CA2198085A1 - Inactivation of pathogens using hydroxymethylamines - Google Patents
Inactivation of pathogens using hydroxymethylaminesInfo
- Publication number
- CA2198085A1 CA2198085A1 CA002198085A CA2198085A CA2198085A1 CA 2198085 A1 CA2198085 A1 CA 2198085A1 CA 002198085 A CA002198085 A CA 002198085A CA 2198085 A CA2198085 A CA 2198085A CA 2198085 A1 CA2198085 A1 CA 2198085A1
- Authority
- CA
- Canada
- Prior art keywords
- acid
- biological fluid
- blood
- hydroxymethyl
- hydroxymethylamine
- 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.)
- Abandoned
Links
- XMYQHJDBLRZMLW-UHFFFAOYSA-N methanolamine Chemical class NCO XMYQHJDBLRZMLW-UHFFFAOYSA-N 0.000 title claims abstract description 23
- 230000002779 inactivation Effects 0.000 title description 24
- 244000052769 pathogen Species 0.000 title description 13
- 239000008280 blood Substances 0.000 claims abstract description 68
- 210000004369 blood Anatomy 0.000 claims abstract description 67
- 238000000034 method Methods 0.000 claims abstract description 60
- 241000700605 Viruses Species 0.000 claims abstract description 54
- 230000000415 inactivating effect Effects 0.000 claims abstract description 17
- 150000003839 salts Chemical class 0.000 claims abstract description 10
- 210000002381 plasma Anatomy 0.000 claims description 51
- WBTIFBJEYFLFFW-UHFFFAOYSA-N 2-(hydroxymethylazaniumyl)acetate Chemical compound OCNCC(O)=O WBTIFBJEYFLFFW-UHFFFAOYSA-N 0.000 claims description 36
- 239000013060 biological fluid Substances 0.000 claims description 36
- -1 hydroxymethyl- Chemical group 0.000 claims description 30
- 239000002253 acid Substances 0.000 claims description 28
- ZRPDXDBGEYHEBJ-UHFFFAOYSA-N alpha-(hydroxymethyl)serine Chemical compound OCC(N)(CO)C(O)=O ZRPDXDBGEYHEBJ-UHFFFAOYSA-N 0.000 claims description 12
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 claims description 12
- 210000003743 erythrocyte Anatomy 0.000 claims description 11
- 241000894006 Bacteria Species 0.000 claims description 10
- 239000004471 Glycine Substances 0.000 claims description 10
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 9
- 125000000217 alkyl group Chemical group 0.000 claims description 9
- 125000003368 amide group Chemical group 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 8
- 244000005700 microbiome Species 0.000 claims description 8
- ZIDILUZYDYKOJK-VKHMYHEASA-N (2r)-2-(hydroxymethylamino)-3-sulfanylpropanoic acid Chemical compound OCN[C@@H](CS)C(O)=O ZIDILUZYDYKOJK-VKHMYHEASA-N 0.000 claims description 7
- JPCXPEOMNMVSNA-VKHMYHEASA-N (2s)-2-(hydroxymethylamino)butanedioic acid Chemical compound OCN[C@H](C(O)=O)CC(O)=O JPCXPEOMNMVSNA-VKHMYHEASA-N 0.000 claims description 6
- LINDVRSYQDJAIC-BYPYZUCNSA-N (2s)-2-(hydroxymethylamino)pentanedioic acid Chemical compound OCN[C@H](C(O)=O)CCC(O)=O LINDVRSYQDJAIC-BYPYZUCNSA-N 0.000 claims description 6
- 125000003118 aryl group Chemical group 0.000 claims description 6
- 125000000547 substituted alkyl group Chemical group 0.000 claims description 6
- 125000003107 substituted aryl group Chemical group 0.000 claims description 6
- OVTOIBSHJOZANW-YFKPBYRVSA-N (2s)-5-amino-2-(hydroxymethylamino)pentanoic acid Chemical compound NCCC[C@@H](C(O)=O)NCO OVTOIBSHJOZANW-YFKPBYRVSA-N 0.000 claims description 5
- UEAZPMMELVEHMT-UHFFFAOYSA-N 1-(hydroxymethylamino)ethanol Chemical compound CC(O)NCO UEAZPMMELVEHMT-UHFFFAOYSA-N 0.000 claims description 5
- LTGSEYCAANRRBN-UHFFFAOYSA-N 2-[2-hydroxyethyl(hydroxymethyl)amino]ethanol Chemical compound OCCN(CO)CCO LTGSEYCAANRRBN-UHFFFAOYSA-N 0.000 claims description 5
- 150000001408 amides Chemical class 0.000 claims description 5
- 239000001257 hydrogen Substances 0.000 claims description 5
- 229910052739 hydrogen Inorganic materials 0.000 claims description 5
- HRTUATFMHZMKRU-LURJTMIESA-N (2s)-6-amino-2-(hydroxymethylamino)hexanoic acid Chemical compound NCCCC[C@@H](C(O)=O)NCO HRTUATFMHZMKRU-LURJTMIESA-N 0.000 claims description 4
- XBVIINKYUWVQBG-UHFFFAOYSA-N 2-(hydroxymethylamino)ethylphosphonic acid Chemical compound OCNCCP(O)(O)=O XBVIINKYUWVQBG-UHFFFAOYSA-N 0.000 claims description 4
- WFHMRVKTBJDPRK-UHFFFAOYSA-N 2-(hydroxymethylamino)pent-4-ynoic acid Chemical compound OCNC(CC#C)C(=O)O WFHMRVKTBJDPRK-UHFFFAOYSA-N 0.000 claims description 4
- 102000004506 Blood Proteins Human genes 0.000 claims description 4
- 108010017384 Blood Proteins Proteins 0.000 claims description 4
- 239000012503 blood component Substances 0.000 claims description 4
- 150000007942 carboxylates Chemical class 0.000 claims description 4
- 239000012141 concentrate Substances 0.000 claims description 4
- 239000004023 fresh frozen plasma Substances 0.000 claims description 4
- 125000000524 functional group Chemical group 0.000 claims description 4
- LEBOTKYUHRKYRQ-HQVZTVAUSA-N (2S)-2-[[4-[(2-amino-4-oxo-3H-pteridin-6-yl)methylamino]benzoyl]amino]-4-(hydroxymethyl)pentanedioic acid Chemical compound OCC(C(=O)O)C[C@@H](C(=O)O)NC(=O)C1=CC=C(NCC2=CN=C3N=C(N)NC(=O)C3=N2)C=C1 LEBOTKYUHRKYRQ-HQVZTVAUSA-N 0.000 claims description 3
- NPLNOZOSLYIREY-YFKPBYRVSA-N (2s)-2-(hydroxymethylamino)-3-methylbutanoic acid Chemical compound CC(C)[C@@H](C(O)=O)NCO NPLNOZOSLYIREY-YFKPBYRVSA-N 0.000 claims description 3
- ZYRQBDPRQPIESZ-UHFFFAOYSA-N 2-(hydroxymethylamino)-2-phenylacetic acid Chemical compound OCNC(C(O)=O)C1=CC=CC=C1 ZYRQBDPRQPIESZ-UHFFFAOYSA-N 0.000 claims description 3
- TWKQLDDTDYYOLU-UHFFFAOYSA-N 2-(hydroxymethylamino)acetamide Chemical compound NC(=O)CNCO TWKQLDDTDYYOLU-UHFFFAOYSA-N 0.000 claims description 3
- HPHASGOJNFCVQR-UHFFFAOYSA-N 2-(hydroxymethylamino)ethyl hydrogen sulfate Chemical compound OCNCCOS(O)(=O)=O HPHASGOJNFCVQR-UHFFFAOYSA-N 0.000 claims description 3
- PXIKUUUEECATBX-UHFFFAOYSA-N 2-aminoethylsulfanylmethanol Chemical compound NCCSCO PXIKUUUEECATBX-UHFFFAOYSA-N 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- NWNPLGLJNZEMTE-MDTVQASCSA-L calcium;(2s)-2-(hydroxymethylamino)-4-methylsulfanylbutanoate Chemical compound [Ca+2].CSCC[C@@H](C([O-])=O)NCO.CSCC[C@@H](C([O-])=O)NCO NWNPLGLJNZEMTE-MDTVQASCSA-L 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 210000004623 platelet-rich plasma Anatomy 0.000 claims description 3
- 230000009467 reduction Effects 0.000 claims description 3
- 239000003634 thrombocyte concentrate Substances 0.000 claims description 3
- FDKHGAJEBIMBKB-UHFFFAOYSA-N 2-(hydroxymethylamino)hexanedioic acid Chemical compound OCNC(C(O)=O)CCCC(O)=O FDKHGAJEBIMBKB-UHFFFAOYSA-N 0.000 claims description 2
- AFQNYHNKUBIJBR-UHFFFAOYSA-N 2-[(4-aminobenzoyl)amino]-3-hydroxypropanoic acid Chemical compound NC1=CC=C(C(=O)NC(CO)C(O)=O)C=C1 AFQNYHNKUBIJBR-UHFFFAOYSA-N 0.000 claims description 2
- JUWJBACXGXJQIQ-UHFFFAOYSA-N 2-aminobutane-1,2-diol Chemical compound CCC(N)(O)CO JUWJBACXGXJQIQ-UHFFFAOYSA-N 0.000 claims description 2
- CNCOEDDPFOAUMB-UHFFFAOYSA-N N-Methylolacrylamide Chemical compound OCNC(=O)C=C CNCOEDDPFOAUMB-UHFFFAOYSA-N 0.000 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 claims description 2
- 125000000623 heterocyclic group Chemical group 0.000 claims description 2
- JRFKIOFLCXKVOT-UHFFFAOYSA-N hydroxymethylnicotinamide Chemical compound OCNC(=O)C1=CC=CN=C1 JRFKIOFLCXKVOT-UHFFFAOYSA-N 0.000 claims description 2
- 229950005422 hydroxymethylnicotinamide Drugs 0.000 claims description 2
- 210000002751 lymph Anatomy 0.000 claims description 2
- 229960001639 penicillamine Drugs 0.000 claims description 2
- UEZVMMHDMIWARA-UHFFFAOYSA-M phosphonate Chemical compound [O-]P(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-M 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 210000003296 saliva Anatomy 0.000 claims description 2
- 210000000582 semen Anatomy 0.000 claims description 2
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 claims description 2
- JYAGTWWDLKTRNC-YFKPBYRVSA-N (2s)-1-(hydroxymethyl)pyrrolidine-2-carboxylic acid Chemical compound OCN1CCC[C@H]1C(O)=O JYAGTWWDLKTRNC-YFKPBYRVSA-N 0.000 claims 1
- VZQNWPMWABFUOZ-VIFPVBQESA-N (2s)-2-(hydroxymethylamino)-3-phenylpropanoic acid Chemical compound OCN[C@H](C(O)=O)CC1=CC=CC=C1 VZQNWPMWABFUOZ-VIFPVBQESA-N 0.000 claims 1
- PTBXRYBPQJHMQS-VKHMYHEASA-N (2s)-2-(hydroxymethylamino)propanoic acid Chemical compound OC(=O)[C@H](C)NCO PTBXRYBPQJHMQS-VKHMYHEASA-N 0.000 claims 1
- DZNJSBHVDXLTIK-UHFFFAOYSA-N 1-(hydroxymethyl)imidazolidine-2,4-dione Chemical compound OCN1CC(=O)NC1=O DZNJSBHVDXLTIK-UHFFFAOYSA-N 0.000 claims 1
- AUBJMCRWFVUBJK-UHFFFAOYSA-N 2-[hydroxymethyl(phosphonomethyl)amino]acetic acid Chemical compound OC(=O)CN(CO)CP(O)(O)=O AUBJMCRWFVUBJK-UHFFFAOYSA-N 0.000 claims 1
- KALSVYGPUQRNAR-UHFFFAOYSA-N OCNOP(=O)(O)C(C(=O)O)CC Chemical compound OCNOP(=O)(O)C(C(=O)O)CC KALSVYGPUQRNAR-UHFFFAOYSA-N 0.000 claims 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims 1
- 239000010836 blood and blood product Substances 0.000 abstract description 27
- 229940125691 blood product Drugs 0.000 abstract description 24
- 238000011534 incubation Methods 0.000 description 26
- 229940071206 hydroxymethylglycinate Drugs 0.000 description 25
- 239000000203 mixture Substances 0.000 description 23
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 21
- 239000011550 stock solution Substances 0.000 description 21
- 210000004027 cell Anatomy 0.000 description 19
- 239000007787 solid Substances 0.000 description 16
- 239000002609 medium Substances 0.000 description 15
- 239000000243 solution Substances 0.000 description 14
- 230000003253 viricidal effect Effects 0.000 description 14
- 230000003612 virological effect Effects 0.000 description 14
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 229960002449 glycine Drugs 0.000 description 9
- 239000012472 biological sample Substances 0.000 description 8
- 239000003112 inhibitor Substances 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 239000000126 substance Substances 0.000 description 7
- 239000000872 buffer Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 6
- 230000001717 pathogenic effect Effects 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 208000030507 AIDS Diseases 0.000 description 5
- 125000004432 carbon atom Chemical group C* 0.000 description 5
- 239000003599 detergent Substances 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 230000002411 adverse Effects 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 125000004356 hydroxy functional group Chemical group O* 0.000 description 4
- 210000004779 membrane envelope Anatomy 0.000 description 4
- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 description 4
- 229960000907 methylthioninium chloride Drugs 0.000 description 4
- 244000309711 non-enveloped viruses Species 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 238000009928 pasteurization Methods 0.000 description 4
- 239000003755 preservative agent Substances 0.000 description 4
- VIIIJFZJKFXOGG-UHFFFAOYSA-N 3-methylchromen-2-one Chemical compound C1=CC=C2OC(=O)C(C)=CC2=C1 VIIIJFZJKFXOGG-UHFFFAOYSA-N 0.000 description 3
- 241000991587 Enterovirus C Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- QIAFMBKCNZACKA-UHFFFAOYSA-N N-benzoylglycine Chemical compound OC(=O)CNC(=O)C1=CC=CC=C1 QIAFMBKCNZACKA-UHFFFAOYSA-N 0.000 description 3
- RWRDLPDLKQPQOW-UHFFFAOYSA-N Pyrrolidine Chemical compound C1CCNC1 RWRDLPDLKQPQOW-UHFFFAOYSA-N 0.000 description 3
- 239000000853 adhesive Substances 0.000 description 3
- 230000001070 adhesive effect Effects 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 239000003899 bactericide agent Substances 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 239000003139 biocide Substances 0.000 description 3
- 210000001124 body fluid Anatomy 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- BTXNYTINYBABQR-UHFFFAOYSA-N hypericin Chemical compound C12=C(O)C=C(O)C(C(C=3C(O)=CC(C)=C4C=33)=O)=C2C3=C2C3=C4C(C)=CC(O)=C3C(=O)C3=C(O)C=C(O)C1=C32 BTXNYTINYBABQR-UHFFFAOYSA-N 0.000 description 3
- 229940005608 hypericin Drugs 0.000 description 3
- PHOKTTKFQUYZPI-UHFFFAOYSA-N hypericin Natural products Cc1cc(O)c2c3C(=O)C(=Cc4c(O)c5c(O)cc(O)c6c7C(=O)C(=Cc8c(C)c1c2c(c78)c(c34)c56)O)O PHOKTTKFQUYZPI-UHFFFAOYSA-N 0.000 description 3
- KQSSATDQUYCRGS-UHFFFAOYSA-N methyl glycinate Chemical compound COC(=O)CN KQSSATDQUYCRGS-UHFFFAOYSA-N 0.000 description 3
- 230000002335 preservative effect Effects 0.000 description 3
- SSKVDVBQSWQEGJ-UHFFFAOYSA-N pseudohypericin Natural products C12=C(O)C=C(O)C(C(C=3C(O)=CC(O)=C4C=33)=O)=C2C3=C2C3=C4C(C)=CC(O)=C3C(=O)C3=C(O)C=C(O)C1=C32 SSKVDVBQSWQEGJ-UHFFFAOYSA-N 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 229950003937 tolonium Drugs 0.000 description 3
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 3
- VOMVAXPNAOVCLH-ZDUSSCGKSA-N (2S)-2-[[4-[(2-amino-4-oxo-3H-pteridin-6-yl)methylamino]benzoyl]amino]-5-(hydroxymethoxy)-5-oxopentanoic acid Chemical compound OCOC(CC[C@@H](C(=O)O)NC(=O)C1=CC=C(NCC2=CN=C3N=C(N)NC(=O)C3=N2)C=C1)=O VOMVAXPNAOVCLH-ZDUSSCGKSA-N 0.000 description 2
- VBMKLMZAAICVJG-DMTCNVIQSA-N (2s,3r)-3-hydroxy-2-(hydroxymethylamino)butanoic acid Chemical compound C[C@@H](O)[C@@H](C(O)=O)NCO VBMKLMZAAICVJG-DMTCNVIQSA-N 0.000 description 2
- LWSDPWMDHBXZAB-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)hexanedioic acid Chemical compound C(CC(=O)O)CC(CO)(C(=O)O)N LWSDPWMDHBXZAB-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 2
- 206010019799 Hepatitis viral Diseases 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 241000125945 Protoparvovirus Species 0.000 description 2
- 241000702263 Reovirus sp. Species 0.000 description 2
- 108010077895 Sarcosine Proteins 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 125000003545 alkoxy group Chemical group 0.000 description 2
- 125000005907 alkyl ester group Chemical group 0.000 description 2
- 125000004414 alkyl thio group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000000234 capsid Anatomy 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- YMGUBTXCNDTFJI-UHFFFAOYSA-N cyclopropanecarboxylic acid Chemical compound OC(=O)C1CC1 YMGUBTXCNDTFJI-UHFFFAOYSA-N 0.000 description 2
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 description 2
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 2
- 229960003964 deoxycholic acid Drugs 0.000 description 2
- 229960001083 diazolidinylurea Drugs 0.000 description 2
- 238000005553 drilling Methods 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- NTNZTEQNFHNYBC-UHFFFAOYSA-N ethyl 2-aminoacetate Chemical compound CCOC(=O)CN NTNZTEQNFHNYBC-UHFFFAOYSA-N 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 2
- 125000001072 heteroaryl group Chemical group 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229960003151 mercaptamine Drugs 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 108700019599 monomethylolglycine Proteins 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- WOMAZEJKVZLLFE-UHFFFAOYSA-N propionylglycine Chemical compound CCC(=O)NCC(O)=O WOMAZEJKVZLLFE-UHFFFAOYSA-N 0.000 description 2
- 230000005180 public health Effects 0.000 description 2
- 238000009877 rendering Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 2
- 229940101011 sodium hydroxymethylglycinate Drugs 0.000 description 2
- CITBNDNUEPMTFC-UHFFFAOYSA-M sodium;2-(hydroxymethylamino)acetate Chemical compound [Na+].OCNCC([O-])=O CITBNDNUEPMTFC-UHFFFAOYSA-M 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 201000001862 viral hepatitis Diseases 0.000 description 2
- RDCSJBMWDXSFRP-UHFFFAOYSA-N (1h-pyrazol-5-ylamino)methanol Chemical compound OCNC1=CC=NN1 RDCSJBMWDXSFRP-UHFFFAOYSA-N 0.000 description 1
- PFAXWXXXDNWFOQ-LMECJBHSSA-N (2S)-5-(diaminomethylideneamino)-2-(hydroxymethylamino)pentanoic acid (2S)-2-(hydroxymethylamino)-3-(4-hydroxyphenyl)propanoic acid Chemical compound OCN[C@@H](CC1=CC=C(C=C1)O)C(=O)O.OCN[C@@H](CCCNC(N)=N)C(=O)O PFAXWXXXDNWFOQ-LMECJBHSSA-N 0.000 description 1
- RCBKINCFGZXYCL-LURJTMIESA-N (2s)-2-(hydroxymethylamino)-4-methylpentanoic acid Chemical compound CC(C)C[C@@H](C(O)=O)NCO RCBKINCFGZXYCL-LURJTMIESA-N 0.000 description 1
- GTLPWMXEGPCOQX-VPCXQMTMSA-N 1-[(2r,3r,4s,5r)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]pyrimidine-2,4-dione Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)N1C(=O)NC(=O)C=C1 GTLPWMXEGPCOQX-VPCXQMTMSA-N 0.000 description 1
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Abstract
A method of inactivating viruses that may be present in a whole blood or blood product intended for administration to an individual is disclosed. The whole blood or blood product sample is treated with an effective quantity of a hydroxymethylamine of formula (I). An exemplary hydroxymethylamine is Nhydroxymethylglycine and salts thereof.
Description
~ W092/02028 ~J;~ 5 PCT1US96111152 INACTIVATION OF PATHOGENS USING ~Y~RO~Y~Y
FIE~D OF THE INVENTION
This invention relates to methods for inactivation of pathogens.
BACKGROUND OF THE INVENTION
Spread of infectious disease resulting from transfusion of cont~m;n~te~ blood, administration of contaminated blood products or handling or usage ~f objects that have come into contact with contaminated blood and/or blood products,has been well documented and is recognized as a major public health concern.
Most notably, tr~n~ q;on of viral hepatitis and/or Acquired Immune Deficiency Syndrome (AIDS) through contaminated blood and blood products has received widespread attention. However, viral hepatitis and AIDS are only two of the many diseases that can be spread through use of contaminated blood and blood produc~s. ~esser known pathogens, such as T-cell lymphotropic vlruses ~Types I and II), cytomegalovirus, Epstein-Barr virus, the parvoviruses and Plasmodi~,m~ (malaria-causing) protozoa, may also be spread through contaminated blood and blood products. In addition, still other microorganisms that have not yet even been identified or recognized as being pathogeni~ may be transmitted through c~n~m;n~ted blood and blood products and, therefore, similarly pose a serious public health risk. The HIV
virus is illustrative of a pathogen that, until ~ recently, was not even recognized. Today, there are over l0 million people worldwide who have contracted AIDS, many of these people having contracted the disease throush use of infected blood or blood products; however, less than two decades ago, AIDS
WO97l~28 ~ PCT~S96/11152 .r) was not even a recognized disease. Thus, it is clear that there is a great need for a method for effectively inactivating pathogens in blood and b~ood product6.
In response to this need a number of techniques have been devised for inactivating pathogens, particularly infectious viral agents, in blood and/or blood products. A review of many of ~hese techniques is presented in Suomela, "Inactivation of Viruses-in lC Blood and Plasma Products," T~ans~usion ~edicine ~eviews, Vol. VII, No. l, pp. 42-57 (~anuary l9g3~, which i9 incorporated herein by reference.
One such technique which has been used to inactivate viruEes in blood and/or blood products is pasteurization. [See Burnouf-Radosevich et al., "A
Pasteurized Therapeutic Plasma, " ~nfusionstheraDie, 19:9l-94 (l992~] The pasteurization of blood and/or blood products is most often effected by heating them in the liquid state for lO hours at 60OC. A small amount of protein stabilizer, such as caprylate or tryptophanate, is often added to the preparation.
After pasteurization has been completed, the stabilizer typically must be removed from the preparation prior to its clinical use. As is the case with many of the existing viral inactivation techniques discussed herein, pasteurization is more effective in inactivating enveloped viruses (i.e., viruses having a lipid envelope surrounding the viral capsid) than in inactivating non-enveloped viruses ~i.e., viruses which lack a lipid envelope surrounding the viral capsid).
W097/020~ 5 PCT~S96/11152 Another technique which has been used to inactivate viruses in blood and/or blo~d products is the solvent/detergent (S/D) method. [See, for example, Hellstern et al., "Manufacture and in vitro S Characterization of a Solvent/Detergent-Treated Human Plasma, " Vox Sanq, 63:178-185 (1992); Horowitz et al., "Solvent/Detergent-Treated Plasma: A Virus-Inactivated substitute for Fresh Frozen Plasma,ll Blood 79:826-831 (1992); and Piquet et al., "Virus Inactivation of Fresh Frozen Plasma by a Solvent Detergent Procedure: Biological Results, Vox Sanc, 63:251-256 ~1992).] The S/~D wethod, which is limited to use in inactivating enveloped viruses, involves treating a blood preparation with an organic mixture which disrupts the lipid envelope of enveloped viruses. The disruption of the lipid envelope leads either to complete structural disruption of the virus or to destruction of the cell receptor recognition site on the virus. In either case, the virus is rendered noninfectious. The solvent used in the S/D
method is most often tri-(n-butyl~phosphate ~T~BP), and the detergent is either Tween 80 , Triton X-100' or sodium deoxycholate. Temperature and time influence the efficacy of the S/D method, typical temperatures being in the range of 24~ C to 37~C, and the typical duration of treatment being at least 6 hours.
Still another technique which has been used to inactivate viruses in blood and/or blood products is photochemical inactivation. [See Mohr et al., ~Virus Inactivated Single-Donor Fresh Plasma Preparations,~
Infusionthera~ie, 19:79-83 (1993); Wagner et al., "Differential sensitivities of viruses in red cell suspensions to methylene blue photosensitization,~
_ _ _ _ _ _ _ _ _ _ , _ _ . _ _ _ _, _ _ , , .. .. . . . , _ . ...
WO97/02028 PCT~96111152 3 ~ ~ ~
Transfusion, 34~6):521-526 (19~4); Wagner et al., ~Red cell alterations associated with virucidal methyler.e blue phototreatment," Transfus_s~ 30-36 ~1993); Mohr et al., "No evidence for neoantigens in human plasma after photomhPm;r~l virus inactivation,"
Ann. Hematol.. ~5:224-229 ~1992); Lanbrecht et al., PhotoinactivatiOn of Viruses in Human ~resh Plasma by Phenothi~7in~ Dyes in ~ n with Visible Light," Yox San~, 60:207-213 (19911, Goodrich et al., "Selective inactivation of viruses in the presence of human platelets: ~V sensitization with psoralen derivatives," Proc. Nat. Acad. Sci. USA. 91:5S52-555 (1994); ~irus Inactivation in Pl~r~ Products, J.-J
Morgenthaler, ed. Xarger, NY (1989); and BioWorld Today. Vol. 4, Nc. 229, pages 1 and 4 (November 24, 1993).] The photochemical inactivation of a blood preparation typically involves treating the blood preparation with a photoactivatable chemical and then irr~iA~in~ the preparation with light of a sufficient wavelength to actlvate the photoactivatable mh~m;C~l, Examples of photoactivatable rhPm; ~1 s used in the photochemical inactivation of viruses preaent in ~lood preparations include psoralens, hypericin, methylene blue and toluidine blue. It is believed that psoralens, which have an affinity for nucleic acids, inactivate viruses by intercalating between viral nucleic acid base pairs and, in the presence of WA light, ~orming a covalent bond with the viral nucleic acid, thereby preventing its transcription and/or replication The nanner in which hypericin, methylene blue and toluidine blue inactivate viruses is not as well defined as that for psoralens. However, it is believed that these chemicals, when photoactlvated, generate the highly reactive entity, singlet oxygen, ~ W097102028 PCT~S9~11152 ~ ~ 17'8l}~5 which then attacks the cellular structure ~e.g. viral ~ envelope) of the virus.
Whereas photochemical inactivation has been largely successful in inactivating enveloped viruses, it has been largely unsuccessful in inactivating non-enveloped viruses. The failure of photochemical inactivation to inactivate non-enveloped viruses is significant since Poliovirus, Adenovirus, Hepatitis A
and Parvovirus (Parvo Bl9) are among those non-enveloped viruses that are pathogenic to humans.
It should be noted that photochemicalinactivation of the type described above has been most successful. when applied to inactivating viruses in blood preparations lacking red blood cells (e.g., plasma). This is because blood preparations that include red blood cells typically absorb light at the same wavelengths used to photoactivate the chemicals.
Viral inactivation agents are substances that render viruses incapable of replication and proliferation. From the literature discussed above, one may conclude that viral inactivating compounds have been ident.ified which are specifically toxic to blood borne viruses such that cells and proteins are not adversely affected. Still it is important to limit exposure of biological samples to viral inactivation agents, such as, for example, psoralens, hypericin, methylene blue, toluidine blue or a combination of tri-(n-butyl) phosphate and a detergent such as Tween 80, Triton X-lO0 or sodium deoxycholate to the minimum extent necessary to reduce potentiall~ signification interactions that could lead to undesirable side effects.
~097~ PCT~S~/11152 'u' g 5 U.S. Patent 4,337,269 (Berke et al.) disclosed a biocidal composition containing a compound, hydroxymethylaminoacetate talso referred to here~n as hydroxymethylslycinate), which is produced by the reaction of glycine or a salt of glycine with formaldehyde. In the aforementioned patent, hydroxymethylglycinate is said to be effective at inhibiting the growth of bacteria, yeasts and molds in a variety of substances susceptible to microbial ~nt~m;n~tion, such as cosmetics, foodstuffs, pharmaceuticals, paintst cutting oils or fluids, agricultural products, oil.drilling fluids, paper industry, embalming solutions, cold steril-i~ation medical and dental equipment, cooling towers, fabric impregnation, latexes, swimming pools, inks, household disinfectants, waxes and polishes, toilet bowl cleaners, bathroom cleaners, laundry detergents, soaps, wood preservatives, hospital and medical antiseptics and adhesives.
Sodium hydroxymethylglycinate is the active ingredient in the preservative SUTTOCID~A, which i~
commercially available from Sutton ~aboratories, Chatham, New Jersey. In certain promotional literat.ure published by Sutton Laboratories, 2~ SUTTOCID~A:is said to be active against Gram-negative and Gram-positive bacteria, yeast and mold and is suggested for use. as a preservative in shampoos, hair conditioners and ~facial treatments.
U.S. Patent 4,900,l~6 ~Berke et al.) disclosed a composition c~nt~;ning one or more 3-isothiazolones and a compound which is a member selected from the group consisting of hydroxymethyl-aminoacetic acid, its salts and lower alkyl esters. The aforementioned W097/02028 ~ 7 8 o g 5 PCT~S96/11152 composition is described in the patent as being effective against bacteria, yeasts and molds.
Suggested applications in the patent for the above-described composition inolude use as a preservative .~ in cosmetics, toiletries and household cleaning products, use as a biocide for synthetic latexes, emulsion paints and other coatings, adhesives, polishes, carpet backing compositions, surfactants, metalworking fluids, industrial and domestic water treatment including cooling tower systems and swimming pools, adhesive mats, drilling mud formulations, painting pastes, spin finish emulsions, polymer dispersions and fuels and as a slimicide for slime control in the manufacture of paper from wood pulp.
It is to be noted that nowhere in the foregoing patents or publications is it taught that the biocidal activity of hydroxymethylaminoacetic acid, its salts and/or low~r alkyl esters can be extended beyond bacteria, yeasts and molds to include viruses.
Moreover, because most biocidal agents that are effective against bacteria, yeasts and molds are not effective against viruses, the foregoing patents and publications do not provide any reasonable basis for one of ordinary skill in the art to expect that hydroxymethylaminoacetic acid and~or its derivatlves would be effective in inactivating viruses.
It is also to be noted that nowhere in the ~ foregoing patents or publications is it taught that hydroxymethylaminoacetic acid and/or its derivatives can be used in blood and/or blood products.
Moreover, because most biocidal agents that are effective against bacteria, yeast and/or molds cannot _ _ _ _ _ _ _ _ _ _ _ _ _ . . ... . . _ _ ..... , . . , . .. _ W097~020~ ~ PCTA~96/~1152 be used to inactivate such r~thn~r~q in blood an/or blood products without adversely affecting the suitability of the treated blood andjor blood product for subse~uent adoinistration to a patient ldue to their toxicity and~or their reactivity with plasma proteins and certain ot.her blood constituents), the foregoing patents and publications do not provide any reasonable basis for one of ordinary skill in the art to expect that hydroxymethyl~m;nna~Ptic acid and/or its derivatives could be used in blood and/or blood products without rendering the treated blood and~or blood product unsuitable for subsequent administration to a patient in need thereof.
A180 of interest is Japanese p-lh ;~h~d Application No. 62-195304, which disclosed that paraform (84~, 25g) was added to 98~ die~h~nnl~m;
(332g~ at 40 degrees, and then stirred at 5C-60 degrees for l hour to yield hydroxymethyldi-~thanolamine. It is also disclosed that hydroxymethyl-diethanolamine, at 300-500ppm, controlled Escherichia coli, Staphylococcus aureu6, Pgeu' .5 aeruginosa, ~acillus subtill6, Proteu6 vulgari6 and P. mirabilis on nutrient agar plates.
It is to be noted, howe~rer, that nowhere in the foregoing Japanese application is it taught or suggested t:hat hydroxymethyldiethanolamine has virucidal activity or that hydroxymethyldiethanol-amine could be used in blood and/or blood products without adversely affecting the suitability of the treated blood and/or blood product for subsequent administration to a patient in need thereof.
Therefore, a need exits for a method for treating blood which inactivates viruses without _ _ , _ _ _ _ _ _ _ . . . . . . . _ _ _ . . .
~ W097102028 ~ 9 8 ~ PCT~S96/11152 _ g _ adversely affecting the suitability of the treated blood and/or blood product for subsequent administration to an individual in need thereof.
SUMMARY OF THE INVENTION
The present invention is a method of inactivating a virus in a biological fluid, such as blood. In the method, the biological fluid is contacted with a hydroxymethylamine (HMA) in sufficient quantity to inactivate the virus ~i.e.-, an l~ effective amount). The biological fluid can be of any type including, but not limited to, whole blood and a wide variety of blood components, including, but not limited to, red blood cells, red blood cell concentrate, platelets, platelet concentrate, platelet rich plasma, platelet poor plasma, source plasma (plro-ar~re5is plasma), fresh frozen plasma, plasma proteins (e.g., clotting factors VIII, ~, etc.), and other body fluids, such as lymph, cerebrospinal fluid, semen, saliva, etc. While targeted at the inactivation of viruses, the method is effective against other microorganisms as well.
These microorganisms can be pathogenic or nonpathogenic and include bacteria, yeasts, molds and protozoa.
In one embodiment, this invention provides a method for inactivating a microorganism contained in a biological fluid. The method comprises the step of contacting the mi~Luu~y~llism with an effective amount of a hydLu~yl~rth~rlamine (HMA). Suitable hydroxy-methylamines include compounds of Formula (I) W097~ PCT~S96/11152 N-CH2-OH ~I~
RL
wherein:
R is chosen from the group consisting of hYdL~L, alkyl, aryl/ substituted alkyl, substituted aryl; and R1 is chosen from the group consisting of acid-, amide-, hydroxy- or mercapto-functional alkyl; acid-, amide-, or hydroxy-functional aryl; acid-, amide-, or hydroxy-functional substituted alkyl; and acid-, amide-, or hydroxy-functional substituted aryl;
or R and R1 may be joined together to form an acid, amide or hydroxy-functional heterocyclic structure.
Preferred XMA~ are those in which the functional group is an amide or an acid selected from the group consisting of carboxylater phosphate, phosphonate, sulfate and sulfonate. Carboxylic acids are particularly preferred.
Preferred individual hydroxymethylamines having the amide functionality include hydroxymethylglycin-amide, hydroxymethylpenicil1;n~m;~ hydroxymethyl-leucin~mlfl~, hydroxymethylacrylamide and hydroxy-methylnicotinamide. Preferred hydroxymethylamineshaving the acid functionality include hydroxymethyl-glycine, hydroxymethylphosphonomethylglycine, hydroxymethyl-p-aminohippuric acid, hydroxy-methylpropargylglycine, hydroxymethyl-o-3~ phosphothreonine, hydLu~y -thyl~m;n~;pic acid, hydroxymethyl-o-phospho5erine, hydroxymethylamino-ethylphosphonic acid, hydroxymethylleucine, hydroxymethyl-~-alanine, hydroxymethylcysteine, hydroxymethylfolic acid, hydroxymethylamino-~ W097~2028 7 1 ? ~ ~ g 5 PCT~S96/11152 phosphonobutyric acid, hydroxymethylphenyl~l~n;n~,hydroxymethylami.nophenylacetic acid, h~droxymethyl-o-phosphorylethanolamine, hydroxymethyl~1 ~ni ne, hydroxymethylserine, hydroxymethylvaline, hydroxymethylmethionine, hydroxymethylglutamic acid, hydroxymethylaspartic acid, hydroxymethyllysine, hydLo~yl-thylproline~ hydroxymethylmercaptopropionyl-glycine, hydroxymethylaminoethyl hydrogen sulfate, hydroxymethylpenicillamine, hydroxymethylornithine, and hydroxymethylcysteine. Preferred hydroxymeth'yl-amines having neither an acid nor an amide functionality include hydroxymethylmercaptoethyl-amine, hydroxymethylaminoethanol, hydroxymethyl-aminopropanol and hydroxymethyldiethanolamine.
A particularly preferred HMA is hydroxymethyl-glycine or a salt thereof.
According to the method of the invention, the hydroxymethylamine and biological fluid are preferably combined to produce a final concentration 2G of IlYdLC~Y.. thylamine of approximately 0.05 ~ - 3.0 by weight; the contact time is from 0.5 hours to 4 hours, and the t.emperature is maintained between about 4~ C and about 30~ C.
In another aspect, the invention relates to a method of processing a biological fluid intended for administration to an individual in need thereof. The method comprises the steps of: (a) treatins the biological fluid with an effective amount of a pathogen-inactivating hydroxymethylamine, thereby 3G producing a treated biological fluid; and ib) then removing free hydroxymethylamine from the treated biological fluid.
W097~2028 ~ PCT~96111152 ;~ ~ 9~3~
In another aspect the invention relates to a method of treating an individual in need of a biological fluid. The method comprises the stepsrof:
(a~ treating the biological fluid with an effective amount of a pathogen-inactivating hydroxymethylamine~
thereby producing a treated biological fluid; and (b) administering the treated biological fluid to the individual in need thereof.
In another aspect, the invention relates to a method of treating a biological fluid. The method comprises combining an effe~tive amount of a virus-inactivatin~ hydLu~y,-,ethylamine with the bio~ogical fluid, whereby at least about a lO-fold reduction in plaque forming units of virus is realized.
Subsequently, the virus-inactivating compound can be removed from the biological fluid prior to its administration to an individual. If the biological fluid is blood, the treated blood can be returned to the individual ~rom whom it was obtained.
Alternatively, the treated blood can be stored and administered later in time to the same individual or another individual in need thereof.
The me~hod of the invention can also be used to inactivate pathogens present in bodily fluids other than blood, and to disinfect medical instruments and analytical equipment that have come into contact with potentially cont~m;n~tP~i blood. Similarly, the method of the invention can also be used to disinfect blood samples that are not i nt~n~d for subsequent administration to an individual, but rather, are intended for subsequent chemical analysis. Other possible applications of the invention are apparent to those skilled in the art.
W097~2028 ~ 9 ~$~ PCT~S~111152 Additional objects, as well as features and advantages, of the present invention will be set forth in part in the detailed description which follows, and in part will be obvious from the s detailed description or may be learned by practice of the invention. Various Pmho~;l ts of the inventions will be described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be under9tood that other embodiments may 10 be utilized and that changes may be made without departing from the scope of the invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is best defined by the appended claims.
DETAI~ED D~SCRIPTION OF THE INVENTION
The present invention relates to the method of use of HMA's as pathogen-inactivating agents, particularly viral-inactivating a~ents, in biological samples, such as blood or blood products.
Use of HMA for treatment of ~athoqens The present invention is based, in part, or the unexpected discovery that a HMA can be used to inactivate viruses in biological samples. The ~resent invention is also ba9ed, in part, on the unexpected discovery that hydroxymethylamines such as those of Formula (I), can be used to inactivate viruses in blood and/or blood products, without rendering the treated blood andfor blood products unsuitable for - 30 subsequent administration to an individual.
WO 97/02028 ~ 3 {3 S; r PCTIUS96111152 Suitable pathogen-inactivating ~e.g., ~irus-inactivating) HMA's for use in the invention can be readily selected by those skilled in the art using art-recognized methods and the test methodology set forth ir. the accompanying Examples. For the purposes of the specification and claims, an HMA that reduces the number of plaque-forming units ~PFU) in a biological fluid by a factor of at least iO (i.e. one log reduction) is a pathogen-inactivating HMA. The ability to reduce the number of PFUs can be assessed using art-recognized methods, one example of which is described in Example l. If an HMA reduces the number of PFUs by at least one log unit, as assessed by the method described in Example l, it is a pathogen~
inactivating XMA. The term "hydroxymethyl~' when used herein in connection with a particular amino compound, designates that the rr-ro-ln~ has a -CH20X
substituent on the amino group.
SuitablQ alkyl groups in compounds of Formula (I~ include straight chain, branched chain, and cyclic alkyl groups, crn~ininrJ one to about 22 carbon atoms, more preferably one to about 12 carbon atoms. ~hen the alkyl group is a cyclic alkyl, 3-, 4- 5-, 6-, and 7- membered rings are preferred.
Suitable aryl groups in compounds of Formula ~I) inc}ude hydrocarbon aryl groups r~nt~ining a 6-membered aromatic ring, such as phenyl~ fused bicyclic systems such as ~- and ~2-naphthyl, histidine, indenyl, tetralinyl, and the like, and monocyclic and polycyclic heteroaryl groups crnt~ining a 5- or 6- m~mbeL~d heteroaromatic ring, e.g., pyridyl, pyrimidinyl, quinolinyl, furanyl, ~ wo g"02028 2 1 9 ~ PCT~S~111152 thienyl, isothiazolyl, isoxazolyl, imidazolyl, lH-pyrrolyl, indolyl, purinyl, and the like.
Suitable substituents in a substituted alkyl or substituted aryl group include halogen ~e.g., fluoro, chloro, bromo), hydroxy, alkoxy (e.g., alkoxy containing one to 8 carbon atoms), alkylthio (e.g., alkylthio wherein the alkyl group contains one to 8 carbon atoms), lower alkyl (i.e., alkyl cont~ining one to four carbon atoms), cycloalkyl (e.g., cyclopropyl, cyclopentyl, cyclohexyl~, phenyl, benzyl, benzo, mercapto, or combinations thereof.
R and R~ of Formula (I) can together form an ~-heterocyclic ~tructure (i.e., a cvclic structure wherein the hydroxymethylated nitrogen is an atom in the cyclic structure), such as a proline, pyrrolidine, piperidine, 2-pyrroline, indoline, aziridine, azetidine, and the like.
In a preferred ~mho~im~nt, the HMA contains a carboxylic acid portion as part of at least one of the substituted alkyl or substituted aryl groups, so that the HMA falls within the broad class of N-hydroxymethylated aminoacids. The aminoacid is not restricted to naturally occurring ~-aminoacids, although they provide particularly convenient starting materials, and their residues are optimally biocompatible.
When the functional group is acidic, salts of the HMA, such as metal salts ~e.g. sodium and potassium salts), and ammonium salts may be used.
W09~20~ ~ PCT~S~11152 ~i s ~ j ~ f~
Preferred ~MAs includ~ l-ydL~y-..cthylglycine, hydroxymethylphosphono~ethylgiycine, hyd;oxymethyl-p-aminohippurate, hydroxymethylpropargylglycine, hydroxymethyl-o-phosphothreonine, hydroxymethyl-aminoadipate, hydroxymethyl-o-ph~crhrqerine, hydroxymethylamino-ethylphosphonic acid, hydroxymethylleucine, ~ydL~y ~hyl-~-alanine hydroxymethylcysteine, hydroxymethylfolate~
hydroxymethylaminophob~ obutyric acid, and hydro-xymethylpheny~ n;n~ and their .~L,e~ dlng sa~ts.~ydroxymethylglycine, hydroxymethylfolate, hydroxymethylAminf;rh~h~ uLyLic acid, hydroxymethylpropargylg.lycine and hydroxymethyl-o-phosphothreonine are particularly preferred.
Other suitable HMAs include hydroxymethylamino-phenylacetic acid, hydroxymethyl-o-phosphorylethanol-amine, hydroxymethyl~l An i n~ ~ hydL~y. Lhylserine, IIYdL~YI Lhylvaline, hydroxymethylmethionine, hydroxymethylglutamate, hydroxymethylaspartate, hydroxymethyllysine, hydLo~y~ thylproline, hydroxymethyl~- ~p~opropionylglycine, hydroxymethyl-mercapto-ethylamine, hydroxymethylaminoethyl hydrogen ~ulfate, hydroxymethylamino-ethanol, hydroxymethyl-penicillamine, hydroxymethylhydantion, hydroxymethyl-ornithine, hydroxymethylcysteine, hydroxymethylamino~propanol, hydroxy~ethyldiethanolamine~ and their corr~.cron~ing salts.
HMAs can be readily synthesized by those skilled in the art. For example, to synthesize a hydroxymethyl-Amin~arid~ one equivaler.t each of the corresponding amino acid, formalin and sodium hydroxide are combined.
W097~ZOZ8 ~i ~ 8 a ~ ~ PCT~S~111152 One application of this invention is a method of inactivating a pathogen present in a biological sample, such as blood or a blood product intended~for administration to an individual in need thereof, said method comprising the step of treating the biological sample, such as blood or a blood product with an effective amount of a pathogen-inactivating acid- or hydroxy-functional HMA.
In accordance with the ~ hi ngS of the present invention, treatment of a biological fluid, such as blood and/or a blood product~comprises combining an appropriate quantity of a pathogen-inactivating HMA
with the biological sample and then allowing the sample to incubate for an appropriate period of time at a suitable temperature. The final concentration of the HMA in the sample is preferably approximately 0.05~-3.0~, more preferably approximately 0.5~. The incubation period is sufficiently long to inactivate pathogen in the sample, commonly from about 0.5 hour to about 4 hours, conventiently about l hour, and the incubation temperature is about 18'C to about 37 C, more preferably about 20 C to about 30 C. In a specific embodiment, the treatment of a blood and/or blood product. comprises combining sodium hydroxy-methylglycinate with the sample to give a finalsodium hydroxymethylglycinate concentration of 0.5~, then allowing the sample to incubate for approximately 60 minutes at a temperature of about 30 C.
The above-described method can be used to ~ inactivate viruses, bacteria, molds, yeasts, protozoa and other pathogens in biological samples, such as whole blood and a wide variety of blood components, W097/o~ PCT~S9~11S2 2~ J Q ~
lncluding, but not limited to, whole blood, red blood cell component, red blood cell concentrate, platelet component, platelet concentrate, platelet rich plasma, platelet poor plasma source plasma, fresh ', frozen plasma and plasma proteins. As mentioned above, one advantageous aspect of the present method is that it does not render a biolo~ical sample unsuitable for subsequent administration (e.g., transfusion) to an individual.
It i9 believed that HMA's inactivate viruses either by reacting ~ith the,protein coat or with the t nucleic acids.
The method of the invention can alr~o be used to inactivate pathogens present in bodily fluids other than blood, and to disinfect medical instruments and analytical equipment that have come into contact with potentially contaminated biological samples, such as blood. Sienilarly~ the method of the invention can be used to disinfect blood samples that are not intended for subsequent administration to an individual, but rather, are intended for subsequent chemical analysis. Cther possible applications of the invention are apparent to those skilled in the art.
The following examples are illustrative only and 2~ should in no way limit the scope of the present invention.
EXAMP~E 1 Five microliters of T4 (American Type Culture Collection No. 11303-~4~ virus stock and 14 ~L of blood plasma were added to each of four tubes. 1 ~L
of a 50~ stock solution of sodium ~ W097~2028 ~ 9 8 ~ PCT~Sg6/lll52 hydroxymethylglycinate ~International Specialty Products, Bound Brook, New ~ersey) was ~ded to a first tube, 1 ~L of a 10 mg/mL stock solution of-diazolidinyl urea (a bactericide) was added to a second tube, 1 ~L of a 10 mg/m~ stock solution of imidazolidinyl urea ~a bactericide) was added to a third tube, and 1 ~L of Phosphate Buffered Saline (PBS) was added to the fourth tube. The mixtureS
were incubated at room temperature for 1 hour before sampling, diluting, mixing with host cells in sof-t agar and pouring onto solid medium. After overnight incubation at 37 C, the plaques were counted. The results are summarized below in T~3BE I.
TABLE I
15S ~ pl- Inhibitor 2 ' ~t on PFIJ~ (Plaque in Mixture Forming ~nita) 1~dh~. 'hylglycinate2.5~ 22xlO~
2Diazo].idinyl nrea0.5 mg~mL14xlO~
3Tmidazolidinyl urea0.5 mg/mL42xlO~
4 None 0 66xlO~
The above results show that, whereas the bactericides diazolidinyl urea and imidiazolidinyl urea were ineffective at the above-indicated concentrations at inactivating T4 virus, hydroxymethylglycinate exhibited strong viricidal activity.
Five microliters of T4 virus stock were added to each of fourteen tubes. Fourteen microliters of blood plasma were added to each of seven of the tubes, and 14 ~L of whole blood were added to each of the remaining seven tubes. One microliter of an appropriate hydroxymethylglycinate stock solution was W097l~2028 ~ 5 PCTn~96~111S2 added to each of six of the plasma-containing tubes and to each:of fiiX of the whole blood-containing tubes to gi~e the below-listed concentrations. Qne microliter of P}3S was added to each of the r~ ; ni ng S tubes. After incubation at room t~ e~Lur~ for 1 hour, samples from each tube were taken, diluted, mixed with host cells, and overlaid onto solid medium. After overnight incubation at 37'C, plaques were counted. The results are summarized below in TAS~E II.
TAB~E II
S~mple Eyl~ thylglycinate Plaama PFU
Concentration or Whole ~lood 1 2.5~ Plasma 21x104 2 1.25% Plasma 71x104 lS 3 0.625~ Plasma 70x104 4 0.25~5 Plasma 58x105 0.125~ _ Plasma 36X106 6 0.063~ Plasma 18x107 7 o~ Plasma 60x103 8 2.S% Whole 29x104 Blood 9 1.25~- Whole 50xlO~
Blood 0.625~ Whole 76xlO' Blood 11 0.25% Whole 43x105 ~ Blood 12 0.125% Whole 74xlO~
~lood 13 0.063% Whole 21xlO~
Blood 14 0~ Whole 67xlO~
~lood ~ wo g7~2028 ~ d a ~ 5 PCT~S96/11152 , ~
-2~-As can be seen from the results above, the inactivation of T4 was dependent upon the concentration of hydroxymethylglycinate. A
concentration of 0.625~ hydroxymethylglycinate led to a 4 log decrease in T4 over a period o~ l hour at room temperature.
Two hundred twenty-five microliters of T4 stock were added to each of twelve tubes. 4.75 ~L of whole blood were added to three of the tubes, 4.75 ~L o~
plasma were added to another three of the tubes and 4.75 ~L of PBS were added to still another three of the tubes, the remaining three tubes serving as controls 25 ~L of a 50~ solution of hydroxymethylglycinate were added to each of the tubes, except for the controls. The various mixtures were incubated at room temperature, and at the times indicated below samples were taken, diluted, mixed with h~st cells and overlaid onto solid medium.
After overnight incubation at 37 C, plaques were counted. The results are summarized below in TAB~E
III.
WO 97~2U28 ~ ' PCT/U596~1152 2 ~ 7 0 3 i ~
2' TP~3L~ I
Sample ~r~LVA1--Lhylslyclnate Tn~ An PF~8 Pr-~bnt ~i~e Whole Yes lS minutes 27x107 Blood 5Plasma Yes lS minutes 9xlO' PBS Yes 15 minutes 25xlO' Control Uo lS minutes ~
Whole Yes 30 minutes 9xlO' Blood 10Plasma Yes 30 minutes 23xlO' PBS Yes 30 miuutes 40xlOs Control ~o 30 minute~ ~D
Whole Yes 60 minutes 14xlO~
Blood 15Plasma Yes 60 minutes 25x105 PBS Yes 60 minutes 14xlO' Control ~'o 60 minutes soxlo7 As can ~e. seen from the results above, higher levels of hydroxymethylglycinate are required to ~0 inactivate T4 in blood or in plasma than in buffer solution. As can also be seen, the efficac~ of hydroxymethylglycinate appears to increase as incubation time increases.
Thirty microliters of T4 stock and 465 ~LL of blood plasma were added to each of ten tubes. 5 ~L
of a 50~ stock solution of hydroxymethylglycinate were added to each of eight of the ten tubes to give a final hydroxy-methylglycinate concentration of O.S~. The mixtures were incubated at the temperatures indicated below. At the times indicated below, samples were taken from each tube, diluted, mixed with host cells and overlaid onto solid medium.
W097~20~~ ~31 ~Q~5 PCT~3S96~11152 After overnight incubation, plaques were counted.
The results are summarized below in TABLE IV.
TABLE IV
Tn~h~tinnElr~ slycil~at-- Tnn~h:~t;~n PF~'--5 TiIe PresentTemperature 30 mlnutes Yes 5~C llxlO' 30 minutes Yes 21~C 103~c10' 30 minutes Yes 30~C 16X10 30 minutes Yes 37~C 22Xl03 1030 minutes No 37~C ND
60 minutes Yes 5~C 12xlO' 60 minutes Yes 21~C 23xlO' 60 minutes Yes 30~C < 103 60 minutes Yes 37~C < 103 1560 minutes 3-~o 37~C ôOxlO7 As can be seen from the above results, the inactivation of T4 in blood plasma using 0.5~
hydroxymethylglycinate is temperature dependent.
Significantly greater viral toxicity was seen at 30'C
than at room temperature, and at 5-C only a l log drop in viable virus was observed.
Thirty microliters of T4 stock and 465 ~L of packed red blood cells were added to each of ten tubes. 5 IlL of a 50% stock solution of hydroxymethylglycinate were added to each of eight of the ten tubes to give a final hydroxymethylglycinate concentration of 0. 59~. The mixtures were incubated at the temperatures indicated below. At the simes indicated below, samples were taken from each tube, diluted, mixed with host cells and overlaid onto solid medium. After overnight incubation at 37~C, WO97~28 ~ g~ PCT~Ss~ll152 plaques were counted. The results are su~marized below in T~3LE V.
TABLE V
r~ t;,~ lr~ . ~ ~157lycin~lt T~ PFtl'8 5Ti~3~ nt 30 minutes Yes 5~C 15X1()7 30 miuutes Yes 21~C 20xlO' 30 minutes Yes 30OC llx1oS
30 minutes Yes 37~C 21xlO' 1030 mi3~utes No 37OC ND
60 minute~ Yes 5~C 44xlO' 60 mirlutes Yes 21~C lOxlOs 60 minutes Yes 30OC 24xlO' 60 minutes Yes 37OC c 103 1560 minutes No 37~3c llxlo' A8 can be seen from the above results, the inactivation of T4 in packed whole blood is also temperature ~r~n~nt. At 37~C, over a 5 log drop in viral viability was observed over the time of the experi~ent. At 5~C~ the decrea~3e in viral ~iability was only 2 logs.
Thirty microliters of T4 stock and 440 ~ of blood plasma were added to each of twelve tubes. 30 ~L of a 50~ stcck solution of hydroxymethylglycinate were added ts each of ten of the twelve tubes to give a final hyd~v~y ~thylglycinate concentration of 3~.
The mixtures were incubated at the temperatures indicated below. At the times indicated below, samples were taken from each tube, diluted, mixed with host cells and overlaid onto solid medium.
After overnight incubation at 37~C, plaques were ~ W097~2028 2 ~ ~ 8 ~ ~ 5 PCT~S96111152 counted. The results are summarized below in Table VI.
TABLE VI
T_~h7~ Y ~ hY1g1YCLU~te T~ h~A_ PF~'6 5Tlme Pre~ent ~ - , 30 minutes Yes 0~C 29xlO' 30 minutes Yes S~C 72X10' 30 minutes Yes 21~C 69xlOs 30 minutes Yes 30~C < 103-1030 minutesYes 37~C ~ 103 30 minutes No 37~C ND
60 minutes Yes 0~C 28xlO' 60 minutes Yes 5~C 35xl06 60 minutes Yes 21~C 75X103 1560 minutesYes 30~C < 10' 60 minutes Yes 37~C < 10~
60 minutes No 37~C 117X107 As can be seen by comparing the above results to those obtained in Examples 4 and 5, 3~6 hydroxymethylglycinate possesses greater viricidal activity than does 0.5~ hydroxymethylglycinate. The above results also indicate that viral inactivation by 3~ hydroxymethylglycinate is temperature dependent. For example, a greater than 5 log 2 5 decrease in viable virus was observed when the incubation temperature was increased from 0~C to 30~C
ExAMeLE 7 Fifty microliters of an o~ernight culture of E.
30 coli and 465 1l~ of blood plasma were added to each of ten tubes. 5 I:LL of a 50~ stock solution of hydroxymethylglycinate were added to each of eight of W097~20Z8 ~ ~ PCT~S~11152 2 ~
-2~-the ten tubes to give a final hydroxymethylglycinate concentration of 0.5~. The mixtures were incubated at the temperatures indicated below. At the times indicated below, samples were taken fro~ each tube, diluted, and plated. After overnight incubation at 37~C, colonies were counted. The results are summarized below in TABLE ~II.
TA3~E VII
T77.-.. h~ti~Ar tLrlg1ycimlte Inc7hst~0n Colonles lOTime 7~7re~t ~ rL~
30 minutes Yes 5~C 7X107 30 minutes Ye~ 21~C llx107 30 minutes Ye5 30~C l5XlOs 30 minutes Yes 370c c lo' 1530 minutes ~70 370c ND
60 minute~ Yes 5~C 41xlO' 60 minutea Yes 1l~7C 7xlO' 60 minutes Yes 300C c lo' 60 minutes Yes 37CC c 10' 2060 mirlUtes No 370c 35xlo' The above results indicate that hydroxy~7~ethylglycinate inactivates bacteria, as well as viruses, in blood pla3ma. The above results also indicate that the inactivation is temperature dependent.
Four hundred sixty-five microliters of blood plasma and 270 ~ of T4 stock were added to each of eight tubes. 5 ~ of 50% stock solution of hydroxy~ethylglycinate~ pH 9.0, were added to each of two of the elght tubes. A sample of the aforementioned hydroxymethylglycinate stock solution ~ W097l02028 p~l 9 ~ ~ 8 5 c was adjusted to pH 7.8 with solid sodium phosphate, and 5 ~L of the pH-adjusted hydroxymethylslycinate solution were added to another two of the eight tubes. 5 ~.7. of buffer, pH 9.0/ were added to another two OL- the eight tubes, tlhe re-~ining two tubes serving as controls. The mixtures were incubated at 30'C. At the times indicated below, samples were taken, diluted, mixed with host cells and overlaid onto solid medium. After overnight incubation at 37~C, plaques were counted. The results are summarized below in TABLE VIII.
T~3LE VIII
ADDITIYE ~ 3'1'~.~ TIIDS ~F~s Eydroxymethylglycinate30 minutes 29x103 pE 7.8 .7.7~L~ hylglycinate 30 minutes 18xlO' pX 9.0 Buffer 30 minutes 22x107 pX 9.0 None -- 30 minutes ~
EydroxymethylglycinateG0 minutes c103 pE 7.8 X~dL~I 'hylglycinate 60 minutes lOx103 pE 9.0 Buffer 60 minutes 78x107 pE 9.0 Uone 60 minutes 26x107 EXAMPLE g Each of the hydroxymethyl derivatives listed below was synthesized along the lines described in U.S. Patent No. 4,337,269 by mixing 10 mmoles of the corresponding L-amino acid with lo mmoles of 509 aqueous sodium hydroxide and 10 mmoles of 37~
formaldehyde. After overnight incubation at room W097~2028 ~ PCT~S96/11152 temperature, no free formaldehyde could be detected in any of the reaction mixtures.
~ 65 ~L of blood plasma and 30 ~L of T4 virus were added to each of nine tubes. In addition, to each tube was added 5 ~L of one oE the hYdL u~y,.,ethyl derivatives listed below, the re-~;n;nq tube having no hydroxymeth~l derivative and serving as a control.
The mixtures were incubated at 30-C for 60 minutes.
Samples were then taken, diluted, mixed with host cells and overlaid onto solid medium. After overnight incubation at 37'C, plaques were counted.
The results are summarized below in TABLE IX.
TABLE IX
~ K PE'U' 13 HYdLU~I ~ thylglycine ~103 Hydroxymethyl~l ~n;n~ 14X103 Hydroxymethylaspartate 7xlO~
Hy~Lv~ thyllysine 28xlOi Hydroxymethylornithine 33xlOs HYdLU~I thylproline 36xlOi HydrcXymethylserine 20x103 HydrcXymethylvaline 54xlO3 None 6xlO3 10 ~L of T4 virus stock and 8~ ~L of olood plasma were added to each of seven tubes. l ~I. of an appropriate hydroxymethy].valine stock solution was added to each of three of the plasma-containing tubes and l ~L of an appropriate hydrox-ymethylaspartate WO 97/02û2~ 7 ~ 5 PCTIUS96l11152 stock solution was added to each of another three of the plasma-containing tubes to give the below-listed concentrations. 1 ~LL of a buffer solution was added to the re-~;n;ng tube as a control. A~ter incubation 5 at 30 C for 1 hour, samples from each tube were taken, diluted, mixed with host cells, and overlaid onto solid medium. After overnight incubation at 37~C, plaques were counted. The results are summarized below in TAPLE X.
TAPLE X
lN~l~l~Ok ~uNu~.lL~ATION PFU's HydLu~y,.,ethylvaline O. 259~ 35xlOs Hydroxymethylaspartate 0. 25~ 12xlOs Hydroxymethylvaline 0;1% 42xlOs 15Hydroxymethylaspartate O .19~ 13X106 HYdL~Y. thylvaline 0.05~ 57X106 Hydroxymethylaspartate 0. 05% 20X106 None 0~ 29xlO' T4 virus stoclk and blood plasma were added to each of six tubes in the manner described above.
Hydroxymethyl-o-phosphorylethanolamine was added to each of two tubes, hydroxymethyl-trp-gly-gly was added to each of another two tubes, and a buffer containing no inhibitor was added to each of the remaining two tubes. The mixtures were incubated at 30 C. At the times indicated below, samples were taken from each tube, diluted, and plated. After overnight incubation at 37 C, plaques were counted.
The results are summarized below in TABLE Y.I.
W 0 97102028 ~ ;~ P~rAUS~6/1115 TP~3LE XI
I ihibltor Inccibation PF~'n Period Hydroxymethyl-o- 30 minutes llxlO' phosphoryI~thanolamine ~ydroxymeth~-trp-gly-gly 30 minutes 17xlO~
~ ne 30 minutes 7.~D
Hydroxylethyl-o- 60 minutes 40x103 ~ho~yllolylethanolamine Hyd-u~ ~thyl-trp-gly-gly 60 minutes 18xlO9 None 60 minutes 25x107 EX~MPLE 12 465 ~L of blood plasma, 30 ~L7.. of T4 stock solution were added to each of ten tubes. 5 ~L of a 50~ solution of l~yd~v~y .~ thylaminopropanol were added to each of t~o tube~. 5 ~L of a 50~ solution of hydLu~y~ Lhylpenicillinamine were added to each of another two tubes. 5 ~lL of a 50% ~301ution of hydroxymethylcysteine ethyl ester were added to each of another two tubes. 5 I~L of PB9 were added to each of another two tubes. 5 ~LL of a solution containing L~ r amount9 of NaOH and formaldehyde incubated overnight at room temperature were added to each of the two L. - i n i ng tubes. The mixtures were incubated at 30~C. At the times indicated below, samples were taken, diluted, mixed with host cells and overlaid onto solid medium. After overnight incubation at 37~C, plaques were counted. The results are summarized below in TA-BLE XII.
TP~3LE XII
}~hi~itor T~ ; rn PFU' 8 P-ri ~d }~y~L~ 'ylar~inopropanol 30 minu~.es llXlO' ~ydroxymethylp~niri1~i 'n~ 30 minu-es 8xlo' E~ydLu~ hylcysteine ethy:L 80 minu~es 6x107 ester ~ WO971020~ ~ f 8 ~ 8 5 PCT~S96/11152 Inhibitor Incubation PF~s - P~ri d None m.nu-es ND
~1~A1 jn;7~ formaldehyde ~ m.nu-es ND
HYdL~ hylaminopropanol m.nu-es SxlO~
E.'ydroxymethylr~ni~ill; 'n~ m-nu es 24xIOs 5HydL~ Lhylcysteine ethyl ~o m_nu-es 6x107 ester None 60 minutes 16xlO' ~1~A1 ;ni7~ formaldehyde 60 minutes 14xlO' 10465 IlL of blood plasma, 30 ~L of T4 stock solution were added to each of eight tubes. 5 ,UL of a 50% solution of hydroxymethylcysteine were added to each of two tubes. 5 ~LL of a 50~ solution of hydro-xymethyl-aminophenyl acetic acid were added to each of another two tubes. 5 ~LL of a 5~9~ solution of hydroxymethylaminoethanol were added to each of another two tubes. 5 I~L of a buffer solution serving as a control were added to each of the two Ll i n; ng tubes. The mixtures were incubated at 30 C. At the times indicated below, samples were taken, diluted, mixed with host cells and overlaid onto solid medium.
After overnight incubation at 37'C, plaques were counted. The results are summarized below in TABLE
XIII.
W0 97t02028 ~ r rCT/US96~ 5~ ~
T~3hE XIII
IN~IBITOR ~ ERIOD PFV'9 E}y~lL~ hylcysteine 30 minutes 7x107 llyd~ Lhyl- 30 Ininutes 23x10' 5aminophenyl ~cetic acid lamino- 30 minute~ 12X1o' ethanol None 30 minutes ND
~y~lL~ hyl ~ysteine 60 minutes 34x10' 10l~ydLv~. ''yl- 60 minutes ~3X10 aminophenyl acetic acid HYIL~ -hylamino- 50 minutes 11X1 et ~ ol None 60 minutes 29X10 465 ~ of blood plasma, 30 ~h of T4 stock solution were added to each of eight tubes. 5 ~L of a 50~ solution of hydL~yll~ethylr ~Lopropionylglycina were added to each of two tubes. 5 ~h of a 50~ solution of hydroxymethylmercaptoethylamine were added to each of another two tubes. 5 ~L of a 50~ solution of hydroxymethylaminoethyl hydrogen sulfate were added to each of another two tubes. 5 ~L of a buffer solution ser~ing as a control were added to each of the two i, -i n i ng tubes. The mixtures were incubated at 30 C. At the times indicated below, samples were taken, diluted, mixed with host cells and overlaid onto solid medium. After overnicht incubation at 37 C, plaques were counted. The results are summarized below in TABhE XIV.
~ W097~202~ 5 PCT~S96/ll~s2 '.i,~; ~;
T~3LE XIV
INSIBITOR INCUBATION PERIOD PFU o Hydroxymethylmercapto 30 minutes lSx10 propionylglycine HYdL~A~. hyl- l0 minutes 20xl0 mercaptoethylamine HvdroxymethylaminO- 30 mlnutes 23xl0' ethyl hydrogen sulLate None 30 minutes ND
10HydL~ hylmercapto- 60 minutes 36xl05 propionylglycine Hy~L~.. _th;l- 60 minutes 22x105 mercaptoethylamine HyvL~ -thylamino- 60 minutes 20X105 15ethyl hydrogen suliate None 60 minutes 17x10' EXAMP~E 15 ~ he virucidal activities of hydroxymethyl-p-aminohippurate, hydroxymethylpropargyl-glycine, and hydroxymethyl-o-phosphothreonine, respectively, were tested in the manner descri~ed above. The results are summarized below in T~3~E XV.
TAB~E XV
I.~3IBITOR T~rT7~7 ~T~ PERIOD PF~o 25HyvLv~ thyl-p-amino- 30 minutes <l0' hippurate HYdLV~ ~hylpropargyl_ 30 minutes ~l0' glycine H.ydLv~. 'hyl-o- 30 minutes lx10' 3 Gphospho-threonine None 30 minutes ND
HydLv~.. _thyl-p-amino- 60 minutes c10 hippurate HY~LV~. ~hylpropargyl- 60 minutesc10 35 glycine HydL~.. _thyl-o- 60 minutes cl0' phospho-tùreonine W097/02028 ... ~ PCT~S96/11152 ~ i~ 2 1 '7~0l~l5 ¦ I~LIBITOR ¦ TUrn~T~ PERIOD ¦ PF~
None 60 minute~3 4x107 The virucidal activities oi hydroxymethyl-aminoadipate, hydroxymethyl-o-phosphoserine, and hydroxymethylaminoethylphosphonic acid, respectively were tested in the manner described abo~e. The results are summarized below in TALLE XVI.
TA6Bi3 XVI
Ii~}Bl~OR INC~BATION PFRIOD Pi~0'-Hyd.u~3.. ~Lhylamino-30 min~tes sxl~3 adipate Hyvlv~ 'hyl_o- 30 minutes ~103 rh~crh~~~rine i~y h~ Llly1amino- 30 minUteB 2x103 15ethylrh~crh~ric acld None 30 minutes ~
~y~h~. L' ylamino- 60 minutes 103 adipdte ~y~L~ '- yl-o- 60 minutes <103 20i~ ' ine ~iy~iJv~ BLylamino- 60 minutes c10 ethylph~cFh~r;c acid U-one 60 m1nutes 24x10 EXAMiPL3 17 465 ~L of blood plasma and 30 ~L of T4 were added to each of six tubes. 5 ~L of hydroxymethyl-~h~Cph~ ~thylglyclne were added to each of two tubes, and 5 ~L of hydroxymethylmethylhydantion were added to each of another two tubes. 5 ~I- of buffer were added to each of the remaining two tubes. The W097~20~ ~I 9~ PCT~59~11152 mixtures were incubated at 30 C. At the times indicated below, samples were taken, diluted, mixed with host cells and overlaid onto solid medium.
After overnight incubation at 37 C, plaques were counted. The results are summarized below in TA~3LE
XVII.
T~3LB XVII
I~hibltor T~l.h.~; n~ pp~ J
P-riod EPdL~. hylrhnQrh~nn_ 30 minutes 28xlO~
methylglycine Hydroxymethylmethylhydantion 30 minutes i3xl06 None 30 minutes ND
Hydroxymethylphn.Qrhn~n 60 minutes ~ lo~
methylglycine H~dL~. thylmethylhydantion 60 minutes 37x105 None 60 minutes 25X106 EXAMP~E 18 The virucidal activities of hydroxymethylglycine 7-amido-~-methylcoumarin, hydroxymethyl-vinylglycinate, hydroxymethylfolic acid, hydroxymethyltaurine and hydroxymethyl-aminoethyl trimethyl ammor.ium chloride, respectively, were tested.in the manner described above at the concentrations indicated below. The resulte are summarized below in TAB~E XVIII
W0 97~2028 ~ 3'i~ ~ PCT/US96111152 S
: -36-TA~3~E XVIII
I~hibltor Inh~bitorPF-7'~.
conc-ntration NYdL~ rlglycine-7-amido-4- 0~5~ 12xloS
methylcoumarin NY~L~ h~lyinylg1yCinate O.SL, ~ 103 N~dL~ hylfolic acid 0.5% ~ 103 NY~LJ~ hyltaurine 0.55S 25xlo' NY~L~. hyl ~ mo~hyl trimethyl o~s% 40xlo' ammonium chloride NydL~r.~._LI~ylglycine-7-amido-4- 0.2s~ 21x105 methylcoumarin ~ydu~l. hyl~inylglycinate 0.25~ 88xl0' N~dL~. hylfolic acid 0.2s% ~ 103 NYdL~r. hyltaurine 0.2s~ 22xloS
XY;L~ hylaminoethyl trimethyl 0.2s~ 56xl05 ammonium chlor3,de ~y~lL~ l.rlglycine-7-amido-4- 0.13~ l9X105 methylcoumarin Rydroxymethyl~inylglyCinate o.l~ 67xl0' ilydL~ lfOlic acid Q.l~ lOXlO~
IIYdL~ thyltaurine o.l~ 30x105 -hylaminoethyl t3imethyl 0.1~ 38xlo~
ammonium chl~oride ~one - lsx1o3 EXl~MPLE 19 The virucidal activities of hydroxymethylglycinate, hydroxymethylaminoadipate, hydroxymethylaminoethyl.phosphonic acid, 3nd hydrox,vmethyl-o-phosphoserine, respectively, were tested in the manner described above at the con-centratione indicated helow~ The results are summarized below in TABLE XIX.
W097~2028 ~ 9 ~ PCT~S96111152 -3,~-TAB~E XIX
InhibitorInhibitor PF~ 5 Conc-ntration Hydroxymethy1glyCinate 0.25~ 9xlOs HYIL~, Lhyl. 'nn~S1rRt~ 0.25~ 14x10 EydL~ ulylaminoethylrhnsrhnni~ 0.25~ 22x105 acid H~dL~. ~hy1-o-l.l7.~ ~.ine 0.25t 32xlOs Hyd~ , 'hylglycinate 0.1~ 16xl05, Eydlv~."_t¦lyl; nnR~;rRt~ 0.1% 20x10 0 Hydroxymethylalllinoethylrhncrhnnic 0.1~6 7X106 acld Hyd~ ' hyl-o~ mSllln~ ine 0.li; 14X106 Hydroxymethylg1ycinate 0~055 2xl06 Hydroxymethyl 'nnR~irRt~ o,oS~ 7xlO' lS Hyd~ hylaminoethylrhnsrhnnic O.OS~31x10 acid Hydroxymethy1-o-~h~h~ Line O.OS~ 20X10 None - SxlO' The virucidal activities of hydroxymethylphospho-nomethylglycinate and hydroxymethylglycinamide, respectively, were tested in the manner described above at the concentrations indicated below. The results are summari2ed below in TABLE XX.
W097~20~ ~ a ~ ~ PCT~596/11152 nh oi tor Inhib~ tor PF17~ n n i- rA r i nn Hydroxymethylrhncrhnnn_ 0, St ND
methylglycinate Xydroxymethylglycinamide o.s~ 99xlQ~
Xyd.~ , thylrhncrhnnn- 0.25t 49xlO~
methylglycinnte Hy~ Lhylglycinamlde 0.25t l5xlO~
Xy~ . hylrhnsrhnnn- 0.1% 34X106 methylglycinate Xyd~ . thyl~lycinzlmide O.1~ 72xlO~
XY~ Ylrhnqrhnnn_ 0.05% SSxlO~
methylglycinate Xyd~ hylglycirlamide 0.05~
None - SSxlO' EXAMP~E 2l The virucidal. activities of hydroxymethylglycinate, hyd~y~ thy~m i n~hippurate, hydroxymethylpropargylglycine and hydroxymethyl-o-phosphothreonine, respectively, were tested in themanner described abo~e at the c~nc~ntrations indi-cated below. The results are summari~ed below in TAB~E XXI.
W097/02028 21 ~85 PCT/US96/11152 -3~-TABLE XXI
Inhihitor Inhibitor P
C~.~c~..L~tion HydL~ Lhylglyclnate 0.25'~ lSxlO' HYdL~" 3 Lhylaminohippnrate o.2SS6 54xl06 Hyd~ . hylpropargylglycine 0.259f 27x103 Hydroxymethyl-0-~ h~h~3.,.~ine 0.2S'6 lOxlO~
Hy~ Lhylglycinate O.lt 7xlO~
Hyd~ . 'hyl: 'n~hirp~r~ F. 0.1~ SX1O~ _ HydL~ . hylpropargylglycine 0.15~ 41xlOs Hydroxymethyl-0-rhnerh~3threonine 0.1~ 36xlO' HydL~ hylglycinate 0.056s 43x103 Hy~lL~ Lhyl: n~hirrl~r~r~ 0.051: 44Xl06 Hydroxymethylpropargylglycine o.OSt 29xlOC
HydLw~. '' yl-0-phosphothreonine O.OSt lSxlOs None - 32x107 The virucidal activities o~
hydroxymethylthreonine, hydroxymethylphos-phothreonine, hydroxymethylserine and hydro-20 xymethylphosphoserine, respective~y, were tested inthe manner described above at the concentrations indicated below. The results are summarized below in TABLE XXII .
W 0 9~2028 ~ 8 5 rCT/US96/11152 ~ TABLE XXII
I~ibltor Inh~bitor PF~7 EY~L~I. thylthreonine 0.25~ a~X10 dydroxyLnethylphoEphothreonine 0.25t 75xlO~
~;Y~L~/~ thylserine 0.2s~ 14xlO' Hydroxymethylrh~crhne~rine 0.25~ 42xlOs Hydroxymethylthreonine 0.1~ 45xlO~
HY~ILVA~, hyl~rh~ h~threonllle O~l~o 30xlOs Xy~.v~ thylserine 0.1~ 22Y10 HY~L~ tX~ h~ 7-~ine 0.17, 21X10 XyJ~ . chylthreonine O.05~ SX10C
HY.1L~, ~hr1Ph~~l10t}L.~I.1ne 0.05~ 43X107 XY~L~/ "hr1ECrine O.OS'L. 17X107 HY~L~. ChY ~.1.. _L';1'~'._Line 0.057, 51X10' 1~ None 42X107 EXPjMP LE 2 3 The virucidal activities of hydroxymethyl-MTh glycine, hydr~yl~ethyl-l-amino-l-cyclopropane carboxylic acid, hydroxymethyl-d,1~2-aminophosphonopropionic acid, hydroxy-methyl-p-aminobenzoic acid, hydroxymethylamino-b~tyrolactorle, hydroxymethyl-d,1--L~minoph~sphonobutyric acid, hydroxymethylaminopyrazole carboxylic acid, hydroxymethylazetidine carboxylate and hydroxymethyldiaminobutyric acid, respectively, were tested in the manner described above at the concentrations indicated below. The results are sumwarized below in TABLE XXIII.
W 0 97/02028 ~ PCTnJs96/lll52 -41~
TA;3LE XXIII
Inhibitor InhibitorPFiJ's -Cono_..LL~.Lion ayd-u~J. 'hyl-MTH-glycine O.S't 13X106 Hydroxymethyl-l-amino-l- o 5~ 17xlO' cyclopropane carboxylic acid HydLu~y~ LIIyl-d~1-2- 0.S~ c 10' I ' n~ hnb~ u~ionic acid HydLu~y.. _Lhyl-p-l nnh''n7OiC O.55~ sxlo3 acid Hyd-u~;.. _Lhylaminobutyrolactone 0.5~ 30xlO~
HydLu~.. _Lhyl-d,l- O.S~ c 10' ~m1n..l.l..~.,1.,.,.~.i...lyLiC acid Hydroxymethylaminopyrazole O.Sl 45xlO' i5 Hydroxymethyl azetidine 0. St 7xlO' carboxylate Hydroxymethylril: nnh~yric acid O.Ssc ~ 10~
HydLu~/ Lhyl-MTH-glycine 0.25~ lsx107 Hydroxymethyl-l-amino-l- 0.25~ 18xlO' cyclopropane carboxylic acid HydLu~J.. _Lhyl-d,1-2- 0.25~ llxlO' ~ iOniC acid HydLu~J, 'hyl-p nnh~n7~1c 0.25~ 30xlO' acld Hyd~u~.. _LLylaminobutyrolactone 0.255. ND
Hydroxymethyl-d,1- 0.25~ c 10' ".. l,1\.. ~l.h.. :uLyLiC acid Carboxyïic aciadmin~PYrazole 0.25% 21xlO~
Hydroxymethyl azetid-n,e 0.25~ 16xlO' carboxylate HydLu~.. Lhylri; nnh-l~yric acid 0.25~ 40xlO' Hydroxymethyl-MTH-glycine O.li 18xlO' Hydroxymethylglutamate 0.2si~ 60xlO' HydLu~",_Lhy1m~hlnn;n~ 0.25~ 68xlOs HydLu~ hylserine 0.25~ 25xlO' Hy~L~J~-Lhylglutamate 0.1$ 12xlO~
Hydroxymethyll ~hinnllle O,l~Lo 28X106 Hydroxymethylserine 0.1~ 27xlOs W 0 97~2028 ~ ~ rCT/US96/11152 ~ 3 q~85 -42-In~ibitor Inhibitor PFV'~
Concentrution Hydroxymethylglutam~ate O.OSt 9X10' Hydroxymethy1mpthimninp o 05~ 7xlO' Hy~ , 'h~yl~erine 0.05t 7xlO~
~one - 9xlO' 8~ /~L of blood plasma and 10 ~L of T4 stock solution were added to each of ten tubes. 1 ~ of an appropriate hydroxymethylglutamate stock solution was added to each of three tubes to give the concentrations indicated below. 1 ~L of an appropriate hydroxymethylmethinnlnp ~tock solution was added to each of another three tubes to give the concentratians indicated below. 1 ~L of an appropriate hydroxymethylserine stock solution was added to each of another three tubes to give the concentrations indicated below. 1 ~L of buf~er was added to the r~ -1n;ng tube, which ~erved as a control. The mixtures were incubated at 30-C. After 1 hour, sam.~ples were taken, diluted, mixed with host cells and o~erlaid onto solid medium. After overnight incubation at 37 C, plaques were counted.
The results are summarized below in T~3LE XXIV.
W097/02028 ~1 9~ 5 PCT~S96~11152 TABLE XXIV
Inhibitor InhibitorPF~'s EYdLU~Y~ ~hylglutamate 0.25'L.60xlO~
Hydlu~. -hy1methionine 0.25%65xlOs HY~LU~. ''ylserine 0.255, 25xlO~
Hydroxymethylglutamate 0.1~12X10~
HY~LU~ 'hylm~hinnin~ O.lt 28xlO' HY~LU~Y thylLerine O ~ lt 27xlOs HYdLU~ ~hylglutamate 0~0S~L.9xlO' Hydroxymethyl~6~hinn;nP 0 OS'L 7xlO~
HydLu~ thylserine 0.05~ 7x106 None - 9xlO' 89 ~L of blood plasma and lO ~L of T4 stock solution were added to each of ten tuhes. l ~L of an appropriate hydroxymethylserine stock solution was added to each of three tubes to give the concentrations indicated below. l ~B of an appropriate hydroxymethyl-~-alanine stock solution was added to each of another three tubes to give the concentrations indicated below. l ~L of an appropriate hydroxymethylglycine stock solution was added to each of another three tubes to give the concentrations indicated below. l ~L of buffer was added to the r~ ;ning tube, which served as a control. The mixtures were incubated at 3G C. After l hour, samples were taken, diluted, mixed with host cells and overlaid onto solid medium. After overnight incubation at 37 C, pla~ues were counted.
The results are summarized below in TABBE XXV.
W097~2028 ~ PCT~S9~11~2 TA33T~E XXV
Inhibitor IDhlbitor P~5'5 }}yd.~ 'hyl~erine 0.25Y~ 8xlO' Hydroxymethyl-B-alanine 0.25~ lSxlO~
Hy.l~ glycine 0.259s 9xlO' H~ hyls~rin~ 0 . 156 14xlOs Hy~ hyl-B-alanine o.l~ 83xl05 Hydroxymethylglycir~e o.lt 8xlO' HY~L~. 'ylserine o.os~21X106 HY~L~. hyl-6-alanino o.os~3~xlO~
HydL~.r.c~11y1~lYCine o.os~ 26xlOs None - 1 2xl 0 ' The following is a listing of virucidal activities observed by the present inventors for a variety oi- hydroxymethyl derivatives ~virucidal activity being expressed in terms o~ PFIJ~s~:
Hydroxymethylglycine Hydroxymethylphosphonomethylglycine 20 Hydroxymethyl-p-~m; n~hi ppurate Hydroxymethylpropargylglycine ~ydroxymethyl-o-pho~phoserine HydrDxymethylaminoethyl-phosphonic acid Hydroxymethylleucine ~5 Hydroxymethyl-~-alanine Hydroxymethylcysteine Hydroxymethylphenylalanine ~ W097/0~8 7 I f ~ C, ~ 5 PCT~Sg6111~52 lo3 Hydroxymethylaminophenylacetic acid ~ydroxymethyl-o-phosphorylethanolamine Hydroxymethylalanine Hydroxymethylserine Hydroxymethylvaline Hydroxymethylmethionine Hydroxymethylglutamate Hydroxymethylaspartate Hydroxymethyllysine Hydroxymethylproline Hydroxymethylmercaptopropionylglycine Hydroxymethylmercaptoethylamine Hydroxymethylaminoethyl hydrogen sul~ate Hydroxymethylaminoethanol Hydroxymethylpenicillamide Hydroxymethylhydantior.
2G Hydroxymethylornithine ~ydroxymethylthreonine Hydroxymethylcysteine Hydroxymethylaminopropanol W097/02028 ~ PCT~5~1111~2 2 1 ~0~
-4Ç-1~7 Hydroxymethyluridine Hydroxymethylphth~7; m; de Dimethylurea S Hydroxymethylcysteineethyl ester Hydroxymethylleucinamide Hydroxymethylarginine Hydroxymethyltyrosine 10~
~ Hydroxymethyldeoxyuridine 4-hydroxymethylimidazole 6-hydroxymethylpter~n Hydroxymethylacrylamide Hydroxymethylcytosine 1.S Hydroxymethyl-Ç-methyluracil Hydroxymethylnico~;n~mi~7~
Hydroxymethyl-trp-gly-gly Hydroxymethylglutamine Diazolidinylurea Imidazolidinylurea ~ W097102028 2 ~ q 8 0 8 ~ PCT~S96/11152 Kinetic Studies A kinetic model was developed to mon tor the complexation between hydroxymethylglycinate (HMGi and a virus. In the kinetic model it was assumed that the reaction of one molecule of HMG with a virus was sufficient to inactivate the virus. Therefore, the overall reaction can be expressed by the simple rate law:
HMG + V ~ VHMG
where "V" is a virus. A large excess of HMG was present in the reaction conditions and therefore a pseudo first order equation could be used.
Furthermore, it was assumed that in a blood bag, no mass transfer effects exist which would affect the reaction kinetics.
It was found that the rate of inactivation follows the model prediction and that the virus can be inactivated in plasma, whole blood and red blood cell concentrate (RBC). The rate is also increased as the temperature of the reaction conditions is increased.
Inactivation of SV4-0, Reovirus, Porcine Parvo virus and Polio virus were demonstrated. For SV4-0, Reovirus and Polio virus, the number of viable cells ~ 25 was reduced below detectable limits. Plasma hemoglobin, potassium, sodium, 2,3-~PG, ATP and lactate were unchanged with reference to a control when treated with HMG at a final concentration of 1600 ppm.
W097~20tX ~ PCT~Sg~lllSt 2 ~ 8 ~ 8 5 The e~bodiments of the present invention recited herein are intended to be merely exemplary and those skilled in the art will be able to make numerous variations and modifications to it without departing from the spirit of the present invention. All such variations and modificstions are intended to be within the scope of the present invention as defined by the claiws appended hereto.
FIE~D OF THE INVENTION
This invention relates to methods for inactivation of pathogens.
BACKGROUND OF THE INVENTION
Spread of infectious disease resulting from transfusion of cont~m;n~te~ blood, administration of contaminated blood products or handling or usage ~f objects that have come into contact with contaminated blood and/or blood products,has been well documented and is recognized as a major public health concern.
Most notably, tr~n~ q;on of viral hepatitis and/or Acquired Immune Deficiency Syndrome (AIDS) through contaminated blood and blood products has received widespread attention. However, viral hepatitis and AIDS are only two of the many diseases that can be spread through use of contaminated blood and blood produc~s. ~esser known pathogens, such as T-cell lymphotropic vlruses ~Types I and II), cytomegalovirus, Epstein-Barr virus, the parvoviruses and Plasmodi~,m~ (malaria-causing) protozoa, may also be spread through contaminated blood and blood products. In addition, still other microorganisms that have not yet even been identified or recognized as being pathogeni~ may be transmitted through c~n~m;n~ted blood and blood products and, therefore, similarly pose a serious public health risk. The HIV
virus is illustrative of a pathogen that, until ~ recently, was not even recognized. Today, there are over l0 million people worldwide who have contracted AIDS, many of these people having contracted the disease throush use of infected blood or blood products; however, less than two decades ago, AIDS
WO97l~28 ~ PCT~S96/11152 .r) was not even a recognized disease. Thus, it is clear that there is a great need for a method for effectively inactivating pathogens in blood and b~ood product6.
In response to this need a number of techniques have been devised for inactivating pathogens, particularly infectious viral agents, in blood and/or blood products. A review of many of ~hese techniques is presented in Suomela, "Inactivation of Viruses-in lC Blood and Plasma Products," T~ans~usion ~edicine ~eviews, Vol. VII, No. l, pp. 42-57 (~anuary l9g3~, which i9 incorporated herein by reference.
One such technique which has been used to inactivate viruEes in blood and/or blood products is pasteurization. [See Burnouf-Radosevich et al., "A
Pasteurized Therapeutic Plasma, " ~nfusionstheraDie, 19:9l-94 (l992~] The pasteurization of blood and/or blood products is most often effected by heating them in the liquid state for lO hours at 60OC. A small amount of protein stabilizer, such as caprylate or tryptophanate, is often added to the preparation.
After pasteurization has been completed, the stabilizer typically must be removed from the preparation prior to its clinical use. As is the case with many of the existing viral inactivation techniques discussed herein, pasteurization is more effective in inactivating enveloped viruses (i.e., viruses having a lipid envelope surrounding the viral capsid) than in inactivating non-enveloped viruses ~i.e., viruses which lack a lipid envelope surrounding the viral capsid).
W097/020~ 5 PCT~S96/11152 Another technique which has been used to inactivate viruses in blood and/or blo~d products is the solvent/detergent (S/D) method. [See, for example, Hellstern et al., "Manufacture and in vitro S Characterization of a Solvent/Detergent-Treated Human Plasma, " Vox Sanq, 63:178-185 (1992); Horowitz et al., "Solvent/Detergent-Treated Plasma: A Virus-Inactivated substitute for Fresh Frozen Plasma,ll Blood 79:826-831 (1992); and Piquet et al., "Virus Inactivation of Fresh Frozen Plasma by a Solvent Detergent Procedure: Biological Results, Vox Sanc, 63:251-256 ~1992).] The S/~D wethod, which is limited to use in inactivating enveloped viruses, involves treating a blood preparation with an organic mixture which disrupts the lipid envelope of enveloped viruses. The disruption of the lipid envelope leads either to complete structural disruption of the virus or to destruction of the cell receptor recognition site on the virus. In either case, the virus is rendered noninfectious. The solvent used in the S/D
method is most often tri-(n-butyl~phosphate ~T~BP), and the detergent is either Tween 80 , Triton X-100' or sodium deoxycholate. Temperature and time influence the efficacy of the S/D method, typical temperatures being in the range of 24~ C to 37~C, and the typical duration of treatment being at least 6 hours.
Still another technique which has been used to inactivate viruses in blood and/or blood products is photochemical inactivation. [See Mohr et al., ~Virus Inactivated Single-Donor Fresh Plasma Preparations,~
Infusionthera~ie, 19:79-83 (1993); Wagner et al., "Differential sensitivities of viruses in red cell suspensions to methylene blue photosensitization,~
_ _ _ _ _ _ _ _ _ _ , _ _ . _ _ _ _, _ _ , , .. .. . . . , _ . ...
WO97/02028 PCT~96111152 3 ~ ~ ~
Transfusion, 34~6):521-526 (19~4); Wagner et al., ~Red cell alterations associated with virucidal methyler.e blue phototreatment," Transfus_s~ 30-36 ~1993); Mohr et al., "No evidence for neoantigens in human plasma after photomhPm;r~l virus inactivation,"
Ann. Hematol.. ~5:224-229 ~1992); Lanbrecht et al., PhotoinactivatiOn of Viruses in Human ~resh Plasma by Phenothi~7in~ Dyes in ~ n with Visible Light," Yox San~, 60:207-213 (19911, Goodrich et al., "Selective inactivation of viruses in the presence of human platelets: ~V sensitization with psoralen derivatives," Proc. Nat. Acad. Sci. USA. 91:5S52-555 (1994); ~irus Inactivation in Pl~r~ Products, J.-J
Morgenthaler, ed. Xarger, NY (1989); and BioWorld Today. Vol. 4, Nc. 229, pages 1 and 4 (November 24, 1993).] The photochemical inactivation of a blood preparation typically involves treating the blood preparation with a photoactivatable chemical and then irr~iA~in~ the preparation with light of a sufficient wavelength to actlvate the photoactivatable mh~m;C~l, Examples of photoactivatable rhPm; ~1 s used in the photochemical inactivation of viruses preaent in ~lood preparations include psoralens, hypericin, methylene blue and toluidine blue. It is believed that psoralens, which have an affinity for nucleic acids, inactivate viruses by intercalating between viral nucleic acid base pairs and, in the presence of WA light, ~orming a covalent bond with the viral nucleic acid, thereby preventing its transcription and/or replication The nanner in which hypericin, methylene blue and toluidine blue inactivate viruses is not as well defined as that for psoralens. However, it is believed that these chemicals, when photoactlvated, generate the highly reactive entity, singlet oxygen, ~ W097102028 PCT~S9~11152 ~ ~ 17'8l}~5 which then attacks the cellular structure ~e.g. viral ~ envelope) of the virus.
Whereas photochemical inactivation has been largely successful in inactivating enveloped viruses, it has been largely unsuccessful in inactivating non-enveloped viruses. The failure of photochemical inactivation to inactivate non-enveloped viruses is significant since Poliovirus, Adenovirus, Hepatitis A
and Parvovirus (Parvo Bl9) are among those non-enveloped viruses that are pathogenic to humans.
It should be noted that photochemicalinactivation of the type described above has been most successful. when applied to inactivating viruses in blood preparations lacking red blood cells (e.g., plasma). This is because blood preparations that include red blood cells typically absorb light at the same wavelengths used to photoactivate the chemicals.
Viral inactivation agents are substances that render viruses incapable of replication and proliferation. From the literature discussed above, one may conclude that viral inactivating compounds have been ident.ified which are specifically toxic to blood borne viruses such that cells and proteins are not adversely affected. Still it is important to limit exposure of biological samples to viral inactivation agents, such as, for example, psoralens, hypericin, methylene blue, toluidine blue or a combination of tri-(n-butyl) phosphate and a detergent such as Tween 80, Triton X-lO0 or sodium deoxycholate to the minimum extent necessary to reduce potentiall~ signification interactions that could lead to undesirable side effects.
~097~ PCT~S~/11152 'u' g 5 U.S. Patent 4,337,269 (Berke et al.) disclosed a biocidal composition containing a compound, hydroxymethylaminoacetate talso referred to here~n as hydroxymethylslycinate), which is produced by the reaction of glycine or a salt of glycine with formaldehyde. In the aforementioned patent, hydroxymethylglycinate is said to be effective at inhibiting the growth of bacteria, yeasts and molds in a variety of substances susceptible to microbial ~nt~m;n~tion, such as cosmetics, foodstuffs, pharmaceuticals, paintst cutting oils or fluids, agricultural products, oil.drilling fluids, paper industry, embalming solutions, cold steril-i~ation medical and dental equipment, cooling towers, fabric impregnation, latexes, swimming pools, inks, household disinfectants, waxes and polishes, toilet bowl cleaners, bathroom cleaners, laundry detergents, soaps, wood preservatives, hospital and medical antiseptics and adhesives.
Sodium hydroxymethylglycinate is the active ingredient in the preservative SUTTOCID~A, which i~
commercially available from Sutton ~aboratories, Chatham, New Jersey. In certain promotional literat.ure published by Sutton Laboratories, 2~ SUTTOCID~A:is said to be active against Gram-negative and Gram-positive bacteria, yeast and mold and is suggested for use. as a preservative in shampoos, hair conditioners and ~facial treatments.
U.S. Patent 4,900,l~6 ~Berke et al.) disclosed a composition c~nt~;ning one or more 3-isothiazolones and a compound which is a member selected from the group consisting of hydroxymethyl-aminoacetic acid, its salts and lower alkyl esters. The aforementioned W097/02028 ~ 7 8 o g 5 PCT~S96/11152 composition is described in the patent as being effective against bacteria, yeasts and molds.
Suggested applications in the patent for the above-described composition inolude use as a preservative .~ in cosmetics, toiletries and household cleaning products, use as a biocide for synthetic latexes, emulsion paints and other coatings, adhesives, polishes, carpet backing compositions, surfactants, metalworking fluids, industrial and domestic water treatment including cooling tower systems and swimming pools, adhesive mats, drilling mud formulations, painting pastes, spin finish emulsions, polymer dispersions and fuels and as a slimicide for slime control in the manufacture of paper from wood pulp.
It is to be noted that nowhere in the foregoing patents or publications is it taught that the biocidal activity of hydroxymethylaminoacetic acid, its salts and/or low~r alkyl esters can be extended beyond bacteria, yeasts and molds to include viruses.
Moreover, because most biocidal agents that are effective against bacteria, yeasts and molds are not effective against viruses, the foregoing patents and publications do not provide any reasonable basis for one of ordinary skill in the art to expect that hydroxymethylaminoacetic acid and~or its derivatlves would be effective in inactivating viruses.
It is also to be noted that nowhere in the ~ foregoing patents or publications is it taught that hydroxymethylaminoacetic acid and/or its derivatives can be used in blood and/or blood products.
Moreover, because most biocidal agents that are effective against bacteria, yeast and/or molds cannot _ _ _ _ _ _ _ _ _ _ _ _ _ . . ... . . _ _ ..... , . . , . .. _ W097~020~ ~ PCTA~96/~1152 be used to inactivate such r~thn~r~q in blood an/or blood products without adversely affecting the suitability of the treated blood andjor blood product for subse~uent adoinistration to a patient ldue to their toxicity and~or their reactivity with plasma proteins and certain ot.her blood constituents), the foregoing patents and publications do not provide any reasonable basis for one of ordinary skill in the art to expect that hydroxymethyl~m;nna~Ptic acid and/or its derivatives could be used in blood and/or blood products without rendering the treated blood and~or blood product unsuitable for subsequent administration to a patient in need thereof.
A180 of interest is Japanese p-lh ;~h~d Application No. 62-195304, which disclosed that paraform (84~, 25g) was added to 98~ die~h~nnl~m;
(332g~ at 40 degrees, and then stirred at 5C-60 degrees for l hour to yield hydroxymethyldi-~thanolamine. It is also disclosed that hydroxymethyl-diethanolamine, at 300-500ppm, controlled Escherichia coli, Staphylococcus aureu6, Pgeu' .5 aeruginosa, ~acillus subtill6, Proteu6 vulgari6 and P. mirabilis on nutrient agar plates.
It is to be noted, howe~rer, that nowhere in the foregoing Japanese application is it taught or suggested t:hat hydroxymethyldiethanolamine has virucidal activity or that hydroxymethyldiethanol-amine could be used in blood and/or blood products without adversely affecting the suitability of the treated blood and/or blood product for subsequent administration to a patient in need thereof.
Therefore, a need exits for a method for treating blood which inactivates viruses without _ _ , _ _ _ _ _ _ _ . . . . . . . _ _ _ . . .
~ W097102028 ~ 9 8 ~ PCT~S96/11152 _ g _ adversely affecting the suitability of the treated blood and/or blood product for subsequent administration to an individual in need thereof.
SUMMARY OF THE INVENTION
The present invention is a method of inactivating a virus in a biological fluid, such as blood. In the method, the biological fluid is contacted with a hydroxymethylamine (HMA) in sufficient quantity to inactivate the virus ~i.e.-, an l~ effective amount). The biological fluid can be of any type including, but not limited to, whole blood and a wide variety of blood components, including, but not limited to, red blood cells, red blood cell concentrate, platelets, platelet concentrate, platelet rich plasma, platelet poor plasma, source plasma (plro-ar~re5is plasma), fresh frozen plasma, plasma proteins (e.g., clotting factors VIII, ~, etc.), and other body fluids, such as lymph, cerebrospinal fluid, semen, saliva, etc. While targeted at the inactivation of viruses, the method is effective against other microorganisms as well.
These microorganisms can be pathogenic or nonpathogenic and include bacteria, yeasts, molds and protozoa.
In one embodiment, this invention provides a method for inactivating a microorganism contained in a biological fluid. The method comprises the step of contacting the mi~Luu~y~llism with an effective amount of a hydLu~yl~rth~rlamine (HMA). Suitable hydroxy-methylamines include compounds of Formula (I) W097~ PCT~S96/11152 N-CH2-OH ~I~
RL
wherein:
R is chosen from the group consisting of hYdL~L, alkyl, aryl/ substituted alkyl, substituted aryl; and R1 is chosen from the group consisting of acid-, amide-, hydroxy- or mercapto-functional alkyl; acid-, amide-, or hydroxy-functional aryl; acid-, amide-, or hydroxy-functional substituted alkyl; and acid-, amide-, or hydroxy-functional substituted aryl;
or R and R1 may be joined together to form an acid, amide or hydroxy-functional heterocyclic structure.
Preferred XMA~ are those in which the functional group is an amide or an acid selected from the group consisting of carboxylater phosphate, phosphonate, sulfate and sulfonate. Carboxylic acids are particularly preferred.
Preferred individual hydroxymethylamines having the amide functionality include hydroxymethylglycin-amide, hydroxymethylpenicil1;n~m;~ hydroxymethyl-leucin~mlfl~, hydroxymethylacrylamide and hydroxy-methylnicotinamide. Preferred hydroxymethylamineshaving the acid functionality include hydroxymethyl-glycine, hydroxymethylphosphonomethylglycine, hydroxymethyl-p-aminohippuric acid, hydroxy-methylpropargylglycine, hydroxymethyl-o-3~ phosphothreonine, hydLu~y -thyl~m;n~;pic acid, hydroxymethyl-o-phospho5erine, hydroxymethylamino-ethylphosphonic acid, hydroxymethylleucine, hydroxymethyl-~-alanine, hydroxymethylcysteine, hydroxymethylfolic acid, hydroxymethylamino-~ W097~2028 7 1 ? ~ ~ g 5 PCT~S96/11152 phosphonobutyric acid, hydroxymethylphenyl~l~n;n~,hydroxymethylami.nophenylacetic acid, h~droxymethyl-o-phosphorylethanolamine, hydroxymethyl~1 ~ni ne, hydroxymethylserine, hydroxymethylvaline, hydroxymethylmethionine, hydroxymethylglutamic acid, hydroxymethylaspartic acid, hydroxymethyllysine, hydLo~yl-thylproline~ hydroxymethylmercaptopropionyl-glycine, hydroxymethylaminoethyl hydrogen sulfate, hydroxymethylpenicillamine, hydroxymethylornithine, and hydroxymethylcysteine. Preferred hydroxymeth'yl-amines having neither an acid nor an amide functionality include hydroxymethylmercaptoethyl-amine, hydroxymethylaminoethanol, hydroxymethyl-aminopropanol and hydroxymethyldiethanolamine.
A particularly preferred HMA is hydroxymethyl-glycine or a salt thereof.
According to the method of the invention, the hydroxymethylamine and biological fluid are preferably combined to produce a final concentration 2G of IlYdLC~Y.. thylamine of approximately 0.05 ~ - 3.0 by weight; the contact time is from 0.5 hours to 4 hours, and the t.emperature is maintained between about 4~ C and about 30~ C.
In another aspect, the invention relates to a method of processing a biological fluid intended for administration to an individual in need thereof. The method comprises the steps of: (a) treatins the biological fluid with an effective amount of a pathogen-inactivating hydroxymethylamine, thereby 3G producing a treated biological fluid; and ib) then removing free hydroxymethylamine from the treated biological fluid.
W097~2028 ~ PCT~96111152 ;~ ~ 9~3~
In another aspect the invention relates to a method of treating an individual in need of a biological fluid. The method comprises the stepsrof:
(a~ treating the biological fluid with an effective amount of a pathogen-inactivating hydroxymethylamine~
thereby producing a treated biological fluid; and (b) administering the treated biological fluid to the individual in need thereof.
In another aspect, the invention relates to a method of treating a biological fluid. The method comprises combining an effe~tive amount of a virus-inactivatin~ hydLu~y,-,ethylamine with the bio~ogical fluid, whereby at least about a lO-fold reduction in plaque forming units of virus is realized.
Subsequently, the virus-inactivating compound can be removed from the biological fluid prior to its administration to an individual. If the biological fluid is blood, the treated blood can be returned to the individual ~rom whom it was obtained.
Alternatively, the treated blood can be stored and administered later in time to the same individual or another individual in need thereof.
The me~hod of the invention can also be used to inactivate pathogens present in bodily fluids other than blood, and to disinfect medical instruments and analytical equipment that have come into contact with potentially cont~m;n~tP~i blood. Similarly, the method of the invention can also be used to disinfect blood samples that are not i nt~n~d for subsequent administration to an individual, but rather, are intended for subsequent chemical analysis. Other possible applications of the invention are apparent to those skilled in the art.
W097~2028 ~ 9 ~$~ PCT~S~111152 Additional objects, as well as features and advantages, of the present invention will be set forth in part in the detailed description which follows, and in part will be obvious from the s detailed description or may be learned by practice of the invention. Various Pmho~;l ts of the inventions will be described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be under9tood that other embodiments may 10 be utilized and that changes may be made without departing from the scope of the invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is best defined by the appended claims.
DETAI~ED D~SCRIPTION OF THE INVENTION
The present invention relates to the method of use of HMA's as pathogen-inactivating agents, particularly viral-inactivating a~ents, in biological samples, such as blood or blood products.
Use of HMA for treatment of ~athoqens The present invention is based, in part, or the unexpected discovery that a HMA can be used to inactivate viruses in biological samples. The ~resent invention is also ba9ed, in part, on the unexpected discovery that hydroxymethylamines such as those of Formula (I), can be used to inactivate viruses in blood and/or blood products, without rendering the treated blood andfor blood products unsuitable for - 30 subsequent administration to an individual.
WO 97/02028 ~ 3 {3 S; r PCTIUS96111152 Suitable pathogen-inactivating ~e.g., ~irus-inactivating) HMA's for use in the invention can be readily selected by those skilled in the art using art-recognized methods and the test methodology set forth ir. the accompanying Examples. For the purposes of the specification and claims, an HMA that reduces the number of plaque-forming units ~PFU) in a biological fluid by a factor of at least iO (i.e. one log reduction) is a pathogen-inactivating HMA. The ability to reduce the number of PFUs can be assessed using art-recognized methods, one example of which is described in Example l. If an HMA reduces the number of PFUs by at least one log unit, as assessed by the method described in Example l, it is a pathogen~
inactivating XMA. The term "hydroxymethyl~' when used herein in connection with a particular amino compound, designates that the rr-ro-ln~ has a -CH20X
substituent on the amino group.
SuitablQ alkyl groups in compounds of Formula (I~ include straight chain, branched chain, and cyclic alkyl groups, crn~ininrJ one to about 22 carbon atoms, more preferably one to about 12 carbon atoms. ~hen the alkyl group is a cyclic alkyl, 3-, 4- 5-, 6-, and 7- membered rings are preferred.
Suitable aryl groups in compounds of Formula ~I) inc}ude hydrocarbon aryl groups r~nt~ining a 6-membered aromatic ring, such as phenyl~ fused bicyclic systems such as ~- and ~2-naphthyl, histidine, indenyl, tetralinyl, and the like, and monocyclic and polycyclic heteroaryl groups crnt~ining a 5- or 6- m~mbeL~d heteroaromatic ring, e.g., pyridyl, pyrimidinyl, quinolinyl, furanyl, ~ wo g"02028 2 1 9 ~ PCT~S~111152 thienyl, isothiazolyl, isoxazolyl, imidazolyl, lH-pyrrolyl, indolyl, purinyl, and the like.
Suitable substituents in a substituted alkyl or substituted aryl group include halogen ~e.g., fluoro, chloro, bromo), hydroxy, alkoxy (e.g., alkoxy containing one to 8 carbon atoms), alkylthio (e.g., alkylthio wherein the alkyl group contains one to 8 carbon atoms), lower alkyl (i.e., alkyl cont~ining one to four carbon atoms), cycloalkyl (e.g., cyclopropyl, cyclopentyl, cyclohexyl~, phenyl, benzyl, benzo, mercapto, or combinations thereof.
R and R~ of Formula (I) can together form an ~-heterocyclic ~tructure (i.e., a cvclic structure wherein the hydroxymethylated nitrogen is an atom in the cyclic structure), such as a proline, pyrrolidine, piperidine, 2-pyrroline, indoline, aziridine, azetidine, and the like.
In a preferred ~mho~im~nt, the HMA contains a carboxylic acid portion as part of at least one of the substituted alkyl or substituted aryl groups, so that the HMA falls within the broad class of N-hydroxymethylated aminoacids. The aminoacid is not restricted to naturally occurring ~-aminoacids, although they provide particularly convenient starting materials, and their residues are optimally biocompatible.
When the functional group is acidic, salts of the HMA, such as metal salts ~e.g. sodium and potassium salts), and ammonium salts may be used.
W09~20~ ~ PCT~S~11152 ~i s ~ j ~ f~
Preferred ~MAs includ~ l-ydL~y-..cthylglycine, hydroxymethylphosphono~ethylgiycine, hyd;oxymethyl-p-aminohippurate, hydroxymethylpropargylglycine, hydroxymethyl-o-phosphothreonine, hydroxymethyl-aminoadipate, hydroxymethyl-o-ph~crhrqerine, hydroxymethylamino-ethylphosphonic acid, hydroxymethylleucine, ~ydL~y ~hyl-~-alanine hydroxymethylcysteine, hydroxymethylfolate~
hydroxymethylaminophob~ obutyric acid, and hydro-xymethylpheny~ n;n~ and their .~L,e~ dlng sa~ts.~ydroxymethylglycine, hydroxymethylfolate, hydroxymethylAminf;rh~h~ uLyLic acid, hydroxymethylpropargylg.lycine and hydroxymethyl-o-phosphothreonine are particularly preferred.
Other suitable HMAs include hydroxymethylamino-phenylacetic acid, hydroxymethyl-o-phosphorylethanol-amine, hydroxymethyl~l An i n~ ~ hydL~y. Lhylserine, IIYdL~YI Lhylvaline, hydroxymethylmethionine, hydroxymethylglutamate, hydroxymethylaspartate, hydroxymethyllysine, hydLo~y~ thylproline, hydroxymethyl~- ~p~opropionylglycine, hydroxymethyl-mercapto-ethylamine, hydroxymethylaminoethyl hydrogen ~ulfate, hydroxymethylamino-ethanol, hydroxymethyl-penicillamine, hydroxymethylhydantion, hydroxymethyl-ornithine, hydroxymethylcysteine, hydroxymethylamino~propanol, hydroxy~ethyldiethanolamine~ and their corr~.cron~ing salts.
HMAs can be readily synthesized by those skilled in the art. For example, to synthesize a hydroxymethyl-Amin~arid~ one equivaler.t each of the corresponding amino acid, formalin and sodium hydroxide are combined.
W097~ZOZ8 ~i ~ 8 a ~ ~ PCT~S~111152 One application of this invention is a method of inactivating a pathogen present in a biological sample, such as blood or a blood product intended~for administration to an individual in need thereof, said method comprising the step of treating the biological sample, such as blood or a blood product with an effective amount of a pathogen-inactivating acid- or hydroxy-functional HMA.
In accordance with the ~ hi ngS of the present invention, treatment of a biological fluid, such as blood and/or a blood product~comprises combining an appropriate quantity of a pathogen-inactivating HMA
with the biological sample and then allowing the sample to incubate for an appropriate period of time at a suitable temperature. The final concentration of the HMA in the sample is preferably approximately 0.05~-3.0~, more preferably approximately 0.5~. The incubation period is sufficiently long to inactivate pathogen in the sample, commonly from about 0.5 hour to about 4 hours, conventiently about l hour, and the incubation temperature is about 18'C to about 37 C, more preferably about 20 C to about 30 C. In a specific embodiment, the treatment of a blood and/or blood product. comprises combining sodium hydroxy-methylglycinate with the sample to give a finalsodium hydroxymethylglycinate concentration of 0.5~, then allowing the sample to incubate for approximately 60 minutes at a temperature of about 30 C.
The above-described method can be used to ~ inactivate viruses, bacteria, molds, yeasts, protozoa and other pathogens in biological samples, such as whole blood and a wide variety of blood components, W097/o~ PCT~S9~11S2 2~ J Q ~
lncluding, but not limited to, whole blood, red blood cell component, red blood cell concentrate, platelet component, platelet concentrate, platelet rich plasma, platelet poor plasma source plasma, fresh ', frozen plasma and plasma proteins. As mentioned above, one advantageous aspect of the present method is that it does not render a biolo~ical sample unsuitable for subsequent administration (e.g., transfusion) to an individual.
It i9 believed that HMA's inactivate viruses either by reacting ~ith the,protein coat or with the t nucleic acids.
The method of the invention can alr~o be used to inactivate pathogens present in bodily fluids other than blood, and to disinfect medical instruments and analytical equipment that have come into contact with potentially contaminated biological samples, such as blood. Sienilarly~ the method of the invention can be used to disinfect blood samples that are not intended for subsequent administration to an individual, but rather, are intended for subsequent chemical analysis. Cther possible applications of the invention are apparent to those skilled in the art.
The following examples are illustrative only and 2~ should in no way limit the scope of the present invention.
EXAMP~E 1 Five microliters of T4 (American Type Culture Collection No. 11303-~4~ virus stock and 14 ~L of blood plasma were added to each of four tubes. 1 ~L
of a 50~ stock solution of sodium ~ W097~2028 ~ 9 8 ~ PCT~Sg6/lll52 hydroxymethylglycinate ~International Specialty Products, Bound Brook, New ~ersey) was ~ded to a first tube, 1 ~L of a 10 mg/mL stock solution of-diazolidinyl urea (a bactericide) was added to a second tube, 1 ~L of a 10 mg/m~ stock solution of imidazolidinyl urea ~a bactericide) was added to a third tube, and 1 ~L of Phosphate Buffered Saline (PBS) was added to the fourth tube. The mixtureS
were incubated at room temperature for 1 hour before sampling, diluting, mixing with host cells in sof-t agar and pouring onto solid medium. After overnight incubation at 37 C, the plaques were counted. The results are summarized below in T~3BE I.
TABLE I
15S ~ pl- Inhibitor 2 ' ~t on PFIJ~ (Plaque in Mixture Forming ~nita) 1~dh~. 'hylglycinate2.5~ 22xlO~
2Diazo].idinyl nrea0.5 mg~mL14xlO~
3Tmidazolidinyl urea0.5 mg/mL42xlO~
4 None 0 66xlO~
The above results show that, whereas the bactericides diazolidinyl urea and imidiazolidinyl urea were ineffective at the above-indicated concentrations at inactivating T4 virus, hydroxymethylglycinate exhibited strong viricidal activity.
Five microliters of T4 virus stock were added to each of fourteen tubes. Fourteen microliters of blood plasma were added to each of seven of the tubes, and 14 ~L of whole blood were added to each of the remaining seven tubes. One microliter of an appropriate hydroxymethylglycinate stock solution was W097l~2028 ~ 5 PCTn~96~111S2 added to each of six of the plasma-containing tubes and to each:of fiiX of the whole blood-containing tubes to gi~e the below-listed concentrations. Qne microliter of P}3S was added to each of the r~ ; ni ng S tubes. After incubation at room t~ e~Lur~ for 1 hour, samples from each tube were taken, diluted, mixed with host cells, and overlaid onto solid medium. After overnight incubation at 37'C, plaques were counted. The results are summarized below in TAS~E II.
TAB~E II
S~mple Eyl~ thylglycinate Plaama PFU
Concentration or Whole ~lood 1 2.5~ Plasma 21x104 2 1.25% Plasma 71x104 lS 3 0.625~ Plasma 70x104 4 0.25~5 Plasma 58x105 0.125~ _ Plasma 36X106 6 0.063~ Plasma 18x107 7 o~ Plasma 60x103 8 2.S% Whole 29x104 Blood 9 1.25~- Whole 50xlO~
Blood 0.625~ Whole 76xlO' Blood 11 0.25% Whole 43x105 ~ Blood 12 0.125% Whole 74xlO~
~lood 13 0.063% Whole 21xlO~
Blood 14 0~ Whole 67xlO~
~lood ~ wo g7~2028 ~ d a ~ 5 PCT~S96/11152 , ~
-2~-As can be seen from the results above, the inactivation of T4 was dependent upon the concentration of hydroxymethylglycinate. A
concentration of 0.625~ hydroxymethylglycinate led to a 4 log decrease in T4 over a period o~ l hour at room temperature.
Two hundred twenty-five microliters of T4 stock were added to each of twelve tubes. 4.75 ~L of whole blood were added to three of the tubes, 4.75 ~L o~
plasma were added to another three of the tubes and 4.75 ~L of PBS were added to still another three of the tubes, the remaining three tubes serving as controls 25 ~L of a 50~ solution of hydroxymethylglycinate were added to each of the tubes, except for the controls. The various mixtures were incubated at room temperature, and at the times indicated below samples were taken, diluted, mixed with h~st cells and overlaid onto solid medium.
After overnight incubation at 37 C, plaques were counted. The results are summarized below in TAB~E
III.
WO 97~2U28 ~ ' PCT/U596~1152 2 ~ 7 0 3 i ~
2' TP~3L~ I
Sample ~r~LVA1--Lhylslyclnate Tn~ An PF~8 Pr-~bnt ~i~e Whole Yes lS minutes 27x107 Blood 5Plasma Yes lS minutes 9xlO' PBS Yes 15 minutes 25xlO' Control Uo lS minutes ~
Whole Yes 30 minutes 9xlO' Blood 10Plasma Yes 30 minutes 23xlO' PBS Yes 30 miuutes 40xlOs Control ~o 30 minute~ ~D
Whole Yes 60 minutes 14xlO~
Blood 15Plasma Yes 60 minutes 25x105 PBS Yes 60 minutes 14xlO' Control ~'o 60 minutes soxlo7 As can ~e. seen from the results above, higher levels of hydroxymethylglycinate are required to ~0 inactivate T4 in blood or in plasma than in buffer solution. As can also be seen, the efficac~ of hydroxymethylglycinate appears to increase as incubation time increases.
Thirty microliters of T4 stock and 465 ~LL of blood plasma were added to each of ten tubes. 5 ~L
of a 50~ stock solution of hydroxymethylglycinate were added to each of eight of the ten tubes to give a final hydroxy-methylglycinate concentration of O.S~. The mixtures were incubated at the temperatures indicated below. At the times indicated below, samples were taken from each tube, diluted, mixed with host cells and overlaid onto solid medium.
W097~20~~ ~31 ~Q~5 PCT~3S96~11152 After overnight incubation, plaques were counted.
The results are summarized below in TABLE IV.
TABLE IV
Tn~h~tinnElr~ slycil~at-- Tnn~h:~t;~n PF~'--5 TiIe PresentTemperature 30 mlnutes Yes 5~C llxlO' 30 minutes Yes 21~C 103~c10' 30 minutes Yes 30~C 16X10 30 minutes Yes 37~C 22Xl03 1030 minutes No 37~C ND
60 minutes Yes 5~C 12xlO' 60 minutes Yes 21~C 23xlO' 60 minutes Yes 30~C < 103 60 minutes Yes 37~C < 103 1560 minutes 3-~o 37~C ôOxlO7 As can be seen from the above results, the inactivation of T4 in blood plasma using 0.5~
hydroxymethylglycinate is temperature dependent.
Significantly greater viral toxicity was seen at 30'C
than at room temperature, and at 5-C only a l log drop in viable virus was observed.
Thirty microliters of T4 stock and 465 ~L of packed red blood cells were added to each of ten tubes. 5 IlL of a 50% stock solution of hydroxymethylglycinate were added to each of eight of the ten tubes to give a final hydroxymethylglycinate concentration of 0. 59~. The mixtures were incubated at the temperatures indicated below. At the simes indicated below, samples were taken from each tube, diluted, mixed with host cells and overlaid onto solid medium. After overnight incubation at 37~C, WO97~28 ~ g~ PCT~Ss~ll152 plaques were counted. The results are su~marized below in T~3LE V.
TABLE V
r~ t;,~ lr~ . ~ ~157lycin~lt T~ PFtl'8 5Ti~3~ nt 30 minutes Yes 5~C 15X1()7 30 miuutes Yes 21~C 20xlO' 30 minutes Yes 30OC llx1oS
30 minutes Yes 37~C 21xlO' 1030 mi3~utes No 37OC ND
60 minute~ Yes 5~C 44xlO' 60 mirlutes Yes 21~C lOxlOs 60 minutes Yes 30OC 24xlO' 60 minutes Yes 37OC c 103 1560 minutes No 37~3c llxlo' A8 can be seen from the above results, the inactivation of T4 in packed whole blood is also temperature ~r~n~nt. At 37~C, over a 5 log drop in viral viability was observed over the time of the experi~ent. At 5~C~ the decrea~3e in viral ~iability was only 2 logs.
Thirty microliters of T4 stock and 440 ~ of blood plasma were added to each of twelve tubes. 30 ~L of a 50~ stcck solution of hydroxymethylglycinate were added ts each of ten of the twelve tubes to give a final hyd~v~y ~thylglycinate concentration of 3~.
The mixtures were incubated at the temperatures indicated below. At the times indicated below, samples were taken from each tube, diluted, mixed with host cells and overlaid onto solid medium.
After overnight incubation at 37~C, plaques were ~ W097~2028 2 ~ ~ 8 ~ ~ 5 PCT~S96111152 counted. The results are summarized below in Table VI.
TABLE VI
T_~h7~ Y ~ hY1g1YCLU~te T~ h~A_ PF~'6 5Tlme Pre~ent ~ - , 30 minutes Yes 0~C 29xlO' 30 minutes Yes S~C 72X10' 30 minutes Yes 21~C 69xlOs 30 minutes Yes 30~C < 103-1030 minutesYes 37~C ~ 103 30 minutes No 37~C ND
60 minutes Yes 0~C 28xlO' 60 minutes Yes 5~C 35xl06 60 minutes Yes 21~C 75X103 1560 minutesYes 30~C < 10' 60 minutes Yes 37~C < 10~
60 minutes No 37~C 117X107 As can be seen by comparing the above results to those obtained in Examples 4 and 5, 3~6 hydroxymethylglycinate possesses greater viricidal activity than does 0.5~ hydroxymethylglycinate. The above results also indicate that viral inactivation by 3~ hydroxymethylglycinate is temperature dependent. For example, a greater than 5 log 2 5 decrease in viable virus was observed when the incubation temperature was increased from 0~C to 30~C
ExAMeLE 7 Fifty microliters of an o~ernight culture of E.
30 coli and 465 1l~ of blood plasma were added to each of ten tubes. 5 I:LL of a 50~ stock solution of hydroxymethylglycinate were added to each of eight of W097~20Z8 ~ ~ PCT~S~11152 2 ~
-2~-the ten tubes to give a final hydroxymethylglycinate concentration of 0.5~. The mixtures were incubated at the temperatures indicated below. At the times indicated below, samples were taken fro~ each tube, diluted, and plated. After overnight incubation at 37~C, colonies were counted. The results are summarized below in TABLE ~II.
TA3~E VII
T77.-.. h~ti~Ar tLrlg1ycimlte Inc7hst~0n Colonles lOTime 7~7re~t ~ rL~
30 minutes Yes 5~C 7X107 30 minutes Ye~ 21~C llx107 30 minutes Ye5 30~C l5XlOs 30 minutes Yes 370c c lo' 1530 minutes ~70 370c ND
60 minute~ Yes 5~C 41xlO' 60 minutea Yes 1l~7C 7xlO' 60 minutes Yes 300C c lo' 60 minutes Yes 37CC c 10' 2060 mirlUtes No 370c 35xlo' The above results indicate that hydroxy~7~ethylglycinate inactivates bacteria, as well as viruses, in blood pla3ma. The above results also indicate that the inactivation is temperature dependent.
Four hundred sixty-five microliters of blood plasma and 270 ~ of T4 stock were added to each of eight tubes. 5 ~ of 50% stock solution of hydroxy~ethylglycinate~ pH 9.0, were added to each of two of the elght tubes. A sample of the aforementioned hydroxymethylglycinate stock solution ~ W097l02028 p~l 9 ~ ~ 8 5 c was adjusted to pH 7.8 with solid sodium phosphate, and 5 ~L of the pH-adjusted hydroxymethylslycinate solution were added to another two of the eight tubes. 5 ~.7. of buffer, pH 9.0/ were added to another two OL- the eight tubes, tlhe re-~ining two tubes serving as controls. The mixtures were incubated at 30'C. At the times indicated below, samples were taken, diluted, mixed with host cells and overlaid onto solid medium. After overnight incubation at 37~C, plaques were counted. The results are summarized below in TABLE VIII.
T~3LE VIII
ADDITIYE ~ 3'1'~.~ TIIDS ~F~s Eydroxymethylglycinate30 minutes 29x103 pE 7.8 .7.7~L~ hylglycinate 30 minutes 18xlO' pX 9.0 Buffer 30 minutes 22x107 pX 9.0 None -- 30 minutes ~
EydroxymethylglycinateG0 minutes c103 pE 7.8 X~dL~I 'hylglycinate 60 minutes lOx103 pE 9.0 Buffer 60 minutes 78x107 pE 9.0 Uone 60 minutes 26x107 EXAMPLE g Each of the hydroxymethyl derivatives listed below was synthesized along the lines described in U.S. Patent No. 4,337,269 by mixing 10 mmoles of the corresponding L-amino acid with lo mmoles of 509 aqueous sodium hydroxide and 10 mmoles of 37~
formaldehyde. After overnight incubation at room W097~2028 ~ PCT~S96/11152 temperature, no free formaldehyde could be detected in any of the reaction mixtures.
~ 65 ~L of blood plasma and 30 ~L of T4 virus were added to each of nine tubes. In addition, to each tube was added 5 ~L of one oE the hYdL u~y,.,ethyl derivatives listed below, the re-~;n;nq tube having no hydroxymeth~l derivative and serving as a control.
The mixtures were incubated at 30-C for 60 minutes.
Samples were then taken, diluted, mixed with host cells and overlaid onto solid medium. After overnight incubation at 37'C, plaques were counted.
The results are summarized below in TABLE IX.
TABLE IX
~ K PE'U' 13 HYdLU~I ~ thylglycine ~103 Hydroxymethyl~l ~n;n~ 14X103 Hydroxymethylaspartate 7xlO~
Hy~Lv~ thyllysine 28xlOi Hydroxymethylornithine 33xlOs HYdLU~I thylproline 36xlOi HydrcXymethylserine 20x103 HydrcXymethylvaline 54xlO3 None 6xlO3 10 ~L of T4 virus stock and 8~ ~L of olood plasma were added to each of seven tubes. l ~I. of an appropriate hydroxymethy].valine stock solution was added to each of three of the plasma-containing tubes and l ~L of an appropriate hydrox-ymethylaspartate WO 97/02û2~ 7 ~ 5 PCTIUS96l11152 stock solution was added to each of another three of the plasma-containing tubes to give the below-listed concentrations. 1 ~LL of a buffer solution was added to the re-~;n;ng tube as a control. A~ter incubation 5 at 30 C for 1 hour, samples from each tube were taken, diluted, mixed with host cells, and overlaid onto solid medium. After overnight incubation at 37~C, plaques were counted. The results are summarized below in TAPLE X.
TAPLE X
lN~l~l~Ok ~uNu~.lL~ATION PFU's HydLu~y,.,ethylvaline O. 259~ 35xlOs Hydroxymethylaspartate 0. 25~ 12xlOs Hydroxymethylvaline 0;1% 42xlOs 15Hydroxymethylaspartate O .19~ 13X106 HYdL~Y. thylvaline 0.05~ 57X106 Hydroxymethylaspartate 0. 05% 20X106 None 0~ 29xlO' T4 virus stoclk and blood plasma were added to each of six tubes in the manner described above.
Hydroxymethyl-o-phosphorylethanolamine was added to each of two tubes, hydroxymethyl-trp-gly-gly was added to each of another two tubes, and a buffer containing no inhibitor was added to each of the remaining two tubes. The mixtures were incubated at 30 C. At the times indicated below, samples were taken from each tube, diluted, and plated. After overnight incubation at 37 C, plaques were counted.
The results are summarized below in TABLE Y.I.
W 0 97102028 ~ ;~ P~rAUS~6/1115 TP~3LE XI
I ihibltor Inccibation PF~'n Period Hydroxymethyl-o- 30 minutes llxlO' phosphoryI~thanolamine ~ydroxymeth~-trp-gly-gly 30 minutes 17xlO~
~ ne 30 minutes 7.~D
Hydroxylethyl-o- 60 minutes 40x103 ~ho~yllolylethanolamine Hyd-u~ ~thyl-trp-gly-gly 60 minutes 18xlO9 None 60 minutes 25x107 EX~MPLE 12 465 ~L of blood plasma, 30 ~L7.. of T4 stock solution were added to each of ten tubes. 5 ~L of a 50~ solution of l~yd~v~y .~ thylaminopropanol were added to each of t~o tube~. 5 ~L of a 50~ solution of hydLu~y~ Lhylpenicillinamine were added to each of another two tubes. 5 ~lL of a 50% ~301ution of hydroxymethylcysteine ethyl ester were added to each of another two tubes. 5 I~L of PB9 were added to each of another two tubes. 5 ~LL of a solution containing L~ r amount9 of NaOH and formaldehyde incubated overnight at room temperature were added to each of the two L. - i n i ng tubes. The mixtures were incubated at 30~C. At the times indicated below, samples were taken, diluted, mixed with host cells and overlaid onto solid medium. After overnight incubation at 37~C, plaques were counted. The results are summarized below in TA-BLE XII.
TP~3LE XII
}~hi~itor T~ ; rn PFU' 8 P-ri ~d }~y~L~ 'ylar~inopropanol 30 minu~.es llXlO' ~ydroxymethylp~niri1~i 'n~ 30 minu-es 8xlo' E~ydLu~ hylcysteine ethy:L 80 minu~es 6x107 ester ~ WO971020~ ~ f 8 ~ 8 5 PCT~S96/11152 Inhibitor Incubation PF~s - P~ri d None m.nu-es ND
~1~A1 jn;7~ formaldehyde ~ m.nu-es ND
HYdL~ hylaminopropanol m.nu-es SxlO~
E.'ydroxymethylr~ni~ill; 'n~ m-nu es 24xIOs 5HydL~ Lhylcysteine ethyl ~o m_nu-es 6x107 ester None 60 minutes 16xlO' ~1~A1 ;ni7~ formaldehyde 60 minutes 14xlO' 10465 IlL of blood plasma, 30 ~L of T4 stock solution were added to each of eight tubes. 5 ,UL of a 50% solution of hydroxymethylcysteine were added to each of two tubes. 5 ~LL of a 50~ solution of hydro-xymethyl-aminophenyl acetic acid were added to each of another two tubes. 5 ~LL of a 5~9~ solution of hydroxymethylaminoethanol were added to each of another two tubes. 5 I~L of a buffer solution serving as a control were added to each of the two Ll i n; ng tubes. The mixtures were incubated at 30 C. At the times indicated below, samples were taken, diluted, mixed with host cells and overlaid onto solid medium.
After overnight incubation at 37'C, plaques were counted. The results are summarized below in TABLE
XIII.
W0 97t02028 ~ r rCT/US96~ 5~ ~
T~3hE XIII
IN~IBITOR ~ ERIOD PFV'9 E}y~lL~ hylcysteine 30 minutes 7x107 llyd~ Lhyl- 30 Ininutes 23x10' 5aminophenyl ~cetic acid lamino- 30 minute~ 12X1o' ethanol None 30 minutes ND
~y~lL~ hyl ~ysteine 60 minutes 34x10' 10l~ydLv~. ''yl- 60 minutes ~3X10 aminophenyl acetic acid HYIL~ -hylamino- 50 minutes 11X1 et ~ ol None 60 minutes 29X10 465 ~ of blood plasma, 30 ~h of T4 stock solution were added to each of eight tubes. 5 ~L of a 50~ solution of hydL~yll~ethylr ~Lopropionylglycina were added to each of two tubes. 5 ~h of a 50~ solution of hydroxymethylmercaptoethylamine were added to each of another two tubes. 5 ~L of a 50~ solution of hydroxymethylaminoethyl hydrogen sulfate were added to each of another two tubes. 5 ~L of a buffer solution ser~ing as a control were added to each of the two i, -i n i ng tubes. The mixtures were incubated at 30 C. At the times indicated below, samples were taken, diluted, mixed with host cells and overlaid onto solid medium. After overnicht incubation at 37 C, plaques were counted. The results are summarized below in TABhE XIV.
~ W097~202~ 5 PCT~S96/ll~s2 '.i,~; ~;
T~3LE XIV
INSIBITOR INCUBATION PERIOD PFU o Hydroxymethylmercapto 30 minutes lSx10 propionylglycine HYdL~A~. hyl- l0 minutes 20xl0 mercaptoethylamine HvdroxymethylaminO- 30 mlnutes 23xl0' ethyl hydrogen sulLate None 30 minutes ND
10HydL~ hylmercapto- 60 minutes 36xl05 propionylglycine Hy~L~.. _th;l- 60 minutes 22x105 mercaptoethylamine HyvL~ -thylamino- 60 minutes 20X105 15ethyl hydrogen suliate None 60 minutes 17x10' EXAMP~E 15 ~ he virucidal activities of hydroxymethyl-p-aminohippurate, hydroxymethylpropargyl-glycine, and hydroxymethyl-o-phosphothreonine, respectively, were tested in the manner descri~ed above. The results are summarized below in T~3~E XV.
TAB~E XV
I.~3IBITOR T~rT7~7 ~T~ PERIOD PF~o 25HyvLv~ thyl-p-amino- 30 minutes <l0' hippurate HYdLV~ ~hylpropargyl_ 30 minutes ~l0' glycine H.ydLv~. 'hyl-o- 30 minutes lx10' 3 Gphospho-threonine None 30 minutes ND
HydLv~.. _thyl-p-amino- 60 minutes c10 hippurate HY~LV~. ~hylpropargyl- 60 minutesc10 35 glycine HydL~.. _thyl-o- 60 minutes cl0' phospho-tùreonine W097/02028 ... ~ PCT~S96/11152 ~ i~ 2 1 '7~0l~l5 ¦ I~LIBITOR ¦ TUrn~T~ PERIOD ¦ PF~
None 60 minute~3 4x107 The virucidal activities oi hydroxymethyl-aminoadipate, hydroxymethyl-o-phosphoserine, and hydroxymethylaminoethylphosphonic acid, respectively were tested in the manner described abo~e. The results are summarized below in TALLE XVI.
TA6Bi3 XVI
Ii~}Bl~OR INC~BATION PFRIOD Pi~0'-Hyd.u~3.. ~Lhylamino-30 min~tes sxl~3 adipate Hyvlv~ 'hyl_o- 30 minutes ~103 rh~crh~~~rine i~y h~ Llly1amino- 30 minUteB 2x103 15ethylrh~crh~ric acld None 30 minutes ~
~y~h~. L' ylamino- 60 minutes 103 adipdte ~y~L~ '- yl-o- 60 minutes <103 20i~ ' ine ~iy~iJv~ BLylamino- 60 minutes c10 ethylph~cFh~r;c acid U-one 60 m1nutes 24x10 EXAMiPL3 17 465 ~L of blood plasma and 30 ~L of T4 were added to each of six tubes. 5 ~L of hydroxymethyl-~h~Cph~ ~thylglyclne were added to each of two tubes, and 5 ~L of hydroxymethylmethylhydantion were added to each of another two tubes. 5 ~I- of buffer were added to each of the remaining two tubes. The W097~20~ ~I 9~ PCT~59~11152 mixtures were incubated at 30 C. At the times indicated below, samples were taken, diluted, mixed with host cells and overlaid onto solid medium.
After overnight incubation at 37 C, plaques were counted. The results are summarized below in TA~3LE
XVII.
T~3LB XVII
I~hibltor T~l.h.~; n~ pp~ J
P-riod EPdL~. hylrhnQrh~nn_ 30 minutes 28xlO~
methylglycine Hydroxymethylmethylhydantion 30 minutes i3xl06 None 30 minutes ND
Hydroxymethylphn.Qrhn~n 60 minutes ~ lo~
methylglycine H~dL~. thylmethylhydantion 60 minutes 37x105 None 60 minutes 25X106 EXAMP~E 18 The virucidal activities of hydroxymethylglycine 7-amido-~-methylcoumarin, hydroxymethyl-vinylglycinate, hydroxymethylfolic acid, hydroxymethyltaurine and hydroxymethyl-aminoethyl trimethyl ammor.ium chloride, respectively, were tested.in the manner described above at the concentrations indicated below. The resulte are summarized below in TAB~E XVIII
W0 97~2028 ~ 3'i~ ~ PCT/US96111152 S
: -36-TA~3~E XVIII
I~hibltor Inh~bitorPF-7'~.
conc-ntration NYdL~ rlglycine-7-amido-4- 0~5~ 12xloS
methylcoumarin NY~L~ h~lyinylg1yCinate O.SL, ~ 103 N~dL~ hylfolic acid 0.5% ~ 103 NY~LJ~ hyltaurine 0.55S 25xlo' NY~L~. hyl ~ mo~hyl trimethyl o~s% 40xlo' ammonium chloride NydL~r.~._LI~ylglycine-7-amido-4- 0.2s~ 21x105 methylcoumarin ~ydu~l. hyl~inylglycinate 0.25~ 88xl0' N~dL~. hylfolic acid 0.2s% ~ 103 NYdL~r. hyltaurine 0.2s~ 22xloS
XY;L~ hylaminoethyl trimethyl 0.2s~ 56xl05 ammonium chlor3,de ~y~lL~ l.rlglycine-7-amido-4- 0.13~ l9X105 methylcoumarin Rydroxymethyl~inylglyCinate o.l~ 67xl0' ilydL~ lfOlic acid Q.l~ lOXlO~
IIYdL~ thyltaurine o.l~ 30x105 -hylaminoethyl t3imethyl 0.1~ 38xlo~
ammonium chl~oride ~one - lsx1o3 EXl~MPLE 19 The virucidal activities of hydroxymethylglycinate, hydroxymethylaminoadipate, hydroxymethylaminoethyl.phosphonic acid, 3nd hydrox,vmethyl-o-phosphoserine, respectively, were tested in the manner described above at the con-centratione indicated helow~ The results are summarized below in TABLE XIX.
W097~2028 ~ 9 ~ PCT~S96111152 -3,~-TAB~E XIX
InhibitorInhibitor PF~ 5 Conc-ntration Hydroxymethy1glyCinate 0.25~ 9xlOs HYIL~, Lhyl. 'nn~S1rRt~ 0.25~ 14x10 EydL~ ulylaminoethylrhnsrhnni~ 0.25~ 22x105 acid H~dL~. ~hy1-o-l.l7.~ ~.ine 0.25t 32xlOs Hyd~ , 'hylglycinate 0.1~ 16xl05, Eydlv~."_t¦lyl; nnR~;rRt~ 0.1% 20x10 0 Hydroxymethylalllinoethylrhncrhnnic 0.1~6 7X106 acld Hyd~ ' hyl-o~ mSllln~ ine 0.li; 14X106 Hydroxymethylg1ycinate 0~055 2xl06 Hydroxymethyl 'nnR~irRt~ o,oS~ 7xlO' lS Hyd~ hylaminoethylrhnsrhnnic O.OS~31x10 acid Hydroxymethy1-o-~h~h~ Line O.OS~ 20X10 None - SxlO' The virucidal activities of hydroxymethylphospho-nomethylglycinate and hydroxymethylglycinamide, respectively, were tested in the manner described above at the concentrations indicated below. The results are summari2ed below in TABLE XX.
W097~20~ ~ a ~ ~ PCT~596/11152 nh oi tor Inhib~ tor PF17~ n n i- rA r i nn Hydroxymethylrhncrhnnn_ 0, St ND
methylglycinate Xydroxymethylglycinamide o.s~ 99xlQ~
Xyd.~ , thylrhncrhnnn- 0.25t 49xlO~
methylglycinnte Hy~ Lhylglycinamlde 0.25t l5xlO~
Xy~ . hylrhnsrhnnn- 0.1% 34X106 methylglycinate Xyd~ . thyl~lycinzlmide O.1~ 72xlO~
XY~ Ylrhnqrhnnn_ 0.05% SSxlO~
methylglycinate Xyd~ hylglycirlamide 0.05~
None - SSxlO' EXAMP~E 2l The virucidal. activities of hydroxymethylglycinate, hyd~y~ thy~m i n~hippurate, hydroxymethylpropargylglycine and hydroxymethyl-o-phosphothreonine, respectively, were tested in themanner described abo~e at the c~nc~ntrations indi-cated below. The results are summari~ed below in TAB~E XXI.
W097/02028 21 ~85 PCT/US96/11152 -3~-TABLE XXI
Inhihitor Inhibitor P
C~.~c~..L~tion HydL~ Lhylglyclnate 0.25'~ lSxlO' HYdL~" 3 Lhylaminohippnrate o.2SS6 54xl06 Hyd~ . hylpropargylglycine 0.259f 27x103 Hydroxymethyl-0-~ h~h~3.,.~ine 0.2S'6 lOxlO~
Hy~ Lhylglycinate O.lt 7xlO~
Hyd~ . 'hyl: 'n~hirp~r~ F. 0.1~ SX1O~ _ HydL~ . hylpropargylglycine 0.15~ 41xlOs Hydroxymethyl-0-rhnerh~3threonine 0.1~ 36xlO' HydL~ hylglycinate 0.056s 43x103 Hy~lL~ Lhyl: n~hirrl~r~r~ 0.051: 44Xl06 Hydroxymethylpropargylglycine o.OSt 29xlOC
HydLw~. '' yl-0-phosphothreonine O.OSt lSxlOs None - 32x107 The virucidal activities o~
hydroxymethylthreonine, hydroxymethylphos-phothreonine, hydroxymethylserine and hydro-20 xymethylphosphoserine, respective~y, were tested inthe manner described above at the concentrations indicated below. The results are summarized below in TABLE XXII .
W 0 9~2028 ~ 8 5 rCT/US96/11152 ~ TABLE XXII
I~ibltor Inh~bitor PF~7 EY~L~I. thylthreonine 0.25~ a~X10 dydroxyLnethylphoEphothreonine 0.25t 75xlO~
~;Y~L~/~ thylserine 0.2s~ 14xlO' Hydroxymethylrh~crhne~rine 0.25~ 42xlOs Hydroxymethylthreonine 0.1~ 45xlO~
HY~ILVA~, hyl~rh~ h~threonllle O~l~o 30xlOs Xy~.v~ thylserine 0.1~ 22Y10 HY~L~ tX~ h~ 7-~ine 0.17, 21X10 XyJ~ . chylthreonine O.05~ SX10C
HY.1L~, ~hr1Ph~~l10t}L.~I.1ne 0.05~ 43X107 XY~L~/ "hr1ECrine O.OS'L. 17X107 HY~L~. ChY ~.1.. _L';1'~'._Line 0.057, 51X10' 1~ None 42X107 EXPjMP LE 2 3 The virucidal activities of hydroxymethyl-MTh glycine, hydr~yl~ethyl-l-amino-l-cyclopropane carboxylic acid, hydroxymethyl-d,1~2-aminophosphonopropionic acid, hydroxy-methyl-p-aminobenzoic acid, hydroxymethylamino-b~tyrolactorle, hydroxymethyl-d,1--L~minoph~sphonobutyric acid, hydroxymethylaminopyrazole carboxylic acid, hydroxymethylazetidine carboxylate and hydroxymethyldiaminobutyric acid, respectively, were tested in the manner described above at the concentrations indicated below. The results are sumwarized below in TABLE XXIII.
W 0 97/02028 ~ PCTnJs96/lll52 -41~
TA;3LE XXIII
Inhibitor InhibitorPFiJ's -Cono_..LL~.Lion ayd-u~J. 'hyl-MTH-glycine O.S't 13X106 Hydroxymethyl-l-amino-l- o 5~ 17xlO' cyclopropane carboxylic acid HydLu~y~ LIIyl-d~1-2- 0.S~ c 10' I ' n~ hnb~ u~ionic acid HydLu~y.. _Lhyl-p-l nnh''n7OiC O.55~ sxlo3 acid Hyd-u~;.. _Lhylaminobutyrolactone 0.5~ 30xlO~
HydLu~.. _Lhyl-d,l- O.S~ c 10' ~m1n..l.l..~.,1.,.,.~.i...lyLiC acid Hydroxymethylaminopyrazole O.Sl 45xlO' i5 Hydroxymethyl azetidine 0. St 7xlO' carboxylate Hydroxymethylril: nnh~yric acid O.Ssc ~ 10~
HydLu~/ Lhyl-MTH-glycine 0.25~ lsx107 Hydroxymethyl-l-amino-l- 0.25~ 18xlO' cyclopropane carboxylic acid HydLu~J.. _Lhyl-d,1-2- 0.25~ llxlO' ~ iOniC acid HydLu~J, 'hyl-p nnh~n7~1c 0.25~ 30xlO' acld Hyd~u~.. _LLylaminobutyrolactone 0.255. ND
Hydroxymethyl-d,1- 0.25~ c 10' ".. l,1\.. ~l.h.. :uLyLiC acid Carboxyïic aciadmin~PYrazole 0.25% 21xlO~
Hydroxymethyl azetid-n,e 0.25~ 16xlO' carboxylate HydLu~.. Lhylri; nnh-l~yric acid 0.25~ 40xlO' Hydroxymethyl-MTH-glycine O.li 18xlO' Hydroxymethylglutamate 0.2si~ 60xlO' HydLu~",_Lhy1m~hlnn;n~ 0.25~ 68xlOs HydLu~ hylserine 0.25~ 25xlO' Hy~L~J~-Lhylglutamate 0.1$ 12xlO~
Hydroxymethyll ~hinnllle O,l~Lo 28X106 Hydroxymethylserine 0.1~ 27xlOs W 0 97~2028 ~ ~ rCT/US96/11152 ~ 3 q~85 -42-In~ibitor Inhibitor PFV'~
Concentrution Hydroxymethylglutam~ate O.OSt 9X10' Hydroxymethy1mpthimninp o 05~ 7xlO' Hy~ , 'h~yl~erine 0.05t 7xlO~
~one - 9xlO' 8~ /~L of blood plasma and 10 ~L of T4 stock solution were added to each of ten tubes. 1 ~ of an appropriate hydroxymethylglutamate stock solution was added to each of three tubes to give the concentrations indicated below. 1 ~L of an appropriate hydroxymethylmethinnlnp ~tock solution was added to each of another three tubes to give the concentratians indicated below. 1 ~L of an appropriate hydroxymethylserine stock solution was added to each of another three tubes to give the concentrations indicated below. 1 ~L of buf~er was added to the r~ -1n;ng tube, which ~erved as a control. The mixtures were incubated at 30-C. After 1 hour, sam.~ples were taken, diluted, mixed with host cells and o~erlaid onto solid medium. After overnight incubation at 37 C, plaques were counted.
The results are summarized below in T~3LE XXIV.
W097/02028 ~1 9~ 5 PCT~S96~11152 TABLE XXIV
Inhibitor InhibitorPF~'s EYdLU~Y~ ~hylglutamate 0.25'L.60xlO~
Hydlu~. -hy1methionine 0.25%65xlOs HY~LU~. ''ylserine 0.255, 25xlO~
Hydroxymethylglutamate 0.1~12X10~
HY~LU~ 'hylm~hinnin~ O.lt 28xlO' HY~LU~Y thylLerine O ~ lt 27xlOs HYdLU~ ~hylglutamate 0~0S~L.9xlO' Hydroxymethyl~6~hinn;nP 0 OS'L 7xlO~
HydLu~ thylserine 0.05~ 7x106 None - 9xlO' 89 ~L of blood plasma and lO ~L of T4 stock solution were added to each of ten tuhes. l ~L of an appropriate hydroxymethylserine stock solution was added to each of three tubes to give the concentrations indicated below. l ~B of an appropriate hydroxymethyl-~-alanine stock solution was added to each of another three tubes to give the concentrations indicated below. l ~L of an appropriate hydroxymethylglycine stock solution was added to each of another three tubes to give the concentrations indicated below. l ~L of buffer was added to the r~ ;ning tube, which served as a control. The mixtures were incubated at 3G C. After l hour, samples were taken, diluted, mixed with host cells and overlaid onto solid medium. After overnight incubation at 37 C, pla~ues were counted.
The results are summarized below in TABBE XXV.
W097~2028 ~ PCT~S9~11~2 TA33T~E XXV
Inhibitor IDhlbitor P~5'5 }}yd.~ 'hyl~erine 0.25Y~ 8xlO' Hydroxymethyl-B-alanine 0.25~ lSxlO~
Hy.l~ glycine 0.259s 9xlO' H~ hyls~rin~ 0 . 156 14xlOs Hy~ hyl-B-alanine o.l~ 83xl05 Hydroxymethylglycir~e o.lt 8xlO' HY~L~. 'ylserine o.os~21X106 HY~L~. hyl-6-alanino o.os~3~xlO~
HydL~.r.c~11y1~lYCine o.os~ 26xlOs None - 1 2xl 0 ' The following is a listing of virucidal activities observed by the present inventors for a variety oi- hydroxymethyl derivatives ~virucidal activity being expressed in terms o~ PFIJ~s~:
Hydroxymethylglycine Hydroxymethylphosphonomethylglycine 20 Hydroxymethyl-p-~m; n~hi ppurate Hydroxymethylpropargylglycine ~ydroxymethyl-o-pho~phoserine HydrDxymethylaminoethyl-phosphonic acid Hydroxymethylleucine ~5 Hydroxymethyl-~-alanine Hydroxymethylcysteine Hydroxymethylphenylalanine ~ W097/0~8 7 I f ~ C, ~ 5 PCT~Sg6111~52 lo3 Hydroxymethylaminophenylacetic acid ~ydroxymethyl-o-phosphorylethanolamine Hydroxymethylalanine Hydroxymethylserine Hydroxymethylvaline Hydroxymethylmethionine Hydroxymethylglutamate Hydroxymethylaspartate Hydroxymethyllysine Hydroxymethylproline Hydroxymethylmercaptopropionylglycine Hydroxymethylmercaptoethylamine Hydroxymethylaminoethyl hydrogen sul~ate Hydroxymethylaminoethanol Hydroxymethylpenicillamide Hydroxymethylhydantior.
2G Hydroxymethylornithine ~ydroxymethylthreonine Hydroxymethylcysteine Hydroxymethylaminopropanol W097/02028 ~ PCT~5~1111~2 2 1 ~0~
-4Ç-1~7 Hydroxymethyluridine Hydroxymethylphth~7; m; de Dimethylurea S Hydroxymethylcysteineethyl ester Hydroxymethylleucinamide Hydroxymethylarginine Hydroxymethyltyrosine 10~
~ Hydroxymethyldeoxyuridine 4-hydroxymethylimidazole 6-hydroxymethylpter~n Hydroxymethylacrylamide Hydroxymethylcytosine 1.S Hydroxymethyl-Ç-methyluracil Hydroxymethylnico~;n~mi~7~
Hydroxymethyl-trp-gly-gly Hydroxymethylglutamine Diazolidinylurea Imidazolidinylurea ~ W097102028 2 ~ q 8 0 8 ~ PCT~S96/11152 Kinetic Studies A kinetic model was developed to mon tor the complexation between hydroxymethylglycinate (HMGi and a virus. In the kinetic model it was assumed that the reaction of one molecule of HMG with a virus was sufficient to inactivate the virus. Therefore, the overall reaction can be expressed by the simple rate law:
HMG + V ~ VHMG
where "V" is a virus. A large excess of HMG was present in the reaction conditions and therefore a pseudo first order equation could be used.
Furthermore, it was assumed that in a blood bag, no mass transfer effects exist which would affect the reaction kinetics.
It was found that the rate of inactivation follows the model prediction and that the virus can be inactivated in plasma, whole blood and red blood cell concentrate (RBC). The rate is also increased as the temperature of the reaction conditions is increased.
Inactivation of SV4-0, Reovirus, Porcine Parvo virus and Polio virus were demonstrated. For SV4-0, Reovirus and Polio virus, the number of viable cells ~ 25 was reduced below detectable limits. Plasma hemoglobin, potassium, sodium, 2,3-~PG, ATP and lactate were unchanged with reference to a control when treated with HMG at a final concentration of 1600 ppm.
W097~20tX ~ PCT~Sg~lllSt 2 ~ 8 ~ 8 5 The e~bodiments of the present invention recited herein are intended to be merely exemplary and those skilled in the art will be able to make numerous variations and modifications to it without departing from the spirit of the present invention. All such variations and modificstions are intended to be within the scope of the present invention as defined by the claiws appended hereto.
Claims (19)
1. A method of inactivating a microorganism in a biological fluid, comprising contacting said biological fluid with an effective amount of a microorganism-inactivating hydroxymethylamine (HMA).
2. The method of claim 1 wherein said microorganism is selected from the group consisting of bacteria, viruses, yeasts and molds.
3. The method of claim 2, wherein said microorganism is a bacterium.
4. The method of claim 2, wherein said microorganism is a virus.
5. The method of claim 1, wherein said HMA is a compound of Formula (I) wherein:
R is chosen from the group consisting of hydrogen, alkyl, aryl, substituted alkyl, substituted aryl;
R1 is chosen from the group consisting of acid-, amide-, hydroxy- or mercapto-functional alkyl;
acid-, amide-, hydroxy- or mercapto-functional aryl; acid-, amide-, hydroxy- or mercapto-functional substituted alkyl; and acid-, amide-, hydroxy- or mercapto-functional substituted aryl;
or R and R1 may be joined together to form an acid, amide or hydroxy-functional heterocyclic structure.
R is chosen from the group consisting of hydrogen, alkyl, aryl, substituted alkyl, substituted aryl;
R1 is chosen from the group consisting of acid-, amide-, hydroxy- or mercapto-functional alkyl;
acid-, amide-, hydroxy- or mercapto-functional aryl; acid-, amide-, hydroxy- or mercapto-functional substituted alkyl; and acid-, amide-, hydroxy- or mercapto-functional substituted aryl;
or R and R1 may be joined together to form an acid, amide or hydroxy-functional heterocyclic structure.
6. The method of claim 5, wherein said acid functional group is selected from the group consisting of carboxylate, phosphate, phosphonate, sulfate and sulfonate.
7. The method of claim 6, wherein said acid functional group is a carboxylate.
8. The method of claim 5, wherein said hydroxymethylamine is selected from the group consisting of hydroxymethylglycinamide, hydroxy-methylpenicillinamide, hydroxymethylleucinamide, hydroxymethylacrylamide and hydroxymethylnicotinamide.
9. The method of claim 5, wherein said hydroxymethylamine is selected from the group consisting of hydroxymethylglycine, hydroxymethyl-phosphonomethylglycine, hydroxymethyl-p-aminohippuric acid, hydroxymethylpropargylglycine, hydroxymethyl -o-phosphothreonine, hydroxymethylaminoadipic acid, hydroxymethyl-o-phosphoserine, hydroxymethylamino-ethylphosphonic acid, hydroxymethylleucine, hydroxymethyl-.beta.-alanine, hydroxymethylcysteine, hydroxymethylfolic acid, hydroxymethylaminophosphono-butyric acid, hydroxymethylphenylalanine, hydroxymethylaminophenylacetic acid, hydroxymethyl -o-phosphorylethanolamine, hydroxymethylalanine, hydroxymethylserine, hydroxymethylvaline, hydroxymethylmethionine, hydroxymethylglutamic acid, hydroxymethylaspartic acid, hydroxymethyllysine, hydroxymethylproline, hydroxymethylmercaptopropioryl-glycine, hydroxymethylmercaptoethylamine, hydroxymethylaminoethyl hydrogen sulfate, hydroxymethylaminoethanol, hydroxymethyl-penicillamine, hydroxymethylhydantoin, hydroxymethylornithine, hydroxymethylcysteine, hydroxymethylaminopropanol, hydroxymethyldiethanol-amine and salts thereof.
10. The method of claim 9, wherein said hydroxymethylamine is hydroxymethylglycine or a salt thereof.
11. The method of claim 1, wherein said hydroxymethylamine and biological fluid are combined to produce a final concentration of hydroxymethylamine in said biological fluid of approximately 0.05% - 3.0 % by weight.
12. The method of claim 1, wherein said biological fluid and hydroxymethylamine are contacted for a period of time from 0.5 hours to 4 hours.
13. The method of claim 1, wherein said biological fluid is contacted with a hydroxymethylamine at a temperature of between about 4° C and about 30° C.
14. The method of claim 1, wherein said biological fluid is whole blood or blood components.
15. The method of claim 14, wherein said blood components are selected from the group consisting of red blood cells, red blood cell concentrate, platelets, platelet concentrate, platelet rich plasma, platelet poor plasma, source plasma (plasmaphoresis plasma), fresh frozen plasma and plasma proteins.
16. The method of claim 1, wherein said biological fluid is selected from the group consisting of lymph, cerebrospinal fluid, semen and saliva.
17. A method of processing a biological fluid intended for administration to an individual in need thereof, said method comprising the steps of:
(a) treating the biological fluid with an effective amount of a pathogen-inactivating hydroxymethylamine, thereby producing a treated biological fluid; and (b) after said treating step, removing free hydroxymethylamine from the treated biological fluid.
(a) treating the biological fluid with an effective amount of a pathogen-inactivating hydroxymethylamine, thereby producing a treated biological fluid; and (b) after said treating step, removing free hydroxymethylamine from the treated biological fluid.
18. A method of treating an individual in need of a biological fluid, said method comprising the steps of:
(a) treating the biological fluid with an effective amount of a pathogen-inactivating hydroxymethylamine, thereby producing a treated biological fluid; and (b) administering the treated biological fluid to the individual in need thereof.
(a) treating the biological fluid with an effective amount of a pathogen-inactivating hydroxymethylamine, thereby producing a treated biological fluid; and (b) administering the treated biological fluid to the individual in need thereof.
19. A method of treating a biological fluid, said method comprising combining an effective amount of a virus-inactivating hydroxymethylamine with said biological fluid, whereby at least about a 10-fold reduction in plaque forming units of virus is realized.
Applications Claiming Priority (4)
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US49647895A | 1995-06-29 | 1995-06-29 | |
US08/496,478 | 1995-06-29 | ||
US52665895A | 1995-09-11 | 1995-09-11 | |
US08/526,658 | 1995-09-11 |
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CA002198085A Abandoned CA2198085A1 (en) | 1995-06-29 | 1996-07-01 | Inactivation of pathogens using hydroxymethylamines |
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EP (1) | EP0792146A1 (en) |
JP (1) | JPH10506927A (en) |
AU (1) | AU7717396A (en) |
BR (1) | BR9606454A (en) |
CA (1) | CA2198085A1 (en) |
WO (1) | WO1997002028A1 (en) |
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US5691132A (en) | 1994-11-14 | 1997-11-25 | Cerus Corporation | Method for inactivating pathogens in red cell compositions using quinacrine mustard |
US6093725A (en) * | 1997-01-06 | 2000-07-25 | Cerus Corporation | Frangible compounds for pathogen inactivation |
US6514987B1 (en) | 1997-01-06 | 2003-02-04 | Cerus Corporation | Frangible compounds for pathogen inactivation |
DE69826521T2 (en) | 1998-01-06 | 2005-09-29 | Cerus Corp., Concord | PROCESS FOR QUENCHING PATHOGEN INACTIVATORS IN BIOLOGICAL MATERIALS |
WO2001058496A1 (en) * | 2000-02-11 | 2001-08-16 | Allied Therapeutics Limited | System for the extracorporeal treatment of blood |
US6635679B2 (en) * | 2001-05-14 | 2003-10-21 | Akzo Nobel N.V. | Methods and compositions for inactivating viruses |
EP1469729B1 (en) | 2002-01-18 | 2015-10-21 | Lonza AG | Virucidal disinfectant |
DK1838355T3 (en) | 2004-10-29 | 2013-10-28 | Cerus Corp | Improved quenching methods for erythrocyte inactivation method |
AU2009233807B2 (en) | 2008-04-09 | 2014-11-27 | Cerus Corporation | Improved quenching methods for red blood cell pathogen inactivation |
CN101885740B (en) * | 2009-05-11 | 2015-07-01 | 李坚 | New preparation method of herbicide glyphosate |
CN110317230B (en) * | 2018-03-30 | 2023-11-07 | 上海昇悦医药科技有限公司 | Phosphate derivatives and use thereof |
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US3239415A (en) * | 1963-01-14 | 1966-03-08 | Upjohn Co | Methods of reducing virus titers in animals with adenine derivatives |
DE2135542A1 (en) * | 1971-07-16 | 1973-01-25 | Boehringer Mannheim Gmbh | Virustatic benzthiazole-2-thione derivs - active against both dna and rna viruses |
SU490824A1 (en) * | 1974-08-27 | 1975-11-05 | Институт полиомиелита и вирусных энцефалитов АМН СССР | Method of inactivating infectiousness of viruses |
US4337269A (en) * | 1979-07-23 | 1982-06-29 | Sutton Laboratories, Inc. | Preservative compositions |
JPS62195304A (en) * | 1986-02-24 | 1987-08-28 | Dainippon Ink & Chem Inc | Industrial antiseptic |
US4833165A (en) * | 1987-10-07 | 1989-05-23 | Louderback Allan Lee | Method of inactivating HTLV-III virus in blood |
-
1996
- 1996-07-01 WO PCT/US1996/011152 patent/WO1997002028A1/en not_active Application Discontinuation
- 1996-07-01 BR BR9606454A patent/BR9606454A/en not_active Application Discontinuation
- 1996-07-01 AU AU77173/96A patent/AU7717396A/en not_active Abandoned
- 1996-07-01 JP JP9505265A patent/JPH10506927A/en active Pending
- 1996-07-01 EP EP96940235A patent/EP0792146A1/en not_active Withdrawn
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WO1997002028A1 (en) | 1997-01-23 |
AU7717396A (en) | 1997-02-05 |
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