SI9300415A - Improved method for preparing soluble glucans - Google Patents
Improved method for preparing soluble glucans Download PDFInfo
- Publication number
- SI9300415A SI9300415A SI9300415A SI9300415A SI9300415A SI 9300415 A SI9300415 A SI 9300415A SI 9300415 A SI9300415 A SI 9300415A SI 9300415 A SI9300415 A SI 9300415A SI 9300415 A SI9300415 A SI 9300415A
- Authority
- SI
- Slovenia
- Prior art keywords
- glucan
- soluble
- phosphate
- particulate
- dmso
- Prior art date
Links
- 229920001503 Glucan Polymers 0.000 title claims abstract description 147
- 238000000034 method Methods 0.000 title claims abstract description 58
- 239000000203 mixture Substances 0.000 claims description 30
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 26
- 230000008569 process Effects 0.000 claims description 24
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 238000002360 preparation method Methods 0.000 claims description 19
- 239000004202 carbamide Substances 0.000 claims description 13
- 230000007935 neutral effect Effects 0.000 claims description 12
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 10
- 239000011541 reaction mixture Substances 0.000 claims description 10
- 108090000288 Glycoproteins Proteins 0.000 claims description 6
- 102000003886 Glycoproteins Human genes 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 230000003196 chaotropic effect Effects 0.000 claims description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 64
- 229910019142 PO4 Inorganic materials 0.000 description 40
- 235000021317 phosphate Nutrition 0.000 description 40
- 239000010452 phosphate Substances 0.000 description 39
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 37
- 239000000243 solution Substances 0.000 description 21
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- 229920000642 polymer Polymers 0.000 description 12
- 230000000694 effects Effects 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 8
- 239000002244 precipitate Substances 0.000 description 8
- 239000002253 acid Substances 0.000 description 7
- 239000012153 distilled water Substances 0.000 description 7
- 238000001990 intravenous administration Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical group OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 6
- 239000007864 aqueous solution Substances 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 150000001720 carbohydrates Chemical class 0.000 description 5
- 235000014633 carbohydrates Nutrition 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 239000002808 molecular sieve Substances 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 210000002540 macrophage Anatomy 0.000 description 4
- 239000010802 sludge Substances 0.000 description 4
- 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 3
- 229920002307 Dextran Polymers 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 230000000975 bioactive effect Effects 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 235000019253 formic acid Nutrition 0.000 description 3
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 231100000252 nontoxic Toxicity 0.000 description 3
- 230000003000 nontoxic effect Effects 0.000 description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 230000007704 transition Effects 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 239000012901 Milli-Q water Substances 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 206010039491 Sarcoma Diseases 0.000 description 2
- 230000021736 acetylation Effects 0.000 description 2
- 238000006640 acetylation reaction Methods 0.000 description 2
- 230000008512 biological response Effects 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 2
- 229910052794 bromium Inorganic materials 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 229930182478 glucoside Natural products 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 230000003394 haemopoietic effect Effects 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
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- 239000004570 mortar (masonry) Substances 0.000 description 2
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000001698 pyrogenic effect Effects 0.000 description 2
- 230000003307 reticuloendothelial effect Effects 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 208000029483 Acquired immunodeficiency Diseases 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 229920002498 Beta-glucan Polymers 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 208000005189 Embolism Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 229920000869 Homopolysaccharide Polymers 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 229920001491 Lentinan Polymers 0.000 description 1
- 206010073734 Microembolism Diseases 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 208000001388 Opportunistic Infections Diseases 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 241000295644 Staphylococcaceae Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 206010047115 Vasculitis Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000005862 Whey Substances 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
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- 239000003929 acidic solution Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000007059 acute toxicity Effects 0.000 description 1
- 231100000403 acute toxicity Toxicity 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000002249 anxiolytic agent Substances 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000007664 blowing Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 150000001721 carbon Chemical group 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
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- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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- 239000012530 fluid Substances 0.000 description 1
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- 229940096919 glycogen Drugs 0.000 description 1
- 230000033687 granuloma formation Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- NAQMVNRVTILPCV-UHFFFAOYSA-N hexane-1,6-diamine Chemical compound NCCCCCCN NAQMVNRVTILPCV-UHFFFAOYSA-N 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 230000036543 hypotension Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 239000000367 immunologic factor Substances 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000002356 laser light scattering Methods 0.000 description 1
- 229940115286 lentinan Drugs 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000011034 membrane dialysis Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- -1 pachyman * Chemical class 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- 238000005375 photometry Methods 0.000 description 1
- 239000003880 polar aprotic solvent Substances 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 108010001062 polysaccharide-K Proteins 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 244000000040 protozoan parasite Species 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 201000000306 sarcoidosis Diseases 0.000 description 1
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate group Chemical group S(=O)(=O)([O-])[O-] QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
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- 238000000196 viscometry Methods 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
1. PODROČJE IZUMA1. FIELD OF THE INVENTION
Ta izum se nanaša na izpopolnjeno, visoko učinkovito metodo za pripravo topnih glukanov. Metoda se prednostno izogiba uporabe visoko polarnih topil kot so na primer dimetilsulfoksid (DMSO). Topni glukani pripravljeni po metodi tega izuma so netoksični in kažejo izrazit imunološki odziv kadar so administrirani in vivo, najbolj opazno imunostimulacijo aktivnosti makrofagov in stimulacijo hamatopoieticne aktivnosti kostnega mozga. Topni glukani tudi kažejo pomembne učinke proti malignim neoplazmam vključno melanomom in sarkomom.The present invention relates to a sophisticated, highly efficient method for the preparation of soluble glucans. The method preferably avoids the use of highly polar solvents such as dimethylsulfoxide (DMSO). The soluble glucans prepared by the method of the present invention are non-toxic and exhibit a pronounced immune response when administered in vivo, most notably immunostimulating macrophage activity and stimulating bone marrow hamatopoietic activity. Soluble glucans also show significant effects against malignant neoplasms including melanoma and sarcoma.
2. OZADJE IZUMA2. BACKGROUND OF THE INVENTION
Izraz glukan se generično nanaša na vrsto naravnih homopolisaharidov ali poliglukoz, vključno s polimeri kot so : celuloza, amiloza, glikogen, laminariati (vrsta polisaharidov), škrob itd. Glukan zajema razvejane in nerazvejane verige glukoznih enot povezanih z 1-3, 14, in 1-6 glukozidnih vezi, ki so lahko tipa a ali β.The term glucan generally refers to a variety of natural homopolysaccharides or polyglucose, including polymers such as: cellulose, amylose, glycogen, laminariates (a type of polysaccharide), starch, etc. Glucan comprises branched and unbranched chains of glucose units linked to 1-3, 14, and 1-6 glucoside bonds, which may be of type a or β.
Partikularni glukan (agregat v vodi netopnih delcev glukana), kakor je imenovan v tem izumu, je označen kot vodno netopni partikulat (agregat delcev glukana) (velikost 1-3 pm) poliglukoze, pridobljen iz celične ovojnice kvasovke Saccharomyces cerevisiae. Partikulatni glukan je makromolekula in obsega zaprto verigo glukopiranoznih enot, povezanih z vrsto β-1-3 glukozidnih vezi (Hassid et.al., 1941, J.Amer. Chem. Soc. 63:295-298; DiLuzio et al.,1979, Infl J.Cancer 24:773-779). Študij z rentgensko difrakcijo je potrdil, da partikulatni glukani tvorijo trikratno-sučno vijačnico. (Sarko et al., 1983, Biochem. Soc. Trans. 11:139142).Particular glucan (an aggregate of water-insoluble glucan particles) as named in the present invention is referred to as a water-insoluble particle (aggregate of glucan particles) (size 1-3 pm) of polyglucose obtained from the cell envelope of the yeast Saccharomyces cerevisiae. Particulate glucan is a macromolecule and comprises a closed chain of glucopyranose units linked to a series of β-1-3 glucoside bonds (Hassid et.al., 1941, J.Amer. Chem. Soc. 63: 295-298; DiLuzio et al., 1979 , Infl. J. Cancer 24: 773-779). X-ray diffraction studies have confirmed that particulate glucans form a three-fold helix. (Sarko et al., 1983, Biochem. Soc. Trans. 11: 139142).
Partikulatni glukan je močan aktivator vrste makrofag/monocitnih celic, komplementov kakor tudi T in B celičnih limfocitov. Tako ima partikulatni glukan izrazit učinek na retikuloendotelični in imunski sistem. Partikulatni glukan je pokazal zmožnost modificiranja gostiteljeve rezistence za širok spekter infektivnih bolezni (glej pregledni članek DiLuzia, 1983, Trends in Pharmacol. Sci. 4:344-347 in citirane reference ). Dodatno partikulatni glukan tudi inhibira rast tumorjev in podaljša preživetje v prirojenih mišjih tumornih modelih (DiLuzio et.al., 1979, Advances in Exp. Med. Biol. 121A.-269-290). In vitro študija normalnih in tumornih celic inkubiranih s partikulatnim glukanom je pokazala, da glukan kaže citostatični učinek na sarkomske in melanomske celice in proliferativni učinek na normalne jetrne in celice kostnega mozga (VVilliams et al., 1985, Hepatology 5:198-206).Particulate glucan is a potent activator of macrophage / monocyte cell type, complement as well as T and B cell lymphocytes. Thus, particulate glucan has a pronounced effect on the reticuloendothelial and immune systems. Particulate glucan demonstrated the ability to modify host resistance to a wide range of infectious diseases (see review article DiLuzia, 1983, Trends and Pharmacol. Sci. 4: 344-347 and cited references). Additionally, particulate glucan also inhibits tumor growth and prolongs survival in congenital mouse tumor models (DiLuzio et.al., 1979; Advances in Exp. Med. Biol. 121A.-269-290). An in vitro study of normal and tumor cells incubated with particulate glucan showed that glucan exhibits a cytostatic effect on sarcoma and melanoma cells and a proliferative effect on normal liver and bone marrow cells (VVilliams et al. 1985, Hepatology 5: 198-206).
Vkljub koristnim biološkim učinkom partikulatnih glukanov so ti različni stranski učinki preprečili njihovo uporabnost v klinični medicini. Dodajanje partikularnega glukana živalim in vivo, je pokazalo na resne stranske učinke, med katerimi so najbolj opazni: (1) tvorba granuloma (sarkoidoza ); (2) razvoj heptatosplenomegalije; (3) naraščajoča občutljivost za gram-negativne infekcije in endotoksine; (4) aktivacija komplementa (anafilatoksina ); razvijanje pljučnega grnulomatoznega vaskulitisa; razvijanja hipotenzije, ki sledi intravenozni administraciji; in (7) rasti mikroembolije pri dodajanju visoko koncentriranega reagenta.Despite the beneficial biological effects of particulate glucans, these various side effects have prevented their usefulness in clinical medicine. Addition of particulate glucan to animals in vivo has revealed serious side effects, the most notable of which are: (1) granuloma formation (sarcoidosis); (2) development of heptatosplenomegaly; (3) increasing susceptibility to gram-negative infections and endotoxins; (4) complement activation (anaphylatoxin); development of pulmonary granulomatous vasculitis; developing hypotension following intravenous administration; and (7) the growth of microembolism upon the addition of a highly concentrated reagent.
Dodatno so opazili tudi relativno visoko stopnjo akutne toksičnosti pri in vivo dodajanju partikulatnega glukana. V primeru enega samega intravenoznega vbrizga vodne suspenzije partikulatnega glukana, so opazili 20% in 100% smrtnost pri miših, ki so sprejele 250 in 500 mg/kg glukana na telesno težo.In addition, a relatively high level of acute toxicity was observed with in vivo addition of particulate glucan. In the case of a single intravenous injection of particulate glucan aqueous suspension, 20% and 100% mortality was observed in mice receiving 250 and 500 mg / kg glucan per body weight, respectively.
Nadalje je zaradi partikularne narave glukananove priprave (1-3 pm), glukan zelo težko administrirati po intravenozni poti. Za ilustracijo, pacientu je potreben stalen nadzor pri intravenoznem (IV) dodajanju partikulatnega glukana, saj je bistvenenega pomena kontinuirno stresanje IV kapljevinaste stekleničke, da se vzdržuje suspenzija in s tem prepreči embolijo pri pacientu.Furthermore, due to the particular nature of glucanan preparation (1-3 pm), glucan is very difficult to administer via the intravenous route. To illustrate, the patient needs constant monitoring of intravenous (IV) administration of particulate glucan, since continuous shaking of the IV droplet bottle is essential to maintain suspension and thus prevent embolism in the patient.
Četudi so šibko topni nevtralni glukani komercialno dostopni, ti pripravki niso primerni za intravenozno administriranje, ker imajo vodne raztopine zelo veliko viskoznost in še važnejše, ker je njihova uporaba, ko jih administrirajo eksperimentalnim živalim, neizogibno spremljana s precejšno toksičnostjo.Even though weakly soluble neutral glucans are commercially available, these preparations are not suitable for intravenous administration because aqueous solutions have a very high viscosity and, more importantly, their use when administered to experimental animals is inevitably accompanied by considerable toxicity.
Zaradi vseh slabosti β-1,3 glukanov pri in vivo administriranju, so že predhodno intenzivno raziskovali nove poti razvoja β-1,3 poliglukoze, ki naj ne bi bila toksična, ne bi inducirala značilne patologije in kljub temu obdržala pomembno imunološko aktivnost.Due to all the disadvantages of β-1,3 glucan in in vivo administration, new pathways for the development of β-1,3 polyglucose, which is not toxic, would not induce characteristic pathology and still retain significant immunological activity, have been extensively investigated previously.
Nefosforilirani glukanovi pripravki nizke molekularne mase, pripravljeni s hidrolizo partikulatnega glukana z mravljinčno kislino, so pokazali antitumorno aktivnost in učinkovitost proti stafilokokom (DiLuzio et al.,lntemat'l J.Cancer 24:773-779). Žal so nizki izkoristki in različnost frakcij, ki se jih dobi po tej metodi, preprečili uporabo teh pripravkov v profilaktične in terapevtske namene. (Glej DiLuzio, 1983, Trends in Pharmacological Sciences 4:344-347).Non-phosphorylated glucan low molecular weight preparations prepared by hydrolysis of particulate glucan with formic acid have shown antitumor activity and efficacy against staphylococci (DiLuzio et al., Intemat'l J.Cancer 24: 773-779). Unfortunately, the low yields and the diversity of fractions obtained by this method prevented the use of these preparations for prophylactic and therapeutic purposes. (See DiLuzio, 1983, Trends in Pharmacological Sciences 4: 344-347).
Poskusi solubilizacije (topljivosti) partikulatnega gkukana z dodatkom dimetilsulfoksida (DMSO), molekulskega relaksanta so bili prav tako neuspešni. Predpostavljali so, da bo DMSO zrahljal konfiguracijo trojne vijačnice glukanove molekule. Partikulatni glukan se sicer zares raztopi v prisotnosti DMSO. Vsi poskusi, da bi izolirali partikulatni glukan iz DMSO raztopine, pa so se izkazali kot neuspešni. Po razredčenju DMSOglukanove raztopine z različnimi vodnimi mediji, kot so glukozne raztopine ali raztopine soli, se je partikulatni glukan preoblikoval. Po razredčenju DMSO-topnih glukanovih raztopin s slanico, so vse živali, ki so dobile injekcije teh raztopin, takoj umrle zaradi visokih koncentracij DMSO ali preureditve partikulatnega glukana. Po izoborjenju partikulatnega glukana z dodatkom etanola (100%) smo oborino zbrali in jo liofilizirali. Tako liofilizirani glukan se je preoblikoval z dodatkom vode.Attempts to solubilize (solubility) the particulate gucana with the addition of dimethylsulfoxide (DMSO), a molecular relaxant, have also been unsuccessful. DMSO was assumed to loosen the configuration of the triple helix of the glucan molecule. Particulate glucan is indeed dissolved in the presence of DMSO. All attempts to isolate the particulate glucan from the DMSO solution, however, have proved unsuccessful. After diluting the DMSOglucan solution with various aqueous media, such as glucose solutions or salt solutions, the particulate glucan was transformed. After dilution of DMSO-soluble glucan solutions with brine, all animals that received injections of these solutions immediately died due to high concentrations of DMSO or rearrangement of particulate glucan. After the formation of particulate glucan with ethanol (100%), the precipitate was collected and lyophilized. The lyophilized glucan was transformed by the addition of water.
Prav tako so bili neuspešni zgodnji poskusi pretvorbe nevtralnega glukanskega preparata partikulatnega glukana v preparat s polarnim nabojem z dodatkom fosfatnih ali sulfatnih skupin, pa tudi z acetiliranjem. Vsi ti postopki so bili izvedeni po solubilizaciji partikulatnega glukana z DMSO, in v vseh primerih se je partikulatni glukan preoblikoval.Early attempts to convert a neutral glucan preparation of particulate glucan into a polar-charged preparation with the addition of phosphate or sulfate groups, as well as acetylation, were also unsuccessful. All of these procedures were performed after solubilization of the particulate glucan with DMSO, and in all cases the particular glucan was transformed.
Nevtralni preparat partikulatnega glukana je bil uspešno pretvorjen v stabilno obliko imenovano topni fosforilirani glukan (tukaj nadalje imenovan glukan fosfat) s hidrolizo s fosforno kislino z uporabo metode na kratko opisane spodaj. Kot je označeno tukaj, se izraza glukan fosfat in topni fosforilirani glukan nanašata na vrsto giukanov solubiliziranih z dodatkom fosfatnih skupin preko reakcije s fosforno kislino. Ti so enaki ali bistveno podobni substancam opisanim v U.S. patentih No. 4,739,064; 4,761,402; 4,818,752 in 4,833,131. Ta topni fosforilirani glukan ni toksičen, ni imunogen, in bistveno ne-pirogen (glej U.S. patente No. 4,739,064; 4,761,402; 4,818,752 in 4,833,131).A neutral particulate glucan preparation was successfully converted to a stable form called soluble phosphorylated glucan (hereinafter referred to as glucan phosphate) by hydrolysis with phosphoric acid using the method briefly described below. As indicated herein, the terms glucan phosphate and soluble phosphorylated glucan refer to a series of giucans solubilized by the addition of phosphate groups via reaction with phosphoric acid. These are identical or substantially similar to the substances described in U.S. Pat. No. 4,739,064; 4,761,402; No. 4,818,752 and 4,833,131. This soluble phosphorylated glucan is non-toxic, non-immunogenic and substantially non-pyrogenic (see U.S. Patent Nos. 4,739,064; 4,761,402; 4,818,752 and 4,833,131).
Po metodi opisani v U.S. patentu No. 4,739,046, je bil pripravljen glukan fosfat kot sledi: partikulatni glukan ali poiiglukozno proteinski kompleks, sta bila suspendirana v močno polarnem aprotičnem topilu: DMSO. Dadali so močan kaotropičen reagent (snov, ki povzroči denaturiranje proteinov) sečnino in zmes segrevali pri 50-150°C med stalnim mešanjem, kateri so počasi dodajali fosforno kislino. Temperaturo se preferenčno drži pri 100°C od 3-12 ur. S tem se zviša izkoristek bioaktivnega produkta. Produkt se izolira in DMSO, sečnino, glukozo in vso nezreagirano fosforno kislino odstrani. Izkoristek po reakciji (6 ur, 100°C) je okoli 70-90%.According to the method described in U.S. Pat. patent no. No. 4,739,046, glucan phosphate was prepared as follows: Particulate glucan or a polyglucose protein complex were suspended in a strongly polar aprotic solvent: DMSO. A strong chaotropic reagent (a substance that causes protein denaturation) was added to the urea and the mixture was heated to 50-150 ° C under constant stirring, to which phosphoric acid was slowly added. The temperature is preferably kept at 100 ° C for 3-12 hours. This increases the yield of the bioactive product. The product is isolated and DMSO, urea, glucose and all unreacted phosphoric acid removed. The yield after reaction (6 hours, 100 ° C) is about 70-90%.
Naslednja nova vrsta topnih giukanov, kjer so poliglukopiranozne verige pridobile skupino z nabojem iz kisline brez fosforja, je opisana v patentni prijavi s serijsko številko 07/649,572. Topni glukani s skupino, ki vsebuje naboj, vključno tako, kot sta sulfate in nitrate, so tudi zmožni pokazati izrazit imunobiološki učinek, kadar so administrirani in vivo. Ti topni glukani imunostimulirajo aktivnost makrofagov z odgovarjajočo aktivacijo imunoaktivnih celic v retikuloendoteličnem in imunskem sistemu. Ti topni glukani tudi dodatno zvišajo hematopoitično (krvotvorno) aktivnost kostnega mozga.Another new type of soluble gyucan, where the polyglucopyranose chains have obtained a phosphor-free acid group, is described in patent application Serial Number 07 / 649,572. Soluble glucans with a charge-containing group, including such as sulfates and nitrates, are also able to show a pronounced immunobiological effect when administered in vivo. These soluble glucans immunostimulate the activity of macrophages through the corresponding activation of immunoactive cells in the reticuloendothelial and immune systems. These soluble glucans also increase the haematopoietic (hematopoietic) activity of the bone marrow.
Skladno z metodami patentne prijave s serijsko številko 07/649,527, so bili topni glukani pripravljeni kot sledi: partikulatni glukan je bil suspendiran v raztopini DMSO in sečnine; koncentrirana zmes segreta na 50-150°C, dodana koncentrirana kislina, (žveplova ali dušikova), in sicer sama, ali v DMSO in reakcijska zmes segrevana med mešanjem pri 50-150°C. Bioaktivni produkt so izolirali in odstranili DMSO, sečnino, glukozo in vso nezreagirano kislino. Po približno 6 urah pri 100°C je bila uporabljena sama kislina, izkoristek podan kot 37.5%, ko pa je bila uporabljena kislina z dodanim DMSO, je bil izkoristek okoli 98%.In accordance with the methods of patent application serial number 07 / 649,527, soluble glucan was prepared as follows: particulate glucan was suspended in DMSO solution and urea; concentrated mixture heated to 50-150 ° C, concentrated acid added (sulfuric or nitric) alone or in DMSO and the reaction mixture heated while stirring at 50-150 ° C. The bioactive product was isolated and the DMSO, urea, glucose and all unreacted acid were removed. After about 6 hours at 100 ° C, the acid itself was used, the yield being given as 37.5%, but when the acid with DMSO was added, the yield was about 98%.
U.S. patent No. 4,707,471 opisuje vodotopno aminirano β-1-3 vezano D-glukanovo zmes in postopek za pripravo take zmesi. Skladno z vključitvijo ene od metod, je bil β 1,3-D-glukan, prednostno sirast ali luskav, hidroliziran v 90% mravljinčni kislini. Kislina je bila odstranjena z uparevanjem, dodana voda in zmes segreta do refluksa za eno uro. Zmes je bila nato ločena na Sephadex G-50 koloni in izločena frakcija z najvišjo molsko maso, ki je bila nadalje procesirana kot sledi. Hidrolizirani glukan je bil raztopljen v vodi, ki je vsebovala brom pri pH 7, kjer je stal v tej raztopini do popolne porabe broma (24-48 ur). Nato je bil pH uravnan do 5.0 in zmes dializirana v vodo in liofilizirana. Oksidirani glukan je bil dodan v raztopino amonijevega, aceta ali 1,6-diaminoheksana v vodi z pH uravnanim na 7.0 z uporabo ocetne kisline skupaj z natrijevim cianoborhidridom in raztopina puščena stati ob mešanju 7 dni. Aminirani glukan je bil dobljen po dializi in liofiliziran. V drugi inačici postopka so bile hidrolizirane luske raztopljene v DMSO pred acetiliranjem in potem aminirane kot opisano zgoraj.U.S. patent no. No. 4,707,471 describes a water-soluble aminated β-1-3 bound D-glucan mixture and a process for preparing such a mixture. According to the inclusion of one of the methods, β 1,3-D-glucan, preferably cheesy or flaky, was hydrolyzed in 90% formic acid. The acid was removed by evaporation, water was added and the mixture heated to reflux for one hour. The mixture was then separated on a Sephadex G-50 column and the fraction with the highest molar mass separated, which was further processed as follows. The hydrolyzed glucan was dissolved in water containing bromine at pH 7 where it stood in this solution until complete consumption of bromine (24-48 hours). The pH was then adjusted to 5.0 and the mixture dialyzed into water and lyophilized. Oxidized glucan was added to a solution of ammonium, acetate or 1,6-diaminohexane in water at pH adjusted to 7.0 using acetic acid together with sodium cyanoborohydride and the solution allowed to stand with stirring for 7 days. Aminated glucan was obtained after dialysis and lyophilized. In another embodiment, the hydrolyzed scales were dissolved in DMSO prior to acetylation and then aminated as described above.
VVO91/03495 Jamasa opisuje topne preparate nevtralnih polimerov glukana s postopkom, ki vsebuje obdelavo glukanovih delcev z edinstveno sekvenco delovanja kisline in baze. Celotne glukanove delce so suspendirali v kisli raztopini, navadno okoli pH=1-5 pri 20-100° C, prednostno z uporabo organskih kislin kot sta ocetna in mravljinčna kislina; odstranili so v kislini netopni glukan in pH uravnali na 7-14. Sirotko so resuspendirali v vroči bazi (NaOH ali KOH) (0.1-1 ON ) pri 4-120° C. Topni glukan so nato pridobili nazaj in ga dodatno očistili.VVO91 / 03495 Jamasa describes soluble preparations of neutral glucan polymers by a process comprising treating glucan particles with a unique sequence of acid and base action. The whole glucan particles were suspended in an acidic solution, usually around pH = 1-5 at 20-100 ° C, preferably using organic acids such as acetic and formic acid; the acid-insoluble glucan was removed and the pH was adjusted to 7-14. The whey was resuspended in hot base (NaOH or KOH) (0.1-1 ON) at 4-120 ° C. The soluble glucan was then recovered and further purified.
U.S. patent No. 3,883,505 opisuje postopek za izbolšanje topnosti slabo topnih ali v vodi netopnih polisaharidov kot so pachyman*, explain*, lentinan itd., z uporabo močnih, vročih vodnih raztopin sečnine, tiosečnine, guanidina in njihovih nižjih N-alkilnih derivatov.U.S. patent no. No. 3,883,505 describes a process for improving the solubility of poorly soluble or water insoluble polysaccharides such as pachyman *, explain *, lentinan, etc., using strong, hot aqueous solutions of urea, thiourea, guanidine and their lower N-alkyl derivatives.
U.S. patenta No. 3,987,166; in 3,943,247 opisujeta zdravljenje živalskih tumorjev in preprečevanje in zdravljenje bakterijskih infekcij. Popolnoma drugače kot fosforilirani glukani pripravljeni po tem postopku ki so neviskozni, so glukani pripravljeni po teh patentih visoko viskozni in težko pripravljivi v vodnih raztopinah višjih koncentracij kot 0.5%.U.S. patent no. 3,987,166; and 3,943,247 describe the treatment of animal tumors and the prevention and treatment of bacterial infections. Completely unlike phosphorylated glucan prepared by this process which is non-viscous, glucan prepared according to these patents are highly viscous and difficult to prepare in aqueous solutions of higher concentrations than 0.5%.
Kljub zgornjim postopkom, so še vedno potrebni visoko efektivni, hitrejši postopki za pridobivanje bioaktivnih topnih glukanov, ki se jih lahko uporablja kot modifikatorje biološkega odziva. Predstavljeni postopek ustreza tej dolgo zamišljeni potrebi.Despite the above procedures, highly effective, faster processes are still required to obtain bioactive soluble glucans that can be used as biological response modifiers. The process presented meets this long-imagined need.
3. POVZETEK PREDLOŽENEGA IZUMA3. SUMMARY OF THE INVENTION
Predloženi izum predstavlja izboljšano visoko učinkovito metodo priprave vodotopnih glukanov. Postopek se izogne uporabi DMSO.The present invention provides an improved highly efficient method for the preparation of water-soluble glucans. The process avoids the use of DMSO.
Postopek tega izuma za pripravo topnih glukanov vsebuje naslednje stopnje:The process of the present invention for the preparation of soluble glucans comprises the following steps:
(a) mešanje nevtralne poliglukoze ali glikoproteina poliglukoze z močnim kaotropičnim reagentom in mletje zmesi do finega prahu;(a) mixing neutral polyglucose or glycoprotein polyglucose with a strong chaotropic reagent and grinding the mixture to a fine powder;
*ni ustreznega slovenskega izraza (b) reagirati zmes finega prahu z močno raztopino koncentrirane fosforne kisline do tvorjenja topnega glukana in do pridobitve topnega glukana iz zmesi.* there is no relevant Slovenian expression (b) react the fine powder mixture with a strong solution of concentrated phosphoric acid to form soluble glucan and to obtain soluble glucan from the mixture.
4. KRATEK OPIS DIAGRAMOV IN SLIK4. BRIEF DESCRIPTION OF THE DIAGRAMS AND FIGURES
Predloženi izum lahko bolje razumemo z naslednjim natančnim opisom , primeri specifičnih vključkov izuma in dodatnimi skicami v katerih:The present invention can be better understood by the following detailed description, examples of specific embodiments of the invention and additional drawings in which:
Slika 1. (A-B) predstavlja analizo prehoda spiralnega zvitka. Dekstran (70kD) (Δ-Δ) služi kot linearna kontrola. Kongo rdeče v natrijevem hidroksidu (o-o) služi kot negativna kontrola. Slika 1A pomeni analizo prehoda spirale za topne fosforilirane glukane pripravljene po postopku predloženega izuma. SLIKA 1B je analiza prehodnega stanja spirale za topni, fosforilirani glukan, pripravljen po znanih postopkih. Slika 2. (A-B) prikazuje 13C -NMR spektre topnih fosforiliranih glukanov. Slika 2A kaže NMR spekter topnega fosforiliranega glukana pripravljenega po že znani metodi. Slika 2B kaže NMR spekter topnega fosforiliranega glukana pripravljenega po postopku opisanem v predloženem izumu.Figure 1. (AB) presents an analysis of the spiral roll transition. Dextran (70kD) (Δ-Δ) serves as a linear control. Congo red in sodium hydroxide (oo) serves as a negative control. Figure 1A is an analysis of the helix transition for soluble phosphorylated glucan prepared according to the process of the present invention. FIG. 1B is an analysis of the transition state of a helix for soluble, phosphorylated glucan prepared by known methods. Figure 2. (AB) shows the 13 C-NMR spectra of soluble phosphorylated glucan. Figure 2A shows the NMR spectrum of soluble phosphorylated glucan prepared by a method known in the art. Figure 2B shows the NMR spectrum of soluble phosphorylated glucan prepared according to the method described in the present invention.
5. NATANČNI OPIS IZUMA5. DETAILED DESCRIPTION OF THE INVENTION
5.1 POSTOPEK ZA PRIPRAVO TOPNIH GLUKANOV5.1. PROCEDURE FOR PREPARATION OF SOLUBLE GLUCANES
Skladno s postopkom predloženega izuma pripravimo vodni topni glukan iz nevtralne poliglukoze ali glikoproteina, ki ga dobimo iz več mikrobioloških izvorov kot sledi: nevtralno poliglukozo ali glikoprotein zmešamo z močnim kaotropičnim reagentom kot je sečnina, in suho zmes močno mešamo in jo končno stremo v fin prah. Primeren je vsak način mletja. Za pripravo majhnih količin se lahko zadovoljimo s terilnico in pestilom. V praksi zmešamo približno 1-4 g nevtralnega poliglukana ali glikoproteina z okoli 10-20 g sečnine. Dodamo koncentrirano fosforno kislino (5-50 ml, konc. 20-43%) da tvorimo blatno zmes in reakcijsko zmes segrejemo na 60-80° C med stalnim mešanjem. Reakcijsko zmes držimo pri 60-80° C, 1-6 ur dokler se ne tvori oborina topnega fosforiliranega glukana. Dodamo 10 ml destilirane vode, da preoblikujemo blato, držimo reakcijsko zmes pri 60-80° C s konstantnim mešanjem; večkrat ponovimo dodajanje vode (prednostno trikrat), da obdržimo blato in nadaljujemo s segrevanjem. Najbolje je držati temperaturo pri 60-80° C 1-2 uri. V praksi, po dveurni reakciji dobimo 97% izkoristek. Med reakcijo se sprošča amonijak iz sečnine, kar je najbolj opazno med 1-2 uro po pričetku segrevanja.In accordance with the process of the present invention, an aqueous soluble glucan is prepared from neutral polyglucose or glycoprotein, which is obtained from several microbial sources as follows: the neutral polyglucose or glycoprotein is mixed with a strong chaotropic reagent such as urea, and the dry mixture is stirred vigorously and finely powdered . Any grinding method is suitable. To prepare small quantities, we can use a mortar and pestle. In practice, about 1-4 g of neutral polyglucan or glycoprotein is mixed with about 10-20 g of urea. Concentrated phosphoric acid (5-50 ml, conc. 20-43%) was added to form a mud mixture and the reaction mixture was heated to 60-80 ° C with constant stirring. The reaction mixture was kept at 60-80 ° C for 1-6 hours until a precipitate of soluble phosphorylated glucan formed. Add 10 ml of distilled water to transform the sludge, keeping the reaction mixture at 60-80 ° C with constant stirring; Repeat the addition of water (preferably three times) to keep the sludge and continue heating. It is best to keep the temperature at 60-80 ° C for 1-2 hours. In practice, after a two-hour reaction, 97% yield is obtained. During the reaction, ammonia is released from urea, which is most noticeable 1-2 hours after the start of heating.
V enem primeru, zmešamo 1-4 g parti-kulatnega glukana z 10-20 g sečnine in 20-25 ml koncentrirane fosforne kisline, da pripravimo blato, ki ga obdelujemo kot zgoraj.In one case, mix 1-4 g of parti-culate glucan with 10-20 g of urea and 20-25 ml of concentrated phosphoric acid to prepare the sludge treated as above.
Topni fosforilirani glukan izoliramo iz reakcijske zmesi kot sledi: reakcijsko zmes odstranimo iz toplotne kopeli in jo raztopimo v velikem volumnu destilirane vode tako da resuspendiramo oborino. Odgovarjajočo raztopino filriramo skozi grob, srednje velik in fin sinterni filter, da odstranimo vse ostanke oborine. Raztopini nato dodamo molekularna sita, ki odstranijo vse komponente manjše od 10.000 molske mase. Ustrezno odstranimo iz raztopine še sečnino, glukozo in nezreagirano fosforno kislino. Upodabljamo lahko vsako znano metodo, ki uporablja molekularna sita. Za primer lahko predstavimo uporabo sit z membransko dializo preko Spectrapor membranskih cevi in dializiramo zmes proti tekoči destilirani vodi. V drugem primeru uporabljamo Millipor dializator/koncentrator z membranskim filtrom velikim 10.000 daltonov molske mase in velikim volumnom dializne tekočine.Soluble phosphorylated glucan is isolated from the reaction mixture as follows: The reaction mixture is removed from the heat bath and dissolved in a large volume of distilled water by resuspending the precipitate. The appropriate solution is filtered through a coarse, medium and fine sinter filter to remove any residue of the precipitate. Molecular sieves are then added to the solution, removing all components smaller than 10,000 moles. Urea, glucose and unreacted phosphoric acid are appropriately removed from the solution. Any known method that uses molecular sieves can be depicted. As an example, the use of membrane dialysis sieves via Spectrapor membrane tubes can be demonstrated and the mixture dialyzed against liquid distilled water. In the second case, we use a Millipor dialyzer / concentrator with a membrane filter of 10,000 daltons of molar mass and a large volume of dialysis fluid.
Postopek opisan v predloženem izumu je hitrejši in učinkovitejši kot predhodno znani postopki za pripravo topnih fosfatov glukana. Potreben čas smo zmanjšali na manj kot 2 uri v primerjavi s 6 urami. Nov postopek ne potrebuje tako intenzivnega gretja kot predhodno znane metode.The process described in the present invention is faster and more efficient than previously known processes for the preparation of soluble phosphate glucan. We reduced the required time to less than 2 hours compared to 6 hours. The new process does not require as intense heating as the previously known method.
Nevtralna poliglukoza, ki jo uporabljamo v predloženem postopku za pripravo topnega fosforiliranega glukana je lahko partikulatni glukan izoliran iz celičnih ovojnic S. cerevisiae po znanih postopkih ( glej DiLuzio et al., 1979, lnt'l J. Cancer 224:773-779: Hassid et al., 1941, J.Amer. Chem. Soc., 63:295-298).The neutral polyglucose used in the present process for the preparation of soluble phosphorylated glucan may be a particulate glucan isolated from S. cerevisiae cell envelopes by known methods (see DiLuzio et al. 1979, lnt'l J. Cancer 224: 773-779: Hassid et al., 1941, J.Amer. Chem. Soc., 63: 295-298.
Dodatno lahko pripravimo topni fosforilirani glukan iz nevtralne poliglukoze ali glikoproteinov pridobljenih iz različnih mikrobioloških izvorov. V Tabeli 1 predstavljamo neizčrpen seznam teh izvorov po U.S. patentu No. 4,739,046, ki je tukaj vključen kot referenca.Additionally, soluble phosphorylated glucan from neutral polyglucose or glycoproteins obtained from various microbial sources can be prepared. In Table 1, we present a non-exhaustive list of these U.S. sources. patent no. No. 4,739,046, which is incorporated herein by reference.
5.2 PRODUKTI IZ TOPNEGA GLUKANA IN NJIHOVA5.2 SOLUBLE GLUCAN PRODUCTS AND THEIR
UPORABAAPPLICATION
Izdelki topnega glukana pripravljeni po ustreznem postopku tega izuma niso toksični, pirogeni in niso imunogeni, če so testirani z interfacialnim obročnim testom. Kot je prikazano v Primeru 7, so topni fosforilirani glukani pripravljeni po teh izboljšanih postopkih snovno enaki po sestavi tem, ki so pripravljeni po predhodno znanih postopkih.The soluble glucan products prepared by the corresponding process of the present invention are non-toxic, pyrogenic and non-immunogenic when tested by interfacial ring test. As shown in Example 7, soluble phosphorylated glucan prepared by these improved processes are substantially identical in composition to those prepared by previously known methods.
Topni glukani pripravljeni po predloženih postopkih kažejo temeljito izražen biološki odziv, kadar jih administriramo in vivo. Bolj natančno, ker so učinkoviti kot biološki modifikatorji biološkega odziva, so produkti pridobljeni po predloženih postopkih uporabni za profilakso in terapijo infekcijskih bolezni povzročenih z vrsto mikroorganizmov, vključno, vendar ne omejujoče na bakterije, viruse, gljive in protozojske parazite. Dodatno lahko topne glukane uporabljamo za preprečevanje in/ali zdravljenje oportunističnih infekcij pri živalih in ljudeh, ki so imunosupresivne kot rezultat predrojstvene ali pridobljene imunodeficience.The soluble glucans prepared by the methods presented show a thoroughly expressed biological response when administered in vivo. More specifically, because they are effective as biological modifiers of biological response, the products obtained by the following methods are useful for the prophylaxis and therapy of infectious diseases caused by a variety of microorganisms, including but not limited to bacteria, viruses, fungi and protozoan parasites. Additionally, soluble glucans can be used to prevent and / or treat opportunistic infections in animals and humans that are immunosuppressive as a result of prenatal or acquired immunodeficiency.
Zaradi nezmožnosti stimuliranja aktivnosti makrofagov in razmnoževanja lahko uporabljamo topne glukane same, ali v kombinacijski terapiji neoplazm.Due to the inability to stimulate macrophage activity and reproduction, soluble glucans can be used alone or in combination therapy of neoplasms.
Načini administriranja so, vendar ne omejeni, na sledeče: oralno, z vbrizgavanjem, vključujoč, vendar ne omejeni na intravenozno, intraperitoralno, podkožno, intramuskularno in lokalno aplikacijo. Topne glukane lahko dodajamo v kombinaciji z vodo, z vodno raztopino ali z katerimkoli biološko sprejemljivim nosilcem.Administration is, but is not limited to, the following: oral, injectable, including but not limited to intravenous, intraperitoneal, subcutaneous, intramuscular and topical application. Soluble glucans can be added in combination with water, with an aqueous solution or with any biologically acceptable carrier.
Za ilustracijo bomo predstavili naslednjo vrsto primerov, vendar ne kot omejujoče za obseg tega izuma.As an illustration, we will present the following series of examples, but not as limiting to the scope of the present invention.
6. PRIPRAVA TOPNEGA FOSFORILIRANEGA6. PREPARATION OF SOLUBLE Phosphorylated
GLUKANAGLUKANA
Partikulatni glukan smo pripravili iz Saccharomyces cerevisiae ustrezno metodi DiLuzia et al., 1979, Infl J. Cancer 24:773-779. 540 g suhega kvasa (Universal Foods Corp., Milvvaukee, Wl) smo suspendirali v 3% raztopini vodnega natrijevega hidroksida v 6 I posodi do končnega volumna 5 I. Suspenzijo smo postavili v vrelo vodno kopel za 4 ure, jo ohladili preko noči in oddekantirali tekočino. Ta postopek smo ponovili trikrat. Preostanek smo nato nakisali s 5,75% klorovodikovo kislino do končnega volumna 5 I in jo postavili v vrelo vodno kopel za 4 ure. Suspenzijo smo nato pustili stati preko noči in tekočino zopet oddekantirali. Preostanek smo dvakrat razredčili z 3% solno kislino do končnega volumna 5 I pri 100°C. Preostanek smo sprali z vrelo vodo in večkrat oddekantirali dokler ostanek ni postal koloiden. Preostaku smo dodali 1 I etanola, zmes skrbno zmešali in jo pustili stati minimalno 24 ur, da se maksimalno ekstrahira. Temno rdečerjavo alkoholno matičnico smo odnučirali od preostanka in zavrgli. Postopek alkoholne ekstrakcije smo ponavljali dokler nismo dobili brezbarvnega supernatanta. Alkohol smo odstranili s štirikratnim spiranjem preostanka z vročo vodo; pripravek partikulatnega glukana smo nato odločili s centrifugiranjem, zamrznili in liofilizirali.Particulate glucan was prepared from Saccharomyces cerevisiae according to the method of DiLuzia et al., 1979, Infl J. Cancer 24: 773-779. 540 g of dry yeast (Universal Foods Corp., Milvvaukee, Wl) was suspended in 3% aqueous sodium hydroxide solution in a 6 I container until a final volume of 5 I. The suspension was placed in a boiling water bath for 4 hours, cooled overnight and decanted. fluid. We repeated this process three times. The residue was then acidified with 5.75% hydrochloric acid to a final volume of 5 I and placed in a boiling water bath for 4 hours. The suspension was then allowed to stand overnight and the liquid was decanted again. The residue was diluted twice with 3% hydrochloric acid to a final volume of 5 I at 100 ° C. The residue was washed with boiling water and decanted several times until the residue became colloidal. 1 I of ethanol was added to the residue, the mixture was carefully mixed and allowed to stand for a minimum of 24 hours to maximize extraction. The dark red liquor mother liquor was discarded from the rest and discarded. The alcohol extraction process was repeated until a colorless supernatant was obtained. The alcohol was removed by washing the residue four times with hot water; the particulate glucan preparation was then decided by centrifugation, frozen and lyophilized.
Topne fosforilirane glukane smo pripravili v smislu predloženega izuma s solubilizacijo in fosforiliranjem partikulatnega glukana kot sledi:Soluble phosphorylated glucan was prepared according to the present invention by solubilizing and phosphorylating the particulate glucan as follows:
g partikulatnega glukana smo zmešali z 18 g sečnine, zmes zmleli s pestilom v terilnici in tako pripravili fino zdrobljeno praškasto zmes. Počasi smo dodali 25 ml fosforne kisline (43%) in pretvorili prašnato zmes v blato. Zmes smo nato segreli na 60-80° C in jo držali pri tej temperaturi nadalnji 1-2 uri s konstantnim mešanjem. Tvorila se je oborina, ki je postala vidna čez kake 1-1,5 ure in je kasneje narasla. Dodali smo približno 10 ml destilirane vode (Milli-Q- voda ) in nadaljevali s segrevanjem in mešanjem. Dodajanje destilirane vode (10 ml) smo trikrat ponovili in še nadaljevali s segrevanjem (1-2 uri). Zmes smo nato prenehali segrevati, jo ohladili in razredčili s približno 1 I destilirane vode, da smo resuspendirali oborino. S serijo filtrov smo zmes prefiltrirali in s tem odstranili preostali precipitat.g of particulate glucan was mixed with 18 g of urea, the mixture was ground with a pestle in a mortar and thus a finely ground powder mixture was prepared. Slowly added 25 ml of phosphoric acid (43%) and converted the powder mixture to sludge. The mixture was then heated to 60-80 ° C and held at this temperature for a further 1-2 hours with constant stirring. A precipitate formed, which became visible within 1-1.5 hours and subsequently increased. About 10 ml of distilled water (Milli-Q- water) was added and heating and stirring was continued. The addition of distilled water (10 ml) was repeated three times and continued to warm (1-2 hours). The mixture was then stopped warming, cooled and diluted with about 1 I of distilled water to resuspend the precipitate. A series of filters filtered the mixture to remove residual precipitate.
Odgovarjajočo raztopino, ki je vsebovala fosforilirani glukan smo nato obdelali z molekularnimi siti in s tem odstranili frakcije z nizko molekulsko maso, vključno sečnino in glukozo. V tej vrsti eksperimentov smo zmes filtrirali skozi grob (1-3 pm), srednji 0,8 pm, 0,65 pm in fini (0,45 pm) sinterni Millipore filter, da bi odstranili oborino. Raztopino smo nato - ponovno obdelali z molekularnimi siti preko dializator/koncentratorja (Millipore Corp. Bedford ,MA) z membranskim filtrom (10.000 MW ). Nato smo z dializo proti 24-100 I destilirane vode (Milli-Q voda) odstranili spojine z niško molekulsko maso.The corresponding solution containing phosphorylated glucan was then treated with molecular sieves to remove low molecular weight fractions, including urea and glucose. In this type of experiment, the mixture was filtered through a coarse (1-3 pm), a mean 0.8 pm, 0.65 pm and a fine (0.45 pm) sinter Millipore filter to remove the precipitate. The solution was then - re-treated with molecular sieves via a dialyser / concentrator (Millipore Corp. Bedford, MA) with a membrane filter (10,000 MW). Then, dialysis against 24-100 I distilled water (Milli-Q water) eliminated niche molecular weight compounds.
Po obdelanju raztopine, ki je vsebovala topni fosforilirani glukan z molekularnimi siti, smo raztopino koncentrirali in liofilizirali. Izkoristek je bil 97%.After treatment of the solution containing soluble phosphorylated glucan with molecular sieves, the solution was concentrated and lyophilized. The yield was 97%.
7. KARAKTERIZACIJA TOPNIH FOSFORILIRANIH7. CHARACTERIZATION OF SOLUBLE PHOSPHORILATED
GLUKANOV PRIPRAVLJENIH PO PREDLOŽENEMGLUCANS PREPARED AFTER PROPOSED
IZUMU.IZUMU.
Izvršili smo serijo poskusov za primerjavo topnih fosforiliranih glukanov (označenih kot glukan fosfat-no DMSO), pripravljenih s postopkom tega izuma opisanim v odseku 5 zgoraj, s topnim fosforiliranim glukanom (označenim kot glukan fosfat), ki je bil pripravljen po predhodnem znanem postopku, po metodi iz U.S. patenta No. 4.739.046.A series of experiments were performed to compare soluble phosphorylated glucan (labeled glucan phosphate DMSO) prepared by the process of the present invention described in section 5 above with soluble phosphorylated glucan (labeled glucan phosphate) prepared by a prior known method, by the method of US patent no. 4.739.046.
7.1 ELEMENTNA SESTAVA7.1 ELEMENTAL COMPOSITION
Elementna sestava glukan fosfat-no DMSO je bila določena v Galbright Laboratoires v Knoxvillu,TN. Primerjava elementne sestave s sestavo glukan fosfata je podana v Tabeli 1.The elemental composition of glucan phosphate-no DMSO was determined at Galbright Laboratoires in Knoxville, TN. Comparison of elemental composition with glucan phosphate composition is given in Table 1.
Tabela 1Table 1
Kemična sestava topnih glukanovChemical composition of soluble glucans
Elementna sestava glukan fosfata pripravljenega po postopku predloženem v tem izumu ga označuje kot bistveno identičnega v primerjavi z znanim gukan fosfatom. Iz Tabele 1. je razvidno, da je edina vidna razlika med produktoma v stopnji fosforiliranja. Glukan fosfat-no DMSO poseduje eno fosfatno substitucijo na vsake 3 glukozne enote, medtem ko pri glukan fosfat-u zasledimo eno fosfatno substitucijo na 7 glukoznih enot.The elemental composition of glucan phosphate prepared according to the process of the present invention characterizes it as substantially identical to that of known gucan phosphate. Table 1 shows that the only visible difference between the products is the degree of phosphorylation. Glucan phosphate DMSO has one phosphate substitution for every 3 glucose units, whereas for glucan phosphate one phosphate substitution per 7 glucose units is observed.
7. 2 RAZPOREDITEV MOLEKULSKIH MAS7. 2 MOLECULAR WEIGHT DISTRIBUTION
Razporeditev molekulskih mas (polimerov) obeh glukanov je bila določena z vodno gelsko permeabilno kromatografijo (GPC). Osnovni GPC sistem sestoji iz Waters 600E odtočnega sistema topil, U6K ročnega injektorja in komore za ogrevanje kolon (Waters Chromatography Division, Millipore Corp., Milford, MA). Mobilna faza je 0,05 M natrijev nitrit, spravljen v sterilnem rezervarju (Kontess, Vineland, NJ). Pred uporabo pa se skrbno odstrani zrak s prepihavanjem s helijem. Pretok mobilne faze je bil 0,5 ml/min. Tri Ultrahydrogel vodne GPC kolone (Waters Chromatography Division, Milford,Ma) z ekskluzijskimi limitami 2 χ 106 D, 5 χ 105 D in 1,2 x 105 D so povezali v vrsti z dodatno Ultrahydrogel zaščitno kolono. Kolone so držali pri 30°C. Hitrost pretoka, temperatura kolon in delovanje črpalke so kontrolirali z Maxima 820 GPC softverom (Dynamic Solutions, Ventura CA).The molecular weight (polymers) distribution of both glucan was determined by aqueous gel permeable chromatography (GPC). The basic GPC system consists of a Waters 600E solvent drain system, a U6K manual injector and a column heating chamber (Waters Chromatography Division, Millipore Corp., Milford, MA). The mobile phase is 0.05 M sodium nitrite stored in a sterile tank (Kontess, Vineland, NJ). Carefully remove the helium blowing air before use. The mobile phase flow was 0.5 ml / min. Three Ultrahydrogel aqueous GPC columns (Waters Chromatography Division, Milford, Ma) with exclusion limits of 2 χ 10 6 D, 5 χ 10 5 D and 1.2 x 10 5 D were connected in series with an additional Ultrahydrogel protection column. The columns were kept at 30 ° C. Flow rate, column temperature and pump performance were controlled with Maxima 820 GPC software (Dynamic Solutions, Ventura CA).
Sistem so kalibrirali z ozkotrakovnimi polulanskimi in dekstranskimi standardi. Za analizo so glukane raztopili v mobilni fazi pri koncentraciji 2-3 mg/ml ob rahlem stresanju, dokler ni potekla popolna hidratacija (2-3 ure). Pri vseh analizah so uporabljali 200 ml injekcijske volumne.The system was calibrated using narrow-band half-wave and dextran standards. For analysis, glucans were dissolved in the mobile phase at a concentration of 2-3 mg / ml with gentle shaking until complete hydration (2-3 hours) had elapsed. 200 ml injection volumes were used in all analyzes.
Absolutne molekulske mase glukanov so določili z on-line večkotno lasersko svetlobno razprševalno fotometrijo (MALLS), z uporabo Dawn-F MALLS fotometra opremljenega s K5 pretočno celico (Wyatt Technology Corp, Santa Barbara CA). Absolutna porazdelitev molekulskih mas (MW), momenti MW (številčno poprečje MW, Z-poprečje MW, utežno poprečje MW ), vrh MW, polidisperznost in koren sredjega kvadratnega radija momenta so bili določeni z ASTRA softverom (v.2,0). Predpostavljen je bil diferencialni indeks lomnega količnika (dn/dc), vrednosti 0,146 cm3/g. Objavljene molekulske mase pollulanovih in dekstranovih standardov, ki so jih uporabili za preverjanje kalibracij kolon, so pokazale dobro ujemanje z MALLS rezultati.Absolute molecular weights of glucan were determined by on-line multilayer laser light scattering photometry (MALLS), using a Dawn-F MALLS photometer equipped with a K5 flow cell (Wyatt Technology Corp, Santa Barbara CA). The absolute distribution of molecular masses (MW), MW moments (numerical average MW, Z-average MW, weighted average MW), MW peak, polydispersity, and root mean square radius of the torque were determined by ASTRA software (v.2,0). A differential refractive index (dn / dc) of 0.146 cm3 / g was assumed. The published molecular weights of the pollulan and dextran standards used to verify column calibrations showed good agreement with the MALLS results.
Dinamična viskoznost ([η]) polimernih glukanov je bila določena z on-line diferenčno viskozimetrijo (d.v.). Pri določevanju kolonskega eluenta so uporabili pretok le-tega skozi Viscotek Model 200, diferencialnega refraktometra/viskometra in analizirali podatke z Unical softverom (Viscotek, Porter, TX). Določevanje molekulskih mas standardov z uporabo omenjenih tehnik je pokazalo dobro ujemanje z MALLS rezultati. Dinamična viskoznost polulanskih standardov se je tesno ujemala s prejšnimi podatki. Poprečne molske mase, polidisperznost in dinamična viskoznost glukan fosfata brez DMSO je podana v Tabeli 2. Za primerjavo so predstavljeni analogni podatki za glukan fosfat pripravljen po postopku iz patenta U.S. No. 4,739,046.The dynamic viscosity ([η]) of polymer glucan was determined by on-line differential viscometry (d.v.). For the determination of the column eluent, it was used to flow through the Viscotek Model 200, differential refractometer / viscometer, and analyze the data with Unical software (Viscotek, Porter, TX). Molecular weight determination of standards using the above techniques showed good agreement with MALLS results. The dynamic viscosity of the Polulan standards closely matched the previous data. The transverse molar masses, polydispersity and dynamic viscosity of glucan phosphate without DMSO are given in Table 2. For comparison, analogous data for glucan phosphate prepared according to the process of U.S. Pat. No. No. 4,739,046.
Tabela 2Table 2
Karakteristike molekulskih masMolecular mass characteristics
a Mn predstavlja: številčno poprečno MW a M n represents: numerically transverse MW
Mw predstavlja: utežno poprečno MW Mz predstavlja: z-poprečno MWM w represents: mostly transverse MW M z represents: z-transverse MW
Mw RMS radij: utežno-poprečni kvadratni koren radija (nm)M w RMS radius: weight transverse square root of the radius (nm)
Vse MW so izražene v g/mol. η predstavlja dinamično viskoznost b RMS radij ni bilo mogoče določiti za ta del vzorca c Zaradi nizke koncentracije polimerov na vrhu 1 ni bilo mogoče določiti dinamične viskoznosti na tem delu vzorca.All MW are expressed in g / mol. η represents the dynamic viscosity b The RMS radius could not be determined for this part of the sample c Due to the low concentration of polymers at the top 1, it was not possible to determine the dynamic viscosity on this part of the sample.
Tabeli 2 se vidi, da so karakteristike molekulskih mas glukan fosfat-no DMSO in glukan fosfata pripravljenega po predhodni metodologiji izredno podobne, pravzaprav v osnovi identične. Pri uporabi navedenih tehnik sta bila opažena dva vrhova pri obeh pripravljenih fosfatnih glukanih. Večina polimerov v obeh preparatih se nahaja v vrhu 2; v obeh preparatih vsebuje vrh 1 < 2% vseh polimerov. V Tabeli 2. je jasno prikazano, da so Mn, značilen za delež polimerov z nizkimi MW, in Mw, znčilen za delež poprečne MW in Ml, delež značilen za delež polimerov z visoko MW, v osnovi identični pri vrhu 2, v obeh pripravljenih glukan fosfatih. Polidisperznostni indeks dodatno potrjuje identičnost, saj kaže na identično homogenost polimerov. Iz Tabele 2. se končno tudi vidi, da je dinamična viskoznost obeh sestavin dejansko identična.Table 2 shows that the molecular weight characteristics of glucan phosphate DMSO and glucan phosphate prepared according to the previous methodology are extremely similar, in fact essentially identical. Using these techniques, two peaks were observed for both prepared phosphate glucan. Most of the polymers in both preparations are in the top 2; in both preparations the peak contains 1 <2% of all polymers. Table 2 clearly shows that M n characteristic of the fraction of polymers with low MW and M w characteristic of the fraction of transverse MW and Ml, the fraction characteristic of the proportion of polymers with high MW, are essentially identical at the top 2, in of both prepared glucan phosphates. The polydispersity index further confirms the identity, as it indicates the identical homogeneity of the polymers. Table 2 also finally shows that the dynamic viscosity of the two components is actually identical.
7.3 ANALIZA KONFORMACIJSKE STRUKTURE7.3 CONFORMITY STRUCTURE ANALYSIS
Konformacijka struktura je bila ocenjena z uporabo tehnike Ogawe in sodelavcev (Ogawa in Hatana, 1978, Carbohydrate Res. 6Z:527535; Ogawa in Tsurugi, 1973, Carbohydrate Res. 29:397-403). Ta tehnika določa absorpcijske maksimume polimernih raztopin kompleksiranih s Kongo rdečim v prisotnosti različnih koncentracij hidroksilnega iona. Prisotnost spojin s trojno vijačnico, bi bila na primer označena s premikom v absorpcijskem maksimumu raztopine pri povečanju koncentracije natrijevega hidroksida. Vodikove vezi se prekinejo z relaksacijo polimerne spirale, kar ima za posledico kompleksiranje Kongo rdečega z ogljikovim hidratom.The conformational structure was evaluated using the technique of Ogawe et al. (Ogawa and Hatana, 1978; Carbohydrate Res. 6Z: 527535; Ogawa and Tsurugi, 1973; Carbohydrate Res. 29: 397-403). This technique determines the absorption maxima of polymer solutions complexed with Congo red in the presence of different concentrations of hydroxyl ion. For example, the presence of compounds with a triple helix would be indicated by a shift in the absorption maximum of the solution with increasing sodium hydroxide concentration. Hydrogen bonds are broken by polymer helix relaxation, resulting in the complexation of Congo red with carbohydrate.
Že prej so pokazali, da je urejena (vijačna) konformacija bistvena pri ogljikovih hidratih kot je glukan, za tvorbo kompleksa z barvilom Kongo rdeče. Vodne raztopine Kongo rdečega (44 pm) so pripravili pri različnih koncentracijah NaOH (1 mM do 1000 mM). Rezultati so predstavljeni v sliki 1. (A in B) in sicer za glukan fosfat-no DMSO in glukan fosfat.Earlier, they showed that an orderly (helical) conformation is essential for carbohydrates such as glucan to form a complex with Congo dye red. Aqueous solutions of Congo red (44 pm) were prepared at various NaOH concentrations (1 mM to 1000 mM). The results are presented in Figure 1. (A and B) for glucan phosphate DMSO and glucan phosphate, respectively.
Kakor se vidi iz slike 1. (A in B), kaže analiza konformacijske strukture obeh giukanov, glukan fosfata in glukan fosfata-no DMSO urejeno konformacijo trojne vijačnice. V obeh primerih je bil opažen premik absobcijskega maksimuma pri spremembi koncentracije natrijevega hidroksida od 0,1 - okoli 0,4 M.As can be seen from Figure 1. (A and B), the conformational structure analysis of the two giucans, glucan phosphate and glucan phosphate DMSO, show an ordered conformation of the triple helix. In both cases, a shift of the absorption maximum was observed when the sodium hydroxide concentration changed from 0.1 to about 0.4 M.
7.4 NUKLEARNA MAGNETNA RESONANČNA7.4 NUCLEAR MAGNETIC RESONANCE
SPEKTROSKOPIJASPECTROSCOPY
13c jedrska magnetna resonančna spektroskopija (13CNMR) na Bruker 260 MHz NMR spektrometru (Bruker Instruments, INc., Billerica, MA) je bila uporabljena za določanje položaja medverižnih povezav v glukan fosfatu in glukan fosfatu-no DMSO. Topna glukan fosfat ali glukan fosfat-no DMSO sta bila raztopljena v D2O (50 mg/ml) in meritve izvedene pri sledečih pogojih:13c Nuclear Magnetic Resonance Spectroscopy ( 13 CNMR) on a Bruker 260 MHz NMR spectrometer (Bruker Instruments, INc., Billerica, MA) was used to determine the position of the interconnections in glucan phosphate and glucan phosphate DMSO. Soluble glucan phosphate or glucan phosphate DMSO was dissolved in D 2 O (50 mg / ml) and measurements were made under the following conditions:
MOČ POLJA: 50 MHzFIELD POWER: 50 MHz
RELAKSACIJSKI ZAMIK: 1 sekunda,RELAXATION LOCK: 1 second,
VELIKOST PULZA: 15° - 20°,PULSE SIZE: 15 ° - 20 °,
ŠTEVILO PONOVITEV: Glukan fosfat 694 pon.REPEAT NUMBER: Glucan Phosphate 694 Mon.
Glukan fosfat-no DMSO, 14.900 pon.Glucan Phosphate DMSO, 14,900 Mon.
Rezultati so prikazani na Sliki 2. (A-B) in v Tabeli 3.The results are shown in Figure 2. (A-B) and Table 3.
Tabela 3Table 3
13C -NMR kemijski premiki glukanov*13C-NMR chemical shifts of glucans *
* Kemijski premiki v ppm* Chemical shifts in ppm
Vrhova C4^ in C513 opažena pri glukan fosfat-no DMSO, nista opazna pri glukan fosfat-u, kar je edina razlika med glukan fosfat-om in glukan fosfat-no DMSO. Jasno je, da spojine kažejo 13C-NMR spektre, ki so značilni za β-1,3-spojitev (Colson, Carbohydrate Res., 71/265, 1979).Peaks C4 ^ and C5 13 observed with glucan phosphate DMSO are not observed with glucan phosphate, which is the only difference between glucan phosphate and glucan phosphate DMSO. It is clear that the compounds exhibit 13 C-NMR spectra that are characteristic of the β-1,3-coupling (Colson, Carbohydrate Res., 71/265, 1979).
Zahtevani in tukaj pojasneni izum ni omejen v širini s specifičnimi opisanimi predstavitvami, ker so te predstavitve namenjene ilustraciji več aspektov izuma. Vse ekvivalentne predstavitve so omejene znotraj obsega tega izuma. Različne modifikacije tega izuma bodo zares evidentne, dodatno k že opisanim tukaj, za vse strokovnjake. Te modifikacije naj bi tudi sovpadle znotraj obsega dodatnih zahtevkov. Citirane so številne reference, katerih vsebina je vključena celovito.The invention claimed and explained herein is not limited in scope by the specific presentations described, since these representations are intended to illustrate several aspects of the invention. All equivalent representations are limited within the scope of the present invention. Various modifications of the present invention will be truly evident, in addition to those already described herein, for all experts. These modifications should also coincide with the scope of the additional claims. A number of references are cited, the content of which is comprehensively included.
Claims (7)
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| CN (1) | CN1091747A (en) |
| AU (1) | AU4837693A (en) |
| CA (1) | CA2141845A1 (en) |
| MX (1) | MX9304728A (en) |
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| US5622939A (en) * | 1992-08-21 | 1997-04-22 | Alpha-Beta Technology, Inc. | Glucan preparation |
| US5488040A (en) * | 1989-09-08 | 1996-01-30 | Alpha-Beta Technology, Inc. | Use of neutral soluble glucan preparations to stimulate platelet production |
| CA2066172A1 (en) * | 1989-09-08 | 1991-03-09 | Alpha Beta Technology, Inc. | Method for producing soluble glucans |
| US5811542A (en) * | 1989-09-08 | 1998-09-22 | Alpha-Beta Technology, Inc. | Method for producing soluble glucans |
| US5786343A (en) * | 1997-03-05 | 1998-07-28 | Immudyne, Inc. | Phagocytosis activator compositions and their use |
| US6046323A (en) * | 1997-07-29 | 2000-04-04 | The Collaborative Group, Ltd. | Conformations of PPG-glucan |
| US6369216B1 (en) | 1998-09-25 | 2002-04-09 | Biopolymer Engineering Pharmaceutical, Inc. | Very high molecular weight β-glucans |
| AU6261999A (en) | 1998-09-25 | 2000-04-17 | Collaborative Group, Ltd., The | Very high molecular weight beta-glucans |
| US7507724B2 (en) * | 2001-01-16 | 2009-03-24 | Sloan-Kettering Institute For Cancer Research | Therapy-enhancing glucan |
| CN117016797B (en) * | 2023-08-14 | 2024-06-04 | 四川合泰新光生物科技有限公司 | Method for improving dissolution rate of glucan |
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| US3883505A (en) * | 1972-07-17 | 1975-05-13 | Ajinomoto Kk | Method of solubilizing polysaccharides |
| US4739046A (en) * | 1985-08-19 | 1988-04-19 | Luzio Nicholas R Di | Soluble phosphorylated glucan |
| US4946450A (en) * | 1989-04-18 | 1990-08-07 | Biosource Genetics Corporation | Glucan/collagen therapeutic eye shields |
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