
mg／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であるいは緩衝液で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効に影響を与
え、通常0.1以上が好ましく、要すれば食温ある
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PN−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PN−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PN−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61％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、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PD−
01（以下PD−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PD−01（以下PD−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PD−A1の
理化学的性質（Na塩による）
(1) 元素分析（％）：Ｃ：27.31〜31.44 Ｈ：4.13
〜5.79 Ｎ：11.03〜13.55 Ｐ：6.81〜8.22
Na：3.80〜4.81
(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15万付近をピ
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：30.0 アデニン：19.9
シトニン：30.7 チミン ：19.4（方法は化
学分析による）
(12) 降素処理：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（DNase）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カルチノーマに対して本
発明物質は生理食塩液に溶液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（リダレツシヨン活性）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た得られる懸濁液の
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、PH6.8〜7.0に調節する。
Pseudomonas putida，IFO12996株を上記組
成の培地で30℃、24時間振盪培養し、培養液を遠
心分離処理して得た湿菌体40ｇを40ml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、0.5M
NaCl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85ml（以下この懸濁
液をPN−１と称する）を得た。この懸濁液の固
形物濃度は5.3mg／mlであり、湿菌体あたり1.1％
の収率であつた。
次にPN−１全量にNaClを終濃度が0.9％にな
るように加えて撹拌し、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、1M NaCl液15ml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15ml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63
mgを得た。本品全量を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（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44.5mgを得た。以下この
ものをPD−A1と称す。
PD−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
マーマー法による本発明物質の製造：
Pseudomons putida IFO12996株を実施例１の
培地で同様の培養条件によつて得た湿菌体250ｇ
を７倍量の10mMリン酸緩衝液に懸濁した後氷冷
下ダイノミルで破砕し、20000xgで20分間遠心し
て無細胞抽出液を得た。この抽出液からマーマー
（Marmur，Journal of Molecular Biology
３，208，1961）法により粗デオキシリボ核酸画
分を得た。この画分を0.9％食塩液に対して透析
した後、100℃、60分加熱した。冷却後蒸留水に
対して透析、中和後凍結乾燥して得た標品56mgに
対し、実施例１と同様のスケールでリボヌクレア
ーゼT2処理を行ない、精製し、本発明物質41mg
を得た。以下このものをCD−A2と称す。CD−
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
PD−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
PD−A1 10mgを10ml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
PD−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。
【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
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
ストレイン２モルモツトのライン10へパトーマ
に対する抗腫瘍作用
本発明物質のストレイン２モルモツトのライン
10へパトーマに対する抗腫瘍作用を調べた。
１×106個のライン（line）10へパトーマ腫瘍
細胞をストレイン（strain）２モルモツトの皮内
に移植し、移植後７日目に実施例３に示した方法
で調製した製剤0.1mlを腫瘍内に投与した。移植
後70日目に生存例数、治癒例数、所属リンパ節へ
の転移の有無を調べた。対照として生理食塩液を
用いて同様に調製したものを用いた。
結果は第２表に示した。
【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。
PD−A1をデオキシリボ核酸分解酵素DNase
（Sigma Chemicals製）で処理した後得られた分
解物ををクロロホルム処理してDNase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。
【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、PD−A1又はPD−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。
【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PD−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。
【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
25ｇ）にPD−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PD−A1を１群６匹のハ
ートレー系雌性モルモツト（平均体重350ｇ）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PD−
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。
試験例 ８
発熱性：
生物学的製剤基準の一般試験法に準じて、減菌
生理食塩液に溶解したPD−A1を１群３頭の在来
種雄性白色ウサギ（平均体重2.46Kg）に500μｇ／
ml／Kgの投与量で耳静脈内に投与し、投与後３時
間にわたつて直腸体温を測定した。
その結果、投与前の対照体温に対する投与後の
測定体温との差体温の最大値を発熱反応とすると
３頭の発熱反応の和は0.72℃で生物学的製剤基準
の判定基準に従い発熱反応が陰陰性と判定され
た。 DETAILED DESCRIPTION OF THE INVENTION The present invention relates to heat-denatured deoxyribonucleic acid PD-01 having antitumor activity obtained from a bacterium belonging to the genus Pseudomonas. Regarding host-mediated antitumor active substances obtained from bacteria belonging to the genus Pseudomonas, which are Gram-negative rods that do not ferment glucose, lipopolysaccharide (endotoxin) present in the outer membrane of the bacterial body, like other Gram-negative bacteria, )It has been known. However, in addition to host-mediated antitumor activity, lipopolysaccharides produced by Gram-negative bacilli, including bacteria of the genus Pseudomonas, not only exhibit pyrogenicity to living organisms, but also cause severe It is known to induce lethal toxicity or shock, and there are many safety issues as a medicine. Although there is some research into detoxified lipid A, which can be obtained from lipopolysaccharide by chemically removing its toxic effects, the antitumor activity of detoxified lipid A alone cannot be said to be at a sufficient level (Cancer
Immunol, Immunother, 7 , 43, 1979). In addition, OEP (Original
It has been pointed out that endotoxin protein) has host-mediated antitumor activity (J.
Biochemistry, 83 , 711, 1978., GANN, 63 ,
503, 1972., GANN, 64 , 523, 1973), OEP is a complex of about 80% protein and about 20% lipopolysaccharide, so there are problems with side effects including antigenicity, so it cannot be put into practical use. It has not yet reached the point where it can be provided. The present inventors previously conducted extensive studies on antitumor active substances obtained from Mycobacterium tuberculosis, and found that heat-denatured deoxyribonucleic acid obtained from a cell-free extract of Mycobacterium tuberculosis exhibits strong host-mediated antitumor activity. It was discovered for the first time that it possesses a very low toxicity and antigenicity. Furthermore, the present inventors conducted a diligent search for active substances based on their experience with Mycobacterium tuberculosis in order to find novel antitumor active substances in cell extracts of bacteria belonging to the genus Pseudomonas. We found that heat-denatured deoxyribonucleic acid prepared from bacteria belonging to the same group using a method similar to that used in the case of Mycobacterium tuberculosis also exhibits high antitumor activity, has extremely low toxicity, and has almost no pyrogenicity or antigenicity. The invention substance is heat-denatured deoxyribonucleic acid
The present invention was completed under the name PD-01. Hereinafter, the substance of the present invention is often simply referred to as heat-denatured deoxyribonucleic acid, modified deoxyribonucleic acid, deoxyribonucleic acid, or the present substance. The deoxyribonucleic acid obtained by the present invention has strong host-mediated antitumor activity similar to the substance derived from Mycobacterium tuberculosis, and its action is similar to that of the substance derived from animal cells. Antitumor effect based on direct damage to cells (Science
149 , 997, 1965, Cancer Research 27 ,
2342, 1967, Proceedings of the National
Academy of Science 61 , 207, 1968). The substance of the present invention, its production method, and its strong host-mediated antitumor activity were first revealed by the present inventors. That is, the present invention provides heat-denatured deoxyribonucleic acid which has antitumor activity through heat denaturation.
The present invention relates to PD-01 and its salts, and more particularly to heat-denatured deoxyribonucleic acid PD-01 having antitumor activity obtained from bacteria of the genus Pseudomonas, as well as its production method and antitumor agent. Furthermore, the present invention relates to a method for producing heat-denatured deoxyribonucleic acid PD-01 having antitumor activity, which comprises heating a nucleic acid fraction obtained by centrifuging a crushed product of bacteria belonging to the genus Pseudomonas. Furthermore, the present invention provides a method for separating a 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 PD-01 having antitumor activity. Furthermore, the present invention provides deoxyribonucleic acid-containing substances about
This is a method for producing heat-denatured deoxyribonucleic acid PD-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 PD which has antitumor activity through heat denaturation.
It is an antitumor agent containing -01 as an active ingredient. The microorganisms used in the present invention are bacteria belonging to the genus Pseudomonas, and preferred ones include:
Pseudomonas (Pseudomonas)
putida) IFO12996 strain and Pseudomonas aeruginosa (Pseudomonas aeruginosa) ATCC-
Examples include 10145 stocks. 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 Pseudomonas putida, peptone yeast extract medium or ordinary bouillon medium is used.
A good culture can be obtained by aerating or statically culturing at a temperature of about 30 to 37°C for 24 hours, which can be centrifuged or filtered to obtain bacterial cells. The obtained bacterial cells were thoroughly mixed with water, preferably an appropriate buffer solution, and placed in a Dynomill while cooling on ice.
-Crush the bacterial cells using a mill or French press to obtain a suspension of crushed bacterial cells.
This suspension is centrifuged to obtain a cell-free extract, but the centrifugation conditions at this time are normal centrifugation that can remove unbroken bacterial cells, partially crushed bacterial cells, cell wall fractions, etc. as sediment. Power is fine, for example 5000xg,
Centrifuge 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 is a method to obtain a nucleic acid fraction by directly treating the cell-free extract with an organic solvent (for example, the Marmur method, the phenol method, etc., 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 a method suitable for obtaining the substance of the present invention on a small scale (usually 1 g or less), and the resulting nucleic acid fraction (hereinafter referred to as PD 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 PD 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 PD nucleic acid fraction and the purified deoxyribonucleic acid fraction are the same as those for the preparation method described later, and 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 impurity removal, facilitate formulation, simultaneously increase solubility, and improve anti-inflammatory properties. It is an object of the present invention to obtain a substance of the present invention that has high tumor 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 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. The suspension containing the nucleic acid fraction is dialyzed against a salt-containing buffer and then, if necessary, dialyzed against water to remove salts. The suspension containing the dialyzed nucleic acid fraction is
-1 fraction. The procedure for obtaining the PN-1 fraction is as follows:
All operations were performed under cooling to avoid unnecessary degradation of the nucleic acid fraction.
Prevent degeneration. The cooling temperature is preferably 0 to 10°C. Two methods are possible to obtain the substance of the invention from the PN-1 fraction. One method is to separate the nucleic acid fraction from the PN-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 PN-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 PN-1 fraction and the pH and salt concentration of the aqueous solvent, the PN-1 fraction 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 the 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 PN-1 fraction when separating the soluble fraction from the PN-1 fraction is 1 to 15
mg/ml. The pH of the aqueous solvent is preferably around neutral, and usually acids, bases, or buffers are used to
to 8. The ionic strength of the aqueous solvent affects the effectiveness of removing precipitates during centrifugation, etc., and is usually preferably 0.1 or more, and if necessary, the ionic strength may be adjusted by adjusting the food temperature or adding potassium chloride or the like. The PN-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 subjecting the deoxyribonucleic acid fraction thus obtained to ribonuclease treatment followed by heat treatment, or by heat treatment followed by ribonuclease treatment, but the heating conditions are the same as in the latter method. Details will be explained later. In the latter method, the PN-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 PN-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 substance of the present invention obtained by these methods can be used as it is as a raw material for pharmaceutical preparations, or can be freeze-dried to form a dry powder. The molecular weight of deoxyribonucleic acid in the substance of the present invention is 3
Distributed between 10,000 and 1,000,000, and the guanine and cytosine (GC) content is approximately 61%. The transition temperature Tm of the substance of the present invention does not show a clear value, and 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 ribonucleic acids, proteins, sugars, etc. in addition to heat-denatured deoxyribonucleic acids, but these can only be considered contaminants, and if necessary These can be removed by further purification. Next, PD- obtained by purification in Example 1
We investigated the physical and chemical properties of 01 (hereinafter referred to as PD-A1) and present the results. Note that the purified product of PD-01 (hereinafter referred to as PD-A2) obtained in Example 2 also exhibits similar physical and chemical properties. Physical and chemical properties of this substance, heat-denatured deoxyribonucleic acid PD-A1 (by Na salt) (1) Elemental analysis (%): C: 27.31-31.44 H: 4.13
~5.79 N: 11.03~13.55 P: 6.81~8.22
Na: 3.80-4.81 (2) Molecular weight: 30,000-1,000,000 Sepharose CL-
According to the gel filtration method using 6B, it is distributed from 30,000 to 1,000,000 with a peak around 150,000. (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 fractions fractionated by STS method. (C) Ninhydrin reaction: Negative at a concentration of 10 μg/ml of this substance. (D) Anthrone reaction: Negative at a concentration of 10 μg/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: 30.0 Adenine: 19.9 Cytonine: 30.7 Thymine: 19.4 (method is based on chemical analysis) (12) Degradation treatment: When this substance is treated with deoxyribonuclease I (DNase I), it has 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. 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 (DNase), 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, which is a syngeneic tumor in mice, the substance of the present invention can be applied to suppress tumor engraftment (suppression activity) by contacting the tumor cells in a physiological saline solution and then implanting it into the animal body. When administered into tumor tissue that had engrafted in the body, the antitumor activity (redaction activity) 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 the resulting suspension using streptomycin Peptone 10g Yeast extract 10g NaCl 5g Water was added to bring the pH to 1 and the pH was adjusted to 6.8 to 7.0. Pseudomonas putida, IFO12996 strain was cultured with shaking at 30℃ for 24 hours in a medium with the above composition, and the culture solution was centrifuged. 40g of wet bacterial cells were suspended in 40ml of 10mM phosphate buffer (PH7.0). After that, 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, and 0.5M
It was suspended in 10mM phosphate buffer (PH7.0) containing NaCl. This suspension was packed in a cellophane tube and dialyzed against the same buffer, followed by dialysis against distilled water to obtain 85 ml of a suspension containing the nucleic acid fraction (hereinafter this suspension is referred to as PN-1). ) was obtained. The solid concentration of this suspension was 5.3 mg/ml, which was 1.1% per wet bacterial cell.
The yield was . Next, NaCl was added to the entire amount of PN-1 to a final concentration of 0.9%, stirred, and heat denatured (100℃,
60 minutes), and then centrifuged at 20,000xg for 20 minutes to obtain a supernatant. After adding NaCl to this supernatant to a final concentration of 0.4M and dissolving it, cetyltrimethylammonium bromide (hereinafter referred to as CTAB)
It is called. manufactured by Tokyo Kasei Co., Ltd.) with a final concentration of 0.2%.
(W/V), stirred thoroughly, and left at room temperature for 30 minutes. The generated precipitate was collected by centrifugation, dissolved in 15 ml of 1M NaCl solution, and then added with an equal volume of chloroform-isoamyl alcohol (24:1).
A mixed solution was added, shaken, and centrifuged 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. The generated precipitate was collected by centrifugation, dissolved in 15 ml of distilled water, and then dialyzed against distilled water to obtain a purified nucleic acid solution. After neutralizing this solution with 1N NaOH, freeze-dry the dried sample 63.
I got mg. Add the entire amount of this product to 0.05M acetate buffer (PH4.5).
After dissolving in 10 ml, add 200 U of ribonuclease T 2 (manufactured by Sankyo Co., Ltd.) dissolved in 2 ml of the same buffer.
The reaction was carried out 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 total amount of water layer is
Sephadex G-100 with a diameter of 2.5 cm and a length of 90 cm, which was cleaned in advance with 0.5M ammonium bicarbonate solution.
(manufactured by Pharmacia Fine Chemicals) column, and after elution with the same solution, a fraction containing deoxyribonucleic acid was obtained as the first eluted fraction. After dialyzing this fraction against water and neutralizing it with NaOH,
Freeze-drying yielded 44.5 mg of the substance of the present invention. Hereinafter, this product will be referred to as PD-A1. The content (purity) of deoxyribonucleic acid in PD-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 by the marmer method: 250 g of wet bacterial cells obtained by culturing Pseudomons putida IFO12996 strain in the medium of Example 1 under the same conditions.
was suspended in 7 times the amount of 10mM phosphate buffer, crushed with a Dynomill under ice cooling, and centrifuged at 20,000xg for 20 minutes to obtain a cell-free extract. This extract was extracted from Marmur, Journal of Molecular Biology.
3, 208, 1961) to obtain a crude deoxyribonucleic acid fraction. This fraction was dialyzed against 0.9% saline and then heated at 100°C for 60 minutes. After cooling, dialysis against distilled water, neutralization, and freeze-drying were performed to obtain 56 mg of the sample, which was purified by ribonuclease T 2 treatment on the same scale as in Example 1, resulting in 41 mg of the substance of the present invention.
I got it. Hereinafter, this product will be referred to as CD-A2. CD-
The deoxyribonucleic acid content of A2 was analyzed in the same manner as in Example 1 and was found to be 98.1%. Example 3 10 mg of liquid PD-A1 was dissolved in 10 ml of PBS minus solution (manufactured by Eiken Chemical Co., Ltd.), and Nyukuripore 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 preparation PD-A1 10mg was dissolved in 10ml of distilled water for injection,
Then, after adding and dissolving 500 mg of mannitol,
It was sterilized using Nyukrypore 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 4 mg of emulsion PD-A1 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. 5 × 10 5 IMC carcinoma cells were intradermally transplanted into CDF1 female mice, and 0.1 ml of the preparation prepared by the method of Example 3 was injected into the tumor every other day for a total of 6 times from the first day after transplantation. Administered intravenously. 35 after transplant
The tumor was excised on the same day, and its weight was measured. The results are shown in Table 1. [Table] Student's 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 Strain 2 guinea pig line 10 Antitumor effect on pathoma Line 1 of strain 2 guinea pig of the substance of the present invention
The antitumor effect against 10hepatoma was investigated. Pathoma tumor cells were transplanted intradermally into strain 2 guinea pigs into line 10 of 1×10 6 cells, and 0.1 ml of the preparation prepared by the method shown in Example 3 was added to the tumor on the 7th day after transplantation. Administered intravenously. Seventy days after transplantation, we examined the number of surviving cases, the number of cured cases, and the presence or absence of metastasis to regional lymph nodes. As a control, a sample prepared in the same manner using physiological saline was used. The results are shown in Table 2. [Table] Test Example 3 Antitumor activity of a substance treated with a nucleolytic enzyme The substance of the present invention was treated with a nuclease to examine the substance of the antitumor activity of the substance of the present invention. The test method is as follows. PD-A1 is a deoxyribonuclease DNase
(manufactured by Sigma Chemicals), the resulting decomposition product was treated with chloroform to remove DNase,
Next, 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. 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 PD-A1 or PD-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. [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. PD-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. [Table] Test Example 6 Acute Toxicity 10 5-week-old ddY male mice per group (average weight
Dissolve PD-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 PD-A1 dissolved in physiological saline was intradermally administered to six Hartley female guinea pigs (average weight 350 g) per group at a dose of 1 mg per animal, three times a week for a total of six times to sensitize them. . PD- after 2 weeks from the last sensitization date
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. Test Example 8 Pyrogenicity: According to the general test method of biological product standards, 500 μg of PD-A1 dissolved in sterile physiological saline was administered to a group of 3 native male white rabbits (average weight 2.46 kg). /
The drug was administered into the ear vein at a dose of ml/Kg, and rectal body temperature was measured over 3 hours after administration. As a result, if the maximum difference in body temperature between the control body temperature before administration and the measured body temperature after administration is defined as the exothermic response, the sum of the exothermic responses of the three animals was 0.72°C, and the exothermic responses were negative and negative according to the criteria for biological products. It was determined that it was sex.
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 diagram 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PD−01及びその塩。 (1) 元素分析（％）： Ｃ：27.31〜31.44 Ｈ：4.13〜5.79 Ｎ：11.03〜13.55 Ｐ：6.81〜8.22 Na：3.80〜4.81 (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15万付近をピ
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) 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。 (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：30.0 アデニン：19.9 シトシン：30.7 チミン ：19.5（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 シユードモナ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PD−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PD−01が製造できるシユードモナス属細菌
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PD−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PD−01を分離することができるシユー
ドモナス属細菌を使用し、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PD−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PD−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PD−01がシユ
ードモナ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 PD-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: 27.31-31.44 H: 4.13-5.79 N: 11.03-13.55 P: 6.81-8.22 Na: 3.80-4.81 (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 from 30,000 to 1,000,000 with a peak around 150,000. (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) 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 . (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 fractions fractionated by STS method. (C) Ninhydrin reaction: Negative at a concentration of 10 μg/ml of this substance. (D) Anthrone reaction: Negative at a concentration of 10 μg/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: 30.0 Adenine: 19.9 Cytosine: 30.7 Thymine: 19.5 (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. Heat-denatured deoxyribonucleic acid PD-01 according to claim 1, which is obtained from a Pseudomonas bacterium. 3 Using a Pseudomonas bacterium that can produce PD-01 by heating and denaturing the nucleic acid fraction obtained from the cell-free extract obtained by centrifuging the disrupted product, the disrupted product is subjected to these treatments. A method for producing heat-denatured deoxyribonucleic acid PD-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 PD-01 from the supernatant. 1. A method for producing heat-denatured deoxyribonucleic acid PD-01 having antitumor activity, which comprises using a Pseudomonas bacterium that is capable of producing sterilized Pseudomonas bacteria, 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 anti-tumor agent containing heat-denatured deoxyribonucleic acid PD-01 as an active ingredient, which has anti-tumor activity through heat denaturation. 10. The antitumor agent according to claim 9, wherein the heat-denatured deoxyribonucleic acid PD-01 is obtained from a Pseudomonas bacterium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57142662A JPS5933226A (en) | 1982-08-19 | 1982-08-19 | Antitumor substance pd-01, its preparation and pharmaceutical preparation containing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57142662A JPS5933226A (en) | 1982-08-19 | 1982-08-19 | Antitumor substance pd-01, its preparation and pharmaceutical preparation containing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5933226A JPS5933226A (en) | 1984-02-23 |
| JPH0369917B2 true JPH0369917B2 (en) | 1991-11-05 |
Family
ID=15320571
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP57142662A Granted JPS5933226A (en) | 1982-08-19 | 1982-08-19 | Antitumor substance pd-01, its preparation and pharmaceutical preparation containing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5933226A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2800005B2 (en) * | 1987-11-18 | 1998-09-21 | 有機合成薬品工業株式会社 | Method for producing deoxyribonucleic acid |
-
1982
- 1982-08-19 JP JP57142662A patent/JPS5933226A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5933226A (en) | 1984-02-23 |
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